11 Biological Chemistry Part (i) Enzyme Chemistry By N. J. TURNER Department of Chemistry University of Exeter Exeter EX4 400 1 Introduction This review covers the literature relevant to the use of enzymes for organic synthesis. The advantages and limitations of using enzymes and whole cells for synthesis have been described in a number of previous reviews.’-7 It is readily apparent from these reports that the oxidoreductases (e.g. baker’s yeast alcohol dehydrogenases) and hydrolytic enzymes (e.g. microbial lipases pig-liver esterase) have found the most widespread application owing to a combination of ease of use broad spectrum of substrates tolerated and predictable stereoselectivity of the biotransformation. In this review the objective has been to cover a wide range of enzyme-catalysed reactions that are either currently being applied to synthesis or have the potential to provide synthetic methodology in areas where chemical-based approaches are inefficient.2 Hydrolytic Enzymes Reactions in Aqueous Media.-Jones has found that the stereospecificities of the isozyme components of commercially available pig-liver esterase are essentially the same towards representative monocyclic and acyclic diester substrates.8 Thus pig- liver esterase can be exploited synthetically as a chiral catalyst with confidence that it will behave as if it were a single protein. Schneider has described the use of a highly selective lipase from a species of Pseudornon~s.~ Various enantiomerically pure secondary alcohols (e.g.PhCHMeOH PhCHCF,OH PhCH,CHMeOH) were prepared in high chemical and optical yields by lipase-catalysed hydrolysis of the ’ G. M. Whitesides and C.-H. Wong Angew. Chem. Int. Ed. Engl. 1985 24 617. J. B. Jones Tetrahedron 1986 42 3351. S. Butt and S. M. Roberts Nut. Prod. Rep. 1986 489. M. P. Schneider and E. H. Reimerdes Forum MikrobioL 1987 10 307. J. B. Jones Proc. 3rd FECS Int. Conf. Chem. Biotechnol. Biol. Act. Nat. Prod. 1985 (publ. 1987) Vol. 1 p. 18. P. E. Sonnet CHEMTECH 1988 18 94. ’H. G. Davies R. H. Green D. R. Kelly and S. M. Roberts ‘Biotransformations in Preparative Organic Chemistry The Use of Isolated Enzymes and Whole Cell Systems in Synthesis’ Academic Press London 1989. L. K. P. Lam C. M. Brown B. De Jeso L. Lym E. J. Toone and J.B. Jones J. Am. Chem. SOC.,1988 110 4409. K. Laumen and M. P. Schneider J. Chem. SOC.,Chem. Commun. 1988 598. 307 308 N. J. Turner corresponding racemic esters. The rate-determining step was postulated to be hydro- lysis of the acyl-enzyme intermediate since different acetates (e.g. OCOCH, OCOCF,) were hydrolysed at different rates. The stereoselectivity of Mucor miehei lipase with various substrates including phospholipids has been reviewed." A study has been made of the preferred orientation of ester groups in hydrolyses catalysed by pig-liver esterase using conformationally rigid substrates.' 'It was shown conclusively that cyclohexane rings with methoxycarbonyl groups in the equatorial (1) rather than axial (2) position are preferentially hydrolysed by pig-liver esterase by a factor of four to seven (Scheme 1).This result is in agreement with previous work by Tamm.'