Synthetic methods Part (iii) Enzyme chemistry 2 A. J. Carnell Department of Chemistry Robert Robinson Laboratories University of Liverpool Liverpool UK L69 7ZD 1 Introduction The aim of this review is to provide the organic chemist with highlights of the literature in biocatalysis from the past year. It is by no means a comprehensive survey but represents a selection of transformations using novel or known biotransformations which might be of general interest to the synthetic organic chemist. Several general reviews have appeared,— in addition to reviews describing methods for improving biocatalyst performance and selectivity using cross-linked enzyme crystals (CLEC’s), immobilized and encapsulated biocatalysts. The preparation of highly sensitive biomolecules for the study of important biological mechanisms has been facilitated by some useful protecting group strategies which have been devised using hydrolytic enzymes. Speci.c reviews have appeared on Baeyer Villiger monooxygenases, — glycosidases and glycosyl transferases, enzymatic C—C bond formation, thiamine-dependent enzymes, and the application of -keto acid decarboxylases.2 Hydrolytic enzymes There seems to be almost no limit to the use of lipases for resolution of alcohols and carboxylic acids. All that is required is access to enough enzymes and the patience to test them under a variety of conditions in the hydrolysis and ester forming modes in aqueous organic or mixed solvents. For example Hiyama tested 84 commercially available lipases for the resolution of the acetoxyketone 1 (Scheme 1).The product hydroxyketone 2 can be transformed into 1-aminoindan-2-ol through oxime formation and diastereoselective hydrogenation. Of those tested Amano PS Meito QLand Fluka 62312 lipases were selected as optimal in terms of their E-values. Hydrolysis in phosphate bu.er (pH 7)—acetonitrile with Amano PS lipase led to highest selectivity (E250). By separation of the resolution products and hydrolysis of the acetoxyketone 1 using catalytic scandium tri.ate in order to avoid racemisation both 39 Annu. Rep. Prog. Chem. Sect. B 1999 95 39—58 Scheme 1 Scheme 2 enantiomers could be prepared in high yield and enantiomeric purity (45—47% 94—96% e.e.). A continuous chemoenzymatic process for the preparation of cypermethrine 6 involving four steps has been described by E.enberger (Scheme 2). The .rst step involves resolution of ( )-3-phenoxybenzaldehyde cyanohydrin acetate 3 with Lipase P in hexane using n-BuOH for the transesteri.cation.Highest selectivity was achieved using Celite-immobilized lipase where the immobilization had been performed at pH 4.5. The mixture of acetate 3 and alcohol 4 was then reacted with enantiomerically enriched acid chloride 5 to give a high yield of (1R,cis,S)-cypermethrine 6 with a 90% d.e. The unreacted acetate 3 could be recycled by distillation and racemization with triethylamine. The enantioselectivity of the enzyme was not a.ected by recycling although the activity diminished signi.cantly the reaction taking twice as long (15 h) on the fourth cycle as on the .rst.Adam and co-workers have carried out a comparative study of transesteri.cation 40 Annu. Rep. Prog. Chem. Sect. B 1999 95 39—58 Scheme 3 Scheme 4 versus hydrolysis for the resolution of synthetically useful threo-allylic diols (Scheme 3). Best results were obtained using Candida antarctica lipase fraction B (CAL-B) hydrolysis to a.ord enantiopure regioisomeric acetates 8 and 9 and highly enriched unreacted diacetate 7. In the nucleoside arena highly regio- and stereoselective deacylation of carbocyclic 3,5-di-O-acyloxetanocins has been achieved using lipases (Scheme 4). Treatment of the dibenzoyl derivative 10 with Lipase MY gave 3-monoprotected compound 12 with high regioselectivity and treatment of the diacetyl starting material 11 with lipase Type XIII from Sigma gave the 5-monoacylated derivative 13 with high enantioselectivity.These derivatives obviously have application in the synthesis of oligonucleotides. Understanding the roles of lipidated proteins in cell signalling processes is at the forefront of biological research. Waldmann has developed a clever and e.cient enzyme-labile blocking group strategy for assembling acid- and base-labile peptide conjugates containing palmitoyl thioesters and farnesyl thioethers. The N-terminus of a given peptide intermediate is protected as its p-acetoxybenzyloxycarbonyl (AcOZ) urethane derviative 14 (Scheme 5). Cleavage can be carried out under neutral conditions with acetyl esterase from oranges or Mucor miehei lipase.The lipase