10 Enzyme Chemistry By S. J. FAULCONBRIDGE K. E. HOLT J.S. PARRATT" S. P. SAVAGE and S. J. C. TAYLOR Chiroscience Ltd. 283 Cambridge Science Park Milton Road Cambridge CB4 4 WE UK 1 Introduction Academic and industrial researchers in the field of organic chemistry are faced with increasingly complex and sophisticated targets to synthesize. In particular the requirement for control of chirality and selective manipulation of similar functional groups frequently necessary under mild conditions encourages the synthetic chemist to explore every opportunity available. Biotransformations represent an important area in the set of methodologies available though a total review of the field is now only realistic in book form. A particularly useful review in this respect was written by Wong and Whitesides,' who tackled the subject from an applied and practical point of view.It is the intention of this review to follow in the footsteps of previous Annual Reports covering enzyme chemistry such as those written by Sutherland.2 Thus this report is broadly divided into the various types of enzyme-catalysed reaction highlighting biotransformations which fulfil the requirements above namely stereo- regio- and chemoselective reactions. 2 Hydrolysis and Condensation Reactions Transesterification of Complex Alcohols and Acids.-The most studied area of biotransformations is still lipase-catalysed hydrolysis and transesterification reactions. Routinely chemists can produce a wide range of low molecular weight enantiopure synthons using such technology.The regio- and stereoselective properties of these enzyme processes are exemplified by Pseudornonas cepacia lipase (Amano Lipase PS) (Scheme l) which was shown to transesterify both diols (1)3 and (2)4 in organic solvent. Such examples typify enzymic resolution technology widely reported in the literature. Other synthetically useful processes include the kinetic resolution of substituted 1,2-diols (Scheme 2). The aryloxy diol (3) was resolved using Lipase PS by an initial non-stereoselective acetylation of the primary hydroxy function followed by a C.-H. Wong and G.M. Whitesides 'Enzymes in Synthetic Organic Chemistry' Tetrahedron Organic Chemistry Series Volume 12,Elsevier Oxford UK,1994. A.G. Sutherland Ann. Rep. Prog.Chem. Sect. B Org. Chem. 1993,90 299. D.M.Coe A. Garofalo S. M. Roberts R. Storer and A. J. Thorpe J. Chem. SOC.,Perkin Trans. I 1994 3061. M.P.Sibi and J.-L. Lu Tetrahedron Lett. 1994,35 4915. 323 S.J. Faulconbridge et al. -q I OAc OAc (72% e.e.) (95% e.e.) li ?f -(R,R) 88% 8.e. (S,S)78%8.8. (S,S)>99% 8.8. 50% yield 21% yield 29% yieM Reagents:i Lipase PS vinyl acetate 30 "C,26 hours; ii Lipase PS vinyl acetate hexane room temperature 24 hours Scheme 1 94% 8.8. (R)96% e.e. (S) 48% yield 52% yield PhLph 2 /Ph .. PhYT + HO OH HO OH HO OAc (*I441 (S)~99%e.e. (R)93% 8.8. Reagents i Lipase PS vinyl acetate THF/NEt, 28 hours (E > LOO); ii Pseudomonas sp. vinyl acetate diisopropyl ether 2 days room temperature (E > 200) Scheme 2 sequential and highly S-selective acetylation of the secondary alcoh01.~ In contrast the resolution of the a,a-disubstituted diol(4) was achieved by an R-selective esterification specifically at the primary hydroxyl catalysed by Pseudomonas sp.lipase.6 The second F. The& J. Weidner S. Ballschuh A. Kunath and H. Schick J. Org. Chem. 1994 59 388. R. P. Hof and R. M. Kellogg Tetrahedron Asymmetry 1994 5 565. Enzyme Chemistry example illustrates a method for generating optically active tertiary alcohols which are regarded generally as being troublesome compounds to obtain directly. The use of lipases in organic solvents has also been extensively studied with regard to the resolution of monohydroxy compounds.The exploitation of such methodology allows access to homochiral synthons which have applications in the synthesis of important pharmaceutical agents such as carbovir7 and teleomycin antibiotim8 PseudomonasJIuorescenslipase was also shown to be of use in the synthesis of the much studied (R)-and (S)-mevalonolactone [by resolution of the racemic 2-(3-methylbut-2- enyl) oxiranemethanol intermediateJg as well as in the resolution of glycerol-2,3-carbonate (a C synthon of much potential)." With the emphasis placed on hydantoinase-catalysed resolution of racemic hydantoin moieties it is refreshing to note that this is not the only methodology of use in this area. A lipase-catalysed transacetylation of 5,5-disubstituted hydantoins has also been described (Scheme 3).' Although the selectivity of the reaction is only moderate this is still an interesting approach to the formation of optically active a,a-disubstituted amino acids.