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Biotechnology Enzymatic C-C coupling in the s Dr Pascal Dünkelmann of Evocatal and Professor Michael Müller of the University of Freiburg advocate biocatalytic C-C bond formation and cleavage to produce enantiopure hydroxy-ketones effiiciently Another example is the application of hydroxynitrile lyases that catalyse the formation of hydrogen cyanide in plants as a protection against herbivores. In synthetic chemistry, these catalysts can be used to form the useful hydroxynitriles in an asymmetric manner, such as in the formation of (R)-mandelic acid using an enzyme derived from almonds. A family of enzymes that has underestimated potential, in terms of technical applications is the thiamine pyrophosphate (TPP)-dependant enzymes.2 These enzymes exhibit a highly interesting synthetic potential They catalyse the formation or cleavage of CC bonds starting from carbonyl compounds. An activated aldehyde intermediate, generated by adding the carbonyl compound to the TPP molecule, represents the activated species in these conversions. (Figure 1) any examples of where enzymes or whole cells are applied efficiently to produce high value chemicals have been introduced in the past decade.1 Among these, hydrolases are the most prominent. These enzymes cleave or form ester or amide bonds by catalysing a saponification or the corresponding back reaction. One example is the racemic resolution of amino acids like methionine to the desired enantiopure L-compounds. Another class of enzymes that can be found regularly in commercialised processes are the so-called oxidoreductases, enzymes that perform oxidation or reduction reactions. A well-known example is Eli Lilly’s whole-cell transformation to generate (S)-(3,4-methylenedioxyphenyl)-2-propanol, the key intermediate in the synthesis of 3,4-methylendioxy-N-methylamphetamine (Tenamfetamine). Various industrial applications have also been established for other enzyme classes. In many examples, stereoselectivity plays an outstanding role. However, in most of these reactions, bonds between carbon and hetero-atoms are formed, cleaved or modified. Even though enzymes are already known to catalyse C-C bond formations, there are only a few examples of their industrial application. Enzymatic aldol reactions catalysed by the enzyme 2-deoxyribose-5phosphate-aldolase (DERA) should be mentioned in this context. M Formation of α-hydroxy-ketones The usefulness of thiazolium-catalysts for C-C-bondforming reactions via ‘Umpolung’ was described by Ugai et al. in a Japanese journal back in 1943.3 Since then, a broad variety of different synthetic applications of thiazolium- or derived catalysts have been shown, in many cases as alternatives to known hydrogen cyanide-catalysed ‘Umpolung’ reactions. In 1966, Shehan and Hunneman introduced the first asymmetric variant of the benzoin condensation. Then, in the 1970s, Stetter et al. published intensive OH CH3 O CH3 O R1 R2 N+ (R)-Phenylacetylcarbinol (PAC) O Pyruvate S (Donor) H R1 CH3 Thiamine pyrophosphate (TPP) CH3 R1 R2 N+ R2 N+ S S HO O- H3C HO CH3 CH3 O- O- O Ph O H Benzaldehyde R1 R2 N+ R1 R2 N S S (Acceptor) CO2 CH3 CH3 HO HO C- CH3 CH3 Activated aldehyde Note: Shown as the formation of (R)-PAC starting from benzaldehyde & pyruvate Figure 1 - Catalytic Cycle of thiamine pyrophosphate-dependant enzymes shown 16 studies on the condensation of aldehydes with α,βunsaturated carbonyl species, the so-called Stetter reactions. During the last two decades, interest in this kind of transformation grew further. In this context, the transformations catalysed by triazolium compounds and the investigations by Knight and Leeper who introduced novel bicyclic thiazolium salts for asymmetric CC bond formations, should be mentioned. Many efforts have also been made to use such catalysts in the synthesis of pharmaceutical compounds. One example is the application of a thiazolium derivative catalysing a Stetter reaction, leading to a key intermediate in the synthesis of atorvastatin.4 What is mostly overlooked in this context is that all these