The biocatalytic reduction of oximes may offer a potential alternative route to primary amines. However, previous studies on biocatalytic oxime reduction using α-oximo-β-keto ester substrates led to an amine intermediate which spontaneously dimerized to the corresponding pyrazine. In this study, to access the primary amine, the reduction of α-oximo malonic esters catalyzed by ene-reductases (EREDs) was explored as a promiscuous activity. Thus, a panel of 14 oximes activated by two adjacent ester moieties was evaluated, employing both wild-type and engineered EREDs. The reduction of these substrates indeed led to the corresponding stable primary amine functionality. Moreover, the unprecedented ERED-catalyzed reduction of the nitro functionality of dimethyl nitromalonate and a hydrazone functionality to the corresponding amines was demonstrated. The nitro reduction is proposed to proceed via the oxime and subsequently imine intermediates in analogy to the reduction of the oxime moiety. These results highlight the synthetic potential of EREDs in the reduction of diverse nitrogenous functionalities toward valuable amine compounds.

Ene reductases known for the reduction of C═C double bonds were shown to reduce C═N bonds, thus (a) selected oximes bearing two ester groups attached to the oximo moiety were reduced to the aliphatic amine moiety, as well as (b) a nitro group and a (c) hydrazone moiety was accepted as well.

doi: 10.1002/cctc.202501696

Strictosidine synthase (STR) is inactive toward N1-methyltryptamine due to steric clashes in the active site that raise reaction barriers. Computational insights guided the use of short-chain aliphatic aldehydes to relieve these clashes, enabling the asymmetric enzymatic synthesis of N9-methyl-tetrahydro-β-carboline derivatives.

Strictosidine synthase (STR) catalyzes in nature the enantioselective Pictet–Spengler condensation of secologanin and tryptamine to form (S)-strictosidine, a key tetrahydro-β-carboline intermediate in the monoterpene indole alkaloid biosynthesis. Extensive studies have revealed that STR exhibits a broad substrate scope, being capable of accepting short-chain aliphatic and aromatic aldehydes and tryptamine derivatives with substitutions at different carbon positions. However, the activity toward N1-substituted tryptamine derivatives remained unexplored. To address this gap, in the present study, molecular dynamics simulations and quantum mechanical calculations were performed to identify the reasons responsible for the previously reported inability of STR in catalyzing the reaction of 1-methyltryptamine with secologanin. It was revealed that this inactivity originates from kinetically prohibitive catalytic steps, caused mainly by the steric clashes in the active site introduced by the N1-methyl group, rather than substrate binding limitations. Guided by the structural insights, short-chain aliphatic aldehydes were predicted to alleviate these steric constraints, supported by calculated feasible reaction barriers. Experimental validation confirmed this prediction, enabling the successful asymmetric synthesis of multiple N9-methyl-tetrahydro-β-carboline derivatives. This work not only advances the fundamental understanding of STR catalysis but also establishes a combined computational-experimental strategy for exploring and extending the enzyme substrate scope.

doi: 10.1002/cctc.202501813

An acyltransferase from Chromobacterium sphagni (CsATase) was identified that catalyzes the regioselective formylation of resorcinol substrates. The formylation of substituted resorcinol derivatives yielded mono-formylated products with up to 99% conversion and up to 74% isolated yield. The structure of CsATase was elucidated by X-ray crystallography, providing insight into its active site.

Although aromatic formylation reactions are highly valuable from a synthetic perspective, a biocatalytic version has not yet been reported. Here, the cofactor-independent multimeric three-component acyltransferase from Chromobacterium sphagni (CsATase) was identified to enable the nonnatural promiscuous regioselective C-formylation of polyphenolic substrates, especially resorcinol derivatives, and thus extending the reaction scope of acyltransferases. Formylation of 4- and 5-substituted resorcinol derivatives gave access to regioselectively mono-formylated products with up to 99% conversion and up to 74% isolated yield. Formylation of phloroglucinol led to the di-formylated product with 99% conversion, outperforming chemical methods. Structural analysis of CsATase by X-ray crystallography provided insights into its active site.

doi: 10.1002/anie.202519387

Carboxylic acids are central building blocks in fine chemical synthesis. Although the direct oxidation of primary alcohols to carboxylic acids appears straightforward from a retrosynthetic perspective, it is often associated with significant challenges. In this Perspective, we discuss concepts of one-pot approaches for the oxidation of primary alcohols to carboxylic acids. These approaches are grouped into chemical and biocatalytic concepts, which are structured according to the stoichiometric oxidant employed. The evolution of these methods is traced from traditional chemical oxidations to modern catalytic and biocatalytic strategies, underscoring the parallel shift in oxidant selection and the resulting improvements in selectivity, practical utility, and sustainability.

doi: 10.1021/jacsau.5c01452

Amide formation represents a key reaction in pharmaceutical synthesis, for which more environmentally friendly methods are sought. In Science, Gao et al. report a biocatalytic oxidative strategy exploiting a redesigned aldehyde dehydrogenase that allows the coupling of aldehydes with amines at the expense of an oxidant [NAD(P)+, molecular oxygen].

doi: 10.1016/j.chempr.2026.102994

Herein, we report a synergistic synthetic strategy to integrate photoredox catalysis and biocatalysis in a one-pot/two-step linear cascade synthesis of enantiomerically pure amines starting from racemic carboxylic acids utilizing visible light and transaminases. This transformation was achieved via quantitative decarboxylative oxidation (>99% conv.) of racemic aryl-alkyl carboxylic acids followed by stereoselective reductive amination of the corresponding dehomologated carbonyl intermediates. The initial photocatalytic step employs blue LED irradiation (427 nm) and molecular oxygen (O2) as the terminal oxidant in the presence of 2.5 mol % of sodium anthraquinone-2-sulfonate (SAS) as a water-soluble, metal-free photocatalyst, operating in an aqueous medium containing 2% v/v of acetonitrile (MeCN) or dimethyl sulfoxide (DMSO) as the respective cosolvents. The subsequent transaminase-catalyzed reductive amination of the in situ-generated ketones furnished the desired optically active amines in a stereocomplementary manner with enantiomeric excesses ranging from 93% to 99.9% and up to >99% conversions after two steps. Among the tested substrates, three representative nonsteroidal anti-inflammatory drugs (NSAIDs), including ibuprofen, flurbiprofen, and naproxen, were successfully transformed into dehomologated NSAID-derived nonracemic amines with 62–>99% ee, respectively. Furthermore, upscaling of the photobiocatalytic process employing 2-(naphthalen-1-yl)propanoic acid (1.0 mmol) as the substrate enabled the synthesis of (R)-1-(naphthalen-1-yl)ethan-1-amine with 92% product formation and >99% ee. Finally, a one-step transformation of the aforementioned amine using a recently developed betaine-based Mitsunobu reagent furnished the enantiomerically pure calcimimetic drug cinacalcet (>99% ee) in 76% isolated yield, without the need for transition metal catalysts and/or hazardous and flammable hydrogen gas.

doi: 10.1021/acsomega.5c10958

Merging photocatalysis with biocatalysis has recently emerged as a powerful strategy in chemical synthesis. Whereas photocatalysis enables the generation of reactive intermediates under mild conditions, biocatalysis provides exceptional selectivity with minimal byproducts. Herein, we report a one-pot/two-step photo-biocatalytic oxidation–reductive amination cascade that transforms α-methyl terminal alkenes into enantiomerically pure amines. In the initial step, 1,1-disubstituted (hetero)aryl alkyl olefins underwent oxidative cleavage to prochiral ketones using molecular oxygen under blue-light irradiation (427 nm) with 9-fluorenone as a transition-metal-free photocatalyst in dimethyl sulfoxide (DMSO), which simultaneously serves as solvent and quencher of in situ generated H2O2. Subsequent sequential combination with transaminase-catalyzed reductive amination furnished the targeted nonracemic amines in good-to-excellent overall conversions (52%–95%) and high-to-excellent enantiomeric excesses (90%–99.9%) in a stereocomplementary manner. The photo-biocatalytic cascade was successfully scaled up with α-methylstyrene (1.0 mmol), enabling the synthesis of the pharmaceutically relevant chiral precursor (R)-(+)-1-phenylethylamine in 93% conversion, 61% isolated yield, and >99% ee. Further derivatization of this key intermediate delivered the L-type calcium channel blocker fendiline in 77% isolated yield and 99% ee, without the need for costly transition metal catalysts, hazardous hydrogen gas, and high pressure.

doi: 10.1002/adsc.70219

Enzymes find broad use as biocatalysts in industry and medicine owing to their exquisite selectivity, efficiency and mild reaction conditions. Custom-designed enzymes can produce tailor-made biocatalysts with potential applications that extend beyond natural reactions. However, current design methods require testing a large number of designs and mostly produce de novo enzymes with low catalytic activities1,2,3. As a result, they require costly experimental optimization and high-throughput screening to be industrially viable4,5. Here we present rotamer inverted fragment finder–diffusion (Riff-Diff), a hybrid machine learning and atomistic modelling strategy for scaffolding catalytic arrays in de novo proteins. We highlight the general applicability of Riff-Diff by designing enzymes for two mechanistically distinct chemical transformations, the retro-aldol reaction and the Morita–Baylis–Hillman reaction. We show that in both cases, it is possible to generate catalysts that exhibit activities rivalling those optimized by in vitro evolution, along with exquisite stereoselectivity. High-resolution structures of six of the designs revealed near-atomic active site design precision. The design strategy can, in principle, be applied to any catalytically competent amino acid array. These findings lay the basis for practical applicability of de novo protein catalysts in synthesis and describe fundamental principles of protein design and enzyme catalysis.

doi: 10.1038/s41586-025-09747-9

The turnover number for decarboxylation catalyzed by UndB was taken to the next level. Still 3 orders of magnitude to go for industrial applications.

doi: 10.1021/acscentsci.5c02102

Alcohol dehydrogenases have become invaluable tools in asymmetric reduction reactions and are frequently encountered in industrial processes. Their exquisite stereoselectivity allows the formation of chiral alcohols in enantiopure form. These enzymes are combined with chemical or additional enzymatic steps to access multifunctionalized compounds in cascades. Protein engineering strategies can efficiently tailor the stereoselectivity toward a given target product and provide robust biocatalysts for reactions under process-relevant conditions. Emerging areas include the atroposelective bioreduction of axially chiral molecules and the exploitation of the catalytic promiscuity of alcohol dehydrogenases in the context of photo-mediated radical catalysis.

doi: 10.1016/B978-0-323-96025-0.00135-6

Nitro compounds are key synthetic intermediates used as enabling tools in synthesis and found in a large range of essential compounds, including pharmaceuticals, pesticides, and various organic dyes. Despite recent methodological developments, the industrial preparation of nitro compounds still suffers from harsh reaction conditions, along with poor selectivity and a problematic environmental footprint. Although biological enzymatic methods exist, mild approaches for bionitration are still underexplored. Enzymes, with their exquisite selectivity and compatibility with mild reaction conditions, have the potential to revolutionize the way nitro compounds are prepared. In this perspective, we systematically analyze currently available biological/enzymatic methods, including the oxidation of an amine precursor or methods consisting of direct oxidative nitration and non-oxidative nitration. By examining both the scope and mechanism of these reactions, we aim to present an update on the state-of-the-art while highlighting current challenges in this emerging field. The goal of this perspective is to inspire innovation in enzymatic nitration for sustainable organic synthesis, providing chemists with a valuable guide.

doi: 10.1021/jacsau.4c00994

Industrial biotechnology stands at a turning point. While enzymes have long promised green, efficient catalysis, their widespread adoption has been held back by practical limitations. In this work, we introduce an innovative family of sustainable carrier materials that redefine enzyme immobilization. These materials are synthesized via a controlled solvothermal process, using renewable sources of sugars and polyphenols to create an interconnected porous structure with tunable surface chemistry. This design enables strong enzyme binding through mechanisms such as metal complexation, hydrophobic interactions, and hydrogen bonding, offering high loading capacities and exceptional operational stability. Developed with sustainability at their core, these materials set new standards for both performance and environmental responsibility, bridging the gap between laboratory research and industrial applications in biocatalysis.

doi: 10.5802/crchim.397

Iván Lavandera García, professor of organic chemistry at the University of Oviedo, Spain, passed away after a long battle with cancer on February 13, 2025, at the age of only 48. Iván was a fantastic friend, colleague, and a dedicated mentor to many students, who profited from his innovative, forward-thinking, and team-oriented spirit which pushed the boundaries of biocatalysis and bioorganic chemistry. He will be remembered as one of the most cheerful and passionate chemists, a fantastic colleague, and, most of all, a dear friend.

doi: 10.1002/anie.202504745

doi: 10.1038/d41586-025-00111-5

An artificial enzymatic cascade to access natural vanillin from eugenol/clove oil was developed by revisiting the very first vanillin synthetic process from 19th century. The biosynthetic route consists of just two biocatalysts used as cell-free preparation and molecular oxygen as oxidizing agent, outperforming also the natural microbial pathway which requires 5 steps.

Vanillin is one of the most important aroma compounds, naturally occurring in vanilla pods. Many routes to access natural vanillin from various renewables have been investigated, including a natural five-step microbial transformation of eugenol to vanillin. Readily available eugenol was also the starting material for a chemical two-step sequence to vanillin employed in the 19th century. Here we show that a two-step sequence can also be realized using biocatalysts only and run it in one-pot simultaneously. This was achieved by isomerizing the C=C double bond of eugenol by oxidation to coniferyl alcohol followed by oxidative C=C cleavage catalyzed by newly identified enzymes. Thus, two oxidative steps catalyzed by two different biocatalysts - one containing flavin and the other a non-heme iron(II) cofactor - were successfully run simultaneously just requiring molecular oxygen as oxidant for each step. Using natural eugenol sources, e. g. clove oil, vanillin was obtained with 91 % product formation. This study shows that natural pathways like the microbial transformation of eugenol to vanillin involving five steps can be shortened, hereto just two simultaneous steps, by exploiting and combining the repertoire of promiscuous enzymatic activities present in different organisms leading to new-to-nature cascades.

doi: 10.1002/cssc.202500387

A single protein that catalyzes two different reactions at two distinct active sites within one structural domain may be beneficial for cascade reactions. We explored this approach by merging a tripeptide catalyst with an alcohol dehydrogenase as a case study to bring together the strengths of enzymatic redox chemistry and peptide-catalyzed carbon–carbon bond formation. A proline-based peptide catalyst for C─C bond formation relying on an enamine intermediate was successfully merged to the N-terminus of an alcohol dehydrogenase to catalyze a cascade involving alcohol oxidation followed by C─C bond formation. It turned out that the reaction speed of the two catalytic sites diverged, and the ε-amino group of lysine residues present in the enzyme interfered with the organocatalytic proline activity. Removing/exchanging lysine residues within the enzyme reduced the background reaction but also the native redox activity. Interestingly, exchanging the N-terminal proline with a histidine switched the stereopreference. The simultaneous cascade reaction of alcohol oxidation to the aldehyde and C─C bond formation with nitrostyrene presents a first proof-of-concept for bringing peptide catalysis together with enzyme catalysis and creating a bi-active site catalyst within a single domain.

A dual-functional catalyst that unites peptide catalysis and biocatalysis within a single protein scaffold at two distinct active sites offers a promising strategy for cascade catalysis. This hybrid catalyst enabled here a one-pot transformation of alcohols to C─C coupled products via enamine activation, revealing challenges such as mismatched reaction rates and interference by lysine residues.

doi: 10.1002/cctc.202500539

The demand for novel enzyme-catalyzed reactions in chemical synthesis has spurred the development of many new-to-nature reactions. Additionally, detailed analysis of biosynthetic pathways can uncover unprecedented chemical/enzymatic mechanisms. In this study, we revisited the catalytic mechanism of the 2-oxoglutarate-dependent dioxygenase Pt2OGD-1, involved in the biosynthesis of huperzine alkaloids. Our experimental and computational investigations uncovered a previously unknown enzymatic C–C bond cleavage in the piperidine ring of the alkaloid scaffold, resembling an oxidative retro-aza-Prins reaction. Here, this transformation is initiated by hydrogen abstraction, followed by electron transfer at the 4-position of the heterocycle, triggering ring opening and finally resulting in the loss of a carbon atom as formaldehyde. This discovery expands the toolbox of reactions, enhances our understanding of these enzymes, and may facilitate their application in the biotechnological production of pharmaceutically relevant alkaloid scaffolds as well as the development of biocatalysts with similar activities.

doi: 10.1021/jacs.4c10410

α-Chiral amines are versatile building blocks for the asymmetric synthesis of optically pure high-added-value chemicals, including pharmaceuticals, agrochemicals, and natural products. Herein, we report a one-pot, two-step sequential deracemization of racemic sec-alcohols to optically enriched primary amines throught a photo-biocatalytic oxidation-reductive amination linear cascade. In the first step, near-to-quantitative oxidation of a set of racemic (hetero)benzylic alcohols into prochiral ketones was accomplished using visible light photoredox catalysis relying on 440 nm blue LEDs irradiation, 9-fluorenone as a transition-metal-free photocatalyst, and O2-saturated dimethyl sulfoxide (DMSO) as the reaction medium and a quencher of the in situ generated hydrogen peroxide (H2O2). The intermediary ketones were further converted into the corresponding non-racemic amines with up to 99% conversion, high-to-excellent optical purity (90–99.9% ee), and complementary absolute configuration via a stereoselective reductive amination catalyzed by lyophilized E. coli cells containing the respective (R)- or (S)-selective recombinant transaminases (E. coli/TAs) in an aqueous KPi buffer in the presence of pyridoxal-5′-phosphate (PLP) as cofactor and isopropylamine (iPrNH2) as an amino group donor. The developed photo-biocatalytic system was scaled up for racemic 1-phenylethanol (1.0 mmol), furnishing (S)-(−)-1-phenylethylamine with 91% conv., 82% isolated yield and 99% ee. An exemplary functionalization of the obtained (S)-α-methylbenzylamine with 6-chloro-3-isopropylpyrimidine-2,4(1H,3H)-dione led to afford an enantiomerically enriched newly launched cardiac-specific myosin inhibitor mavacamten in 76% yield and 99% ee. In addition, a single-crystal X-ray diffraction (XRD) analysis was performed, furnishing the first crystal structure for the titled API.

doi: 10.1002/adsc.202500250

Dynamic kinetic resolution (DKR) is a key method used to prepare optically pure compounds in 100 % theoretical yield starting from racemic substrates by combining the interconversion of substrate enantiomers with an enantioselective transformation. Various chemoenzymatic DKR approaches have been developed to deracemize secondary alcohols, typically requiring an organic solvent to facilitate enantioselective acylation, primarily catalyzed by lipases, alongside racemization mediated by an achiral, non-enzymatic catalyst. Achieving both steps in an aqueous solution remained elusive. Herein, we report a DKR of racemic sec-alcohols in an aqueous solution requiring only two biocatalysts. The first key to success was to achieve fast racemization in a buffer employing a non-stereoselective variant of an alcohol dehydrogenase (Lk-ADH-Prince) via a hydrogen-borrowing oxidation-reduction sequence. Engineered variants of the acyltransferase from Mycobacterium smegmatis (MsAcT) enabled enantioselective acyl transfer in water. Besides the appropriate choice of the enzymes, identifying a suitable acyl donor was a second key to the success. The DKR was successfully demonstrated using (R)-selective MsAcT variants for a broad range of racemic (hetero)benzylic alcohols with 2,2,2-trifluoroethyl acetate as the acyl donor, yielding (R)-acetates with up to >99 % conv. and high-to-excellent optical purity (83-99.9 % ee). The (S)-acetates were accessible using a stereocomplementary (S)-selective MsAcT variant. Notably, substrate concentrations of up to 400 mM were tolerated in selected cases.

doi: 10.1002/anie.202420133

Photocatalysis and biocatalysis represent powerful efficient tools in synthetic chemistry. While, both have individually shown promising results, their integration remains challenging, particularly in continuous flow processes. This work presents a semicontinuous setup, combining photo- and biocatalysis in a multistep synthesis for the production of optically pure (S)-3,3,3-trifluoro-1-phenylpropan-1-ol. Initially, a photocatalytic trifluoromethylation of a methyl ketone in α-position in a self-made photoreactor was tested in flow, followed by enzymatic ketone reduction catalyzed by an alcohol dehydrogenase (variant of an ADH from Lactobacillus kefir). The study addresses the challenge of enzyme stability in aggressive solvents, developing a robust immobilization approach for the selected ADH with a PVA/PEG cryogel matrix. This strategy has been investigated in this work to ensure enzyme stability in THF, marking a notable advance in compatibility for continuous cascades. The separate process steps were finally combined in a semicontinuous flow system, achieving a space–time yield for the photocatalytic step of 39.8 g L−1 h−1 and of 1.12 g L−1 h−1 for the enzymatic step. The study signifies one of the first instances of combining photo- and biocatalysis in continuous cascades, offering an innovative approach to synthesizing chiral 3,3,3-trifluoro-1-phenylpropan-1-ol.

doi: 10.1007/s11696-024-03649-2

A cyclic deracemization process that utilizes alternately a biocatalytic enantioselective reduction employing (S)-selective methionine sulfoxide reductase from Pseudomonas alcaliphila (paMsr) supplemented with DTT as external reducing equivalent, and a stereo-unselective photocatalytic oxidation reaction using the readily accessible Eosin Y as photocatalyst to achieve chiral sulfoxides is presented. Evaluation of the substrate scope demonstrates a general applicability of this modular system.

The synergistic combination of biocatalysis and photocatalysis is emerging as powerful tool for the development of sustainable and atom-efficient synthetic concepts facilitating an enormous portfolio of possible reactions which even goes beyond the capabilities found in nature. Here, a cyclic deracemization process is presented tailored for the synthesis of optically pure sulfoxides which are versatile structural motifs in asymmetric synthesis as well as in bioactive compounds. Enantioselective enzyme-catalyzed reduction of rac-sulfoxides was combined with a stereo-unselective photocatalytic oxidation of the corresponding sulfide intermediate. The utilization of the readily accessible and rather inexpensive photocatalyst Eosin Y increases the usability of this synthetic method. To overcome the incompatibility between the photocatalyst Eosin Y and the biocatalytic step, the cyclic deracemization process was performed in a step-wise fashion via alternated reduction in the darkness and oxidation under illumination. This modular system allowed precise adjustments of reaction parameters yielding the desired sulfoxide targets with up to >99 % ee. Evaluation of the substrate scope including a range of structurally diverse molecules demonstrated its broad applicability.

doi: 10.1002/cctc.202400073

Fatty acid photodecarboxylases (FAPs) show biotechnological promise by producing alka(e)nes from natural fatty acids. Using a cavity based search, 30 distant or unrelated enzymes with potential FAP-activity were identified either from the PDB‘s flavoproteins of from homology models. Docking experiments and in vitro characterization led to the discovery of four photodecarboxylases.

Light-dependent fatty acid photodecarboxylases (FAPs) hold significant potential for biotechnology, due to their capability to produce alka(e)nes directly from the corresponding (un)saturated natural fatty acids requiring light as the only reagent. This study expands the family of FAPs through cavity-based enzyme discovery methods. Thirty enzyme candidates with potential photodecarboxylation activity were identified by matching the cavities of four related template structures against the Protein Data Bank's flavoproteins, a library of proteins identified via the Foldseek Search Server, and homology models of sequences resulting from BLAST. Subsequent docking experiments narrowed this library to ten promising enzymes, which were expressed and assessed in vitro, identifying four photodecarboxylases. Out of these enzymes, the GMC oxidoreductase from Coccomyxa sp. Obi (CoFAP) was characterized in detail, which revealed high activity in the decarboxylation reactions of palmitic acid and octanoic acid and a broad pH tolerance (pH 6.5–9.5).

doi: 10.1002/cbic.202400631

Many relevant metabolites, as well as chemical commodities, contain at least one sulfate ester group. Consequently, biocatalytic strategies to attach sulfate to a molecule under mild conditions are of high interest. In order to expand the enzymatic toolbox available, five new arylsulfate sulfotransferases (ASSTs) were identified in this study. Overexpression in Escherichia coli and enzyme purification resulted in soluble proteins which catalyzed the sulfate transfer to an acceptor substrate using p-nitrophenyl sulfate (pNPS) as sulfate donor. Optimal reaction conditions were established with respect to temperature and pH, as well as their tolerance to organic co-solvents and melting temperature. Additionally, the kinetic parameters (Vmax, KM, and kcat) were determined. The substrate scope for the acceptor showed that a structurally diverse spectrum of alcohols is accepted. The substrates included phenolic alcohols with one, two, and three hydroxy groups, linear and cyclic aliphatic alcohols, and amines. The phenolic substrates were accepted reaching activities of up to 154 U/mg purified enzyme. Additionally, also the aliphatic alcohols (both linear and cyclic) were accepted at reduced activity, showing that these enzymes are not limited to phenolic alcohols. Moreover, catalytic activity was detected when using aniline as an acceptor substrate implying their ability to sulfate also amino groups. Finally, the consecutive sulfation of di- and trihydroxy compounds was observed, resulting in the detection of the corresponding disulfated molecules. KEY POINTS: • Five novel arylsulfate sulfotransferases were identified and characterized. • Accepted substrates included aromatic and aliphatic alcohols, as well as aniline. • Disulfation of di- and trihydroxy aromatic compounds was studied and confirmed.

doi: 10.1007/s00253-024-13354-5

Deep eutectic solvents (DESs) have emerged as green solvents with versatile applications, demonstrating significant potential in biocatalysis. They often increase the solubility of poorly water-soluble substrates, serve as smart co-substrates, modulate enzyme stereoselectivity, and potentially improve enzyme activity and stability. Despite these advantages, screening for an optimal DES and determining the appropriate water content for a given biocatalytic reaction remains a complex and time-consuming process, posing a significant challenge.

doi: 10.3389/fchem.2024.1467810

The reduction of oximes was recently identified as a promiscuous activity of Old Yellow Enzymes (OYEs). This reaction involves a two-step reduction of α-oxime-ß-ketoesters to the corresponding amines, which spontaneously dimerise to yield pyrazine derivatives. This biotransformation is currently limited to substrates with small substituents like methyl/ethyl on the keto moiety. We used a structure-based approach to engineer 12-oxophytodienoate reductase 3 (OPR3) from Solanum lycopersicum as a prototypical OYE to accept oximes with bulkier substituents. To this end, three single and two double variants were prepared and tested on six oxime substrates. The engineered variants indeed showed activity on some of the bulkier substrates, which had not been converted at all by the wild-type enzyme, including the diester compound diethyl-2-(hydroximino) malonate. While we were unable to identify variants capable of converting substrates with branched and aromatic substituents, the results demonstrate the validity of our engineering approach, suggesting potential pathways for expanding the substrate scope of OYEs.

Based on structural models of substrate complexes, variants of Old Yellow Enzymes (OYEs) are engineered to expand the substrate scope for the biocatalytic reduction of bulky α-oximo-β-keto esters. These variants demonstrate the ability to reduce substrates with larger substituents that the wild-type enzyme cannot convert, thus extending the potential applications of OYEs in biocatalysis.

doi: 10.1002/cctc.202400642

Stabilized enzymes are crucial for the industrial application of biocatalysis due to their enhanced operational stability, which leads to prolonged enzyme activity, cost-efficiency and consequently scalability of biocatalytic processes. Over the past decade, numerous studies have demonstrated that deep eutectic solvents (DES) are excellent enzyme stabilizers. However, the search for an optimal DES has primarily relied on trial-and-error methods, lacking systematic exploration of DES structure-activity relationships. Therefore, this study aims to rationally design DES to stabilize various dehydrogenases through extensive experimental screening, followed by the development of a straightforward and reliable mathematical model to predict the efficacy of DES in enzyme stabilization. A total of 28 DES were tested for their ability to stabilize three dehydrogenases at 30°C: (S)-alcohol dehydrogenase from Rhodococcus ruber (ADH-A), (R)-alcohol dehydrogenase from Lactobacillus kefir (Lk-ADH) and glucose dehydrogenase from Bacillus megaterium (GDH). The residual activity of these enzymes in the presence of DES was quantified using first-order kinetic models. The screening revealed that DES based on polyols serve as promising stabilizing environments for the three tested dehydrogenases, particularly for the enzymes Lk-ADH and GDH, which are intrinsically unstable in aqueous environments. In glycerol-based DES, increases in enzyme half-life of up to 175-fold for Lk-ADH and 60-fold for GDH were observed compared to reference buffers. Furthermore, to establish the relationship between the enzyme inactivation rate constants and DES descriptors generated by the Conductor-like Screening Model for Real Solvents, artificial neural network models were developed. The models for ADH-A and GDH showed high efficiency and reliability (R2 > 0.75) for in silico screening of the enzyme inactivation rate constants based on DES descriptors. In conclusion, these results highlight the significant potential of the integrated experimental and in silico approach for the rational design of DES tailored to stabilize enzymes.

doi: 10.3389/fchem.2024.1436049

The oxidation of sec-alcohols is a common reaction in organic chemistry. We report a biocatalytic approach for oxidizing racemic (hetero)aromatic sec-alcohols to the corresponding ketones. The reaction relies on employing freeze-dried E. coli cells containing a recombinant variant of an alcohol dehydrogenase deduced from Lactobacillus kefir (E. coli/Lk-ADH Prince) and vinyl acetate as an in situ acetaldehyde surrogate as oxidant. Biooxidations of a set of 20 racemic (hetero)aromatic alcohols were carried out in the presence of a catalytic amount of NADP+ cofactor, the biocatalyst, and vinyl acetate in an aqueous medium to generate the corresponding ketones with up to >99 % conv. Preparative scale (1.0 mmol; 100 mM in 10 mL) reactions led to obtaining ketones in the 56–83 % isolated yield range.

Herein, we show that an engineered variant of an alcohol dehydrogenase deduced from Lactobacillus kefir (Lk-ADH Prince) serves as an efficient biocatalyst for biooxidation of sec-alcohols under mild conditions employing vinyl acetate as an acetaldehyde surrogate, which takes up the hydride, allowing the synthesis of variously functionalized ketones quantitively. The merger of the Lk-ADH Prince-catalyzed biooxidation with transaminase-catalyzed amination in a one-pot, two-step sequential manner led to optically active amines. This work should provide useful input to the field of redox biotransformations.

doi: 10.1002/cctc.202400803

As water miscible organic co-solvents are often required for enzyme reactions to improve e.g., the solubility of the substrate in the aqueous medium, an enzyme is required which displays high stability in the presence of this co-solvent. Consequently, it is of utmost importance to identify the most suitable enzyme or the appropriate reaction conditions. Until now, the melting temperature is used in general as a measure for stability of enzymes. The experiments here show, that the melting temperature does not correlate to the activity observed in the presence of the solvent. As an alternative parameter, the concentration of the co-solvent at the point of 50% protein unfolding at a specific temperature T in short \({c}_{{U}_{50}}^{T}\) is introduced. Analyzing a set of ene reductases, \({c}_{{U}_{50}}^{T}\) is shown to indicate the concentration of the co-solvent where also the activity of the enzyme drops fastest. Comparing possible rankings of enzymes according to melting temperature and \({c}_{{U}_{50}}^{T}\) reveals a clearly diverging outcome also depending on the specific solvent used. Additionally, plots of \({c}_{{U}_{50}}\) versus temperature enable a fast identification of possible reaction windows to deduce tolerated solvent concentrations and temperature.

doi: 10.1038/s41467-024-49774-0

5-(Hydroxymethyl)furfural (HMF) is a key platform chemical derived from renewable biomass sources, holding great potential as starting material for the synthesis of valuable compounds, thereby replacing petrochemical-derived counterparts. Among these valorised compounds, 2,5-furandicarboxylic acid (FDCA) has emerged as a versatile building block. Here we demonstrate the biocatalytic synthesis of FDCA from HMF via a one-pot three-step oxidative cascade performed via two operative steps under mild reaction conditions employing two unspecific peroxygenases (UPOs) using hydrogen peroxide as the only oxidant. The challenge of HMF oxidation by UPOs is the chemoselectivity of the first step, as one of the two possible oxidation products is only a poor substrate for further oxidation. The unspecific peroxygenase from Marasmius oreades (MorUPO) was found to oxidize 100 mM of HMF to 5-formyl-2-furoic acid (FFCA) with 95 % chemoselectivity. In the sequential one-pot cascade employing MorUPO (TON up to 13535) and the UPO from Agrocybe aegerita (AaeUPO, TON up to 7079), 100 mM of HMF were oxidized to FDCA reaching up to 99 % conversion and yielding 861 mg isolated pure crystalline FDCA, presenting the first example of a gram scale biocatalytic synthesis of FDCA involving UPOs.

A scalable one-pot cascade for the oxidation of 5-(hydroxymethyl)furfural (HMF) and subsequent isolation of 2,5-furandicarboxylic acid (FDCA) was successfully performed using two unspecific peroxygenases (UPOs). The UPO from Marasmius oreades (MorUPO) oxidized HMF to 5-formyl-2-furoic acid (FFCA) with 95 % chemoselectivity. In a sequential step, the UPO from Agrocybe aegerita (AaeUPO) completed the oxidation-cascade, achieving up to 99 % conversion to FDCA and yielding 861 mg isolated pure crystalline FDCA.

doi: 10.1002/cssc.202400156

The amide moiety belongs to the most common motives in pharmaceutical chemistry, present in many prescribed small-molecule pharmaceuticals. Methods for its manufacture are still in high demand, especially using water/buffer as a solvent and avoiding stoichiometric amounts of activation reagents. Herein, we identified from a library of lipases/esterases/acyltransferases and variants thereof a lipase originating from Sphingomonas sp. HXN-200 (SpL) able to form amides in aqueous solution starting from a broad scope of sterically demanding heteroaromatic ethyl esters as well as aliphatic amines, reaching isolated yields up to 99% on preparative scale and space time yields of up to 864 g L–1 d–1; thus, in selected cases, the amide was formed within minutes. The enzyme features an aspartate next to the canonical serine of the catalytic triad, which was essential for amide formation. Furthermore, the enzyme structure revealed two tunnels to the active site, presumably one for the ester and one for the amine, which permit the bringing together of the sterically demanding heteroaromatic esters and the amine in the active site. This work shows that biocatalytic amide formation starting from various five- and six-membered heteroaromatic ethyl esters in the buffer can serve as a platform for preparative amide synthesis.

doi: 10.1021/acscatal.4c01268

Biocatalysis has gained increasing importance as an eco-friendly alternative for the production of bulk and fine chemicals. Within this paradigm, Baeyer Villiger monoxygenases (BVMOs) serve as enzymatic catalysts that provide a safe and sustainable route to the conventional synthesis of lactones, such as caprolactone, which is employed for the production of polycaprolactone (PCL), a biocompatible polymer for medicinal applications. In this work, we present a three-step, semi-continuous production of PCL using an entirely biocatalytic process, highlighting the merits of continuous manufacturing for enhancing biocatalysis. First, caprolactone is produced in batch from cyclohexanol using a coenzymatic cascade involving an alcohol dehydrogenase (ADH) and BVMO. Different process parameters and aeration modes were explored to optimize the cascade's productivity. Secondly, the continuous extraction of caprolactone into an organic solvent, needed for the polymerization step, was optimized. 3D-printed mixers were applied to enhance the mass transfer between the organic and the aqueous phases. Lastly, we investigated the ring-opening polymerization of caprolactone to PCL catalyzed by Candida antarctica lipase B (CAL-B), with a focus on eco-friendly solvents like cyclopentyl-methyl-ether (CPME). Space–time-yields up to 58.5 g L−1 h−1 were achieved with our overall setup. By optimizing the individual process steps, we present an efficient and sustainable pathway for PCL production.

doi: 10.1039/d3re00536d

Medium-sized 5- and 6-membered ring lactams are molecules with remarkable stability, in contrast to smaller β-lactams. As monomers, they grant access to nylon-4 and nylon-5, which are alternative polyamides to widespread caprolactam-based nylon-6. Chemical hydrolysis of monocyclic γ- and δ-lactams to the corresponding amino acids requires harsh reaction conditions and up to now, no mild (enzymatic) protocol has been reported. Herein, the biocatalytic potential of a pair of heterologously expressed bacterial ATP-dependent oxoprolinases – OplA and OplB – was exploited. Strong activity in the presence of excess of ATP was monitored on δ-valerolactam and derivatives thereof, while trace activity was detected on γ-butyrolactam. An ATP recycling system based on cheap Graham's salt (sodium metaphosphate) and a polyphosphate kinase allowed the use of catalytic amounts of ATP, leading to up to full conversion of 10 mM δ-valerolactam at 30 °C in aqueous medium. Further improvements were obtained by co-expressing OplA and OplB using the pETDuet1 vector, a strategy which enhanced the soluble expression yield and the protein stability. Finally, a range of phosphodonors was investigated in place of ATP. With acetyl phosphate and carbamoyl phosphate, turnover numbers up to 176 were reached, providing hints on a possible mechanism, which was studied by 31P-NMR.

doi: 10.1039/D3GC04434C

NAD(P)H-dependent reductases have been established as chemoselective biocatalysts for the asymmetric reduction of C=O, C=N, and C=C bonds. Serendipity, screening, and engineering unearthed that the ability to convert distinct electrophiles is shared by numerous enzyme families. This review sheds light on this underexplored facet of NAD(P)H-dependent reductases and highlights the catalytic versatility of these enzymes.

The asymmetric reduction of double bonds using NAD(P)H-dependent oxidoreductases has proven to be an efficient tool for the synthesis of important chiral molecules in research and on industrial scale. These enzymes are commercially available in screening kits for the reduction of C=O (ketones), C=C (activated alkenes), or C=N bonds (imines). Recent reports, however, indicate that the ability to accommodate multiple reductase activities on distinct C=X bonds occurs in different enzyme classes, either natively or after mutagenesis. This challenges the common perception of highly selective oxidoreductases for one type of electrophilic substrate. Consideration of this underexplored potential in enzyme screenings and protein engineering campaigns may contribute to the identification of complementary biocatalytic processes for the synthesis of chiral compounds. This review will contribute to a global understanding of the promiscuous behavior of NAD(P)H-dependent oxidoreductases on C=X bond reduction and inspire future discoveries with respect to unconventional biocatalytic routes in asymmetric synthesis.

doi: 10.1002/anie.202314740

Regio- and stereoselective functionalisation reactions like C–H oxidation are of high importance for instance for the valorization of renewables like fatty acids by α-hydroxylation. Here, peroxygenases were envisioned to be of high interest as they require common hydrogen peroxide as the only oxidant generating water as the sole side product. As the unspecific peroxygenase from Hypoxylon sp. (HspUPO) turned out to be not selective for α-hydroxylation, various bacterial peroxygenases from the CYP152 family were tested for the stereoselective α-hydroxylation of medium chain fatty acids (C6, C8, C10). The enzyme P450Exα proved to be highly suitable for the conversion of caproic acid (C6) (95% conv.) and showed high regioselectivity to give the α-hydroxylated product (α : β-selectivity = 14 : 1). Additionally, P450Exα successfully converted the dicarboxylic acids azelaic acid (C9) and sebacic acid (C10) exclusively to the corresponding α-monohydroxylated product (up to >99% conversion). P450Spα hydroxylated the fatty acids C6, C8 and C10 preferentially in α-position giving the optically pure or optically enriched (S)-enantiomer [ee 95–>99% (S)] with up to 99% conversion. Both enzymes were used for preparative synthesis of α-hydroxylated fatty acids at up to 150 mM substrate concentration on 50 mL scale giving for instance 2-hydroxyoctanoic acid with 87% yield on gram scale (1260 mg) reaching TONs up to 42 000.

doi: 10.1039/d3gc04593e

Chiral alcohols are versatile building blocks and are of particular interest in the asymmetric synthesis of nonracemic active pharmaceutical ingredients, agrochemicals, fragrances, flavors, natural products, etc. Herein, we report on a “one-pot sequential two-step” concurrent oxidation–reduction photobiocatalytic process to synthesize enantiomerically enriched alcohols. In this regard, an efficient photocatalytic system based on irradiation with 440 nm blue LEDs in the presence of 9-fluorenone as a metal-free photocatalyst and molecular oxygen as the terminal oxidant in dry DMSO as the hydrogen peroxide-neutralizing agent was used to oxidize a broad range of racemic (hetero)benzylic alcohols into prochiral ketones quantitively (>99% conv.). The in situ formed carbonyl compounds were subsequently converted into the corresponding chiral alcohols via a sequential biocatalytic transhydrogenation catalyzed by lyophilized E. coli cells overexpressing highly stereoselective and stereocomplementary recombinant alcohol dehydrogenases (ADHs) originated from Rhodococcus ruber (E. coli/ADH-A) or Rhodococcus erythropolis (E. coli/ReADH) to obtain (S)-alcohols and Lactobacillus kefir (E. coli/Lk-ADH) or KRED-110 to obtain (R)-alcohols, respectively. Overall, the elaborated photobiocatalytic deracemization of racemic alcohols using a 9-fluorenone-O2-blue LED-DMSO-E. coli/ADH system carried out on a semipreparative scale (0.25 mmol; 63 mM final conc. in 4 mL) at room temperature yielded nonracemic aryl alcohols with 82–99.9% conv., in up to 92% isolated yield, with 97–99.9% ee and complementary chirality.

doi: 10.1021/acscatal.3c05100

An enzyme and a peptide catalyze—in an aqueous buffer—a two-step cascade reaction with high chemo- and stereoselectivity in one pot. The optimization of the modular peptide catalyst and the identification of common reaction conditions were key for bringing the two worlds of enzyme and peptide catalysis together.

Enzymes and peptide catalysts consist of the same building blocks but require vastly different environments to operate best. Herein, we show that an enzyme and a peptide catalyst can work together in a single reaction vessel to catalyze a two-step cascade reaction with high chemo- and stereoselectivity. Abundant linear alcohols, nitroolefins, an alcohol oxidase, and a tripeptide catalyst provided chiral γ-nitroaldehydes in aqueous buffer. High yields (up to 92 %) and stereoselectivities (up to 98 % ee) were achieved for the cascade through the rational design of the peptide catalyst and the identification of common reaction conditions.

doi: 10.1002/anie.202319457

Density functional theory (DFT) calculations are employed to uncover the detailed reaction mechanism for the Fries rearrangement of 3-hydroxyphenyl acetate to 2′,4′-dihydroxyacetophenone catalyzed by the acyltransferase from Pseudomonas protegens (PpATase). Relative binding energies of other acetyl acceptors are also calculated to evaluate the possibility of PpATase catalyzing an intermolecular Fries rearrangement.

The acyltransferase from Pseudomonas protegens (PpATase) catalyzes in nature the reversible transformation of monoacetylphloroglucinol to diacetylphloroglucinol and phloroglucinol. Interestingly, this enzyme has been shown to catalyze the promiscuous transformation of 3-hydroxyphenyl acetate to 2′,4′-dihydroxyacetophenone, representing a biological version of the Fries rearrangement. In the present study, we report a mechanistic investigation of this activity of PpATase using quantum chemical calculations. A detailed mechanism is proposed, and the energy profile for the reaction is presented. The calculations show that the acylation of the enzyme is highly exothermic, while the acetyl transfer back to the substrate is only slightly exothermic. The deprotonation of the C6−H of the substrate is rate-limiting, and a remote aspartate residue (Asp137) is proposed to be the general base group in this step. Analysis of the binding energies of various acetyl acceptors shows that PpATase can promote both intramolecular and intermolecular Fries rearrangement towards diverse compounds.

doi: 10.1002/open.202300256

The reduction of C═X (X = N, O) bonds is a cornerstone in both synthetic organic chemistry and biocatalysis. Conventional reduction mechanisms usually involve a hydride ion targeting the less electronegative carbon atom. In a departure from this paradigm, our investigation into Old Yellow Enzymes (OYEs) reveals a mechanism involving transfer of hydride to the formally more electronegative nitrogen atom within a C═N bond. Beyond their known ability to reduce electronically activated C═C double bonds, e.g., in α, β-unsaturated ketones, these enzymes have recently been shown to reduce α-oximo-β-ketoesters to the corresponding amines. It has been proposed that this transformation involves two successive reduction steps and proceeds via imine intermediates formed by the reductive dehydration of the oxime moieties. We employ advanced quantum mechanics/molecular mechanics (QM/MM) simulations, enriched by a two-tiered approach incorporating QM/MM (UB3LYP-6-31G*/OPLS2005) geometry optimization, QM/MM (B3LYP-6-31G*/amberff19sb) steered molecular dynamics simulations, and detailed natural-bond-orbital analyses to decipher the unconventional hydride transfer to nitrogen in both reduction steps and to delineate the role of active site residues as well as of substituents present in the substrates. Our computational results confirm the proposed mechanism and agree well with experimental mutagenesis and enzyme kinetics data. According to our model, the catalysis of OYE involves hydride transfer from the flavin cofactor to the nitrogen atom in oximoketoesters as well as iminoketoesters followed by protonation at the adjacent oxygen or carbon atoms by conserved tyrosine residues and active site water molecules. Two histidine residues play a key role in the polarization and activation of the C═N bond, and conformational changes of the substrate observed along the reaction coordinate underline the crucial importance of dynamic electron delocalization for efficient catalysis.

doi: 10.1021/acscatal.3c04362

In Saccharomyces kluyveri, dihydropyrimidinase (DHPaseSK) is involved in the pyrimidine degradation pathway, which includes the reversible ring cleavage between nitrogen 3 and carbon 4 of 5,6-dihydrouracil. In this study, DPHaseSK was successfully cloned and expressed in E. coli BL-21 Gold (DE3) with and without affinity tags. Thereby, the Strep-tag enabled fastest purification and highest specific activity (9.5 ± 0.5 U/mg). The biochemically characterized DHPaseSK_Strep had similar kinetic parameters (Kcat/Km) on 5,6-dihydrouracil (DHU) and para-nitroacetanilide respectively, with 7,229 and 4060 M-1 s-1. The hydrolytic ability of DHPaseSK_Strep to polyamides (PA) was tested on PA consisting of monomers with different chain length (PA-6, PA-6,6, PA-4,6, PA-4,10 and PA-12). According to LC-MS/TOF analysis, DHPaseSK_Strep showed a preference for films containing the shorter chain monomers (e.g., PA-4,6). In contrast, an amidase from Nocardia farcinica (NFpolyA) showed some preference for PA consisting of longer chain monomers. In conclusion, in this work DHPaseSK_Strep was demonstrated to be able to cleave amide bonds in synthetic polymers, which can be an important basis for development of functionalization and recycling processes for polyamide containing materials.

doi: 10.3389/fbioe.2023.1158226

The enzymatic formal reduction of α-angelica lactone to both enantiomers of γ-valerolactone was achieved in a biocatalytic one-pot cascade combining an asymmetric isomerization step and a reduction step. A fusion protein linking two Old Yellow Enzymes with complementary activities showed excellent (R)-selectivity, while the (S)-enantiomer was obtained using a single enzyme for both steps of the cascade.