(R)80% 8.8. 31% yield (S)43% 8.8. 56% yield Reagents i Pseudomonas sp. vinyl acetate 5 hours acetonitrile 0 "C scheme 3 Biotransformations are still finding a useful role within the realms of organometallic chemistry. Moreover lipases have been shown to be most effective as catalysts for the kinetic resolution of planar-chiral ferrocenes in transesterification mode.' Such resolutions tend to make use of hydroxymethyl groups situated on unsaturated ligands as illustrated in Scheme 4. In this example Pseudomonas sp. lipase catalyses the acetylation reaction using isoprenyl acetate to resolve the racemic chromium species (5) with extremely high selectivity (E > 500).13 The transesterification of complex acids has also been an area of research widely investigated with many diverse substrates being utilized.Much of this effort has focused upon the area of 2-arylpropionic acid resolution with a view to producing the well known Profen family of antiinflammatory drugs. 14*1 The regioselective behav- iour of lipases towards complex acid moieties has also been explored with one spectacular example being the enzyme-mediated synthesis of diester crown ethers. ' H. Nakano Y. Okuyama K. Iwasa and H. Hongo Tetrahedron Asymmetry 1994 5 1155. H. Sundram A. Golebiowski and C. R. Johnson Tetrahedron Lett. 1994,35 6975. P. Ferraboshi P. Grisenti S.Casati and E. Santaniello SYNLETT 1994 754. lo M. Pallavicini E. Valoti L. Villa and 0.Piccolo J. Org. Chem. 1994 59 1751. '' E. Mizuguchi K. Achiwa H. Wakamatsu and Y. Terao Tetrahedron Asymmetry 1994 5 1407. G. Nicolosi A. Patti R. Morrone and M. Piattelli Tetrahedron Asymmetry 1994 5 1275 l3 M. Uemura H. Nishimura S. Yammada and Y. Hayashi Tetrahedron Asymmetry 1994 5 1673. l4 M. Arroyo and J. V. Sinisterra J. Org. Chem. 1994 59 4410. S.-W. Tsai and H.-J. Wei Enzyme Microb. Technol. 1994 16 328. S.J. Faulconbridge et al. (1R ,2S)>99% 8.8. (1S,2R)98%e.e. 47% yield 48% yield Reagents i Pseudomonas sp. lipase 25 "C 3 hours isoprenyl acetate Scheme 4 Polyethylene Recovered Acyclic Cyclic glycol (6) (8a)-(8c)% (9a)-(9c)% (7a) n = 1 57 12 9 (7b) n = 2 14 3 5 (7~) n = 3 42 7 7 Reagents i Mucor miehei lipase toluene 6O"C 12 days Scheme 5 Conde and coworkers employed Mucor miehei lipase together with a range of polyethylene glycol nucleophiles (7at(7c) to transesterify 1,3-bis(3,5-diethoxycar-bonyl- 1H-pyrazol-1-y1)propane (6) (Scheme 5).16 Despite the poor isolated yields of (8at(8c) and (9at(9c) notable regiocontrol is exhibited with no byproducts detected when using nucleophiles (7b) and (7c) and less than 1 YOisolated when the reaction was l6 M.Fierros M. I. Rodriguez-Franco P. Navarro and S. Conde Bioorg.Med. Chem. Lett. 1994,4,2523. Enzyme Chemistry MeoXco2Me H C02Me Reagents i CCL hexane 3.5 hours 40 "C,BnOH (53% conversion); ii H,/Pd toluene/hexane; iii BH, THF Scheme 6 performed with (7a).Such biocatalytic methodology is far from well characterized; however such examples can only encourage enzymic methodology for the preparation of macromolecules in the future. Shapira and Gutman reported a strategy for the formation of a series of hitherto unknown chiral monosubstituted malonate diesters via a lipase-mediated transes- terification of the corresponding prochiral dimethyl malonate esters. Candida cylin-dracea lipase (CCL),with benzyl alcohol as the nucleophile provided the best results in terms of product e.e. when using the methoxy malonate (10) (Scheme 6).17The solvent of choice was hexane selected for its well-known compatibility with enzymes and its high hydrophobicity minimizing the lability of the malonic hydrogen (thus reducing undesirable product racemization).This approach also allowed for the isolation of the unstable malonate half ester (12) easily effected by the catalytic hydrogenation of the benzyl ester (1 1). Further synthetic manipulation accessed the hydroxy ester (13) with an e.e. of 70%. The adaptation of reversible enzyme reactions to provide irreversible