was used in cases where an organic co-solvent was required to solubilize the peptide in which case the esterase was inactivated. After cleavage of the acetate the resulting quinomethane 15 spontaneously fragments liberating the desired peptide conjugate 16. Characteristic S-palmitoylated and S-farnesoylated C-terminus peptides of the human N-Ras protein were synthesized using this very clean deprotection method. Combination with classical methodology allowed the synthesis of various .uorescent and modi.ed Ras 41 Annu. Rep. Prog. Chem. Sect. B 1999 95 39—58 Scheme 5 lipopeptides which were used for in vivo cell biology experiments. These experiments resulted in a model for the targeting of lipidated peptides and proteins to the plasma membrane by S-palmitoylation.In principle this enzymatic deblocking methodology is general since other acyl groups can be used with the appropriate hydrolytic enzyme. Waldmann has previously used PhAcOZ as a protecting group in phosphoglycopeptide synthesis cleaving with penicillin G acylase. In a highly e.cient approach to the synthesis of racemic and ()- and ()- conduritol C Ba� ckvall has used sequential palladium-catalysed 1,4-diacetoxylation and enzyme hydrolysis (Scheme 6). The palladium reaction used to convert the microbially derived diene 17 gave high trans selectivity for the production of the diacetate 18 when using phthalocyanine—O rather than the alternative MnO Chemical hydrolysis gave racemic conduritol C.Hydrolysis of the diacetate with Candida rugosa lipase gave a near perfect resolution. The diol 19 and diacetate 18 were then deprotected to give ()- and ()-conduritol C respectively. Key to the success of this strategy was the enzyme’s ability to recognize two acetates in one enantiomer of the substrate simultaneously. . N-Stearoyl-C -erythro-sphinganine diacetate 20 has been successfully resolved with regioselective primary acetate hydrolysis using immobilized Burkholderia cepacia lipase (SC lipase A) in a biphasic system (decane—phosphate bu.er; 10 1) in the presence of Triton X-100 (Scheme 7). The resemblance of this substrate to the natural triacylglycerol lipase substrates is noteworthy. The e.e. of the recovered (natural) ()-diacetate 20 could be enhanced by repeating the resolution.The native nonimmobilized enzyme showed low enantioselectivity but the high regioselectivity was 42 Annu. Rep. Prog. Chem. Sect. B 1999 95 39—58 Scheme 6 harnessed to a.ord the natural ()-monoacetate 21 from the ()-diacetate 20 which then could be selectively glycosylated at the primary position to form a glycosphingolipid. Due to the combination and relationship between functional groups in products of the Bayliss—Hillman reaction these materials provide valuable synthetic building blocks. Alcohols such as 22 and 23 have been successfully resolved using lipase PS acylation in acetonitrile to a.ord (R)-acetates 24 and 25 and unreacted (S)-alcohols. The acyl donor was isopropenyl acetate for 22 and vinyl acetate for 23.The E-values (349 for 22 and 424 for 23) obtained here were exceptionally high (Scheme 8). The lipase from Pseudomonas fragii has been little used in synthetic biotransformations. Crout has demonstrated its application in the preparation of a key intermediate 26 for the synthesis of carba-sugars (Scheme 9). Lipase catalysed acylation of alcohol 43 Annu. Rep. Prog. Chem. Sect. B 1999 95 39—5Scheme 7 Scheme 8 Scheme 9 26 in a carefully selected solvent mixture (BuOMe containing 11% acetone) gave best results. The enantiopure acetate 27 could be obtained from the 91% e.e. material by recrystallizing the minor enantiomer from hot ethanol. 3Nitrile and epoxide hydrolysis Aliphatic ,-dinitriles 28 have been converted into -cyanocarboxylic acid ammonium salts 29 using either Acidovorax facalis 72W ATCC 55746 which contains a nitrilase or Comamonas testosteroni 5-M GAM ATCC 55744 containing nitrile hydratase and amidase activities.The acid salts were then converted directly to the lactams 30 by hydrogenation in aqueous solution without isolation of the intermediates. Only one of the two possible lactam products was produced from -alkylsubstituted ,-dinitriles resulting from -cyano group hydrolysis (Scheme 10). Conversions for both steps were generally high (80%) and no inactivation of the hydrogenation catalysts by microbial contaminants was observed. The microorgan- 44 Annu. Rep. Prog. Chem. Sect. B 1999 95 39—58 Scheme 10 ism Acidovorax facalis was heat treated (50 °C) for 1 hour before use in order to destroy an unwanted endogenous nitrile hydratase activity