efforts only mimic what nature does very well, after millions of years of evolution. Although the structure of TPP, the natural equivalent to the above mentioned heterocyclic catalysts, was not elucidated before 1937, a biotechnological process applying the synthetic principle, in which pyruvate derived from the central metabolism of Saccharomyces cerevisiae and benzaldehyde are coupled to give (R)-phenyl-acetylcarbinol, a precursor in the synthesis of Ephedrine, had already been established in 1921. Figure 1 depicts the catalytic cycle of this reaction. The first step is the addition of pyruvate, acting as the donor, to the catalyst TPP. After the elimination of CO2, an enamine complex, the so-called activated aldehyde, is formed. To this, the second aldehyde, the acceptor, is bound. Afterwards, the final 2-hydroxy ketone is released. This reaction was established as a whole-cell transformation using baker’s yeast. It is still in use and represents the only commercialised process applying a TPP-dependent enzyme. Enzymes that have already been studied intensively because of their high synthetic potential include various pyruvate decarboxylases from bacteria or yeasts, benzoylformate decarboxylase from the microorganism Pseudomonas putida and the branched-chain 2keto acid decarboxylase from Lactococcus lactis. These proteins catalyse the non-oxidative decarboxylation of 2-keto acids to aldehydes as a main reaction. The benzoin condensation-like C-C bond formation occurs as a side reaction. It is important to mention here that these enzymes are able to accept aldehydes as donors directly. This is, of course, beneficial, as the aldehydes are much cheaper and more available than the corresponding 2-oxo-acids. Typical products that result from these conversions are aliphatic and aromatic 2-hydroxy-ketones. Among these are 2-hydroxy-1-phenylpropanone (HPP) and phenyl-acetylcarbinol (PAC). While the (S)-enantiomer of HPP represents a key intermediate for the API bupropione, the (R)-enantiomer of PAC can efficiently be used in the synthesis of ephedrine, as described above. November 2011 Speciality Chemicals Magazine www.specchemonline.com Biotechnology synthesis of fine chemicals CH3 CH3 R R O R' O OH OH CH3 CH3 H3C OH R R' Enzyme-catalysed Stetter reaction O CH3 OH However, attempts to solve this problem by changing the structure of the enzyme, as well as by identifying homologues, are under investigation. In this context, the elucidation of the three-dimensional structure of YerE, which is not yet known, will be important. O O OH CH3 OH R O O R O O R CH3 H + H O R + O TPP CH3 OH O OH OH O CH3 R CH3 O O CH3 CH3 CH3 H3C O OH OH O OH CH3 R Figure 2 - Chiral 2-hydroxy ketones accessible via TPP-catalysed syntheses For other TPP-dependent enzymes the C-C bond formation and cleavage represents the main reaction. Enzymes that have been studied in this context are, for example, transketolases which form D-seduheptulose7-phosphate by transferring 2-hydroxy-acetyl-residue to D-ribose-5-phosphate and benzaldehyde lyase isolated from Pseudomomas fluorescens. The latter catalyses the reversible cleavage of one molecule of (R)benzoin into two molecules of benzaldehyde. From a synthetic standpoint, it is more interesting that this enzyme also catalyses the C-C bond formation highly efficiently. Not only (R)-benzoin but also a vast variety of benzoin derivatives, including mixed benzoins, plus (R)-HPP derivatives and aliphatic 2hydroxy ketones are accessible using this enzyme. In addition to the (R)-enantiomer, the (S)-enantiomers are also available from these reactions, by resolution of the racemic compounds. In this case, the enantiomerically pure (S)-compounds remain untouched in the reaction while the (R)-enantiomer is cleaved into the corresponding aldehydes. Not surprisingly for enzymatic conversions the described ligase and lyase reactions are, in most cases, highly stereoselective. The number of compounds that can be synthesised in such reactions is constantly increasing. This is due to the fact that in many cases the enzymes do not only catalyse the conversion