Bioorganic Chemistry

Publications

201720162015201420132012201120102009200820072006200520042003200220012000

Publications from years earlier than 2000 are listed in the ‘Performance Records’ of Kurt Faber and Wolfgang Kroutil at UNIGRAZonline.

2017

Full Paper

Stereoselective Synthesis of Functionalized Bicyclic Scaffolds by Passerini 3-Center-2-Component Reactions of Cyclic Ketoacids

R. C. Cioc, V. Estévez, D. J. van der Niet, C. M. L. Vande Velde, N. G. Turrini, M. Hall, K. Faber, E. Ruijter, R. V. A. Orru

Eur. J. Org. Chem. 2017, 1262–1271.

Graphical Abstract

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

Full Paper

Asymmetric Biocatalytic Synthesis of Fluorinated Pyridines through Transesterification or Transamination: Computational Insights into the Reactivity of Transaminases

M. López-Iglesias, D. González-Martínez, M. Rodríguez-Mata, V. Gotor, E. Busto, W. Kroutil, V. Gotor-Fernández

Adv. Synth. Catal. 2017, 359, 279–291.

Graphical Abstract

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

2016

Full Paper

Regio- and Enantioselective Sequential Dehalogenation of rac-1,3-Dibromobutane by Haloalkane Dehalogenase LinB

J. Gross, Z. Prokop, D. Janssen, K. Faber, M. Hall

ChemBioChem 2016, 17, 1437–1441.

Graphical Abstract

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

Review

Building Bridges: Biocatalytic C–C-Bond Formation toward Multifunctional Products

N. G. Schmidt, E. Eger, W. Kroutil

ACS Catal. 2016, 6, 4286–4311.

Graphical Abstract

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

Full Paper

Biocatalytic Transamination for the Asymmetric Synthesis of Pyridylalkylamines. Structural and Activity Features in the Reactivity of Transaminases

M. López-Iglesias, D. González-Martínez, V. Gotor, E. Busto, W. Kroutil, V. Gotor-Fernández

ACS Catal. 2016, 6, 4003–4009.

Graphical Abstract

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

Communication

Enzymatic Oxidative Tandem Decarboxylation of Dioic Acids to Terminal Dienes

A. Dennig, S. Kurakin, M. Kuhn, A. Dordic, M. Hall, K. Faber

Eur. J. Org. Chem. 2016, 3473–3477.

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

Communication

Sequential Enzymatic Conversion of α-Angelica Lactone to γ-Valerolactone through Hydride-Independent C=C Bond Isomerization

N. G. Turrini, E. Eger, T. C. Reiter, K. Faber, M. Hall

ChemSusChem 2016, 9, 3393–3396.

Graphical Abstract

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

Communication

Biocatalytic trifluoromethylation of unprotected phenols

R. C. Simon, E. Busto, N. Richter, V. Resch, K. N. Houk, W. Kroutil

Nat. Commun. 2016, 7, 13323.

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

Full Paper

Engineering of TM1459 from Thermotoga maritima for Increased Oxidative Alkene Cleavage Activity

M. Fink, S. Trunk, M. Hall, H. Schwab, K. Steiner

Front. Microbiol. 2016, 7, 1511.

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

Communication

Biocatalytic functionalization of hydroxyalkyl acrylates and phenoxyethanol via phosphorylation

G. Tasnádi, M. Hall, K. Baldenius, K. Ditrich, K. Faber

J. Biotechnol. 2016, 233, 219–227.

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

Full Paper

Biocatalytic Characterization of Human FMO5: Unearthing Baeyer–Villiger Reactions in Humans

F. Fiorentini, M. Geier, C. Binda, M. Winkler, K. Faber, M. Hall, A. Mattevi

ACS Chem. Biol. 2016, 11, 1039–1048.

Graphical Abstract

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

Letter

One-Pot, Two-Module Three-Step Cascade To Transform Phenol Derivatives to Enantiomerically Pure (R)- or (S)-p-Hydroxyphenyl Lactic Acids

E. Busto, R. C. Simon, N. Richter, W. Kroutil

ACS Catal. 2016, 6, 2393–2397.

Graphical Abstract

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

Full Paper

Enantioselective Reduction of Ethyl 3-Oxo-5-phenylpentanoate with Whole-Cell Biocatalysts

A. Zadlo, J. H. Schrittwieser, D. Koszelewski, W. Kroutil, R. Ostaszewski

Eur. J. Org. Chem. 2016, 1007–1011.

Graphical Abstract

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

Communication

Catalytic Promiscuity of Transaminases: Preparation of Enantioenriched β-Fluoroamines by Formal Tandem Hydrodefluorination/Deamination

A. Cuetos, M. García-Ramos, E.-M. Fischereder, A. Díaz-Rodríguez, G. Grogan, V. Gotor, W. Kroutil, I. Lavandera

Angew. Chem. Int. Ed. 2016, 55, 3144–3147.

Graphical Abstract

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

Full Paper

Regio- and Stereoselective Biocatalytic Monoamination of a Triketone Enables Asymmetric Synthesis of Both Enantiomers of the Pyrrolizidine Alkaloid Xenovenine Employing Transaminases

S. E. Payer, J. H. Schrittwieser, B. Grischek, R. C. Simon, W. Kroutil

Adv. Synth. Catal. 2016, 358, 444–451.

Graphical Abstract

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

Full Paper

Stereoselective Cascade to C3-Methylated Strictosidine Derivatives Employing Transaminases and Strictosidine Synthases

E.-M. Fischereder, D. Pressnitz, W. Kroutil

ACS Catal. 2016, 6, 23–30.

Graphical Abstract

(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

Communication

Exploiting Acid Phosphatases in the Synthesis of Phosphorylated Monoalcohols and Diols

G. Tasnádi, M. Lukesch, M. Zechner, W. Jud, M. Hall, K. Ditrich, K. Baldenius, A. F. Hartog, R. Wever, K. Faber

Eur. J. Org. Chem. 2016, 45–50.

Graphical Abstract

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

2015

Communication

Enantioselective Oxidative Aerobic Dealkylation of N-Ethyl Benzylisoquinolines by Employing the Berberine Bridge Enzyme

S. Gandomkar, E.-M. Fischereder, J. H. Schrittwieser, S. Wallner, Z. Habibi, P. Macheroux, W. Kroutil

Angew. Chem. Int. Ed. 2015, 54, 15051–15054.

Graphical Abstract

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

Letter

Biocatalytic One-Pot Synthesis of L-Tyrosine Derivatives from Monosubstituted Benzenes, Pyruvate, and Ammonia

A. Dennig, E. Busto, W. Kroutil, K. Faber

ACS Catal. 2015, 5, 7503–7506.

Graphical Abstract

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

Microreview

The Industrial Age of Biocatalytic Transamination

M. Fuchs, J. E. Farnberger, W. Kroutil

Eur. J. Org. Chem. 2015, 6965–6982.

Graphical Abstract

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

Full Paper

Oxidative Alkene Cleavage Catalysed by Manganese-Dependent Cupin TM1459 from Thermotoga maritima

I. Hajnal, K. Faber, H. Schwab, M. Hall, K. Steiner

Adv. Synth. Catal. 2015, 357, 3309–3316.

Graphical Abstract

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

Communication

Chemoenzymatic Synthesis towards the Active Agent Travoprost

C. Holec, D. Sandkuhl, D. Rother, W. Kroutil, J. Pietruszka

ChemCatChem 2015, 7, 3125–3130.

Graphical Abstract

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

doi: 10.1002/cctc.201500587

Communication

Amination of ω-Functionalized Aliphatic Primary Alcohols by a Biocatalytic Oxidation–Transamination Cascade

M. Pickl, M. Fuchs, S. M. Glueck, K. Faber

ChemCatChem 2015, 7, 3121–3124.

Graphical Abstract

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

Communication

Vinylation of Unprotected Phenols Using a Biocatalytic System

E. Busto, R. C. Simon, W. Kroutil

Angew. Chem. Int. Ed. 2015, 54, 10899–10902.

Graphical Abstract

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

Mini-Review

The substrate tolerance of alcohol oxidases

M. Pickl, M. Fuchs, S. M. Glueck, K. Faber

Appl. Microbiol. Biotechnol. 2015, 99, 6617–6642.

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

Communication

Oxidative Decarboxylation of Short-Chain Fatty Acids to 1-Alkenes

A. Dennig, M. Kuhn, S. Tassoti, A. Thiessenhusen, S. Gilch, T. Bülter, T. Haas, M. Hall, K. Faber

Angew. Chem. Int. Ed. 2015, 54, 8819–8822.

Graphical Abstract

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

Full Paper

ω-Transaminases for the amination of functionalised cyclic ketones

N. Richter, R. C. Simon, H. Lechner, W. Kroutil, J. M. Ward, H. C. Hailes

Org. Biomol. Chem. 2015, 13, 8843–8851.

Graphical Abstract

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

Full Paper

Investigation of Structural Determinants for the Substrate Specificity in the Zinc-Dependent Alcohol Dehydrogenase CPCR2 from Candida parapsilosis

C. Loderer, G. V. Dhoke, M. D. Davari, W. Kroutil, U. Schwaneberg, M. Bocola, M. B. Ansorge-Schumacher

ChemBioChem 2015, 16, 1512–1519.

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 kcat·KM−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

Full Paper

Regioselective Enzymatic β-Carboxylation of para-Hydroxystyrene Derivatives Catalyzed by Phenolic Acid Decarboxylases

C. Wuensch, T. Pavkov-Keller, G. Steinkellner, J. Gross, M. Fuchs, A. Hromic, A. Lyskowski, K. Fauland, K. Gruber, S. M. Glueck, Kurt Faber

Adv. Synth. Catal. 2015, 357, 1909–1918.

Graphical Abstract

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

Letter

Asymmetric Biocatalytic Amination of Ketones at the Expense of NH3 and Molecular Hydrogen

A. K. Holzer, K. Hiebler, F. G. Mutti, R. C. Simon, L. Lauterbach, O. Lenz, W. Kroutil

Org. Lett. 2015, 17, 2431–2433.

Graphical Abstract

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

Full Paper

Enzymatic Synthesis of Optically Active Lactones via Asymmetric Bioreduction using Ene-Reductases from the Old Yellow Enzyme Family

N. G. Turrini, M. Hall, K. Faber

Adv. Synth. Catal. 2015, 357, 1861–1871.

Graphical Abstract

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

Full Paper

Biocatalytic Asymmetric Synthesis of Optically Pure Aromatic Propargylic Amines Employing ω-Transaminases

N. G. Schmidt, R. C. Simon, W. Kroutil

Adv. Synth. Catal. 2015, 357, 1815–1821.

Graphical Abstract

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

Full Paper

A system for ω-transaminase mediated (R)-amination using L-alanine as an amine donor

N. Richter, J. E. Farnberger, D. Pressnitz, H. Lechner, F. Zepeck, W. Kroutil

Green Chem. 2015, 17, 2952–2958.

Graphical Abstract

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

Review

Biocatalytic Imine Reduction and Reductive Amination of Ketones

J. H. Schrittwieser, S. Velikogne, W. Kroutil

Adv. Synth. Catal. 2015, 357, 1655–1658.

Graphical Abstract

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

Commentary

Engineering Biocatalysts for Synthesis Including Cascade Processes

K. Faber, W.-D. Fessner, N. J. Turner

Adv. Synth. Catal. 2015, 357, 1565–1566.

Full Paper

Anthranoyl-CoA monooxygenase/reductase from Azoarcus evansii possesses both FMN and FAD in two distinct and independent active sites

T. Bergner, T. Pavkov-Keller, K. Kreuzer, J. Kowaliuk, M. Plank, K. Runggatscher, N. G. Turrini, B. Zucol, S. Wallner, K. Faber, K. Gruber, P. Macheroux

Biochim. Biophys. Acta 2015, 1854, 890–896.

Graphical Abstract

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

Review

Biocatalysts for the formation of three- to six-membered carbo- and heterocycles

H. Lechner, D. Pressnitz, W. Kroutil

Biotechnol. Adv. 2015, 33, 457–480.