which was found to produce dicarboxylic acids.Rhone-Poulenc has patented the use of an immobilized recombinant E. coli strain containing a nitrilase from Alcaligenes faecalis for the enantioselective hydrolysis of 2-hydroxy-4-methylthiobutyronitrile to give the corresponding carboxylic acid. Furstoss discovered a surprising enantioselectivity enhancement e.ect when using a two-phase system for the enantioselective hydrolysis of p-bromo--methylstyrene epoxide 31 with Aspergillus niger crude cell extract. When carrying out the reaction in phosphate bu.er below the saturation point of the substrate the E value for the transformation was 20.However on using a substrate concentration way above the saturation point such that the substrate formed a second phase the E value increased 13-fold to 260 for the production of the (S)-epoxide 31 and (R)-diol 32 (Scheme 11). The authors ruled out the possibility of non-selective spontaneous hydrolysis occurring in the dilute system which might be lessened under biphasic conditions and so far have no explanation for the e.ect. The reaction was successfully carried out on 6 g of epoxide using only 75 ml of bu.er and 350mg of crude cell extract. 4 Oxidations Regioselective and asymmetric hydroxylations catalysed by enzymes continue to draw attention since these transformations are in many cases di.cult to control in chemical processes.Asymmetric -hydroxylation of long chain carboxylic acids 33 has been achieved using molecular oxygen catalysed by the -oxidase from peas (Pisum sativum). (R)-Hydroxyacids 34 were produced enantiomerically pure on up to a 1 mmol scale. Double and triple bonds oxygen and sulfur atoms were tolerated in the sidechain as long as they were at least three carbons away from the carboxylic acid group (Scheme 12). The major by-product was the next lower aldehyde formed presumably by decarboxylation of the intermediate -peroxyacid. Turner and Flitsch have shown that Cbz-protected piperidines are biohydroxylated with greater regioselectivity than the corresponding N-benzoyl analogues when incubated with the fungus Beauvaria bassiana ATCC 7159 (Scheme 13). Most substrates were regioselectively hydroxylated in the 4-position giving compounds 35 except N-Cbz-3-methylpiperidine and N-Cbz-2-methylpiperidine which underwent hydroxylation in both the 3- and 4-positions.Previous models proposed 45 Annu. Rep. Prog. Chem. Sect. B 1999 95 39—58 Scheme 11 Scheme 12 Scheme 13 for this organism emphasised the importance of a distance of 3.3—6.2Åbetween the site of hydroxylation and the carbonyl group which is the same for both N-Cbz and N-benzoyl protected piperidines. These results suggest that the distance to the aromatic side-chain is also important and that the active site of the enzyme contains a de.ned aromatic binding pocket. All of the biotransformations were run to completion leading the authors to suggest that enantioselectivity was unlikely.The commercially available chloroperoxidase is now showing promise for hydroxylation reactions. In combination with hydrogen peroxide or tert-butyl hydroperoxide it has been possible to hydroxylate a range of substituted alkynes 36 (Scheme 14). For R larger than methyl the e.e.’s of the product propargylic alcohols 37 were high (78—95%) although yields were in most cases disappointing due to substantial overoxidation of these products to the corresponding ketones 38. Two exceptions were RAcOCH and BrCH where alcohol yields were 52 and 65% respectively. Enantioselectivities with BuOOH as the oxidant were slightly lower although in contrast to hydrogen peroxide which must be added slowly owing to the catalase activity of the enzyme it has the advantage that it can be added at the beginning of the reaction.Stewart et al. have recently used their recombinant baker’s yeast strain which expresses cyclohexanone monooxygenase to examine 2- and 3-substituted cyclopen- 46 Annu. Rep. Prog. Chem. Sect. B 1999 95 39—58 Scheme 14 Scheme 15 tanones as substrates (Scheme 15). In contrast to 2-alkylcyclohexanones which from a previous study display high enantioselectivity where RMe or larger the 2-alkyl substituted cyclopentanones 39 required at least a four carbon substituent in which case optically pure (R)-ketone 39 and (S)-lactone 40 could be isolated from a single biotransformation (E200). As with the cyclohexanones the 3-substituted cyclopentanones 41 where RMe Et n-Pr or allyl were oxidized to give both regioisomeric lactones 42 and 43.However side-chains of n-butyl or larger gave regioselectively the 5-alkyl lactone 42 but the