* Reagents i pig-liver esterase 5% MeOH Scheme 1 The esterase- (steapsin) catalysed hydrolysis of racemic isobutyl3,4-epoxybutyrate occurred with good stereoselectivity yielding the unreacted ester (3) having the R configuration (> 95% enantiomeric excess). This intermediate was subsequently hydrolysed non-selectively with alcalase to give (4)and converted into (R)-(-)-carnitine chloride (5).' (3) (4) A strategy for enhancing the enantiomeric specificity of lipase-catalysed hydrolysis of esters has been developed by Sih.I4 Thus in the hydrolysis of the substrates (6) little or no enantioselectivity was observed using lipase from a species of Pseudomonas.However the introduction of a non-hydrolysable ester-protecting group for the carboxylic acid (7) led to hydrolysis of the acetate group yielding the corresponding (R)-hydroxy t-butyl esters with >99% e.e. Porcine pancreatic lipase has been used to prepare the chiral intermediate (8) from the corresponding racemic acetate in enantiomerically pure form. This key lo P. E. Sonnet J. Am. Oil Chem. SOC. 1988 65 900. L. K. P. Lam and J. B. Jones J. Org. Chem. 1988 53 2673. l2 P. Mohr N. Waespe-SarEeviE C. Tamm K. Gawronska and J. G. Gawronski Helu. Chim. Acta 1983 66 2501. l3 D. Bianchi W. Cabri P. Cesti F. Francalanci and M. Ricci J. Org. Chem. 1988 53 104. A. Scilimati T. K. Ngooi and C. J. Sih Tetrahedron Lett.1988 29 4927. 309 Biological Chemistry-Part (i) Enzyme Chemistry 0 0 TBDMSO OAc C02Me Cl+ chiral building block was used for a convergent synthesis of the anti-tumour marine prostanoid punaglandin 4 (9) in overall yield of 1.5%.I5 y- 8- and E-lactones undergo ring opening with concomitant kinetic resolution in the presence of porcine pancreatic lipase horse-liver esterase or pig-liver esterase. Yields are in the region 60-90% with 35-92% e.e.I6 Crout has provided an interesting example of the tolerance of pig-liver esterase for unusual substrates by enzymically resolving the racemic organometallic ester 2-ethoxycarbonylbuta-1,3-dienetricarbonyliron (10). The enantiomeric excesses of both the product (11) and unreacted ester (10) were 85% and could be raised to 98% by one recry~tallization.’~ (c0)37e ,C02Et pig-liver esterase ____* 20% MeOH pH 7,40 h Reactions in Organic Solvents.-Several reviews have been published dealing with the use of enzymes particularly proteases esterases and lipases in organic sol-vent~.’*”~ Pancreatin in dry tetrahydrofuran-triethylamine (25 :1) catalyses the highly enantioselective esterification of the meso-diol (12) with 2,2,2-trichloroethyl acetate to yield the monoacetate (13) in 48% yield and 95% e.e.The diacetate (14) formed in 48% yield could be hydrolysed and reused. In the absence of triethyl-amine the transesterification was very slow.2o l5 K. Mori and T. Takeuchi Tetrahedron 1988 44 333. 16 L. Bianco E.Guibe-Jampel and G. Rousseau Tetrahedron Lett. 1988 29 1915. 17 N. W. Alcock D. H. G. Crout C. M. Henderson and S. E. Thomas J. Chem. Soc. Chem. Commun. 1988 746. la A. M. Klibanov CHEMTECH 1986 354. 19 A. Zaks and A. M. Klibanov Science 1984 224 1249. 20 F. Theil S. Ballschuh H. Schick M. Haupt B. Hafner and S. Schwarz Synthesis 1988 540. 310 N. J. Turner Two groups have prepared macrocyclic lactones by the use of lipases in organic solvents. The long-chain hydroxy esters (15) underwent lactonization in the presence of lipase to give the desired product (16) and the diolide (17). The ratio of (16) to (17) was found to be 2.7 1 when n = 15 falling to 1.5:1 when n = 12. The il 0 enantioselectivity of the reaction was demonstrated by the conversion of racemic methyl 10-hydroxyundecanoate into exclusively the (R)-isomer of the corresponding lactone.2’ An alternative approach relied on direct condensation of diacids with diols in non-aqueous media (e.g.iso-octane CC14 PhMe hexane and EtOH) in the presence of lipases from Cundidu cylundriceu Pseudomonus species and porcine pancreas. Best results were obtained with ring sizes of 24-28.22 Papain in a biphasic system consisting