The formal asymmetric and stereodivergent enzymatic reduction of α-angelica lactone to both enantiomers of γ-valerolactone was achieved in a one-pot cascade by uniting the promiscuous stereoselective isomerization activity of Old Yellow Enzymes with their native reductase activity. In addition to running the cascade with one enzyme for each catalytic step, a bifunctional isomerase-reductase biocatalyst was designed by fusing two Old Yellow Enzymes, thereby generating an unprecedented case of an artificial enzyme catalyzing the reduction of nonactivated C=C bonds to access (R)-valerolactone in overall 41 % conversion and up to 91 % ee. The enzyme BfOYE4 could be used as single biocatalyst for both steps and delivered (S)-valerolactone in up to 84 % ee and 41 % overall conversion. The reducing equivalents were provided by a nicotinamide recycling system based on formate and formate dehydrogenase, added in a second step. This enzymatic system provides an asymmetric route to valuable chiral building blocks from an abundant bio-based chemical.

doi: 10.1002/cbic.202300146

Hydrogen transfer biocatalysts to prepare optically pure alcohols are in need, especially when it comes to sterically demanding ketones, whereof the bioreduced products are either essential precursors of pharmaceutically relevant compounds or constitute APIs themselves. In this study, we report on the biocatalytic potential of an anti-Prelog (R)-specific Lactobacillus kefir ADH variant (Lk-ADH-E145F-F147L-Y190C, named Lk-ADH Prince) employed as E. coli/ADH whole-cell biocatalyst and its characterization for stereoselective reduction of prochiral carbonyl substrates. Key enzymatic reaction parameters, including the reaction medium, evaluation of cofactor-dependency, organic co-solvent tolerance, and substrate loading, were determined employing the drug pentoxifylline as a model prochiral ketone. Furthermore, to tap the substrate scope of Lk-ADH Prince in hydrogen transfer reactions, a broad range of 34 carbonylic derivatives was screened. Our data demonstrate that E. coli/Lk-ADH Prince exhibits activity toward a variety of structurally different ketones, furnishing optically active alcohol products at the high conversion of 65–99.9% and in moderate-to-high isolated yields (38–91%) with excellent anti-Prelog (R)-stereoselectivity (up to >99% ee) at substrate concentrations up to 100 mM.

doi: 10.1038/s42004-023-01013-1

Identifying (bio)catalysts displaying high enantio-/stereoselectivity is a fundamental prerequisite for the advancement of asymmetric catalysis. Herein, a high-throughput, stereoselective screening assay is reported that gives information on enantioselectivity, stereopreference and activity as showcased for peroxygenase-catalyzed hydroxylation. The assay is based on spectrophotometric analysis of the simultaneous formation of NAD(P)H from the alcohol dehydrogenase catalyzed enantioselective oxidation of the sec-alcohol product formed in the peroxygenase reaction. The assay was applied to investigate a library comprising 44 unspecific peroxygenases (UPOs) containing 25 UPOs not reported yet. Thereby, previously non-described wild-type UPOs displaying (S)- as well as (R)-stereoselectivity for the hydroxylation of representative model substrates were identified, reaching up to 98 % ee for the (R)- and 94 % ee for the (S)-enantiomer. Homology models with concomitant docking studies indicated the structural reason for the observed complementary stereopreference.

A high-throughput assay was established to assess stereoselectivity, stereopreference, and activity in peroxygenase-catalyzed hydroxylation. The assay accurately predicts the ee value of the product with ±11 % accuracy. It enabled the discovery of novel wild-type fungal peroxygenases with remarkable enantioselectivity for both (R)- and (S)-alcohols. Homology models and docking studies supported the observed stereopreferences.

doi: 10.1002/anie.202312721

Unspecific peroxygenases have attracted interest in synthetic chemistry, especially for the oxidative activation of C−H bonds, as they only require hydrogen peroxide (H2O2) instead of a cofactor. Due to their instability in even small amounts of H2O2, different strategies like enzyme immobilization or in situ H2O2 production have been developed to improve the stability of these enzymes. While most strategies have been studied separately, a combination of photocatalysis with immobilized enzymes was only recently reported. To show the advantages and limiting factors of immobilized enzyme in a photobiocatalytic reaction, a comparison is made between free and immobilized enzymes. Adjustment of critical parameters such as (i) enzyme and substrate concentration, (ii) illumination wavelength and (iii) light intensity results in significantly increased enzyme stabilities of the immobilized variant. Moreover, under optimized conditions a turnover number of 334,500 was reached.

The right light for peroxizymes: Increased stability of peroxygenases can be achieved by a combination of enzyme immobilization and photocatalytic in situ hydrogen peroxide supply. Therefore, oxygen reduction over illuminated potassium phosphate-doped graphitic carbon nitride was cascaded with the unspecific peroxygenase from Agrocybe aegerita immobilized on an amino methacrylate carrier to hydroxylate 4-ethylbenzoic acid.

doi: 10.1002/cssc.202300613

Efficient access to the morphinan scaffold remains a major challenge in both synthetic chemistry and biotechnology. Here, a biomimetic chemo-enzymatic strategy to synthesize the natural promorphinan intermediate (+)-salutaridine is demonstrated. By combining early-stage organic synthesis with enzymatic asymmetric key step transformations, the prochiral natural intermediate 1,2-dehydroreticuline was prepared and subsequently stereoselectively reduced by the enzyme 1,2-dehydroreticuline reductase obtaining (R)-reticuline in high ee and yield (>99% ee, up to quant. conversion, 92% isol. yield). In the final step, membrane-bound salutaridine synthase was used to perform the selective ortho-para phenol coupling to give (+)-salutaridine. The synthetic route shows the potential of combining early-stage advanced organic chemistry to minimize protecting group techniques with late-stage multi-step biocatalysis to provide an unprecedented access to the medicinally important compound class of promorphinans.

doi: 10.1039/d3sc02304d

We report on novel chemoenzymatic routes toward tenofovir using low-cost starting materials and commercial or homemade enzyme preparations as biocatalysts. The biocatalytic key step was accomplished either via stereoselective reduction using an alcohol dehydrogenase or via kinetic resolution using a lipase. By employing a suspension of immobilized lipase from Burkholderia cepacia (Amano PS-IM) in a mixture of vinyl acetate and toluene, the desired (R)-ester (99% ee) was obtained on a 500 mg scale (60 mM) in 47% yield. Alternatively, stereoselective reduction of 1-(6-chloro-9H-purin-9-yl) propan-2-one (84 mg, 100 mM) catalyzed by lyophilized E. coli cells harboring recombinant alcohol dehydrogenase (ADH) from Lactobacillus kefir (E. coli/Lk-ADH Prince) allowed one to reach quantitative conversion, 86% yield and excellent optical purity (>99% ee) of the corresponding (R)-alcohol. The key (R)-intermediate was transformed into tenofovir through “one-pot” aminolysis–hydrolysis of (R)-acetate in NH3-saturated methanol, alkylation of the resulting (R)-alcohol with tosylated diethyl(hydroxymethyl) phosphonate, and bromotrimethylsilane (TMSBr)-mediated cleavage of the formed phosphonate ester into the free phosphonic acid. The elaborated enzymatic strategy could be applicable in the asymmetric synthesis of tenofovir prodrug derivatives, including 5′-disoproxil fumarate (TDF, Viread) and 5′-alafenamide (TAF, Vemlidy). The molecular basis of the stereoselectivity of the employed ADHs was revealed by molecular docking studies.

doi: 10.1021/acs.joc.3c01005

Enzymatic carbon dioxide fixation is one of the most important metabolic reactions as it allows the capture of inorganic carbon from the atmosphere and its conversion into organic biomass. However, due to the often unfavorable thermodynamics and the difficulties associated with the utilization of CO2, a gaseous substrate that is found in comparatively low concentrations in the atmosphere, such reactions remain challenging for biotechnological applications. Nature has tackled these problems by evolution of dedicated CO2-fixing enzymes, i.e., carboxylases, and embedding them in complex metabolic pathways. Biotechnology employs such carboxylating and decarboxylating enzymes for the carboxylation of aromatic and aliphatic substrates either by embedding them into more complex reaction cascades or by shifting the reaction equilibrium via reaction engineering. This review aims to provide an overview of natural CO2-fixing enzymes and their mechanistic similarities. We also discuss biocatalytic applications of carboxylases and decarboxylases for the synthesis of valuable products and provide a separate summary of strategies to improve the efficiency of such processes. We briefly summarize natural CO2 fixation pathways, provide a roadmap for the design and implementation of artificial carbon fixation pathways, and highlight examples of biocatalytic cascades involving carboxylases. Additionally, we suggest that biochemical utilization of reduced CO2 derivates, such as formate or methanol, represents a suitable alternative to direct use of CO2 and provide several examples. Our discussion closes with a techno-economic perspective on enzymatic CO2 fixation and its potential to reduce CO2 emissions.

doi: 10.1021/acs.chemrev.2c00581

Substituted piperidine rings are a common motif in natural products and pharmaceutical drugs. The asymmetric synthesis of piperidines bearing multiple stereocentres remains a challenge, and current approaches often rely on lengthy reaction sequences and ‘chiral pool’ strategies. Herein, we report multi-enzymatic and chemo-enzymatic methods that allow the preparation of piperidines with three chirality centres in only two steps from achiral diketoester precursors. Stereocontrol is achieved by a highly enantioselective transamination leading to optically pure (ee >99%) enamine or imine intermediates, followed by diastereoselective reduction of these unsaturated N-heterocycles using either platinum(0)-catalysed flow hydrogenation or enzymatic imine reduction. In the latter case, coupling of the two biocatalytic reactions in a concurrent one-pot process is possible, thus reducing the synthetic sequence to a single biotransformation. In total, nine trisubstituted piperidines were prepared in high stereoisomeric purities (dr ≥98:2) and isolated yields of up to 73%. Lead-likeness analysis of five representative products using an open-source webtool suggests that these compounds possess considerable application potential as building blocks in drug discovery.

doi: 0.1002/adsc.202300050

Although optical pure amino alcohols are in high demand due to their widespread applicability, they still remain challenging to synthesize, since commonly elaborated protection strategies are required. Here, a multi-enzymatic methodology is presented that circumvents this obstacle furnishing enantioenriched 1,3-amino alcohols out of commodity chemicals. A Type I aldolase forged the carbon backbone with an enantioenriched aldol motif, which was subsequently subjected to enzymatic transamination. A panel of 194 TAs was tested on diverse nine aldol products prepared through different nucleophiles and electrophiles. Due to the availability of (R)- and (S)-selective TAs, both diastereomers of the 1,3-amino alcohol motif were accessible. A two-step process enabled the synthesis of the desired amino alcohols with up to three chiral centers with de up to >97 in the final products.

doi: 10.1002/adsc.202300201

The recent increase of interest in photocatalysis spread to biocatalysis and triggered a rush for the development of light-dependent enzyme-mediated or enzyme-coupled processes. After several years of intense research on photobiocatalysis, it is time to evaluate the state of the field in a structured manner. In this Perspective, we suggest to group photobiocatalysis into distinct disciplines and provide principal guidelines and standards for the reporting of photobiocatalytic research results as well as advice on performing photobiocatalytic reactions. Overall, we assess that the field contributes to the diversity of biocatalytic reactions while offering the selectivity of enzymes to photocatalysis. We foresee that the ongoing excitement for light-dependent enzymatic processes will lead to the discovery of novel photobiocatalytic mechanisms to complement biocatalysis with new bond-forming reactions and will provide additional innovative strategies to utilize light as a possible benign energy source.

doi: 10.1002/cptc.202200325

Mass spectrometry-based high-throughput screening methods combine the advantages of photometric or fluorometric assays and analytical chromatography, as they are reasonably fast (throughput ≥1 sample/min) and broadly applicable, with no need for labelled substrates or products. However, the established MS-based screening approaches require specialised and expensive hardware, which limits their broad use throughout the research community. We show that a more common instrumental platform, a single-quadrupole HPLC-MS, can be used to rapidly analyse diverse biotransformations by flow-injection mass spectrometry (FIA-MS), that is, by automated infusion of samples to the ESI-MS detector without prior chromatographic separation. Common organic buffers can be employed as internal standard for quantification, and the method provides readily validated activity and selectivity information with an analytical run time of one minute per sample. We report four application examples that cover a broad range of analyte structures and concentrations (0.1–50 mM before dilution) and diverse biocatalyst preparations (crude cell lysates and whole microbial cells). Our results establish FIA-MS as a versatile and reliable alternative to more traditional methods for screening enzymatic reactions.

Separation not required: Mass spectrometry without prior chromatographic separation, carried out on a single-quadrupole HPLC-MS, can be used for the qualitative and quantitative analysis of diverse biotransformations. This flow-injection analysis mass spectrometry (FIA-MS) approach represents an attractive alternative to more traditional photometric, fluorometric, and chromatographic methods for screening enzymatic reactions.

doi: 10.1002/cbic.202300170

Oxidative alkene cleavage is a highly interesting reaction to obtain aldehydes and ketones. The Mn-dependent protein TM1459 from Thermotoga maritima can catalyse alkene cleavage of styrene derivatives in the presence of tert-butyl hydroperoxide. Despite the high thermal stability of the enzyme, it gets inactivated during the reaction. The data reported here indicate that auto-oxidation is responsible for the low stability of TM1459 in the oxidative environment required for the alkene cleavage reaction. By targeting the exchange of residues prone to oxidation, this phenomenon was successfully prevented. Importantly, the stability to oxidation conveyed by the amino acid exchanges led to increased enzyme activity. However, the exchanges resulted in slightly modified positions of two of the four metal-binding amino acids, thereby strongly impacting metal binding.

doi: 10.1002/cite.202200176

Lignin-derived aryl methyl ethers (e.g. coniferyl alcohol, ferulic acid) are expected to be a future carbon source for chemistry. The well-known P450 dependent biocatalytic O-demethylation of these aryl methyl ethers is prone to side product formation especially for the oxidation sensitive catechol products which get easily oxidized in the presence of O2. Alternatively, biocatalytic demethylation using cobalamin dependent enzymes may be used under anaerobic conditions, whereby two proteins, namely a methyltransferase and a carrier protein are required. To make this approach applicable for preparative transformations, fusion proteins were designed connecting the cobalamin-dependent methyltransferase (MT) with the corrinoid-binding protein (CP) from Desulfitobacterium hafniense by variable glycine linkers. From the proteins created, the fusion enzyme MT-L5-CP with the shortest linker performed best of all fusion enzymes investigated showing comparable and, in some aspects, even better performance than the separated proteins. The fusion enzymes provided several advantages like that the cobalamin cofactor loading step required originally for the CP could be skipped enabling a significantly simpler protocol. Consequently, the biocatalytic demethylation was performed using Schlenk conditions allowing the O-demethylation e.g. of the monolignol coniferyl alcohol on a 25 mL scale leading to 75% conversion. The fusion enzyme represents a promising starting point to be evolved for alternative demethylation reactions to diversify natural products and to valorize lignin.

doi: 10.1039/D2RA08005B

The biocatalytic reduction of the oxime moiety to the corresponding amine group has only recently been found to be a promiscuous activity of ene-reductases transforming α-oximo β-keto esters. However, the reaction pathway of this two-step reduction remained elusive. By studying the crystal structures of enzyme oxime complexes, analyzing molecular dynamics simulations, and investigating biocatalytic cascades and possible intermediates, we obtained evidence that the reaction proceeds via an imine intermediate and not via the hydroxylamine intermediate. The imine is reduced further by the ene-reductase to the amine product. Remarkably, a non-canonical tyrosine residue was found to contribute to the catalytic activity of the ene-reductase OPR3, protonating the hydroxyl group of the oxime in the first reduction step.

doi: 10.1021/acscatal.2c06137

Native and promiscuous catalytic activities of flavin-dependent Old Yellow Enzymes (OYEs) reported to date are initiated by the reduced flavin upon electron transfer. As a rare exception, the isomerization of a nonactivated C═C bond was shown to be hydride-independent with two nonstereoselective yeast OYEs. Here, we report the asymmetric isomerization of a prochiral model substrate, γ-methyl β,γ-butenolide, to the corresponding (R)- and (S)-enantiomers of the γ-methyl α,β-butenolide in up to >99% ee by two stereocomplementary OYEs of algal and fungal origin, respectively, which operate by asymmetric proton transfer. Mechanistic studies based on two newly solved crystal structures, along with soaking experiments and site-directed mutagenesis, support the crucial role of partially nonconserved tyrosine residues for the activity and stereoselectivity of both (R)- and (S)-isomerases. This study offers a unique view on the potential of flavoproteins in nonredox catalysis and provides hints for scouting olefin isomerases in likely stereodivergent classes of OYEs.

doi: 10.1021/acscatal.2c01110

Alcohol dehydrogenases (ADHs; EC 1.1.1.1) have been widely used for the reversible redox reactions of carbonyl compounds (i.e., aldehydes and ketones) and primary or secondary alcohols, often resulting in optically pure hydroxyl products with high added value. In this work, we report a concise chemoenzymatic route toward xanthine-based enantiomerically pure active pharmaceutical ingredients (API) – proxyphylline, xanthinol, and diprophylline employing various recombinant short-chain ADHs with (R)- or (S)-selectivity as key biocatalysts. By choosing the appropriate ADH, the (R)- as well as the (S)-enantiomer of proxyphylline was prepared in excellent enantiomeric excess (99–99.9% ee), >99% conversion, and the isolated yield ranging from 65% to 74%, depending on the used biocatalyst (ADH-A from Rhodococcus ruber or a variant derived from Lactobacillus kefir, Lk-ADH-Lica). In turn, E. coli/ADH-catalyzed bioreduction of the carbonylic precursor of xanthinol and diprophylline furnished the corresponding (S)-chlorohydrin in >99% ee, >99% conversion, and 80% yield (in the case of Lk-ADH-Lica); while the (R)-counterpart was afforded in 94% ee, 64% conversion, and 41% yield (in the case of SyADH from Sphingobium yanoikuyae). After further chemical functionalization of the key (S)-chlorohydrin intermediate, the desired homochiral (R)-xanthinol (>99% ee) was obtained in 97% yield and (S)-diprophylline (>99% ee) in 90% yield. The devised biocatalytic method is straightforward and thus might be considered practical in the manufacturing of title pharmaceuticals.

doi: 10.1016/j.bioorg.2022.105967

Efficient amide formation is of high importance for the chemical and pharmaceutical industry. The direct biocatalytic one-pot transformation of acids into amides without substrate activation is a highly desirable but highly challenging reaction; this is why in general the acid is activated using additional reagents before amide formation occurs. In particular, amidation of α-amino acids is challenging and in general requires protection strategies for the amino functionality. A further challenge is the low solubility of the unprotected amino acids in organic solvents. Furthermore, the amidation process is prone to racemisation as observed for the acyl chloride derivative. These three challenges may be addressed using biocatalysis. Here the enzyme catalyzed, racemization-free amidation of unprotected L-proline with ammonia in an organic solvent is described. Comprehensive reaction, solvent and enzyme engineering allowed obtaining high L-prolinamide concentrations. For instance at 145 mM substrate concentration, 80% conversion was achieved employing an immobilized CalB variant and ammonia in 2-methyl-2-butanol at 70 °C. A two-fold increase in L-prolinamide formation was achieved employing the immobilized and engineered enzyme variant CalBopt-24 T245S compared to wild type CalB. In contrast to chemical processes, racemization, halogenated solvents and waste are avoided/minimized and atom efficiency is significantly improved from 45.5% to 86.4%. The excellent optical purity of the obtained product (ee >99%) and the stability of immobilized CalB pave the way for an innovative industrial process to produce L-prolinamide, a key intermediate in drug synthesis.

doi: 10.1039/d2gc00783e

Lisofylline (LSF) is a synthetic methylxanthine active agent exhibiting potent anti-inflammatory and immunomodulatory properties; therefore, it has been widely investigated as a promising drug candidate for treating various autoimmune disorders, including type 1 diabetes. In this study, we report on developing a sequential chemoenzymatic one-pot two-step deracemization protocol for racemic LSF. This task was accomplished in a stereo-complementary manner via a tandem bi-enzymatic oxidation–reduction reaction sequence composed of (i) non-selective chemoenzymatic aerobic oxidation of LSF to pentoxifylline (PTX) catalyzed by commercially available laccase from Trametes versicolor (LTv) and 2,2,6,6-tetramethylpiperidinyloxy radical (TEMPO) as a redox mediator, and (ii) stereoselective bioreduction of in situ formed PTX to give enantiomeric LSF, which was catalyzed by home-made lyophilized E. coli cells harboring overexpressed alcohol dehydrogenases (ADHs) with complementary stereospecificity. Firstly, a multi-step optimization procedure of LTv/TEMPO-catalyzed oxidation of LSF allowed achieving dramatic improvement of the conversion rates from an initial 16% up to 95%, demonstrating the high synthetic potency of this method compared to traditional chemical reactions requiring toxic oxidants used in stoichiometric amounts. In turn, separate stereoselective bioreductions of PTX using recombinant ADHs from Rhodococcus ruber (E. coli/ADH-A) and Lactobacillus kefiri (E. coli/LK-ADH Prince) furnished both LSF enantiomers (>99% ee) with high 93–94% conversion and in 65–67% yield range, respectively. The coupling of the above-mentioned chemoenzymatic steps afforded both antipodes of LSF on a preparative scale (0.16 mmol of racemic LSF) in the range of 56–67% yield and 94% ee depending on the employed ADHs.

doi: 10.1039/d2cy00145d

Efficient chemoenzymatic routes toward the synthesis of both enantiomers of adrenergic β-blockers were accomplished by identifying a central chiral building block, which was first prepared using lipase-catalyzed kinetic resolution (KR, Amano PS-IM) as the asymmetric step at a five gram-scale (209 mM conc.). The enantiopure (R)-chlorohydrin (>99% ee) subsequently obtained was used for the synthesis of a series of model (R)-(+)-β-blockers (i.e., propranolol, alprenolol, pindolol, carazolol, moprolol, and metoprolol), which were produced with enantiomeric excess in the range of 96–99.9%. The pharmaceutically relevant (S)-counterpart, taking propranolol as a model, was synthesized in excellent enantiomeric purity (99% ee) via acetolysis of the respective enantiomerically pure (R)-mesylate by using cesium acetate and a catalytic amount of 18-Crown-6, followed by acidic hydrolysis of the formed (S)-acetate. Alternatively, asymmetric reduction of a prochiral ketone, namely 2-(3-chloro-2-oxopropyl)-1H-isoindole-1,3(2H)-dione, was performed using lyophilized E. coli cells harboring overexpressed recombinant alcohol dehydrogenase from Lactobacillus kefir (E. coli/Lk-ADH-Lica) giving the corresponding chlorohydrin with >99% ee. Setting the stereocenter early in the synthesis and performing a 4-step reaction sequence in a ‘one-pot two-step’ procedure allowed the design of a ‘step-economic’ route with a potential dramatic improvement in process efficiency. The synthetic method can serve for the preparation of a broad scope of enantiomerically enriched β-blockers, the chemical structures of which rely on the common α-hydroxy-N-isopropylamine moiety, and in this sense, might be industrially attractive.

doi: 10.1039/d2ra04302e

Many biocatalytic redox reactions depend on the cofactor NAD(P)H, which may be provided by dedicated recycling systems. Exploiting light and water for NADPH-regeneration as it is performed, e.g. by cyanobacteria, is conceptually very appealing due to its high atom economy. However, the current use of cyanobacteria is limited, e.g. by challenging and time-consuming heterologous enzyme expression in cyanobacteria as well as limitations of substrate or product transport through the cell wall. Here we establish a transmembrane electron shuttling system propelled by the cyanobacterial photosynthesis to drive extracellular NAD(P)H-dependent redox reactions. The modular photo-electron shuttling (MPS) overcomes the need for cloning and problems associated with enzyme- or substrate-toxicity and substrate uptake. The MPS was demonstrated on four classes of enzymes with 19 enzymes and various types of substrates, reaching conversions of up to 99 % and giving products with >99 % optical purity.

doi: 10.1002/ange.202207971

The challenges of light-dependent biocatalytic transformations of lipophilic substrates in aqueous media are manifold. For instance, photolability of the catalyst as well as insufficient light penetration into the reaction vessel may be further exacerbated by a heterogeneously dispersed substrate. Light penetration may be addressed by performing the reaction in continuous flow, which allows two modes of applying the catalyst: (i) heterogeneously, immobilized on a carrier, which requires light-permeable supports, or (ii) homogeneously, dissolved in the reaction mixture. Taking the light-dependent photodecarboxylation of palmitic acid catalyzed by fatty-acid photodecarboxylase from Chlorella variabilis (CvFAP) as a showcase, strategies for the transfer of a photoenzyme-catalyzed reaction into continuous flow were identified. A range of different supports were evaluated for the immobilization of CvFAP, whereby Eupergit C250 L was the carrier of choice. As the photostability of the catalyst was a limiting factor, a homogeneous system was preferred instead of employing the heterogenized enzyme. This implied that photolabile enzymes may preferably be applied in solution if repair mechanisms cannot be provided. Furthermore, when comparing different wavelengths and light intensities, extinction coefficients may be considered to ensure comparable absorption at each wavelength. Employing homogeneous conditions in the CvFAP-catalyzed photodecarboxylation of palmitic acid afforded a space-time yield unsurpassed by any reported batch process (5.7 g·L–1·h–1, 26.9 mmol·L–1·h–1) for this reaction, demonstrating the advantage of continuous flow in attaining higher productivity of photobiocatalytic processes.

doi: 10.1021/acscatal.2c04444

The enzymatic kinetic resolution (EKR) of racemic alcohols or esters is a broadly recognized methodology for the preparation of these compounds in optically active form. Although EKR approaches have been developed for the enantioselective transesterification of a vast number of secondary alcohols or hydrolysis of their respective esters, to date, there is no report of bio- or chemo-catalytic asymmetric synthesis of non-racemic alcohols possessing 1,2,3,4-tetrahydroquinoline moiety, which are valuable building blocks for the pharmaceutical industry. In this work, the kinetic resolution of a set of racemic 1,2,3,4-tetrahydroquinoline-propan-2-ols was successfully carried out in neat organic solvents (in the case of CAL-B and BCL) or in water (in the case of MsAcT single variants) using immobilized lipases from Candida antarctica type B (CAL-B) and Burkholderia cepacia (BCL) or engineered acyltransferase variants from Mycobacterium smegmatis (MsAcT) as the biocatalysts and vinyl acetate as irreversible acyl donor, yielding enantiomerically enriched (S)-alcohols and the corresponding (R)-acetates with E-values up to 328 and excellent optical purities (>99% ee). In general, higher ee-values were observed in the reactions catalyzed by lipases; however, the rates of the reactions were significantly better in the case of MsAcT-catalyzed enantioselective transesterifications. Interestingly, we have experimentally proved that enantiomerically enriched 1-(7-nitro-3,4-dihydroquinolin-1(2H)-yl)propan-2-ol undergoes spontaneous amplification of optical purity under achiral chromatographic conditions.

doi: 10.3390/catal12121610

Regioselective carbon−carbon bond formation belongs to the challenging tasks in organic synthesis. In this context, C−C bond formation catalyzed by 4-dimethylallyltryptophan synthases (4-DMATSs) represents a possible tool to regioselectively synthesize C4-prenylated indole derivatives without site-specific preactivation and circumventing the need of protection groups as used in chemical synthetic approaches. In this study, a toolbox of 4-DMATSs to produce a set of 4-dimethylallyl tryptophan and indole derivatives was identified. Using three wild-type enzymes as well as variants, various C5-substituted tryptophan derivatives as well as N-methyl tryptophan were successfully prenylated with conversions up to 90 %. Even truncated tryptophan derivatives like tryptamine and 3-indole propanoic acid were regioselectively prenylated in position C4. The acceptance of C5-substituted tryptophan derivatives was improved up to 5-fold by generating variants (e. g. T108S). The feasibility of semi-preparative prenylation of selected tryptophan derivatives was successfully demonstrated on 100 mg scale at 15 mM substrate concentration, allowing to reduce the previously published multistep chemical synthetic sequence to just a single step.

doi: 10.1002/cbic.202200311

Exploiting light to drive redox reactions is currently a hot topic since light is considered as an environmentally friendly source of energy. Consequently, cyanobacteria, which can use light e.g., for generating NADPH, are in the focus of research. Previously, it has been shown that various heterologous redox enzymes could be expressed in these microorganisms. Here we demonstrated the successful inducer-free expression of α-keto-acid dehydrogenases (L-HicDH and D-HicDH) from Lactobacillus confusus DSM 20196 and Lactobacillus paracasei DSM 20008 in Synechocystis sp. PCC 6803 ΔhoxYH mutant using replicative plasmids. While the L-HicDH showed poor activity limited by the amount of expressed enzyme, the D-HicDH was applied both in vivo and in vitro, transforming the selected α-keto acids to the corresponding optically pure (R)-α-hydroxy acids (ee >99%) in up to 53% and 90% conversion, respectively.

doi: 10.1016/j.engmic.2021.100008

Kurt Faber, a synthetic organic chemist by training, strongly influenced the field of biocatalysis throughout the course of his four-decade career (from the late 1980s to the early 2020s). The evolution of his career has gone hand in hand with the development of biocatalysis into a mature and versatile discipline originally deep-rooted in organic chemistry that quickly integrated techniques from molecular biology. His work has provided numerous grounds for recognizing natural catalysts as essential tools in organic synthesis, complementing the more chemical approaches. An important aspect of Kurt Faber’s career encompasses teaching, mentoring, and encouraging the development of younger scientists worldwide. His book, Biotransformations in Organic Chemistry, now in its seventh edition, has become a recognized tool for training generations of chemists and biotechnologists and remains a reference worldwide. At Biotrans 2021, the 15th International Symposium on Biocatalysis and Biotransformations (originally scheduled to be held in Graz, Austria, and eventually held online due to the COVID-19 pandemic), Kurt Faber received the Biotrans senior award in recognition of his innovative contributions to the field. In this Account, we would like to acknowledge his contributions to the field of biocatalysis, highlighting both the growing importance of this discipline in many (industrial) sectors and the profound changes that have occurred in recent decades, while reflecting on some of Kurt Faber’s most important discoveries and his legacy to this field.

doi: 10.1021/acscatal.2c00579

The concurrent operation of chemical and biocatalytic reactions in one pot is still a challenging task, and, in particular for chemical photocatalysts, examples besides simple cofactor recycling systems are rare. However, especially due to the complementary chemistry that the two fields of catalysis promote, their combination in one pot has the potential to unlock intriguing, unprecedented overall reactivities. Herein we demonstrate a concurrent biocatalytic reduction and photocatalytic oxidation process. Specifically, the enantioselective biocatalytic sulfoxide reduction using (S)-selective methionine sulfoxide reductases was coupled to an unselective light-dependent sulfoxidation. Protochlorophyllide was established as a new green photocatalyst for the sulfoxidation. Overall, a cyclic deracemization process to produce nonracemic sulfoxides was achieved and the target compounds were obtained with excellent conversions (up to 91%) and superb optical purity (>99% ee).

doi: 10.1002/anie.202117103

doi: 10.1002/ange.202117103

The regio- and stereoselective mono-reduction of a particular C=C bond of conjugated C=C double bonds is a very challenging task. Here the regio- and stereoselective 1,4-reduction of pseudoionone, an α,β,γ,δ-bisunsaturated ketone, was demonstrated to give geranylacetone, an industrially relevant molecule. OYE1 from Saccharomyces pastorianus was identified as the most suitable biocatalyst for this reaction. Elevated substrate concentrations of up to 200 mM were tolerated allowing still to reach excellent conversions (>99% and 80% for 100 or 200 mM pseudoionone concentration, respectively). Interestingly, the organic cosolvent often required for substrate solubilization in aqueous buffer can be avoided for pseudoionone when using permeabilized E. coli cells containing the overexpressed enzyme instead of purified enzyme, reaching still >99 % conversion at 100 mM (19.2 g/L) substrate concentration. Performing this reaction at a 0.5 g scale allowed to run the reaction to completion (>99 %) and pure product was isolated with 80 % yield. Additionally, the bis-unsaturated ketone 6-methyl-3,5-heptadien-2-one was transformed under similar conditions giving the floral compound sulcatone with excellent conversion (97 %) and 77 % isolated yield. Finally, the stereoselective reduction of the (E,E)- over the (E,Z)-pseudoionone isomer was enabled by the ene-reductase from Zymomonas mobilis (NCR). Thus, both (E)-geranylacetone and (E,Z)-pseudoionone were obtained with isomeric excess above 60 %.

doi: 110.1002/cctc.202101557

Biocatalysis, using enzymes for organic synthesis, has emerged as powerful tool for the synthesis of active pharmaceutical ingredients (APIs). The first industrial biocatalytic processes launched in the first half of the last century exploited whole-cell microorganisms where the specific enzyme at work was not known. In the meantime, novel molecular biology methods, such as efficient gene sequencing and synthesis, triggered breakthroughs in directed evolution for the rapid development of process-stable enzymes with broad substrate scope and good selectivities tailored for specific substrates. To date, enzymes are employed to enable shorter, more efficient, and more sustainable alternative routes toward (established) small molecule APIs, and are additionally used to perform standard reactions in API synthesis more efficiently. Herein, large-scale synthetic routes containing biocatalytic key steps toward >130 APIs of approved drugs and drug candidates are compared with the corresponding chemical protocols (if available) regarding the steps, reaction conditions, and scale. The review is structured according to the functional group formed in the reaction.

doi: 10.1021/acs.chemrev.1c00574

Efficient synthetic techniques for the diversification of natural products are incremental for drug discovery processes of the pharmaceutical industry because these complex bioactive compounds often require an adjustment of properties. Human liver P450 3A4, key player of the body's detoxification system and decisive factor of a drug's metabolic fate, is renowned for its broad substrate scope including many natural products. In this study, we investigated the synthetic potential of human P450 3A4 for the diversification of natural product classes and isolated the produced metabolites of six selected natural products at a preparative 100-mg scale. Aided by efficient expression levels in P. pastoris, this whole-cell biocatalyst was found to be highly effective at the intended job allowing the identification of a total of 31 authentic human metabolites, many of them for the first time. By revealing an unprecedented degree of diversification, this study extends the synthetic repertoire for efficient enzymatic natural product modification in a one-step fashion and adds a completely new view to an old enzyme traditionally used for inhibition and toxicology studies.

doi: 10.1002/cctc.202101564

The biocatalytic conversion of fatty acids to α-ketoacids was accomplished by the action of two enzymes combined in a simultaneous one-pot two-step cascade. In the first step, P450 monooxygenase from Sphingomonas paucimobilis used hydrogen peroxide in the so-called peroxygenase mode for the regio- and enantioselective formation of α-hydroxyacids. In the next step, these hydroxyacid intermediates were further oxidized to the corresponding α-ketoacids by an α-hydroxyacid oxidase from Aerococcus viridans at the expense of molecular oxygen, thereby regenerating hydrogen peroxide used in the first step. Overall, the cascade was designed to employ catalytic quantities of hydrogen peroxide and proceeded at room temperature in dilute aqueous H2O2 solutions (≤0.01%). This setup could be applied to the conversion of a range of fatty acids (C6:0 to C10:0) and was scaled up to allow the production of 2-oxooctanoic acid in 91% isolated yield.

doi: 10.1007/978-1-0716-1826-4_16

Biocatalysis presents several advantages as synthetic methodology. Oxidoreductases in particular have become highly valuable enzymes to access optically active products in high enantiopurity. Well-established enzymes in industry as well as emerging biocatalysts are herein discussed and include alcohol dehydrogenases, ene-reductases, monooxygenases, and peroxygenases. The chapter provides practical guidelines for the selection and use of these enzymes in synthesis, with a strong focus on the generation of enantiopure molecules. Synthetic protocols are detailed for selected examples applied to asymmetric synthesis.

doi: 10.1002/9783527824465.ch7

Enzymes, at the turn of the 21st century, are gaining a momentum. Especially in the field of synthetic organic chemistry, a broad variety of biocatalysts are being applied in an increasing number of processes running at up to industrial scale. In addition to the advantages of employing enzymes under environmentally friendly reaction conditions, synthetic chemists are recognizing the value of enzymes connected to the exquisite selectivity of these natural (or engineered) catalysts. The use of hydrolases in enantioselective protocols paved the way to the application of enzymes in asymmetric synthesis, in particular in the context of biocatalytic (dynamic) kinetic resolutions. After two decades of impressive development, the field is now mature to propose a panel of catalytically diverse enzymes for (i) stereoselective reactions with prochiral compounds, such as double bond reduction and bond forming reactions, (ii) formal enantioselective replacement of one of two enantiotopic groups of prochiral substrates, as well as (iii) atroposelective reactions with noncentrally chiral compounds. In this review, the major enzymatic strategies broadly applicable in the asymmetric synthesis of optically pure chiral compounds are presented, with a focus on the reactions developed within the past decade. This journal is © The Royal Society of Chemistry.

doi: 10.1039/d1cb00080b

The hydrodefluorination of various α-fluoroketones has been performed under mild reaction conditions and in aqueous medium taking advantage of the promiscuous activity shown by transaminases using a stoichiometric amount of, e. g. 2-propylamine, releasing in this case acetone, ammonia and hydrogen fluoride as by-products. The process could be done in a regio- or even in a stereoselective manner.

doi: 10.1002/cctc.202100901

Unspecific peroxygenases (UPOs) are glycosylated fungal enzymes that can selectively oxidize C–H bonds. UPOs employ hydrogen peroxide as the oxygen donor and reductant. With such an easy-to-handle cosubstrate and without the need for a reducing agent, UPOs are emerging as convenient oxidative biocatalysts. Here, an unspecific peroxygenase from Hypoxylon sp. EC38 (HspUPO) was identified in an activity-based screen of six putative peroxygenase enzymes that were heterologously expressed in Pichia pastoris. The enzyme was found to tolerate selected organic solvents such as acetonitrile and acetone. HspUPO is a versatile catalyst performing various reactions, such as the oxidation of prim- and sec-alcohols, epoxidations, and hydroxylations. Semipreparative biotransformations were demonstrated for the nonenantioselective oxidation of racemic 1-phenylethanol rac-1b (TON = 13 000), giving the product with 88% isolated yield, and the oxidation of indole 6a to give indigo 6b (TON = 2800) with 98% isolated yield. HspUPO features a compact and rigid three-dimensional conformation that wraps around the heme and defines a funnel-shaped tunnel that leads to the heme iron from the protein surface. The tunnel extends along a distance of about 12 Å with a fairly constant diameter in its innermost segment. Its surface comprises both hydrophobic and hydrophilic groups for dealing with substrates of variable polarities. The structural investigation of several protein–ligand complexes revealed that the active site of HspUPO is accessible to molecules of varying bulkiness with minimal or no conformational changes, explaining the relatively broad substrate scope of the enzyme. With its convenient expression system, robust operational properties, relatively small size, well-defined structural features, and diverse reaction scope, HspUPO is an exploitable candidate for peroxygenase-based biocatalysis.

doi: 10.1021/acscatal.1c03065

The substrate scope of the asymmetric allylation with zinc organyls catalyzed by 3,3-bis(2,4,6-triisopropylphenyl)-1,1-binaphthyl-2,2-diyl hydrogenphosphate (TRIP) has been extended to non-cyclic ester organozinc reagents and ketones. Tertiary chiral alcohols are obtained with ee's up to 94% and two stereogenic centers can be created. Compared to the previous lactone reagent the stereopreference switches almost completely, proving the fact that the nature of the organometallic compound is of immense importance for the asymmetry of the product.

doi: 10.1002/adsc.202100037

Alkaloids are a group of natural products with interesting pharmacological properties and a long history of medicinal application. Their complex molecular structures have fascinated chemists for decades, and their total synthesis still poses a considerable challenge. In a previous review, we have illustrated how biocatalysis can make valuable contributions to the asymmetric synthesis of alkaloids. The chemo-enzymatic strategies discussed therein have been further explored and improved in recent years, and advances in amine biocatalysis have vastly expanded the opportunities for incorporating enzymes into synthetic routes towards these important natural products. The present review summarises modern developments in chemo-enzymatic alkaloid synthesis since 2013, in which the biocatalytic transformations continue to take an increasingly ‘central’ role.

doi: 10.1039/d1ra04181a

Demethylating methyl phenyl ethers is challenging, especially when the products are catechol derivatives prone to follow-up reactions. For biocatalytic demethylation, monooxygenases have previously been described requiring molecular oxygen which may cause oxidative side reactions. Here we show that such compounds can be demethylated anaerobically by using cobalamin-dependent methyltransferases exploiting thiols like ethyl 3-mercaptopropionate as a methyl trap. Using just two equivalents of this reagent, a broad spectrum of substituted guaiacol derivatives were demethylated, with conversions mostly above 90%. This strategy was used to prepare the highly valuable antioxidant hydroxytyrosol on a one-gram scale in 97% isolated yield.

doi: 10.1002/anie.202104278

Controlling the selectivity of a chemical reaction with external stimuli is common in thermal processes, but rare in visible-light photocatalysis. Here we show that the redox potential of a carbon nitride photocatalyst (CN-OA-m) can be tuned by changing the irradiation wavelength to generate electron holes with different oxidation potentials. This tuning was the key to realizing photo-chemo-enzymatic cascades that give either the (S)- or the (R)-enantiomer of phenylethanol. In combination with an unspecific peroxygenase from Agrocybe aegerita, green light irradiation of CN-OA-m led to the enantioselective hydroxylation of ethylbenzene to (R)-1-phenylethanol (99% ee). In contrast, blue light irradiation triggered the photocatalytic oxidation of ethylbenzene to acetophenone, which in turn was enantioselectively reduced with an alcohol dehydrogenase from Rhodococcus ruber to form (S)-1-phenylethanol (93% ee).

doi: 10.1002/anie.202100164

The asymmetric amination of secondary racemic allylic alcohols bears several challenges like the reactivity of the bi-functional substrate/product as well as of the α,β-unsaturated ketone intermediate in an oxidation-reductive amination sequence. Heading for a biocatalytic amination cascade with a minimal number of enzymes, an oxidation step was implemented relying on a single PQQ-dependent dehydrogenase with low enantioselectivity. This enzyme allowed the oxidation of both enantiomers at the expense of iron(III) as oxidant. The stereoselective amination of the α,β-unsaturated ketone intermediate was achieved with transaminases using 1-phenylethylamine as formal reducing agent as well as nitrogen source. Choosing an appropriate transaminase, either the (R)- or (S)-enantiomer was obtained in optically pure form (>98 % ee). The enantio-convergent amination of the racemic allylic alcohols to one single allylic amine enantiomer was achieved in one pot in a sequential cascade.

doi: DOI: 10.1002/cctc.202001707

Biocatalysis, using defined enzymes for organic transformations, has become a common tool in organic synthesis, which is also frequently applied in industry. The generally high activity and outstanding stereo-, regio-, and chemoselectivity observed in many biotransformations are the result of a precise control of the reaction in the active site of the biocatalyst. This control is achieved by exact positioning of the reagents relative to each other in a fine-tuned 3D environment, by specific activating interactions between reagents and the protein, and by subtle movements of the catalyst. Enzyme engineering enables one to adapt the catalyst to the desired reaction and process. A well-filled biocatalytic toolbox is ready to be used for various reactions. Providing nonnatural reagents and conditions and evolving biocatalysts enables one to play with the myriad of options for creating novel transformations and thereby opening new, short pathways to desired target molecules. Combining several biocatalysts in one pot to perform several reactions concurrently increases the efficiency of biocatalysis even further.

doi: 10.1021/acscentsci.0c01496

Broad substrate tolerance and excellent regioselectivity, as well as independence from sensitive cofactors have established benzoic acid decarboxylases from microbial sources as efficient biocatalysts. Robustness under process conditions makes them particularly attractive for preparative-scale applications. The divalent metal-dependent enzymes are capable of catalyzing the reversible non-oxidative (de)carboxylation of a variety of electron-rich (hetero)aromatic substrates analogously to the chemical Kolbe-Schmitt reaction. Elemental mass spectrometry supported by crystal structure elucidation and quantum chemical calculations verified the presence of a catalytically relevant Mg²⁺ complexed in the active site of 2,3-dihydroxybenoic acid decarboxylase from Aspergillus oryzae (2,3-DHBD_Ao). This unique example with respect to the nature of the metal is in contrast to mechanistically related decarboxylases, which generally have Zn²⁺ or Mn²⁺ as the catalytically active metal.

doi: 10.1002/cbic.202000600

Flavoenzymes are broadly employed as biocatalysts for a large variety of reactions, owing to the chemical versatility of the flavin cofactor. Oxidases set aside, many flavoenzymes require a source of electrons in form of the biological reductant nicotinamide NAD(P)H in order to initiate catalysis via the reduced flavin. Chemists can take advantage of the reactivity of reduced flavins with oxygen to carry out monooxygenation reactions, while the reduced flavin can also be used for formal hydrogenation reactions. The main advantage of these reactions compared to chemical approaches is the frequent regio-, chemo- and stereo-selectivity of the biocatalysts, which allows the synthesis of chiral molecules in optically active form. This chapter provides an overview of the variety of biocatalytic processes that have been developed with flavoenzymes, with a particular focus on nicotinamide-dependent enzymes. The diversity of molecules obtained is highlighted and in several cases, strategies that allow control of the stereochemical outcome of the reactions are reviewed.

doi: 10.1016/bs.enz.2020.05.001

doi: 10.1002/9781119487043.ch9

A self-sufficient nicotinamide-dependent intramolecular bio-Tishchenko-type reaction was developed. The reaction is catalyzed by alcohol dehydrogenases and proceeds through formal intramolecular hydride transfer on dialdehydes to deliver lactones. Regioselectivity on [1,1′-biphenyl]-2,2′-dicarbaldehyde substrates could be controlled via the electronic properties of the substituents. Preparative scale synthesis provided access to substituted dibenzo[c,e]oxepin-5(7H)-ones.

doi: 10.1039/D0CC02509G

Looking for new ene-reductases with uncovered features beneficial for biotechnological applications, by mining genomes of photosynthetic extremophile organisms, we identified two new Old Yellow Enzyme homologues: CtOYE, deriving from the cyanobacterium Chroococcidiopsis thermalis, and GsOYE, from the alga Galdieria sulphuraria. Both enzymes were produced and purified with very good yields and displayed catalytic activity on a broad substrate spectrum by reducing α,β-unsaturated ketones, aldehydes, maleimides and nitroalkenes with good to excellent stereoselectivity. Both enzymes prefer NADPH but demonstrate a good acceptance of NADH as cofactor. CtOYE and GsOYE represent robust biocatalysts showing high thermostability, a wide range of pH optimum and good co-solvent tolerance. High resolution X-ray crystal structures of both enzymes have been determined, revealing conserved features of the classical OYE subfamily as well as unique properties, such as a very long loop entering the active site or an additional C-terminal alpha helix in GsOYE. Not surprisingly, the active site of CtOYE and GsOYE structures revealed high affinity toward anions caught from the mother liquor and trapped in the anion hole where electron-withdrawing groups such as carbonyl group are engaged. Ligands (para-hydroxybenzaldehyde and 2-methyl-cyclopenten-1-one) added on purpose to study complexes of GsOYE were detected in the enzyme catalytic cavity, stacking on top of the FMN cofactor, and support the key role of conserved residues and FMN cofactor in the catalysis.

doi: 10.1007/s00253-019-10287-2

An immobilized bi-functional redox biocatalyst was designed for the asymmetric reduction of alkenes by nicotinamide-dependent ene-reductases. The biocatalyst, which consists of co-immobilized ene-reductase and glucose dehydrogenase, was implemented in biotransformations in the presence of glucose as source of reducing equivalents and catalytic amounts of the cofactor. Enzyme co-immobilization employing glutaraldehyde activated Relizyme HA403/M as support material was performed directly from the crude cell-free extract obtained after protein overexpression in E. coli and cell lysis, avoiding enzyme purification steps. The resulting optimum catalyst showed excellent level of activity and stereoselectivity in asymmetric reduction reactions using either OYE3 from Saccharomyces cerevisiae or NCR from Zymomonas mobilis in the presence of organic cosolvents in up to 20 vol%. The bi-functional redox biocatalyst, which demonstrated remarkable reusability over several cycles, was applied in preparative-scale synthesis at 50 mM substrate concentration and provided access to three industrially relevant chiral compounds in high enantiopurity (ee up to 97 %) and in up to 42 % isolated yield. The present method highlights the potential of (co-)immobilization of ene-reductases, notorious for their poor scalability, and complements the few existing methods available for increasing productivity in asymmetric bioreduction reactions.

doi: 10.1016/j.jbiotec.2020.08.005

Glycerol is a byproduct of biodiesel production and is generated in large amounts, which has resulted in an increased interest in its valorization. In addition to its use as an energy source directly, the chemical modification of glycerol may result in value-added derivatives. Herein, acid phosphatases employed in the synthetic mode were evaluated for the enzymatic phosphorylation of glycerol. Nonspecific acid phosphatases could tolerate glycerol concentrations up to 80 wt % and pyrophosphate concentrations up to 20 wt % and led to product titers up to 167 g L⁻¹ in a kinetic approach. In the complementary thermodynamic approach, phytases were able to condense glycerol and inorganic monophosphate directly. This unexpected behavior enabled the simple and cost-effective production of rac-glycerol-1-phosphate from crude glycerol obtained from a biodiesel plant. A preparative-scale synthesis on a 100-mL-scale resulted in the production of 16.6 g of rac-glycerol-1-phosphate with a reasonable purity (≈75%).

doi: 10.1002/cssc.201903236

Biocatalysis is increasingly used in combination with light to develop new and more sustainable synthetic methods. Thereby, mostly a chemical photocatalyst harvesting the light energy is combined with an established enzymatic reaction, thus the biocatalyst itself does not require the light for its specific reaction. Here we expand the library of an enzyme which requires light for its natural reaction, namely the light-dependent protochlorophyllide oxidoreductase (LPOR). This enzyme catalyzes the NADPH-dependent reduction of a C=C in a N-heterocycle. Out of five LPORs identified by sequence search, four were found to be well expressible in E. coli and active. Investigating the light intensity, which is an important parameter describing energy input and subsequently may enable fast reaction, it turned out that the four LPORs can stand the maximum light intensity reachable with the equipment used (1450 μmol photons m^–2 s^–1). However, the natural substrate and product were degraded at these conditions, allowing only 15 % of the maximum input (211 μmol photons m^–2 s^–1). Furthermore, the LPORs accepted seven different water miscible solvents with a solvent content of up to 20% v/v and were active at a pH from 6 to 10. While all LPORs known to date are exclusively NADPH dependent, two LPORs identified here were active also with NADH. The cofactor selectivity could be pinned to three amino acid residues, which interestingly do not directly bind to the cofactor.

doi: 10.1002/cctc.202000561.