e.e.’s were low and absolute con.gurations were not determined. The di.erences between behaviour of cyclopentanones and cyclohexanones were ascribed to the relatively low energy barriers between alternative cyclopentane conformations and the accessibility of half-chair structures in this ring system. Hence for the 2-substituted series a larger side-chain which can be involved in additional active site interactions is needed for the cyclopentanones where energy di.erences between substituents in pseudoaxial and pseudoequatorial postions in the twist boat conformation are minimal.These additional interactions of the larger side-chains also appeared to be responsible for regioselectivity in the 3-substituted cyclopentanones. Brosa and co-workers have carried out microbial Baeyer Villiger reactions in 47 Annu. Rep. Prog. Chem. Sect. B 1999 95 39—58 Scheme 16 organic solvents and biphasic conditions. In phosphate bu.er the model substrate norbornanone 44 normally gives the regioisomeric lactones 45 and 46 in a 7 1 ratio with Pseudomonas putida ATCC 10007 a bacterium known to contain two types of monooxygenase enzymes MO1 and MO2. However on using 1 1 mixtures of bu.er and immiscible solvents the ratio of 45 46 was reduced to 2 1 (Scheme 16). Although reaction time was not greatly a.ected using water/octane use of toluene as cosolvent required longer reaction time (11 h).Conversion and reaction time using decanol either as a biphasic system or as a single solvent were not useful. The biotransformation also ran at a similar rate in toluene or octane and formation of lactone 45 was favoured being exclusive in the latter solvent. Chemical Baeyer Villiger reaction using tri.uoroperacetic acid gave a 14:1 ratio of lactones in favour of 45.No enantioselectivities were reported from this study. A chemoenzymatic method for enantioselective Baeyer Villiger reaction using a lipase enzyme Novozyme 435 has been devised by Guibe� -Jampel et al. The process is autocatalytic and uses dry media and urea—hydrogen peroxide as the primary oxidant. Lipase catalysed perhydrolysis of an -substituted cyclohexanone is enantioselective generating 6-substituted caprolactones and the corresponding hydroxyacids in enantiomericall enriched form. The intermediate hydroxyperacids generated by the lipase perhydrolysis are themselves oxidants for the Baeyer Villiger reaction making the process autocatalytic.The most widely studied Baeyer Villiger enzyme is cyclohexanone monooxygenase which has also been used for asymmetric sulfoxidation of aryl—aryl dialkyl sul.des and 1,3-dithioacetals in the past. A recent development has shown it to be e.ective for promoting enantioselective oxidation of organic cyclic sul.tes to sulfates. The requisite redox cofactor NADPH was recycled using the established glucose-6-phosphate dehydrogenase system.cis- and trans-4-benzyloxymethyl-1,3,2-dioxathiolane- 2-oxides 47 were resolved by CHMO both diastereoisomers giving the (4R)-cyclic sulfate 48 although the trans diastereomer reacted more slowly and with lower enantioselectivity. 48 Annu. Rep. Prog. Chem. Sect. B 1999 95 39—58 Scheme 17 Interestingly the relative rate and sense of enantioselectivity were reversed for the oxidation of 4-methyl-1,3,2-dioxathiane 2-oxides 49. trans sul.tes reacted faster than cis sul.tes and showed higher enantioselectivity (Scheme 17). A wide variety of alkyl aryl sul.des have been oxidized using the toluene dioxygenase (TDO) or naphthalene dioxygenase (NDO) systems contained in strains of Pseudomonas putida in order to provide more comprehensive data on this reaction.Notably the bioxidation of phenyl methyl sul.de with Pseudomonas putida UV4 was scaled up to give ca. 8 g (90% yield) of enantiopure (S)-sulfoxide. In general enantiocomplementary results were observed with TDO-catalysed (UV4) oxidation favouring the (R)-enantiomer and NDO (P. putida NCIMB 8859) favouring the (S)-enantiomer. Microbial cis dihydroxylation of azulene and non-aromatic polyenes has been demonstrated using P. putida UV4 (Scheme 18). Azulene 51 gave the enantiomerically pure (4R,5S)-diol 52 in 20% yield. A range of other trienes and dienes 53–56 gave (1R,2S)-diols 57–60 in around 30% yield all enantiomerically pure except the diol derived from cyclopentadiene which had a 20% e.e. Oxidation of cycloheptatriene gave dienediol 57 and achiral dienediol 61 in a 2 1 ratio.Interestingly dihydroxylation of these substrates with the Sharpless AD-mix- reagent gave much lower selectivities 5—40% e.e. NDO in P. putida strains 8859 or 9816/11 gave the same selectivity