10 Enzyme Chemistry By A. G.SUTHERLAND School of Applied Chemistry University of North London London N7 $08,UK 1 Introduction As some biotransformation methods particularly the kinetic resolution of carboxylate esters by hydrolysis or condensation are increasingly thought of as a standard weapon in a synthetic chemist’s armoury there has been a marked tendency for specialists in the enzyme chemistry field to move on to new areas. Thus the past year has seen an increase in the investigation of areas such as amide and epoxide hydrolysis and the use of oxidoreductase systems in resoIution processes. Accordingly in line with the trend of last year,’ this report will deal in greater detail with these emerging methods rather than attempting to reflect the relative volume of papers published on each of the topics below.The rate of publication on enzymes in synthesis has now made the publication of a full review of the subject a virtual impossibility but a Iarge symposium-in-print goes much of the way to delineating the state of the art.’ A review of the use of enzymes in the synthesis ofchiral drugs provides a different ~iewpoint.~ Two doyens of the field have written perspectives on their recent work thus Jones has discussed his seminal research on the prediction and control of enzyme ~pecificity,~ while Wong has summarized his syntheses of aza sugars using several enzyme systems.’ 2 Hydrolysis and Condensation Reactions Alcohols Carboxylic Acids and Esters-The resolution of racemic typically second- ary alcohols (or their esters) by acylation (or by hydrolysis or transesterification) forms the bulk of the current literature on biotransformations and as a relatively routine technique will only be covered briefly here.The transesterification methods have been reviewed6 and compared with similar chemical methods.’ Secondary alcohol resolutions are at their most powerful when used to resolve small highly functionalized and readily available starting materials such as the buten01 ’ A.G. Sutherland Ann. Rep. Prog. Chem. Sect. 3,Org. Chem. 1992 89 281. Tetruhedron:Asymmetry 1993,4 757-1396. A. L. Margolin Enzyme Microb. Technol.,1993 15 266. J. B. Jones Can. J. Chem. 1993 71 1273. G.C. Look C.H.Fotsch and C.-H. Wong Acc. Chem. Res. 1993,24 182. E.Santaniello P. Ferraboschi and P. Grisenti Enzyme Micrab. Technol. 1993 15 367. ’ .I.Otera Chem. Reo. 1993 93 f449. 299 A. G. Sutherland 0 0 (1) 32% 98%e.e. 40%,98% e.e. (2) > 95% e.e. > 95% e.e. (40% conversion) (60% conversion) Reagents i Pseudomonas sp. lipase toluene H,O; ii Pseuodomonas puorescens lipase vinyl acetate Scheme 1 derivative or the lactone (2)9 (Scheme 11 the last of which has been used to synthesize the anti-HIV carbocyclic nucleoside precursor carbovirg and also the key hydroxy-lactone moiety of the HMG CoA reductase inhibitors based on compactin." Other notable and extremely efficient resolutions include that of a general inositol precursor' and the chiral auxiIiary trans-2-(cc-cumyl )cyclohexanoI.' * Dasrhadi and O'Hagan have explored the resolution of diarylmethanols and found that the extent of discrimination between relatively similar rings @.g.2,5-difluorophenyI versus phenyl) can be surprisingly high.I3 The past year has seen more examples of successful resolutions of tertiary alcohols coming to light. Thus hydrolysis at the more hindered carbonyl of the oxalate (3) gave the resulting norbornanol in good enantiomeric excess (Scheme 2),14 while the enantiomers of bridgehead alcohols in a seriesof c4.1.O]heptanes have been resolved by hydrolysis of the corresponding chloroacetates.' OH 0 0 42%,90% e.e. 338 88% e.e. (3) Reagents i porcine pancreatic lipase Bu'OH H,O Scheme 2 8 M. Bazinger G.J. Griffiths and J. F. McGarrity Tetrahedron Asymmetry 1993,4 723.9 R. A. MacKeith R. McCague H.F. Olivo C. F. Palmer and S. M. Roberts J. Chem. Soc. Perkin Trans. 