of McIlvaine buffer and ethyl acetate- triethylamine catalyses the condensation of [( p-methoxybenzyl)oxy]carbonyl-L-aspartic acid and L-phenylalanine methyl ester to give (18) -a precursor for the artificial sweetener aspartame -in 45% yield. This enzymic process offers the advantage over the chemical method that no protection of the side-chain carboxyl group of the aspartyl residue is required.23 By the use of organic solvents the amidase activities of certain serine and cysteine proteases (e.g.trypsin chymotrypsin papain and subtilisin) can be reduced whilst still retaining significant esterase activities. Under these conditions the enzymes can ’’ A. Makita T. Nihira and Y. Yarnada Terrahedron Leu. 1987 28 805. 22 Z. W. Wei and C. J. Sih J. Am. Chem. SOC.,1988 110 1999. 23 S. T. Chen and K. T. Wang J. Org. Chem. 1988 53 4589. 311 Biological Chemistry- Part (i) Enzyme Chemistry be used as peptide ligases in a kinetically controlled approach for the stepwise synthesis and fragment coupling of peptides. Importantly the products are free from the secondary hydrolysis that normally accompanies protease-catalysed peptide synthesis.This methodology was used to prepare peptides containing both D-and L-amino acids.24 When papain was modified with 2,4-bis-[ 0-methoxypoly(ethy1ene glycol)]-6- chloro-s- triazine [activated poly( ethylene glycol)] the resulting immobilized bio- catalyst was soluble in benzene and retained catalytic activity. To demonstrate its use N-benzoyl-L-alanine alkylamides were synthesized from N-benzoyl-L-alanine methyl ester and various alkylamine~.~~ The result of modifying thermolysin with poly(ethy1ene glycol) was to alter its specificity towards substrates. Thus in organic solvents hydrophilic as well as acidic amino acids were better carboxyl donors than hydrophobic residues contrary to what is observed in both the enzyme-catalysed synthesis and hydrolysis of peptide bonds in water.26 An interesting pointer to the future use of enzymes in environments other than water/organic solvents is provided by the studies on the interesterification reactions catalysed by lipases in supercritical carbon dioxide.Thus lipases from Rhizopus delemar R. japonicus and M. rniehei were found to catalyse interesterification of triolein and stearic acid in supercritical carbon dioxide at 50 "C and 29.4 MPa.27 3 Oxidoreductases Baker's yeast reduction of the N-protected keto proline (19) gave the (+)-cis-(2R 3S)-3-hydroxyproline (20) (80% e.e. 99% d.e.). Subsequent chemical trans- formations converted (20) into (-)-( 1S 5s)-2-oxa-6-azabicyclo[3.3.0]octan-3-one (Geissman- Waiss lactone) (21) and also provided for formal total syntheses of (-)-retronecine and related pyrrolizidine alkaloids.28 HO H (s-.'02Et baker'syeast ___ H c1- \ BOC BOC The keto alcohol (22) has been prepared on an 8 g scale by reduction of the corresponding prochiral diketone using Pichia terricola (>99% e.e.).(22) was then converted into the corresponding 3,5-dinitrobenzoate which was recrystallized to purity. This was then used as the enantiomerically pure starting material for the synthesis of the naturally occurring isomers of juvenile hormones I (23) and I1 (24). Overall yields were 2.7 and 1.2% re~pectively.~~ 24 C. F. Barbas 111 J. R. Matos J. B. West and C.-H. Wong J. Am.Chem. Soc. 1988 110 5162. 25 H. Lee K. Takahashi Y. Kodera K. Owada T. Tsuzuki A. Matsushima and Y. Inada Biotechnol. Lett. 1988 10 403. 26 A. Ferjancic A. Puigserver and H. Gaertner Biotechnol. Lett. 1988 10 101. 27 Y. M. Chi K. Nakamura and T. Yano Agric. Biol. Chem. 1988 52 1541. 