processes has been reviewed with particular reference to strategies applicable to the synthesis not only of esters but also of peptides nucleosides and carbohydrates." However another publication examined the use of mixed carboxylic carbonic anhydrides as irreversible acyl transfer reagents for the lipozyme-catalysed resolution of carboxylic acids.'' Such acyl transfer reactions were reported to be rapid (20 minutes to 2 hours) and due to the liberation of carbon dioxide the equilibrium position was shifted completely towards the products (Scheme 7).Furthermore the other side product isopropanol is apparently inert towards side reactions such as subsequent trans- esterification of the ester product. 0 0 0 R\roYop+ + PrOH ,-f+ phpopr + Pr'OH Me co2 Me 90% 8.8. (E = 20) (absolute stereochemistry not reported) Reagents i Lipozyme room temperature Bu'OMe 60 minutes 55% conversion Scheme 7 M. Shapira and A. L. Gutman Tetrahedron Asymmetry 1994 5 1689. J.-M. Fang and C.-H. Wong SYNLETT 1994 393. l9 E. Guibe-Jampel and M. Bassir Tetrahedron Lett. 1994 35 421. S.J. Faulconbridge et al.-i Et02C CO2Et H02C CO2Et Reagents i PLE pH 7.8 27 "C Scheme 8 Ad Ad ,Ph i t 0gPh 0g" -(14) (i)cis (3R,2S)acetate>99°/o8.8. (3S,2R)alcohol>99?h e.e. Reagents i Lipase PS/Lipase BMS (Bristol-Myers Squibb) Scheme 9 Hydrolysis of Complex Acids and Alcohols.-A worthy example of the versatility of lipase enzymes in the hydrolytic mode was reported by Adamczyk et aL2* Both benzyl and methyl esters of rapamycin 42-hemisuccinate were cleaved under mild conditions using Pseudomonas sp. lipase. Indeed it was claimed that the deprotection of benzyl esters in the presence of other easily reducible groups could well be considered equivalent to a selective reduction. Such mild hydrolytic conditions coupled with the the fact that peptide linkages are inert to lipase attack have also found application within the realms of glycopeptide chemistry.2 The useful regioselectivity of lipases was demonstrated in the hydrolysis of diethyl itaconate to the monoester (Scheme 8);22 chemical esterification of itaconic acid gave the regioisomer.Enzymes are increasingly being used by pharmaceutical companies for the synthesis of key intermediates (Scheme 9).23The azetidinone (14) is an intermediate in the synthesis of the anticancer compound paclitaxel (Taxolo). Immobilization of the lipase on polypropylene facilitated reuse of the enzyme for ten successive cycles without loss of activity or selectivity. Hydrolases continue to appear from unusual sources thus providing alternatives to the available set of commercial lipases (Scheme A variety of homochiral trans-2-arylcyclohexan- 1-01s were thus synthesized using crude chicken liver esterase.The creation of a sole centre of chirality on a heteroatom in good e.e. has been reported (Scheme 1 l).25In this example the prochiral sulfoxides (15) and (16) were converted into the ester acids (17) and (18) respectively. In the case of (17) the absolute 2o M. Adarnczyk J. C. Gebler and P. G. Mattingly Tetrahedron Lett. 1994 35 1019. 2' H. Kunz D. Kowalczyk P. Braun and G. Braurn Angew. Chem. Int. Ed. Engl. 1994 33 336. 22 P. Ferraboschi S. Casati P. Grisenti and E. Santaniello Tetrahedron 1994 50 3251. 23 R. N. Patel A. Banerjee R. Y. KO,J. M. Howell W.-S. Li F.T. Comezoglu R. A. Partyka and L. Szarka Biotechnol.Appl. Biochem. 1994 20 23. 24 D. Basavaiah and P. Dharrna Rao Tetrahedron Asymmetry 1994 5 223. 25 M. Mikolajczyk P. Kielbasinski R. Zurawinski W. Wieczorek and J. Blaszczyk SYNLETT 1994 127. Enzyme Chemistry Ar Ar Ar = phenyl 1-naphthyl >99% e .e . 4-met hylphenyl 2,4,64rimethylphenyl Reagents i Crude chicken liver esterase Scheme 10 (15) (R)-(17) 9290 e.e. 63% yieM (16) (R)-(18) 67% e.e. 70% yield Reagents i cr-chymotrypsin 16 hours pH 7.5; ii PLE 40 hours pH 7.5 Scheme 11 A& 0 + I &!\Ph Me U MPh e (S)-(19) 49% 8.8. (R)-(20)53% 8.8. 