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

Review

Cobalamin-dependent enzymatic O-, N-, and S-demethylation

N. Richter, F. Zepeck, W. Kroutil

Trends Biotechnol. 2015, 33, 371–373.

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

Editorial Review

(Chemo)enzymatic cascades—Nature's synthetic strategy transferred to the laboratory

E. García-Junceda, I. Lavandera, D. Rother, J. H. Schrittwieser

J. Mol. Catal. B: Enzym. 2015, 114, 1–6.

Graphical Abstract

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

Full Paper

Chiral Chlorohydrins from the Biocatalyzed Reduction of Chloroketones: Chiral Building Blocks for Antiretroviral Drugs

A. S. de Miranda, R. C. Simon, B. Grischek, G. C. de Paula, B. A. C. Horta, L. S. M. de Miranda, W. Kroutil, C. O Kappe, R. O. M. A. de Souza

ChemCatChem 2015, 7, 984–992.

Graphical Abstract

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

Communication

A synthetic biology approach for the transformation of L-α-amino acids to the corresponding enantiopure (R)- or (S)-α-hydroxy acids

G. Gourinchas, E. Busto, M. Killinger, N. Richter, B. Wiltschi, W. Kroutil

Chem. Commun. 2015, 51, 2828–2831.

Graphical Abstract

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

Communication

Deracemisation of profenol core by combining laccase/TEMPO-mediated oxidation and alcohol dehydrogenase-catalysed dynamic kinetic resolution

A. Díaz-Rodríguez, N. Ríos-Lombardía, J. H. Sattler, I. Lavandera, V. Gotor-Fernández, W. Kroutil, V. Gotor

Catal. Sci. Technol. 2015, 5, 1443–1446.

Graphical Abstract

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

2014

Full Paper

Identification and Application of Enantiocomplementary Lactamases for Vince Lactam Derivatives

Z. Assaf, E. Eger, Z. Vitnik, W. M. F. Fabian, D. Ribitsch, G. M. Guebitz, K. Faber, M. Hall

ChemCatChem 2014, 6, 2517–2521.

Graphical Abstract

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

Full Paper

Dynamic Kinetic Resolution of 2-Phenylpropanal Derivatives to Yield β-Chiral Primary Amines via Bioamination

C. S. Fuchs, M. Hollauf, M. Meissner, R. C. Simon, T. Besset, J. N. H. Reek, W. Riethorst, F. Zepeck, W. Kroutil

Adv. Synth. Catal. 2014, 356, 2257–2265.

Graphical Abstract

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

Full Paper

Stereochemistry and Mechanism of Enzymatic and Non-Enzymatic Hydrolysis of Benzylic sec-Sulfate Esters

M. Toesch, M. Schober, R. Breinbauer, K. Faber

Eur. J. Org. Chem. 2014, 3930–3934.

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 SN2-type nucleophilic attack of [OH] at the benzylic carbon represents the major pathway, whereas (ii) SN1-type formation of a planar benzylic carbenium ion leading to racemization was a minor event, and (iii) Retaining SN2-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

Communication

Introducing an In Situ Capping Strategy in Systems Biocatalysis To Access 6-Aminohexanoic acid

J. H. Sattler, M. Fuchs, F. G. Mutti, B. Grischek, P. Engel, J. Pfeffer, J. M. Woodley, W. Kroutil

Angew. Chem. Int. Ed. 2014, 53, 14153–14157.

Graphical Abstract

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

Letter

Continuous Flow Synthesis of Chiral Amines in Organic Solvents: Immobilization of E. coli Cells Containing Both ω-Transaminase and PLP

L. H. Andrade, W. Kroutil, T. F. Jamison

Org. Lett. 2014, 16, 6092–6095.

Graphical Abstract

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

Communication

Stereoselective synthesis of γ-hydroxynorvaline through combination of organo- and biocatalysis

R. C. Simon, E. Busto, J. H. Schrittwieser, J. H. Sattler, J. Pietruszka, K. Faber, W. Kroutil

Chem. Commun. 2014, 50, 15669–15672.

Graphical Abstract

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

Full Paper

Synthesis of (R)- or (S)-valinol using ω-transaminases in aqueous and organic media

C. S. Fuchs, R. C. Simon, W. Riethorst, F. Zepeck, W. Kroutil

Bioorg. Med. Chem. 2014, 22, 5558–5562.

Graphical Abstract

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

Full Paper

Engineering strictosidine synthase: Rational design of a small, focused circular permutation library of the β-propeller fold enzyme

E. Fischereder, D. Pressnitz, W. Kroutil, S. Lutz

Bioorg. Med. Chem. 2014, 22, 5633–5637.

Graphical Abstract

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

Full Paper

Deracemisation of benzylisoquinoline alkaloids employing monoamine oxidase variants

J. H. Schrittwieser, B. Groenendaal, S. C. Willies, D. Ghislieri, I. Rowles, V. Resch, J. H. Sattler, E.-M. Fischereder, B. Grischek, W.-D. Lienhart, N. J. Turner, W. Kroutil

Catal. Sci. Technol. 2014, 4, 3657–3664.

Graphical Abstract

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

Full Paper

Biocontrolled Formal Inversion or Retention of L-α-Amino Acids to Enantiopure (R)- or (S)-Hydroxyacids

E. Busto, N. Richter, B. Grischek, W. Kroutil

Chem. Eur. J. 2014, 20, 11225–11228.

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

Full Paper

Minireactor-based high-throughput temperature profiling for the optimization of microbial and enzymatic processes

M. Kunze, C. Lattermann, S. Diederichs, W. Kroutil, J. Büchs

J. Biol. Eng. 2014, 8, 22.

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

Communication

Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations

G. Steinkellner, C. C. Gruber, T. Pavkov-Keller, A. Binter, K. Steiner, C. Winkler, A. Łyskowski, O. Schwamberger, M. Oberer, H. Schwab, K. Faber, P. Macheroux, K. Gruber

Nat. Commun. 2014, 5, 4150.

Graphical Abstract

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

Communication

Cutting Short the Asymmetric Synthesis of the Ramatroban Precursor by Employing ω-Transaminases

E. Busto, R. C. Simon, B. Grischek, V. Gotor-Fernández, W. Kroutil

Adv. Synth. Catal. 2014, 356, 1937–1942.

Graphical Abstract

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

Communication

Synthesis of pharmaceutically relevant 17-α-amino steroids using an ω-transaminase

N. Richter, R. C. Simon, W. Kroutil, J. M. Ward, H. C. Hailes

Chem. Commun. 2014, 50, 6098–6100.

Graphical Abstract

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

Update

Nitrile as Activating Group in the Asymmetric Bioreduction of β-Cyanoacrylic Acids Catalyzed by Ene-Reductases

C. K. Winkler, D. Clay, N. G. Turrini, H. Lechner, W. Kroutil, S. Davies, S. Debarge, P. O'Neill, J. Steflik, M. Karmilowicz, J. W. Wong, K. Faber

Adv. Synth. Catal. 2014, 356, 1878–1882.

Graphical Abstract

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

Full Paper

Organic Solvent Tolerance of Retro-Friedel–Crafts Hydrolases

E. Siirola, W. Kroutil

Top. Catal. 2014, 57, 392–400.

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

Full Paper

Mutational Analysis of the C–C Bond Cleaving Enzyme Phloretin Hydrolase from Eubacterium ramulus

A. Frank, E. Siirola, W. Kroutil, G. Grogan

Top. Catal. 2014, 57, 376–384.

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

Letter

Bioreduction and disproportionation of cyclohex-2-enone catalyzed by ene-reductase OYE-1 in ‘micro-aqueous’ organic solvents

D. Clay, C. K. Winkler, G. Tasnádi, K. Faber

Biotechnol. Lett. 2014, 36, 1329–1333.

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 (Kphenol). As a predictive parameter, the ratio of logP/Kphenol should be preferably ≥100.

doi: 10.1007/s10529-014-1494-5

Full Paper

Regioselective ortho-carboxylation of phenols catalyzed by benzoic acid decarboxylases: a biocatalytic equivalent to the Kolbe–Schmitt reaction

C. Wuensch, J. Gross, G. Steinkellner, A. Lyskowski, K. Gruber, S. M. Glueck, K. Faber

RSC Adv. 2014, 4, 9673–9679.

Graphical Abstract

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

Communication

Deracemization By Simultaneous Bio-oxidative Kinetic Resolution and Stereoinversion

J. H. Schrittwieser, B. Groenendaal, V. Resch, D. Ghislieri, S. Wallner, E.-M. Fischereder, E. Fuchs, B. Grischek, J. H. Sattler, P. Macheroux, N. J. Turner, W. Kroutil

Angew. Chem. Int. Ed. 2014, 53, 3731–3734.

Graphical Abstract

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

Communication

Aerobic oxidation of isosorbide and isomannide employing TEMPO/laccase

J. Gross, K. Tauber, M. Fuchs, N. G. Schmidt, A. Rajagopalan, K. Faber, W. M. F. Fabian, J. Pfeffer, T. Haas, W. Kroutil

Green Chem. 2014, 16, 2117–2121.

Graphical Abstract

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

Full Paper

Expanding the Scope of Alcohol Dehydrogenases towards Bulkier Substrates: Stereo- and Enantiopreference for α,α-Dihalogenated Ketones

K. Kędziora, F. R. Bisogno, I. Lavandera, V. Gotor-Fernández, J. Montejo-Bernardo, S. García-Granda, W. Kroutil, V. Gotor

ChemCatChem 2014, 6, 1066–1072.

Graphical Abstract

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

Full Paper

Enzymatic asymmetric synthesis of the silodosin amine intermediate

R. C. Simon, J. H. Sattler, J. E. Farnberger, C. S. Fuchs, N. Richter, F. Zepeck, W. Kroutil

Tetrahedron: Asymmetry 2014, 25, 284–288.

Graphical Abstract

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

Full Paper

Steric vs. electronic effects in the Lactobacillus brevis ADH-catalyzed bioreduction of ketones

C. Rodríguez, W. Borzecka, J. H. Sattler, W. Kroutil, I. Lavandera, V. Gotor

Org. Biomol. Chem. 2014, 12, 673–681.

Graphical Abstract

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

Review

Recent Developments of Cascade Reactions Involving ω-Transaminases

R. C. Simon, N. Richter, E. Busto, W. Kroutil

ACS Catal. 2014, 4, 129–143.

Graphical Abstract

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

Full Paper

NAD(P)H-Independent Asymmetric C=C Bond Reduction Catalyzed by Ene Reductases by Using Artificial Co-substrates as the Hydrogen Donor

C. K. Winkler, D. Clay, M. Entner, M. Plank, K. Faber

Chem. Eur. J. 2014, 20, 1403–1409.

Graphical Abstract

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

Full Paper

Chemoenzymatic Synthesis of Enantiomerically Pure syn-Configured 1-Aryl-3-methylisochroman Derivatives

R. C. Simon, E. Busto, N. Richter, F. Belaj, W. Kroutil

Eur. J. Org. Chem. 2014, 2014, 111–121.

Graphical Abstract

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

Review

Microbial alkyl- and aryl-sulfatases: mechanism, occurrence, screening and stereoselectivities

M. Toesch, M. Schober, K. Faber

Appl. Microbiol. Biotechnol. 2014, 4, 1485–1496.

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

2013

Full Paper

Asymmetric reductive amination of boron-containing aryl-ketones using ω-transaminases

J. S. Reis, R. C. Simon, W. Kroutil, L. H. Andrade

Tetrahedron: Asymmetry 2013, 24, 1495–1501.

Graphical Abstract

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

Full Paper

Escherichia coli/ADH-A: An All-Inclusive Catalyst for the Selective Biooxidation and Deracemisation of Secondary Alcohols

C. E. Paul, I. Lavandera, V. Gotor-Fernández, W. Kroutil, V. Gotor

ChemCatChem 2013, 5, 3875–3881.

Graphical Abstract

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

Full Paper

One-pot combination of enzyme and Pd nanoparticle catalysis for the synthesis of enantiomerically pure 1,2-amino alcohols

J. H. Schrittwieser, F. Coccia, S. Kara, B. Grischek, W. Kroutil, N. d'Alessandro, F. Hollmann

Green Chem. 2013, 15, 3318–3331.