28 J. Cooper P. T. Gallagher and D. W. Knight J. Chem. Soc. Chem. Cownun. 1988 509. 29 K. Mori and K. Fujiwhara Tetrahedron 1988 44 343. 312 N. J. Turner H (22) (23) R = Me (24) R = H Practical procedures have been developed for the enantioselective reduction of 2-acetylfuran and 2-trifluoroacetylfuran to the corresponding carbinols (S)-2-acetylfuran and (S)-2- trifluoroacetylfuran with 88-90% e.e. by using Ther-moanaerobium brockii alcohol dehydrogenase coupled with an NADPH regeneration system.30 Preparative scale horse-liver alcohol dehydrogenase-catalysed reductions of prochiral meso and racemic cis-and trans-decalindiones occur with concurrent regio- and stereospecificity to give good yields of enantiomerically pure keto alcohol products.In each case the reduction occurs to give the (S)-alcohol in a manner that is completely predicted by the cubic section active site model. The chiral synthon utility of such keto alcohols is illustrated by a direct and efficient synthesis of (+)-(4R)-twistanone (25) from (26) in 51% overall yield.31 Baker’s yeast is capable of catalysing transformations other than alcohol-ketone conversion. Thus an approach to the asymmetric synthesis of remote-functionalized sterols based on the enantioselective enzymic cyclization of the C 1 hydroxylated surrogate substrate (27) with Saccharomyces cerevisiae has been described.Incuba- tion of the functionalized squalene analogue (27) with baker’s yeast in phosphate buffer leads to stereospecific formation of the C28 hydroxylated sterol (+)-(28) in 30 D. G. Drueckhammer C. F. Barbas 111 K. Nozaki C.-H. Wong C. Y. Wood and M. A. Ciufolini J. Org. Chem. 1988 53 1607. 31 D. R. Dodds and J. B. Jones J. Am. Chem. SOC.,1988 110 577. Biological Chemistry- Part (i) Enzyme Chemistry 40.5% yield.32 Previous work by the same group used analogous cyclizations to prepare the cytotoxic agent ganoderic Z.33 Baker’s yeast has also been used to catalyse dehydrogenation of a thia analogue of stearic acid (29) to yield (30).Subsequent treatment with a second organism Lactobacillus plantarum stereospecifically converted (30) into the cyclopropyl com- pound (31).34 LS 1S. cerevisiae HH IL. piantarum 4 Carbon-Carbon Bond Formation Fructose 1,6-diphosphate aldolase from rabbit muscle has proved to be a useful catalyst for the synthesis of carbon-carbon bonds. In nature this enzyme mediates the condensation of D-glyceraldehyde 3-phosphate (32) and dihydroxyacetone phos- phate (33) to give fructose 1,6-diphosphate (34). The enzyme is highly specific for dihydroxyacetone phosphate as the nucleophilic component but accepts a number of achiral and chiral (both D and L) aldehydes as the electrophile. Recently it has been shown to catalyse aldol reactions of nitrogen-containing aldehydes providing several novel nitrogen-containing and C-alkyl sugars on 4-20 mmol scales.35 Two groups have used rabbit-muscle aldolase for efficient syntheses of the glycosidase inhibitors 1 -deoxymannojirimycin (35) and 1 -deoxynojirimycin (36).36,37 In both cases the aldolase was used in the key step to catalyse the condensation of dihy- droxyacetone phosphate and (KS)-3-azido-2-hydroxypropanal(37) to give the aldol product (38).Subsequent cleavage of the phosphate group using acid phos- phatase followed by hydrogenation yielded the desired compounds (35) and (36). Wong has investigated the substrate specificity of N-acetylneuraminic acid aldolase. By replacing the natural substrate N-acetylmannosamine with 6-0-acetyl- N-acetylmannosamine the aldolase-catalysed condensation with pyruvate gave the 32 J.C. Medina and K. S. Kyler J. Am. Chem. Soc. 1988 110,4818. 