1 1993,313. 10 R. McCague H. F. Olivo and S. M. Roberts Tetrahedron Lett. 1993,34 3785. I1 L. Ling and S. Ozaki Tetrahedron Lett. 1993 34 2501. 12 D. L. Cornins and J. M. Salvador Tetrahedron Lett. 1993,34 801. I3 L. Dasradhi and ID. O'Hagan Bioorg. Med. Chem. Lett. 1993,3 1655. I4 I. Brackenridge,R. McCague S. M. Roberts and N. J. Turner J. Chem.Soc. Perkin Trans. I 1993 1093. 15 J. P. Barnier L. Blanco G. Rousseau E. Guibe-Jampel and I. Fresse J. Urg. Chem. 1993 58 1570. Enzyme Chemistry 301 The desymmetrization of mew diols (the 'meso trick') by lipase-catalyzed acylation remains a popular approa~h.'~.~' This technique has been extended by Uguen and coworkers to a tetra01 (4) with two-fold meso symmetry.'* Acylation gave a mixture of C,-symmetrica1 and meso diacetates (Scheme 31 which after conversion into the corresponding bis(phenylsulfides) were separated to give the chiraI product (5)as one enantiomer.An elegant exploitation of the meso trick in combination with a kinetic resolution has been used to deracemize and dediastereoisomerize the mixture of isomers of hexan-2,4-diol (Scheme 3)'' It was shown that acylation of these substrates only occurs at the S alcohol positions so that Mitsonobu inversion of the crude mixture followed by hydrolysis gives only the S,S enantiomer. 78% combined (5) 73% 8% t iii,vi v JJY OH OH > 98%e.e. > 98%d.e.66%,> 98%e.e. > 72% d.e. Reagents i Pseudomonas fluorescens lipase vinyl acetate; ii PhSSPh PBu, CH,CI,; iii Pseudomonas sp. lipase vinyl acetate Bu'OMe; iv DEAD p-nitrobenzoic acid PPh,; v LiOH; vi separation Scheme 3 l6 S. Takano M. Moriya Y. Higashi and K. Ogasawara,J. Chem. Soc. Chem. Commun. 1993 177. " N. Toyooka A. Nishino and T. Mornose Tetrahedron Lett. 1993,34 4539. P. BreuiIles T. Schmittberger and D. Uguen TetrahedronLett.,1993 34,4205. '' M.-J. Kim and I.S. Lee SYNLETT 1993 767. A. G. Sutherlaand Over recent years lipases have become popular as catalysts for regioselective ester hydrolysis (or condensation) and further examples of this use in the carbohydrate2’.*’ and phosph~lipid~’*~~ fields continue to be reported.This methodology has now been extended to poIyhydroxybenz~pyran,~~ and other aromaticz6 ben~opyranone,~~ systems with considerable success. The kinetic resolution of racemic carboxylic acids largely by hydrolysisof a simple alkyI ester also maintains its popularity -notably in the resolution of non-natural amino a~ids.’~.~’ A particularIy topical resolution is the transesterification of methyf trans-8-phenylglycidate under Mucor miehei lipase catalysis. Both enantiornerically pure products can be converted enantioconvergently into the taxol C-13 side chain (6) (Scheme 4).29 Reagents i Mucor miehei lipase Bu’OH hexane Scheme 4 A striking example of the use of the meso trick in the context of complex carboxylic acids has been provided by the direct enantioselective synthesis of the calcium antagonist (7) (Scheme 5).30 Pig liver esterase has been shown to perform diastereoselective hydrolyses of arylidene and dialkylidene malonate diester~.~’~~’ In the examples studied reaction tended to occur at the 2 position ester with moderate selectivity e.g.(8) (R’ = n-2a G. B. Oguntimein H. Erdrnann and R.D. Schmid Biotechnol. Lett. 1993 IS,175. 2* R. Lopez C. Perez A. Fernandez-Mayoralas and S. Conde J. Carbobydr. Chem. 1993 12 165. 22 G. Lin F.-C. Wu and S.-H. Liu Tetrahedron Lett. 1993 34 1959. 