but lower yields for all substrates except for azulene and cycloheptatriene which were not transformed. Eupergit immobilized nucleoside oxidase from Stenotrophomonas maltophila was used to generate 5-carboxylic acid derivatives of nucleoside analogues. The enzyme had a broad speci.city for unnatural nucleosides and aristeromycin and neplanocin A were also good substrates. Substrates with a methyl group in the 2-position of the ribose ring or 2,3-acetonides of natural substrates were not accepted. The reaction 49 Annu. Rep. Prog.Chem. Sect. B 1999 95 39—58 Scheme 18 was scaled up to 20 gL and addition of quinol at 1 gL served to stabilize the enzyme enabling it to be recycled. 5 Reductions A novel cofactor recycling system for NADH and FMNH has been devised by Bhaduri et al. using H as the terminal reductant. This was coupled to lactate dehydrogenase and the conversion of pyruvate to lactate demonstrated with NADH being regenerated. The strategy is based on the relay of electrons from hydrogen via a platinum carbonyl cluster and the redox dye safranine O (Saf) 62 to NAD (Scheme 19). In order to overcome solubility and stability problems associated with cofactors and the cluster respectively a biphasic system was employed where the safranine O shuttled between the two phases.The salt [Saf] [Pt (CO) ] was prepared and used to supply the two redox components to the system. An enzyme called pyridine nucleotide transhydrogenase from Pseudomonas .uorescens NCIMB 9815 was cloned sequenced and overexpressed in E. coli allowing relatively easy preparation of large amounts of the enzyme. This enzyme catalyses transfer of reducing equivalents 50 Annu. Rep. Prog. Chem. Sect. B 1999 95 39—58 Scheme 19 between NAD(H) and NADP(H) and can be used to enhance a biotransformation process and in enzyme based analytical assays. Adam has obtained new isolates from soil for the enantioselective reduction of alkyl hydroperoxides. The organisms were selected using hydrogen peroxide to induce peroxidase transcription followed by addition of the hydroperoxide substrate.Of the strains isolated the best in terms of enantioselectivity was Bacillus subtilis which converted 1-phenylethyl hydroperoxide with modest enantioselectivity to a.ord the (S)-alcohol (30% e.e.) and the (R)-hydroperoxide (88% e.e.). Fungal systems Aspergillus niger Botrytis cinerea and Penicillium verrucosum carried out the conversion with opposite selectivity as does the previously used horseradish peroxidase which generally gives much higher e.e.’s. Fujisawa et al. have reported enantiocomplementary biocatalysts for the reduction of tri.uoroacetyl biphenyl derivatives 63 (Scheme 20). The chiral alcohol products have application in liquid crystals. Baker’s yeast in water—ethanol gave variable yields and e.e.’s depending on the R group in 63 for formation of the (R)-con.gured alcohols 64 resulting from Prelog selectivity.Geotrichum candidum acetone powder in phosphate bu.er—alcohol with supplementalNAD a.orded variable yields and high e.e.’s for formation of the (S)-con.gured alcohols 64. In most cases the alcohol co-solvent was octan-2-ol but where ROH or OMe propan-2-ol was optimal. A similar complementary set of biocatalysts has been determined by Fogagnolo and co-workers for accessing either enantiomer of a wide selection of 1-heteroaryl- and 1-arylpropan- 2-ols in high enantiomeric purity using microbial redox reactions (Scheme 21). Stereoselective oxidation of racemic alcohols 65 was achieved with Pseudomonas paucimobilis giving 40—47% yields of the (R)-alcohols in high enantiomeric purity (90—100% e.e.).The corresponding ketones 66 could be reduced using one of three microorganisms again giving high yields and exclusive selectivity for formation of the (S)-alcohols. The range of heteroaromatic groups in these substrates accepted by these organisms is particularly noteworthy. The use of redox enzymes for the deracemization of alcohols is an attractive approach. One can employ isolated enzymes with matched selectivity and the requisite cofactors in a two step procedure or a whole cell system which contains the necessary enzymes and cofactors to drive the deracemization to completion. Adam has reported the combination of glycolate oxidase from spinach and .