33 J. Bujons R. Guajardo and K. S. Kyler J. Am. Chem. Soc. 1988 110 604. 34 P. H. Buist and H. G. Dallmann Tetrahedron Lett. 1988 29 285. 3s J. R. Durrwachter and C.-H. Wong J. Org. Chem. 1988 53 4175. 36 T. Ziegler A. Straub and F. Effenberger Angew. Chem. Int. Ed. Engl. 1988 27 716. 37 R. L. Pederson M. J. Kim and C.-H. Wong Tetrahedron Lett. 1988 29 4645. 314 N. J. Turner R2 H (32) R' = OH R2 = CH,0P0,H2 (33) (34) R' = OH R2 = CH20P03H2 (37) R' = OH R2 = N (38) R' = OH R = N OH OH HO,. Ho+/*-oH N AC H20H ~ H 120H H compound (39) in 59% overall yield.6- O-Acetyl-N-acetylmannosamine was itself prepared enzymatically by regiospecific acylation of the 6-OH group of N-acetyl- mannosamine with isopropenyl acetate catalysed by protease N.38 AcOdoH ?H AcHN W C 0 2 H The mandelonitrilase from bitter almonds has previously been shown to be a useful catalyst for the synthesis of optically active cyanohydrii~s.~~ In a recent report benzaldehyde was converted into the (R)-cyanohydrin (40) in 94% yield and >98% e.e. The conditions involve the use of a potassium cyanide-acetic acid buffer (pH 5.4) with ethanol at 0 "C. Reactions could be performed on a 30-50 g scale.4o A novel catalytic activity of lipases has been discovered namely asymmetric Michael addition to provide optically active trifluoromethylated compound^.^'^^^ Lipases from species of Alcaligenes and Achromobacter catalysed the addition of various thiols and dialkylamines in organic solvents to (E)-ethyl 3-(trifluoromethy1)- and 2-(trifluoromethy1)-propenoate,yielding the corresponding Michael adducts.38 M. J. Kim W. J. Hennen H. M. Sweers and C.-H. Wong J. Am. Chem. SOC.,1988 110 6481. 39 F. Effenberger T. Ziegler and S. Forster Angew. Chem. Znt. Ed. Engl. 1987 26,458. 40 J. Brussee E. C. Roos and A. Van der Gen Tetrahedron Lett. 1988 29,4485. 4' T. Kitazurne T. Ikeya and K. Murata J. Chem. SOC.,Chem. Commun. 1986 1331. 42 T. Kitazume K. Murata Y. Kokusho and S. Iwasaki J. Fluorine Chem. 1988 39,75. Biological Chemistry- Part (i) Enzyme Chemistry 5 Enzymes Acting on Carbohydrates and Oligosaccharides Enzymes are being used to great effect to overcome the problem of selective protection in carbohydrate synthesis.Thus glucose can be regioselectively acylated at the 6-OH using 2,2,2-trichloroethyl butyrate in anhydrous DMF and subtilisin as the catalyst. Similar esterifications were carried out on the disaccharides maltose and cellobiose both giving >95% selectivity. Interestingly with sucrose subtilisin acylates the C1 hydroxy group to give (41) whereas in the chemical acylation the most reactive -OH is at the C6 position. The reactions could be carried out on gram scales with isolated yields of approximately 50% .43 0 CHzOk roH I OH OH (41) Related work has been carried out by Wong.44 Thus with the methyl furanosides of D-ribose D-arabinose D-xylose and 2-deoxy-~-ribose acetylation of the primary hydroxy functions was achieved with porcine pancreatic lipase in THF using 2,2,2- trifluoroethyl acetate as the acyl donor.In aqueous media (containing 10% DMF) selective deacylation of the corresponding peracetylated series was achieved using Candida cy Iandricea lipase. Cytidine 5’-monophospho-N-acetylneuraminic acid (CMP-NeuAc) (42) is an important intermediate in the biosynthesis of certain glycoproteins. It has been prepared by two groups each using a multi-enzyme approach (Scheme 2)?47 The key difference between the two systems is the method used to generate the expensive cytidine 5’-triphosphate (CTP) in situ from the relatively cheap cytidine 5’-monophosphate (CMP).white side^^^.