of their natural substrate but also that of a broad variety of compounds with similar functionality. In addition, scientists also take advantage of the fact that more and more variants of these enzymes are becoming available. Figure 2 shows various chiral compounds that can be obtained by applying the described TPP-dependant enzymes. Synthetic route to tertiary alcohols One TPP-dependent enzyme with a tremendous synthetic potential that has been studied in the recent past, is a lyase that was isolated from Yersinia pseudotuberculosis.5 The application of this protein allows the synthesis of tertiary alcohols in an asymmetric manner, by catalysing the C-C bond formation between a ketone and pyruvate or acetaldehyde, whereas the synthesis of such compounds by classical organo-synthetic means is difficult to achieve. A broad variety of carbonyl compounds were accepted as acceptor in this conversion. Among these acceptors are small cyclic aliphatic compounds like cyclohexanone derivatives, open chain ketones, 1,2diketones as well as α- and β-keto esters (Figure 3). The tertiary alcohols obtained in these reactions represent valuable building blocks for the asymmetric synthesis of 1,2-diols vicinal amino alcohols or two contiguous tertiary alcohols. Unfortunately, the ee’s that can be achieved in these reactions are not yet satisfactory. O O Although the TPP-dependent enzymes catalyse the stereoselective formation or cleavage of α-hydroxyketones, this class of enzymes is not limited to these transformations. Recent studies have revealed that the Stetter reaction also can be carried out biocatalytically by using the appropriate enzyme-catalyst. In the 1970s, Stetter and others showed that 1,4diketones are accessible by coupling an aldehyde to an α,β-unsaturated ketone. Cyanide can be used in this reaction as a catalyst, as can various thiazolium or triazolium salts. Different studies have been published that dealt with stereoselective variants of this reaction. However, whilst some major improvements have been achieved with regard to the conversion and yield, the enantioselectivity is still a difficult issue. Enzymes can be efficient alternatives in this respect. It can be shown that various enzymatically catalysed couplings, between α,β-unsaturated ketones and mainly pyruvates, are catalysed with high enantioselectivity by applying the enzyme PigD (Figure 4).6 This enzyme was isolated from the microorganism Serratia marcescens and is involved in the biosynthesis of the red pigment prodigiosin. It is important to note that the formation of 1,2adducts, with ketones as acceptor substrates was not observed, which nicely complements the aforementioned YerE-catalysed transformations. PigD catalyses only the 1,4-addition. Interestingly, not only aliphatic but also aromatic and some heterocyclic α,β-unsaturated ketones selectively reacted with pyruvate in the presence of PigD. Larger substituents, even on both sides of the carbonyl group, did not sterically affect the Stetter reaction. Pyruvate is the preferred donor substrate, but 2-oxobutanoate could also be employed. The yields of isolated products are not yet satisfactory, but one should be aware that all ketone compounds are non-physiological substrates of the wild-type enzyme PigD. O HO + CH3 H CH3 CH3 O YerE, TPP CH3 O Acceptor Examples of additional acceptors: O Donor Conversion after 25 hours: 20 % ee = 96% O O O O H3C CH3 O O O CH3 O Figure 3 - Examples of the synthesis of chiral tertiary alcohols accessible via TPP-catalysed reactions Speciality Chemicals Magazine November 2011 17 www.specchemonline.com Biotechnology O O H3C CH3 + H3C O O - - CO2 H3C O References: O PigD, TPP H3C 1. C. Wandrey, A. Liese & D. Kihumbu, 2000 Org. Proc. Res. Dev. 2000, 4, 286-290; M. Hall & A. S. Bommarius, Chem. Rev. 2011, 111, 4088-4110 2. M. Müller, D. Gocke & M. Pohl, FEBS 2009, 276, 28942904; D. Gocke, T. Graf, H. Brosi, I. Frindi-Wosch, L. Walter, M. Müller & M. Pohl, J. Mol. Catal. B: Enzymatic 2009, 61, 30-35; P. Domínguez de María, M. Pohl, D. Gocke, H. Gröger, H. Trauthwein, T. Stillger, L. Walter & M. Müller, Eur. J. Org. Chem. 2007, 2940-2944; P. Hoyos, J.