Stereoselective catalysts for the Pictet–Spengler reaction of tryptamines and aldehydes may allow a simple and fast approach to chiral 1-substituted tetrahydro-β-carbolines. Although biocatalysts have previously been employed for the Pictet–Spengler reaction, not a single one accepts benzaldehyde and its substituted derivatives. To address this challenge, a combination of substrate walking and transfer of beneficial mutations between different wild-type backbones was used to develop a strictosidine synthase from Rauvolfia serpentina (RsSTR) into a suitable enzyme for the asymmetric Pictet–Spengler condensation of tryptamine and benzaldehyde derivatives. The double variant RsSTR V176L/V208A accepted various ortho-, meta- and para-substituted benzaldehydes and produced the corresponding chiral 1-aryl-tetrahydro-β-carbolines with up to 99% enantiomeric excess.

doi: 10.1002/chem.202004449

Regioselective reactions represent a significant challenge for organic chemistry. Here the regioselective methylation of a single hydroxy group of 4-substituted catechols was investigated employing the cobalamin‐dependent methyltransferase from Desulfitobacterium hafniense. Catechols substituted in position four were methylated either in meta- or para-position to the substituent depending whether the substituent was polar or apolar. While the biocatalytic cobalamin dependent methylation was meta‐selective with 4‐substituted catechols bearing hydrophilic groups, it was para‐selective for hydrophobic substituents. Furthermore, the presence of water miscible co‐solvents had a clear improving influence, whereby THF turned out to enable the formation of a single regioisomer in selected cases. Finally, it was found that also the pH led to an enhancement of regioselectivity for the cases investigated.

doi: 10.1002/cctc.202001296

Although enzymes have been found for many reactions, there are still transformations for which no enzyme is known. For instance, not a single defined enzyme has been described for the reduction of the C═N bond of an oxime, only whole organisms. Such an enzymatic reduction of an oxime may give access to (chiral) amines. By serendipity, we found that the oxime moiety adjacent to a ketone as well as an ester group can be reduced by ene-reductases (ERs) to an intermediate amino group. ERs are well-known enzymes for the reduction of activated alkenes, as of α,β-unsaturated ketones. For the specific substrate used here, the amine intermediate spontaneously reacts further to tetrasubstituted pyrazines. This reduction reaction represents an unexpected promiscuous activity of ERs expanding the toolkit of transformations using enzymes.

doi: 10.1021/acscatal.0c03755

The acyltransferase from Mycobacterium smegmatis (MsAcT) complements the well-established acylation activity of hydrolases in organic solvents with its activity to perform acylation reactions (among other reactions) in an aqueous environment. The enzyme’s potential is however limited, due to its poor regio- and stereoselectivity with enantioselectivities (E-values) below 20 for bulky (aromatic) substrates. By applying computer-guided rational design, a library of single variants was designed that allowed conversion of a set of previously challenging substrates with good activity and E-values up to >200. The computational predictions were found to be in agreement with experimental data, which in turn allowed for the generation of even more active and selective double variants. Overall, the produced set of variants provides a toolbox for the enantioselective acylation of challenging alcohols in water, effectively contributing to an alternative to reactions in organic solvents.

doi: 10.1021/acscatal.0c02981

The cleavage of aryl methyl ethers is a common reaction in chemistry requiring rather harsh conditions; consequently, it is prone to undesired reactions and lacks regioselectivity. Nevertheless, O-demethylation of aryl methyl ethers is a tool to valorize natural and pharmaceutical compounds by deprotecting reactive hydroxyl moieties. Various oxidative enzymes are known to catalyze this reaction at the expense of molecular oxygen, which may lead in the case of phenols/catechols to undesired side reactions (e.g., oxidation, polymerization). Here an oxygen-independent demethylation via methyl transfer is presented employing a cobalamin-dependent veratrol-O-demethylase (vdmB). The biocatalytic demethylation transforms a variety of aryl methyl ethers with two functional methoxy moieties either in 1,2-position or in 1,3-position. Biocatalytic reactions enabled, for instance, the regioselective monodemethylation of substituted 3,4-dimethoxy phenol as well as the monodemethylation of 1,3,5-trimethoxybenzene. The methyltransferase vdmB was also successfully applied for the regioselective demethylation of natural compounds such as papaverine and rac-yatein. The approach presented here represents an alternative to chemical and enzymatic demethylation concepts and allows performing regioselective demethylation in the absence of oxygen under mild conditions, representing a valuable extension of the synthetic repertoire to modify pharmaceuticals and diversify natural products.

doi: 10.1021/acscatal.0c02790

3,3-Bis(2,4,6-triisopropylphenyl)-1,1-binaphthyl-2,2-diyl hydrogenphosphate (TRIP) catalyzes the asymmetric allylation of aldehydes with organozinc compounds, leading to highly valuable structural motifs, like precursors to lignan natural products. Our previously reported mechanistic proposal relies on two reaction intermediates and requires further investigation to really understand the mode of action and the origins of stereoselectivity. Detailed ab initio calculations, supported by experimental data, render a substantially different mode of action to the allyl boronate congener. Instead of a Brønsted acid-based catalytic activation, the chiral phosphate acts as a counterion for the Lewis acidic zinc ion, which provides the activation of the aldehyde.

doi: 10.1021/acs.joc.0c00992

An efficient stereoselective syntheses of a series of functionalized optically active γ-aryl-γ-butyrolactones is achieved by enzymatic asymmetric reduction of the corresponding sterically demanding γ-keto esters employing wild-type and recombinant alcohol dehydrogenases. The best stereoselectivities for the reduction via hydrogen transfer was obtained with two short chain dehydrogenases (SDRs) of complementary stereospecificity from Aromatoleum aromaticum, namely the Prelog-specific NADH-dependent (S)-1-phenylethanol dehydrogenase [(S)-PED] and the anti-Prelog-specific (R)-1-(4-hydroxyphenyl)-ethanol dehydrogenase [(R)-HPED], respectively. Biotransformations catalyzed by both enzymes, followed by TFA-catalyzed cyclization of the resulting γ-hydroxy esters, furnished the respective (S)- and (R)-configured products with exquisite optical purity (up to >99% ee). The synthetic value was demonstrated on preparative scale for the asymmetric bioreduction of the model compound, methyl 4-oxo-4-phenylbutanoate, affording optically pure (S)-γ-phenyl-γ-butyrolactone (>99% ee) in 67–74% isolated yield at 89–95% conversion depending on the applied scale.

doi: 10.1002/adsc.201901483

Dihydropinidine is a piperidine alkaloid found in spruce needles that has shown promising antifeedant activity against the large pine weevil, a widespread and economically relevant pest of coniferous tree plantations. Chemo-enzymatic approaches have previously been shown to enable a step-economic access to both enantiomers of this alkaloid, but the scalability of these syntheses is limited. Herein, we report a chemo-enzymatic route to dihydropinidine that is dominated by biocatalytic steps and affords the target alkaloid in excellent stereoisomeric purity (>99% ee and de) and high yield (57% overall) on multigram scale. Our synthesis makes use of a solvent-free, Lewis acid-catalyzed Michael addition and a biocatalytic alternative to Krapcho dealkoxycarbonylation, achieved by pig liver esterase (PLE)-catalyzed ester hydrolysis and acidification, and specifically developed for this purpose, to access a key intermediate, nonane-2,6-dione. This diketone is then converted into dihydropinidine by a concurrent one-pot (cascade) biotransformation catalyzed by a transaminase, an imine reductase, and an alcohol dehydrogenase. High yields and excellent regio- and stereoselectivities of the individual transformations render chromatographic purification of intermediates unnecessary and make it possible to carry out the entire sequence with a final hydrochloride precipitation of the target alkaloid as the sole purification step.

doi: 10.1021/acscatal.9b04611

Light-driven biotransformations in recombinant cyanobacteria allow to employ photosynthetic water-splitting for cofactor-regeneration and thus, to save the use of organic electron donors. The genes of three recombinant imine reductases (IREDs) were expressed in the cyanobacterium Synechocystis sp. PCC 6803 and eight cyclic imine substrates were screened in whole-cell biotransformations. While initial reactions showed low to moderate rates, optimization of the reaction conditions in combination with promoter engineering allowed to alleviate toxicity effects and achieve full conversion of prochiral imines with initial rates of up to 6.3 mM h^–1. The high specific activity of up to 22 U gCDW^–1 demonstrates that recombinant cyanobacteria can provide large amounts of NADPH during whole cell reactions. The excellent optical purity of the products with up to >99% ee underlines the usefulness of cyanobacteria for the stereoselective synthesis of amines.

doi: 10.1002/cctc.201901592

Having identified inconsistencies when repeating literature examples of photochemical transformations and difficulties recreating experimental setups, we devised several criteria that an ideal lab-scale reactor should achieve. Herein, we introduce a versatile photoreactor for high-throughput screening, preparative-scale batch reactions and continuous processing, all with a single light source. The reactor utilizes interchangeable arrays of pseudo‐monochromatic high-power LEDs in a range of synthetically useful wavelengths, combined with excellent temperature control. Moreover, light intensity can be modulated in an accurate and straightforward manner. This system has subsequently been tested on a range of literature methodologies.

doi: 10.1002/cptc.201900203

The Pictet–Spengler reaction is a valuable route to 1,2,3,4-tetrahydro-β-carboline (THBC) and isoquinoline scaffolds found in many important pharmaceuticals. Strictosidine synthase (STR) catalyzes the Pictet–Spengler condensation of tryptamine and the aldehyde secologanin to give (S)-strictosidine as a key intermediate in indole alkaloid biosynthesis. STRs also accept short-chain aliphatic aldehydes to give enantioenriched alkaloid products with up to 99% ee STRs are thus valuable asymmetric organocatalysts for applications in organic synthesis. The STR catalysis of reactions of small aldehydes gives an unexpected switch in stereopreference, leading to formation of the (R)-products. Here we report a rationale for the formation of the (R)-configured products by the STR enzyme from Ophiorrhiza pumila (OpSTR) using a combination of X-ray crystallography, mutational, and molecular dynamics (MD) studies. We discovered that short-chain aldehydes bind in an inverted fashion compared to secologanin leading to the inverted stereopreference for the observed (R)-product in those cases. The study demonstrates that the same catalyst can have two different productive binding modes for one substrate but give different absolute configuration of the products by binding the aldehyde substrate differently. These results will guide future engineering of STRs and related enzymes for biocatalytic applications.

doi: 10.1021/jacs.9b08704

The biocatalytic Friedel–Crafts acylation has been identified recently for the acetylation of resorcinol using activated acetic acid esters for the synthesis of acetophenone derivatives catalyzed by an acyltransferase. Because the wild-type enzyme is limited to acetic and propionic derivatives as the substrate, variants were designed to extend the substrate scope of this enzyme. By rational protein engineering, the key residue in the active site was identified which can be replaced to allow binding of bulkier acyl moieties. The single-point variant F148V enabled the transformation of previously inaccessible medium chain length alkyl and alkoxyalkyl carboxylic esters as donor substrates with up to 99% conversion and up to >99% isolated yield.

doi: 10.1021/acscatal.9b04617

The biocatalytic activity of a so far underexploited alkaline phosphatase, PhoK from Sphingomonas sp. BSAR-1, was extensively studied in transphosphorylation and hydrolysis reactions. The use of high-energy phosphate donors and oligophosphates as suitable phosphate donors was evaluated, as well as the hydrolytic activity on a variety of phosphate monoesters. While substrates bearing free hydroxy group displayed only moderate reactivity as acceptors for transphosphorylation by PhoK, strong hydrolytic activity on a broad variety of phosphate monoesters under alkaline conditions was observed. Site-directed mutagenesis of selected amino acid residues in the active site provided valuable insights on their involvement in enzyme catalysis. The key residue Thr89 so far postulated to engage in enzyme phosphorylation was confirmed to be crucial for catalysis and could be replaced by serine, albeit with much lower catalytic efficiency.

doi: 10.1016/j.bbapap.2019.140291

Acyltransferases isolated from Pseudomonas protegens (PpATase) and Pseudomonas fluorescens (PfATase) have recently been reported to catalyze the Friedel–Crafts acylation, providing a biological version of this classical organic reaction. These enzymes catalyze the cofactor-independent acylation of monoacetylphloroglucinol (MAPG) to diacetylphloroglucinol (DAPG) and phloroglucinol (PG) and have been demonstrated to have a wide substrate scope, making them valuable for potential applications in biocatalysis. Herein, we present a detailed reaction mechanism of PpATase on the basis of quantum chemical calculations, employing a large model of the active site. The proposed mechanism is consistent with available kinetics, mutagenesis, and structural data. The roles of various active site residues are analyzed. Very importantly, the Asp137 residue, located more than 10 Å from the substrate, is predicted to be the proton source for the protonation of the substrate in the rate-determining step. This key prediction is corroborated by site-directed mutagenesis experiments. Based on the current calculations, the regioselectivity of PpATase and its specificity toward non-natural substrates can be rationalized.

doi: 10.1021/acscatal.9b04208

A number of self-sufficient hydride transfer processes have been reported in biocatalysis, with a common feature being the dependence on nicotinamide as a cofactor. This cofactor is provided in catalytic amounts and serves as a hydride shuttle to connect two or more enzymatic redox events, usually ensuring overall redox neutrality. Creative systems were designed to produce synthetic sequences characterized by high hydride economy, typically going in hand with excellent atom economy. Several redox enzymes have been successfully combined in one-pot one-step to allow functionalization of a large variety of molecules while preventing by-product formation. This review analyzes and classifies the various strategies, with a strong focus on efficiency, which is evaluated here in terms of the hydride economy and measured by the turnover number of the nicotinamide cofactor(S). The review ends with a critical evaluation of the reported systems and highlights areas where further improvements might be desirable.

doi: 10.1039/c8cs00903a

Regioselective hydroxylation on inactivated C−H bonds is among the dream reactions of organic chemists. Cytochrome P450 enzymes (CYPs) perform this reaction in general with high regio- and stereoselectivity (e. g. for steroids as substrates). Furthermore, enzyme engineering may allow to tune the properties of the enzyme. Regioselective hydroxylation of shorter or linear molecules (fatty acids), however, remains challenging even with this enzyme class, due to the high similarity of the substrate's backbone carbons and their conformational flexibility. CYPs hydroxylating fatty acids selectively in the chemically more distinct α- or ω-position are well described. In contrast, selective in-chain hydroxylation of fatty acids lacks precedence. The peroxygenase CYP152A1 (P450Bsβ) is a family member that displays fatty acid hydroxylation at both, the α- and β-position, with preference for the α-position. Herein we report the influence of hydrophobic active site residues on the hydroxylation pattern of this enzyme. By site directed mutagenesis and combination of the libraries, double and triple mutation variants were identified, which hydroxylated decanoic acid (C₁₀) with improved regio‐selectivity in the β-position. Variants were identified with a 10-fold increase of the β-regioselectivity (expressed as α/β-ratio) compared to the wild type. In total 103 variants of CYP152A1 (P450Bsβ) were investigated.

doi: 10.1002/cctc.201901679

The oxidation of allylic alcohols is challenging to perform in a chemo- as well as stereo-selective fashion at the expense of molecular oxygen using conventional chemical protocols. Here, we report the identification of a library of flavin-dependent oxidases including variants of the berberine bridge enzyme (BBE) analogue from Arabidopsis thaliana (AtBBE15) and the 5-(hydroxymethyl)furfural oxidase (HMFO) and its variants (V465T, V465S, V465T/W466H and V367R/W466F) for the enantioselective oxidation of sec-allylic alcohols. While primary and benzylic alcohols as well as certain sugars are well known to be transformed by flavin-dependent oxidases, sec-allylic alcohols have not been studied yet except in a single report. The model substrates investigated were oxidized enantioselectively in a kinetic resolution with an E-value of up to >200. For instance HMFO V465S/T oxidized the (S)-enantiomer of (E)-oct-3-en-2-ol (1 a) and (E)-4-phenylbut-3-en-2-ol with E>200 giving the remaining (R)-alcohol with ee>99% at 50% conversion. The enantioselectivity could be decreased if required by medium engineering by the addition of cosolvents (e.g. dimethyl sulfoxide).

doi: 10.1002/adsc.201900921

Regioselective reactions allow the differentiation between two or more chemically identical reactive centers within the same molecule. They are highly desirable transformations in organic synthesis, as they avoid additional chemical operations and sophisticated protection/deprotection strategies. In this context, enzymes, which present exquisite selectivity and reactivity, have been widely employed as catalysts in numerous regioselective transformations. This review focuses on two recently developed biocatalytic processes that present outstanding regioselectity: the transaminase-catalyzed asymmetric amination of di- and triketo compounds, and the stereoselective C–C coupling between phenol derivatives, ammonia and pyruvate for the synthesis of tyrosine analogues, catalyzed by tyrosine phenol lyases. Additionally, elegant and straightforward cascades that have combined the aforementioned biotransformations with other enzymatic and/or chemocatalytic processes are compiled in this contribution. Overall, this review aims to provide a general view of the synthetic possibilities that two relatively recently described regio- and stereoselective biotransformations can provide.

doi: 10.1007/s11244-018-1054-7

Indirubin is a biologically active compound found in Danggui Longhui Wan, which is a traditional Chinese medicine for chronic myelocytic leukemia. In the biosynthesis of indirubin, the formation of indigo, which is a stereoisomer of indirubin, is a major side reaction. Recent findings have suggested that cysteine supplementation shifts product selectivity from indigo to indirubin. Here, we disclose how cysteine is involved in enhancing the product selectivity in the synthesis of indirubin using a flavin-containing monooxygenase from Methylophaga aminisulfidivorans (MaFMO). First, cysteine reacts with indoxyl to synthesize 2-cysteinylindoleninone, inhibiting the dimerization of indoxyl. Second, the reducing power of cysteine allows MaFMO to additionally hydroxylate indoxyl toward isatin, overcoming the problem in biased distribution of two different precursors. Third, cysteine activates isatin to react with 2-cysteinylindoleninone to form indirubin. Based on this revealed mechanism, indirubin derivatives with different indole ring components were synthesized.

doi: 10.1021/acscatal.9b02613

Due to its function as a vasopressor, the vicinal amino alcohol methoxamine is a potential candidate for the treatment of hypotension and incontinence or has applications in ophthalmology. In this study, a biocatalytic method was developed to produce each of the four stereoisomers of methoxamine in a sequential 1-pot 2-step cascade reaction, starting from readily available pyruvate and 2,5-dimethoxybenzaldehyde without intermediate isolation. All four isomers are accessible with high conversions and very good stereoselectivities through the modular combination of carboligases and transaminases with high stereoselectivities. The development of these cascades was made possible, among other factors, by the integration of a recently engineered triple variant of the pyruvate decarboxylase from Acetobacter pasteurianus (ApPDC-E469G-I468A-W543F), which provided access to the intermediate (S)-1-hydroxy-1-(2,5-dimethoxyphenyl)propan-2-one with a high enantiomeric excess of 98%. For the amination of these sterically demanding 2-hydroxy ketones, Bacillus megaterium transaminase in particular has proven to be a highly active and selective catalyst. All four methoxamine stereoisomers were achieved with isomeric contents between 94% and 99% and total conversions of both steps between 59% and 80%. Separation of each isomer without derivatizing was possible using supercritical fluid chomratographie with two columns connected in a row. A preparative scale (75 mL) of the 1-pot 2-step cascade to (1S,2R)-methoxamine including nonoptimized product isolation showed 85 mg HCl salt (46% isolated yield) with 94% purity (NMR) and an isomeric content of 98%.

doi: 10.1021/acscatal.9b01081

Podophyllotoxin is probably the most prominent representative of lignan natural products. Deoxy-, epi-, and podophyllotoxin, which are all precursors to frequently used chemotherapeutic agents, were prepared by a stereodivergent biotransformation and a biocatalytic kinetic resolution of the corresponding dibenzylbutyrolactones with the same 2-oxoglutarate-dependent dioxygenase. The reaction can be conducted on 2 g scale, and the enzyme allows tailoring of the initial, "natural" structure and thus transforms various non-natural derivatives. Depending on the substitution pattern, the enzyme performs an oxidative C−C bond formation by C−H activation or hydroxylation at the benzylic position prone to ring closure.

doi: 10.1002/anie.201900926

The utilization of carbon dioxide as a C₁-building block for the production of valuable chemicals has recently attracted much interest. Whereas chemical CO₂ fixation is dominated by C−O and C−N bond forming reactions, the development of novel concepts for the carboxylation of C-nucleophiles, which leads to the formation of carboxylic acids, is highly desired. Beside transition metal catalysis, biocatalysis has emerged as an attractive method for the highly regioselective (de)carboxylation of electron-rich (hetero)aromatics, which has been recently further expanded to include conjugated α,β-unsaturated (acrylic) acid derivatives. Depending on the type of substrate, different classes of enzymes have been explored for (i) the ortho-carboxylation of phenols catalyzed by metal-dependent ortho-benzoic acid decarboxylases and (ii) the side-chain carboxylation of para-hydroxystyrenes mediated by metal-independent phenolic acid decarboxylases. Just recently, the portfolio of bio-carboxylation reactions was complemented by (iii) the para-carboxylation of phenols and the decarboxylation of electron-rich heterocyclic and acrylic acid derivatives mediated by prenylated FMN-dependent decarboxylases, which is the main focus of this review. Bio(de)carboxylation processes proceed under physiological reaction conditions employing bicarbonate or (pressurized) CO2 when running in the energetically uphill carboxylation direction. Aiming to facilitate the application of these enzymes in preparative-scale biotransformations, their catalytic mechanism and substrate scope are analyzed in this review.

doi: 10.1002/adsc.201900275

No Abstract

doi: 10.1002/adsc.201900610

The ether functionality represents a very common motif in organic chemistry and especially the methyl ether is commonly found in natural products. Its formation and cleavage can be achieved via countless chemical procedures. Nevertheless, since in particular the cleavage often involves harsh reaction conditions, milder alternatives are highly demanded. Very recently, we have reported on a biocatalytic shuttle catalysis concept for reversible cleavage and formation of phenolic O-methyl ethers employing a corrinoid-dependent methyl transferase system from the anaerobic organism Desulfitobacterium hafniense. Here we report the technical study of this system, focusing on the demethylation of guaiacol as model reaction. The optimal buffer-, pH-, temperature- and cofactor-preferences were determined as well as the influence of organic co-solvents. Beside methyl cobalamin also hydroxocobalamin turned out to be a suitable cofactor species, although the latter required activation. Various O-methyl phenyl ethers were successfully demethylated with conversions up to 82% at 10 mM substrate concentration.

doi: 10.1002/adsc.201801590

Isomerization is a fundamental reaction in chemistry. However, isomerization of phenyl methyl ethers has not been described yet. Using a cobalamin-dependent methyl transferase, a reversible shuttle concept was investigated for isomerization of catechol monomethyl ethers. The methyl ether of substituted catechol derivatives was successfully transferred onto the adjacent hydroxy moiety. For instance, the cobalamin-dependent biocatalyst transformed isovanillin to its regioisomer vanillin with significant regioisomeric excess (68% vanillin). To the best of our knowledge, isomerization by methyl transfer employing a methyl transferase has not been reported before.

doi: 10.1021/acscatal.8b05072

Light has received increased attention for various chemical reactions but also in combination with biocatalytic reactions. Because currently only a few enzymatic reactions are known, which per se require light, most transformations involving light and a biocatalyst exploit light either for providing the cosubstrate or cofactor in an appropriate redox state for the biotransformation. In selected cases, a promiscuous activity of known enzymes in the presence of light could be induced. In other approaches, light-induced chemical reactions have been combined with a biocatalytic step, or light-induced biocatalytic reactions were combined with chemical reactions in a linear cascade. Finally, enzymes with a light switchable moiety have been investigated to turn off/on or tune the actual reaction. This Review gives an overview of the various approaches for using light in biocatalysis.

doi: 10.1021/acscatal.9b00656

Immobilization of transaminases creates promising biocatalysts for production of chiral amines in batch or continuous-flow mode reactions. E. coli cells containing overexpressed transaminases of various selectivities and hollow silica microspheres as supporting agent were immobilized by an improved sol-gel process to produce immobilized transaminase biocatalysts with suitable stability and mechanical properties for continuous-flow applications. The immobilized cell-based transaminase biocatalyst proved to be durable and easy-to-use in kinetic resolution of four racemic amines 1a–d. The batch and continuous-flow mode kinetic resolutions with transaminase biocatalyst of opposite stereopreference provided access to both enantiomers of the corresponding amines. By using the most suitable immobilized transaminase biocatalysts, this study describes the first transaminase-based approach for the production of both pure enantiomers of 1-(3,4-dimethoxyphenyl)ethan-1-amine 1d.

doi: 10.3390/catal9050438

Phenolic acid decarboxylase from Bacillus subtilis (BsPAD) converts several p-hydroxycinnamic acid derivatives into the corresponding p-hydroxystyrenes, which are considered to be promising bio-based aromatic chemicals. Despite the enzyme's high activity and stability, the low solubility of its substrates presents severe limitations for the establishment of industrial processes. Accordingly, deep eutectic solvents (DESs) have emerged as interesting alternatives to aqueous or organic solvents and biphasic systems as they offer unique reaction conditions while remaining biocompatible and biodegradable. Herein, we show that BsPAD could tolerate choline chloride (ChCl)-based eutectic solvents with 0–50% water content, which allowed conversion to the corresponding p-hydroxystyrene derivatives (>99%) at substrate loadings of up to 300 mM due to the exceptional solubilizing properties of this solvent. As the enzyme showed some remarkable reactivity differences in DES and water, we further explored the substrate scope of the enzyme and a mutant with increased space in the active site. The comparison of substrates with different substituents on the aryl group indicated that the substrate preference is determined by steric, rather than electronic effects. Furthermore, we report that the choice of the solvent influences the acceptance of different substrates as evidenced by the fact that DES strongly favored the conversion of caffeic acid, which is only poorly converted in aqueous media.

doi: 10.1021/acssuschemeng.9b03455

Light-driven biotransformations in recombinant cyanobacteria allow to employ photosynthetic water-splitting for cofactor-regeneration and thus, to save the use of organic electron donors. The genes of three recombinant imine reductases (IREDs) were expressed in the cyanobacterium Synechocystis sp. PCC 6803 and eight cyclic imine substrates were screened in whole-cell biotransformations. While initial reactions showed low to moderate rates, optimization of the reaction conditions in combination with promoter engineering allowed to alleviate toxicity effects and achieve full conversion of prochiral imines with initial rates of up to 6.3 mM h⁻¹. The high specific activity of up to 22 U g_CDW⁻¹ demonstrates that recombinant cyanobacteria can provide large amounts of NADPH during whole cell reactions. The excellent optical purity of the products with up to >99 %ee underlines the usefulness of cyanobacteria for the stereoselective synthesis of amines.

doi: 10.1002/cctc.201901592

Establishing causal links between bacterial metabolites and human intestinal disease is a significant challenge. This study reveals the molecular basis of antibiotic-associated hemorrhagic colitis (AAHC) caused by intestinal resident Klebsiella oxytoca. Colitogenic strains produce the nonribosomal peptides tilivalline and tilimycin. Here, we verify that these enterotoxins are present in the human intestine during active colitis and determine their concentrations in a murine disease model. Although both toxins share a pyrrolobenzodiazepine structure, they have distinct molecular targets. Tilimycin acts as a genotoxin. Its interaction with DNA activates damage repair mechanisms in cultured cells and causes DNA strand breakage and an increased lesion burden in cecal enterocytes of colonized mice. In contrast, tilivalline binds tubulin and stabilizes microtubules leading to mitotic arrest. To our knowledge, this activity is unique for microbiota-derived metabolites of the human intestine. The capacity of both toxins to induce apoptosis in intestinal epithelial cells—a hallmark feature of AAHC—by independent modes of action, strengthens our proposal that these metabolites act collectively in the pathogenicity of colitis.

doi: 10.1073/pnas.1819154116

In contrast to many chemical dihydroxylation methods, enzymatic epoxide hydrolysis provides an environmentally benign route to vicinal diols, which are important intermediates in the synthesis of fine chemicals and pharmaceuticals. Using epoxide hydrolases, enantiopure diols are accessible under mild conditions. In order to assess the selectivity of epoxide hydrolases on geraniol-derived oxiranes, a range of derivatives were screened against a large variety of enzyme preparations. For nearly all substrates, a matching hydrolase with excellent enantioselectivity (≥95% ee) could be found. In addition, a chemoenzymatic approach for the stereoselective synthesis of furanoid linalool oxide was developed. Combination of enzymatic enantioselective hydrolysis with stereoselective Tsuji‐Trost reaction granted diastereoselective access to trans-(2R,5R)-configured linalool oxide with high diastereomeric and enantiomeric excess (97% de and 97% ee).

doi: 10.1002/adsc.201801094

bis-Tetrahydrofuran acetogenins are a class of natural products displaying highly potent and selective anti-tumor activity. Herein we report a new route to precursors of these natural products, utilizing the pseudo C₂-symmetry in the central bis-tetrahydrofuran fragment. Key steps of our stereoselective chemoenzymatic strategy include the epoxide hydrolase-mediated desymmetrization of meso-epoxides and a cascade cyclization in either "inside-out" or "outside-in" direction, providing stereoselective access to the cores of both bullatacin- and rolliniastatin 1-type acetogenins with 6 stereocenters each from a common mono-epoxide precursor.

doi: 10.1002/ejoc.201801562

Functionalization of aromatic compounds by acylation has considerable significance in synthetic organic chemistry. As an alternative to chemical Friedel-Crafts acylation, the C-acyltransferase from Pseudomonas protegens has been found to catalyze C−C bond formation with non-natural resorcinol substrates. Extending the scope of acyl donors, it is now shown that the enzyme is also able to catalyze C−S bond cleavage prior to C−C bond formation, thus aliphatic and aromatic thioesters can be used as acyl donors. It is worth to mention that this reaction can be performed in aqueous buffer. Identifying ethyl thioacetate as the most suitable acetyl donor, the products were obtained with up to >99% conversion and up to 88% isolated yield without using additional base additives; this represents a significant advancement to prior protocols.

doi: 10.1002/cctc.201801856

Artificial cascade reactions involving biocatalysts have demonstrated a tremendous potential during the recent years. This review just focuses on selected examples of the last year and putting them into context to a previously published suggestion for classification. Subdividing the cascades according to the number of catalysts in the linear sequence, and classifying whether the steps are performed simultaneous or in a sequential fashion as well as whether the reaction sequence is performed in vitro or in vivo allows to organise the concepts. The last year showed, that combinations of in vivo as well as in vitro are possible. Incompatible reaction steps may be run in a sequential fashion or by compartmentalisation of the incompatible steps either by using special reactors (membrane), polymersomes or flow techniques.

doi: 10.1002/cctc.201801063

The majority of cytochrome P450 enzymes (CYPs) predominantly operate as monooxygenases, but recently a class of P450 enzymes was discovered, that can act as peroxygenases (CYP152). These enzymes convert fatty acids through oxidative decarboxylation, yielding terminal alkenes, and through α- and β-hydroxylation to yield hydroxy-fatty acids. Bioderived olefins may serve as biofuels, and hence understanding the mechanism and substrate scope of this class of enzymes is important. In this work, we report on the substrate scope and catalytic promiscuity of CYP OleT_JE and two of its orthologues from the CYP152 family, utilizing α-monosubstituted branched carboxylic acids. We identify α,β-desaturation as an unexpected dominant pathway for CYP OleT_JE with 2-methylbutyric acid. To rationalize product distributions arising from α/β-hydroxylation, oxidative decarboxylation, and desaturation depending on the substrate's structure and binding pattern, a computational study was performed based on an active site complex of CYP OleT_JE containing the heme cofactor in the substrate binding pocket and 2-methylbutyric acid as substrate. It is shown that substrate positioning determines the accessibility of the oxidizing species (Compound I) to the substrate and hence the regio- and chemoselectivity of the reaction. Furthermore, the results show that, for 2-methylbutyric acid, α,β-desaturation is favorable because of a rate-determining α-hydrogen atom abstraction, which cannot proceed to decarboxylation. Moreover, substrate hydroxylation is energetically impeded due to the tight shape and size of the substrate binding pocket.

doi: 10.1021/acscatal.8b03733

C−C bond-forming reactions are key transformations for setting up the carbon frameworks of organic compounds. In this context, Friedel–Crafts acylation is commonly used for the synthesis of aryl ketones, which are common motifs in many fine chemicals and natural products. A bacterial multicomponent acyltransferase from Pseudomonas protegens (PpATase) catalyzes such Friedel–Crafts C-acylation of phenolic substrates in aqueous solution, reaching up to >99% conversion without the need for CoA-activated reagents. We determined X-ray crystal structures of the native and ligand-bound complexes. This multimeric enzyme consists of three subunits: PhlA, PhlB, and PhlC, arranged in a Phl(A₂C₂)₂B₄ composition. The structure of a reaction intermediate obtained from crystals soaked with the natural substrate 1-(2,4,6-trihydroxyphenyl)ethanone together with site-directed mutagenesis studies revealed that only residues from the PhlC subunits are involved in the acyl transfer reaction, with Cys88 very likely playing a significant role during catalysis. These structural and mechanistic insights form the basis of further enzyme engineering efforts directed towards enhancing the substrate scope of this enzyme.

doi: 10.1002/cbic.201800462

Fluorination and trifluoromethylation are indispensable tools in the preparation of modern pharmaceuticals and APIs. Herein we present a concept for the introduction of a trifluoromethyl group into unprotected phenols employing catalytic copper(I) iodide and hydroquinone, tBuOOH, and the Langlois' reagent. The method proceeds under mild conditions and exhibits an extended substrate scope compared to the biocatalytic trifluoromethylation using laccase from Agaricus bisporus. Various functional groups such as aldehydes, esters, ethers, ketones and nitriles were tolerated. The hydroquinone-mediated trifluoromethylation reaction allowed accessing trifluoromethylated phenols, which are cumbersome to prepare via previously known chemical methods.

doi: 10.1002/ejoc.201801111

The acyl transferase from Mycobacterium smegmatis (MsAcT) catalyzes the acyl transfer between a range of primary and secondary alcohols, whereby its outstanding ability is to perform this reaction in aqueous solution. Therefore, MsAcT opens different options for acylation reactions enabling alternatives for many conventionally hydrolytic enzymes used in biocatalysis. Nevertheless, hydrolysis is still a major side reaction of this enzyme. To provide a detailed understanding of the competition between hydrolysis and transesterification reactions, a combination of density functional theory and free energy perturbation methods have been employed. The relative binding free energies and the energy profiles of the chemical steps involved in the reaction were calculated for a number of substrates. The calculations show that the enzyme active site exhibits a higher affinity for substrates with an aromatic ring. The rate-determining step corresponds to the collapse of a negatively charged tetrahedral intermediate in the substrate acylation half-reaction. The intrinsic barriers of the transesterification and hydrolysis half-reactions are calculated to be of similar heights, suggesting that the determining factor in the MsAcT specificity is the higher binding affinity of the active site for the alcohol substrates relative to water. Finally, the influence of the acyl donor on the MsAcT-catalyzed reaction is also investigated by considering different esters in the calculations.

doi: 10.1021/acscatal.8b03360

Eine Wertschöpfung aus natürlichen Kohlenstoffquellen erfordert die Funktionalisierung von biobasierten Chemikalien. Eine atomeffiziente biokatalytische oxidative Kaskade wurde entwickelt, um aus gesättigten Fettsäuren α-Ketosäuren herzustellen. Dafür wurde ein Cytochrom P450 im Peroxygenase‐Modus für die regioselektive α-Hydroxylierung der Fettsäuren gemeinsam mit α-Hydroxysäure-Oxidase(n) für die nachfolgende enantioselektive Oxidation eingesetzt, was zu einer internen Regeneration des Oxidationsmittels H₂O₂ führte. Dadurch konnte die Zersetzung der Ketosäure unterdrückt werden, und die Lebensdauer des Biokatalysators wurde wenig beeinflusst. Die O₂-abhängige Kaskade beruht auf katalytischen Mengen an H₂O₂ und bildet Wasser als einziges Nebenprodukt. Aus Octansäure konnte unter milden Bedingungen 2-Oxooctansäure in einer simultanen zweistufigen Eintopf-Kaskade in wässrigem Puffer mit bis zu >99 % Umsatz ohne Anreicherung der Hydroxysäure hergestellt werden. Das Produkt wurde in präparativem Maßstab mit 91 % Ausbeute isoliert, und die Kaskade wurde für Fettsäuren mit verschiedenen Kettenlängen angewendet (C6:0 bis C10:0).

doi: 10.1002/ange.201710227

Selenoneine, a naturally occurring form of selenium, is the selenium analogue of ergothioneine, a sulfur species with health relevance not only as a purported antioxidant but likely also beyond. Selenoneine has been speculated to exhibit similar effects. To study selenoneine's health properties as well as its metabolic transformation, the pure compound is required. Chemical synthesis of selenoneine, however, is challenging and biosynthetic approaches have been sought. We herein report the biosynthesis and isolation of selenoneine from genetically modified fission yeast Schizosaccharomyces pombe grown in a medium containing sodium selenate. After cell lysis and extraction with methanol, selenoneine was purified by three consecutive preparative reversed-phase HPLC steps. The product obtained at the mg level was characterised by high resolution mass spectrometry, NMR and HPLC/ICPMS. Biosynthesis was found to be a promising alternative to chemical synthesis, and should be suitable for upscaling to produce higher amounts of this important selenium species in the future.

doi: 10.1039/c8mt00200b

The operability and substrate scope of a redesigned vinylphenol hydratase as a single biocatalyst or as part of multienzyme cascades using either substituted coumaric acids or phenols as stable, cheap, and readily available substrates are reported.

doi: 10.1021/acs.orglett.8b02058

Fungal ferulic acid decarboxylases (FDCs) belong to the UbiD-family of enzymes and catalyse the reversible (de)carboxylation of cinnamic acid derivatives through the use of a prenylated flavin cofactor. The latter is synthesised by the flavin prenyltransferase UbiX. Herein, we demonstrate the applicability of FDC/UbiX expressing cells for both isolated enzyme and whole-cell biocatalysis. FDCs exhibit high activity with total turnover numbers (TTN) of up to 55000 and turnover frequency (TOF) of up to 370 min⁻¹. Co‐solvent compatibility studies revealed FDC's tolerance to some organic solvents up 20% v/v. Using the in-vitro (de)carboxylase activity of holo-FDC as well as whole-cell biocatalysts, we performed a substrate profiling study of three FDCs, providing insights into structural determinants of activity. FDCs display broad substrate tolerance towards a wide range of acrylic acid derivatives bearing (hetero)cyclic or olefinic substituents at C3 affording conversions of up to >99%. The synthetic utility of FDCs was demonstrated by a preparative-scale decarboxylation.