^^ employs adenylate kinase pyruvate kinase and phosphoenol pyruvate (PEP) as the ultimate phosphate donor to convert CMP into CTP this reaction being carfied out on a gram scale. An alternative approach uses nucleoside monophosphokinase instead of adenylate kina~e.~’ Thereafter both methods use the enzyme CMP-NeuAc synthetase to couple CTP with N-acetyl- neuraminic acid (NeuAc) generating CMP-NeuAc. Sialyl transferases are a group of enzymes that catalyse the transfer of NeuAc from CMP-NeuAc to oligosaccharide chains of glycopeptides. Two representatives of this group have been used to construct a disialylated tetrasaccharide analogous to M and N blood group determinants of glycophorin A.48Thus chemically synthe- 43 S.Riva J. Chopineau A. P. G. Kieboom and A. M. Klibanov J. Am. Chem. SOC.,1988 110 584. 44 W. J. Hennen H. M. Sweers Y.-F. Wang and C.-H. Wong J. Org. Chem. 1988 53 4939. 45 E. S. Simon M. D. Bednarski and G. M. Whitesides Tetrahedron Lett. 1988 29 1123. 46 E. S. Simon M. D. Bednarski and G. M. Whitesides J. Am. Chem. Soc. 1988 110 7159. 47 C. Auge and C. Gautheron Tetrahedron Lett. 1988 29 789. 48 H. T. De Heij M. Kloosterman P. L. Koppen J. H. Van Boom and D. H. Van den Eijnden J. Carbohydr. Chem. 1988 7 209. 3 16 N. J. Turner AcHN MeCOC0,H OH -PEP -CTP CMP CDP i ix CTPi i g D p 0 I1y!--;:0 AcHN PYr PEP I I OH OH Reagents i NeuAc aldolase; ii adenylate kinase or nucleoside monophosphokinase; iii pyruvate kinase; iv CMP-NeuAc synthetase Scheme 2 sized phenyl 2-acetamido-2-deoxy-3- 0-p-D-galactopyranosyl-a-D-galactopyrano-side was treated sequentially with CMP-[’4C]-NeuAc in the presence of sialyl transferase from human placenta followed by CMP-[3H]-NeuAc and a sialyl trans- ferase from rat-liver microsomes to give the desired product in 10% yield.The use of glycosidases rather than glycosyl transferases offers a complementary method for constructing glycosidic bonds with high regioselectivity. Two approaches are possible (a) ‘transglycosidation’ using an activated sugar e.g. o-nitrophenyl sugars and (b) ‘reverse’ hydrolysis. An example of the first approach is given by the synthesis of monoacyl galactoglycerides (43) by P-galactosidase-catalysed trans-glycosidation of lactose or a-nitrophenyl galactopyranoside with 2,3-epoxypropanol and subsequent opening of the so formed 1-O-p-D-galaCtOpyranOSyl-2,3-epoxypropanol with a fatty acid.49 Yields for the initial glycoside formation were in the region 25-3070.Similarly fructose has been transferred by invertase-catalysed alcoholysis of sucrose in water-primary alcohol mixtures containing -do% organic solvent giving 540% alkyl P-D-fructofuranoside formation. No formation of fructosides was observed under anhydrous conditions. 6-Ketose formation due to surcrose itself acting as acceptor could be suppressed by increasing the concentration F. Bjoerkling and S. E. Godtfredsen Tefrahedron 1988 44 2957.317 Biological Chemistry- Part (i) Enzyme Chemistry OH HO / of aliphatic alcohol.50 By contrast a-D-glycosyl-D-fructoses were synthesized by use of a ‘reversed’ hydrolysis activity of a-glucosidase from Saccharomyces species. Using a simple batch method the major product was cy-D-glucosyl-(~+ 1)-D-fructose (44) with smaller amounts of a-(1+ 4)- a-(1 + 5)- and a-(1+ 6)-roH OH (44) linked products. With an immobilized a-glucosidase column and an activated carbon column (to remove product disaccharides thereby shifting the equilibrium in the synthesis direction) in series the same saccharides were formed (although in con- siderably lower yield) but the major product was a-D-glyCOSyl-(l + 4)-D-frUCt0Se.” 6 Miscellaneous Biotransformations The conversion of benzene (and analogues) by Pseudomonas putida into cyclo- hexadienediols [e.g.