Graphical Abstract

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

Full Paper

Overcoming co-product inhibition in the nicotinamide independent asymmetric bioreduction of activated C=C-bonds using flavin-dependent ene-reductases

C. K. Winkler, D. Clay, E. van Heerden, K. Faber

Biotechnol. Bioeng. 2013, 110, 3085–3092.

Graphical Abstract

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

Communication

Enzymatic Aerobic Alkene Cleavage Catalyzed by a Mn3+-Dependent Proteinase A Homologue

A. Rajagopalan, M. Schober, A. Emmerstorfer, L. Hammerer, A. Migglautsch, B. Seisser, S. M. Glueck, F. Niehaus, J. Eck, H. Pichler, K. Gruber, W. Kroutil

ChemBioChem 2013, 14, 2427–2430.

Graphical Abstract

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

Review

Oxidative Alkene Cleavage by Chemical and Enzymatic Methods

A. Rajagopalan, M. Lara, W. Kroutil

Adv. Synth. Catal. 2013, 355, 3321–3335.

Graphical Abstract

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

Full Paper

Pushing the equilibrium of regio-complementary carboxylation of phenols and hydroxystyrene derivatives

C. Wuensch, N. Schmidt, J. Gross, B. Grischek, S. M. Glueck, K. Faber

J. Biotechnol. 2013, 168, 264–270.

Graphical Abstract

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

Communication

Asymmetric Synthesis of β-Substituted α-Methylenebutyrolactones via TRIP-Catalyzed Allylation: Mechanistic Studies and Application to the Synthesis of (S)-(–)-Hydroxymatairesinol

M. Fuchs, M. Schober, A. Orthaber, K. Faber

Adv. Synth. Catal. 2013, 355, 2499–2505.

Graphical Abstract

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

Review

Inverting hydrolases and their use in enantioconvergent biotransformations

M. Schober, K. Faber

Trends Biotechnol. 2013, 31, 468–478.

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

Communication

Asymmetric Biocatalytic Cannizzaro-Type Reaction

C. Wuensch, H. Lechner, S. M. Glueck, K. Zangger, M. Hall, K. Faber

ChemCatChem 2013, 5, 1744–1748.

Graphical Abstract

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

Review

Unusual reactions mediated by FMN-dependent ene- and nitro-reductases

K. Durchschein, M. Hall, K. Faber

Green Chem. 2013, 15, 1764–1772.

Graphical Abstract

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

Full Paper

Controlling stereoselectivity by enzymatic and chemical means to access enantiomerically pure (1S,3R)-1-benzyl-2,3-dimethyl-1,2,3,4-tetrahydroisoquinoline derivatives

A. A. Orden, J. H. Schrittwieser, V. Resch, F. G. Mutti, W. Kroutil

Tetrahedron: Asymmetry 2013, 24, 744–749.

Graphical Abstract

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

Communication

Asymmetric Synthesis of 3-Substituted Cyclohexylamine Derivatives from Prochiral Diketones via Three Biocatalytic Steps

E. Siirola, F. G. Mutti, B. Grischek, S. F. Hoefler, W. M. F. Fabian, G. Grogan, W. Kroutil

Adv. Synth. Catal. 2013, 355, 1703–1708.

Graphical Abstract

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

Review

C–C hydrolases for biocatalysis

E. Siirola, A. Frank, G. Grogan, W. Kroutil

Adv. Synth. Catal. 2013, 355, 1677–1691.

Graphical Abstract

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

Full Paper

Biochemical characterization of an alcohol dehydrogenase from Ralstonia sp.

J. Kulig, A. Frese, W. Kroutil, M. Pohl, D. Rother

Biotechnol. Bioeng. 2013, 110, 1838–1848.

Graphical Abstract

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 alpha-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

Full Paper

The Structure of Glycerol Trinitrate Reductase NerA from Agrobacterium radiobacter Reveals the Molecular Reason for Nitro- and Ene-Reductase Activity in OYE Homologues

G. Oberdorfer, A. Binter, S. Wallner, K. Durchschein, M. Hall, K. Faber, P. Macheroux, K. Gruber

ChemBioChem 2013, 14, 836–845.

Graphical Abstract

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

Review

Asymmetric Preparation of prim-, sec-, and tert-Amines Employing Selected Biocatalysts

W. Kroutil, E.-M. Fischereder, C. S. Fuchs, H. Lechner, F. G. Mutti, D. Pressnitz, A. Rajagopalan, J. H. Sattler, R. C. Simon, E. Siirola

Org. Process Res. Dev. 2013, 17, 751–759.

Graphical Abstract

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

Full Paper

Concise Chemoenzymatic Three-Step Total Synthesis of Isosolenopsin through Medium Engineering

R. C. Simon, C. S. Fuchs, H. Lechner, F. Zepeck, W. Kroutil

Eur. J. Org. Chem. 2013, 3397–3402.

Graphical Abstract

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

Full Paper

Asymmetric Amination of Tetralone and Chromanone Derivatives Employing ω-Transaminases

D. Pressnitz, C. S. Fuchs, J. H. Sattler, T. Knaus, P. Macheroux, F. G. Mutti, W. Kroutil

ACS Catal. 2013, 3, 555–559.

Graphical Abstract

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

Review

Biocatalytic synthesis of enantiopure building blocks for pharmaceuticals

R. C. Simon, F. G. Mutti, W. Kroutil

Drug Discovery Today: Technologies 2013, 10, e37–e44.

Graphical Abstract

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

Full Paper

Artificial Multi-Enzyme Networks for the Asymmetric Amination of sec-Alcohols

K. Tauber, M. Fuchs, J. H. Sattler, J. Pitzer, D. Pressnitz, D. Koszelewski, K. Faber, J. Pfeffer, T. Haas, W. Kroutil

Chem. Eur. J. 2013, 19, 4030–4035.

Graphical Abstract

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

Full Paper

Alkene cleavage by white-rot Trametes hirsuta: Inducing enzyme activity by a fungicide

A. Rajagopalan, B. Seisser, F. G. Mutti, M. Schober, W. Kroutil

J. Mol. Catal. B: Enzym. 2013, 90, 118–122.

Graphical Abstract

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

Full Paper

Chemoenzymatic Synthesis of All Four Diastereomers of 2,6-Disubstituted Piperidines through Stereoselective Monoamination of 1,5-Diketones

R.C. Simon, M. F. Zepeck, W. Kroutil

Chem. Eur. J. 2013, 19, 2859–2865.

Graphical Abstract

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

Communication

One-pot Deracemisation of sec-Alcohols via Enantioconvergent Enzymatic Hydrolysis of Alkyl Sulfates Using Stereo-Complementary Sulfatases

M. Schober, M. Toesch, T. Knaus, G. A. Strohmeier, B. van Loo, M. Fuchs, F. Hollfelder, P. Macheroux, K. Faber

Angew. Chem. Int. Ed. 2013, 52, 3277–3279.

Graphical Abstract

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

Communication

Asymmetric Enzymatic Hydration of Hydroxystyrene Derivatives

C. Wuensch, J. Gross, G. Steinkellner, K. Gruber, S. M. Glueck, K. Faber

Angew. Chem. Int. Ed. 2013, 52, 2293–2297.

Graphical Abstract

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

Full Paper

Chemoenzymatic Asymmetric Total Synthesis of (R)-Lasiodiplodin Methyl Ether through a Sulfatase-Based Deracemization Process

M. Fuchs, M. Toesch, M. Schober, C. Wuensch, K. Faber

Eur. J. Org. Chem. 2013, 2013, 356–361.

Graphical Abstract

(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

2012

Full Paper

Structure and Mechanism of the First Inverting Alkylsulfatase Specific for Secondary Alkyl Sulfates

T. Knaus, M. Schober, B. Kepplinger, M. Faccinelli, J. Pitzer, K. Faber, P. Macheroux, U. Wagner

FEBS J. 2012, 279, 4374–4384.

Graphical Abstract

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

Full Paper

Unusual C=C-Bond Isomerization of an alpha,beta-Unsaturated gamma-Butyrolactone Catalyzed by Flavoproteins from the Old Yellow Enzyme Family

K. Durchschein, S. Wallner, P. Macheroux, K. Zangger, W. M. F. Fabian, K. Faber

ChemBioChem 2012, 13, 2346–2351.

Graphical Abstract

An unexpected, redox-neutral C=C bond isomerization of a γ-butyrolactone bearing an exo-methylene unit to the thermodynamically more favoured endo isomer (kcat=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

Full Paper

Nicotinamide-Dependent Ene-Reductases as Alternative Biocatalysts for the Reduction of Activated Alkenes

K. Durchschein, S. Wallner P. Macheroux, W. Schwab, T. Winkler, W. Kreis, K. Faber

J. Org. Chem. 2012, 2012, 4963–4968.

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

Full Paper

Improved Chemoenzymatic Asymmetric Synthesis of (S)-Rivastigmine

M. Fuchs, D. Koszelewski, K. Tauber, J. Sattler, W. Banko, A. K. Holzer, M. Pickl, W. Kroutil, K. Faber

Tetrahedron 2012, 68, 7691–7694.

Graphical Abstract

(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

Full Paper

Amination of Benzylic and Cinnamic Alcohols via a Biocatalytic Aerobic Oxidation-Transamination-Cascade

M. Fuchs, K. Tauber, J. Sattler, H. Lechner, J. Pfeffer, W. Kroutil, K. Faber

RSC Adv. 2012, 2, 6262–6265.

Graphical Abstract

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

Full Paper

Inverting the Regioselectivity of the Berberine Bridge Enzyme by Employing Customized Fluorine-Containing Substrates

V. Resch, H. Lechner, J. H. Schrittwieser, S. Wallner, K. Gruber, P. Macheroux, W. Kroutil

Chem. Eur. J. 2012, 18, 13173–13179.

Graphical Abstract

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

Full Paper

A Substrate-Driven Approach to Determine Reactivities of alpha,beta-Unsaturated Carboxylic Esters Towards Asymmetric Bioreduction

G. Tasnádi, C. K. Winkler, D. Clay, N. Sultana, W. M. F. Fabian, M. Hall, K. Ditrich, K. Faber

Chem. Eur. J. 2012, 18, 10362–10367.

Graphical Abstract

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

Account

Stereocontrol Strategies in the Asymmetric Bioreduction of Alkenes

G. Oberdorfer, K. Gruber, K. Faber, M. Hall

Synlett 2012, 23, 1857–1864.

Graphical Abstract

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

Review

Asymmetric Bioreduction of Activated Alkenes to Industrially Relevant Optically Active Compounds

C. K. Winkler, G. Tasnádi, D. Clay, M. Hall, K. Faber

J. Biotechnol. 2012, 162, 381–389.

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

Communication

Redox Self-Sufficient Biocatalyst Network for the Amination of Primary Alcohols

J. H. Sattler, M. Fuchs, K. Tauber, F. G. Mutti, K. Faber, J. Pfeffer, T. Haas, W. Kroutil

Angew. Chem. Int. Ed. 2012, 51, 9156–9159.

Graphical Abstract

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

Communication

Reductive dehalogenation of β-haloacrylic ester derivatives mediated by ene-reductases

G.Tasnádi ,C. K. Winkler ,D. Clay ,M. Hall,K. Faber

Catal. Sci. Technol. 2012, 2, 1548–1552.

Graphical Abstract

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

Full Paper

Stereoselective synthesis of bulky 1,2-diols with alcohol dehydrogenases

J. Kulig, R. C. Simon, C. A. Rose, S. M. Husain, M. Häckh, S. Lüdeke, K. Zeitler, W. Kroutil, M. Pohl, D. Rother

Catal. Sci. Technol. 2012, 2, 1580–1589.

Graphical Abstract

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

Review

Electrophilic and Nucleophilic Enzymatic Cascade Reactions in Biosynthesis

B. T. Ueberbacher, M. Hall, K. Faber

Nat. Prod. Rep. 2012, 29, 337–350.

Graphical Abstract

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

Full Paper

Amination of ketones by employing two new (S)-selective ω-transaminases and the His-tagged ω-TA from Vibrio fluvialis

F. G. Mutti, C. S. Fuchs, D. Pressnitz, N. G. Turrini, J. H. Sattler, A. Lerchner, A. Skerra, W. Kroutil

Eur. J. Org. Chem. 2012, 2012, 1003–1007.

