Bioorganic Chemistry

Our Research

Graphical Abstract

The art to make ‘impossible’ reactions work by using the most powerful catalysts on earth – enzymes – is known as biocatalysis. There is one driving force behind our projects, i.e. cutting tedious asymmetric syntheses short. During the 1980s we have mainly been using industrially produced enzymes (such as lipases, esterases and proteases), which offers the advantage that the chiral catalyst comes in a nice parcel by mail. On the other hand, other chemists do the same. Confronted by some tough competition we went into Nature's supermarket (we usually prefer the NCIMB-, or IFO- and DSM-branch) in search for novel enzymes. Other people breed dogs, we subsequently started to grow our own ‘pet’ biocatalysts. This, of course, proceeded numerous heart-felt warnings by the microbiologists next door, who expressed their unshakeable belief that organic chemists are too stupid to grow bugs. Fortunately, the bugs did not share this prejudice. They grew well, because we fed them tasty goodies. After all, even microorganisms do not bite the hand that feeds them. Meanwhile our culture collection (where FCC stands for ‘Fab-Crew-Collection’) contains some 300 microbial strains encompassing bacteria, lower and higher fungi and, most recently, also a selected set of extremophiles.

For an overview on biotransformations and a textbook see:
  • K. Faber, W. Kroutil, Curr. Opin. Chem. Biol. 2005, 9, 181.
  • K. Faber, Pure Appl. Chem. 1997, 69, 1613.
  • K. Faber, Biotransformations in Organic Chemistry, 6th revised edn., Springer, Heidelberg, 2011; 423 pages, 29 tables, 286 schemes, 38 figures, ca. 2600 references

Research Topics

Green redox chemistry employing alcohol dehydrogenases

A large number of redox-reactions in organic chemistry are still based on (heavy) metals, many of which are (i) expensive, (ii) toxic, (iii) environmentally inacceptable (in particular when required in molar amounts) and (iv) difficult to remove from the product(s). As a consequence, any metal-independent method to perform redox reactions would present a viable alternative.

Alcohol dehydrogenases (ADHs) have long been used for the bioreduction of carbonyl compounds and (to a lesser extent) for the biooxidation of alcohols. The merits of these systems are that (i) the reaction is catalysed by an innocuous enzyme and (ii) the hydride-donors (the reductant) or -acceptors (the oxidant) are cheap, safe and innocuous. The main reason for the limited number of large-scale applications based on ADHs is the fact that ADHs in general are quite sensitive enzymes and – most importantly – they require a cofactor which has to be recycled. Whereas NADH-recycling is feasible (but not trivial), the regeneration of NADPH is still a complicated task, in particular on a large-scale.

Graphical Abstract

In order to circumvent these limitations, we have recently identified a bacterial ADH which seems to be perfectly suited for this task. For instance, the enzyme can oxidise a sec-alcohol enantioselectively at the expense of acetone as the oxidant going in hand with an oxidative kinetic resolution. The reverse reaction – the asymmetric reduction of a ketone – is accomplished using 2-propanol as reductant using the same enzyme. The enzyme seems to be a tough cookie, as it tolerates exceptionally high concentrations of organic cosolvents, co-substrates (such as acetone or 2-propanol) and substrate.

Further reading:

  • W. Stampfer, B. Kosjek, C. Moitzi, W. Kroutil, K. Faber, Angew. Chem. Int. Ed. 2002, 41, 1014.
  • W. Stampfer, B. Kosjek, W. Kroutil, K. Faber, Biotechnol. Bioeng. 2003, 81, 865.
  • W. Stampfer, B. Kosjek, K. Faber, W. Kroutil, J. Org. Chem. 2003, 68, 402.
  • W. Stampfer, B. Kosjek, K. Faber, W. Kroutil, Tetrahedron: Asymmetry 2003, 14, 275.
  • B. Kosjek, W. Stampfer, S. M. Glueck, M. Pogorevc, U. Ellmer, S. R. Wallner, M. F. Koegl, T. M. Poessl, S. F. Mayer, B. Ueberbacher, K. Faber, W. Kroutil, J. Mol. Catal. B: Enzym. 2003, 22, 1.
  • B. Kosjek, W. Stampfer, R. van Deursen, K. Faber, W. Kroutil, Tetrahedron 2003, 59, 9517.
  • B. Kosjek, W. Stampfer, M. Pogorevc, W. Goessler, K. Faber, W. Kroutil, Biotechnol. Bioeng. 2004, 86, 55.
  • R. van Deursen, W. Stampfer, K. Edegger, K. Faber, W. Kroutil, J. Mol. Catal. B: Enzym. 2004, 31, 159.