0 ! -11 I1 ! MeO-P-We MeO-P-OH + MeO-P-SMe I I I NHCOMe NHCOMe NHCOMe (achiral) Reagents i Cholesterol esterase; ii Phosphotriesterase from Pseudomonas diminuta or Flauobacterium sp.Scheme 12 stereochemistry was successfully established by single crystal X-ray diffraction. Compounds containing a phosphorus stereocentre have also been obtained using hydrolytic enzymes (Scheme 12). Hence (+)-(19) was resolved uia a cholesterol esterase-catalysed hydrolysis of the remote acetate functionality yielding the pheno1 S.J. Faulconbridge et al. (4(22) (23)61% 8.8. (22) 18% 8.8. Reagents i CCL 34 hours 40% conversion Scheme 13 i R (24) R = H (25) R=NOp Reagents i fi-glucosidase Scheme 14 (R)-(20).26 In an alternative approach phosphotriesterase was found to hydrolyse the P-S bond S-selectively in the organophosphate triester ( & )-(21).27 The resolution of tertiary alcohols has been explored using Candida cylindracea lipase.The racemic t-acetylenic alcohol (22) was resolved by hydrolysing the acetate function (Scheme 13) albeit with poor e.e. to furnish the t-alcohol (23).28 Glycosidation Reactions.-A wide variety of glycosides have been synthesized using P-glycosidases and transfera~es.~’~~ The diastereoselective cleavage of sulfoxides (24) and (25) was demonstrated using P-glycosidases (Scheme 14).32 This represents the first example of the diastereoselective hydrolysis of sulfoxides by an enzyme and gave diastereoisomerically pure recovered sulfoxides. An elegant synthesis of aleppotriolo-side a naturally occurring glucoside was achieved in which the two key reactions were a Pseudornonas JEuorescens-catalysed enantioselective resolution and transglucosyla- tion of the intermediate by a thermophilic P-glycosidase from Sulfolobus solfutaricus (Scheme 15).33 Nitrile and Epoxide Hydrolysis.-Interest in biocatalytic nitrile hydrolysis is growing steadily.The area has been spotlighted as a mini review in a keynote article by Turner 26 A.N. Serreqi and R. J. Kazlauskas J. Org. Chem. 1994 59 7609. 2’ M.-Y. Chae J. F. Postula and F. M. Raushel Bioorg. Med. Chem. Lett. 1994 4 1473. 28 D. O’Hagan and N. A. Zaidi Tetrahedron Asymmetry 1994 5 11 11. 29 W. H. Binder H. Kahlig and W. Schmid Tetrahedron 1994 50 10407. ’O A. Baker N. J. Turner and M. C. Webberley Tetrahedron Asymmetry 1994 5 2517. G.F. Herrmann P. Wang G.-J. Shen and C.-H. Wong Angew. Chem. Int. Ed. Engl.1994 33 1241. 32 0.Karthaus S. Shoda and S. Kobayashi Tetrahedron Asymmetry 1994 5 2213. 33 A. Trincone E. Pagnotta and G. Sodano Tetrahedron Lett. 1994 35 1415. Enzyme Chemistry 331 OAc OAc OH \0' HO*LG HO '* OH aleppotridoside Reagents i Pseudomonas fluorexens lipase pH 7 6 days; ii CH,MgBr Et,O; iii Sulfolobus solfataricus homogenate phenyl-fl-D-glucoside Scheme 15 and coworkers.34 Also reported here were the first examples of biocatalytic regioselective nitrile hydrolysis effected by an immobilized whole-cell preparation (derived from Rhodococcus sp.) containing both nitrile hydratase and amidase enzymes. As Scheme 16 illustrates both hydrolytic reactions proceed selectively to the nitrile-amide compounds (28) and (29) with no other products detected.Interestingly the non-fluorinated analogues of (26) and (27) were biotransformed with little or no regiocontrol and furthermore the products observed were nitrile-acids and not nitrile-amides. Other groups working in this field have further investigated the enantioselectivity of nitrile biohydrolysis. Racemic 2-arylpropionitriles have been studied using enriched bacterial isolates; such methods include a highly selective process for the synthesis of enantiopure (S)-napr~xen.~~.~~ Furthermore Knowles and coworkers demonstrated the enantioselective behaviour of a nitrilase enzyme from R. rhodochrous that catalyses the single step process of transforming nitriles directly into carboxylic acids. Both racemic 2-methylbutyronitrile and 2-methylhexanitrile were hydrolysed to their corresponding carboxylic acids in an S-selective fashion and with high e.e.37,38 The 34 J.Crosby J. Moilliet J. S. Parratt and N. J. Turner J. Chem. Soc. Perkin Trans. I 1994 1679. 35 R. Bauer B. Hirrlinger N. Layh A. Stolz and H.-J. Knackmuss Appl. Microbiol. Biotechnol. 1994,42 1. 36 N. Layh A. Stolz J. Bohme F. Effenberger and H.-J. Knackmuss J. Biotechnol. 1994 33 175. 37 M.L. Gradley C. J. F. Derverson and C.J. Knowles Arch. Microbiol. 1994 161 246. 38 M. L. Gradley and C.J. Knowles Biotechnol. Lett. 1994 16 41. S.J. Faulconbridge et al. Reagents i Nitrilase SP361 phosphate buffer 7G115 hours 30 "C Scheme 16 OH (S)-(31) >90% e.8. AN3 (R)-(32),>60% 8.8. Reagents i SP409 TRIS buffer N; room temperature Scheme 17 enantioselective potential of biocatalytic prochiral 3-0-substituted glutaronitrile hydrolysis has been further reported this year with the production of enantiomerically pure (S)-cyano-acids using Brevibacteriurn SP.