doi: 10.1002/cctc.201800643

An easy to use method combining the selectivity of metal chelate affinity binding with strong covalent linking was developed for immobilization of non-specific acid phosphatases bearing a His-tag from crude cell lysate. Silica nanoparticles were grafted with aminopropyl functions which were partially transformed further with EDTA dianhydride to chelators. The heterofunctionalized nanoparticles charged with Ni²⁺ as the most appropriate metal ion were applied as support. First, the His-tagged phosphatases were selectively bound to the metal-chelate functions of the support. Then, the enzyme-charged silica nanoparticles were further stabilized by forming a covalent linkage between nucleophilic moieties at the enzyme surface and free amino groups of the support using neopentylglycol diglycidylether as the most effective bifunctional linking agent. The phosphatase biocatalysts obtained by this method exhibited better phosphate transfer activity with a range of alcohols and PP_i as phosphate donor in aqueous medium applying batch and continuous-flow modes than the ones immobilized on conventional supports. Furthermore, this novel strategy opens up novel possibility for efficient immobilization of other His-tagged recombinant enzymes.

doi: 10.1002/cctc.201800405

Imine reductases (IREDs) have recently become a primary focus of research in biocatalysis, complementing other classes of amine-forming enzymes such as transaminases and amine dehydrogenases. Following in the footsteps of other research groups, we have established a set of IRED biocatalysts by sequence-based in silico enzyme discovery. In this study, we present basic characterisation data for these novel IREDs and explore their activity and stereoselectivity using a panel of structurally diverse cyclic imines as substrates. Specific activities of >1 U/mg and excellent stereoselectivities (ee>99%) were observed in many cases, and the enzymes proved surprisingly tolerant towards elevated substrate loadings. Co-expression of the IREDs with an alcohol dehydrogenase for cofactor regeneration led to whole-cell biocatalysts capable of efficiently reducing imines at 100 mM initial concentration with no need for the addition of extracellular nicotinamide cofactor. Preparative biotransformations on gram scale using these 'designer cells' afforded chiral amines in good yield and excellent optical purity.

doi: 10.1002/cctc.201800607

Stereoselective methods for the synthesis of tetrahydro-β-carbolines are of significant interest due to the broad spectrum of biological activity of the target molecules. In the plant kingdom, strictosidine synthases catalyze the C−C coupling through a Pictet–Spengler reaction of tryptamine and secologanin to exclusively form the (S)-configured tetrahydro-β-carboline (S)-strictosidine. Investigating the biocatalytic Pictet–Spengler reaction of tryptamine with small-molecular-weight aliphatic aldehydes revealed that the strictosidine synthases give unexpectedly access to the (R)-configured product. Developing an efficient expression method for the enzyme allowed the preparative transformation of various aldehydes, giving the products with up to >98% ee. With this tool in hand, a chemoenzymatic two-step synthesis of (R)-harmicine was achieved, giving (R)-harmicine in 67% overall yield in optically pure form.

doi: 10.1002/anie.201803372

The biocatalytic asymmetric disproportionation of aldehydes catalyzed by horse liver alcohol dehydrogenase (HLADH) was assessed in detail on a series of racemic 2-arylpropanals. Statistical optimization by means of design of experiments (DoE) allowed the identification of critical interdependencies between several reaction parameters and revealed a specific experimental window for reaching an 'optimal compromise' in the reaction outcome. The biocatalytic system could be applied to a variety of 2-arylpropanals and granted access in a redox‐neutral manner to enantioenriched (S)-profens and profenols following a parallel interconnected dynamic asymmetric transformation (PIDAT). The reaction can be performed in aqueous buffer at ambient conditions, does not rely on a sacrificial co-substrate, and requires only catalytic amounts of cofactor and a single enzyme. The high atom-efficiency was exemplified by the conversion of 75 mM of rac-2-phenylpropanal with 0.03 mol% of HLADH in the presence of ∼0.013 eq. of oxidized nicotinamide adenine dinucleotide (NAD⁺), yielding 28.1 mM of (S)-2-phenylpropanol in 96% ee and 26.5 mM of (S)-2-phenylpropionic acid in 89% ee, in 73% overall conversion. Isolated yield of 62% was obtained on 100 mg-scale, with intact enantiopurities.

doi: 10.1002/adsc.201800541

The formation of C-C bonds by using CoA independent acyltransferases may have significant impact for novel methods for biotechnology. We report the identification of Pseudomonas strains with CoA-independent acyltransferase activity as well as the heterologous expression of the enzyme in E. coli. The cloning strategies and selected expression studies are discussed. The recombinant acyltransferases were characterized with regard to thermal and storage stability, pH,- and co-solvent tolerance. Moreover, the impact of bivalent metals, inhibitors, and other additives was tested. Careful selection of expression and working conditions led to obtain recombinant acyltransferase form Pseudomonas protegens with up to 11 U mL⁻¹ activity.

doi: 10.1007/s00253-018-9052-z

Undesired product hydrolysis along with large amounts of waste in form of inorganic monophosphate by‐product are the main obstacles associated with the use of pyrophosphate in the phosphatase‐catalyzed synthesis of phosphate monoesters on large scale. In order to overcome both limitations, we screened a broad range of natural and synthetic organic phosphate donors with several enzymes on a broad variety of hydroxyl-compounds. Among them, acetyl phosphate delivered stable product levels and high phospho-transfer efficiency at the lower functional pH-limit, which translated into excellent productivity. The protocol is generally applicable to acid phosphatases and compatible with a range of diverse substrates. Preparative-scale transformations using acetyl phosphate synthesized from cheap starting materials yielded multiple grams of various sugar phosphates with up to 433 g L^–1 h^–1 space-time yield and 75% reduction of barium phosphate waste.

doi: 10.1002/adsc.201800306

Several chemoenzymatic routes have been explored for the preparation of cinacalcet, a calcimimetic agent. Transaminases (TAs) and ketoreductases (KREDs) turned out to be useful biocatalysts for the preparation of key optically active precursors. Thus, the asymmetric amination of 1-acetonaphthone yielded an enantiopure (R)-amine, which can be alkylated in one step to yield cinacalcet. Alternatively, the bioreduction of the same ketone resulted in an enantiopure (S)-alcohol, which was easily converted into the previous (R)-amine. In addition, the reduction was efficiently performed with the KRED and its cofactor co-immobilized on the same porous surface. This self-sufficient heterogeneous biocatalyst presented an accumulated total turnover number (TTN) for the cofactor of 675 after 5 consecutive operational cycles. Finally, in a preparative scale synthesis the TA-based approach was performed in aqueous medium and led to enantiopure cinacalcet in two steps and 50% overall yield.

doi: 10.1002/adsc.201701485

α-, β-, and ω-Hydroxy acids, amino acids, and lactones represent common building blocks and intermediates for various target molecules. This review summarizes artificial cascades published during the last 10 years leading to these products. Renewables as well as compounds originating from fossil resources have been employed as starting material. The review provides an inspiration for new cascade designs and may be the basis to design variations of these cascades starting either from alternative substrates or extending them to even more sophisticated products.

doi: 10.3390/catal8050205

The biocatalytic reduction of activated C=C-bonds is dominated by ene-reductases from the Old Yellow Enzyme family, which gained broad practical use owing to exquisite stereoselectivity combined with wide substrate scope. Protein diversity is fostered by mining distinct protein classes and by implementing protein engineering techniques. Recent efforts are focusing on expanding the chemical complexity of the product portfolio, either through substrate functionalization or design of multi-step reactions. This review also highlights unusual chemistries catalyzed by ene-reductases and presents emerging methodologies developed to bypass the need of natural nicotinamide cofactors.

doi: 10.1016/j.cbpa.2017.12.003

Hydrolysis of organic sulfate esters proceeds by two distinct mechanisms, water attacking at either sulfur (S–O bond cleavage) or carbon (C–O bond cleavage). In primary and secondary alkyl sulfates, attack at carbon is favored, whereas in aromatic sulfates and sulfated sugars, attack at sulfur is preferred. This mechanistic distinction is mirrored in the classification of enzymes that catalyze sulfate ester hydrolysis: arylsulfatases (ASs) catalyze S–O cleavage in sulfate sugars and arylsulfates, and alkyl sulfatases break the C–O bond of alkyl sulfates. Sinorhizobium meliloti choline sulfatase (SmCS) efficiently catalyzes the hydrolysis of alkyl sulfate choline-O-sulfate (k_cat/K_M = 4.8 × 103 s⁻¹ M⁻¹) as well as arylsulfate 4-nitrophenyl sulfate (k_cat/K_M = 12 s⁻¹ M⁻¹). Its 2.8-Å resolution X-ray structure shows a buried, largely hydrophobic active site in which a conserved glutamate (Glu386) plays a role in recognition of the quaternary ammonium group of the choline substrate. SmCS structurally resembles members of the alkaline phosphatase superfamily, being most closely related to dimeric ASs and tetrameric phosphonate monoester hydrolases. Although >70% of the amino acids between protomers align structurally (RMSDs 1.79–1.99 Å), the oligomeric structures show distinctly different packing and protomer–protomer interfaces. The latter also play an important role in active site formation. Mutagenesis of the conserved active site residues typical for ASs, H₂¹⁸O-labeling studies and the observation of catalytically promiscuous behavior toward phosphoesters confirm the close relation to alkaline phosphatase superfamily members and suggest that SmCS is an AS that catalyzes S–O cleavage in alkyl sulfate esters with extreme catalytic proficiency.

doi: 10.1016/j.jmb.2018.02.010

Various flavoprotein oxidases were recently shown to oxidize primary thiols. Herein, this reactivity is extended to sec-thiols by using structure-guided engineering of 5-(hydroxymethyl)furfural oxidase (HMFO). The variants obtained were employed for the oxidative kinetic resolution of racemic sec-thiols, thus yielding the corresponding thioketones and nonreacted R-configured thiols with excellent enantioselectivities (E≥200). The engineering strategy applied went beyond the classic approach of replacing bulky amino acid residues with smaller ones, as the active site was additionally enlarged by a newly introduced Thr residue. This residue established a hydrogen-bonding interaction with the substrates, as verified in the crystal structure of the variant. These strategies unlocked HMFO variants for the enantioselective oxidation of a range of sec-thiols.

doi: 10.1002/anie.201713189

The promiscuous regio- and stereoselective hydration of 4-hydroxystyrenes catalyzed by ferulic acid decarboxylase from Enterobacter sp. (FDC_Es) depends on bicarbonate bound in the active site, which serves as a proton relay activating a water molecule for nucleophilic attack on a quinone methide electrophile. This "cofactor" is crucial for achieving improved conversions and high stereoselectivities for (S)-configured benzylic alcohol products. Similar effects were observed with simple aliphatic carboxylic acids as additives. A rational redesign of the active site by replacing the bicarbonate or acetate "cofactor" with a newly introduced side-chain carboxylate from an adjacent amino acid yielded mutants that efficiently acted as C=C hydratases. A single-point mutation of valine 46 to glutamate or aspartate improved the hydration activity by 40% and boosted the stereoselectivity 39-fold in the absence of bicarbonate or acetate.

doi: 10.1021/acscatal.7b04293

Cytochrome P450 enzymes catalyze a broad set of C–H activation reactions, the most prominent being hydroxylation. This review provides an overview of the regioselectivity (CH₃-terminal, in-chain, and carboxylate-terminal) and the optical purity of the hydroxylation products obtained from fatty acids as far as described, focusing on systems close to preparative application.

doi: 10.1007/s10562-017-2273-4

Norcoclaurine synthases (NCS), catalyzing a Pictet–Spengler reaction in plants as one of the first enzymes in the biosynthetic benzylisoquinoline pathway, are investigated for biocatalytic transformations. The library of NCS available is extended by two novel NCSs from Argemone mexicana (AmNCS1, AmNCS2) and one new NCS from Corydalis saxicola (CsNCS); furthermore, it is shown that the NCS from Papaver bracteatum (PbNCS) is a highly productive catalyst leading to the isoquinoline product with up to >99% e.e. Under certain conditions lyophilized whole Escherichia coli cells containing the various overexpressed NCS turned out to be suitable catalysts. The reaction using dopamine as substrate bears several challenges such as the spontaneous non-stereoselective background reaction and side reactions. The PbNCS enzyme is successfully immobilized on various carriers whereby EziG3 proved to be the best suited for biotransformations. Dopamine showed limited stability in solution resulting in the coating of the catalyst over time, which could be solved by the addition of ascorbic acid (e.g., 1 mg ml⁻¹) as antioxidant.

doi: 10.1002/biot.201700542

Racemization in combination with a kinetic resolution is the base for a dynamic kinetic resolution (DKR). Biocatalytic racemization was successfully performed for a broad scope of sec-alcohols by employing a single alcohol dehydrogenase (ADH) variant from Thermoanaerobacter pseudoethanolicus (formerly T. ethanolicus; TeSADH W110A I86A C295A). The catalyst employed as a lyophilized whole cell preparation or cell free extract, which tolerated various non‐water miscible organic solvents under micro-aqueous or two-phase conditions, whereby cyclohexane and n-hexane suited best. Various concepts for combining the enzymatic racemization with an enzymatic kinetic resolution to achieve overall a bis-enzymatic DKR were evaluated. A proof of concept showed a successful DKR with racemization in aqueous phase combined with acylation in the organic phase.

doi: 10.1002/cctc.201701395

Over the last decades biocatalysis has emerged as an indispensable and versatile tool for the asymmetric synthesis of active pharmaceutical ingredients (APIs). In this context, especially transaminases (TAs) have been successfully used for the preparation of numerous α-chiral, optically pure amines, serving as important building blocks for APIs. Here we elaborate on the development of transaminases recognizing the α-chiral centre adjacent to an aldehyde moiety with aliphatic residues, opening up concepts for novel synthetic routes to the antiepileptic drugs Brivaracetam and Pregabalin. The transformation proceeded via dynamic kinetic resolution (DKR) based on the bio-induced racemisation of the aldehyde enantiomers, enabling the amination of the racemic substrates with quantitative conversions. Medium, substrate as well as enzyme engineering gave access to both (R)- and (S)-enantiomers of the amine precursors of the stereocomplementary drugs in high optical purity, representing a short route to mentioned APIs.

doi: 10.1002/adsc.201701449

The review compiles artificial cascades involving enzymes with a focus on the last 10 years. A cascade is defined as the combination of at least two reaction steps in a single reaction vessel without isolation of the intermediates, whereby at least one step is catalyzed by an enzyme. Additionally, cascades performed in vivo and in vitro are discussed separately, whereby in vivo cascades are defined here as cascades relying on cofactor recycling by the metabolism or on a metabolite from the living organism. The review introduces a systematic classification of the cascades according to the number of enzymes in the linear sequence and differentiates between cascades involving exclusively enzymes and combinations of enzymes with non-natural catalysts or chemical steps. Since the number of examples involving two enzymes is predominant, the two enzyme cascades are further subdivided according to the number, order, and type of redox steps. Furthermore, this classification differentiates between cascades where all reaction steps are performed simultaneously, sequentially, or in flow.

doi: 10.1021/acs.chemrev.7b00033

The functionalization of bio-based chemicals is essential to allow valorization of natural carbon sources. An atom-efficient biocatalytic oxidative cascade was developed for the conversion of saturated fatty acids to α-ketoacids. Employment of P450 monooxygenase in the peroxygenase mode for regioselective α-hydroxylation of fatty acids combined with enantioselective oxidation by α-hydroxyacid oxidase(S) resulted in internal recycling of the oxidant H₂O₂, thus minimizing degradation of ketoacid product and maximizing biocatalyst lifetime. The O₂-dependent cascade relies on catalytic amounts of H₂O₂ and releases water as sole by-product. Octanoic acid was converted under mild conditions in aqueous buffer to 2-oxooctanoic acid in a simultaneous one-pot two-step cascade in up to >99% conversion without accumulation of hydroxyacid intermediate. Scale-up allowed isolation of final product in 91% yield and the cascade was applied to fatty acids of various chain lengths (C6:0 to C10:0).

doi: 10.1002/anie.201710227

The utilization of gaseous carbon dioxide instead of bicarbonate would greatly facilitate process development for enzyme catalyzed carboxylations on a large scale. As a proof-of-concept, 1,3-dihydroxybenzene (resorcinol) was carboxylated in the ortho-position using pressurized CO₂ (∼30–40 bar) catalyzed by ortho-benzoic acid decarboxylases with up to 68% conversion. Optimization studies revealed tight pH-control and enzyme stability as the most important determinants.

doi: 10.1039/c8gc00008e

A series of functionalized α-chiral phosphonates was obtained by enzymatic reduction of the corresponding β-activated vinylphosphonate derivatives employing several ene-reductases of the Old Yellow Enzyme family as biocatalysts. The insufficient activation of the phosphonate group alone was compensated by introduction of an electron-withdrawing group at the β-position, which resulted in high levels of conversion (up to >99%). All active enzymes consistently furnished (R)-configurated products with exquisite stereoselectivity, thereby providing a stereo-complementary approach to current asymmetric hydrogenation protocols on similar compounds. Preparative-scale syntheses performed in aqueous buffer using formate/formate dehydrogenase for recycling of the nicotinamide cofactor granted access to β-keto-, cyano- and ester phosphonates from the (E)-isomers of α,β-unsaturated phosphonates in up to 72% isolated yield and >99% ee.

doi: 10.1002/adsc.201700716

The oxidation of alcohols to the corresponding carbonyl or carboxyl compounds represents a convenient strategy for the selective introduction of electrophilic carbon centres into carbohydrate-based starting materials. The O₂-dependent oxidation of prim-alcohols by flavin-containing alcohol oxidases often yields mixtures of aldehyde and carboxylic acid, which is due to "over-oxidation" of the aldehyde hydrate intermediate. In order to directly convert alcohols into carboxylic acids, rational engineering of 5-(hydroxymethyl)furfural oxidase was performed. In an attempt to improve the binding of the aldehyde hydrate in the active site to boost aldehyde-oxidase activity, two active-site residues were exchanged for hydrogen-bond-donating and -accepting amino acids. Enhanced over-oxidation was demonstrated and Michaelis–Menten kinetics were performed to corroborate these findings.

doi: 10.3390/molecules22122205

The nonribosomal enterotoxin tilivalline was the first naturally occurring pyrrolobenzodiazepine to be linked to disease in the human intestine. Since the producing organism Klebsiella oxytoca is part of the intestinal microbiota and the pyrrolobenzodiazepine causes the pathogenesis of colitis it is important to understand the biosynthesis and regulation of tilivalline activity. Here we report the biosynthesis of tilivalline and show that this nonribosomal peptide assembly pathway initially generates tilimycin, a simple pyrrolobenzodiazepine with cytotoxic properties. Tilivalline results from the non-enzymatic spontaneous reaction of tilimycin with biogenetically generated indole. Through a chemical total synthesis of tilimycin we could corroborate the predictions made about the biosynthesis. Production of two cytotoxic pyrrolobenzodiazepines with distinct functionalities by human gut resident Klebsiella oxytoca has important implications for intestinal disease.

doi: 10.1002/anie.201707737

The Friedel–Crafts acylation is a broadly applied reaction that can be conducted using various types of catalyst. However, a biocatalytic alternative has only been reported recently. In this study, the scope of acetyl donors is described, showing that, in addition to vinyl acetate derivatives, phenyl esters are also suitable donors. Furthermore, it was found that various amines enhance the reaction, whereby the effect do not seem to be correlated to the pH but to the structure of the donor. For instance, 1,4‐diazabicyclo[2.2.2]octane (DABCO) turned out to be a viable alternative to imidazole; however the former performed best at pH 9.85, whereas the latter performed best at pH 8.3.

doi: 10.1002/ejoc.201701079

The utilization of CO₂ as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme-catalyzed para-carboxylation of catechols, employing 3,4-dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMN^iminium species. This study reports on the in vitro reconstitution and activation of a prFMN-dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN-associated 1,3-dipolar cycloadditions in related enzymes.

doi: 10.1002/anie.201708091

The major drawback of using phosphatases for transphosphorylation reactions lies in product depletion caused by the natural hydrolytic activity of the enzymes. Variants of PhoC-Mm from Morganella morganii and NSAP-Eb from Escherichia blattae were studied for their ability to maintain a high product level in the transphosphorylation of various primary alcohols. A single amino acid exchange delivered phosphatase variant PhoC-Mm G92D, which was able to catalyze the phosphorylation of primary alcohols without any major hydrolysis of the formed phosphate esters. The mutation mostly improved the affinity of the enzyme for alcohols, while rate constants of transphosphorylation and hydrolysis were decreased, overall resulting in a superior catalytic efficiency in transphosphorylation compared to hydrolysis. The presence of residual substrate alcohol at a given concentration was crucial to suppress phosphate ester hydrolysis. The present work extends the synthetic applicability of phosphatase variants beyond the previously reported nucleosides and allows preparative-scale production of various primary phosphate esters (yields up to 42%) with high enzyme productivity (TONs up to ∼66,000).

doi: 10.1002/bit.26352

Flavin-containing monooxygenases (FMOs) are emerging as effective players in oxidative drug metabolism. Until recently, the functions of the five human FMO isoforms were mostly linked to their capability of oxygenating molecules containing soft N- and S-nucleophiles. However, the human FMO isoform 5 was recently shown to feature an atypical activity as Baeyer–Villiger monooxygenase. With the aim of evaluating such an alternative entry point in the metabolism of active pharmaceutical ingredients, we selected and tested drug molecules bearing a carbonyl group on an aliphatic chain. Nabumetone and pentoxifylline, two widely used pharmaceuticals, were thereby demonstrated to be efficiently oxidized in vitro by FMO5 to the corresponding acetate esters with high selectivity. The proposed pathways explain the formation of a predominant plasma metabolite of pentoxifylline as well as the crucial transformation of the pro-drug nabumetone into the pharmacologically active compound. Using the recombinant enzyme, the ester derivatives of both drugs were obtained in milligram amounts, purified, and fully characterized. This protocol can potentially be extended to other FMO5 candidate substrates as it represents an effective and robust bench-ready platform applicable to API screening and metabolite synthesis.

doi: 10.1021/acschembio.7b00470

Background: Transaminases have become a key tool in biocatalysis to introduce the amine functionality into a range of molecules like prochiral α-ketoacids and ketones. However, due to the necessity of shifting the equilibrium towards the product side (depending on the amine donor) an efficient amination system may require three enzymes. So far, this well-established transformation has mainly been performed in vitro by assembling all biocatalysts individually, which comes along with elaborate and costly preparation steps. We present the design and characterization of a flexible approach enabling a quick set-up of single-cell biocatalysts producing the desired enzymes. By choosing an appropriate co-expression strategy, a modular system was obtained, allowing for flexible plug-and-play combination of enzymes chosen from the toolbox of available transaminases and/or recycling enzymes tailored for the desired application.
Results: By using a two-plasmid strategy for the recycling enzyme and the transaminase together with chromosomal integration of an amino acid dehydrogenase, two enzyme modules could individually be selected and combined with specifically tailored E. coli strains. Various plug-and-play combinations of the enzymes led to the construction of a series of single-cell catalysts suitable for the amination of various types of substrates. On the one hand the fermentative amination of α-ketoacids coupled both with metabolic and non-metabolic cofactor regeneration was studied, giving access to the corresponding α-amino acids in up to 96% conversion. On the other hand, biocatalysts were employed in a non-metabolic, "in vitro-type" asymmetric reductive amination of the prochiral ketone 4-phenyl-2-butanone, yielding the amine in good conversion (77%) and excellent stereoselectivity (ee = 98%).
Conclusions: The described modularized concept enables the construction of tailored single-cell catalysts which provide all required enzymes for asymmetric reductive amination in a flexible fashion, representing a more efficient approach for the production of chiral amines and amino acids.

doi: 10.1186/s12934-017-0750-5

The catalytic promiscuity of a ferulic acid decarboxylase from Enterobacter sp. (FDC_Es) and phenolic acid decarboxylases (PADs) for the asymmetric conjugate addition of water across the C=C bond of hydroxystyrenes was extended to the N-, C- and S-nucleophiles methoxyamine, cyanide and propanethiol to furnish the corresponding addition products in up to 91% ee. The products obtained from the biotransformation employing the most suitable enzyme/nucleophile pairs were isolated and characterized after optimizing the reaction conditions. Finally, a mechanistic rationale supported by quantum mechanical calculations for the highly (S)-selective addition of cyanide is proposed.

doi: 10.1002/adsc.201700247

Alcohol dehydrogenases are well‐established catalysts for various reduction reactions. However, the reduction of carboxylic acid derivatives has not yet been reported with these enzymes. In this contribution, we demonstrated that carboxylic acid thioesters could be readily reduced by a range of alcohol dehydrogenases, albeit at significantly reduced rates relative to those observed for corresponding ketones. A molecular explanation, especially for the lower turnover rates for thioesters relative to those obtained for ketones, is presented, as is a preliminary substrate scope.

doi: 10.1002/cctc.201700165

The transformation of (abundant) oxygenated biomass-derived building blocks via chemo-enzymatic methods is a valuable concept for accessing useful compounds, as it combines the high selectivity of enzymes and the versatility of chemical catalysts. In this work, we demonstrate a straightforward combination of a phenolic acid decarboxylase (PAD) and palladium on charcoal (Pd/C) that affords the flavor compound 4-ethylguaiacol from ferulic acid. The use of a two-phase system proved to be advantageous in terms of enzyme activity, stability, and volumetric productivity and allows us to carry out the hydrogenation step directly in the organic layer containing exclusively the intermediate, vinylguaiacol. The enzymatic decarboxylation step in the biphasic system afforded 89% conversion of 100 mM (19 g L^–1) ferulic acid with an isolated yield of 75%. By extracting 4-vinylguaiacol continuously into the organic phase, conversion was enhanced to 92% using 170 mM (33 g L^–1) ferulic acid, which was only possible in the continuous extraction and distillation setup developed. The reaction cascade (PAD–Pd/C) is demonstrated at gram scale, affording the target product 4-ethylguaiacol (1.1 g) in 70% isolated yield in a two-step two-pot process. The enzymatic step was characterized in detail to overcome major constraints, and the process favorably compares in terms of the environmental impact with traditional approaches.

doi: 10.1021/acs.oprd.6b00362

The biooxidation of benzylic alcohols catalyzed by (5-hydroxymethyl)furfural oxidase (HMFO) to the corresponding benzaldehydes or benzoic acids was investigated in the presence of various organic (co)solvents. Whereas the enzyme activity decreased at elevated concentrations of water-miscible polar solvents, the presence of (halogenated) hydrocarbons was tolerated up to 90% (v/v), which led to drastically improved conversions of up to >99% in case of hexafluorobenzene. This remarkable effect was correlated with the improved solubility of O₂ in the employed solvents. Reaction rates were boosted by O₂-saturation of solvents.

doi: 10.1016/j.tet.2017.07.038

The asymmetric bioreduction of α,β-unsaturated γ-keto esters using ene-reductases from the Old Yellow Enzyme family proceeds with excellent stereoselectivity and high conversion levels, covering a broad range of acyclic and cyclic derivatives. Various strategies were employed to provide access to both enantiomers, which are versatile precursors of bioactive molecules. The regioselectivity of hydride addition on di-activated alkenes was elucidated by isotopic labeling experiments and showed strong preference for the keto moiety as activating/binding group as opposed to the ester. Finally, chemoenzymatic synthesis of (R)-2-(2-oxocyclohexyl)acetic acid was achieved in high ee on a preparative scale combining enzymatic reduction followed by ester hydrogenolysis.

doi: 10.1039/C6GC02493A

Chemoenzymatic and enzymatic cascade reactions enable the synthesis of complex stereocomplementary 1,3,4-trisubstituted tetrahydroisoquinolines (THIQs) with three chiral centers in a step-efficient and selective manner without intermediate purification. The cascade employs inexpensive substrates (3-hydroxybenzaldehyde and pyruvate), and involves a carboligation step, a subsequent transamination, and finally a Pictet–Spengler reaction with a carbonyl cosubstrate. Appropriate selection of the carboligase and transaminase enzymes enabled the biocatalytic formation of (1R,2S)-metaraminol. Subsequent cyclization catalyzed either enzymatically by a norcoclaurine synthase or chemically by phosphate resulted in opposite stereoselectivities in the products at the C1 position, thus providing access to both orientations of the THIQ C1 substituent. This highlights the importance of selecting from both chemo- and biocatalysts for optimal results.

doi: 10.1002/anie.201705855

The Friedel–Crafts acylation is commonly used for the synthesis of aryl ketones, and a biocatalytic version, which may benefit from the chemo- and regioselectivity of enzymes, has not yet been introduced. Described here is a bacterial acyltransferase which can catalyze Friedel–Crafts C-acylation of phenolic substrates in buffer without the need of CoA-activated reagents. Conversions reach up to >99%, and various C- or O-acyl donors, such as DAPG or isopropenyl acetate, are accepted by this enzyme. Furthermore the enzyme enables a Fries rearrangement-like reaction of resorcinol derivatives. These findings open an avenue for the development of alternative and selective C−C bond formation methods.

doi: 10.1002/anie.201703270

A biocatalytic approach was employed for the asymmetric reduction of sterically demanding ketones to prepare 3-hydroxy-5-oxo-5-phenylpentanoates and 5-hydroxy-3-oxo-5-phenylpentanoates. Screening a collection of microorganisms led to the identification of stereocomplementary microbial strains that provide access to both enantiomers of 3-hydroxy-5-oxo-5-phenylpentanoates and 5-hydroxy-3-oxo-5-phenylpentanoates with high enantiomeric excess (up to 99% ee). Moreover, the application of Saccharomyces cerevisiae gave two diastereomers of 3,5-dihydroxy-5-phenylpentanoates with high enantiomeric excess (up to 99% ee). The applicability of the identified strains was demonstrated by transforming the obtained dihydroxy ester into the chemically valuable lactone (4S,6R)-tetrahydro-4-hydroxy-6-phenyl-pyran-2-one.

doi: 10.1016/j.tetasy.2017.05.005

In order to extend the applicability of the regioselective enzymatic carboxylation of phenols, the substrate scope of o-benzoic acid (de)carboxylases has been investigated towards complex molecules with an emphasis on flavouring agents and polyphenols possessing antioxidant properties. o-Hydroxycarboxylic acid products were obtained with perfect regioselectivity, in moderate to excellent yields. The applicability of this method was proven by the regioselective bio-carboxylation of resveratrol on a preparative scale with 95% yield.

doi: 10.1002/adsc.201601046

Transaminases (TAs) have recently been established as catalysts for the asymmetric, reductive amination of prochiral ketones. Depending on the ketone substrate and the amine donor (the cosubstrate), equilibrium constants may limit high conversions; thus, methods to overcome this limitation are required. Removal of the co-product from the reaction equilibrium through spontaneous, intramolecular reactions has provided a successful solution to this problem; therefore, these amine donors have been named "smart cosubstrates". Here, we present a comparison of various bifunctional amine donors including vicinal diamines as potential structural cosubstrate motifs. Upon TA-catalyzed deamination of 1,2-diamines, spontaneous dimerization of the resulting α-aminoketones and oxidation gave heteroaromatic pyrazines.

doi: 10.1002/ejoc.201700253

Alcohol dehydrogenases are well-established catalysts for various reduction reactions. However, the reduction of carboxylic acid derivatives has not yet been reported with these enzymes. In this contribution, we demonstrated that carboxylic acid thioesters could be readily reduced by a range of alcohol dehydrogenases, albeit at significantly reduced rates relative to those observed for corresponding ketones. A molecular explanation, especially for the lower turnover rates for thioesters relative to those obtained for ketones, is presented, as is a preliminary substrate scope.

doi: 10.1002/cctc.201700165

We report the use of bifunctional starting materials (ketoacids) in a diastereoselective Passerini three-center-two-component reaction. Study of the reaction scope revealed the required structural features for stereoselectivity in the isocyanide addition. In this system, an interesting isomerization of the primary Passerini product – the α-carboxamido lactone – into an atypical product, an α-hydroxy imide, was found to occur under acidic conditions. Furthermore, enantioenriched Passerini products can be generated from an enantioenriched ketoacid obtained by chemoenzymatic synthesis.

doi: 10.1002/ejoc.201601432

The synthesis of a family of pyridines bearing a fluorinated substituent on the aromatic ring has been carried out through two independent and highly stereoselective chemoenzymatic strategies. Short chemical synthetic routes toward fluorinated racemic amines and prochiral ketones have been developed, which served as substrates to explore the suitability of lipases and transaminases in asymmetric biotransformations. The lipase-catalyzed kinetic resolution via acylation of racemic amines proceeded smoothly giving conversions close to 50% and excellent enantioselectivities. Alternatively, the biotransamination of the corresponding prochiral ketones was investigated giving access to both optically pure amine enantiomers using transaminases with complementary selectivity. High to quantitative conversion values were achieved, which allowed the isolation of the amines in moderate to high yields (40–88%). A deeper understanding of the latter process was enabled by performing theoretical calculations on thermodynamic and mechanistic aspects. Calculations showed that the biotransamination reactions are highly favoured by the presence of fluorine atoms and the pyridine ring.

doi: 10.1002/adsc.201600835

The hydrolytic dehalogenation of rac-1,3-dibromobutane catalyzed by the haloalkane dehalogenase LinB from Sphingobium japonicum UT26 proceeds in a sequential fashion: initial formation of intermediate haloalcohols followed by a second hydrolytic step to produce the final diol. Detailed investigation of the course of the reaction revealed favored nucleophilic displacement of the sec-halogen in the first hydrolytic event with pronounced R enantioselectivity. The second hydrolysis step proceeded with a regioselectivity switch at the primary position, with preference for the S enantiomer. Because of complex competition between all eight possible reactions, intermediate haloalcohols formed with moderate to good ee ((S)-4-bromobutan-2-ol: up to 87%). Similarly, (S)-butane-1,3-diol was formed at a maximum ee of 35% before full hydrolysis furnished the racemic diol product.

doi: 10.1002/cbic.201600227

Carbon–carbon bond formation is the key reaction for organic synthesis to construct the carbon framework of organic molecules. The review gives a selection of biocatalytic C–C-bond-forming reactions which have been investigated during the last 5 years and which have already been proven to be applicable for organic synthesis. In most cases, the reactions lead to products functionalized at the site of C–C-bond formation (e.g., α-hydroxy ketones, aminoalcohols, diols, 1,4-diketones, etc.) or allow to decorate aromatic and heteroaromatic molecules. Furthermore, examples for cyclization of (non)natural precursors leading to saturated carbocycles are given as well as the stereoselective cyclopropanation of olefins affording cyclopropanes. Although many tools are already available, recent research also makes it clear that nature provides an even broader set of enzymes to perform specific C–C coupling reactions. The possibilities are without limit; however, a big library of variants for different types of reactions is required to have the specific enzyme for a desired specific (stereoselective) reaction at hand.

doi: 10.1021/acscatal.6b00758

A set of transaminases has been investigated for the biocatalytic amination of 1-(4-chloropyridin-2-yl)alkan-1-ones. The influence of the chain length of the n–1-alkanone at the C-2 position of the pyridine has been studied in the reaction with different (R)- and (S)-selective transaminases. Thus, enantiopure amines were isolated with high purity starting from a wide selection of prochiral ketones. On the one hand, excellent yields (from 97 to >99% conversion, up to 93% isolated yield) and stereoselectivity values (>99% ee for both amine enantiomers) were found for n–1-alkanone linear short chain substituents such as ethanone or propanone. On the other hand, more hindered substrates were accepted only when using evolved enzymes such as an evolved variant of (R)-Arthrobacter (ArRmut11-TA). An initial common structural feature was the presence of a chlorine atom on the C-4 position of the pyridine core, which was found to increase the reactivity of the starting ketone, giving extra versatility for the introduction of other chemical functionalities toward more complex and applicable organic molecules. In order to gain a deeper understanding about the substrate specificity of different transaminases, additional structural features were considered by variation of the acetyl group position on the pyridine ring and the use of related acetophenone derivatives.

doi: 10.1021/acscatal.6b00686

The biocatalytic oxidative tandem decarboxylation of C₇–C₁₈ dicarboxylic acids to terminal C₅–C₁₆ dienes was catalyzed by the P450 monooxygenase OleT with conversions up to 29% for 1,11-dodecadiene (0.49 g L^–1). The sequential nature of the cascade was proven by the fact that decarboxylation of intermediate C₆–C₁₁ ω-alkenoic acids and heptanedioic acid exclusively gave nonconjugated 1,4-pentadiene; scale-up allowed the isolation of 1,15-hexadecadiene and 1,11-dodecadiene; the system represents a short and green route to terminal dienes from renewable dicarboxylic acids.

doi: 10.1002/ejoc.201600358

A case of hydride-independent reaction catalyzed by flavin-dependent ene-reductases from the Old Yellow Enzyme (OYE) family was identified. α-Angelica lactone was isomerized to the conjugated β-isomer in a nicotinamide-free and hydride-independent process. The catalytic cycle of C=C bond isomerization appears to be flavin-independent and to rely solely on a deprotonation–reprotonation sequence through acid–base catalysis. Key residues in the enzyme active site were mutated and provided insight on important mechanistic features. The isomerization of α-angelica lactone by OYE2 in aqueous buffer furnished 6.3 mM β-isomer in 15 min at 30 °C. In presence of nicotinamide adenine dinucleotide (NADH), the latter could be further reduced to γ-valerolactone. This enzymatic tool was successfully applied on semi-preparative scale and constitutes a sustainable process for the valorization of platform chemicals from renewable resources.

doi: 10.1002/cssc.201601363

Organofluorine compounds have become important building blocks for a broad range of advanced materials, polymers, agrochemicals, and increasingly for pharmaceuticals. Despite tremendous progress within the area of fluorination chemistry, methods for the direct introduction of fluoroalkyl-groups into organic molecules without prefunctionalization are still highly desired. Here we present a concept for the introduction of the trifluoromethyl group into unprotected phenols by employing a biocatalyst (laccase), tBuOOH, and either the Langlois' reagent or Baran's zinc sulfinate. The method relies on the recombination of two radical species, namely, the phenol radical cation generated directly by the laccase and the CF₃-radical. Various functional groups such as ketone, ester, aldehyde, ether and nitrile are tolerated. This laccase-catalysed trifluoromethylation proceeds under mild conditions and allows accessing trifluoromethyl-substituted phenols that were not available by classical methods.

doi: 10.1038/ncomms13323

Oxidative cleavage of alkenes is a widely employed process allowing oxyfunctionalization to corresponding carbonyl compounds. Recently, a novel biocatalytic oxidative alkene cleavage activity on styrene derivatives was identified in TM1459 from Thermotoga maritima. In this work we engineered the enzyme by site-saturation mutagenesis of active site amino acids to increase its activity and to broaden its substrate scope. A high-throughput assay for the detection of the ketone products was successfully developed. Several variants with up to twofold improved conversion level of styrene derivatives were successfully identified. Especially, changes in or removal of the C-terminus of TM1459 increased the activity most significantly. These best variants also displayed a slightly enlarged substrate scope.

doi: 10.3389/fmicb.2016.01511

The enzymatic phosphorylation of phenoxyethanol, 2-hydroxyethyl acrylate and 4-hydroxybutyl acrylate catalyzed by acid phosphatases PhoN-Sf and PiACP at the expense of inorganic di-, tri-, hexameta- or polyphosphate was applied to the preparative-scale synthesis of phosphorylated compounds. The reaction conditions were optimized with respect to enzyme immobilization, substrate concentration, pH and type of phosphate donor. The mild reaction conditions prevented undesired polymerization and hydrolysis of the acrylate ester moiety. Application of a continuous flow system allowed facile scale-up and mono-phosphates were obtained in up to 26% isolated yield with space-time yields of 0.89 kg L⁻¹ h⁻¹.

doi: 10.1016/j.jbiotec.2016.07.009

Flavin-containing mono-oxygenases are known as potent drug-metabolizing enzymes, providing complementary functions to the well-investigated cytochrome P450 mono-oxygenases. While human FMO isoforms are typically involved in the oxidation of soft nucleophiles, the biocatalytic activity of human FMO5 (along its physiological role) has long remained unexplored. In this study, we demonstrate the atypical in vitro activity of human FMO5 as a Baeyer–Villiger mono-oxygenase on a broad range of substrates, revealing the first example to date of a human protein catalyzing such reactions. The isolated and purified protein was active on diverse carbonyl compounds, whereas soft nucleophiles were mostly non- or poorly reactive. The absence of the typical characteristic sequence motifs sets human FMO5 apart from all characterized Baeyer–Villiger mono-oxygenases so far. These findings open new perspectives in human oxidative metabolism.

doi: 10.1021/acschembio.5b01016

Readily available phenol derivatives were substituted in para-position via a C–C bond formation to give enantiomerically pure (R)- or (S)-3-(para-hydroxyphenyl) lactic acids. The transformation was achieved by designing a biocatalytic cascade consisting of three linear steps, namely, (i) the C–C coupling of the phenol and pyruvate in the presence of ammonia to afford the corresponding L-tyrosine derivative, followed by (ii) oxidative deamination and (iii) enantioselective reduction. Compatibility analysis showed that the reaction rate of the first step is slowed in the presence of the product of the third step; consequently, the three-step cascade was subdivided in two modules (module 1 = step 1; module 2 = steps 2 and 3), which were run in one pot sequentially. Because of the exquisite selectivity achieved in the C–C coupling step, para-isomers were obtained exclusively. By choosing the appropriate alcohol dehydrogenase, the (R)- as well as the (S)-isomer were isolated in enantiopure form. Preparative transformations of 2-, 3-, and 2,3-disubstituted phenols (23–96 mM) afforded the corresponding (R)- and (S)-para-hydroxyphenyl lactic acids in high yield (58%–85%) and enantiopure form (ee > 97%).

doi: 10.1021/acscatal.6b00030

The biocatalytic stereoselective synthesis of a sterically demanding sec-alcohol (ethyl 3-hydroxy-5-phenylpentanoate) was investigated by starting from the corresponding prochiral ketone. Screening of a collection of microorganisms led to the identification of stereocomplementary catalysts suitable for accessing both enantiomers of the target compound. Cofactors, recycling systems and 2-propanol amounts were optimized for selected biocatalysts, leading to excellent enantiomeric excesses for the obtained hydroxy ester with up to 99% ee. The utility of the identified strains was showcased by using preparative-scale reactions.

doi: 10.1002/ejoc.201501460

Transaminases are valuable enzymes for industrial biocatalysis and enable the preparation of optically pure amines. For these transformations they require either an amine donor (amination of ketones) or an amine acceptor (deamination of racemic amines). Herein transaminases are shown to react with aromatic β-fluoroamines, thus leading to simultaneous enantioselective dehalogenation and deamination to form the corresponding acetophenone derivatives in the absence of an amine acceptor. A series of racemic β-fluoroamines was resolved in a kinetic resolution by tandem hydrodefluorination/deamination, thus giving the corresponding amines with up to greater than 99% ee. This protocol is the first example of exploiting the catalytic promiscuity of transaminases as a tool for novel transformations.

doi: 10.1002/anie.201510554

The (+)- as well as the (−)-enantiomer of the pyrrolizidine alkaloid xenovenine were prepared within five steps with 17 and 30% overall yields, respectively, in optically pure form, >99% ee as well as >99% de. In the asymmetric key step a transaminase performed a regio- and stereoselective monoamination of a triketone. By employing two enantiocomplementary transaminases from Arthrobacter sp. both enantiomers were accessible. The triketone was readily prepared via two steps starting from commercially available, achiral 2-(n-heptyl)furan. In the final catalytic hydrogenation step, the newly introduced chiral centre directed hydrogen addition to form preferentially the desired (5Z,8E)-diastereomer. The regio- and stereoselective amination of a single ketone moiety out of three allowed the performance of the shortest and highest yielding total synthesis of the bicyclic showcase pyrrolizidine alkaloid without the need for protecting strategies.

doi: 10.1002/adsc.201500781

(S)-Strictosidine represents the first key intermediate in the biosynthesis of several pharmaceutically relevant monoterpenoid indole alkaloids. Optically pure C3-methyl-substituted strictosidine derivatives were prepared by setting up the two stereogenic centers at the β-carboline core via two enzymatic steps catalyzed by the enzymes transaminase and strictosidine synthase in a one-pot cascade fashion. The two enzymatic steps were performed simultaneously as well as in a stepwise fashion. The amination of the prochiral ketones led to optically pure amines with up to >98% enantiomeric excess. Depending on the enzyme used, the (S)- and (R)-enantiomers were prepared in most cases. Selected amines were then condensed with secologanin in a Pictet–Spengler reaction catalyzed by strictosidine synthase leading to diastereomerically pure products (>98% diastereomeric excess).

doi: 10.1021/acscatal.5b01839

A set of phosphatases was evaluated for their potential to catalyze the regio- and stereoselective phosphorylation of alcohols using a high-energy inorganic phosphate donor, such as di-, tri- and polyphosphate. Parameters such as type and amount of phosphate donor and pH of the reaction were investigated in order to minimize the thermodynamically favored hydrolysis of the phosphate donor and the formed phosphate ester. Diols were monophosphorylated with high selectivities. This biocatalytic phosphorylation method provides selectively activated and/or protected synthetic intermediates for further chemical and/or enzymatic transformations and is applicable to a large scale (6.86 g) in a flow setup with immobilized phosphatase.