Deracemization techniques

Although the biocatalytic literature is full of kinetic resolutions using hydrolytic enzymes such as lipases, esterases and proteases, there are several drawbacks associated with those reactions: The reaction has to be terminated at 50% conversion, yielding a 1:1-mixture of formed product and remaining substrate, which have to be separated (usually by boring by flash chromatography). As the maximum theoretical yield of substrate and product is 50% each and there is only need for one stereoisomer, the other has to be discarded. Since this is totally inacceptable for our industrial sponsors who generously pay our bills, we had to think of some solutions.

Since the theoretically possible number of racemates will always be larger than that of prochiral and meso-compounds, the question is not how to avoid kinetic resolution but how to transform it into a process which leads to a single stereoisomeric product in 100% theoretical yield from a racemate. These protocols are generally denoted as ‘deracemization’. Since several years, we pursue the following strategies:

Graphical Abstract

Modified variations of these concepts were applied for example for the enantioselective synthesis of secondary alcohols. An example would be an oxidation–reduction sequence combining different alcohol dehydrogenases and cofactor recycling systems, resulting in the synthesis of a single enantiomer starting from a racemic substrate mixture.

Graphical Abstract

Another example for a deracemisation process – combining again a concurrent oxidative and reductive step – would be the combination of the mandelate dehydrogenase and an amino acid dehydrogenase. In addition mandelate racemase was used to continuously provide the preferred enantiomer for the D-selective mandelate dehydrogenase. Using this system, racemic mandelic acid was converted into enantiomerically pure phenyl glycine. Another advantage of this system is the internal cofactor recycling.

Graphical Abstract

Further reading:

  • H. Stecher, K. Faber, Synthesis 1997, 1.
  • U. T. Strauss, U. Felfer, K. Faber, Tetrahedron: Asymmetry 1999, 10, 107.
  • Faber, Chem. Eur. J. 2001, 7, 5004.
  • C. V. Voss, C. C. Gruber, W. Kroutil, Synlett 2010, 7, 991.
  • C. V. Voss, C. C. Gruber, K. Faber, T. Knaus, P. Macheroux, W. Kroutil, J. Am. Chem. Soc 2008, 42, 13969.
  • C. V. Voss, C. C. Gruber, W. Kroutil, Angew. Chem., Int. Ed. 2008, 47, 741.
  • C. V. Voss, C. C. Gruber, W. Kroutil, Tetrahedron: Asymmetry 2007, 18, 276.
  • V. Resch, W.M.F. Fabian, W. Kroutil, Adv. Synth. Catal. 2010, 352, 993.

Alkyl Sulfatases

In order to keep things simple, we envisaged to transform both enantiomers of a racemate via independent and stereochemically matching pathways directly into a single stereoisomeric product. In order to meet this goal, the two enantiomers have to be processed with retention and inversion of configuration, respectively. As a consequence, the catalyst(s) required do not only have to be enantio-, but also stereoselective at the same time – a rather difficult task, but a piece of cake for enzymes! Alkyl sulfatases, which can perform this task, are being investigated in this context.

Enantioconvergent enzymatic hydrolysis of sulfate esters:

A group of enzymes that may act through retention and inversion of configuration has been completely overlooked by the majority of our competitors: sulfatases. Since we felt very happy having a whole group of enzymes at our disposal with no competitors around, sulfatases clearly became our most recent pets.