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

Communication

Expanding the regioselective enzymatic repertoire: oxidative mono-cleavage of dialkenes catalyzed by Trametes hirsuta

C. E. Paul, A. Rajagopalan, I. Lavandera, V. Gotor-Fernández,W. Kroutil, V. Gotor

Chem. Commun. 2012, 48, 3303–3305.

Graphical Abstract

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

Communication

Experimental Determination of Thermodynamic Equilibrium in Biocatalytic Transamination

P. Tufvesson, J. S. Jensen, W. Kroutil, J. M. Woodley

Biotechnol. Bioeng. 2012, 109, 2159–2162.

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

Full Paper

Access to Enantiopure α-Alkyl-ß-hydroxy Esters through Dynamic Kinetic Resolutions Employing Purified/Overexpressed Alcohol Dehydrogenases

A.Cuetos, A. Rioz-Martínez, F. R. Bisogno, B. Grischek, I. Lavandera, G. de Gonzalo, W. Kroutil, V. Gotor

Adv. Synth. Catal. 2012, 354, 1743–1749.

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

Full Paper

The Substrate Spectrum of the Inverting sec-Alkylsulfatase Pisa1

M.Schober,T. Knaus, M.Toesch, P. Macheroux, U. Wagner, K. Faber

Adv. Synth. Catal. 2012, 354, 1737–1742.

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

Letter

Regioselective Enzymatic Carboxylation of Phenols and Hydroxystyrene Derivatives

C. Wuensch, S. M. Glueck, J. Gross, D. Koszelewski, M.Schober, K. Faber

Org. Lett. 2012, 14, 1974–1977.

Graphical Abstract

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

2011

Communications

A highly efficient ADH-coupled NADH-recycling system for the asymmetric bioreduction of carbon-carbon double bonds using enoate reductases

K. Tauber, M. Hall, W. Kroutil, W. M. F. Fabian, K. Faber, S. M. Glueck

Biotechnol. Bioeng. 2011, 108, 1462–1467.

Graphical Abstract

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

Highlight

Creating a biocatalyst for the production of an optically pure sterically hindered amine

F. G. Mutti, J. Sattler, K. Tauber, W. Kroutil

ChemCatChem 2011, 3, 109–111.

Graphical Abstract

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

Communication

Biocatalytic Enantioselective Oxidative C–C Coupling by Aerobic C–H Activation

J. H. Schrittwieser, V. Resch, J. H. Sattler, W.-D. Lienhart, K. Durchschein, A. Winkler, K. Gruber, P. Macheroux, W. Kroutil

Angew. Chem. Int. Ed. 2011, 50, 1068–1071.

Graphical Abstract

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

Full Paper

Bicontinous microemulsion as reaction medium for ω-transaminase catalysed biotransformations

M. Laupheimer, S. Engelskirchen, K. Tauber, W. Kroutil, C. Stubenrauch

Tenside Surfact. Det. 2011, 48, 28-32.

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

Review

Novel carbon–carbon bond formations for biocatalysis

V. Resch, J. H. Schrittwieser, E. Siirola, W. Kroutil

Curr. Opin. Biotechnol. 2011, 22, 793–799.

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

Communication

Old yellow enzyme-catalyzed dehydrogenation of saturated ketones

M. Schittmayer, A. Glieder, M. K. Uhl, A. Winkler, Si. Zach, J. H. Schrittwieser, W. Kroutil, P. Macheroux, K. Gruber, S. Kambourakis, J. D. Rozzell, M. Winkler

Adv. Synth. Catal. 2011, 353, 268–274.

Graphical Abstract

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

Review

Recent biocatalytic oxidation–reduction cascades

J. H. Schrittwieser, J. Sattler, V. Resch, F. G. Mutti, W. Kroutil

Curr. Opin. Chem. Biol. 2011, 15, 249–256.

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

Review

Biocatalytic reactions: selected highlights

A. Rajagopalan, W. Kroutil

Materials Today 2011, 14, 144–152.

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

Letter

Chemoselective aerobic oxidation of 4-allylanisol by Fe(III) porphyrins in an aqueous system

O. Vakuliuk, F. G. Mutti, M. Lara, D. T. Gryko, W. Kroutil

Tetrahedron Lett. 2011, 52, 3555–3557.

Graphical Abstract

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

Full Paper

Biocatalytic Organic Synthesis of Optically Pure (S)-Scoulerine and Berbine and Benzylisoquinoline Alkaloids

J. H. Schrittwieser, V. Resch, S. Wallner, W.-D. Lienhart, J. H. Sattler, J. Resch, P. Macheroux, W. Kroutil

J. Org. Chem. 2011, 76, 6703–6714.

Graphical Abstract

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

Full Paper

Biocatalytic Oxidative C–C Bond Formation Catalysed by the Berberine Bridge Enzyme: Optimal Reaction Conditions

F. G. Mutti, C. S. Fuchs, D. Pressnitz, J. H. Sattler, W. Kroutil

Adv. Synth. Catal. 2011, 353, 2377–2383.

Graphical Abstract

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

Full Paper

Tolerance of β-diketone hydrolases as representatives of the crotonase superfamily towards organic solvents

E. Siirola, B. Grischek, D. Clay, A. Frank, G. Grogan, W. Kroutil

Biotechnol. Bioeng. 2011, 108, 2815–2822.

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

Update

Stereoselectivity of four (R)-selective transaminases for the asymmetric amination of ketones

F. G. Mutti, C. S. Fuchs, D. Pressnitz, J. H. Sattler, W. Kroutil

Adv. Synth. Catal. 2011, 353, 3227–3233.

Graphical Abstract

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

Full Paper

Stereocomplementary asymmetric bioreduction of boron-containing ketones mediated by alcohol dehydrogenases

T. Barcellos, K. Tauber, W. Kroutil, L. H. Andrade

Tetrahedron: Asymmetry 2011, 22, 1772–1777.

Graphical Abstract

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

Communication

Homoallylic Alcohols via a Chemo-Enzymatic One-Pot Oxidation–Allylation Cascade

M. Fuchs, M. Schober, J. Pfeffer, W. Kroutil, R. Birner-Gruenberger, K. Faber

Adv. Synth. Catal. 2011, 353, 2354–2358.

Graphical Abstract

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

Letter

A Stereoselective Inverting sec-Alkylsulfatase for the Deracemisation of sec-Alcohols

M. Schober, P. Gadler, T. Knaus, H. Kayer, R. Birner-Grünberger, C. Gülly, P. Macheroux, U. Wagner, K. Faber

Org. Lett. 2011, 13, 4296–4299.

Graphical Abstract

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

Communication

Stereopreferences of Old Yellow Enzymes: Structure Correlations and Sequence Patterns in Enoate Reductases

G. Oberdorfer, G. Steinkellner, C. Stueckler, K. Faber, K. Gruber

ChemCatChem 2011, 3, 1562–1566.

Graphical Abstract

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

Letter

Asymmetric Bioreduction of Alkenes Using Ene-Reductases YersER and KYE1, and Effects of Organic Solvents

Y. Yanto, C. K. Winkler, S. Lohr, M. Hall, K. Faber, A. S. Bommarius

Org. Lett. 2011, 13, 2540–2543.

Graphical Abstract

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

Update

Stereo-Controlled Asymmetric Bioreduction of alpha,beta-Dehydroamino Acid Derivatives

C. Stueckler, C. K. Winkler, M. Hall, B. Hauer, M. Bonnekessel, K. Zangger, K. Faber

Adv. Synth. Catal. 2011, 353, 1169–1173.

Graphical Abstract

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

Full Paper

Reductive Biotransformation of Nitroalkenes via Nitroso-intermediates to Oxazetes Catalyzed by Xenobiotic Reductase A (XenA)

K. Durchschein, W. M. F. Fabian, P. Macheroux, K. Zangger, G. Trimmel, K. Faber

Org. Biomol. Chem. 2011, 9, 3364–3369.

Graphical Abstract

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

Review

Multi-Enzymatic Cascade Reactions: Overview and Perspectives

E. Ricca, B. Brucher, J. H. Schrittwieser

Adv. Synth. Catal. 2011, 353, 2239–2262.

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

Full Paper

Enzymatic Racemisation of Amines Catalysed by Enantiocomplementary ω-Transaminases

D. Koszelewski, B. Grischek, S. M. Glueck, W. Kroutil, K. Faber

Chem. Eur. J. 2011, 17, 378–383.

Graphical Abstract

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

2010

Full Paper

Synthesis of Optically Active Amines Employing Recombinant ω-Transaminases in E. coli Cells

D. Koszelewski, M. Göritzer, D. Clay, B. Seisser, W. Kroutil

ChemCatChem 2010, 2, 73–77.

Graphical Abstract

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

Full Paper

Cutting Long Syntheses Short: Access to Non-Natural Tyrosine Derivatives Employing an Engineered Tyrosine Phenol Lyase

B. Seisser, R. Zinkl, K. Gruber, F. Kaufmann, A. Hafner, W. Kroutil

Adv. Synth. Catal. 2010, 352, 731–736.

Graphical Abstract

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

Account

Deracemisation of Secondary Alcohols via Biocatalytic Stereoinversion

C. V. Voss, C. C. Gruber, W. Kroutil

Synlett 2010, 991–998.

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

Full Paper

ω-Transaminases as efficient biocatalysts to obtain novel chiral selenium-amine ligands for Pd-catalysis

L. H. Andrade, A. V. Silva, P. Milani, D. Koszelewski, W. Kroutil

Org. Biomol. Chem. 2010, 8, 2043–2051.

Graphical Abstract

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

Communication

Deracemisation of Mandelic Acid to Optically Pure Non-Natural L-Phenylglycine via a Redox-Neutral Biocatalytic Cascade

V. Resch, W. M. F. Fabian, W. Kroutil

Adv. Synth. Catal. 2010, 352, 993–997.

Graphical Abstract

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

Communication

Highly Recyclable Chemo-/Biocatalyzed Cascade Reactions with Ionic Liquids: One-Pot Synthesis of Chiral Biaryl Alcohols

V. Gauchot, W. Kroutil, A. R. Schmitzer

Chem. Eur. J. 2010, 16, 6748–6751.

Graphical Abstract

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

Full Paper

Biooxidation of Primary Alcohols to Aldehydes through Hydrogen Transfer Employing Janibacter terrae

T. Orbegozo, J. G. de Vries, W. Kroutil

Eur. J. Org. Chem. 2010, 3445–3448.

Graphical Abstract

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

Full Paper

Testing of microorganisms for ω-transaminase activity

D. Clay, D. Koszelewski, B. Grischek, J. Gross, I. Lavandera, W. Kroutil

Tetrahedron: Asymmetry 2010, 21, 2005–2009.

Graphical Abstract

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

Communication

Structural insights into substrate specificity and solvent tolerance in alcohol dehydrogenase ADH-‘A’ from Rhodococcus ruber DSM 44541

M. Karabec, A. Lyskowski, K. C. Tauber, G. Steinkellner, W. Kroutil, G. Grogan, K. Gruber

Chem. Commun. 2010, 46, 6314–6316.

Graphical Abstract

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

Full Paper

Ketone–Alcohol Hydrogen-Transfer Equilibria: Is the Biooxidation of Halohydrins Blocked?

F. R. Bisogno, E. García-Urdiales, H. Valdés, I. Lavandera, W. Kroutil, D. Suárez, V. Gotor

Chem. Eur. J. 2010, 16, 11012–11019.

Graphical Abstract

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

Communication

Simultaneous iridium catalysed oxidation and enzymatic reduction employing orthogonal reagents

F. G. Mutti, A. Orthaber, J. H. Schrittwieser, J. G. de Vries, R. Pietschnig, W. Kroutil

Chem. Commun. 2010, 46, 8046–8048.

Graphical Abstract

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

Full Paper

Ostensible Enzyme Promiscuity: Alkene Cleavage by Peroxidases

F. G. Mutti, M. Lara, M. Kroutil, W. Kroutil

Chem. Eur. J. 2010, 16, 14142–14148.