-V. Sinisterra, F. Molinari, A. Alcantara & P. Dominguez de Maria, Acc. Chem. Res. 2010, 43, 288299 3. T. Ugai, R. Tanaka & T. Dokawa, J. Pharm. Soc. Jpn. 1943, 63, 296 4. K. L. Baumann, D.E. Butler, C. F. Deering, K.E. Mennen, A. Millar, T.N. Nanninga, C.W. Palmer & B.D. Roth, Tetrahedron Lett. 1992, 33, 2283-2284 5. P. Lehwald, M. Richter, C. Röhr, H.-W. Liu & M. Müller, Angew. Chem. 2010, 122, 2439 -2442 6. C. Dresen, M. Richter, M. Pohl, S. Lüdeke & M. Müller, Angew. Chem. 2010, 122, 6750 -6753 CH3 Yield: 38 % ee > 99 % Examples of additional acceptors: O O H2C O CH3 O CH3 CH3 O OH Figure 4 - Examples of asymmetric TPP-catalysed Stetter reactions Catalyst optimisation be optimised, for instance, for pH- and temperaturestability and solvent tolerance, and also for regio- or stereoselectivity. The common way to identify the appropriate enzyme for a conversion, carried out by enzyme optimisation specialists is to screen enzyme libraries consisting of variants generated by molecular biology mutation techniques. The enzyme variant showing the best performance might be used as the catalyst or might undergo additional mutation rounds until sufficient performance is obtained. Various enzyme classes, especially those mentioned in the introduction, are also commercially available as ready-to-use test kits allowing every scientist to study the viability of enzyme catalysis for the conversion of interest. The TPP-dependant enzymes have not been commercially available so far. Evocatal recently launched a screening kit with a selection of these enzymes so that they are now easily accessible for academic and industrial research. The increasing acceptance of biocatalysis as a tool in organic synthesis which is, of course, growing with every commercialised process, has also resulted in a strong stimulation of different kinds of investigation into how to make enzymes available for any application that goes beyond proof-of-principle. It is, nowadays, state of the art to produce biocatalysts above in special recombinant production organisms like Eschericha, Bacillus or Pichia strains. Such organisms in combination with special microbiological protein-production tools, allow the production of the desired protein at an almost unlimited scale. As a result, the costs for these catalysts can be reduced below those of the classical non-enzymatic chiral catalysts. Moreover, due to the increasing number and improving capabilities of evolution techniques, enzymes can be adapted perfectly to the requirements of the needed conversion. The catalyst might OH OHOHO OH OH OH O OH O OH OH O OH OH OH OH O OH OH OH O OH OH O OH O OH OH O O OH O OH OH O OH Cl OH O OH O O OH OH O OH OH OH O OH O OH O OH O OH O OH OH OH OH OOH OH OH O OH O O OH OH O OH Cl OH O OH OH OH OH OH OH O OH O O O OH OH O O O OH OH O OH OH O O O OH OH O OH Cl OH Cl O O OH OH O OH OH OH O OH OH OH O OH OHOHO OH O O OH O OH OH O Cl OH OHOH O OH OHO O OH Cl OH OH O OH O O OH OH O OH Cl O OH O OH OH O OH OH OH OH OH O Cl O OH OH Cl O O OH O O OH OH O OH O Cl O OH O OH O OH O OH OH O OH O OH O OH O OH O OH OH OH O OH O O OH O OH Cl OH OH O OH O OH O OH OH OH OH O Cl OH Cl OH OH OH OH OH OH OH O OH OH O OH O O OH O Cl O evocatal – the art of chiral purity evocatal develops and produces high performance biocatalysts for the chemical and pharmaceutical industries. Our customers gZ\VgYjhVhVegZb^jbegdk^YZgd[]^\]fjVa^in!ÂcZX]^gVaWj^aY" ing blocks: from a few grams to several tons. If you are interested in our products, feel free to visit our website and download our catalogue. g t www.evocatal.com · +49 211 1576095-0 O OH OH O O OH OH OH O O OH OH OH OH OH O O OH O OOH OH OH OH O OH OH Cl OH O OH O OH OH O OH OH O O OH O OH OH OH OH OO O OH OH O OH O OH O OH OH OO OH Cl OH O OH OH OH O OH OH O OH O OH OH O OH OH O OH O OH OH O OH O OH O O OH O OH Cl For more information, please contact: Dr Pascal Dünkelmann Director of Commercialisation Evocatal GmbH Merowingerplatz 1a D-40225 Düsseldorf Germany Tel: +49 211 157 6095-0 E-mail: [email protected] Website: www.evocatal.com