These enzymes catalyse the hydrolysis of sulfate esters and – depending on their biological source – they may act either with inversion – such as sulfatases from Rhodococcus ruber DSM 44541 and Sulfolobus acidocaldarius DSM 639 – or retention of configuration – such as an enzyme from the marine planctomycete Rhodopirellula baltica DSM 10527. In order to keep extremophiles happy – they love temperatures close to 100°C and an awfully acidic pH – we grow them in a conventional drying oven and feed them with yellow sulfur powder.

Graphical Abstract

Further reading:

  • W. Stampfer, B. Kosjek, C. Moitzi, W. Kroutil, K. Faber, Angew. Chem. Int. Ed. Engl. 2002, 41, 1014.
  • W. Stampfer, B. Kosjek, W. Kroutil, K. Faber, Biotechnol. Bioeng. 2003, 81, 865.
  • W. Stampfer, B. Kosjek, K. Faber, W. Kroutil, J. Org. Chem. 2003, 68, 402.
  • W. Stampfer, B. Kosjek, K. Faber, W. Kroutil, Tetrahedron: Asymmetry 2003, 14, 275.
  • B. Kosjek, W. Stampfer, S. M. Glueck, M. Pogorevc, U. Ellmer, S. R. Wallner, M. F. Koegl, T. M. Poessl, S. F. Mayer, B. Ueberbacher, K. Faber, W. Kroutil, J. Mol. Catal. B: Enzym. 2003, 22, 1.
  • B. Kosjek, W. Stampfer, R. van Deursen, K. Faber, W. Kroutil, Tetrahedron 2003, 59, 9517.
  • B. Kosjek, W. Stampfer, M. Pogorevc, W. Goessler, K. Faber, W. Kroutil, Biotechnol. Bioeng. 2004, 86, 55.
  • R. van Deursen, W. Stampfer, K. Edegger, K. Faber, W. Kroutil, J. Mol. Catal. B: Enzym. 2004, 31, 159.

Biocatalytic cascade reactions

Design of artificial metabolism/cascades

By creating an artificial cascade, transformations can be established that are not easily realized by using chemical methodology. For instance, the deracemisation of racemic sec-alcohols was accomplished, yielding a single alcohol enantiomer with up to >99% yield and >99% optical purity in one pot via an enantioselective oxidation and a stereoselective reduction (Angew. Chem. Int. Ed. 2008, 47, 741-745; J. Am. Chem. Soc. 2008, 130, 13969–13972):

Graphical Abstract

By biocatalytic formal reductive amination ketones were transformed in an asymmetric fashion to the corresponding optically pure α-chiral primary amines employing three enzymes (Angew. Chem. Int. Ed. 2008, 47, 9337–9340). With this approach various optically pure amines could be prepared which serve as intermediates for the preparation of bioactive compounds (Org. Lett. 2009, 11, 4810-4812; ChemCatChem 2010, 2, 73-77; Adv. Synth. Catal. 2011, 353, 3227-3233; Eur. J. Org. Chem. 2012, 1003–1007).

Graphical Abstract

For the preparation of a non-natural amino-acid – phenyl glycine – from racemic mandelic acid a redox-neutral metabolism was designed employing three different enzymes. The cascade consisted of three steps: a racemisation, an enantioselective oxidation and a stereoselective reductive amination (Adv. Synth. Catal. 2010, 352, 993–997). It has to be noted that although the metabolism involves two redox-steps, the overall transformation is redox-neutral, since the NADH formed in the oxidation step is consumed in the reductive amination; thus no external redox-reagents are required.

Graphical Abstract

Simultaneously combining redox enzymes in artificial metabolism is in most cases easier than combining a redox-active metal catalyst with a redox enzyme. Nevertheless, for the proof of concept an Ir-catalyst was combined with an alcohol dehydrogenase to achieve deracemization of chlorohydrins. A challenge for the system was the identification of a suitable oxidation agent for the oxidation step which could be combined with the ADH without interference (Chem. Commun. 2010, 46, 8046–8048).