Graphical Abstract

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

Full Paper

Asymmetric Synthesis of O-Protected Acyloins Using Enoate Reductases: Stereochemical Control through Protecting Group Modification

C. K. Winkler, C. Stueckler, N. J. Mueller, D. Pressnitz, K. Faber

Eur. J. Org. Chem. 2010, 6354–6358.

Graphical Abstract

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

Update

Asymmetric Synthesis of (R)-3-Hydroxy-2-methylpropanoate (‘Roche Ester’) and Derivatives via Biocatalytic C=C-Bond Reduction

D. Koszelewski, K. Tauber, K. Faber, W. Kroutil

Adv. Synth. Catal. 2010, 352, 2663–2666.

Graphical Abstract

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

Communication

Chemoenzymatic asymmetric total synthesis of (S)-Rivastigmine using ω-transaminases

M. Fuchs, D. Koszelewski, K. Tauber, W. Kroutil, K. Faber

Chem. Commun. 2010, 46, 5500–5502.

Graphical Abstract

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

Review

ω-Transaminases for the synthesis of non-racemic α-chiral primary amines

D. Koszelewski, K. Tauber, K. Faber, W. Kroutil

Trends Biotechnol. 2010, 28, 324–332.

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

Full Paper

Bioreduction of α-methylcinnamaldehyde derivatives: chemo-enzymatic asymmetric synthesis of Lilial™ and Helional™

C. Stueckler, N. J. Mueller, C. K. Winkler, S. M. Glueck, K. Gruber, G. Steinkellner, K. Faber

Dalton Trans. 2010, 39, 8472–8476.

Graphical Abstract

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

Full Paper

The Substrate Spectra of Pentaerythritol Tetranitrate Reductase, Morphinone Reductase, N-Ethylmaleimide Reductase and Estrogen-Binding Protein in the Asymmetric Bioreduction of Activated Alkenes

N. J. Mueller, C. Stueckler, B. Hauer, N. Baudendistel, H. Housden, N. C. Bruce, K. Faber

Adv. Synth. Catal. 2010, 352, 387–394.

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

Full Paper

The flavoprotein-catalyzed reduction of aliphatic nitro-compounds represents a biocatalytic equivalent to the Nef-reaction

K. Durchschein, B. Ferreira-da Silva, S. Wallner, P. Macheroux, W. Kroutil, S. M. Glueck, K. Faber

Green Chem. 2010, 12, 616–619.

Graphical Abstract

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

Full Paper

Immobilization of ω-transaminases by encapsulation in a sol–gel/celite matrix

D. Koszelewski, N. Müller, J. H. Schrittwieser, K. Faber, W. Kroutil

J. Mol. Catal. B: Enzym. 2010, 63, 39–44.

Graphical Abstract

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

Full Paper

Nicotinamide-independent asymmetric bioreduction of C=C-bonds via disproportionation of enones catalyzed by enoate reductases

C. Stueckler, T. C. Reiter, N. Baudendistel, K. Faber

Tetrahedron 2010, 66, 663–667.

Graphical Abstract

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

Full Paper

Chemo-promiscuity of alcohol dehydrogenases: reduction of phenylacetaldoxime to the alcohol

B. Ferreira-Silva, I. Lavandera, A. Kern, K. Faber, W. Kroutil

Tetrahedron 2010, 66, 3410–3414.

Graphical Abstract

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

Review

Biocatalytic Carboxylation

S. M. Glueck, S. Gümüs, W. M. F. Fabian, K. Faber

Chem. Soc. Rev. 2010, 39, 313–328.

Graphical Abstract

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

2009

Full Paper

Shifting the equilibrium of a biocatalytic cascade synthesis to enantiopure epoxides using anion exchangers

J. H. Schrittwieser, I. Lavandera, B. Seisser, B. Mautner, J. H. Lutje Spelberg, W. Kroutil

Tetrahedron: Asymmetry 2009, 20, 483–488.

Graphical Abstract

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

Communication

Oxidative Enzymatic Alkene Cleavage: Indications for a Nonclassical Enzyme Mechanism

M. Lara, F. G. Mutti, S. M. Glueck, W. Kroutil

J. Am. Chem. Soc. 2009, 131, 5368–5369.

Graphical Abstract

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

Full Paper

Deracemisation of alpha-Chiral Primary Amines by a One-Pot, Two-Step Cascade Reaction Catalysed by omega-Transaminases

D. Koszelewski, D. Clay, D. Rozzell, W. Kroutil

Eur. J. Org. Chem. 2009, 14, 2289–2292.

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

Full Paper

Biocatalytic Cascade for the Synthesis of Enantiopure beta-Azidoalcohols and beta-Hydroxynitriles

J. H. Schrittwieser, I. Lavandera, B. Seisser, B. Mautner, W. Kroutil

Eur. J. Org. Chem. 2009, 14, 2293–2298.

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

Full Paper

Biocatalytic oxidation of benzyl alcohol to benzaldehyde via hydrogen transfer

T. Orbegozo, I. Lavandera, W. M. F. Fabian, B. Mautner, J. G. de Vries, W. Kroutil

Tetrahedron 2009, 65, 6805–6809.

Graphical Abstract

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

Full Paper

Kinetic Resolution of rac-1-Phenylethanol with Immobilized Lipases: A Critical Comparison of Microwave and Conventional Heating Protocols

R. O. M. A. de Souza, O. A. C. Antunes, W. Kroutil, C. O. Kappe

J. Org. Chem. 2009, 74, 6157–6162.

Graphical Abstract

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

Full Paper

Enzymatic surface hydrolysis of poly(ethylene terephthalate) and bis(benzoyloxyethyl) terephthalate by lipase and cutinase in the presence of surface active molecules

A. Eberl, S. Heumanna, T. Brückner, R. Araujod, A. Cavaco-Paulod, F. Kaufmanne,W. Kroutil, G. M. Guebitz

J. Biotechnol. 2009, 143, 207–212.

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

Full Paper

Asymmetric synthesis of chiral 2-hydroxy ketones by coupled biocatalytic alkene oxidation and C-C bond formation

N. Kurlemann, M. Lara, M. Pohl,W. Kroutil, A. Liese

J. Mol. Catal. B: Enzym. 2009, 61, 111–116.

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

Full Paper

Deracemization of Mexiletine Biocatalyzed by omega-Transaminases

D. Koszelewski, D. Pressnitz, D. Clay, W. Kroutil

Org. Lett. 2009, 11, 4810–4812.

Graphical Abstract

(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

Full Paper

Synthesis of 4-Phenylpyrrolidin-2-one via Dynamic Kinetic Resolution Catalyzed by omega-Transaminases

D. Koszelewski, D. Clay, K. Faber. W. Kroutil

J. Mol. Catal. B, Enzym 2009, 60, 191–194.

Graphical Abstract

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

Full Paper

Epoxide-Hydrolase-Initiated Hydrolysis/Rearrangement Cascade of a Methylene-Interrupted Bis-Epoxide Yields Chiral THF-Moieties Without Involvement of a ‘Cyclase’

B. T. Ueberbacher, G. Oberdorfer, K. Gruber, K. Faber

ChemBioChem 2009, 10, 1697–1704.

Graphical Abstract

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

Full Paper

Biocatalytic Racemisation of alpha-Hydroxycarboxylic Acids Using a Stereo-complementary Pair of alpha-Hydroxycarboxylic Acid Dehydrogenases

A. Bodlenner, S. M. Glueck, B. M. Nestl, C. C. Gruber, N. Baudendistel, B. Hauer, W. Kroutil, K. Faber

Tetrahedron 2009, 65, 7752–7755.

Graphical Abstract

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

Full Paper

Enantio-Complementary Inverting sec-Alkylsulfatase Activity in Cyano- and Thio-Bacteria Synechococcus and Paracoccus spp.: Selectivity-Enhancement by Medium-Engineering

P. Gadler, T. C. Reiter, K. Hoelsch, D. Weuster-Botz, K. Faber

Tetrahedron: Asymmetry 2009, 20, 115–118.

Graphical Abstract

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

Full Paper

Enantio-complementary Chemo-enzymatic Asymmetric Synthesis of (R)- and (S)-Chromanmethanol

M. Fuchs, Y. Simeo, B. T. Ueberbacher, T. Netscher, K. Faber

Eur. J. Org. Chem. 2009, 6, 833–840.

Graphical Abstract

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

Full Paper

Epoxidation of Conjugated C=C-Bonds and Sulfur-Oxidation of Thioethers Mediated by NADH:FMN-Dependent Oxidoreductases

N. J. Mueller, C. Stueckler, M. Hall, P. Macheroux, K. Faber

J. Org. Biomol. Chem. 2009, 7, 1115–1119.

Graphical Abstract

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

2008

Communication

Deracemisation of sec-Alcohols Through a Concurrent Tandem Biocatalytic Oxidation and Reduction

C. V. Voss, C. C. Gruber, W. Kroutil

Angew. Chem. Int. Ed. 2008, 47, 714–745.

Graphical Abstract

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

Communication

Formal Asymmetric Biocatalytic Reductive Amination

D. Koszelewski, I. Lavandera, D. Clay, G.M. Guebitz, D. Rozzell, W. Kroutil

Angew. Chem. Int. Ed. 2008, 47, 9337–9340.

Graphical Abstract

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

Full Paper

Asymmetric Bioreduction of C=C Bonds using Enoate Reductases OPR1, OPR3 and YqjM: Enzyme-Based Stereocontrol

M. Hall, C. Stueckler, H. Ehammer, E. Pointner, G. Oberdorfer, K. Gruber, B.Hauer, R. Stuermer, W. Kroutil, P. Macheroux, K. Faber

Adv. Synth. Catal. 2008, 350, 441–418.

Graphical Abstract

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

Full Paper

Asymmetric Bioreduction of Activated C=C Bonds Using Zymomonas mobilis NCR Enoate Reductase and Old Yellow Enzymes OYE 1–3 from Yeasts

M. Hall, C. Stueckler, B. Hauer, R. Stuermer, T. Friedrich, M. Breur, W. Kroutil, K. Faber

Eur. J. Org. Chem. 2008, , 1511–1516.

Graphical Abstract

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

Full Paper

Stereocomplementary Asymmetric Reduction of Bulky–Bulky Ketones by Biocatalytic Hydrogen Transfer

I. Lavandera, G. Oberdorfer, J. Gross, S. de Wildeman, W. Kroutil

Eur. J. Org. Chem. 2008, , 2539–2543.

Graphical Abstract

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

Full Paper

An Exceptionally DMSO-Tolerant Alcohol Dehydrogenase for the Stereoselective Reduction of Ketones

I. Lavandera, A. Kern, M. Schaffenberger, J. Gross, A. Glieder, S. de Wildeman, W. Kroutil

ChemSusChem 2008, 1, 461–463.

Graphical Abstract

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

Letter

One-Way Biohydrogen Transfer for Oxidation of sec-Alcohols

I. Lavandera, A. Kern, V. Resch, B. Ferreira-Silva, A. Glieder, W. M. F. Fabian, S. de Wildeman, W. Kroutil

Org. Lett. 2008, 10, 2155–2158.

Graphical Abstract

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

Full Paper

Novel Immobilization Routes for the Covalent Binding of an Alcohol Dehydrogenase from Rhodococcus ruber DSM 44541

K. Goldberg, A. Krueger, T. Meinhardt, W. Kroutil, B. Mautner, A. Liese

Tetrahedron: Asymmetry 2008, 19, 1171–1173.

Graphical Abstract

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

Full Paper

Biocatalytic Cleavage of Alkenes with O2 and Trametes hirsuta G FCC 047

M. Lara, F. G. Mutti, S. M. Glueck, W. Kroutil

Eur. J. Org. Chem. 2008, , 3668–3672.

Graphical Abstract

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

Communication

Stereoselective Bioreduction of Bulky-Bulky Ketones by a Novel ADH from Ralstonia sp.