23 T. Morimoto N. Murakami A. Nagatsu and 3. Sakakibara Tetrahedron Lett. 1993,34 2487. 24 D. Lambusta G. Nicolosi A. Patti and M. Piattelli Synthesis 1993 1155. ” V. S. Parmar A. K. Prasad N.K. Sharma A.Vardhan H. N. Pati S. K. Sharma and K.S Bisht J. Chem. SOC.,Chem. Commun. 1993,27. 26 G. NicoIosi M. Piattelli and C. Sanfilippo Tetrahedron 1993 49 3143. 27 B. NieIsen H. Fisker B. Bjarke U. Madsen D. R. Curtis P. Krogsgaard-Larsen and J. J. Hansen Bioorg. Med. Chem. Lett. 1993 3 107. B. Imperiali T.J. Prins and S. L. Fisher 1. Org. Chern. 1993 58 1613. 29 Gou,Y.-C. Liu and C. S. Chen. J. Org. Chem. 1993 58 1287. D.-M. 30 T. Adachi M. Ishii Y. Ohta T. Ota T. Ogawa and K. Hanada Tetrahedron Asymmetry 1993,4,2041. 31 T. Schirmeister and H.-H. Otto J. Org. Chem. 1993 58 4819. 32 T. Schirmeister and H.-H. Otto Angew. Chem. fnt. Ed. Engl. 1993,32 572. Enzyme Chemistry ('7) 7745,> 99% e.e. Reagents i Aspergillus metieus protease 3-nitrooxypropan- 1-01 H,O Scheme 5 C,H 1 R2= CH3) 35% de,31to complete selectivity e.g.(8) (R'= Ph R2= H) and (9).3t,32 Amines Amides and Lactams.-The past year has seen a considerable upsurge in interest in the enzyme-catalysed hydrolysis and condensation reactions of amides. An example of this which could prove to be of some significance is the report by Sih and coworkersof the lipase-catalysed rnethanolysis of the /3-lactam (10)(Scheme6),which provides direct access to the C-13 side chain of taxol (6).33 0 Ad* ,I Ph A*. ,mo ii,K,H 0 (*I0pyL.p;Ka OMe Ph40H Ph OH (10) 42% > 99.5% e.e. (61 Reagents i Pseudomonos sp. lipase Bu'OMe MeOH; it NaOH(aq.) Scheme 6 33 R. Brieva J.Z. Crich and C.J. Sih 1.Org. Chem. 1993 58 1068.A. G.Sutherland The penicillin acylase from Escherichia coli has emerged in the important new application of the kinetic resolution of racemic amines via the hydrolysis of the corresponding phenylacetamides. This protocol has been used to synthesize both fl-34 and y-amino acids35 in high enantiomeric purity. The refinement of using p-hydroxyphenylacetamides in these reactions which often provides an increase in enantiosefectivity,also looks promising.36 In the opposite reaction direction Sheldon and coworkers have demonstrated that Iipases can catalyse the conversion of carboxylic esters into the corresponding primary amides (in the presence of and have further observed that this process can display a higher enantioselectivity than the analogous aqueous hydrdysis (Scheme 7).37Gotor and coIIeagues have shown that this type of procedure can be extended to more functionalized amine systems,38 while it has also been demonstrated that the protease 'alcalase' can catalyse the conversion of a peptide C-terminal ester into the analogous primary amide in a similar process.39 Proteases have seen further use in the incorporation of (protected) amino acid 96%e.e.Reagents i Candida antartica lipase Bu'OH NH Scheme 7 residues into peptides4' (and also peptide iso~feres~~) where the use of C-terminal oxazolones as acyl donors has proved to be effective as the enzyme only utilizes the 'naturaI' L enantiomer or diastereoisomer of the interconverting Epoxides.-The use of epoxide hydrolases to provide enantiomerically pure epoxides or vicinal diols has always seemed attractive.This approach has been limited by the fact that the well-characterized enzymes in this class usually from mammalian sources,are available in only very small quantities (although elegant uses of these systems are still appearing in the literat~re).~~.~~ However this situation is now likely to change as a 34 V. A. Soloshonok Y. K.Svedas V. P. Kukhar A.G. Kirilenko A. V. Rybakova V. A. SoIdenko N. A. Fokina,O. V,Kogut,I. Yu.Galaev,E. V. Kozlova,I. P.Shishkina,andS. V.Galushko,S YNLETT,1993,339. 