Graphical Abstract

Further reading:

  • J. H. Schrittwieser, J. Sattler, V. Resch, F. G. Mutti, W. Kroutil, Curr. Opin. Chem. Biol 2011, 15, 249.
  • E. Ricca, B. Brucher, J. H. Schrittwieser, Adv. Synth. Catal. 2011, 353;2239.

Enzyme-triggered domino reactions:

Reactions proceeding through more than a single step in a concurrent fashion are generally denoted as domino or cascade reactions. Despite the fact that they may proceed via a highly reactive, unstable intermediate (which often eludes isolation), the final product often can be isolated in good yields. This is due to the fact that decomposition of the intermediate is largely suppressed since it is transformed in the same instant as it appears and is never present in measurable concentrations.

By making use of the stereoselectivity of enzymes, cascade reactions can be rendered asymmetric, if the first reaction step within the cascade is ‘triggered’ by a biocatalyst. Thus, the final product can be obtained in nonracemic form. In an exploratory project we have investigated the possibility to use enzymes for the initiation of asymmetric cascade reactions. In the meantime, we have managed to control a biohydrolysis–rearrangement cascade of haloalkyl- and bis-epoxides. The cascade was triggered by an epoxide hydrolase to furnish the corresponding vic-diol, which spontaneously underwent cyclisation to form an epoxy-alcohol or a tetrahydrofurane in high d.e. and e.e. as the sole product. Depending on the type of substrate, the ‘generation’ of up to four chiral centers could be simultaneously controlled. Traditional methodology can only dream of such highly complex reactions!

Graphical Abstract

Fascinated by the simplicity to generate highly functionalised molecules, we employed this methodology to the chemo-enzymatic asymmetric total synthesis of several bioactive molecules, such as terpenoids and anti-tumor agents, etc. The next goal is the synthesis of the central core of a large group of bioactive agents (termed ‘Annonaceous acetogenins’) from tropical plants.

Further reading:

  • S. F. Mayer, W. Kroutil, K. Faber, Chem. Soc. Rev. 2001, 30, 332–339.
  • S. F. Mayer, A. Steinreiber, R. V. A. Orru, K. Faber, Eur. J. Org. Chem. 2001, 4537–4542.
  • S. F. Mayer, A. Steinreiber, R. V. A. Orru, K. Faber, Tetrahedron: Asymmetry 2001, 12, 41–43.
  • S. F. Mayer, A. Steinreiber, M. Goriup, R. Saf, K. Faber, Tetrahedron: Asymmetry 2002, 13, 523–528.
  • S. F. Mayer, A. Steinreiber, R. V. A. Orru, K. Faber, J. Org. Chem. 2002, 67, 9115–21.
  • S. M. Glueck, W. M. F. Fabian, K. Faber, S. F. Mayer, Chem. Eur. J. 2004, 10, 3467–78.

Alkene C=C-cleavage via bio-ozonisation

A large number of redox-reactions in organic chemistry are still based on (heavy) metals, many of which are (i) expensive, (ii) toxic, (iii) environmentally inacceptable (in particular when required in molar amounts) and (iv) difficult to remove from the product(s). As a consequence, any metal-independent method to perform redox-reactions would present a viable alternative.

The development of “green” chemical oxidation processes belongs to the burning hot topics in organic chemistry. Ozonisation of C=C double bonds giving access to carbonyl compounds (under reductive conditions) is one of the most used oxidation methods for C=C cleavage. However, safety hazards and the need for special equipment as well as reducing reagents in molar amounts complicate this reaction. Other C=C cleaving protocols require stoichiometric amounts of oxidants such as metal-based reagents (e.g. NaIO4, Cr-, Ru-salts). By serendipity we identified a simple biocatalytic alternative employing a cell extract from Trametes sp. in buffer and consumption of molecular oxygen without production of waste.

Graphical Abstract

A broad variety of substituted styrene derivatives can be cleaved to the corresponding aldehyde or ketone, depending on the substitution pattern. The method does not require specialized equipment (ozonizer) or chemicals (reducing agents, oxidizing salts). It requires the most innocuous oxidant, namely oxygen. The highest possible achievable atom efficiency for alkene cleavage can only be reached by using molecular oxygen, which was achieved by the presented biocatalytic protocol. We have demonstrated that the repertoire of biocatalytic reactions applied for organic synthesis can be extended by the described biocatalytic alkene cleavage.