(45) and (46)] has provided organic chemists with a valuable synthon for natural product synthesis. Ley has used this transformation as the key step in the synthesis of the cellular secondary messenger myo-inositol 1,4,5-triphos- phate (47) starting from (45).52 Hudlicky has utilized the corresponding chiral compound (46) derived from enantioselective oxidation of toluene as a versatile starting material for the preparation of several prostaglandin and terpene ~ynthons.~~ Another potentially useful oxygenase that is beginning to be studied is cycloalk- anone oxygenase. Using an extract from Acinetobacter NCIB 9871 the ring 50 H. Fujimoto and K.Ajisaka Biotechnol. Lett. 1988 10 107. 51 A. J. J. Straathof J. P. Vrijenhoef E. P. A. T. Sprangers H. Van Bekkum and A. P. G. Kieboom J. Carbohydr. Chem. 1988 7 223. 52 S. V. Ley and F. Sternfeld Tetrahedron Lett. 1988 29 5305. 53 T. Hudlicky H. Luna G. Barbieri and L. D. Kwart J. Am. Chem. SOC.,1988 110 4735. 318 N. J. Turner H203p0v0H HO' OH (45) R = H OP03H2 (46) R = Me (47) expansion of a number of meso-ketones could be carried out to give the correspond- ing lac tone^.^^ Oxidation of the ketone (48) resulted in the five-membered ring lactone (49) from hydrolysis and relactonization of the initially formed seven-membered ring. ...Q,,* ?H cycloalkanone o+ oxygenase b OH 0 (48) (49) Microbial hydroxylation represents an area in which enzymes are likely to prove superior to traditional chemical methods.In most cases the enzymic reactions proceed with moderate to high regioselectivity but poor enantioselectivity. Furstoss and his group have recently claimed the first example of a highly enantioselective hydroxylation process. Thus the racemic lactam (50) underwent hydroxylation with Beauvaria sulfurescens to give a mixture of products the major component (30% yield) being (51)with an optical purity Of 90% .55 Similar enantioselectivity has been demonstrated using dopamine-P- hydroxylase which was used as the catalyst in the hydroxylation of 2-( l-cyclohexenyl)-2-aminoethanolat the pro-R position to give (R)-1-(cyclohexenyl)-2-aminoethanol(52).56 Beauvaria sulfurescens _____ eNH2 &H NYPh &NKPh 0 0 The hydrocarbon monooxygenase from Pseudomonas oleouqruns is a prototypical w-hydroxylase known to carry out hydroxylation at the terminal methyl group of alkanes as well as epoxidation of terminal olefins.It has now been shown that this enzyme system catalyses stereospecific sulphoxidation of methyl thioether substrates 54 M. J. Taschner and D. J. Black J. Am. Chem. Soc. 1988 110 6892. 55 A. Archelas J.-D. Fourneron R. Furstoss M. Cesario and C. Pascard J. Org. Chem. 1988 53 1797. 56 S. R.Sirimanne and S. W. May J. Am. Chem. SOC.,1988 110 7560. Biological Chemistry- Part (i) Enzyme Chemistry representing the first clear example of oxygenase-produced chiral aliphatic sul- phoxides yet reported.In addition this enzyme catalyses oxidative 0-demethylation of branched alkyl methyl and branched vinyl methyl ethers to secondary alcohols and ketones re~pectively.~’ Stereoselective alkene epoxidation has also been achieved using strains of species of Mycobacterium and Nor~ardia.~~ Among the various substrates and organisms tried the best results were achieved with but-3-en- 1 -01 and a Norcardia strain IPl yielding the corresponding (R)-epoxide in 13% yield and 84% e.e. A rabbit microsomal epoxide hydrolase is capable of enantioselectively opening a racemic ep~xide.