35 A. L. Margolin Tetrahedron Lett. 1993 34 1239. 36 A. Guy A Dumant and P.Sziraky Bioorg. Med. Chem. Lett. 1993 3 1041. 37 M.C. de Zoete A. C. Kock-van Dalen F. van Rantwijk and R. A. Sheldon J.Chern.SOC.,Cbem. Commun. 1993 1831. 38 S. Puertas R. Brieva F. Rebolledo and Y. Gotor Tetrahedron 1993 49 4007. 39 S.-T.Chen M.-K. Jang and K.-T. Wang Synthesis 1993,858. S.-T. Chen C.-C. Tu and K.-T. Wang Bioorg. Med. Chem. Lert. 1993 3 539. 41 M. Schuster B. Munoz W. Yuan and C.-H. Wong Tetrahedron Lett. 1993,34 1247. 42 B.K. Hwang Q.-M. Gu and C.J. Sih J. Am. Chern. SOC. 1993 115 7912. 43 3.Borhan J. Nourooz-Zadeh,T. Uemetsu €3. D. Hammock,and M.J. Kurth Tetrahedron I993,49,2601. ''P. Barili G. Berti and E. Mastrorilli Tetrahedron 1993,49 6263. Enzyme Chemistry 305 number of microbial sources ofthese enzymes have come to light in the past year. While some of these systems such as those from Saccharomyces cereuisiae4’ and Rhodococcus SP.,~‘have yet to be shown to have potential in enantioselective reactions others from Aspergillus niger4’ and Beauusria sulfurescens,48 have considerabIe promise in this context.The reaction of the last two systems with racemic styrene oxide is of particular interest (Scheme 8).48 Thus A. niger hydrolyses the R epoxide seIectively to give the R diol while in contrast B. sulfurescens hydrolyses the S epoxide but with inversion of configuration at the benzylic position again to give the R diol. Therefore when this sequence is taken to its logical conclusion and the racernic epoxide is treated with a mixture of the two organisms both epoxide enantiomers ate converted into the R diol in high yield and enantioselectivity (Scheme 8). 23%,96%e.e. 54% 51% e.e.Pb/+A+ PhLo. PI3 19%,98% e.e. 47% 83% e.e. 92% 89%e.e. Reagents i Aspergillus niger; ii Beauvaria sulfllrescens Scheme 8 3 Oxidation Reactions Boyd at a!. have studied the cis dihydroxylation of benzo-fused unsaturated heterocycles by the dioxygenase of Pseudornonas putida (in whole cell form).49 A clear pattern emerged (Scheme 9) where non-aromatic heterocyclic aIkenes such as the chromene (ll) were oxidized to give an S-configuration alcohol at the benzylic position while heteroarenes e.g. benzothiophene were oxidized to give an R configuration. Other products were obtained from the heteroarenes through mutarota- tion and through oxidation of the benzenoid ring. The products of this type of dihydroxylation reaction have seen many applications in asymmetric synthesis in recent years and this trend continues for example in the Hudlicky group’s versatile synthesis of a range of biologically active aza sugars.50 ’’ G.Fauche R. M. Horak and 0.Meth-Cohn J. Chem. Soc. Chem. Cornmun. 1993 119. *‘ P. Hechtberger,G.Wirnberger M. Mischitz N. Kiempier and K. Faber Terrahedron Asymmetry 1993,4 1161. ‘’ X.-J. Chen A. Archelas and R. Furstoss J. Org. Chem. 1993 58 5528. 48 S. Pedragosa-Moreau A. Archelas and R. Furstoss J. Org. Chem. 1993 58 5533. 49 D. R. Boyd N. D. Sharma R. Boyle B.T. McMurray T.A. Evans J. F. Malone H. Dalton 3. Chima and G.N. Sheldrake J. Chem. Soc. Chem. Commtm. 