Further reading:

  • H. Mang, J. Gross, M. Lara, C. Goessler, G. M. Guebitz, W. Kroutil, Angew. Chem., Int. Ed. 2006, 45, 5201–5203.
  • H. Mang, J. Gross, M. Lara, C. Goessler, H. E. Schoemaker, G. M. Guebitz, W. Kroutil, Tetrahedron 2007, 63, 3350–3354.
  • M. Lara, F. G. Mutti, S. M. Glueck, W. Kroutil, Eur. J. Org. Chem. 2008, 3668–3672.
  • M. Lara, F. G. Mutti, S. M. Glueck, W. Kroutil, J. Am. Chem. Soc. 2009, 131, 5368–5369.
  • C. E. Paul, A. Rajagopalan, I. Lavandera, V. Gotor-Fernández, W. Kroutil, V. Gotor, Chem. Commun. 2012, 48, 3303.

Asymmetric conjugate reduction of activated C=C bonds

The asymmetric bioreduction of alkenes bearing an electron-withdrawing group leads to the creation of up to two chiral carbon centers. The enzymes responsible for this useful transformation are flavoproteins from the ‘old-yellow-enzyme family’ (often denoted as enoate- or ene-reductases), which ultimately derive their reduction equivalents from of NAD(P)H.

Although this biotransformation has been known for decades, its practical use has been impeded by the requirement of whole microbial cells, most prominent baker's yeast: Although it is simple to use, it generally gives lousy yields, variable e.e.'s and a messy workup. On the other hand, anaerobic bacteria, such as Clostridium spp. from the intestine of ruminants were no attractive alternative, since their enoate reductases are extremely sensitive towards traces of O2. It is therefore not surprising, that the number of practical-scale applications of these enzymes is extremely limited.

Graphical Abstract

In search for a suitable alternative, we turned our attention to ene-reductases from plants and bacteria: Three extremely promising candidates were identified with the help of our friends from the biochemistry department: YqjM from Bacillus subtilis and isoenzymes OPR1 and OPR3 from tomato (don't worry about the unspeakable term YqjM, the letters have the same meaning as those of a car's licence plate: practically none). These three candidates turned out to be a gold mine: they are not fuzzy about the cofactor by taking cheap NADH about equally well as the more expensive NADPH, and they tolerate a large variety of electron-withdrawing (activating) groups, such as aldehyde, ketone, carboxylic acid & ester, imide, nitro and (we presume) a lot more. Since these enzymes are overall extremely stereoselective, tables of substrate-selectivitiy data are usually boring to read with a lot of ‘ee >99%’-entries. However, this is very much appraciated by our industrial partners from BASF AG, who finance this project.

Further reading:

  • R. Stuermer, B. Hauer, M. Hall, K. Faber, Curr. Opin. Chem. Biol. 2007, 11, 203.
  • M. Hall, C. Stueckler, W. Kroutil, P. Macheroux, K. Faber, Angew. Chem. Int. Ed. 2007, 46, 3934.
  • N. J. Mueller, C. Stueckler, B. Hauer, N. Baudendistel, H. Housden, N. C. Bruce, K. Faber, Adv. Synth. Catal. 2010, 352, 387.
  • C. Stueckler, N. J. Mueller, C. K. Winkler, S. M. Glueck, K. Gruber, G. Steinkellner, K. Faber, J. Chem. Soc., Dalton Trans. 2010, 39, 8472.
  • C. Stueckler, C. K. Winkler, M. Bonnekessel, K. Faber, Adv. Synth. Catal. 2010, 352, 2663.
  • C. K. Winkler, C. Stueckler, N. J. Mueller, D. Pressnitz, K. Faber, Eur. J. Org. Chem. 2010, 6354.
  • K. Tauber, M. Hall, W. Kroutil, W. M. F. Fabian, K. Faber, S. M. Glueck, Biotechnol. Bioeng. 2011, 108, 1462.
  • C. Stueckler, C. K. Winkler, M. Hall, B. Hauer, M. Bonnekessel, K. Zangger, K. Faber, Adv. Synth. Catal. 2011, 353, 1169.
  • G. Oberdorfer, G. Steinkellner, C. Stueckler, K. Faber, K. Gruber, ChemCatChem 2011, 3, 1562.