~~ Thus treatment of 100 mg of the racemic epoxide (53) until 30% conversion resulted in a 60% e.e. for the (-)-(lR,2S,3R)-diol (54) and a 30% e.e.for the unreacted (-)-( 1 R,2R,3S)-epoxide. Br Br 7 Novel Biocatalysts Since the first demonstrations that antibodies could be successfully elicited and used to catalyse predefined reactions recent work has focused on increasing their efficiency and extending the range of reactions capable of being catalysed. Lerner has shown that phosphonate esters can be used to mimic a carboxyl esterolytic transition state.60 Among the twenty antibodies that were raised and screened for hydrolysis of the carboxylic ester five were found to be esterases. In addition the transition state analogue was a specific inhibitor of the esterase activity. One antibody accelerated the hydrolysis of a related substrate with a catalytic constant (k,,,) of 20 sC1 and a K of 1.5 mM at pH 8.0.This represents an acceleration of several million fold over the rate of spontaneous hydrolysis. Two groups have raised antibodies against a putative transition state (55)for the Claisen rearrangement of chorismate (56) to prephenate (57).6‘762The kinetic data co; OR OH OH (55) (56) (57) 57 A. G. Katapodis H. A. Smith and S. W. May J. Am. Chem. SOC.,1988 110 897. 50 A. Archelas S. Hartmans and J. Tramper Biocatalysis 1988 1 283. 59 G. Bellucci M. Ferretti A. Lippi and F. Marioni J. Chem. Soc. Perkin Trans. 1 1988 2715. 60 A. Tramontano A. A. Ammann and R. A. Lerner J. Am. Chem. SOC.,1988 110 2282. 61 D. Hilvert and K. D. Nared J. Am. Chem. SOC.,1988 110 5593. 62 D. Y. Jackson J. W. Jacobs R. Sugasawara S. H. Reich P.A. Bartlett and P. G. Schultz J. Am. Chem. Soc. 1988 110 4841. 320 N. J. Turner obtained62 showed that one of the antibodies against (55) could effectively catalyse the conversion of (56) into (579 (k,,,/k,,,, = 1 x lo4 compared with 3 x lo6 for chorismate mutase from Escherichia coli). Schultz has begun to extend the concept of antibody catalysis by an elegant e~periment.~~ He has raised an antibody to the cis-syn-thymine dimer (58) that catalyses the photocleavage of (59) as well as (58). The principle being exploited I R R (58) R = OH (59) R = NHCH,C02H here is that indoles are well known sensitizers of the photoreversal of pyrimidine dimers and it was expected that an antibody binding site specific for the polarized .rr-system of a thymine dimer might contain a complementary tryptophan residue.The wavelength dependence of the antibody-catalysed reaction as well as the quenching of antibody fluoresence upon substrate binding are indicative of a binding-site tryptophan. This study represents the extension of antibody catalysis to a new class of reactions namely C-C bond cleavage. The idea of preparing mbdified enzymes by chemical derivatization of existing enzymes is another potentially powerful strategy for designing new biocatalysts. A thermophilic semisynthetic flavoenzyme was prepared by alkylation of cysteine-149 at the active site of glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus with 7-(a-bromoacetyl)-l0-methylisoalloxazine. The initially for- med tetrameric flavoprotein irreversibly dissociated into dimers.These dimers were very stable and served as catalysts for the oxidation of 1,4-dihydronicotinamidesat temperatures as high as 55 "C. NADH was the best substrate examined under these condition^.^^ 63 A. G. Cochran R. Sugasawara and P. G. Schultz J. Am. Chem. Soc. 1988 110 7888. 64 D. Hilvert Y. Hatanaka and E. T. Kaiser J. Am. Chem. Soc. 1988 110 682.