I. Lavandera, A. Kern, B. Ferreira-Silva, A. Glieder, S. de Wildeman, W. Kroutil

J. Org. Chem. 2008, 73, 6003–6005.

Graphical Abstract

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

Full Paper

Asymmetric anti-Prelog reduction of ketones catalysed by Paracoccus pantotrophus and Comamonas sp. cells via hydrogen transfer

I. Lavandera, B. Höller, A. Kern, U. Ellmer, A. Glieder, S. de Wildeman, W. Kroutil

Tetrahedron: Asymmetry 2008, 19, 1954–1958.

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

Full Paper

Asymmetric anti-Prelog reduction of ketones catalysed by Paracoccus pantotrophus and Comamonas sp. cells via hydrogen transfer

I. Lavandera, B. Höller, A. Kern, U. Ellmer, A. Glieder, S. de Wildeman, W. Kroutil

Tetrahedron: Asymmetry 2008, 19, 1954–1958.

Graphical Abstract

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

Full Paper

Stereoselective anti-Prelog Reduction of Ketones by Whole Cells of Comamonas testosteroni in a ‘Substrate-Coupled’ Approach

S. R. Wallner, I. Lavandera, S. F. Mayer, R. Öhrlein, A. Hafner, K. Edegger, K. Faber, W. Kroutil

J. Mol. Catal. B: Enzym. 2008, 55, 126–129.

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

Full Paper

Orchestration of Concurrent Oxidation and Reduction Cycles for Stereoinversion and Deracemisation of sec-Alcohols

C. V. Voss, C. C. Gruber, K. Faber, T. Knaus, P. Macheroux, W. Kroutil

J. Am. Chem. Soc. 2008, 130, 13969–13972.

Graphical Abstract

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

Minireview

Enantiocomplementary Enzymes: Classification, Molecular Basis for their Reversed Enantiopreference and Prospects for Mirror-Image Biotransformations

P. Mugford, U. Wagner, Y. Jiang, K. Faber, R. Kazlauskas

Angew. Chem. Int. Ed. 2008, 47, 8782–8793.

Graphical Abstract

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

Concept

De-racemization of Enantiomers versus De-Epimerization of Diastereomers: Classification of Novel Types of DYKAT

J. Steinreiber, K. Faber, H. Griengl

Chem. Eur. J. 2008, 14, 8060–8072.

Graphical Abstract

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

2007

Full Paper

A Biocatalytic One-Pot Oxidation/Reduction Sequence for the Deracemisation of a sec-Alcohol

C. V. Voss, C. C. Gruber, W. Kroutil

Tetrahedron: Asymmetry 2007, 18, 276–281.

Graphical Abstract

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

Full Paper

Optimization of a Biocatalytic Single-step Alkene Cleavage of Aryl Alkenes

H. Mang, ,J. Gross, M. Lara, C. Goessler, H. E. Schoemaker, G. M. Guebitz, W. Kroutil

Tetrahedron 2007, 63, 3350–3354.

Graphical Abstract

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

Letter

Enzymatic Reduction of Ketones in "Micro-Aqueous" Media Catalyzed by ADH-A from Rhodococcus ruber

G. de Gonzalo, I. Lavandera, K. Faber, W. Kroutil

Org. Lett. 2007, 9, 2163–2166.

Graphical Abstract

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

Communication

Asymmetric Bioreduction of Activated Alkenes Using Cloned 12-Oxophytodienoate Reductase Isoenzymes OPR-1 and OPR-3 from Lycopersicon esculentum (Tomato): A Striking Change of Stereoselectivity

M. Hall, C. Stueckler, W. Kroutil, P. Macheroux, K. Faber

Angew. Chem. Int. Ed. 2007, 46, 3934–3937.

Graphical Abstract

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

Full Paper

Stereo-Complementary Two-Step Cascades Using a Two-Enzyme System Leading to Enantiopure Epoxides

B. Seisser, I. Lavandera, K. Faber, H. J. Lutje Spelberg, W. Kroutil

Adv. Synth. Catal. 2007, 349, 1399–1404.

Graphical Abstract

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

Full Paper

Biocatalytic Racemization of Synthetically Important Functionalized alpha-Hydroxyketones Using Microbial Cells

B. M. Nestl, A. Bodlenner, R. Stuermer, B. Hauer, W. Kroutil, K. Faber

Tetrahedron: Asymmetry 2007, 18, 1465–1474.

Graphical Abstract

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

Full Paper

An Algorithm for the Deconvolution of Mass Spectrosopic Patterns in Isotope Labeling Studies. Evaluation for the Hydrogen-Deuterium Exchange Reaction in Ketones

C. C. Gruber, G. Oberdorfer, C. V. Voss, J. M. Kremsner, C. O. Kappe, W. Kroutil

J. Org. Chem. 2007, 72, 5778–5783.

Graphical Abstract

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

Full Paper

Hydrolytic Resolution of (R,S)-2-Hydroxycarboxylic Acid Esters in Biphasic Media: Implication for Rate-Limiting Formation or Breakdown of Tetrahedral Intermediates in Acylation Step

P.-Y Wang, T.-L. Chen, S.-W. Tsai, W. Kroutil

Biotechnol. Bioeng. 2007, 98, 30–38.

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

Full Paper

Biocatalytic Racemization of sec-Alcohols and alpha-Hydroxyketones using Lyophilized Microbial Cells

B. M. Nestl, C. V. Voss, A. Bodlenner, U. Ellmer-Schaumberger, W. Kroutil, K. Faber

Appl. Microbiol. Biotechnol. 2007, 76, 1001–1008.

Graphical Abstract

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

Full Paper

Emulation of Racemase Activity by Employing a Pair of Stereocomplementary Biocatalysts

G. de Gonzalo, I. Lavandera, K. Durchschein, D. Wurm, K. Faber, W. Kroutil

Chem. Eur. J. 2007, 13, 8271–8276.

Graphical Abstract

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

Full Paper

Asymmetric Biocatalytic Reduction of Ketones using Hydroxyfunctionalised Water-Miscible Ionic Liquids as Solvents

G. de Gonzalo, I. Lavandera, K. Durchschein, D. Wurm, K. Faber, W. Kroutil

Tetrahedron: Asymmetry 2007, 18, 2541–2546.

Graphical Abstract

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

Letter

Stereocomplementary Bioreduction of alpha,beta-Unsaturated Dicarboxylic Acids and Dimethyl Esters using Enoate Reductases: Enzyme- and Substrate-Based Stereocontrol

C. Stueckler, M. Hall, H. Ehammer, W. Pointner, W. Kroutil, P. Macheroux, K. Faber

Org. Lett. 2007, 9, 5409–5411.

Graphical Abstract

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

Review

New Enzymes for Biotransformations: Microbial Alkyl Sulfatases Displaying Stereo- and Enantioselectivity

P. Gadler, K. Faber

Trends Biotechnol. 2007, 25, 83–88.

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

Review

Asymmetric Bioreduction of Activated C=C Bonds Using Enoate Reductases from the Old Yellow Enzyme Family

B. Hauer, R. Stuermer, M. Hall, K. Faber

Curr. Opin. Chem. Biol. 2007, 11, 203–213.

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

Full Paper

Highly Enantioselective Biohydrolysis of sec-Alkyl Sulfate Esters with Inversion of Configuration Catalysed by Pseudomonas spp.

P. Gadler, K. Faber

Eur. J. Org. Chem. 2007, 2007, 5227–5530.

Graphical Abstract

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

2006

Full Paper

Enantio-complementary deracemization of (±)-2-hydroxy-4-phenylbutanoic acid and (±)-3-phenyllactic acid using lipase-catalyzed kinetic resolution combined with biocatalytic racemization

B. Larissegger-Schnell, S. M. Glueck, W. Kroutil, K. Faber

Tetrahedron 2006, 62, 2912–2916.

Graphical Abstract

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

Review

Biocatalytic approaches for the quantitative production of single stereoisomers from racemates

P. Gadler, S. M. Glueck, W. Kroutil, B. M. Nestl, B. Larissegger-Schnell, B. T. Ueberbacher, S. R. Wallner, K. Faber

Biochem. Soc. Trans. 2006, 34, 296–300.

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

Full Paper

Regio- and Stereoselective Reduction of Diketones and Oxidation of Diols by Biocatalytic Hydrogen Transfer

K. Edegger, W. Stampfer, B. Seisser, K. Faber, S. F. Mayer, R. Oehrlein, A. Hafner, W. Kroutil

Eur. J. Org. Chem. 2006, 1904–1909.

Graphical Abstract

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

Full Paper

Optimization of Reaction Parameters and Cultivation Conditions for Biocatalytic Hydrogen Transfer Employing Overexpressed ADH-‘A’ from Rhodococcus ruber DSM 44541 in E. coli

K. Edegger, C. C. Gruber, K. Faber, A. Hafner, W. Kroutil

Eng. Life Sci. 2006, 6, 149–154.

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

Communication

Biocatalytic Racemization of α-Hydroxy Ketones (Acyloins) at Physiological Conditions using Lactobacillus paracasei DSM 20207

B. M. Nessl, W. Kroutil, K. Faber

Adv. Synth. Catal. 2006, 348, 873–876.

Graphical Abstract

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

Full Paper

Biocatalytic oxidation of sec-alcohols via hydrogen transfer

K. Edegger, H. Mang, K. Faber, J. Gross, W. Kroutil

J. Mol. Catal. A: Chem. 2006, 251, 66–70.

Graphical Abstract

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

Communication

Biocatalytic deuterium- and hydrogen-transfer using over-expressed ADH-‘A’: enhanced stereoselectivity and 2H-labeled chiral alcohols

K. Edegger, C. C. Gruber, T. M. Poessl, S. R. Wallner, I. Lavandera, K. Faber, F. Niehaus, J. Eck, R. Oehrlein, A. Hafner, W. Kroutil

Chem. Commun. 2006, 2402–2404.

Graphical Abstract

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

Communication

Biocatalytic Single-Step Alkene Cleavage from Aryl Alkenes: An Enzymatic Equivalent to Reductive Ozonization

H. Mang, J. Gross, M. Lara, C. Goessler, H. E. Shoemaker, G. M. Guebitz, W. Kroutil

Angew. Chem. Int. Ed. 2006, 45 5201–5203.

Graphical Abstract

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

Full Paper

Overcoming the thermodynamic limitation in asymmetric hydrogen transfer reactions catalyzed by whole cells

K. Goldberg, K. Edegger, W. Kroutil, A. Liese

Biotechnol. Bioeng. 2006, 95 192–198.

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

Review

From a Racemate to a Single Enantiomer: Deracemization by Stereoinversion

C. C. Gruber, I. Lavandera, K. Faber, W. Kroutil

Adv. Synth. Catal. 2006, 348 1789–1805.

Graphical Abstract

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

Full Paper

Biocatalytic Racemization of (Hetero)Aryl-aliphatic α-Hydroxycarboxylic Acids by Lactobacillus spp. Proceeds via an Oxidation–Reduction Sequence

B. M. Nestl, S. M. Glueck, M. Hall, W. Kroutil, R. Stuermer, B. Hauer, K. Faber

Eur. J. Org. Chem. 2006, 4573–4577.

Graphical Abstract

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

Full Paper

Evidence that a Novel Thioesterase is Responsible for Polyketide Chain Release during Biosynthesis of the Polyether Ionophore Monensin

B. M. Harvey, H. Hong, M. A. Jones, Z. A. Hughes-Thomas, R. M. Goss, M. L. Heathcote, V. M. Bolanos-García, W. Kroutil, J. Staunton, P. F. Leadley, J. B. Spencer

ChemBioChem 2006, 7, 1435–1442.

Graphical Abstract

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

Full Paper

Asymmetric whole-cell bioreduction of an α,β-unsaturated aldehyde (citral): competing prim-alcohol dehydrogenase and C–C lyase activities

M. Hall, B. Hauer, R. Stuermer, W. Kroutil, K. Faber

Tetrahedron: Asymmetry 2006, 17, 3058–3062.

Graphical Abstract

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

Full Paper

Selectivity enhancement of enantio- and stereo-complementary epoxide hydrolases and chemo-enzymatic deracemization of (±)-2-methylglycidyl benzyl ether

Y. Simeó, K. Faber

Tetrahedron: Asymmetry 2006, 17, 402–409.