Asymmetric amination employing ω-transaminases

ω-Transaminases employ pyridoxal-5'-phosphate (PLP) as cofactor to transfer formally ammonia and electrons from an amino donor to a ketone acceptor. In contrast to α-amino acid aminotransferases, ω-transaminases possess the capability to convert also substrates lacking an α-carboxylic moiety, making them therefore very attractive for organic synthesis. The advantage of using ω-transaminases compared to chemical methods (i.e., Rh-catalysed hydrogenation of enamines) stems from the milder reaction conditions (i.e., atmospheric pressure, ambient temperature), high enantioselectivity, broad substrate specificity and the absence of heavy metal contaminants.

In our group, we are employing these enzymes to carry out a formal asymmetric reductive amination of prochiral ketones, which leads theoretically to quantitative conversion. However, the equilibrium for the amination lies on the side of the ketone when using alanine as the amine donor (Keq 10–4 – 10–5 for acetophenone). Thus, an excess of alanine is required (5 eq.) and simultaneously the co-product pyruvate is removed employing either LDH or AlaDH. Continuing our studies, various ketones were converted to chiral amines with excellent conversion and enantioselectivity (>99% ee) using (R)- or (S)-selective ω-transaminases of known amino acid sequence. Furthermore, a variant originated from Arthrobacter sp. was employed using an excess of 2-propylamine as amino donor to convert more sterically hindered substrates.

The obtained optically pure amines are precursors of a plethora of bioactive compounds, especially for pharmaceutical and agrochemical applications. For instance, amines (R)-2a, (R)-2b, (R)-2c are building blocks for dilevalol, (R,R)-formoterol and (R)-mexiletine, respectively. (S)-2e is also precursor of the herbicides (S)-metolachlor (Dual) and (S)-dimethenamide (Outlook).

Graphical Abstract

Further reading:

  • 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, 1003.
  • F. G. Mutti, C. S. Fuchs, D. Pressnitz, J. H. Sattler,W. Kroutil, Adv. Synth. Catal. 2011, 353, 3227.
  • F. G. Mutti, J. Sattler, K. Tauber,W. Kroutil. ChemCatChem 2011, 3, 109.
  • M. Fuchs, D. Koszelewski, K. Tauber, W. Kroutil, K. Faber, Chem. Commun. 2010, 5500.
  • D. Koszelewski, M. Göritzer, D. Clay, B. Seisser, W. Kroutil. ChemCatChem 2010, 2, 73.
  • D. Koszelewski, I. Lavandera, D. Clay, G. M. Guebitz, D. Rozzell, W. Kroutil. Angew. Chem. Int. Ed: 2008, 47, 9337.
  • 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.
  • D. Koszelewski, I. Lavandera, D. Clay, D. Rozzell, W. Kroutil. Adv. Synth. Catal. 2008, 350, 2761.

Computational modeling of biocatalytic reactions

Many biocatalytic reactions are proceeding via more than one step, and the kinetics of these processes are usually a nightmare for the organic chemist – just imagine that the product from step 1 constitutes the substrate for step 2. In order to facilitate the understanding of complex kinetics, we have developed several computer programs which have a nice and user-friendly surface while all the dry and boring math is hidden behind the screen. All programs are available free of charge from our ftp-server for both Windows and MacOS.

Graphical Abstract

Selectivity:

This program is used for the calculation of the selectivity of kinetic resolutions – expressed as the Enantiomeric Ratio (E).
Download at ftp://biocatalysis.uni-graz.at or use the online tool.