~' Faber et al.have continued their investigations into the use of an immobilized whole-cell preparation SP409 derived from Rhodococcus sp. to effect enantioselective epoxide hydr~lysis.~' The racemic epoxide (30) (Scheme 17) was exposed to the biocatalyst in the presence of azide to form both the (S)-diol(31) (>90% e.e.) and the (R)-azido-alcohol (32) (>60% e.e.). Careful monitoring of the reaction in terms of optical purities of the reaction components against time led to the conclusion that the epoxide ring-opening by azide was enzyme catalysed and was not a spontaneous 39 A.Kerridge J. S. Parratt S. M. Roberts F. Theil N. J. Turner and A. J. Willetts Bioorg. Med. Chem.,1994 2 447. 40 M. Mischitz and K. Faber Tetrahedron Lett. 1994 35 81. Enzyme Chemistry 333 reaction. No conclusions were drawn however as to whether the epoxide hydrolase accepted azide as a nucleophile or if another enzyme was involved. Other work reported in this field includes the exploitation of cytosolic and microsomal epoxide hydrolases from rabbit liver.41 It was demonstrated that microsomal epoxide hydrolase was able regio- and enantioselectively to ring open both styrene oxide and trans- 1-phenylpropene oxide at the non-benzylic oxirane carbon.However in contrast cytosolic epoxide hydrolase effects a non-selective hydrolytic attack of styrene oxide and a regioselective but non-enantioselective reaction at the benzylic carbon of trans-1-phenylpropene oxide. Amide Hydrolysis and Condensation.-The ability of enzymes to hydrolyse amide functionalities has been further de~cribed.~~,~~ However the much-studied penicillin acylase still appears to be at the forefront with regard to synthetic versatility. The substrate specificity of microbial penicillin acylase towards a variety of N-phenylacetyl-P-fluoroalkyl-P-aminoacids has been in~estigated.~~ The results pub- lished suggested that the high level of enantioselectivity exhibited was not influenced by the length of the fluoroalkyl chain.In contrast however it was reported that modification of the phenylacetyl moiety at the a-position with a range of different substituents does affect the activity of immobilized penicillin-(; acylase towards penicillin and cephalosporin derivative^.^^ Furthermore the insertion or removal of atoms between the aromatic nucleus of the phenylacetyl fragment and the centre of hydrolysis lead to molecules which are no longer substrates for the enzyme. The continued use of Alcalase a proteolytic catalyst whose major component is subtilisin Carlsberg in peptide synthesis has yielded some significant results particularly in the preparation of a variety of biologically active intermediate^.^^.^^ The thiol protease papain has also been utilized with Sih and coworkers employing 5(4H)-oxazolinones as acyl donors in the synthesis of peptide segments.48 This methodology brought about the successful coupling of the oxide insulin B chain to angiotensin I11 in an impressive 59% yield.3 Reduction Reactions Wholecell Reductions.-The enantioselective conversion of ketones into chiral alcohols continues to be the most active domain in whole-cell reductions where bakers’ yeast remains the most popular choice of biocatalyst. Buisson et al. observed high diastereoselectivity and enantioselectivity in reduction of the a-substituted /3-ketoester (33) with a selection of microorganisms (Scheme 18). In most cases cis:trans ratios of the product (34) were reported to exceed 99 1. The (1R,2R)-enantiomer was produced by fungal strains such as Mucor racemosus Rhizopus 41 G.Bellucci C. Chiappe A. Cordoni and F. Marioni Tetrahedron Lett. 1994 35 4219. 42 B. Kaptein H. M. Moody Q. B. Broxterman and J. Kamphuis J. Chem. Soc. Perkin Trans. I 1994 1495. 43 J. Ogawa M.C.-M. Chung S. Hida H. Yamada and S. Shimizu J. Biotechnol. 1994 38 11. 44 V.A. Soloshonok A.G. Kirilenko N. A. Fokina I. P. Shishkina S.V. Galushko V. P. Kukhar V. K. Svedas and E. V. Kozlova Tetrahedron Asymmetry 1994 5 11 19. 45 M. van der Mey and E. de Vroom Bioorg. Med. Chem. Lett. 1994 4 345. 46 S.-T. Chen S.-Y. Chen C.-L. Kao and K.-T. Wang Biotechnol. Lett. 1994 16 1075. 4’ S.-T. Chen S.-Y. Chen C.-L. Kao and K.