doi: 10.1002/ejoc.201501306

N-Dealkylation methods are well described for organic chemistry and the reaction is known in nature and drug metabolism; however, to our knowledge, enantioselective N-dealkylation has not been yet reported. In this study, exclusively the (S)-enantiomers of racemic N-ethyl tertiary amines (1-benzyl-N-ethyl-1,2,3,4-tetrahydroisoquinolines) were dealkylated to give the corresponding secondary (S)-amines in an enantioselective fashion at the expense of molecular oxygen. The reaction is catalyzed by the berberine bridge enzyme, which is known for C–C bond formation. The dealkylation was demonstrated on a 100 mg scale and gave optically pure dealkylated products (ee > 99%).

doi: 10.1002/anie.201507970

L-Tyrosine derivatives were obtained in >97% ee via a biocatalytic one-pot two-step cascade using substituted benzenes, pyruvate, and NH₃ as starting materials. In the first step, monosubstituted arenes were regioselectively hydroxylated in the o-position by monooxygenase P450 BM3 (using O₂ as oxidant with NADPH-recycling) to yield the corresponding phenols, which subsequently underwent C–C coupling and simultaneous asymmetric amination with pyruvate and NH₃ using tyrosine phenol lyase to furnish L-DOPA surrogates in up to 5.2 g L^–1. Instead of analytically pure arenes, crude aromatic gasoline blends containing toluene were used to yield 3-methyl-L-tyrosine in excellent yield (2 g L^–1) and >97% ee.

doi: 10.1021/acscatal.5b02129

During the last decade the use of ω-transaminases has been identified as a very powerful method for the preparation of optically pure amines from the corresponding ketones. Their immense potential for the preparation of chiral amines, together with their ease of use in combination with existing biocatalytic methods, have made these biocatalysts a competitor to any chemical methodology for (asymmetric) amination. An increasing number of examples, especially from industry, shows that this biocatalytic technology outmaneuvers existing chemical processes by its simple and flexible nature. In the last few years numerous publications and patents on synthetic routes, mainly to pharmaceuticals, involving ω-transaminases have been published. The review gives an overview of the application of ω-transaminases in organic synthesis with a focus on active pharmaceutical ingredients (APIs) and the developments during the last few years.

doi: 10.1002/ejoc.201500852

A novel biocatalytic oxidative alkene cleavage activity was identified in protein TM1459 from Thermotoga maritima, a so far uncharacterised metalloprotein with a cupin fold, which preferentially binds manganese (over iron and zinc). Various styrene derivatives were converted with high chemoselectivity to the corresponding carbonyl compounds by the manganese-containing protein, using organic hydroperoxide and molecular oxygen as oxidant. 4-Chloroacetophenone could be obtained in 40% conversion from 4-chloro-α-methylstyrene (50 mM) in a biphasic system using ethyl acetate as organic cosolvent (5% v/v), while 76% conversion was obtained at a lower substrate concentration (10 mM). This novel biocatalyst can be easily over-expressed in Escherichia coli in exceptionally high yield and purified, and thus may offer a valuable and safer alternative in oxidative C=C bond cleavage reactions for synthetic applications.

doi: 10.1002/adsc.201500608

The antiglaucoma agent travoprost, which is an analogue of the prostaglandin PGF_2α, was synthesized by means of a three-component coupling utilizing chemoenzymatically generated building blocks in high enantiopurity.

doi: 10.1002/cctc.201500587

Amination of non-activated aliphatic fatty alcohols to the corresponding primary amines was achieved through a five-enzyme cascade reaction by coupling a long-chain alcohol oxidase from Aspergillus fumigatus (LCAO_Af) with a ω-transaminase from Chromobacterium violaceum (ω-TA_Cv). The alcohol was oxidized at the expense of molecular oxygen to yield the corresponding aldehyde, which was subsequently aminated by the PLP-dependent ω-TA to yield the final primary amine product. The overall cascade was optimized with respect to pH, O₂ pressure, substrate concentration, decomposition of H₂O₂ (derived from alcohol oxidation), NADH regeneration, and biocatalyst ratio. The substrate scope of this concept was investigated under optimized conditions by using terminally functionalized C₄–C₁₁ fatty primary alcohols bearing halogen, alkyne, amino, hydroxy, thiol, and nitrile groups.

doi: 10.1002/cctc.201500589

Readily available substituted phenols were coupled with pyruvate in buffer solution under atmospheric conditions to afford the corresponding para-vinylphenol derivatives while releasing only one molecule of CO₂ and water as the by-products. This transformation was achieved by designing a biocatalytic system that combines three biocatalytic steps, namely the C–C coupling of phenol and pyruvate in the presence of ammonia, which leads to the corresponding tyrosine derivative, followed by deamination and decarboxylation. The biocatalytic transformation proceeded with high regioselectivity and afforded exclusively the desired para products. This method thus represents an environmentally friendly approach for the direct vinylation of readily available 2-, 3-, or 2,3-disubstituted phenols on preparative scale (0.5 mmol) that provides vinylphenols in high yields (65–83%).

doi: 10.1002/anie.201505696

Alcohols are a rich source of compounds from renewable sources, but they have to be activated in order to allow the modification of their carbon backbone. The latter can be achieved via oxidation to the corresponding aldehydes or ketones. As an alternative to (thermodynamically disfavoured) nicotinamide-dependent alcohol dehydrogenases, alcohol oxidases make use of molecular oxygen but their application is under-represented in synthetic biotransformations. In this review, the mechanism of copper-containing and flavoprotein alcohol oxidases is discussed in view of their ability to accept electronically activated or non-activated alcohols and their propensity towards over-oxidation of aldehydes yielding carboxylic acids. In order to facilitate the selection of the optimal enzyme for a given biocatalytic application, the substrate tolerance of alcohol oxidases is compiled and discussed: Substrates are classified into groups (non-activated prim- and sec-alcohols; activated allylic, cinnamic and benzylic alcohols; hydroxy acids; sugar alcohols; nucleotide alcohols; sterols) together with suitable alcohol oxidases, their microbial source, relative activities and (stereo)selectivities.

doi: 10.1007/s00253-015-6699-6

The enzymatic oxidative decarboxylation of linear short-chain fatty acids (C4:0–C9:0) employing the P450 monooxygenase OleT, O₂ as the oxidant, and NAD(P)H as the electron donor gave the corresponding terminal C₃ to C₈ alkenes with product titers of up to 0.93 g L⁻¹ and TTNs of >2000. Key to this process was the construction of an efficient electron-transfer chain employing putidaredoxin CamAB in combination with NAD(P)H recycling at the expense of glucose, formate, or phosphite. This system allows for the biocatalytic production of industrially important 1-alkenes, such as propene and 1-octene, from renewable resources for the first time.

doi: 10.1002/anie.201502925

The potential of a number of enantiocomplementary ω-transaminases (ω-TAms) in the amination of cyclic ketones has been investigated. After a preliminary screening of several compounds with increasing complexity, different approaches to shift the equilibrium of the reaction to the amine products were studied, and reaction conditions (temperature and pH) optimised. Interestingly, 2-propylamine as an amine donor was tolerated by all five selected ω-TAms, and therefore used in further experiments. Due to the higher conversions observed and interest in chiral amines studies then focused on the amination of α-tetralone and 2-methylcyclohexanone. Both ketones were aminated to give the corresponding amine with at least one of the employed enzymes. Moreover, the amination of 2-methylcyclohexanone was investigated in more detail due to the different stereoselectivities observed with TAms used. The highest yields and stereoselectivities were obtained using the ω-TAm from Chromobacterium violaceum (CV-TAm), producing 2-methylcyclohexylamine with complete stereoselectivity at the (1S)-amine position and up to 24:1 selectivity for the cis:trans [(1S,2R):(1S,2S)] isomer.

doi: 10.1039/c5ob01204j

Zinc-dependent alcohol dehydrogenases (ADHs) are a class of enzymes applied in different biocatalytic processes ranging from lab to industrial scale. However, one drawback is the limited substrate range, necessitating a whole array of different ADHs for the relevant substrate classes. In this study, we investigated structural determinants of the substrate spectrum in the zinc-dependent ADH carbonyl reductase 2 from Candida parapsilosis (CPCR2), combining methods of mutational analysis with in silico substrate docking. Assigned active site residues were genetically randomized, and the resulting mutant libraries were screened with a selection of challenging carbonyl substrates. Three variants (C57A, W116K, and L119M) with improved activities toward different substrates were detected at neighboring positions in the active site. Thus, all possible combinations of the mutations were generated and characterized for their substrate specificity, yielding several improved variants. The most interesting were a C57A variant, with a 27-fold increase in specific activity for 4´-acetamidoacetophenone, and the double mutant CPCR2 B16-(C57A, L119M), with a 45-fold improvement in the k_cat·K_M⁻¹ value. The obtained variants were further investigated by in silico docking experiments. The results indicate that the mentioned residues are structural determinants of the substrate specificity of CPCR2, being major players in the definition of the active site. Comparison of these results with closely related enzymes suggests that these might even be transferred to other ADHs.

doi: 10.1002/cbic.201500100

We report on a ‘green’ method for the utilization of carbon dioxide as C1 unit for the regioselective synthesis of (E)-cinnamic acids via regioselective enzymatic carboxylation of para-hydroxystyrenes. Phenolic acid decarboxylases from bacterial sources catalyzed the β-carboxylation of para-hydroxystyrene derivatives with excellent regio- and (E/Z)-stereoselectivity by exclusively acting at the β-carbon atom of the C=C side chain to furnish the corresponding (E)-cinnamic acid derivatives in up to 40% conversion at the expense of bicarbonate as carbon dioxide source. Studies on the substrate scope of this strategy are presented and a catalytic mechanism is proposed based on molecular modelling studies supported by mutagenesis of amino acid residues in the active site.

doi: 10.1002/adsc.201401028

A biocatalytic system is presented for the stereoselective amination of ketones at the expense of NH₃ and molecular hydrogen. By using a NAD⁺-reducing hydrogenase, an alanine dehydrogenase, and a suitable ω-transaminase, the R- as well as the S-enantiomer of various amines could be prepared with up to >99% ee and 98% conversion..

doi: 10.1021/acs.orglett.5b01154

In contrast to the widely studied asymmetric bioreduction of α,β-unsaturated carboxylic acid esters catalyzed by ene-reductases, the reaction applied to lactones remains unexplored. A broad set of ene-reductases was found to reduce various α-, β- and γ-substituted α,β-unsaturated butyrolactones to yield the corresponding saturated non-racemic lactones. Substitution patterns greatly influenced activities and stereoselectivities and lactone products were obtained in moderate to excellent yields; importantly, enzyme-based stereocontrol allowed access to both enantiomers in up to >99% ee. Chiral recognition of a distant γ-center led to kinetic resolution with remarkable enantioselectivities (E values up to 49). An unprecedented case of dynamic kinetic resolution was observed with 3-methyl-5-phenylfuran-2(5H)-one, whereby spontaneous racemization of the substrate furnished the product in up to 73% conversion and >99% ee and 96% de.

doi: 10.1002/adsc.201500094

The asymmetric reductive bio-amination of prochiral aromatic propargyl ketones led to the corresponding amines in optically pure form (ee >99%). The (R)- as well as the (S)-enantiomers of the propargylic amines were obtained, employing either (R)-selective ω-transaminases (ω-TAs) originating from Arthrobacter sp. and Aspergillus terreus or an (S)-selective ω-TA from Chromobacterium violaceum. The product propargylic amines were obtained with high conversions (up to 99%). To simplify product isolation, protection of the free amino group to the corresponding acetamides or benzamides was performed without loss of optical puritiy. The final products were isolated in moderate to good yields (33–67% over two steps) in optical pure form without additional purification steps. Although propargyl ketones are described in the literature to be irreversible inhibitors for aminotransferases, suitable ω-transaminases were identified for the amination of these compounds.

doi: 10.1002/adsc.201500086

Chiral amines are important building blocks for fine chemicals and pharmaceuticals. Consequently, various biocatalytic routes in particular using ω-transaminases (ω-TAs) have been developed recently. Although catalysts for the synthesis of both enantiomers are available, the application of alanine dependent (R)-selective ω-TAs is less favourable due to the requirement of the more expensive D-alanine as an amine donor. Here we describe an efficient method for (R)-amination using ω-TAs in combination with an alanine racemase (AlaR). In this case, the readily available L-alanine can be used as an amine donor leading to improved atom efficiency and significantly reduced costs.

doi: 10.1039/c4gc02363c

Chiral amines represent a prominent functional group in pharmaceuticals and agrochemicals and are hence attractive targets for asymmetric synthesis. Since the pharmaceutical industry has identified biocatalysis as a valuable tool for synthesising chiral molecules with high enantiomeric excess and under mild reaction conditions, enzymatic methods for chiral amine synthesis are increasing in importance. Among the strategies available in this context, the asymmetric reduction of imines by NAD(P)H-dependent enzymes and the related reductive amination of ketones have long remained underrepresented. However, recent years have witnessed an impressive progress in the application of natural or engineered imine-reducing enzymes, such as imine reductases, opine dehydrogenases, amine dehydrogenases, and artificial metalloenzymes. This review provides a comprehensive overview of biocatalytic imine reduction and reductive amination of ketones, highlighting the natural roles, substrate scopes, structural features, and potential application fields of the involved enzymes.

doi: 10.1002/adsc.201500213

doi: 10.1002/adsc.201500450

Anthranoyl-CoA monooxygenase/reductase (ACMR) participates in an unusual pathway for the degradation of aromatic compounds in Azoarcus evansii. It catalyzes the monooxygenation of anthranoyl-CoA to 5-hydroxyl-2-aminobenzoyl-CoA and the subsequent reduction to the dearomatized product 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. The two reactions occur in separate domains, termed the monooxygenase and reductase domain. Both domains were reported to utilize FAD as a cofactor for hydroxylation and reduction, respectively. We have heterologously expressed ACMR in Escherichia coli BL21 and found that the monooxygenase domain contains FAD. However, the reductase domain utilizes FMN and not FAD for the reduction of the intermediate 5-hydroxyl-2-aminobenzoyl-CoA. A homology model for the reductase domain predicted a topology similar to the Old Yellow Enzyme family, which exclusively bind FMN, in accordance with our results. Binding studies with 2-aminobenzoyl-CoA (AbCoA) and p-hydroxybenzaldehyde (pHB) as probes for the monooxygenase and reductase domain, respectively, indicated that two functionally distinct and independent active sites exist. Given the homodimeric quartenary structure of ACMR and the compact shape of the dimer as determined by small-angle X-ray scattering experiments we propose that the monooxygenase and reductase domain of opposite peptide chains are involved in the transformation of anthranoyl-CoA to 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA.

doi: 10.1016/j.bbapap.2015.03.011

During the last decade, the number of different types of enzymes applicable for organic synthesis as biocatalysts has significantly increased. Consequently, the spectrum of reactions has significantly expanded also for cyclisations. This review highlights heterologously expressable biocatalysts transforming non-natural substrates for the formation of three- to six-membered carbo- and heterocycles, excluding terpene cyclases as well as SAM-dependent enzymes. The review focuses on the non-natural substrate scope and the mechanism of the selected enzymes.

doi: 10.1016/j.biotechadv.2015.01.012

Cobalamine cofactors (vitamin B12) are complex organometallic molecules that are crucial for the activity of a variety of different interesting enzymes such as isomerases, methyltransferases, and dehalogenases. Developments in understanding the structure, mechanism, and role in nature of methylcobalamin-dependent methyltransferases make them excellent candidates for biotechnological applications such as biocatalytic dealkylation.

doi: 10.1016/j.tibtech.2015.03.011

The astonishing efficiency with which living organisms build complex molecules from simple starting materials has inspired chemists for centuries. Among the synthetic strategies that nature uses to achieve this efficiency, the combination of several enzymatic transformations in cascading sequences is of outstanding importance. With the rise of biocatalysis, researchers now have the tools at hand to mimic this strategy and develop artificial enzyme cascades of impressive complexity. This editorial review aims to introduce the reader to some key aspects of (chemo)enzymatic cascades, as well as to put the submissions to the present Special Issue into a broader context.

doi: 10.1016/j.molcatb.2014.12.007

E. coli cells that contain overexpressed alcohol dehydrogenases (ADHs) were screened as biocatalysts for the stereoselective reduction of chloroketones 5a–d, the corresponding halohydrins 6a–d of which are building blocks in the synthesis of antiretroviral drugs. Among them, ADH from Sphingobium yanoikuyae was found to reduce chloroketone 5c with a high stereoselectivity (90% de) and conversion (85%) to furnish threo halohydrin (R,S)-6c. ADH from Ralstonia sp. (RasADH) was able to reduce 5a and 5b with complementary diastereoselectivity to provide access to both threo and erythro halohydrins through “substrate-based” stereocontrol. The RasADH-catalyzed reductions were optimized to provide (R,S)-6a with 98% conversion and 84% diastereomeric excess (de) and (S,S)-6b with 95% conversion and 86% de. Molecular modeling studies showed that 5b, which features a carboxybenzyl protecting group, is able to bind to the enzyme catalytic site in an “inverted” mode in comparison to tert-butyloxycarbonyl- and methyloxycarbonyl-protected substrates 5a and 5c, which sheds light on the observed switching of the stereopreference. RasADH-catalyzed reductions were optimized to provide (R,S)-6a with 98% conversion and 84% de and (S,S)-6b with 95% conversion and 86% de.

doi: 10.1002/cctc.201403023

Combinatorial assembly and variation of promoters on a single expression plasmid allowed the balance of the catalytic steps of a three enzyme (L-AAD, HIC, FDH) cascade in E. coli. The designer cell catalyst quantitatively transformed L-amino acids to the corres- ponding optically pure (R)- and (S)-α-hydroxy acids at up to 200 mM substrate concentration.

doi: 10.1039/c4cc08286a

A mild one-pot methodology has been developed to deracemise rac-2-phenyl-1-propanol by combining the use of non-selective laccase/TEMPO-mediated oxidation with enantioselective bioreduction of the racemic aldehyde intermediate under dynamic conditions. The process was easily scalable and stereocontrollable by selecting the suitable biocatalyst.

doi: 10.1039/c4cy01351d

Four enzymes showing hydrolytic activity on derivatives of 2-azabicyclo[2.2.1]hept-5-en-3-one (Vince lactam) were successfully identified through analysis of protein crystal structure and amino acid sequence alignments. Enantiocomplementary activities were observed on Vince lactam and its saturated analog 2-azabicyclo[2.2.1]heptan-3-one with non-heme chloroperoxidase (CPO-T) from Streptomyces aureofaciens, cyclic imide hydrolase (CIH) from Pseudomonas putida, polyamidase (NfpolyA) from Nocardia farcinica, and amidase (AMI) from Rhodococcus globerulus, and perfect kinetic resolution was achieved (E>200). Computational analysis of amide bond resonance stabilization in lactams correlated well with the overall reactivity pattern of the lactams as a function of ring size and strain. The biocatalysts cloned and investigated in this study could be of interest for the synthesis of enantiopure carbocyclic nucleoside analogues.

doi: 10.1002/cctc.201402077

The amination of racemic α-chiral aldehydes, 2-phenylpropanal derivatives, was investigated employing ω-transaminases. By medium and substrate engineering the optical purity of the resulting β-chiral chiral amine could be enhanced to reach optical purities up to 99% ee. Using enantiocomplementary ω-transaminases allowed us to access the (R)- as well as the (S)-enantiomer in most cases. It is important to note that the stereopreference of the ω-transaminases found for α-chiral aldehydes did not correlate with the stereopreference previously observed for the amination of methyl ketones. In one case the stereopreference switched even upon exchanging a methyl substituent to a methoxy group.

doi: 10.1002/adsc.201400217

The substrate scope of inverting alkylsulfatase Pisa1 was extended towards benzylic sec-sulfate esters by suppression of competing non-enzymatic autohydrolysis by addition of dimethyl sulfoxide as co-solvent. Detailed investigation of the mechanism of autohydrolysis in ¹⁸O-labeled buffer by using an enantiopure sec-benzylic sulfate ester as substrate revealed that from the three possible pathways (i) inverting S_N2-type nucleophilic attack of [OH^–] at the benzylic carbon represents the major pathway, whereas (ii) S_N1-type formation of a planar benzylic carbenium ion leading to racemization was a minor event, and (iii) Retaining S_N2-type nucleophilic attack at sulfur took place at the limits of detection. The data obtained are interpreted by analysis of Hammett constants of meta substituents.

doi: 10.1002/ejoc.201402211

The combination of two cofactor self-sufficient biocatalytic cascade modules allowed the successful transformation of cyclohexanol into the nylon-6 monomer 6-aminohexanoic acid at the expense of only oxygen and ammonia. A hitherto unprecedented carboxylic acid capping strategy was introduced to minimize the formation of the dead-end intermediate 6-hydroxyhexanoic acid. For this purpose, the precursor ε-caprolactone was converted in aqueous medium in the presence of methanol into the corresponding methyl ester instead of the acid. Hence, it was shown for the first time that esterases—specifically horse liver esterase—can perform the selective ring-opening of ε-caprolactone with a clear preference for methanol over water as the nucleophile.

doi: 10.1002/anie.201409227

E. coli cells containing overexpressed (R)-selective ω-transaminase and the cofactor PLP were immobilized on methacrylate beads suitable for continuous flow applications. The use of an organic solvent suppresses leaching of PLP from the cells; no additional cofactor was required after setting up the packed-bed reactor containing the biocatalyst (ω-TA-PLP). Non-natural ketone substrates were transformed in flow with excellent enantioselectivity (>99% ee). Features of this novel system include high-throughput (30–60 min residence time), clean production (no quench, workup, or purification required), high enzyme stability (the packed-bed reactor can be continuously operated for 1–10 days), and excellent mass recovery.

doi: 10.1021/ol502712v

An efficient route for the synthesis of all four diastereomers of PMP-protected α-amino-γ-butyrolacton to access γ-hydroxynorvaline was established. The asymmetric key steps comprise an organocatalytic Mannich reaction and an enzymatic ketone reduction. Three reaction steps could be integrated in a one-pot process, using 2-PrOH both as solvent and as reducing agent. The sequential construction of stereogenic centres gave access to each of the four stereoisomers in high yield and with excellent stereocontrol.

doi: 10.1039/c4cc06230b

Valinol is part of numerous pharmaceuticals and has various other important applications. Optically pure valinol (ee >99%) was prepared employing different ω-transaminases from the corresponding prochiral hydroxy ketone. By the choice of the enzyme the (R)- as well as the (S)-enantiomer were accessible. Reductive amination was performed in organic solvent (MTBE) using 2-propyl amine as amine donor whereas alanine was applied in aqueous medium. Transformations in phosphate buffer were successfully performed even at 200 mM substrate concentration (20.4 g/L) leading to 99% (R) and 94% (S) conversion with perfect optical purity (>99% ee).

doi: 10.1016/j.bmc.2014.05.055

Strictosidine synthases catalyze the formation of strictosidine, a key intermediate in the biosynthesis of a large variety of monoterpenoid indole alkaloids. Efforts to utilize these biocatalysts for the preparation of strictosidine analogs have however been of limited success due to the high substrate specificity of these enzymes. We have explored the impact of a protein engineering approach called circular permutation on the activity of strictosidine synthase from the Indian medicinal plant Rauvolfia serpentina. To expedite the discovery process, our study departs from the usual process of creating a random protein library, followed by extensive screening. Instead, a small, focused library of circular permutated variants of the six bladed β-propeller protein was prepared, specifically probing two regions which cover the enzyme active site. The observed activity changes suggest important roles of both regions in protein folding, stability and catalysis.

doi: 10.1016/j.bmc.2014.06.023

Chemo-enzymatic deracemisation was applied to obtain the (S)-enantiomer of 1-benzylisoquinolines from the racemate in high isolated yield (up to 85%) and excellent optical purity (ee > 97%). The one-pot deracemisation protocol encompassed enantioselective oxidation by a monoamine oxidase (MAO-N) and concomitant reduction of the resulting iminium species by ammonia-borane. The challenge was the oxidation at the sterically demanding chiral centre. Recently developed variants of MAO-N, featuring an enlarged active-site pocket, turned out to be suitable biocatalysts for these substrates. In contrast to previous MAO-N variants, which preferentially converted the (S)-enantiomer, the MAO-N variant D11 used in the present study was found to oxidise all tested benzylisoquinoline substrates with (R)-enantiopreference. The structural determinants of enantioselectivity were investigated by means of protein–ligand docking simulations. The applicability of the deracemisation system was demonstrated on preparative scale (150 mg) for three benzylisoquinoline alkaloids (natural as well as non-natural), including the hypotensive and antispasmodic agent (S)-reticuline.

doi: 10.1039/c4cy00642a

Natural L-α-amino acids and L-norleucine were transformed to the corresponding α-hydroxy acids by formal biocatalytic inversion or retention of absolute configuration. The one-pot transformation was achieved by a concurrent oxidation reduction cascade in aqueous media. A representative panel of enantiopure (R)- and (S)-2-hydroxy acids possessing aliphatic, aromatic and heteroaromatic moieties were isolated in high yield (67–85%) and enantiopure form (>99% ee) without requiring chromatographic purification.

doi: 10.1002/chem.201403195

Background: Bioprocesses depend on a number of different operating parameters and temperature is one of the most important ones. Unfortunately, systems for rapid determination of temperature dependent reaction kinetics are rare. Obviously, there is a need for a high-throughput screening procedure of temperature dependent process behavior. Even though, well equipped micro-bioreactors are a promising approach sufficient temperature control is quite challenging and rather complex.
Results: In this work a unique system is presented combining an optical on-line monitoring device with a customized temperature control unit for 96 well microtiter plates. By exposing microtiter plates to specific temperature profiles, high-throughput temperature optimization for microbial and enzymatic systems in a micro-scale of 200 μL is realized. For single well resolved temperature measurement fluorescence thermometry was used, combining the fluorescent dyes Rhodamin B and Rhodamin 110. The real time monitoring of the microbial and enzymatic reactions provides extensive data output. To evaluate this novel system the temperature optima for Escherichia coli and Kluyveromyces lactis regarding growth and recombinant protein production were determined. Furthermore, the commercial cellulase mixture Celluclast as a representative for enzymes was investigated applying a fluorescent activity assay.
Conclusion: Microtiter plate-based high-throughput temperature profiling is a convenient tool for characterizing temperature dependent reaction processes. It allows the evaluation of numerous conditions, e.g. microorganisms, enzymes, media, and others, in a short time. The simple temperature control combined with a commercial on-line monitoring device makes it a user friendly system.

doi: 10.1186/1754-1611-8-22

The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites (‘catalophores’). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C–C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts.

doi: 10.1038/ncomms5150

Starting from an adequate ketone precursor previous reports required three steps for the preparation of (R)-2,3,4,9-tetrahydro-1H-carbazol-3-amine, a key intermediate for the synthesis of the antiallergic drug ramatroban. A single biocatalytic step was sufficient to prepare the target amine with >97% ee (HPLC) via reductive amination of the corresponding ketone using an ω-transaminase as biocatalyst. Since the ketone was barely soluble under the reaction conditions employed, it was provided as a solid and still the reaction went to completion within 4 h at 50 mM substrate concentration. Although 2-propylamine is regarded as an ideal amine donor, it turned out to be detrimental for the specific ketone precursor leading to the formation of various side products. These could be avoided by using (R)-1-phenylethylamine as the best suited amine donor. An alternative work-up was developed via freeze-drying of the reaction mixture, enabling the isolation of the desired (R)-amine in excellent yield (96%) and enantiopure form on a preparative scale (500 mg). No purification steps (e.g., column chromatography, crystallisation) were required.

doi: 10.1002/adsc.201300993

An efficient and sustainable biocatalytic route for the synthesis of important 17-α-amino steroids has been developed using an ω-transaminase variant from Arthrobacter sp. Optimisation of the reaction conditions facilitated the synthesis of these valuable synthons on a preparative scale, affording excellent isolated yields and stereocontrol.

doi: 10.1039/c3cc49080g

Asymmetric bioreduction of an (E)-β-cyano-2,4-dienoic acid derivative by ene-reductases allowed a shortened access to a precursor of pregabalin [(S)-3-(aminomethyl)-5-methylhexanoic acid] possessing the desired configuration in up to 94% conversion and >99% ee. Deuterium labelling studies showed that the nitrile moiety was the preferred activating/anchor group in the active site of the enzyme over the carboxylic acid or the corresponding methyl ester.

doi: 10.1002/adsc.201301055

Retro-Friedel–Crafts hydrolases are co-factor independent enzymes with unusual reactivity and selectivity. These unique hydrolases are scarcely studied for biocatalytical applications in organic chemistry yet, although many other hydrolytic enzymes (e.g. lipases) are commonly applied as catalysts. Two Friedel–Crafts hydrolases were selected, namely 2,6-diacetylphloroglucinol hydrolase (PhlG) from Pseudomonas fluorescens and phloretin hydrolase from Eubacterium ramulus (Phy), to test the suitability of these enzymes in synthetic applications. The activity and stability of PhlG and Phy as lyophilized cells or lyophilized crude extracts were investigated in the presence of organic co-solvents. It was shown, that by careful selection of the co-solvent the enzymes catalyse C–C hydrolysis in a buffer solvent mixture with improved conversions at 50 mM substrate concentration. However, attempts to catalyze C–C-bond formation in organic solvents were unsuccessful.

doi: 10.1007/s11244-013-0193-0

Phloretin hydrolase from Eubacterium ramulus (Phy) catalyzes the hydrolysis of the dihydrochalcone phloretin to phloroglucinol and phloretic acid, performing a formal retro- Friedel–Crafts acylation reaction on its substrate. Its closest sequence homolog, of 25% amino acid sequence identity, is diacetyl phloroglucinol hydrolase (Phlg) from Pseudomonas fluorescens, which catalyses a similar, hydrolytic, de-acylation of its substrate. The structure of Phlg has been determined and a catalytic mechanism proposed (J Biol Chem 285:4603–4611, 2010). In order to compare the catalytic characteristics of Phy with Phlg, the gene encoding Phy was expressed and the enzyme purified and crystallised. An X-ray fluorescence scan identified zinc within the crystals. A homology model of Phy, based on the structure of Phlg (PDB code 3HWP), informed the construction of a point mutant library of the enzyme, targeting residues shared with Phlg that are thought to be involved in zinc binding and the recognition of acyl and phenol functionality on the aromatic ring of the substrates. Mutation of His123, His251, Glu154 and Glu255 (conserved zinc binding residues) resulted in variants that were either poorly expressed, or of much reduced activity; Mutation of Tyr115 and His203, thought to bind the phenol groups in the 1-and 3-positions of the phloroglucinol ring respectively, resulted in variants of 15-fold reduced activity and an inactive variant. These results are suggestive of conservation of some aspects of mechanism and substrate recognition between Phy and Phlg, and of the catalytic characteristics of Zn-dependent C–C hydrolases of this type in general.

doi: 10.1007/s11244-013-0196-x

The bioreduction and disproportionation of cyclohex-2-enone catalyzed by Old Yellow Enzyme 1 was investigated in presence of organic (co)solvents. Whereas the NADH-dependent bioreduction activity strongly decreased at elevated co-solvent concentrations due to the insolubility of the nicotinamide-cofactor, the NADH-free disproportionation was significantly improved in water-immiscible organic co-solvents at 90 % (v/v) with near-quantitative conversion. This positive effect was attributed to removal of the inhibiting co-product, phenol, from the enzyme's active site. The best co-solvents show high lipophilicity (logP) and a high potential to solubilize phenol (K_phenol). As a predictive parameter, the ratio of logP/K_phenol should be preferably ≥100.

doi: 10.1007/s10529-014-1494-5

The enzyme catalyzed carboxylation of electron-rich phenol derivatives employing recombinant benzoic acid decarboxylases at the expense of bicarbonate as CO₂ source is reported. In contrast to the classic Kolbe–Schmitt reaction, the biocatalytic equivalent proceeded in a highly regioselective fashion exclusively at the ortho-position of the phenolic directing group in up to 80% conversion. Several enzymes were identified, which displayed a remarkably broad substrate scope encompassing alkyl, alkoxy, halo and amino-functionalities. Based on the crystal structure and molecular docking simulations, a mechanistic proposal for 2,6-dihydroxybenzoic acid decarboxylase is presented.

doi: 10.1039/c3ra47719c

Deracemization, that is, the transformation of a racemate into a single product enantiomer with theoretically 100% conversion and 100% ee, is an appealing but also challenging option for asymmetric synthesis. Herein a novel chemo-enzymatic deracemization concept by a cascade is described: the pathway involves two enantioselective oxidation steps and one non-stereoselective reduction step, enabling stereoinversion and a simultaneous kinetic resolution. The concept was exemplified for the transformation of rac-benzylisoquinolines to optically pure (S)-berbines. The racemic substrates were transformed to optically pure products (ee>97%) with up to 98% conversion and up to 88% yield of isolated product.

doi: 10.1002/anie.201400027

The oxidation of the renewable diols isosorbide and isomannide was successfully achieved using a TEMPO/laccase system. Furthermore, various TEMPO-derivatives were tested leading to conversions of up to >99% for the oxidation of isosorbide, isomannide, indanol and a halohydrin to the corresponding ketone.

doi: 10.1039/c3gc41855c

Alcohol dehydrogenases (ADHs) were identified as suitable enzymes for the reduction of the corresponding α,α-dihalogenated ketones, obtaining optically pure β,β-dichloro- or β,β-dibromohydrins with excellent conversions and enantiomeric excess. Among the different biocatalysts tested, ADHs from Rhodococcus ruber (ADH-A), Ralstonia sp. (RasADH), Lactobacillus brevis (LBADH), and PR2ADH proved to be the most efficient ones in terms of activity and stereoselectivity. In a further study, two racemic α-substituted ketones, namely α-bromo- α-chloro- and α-chloro-α-fluoroacetophenone were investigated to obtain one of the four possible diastereoisomers through a dynamic kinetic process. In the case of the brominated derivative, only the (1R)-enantiomer was obtained by using ADH-A, although with moderate diastereomeric excess (>99% ee, 63% de), whereas the fluorinated ketone exhibited a lower stereoselectivity (up to 45% de).

doi: 10.1002/cctc.201300834

Various enantiocomplementary ω-transaminases (ωTAs) were investigated in kinetic resolution and asymmetric reductive amination reactions to prepare silodosin amine. Whilst the enzymatic kinetic resolution gave moderate to good results with respect to the yield and enantioselectivity, the asymmetric reductive amination proved to be superior. The best results were obtained with the ωTA originating from (R)-Arthrobacter sp. which afforded the desired bioactive (R)-enantiomer in enantiomerically pure form (ee >97%) at excellent conversion (conv. >97%) under mild and benign reaction conditions.

doi: 10.1016/j.tetasy.2013.12.012

Lactobacillus brevis ADH (LBADH) is an alcohol dehydrogenase that is commonly employed to reduce alkyl or aryl ketones usually bearing a methyl, an ethyl or a chloromethyl as a small ketone substituent to the corresponding (R)-alcohols. Herein we have tested a series of 24 acetophenone derivatives differing in their size and electronic properties for their reduction employing LBADH. After plotting the relative activity against the measured substrate volumes we observed that apart from the substrate size other effects must be responsible for the activity obtained. Compared to acetophenone (100% relative activity), other small substrates such as propiophenone, α,α,α-trifluoroacetophenone, α-hydroxyacetophenone, and benzoylacetonitrile had relative activities lower than 30%, while medium-sized ketones such as α-bromo-, α,α-dichloro-, and α,α-dibromoacetophenone presented relative activities between 70% and 550%. Moreover, the comparison between the enzymatic activity and the obtained final conversions using an excess or just 2.5 equiv. of the hydrogen donor 2-propanol, denoted again deviations between them. These data supported that these hydrogen transfer (HT) transformations are mainly thermodynamically controlled. For instance, bulky α-halogenated derivatives could be quantitatively reduced by LBADH even employing 2.5 equiv. of 2-propanol independently of their kinetic values. Finally, we found good correlations between the IR absorption band of the carbonyl groups and the degrees of conversion obtained in these HT processes, making this simple method a convenient tool to predict the success of these transformations.

doi: 10.1039/c3ob42057d

Enzymatic cascade reactions experience tremendous attention by cutting short conventional step-by-step synthesis in a highly efficient and elegant fashion. Focusing on ω-transaminases, this review provides an overview of different biocatalytic strategies to afford a variety of (chiral) amines employing diverse cascade systems: Cascades to shift the reaction equilibrium as well as cascades for the amination of alcohols and nonactivated C–H bonds are discussed. Cascades enable the deracemization of rac-amines, other ones involve biocatalyzed C–C bond formation and C–C bond hydrolysis. Finally, the potential of spontaneous ring closure reactions initiated by ω-transaminases is illustrated.

doi: 10.1021/cs400930v

To develop a nicotinamide-independent single flavoenzyme system for the asymmetric bioreduction of C=C bonds, four types of hydrogen donor, encompassing more than 50 candidates, were investigated. Six highly potent, cheap, and commercially available co-substrates were identified that (under the optimized conditions) resulted in conversions and enantioselectivities comparable with, or even superior to, those obtained with traditional two-enzyme nicotinamide adenine dinucleotide phosphate (NAD(P)H)-recycling systems.

doi: 10.1002/chem.201303897

A two-step synthesis of various enantiomerically pure 1-aryl-3-methylisochroman derivatives was accomplished through asymmetric biocatalytic ketone reduction followed by an oxa-Pictet–Spengler reaction. The compounds were obtained in good to excellent yield (47–92%) in favor of the syn diastereomers [dr (syn/anti) up to 99:1]. Enantiopure arylpropanols serving as pronucleophiles for the C–C bond-formation step were obtained by biocatalytic reduction by employing enantiocomplementary alcohol dehydrogenases, which gave access to the (S) and (R) enantiomer with up to >99% conversion and up to >99% ee.

doi: 10.1002/ejoc.201301429

This review gives an overview on the occurrence of sulfatases in Prokaryota, Eukaryota and Archaea. The mechanism of enzymes acting with retention or inversion of configuration during sulfate ester hydrolysis is discussed taking two complementary examples. Methods for the discovery of novel alkyl sulfatases are described by way of sequence-based search and enzyme induction. A comprehensive list of organisms with their respective substrate scope regarding prim- and sec-alkyl sulfate esters allows to assess the capabilities and limitations of various biocatalysts employed as whole cell systems or as purified enzymes with respect to their activities and enantioselectivities. Methods for immobilization and selectivity enhancement by addition of metal ions or organic (co)solvents are summarised.

doi: 10.1007/s00253-013-5438-0

The asymmetric reductive amination of aryl-ketones bearing various boron-functionalities (acid, ester or potassium trifluoroborates) was investigated employing enantiocomplementary ω-transaminases as catalysts. Under the optimized conditions, high conversions (up to 94%) and excellent ee’s (up to >99%) were obtained providing access to both (R)- and (S)-configured amino-aryl boronates under mild reaction conditions.

doi: 10.1016/j.tetasy.2013.10.004

The nicotinamide adenine dinucleotide regeneration system present in Escherichia coli cells was exploited for the oxidation and deracemisation of secondary alcohols with the overexpressed alcohol dehydrogenase from Rhodococcus ruber DSM 44541 (E. coli/ADH-A). Thus, various racemic alcohols were selectively oxidised with lyophilised or resting E. coli/ADH-A cells without need for an external cofactor or co-substrate. The addition of these substrates to the E. coli/ADH-A cells in buffer afforded the corresponding ketones and the remaining enantioenriched (R)-alcohols. This methodology was used for the desymmetrisation of a meso-diol and for the synthesis of the highly valuable raspberry ketone. Moreover, a biocatalytic concurrent process was developed with the resting cells of E. coli/ADH-A, ADH from Lactobacillus brevis, and glucose dehydrogenase for the deracemisation of various secondary alcohols, which afforded the desired enantiopure alcohols in more than 99% ee starting from the racemic mixture. The reaction time of deracemisation of 1-phenylethanol was estimated to be less than 30 min. The stereoinversion of (S)-1-phenylethanol to its pure (R)-enantiomer was also achieved, which provided a biocatalytic alternative to the chemical Mitsunobu inversion reaction.

doi: 10.1002/cctc.201300409

One-pot combinations of sequential catalytic reactions can offer practical and ecological advantages over classical multi-step synthesis schemes. In this context, the integration of enzymatic and chemo-catalytic transformations holds particular potential for efficient and selective reaction sequences that would not be possible using either method alone. Here, we report the one-pot combination of alcohol dehydrogenase-catalysed asymmetric reduction of 2-azido ketones and Pd nanoparticle-catalysed hydrogenation of the resulting azido alcohols, which gives access to both enantiomers of aromatic 1,2-amino alcohols in high yields and excellent optical purity (ee >99%). Furthermore, we demonstrate the incorporation of an upstream azidolysis and a downstream acylation step into the one-pot system, thus establishing a highly integrated synthesis of the antiviral natural product (S)-tembamide in 73% yield (ee >99%) over 4 steps. Avoiding the purification and isolation of intermediates in this synthetic sequence leads to an unprecedentedly low ecological footprint, as quantified by the E-factor and solvent demand.

doi: 10.1039/C3GC41666F

Eleven flavoproteins from the old yellow enzyme family were found to catalyze the disproportionation (“dismutation”) of conjugated enones. Incomplete conversions, which were attributed to enzyme inhibition by the co-product phenol could be circumvented via in situ co-product removal by scavenging the phenol using the polymeric adsorbent MP-carbonate. The optimized system allowed to reduce an alkene activated by ester groups in a “coupled-substrate” approach via nicotinamide-free hydrogen transfer with >90% conversion and complete stereoselectivity.

doi: 10.1002/bit.24981

New clothes for Mn³⁺: Aerobic alkene cleavage of styrene-type substrates by Trametes hirsuta is attributed to an enzyme that is dependent on manganese in oxidation state three. The enzyme has a proteinase backbone and binds Mn³⁺ exclusively via oxygen atoms, in contrast to all known Mn³⁺ enzymes.

doi: 10.1002/cbic.201300601

The cleavage of alkenes to the corresponding carbonyl products is a widely employed method in organic synthesis, especially to introduce oxygen functionalities into molecules, remove protecting groups and tailor large molecules. Chemical methods available for alkene cleavage include, for instance, ozonolysis, several metal-based variants (KMnO₄, OsO₄, RuO₄, etc.), electrochemical alternatives, singlet oxygen, hypervalent iodine and organic molecules in combination with oxygen. Furthermore, several enzymatic methods for alkene cleavage have been described to establish safe, mild and selective oxidation methods. Various heme and non-heme iron-dependent enzymes catalyse the alkene cleavage at ambient temperature and atmospheric pressure in an aqueous buffer, showing good chemo- and regioselectivities in selected cases. Quite recently some Cu-, Mn- and Ni-dependent enzymes have been identified for this reaction. This review gives an overview of the different chemical and enzymatic methods available for the cleavage of alkenes.

doi: 10.1002/adsc.201300882

The enzymatic carboxylation of electron-rich aromatics, which represents a promising ‘green’ equivalent to the chemical Kolbe–Schmitt reaction, is thermodynamically disfavored and is therefore impeded by incomplete conversions. Optimization of the reaction conditions, such as pH, temperature, substrate concentration and the use of organic co-solvents and/or ionic liquids allowed to push the conversion in favor of carboxylation by a factor of up to 50%. Careful selection of the type of bicarbonate salt used as CO₂ source was crucial to ensure optimal activities. Among two types of carboxylases tested with their natural substrates, benzoic acid decarboxylase from Rhizobium sp. proved to be significantly more stable than phenolic acid decarboxylase from Mycobacterium colombiense; it tolerated reaction temperatures of up to 50 °C and substrate concentrations of up to 100 mM and allowed efficient biocatalyst recycling.

doi: 10.1016/j.jbiotec.2013.07.017

Asymmetric allylation of (hetero)aromatic aldehydes by a zinc(II)-allylbutyrolactone species catalyzed by a chiral BINOL-type phosphoric acid gave β-substituted α-methylenebutyrolactones in 68 to >99% ee and 52–91% isolated yield. DFT studies on the intermediate Zn²⁺-complex – crucial for chiral induction – suggest a six-membered ring intermediate, which allows the phosphoric acid moiety to activate the aldehyde. The methodology was applied to the synthesis of the antitumour natural product (S)-(–)-hydroxymatairesinol.

doi: 10.1002/adsc.201300392

Owing to the more abundant occurrence of racemic compounds compared to prochiral or meso forms, most enantiomerically pure products are obtained via racemate resolution. This review summarizes (chemo)enzymatic enantioconvergent processes based on the use of hydrolytic enzymes, which are able to invert a stereocenter during catalysis that can overcome the 50%-yield limitation of kinetic resolution. Recent developments are presented in the fields of inverting or retaining sulfatases, epoxide hydrolases and dehalogenases, which allow the production of secondary alcohols or vicinal diols at a 100% theoretical yield from a racemate via enantioconvergent processes.

doi: 10.1016/j.tibtech.2013.05.005

Blowing out of disproportion: Alcohol dehydrogenases were found to catalyze a bio-Cannizzaro-type reaction. The disproportionation of various aldehydes into their corresponding alcohols and carboxylic acids was achieved in a redox-neutral process. The asymmetric variant proceeded with high stereoselectivities.

doi: 10.1002/cctc.201300028

Due to the chemical versatility of the flavin cofactor, FMN-dependent ene-reductases and nitro-reductases can catalyze or mediate a diverse spectrum of chemical reactions. Among them, two-electron transfer reactions dominate, which may proceed via sequential hydride transfer at the same or at alternate reactive sites. In addition, highly reactive intermediates are often formed, which undergo subsequent spontaneous (non-enzymatic) reactions leading to further enzymatic transformations in a cascade. Besides the well-known reductive processes involving alkenes and nitro groups at the expense of a reduced flavin cofactor, redox-neutral processes including disproportionation and C=C-bond isomerization reactions are catalyzed by OYE homologues. Unusual flavin-dependent biotransformations are reviewed with a special focus on the OYE family of flavoproteins (ene-reductases) and oxygen-insensitive FMN-dependent nitro-reductases.