Selectivity-KReSH

In case you observe some undesired spontaneous background reaction in a kinetic resolution, which is messing up your selectivity, you can calculate the maximum of selectivity, which you would obtain, if you could suppress the background reaction.
Download at ftp://biocatalysis.uni-graz.at

SeKiRe

Two types of sequential reactions of a bifunctional starting material, i.e. (i) asymmetrization followed by kinetic resolution, and (ii) sequential kinetic resolution can be modelled and analyzed by using this program.
Download at ftp://biocatalysis.uni-graz.at

Cyclo

Cyclic deracemization of compounds possessing a sec-alcohol or sec-amino group proceeding via a cyclic sequential oxidation-reduction process can be dealt with this program.
Download at ftp://biocatalysis.uni-graz.at

Further reading:

  • W. Kroutil, A. Kleewein, K. Faber, Tetrahedron: Asymmetry 1997, 8, 3251.
  • W. Kroutil, A. Kleewein, K. Faber, Tetrahedron: Asymmetry 1997, 8, 3263.
  • W. Kroutil, K. Faber, Tetrahedron: Asymmetry 1998, 9, 2901.

Enzymatic carboxylation

In order to alleviate the predominant dependence of the chemical industry on petroleum-based platform intermediates, the development of CO2-fixation reactions represents a major challenge in synthetic organic chemistry, which would allow to convert a problematic waste gas into a useful carbon source for the production of chemicals. Since Nature is fixing CO2 on large scale, we are trying to copy some tricks for the synthesis of carboxylic acids. Several benzoic acid decarboxylases were able to catalyze the reverse carboxylation of phenols in carbonate buffer with high regioselectivity. Surprisingly, carboxylation of the terminal vinyl moiety of hydroxystyrenes yielding cinnamic acids was accomplished by phenolic acid decarboxylases. For the latter transformation no chemical counterpart is known.

Graphical Abstract

For further reading see:

  • S. M. Glueck, S. Gümüs, W. M. F. Fabian, K. Faber Chem. Soc. Rev. 2010, 39, 313.

Cyclases in biosynthesis

The biosynthesis of complex natural products is not carried out in a stepwise fashion, but through enzyme-initiated cascade processes. Analysis of the underlying principles revealed that cyclic terpenoids are generally obtained by electrophilic cascades (via intermediate carbenium ions), whereas cyclic polyethers are formed by nucleophilic cascade reactions from (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. Although the essential role of (cyclase) 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.

Graphical Abstract

Further reading:

  • B. T. Ueberbacher, M. Hall, K. Faber, Nat. Prod. Rep. 2012, 29, 337.

C–C bond hydrolases in organic synthesis

Although hydrolases cleaving carbon-heteroatom bonds have been successfully used as biocatalysts in asymmetric reactions for a long time, the potential applications of C–C-bond hydrolases in biotransformations have not been widely investigated. C–C-bond hydrolases are enzymes capable of catalysing the hydrolytic cleavage of selected ketonic substances, such as β-diketones (retro-Claisen reaction).

Graphical Abstract

6-Oxocamphor hydrolases (OCH) are examples for β-diketone hydrolases, and members of the crotonase superfamily. These enzymes have been applied to enantioselective biotransformations for desymmetrization of prochiral β-diketones.2 To further investigate the applicability of the C–C bond hydrolases in organic synthesis, we are collaborating with the group of Gideon Grogan (University of York).

Our study on OCH enzymes represents the first investigation of organic solvent stability for members of the crotonase superfamily, with a view to their process suitability. We are currently exploiting the organic solvent stability of these interesting biocatalysts in further organic syntheses.

Further reading:

  • G. Grogan, J. Graf, A. Jones, S. Parsons, N. J. Turner, S. L. Flitsch, Angew. Chem. Int. Ed. 2001, 40, 1111.
  • C. L. Hill, L. C. Hung, D. J. Smith, C. S. Verma, G. Grogan, Adv. Synth. Catal., 2007, 349, 1353.
  • C. L. Hill, C. S. Verma, G. Grogan, Adv. Synth. Catal., 2007, 349, 916.
  • E. Siirola, B. Grischek, D. Clay, A. Frank, G. Grogan, W. Kroutil, Biotechnol. Bioeng., 2011, 108, 2815.