-T. Wang Bioorg. Med. Chem. Lett. 1994,4,443. 48 B.K. Hwang Q.-M. Gu and C. J. Sih Tetrahedron Lett.1994 35 2317. S.J. Faulconbridge et al. 0 OH (33) (34) Scheme 18 (35) (36)72% yield Reagents i Bakers' yeast Scheme 19 0 0 X X X = Halogen NO2,CHO COCH3 COPh Reagents i Bakers' yeast NaOH H,O-MeOH 7G80 "C Scheme 20 arrhizus and Sporotrichum exile in >99% e.e. In contrast the complementary enantio activity was demonstrated by bakers' yeast affording the (lS,2S)-enantiomer in >95% e.e.49 A topic of current therapeutic interest is the synthesis of paclitaxel a compound possessing significant antitumour activity. A precursor of its C-13 side chain (2R,3S)-phenylisoserine (36) has been prepared by bakers' yeast reduction of the a-ketoester (35) furnishing the correct stereochemistry at the new chiral centre (Scheme 19).50 In addition to the synthesis of chiral alcohols bakers' yeast enjoys widespread application to a host of other reductions.One such reaction is the reduction of aromatic nitro compounds an area which has attracted only limited study. Baik et al. report selective reduction of substituted nitrobenzenes to the corresponding anilines (Scheme 20).51 In examples where the substituent contains carbonyl functionality (e.g. X = CHO COMe COPh) the amino derivative was selectively obtained in high yield without further carbonyl reduction. 49 D. Buisson R. Cecchi J. A. Laffitte U. Guzzi and R. Azerad Tetrahedron Lett. 1994 35,3091. J. Kearns and M. M. Kayser Tetrahedron Lett. 1994 35,2845. '' W.Baik J. L. Han K.-C. Lee N.-H. Lee B.-H. Kim and J.T. Hahn Tetrahedron Lett.1994 35,3965. Enzyme Chemistry Bakers' yeast 56% 10% Enzyme Enzyme NADH NAD' 1 Scheme 21 (41a) R=H (42a) R= H (43a) R = H (41b) R=Me (42b) R = Me (43b) R=Me Reagents i Bakers' yeast 7G72 hours 33 "C Scheme 22 Interestingly in a parallel study bakers' yeast reduction of 2-nitrobenzonitrile (37) furnished 2-aminobenzamide (38) as the major product. The mechanism (Scheme 21) postulated involved initial reduction to the oxime (39) followed by intramolecular attack of the hydroxyl group onto the nitrile (40). A subsequent two-electron reduction of (40) would furnish the benzamide (38).52 Reduction of the (E)-nitrophenyl nitroalkenes (Scheme 22) also catalysed by bakers' yeast demonstrated a degree of chemoselectivity dependent upon the nitroalkene substituent R.The substrate (41a) produced almost exclusively the nitroalkane (42a). In contrast where the substituent was methyl (41b) the aniline derivative (43b) was obtained in preference to the nitroalkane (42b).53 The unsaturated b-lactone (44) underwent kinetic resolution with bakers' yeast supplying (+ )-(R)-(44) in 99% e.e. (Scheme 23). Mechanistic considerations based on analogous work proposed hydride delivery in the /?position from the upper re face of the lactone framework affording selective reduction of the S enanti~mer.~~ 52 C. L. Davey L. W. Powell N. J. Turner and A. Wells Tetrahedron Lett. 1994 35,7867. 53 M. Takeshita S. Yoshida and Y. Kohno Heterocycles 1994 37 553. 54 C. Fuganti G. Pedrocchi-Fantone A.Sarra and S. Servi Tetrahedron Asymmetry 1994 5 1135. S.J. Faulconbridge et al. (44) (R)-(44),99% e.e. Reagents i Bakers' yeast D-glucose 36 "C,DMSO 6 hours Scheme 23 i C02Et -4-"CO2Et (45) (46)100%e.e Reagents i Enzyme from Catharanthus roseus NADPH Scheme 24 (47) (48) 97V0e.e. Reagents i DMSO reductase benzyl viologen 25 "C Scheme 25 Isolated-enzyme Reductions.-On the whole most isolated reductases have been extracted from either animal tissue or microbes while enzymes from plant cell cultures are comparatively rare. One such enzyme purified from the membrane portion of Catharanthus roseus reduced the cr-keto ester (45) enantioselectively to the (R)-cr-hydroxy ester (46) in 100% e.e. (Scheme 24).5s A DMSO reductase from a mutant of Rhodobacter sphaeroides f.s.denitrijcans exclusively reduced the (S)-sulfoxide (47) to its sulfide (Scheme 25) leaving the optically active R enantiomer (48).56 The development and application of reductions effected by isolated enzymes continue to be hindered by cofactor recycling. Traditionally this problem has been tackled albeit inefficiently by enzymic or chemical means. Another approach involves direct electrochemical regeneration of the cofactor a method which suffers from the formation of isomers and dimers. Yun et al. however report a promising system for NADH regeneration employing a diaphragm composed of an anion-charged 55 H. Hamada N. Nakajima Y. Shisa M. Funahashi and K. Nakamura Bioorg. Med. Chem. Lett. 1994,4 907.56 M. Abo M. Tachibana A. Okubo and S. Yamazaki Biosci Biotech. Biochem. 