doi: 10.1039/C3GC40588E

A chemoenzymatic strategy for the synthesis of enantiomerically pure novel alkaloids (1S,3R)-1-benzyl-2,3-dimethyl-1,2,3,4-tetrahydroisoquinolines is presented. The key steps are the biocatalytic stereoselective reductive amination of substituted 1-phenylpropan-2-one derivatives to yield chiral amines employing microbial ω-transaminases, and the diastereoselective reduction of a Bischler–Napieralski imine intermediate by catalytic hydrogenation in the presence of palladium on charcoal, leading exclusively to the desired cis-isomer.

doi: 10.1016/j.tetasy.2013.05.003

Prochiral bicyclic diketones were transformed to a single diastereomer of 3-substituted cyclohexylamine derivatives via three consecutive biocatalytic steps. The two chiral centres were set up by a C-C hydrolase (6-oxocamphor hydrolase) in the first step and by an ω-transaminase in the last step. The esterification of the intermediate keto acid was catalysed by a lipase in the second step if possible. For two substrates the C-C hydrolytic step as well as the esterification could be run simultaneously in a one-pot cascade in an organic solvent. In one example, the reaction mixture of the first two steps could be directly subjected to bio-amination in an organic solvent without the need to change the reaction medium. Depending on the choice of the ω-transaminase employed and the substrate the cis- as well as the trans-diastereomers could be obtained in optically pure forms.

doi: 10.1002/adsc.201201057

Although C-C bond hydrolases are distributed widely in Nature, they has as yet have received only limited attention in the area of biocatalysis compared to their counterpart the C-heteroatom hydrolases, such as lipases and proteases. However, the substrate range of C-C hydrolases, and their non-dependence on cofactors, suggest that these enzymes may have considerable potential for applications in synthesis. In addition, hydrolases such as the β-diketone hydrolase from Rhodococcus (OCH) are known, that catalyse the formation of interesting chiral intermediates. Further enzymes, such as kynureninase and a meta-cleavage product hydrolase (MhpC), are able to catalyse carbon-carbon bond formation, suggesting wider applications in biocatalysis than previously envisaged. In this review, the distribution, catalytic characteristics and applications of C-C hydrolases are described, with a view to assessing their potentialfor use in biocatalytic processes in the future.

doi: 10.1002/adsc.201300232

Stereoselective reduction towards pharmaceutically potent products with multi-chiral centers is an ongoing hot topic, but up to now catalysts for reductions of bulky aromatic substrates are rare. The NADPH-dependent alcohol dehydrogenase from Ralstonia sp. (RADH) is an exception as it prefers sterically demanding substrates. Recent studies with this enzyme indicated outstanding potential for the reduction of various α-hydroxy ketones, but were performed with crude cell extract, which hampered its detailed characterization. We have established a procedure for the purification and storage of RADH and found a significantly stabilizing effect by addition of CaCl₂. Detailed analysis of the pH-dependent activity and stability yielded a broad pH-optimum (pH 6–9.5) for the reduction reaction and a sharp optimum of pH 10–11.5 for the oxidation reaction. The enzyme exhibits highest stability at pH 5.5–8 and 8–15°C; nevertheless, biotransformations can also be carried out at 25°C (half-life 80 h). Under optimized reaction parameters a thorough study of the substrate range of RADH including the reduction of different aldehydes and ketones and the oxidation of a broad range of alcohols was conducted. In contrast to most other known alcohol dehydrogenases, RADH clearly prefers aromatic and cyclic aliphatic compounds, which makes this enzyme unique for conversion of space demanding substrates. Further, reductions are catalyzed with extremely high stereoselectivity (>99% enantio- and diastereomeric excess). In order to identify appropriate substrate and cofactor concentrations for biotransformations, kinetic parameters were determined for NADP(H) and selected substrates. Among these, we studied the reduction of both enantiomers of 2-hydroxypropiophenone in more detail.

doi: 10.1002/bit.24857

In recent years, Old Yellow Enzymes (OYEs) and their homologues have found broad application in the efficient asymmetric hydrogenation of activated C=C bonds with high selectivities and yields. Members of this class of enzymes have been found in many different organisms and are rather diverse on the sequence level, with pairwise identities as low as 20%, but they exhibit significant structural similarities with the adoption of a conserved (αβ)8-barrel fold. Some OYEs have been shown not only to reduce C=C double bonds, but also to be capable of reducing nitro groups in both saturated and unsaturated substrates. In order to understand this dual activity we determined and analyzed X-ray crystal structures of NerA from Agrobacterium radiobacter, both in its apo form and in complex with 4-hydroxybenzaldehyde and with 1-nitro-2-phenylpropene. These structures, together with spectroscopic studies of substrate binding to several OYEs, indicate that nitro-containing substrates can bind to OYEs in different binding modes, one of which leads to C=C double bond reduction and the other to nitro group reduction.

doi: 10.1002/cbic.201300136

This account focuses on the application of ω-transaminases, lyases, and oxidases for the preparation of amines considering mainly work from our own lab. Examples are given to access α-chiral primary amines from the corresponding ketones as well as terminal amines from primary alcohols via a two-step biocascade. 2,6-Disubstituted piperidines, as examples for secondary amines, are prepared by biocatalytical regioselective asymmetric monoamination of designated diketones followed by spontaneous ring closure and a subsequent diastereoselective reduction step. Optically pure tert-amines such as berbines and N-methyl benzylisoquinolines are obtained by kinetic resolution via an enantioselective aerobic oxidative C–C bond formation.

doi: 10.1021/op4000237

A short and efficient total synthesis of the alkaloid isosolenopsin and its enantiomer has been achieved. The key step was a ω-transaminase-catalysed regioselective monoamination of the diketone pentadecane-2,6-dione, which was obtained in a single step through the application of a Grignard reaction. Initial low conversions in the biotransformation could be overcome by optimisation of the reaction conditions employing suitable cosolvents. In the presence of 20 vol.-% N,N-dimethylformamide (DMF) or n-heptane the best results were obtained by employing two enantiocomplementary ω-transaminases originating from Arthrobacter at 30–40 °C; under these conditions, conversions of more than 99% and perfect stereocontrol (ee > 99%) were achieved. Diastereoselective chemical reduction (H₂/Pd/C) of the biocatalytic product gave the target compound. The linear three-step synthesis provided the natural product isosolenopsin in diastereomerically pure form (ee > 99%, dr = 99:1) with an overall yield of 64%.

doi: 10.1002/ejoc.201300157

Various (S)-selective and (R)-selective ω-transaminases were investigated for the amination of 1- and 2-tetralone and derivatives as well as of 3- and 4-chromanone. All ketones tested were aminated to give the corresponding enantiopure amines (ee > 99%) employing at least one of the enzymes investigated. In most of the cases the (S)- as well as the (R)-enantiomer was obtained in optically pure form. The amination of 3-chromanone was performed on a 100 mg scale leading to optically pure (R)-3-aminochromane (ee > 99%) with complete conversion and 78% isolated yield.

doi: 10.1021/cs400002d

Biocatalytic transformations have emerged as a viable alternative to other asymmetric chemical methods due to the intrinsic high stereoselectivity of the enzymes and the mild reaction conditions. Just a decade ago, the reaction scope of applicable biotransformations for organic synthesis was limited to a handful of reaction types. Tremendous progress has been made in the meantime so that this review presents only a small selection of the broad range of possible biotransfromations for organic synthesis available today. Lyases (hydroxynitrile lyase, aldolases) and redox enzymes like alcohol dehydrogenases, Baeyer–Villiger monooxygenase, dioxygenases, ene reductases, berberine bridge enzyme and ω-transaminases are discussed besides hydrolases.

doi: 10.1016/j.ddtec.2012.08.002

Various artificial network designs that involve biocatalysts were tested for the asymmetric amination of sec-alcohols to the corresponding α-chiral primary amines. The artificial systems tested involved three to five redox enzymes and were exemplary of a range of different sec-alcohol substrates. Alcohols were oxidised to the corresponding ketone by an alcohol dehydrogenase. The ketones were subsequently aminated by employing a ω-transaminase. Of special interest were redox-neutral designs in which the hydride abstracted in the oxidation step was reused in the amination step of the cascade. Under optimised conditions up to 91% conversion of an alcohol to the amine was achieved.

doi: 10.1002/chem.201202666

Alkene cleavage is a widely employed oxidation reaction in organic chemistry. An enzyme preparation of the wood degrading fungus Trametes hirsuta is known to cleave the C=C double bond adjacent to an aromatic ring to give the corresponding carbonyl compound at the expense of molecular oxygen as the sole oxidant. Lab-grown fungus cultures displayed varied activity and lost their alkene cleavage activity over generations of growth. t-Anethole, which is the best accepted substrate by the enzyme, is described as a major component of essential oils produced by certain plants with powerful fungicidal property. We could now show that the alkene cleaving activity was improved by the addition of the fungicide t-anethole during culture growth which represented to be an efficient method to produce cells possessing a consistent level of high alkene cleavage activity.

doi: 10.1016/j.molcatb.2013.02.002

The regioselectivity of various enantiocomplementary ω-transaminases was evaluated for the stereoselective monoamination of designated 1,5-diketones; excellent conversions, enantio- and regioselectivities were observed. The resulting amino-ketones underwent spontaneous intramolecular ring closure to afford Δ1-piperideines, which served as precursors for the cis- and anti-piperidine scaffold as demonstrated for the synthesis of the alkaloids dihydropinidine and epi-dihydropinidine. Key to the success of accessing the trans-piperidines was a Lewis acid mediated conformational change of the Δ1-piperideines in the reduction step. Thus, all four diastereomers of 2,6-disubstituted piperidines could successfully be prepared.

doi: 10.1002/chem.201202793

Hand in hand: The title transformation was achieved using a pair of sulfatases acting through inversion and retention of configuration on opposite substrate enantiomers. Using Pseudomonas aeruginosa arylsulfatase PAS with alkylsulfatase PISA1 in one-pot leads to sec-alcohols (80 to >99% conversion) with 91 to greater than 99% ee.

doi: 10.1002/anie.201209946

More than one activity: Owing to their hydratase activity, phenolic acid decarboxylases catalyze the regio- and stereoselective addition of H₂O across the C=C double bond of hydroxystyrene derivatives yielding (S)-4-(1-hydroxyethyl)phenols with up to 82% conversion and 71% ee.

doi: 10.1002/anie.201207916

(R)-Lasiodiplodin methyl ether, a precursor of the antileukemic agent lasiodiplodin, was synthesized through a seven-step linear sequence. Chirality was introduced through a sulfatase-based deracemization process, in which a functionalized (rac)-sec-sulfate ester was enzymatically hydrolyzed with inversion of the stereocenter using an alkyl sulfatase. The remaining sulfate ester enantiomer was hydrolyzed with retention of configuration under acidic conditions, yielding the chiral key building block as the sole product in 93% ee The total synthesis was completed through Negishi cross-coupling and ring-closing metathesis.

doi: 10.1002/ejoc.201201296

A highly enantioselective and stereoselective secondary alkylsulfatase from Pseudomonas sp. DSM6611 (Pisa1) was heterologously expressed in Escherichia coli BL21, and purified to homogeneity for kinetic and structural studies. Structure determination of Pisa1 by X-ray crystallography showed that the protein belongs to the family of metallo-β-lactamases with a conserved binuclear Zn²⁺ cluster in the active site. In contrast to a closely related alkylsulfatase from Pseudomonas aeruginosa (SdsA1), Pisa1 showed a preference for secondary rather than primary alkyl sulfates, and enantioselectively hydrolyzed the (R)-enantiomer of rac-2-octyl sulfate, yielding (S)-2-octanol with inversion of absolute configuration as a result of C–O bond cleavage. In order to elucidate the mechanism of inverting sulfate ester hydrolysis, for which no counterpart in chemical catalysis exists, we designed variants of Pisa1 guided by three-dimensional structure and docking experiments. In the course of these studies, we identified an invariant histidine (His317) near the sulfate-binding site as the general acid for crucial protonation of the sulfate leaving group. Additionally, amino acid replacements in the alkyl chain-binding pocket generated an enzyme variant that lost its stereoselectivity towards rac-2-octyl sulfate. These findings are discussed in light of the potential use of this enzyme family for applications in biocatalysis.

doi: 10.1111/febs.12027

An unexpected, redox-neutral C=C bond isomerization of a γ-butyrolactone bearing an exo-methylene unit to the thermodynamically more favoured endo isomer (k_cat=0.076 s⁻¹) catalysed by flavoproteins from the Old Yellow Enzyme family was discovered. Theoretical calculations and kinetic data support a mechanism through which the isomerization proceeds through FMN-mediated hydride addition onto exo-Cβ, followed by hydride abstraction from endo-Cβ’, which is in line with the well-established C=C bond bioreduction of OYEs. This new isomerase activity enriches the catalytic versatility of ene-reductases.

doi: 10.1002/cbic.201200475

Four NAD(P)H-dependent non-flavin ene reductases have been investigated for their ability to reduce activated C=C bonds in an asymmetric fashion by using 20 structurally diverse substrates. In comparison with flavin-dependent Old Yellow Enzyme homologues, a higher degree of electronic activation was required, because the best activities were obtained with enals and nitroalkenes rather than enones and carboxylic esters. Although FaEO from Fragaria x ananassa (strawberry) and its homologue SlEO from Solanum lycopersicum (tomato) exhibited a narrow substrate spectrum, progesterone 5β-reductase (At5β-StR) from Arabidopsis thaliana (thale cress) and leukotriene B4 12-hydroxydehydrogenase (LTB4DH/PGR) from Rattus norvegicus (rat) appear to be promising candidates, in particular for the asymmetric bioreduction of open-chain enals, nitroalkenes and α,β-unsaturated γ-butyrolactones. Competing nitro reduction and non-enzymatic Weitz–Scheffer epoxidation were largely suppressed.

doi: 10.1002/ejoc.201200776

(S)-Rivastigmine [(S)-1] was obtained via a four-step synthesis using an asymmetric enzymatic transamination protocol as the key step. An early introduction of the carbamate pharmacophore side chain avoided the use of protective group strategies and hence led to a considerable shortcut. This strategy required a novel ω-transaminase from Paracoccus denitrificans, which could transform the highly polar key substrate 3-acetylphenyl ethyl(methyl)carbamate (4) to the corresponding amine (S)-5 in 99% ee and >80% conversion.

doi: 10.1016/j.tet.2012.06.031

The amination of benzylic and cinnamic alcohols was achieved via a biocatalytic, one-pot oxidation–transamination cascade in aqueous medium at physiological conditions. Alcohol oxidation by galactose oxidase at the expense of O₂ furnished the corresponding aldehydes, which were aminated using ω-transaminases in situ. The applicability of this method was demonstrated by a short synthesis of the antifungal agent naftifine.

doi: 10.1039/C2RA20800H

Fluorine is commonly applied in pharmaceuticals to block the degradation of bioactive compounds at a specific site of the molecule. Blocking of the reaction center of the enzyme-catalyzed ring closure of 1,2,3,4-tetrahydrobenzylisoquinolines by a fluoro moiety allowed redirecting the berberine bridge enzyme (BBE)-catalyzed transformation of these compounds to give the formation of an alternative regioisomeric product namely 11-hydroxy-functionalized tetrahydroprotoberberines instead of the commonly formed 9-hydroxy-functionalized products. Alternative strategies to change the regioselectivity of the enzyme, such as protein engineering, were not applicable in this special case due to missing substrate–enzyme interactions. Medium engineering, as another possible strategy, had clear influence on the regioselectivity of the reaction pathway, but did not lead to perfect selectivity. Thus, only substrate tuning by introducing a fluoro moiety at one potential reactive carbon center switched the reaction to the formation of exclusively one regioisomer with perfect enantioselectivity.

doi: 10.1002/chem.201201895

The degree of C=C bond activation in the asymmetric bioreduction of α,β-unsaturated carboxylic esters by ene-reductases was studied, and general recommendations to render these “borderline-substrates” more reactive towards enzymatic reduction are proposed. The concept of “supported substrate activation” was developed. In general, an additional α-halogenated substituent proved to be beneficial for enzymatic activity, whereas β-alkyl or β-aryl substituents were detrimental for the reactivity of nonhalogenated substrates, and α-cyano groups showed little effect. The alcohol moiety of the ester functionality was found to have a strong influence on the reaction rate. Overall, activities were determined by both steric and electronic effects.

doi: 10.1002/chem.201200990

The asymmetric bioreduction of prochiral conjugated alkenes using ene-reductases allows powerful strategies to access both enantiomers of the product with high stereoselectivity. This may be achieved (i) by using pairs of (iso)enzymes, which bind the alkene moiety in mirror-image orientations to affect hydride attack from opposite sides, (ii) via a switch in the (E/Z)-geometry of the alkene unit, or (iii) by changing the size of the protective groups of the substrate, which enforces a flipped orientation in the active site. Modeling studies provide a rationale for the molecular basis of substrate binding and allow the prediction of the stereochemical outcome of this useful bioreduction.

doi: 10.1055/s-0032-1316591

Ene-reductases from the ‘Old Yellow Enzyme’ family of flavoproteins catalyze the asymmetric reduction of various α,β-unsaturated compounds at the expense of a nicotinamide cofactor. They have been applied to the synthesis of valuable enantiopure products, including chiral building blocks with broad industrial applications, terpenoids, amino acid derivatives and fragrances. The combination of these highly stereoselective biocatalysts with a cofactor recycling system has allowed the development of cost-effective methods for the generation of optically active molecules, which is strengthened by the availability of stereo-complementary enzyme homologues.

doi: 10.1016/j.jbiotec.2012.03.023

Driving the machinery: A biocatalytic redox-neutral cascade for the preparation of terminal primary amines from primary alcohols at the expense of ammonia has been established in a one-pot one-step method (see picture). Applying this artificial biocatalyst network, long-chain 1,ω-alkanediols were converted into diamines, which are building blocks for polymers, in up to 99% conversion.

doi: 10.1002/anie.201204683

The enzymatic bioreduction of β-halo-α,β-unsaturated carboxylic esters proceeded via sequential enzymatic C=C bond reduction—β-elimination to afford saturated carboxylic esters. This novel biodegradation pathway combines the reductive activity of ene-reductases with the spontaneous β-elimination of hydrohalous acid from the unstable (saturated) intermediates. Both enantiomers of methyl 2-chloro-, 2-bromo- and 2-iodopropionate were obtained in good to excellent enantiopurity via enzyme-based stereocontrol using various members of the ‘Old Yellow Enzyme’ family of flavoproteins. Overall, this pathway resembles a reductive dehalogenation of β-halogenated acrylic esters.

doi: 10.1039/C2CY20079A

Although biotransformations implementing alcohol dehydrogenases (ADHs) are widespread, enzymes which catalyse the reduction and oxidation of sterically demanding substrates, especially 2-hydroxy ketones, are still rare. To fill this gap eight ADHs were investigated concerning their potential to reduce bulky 2-hydroxy ketones. All of these enzymes showed good activities along with excellent enantio- (ee > 99%) and diastereoselectivities (de > 99%). Due to their differences in substrate preferences and stereoselectivity a broad range of diastereomerically pure 1,2-diols is now accessible via biotransformation. Best results were obtained using the alcohol dehydrogenase from Ralstonia sp. (Cupriavidussp.) (RADH), which showed a broad substrate range, especially for sterically demanding compounds. Araliphatic 2-hydroxy ketones, like (R)-2-hydroxy-1-phenylpropan-1-one ((R)-2-HPP), were reduced much faster than aliphatic or aromatic aldehydes (e.g. benzaldehyde) under the applied conditions. Additionally (R)- as well as (S)-2-hydroxy ketones were converted with high diastereoselectivities (de > 99%). RADH, which was up to now only studied as a whole cell biocatalyst overexpressed in E. coli, was purified and thoroughly characterised concerning its catalytic properties.

doi: 10.1039/C2CY20120H

The biosynthesis of cyclic terpenoids and polyethers involves enzyme-initiated cascade reactions for ring formation. While the former are obtained by electrophilic cascades through carbenium ions as intermediates, cyclic polyethers are formed by nucleophilic cascade reactions of (poly)epoxide precursors. These mechanistically complementary pathways follow common principles via (i) triggering of the cascade by forming a reactive intermediate (‘initiation’), (ii) sequential ‘proliferation’ of the cyclization and finally (iii) ‘termination’ of the cascade. As analyzed in this concept paper, the multiplicity of precursors, combined with various initiation and termination routes and kinetically favored or disfavored cyclization modes accounts for the enormous diversity in cyclic terpenoid and polyether scaffolds. Although the essential role of enzymes in the triggering of these cascades is reasonably well understood, remarkably little is known about their influence in proliferation reactions, especially those implying kinetically disfavored (anti-Markovnikov and anti-Baldwin) routes. Mechanistic analysis of enzymatic cascade reactions provides biomimetic strategies for natural product synthesis.

doi: 10.1039/C2NP00078D

Two recently identified (S)-selective ω-transaminases (ω-TAs) that originate from Paracoccus denitrificans (Strep-PD-ωTA, cloned with an N-terminal Strep-tag II) and Pseudomonas fluorescens (PF-ωTA) were employed for the asymmetric amination of selected prochiral ketones. The substrates tested were transformed into optically pure amines (>99% ee) with high conversion (up to >99%). The ω-TAs led to higher conversion in the absence of dimethyl sulfoxide as a cosolvent than in its presence (15%, v/v). Additionally, it was shown that a His-tagged recombinant transaminase from Vibrio fluvialis (His-VF-ωTA, cloned with an N-terminal His₆-tag) showed for a single substrate, ethyl acetoacetate, significantly higher stereoselectivity for the amination compared to the corresponding commercial enzyme preparation (>99 vs. 50%).

doi: 10.1002/ejoc.201101476

The first report of a biocatalytic regioselective oxidative mono-cleavage of dialkenes was successfully achieved employing a cell-free enzyme preparation from Trametes hirsuta at the expense of molecular oxygen. Selected reactions were performed on a preparative scale affording high to excellent conversions and chemoselectivities.

doi: 10.1002/bit.24472

The equilibrium constant is a critical parameter for making rational design choices in biocatalytic transamination for the synthesis of chiral amines. However, very few reports are available in the scientific literature determining the equilibrium constant (K) for the transamination of ketones. Various methods for determining (or estimating) equilibrium have previously been suggested, both experimental as well as computational (based on group contribution methods). However, none of these were found suitable for determining the equilibrium constant for the transamination of ketones. Therefore, in this communication we suggest a simple experimental methodology which we hope will stimulate more accurate determination of thermodynamic equilibria when reporting the results of transaminase-catalyzed reactions in order to increase understanding of the relationship between substrate and product molecular structure on reaction thermodynamics.

doi: 10.1039/C2CC17572J

α-Alkyl-β-hydroxy esters were obtained via dynamic kinetic resolution (DKR) employing purified or crude E. coli overexpressed alcohol dehydrogenases (ADHs). ADH-A from R. ruber, CPADH from C. parapsilosis and TesADH from T. ethanolicus afforded syn-(2R,3S) derivatives with very high selectivities for sterically not impeded ketones (‘small-bulky’ substrates), while ADHs from S. yanoikuyae (SyADH) and Ralstonia sp. (RasADH) could also accept bulkier keto esters (‘bulky-bulky’ substrates). SyADH also provided preferentially syn-(2R,3S) isomers and RasADH showed in some cases good selectivity towards the formation of anti-(2S,3S) derivatives. With anti-Prelog ADHs such as LBADH from L. brevis or LKADH from L. kefir, syn-(2S,3R) alcohols were obtained with high conversions and diastereomeric excess in some cases, especially with LBADH. Furthermore, due to the thermodynamically favoured reduction of these substrates, it was possible to employ just a minimal excess of 2-propanol to obtain the final products with quantitative conversions.

doi: 10.1002/adsc.201200139

The substrate spectrum of the inverting alkylsulfatase Pisa1 was investigated using a range of sec-alkyl sulfate esters bearing aromatic, olefinic and acetylenic moieties. Perfect enantioselectivities were obtained for substrates bearing groups of different size adjacent to the sulfate ester moiety. Insufficient selectivities could be doubled by using dimethyl sulfoxide (DMSO) as co-solvent. Hydrolytically unstable benzylic sulfate esters could be sufficiently stabilised by introduction of electron-withdrawing substituents. Overall, Pisa1 appears to be a very useful inverting alkylsulfatase for the deracemisation of rac-sec-alcohols via enzymatic hydrolysis of their corresponding sulfate esters, which furnishes homochiral products possessing the ‘anti-Kazlauskas’ configuration.

doi: 10.1002/adsc.201100864

The enzymatic carboxylation of phenol and styrene derivatives using (de)carboxylases in carbonate buffer proceeded in a highly regioselective fashion: Benzoic acid (de)carboxylases selectively formed o-hydroxybenzoic acid derivatives, phenolic acid (de)carboxylases selectively acted at the β-carbon atom of styrenes forming (E)-cinnamic acids.

doi: 10.1021/ol300385k

The asymmetric bioreduction of activated alkenes catalyzed by flavin-dependent enoate reductases from the OYE-family represents a powerful method for the production of optically active compounds. For its preparative-scale application, efficient and economic NADH-recycling is crucial. A novel enzyme-coupled NADH-recycling system is proposed based on the concurrent oxidation of a sacrificial sec-alcohol catalyzed by an alcohol dehydrogenase (ADH-A). Due to the highly favorable position of the equilibrium of ene-reduction versus alcohol-oxidation, the cosubstrate is only required in slight excess.

doi: 10.1002/bit.23078

Amine for the top: A biocatalyst able to perform transamination of less hindered ketones is modified to transform a sterically demanding ketone substrate to the corresponding optically pure amine. This goal is achieved by rational analysis and design of the catalyst’s structure as well as other techniques of directed evolution. The biocatalyst is successfully adapted to the process and not vice versa.

doi: 10.1002/cctc.201000349

Bridging the gap: The berberine bridge enzyme (BBE) was employed for the first preparative oxidative biocatalytic C-C coupling that leads to a new intramolecular bond. This unique transformation requires O₂ as sole stoichiometric oxidant and gives access to novel optically pure (S)-berbine 2 and (R)-1-benzyl-1,2,3,4-tetrahydroisoquinoline 1 alkaloid derivatives by kinetic resolution.

doi: 10.1002/anie.201006268

Due to their high enantioselectivity biotransformations, i.e. enzyme-catalyzed conversion of organic compounds, are extremely attractive reactions.  However, a limiting factor for choosing substrates is the enzyme-substrate incompatibility.  This occurs when a hydrophilic enzyme which naturally resides in the aqueous cell cytoplasm is supposed to convert a hydrophobic substrate.  In this context bicontinuous microemulsions appear to be a beneficial reaction medium for biotransformations, particularly due to their large interfacial area between a hydrophilic and a hydrophobic compound.  As a ‘proof of concept’ we performed ω-transaminase (EC 2.6.1.18) catalyzed model reactions in a bicontinuous microemulsion of the type phosphate buffer/NaCl - n-octane - pentaethylene glycol monodecyl ether.

doi: 10.3139/113.110100

Carbon–carbon bond formation is the key transformation in organic synthesis to set up the carbon backbone of organic molecules. However, only a limited number of enzymatic C–C bond forming reactions have been applied in biocatalytic organic synthesis. Recently, further name reactions have been accomplished for the first time employing enzymes on a preparative scale, for instance the Stetter and Pictet–Spengler reaction or oxidative C–C bond formation. Furthermore, novel enzymatic C–C bond forming reactions have been identified like benzylation of aromatics, intermolecular Diels-Alder or reductive coupling of carbon monoxide.

doi: 10.1016/j.copbio.2011.02.002

Enzymes from extremophiles have always been of great interest for biotechnology because of their ruggedness against various stress factors. We have isolated, cloned, heterologously expressed and characterized a thermostable old yellow enzyme (OYE) from Geobacillus kaustophilus. In addition to the expected 'enone' reduction, GkOYE also catalyzes the reverse reaction, i.e., the desaturation of C-C bonds adjacent to a carbonyl to give the corresponding α,β-unsaturated ketone. The reaction proceeds at the expense of molecular oxygen without the need for a nicotinamide cofactor and represents an environmentally benign alternative to known chemical dehydrogenation methods.

doi: 10.1002/adsc.201000862

The combination of an oxidation and a reduction in a cascade allows performing transformations in a very economic and efficient fashion. The challenge is how to combine an oxidation with a reduction in one pot, either by running the two reactions simultaneously or in a stepwise fashion without isolation of intermediates. The broader availability of various redox enzymes nowadays has triggered the recent investigation of various oxidation–reduction cascades.

doi: 10.1016/j.cbpa.2010.11.010

Biocatalytic reactions have been identified as an outstanding option for various applications in material chemistry such as modifying surfaces under mild conditions, preparing polymers, controlling self-assembly systems and manufacturing (chiral) monomers. Mostly driven by research for producing bioactive compounds, ‘novel’ biocatalytic reactions have recently become mature enough to be exploited. While transformations involving lipases and laccases/peroxidases are already widely applied, more recent improved reactions allow the (asymmetric) amination of ketones/aldehydes, the oxidation of amines and alcohols, the asymmetric reduction of ketones, or the hydroxylation of alkanes and fatty acids. Many of these reactions are ready to be exploited for materials science.

doi: 10.1016/S1369-7021(11)70086-4

The allyl moiety of 4-allyl-anisol was oxidized in the presence of a Fe(III) porphyrin derivative to the corresponding α,β-unsaturated aldehyde in an initial oxidation step with perfect chemoselectivity. Molecular oxygen was employed as the sole environmental innocuous oxidant. The reaction was performed in an aqueous buffer/CH₂Cl₂ mixture using the detergent Tween 80 to homogenize the system.

doi: 10.1016/j.tetlet.2011.03.050

A chemoenzymatic approach for the asymmetric total synthesis of the title compounds is described that employs an enantioselective oxidative C–C bond formation catalyzed by berberine bridge enzyme (BBE) in the asymmetric key step. This unique reaction yielded enantiomerically pure (R)-benzylisoquinoline derivatives and (S)-berbines such as the natural product (S)-scoulerine, a sedative and muscle relaxing agent. The racemic substrates rac-1 required for the biotransformation were prepared in 4–8 linear steps using either a Bischler–Napieralski cyclization or a C1–Cα alkylation approach. The chemoenzymatic synthesis was applied to the preparation of fourteen enantiomerically pure alkaloids, including the natural products (S)-scoulerine and (R)-reticuline, and gave overall yields of up to 20% over 5–9 linear steps.

doi: 10.1021/jo201056f

Berberine bridge enzyme (BBE) catalyses the oxidative formation of an intramolecular C–C bond using (S)-reticuline as the natural substrate to form (S)-scoulerine as the product. To allow application of the enzyme on a preparative scale for the synthesis of novel optically pure berbine and isoquinoline derivatives, an organic solvent is required to solubilise the barely soluble substrates. It was shown that BBE tolerates a broad variety of organic co-solvents. Ideally the enzymatic enantioselective oxidative C–C bond formation can be performed in 70% v v⁻¹ toluene concentration, which allowed a soluble substrate concentration of at least 20 g L⁻¹. In addition, the enzyme works in a broad operational window concerning pH and temperature. High conversions can be reached between pH 8 and 11 and from 30 to 50 °C, respectively. The enantioselective oxidative C-C bond formation was demonstrated on a preparative scale (500 mg) in a kinetic resolution leading to optically pure products (>97% ee).

doi: 10.1002/adsc.201100233

Crotonase superfamily enzymes catalyze a wide variety of reactions, including hydrolytic C–C bond cleavage in symmetrical β-diketones by 6-oxo camphor hydrolase (OCH) from Rhodococcus sp. The organic solvent tolerance and temperature stability of OCH and its structurally related ortholog Anabaena β-diketone hydrolase have been investigated. Both enzymes showed excellent tolerance toward organic solvents; for instance, even in the presence of 80% (v/v) THF or dioxane, OCH was still active. In most solvent mixtures, except methanol, the stereospecificity was conserved (>99% e.e. of product), hence neither the type of solvent nor its concentration appeared to have an effect on the stereoselectivity of the enzyme. Attempts to correlate the observed activities with log P, functional solvent group or denaturing capacity (DC) of the solvent were only successful in the case of DC for water miscible solvents. This study represents the first investigation of organic solvent stability for members of the crotonase superfamily.

doi: 10.1002/bit.23275

Four (R)-ω-transaminases originating from Hyphomonas neptunium (HN-ωTA), Aspergillus terreus (AT-ωTA) and Arthrobacter sp. (ArR-ωTA), as well as an evolved transaminase (ArRmut11-ωTA) were successfully employed for the amination of prochiral ketones leading to optically pure (R)-amines. The first three transaminases displayed perfect stereoselectivity for the amination of all substrates tested (ee >99%). Furthermore, the transaminase AT-ωTA led in most cases to better conversion than ArR-ωTA and HN-ωTA using D-alanine as amine donor. α-Tetralone, which was the only substrate not accepted by HN-ωTA, ArR-ωTA, and AT-ωTA, was successfully transformed with perfect enantioselectivity (ee >99%) into the corresponding optically pure amine employing the variant ArRmut11-ωTA.

doi: 10.1002/adsc.201100558

Optically active boron-containing alcohols were prepared via the stereoselective reduction of the corresponding carbonyl compounds by alcohol dehydrogenases. Depending on the substrate, both (R)-alcohols and (S)-alcohols were obtained with excellent enantioselectivity (up to >99% ee) employing either ADH-A or LB-ADH.

doi: 10.1016/j.tetasy.2011.10.012

A chemo-enzymatic one-pot, two-step transformation of (hetero)-benzylic and cinnamic alcohols to yield the elongated homoallylic sec-alcohols in water in up to 96% isolated yield has been developed. The sequence comprised an enzymatic alcohol oxidation using galactose oxidase from Fusarium sp. NRRL 2903 to furnish the corresponding aldehydes, which were subjected directly to allylation via indium(0)-mediated Barbier-type coupling with allyl bromide or by addition of allylboronic acid pinacol ester.

doi: 10.1002/adsc.201100380

A metallo-β-lactamase-type alkylsulfatase was found to catalyze the enantioselective hydrolysis of sec-alkylsulfates with strict inversion of configuration. This catalytic event, which does not have an analog in chemocatalysis, yields homochiral (S)-configurated alcohols and nonreacted sulfate esters. The latter could be converted into (S)-sec-alcohols as the sole product in up to >99% ee via a chemoenzymatic deracemization protocol on a preparative scale.

doi: 10.1021/ol201635y

From active sites and stereospecificity: Active-site structures of old yellow enzymes (OYEs) are correlated with their stereopreferences in the reduction of an aromatic nitroalkene, which leads to the identification of distinct clusters. These structural clusters are mapped onto sequence space, which yields four characteristic sequence motifs that may be used to cluster OYEs on the basis of their primary structure, as well as to predict their structural and biocatalytic properties.

doi: 10.1002/cctc.201100141

Asymmetric trans-bioreduction of activated alkenes by KYE1 from Kluyveromyces lactis and Yers-ER from Yersinia bercovieri, two ene–reductases from the Old Yellow Enzyme family, showed a broad substrate spectrum with a moderate to excellent degree of stereoselectivity. Both substrate- and enzyme-based stereocontrols were observed to furnish opposite stereoisomeric products. The effects of organic solvents on enzyme activity and stereoselectivity were outlined in this study, where two-phase systems hexane and toluene are shown to sustain bioreduction efficiency even at high organic solvent content.

doi: 10.1021/ol200394p

α,β-Dehydroamino acid derivatives proved to be a novel substrate class for ene-reductases from the ‘old yellow enzyme’ (OYE) family. Whereas N-acylamino substituents were tolerated in the α-position, β-analogues were generally unreactive. For aspartic acid derivatives, the stereochemical outcome of the bioreduction using OYE3 could be controlled by variation of the N-acyl protective group to furnish the corresponding (S)- or (R)-amino acid derivatives. This switch of stereopreference was explained by a change in the substrate binding, by exchange of the activating ester group, which was proven by ²H-labelling experiments.

doi: 10.1002/adsc.201100042

TA novel reductive biotransformation pathway for β,β-disubstituted nitroalkenes catalyzed by flavoproteins from the Old Yellow Enzyme (OYE) family was elucidated. It was shown to proceed via enzymatic reduction of the nitro-moiety to furnish the corresponding nitroso-alkene, which underwent spontaneous (non-enzymatic) electrocyclization to form highly strained 1,2-oxazete derivatives. At elevated temperatures the latter lost HCN via a retro-[2+2]-cycloaddition to form the corresponding ketones. This pathway was particularly dominant using xenobiotic reductase A, while pentaerythritol tetranitrate-reductase predominantly catalyzed the biodegradation via the Nef-pathway.

doi: 10.1039/C0OB01216E

Multi-enzymatic cascade reactions, i.e., the combination of several enzymatic transformations in concurrent one-pot processes, offer considerable advantages: the demand of time, costs and chemicals for product recovery may be reduced, reversible reactions can be driven to completion and the concentration of harmful or unstable compounds can be kept to a minimum. This review summarizes the developments in multi-enzymatic cascades employed for the asymmetric synthesis of chiral alcohols, amines and amino acids, as well as for C–C bond formation. In addition, a general classification of biocatalytic cascade systems is provided and bioprocess engineering aspects associated with the topic are discussed.

doi: 10.1002/adsc.201100256

A strategy for the biocatalytic racemization of primary α-chiral amines was developed by employing a pair of stereocomplementary PLP-dependent ω-transaminases. The interconversion of amine enantiomers proceeded through reversible transamination by a prochiral ketone intermediate, either catalyzed by a pair of stereocomplementary ω-transaminases or by a single enzyme possessing low stereoselectivity. To tune the system, the type and concentration of a nonchiral amino acceptor proved to be crucial. Finally, racemization could be achieved by the cross-transamination of two different amines without a requirement for an external amino acceptor. Several synthetically and industrially important amines could be enzymatically racemized under mild reaction conditions.

doi: 10.1002/chem.201001602

Various recombinant ω-transaminases, overexpressed in E. coli cells and employed as whole-cell catalysts, are tested for the synthesis of enantiomerically pure amines from the corresponding prochiral ketones. Optically pure (S)-amines are obtained by formal reductive amination, consuming just ammonia and a cheap reducing agent (formate) with up to 99% ee and 97% yield. The other enantiomer was accessible by employing the same ω-transaminases in a kinetic resolution starting from racemic amines. A ω-transaminase derived from an Arthrobacter species displayed the highest stereoselectivity for all substrates tested, both for the kinetic resolution of rac-amines and for the amination of ketones.

doi: 10.1002/adsc.200900826

The chemical synthesis of 3-substituted tyrosine derivatives requires a minimum of four steps to access optically enriched material starting from commercial precursors. Attempting to short-cut the cumbersome chemical synthesis of 3-substituted tyrosine derivatives, a single step biocatalytic approach was identified employing the tyrosine phenol lyase from Citrobacter freundii. The enzyme catalyses the hydrolysis of tyrosine to phenol, pyruvate and ammonium as well as the reverse reaction, thus the formation of tyrosine from phenol, pyruvate and ammonium. Since the wild-type enzyme possessed a very narrow substrate spectrum, structure-guided, site-directed mutagenesis was required to change the substrate specificity of this C–C bond forming enzyme. The best variant M379V transformed, for example, o-cresol, o-methoxyphenol and o-chlorophenol efficiently to the corresponding tyrosine derivatives without any detectable side-product. In contrast, all three phenol compounds were non-substrates for the wild-type enzyme. Employing the mutant, various L-tyrosine derivatives (3-Me, 3-OMe, 3-F, 3-Cl) were obtained with complete conversion and excellent enantiomeric excess (>97%) in just a single ‘green’ step starting from pyruvate and commercially available phenol derivatives.

doi: 10.1002/adsc.200900826

The development of various deracemisation concepts from our laboratory for secondary alcohols is summarised. The aim was to find alternatives for dynamic kinetic resolution and related deracemisation concepts. In our improved system, deracemisation is achieved via simultaneous enantioselective oxidation and asymmetric reduction, thereby demonstrating a rare example of concurrent oxidation and reduction in preparative organic chemistry. Such concepts could also be exploited for the racemisation of secondary alcohols through omitting the cofactor recycling.

doi: 10.1055/s-0029-1219567

ω-Transaminases have been evaluated as biocatalysts in the reductive amination of organoselenium acetophenones to the corresponding amines, and in the kinetic resolution of racemic organoselenium amines. Kinetic resolution proved to be more efficient than the asymmetric reductive amination. By using these methodologies we were able to obtain both amine enantiomers in high enantiomeric excess (up to 99%). Derivatives of the obtained optically pure o-selenium 1-phenylethyl amine were evaluated as ligands in the palladium-catalyzed asymmetric alkylation, giving the alkylated product in up to 99% ee.

doi: 10.1039/b920946h

A biocatalytic redox-neutral reaction cascade was designed for the deracemisation of racemic mandelic acid to yield optically pure L-phenylglycine employing three enzymes. The cascade consisted of three steps: a racemisation, an enantioselective oxidation and a stereoselective reductive amination. The enantioselective oxidation of D-mandelic acid to the corresponding oxo acid was coupled with the stereoselective reductive amination of the latter; thus the oxidation as well as the reduction reactions were performed simultaneously. The formal hydrogen abstracted in the first step – the oxidation – was consumed in the reductive amination allowing a redox-neutral cascade due to a cascade-internal cofactor recycling. The enantiomers of the starting material were interconverted by a racemase (mandelate racemase) ensuring that in theory 100% of the starting material can be transformed. Using this set-up racemic mandelic acid was transformed to optically pure L-phenylglycine (ee >97%) at 94% conversion without the requirement of any additional redox reagents in stoichiometric amounts.

doi: 10.1002/adsc.200900891

Mix and match: We report a cascade reaction sequence for the highly enantio- and diastereoselective synthesis of biaryl alcohols, employing a Suzuki coupling followed by a biocatalytic reduction in a biphasic system containing ionic liquids (ILs; see figure). We demonstrate the recyclability of the IL phase as well as the aqueous phase up to four cycles with only a negligible deactivation of reactivity and selectivity.

doi: 10.1002/chem.201000302

Chemoselective oxidations still represent a challenge for chemists. Lyophilized cells of Janibacter terrae were employed for the chemoselective oxidation of primary alcohols to the corresponding aldehydes by hydrogen transfer with the use of acetaldehyde as the hydrogen acceptor. Secondary alcohol moieties were transformed at a much slower rate. The substrate spectrum encompasses substituted benzyl alcohols, whereby substrates with a substituent in the meta position were well tolerated, whereas only very small substituents were tolerated in the ortho position. Furthermore, n-alkanols and allylic alcohols were transformed with good conversions. The biocatalyst was compatible with DMSO as a water miscible organic solvent up to 30% v/v.

doi: 10.1002/ejoc.201000260

Various bacterial cells were tested to identify ω-transaminase activity. For this purpose, the kinetic resolution of a rac-amine was chosen as an assay reaction transforming, in the ideal case, one enantiomer into the corresponding ketone and leaving the other enantiomer untouched. Sodium pyruvate was employed as an amino acceptor. To test also for the amination of the prochiral ketone various amino donors were investigated. Alanine proved to be the most suitable amino donor especially when coupled with a pyruvate decarboxylase to shift the reaction equilibrium; however, much lower conversions were achieved compared to the kinetic resolution. Janibacter terrae DSM 13953 was identified as the most suitable microorganism to possess ω-transaminase activity.

doi: 10.1016/j.tetasy.2010.07.009

The structure of the alcohol dehydrogenase ADH-‘A’ from Rhodococcus ruber reveals possible reasons for its remarkable tolerance to organic co-solvents and suggests new directions for structure-informed mutagenesis to produce enzymes of altered substrate specificity or improved selectivity.

doi: 10.1039/c0cc00929f

To ensure the quasi-irreversibility of the oxidation of alcohols coupled with the reduction of ketones in a hydrogen-transfer (HT) fashion, stoichiometric amounts of α-halo carbonyl compounds have been employed as hydrogen acceptors. The reason that these substrates lead to quasi-quantitative conversions has been tacitly attributed to both thermodynamic and kinetic effects. To provide a clear rationale for this behavior, we investigate herein the redox equilibrium of a selected series of ketones and 2-propanol by undertaking a study that combines experimental and theoretical approaches. First, the activity of the (R)-specific alcohol dehydrogenase from Lactobacillus brevis (LBADH) with these substrates was studied. The docking of acetophenone/(R)-1-phenyethanol and α-chloroacetophenone/(S)-2-chloro-1-phenylethanol in the active site of the enzyme confirms that there seems to be no structural reason for the lack of reactivity of halohydrins. This assumption is confirmed by the fact that the corresponding aluminum-catalyzed Meerwein–Ponndorf–Verley–Oppenauer (MPVO) reactions afford similar conversions to those obtained with LBADH, showing that the observed reactivity is independent of the catalyst employed. While the initial rates of the enzymatic reductions and the IR ν(C=O) values contradict the general belief that electron-withdrawing groups increase the electrophilicity of the carbonyl group, the calculated ΔG values of the isodesmic redox transformations of these series of ketones/alcohols with 2-propanol/acetone support the thermodynamic control of the reaction. As a result, a general method to predict the degree of conversion obtained in the HT-reduction process of a given ketone based on the IR absorption band of the carbonyl group is proposed, and a strategy to achieve the HT oxidation of halohydrins is also shown.