1993,49. so T. Hudlicky J. Rouden and H. Luna J. Org. Chem. 1993,58 985. A. G. Sutherland OH 15% > 98%e-e.158 > 98% e.e. 98 > 98% e.e. Reagents i Pseudomonas putida UV4 Scheme 9 The enzymatic Baeyer-Villiger reaction is an attractive way to introduce both extra functionality and optical activity into a molecule. To date attempts to use the requisite monooxygenase obtained from Acinetobacter calcoaceticus in isolated form have been hindered by the difficulty in recycling the cofactor required NADPH. However Roberts and coworkers have obtained a new monooxygenase from fseudomonas putida that requires the easily recyclable NADH cofactor and have demonstrated that 60% e.e. > 95% e-e. Reagents i Pseudomonos putida NCIMB 10007 monooxygenase air NADH formate Condido boidinnii formate dehydrogenase Scheme 10 this enzyme is of considerable potential in these oxidations (Scheme The commercially availabIe enzyme Caldariomyces fumago chloroperoxidase has been shown to catalyse the epoxidation of alkenes by hydrogen peroxide.Although the reaction is generally restricted to the oxidation of cis-disubstituted alkenes the high enantiomeric excesses available make this a very promising discovery (Scheme 11 An interesting deveIopment in the past year has been the use of enzyme systems in ‘enantiosekctive destruction’ processes in which one enantiomer of a racemate is removed by oxidation to an achiral product. Using such a procedure a number of mphenylalanine analogues have been prepared by conversion of the L enantiomer into the keto acid using an L-amino acid oxida~e.’~ Similarly,a number ofenantiomerically G.Grogan S. M. Roberts and A. J. Willetts J. Chem. SOC.,Chem. Commun. 1993 699. 52 E.3. Atlain L.P. Hager L. Deng and E. N. Jacobsen J. Am. Chem. Soc. 1993 115,4415. 53 M.C. Pirrung and N. Krishnamurthy J. Org. Chem. 1993 58,957. Enzyme Chemistry -‘w I. .. 0 78% 96%e.e. Reagents i chloroperoxidase H,O (added slowly) pH 5 acetone Scheme I1 pure 1-arylethanols have been prepared by oxidation of the other enantiomer using a range of whole-cell systems which have included bakers’ yeast (Saccharornyces cere~isiae),~~ although other microorganisms have proved more efficient (Scheme 12).55 OH OH 0 100% e.e. OH 100% e.e. Reagents i Bacillus stearothermophilus; ii Acinetobacter calcoaceticus Scheme I2 4 Reduction Reactions The area of enzyme-mediated reductions continues to be dominated by whole cell transformations of 8-ketoesters.Bakers’ yeast has enduring popularity in this area and continues to prove highly effective for providing either simple chi run^,^^ or more sophisticated targets (Scheme l?).57 75-80% > 99% d,e. >93%e.e. Reagents i Saccharomyces cerevisiae sucrose 30“C Scheme 13 Fuganti et al. have looked at the reduction of B-ketoesters with a range of microorganisms as a potentia1 route to the /I-Iactam antibiotic precursor (12). While 54 G.Fantin M. Fogagnolo,A. Medici P. Pedrini S. Poii,and M. Sinigaglia Tetrahedron Lett. 1993,34,883. 55 G. Fantin,M. Fogagnolo A. Medici P. Pedrini S. PoIi and F. Gardini.Tetrahedron:Asymmetry 1993,4 1607. 56 H. Akita R. Todoroki H. Endo Y. Ikari and T. Oishi Synthesis 1993 513. ” D. W. Knight N. Lewis A.C. Share and D. Haigh Tetrahedron Asymmetry 1993 4 525. A. G. Sutherland the obvious precursor (13) failed to give useful diastereoselectivity the use of the sulfide-substituted analogue (14)gave the required intermediate (after desulfurization) as a single diastereoisomer (Scheme 14).58 00 Reagents i Candida guilfiermondii,gIucose; ii Raney nickel EtOH Scheme 14 Aside from /I-ketoesters remarkable selectivity has been reported in the reduction of a benzophenone where the only differentiation between the two aromatic rings is at the para position and hence remote from the ketone (Scheme 15).59 hi-/d'o \ \ \ \ CI a -50% 100% e.e.Reagents i Debaryomyces marama 30°C Scheme 15 Considerable attention continues to be paid to the reaction conditions of bakers' yeast reductions with the intention of optimizing not only yield and enantioselectivity but also ease of operation. It is now well estabiished that immobilizing