1994 58 596. Enzyme Chemistry 337 membrane coupled with appropriate regulation of the cathodic potential. The system worked favourably as an NADH regenerator in a reaction involving lactate dehydr~genase.~’ An indirect electrochemical approach was developed by Fry et al. in which a long-term stable electroenzymatic system was prepared for NADH regeneration simply by coimmobilizing lipoamide dehydrogenase and a methyl viologen mediator on an electrode under a NafionO film. This approach is superior to a homogeneous two-enzyme system as both enzymes are separated and the biotoxic viologen is kept out of solution.58 4 Oxidation Reactions Biocatalytic oxidation of organic compounds (primarily of carbon or sulfur) is becoming an increasingly useful methodology in organic synthesis.Although most examples target the introduction of chirality useful regioselective oxidations have been noted. Baeyer-Villiger oxidation of ketones is an important transformation for lactone synthesis. The Baeyer-Villiger oxidation of monocyclic and bicyclic ketones by Acinetobacter calcoaceticus NCIMB 987 1 and Pseudomonas putida NCIMB 10007has been studied in some detail.59 For example Acinetobacter calcoaceticus oxidized the racemic dihalogenoketone (49) to optically active lactone (50) and recovered ketone (51) (Scheme 26). The ketone was converted in seven steps to an azidothymidine (AZT) analogue (52). The microbial dioxygenation of substituted benzenes to produce homochiral cis-dihydrodiols has long been recognized and examples of the use of these diols in organic synthesis continue to appear in the literature.Thus a recent example is the chemoenzymatic synthesis of D-erythro-C *-and L-threo-C 8-sphingosines,60 which when combined with carbohydrates and fatty acids constitute glycosphigolipids. These are of much interest due to their diverse biological roles such as protein kinase C inhibition and information transfer agents between developing vertebral cells. The cyclohexadiene-cis-diol from chlorobenzene was used to synthesize (53) and (54) precursors for (55) and (56) (Scheme 27). Selective oxidative metabolism of racemic compounds by microorganisms has been frequently used for the provision of chiral molecules.Thus exposure of Candida parapsilosis to butane- 1,3-diol allowed accumulation of multikilo quantities of (R)-(-)-butane-1,3-diol (Scheme 28).61 Whole cell oxidation of several prochiral diols by Nocardia corallina (Scheme 29) resulted in the formation of chiral lactones of high optical purity.62 In all the examples cited the products were derived from the oxidation of the pro-S hydroxymethylene group. The biotransformation of racemic mandelic acid by a mutant of Pseudomonas putida 57 S. Yun M. Taya and S. Tone Biotechnol. Lett. 1994 16 1053. A. J. Fry S. B. Sobolov M. D. Leonida and K.I. Voivodov Tetrahedron Lett. 1994 35 5607. 59 R. Gagnon G. Grogan M. S. Levitt S. M. Roberts P.W. H. Wan and A. J. Willetts J.Chem. SOC. Perkin Trans. I 1994 2537. 6o T. Hudlicky T. Nugent and W. Griffith J. Org. Chem. 1994 59 7944. 61 A. Matsuyama and Y. Kobayashi Biosci. Biotech. Biochem. 1994 58 1148. 62 H. Luna K. Prasad and 0.RepiE Tetrahedron Asymmetry 1994 5 303. S.J. Faulconbridge et al. (49) (50) (511 e.e.>95% 40% yield e.e.>95% 40% yield 7 steps 1 F Reagents i A. calcoaceticus NCIMB 9871 Scheme 26 OH (53) (55) (Scheme30),defective in muconolactone isomerase provided the highly functionalized muconolactone 5-carboxymethyl-2,5-dihydrofuran-2-one in high enantiomeric purity (97% e.e.).63 D. W. Ribbons and A. G. Sutherland Tetrahedron 1994 50 3587 Enzyme Chemistry 94% 8.8. Reagents i Candida parapsilosis Scheme 28 >!39% 8.8.OH OH 95% 8.8. Reagents i Nocardia Corallina Scheme 29 Reagents i muconate cycloisomerase; ii muconolactone isomerase Scheme 30 Microbial oxidation of sulfur for the preparation of chiral sulfoxides continues to attract interest.64 The fungus Helminthosporiurn species NRRL 467 1 was used to 64 H. L. Holland F. M. Brown and B. G. Larsen