doi: 10.1002/chem.201001233

An iridium catalysed oxidation was coupled concurrently to an asymmetric biocatalytic reduction in one-pot; thus it was shown for the first time that iridium- and alcohol dehydrogenase-catalysed redox reactions are compatible. As a model system racemic chlorohydrins were transformed to enantioenriched chlorohydrins via an oxidation–asymmetric reduction sequence.

doi: 10.1039/c0cc02813d

Enzyme promiscuity is generally accepted as the ability of an enzyme to catalyse alternate chemical reactions besides the ‘natural‘ one. In this paper peroxidases were shown to catalyse the cleavage of a C=C double bond adjacent to an aromatic moiety for selected substrates at the expense of molecular oxygen at an acidic pH. It was clearly shown that the reaction occurs due to the presence of the enzyme; furthermore, the reactivity was clearly linked to the hemin moiety of the peroxidase. Comparison of the transformations catalysed by peroxidase and by hemin chloride revealed that these two reactions proceed equally fast; additional experiments confirmed that the peptide backbone was not obligatory for the reaction and only a single functional group of the enzyme was required, namely in this case the prosthetic group (hemin). Consequently, we propose to define such a promiscuous activity as ‘ostensible enzyme promiscuity’. Thus, we call an activity that is catalysed by an enzyme ‘ostensible enzyme promiscuity’ if the reactivity can be tracked back to a single catalytic site, which on its own can already perform the reaction equally well in the absence of the peptide backbone.

doi: 10.1002/chem.201002265

O-Protected cyclic acyloins were obtained in nonracemic form through asymmetric bioreduction of α,β-unsaturated alkoxy ketones by using 11 different enoate reductases from the "Old Yellow Enzyme" family. The stereochemical outcome of the biotransformation could be switched by variation of the O-protecting group or by the ring size of the substrate, which allows access to both stereoisomers in up to >97% ee. Whereas α-alkoxy enones were readily accepted as substrates, β-analogs were not converted. Overall, α-alkoxy enones represent a novel type of substrate for flavin-dependent ene-reductases.

doi: 10.1002/ejoc.201001042

Enoate reductases from the ‘old yellow enzyme’ family were employed for the asymmetric bioreduction of methyl 2-hydroxymethylacrylate and its O-allyl, O-benzyl and O-TBDMS derivatives to furnish (R)-configurated methyl 3-hydroxy-2-methylpropionate products in up to >99% ee. Variation of the O-protective group had little influence on the stereoselectivity, but a major impact on the reaction rate.

doi: 10.1002/adsc.201000522

A straightforward, high-yielding, chemoenzymatic total synthesis of enantiopure (S)-Rivastigmine was developed using various ω-transaminases for the asymmetric amination of appropriate acetophenone precursors. Optimisation of the biotransformation allowed scale-up and the total synthesis of (S)-Rivastigmine.

doi: 10.1039/c0cc00585a

Optically pure amines are highly valuable products or key intermediates for a vast number of bioactive compounds; however, efficient methods for their preparation are rare. ω-Transaminases (TAs) can be applied either for the kinetic resolution of racemic amines or for the asymmetric synthesis of amines from the corresponding ketones. The latter process is more advantageous because it leads to 100% product, and is therefore a major focus of this review. This review summarizes various methodologies for transamination reactions, and provides an overview of ω-TAs that have the potential to be used for the preparation of a broad spectrum of α-chiral amines. Recent methodological developments as well as some recently identified novel ω-TAs warrant an update on this topic.

doi: 10.1016/j.tibtech.2010.03.003

Nonracemic aryl-substituted α-methyldihydrocinnamaldehyde derivatives employed as olfactory principles in perfumes (Lilial™, Helional™) were obtained via enzymatic reduction of the corresponding cinnamaldehyde precursors using cloned and overexpressed ene-reductases. (R)-Enantiomers were obtained using the old-yellow-enzyme (OYE) homolog YqjM from Bacillus subtilis and 12-oxophytodienoic acid reductase isoenzyme OPR1 from tomato (e.e._max 53%), and (S)-aldehydes were furnished in up to 97% e.e. using isoenzyme OPR3, nicotinamide 2-cyclohexene-1-one reductase NCR from Zymomonas mobilis and yeast OYE isoenzymes 1–3 under optimised reaction conditions in the presence of t-butyl methyl ether as the co-solvent. The stereochemical outcome of the reduction of α-methylcinnamaldehyde using NCR and OYEs 1–3 [previously reported to be (R)] was unambiguously corrected to be (S).

doi: 10.1039/c002971h

Four flavoproteins from the old yellow enzyme (OYE) family, pentaerythritol tetranitrate (PETNR) reductase, N-ethylmaleimide reductase (NEMR), morphinone reductase (MorR) and estrogen-binding protein (EBP1), exhibited a broad substrate tolerance by accepting conjugated enals, enones, imides, dicarboxylic acids and esters, as well as a nitroalkene and therefore can be employed for the asymmetric bioreduction of carbon-carbon double (C=C) bonds. In particular, morphinone reductase and estrogen-binding protein often showed a complementary stereochemical preference in comparison to that of previously investigated OYEs.

doi: 10.1002/adsc.200900832

The bioreduction of aliphatic sec-nitro compounds catalyzed by purified flavoproteins from the old-yellow-enzyme family unexpectedly furnished the corresponding carbonyl compounds instead of the expected amines and thus represents a biocatalytic equivalent to the Nef-reaction. The pathway was shown to proceed via initial reduction of the nitro-group to yield the nitroso-derivative, which spontaneously tautomerized to yield the more stable oxime, which was enzymatically reduced in a second step to furnish a hydrolytically unstable imine-species, which spontaneously hydrolyzed to finally give a carbonyl compound and ammonia.

doi: 10.1039/b922691e

Commercially available ω-transaminases ω-TA-117, -113, and Vibrio fluvialis (Vf-AT) have been immobilized in a sol–gel matrix. Improved results were obtained by employing Celite 545 as additive. The immobilized ω-transaminases ω-TA-117, -113, and V. fluvialis (Vf-AT) were tested in the kinetic resolution of α-chiral primary amines. In contrast to the free enzyme ω-TA-117, the sol–gel/celite immobilized enzyme showed activity even at pH 11. Recycling of the sol–gel/Celite 545 immobilized ω-transaminase ω-TA-117 was performed over five reaction cycles without any substantial loss in enantioselectivity and conversion. Finally, the immobilized ω-TA 117 was employed in a one-pot two-step deracemization of rac-mexiletine and rac-4-phenyl-2-butylamine, two pharmacologically relevant amines. The corresponding optically pure (S)-amines were obtained in up to 95% isolated yield (>99% ee).

doi: 10.1016/j.molcatb.2009.12.001

The asymmetric bioreduction of activated C=C-bonds catalyzed by a single flavoprotein was achieved via direct hydrogen transfer from a sacrificial 2-enone or 1,4-dione as hydrogen donor without requirement of a nicotinamide cofactor. Due to its simplicity, this system has clear advantages over conventional FAD-recycling systems.

doi: 10.1016/j.tet.2009.11.065

The reduction of phenylacetaldoxime was catalysed by alcohol dehydrogenases in the presence of NAD(P)H yielding finally the primary alcohol via the imine and aldehyde intermediates. This suggests that the hydride of the cofactor NAD(P)H is transferred to the N-atom of the oxime moiety and not to the carbon atom, as usual stated. This reaction represents the first example of a catalytic chemo-promiscuity of alcohol dehydrogenases.

doi: 10.1016/j.tet.2010.03.050

Dwindling petroleum feedstocks and increased CO2-concentrations in the atmosphere currently open the concept of using CO₂ as raw material for the synthesis of well-defined organic compounds. In parallel to recent advances in the chemical CO₂-fixation, enzymatic (biocatalytic) carboxylation is currently being investigated at an increased pace. On the one hand, this critical review provides a concise overview on highly specific biosynthetic pathways for CO₂-fixation and, on the other hand, a summary of biodegradation (detoxification) processes involving enzymes which possess relaxed substrate specificities, which allow their application for the regioselective carboxylation of organic substrates to furnish the corresponding carboxylic acids (145 references).

doi: 10.1039/b807875k

Hydroxide-loaded anion exchangers have been successfully employed to shift the equilibrium of a one-pot, two-step, two-enzyme cascade reaction affording enantiopure epoxides starting from prochiral α-chloroketones. The α-chloroketones were asymmetrically reduced employing an alcohol dehydrogenase and then transformed further to the corresponding epoxides employing halohydrin dehalogenases. Each epoxide enantiomer could be obtained with up to 93% conversion in enantiomerically pure form (>99% ee). In contrast to previous studies the amount of hydride donor (2-propanol) could be reduced due to favoured halohydrin formation in the reduction of α-chloroketones.

doi: 10.1016/j.tetasy.2009.02.035

An enzyme preparation of Trametes hirsuta cleaves alkenes following neither the classical dioxygenase mechanism nor via a monooxygenase mechanism. A catalytic cycle for an alternative enzymatic alkene cleavage was proposed, whereby two oxygen atoms derived from two different oxygen molecules are incorporated into the product(S).

doi: 10.1021/ja8097096

Racemic α-chiral primary amines were deracemised to optically pure amines in up to >99 % conversion and >99 % ee within 48 h. The deracemisation was a result of a stereoinversion of one amine enantiomer; the formal stereoinversion was achieved by a one-pot, two-step procedure: in the first step, kinetic resolution of the chiral racemic amine was performed by employing a ω-transaminase to yield an intermediate ketone and the remaining optically pure amine; in the second step, the ketone intermediate was stereoselectively transformed into the amine by employing alanine as the amine donor and a ω-transaminase displaying opposite stereopreference than the ω-transaminase in the first step. In the second step, lactate dehydrogenase was used to remove the side product pyruvate to shift the unfavourable reaction equilibrium to the product side. Depending on the order of the enantiocomplementary enzymes employed in the cascade, the (R), as well as the (S)-enantiomer was accessible.

doi: 10.1002/ejoc.200801265

A three-step, two-enzyme, one-pot reaction sequence starting from prochiral α-chloroketones leading to enantiopure β-azidoalcohols and β-hydroxynitriles is described. Asymmetric bioreduction of α-chloroketones by hydrogen transfer catalysed by an alcohol dehydrogenase (ADH) established the stereogenic centre in the first step to furnish enantiopure chlorohydrin intermediates. Subsequent biocatalysed ring closure to the epoxide and nucleophilic ring opening with azide, N3–, or cyanide, CN–, both catalysed by a nonselective halohydrin dehalogenase (Hhe) proceeded with full retention of configuration to give enantiopure β-azidoalcohols and β-hydroxynitriles, respectively. Both enantiomers of various optically pure β-azidoalcohols and β-hydroxynitriles were synthesised.

doi: 10.1016/j.tet.2009.06.088

Various types of biocatalysts like oxidases, alcohol dehydrogenases, and microbial cells were tested for the oxidation of benzyl alcohol. Oxidases in combination with molecular oxygen led to low conversion. Alcohol dehydrogenases and microbial cells were tested in a hydrogen transfer reaction employing acetaldehyde, chloroacetone, and acetone as hydrogen acceptor. Excellent conversion (95%) could be achieved employing lyophilised cells of Janibacter terrae DSM 13953 at a substrate concentration of 97 mM.

doi: 10.1016/j.tet.2009.06.088

The lipase-catalyzed kinetic resolution of rac-1-phenylethanol with vinyl acetate as acyl donor and cyclohexane as solvent has been investigated applying both microwave dielectric heating and conventional thermal heating in order to probe the existence of nonthermal microwave effects. All transformations were conducted at 40 °C in a dedicated reactor setup that allowed accurate internal reaction temperature measurements with use of fiber-optic probes. Quartz reaction vessels that allow higher levels of microwave power to be administered to the reaction mixture were used for all experiments. For all five studied immobilized lipases, the observed reactivities and enantioselectivities in microwave and oil bath experiments were identical and thus not related to the presence of the microwave field. The effect of magnetic stirring proved critical as too rapid stirring in some instances destroyed the enzyme support structure and led to altered reactivities and selectivities.

doi: 10.1021/jo9010443

A lipase from Thermomyces lanuginosus and cutinases from Thermobifida fusca and Fusarium solani hydrolysed poly(ethylene terephthalate) (PET) fabrics and films and bis(benzoyloxyethyl) terephthalate (3PET) endo-wise as shown by MALDI-Tof-MS, LC–UVD/MS, cationic dyeing and XPS analysis. Due to interfacial activation of the lipase in the presence of Triton X-100, a seven-fold increase of hydrolysis products released from 3PET was measured. In the presence of the plasticizer N,N-diethyl-2-phenylacetamide (DEPA), increased hydrolysis rates of semi-crystalline PET films and fabrics were measured both for lipase and cutinase. The formation of novel polar groups resulted in enhanced dye ability with additional increase in colour depth by 130% and 300% for cutinase and lipase, respectively, in the presence of plasticizer.

doi: 10.1016/j.jbiotec.2009.07.008

Two different biocatalytic reactions – a C=C cleavage and a C-C forming reaction – were evaluated concerning their application in a reaction sequence. In the overall reaction, an aromatic alkene was converted to a chiral 2-hydroxy ketone. In the first step, the olefin trans-anethole was converted to para-anisaldehyde and acetaldehyde by an aqueous extract of the white rot fungus Trametes hirsuta G FCC 047. The selective oxidative cleavage of the carbon–carbon double bond was achieved using molecular oxygen as a substrate. In a second step p-anisaldehyde was ligated to acetaldehyde to yield either (R)- or (S)-2-hydroxy-1-(4-methoxyphenyl)-propanone. The reaction was catalyzed by the enantiocomplementary C-C bond forming enzymes benzaldehyde lyase and benzoylformate decarboxylase, respectively.

doi: 10.1016/j.molcatb.2008.08.009

(S)-as well as (R)-mexiletine [1-(2,6-dimethylphenoxy)-2-propanamine], a chiral orally effective antiarrhythmic agent, was prepared by deracemization starting from the commercially available racemic amine using ω-transaminases in up to >99% ee and conversion with 97% isolated yield by a one-pot two-step procedure. The absolute configuration could be easily switched to the other enantiomer, just by switching the order of the applied transaminases. The cosubstrate pyruvate needed in the first oxidative step was recycled by using an amino acid oxidase.

doi: 10.1021/ol901834x

Enantiomerically enriched 4-phenylpyrrolidin-2-one was prepared within only three steps starting from a commercial compound employing dynamic kinetic resolution (DKR) as the key asymmetric step. To the best of our knowledge, for the first time a DKR was performed involving an enzymatic enantioselective amination reaction catalyzed by ω-transaminases. Careful optimization of co-solvent and pH conditions allowed enhancing the enantioselectivity. The general method allows access to 4-arylpyrrolidin-2-ones derivatives, the cyclic analogues of γ-aminobutyric acid (GABA) derivatives.

doi: 10.1016/j.molcatb.2009.05.006

In contrast with electrophilic enzyme-catalysed cyclisations in terpenoid biosynthesis, cyclisations of tetrahydrofuran moieties found in several groups of natural products, such as annonaceous acetogenins, neurofurans and phytooxylipins, appear to proceed through a nucleophilic cascade mechanism starting from bis-epoxy fatty acid precursors. This hypothesis was verified by epoxide-hydrolase-catalysed hydrolytic ring-opening/cyclisation cascades starting from a methylene-interrupted meso-bis-epoxide model substrate, which furnished the corresponding THF products with excellent de and ee values. Molecular modelling showed that the points of enzyme attack were consistent with the stereospecificities of the enzymes, whereas the stereochemical courses of the cyclisation were solely governed by Baldwin's rules and did not invoke the involvements of a “cyclase”.

doi: 10.1002/cbic.200900176

Biocatalytic racemization of aliphatic, (aryl)aliphatic and aromatic α-hydroxycarboxylic acids was achieved via a reversible oxidation-reduction sequence using a pair of stereo-complementary Prelog- and anti-Prelog D- and L-α-hydroxyisocaproate dehydrogenases from Lactobacillus confusus DSM 20196 and Lactobacillus paracasei DSM 20008, resp., overexpressed in Escherichia coli. The mild reaction conditions ensured essential ‘clean’ isomerization, undesired ‘over-oxidation’ of the substrate forming the α-ketoacid could be suppressed by exclusion of O₂ and adjustment of the NAD⁺/NADH-ratio.

doi: 10.1016/j.tet.2009.06.051

Whole resting cells of cyano- and thio-bacteria Synechococcus and Paracoccus spp. were shown to possess inverting alkylsulfatase activity for a broad spectrum of sec-alkylsulfate esters, which furnished either (R)- or (S)-sec-alcohols from the corresponding rac-sulfate esters in an enantiocomplementary fashion. Low enantioselectivities (E-values 1–4) could be dramatically improved by the addition of lower alcohols (e.g., t-BuOH) or by using a biphasic medium containing t-BuOMe (E >200).

doi: 10.1016/j.tetasy.2009.01.007

A non-lipase-based, enantiocomplementary chemoenzymatic route towards enantiopure (R)- and (S)-chromanemethanol (12), which are the key building blocks for the synthesis of stereoisomerically pure α-tocopherols, has been achieved by the biocatalytic resolution of a racemic 2,2-disubstituted oxirane using an epoxide hydrolase and a halohydrin dehalogenase, which exhibit opposite enantiopreferences. The introduction of chirality at an early stage of the synthesis ensured a high efficiency, leading to total overall yields of 16 and 26% for (R)- and (S)-chromanemethanol (12), respectively.

doi: 10.1002/ejoc.200800950

Three FMN-dependent oxidoreductases, YcnD and YhdA from Bacillus subtilis and Lot6p from Saccharomyces cerevisiae, oxidised α,β-unsaturated carbonyl compounds and a thioether, respectively, to furnish the corresponding racemic epoxides or sulfoxide, respectively. The mechanism of this enzyme-mediated (rather than enzyme-catalysed) oxidation was shown to proceed via the NADH-dependent reduction of O₂, forming H₂O₂, which acted as oxidant in a spontaneous (non-enzymatic) fashion.

doi: 10.1039/b819057g

Breaking the mirror: A purified alcohol dehydrogenase (ADH) for stereoselective reduction and whole cells of a microorganism for enantioselective oxidation operated concurrently to effect the stereoinversion of one enantiomer of a racemic secondary alcohol and provide the optically pure alcohol in >99% yield (see scheme). R,R′=alkyl.

doi: 10.1002/anie.200703296

All for one: A combination of three biocatalysts (ω-transaminase, alanine dehydrogenase, and an enzyme such as formate dehydrogenase for cofactor recycling) catalyze a cascade to achieve the asymmetric transformation of a ketone into a primary α-chiral unprotected amine through a formal stereoselective reductive amination (see scheme). Only ammonia and the reducing agent (formate) are consumed during this reaction.

doi: 10.1002/anie.200803763

Three cloned enoate reductases from the “old yellow enzyme” family of flavoproteins were investigated in the asymmetric bioreduction of activated alkenes. 12-Oxophytodienoate reductase isoenzymes OPR1 and OPR3 from Lycopersicon esculentum (tomato), and YqjM from Bacillus subtilis displayed a remarkably broad substrate spectrum by reducing α,β-unsaturated aldehydes, ketones, maleimides and nitroalkenes. The reaction proceeded with absolute chemoselectivity – only the conjugated C=C bond was reduced, while isolated olefins and carbonyl groups remained intact – with excellent stereoselectivities (ees up to >99%). Upon reduction of a nitroalkene, the stereochemical outcome could be determined via choice of the appropriate enzyme (OPR1 versus OPR3 or YqjM), which furnished the corresponding enantiomeric nitroalkanes in excellent ee. Molecular modelling suggests that this “enzyme-based stereocontrol” is caused by subtle differences within the active site geometries.

doi: 10.1002/adsc.200700458

The asymmetric bioreduction of C=C-bonds bearing an electron-withdrawing group, such as an aldehyde, ketone, imide, nitro, carboxylic acid, or ester moiety by a novel enoate reductase from Zymomonas mobilis and Old Yellow Enzymes OYE 1–3 from yeasts furnished the corresponding saturated products in up to >99 % ee. Depending on the substrate type, stereocontrol was achieved by variation of the substrate structure, by switching the (E/Z) geometry of the alkene or by choice of the appropriate enzyme. This substrate- orenzyme-based stereocontrol allowed access to the opposite enantiomeric products.

doi: 10.1002/ejoc.200701208

Biocatalysts suitable for the reduction of ketones bearing two sterically demanding substituents (bulky–bulky ketones) at elevated substrate concentration (10 g L^–1) were identified. The biocatalysts can be employed in a substrate-coupled approach; thus, a simple alcohol such as ethanol or 2-propanol serves as a hydrogen donor. Both enantiomers are accessible by using either Rhodococcus ruber DSM 44541 and ethanol or Ralstonia sp. DSM 6428 and Sphingobium yanoikuyae DSM 6900 and ethanol or 2-propanol as the hydrogen source. For Rhodococcus ruber DSM 44541, it was found that bulky–bulky ketones were only transformed when ethanol was used as a hydrogen source, whereas no conversion was observed when 2-propanol was employed. From the substrate spectrum, as well as from the cosubstrate preference, it became clear that a different alcohol dehydrogenase than the previously described ADH-“A” is active in the presence of ethanol in Rhodococcus ruber.

doi: 10.1002/ejoc.200800103

A novel short-chain alcohol dehydrogenase from Paracoccus pantotrophus DSM 11072, which is applicable for hydrogen transfer, has been identified, cloned, and overexpressed in E. coli. The enzyme stereoselectively reduces several ketones in a sustainable substrate-coupled approach using 2-propanol (5% v/v) as hydrogen donor. The enzyme maintained its activity in organic co-solvents in biphasic as well as monophasic systems and was even active in micro-aqueous media (1% v/v aqueous buffer). In general, a higher conversion was observed at higher logP values of the solvent, however, DMSO, which exhibits the lowest logP value of all solvents investigated, was not only tolerated but led to a higher conversion and relative activity (110–210%). For example, the conversion after 24 h in 15% v/v DMSO was double that for the reaction performed in buffer. This tolerance to DMSO may be attributed to the ability of the wild-type strain to adapt and grow in media with high sulfur content.

doi: 10.1002/cssc.200800032

Quasi-irreversible oxidation of sec-alcohols was achieved via biocatalytic hydrogen transfer reactions using alcohol dehydrogenases employing selected ketones as hydrogen acceptors, which can only be reduced but not oxidized. Thus, only 1 equiv of oxidant was required instead of a large excess. For the oxidation of both isomers of methylcarbinols a single nonstereoselective short-chain dehydrogenase/reductase from Sphingobium yanoikuyae was identified and overexpressed in E. coli.

doi: 10.1021/ol800549f

Immobilization of the alcohol dehydrogenase ADH-‘A’ from Rhodococcus ruber DSM 44541 has been performed with different amino-functionalized carrier materials. The procedure included the activation of the carrier with glutaraldehyde and subsequent covalent binding to the enzyme. The porous glass beads TRISOPERL® and TRISOPOR®, magnetic particles, and detonation nanodiamonds were used as carriers in these experiments. In all cases, the immobilization was successful with almost quantitative immobilization yields; subsequently the activity for the reduction of acetophenone was lower compared to the activity of the free biocatalyst. Activity yields of 40% and 60% were obtained. The immobilized biocatalysts showed high stabilities in repetitive batches.

doi: 10.1016/j.tetasy.2008.04.034

Alkenes possessing a C=C double bond adjacent to an aromatic ring were cleaved to yield the corresponding carbonyl compounds by use of molecular oxygen as the sole oxidant and a cell-free extract of the wood-degrading fungus Trametes hirsuta FCC 047 as catalyst. The oxygen pressure required was optimized. Special adapted equipment allowed 96 reactions to be performed in parallel under controlled oxygen pressure. A broad spectrum of aryl-alkenes was successfully converted into the corresponding ketones/aldehydes with excellent chemoselectivity under a controlled oxygen atmosphere (2 bar).

doi: 10.1002/ejoc.200800261

Ketones with two bulky substituents, named bulky-bulky ketones, as well as less sterically demanding ketones were successfully reduced to the corresponding optically highly enriched alcohols using a novel identified recombinant short-chain alcohol dehydrogenase RasADH from Ralstonia sp. DSM 6428 overexpressed in E. coli.

doi: 10.1021/jo800849d

A broad range of ketones including methyl-aryl-, methyl-alkyl-, cyclic and sterically hindered ketones were reduced to the corresponding anti-Prelog alcohols with moderate to excellent stereoselectivities by employing lyophilised cells of Paracoccus pantotrophus DSM 11072 and Comamonas sp. DSM 15091 via hydrogen transfer. The reduction equivalents were provided using 2-propanol as a hydride donor. For instance, acetophenone was reduced to the corresponding (R)-enantiomer with >99% ee.

doi: 10.1016/j.tetasy.2008.08.005

A broad range of ketones including methyl-aryl-, methyl-alkyl-, cyclic and sterically hindered ketones were reduced to the corresponding anti-Prelog alcohols with moderate to excellent stereoselectivities by employing lyophilised cells of Paracoccus pantotrophus DSM 11072 and Comamonas sp. DSM 15091 via hydrogen transfer. The reduction equivalents were provided using 2-propanol as a hydride donor. For instance, acetophenone was reduced to the corresponding (R)-enantiomer with >99% ee.

doi: 10.1016/j.tetasy.2008.08.005

Lyophilized cells of the open accessible bacterium Comamonas testosteroni DSM 1455 proved to be an excellent catalyst for the asymmetric reduction of different α-azido, α-bromo, and α-nitro ketones at elevated substrate concentrations (16 g/L) in a ‘substrate-coupled’ approach using 20% (v/v) of 2-propanol as hydrogen donor. Excellent anti-Prelog stereoselectivity was obtained, which is less common found in nature.

doi: 10.1016/j.molcatb.2008.02.009

Black and white are opposites as are oxidation and reduction. Performing an oxidation, for example, of a sec-alcohol and a reduction of the corresponding ketone in the same vessel without separation of the reagents seems to be an impossible task. Here we show that oxidative cofactor recycling of NADP⁺ and reductive regeneration of NADH can be performed simultaneously in the same compartment without significant interference. Regeneration cycles can be run in opposing directions beside each other enabling one-pot transformation of racemic alcohols to one enantiomer via concurrent enantioselective oxidation and asymmetric reduction employing defined alcohol dehydrogenases with opposite stereo- and cofactor-preference. Thus, by careful selection of appropriate enzymes, NADH recycling can be performed in the presence of NADP+ recycling to achieve overall, for example, deracemisation of sec-alcohols or stereoinversion representing a possible concept for a “green” equivalent to the chemical-intensive Mitsunobu inversion.

doi: 10.1021/ja804816a

One often-cited weakness of biocatalysis is the lack of mirror-image enzymes for the formation of either enantiomer of a product in asymmetric synthesis. Enantiocomplementary enzymes exist as the solution to this problem in nature. These enzyme pairs, which catalyze the same reaction but favor opposite enantiomers, are not mirror-image molecules; however, they contain active sites that are functionally mirror images of one another. To create mirror-image active sites, nature can change the location of the binding site and/or the location of key catalytic groups. In this Minireview, X-ray crystal structures of enantiocomplementary enzymes are surveyed and classified into four groups according to how the mirror-image active sites are formed.

doi: 10.1002/anie.200705159

The isolation of single stereoisomers from a racemic (or diastereomeric) mixture by enzymatic or chemical resolution techniques goes in hand with the disposal of 50 % (racemate) or more (diastereomeric mixtures) of the “undesired” substrate isomer(S). In order to circumvent this drawback, dynamic systems have been developed for the de-racemization of enantiomers and the de-epimerizations of diastereomers. Key strategies within this area are discussed and are classified according to their underlying kinetics, that is, dynamic kinetic resolution (DKR), dynamic kinetic asymmetric transformations (DYKAT), and hybrids between both of them. Finally, two novel types of DYKAT are defined.

doi: 10.1002/chem.200701643

Biocatalytic deracemisation via inversion of rac-2-decanol was accomplished by a combined oxidation/reduction sequence using the same ‘single’ catalyst for both steps. Overall, the (R)-alcohol was inverted to the corresponding (S)-alcohol. Lyophilised cells of various Rhodococci spp. were tested for the unselective oxidation of the racemic sec-alcohol using acetone as the hydrogen acceptor in the first step. For the second step, the stereoselective asymmetric reduction of the corresponding ketone, 2-propanol was employed as the hydrogen donor. Employing lyophilised cells of Rhodococcus sp. CBS 717.73 racemic 2-decanol was transformed to (S)-2-decanol with excellent enantiomeric excess (92% ee) and yield (82% isolated yield) in the combined one-pot oxidation/reduction sequence.

doi: 10.1016/j.tetasy.2007.01.013

The oxidative cleavage of a C=C double bond adjacent to an aryl moiety was achieved in the presence of a protein preparation of Trametes hirsuta G FCC 047 to yield the corresponding aldehydes. Molecular oxygen was the only oxidant required. All positive substrates had a C=C bond conjugated to an aromatic system, all other compounds tested not fulfilling this requirement were non-substrates. The optimum reaction conditions are 20 °C, pH 6–6.5, 15% v/v ethanol as co-solvent at an apparent oxygen pressure of 2 bar.

doi: 10.1016/j.tet.2007.02.034

Mono- and biphasic aqueous−organic solvent systems (50% v v^-1) as well as micro-aqueous organic systems (99% v v-1) were successfully employed for the biocatalytic reduction of ketones catalyzed by alcohol dehydrogenase ADH-A from Rhodococcus ruber via hydrogen transfer. A clear correlation between the logP of the organic solvent and the enzyme activity - the higher, the better - was found. The use of organic solvents allowed highly stereoselective enzymatic carbonyl reductions at substrate concentrations close to 2.0 M.

doi: 10.1021/ol070679c

Tomato source: 12-Oxophytodienoate reductase isoenzymes OPR1 and OPR3 from tomato possess a broad substrate spectrum for the asymmetric bioreduction of α,β-unsaturated enals, enones, dicarboxylic acids, and N-substituted maleimides (see scheme). Stereocomplementary behavior of both isoenzymes was observed in the reduction of a nitroalkene that led to the formation of opposite stereoisomers in high enantiomeric excess.

doi: 10.1002/anie.200605168

A novel one-pot, two-step, two-enzyme cascade is described. Pro-chiral α-chloro ketones are stereoselectively reduced to the corresponding halohydrins as an intermediate by a biocatalytic hydrogen transfer process. The intermediate is transformed to the corresponding epoxide by a non-enantioselective halohydrin dehalogenase. Thus, by combining a Prelog- or anti-Prelog alcohol dehydrogenase with a non-selective halohydrin dehalogenase, enantiopure (R)- as well as (S)-epoxides were obtained.

doi: 10.1002/adsc.200700027

Biocatalytic racemization of straight-chain and cyclic acyloins bearing (halo)alkyl, alkenyl and functionalized (hetero)aryl moieties was accomplished using whole resting cells of bacteria, fungi and yeasts. Mild physiological reaction conditions ensured the suppression of undesired side-reactions, such as elimination or condensation. This biocatalytic protocol represents a useful tool for the clean racemization of unwanted enantiomers of synthetically important α-hydroxyketones derived from kinetic resolution.

doi: 10.1016/j.tetasy.2007.06.005

An easy to use computerized algorithm for the determination of the amount of each labeled species differing in the number of incorporated isotope labels based on mass spectroscopic data is described and evaluated. Employing this algorithm, the microwave-assisted synthesis of various α-labeled deuterium ketones via hydrogen−deuterium exchange with deuterium oxide was optimized with respect to time, temperature, and degree of labeling. For thermally stable ketones the exchange of α-protons was achieved at 180 °C within 40−200 min. Compared to reflux conditions, the microwave-assisted protocol led to a reduction of the required reaction time from 75−94 h to 40−200 min. The α-labeled deuterium ketones were reduced by biocatalytic hydrogen transfer to the corresponding enantiopure chiral alcohols and the deconvolution algorithm validated by regression analysis of a mixture of labeled and unlabeled ketones/alcohols.

doi: 10.1021/jo070831o

A thermally stable esterase (SNSM-87) from Klebsiella oxytoca is explored as an enantioselective biocatalyst for the hydrolytic resolution of (R,S)-2-hydroxycarboxylic acid esters in biphasic media, where the best methyl esters possessing the highest enantioselectivity and reactivity are selected and elucidated in terms of the structure–enantioselectivity correlations and substrate partitioning in the aqueous phase. With (R,S)-2-chloromandelates as the model substrates, an expanded Michaelis–Menten mechanism for the rate-limiting acylation step is adopted for the kinetic analysis. The Brønsted slope of 25.7 for the fast-reacting (S)-2-chloromandelates containing a difficult leaving alcohol moiety, as well as that of 4.13 for the slow-reacting (R)-2-chloromandelates in the whole range of leaving alcohol moieties, indicates that the breakdown of tetrahedral intermediates to acyl-enzyme intermediates is rate-limiting. However, the rate-limiting step shifts to the formation of tetrahedral intermediates for the (S)-2-chloromandelates containing an easy leaving alcohol moiety, and leads to an optimal enantioselectivity for the methyl ester substrate.

doi: 10.1002/bit.21394

Biocatalytic racemization of aliphatic and aryl-aliphatic sec-alcohols and α-hydroxyketones (acyloins) was accomplished using whole resting cells of bacteria, fungi, and one yeast. The mild (physiological) reaction conditions ensured the suppression of undesired side reactions, such as elimination or condensation. Cofactor and inhibitor studies suggest that the racemization proceeds through an equilibrium-controlled enzymatic oxidation–reduction sequence via the corresponding ketones or α-diketones, respectively, which were detected in various amounts. Ketone formation could be completely suppressed by exclusion of molecular oxygen.

doi: 10.1007/s00253-007-1071-0

Racemization is the key step to turn a kinetic resolution process into dynamic resolution. A general strategy for racemization under mild reaction conditions by employing stereoselective biocatalysts is presented, in which racemization is achieved by employing a pair of stereocomplementary biocatalysts that reversibly interconvert an sp3 to a sp2 center. The formal interconversion of the enantiomers proceeds via a prochiral sp² intermediate the formation of which is catalyzed either by two stereocomplementary enzymes or by a single enzyme with low stereoselectivity. By choosing appropriate reaction conditions, the amount of the prochiral intermediate is kept to a minimum. This general strategy, which is applicable to redox enzymes (e.g., by acting on R²CHOH and R²CHNHR groups) and lyase-catalyzed addition–elimination reactions, was proven for the racemization of secondary alcohols by employing alcohol dehydrogenases. Thus, enantiopure chiral alcohols were used as model substrates and were racemized either with highly stereoselective biocatalysts or by using (rarely found) non-selective enzymes.

doi: 10.1002/chem.200700528

Bi- and monophasic ionic liquid (IL)/buffer systems were successfully employed for the biocatalytic reduction of ketones catalysed by the alcohol dehydrogenase ADH-‘A’ from Rhodococcus ruber via hydrogen transfer. Two different catalyst preparations were employed, namely recombinant ADH-‘A’ ‘immobilised’ in Escherichia coli and partially purified ADH-‘A’. For biphasic systems conversions were acceptable until 20% v v⁻¹ of IL. In contrast, hydroxy-functionalised ‘Tris-like’-ILs were successfully employed in monophasic systems up to 90% v v⁻¹ IL. The use of these solvents allowed highly stereoselective enzymatic carbonyl reductions at substrate concentrations from 1.2 to 1.5 M.

doi: 10.1016/j.tetasy.2007.10.010

Asymmetric bioreduction of α,β-unsaturated dicarboxylic acids, such as 2-methylmaleic/fumaric and 2-methylenesuccinic acid, as well as the corresponding dimethyl esters, using three cloned enoate reductases furnished 2-methylsuccinic acid or dimethyl 2-methylsuccinate, respectively. Opposite stereoisomeric products were obtained in up to >99% ee either by choice of the enzyme or by using E/Z-configurated substrates. Cofactor-recycling systems (NADH/FDH/formate, NADH/GDH/glucose or NADPH/G6PDH/glucose-6-phosphate) only worked in presence of a divalent metal ion, such as Ca²⁺, Mg²⁺, or Zn²⁺.

doi: 10.1021/ol7019185

The majority of hydrolytic enzymes used in white biotechnology for the production of non-natural compounds – such as carboxyl ester hydrolases, lipases and proteases – show a certain preference for a given enantiomer. However, they are unable to alter the stereochemistry of the substrate during catalysis with respect to inversion or retention of configuration. The latter can be achieved by (alkyl) sulfatases, which can be employed for the enantio-convergent transformation of racemic sulfate esters into a single stereoisomeric secondary alcohol, with a theoretical yield of 100%. This is a major improvement over traditional kinetic resolution processes, which yield both enantiomers, each at 50%.

doi: 10.1016/j.tibtech.2006.11.006

The asymmetric bioreduction of alkenes bearing an electron-withdrawing group using flavin-dependent enzymes from the ‘old yellow enzyme’ family at the expense of NAD(P)H yields the corresponding non-racemic alkanes going in hand with the creation of up to two chiral carbon centres. To avoid external cofactor recycling, this intriguing biotransformation was hitherto performed using whole microbial cells, which frequently showed insufficient stereoselectivities and/or undesired side reactions because of the action of competing enzymatic activities. Co-expression of enoate reductases with the corresponding redox enzymes for NAD(P)H recycling in a suitable host enables to overcome these drawbacks to furnish highly stereoselective and ‘clean’ C=C bioreductions on a preparative scale that are difficult to perform by conventional means.

doi: 10.1016/j.cbpa.2007.02.025

In search of highly enantioselective microbial sec-alkyl sulfatase activity, a broad screening among bacteria, fungi andArchaea revealed several Ralstonia and Pseudomonas spp. as valuable sources, whereas fungi were completely inactive. In particular, Pseudomonas sp. DSM 6611 was able to hydrolyse the (R)-enantiomers of a broad range of rac-sec-alkyl sulfate esters with excellent enantioselectivities (E > 200) to furnish the corresponding inverted (S)-sec-alcohols in high ee's. The substrate range of this organism was remarkably broad and bulky groups were also nicely tolerated.

doi: 10.1002/ejoc.200700637

Deracemization of (±)-3-phenyllactic acid (1) and (±)-2-hydroxy-4-phenylbutanoic acid (2) was accomplished by lipase-catalysed kinetic resolution coupled to biocatalytic racemization of the non-reacting substrate enantiomers using Lactobacillus paracasei DSM 20008. Cyclic repetition of this sequence led to a single enantiomeric product from the racemate. Access to both enantiomers was achieved by switching between lipase-catalysed acyl-transfer and ester hydrolysis reactions. Both products constitute important building blocks for virus protease- and ACE-inhibitors, respectively.

doi: 10.1016/j.tet.2006.01.007

Strategies for the chemoenzymatic transformation of a racemate into a single stereoisomeric product in quantitative yield have been developed. A range of industrially relevant α-hydroxycarboxylic acids was deracemized in a stepwise fashion via lipase-catalysed enantioselective O-acylation, followed by mandelate racemase-catalysed racemization of the remaining non-reacted substrate enantiomer. Alternatively, aliphatic α-hydroxycarboxylic acids were enzymatically isomerized using whole resting cells of Lactobacillus spp. Enantioselective hydrolysis of rac-sec-alkyl sulphate esters was accomplished using novel alkyl sulphatases of microbial origin. The stereochemical path of catalysis could be controlled by choice of the biocatalyst. Whereas Rhodococcus ruber DSM 44541 and Sulfolobus acidocaldarius DSM 639 act through inversion of configuration, stereo-complementary retaining sulphatase activity was detected in the marine planctomycete Rhodopirellula baltica DSM 10527.

doi: 10.1042/bst20060296

The asymmetric reduction of symmetrical and nonsymmetrical diketones as well as the stereoselective oxidation of various diols by biocatalytic hydrogen transfer was investigated by employing lyophilized cells of Rhodococcus ruber DSM 44541 containing alcohol dehydrogense ADH-‘A’. Symmetrical and nonsymmetrical diketones at the (ω-1)- and (ω-2)-positions are reduced to the Prelog product with high stereopreference, while sterically more demanding ketone moieties, for example those at the (ω-3)-position, remain unchanged. For the oxidation mode, differentiation between primary and secondary alcohols is achieved, and the (S)-configured secondary alcohols at the (ω-1)- and (ω-2)-positions are oxidized preferentially.

doi: 10.1002/ejoc.200500839

The alcohol dehydrogenase ADH-‘A’ from Rhodococcus ruber DSM 44541 represents a highly efficient catalyst for biocatalytic hydrogen transfer reactions. Starting from an exceedingly low level of active ADH-‘A’ in Escherichia coli, the apparent specific activity of ADH-‘A’ overexpressed in E. coli cells could be drastically enhanced by a factor of 550 by optimizing the host and induction/growth conditions. The influence of reaction parameters like pH, cosubstrate (2-propanol, acetone) concentration, substrate concentration temperature and additional cofactor on the apparent activity was investigated. In contrast to the purified enzyme, the pH optimum for oxidation and reduction were identical. Due to the employment of whole cells of E. coli/ADH-‘A’ as catalyst lower operational stability was found.

doi: 10.1002/elsc.200620902

Biocatalytic racemization of open-chain and cyclic dialkyl-, alkyl-aryl- and diaryl-substituted acyloins was accomplished using whole resting cells of Lactobacillus paracasei DSM 20207. The mild (physiological) reaction conditions ensured the suppression of undesired side reactions, such as elimination or condensation. This novel biocatalytic isomerization protocol represents an essential tool for the deracemization of pharmacologically important building blocks.

doi: 10.1002/adsc.200606055

Employing acetone as hydrogen acceptor various sec-alcohols can be oxidized to the corresponding ketone in an asymmetric fashion. The enzyme exhibits exclusive regioselectivity for secondary alcohols, primary alcohols remain untouched. This protocol does not only provide a simple ‘green’ oxidation method for organic synthesis at ambient conditions, but also allows the preparation of ketones, which are labeled ‘natural’ for flavor and fragrance applications.

doi: 10.1016/j.molcata.2006.02.007

Employing the over-expressed highly organic solvent tolerant alcohol dehydrogenase ADH-‘A’ from Rhodococcus ruber DSM 44541, versatile building blocks, which were not accessible by the wild type catalyst, were obtained in > 99% e.e.; furthermore, employing d₈-2-propanol as deuterium source, stereoselective biocatalytic deuterium transfer was made feasible to furnish enantiopure deuterium labeled sec-alcohols on a preparative scale employing a single enzyme.

doi: 10.1039/b602487d

O₂can do: Innocuous molecular oxygen O₂ is the only reagent needed to perform highly chemoselective biocatalytic single-step alkene-cleavage reactions (see scheme). The products are analogous to those of (reductive) ozonization and related metal-based methods. In contrast neither special equipment nor an additional reducing agent is required. The biocatalytic reaction can be performed at ambient temperature. Depending on the substrate, aldehydes or ketones are obtained.

doi: 10.1002/anie.200601574

Whole lyophilized cells of an Escherichia coli overexpressing the alcohol dehydrogenase (ADH-'A') from Rhodococcus ruber DSM 44541 were used for the asymmetric reduction of ketones to secondary alcohols. The recycling of the required nicotinamide cofactor (NADH) was achieved in a coupled-substrate process. In the course of the reaction the ketone is reduced to the alcohol and the hydrogen donor 2-propanol is oxidized to acetone by one enzyme. This leads to a thermodynamic equilibrium between all four components determining the maximum achievable conversion. To overcome this limitation an in situ product removal technique (ISPR) for the application with whole cells was developed. In this method the most volatile compound is separated from the reaction vessel by an air flow resulting in a shift of the equilibrium towards the desired secondary alcohol. The so-called stripping process represents a simple and efficient method to overcome the thermodynamic limitation in biocatalytic reactions. Employing this method, the conversion of selected biotransformations was increased up to completeness.

doi: 10.1002/bit.21014

The stereoinversion of one enantiomer into its mirror-image counterpart within a racemate furnishes a single stereoisomeric product in 100% theoretical yield. This extremely efficient type of deracemization, whereby substrate and product possess an identical chemical structure, can be achieved by using bio- or chemo-catalysts or combinations thereof and is applicable to secondary alcohols, amines and α-substituted carboxylic acids. Special emphasis is devoted to the theoretical background of the one-pot, single-step deracemization of sec-alcohols.

doi: 10.1002/adsc.200606158

The biocatalytic racemization of a range of (hetero)aryl- and (di)aryl-aliphatic α-hydroxycarboxylic acids has been achieved by using whole resting cells of Lactobacillus spp. The essentially mild (physiological) reaction conditions ensure the suppression of undesired side reactions, such as elimination, decomposition or condensation. Cofactor/inhibitor studies using a cell-free extract of Lactobacillus paracasei DSM 20207 reveal that the addition of redox cofactors (NAD⁺/NADH) leads to a distinct increase in the racemization rate, while strong inhibition is observed in the presence of Thio-NAD⁺, which suggests that the racemization proceeds by an oxidation–reduction sequence rather than involvement of a "racemase" enzyme.