yeast in or on a solid support (e.g. calcium alginate beads) a relatively simple procedure has considerable benefits with respect to the latter objective. Bhalerao et at. have now shown that using such immobilized systems exquisite and concomitant control of enantioselectivity and yield can be obtained by careful and continuous control of the reaction pH (Scheme 161,a factor largely ignored by others.60 In a remarkably different approach to the same end it has been shown that ethyl acetoacetate can be reduced '' C.Fuganti S. Lanati S. Servi A. Tagliani A. Bedeschi and G. Francheschi,J. Chem. SOC.,Perkin Trans.I 1993,2247. 59 G. Spassov V. Pramatorova R. Vlahov and G. Snatzke Tetrahedron:Asymmetry 1993,4,301. '* U.T. Bhalerao Y. Chandraprakash R. L. Babu and N. W. Fadnavis Synth. Commun. 1993,23 1201. Enzyme Chemistry pH product % e.e. 3 82 4.5 99 6 80 8 35 Reagents i Sacchmomyces cereuisiae Scheme 16 with high enantioselectivity and in good yield by the simple expedient of stirring the substrate with commercial freeze dried yeast in wet light petrole~rn.~' Adam et a!. have reported an unusual reductive kinetic resolution. Treatment of racemic hydroperoxides with horseradish peroxidase in the presence of an oxidation substrate leads to enantioselective reduction of the R hydroperoxide (Scheme I 7).62 > 99% e.e.> 99% e.e. Reagents i Horseradish peroxidase 2-methoxyphenol Scheme 17 5 Carbon-Carbon Bond-forming and Cleaving Reactions Although there is interest in the use of many different carbon-carbon bond-forming enzymes such as ~xynitrilase,~~ and pyruvate decarb~xylase,~~ tran~ketolase,~~ this area is dominated in the literature by reports of aidolase-catalysed reactions. Aldolase research seems evenly divided between the discovery and characterization of new enzymes (usually from a microorganism) as typified by Wong's study of the 3-deoxy-~-manno-2-octulosonic and acid aldolase from Aureobacterium b~rkerei,~~ the detailed investigation of the extremes of substrate specificity of more well-characterized catalysts such as rabbit muscle aldola~e.~~ Whitesides and coworkers have found that N-acetyl neuraminic acid aldolase accepts N-carboxybenzyl mannosamine (15)as a substrate (Scheme 18).This allows a subsequent simple nitrogen deprotection providing ultimately access to the glycoside 61 L.Y.Jayasinghe A. J. Smallridge and M. A. TrewhelIa TetrahedronLett. 1993,34,3949. '* W.Adam U. Hoch C.R.Saba-MolIer and P. Schreier Angew. Chem. int. Ed. EngL 1993,32 1737. 63 M. North SYNLETT 1993,807. '* G.R.Hobbs M. D. LilIy N. J. Turner J. M. Ward A. J. Willetts and J. M. Woodley J. Chem. Soc. Perkin Trans.I 1993 165. 65 V. Kren D.H.G. Crout H. Dalton D. W. Hutchinson W. Konig M. M. Turner G. Dean and N. Thomson J. Chem. Soc. Chem. Commun. 1993 341. 66 T.Sugai G.J. Shen Y.Ichikawa and C.-H. Wong J. Am. Chem. Soc. 1993,115,413. '' W.J. Lees and G. M. Whitesides J. Org. Chem. 1993,58,1887. A. G. Sutheriand Reagents i NeuAc aldolase sodium pyruvate Scheme 18 (16) which is a general precursor to a range of N-acyl neuraminic acid analogues of potential as inhibitors of virus-cell adhesion.68 Herbert et al. have discovered a 8-hydroxy-a-aminoacid aldolase in Streptomyces amakusaensis that is highly selective for the cleavage of the 2S 3R enantiomer (and diastereoisorner) and hence could have considerable potentia1 in the synthesis of the 2R 3s systems by selective degradation of the racemate.69 '' M.A.Sparks,K.W. Williams C. Lukacs A. Schrell G. Priebe A Spakenstein and C.M.Whitesides Tetrahedron 1993,49 1 69 R. B. Herbert B. Wilkinson G.J. Ellames and E. K. Kunec J. Chem. Soe. Chem. Comun. 1993 205.