Tetrahedron Asymmetry 1994 5 1241. S.J. Faulconbridge et al. R = Me Et Pr" Bun (S) 52435% e .e. 42-70%yield Reagents i Helminthosporium NRRL 4671 Scheme 31 Reagents i Pseudomonas acidovorans Scheme 32 biotransform a series of para-alkylbenzyl sulfides (Scheme 3 1) giving moderate yields of sulfoxides with predominantly S chirality at sulfur. Lower yields of the sulfones were also reported.Regiospecific hydroxylation of pyrazine carboxylic acids gives compounds for synthesis of pharmaceutically active substances such as Glipicide an antidiabetic or Pyrazinamide a tuberculostatic agent.65 Of several biotransformations investigated the most promising was the hydroxylation of pyrazine carboxylic acid to 5-hydroxypyrazine-Zcarboxylic acid (Scheme 32). A product concentration of 75 g 1-' in 96% yield was obtained in 24 hours. 5 Carbon-Carbon Bond-forming Reactions The continuing interest in aza sugars and their analogues due to their antiviral and antitumour activities has resulted in several publications relating to their synthesis this year. These routes utilize aldola~e~~.~~ and transketolase68 enzymes to catalyse the key asymmetric aldol addition reaction (Scheme 33).A new route to deoxythiosugars based on aldolases has been de~eloped.~' The use of thioaldehydes as acceptors for fructose 1,6-diphosphate aldolase rhamulose 1-phosphate aldolase fuculose 1-phosphate aldolase and 2-deoxyribose 5-phosphate aldolase demonstrates the synthetic utility of these enzymes. Kragl et al. report new findings in the sialic acid aldolase-catalysed condensation of pyruvate with various sugar substrates (for an example see Scheme 34).70 An apparent relationship between 65 A. Kiener J.-P. Roduit A. Tschech A. Tinschert and K. Heinzmann SYNLETT 1994 814. 66 K. E. Holt F. J. Leeper and S. Handa J. Chem. SOC.,Perkin Trans. I 1994 231. 67 I. Henderson K. Laslo and C.-H.Wong Tetrahedron Lett. 1994 35,359. 68 L. Hecquet M. Lemaire J. Bolte and C. Demuynck Tetrahedron Lett. 1994 35,8791. 69 W.-C. Chou L. Chen J.-M. Fang and C.-H. Wong J. Am. Chem. SOC. 1994 114,6191. 'O U. Kragl A. Godde C. Wandrey N. Lubin and C. Auge J. Chem. SOC.,Perkin Trans. I 1994 119. Enzyme Chemistry 341 OH OH OH "Y, HO OH 1. I1 HOWOH iii_ GO H -N3 OH 0 OH 0 OEt 1 1 OH Reagents i Rabbit muscle aldolase dihydroxyacetone phosphate; ii acid phosphatase; iii 10% Pd/C H, 40 psi; iv BuLi dithiane; v HCI-KCI buffer pH l/EtOH (70/30); vi hydroxypyruvate transketolase Scheme33 OH HO OH O H -Ho9c02H WI HO HO OH HO Dallose Reagents i Sialyl aldolase pyruvate Scheme 34 enzyme stereoselectivity and conformation and stereochemistry at C-3of the substrate is discussed.The enzyme-catalysed asymmetric synthesis of cyanohydrins has been reviewed this year by Effenberger." Elsewhere Kiljunen describes the use of lyophilized powdered and washed Sorghum shoots as an (S)-oxynitrilase source thus eliminating the need for purification and immobilization of this enzyme (Scheme 35).72 71 F. Effenberger Angew. Chem. lnt. Ed. Engl. 1994 33 1555. 72 E. Kiljunen and L.T. Kanerva Tetrahedron Asymmetry 1994 5 311. S.J. Faulconbridge et al. CN I -0""" i c8. 90% yield 90% 8.8. Reagents i Acetone cyanohydrin Sorghum bicolor shoots diisopropyl ether Scheme 35 Antlbody <Ar > 98% 8.8. Scheme 36 86?4e.e. 1.5 g Reagents i H' antibody 20T pH 6 Scheme 37 6 Abzymes Since the first catalytic antibodies (abzymes) were reported in 1986,abzymes catalysing numerous types of reaction have been reported.This year has seen a large amount of literature on the subject including two review^.^^^^^ An interesting discovery by Koch et al. was the enantioselective epoxidation of unfunctionalized alkenes-the first report of an antibody-catalysed oxidation reaction at carbon (for an example see Scheme 36).75 In order for abzymes to be synthetically useful it is essential that their large scale use is possible. Thus Reymond et al. have reported the multigram hydrolysis of enol ether (57) using a reusable catalytic antibody and a very simple laboratory procedure (Scheme 37).76 73 G.M. Blackburn and P. Wentworth The Genetic Engineer and Biotechnologist 1994 14 9 74 H. Suzuki J. Biochem. 1994 115 623. 7s A. Koch J.-L. Reymond and R. A. Lerner J. Am. Chem. Soc. 1994 116 803. 76 J.-L. Reymond J.-L. Reber and R.A. Lerner Angew. Chem. Int. Ed. Engl. 1994,33 475.