Graphical Abstract

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

2005

Full Paper

Esterification of streptol — a cyclitol derivative — by Candida rugosa lipase: influence of the acyl donor on regioselectivity

W. Kroutil, L. Hagmann, T. C. Schuez, V. Jungmann, J. P. Pachlatko

J. Mol. Catal. B: Enzym. 2005, 32, 247–252.

Graphical Abstract

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

Communication

Biocatalytic racemisation of α-hydroxycarboxylic acids at physiological conditions

S. M. Glueck, B. Larissegger-Schnell, K. Csar, W. Kroutil, K. Faber

Chem. Commun. 2005, 1904–1905.

Graphical Abstract

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

Review

New enzymes for biotransformations

K. Faber, W. Kroutil

Curr. Opin. Chem. Biol. 2005, 9, 181–187.

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

Full Paper

Biocatalytic Racemization of Aliphatic, Arylaliphatic, and Aromatic α-Hydroxycarboxylic Acids

S. M. Glueck, M. Pirker, B. M. Nestl, B. T. Ueberbacher, B. Larissegger-Schnell, K. Csar, B. Hauer, R. Stuermer, W. Kroutil, K. Faber

J. Org. Chem. 2005, 70, 4028–4032.

Graphical Abstract

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

Review

The Substrate Spectrum of Mandelate Racemase: Minimum Structural Requirements for Substrates and Substrate Model

U. Felfer, M. Goriup, M. F. Koegl, U. Wagner, B. Larissegger-Schnell, K. Faber, W. Kroutil

Adv. Synth. Catal. 2005, 347, 951–961.

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

Letter

Chemo-Enzymatic Synthesis of (R)- and (S)-2-Hydroxy-4-phenylbutanoic Acid via Enantio-Complementary Deracemization of (±)-2-Hydroxy-4-phenyl-3-butenoic Acid Using a Racemase-Lipase Two-Enzyme System

B. Larissegger-Schnell, W. Kroutil, K. Faber

Synlett 2005, 12, 1936–1938.

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

Full Paper

Non-Racemic Halohydrins via Biocatalytic Hydrogen-Transfer Reduction of Halo-Ketones and One-Pot Cascade Reaction to Enantiopure Epoxides

T. M. Poessl, B. Kosjek, U. Ellmer, C. C. Gruber, K. Edegger, K. Faber, P. Hildebrandt, U. T. Bornscheuer, W. Kroutil

Adv. Synth. Catal. 2005, 347, 1827–1834.

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

Full Paper

Stereoselective hydrolysis of sec-mono-alkyl sulfate esters with retention of configuration

S. R. Wallner, B. M. Nestl, K. Faber

Tetrahedron 2005, 61, 1517–1521.

Graphical Abstract

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

Full Paper

A Chemoenzymatic, Enantioconvergent, Asymmetric Total Synthesis of (R)-Fridamycin E

B. J. Ueberbacher, I. Osprian, S. F. Mayer, K. Faber

Eur. J. Org. Chem. 2005, 1266–1270.

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

Full Paper

Highly enantioselective stereo-inverting sec-alkylsulfatase activity of hyperthermophilic Archaea

S. R. Wallner, B. M. Nestl, K. Faber

Org. Biomol. Chem. 2005, 3, 2652–2656.

Graphical Abstract

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

Communication

Highly Enantioselective sec-Alkyl Sulfatase Activity of the Marine Planctomycete Rhodopirellula baltica Shows Retention of Configuration

S. R. Wallner, M. Bauer, C. Würdemann, P. Wecker, F. O. Gloeckner, K. Faber

Angew. Chem. Int. Ed. 2005, 44, 6381–6384.

Graphical Abstract

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

2004

full paper

Simple Biocatalytic Access to Enantiopure (S)-1-Heteroarylethanols Employing a Microbial Hydrogen Transfer Reaction

W. Stampfer, K. Edegger, B. Kosjek, K. Faber, W. Kroutil

Adv. Synth. Catal. 2004, 346, 57–62.

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

full paper

Purification and Characterization of a Chemotolerant Alcohol Dehydrogenase Applicable to Coupled Redox Reactions

W. Stampfer, B. Kosjek, M. Pogorevc, W. Goessler, K. Faber, W. Kroutil

Biotechnol. Bioeng. 2004, 86, 55–62.

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

Review

Biocatalytic Oxidation of Primary and Secondary Alcohols

W. Kroutil, H. Mang, K. Edegger, K. Faber

Adv. Synth. Catal. 2004, 346, 125–142.

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

review

Recent advances in the biocatalytic reduction of ketones and oxidation of sec-alcohols

W. Kroutil, H. Mang, K. Edegger, K. Faber

Curr. Opin. Chem. Biol. 2004, 8, 120–126.

Graphical Abstract

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

Full Paper

Mandelate racemase activity in ionic liquids: scopes and limitations

N. Kaftzik, W. Kroutil, K. Faber, U. Kragl

J. Mol. Catal. A: Chem. 2004, 214, 107–112.

Graphical Abstract

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 aw > 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

Full Paper

Chemo- and stereo-selective biocatalytic reduction of α,β-unsaturated ketones employing a chemo-tolerant ADH from Rhodococcus ruber DSM 44541

R. van Deursen, W. Stampfer, K. Edegger, K. Faber, W. Kroutil

J. Mol. Catal. B: Enzym. 2004, 31, 159–163.

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

Full Paper

Identification of Various Lipolytic Enzymes in Crude Porcine Pancreatic Lipase Preparations Using Covalent Fluorescent Inhibitors

R. Birner-Gruenberger, H. Scholze, K. Faber, A. Hermetter

Biotechnol. Bioeng. 2004, 85, 147–154.

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

Full Paper

Biocatalytic Enantio-Convergent Preparation of sec-Alcohols Using Sulfatases

S. R. Wallner, M. Pogorevc, H. Trauthwein, K. Faber

Eng. Life Sci. 2004, 4, 512–516.

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

Full Paper

Biocatalytic Asymmetric Rearrangment of a Methylene-Interrupted Bis-Epoxide: Simultaneous Control of Four Asymmetric Centers Through a Bio-Mimetic Reaction Cascade

S. M. Glueck, W. M. F. Fabian, K. Faber, S. F. Mayer

Chem. Eur. J. 2004, 10, 3467–3478.

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

Letter

Highly Enantioselective sec-Alkyl Sulfatase Activity of Sulfolobus acidocaldarius DSM 639

S. R. Wallner, B. M. Nestl, K. Faber

Org. Lett. 2004, 6, 5009–5010.

Graphical Abstract

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

Full Paper

Chemo-enzymatic Enantio-convergent Asymmetric Synthesis of (R)-(+)-Marmin

K. Edegger, S. F. Mayer, A. Steinreiber, K. Faber

Tetrahedron 2004, 60, 583–588.

Graphical Abstract

Asymmetric biohydrolysis of trisubstituted terpenoid oxiranes (rac-1arac-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

2003

Full Paper

Biocatalytic Asymmetric Hydrogen Transfer Employing Rhodococcus ruber DSM 44541

W. Stampfer, B. Kosjek, K. Faber, W. Kroutil

J. Org. Chem. 2003, 68, 402–406.

Graphical Abstract

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

Full Paper

Biocatalytic oxidative kinetic resolution of sec-alcohols: stereocontrol through substrate-modification

W. Stampfer, B. Kosjek, K. Faber, W. Kroutil

Tetrahedron: Asymmetry 2003, 14, 275–280.

Graphical Abstract

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

Full Paper

Optimization of the organic solvent-stable asymmetric hydrogen transfer system of Rhodococcus ruber DSM 44541: an activity-growth study

W. Stampfer, B. Kosjek, S. M. Glueck, M. Pogorevc, U. Ellmer, S. R. Wallner, T. M. Koegl, T. M. Poessl, S. F. Mayer, B. Ueberbacher, K. Faber, W. Kroutil

J. Mol. Catal. B: Enzym. 2003, 22, 1–6.

Graphical Abstract

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

Review

Enzymatic Racemisation and its Application to Synthetic Biotransformations

B. Schnell, K. Faber, W. Kroutil

Adv. Synth. Catal. 2003, 345, 653–666.

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

Full Paper

Efficient production of raspberry ketone via 'green' biocatalytic oxidation

B. Kosjek, W. Stampfer, R. van Deursen, K. Faber, W. Kroutil

Tetrahedron 2003, 59, 9517–9521.

Graphical Abstract

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

Full Paper

Enantioselectivity of Epoxide Hydrolase Catalyzed Oxirane Ring Opening: a 3D QSAR Study

J. Paier, T. Stockner, A. Steinreiber, K. Faber, W. M. F. Fabian

J. Comp.-Aided Mol. Design 2003, 17, 1–11.

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

Full Paper

Purification and Characterization of an Inverting Stereo- and Enantioselective sec-Alkylsulfatase from the Gram-Positive Bacterium Rhodococcus ruber DSM 44541

M. Pogorevc, K. Faber

Appl. Environ. Microbiol. 2003, 69, 2810–2815.

Graphical Abstract

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

Full Paper

Quantitative Structure–Activity Relationships for the Enantioselectivity of Oxirane Ring-Opening Catalyzed by Epoxide Hydrolases

S. Funar-Timofei, T. Suzuki, J. A. Paier, A. Steinreiber, K. Faber, W. M. F. Fabian

J. Chem. Inform. Comput. Sci. 2003, 43, 934–940.

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

Full Paper

Biomimetic approach toward the stereoselective synthesis of acetogenins

R. V. A. Orru, B. Groenendaal, J. van Heyst, M. Hunting, C. Wesseling, R. F. Schmitz, S. F. Mayer, K. Faber

Pure Appl. Chem. 2003, 75, 259–264.

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

Full Paper

Chemo-enzymatic enantio-convergent asymmetric total synthesis of (S)-(+)-dictyoprolene using a kinetic resolution—stereoinversion protocol

A. Wallner, H. Mang, S. M. Glueck, A. Steinreiber, S. F. Mayer, K. Faber

Tetrahedron: Asymmetry 2003, 14, 2427–2432.

Graphical Abstract

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

2002

Letter

Dynamic Kinetic Resolution of γ-Hydroxy Acid Derivatives

A.-B. Runmo, O. Pamies, K. Faber, J.-E. Bäckvall

Tetrahedron Lett. 2002, 43, 2983–2986.

Graphical Abstract

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

communication

Biocatalytic Asymmetric Hydrogen Transfer

W. Stampfer, B. Kosjek, C. Moitzi, W. Kroutil, K. Faber

Angew. Chem. Int. Ed. 2002, 41, 1014–1017.

Graphical Abstract

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

full paper

Stereoselectivity in Biocatalytic Enantioconvergent Reactions and a Computer Program for its Determination

K. Faber, W. Kroutil

Tetrahedron: Asymmetry 2002, 13, 377–382.

Graphical Abstract

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 ki, the enantioselectivity (E) and the RI ratio (RI) can be determined; in addition, processes can be simulated based on assumed ki values.

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

communication

Kinetic Resolution of rac-sec-Alkyl Sulfate Esters via Hydrolysis by an Alkylsulfatase from Rhodococcus ruber DSM 44541 Furnishes Homochiral Products

M. Pogorevc, W. Kroutil, S. R. Wallner, K. Faber

Angew. Chem. Int. Ed. 2002, 41, 4052–4054.

Graphical Abstract

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

full paper

Total Synthesis of (+)-exo-Brevicomin Based on Enantioconvergent Biocatalytic Hydrolysis of an Alkene-functionalized 2,3-Disubstituted Epoxide.

S. F. Mayer, H. Mang, A. Steinreiber, R. Saf, K. Faber

Can. J. Chem. 2002, 80, 362–369.

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

full paper

Chemo-Enzymatic Asymmetric Total Syntheses of Antitumor Agents (3R,9R,10R)- and (3S,9R,10R)-Panaxytriol and (R)- and (S)-Falcarinol from Panax ginseng Using an Enantio-Convergent Enzyme-Triggered Cascade-Reaction

S. F. Mayer, A. Steinreiber, R. V. A. Orru, K. Faber

J. Org. Chem. 2002, 67, 9115–9121.

Graphical Abstract

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

Letter

A Caveat for the Use of logP-Values for the Assessment of the Biocompatibility of Organic Solvents

M. Pogorevc, H. Stecher, K. Faber

Biotechnol. Lett. 2002, 24, 857–860.