doi: 10.1002/ejoc.200600454

Polyether ionophores, such as monensin A, are known to be biosynthesised, like many other antibiotic polyketides, on giant modular polyketide synthases (PKSs), but the intermediates and enzymes involved in the subsequent steps of oxidative cyclisation remain undefined. In particular there has been no agreement on the mechanism and timing of the final polyketide chain release. We now report evidence that MonCII from the monensin biosynthetic gene cluster in Streptomyces cinnamonensis, which was previously thought to be an epoxide hydrolase, is a novel thioesterase that belongs to the α/β-hydrolase structural family and might catalyse this step. Purified recombinant MonCII was found to hydrolyse several thioester substrates, including an N-acetylcysteamine thioester derivative of monensin A. Further, incubation with a hallmark inhibitor of such enzymes, phenylmethanesulfonyl fluoride, led to inhibition of the thioesterase activity and to the accumulation of an acylated form of MonCII. These findings require a reassessment of the role of other enzymes implicated in the late stages of polyether ionophore biosynthesis.

doi: 10.1002/cbic.200500474

Asymmetric bioreduction of (E/Z)-3,7-dimethyl-2,6-octadienal (citral) using the enoate reductase activity of whole cells of yeasts, bacteria and fungi, gave the α,β-saturated aldehyde (R)-3,7-dimethyl-6-octenal (citronellal), which constitutes an important flavour component, in up to >95% ee. Depending on the microorganism, various amounts of prim-alcohols (nerol/geraniol and citronellol) were formed due to the action of competing prim-alcohol dehydrogenases. Citral lyase activity—leading to the loss of a C2-fragment (acetaldehyde) forming sulcatone—and oxidation of the aldehyde moiety yielding the carboxylic acid (geranic/neric acid) were detected as additional metabolic activities.

doi: 10.1016/j.tetasy.2006.11.018

The kinetic resolution of (±)-2-methylglycidyl benzyl ether was achieved via enantioselective biohydrolysis using microbial and plant epoxide hydrolases. Depending on the type of enzyme, opposite enantiopreference and stereo-complementary mode of action (i.e., retention vs inversion of configuration) led to hetero- and homochiral product mixtures. Optimization of the reaction conditions for Rhodococcus sp. R312 led to significantly enhanced enantioselectivity (E >200), which enabled the deracemization of (±)-2-methylglycidyl benzyl ether via biohydrolysis (proceeding with retention of configuration) followed by inverting acid-catalyzed hydrolysis to furnish (R)-1-benzyloxy-2-methylpropane-2,3-diol in >97% ee and 78% yield from the racemate.

doi: 10.1016/j.tetasy.2005.12.018

The influence of the nature of acyl donors on the regioselectivity of Candida rugosa lipase for the esterification of streptol — a cyclitol derivative — was investigated. Excellent regioselectivity for the formation of 3,7-disubstituted derivatives was observed for vinyl butyrate (100% 3,7-derivative, 68% yield) and vinyl propionate (100% 3,7-derivative, 46% yield) as acyl donors. In contrast, for vinyl methacrylate as acyl donor, a mixture of 71% 3,7-derivative and 29% 1,7-derivative was obtained. Varying the chain length, a certain dependency of regioselectivity on the acyl donor was observed, however, no logical correlation satisfying all cases was found. Mono-substituted streptol derivatives were obtained by employing Novozym 243.

doi: 10.1016/j.molcatb.2004.12.012

Biocatalytic racemisation of aliphatic, aryl-aliphatic and aromatic α-hydroxycarboxylic acids was accomplished using whole resting cells of Lactobacillus paracasei DSM 20207; the mild (physiological) reaction conditions ensured an essentially ‘clean’ isomerization in the absence of side reactions, such as elimination or decomposition.

doi: 10.1039/b418786e

Several novel bioprocesses that have little or no counterpart in traditional methodology have recently been reported. The stereoselective and enantioselective hydrolysis of sec-alkyl sulfate esters by alkyl sulfatases proceeds with inversion of configuration and furnishes a homochiral product mixture. Haloalcohol dehalogenases were shown to accept various non-natural nucleophiles, such as azide, cyanide and nitrite for the asymmetric opening of epoxides giving rise to the corresponding azido-, cyano-, and nitro-alcohols as non-natural products. Asymmetric carbon–carbon bond formation via the acyloin- and benzoin-reaction was successfully catalyzed in water by novel lyases, such as benzoylformate decarboxylase and benzaldehyde lyase. New methods for the production of chiral nonracemic α-L-amino acids and amines were recently reported. Enantioselective stereoinversion of racemic α-aryl- and α-aryloxycarboxylic acids via epimerase-catalyzed inversion led to a single stereoisomeric product from the racemate.

doi: 10.1016/j.cbpa.2005.01.001

Biocatalytic racemization of a range of aliphatic, (aryl)aliphatic, and aromatic α-hydroxycarboxylic acids was accomplished by using whole resting cells of a range of Lactobacillus spp. The mild (physiological) reaction conditions ensured an essentially "clean" isomerization in the absence of side reactions, such as elimination or decomposition. Whereas straight-chain aliphatic 2-hydroxycarboxylic acids were racemized with excellent rates (up to 85% relative to lactate), steric hindrance was observed for branched-chain analogues. Good rates were observed for aryl−alkyl derivatives, such as 3-phenyllactic acid (up to 59%) and 4-phenyl-2-hydroxybutanoic acid (up to 47%). In addition, also mandelate and its o-chloro analogue were accepted at a fair rate (45%). This biocatalytic racemization represents an important tool for the deracemization of a number of pharmaceutically important building blocks.

doi: 10.1021/jo050156n

Mandelate racemase (EC 5.1.2.2) is one of the few biochemically well-characterized racemases. The remarkable stability of this cofactor-independent enzyme and its broad substrate tolerance make it an ideal candidate for the racemization of non-natural α-hydroxycarboxylic acids under physiological reaction conditions to be applied in deracemization protocols in connection with a kinetic resolution step. This review summarizes all aspects of mandelate racemase relevant for the application of this enzyme in preparative-scale biotransformations with special emphasis on its substrate tolerance. Collection and evaluation of substrate structure-activity data led to a set of general guidelines, which were used as basis for the construction of a general substrate model, which allows a quick estimation of the expected activity for a given substrate.

doi: 10.1002/adsc.200505012

Deracemization of (±)-2-hydroxy-4-phenylbut-3-enoic acid was accomplished by lipase-catalyzed kinetic resolution ­coupled to mandelate racemase-mediated racemization of the non-reacting substrate enantiomer. Stepwise cyclic repetition of this ­sequence led to a single enantiomeric product, the stereochemical outcome of which could be controlled by switching between lipase-catalyzed acyl-transfer/ester hydrolysis reactions. Both enantio­meric products were easily transformed into (R)- and (S)-2-hydroxy-4-phenylbutanoic acid, important building blocks for ACE-inhibitors.

doi: 10.1055/s-2005-871577

Biocatalytic hydrogen-transfer reduction of α-chloro-ketones furnished non-racemic chlorohydrins by employing either Rhodococcus ruber as lyophilized cell catalyst or an alcohol dehydrogenase preparation from Pseudomonas fluorescens DSM 50106 (PF-ADH). For all substrates investigated, Rhodococcus ruber gave strictly the "Prelog" product, whereas PF-ADH showed scattered stereopreference. One possibility for a follow-up reaction of halohydrins is the ring closure to the corresponding epoxide. A novel "one pot-one step strategy" was employed to obtain the enantiopure epoxide from the αα-chloro-ketone in a cascade like fashion at pH>12 involving biocatalytic hydrogen transfer reduction and in situ chemo-catalyzed ring closure.

doi: 10.1002/adsc.200505094

An optimised method for the stereoselective hydrolysis of sec-alkylsulfate monoesters with absolute retention of configuration was developed. Under optimised conditions, clean hydrolysis of (R)-2-octyl sulfate was achieved in aqueous t-butyl methyl ether (3:97) using 0.6 equiv of p-toluenesulfonic acid as catalyst and 0.33 equiv of dioxane as mediator to give (R)-2-octanol as the sole product in the absence of side reactions, such as racemisation or elimination.

doi: 10.1016/j.tet.2004.11.075

A chemoenzymatic, asymmetric total synthesis of the anti-biotic (R)-fridamycin E has been accomplished following a biocatalytic deracemization procotol. The key step comprises the construction of the chiral side-chain from a functionalized rac-2,2-disubstituted oxirane via a kinetic resolution/stereoinversion sequence without formation of the undesired stereoisomer.

doi: ejoc.200400720

rac-sec-Alkyl sulfate esters 1a–8a were resolved in low to excellent enantioselectivities with E-values up to >200 using whole cells of aerobically-grown hyperthermophilic sulfur-metabolizers, such as Sulfolobus solfataricus DSM 1617, Sulfolobus shibatae DSM 5389 and, most notably, Sulfolobus acidocaldarius DSM 639. Significantly enhanced selectivities were obtained using cells grown on sucrose-enriched Brock-medium. The stereochemical course of this biohydrolysis was shown to proceed with strict inversion of configuration, thus the preferred (R)-enantiomers were converted into the corresponding (S)-sec-alcohols to furnish a homochiral product mixture.

doi: 10.1039/b504883d

Hydrolytic enzymes: The marine planctomycete Rhodopirellula baltica DSM 10527 displays high stereo- and enantioselective alkyl sulfatase activity towards (±)-sec-alkyl sulfates with retention of configuration through cleavage of their S–O bond (see scheme; pathway B), whereas inversion of configuration is observed upon cleavage of the C–O bond (pathway A).

doi: 10.1002/anie.200501955

Lyophilised cells of various Rhodococcus spp. were employed in an efficient hydrogen transfer-like process for the asymmetric bioreduction of heteroaryl methyl ketones using 2-propanol as hydrogen donor. Besides the genus Rhodococcus, only Mycoplana rubra R14 showed a comparable stability towards elevated concentrations of the co-substrate 2-propanol. Among the organisms tested, Rhodococcus ruber DSM 44541 and DSM 43338 showed best activity and selectivity. With these strains, the reaction proceeded with high stereoselectivity (ee 99%) and predictable stereochemical outcome regardless of the nature of the heteroaromatic ring system. The reaction could be performed at the exceptional substrate concentration of up to 0.4 mol L⁻¹ in an environmentally friendly aqueous-organic solvent mixture at room temperature and is easy to handle, thus providing a very practical tool to access enantiopure 1-heteroarylethanols.

doi: 10.1002/adsc.200303210

The purification and characterization of an organic solvent tolerant, NADH-dependent medium-chain secondary alcohol dehydrogenase (denoted sec-ADH "A") from Rhodococcus ruber DSM 44541 is reported. The enzyme can withstand elevated concentrations of organic solvents, such as acetone (up to 50% v/v) and 2-propanol (up to 80% v/v). Thus, it is ideally suited for the preparative-scale enantioselective oxidation of sec-alcohol and the asymmetric reduction of ketones, using acetone and 2-propanol, respectively, as cosubstrates for cofactor-regeneration via a coupled-substrate approach. The homodimeric protein was found to bear tightly bound zinc and displayed a molecular mass of 38 kDa per subunit as determined by SDS gel electrophoresis. The optimal temperature ranged from 30–50°C and the half-life at 50°C was 35 h. In addition, excellent storage stability was found. The pH optimum for reduction is pH 6.5; pH 9.0 is preferred for oxidation. The enzyme followed a sequential reaction mechanism. The substrates are medium chain sec-alcohols or (ω-1)-ketones; primary alcohols or aldehydes are not accepted.

doi: 10.1002/bit.20004

Driven by the immaturity of many organic oxidation reactions and the necessity for ‘green’ chemical processes, biocatalytic redox processes are being investigated with increasing intensity in order to tap the full potential of the excellent chemo-, regio- and enantioselectivity of enzymes. Despite their unmatched advantage in view of environmental aspects, the requirement of cofactors and the availability of redox enzymes able to tolerate high concentrations of organic (co)substrates sets limitations. However, during the past years, an increasing number of applications to the bio-oxidation of primary and secondary alcohols with novel redox enzymes have been developed. This review gives an overview on the different methods and their potential and limits.

doi: 10.1002/adsc.200303177

To improve the efficiency and applicability of biocatalytic redox-reactions for asymmetric ketone-reduction and enantioselective alcohol-oxidation catalyzed by nicotinamide-dependent dehydrogenases/reductases, several achievements for cofactor-recycling have been made during the last two years. First, the use of hydrogenases for NADPH recycling in a two enzyme system. Second, preparative transformations with alcohol dehydrogenases coupled with NADH oxidases for NAD⁺/NADP⁺ recycling. Third, an exceptional chemo-stable alcohol dehydrogenase can efficiently use i-propanol and acetone as cosubstrates for reduction and oxidation, respectively, in a single-enzyme system. Novel carbonyl reductases and dehydrogenases derived from plant cells are particularly suited for sterically demanding substrates.

doi: 10.1016/j.cbpa.2004.02.005

Ionic liquids (IL) offer new possibilities for solvent engineering for biocatalytic reactions. The deracemization of (±)-mandelic acid using a lipase-mandelate racemase two-enzyme system was used to investigate the scopes and limitations of ionic liquids as new reaction media for a dynamic resolution approach. Mandelate racemase [EC 5.1.2.2] from Pseudomonas putida ATCC 12633 was observed to be active in ionic liquids such as 1,3-dimethylimidazolium methylsulfate ([MMIM][MeSO₄]) or 1-butyl-3-methyl-imidazolium octylsulfate ([BMIM][OctSO₄]) at water activities a_w > 0.74. Mandelate racemase activity could also be obtained in a biphasic system consisting of water and 1-octyl-3-methylimidazolium hexafluorophosphate ([OMIM][PF₆]) in a ratio of 1:10.

doi: 10.1016/j.molcata.2003.11.034

Biocatalytic reduction of the keto-moiety of α,β-unsaturated ketones (enones) was achieved with absolute chemo- and stereo-selectivity employing whole lyophilized cells of Rhodococcus ruber DSM 44541 to furnish the corresponding allylic alcohols in e.e. up to >99%. It was shown that a stereocenter in γ-position of the ketone moiety to be reduced is too distant from the reaction center to induce any significant diastereoselectivity, thus no kinetic resolution of an racemic ketone occurred.

doi: 10.1016/j.molcatb.2004.09.004

We developed a specific method for determination and discrimination of lipo-/estero-lytic enzymes in crude lipase preparations. Here we study the composition of commercial porcine pancreatic lipase (PPL), since it is widely used for bioconversions of synthetic and natural substrates. Our method is based on incubation of enzyme samples with fluorescently labeled alkyl- or dialkylglyceryl-phosphonates in an appropriate solvent followed by protein separation by electrophoresis and fluorescence detection with a CCD camera. After incubation with short-chain alkylphosphonate solubilized by taurodeoxycholate, crude PPL preparations showed a very weak band at 50 kDa, which is indicative of low PPL concentrations in these samples. In addition, seven other fluorescent bands were detected. The band at the lowest molecular weight corresponded to α-chymotrypsin. Two intensive fluorescent bands were in the molecular weight range of chymotrypsinogen (26 kDa) and four weak bands were in the range 20–24 kDa. Long-chain dialkylglycerophosphonate labeled two protein bands in crude PPL: α-chymotrypsin and a very intensive band corresponding to the molecular weight of chymotrypsinogen. Detection of cholesterol esterase (98 kDa) in crude PPL preparations depended on addition of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) to the incubation mix, as demonstrated by spiking with cholesterol esterase. Thus, commercial crude PPL preparations contain a variety of estero-/lipo-lytic enzymes in addition to rather low amounts of active PPL, which should be considered when using crude PPL for bioconversions. Our method can also be used to show whether an isolated esterolytic activity corresponds to a single protein or isoenzymes. Here we confirm by 2D-electrophoretic separation of “pure” PPL that PPL exists as isoenzymes in different glycosylated forms.

doi: 10.1002/bit.10894

The deracemization of sec-alcohols via enantioconvergent processes is of high interest in order to overcome the 50% yield limitations of kinetic resolution. As enantioconvergent processes are characterized by two reactions with one crossing the plane of symmetry, one of these reactions has to proceed with inversion of configuration. In lipase catalyzed resolutions the enzymatic step proceeds with retention of configuration, and the remaining enantiomer is converted by the Mitsunobu reaction or activated esters under inversion of configuration to achieve complete deracemization. In contrast, alkyl sulfatases, which catalyze the enantioselective hydrolysis of sulfate esters, are one of the rare hydrolytic enzymes which work under inversion of configuration. In this minireview we give an overview of the state of the art in enantioconvergent processes for the deracemization of sec-alcohols, with the focus on recently developed alkyl-sulfatases from Rhodococcus spp.

doi: 10.1002/elsc.200402151

Asymmetric enzyme-catalyzed hydrolysis of methylene-interrupted bis-epoxides 1a and 1b catalyzed by bacterial epoxide hydrolases furnished tetrahydrofuran derivatives 2a and 2b through a hydrolysis–rearrangement cascade. Whereas racemic bis-oxiranes 1b–d underwent kinetic resolution with moderate stereoselectivities to yield products with up to 92% ee and 66% de: meso-bis-oxirane cis,cis-1a was transformed into (6R,7R,9S,10S)-2a in 94% ee and 89% de at high conversion (85%) by Rhodococcus sp. CBS 717.73 as the major product. The reaction sequence resembles a biomimetic reaction cascade and provides an efficient entry into the structural core of annonaceous acetogenins with simultaneous control of four stereocenters.

doi: 10.1002/chem.200400061

rac-sec-Alkyl sulfate esters 1a−4a were resolved in high enantioselectivities with E-values up to >200 using whole cells of aerobically grown Sulfolobus acidocaldarius DSM 639. The stereochemical course of this biohydrolysis was shown to proceed with strict inversion of configuration; thus, the preferred (R)-enantiomers were converted into the corresponding (S)-sec-alcohols to furnish a homochiral product mixture.

doi: 10.1021/ol0477778

Asymmetric biohydrolysis of trisubstituted terpenoid oxiranes (rac-1a–rac-3a) was accomplished by employing the epoxide hydrolase activity Rhodococcus and Streptomyces spp. Depending on the biocatalyst, the biohydrolysis proceeded in an enantio-convergent fashion and gave the corresponding vic-diols in up to 97% ee at conversions beyond the 50%-threshold. In order to avoid a depletion of the ee of product by further oxidative metabolism, bioconversions had to be conducted in an inert atmosphere with exclusion of molecular oxygen. The synthetic applicability of this method was demonstrated by the asymmetric total synthesis of the monoterpenoid coumarin (R)-(+)-Marmin in 95% ee.

doi: 10.1016/j.tet.2003.10.106

Nonracemic sec-alcohols of opposite absolute configuration were obtained either by asymmetric reduction of the corresponding ketone using 2-propanol as hydrogen donor or by enantioselective oxidation through kinetic resolution of the rac-alcohol using acetone as hydrogen acceptor employing whole lyophilized cells of Rhodococcus ruber DSM 44541. The microbial oxidation/reduction system exhibits not only excellent stereo- and enantioselectivity but also a broad substrate spectrum. Due to the exceptional tolerance of the biocatalyst toward elevated concentrations of organic materials (solvents, substrates and cosubstrates), the process is highly efficient. The simple preparation of the biocatalyst and its ease of handling turns this system into a versatile tool for organic synthesis.

doi: 10.1021/jo026216w

Whole lyophilised cells of Rhodococcus ruber DSM 44541 were employed for the oxidative kinetic resolution of sec-alcohols using acetone as hydrogen acceptor. The enantioselectivity of this process could be controlled effectively by introducing C–C multiple bonds into substrates, which were inefficiently recognised, in particular short-chain (ω-1)-alcohols and (ω-2)-analogs. Thus, the enantioselectivities of rac-2-pentanol (E=16.8) and rac-3-octanol (E=13.3) were significantly improved by introducing a C=C bond adjacent to the alcohol moiety to give racemic (E)-pent-3-en-2-ol and 4-(E)-octen-3-ol, which were resolved with excellent selectivities (E >100 and 50, respectively). In addition, it was found that high stereodifferentiation between the E- and Z-configured double bonds occurred, as the corresponding (Z)-isomers were not converted. Similar selectivity-enhancing effects were observed with acetylenic analogs.

doi: 10.1016/S0957-4166(02)00795-4

The organic solvent-stable redox-system of Rhodococcus ruber DSM 44541, which allows the efficient oxidation/reduction of sec-alcohols/ketones at the expense of acetone/2-propanol, respectively, as cosubstrate was optimized with respect to a maximum of alcohol dehydrogenase activity during cell growth. Comparison of the fermentation of R. ruber DSM 44541 in shake flasks cultures (1 l flask with 250 ml medium) and in a bioreactor (15 l with 10 l working volume) revealed that the desired organic solvent-stable alcohol dehydrogenase activity reached its maximum during the log phase for the bioreactor. In contrast, in shake flasks the maximum of activity was reached during the stationary phase.

doi: 10.1016/S1381-1177(02)00265-5

In contrast to kinetic resolution, where only 50% of the racemic starting material can be converted into the desired product and where the remaining 'wrong' enantiomer has to be considered as waste, so-called deracemisation processes allow the production of a single stereoisomeric product from racemic starting material. In this context, the use of environmentally benign methods for biocatalytic racemisation holds great potential. The small and largely overlooked group of racemases (EC 5.1.X.X), which are increasingly being used for dynamic kinetic resolution or in auxiliary biocatalytic recycling processes, are reviewed with respect to their properties, their substrate tolerance and their biocatalytic potential.

doi: 10.1002/adsc.200303009

For the development of a 'green' oxidation method, the transformation of 4-(p-hydroxyphenyl)butan-2-ol (rhododendrol) into 4-(p-hydroxyphenyl)butan-2-one (raspberry ketone) was used as a model reaction. Different lyophilized cells of Rhodococcus spp. have been screened for their ability to perform the desired oxidation. Rhodococcus equi IFO 3730 and R. ruber DSM 44541 were able to use acetone as a hydrogen acceptor in a hydrogen transfer-like process. The oxidation can be performed at substrate concentrations up to 500 g/L.

doi: 10.1016/j.tet.2003.10.019

A 3D QSAR analysis (quantitative structure activity relationships) of a set of 2,2-disubstituted epoxides, substrates for epoxide hydrolases originating from four different organisms, was conducted by CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis), with respect to the enantioselective ring opening to the corresponding vicinal diol. Structural variations of the substrates include alkyl chains of different lengths, unsaturated moieties ((E)- and (Z)-alkenyl, alkinyl, aryl) and electronegative groups (ether oxygens, halogen atoms) at different locations within the 2-substituent group. Generally, all four organisms, namely Rhodococcus ruber NCIMB 11216, Rhodococcus ruber DSM 43338, Rhodococcus ruber DSM 44540 and Rhodococcus ruber DSM 44539, preferentially react with the (S)-enantiomer of the epoxide. Enantioselectivities (enantiomeric ratio, ln E values) show a rather large variation, ranging from almost no (ln E<1) to nearly complete selectivity (ln E>5.3). In addition, the response of the epoxide hydrolases stemming from the four organisms towards structural modifications of the substrate is different. Models for the enantioselectivity (enantiomeric ratio, ln E values) obtained by CoMFA and CoMSIA are of different but reasonable predictive power, e.g., q² _CV=0.701 and r²=0.937 for the CoMFA model of Rhodococcus ruber DSM 43338. Enantiomeric ratios for the test molecules can be well predicted. Plots of steric and electrostatic CoMFA (CoMSIA) fields allow conclusions to be drawn for the choice of the most suitable organism for a specific type of substrate.

doi: 10.1023/A:1024562326498

Whole cells of Rhodococcus ruber DSM 44541 were found to hydrolyze (±)-2-octyl sulfate in a stereo- and enantiospecific fashion. When growing on a complex medium, the cells produced two sec-alkylsulfatases and (at least) one prim-alkylsulfatase in the absence of an inducer, such as a sec-alkyl sulfate or a sec-alcohol. From the crude cell-free lysate, two proteins responsible for sulfate ester hydrolysis (designated RS1 and RS2) were separated from each other based on their different hydrophobicities and were subjected to further chromatographic purification. In contrast to sulfatase RS1, enzyme RS2 proved to be reasonably stable and thus could be purified to homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band at a molecular mass of 43 kDa. Maximal enzyme activity was observed at 30°C and at pH 7.5. Sulfatase RS2 showed a clear preference for the hydrolysis of linear secondary alkyl sulfates, such as 2-, 3-, or 4-octyl sulfate, with remarkable enantioselectivity (an enantiomeric ratio of up to 21 [23]). Enzymatic hydrolysis of (R)-2-octyl sulfate furnished (S)-2-octanol without racemization, which revealed that the enzymatic hydrolysis proceeded through inversion of the configuration at the stereogenic carbon atom. Screening of a broad palette of potential substrates showed that the enzyme exhibited limited substrate tolerance; while simple linear sec-alkyl sulfates (C₇ to C₁₀) were freely accepted, no activity was found with branched and mixed aryl-alkyl sec-sulfates. Due to the fact that prim-sulfates were not accepted, the enzyme was classified as sec-alkylsulfatase (EC 3.1.6.X).

doi: 10.1128/AEM.69.5.2810-2815.2003

The enantioselective ring-opening catalyzed by epoxide hydrolases originating from seven different sources of a series of 2,2-disubstituted oxiranes containing alkyl chains of different lengths, unsaturated (alkenyl, alkinyl) and aromatic groups as well as electronegative heteroatoms at various positions within the side chain was analyzed by quantitative structure−activity relationships. Models for the enantioselectivity were derived with the aid of multiple linear regression analysis (MLR) using several steric and electronic (quantum chemical) descriptors. On the basis of the models derived by MLR nonlinear modeling with artificial neural networks (ANN) was also done. Good predictive performance was observed for both modeling approaches. The models also indicate that different steric and/or electronic features account for the enantioselectivities observed for the individual epoxide hydrolases.

doi: 10.1021/ci020047z

Acetogenins isolated from the Annonaceae family of tropical trees have drawn considerable attention owing to their broad spectrum of biological activities. They are structurally characterized by the presence of one to three tetrahydrofuran rings in the center of a long (partly hydroxylated) hydrocarbon chain that ends in a (functionalized) butenolide moiety. Here we describe some of our results toward the first asymmetric total synthesis of cis-gigantrionenin, a principal acetogenin. In this approach, an enzyme-catalyzed epoxide hydrolysis and an enzyme-triggered double cyclization are crucial and give stereoselective access to essential chiral building blocks.

doi: 10.1351/pac200375020259

A single enantiomer of a (stereo)chemically labile allylic-homoallylic alcohol was obtained in 91% e.e. and 96% yield from the racemate by employing a lipase-catalysed kinetic resolution coupled to in situ inversion under carefully controlled (Mitsunobu) conditions in order to suppress side reactions, such as elimination and racemisation. This technique was successfully applied to an enantio-convergent asymmetric total synthesis of the algal fragrance component (S)-dictyoprolene.

doi: 10.1016/S0957-4166(03)00481-6

Enzymatic resolution and dynamic kinetic resolution of γ-hydroxy acid derivatives 1 have been investigated. Efficient kinetic resolution was obtained using Pseudomonas cepacia lipase in toluene (E value ∼400). The combination of enzymatic kinetic resolution with a ruthenium-catalyzed racemization resulted in an efficient dynamic kinetic resolution. The use of a hydrogen source to depress ketone formation in the dynamic kinetic resolution yields the corresponding acetates in good yield (up to 93%) and enantioselectivity (up to 99%).

doi: 10.1016/S0040-4039(02)00418-5

Celling chemistry: Ketones (secondary alcohols) can be biocatalytically reduced (oxidized) at a substrate concentration of up to 1.8 mol L⁻¹ in an asymmetric fashion by employing a novel secondary alcohol dehydrogenase from Rhodococcus ruber DSM 44541 (see scheme). Furthermore, the enzyme is exceptionally stable at high cosubstrate concentrations, that is, 2-propanol (50 % v/v) for reduction and acetone (20 % v/v) for oxidation, respectively.

doi: 10.1002/1521-3773(20020315)41:6<1014::AID-ANIE1014>3.0.CO;2-6

For the description of the stereoselectivity of (bio)catalytic asymmetric reactions which may proceed via different regio- or stereo-isomeric pathways (e.g. catalysed by epoxide hydrolases, dehalogenases, sulfatases or glycosidases), a parameter ‘RI’ (Retention–Inversion ratio) was introduced in analogy to the Enantiomeric Ratio (E), which describes enantioselectivity. A computer program was developed for the treatment of the kinetics of such single-step processes, which offer the potential of deracemization, i.e. a single stereoisomeric product is formed from a racemate in an enantioconvergent fashion. By analysis of experimentally determined progress curves of the enantiomeric excess of substrate and product (e.e._S, e.e._P, respectively) and the conversion (c), relative first-order rate constants k_i, the enantioselectivity (E) and the RI ratio (RI) can be determined; in addition, processes can be simulated based on assumed k_i values.

doi: 10.1016/S0957-4166(02)00084-8

The biocatalytic hydrolysis of (±)-sec-alkyl sulfate esters with an alkylsulfatase from Rhodococcus ruber DSM 44541 proceeded with high enantioselectivity (up to 99 % ee) and with absolute stereoselectivity through inversion of configuration. Thus, a rac substrate was converted into homochiral S-configured products.

doi: 10.1002/1521-3773(20021104)41:21<4052::AID-ANIE4052>3.0.CO;2-W

A short total asymmetric synthesis of (+)-exo- and (–)-endo-brevicomin ((+)-exo-3 and (–)-endo-3), which are components of the attracting pheromone system of several bark-beetle species belonging to the genera Dendroctonus and Dryocoetes, was accomplished via a chemoenzymatic protocol. The key step consisted of biocatalytic hydrolysis by bacterial epoxide hydrolases of cis-configured 2,3-disubstituted oxiranes bearing olefinic side chains. This reaction proceeded in an enantioconvergent fashion, by affording a single enantiomeric vic-diol from the rac-epoxide in up to 92% ee and 83% isolated yield.

doi: link

Total asymmetric synthesis of two components of Panax ginseng showing antitumor activity, i.e., (3R,9R,10R)- and (3S,9R,10R)-Panaxytriol and of both enantiomers of Falcarinol was accomplished. Due to the fact that the synthetic strategy was based on enantioconvergent biotransformations, the occurrence of any undesired stereoisomer was entirely avoided. The absolute configuration of naturally occurring Panaxytriol was confirmed to be (3R,9R,10R) on the basis of optical rotation values. It was shown that enzyme-triggered cascade reactions represent a valuable tool for the synthesis of natural products.

doi: 10.1021/jo020073w

The degree of enzyme deactivation for lipases from Candida rugosa and Pseudomonas sp., hydroxynitrile lyase and mandelate racemase upon exposure to organic solvents can be correlated to their respective partition coefficients (log P values). However, three unexpected results were obtained: (1) the deactivation exerted by protic solvents, e.g., methanol, is severely underestimated; (2) little deactivation by an organic solvent cannot neccessarily be correlated to catalytic activity in this medium, and (3) in contrast to other enzymes, hydroxynitrile lyase is exceptionally stable towards deactivation by DMF.

doi: 10.1023/A:1015598523282

A short chemoenzymatic route to two natural products—the first, a constituent of Jamaican rum and the second the (+)-antipode of the gibberelin synergist (−)-Pestalotin—was accomplished based on an enzyme-triggered cascade-reaction. Thus, a racemic halomethyl oxirane was hydrolyzed by bacterial epoxide hydrolases to furnish the corresponding vic-halomethyl-diol, which underwent spontaneous ring-closure to furnish an epoxy alcohol in up to 93% e.e. and ≥99 d.e. Due to the fact that this process was enantioconvergent, the occurrence of the undesired enantiomer was entirely avoided.

doi: 10.1016/S0957-4166(02)00124-6

The constitutive epoxide hydrolase activity of Rhodococcus ruber DSM 44540 strongly depends on the status of the cells and appears to be regulated by a catabolic switch: activity peaked when glucose was exhausted and peptone/yeast extract consumption started. The activity-maximum for the kinetic resolution of a 2,2- and the enantioconvergent asymmetric biohydrolysis of a 2,3-disubstituted oxirane coincided. In order to obtain a maximum yield, cells should be harvested after ca. 17 h.

doi: 10.1016/S1381-1177(02)00083-8

Enantioselective biohydrolysis of sec-alkyl sulfate esters using a bacterial alkylsulfatase from Rhodococcus ruber DSM 44541 proceeded in a stereoselective fashion though inversion of configuration. Thus, from racemic substrates, the corresponding (R)-enantiomers were hydrolyzed selectively to furnish the corresponding sec-alcohol and non-reacted sulfate ester, both of (S)-configuration, which represents a homochiral product mixture. The enantioselectivities were found to depend on the substrate structure and were optimal for sec-sulfate esters in the ω-1 position (up to E=21). Since the enzyme was inactive on prim-sulfate esters, it can be classified as a sec-alkylsulfatase [EC 3.1.6.X].

doi: 10.1016/S0957-4166(02)00362-2

The Enantioselectivity of the biohydrolysis of sec-alkyl sulfate esters using a bacterial alkylsulfatase from Rhodococcus ruber DSM 44541 was dramatically enhanced in presence of additives (‘enhancers’) such as carbohydrates, polyethylene glycol, detergents, metal ions and through enzyme immobilization. In presence of iron, the E value for the kinetic resolution of (±)-3- and (±)-4-octyl sulfate was improved from E=3.9 to ≥200 and E=1.1 to 10, respectively.

doi: 10.1016/S0957-4166(02)00363-4

Organic compounds can be transformed through enzyme-triggered domino (or cascade) reactions via several (inseparable) consecutive steps in an asymmetric fashion to yield nonracemic products. Despite the fact that these sequences often involve the occurrence of highly reactive unstable intermediates, the overall efficiency of these processes can be high, provided that the reaction rates of the individual steps match each other in order to minimize side reactions.

doi: 10.1351/pac200274122253

The biocatalytic degradation of a cyclic trithiocarbonate, 6-amino-5-methoxycarbonyl-thieno[2,3-d]-1,3-dithiole-2-thione 1, is reported. The product of the hydrolysis of the five-membered ring by Pseudomonas chlororaphis ATCC 9447 oxidatively dimerized to form the tetrathiocin derivative 2. Furthermore, we performed the first preparative biocatalytic methylation of an unnatural compound employing Emericella unguis ATCC 10032 by cleaving the dithiole ring 1 followed by methylation of both thiol groups to form the methylated product 4 in 64% isolated yield.

doi: 10.1016/S0040-4020(02)00146-1

A highly efficient chemo-enzymatic asymmetric synthesis of chiral C4-building blocks containing a fully substituted carbon center is reported. The key transformation consists of a deracemization based on an enantioconvergent asymmetric hydrolysis of an epoxide using combined bio- and chemo-catalysis leading to a single enantiomeric product in >98% e.e. A simple switch of steps leads to kinetic resolution giving access to products of opposite configuration.

doi: 10.1055/s-2001-9703

The epoxide hydrolase-catalyzed resolution of (±)-2-methylglycidyl benzyl ether, a versatile chiral building block for the asymmetric synthesis of bioactive compounds, mediated by whole cells of Rhodococcus ruber SM 1789 was accomplished. Among various parameters (such as temperature, buffer type, pH and catalyst/substrate-ratio) an elevated substrate-concentration proved to be particularly sensitive with respect to a significant enhancement of the enantioselectivity.

doi: 10.1023/A:1005636121060

Enzymatic racemization of mandelic acid derivatives modified at the α-hydroxy acid moiety was achieved using mandelate racemase [EC 5.1.2.2]. Whereas α-amino acid derivatives, such as phenyl glycine and mandelic acid hydrazide were not accepted, the mandelic acid amide was racemized at an acceptable rate. The latter was significantly enhanced by an electron-withdrawing substituent in the phenyl moiety. Based on the catalytic mechanism of the enzyme, the relative activities of non-natural substrates could be explained by steric and electronic reasons.

doi: 10.1016/S1381-1177(01)00036-4

Efficient enzymatic racemization of 2-hydroxy-2-heteroaryl-acetic acid derivatives by mandelate racemase under mild conditions is reported for the first time. (i) Steric limitations for aryl-substituted mandelate derivatives were elucidated to be particularly striking for o-substituents, whereas m- and p-analogues were freely accepted, as well as heteroaryl- and naphthyl-analogs. (ii) The electronic character of substituents was found to play an important role: whereas electron-withdrawing substituents dramatically enhanced the racemization rates, electron-donating analogs caused a depletion. This effect could be ascribed to an α-carbanion-stabilization in accordance with the known enzyme mechanism. The latter was modeled by comparison of gas phase deprotonation energies as a useful parameter to describe resonance stabilization. The calculated data nicely correlate with the experimentally observed activities for a specific substrate as long as other parameters, such as steric restrictions, are absent or play a minor role.

doi: 10.1016/S1381-1177(01)00035-2

Asymmetric biohydrolysis of trialkyl oxiranes (±)-1a–3a using the epoxide hydrolase activity of whole bacterial cells proceeded in an enantioconvergent fashion and thus led to the corresponding (R)-configurated vicinal diols 1b–3b in up to 97% enantiomeric excess (e.e.) as the sole product. The mechanism of this enantioconvergence was investigated by ¹⁸O-labelling experiments and it was found that both enantiomers were hydrolysed with opposite regioselectivity.

doi: 10.1016/S0957-4166(01)00256-7

The biocatalytic hydrolysis of the (±)-2,3-disubstituted cis-chloroalkyl epoxides 1a and 2a using resting cells of Rhodococcus sp. did not give the corresponding chloroalkyl vic-diols 1b, and 2b, respectively, but furnished the rearranged products (2R,3R)-1c and (2R,3R)-2c in high e.e. as the sole products via an enzyme-triggered enantio-convergent cascade-reaction.

doi: 10.1016/S0957-4166(01)00010-6

Non-sequential processes which allow the transformation of a racemate into a single stereoisomeric product without the occurrence of an “undesired” isomer are classified according to their underlying stereochemistry. A re-definition of the term “de-racemization” is proposed.

doi: 10.1016/S1381-1177(01)00049-2

Biocatalytic hydrolysis of 2,3-disubstituted rac-cis- and rac-trans-haloalkyl epoxides 1a−8a using the epoxide hydrolase activity of whole bacterial cells furnished the corresponding vicinal diols 1b−8b as intermediates; these (spontaneously) underwent ring closure to yield cyclic products 1c−6c through an enzyme-triggered cascade reaction. In particular, cis-configured substrates (1a, 3a, 5a, 7a) were transformed in an enantioconvergent fashion, which resulted in the formation of single stereoisomeric products in 100% des and up to 92% ees from the racemates.

doi: 10.1002/1099-0690(200112)2001:23<4537::AID-EJOC4537>3.0.CO;2-E

Non-sequential processes which allow the transformation of a racemate into a single stereoisomeric product without the occurrence of an “undesired” isomer are classified according to their underlying stereochemistry. A re-definition of the term “de-racemization” is proposed.

doi: 10.1002/1521-3765(20011203)7:23<5004::AID-CHEM5004>3.0.CO;2-X

An enantioconvergent synthesis of the aggregation pheromones (R)- and (S)-sulcatol (6-methyl-5-hepten-2-ol) is described. Key steps in the deracemization strategy are sequential combinations of enzymatic resolutions and Mitsunobu inversions. Racemization-free Mitsunobu transformations have been carried out within 5 min by microwave irradiation, providing the desired sulcatyl acetates with clean inversion of chirality.

doi: 10.1016/S0040-4039(01)01248-5

Epoxide hydrolases from microbial sources are highly versatile biocatalysts for the asymmetric hydrolysis of epoxides on a preparative scale. Besides kinetic resolution, which furnishes the corresponding vicinal diol and remaining non-hydrolysed epoxide in nonracemic form, enantioconvergent processes are possible: these are highly attractive as they lead to the formation of a single enantiomeric diol from a racemic oxirane. The data accumulated over recent years reveal a common picture of the substrate structure selectivity pattern of microbial epoxide hydrolases and indicate that substrates of various structural types can be selectively hydrolysed with enzymes from certain microbial sources.

doi: 10.1016/S0958-1669(01)00262-2

A short asymmetric total synthesis of the plant constituent myrcenediol [(R)-1], and (S)-7,7-dimethyl-6,8-dioxabicyclo[3.2.1]octane (2), which is a volatile constituent of the aroma of beer was accomplished via a chemoenzymatic protocol. The key step consisted of a biocatalytic hydrolysis of trisubstituted epoxides bearing olefinic side chains which proceeded in an enantioconvergent fashion, i.e., a single enantiomeric vic-diol was obtained from the racemate in up to 91% ee and 92% isolated yield.

doi: 10.1055/s-2001-17713

Domino or cascade reactions involve the transformation of materials through several inseparable steps, which often proceed via highly reactive intermediates. In the case where the reaction sequence is triggered by a biocatalyst, such as an enzyme, the cascade may proceed in a highly chemo- or stereoselective way. In this review, emphasis is laid on biocatalyzed domino reactions of non-natural compounds (rather than natural substrates) which have been aptly denoted as 'enzyme-initiated' (or -'triggered') domino (or cascade) reactions. Biosynthetic pathways involving biological cascade reactions are out of the scope of this review (see, for example, D. E. Cane, Chem. Rev., 1990, 90, 1089).

doi: 10.1039/b105493g

A short chemoenzymatic synthesis of the (2R,5S)- and (2R,5R)-stereoisomer of the bark beetle pheromone Pityol 1 was achieved from (±)-Sulcatol 2 in an enantio- and diastereo-convergent fashion without the formation of any 'unwanted' stereoisomer. The key steps include: (i) lipase-catalyzed deracemization of (±)-2 using kinetic resolution coupled to an in-situ inversion or, alternatively, dynamic resolution using combined lipase- and Ru-catalysis; and (ii) creation of the second stereogenic center by an epoxide hydrolase-catalyzed diastereo-convergent hydrolysis of a haloalkyl oxirane, followed by spontaneous ring closure to form 1 in a stereoselective fashion.

doi: 10.1016/s0957-4166(01)00370-6

A one-pot method for the deracemisation of the enol acetate 1 derived from the prochiral 4,4-disubstituted cyclohexanone 2 has been developed using the combination of Pseudomonas fluorescens lipase and potassium t-butoxide/isopropenyl acetate to give the enantiomerically pure enol acetate (S)-1 in 82% yield. Calculations based on the inherent enantioselectivity of the lipase (E) allowed an estimation of the optimum theoretical conversion for each enzyme step prior to recycling the ketone.

doi: 10.1016/S0040-4039(01)01156-x

Carboxyl esters bearing a fully substituted chiral center adjacent to the ester moiety, i.e., esters of tert-alcohols and of α,α-disubstituted carboxylates, are usually not accepted as substrates for hydrolytic enzymes such as esterases, proteases, and lipases. In order to circumvent this limitation, three strategies, which are reviewed in this paper, have been developed. (i) Several proteases and (still unspecified) microbial esterases are capable of hydrolysing esters of tert-alcohols and α,α-disubstituted carboxylic acids despite their steric bulkiness, but the number of these highly useful enzymes is rather limited. Alternatively, (ii) the use of ‘activated esters’ bearing electron-withdrawing groups enhances the electrophilic properties of the ester moiety (thus increasing the enzymatic reaction rate) may help to overcome slow reaction rates. On the other hand, (iii) spatial separation of the bulky quarternary carbon atom bearing the chiral center from the ester group to be hydrolysed by a spacer moiety led to modified (non-activated) substrates which were readily accepted.

doi: 10.1016/S1381-1177(99)00121-6

Biocatalytic resolution of the tertiary terpene alcohol (±)-linalool was accomplished via hydrolysis of its corresponding acetate ester using two highly enantiospecific enzymes (E > 100). The latter were identified in a crude cell-free extract of Rhodococcus ruber DSM 43338 and could be separated by (partial) protein purification. Since they showed opposite enantiopreference, they were termed (R)- and (S)-linalyl acetate hydrolase (LAH). The activity and selectivity of the enzyme preparations was markedly dependent on the fermentation conditions.

doi: 10.1007/s007060070092

Mandelate racemase [EC 5.1.2.2] from Pseudomonas putida ATCC 12336 was efficiently immobilized through ionic binding onto DEAE- and TEAE 23-cellulose. The activity of the immobilized enzyme was significantly enhanced as compared to the native protein, i.e., 2.7- and 2.5-fold, respectively. DEAE-cellulose-immobilized mandelate racemase could be efficiently used in repeated batch reactions for the racemization of (R)-mandelic acid under mild conditions.

doi: 10.1023/A:1005621021983

A practical and short synthesis of the enantiomerically pure dihydropyrimidone antihypertensive agent (R)-SQ 32926 has been developed. The key step in the synthesis is the enzymatic resolution of an N3-acetoxymethyl-activated dihydropyrimidone precursor by Thermomyces lanuginosus lipase. The absolute configuration of (R)-SQ 32926 was confirmed by circular dichroism spectroscopy.

doi: 10.1016/S0957-4166(00)00081-1

The biohydrolysis of 2,2-disubstituted oxiranes bearing various oxygen functional groups was investigated using the epoxide hydrolase activity of 11 bacterial strains. The results show that the activity and the selectivity strongly depend on the substrate structure and the biocatalyst. Whereas substrates possessing free hydroxyl groups were not transformed, their analogs, protected as ethers, were well accepted. This allowed the convenient modulation of the enantioselectivity by proper choice of the ether group according to size and polarity. It was found that the distance of the ether-oxygen to the stereogenic quaternary carbon center of the oxirane ring had a profound influence on the enantioselectivity, and several oxiranes were resolved with good to excellent selectivities. The enantiomerically enriched epoxides and vicinal diols thus obtained contain a useful “synthetic handle” in their side chain, which allows their use as building blocks in asymmetric synthesis.

doi: 10.1002/1099-0690(200011)2000:22<3703::AID-EJOC3703>3.0.CO;2-3