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

full paper

Chemo-Enzymatic Asymmetric Total Syntheses of a Constituent of Jamaican Rum and of (+)-Pestalotin Using an Enantio-Convergent Enzyme-Triggered Cascade-Reaction

S. F. Mayer, A. Steinreiber, M. Goriup, R. Saf, K. Faber

Tetrahedron: Asymmetry 2002, 13, 523–528.

Graphical Abstract

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

full paper

Preparation of an Epoxide-Hydrolyzing Biocatalyst: Rhodococcus ruber DSM 44540 - an Activity-Growth Study

S. F. Mayer, S. M. Glueck, M. Pogorevc, A. Steinreiber, W. Stampfer, W. Kroutil, K. Faber

J. Mol. Catal. B: Enzym. 2002, 18, 163–168.

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

full paper

Enantioselective Stereoinversion of sec-Alkyl Sulfates by an Alkylsulfatase from Rhodococcus ruber DSM 44541

M. Pogorevc, K. Faber

Tetrahedron: Asymmetry 2002, 13, 1435–1441.

Graphical Abstract

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

full paper

Selectivity-Enhancement in Enantioselective Hydrolysis of sec-Alkyl Sulfates by an Alkylsulfatase from Rhodococcus ruber DSM 44541

M. Pogorevc, U. T. Strauss, T. Riermeier, K. Faber

Tetrahedron: Asymmetry 2002, 13, 1443–1447.

Graphical Abstract

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

full paper

Advances in Biocatalytic Synthesis: Enzyme-Triggered Asymmetric Cascade-Reactions.

S. Glueck, S. F. Mayer, W. Kroutil, K. Faber

Pure Appl. Chem 2002, 74, 2253–2257.

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

full paper

First preparative biocatalytic hydrolysis and S-methylation of cyclic trithiocarbonates

W. Kroutil, A. A. Stampfli, R. Dahinden, M. Jorg, U. Muller, J. P. Pachlatko

Tetrahedron 2002, 58, 2589–2592.

Graphical Abstract

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

2001

letter

Chemo-Enzymatic Enantio-Convergent Synthesis of C4-Building Blocks Containing a Fully Substituted Chiral Carbon Center Using Bacterial Epoxide Hydrolases

A. Steinreiber, H. Hellström, S. F. Mayer, R. V. A. Orru, K. Faber

Synlett, 2001 23, 169–173.

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

Letter

Bacterial Epoxide Hydrolase Catalyzed Resolution of a 2,2-Disubstituted Oxirane: Optimization and Upscaling

H. Hellström, A. Steinreiber, S. F. Mayer, K. Faber

Biotechnol. Lett. 2001, 23, 169–173.

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

full paper

The Substrate-Spectrum of Mandelate Racemase. Part 1: Variation of the α-Hydroxy Acid Moiety

M. Goriup, U. T. Strauss, U. Felfer, W. Kroutil, K. Faber

J. Mol. Catal. B: Enzym. 2001, 15, 207–211.

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

full paper

The Substrate-Spectrum of Mandelate Racemase, Part 2: (Hetero)-Aryl-Substituted Mandelates and Modulation of Activity

U. Felfer, U. T. Strauss, W. Kroutil, W. M. F. Fabian, K. Faber

J. Mol. Catal. B: Enzym. 2001, 15, 213–222.

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

full paper

Biocatalytic Asymmetric and Enantioconvergent Hydrolysis of Trisubstituted Oxiranes

A. Steinreiber, S. F. Mayer, R. Saf, K. Faber

Tetrahedron: Asymmetry 2001, 12, 1519–1528.

Graphical Abstract

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

full paper

An Enzyme-Triggered Enantio-Convergent Cascade-Reaction

S. F. Mayer, A. Steinreiber, R. V. A. Orru, K. Faber

Tetrahedron: Asymmetry 2001, 12, 41–43.

Graphical Abstract

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

full paper

Activation of Mandelate Racemase via Immobilisation in Lyotropic Liquid Crystals for Biocatalysis in Organic Solvents: Application and Modelling

C. Bauer, M. Boy, K. Faber, U. Felfer, H. Voss

J. Mol. Catal. B: Enzym. 2001, 16, 91–100.

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

full paper

Enzyme-Triggered Enantio-Convergent Transformation of Haloalkyl Epoxides

S. F. Mayer, A. Steinreiber, R. V. A. Orru, K. Faber

Eur. J. Org. Chem. 2001, 2001, 4537–4542.

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

review

Non-Sequential Processes for the Transformation of a Racemate into a Single Stereoisomeric Product: Proposal for Stereochemical Classification

K. Faber

Chem. Eur. J. 2001, 7, 5004–5010.

Graphical Abstract

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

letter

High-Speed Microwave-Promoted Mitsunobu Inversions. Application Toward the Deracemization of Sulcatol

A. Steinreiber, A. Stadler, S. F. Mayer, K. Faber, C. O. Kappe

Tetrahedron Lett. 2001, 42, 6283–6286.

Graphical Abstract

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

review

Microbial Epoxide Hydrolases for Preparative Biotransformations

A. Steinreiber, K. Faber

Curr. Opin. Biotechnol. 2001, 12, 552–558.

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

full paper

Asymmetric Total Synthesis of a Beer-Aroma Constituent Based on Enantioconvergent Biocatalytic Hydrolysis of Trisubstituted Epoxides

A. Steinreiber, S. F. Mayer, K. Faber

Synthesis 2001, 13, 2035–2039.

Graphical Abstract

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

Review

Enzyme-Initiated Domino-(Cascade)-Reactions

S. F. Mayer, W. Kroutil, K. Faber

Chem. Soc. Rev. 2001, 30, 332–339.

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

Full Paper

Enantio- and Diastereo-Convergent Synthesis of (2R,5R)- and (2R,5S)-Pityol Through Enzyme-Triggered Ring-Closure

A. Steinreiber, K. Edegger, S. F. Mayer, K. Faber

Tetrahedron: Asymmetry 2001, 12, 2067–2071.

Graphical Abstract

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

Letter

One-pot Deracemization of an Enol Acetate Derived from a Prochiral Cyclohexanone

G. R. Allan, A. J. Carnell, W. Kroutil

Tetrahedron Lett. 2001, 42, 5959–5962.

Graphical Abstract

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

2000

full paper

Biocatalytic Resolution of Sterically Hindered Alcohols, Carboxylic Acids and Esters Containing Fully Substituted Chiral Centers by Hydrolytic Enzymes

M. Pogorevc, K. Faber

J. Mol. Catal. B: Enzym. 2000, 10, 357–376.

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

full paper

Carboxyl Esterase Preparations from Rhodococcus sp. for the Resolution of Linalyl Acetate

M. Pogorevc, U. T. Strauss, M. Hayn, K. Faber

Chem. Monthly 2000, 131, 639–644.

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

Letter

Stabilization and Activity-Enhancement of Mandelate Racemase from Pseudomonas putida ATCC 12336 by Immobilization

U. T. Strauss, A. Kandelbauer, K. Faber

Biotechnol. Lett. 2000, 22, 515–520.

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

full paper

Synthesis of Enantiomerically Pure 4-Aryl-3,4-dihydropyrimidin-2(1H)-ones via Enzymatic Resolution: Preparation of the Antihypertensive Agent (R)-SQ 32926.

B. Schnell, U. T. Strauss, P. Verdino, K. Faber, C. O. Kappe

Tetrahedron: Asymmetry 2000, 11, 1449–1453.

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

full paper

Enantioselective Hydrolysis of Functionalized 2,2-Disubstituted Exiranes with Bacterial Epoxide Hydrolases

A. Steinreiber, I. Osprian, S. F. Mayer, R. V. A. Orru, K. Faber

Eur. J. Org. Chem. 2000, 3703–3711.

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

full paper

Selectivity-Enhancement of Epoxide-Hydrolase Catalyzed Resolution of 2,2-Disubstituted Oxiranes by Substrate Modification

I. Osprian, W. Stampfer, K. Faber

J. Chem. Soc., Perkin Trans. 2000, 1, 3779–3785.

A series of (±)-2,2-disubstituted oxiranes bearing an alkene or alkyne functional group were resolved by bacterial epoxide hydrolases with excellent selectivities. The presence of a carbon–carbon double or triple bond furnished a highly flexible system for substrate modification, which allowed the enantioselectivity to be tuned by rational substrate modification. Thus, a significant selectivity enhancement of more than a ten-fold increase of E-values was achieved by appropriate choice of the C–C multiple bond, i.e. by (i) choosing an alkene or alkyne moiety or by (ii) variation of the E/Z-configuration of olefinic substrates. The enantioenriched epoxides and vicinal diols thus obtained may be easily transformed into ω-functionalized building blocks containing a chiral fully substituted carbon atom by oxidative cleavage of the carbon–carbon multiple bond.

doi: 10.1039/B005203P

Editorial Overview

'Chemical Biotechnology', a Happy Marriage Between Chemistry and Biotechnology: Asymmetric Synthesis via Green Chemistry

K. Faber, R. Patel

Curr. Opin. Biotechnol. 2000, 11, 517–519.

During the past decade, there has been an increasing awareness of the enormous potential of enzymes for the transformation of man-made ‘synthetic’ materials with high chemo-, regio- and enantio-specificity [1] and [2]. This development is most aptly reflected by the fact that an annual average of ∼600 scientific original papers have been published since the late 1980s in this interdisciplinary field between (organic) chemistry and biotechnology (data taken from Database Faber, February 2000). Increasing understanding of the mechanism of drugs on a molecular level has led to the widespread awareness of the importance of chirality as the key to the efficacy of many drugs. In many cases where the switch from racemate drug substance to enantiomerically pure compound is feasible, there is the opportunity to extend the use of an industrial process. The physical characteristics of enantiomers versus racemic compounds in many cases confer processing or formulation advantages [3] and [4].

doi: 10.1016/S0958-1669(00)00157-9

full paper

Synthesis and Reactions of Biginelli-compounds. Part 23. Chemoenzymatic Syntheses of Enantiomerically Pure 4-Aryl-3,4-dihydropyrimidin-2(1H)-ones

B. Schnell, W. Krenn, K. Faber, C. O. Kappe

J. Chem. Soc., Perkin Trans. 2000, 1, 4382–4389.

Enantiomerically pure dihydropyrimidones (DHPMs) were prepared by lipase-catalyzed enzymatic resolution of two types of activated DHPM esters. In the first model series, pivaloyloxymethyl-activated DHPM C5-esters 10a–c were resolved on an analytical scale by various lipases in two different solvent systems with selectivities E < 50. Alternatively, attachment of an acetoxymethyl residue at the N3 position of the DHPM scaffold led to activated ester 15, which was selectively cleaved by Thermomyces lanuginosus lipase (E > 200) to furnish, after deprotection, DHPMs (R)- and (S)-13 on a semi-preparative scale. Treatment of (R)-13 with trichloroacetyl isocyanate produced the antihypertensive agent (R)-SQ 32926.

doi: 10.1039/B006372J

full paper

Selectivity Enhancement of Epoxide Hydrolase Catalyzed Resolution of 2,2-Disubstituted Oxiranes by Substrate Modification

I. Osprian, W. Stampfer, K. Faber

J. Chem. Soc, Perkin Trans. 2000, 1, 3779–3785.

A series of (±)-2,2-disubstituted oxiranes bearing an alkene or alkyne functional group were resolved by bacterial epoxide hydrolases with excellent selectivities. The presence of a carbon–carbon double or triple bond furnished a highly flexible system for substrate modification, which allowed the enantioselectivity to be tuned by rational substrate modification. Thus, a significant selectivity enhancement of more than a ten-fold increase of E-values was achieved by appropriate choice of the C–C multiple bond, i.e. by (i) choosing an alkene or alkyne moiety or by (ii) variation of the E/Z-configuration of olefinic substrates. The enantioenriched epoxides and vicinal diols thus obtained may be easily transformed into ω-functionalized building blocks containing a chiral fully substituted carbon atom by oxidative cleavage of the carbon–carbon multiple bond.

doi: 10.1039/B005203P