-lactate dehydrogenase from Lactobacillus leichmannii for the deracemization of 2-hydroxyacids 67 via the - ketoacids 68 (Scheme 22).The .rst enzyme requires .avin mononucleotide and oxygen to function. The oxygen is reduced to hydrogen peroxide which can be decomposed with catalase in situ. The NADH required by the lactate dehydrogenase was recycled using the well established formate dehydrogenase system. Since it was necessary to run 51 Annu. Rep. Prog. Chem. Sect. B 1999 95 39—58 Scheme 20 Scheme 21 Annu. Rep. Prog. Chem. Sect. B 1999 95 39—58 52 Scheme 22 Scheme 23 the second step (lactate DH) under nitrogen it was not possible to combine both steps and maintain high enantioselectivity (e.e.’s 67—91%) because under these conditions reduction of the ketoacid 68 to the opposite (S)-alcohol by the oxidase enzyme starts to compete.Notably phenyllactic acid was a poor substrate for this system and mandelic acid could not be transformed. Carnell et al. have recently shown that trans or cis indane-1,2-diols 69 and 70 can be deracemized using whole cells of Corynesporia cassiicola. As with the previously 53 Annu. Rep. Prog. Chem. Sect. B 1999 95 39—58 described substrate cyclohexane-1,2-diol the trans isomer reacted faster leading to 83% yield of the optically pure (S,S)-trans diol 69 (Scheme 23). The cis diol 70 underwent a similar transformation with both enantiomers being converted to the (S,S)-trans diol 69. Utaka has puri.ed a reductase enzyme to homogeneity (MW 37 KDa) from baker’s yeast and used it to carry out reduction of a range of typical substrates using the glucose-6-phosphate dehydrogenase system for cofactor recycling.1-Chlorohexan-2- one 1-acetoxyheptan-2-one methyl acetoacetate ethyl pyruvate 1-chloropentane- 2,4-dione and hexane-2,4-dione were all reduced to the corresponding alcohols in high e.e. (98%). Biotransformations with whole cells give signi.cantly lower selectivity in some cases. High speci.city constants (k/Km10—10 sM) and relatively low Michaelis constants (Km10 —10M) were measured for the substrates suggesting broad substrate speci.city of the enzyme. 6 Oligosaccharide synthesis The enzymatic synthesis of complex glycosides for the study of selectin binding continues to highlight the e.ciency of using biotransformations in this area.Thomas et al. have used galactosyl sialyl and fucosyl transferases to synthesize N-linked oligosaccharides terminating in multiple sialyl-Lewis (SLe)and GalNAc-Lewis determinants. Lubineau et al. have synthesized a 3 6 -disulfated Lewis pentasaccharide a candidate for human L-selectin using a chemoenzymatic approach. Wong et al. have synthesized .ve SLe dimers 73 and .ve SLe carboxylic acids 74 by coupling chemoenzymatically synthesized amino substituted SLe derivative 71 to homobifunctional cross-linkers 72 of varying chain length (Scheme 24). These derivatives were used in competitive binding assays to immobilized E- and P-selectin against a sialyl Lewis a polymer in order to probe the importance of multivalent interactions between the selectins and their natural glycosylated glycoprotein ligands.Particularly low IC values were observed for dimers where n3 and 6 and the strongest binding for the carboxylic acid derivative was where n2 for P-selectin. Hence optimal spacing between two SLe moieties could not be identi.ed. For the carboxylate a smaller entropy loss would be associated with n2 upon binding. Wong has also reported the use of a recombinant (13) galactosyl transferase for the synthesis of xenoactive -galactosyl epitopes. The interaction of epitopes bearing a Gal1—3Gal terminus on the surface of animal cells with anti--galactosyl antibodies in human serum is implicated in antibody-mediated hyperacute rejection in xenotransplantation.In a one-pot reaction an -galactosyl pentasaccharide was synthesized using the recombinant -(13) galactosyl transferase and -(14) galactosyl transferase both enzymes utilize the UDP-galactose donor. In situ cofactor recycling was employed to regenerate the expensive nucleotide donor and avoid product inhibition of the transferases. The e.ect of co-solvents on the stability and activity of -(1 4)-galactosoyl transferase from bovine colostrum and its ancillary enzyme UDP-galactose-4-epimerase was determined. Dimethyl sulfoxide and methanol could be used up to 20%v/v whereas tetrahydrofuran inactivated the transferase at 5%v/v. The former solvent mixtures were exploited for the galactosylation of the poorly water soluble coumarinic glucoside fraxin. 54 Annu.Rep. Prog. Chem. Sect. B 1999 95 39—58 Scheme 24 Montero and co-workers found that the regioselectivity of transgalactosylation of - and -xylose catalysed by -galactosidases depends on the source of the enzyme. Enriched mixtures of 4- 3- and 2-O--galactopyranosyl-xylose were obtained using the enzyme from E. coli bovine testes or Aspergillus oryzae respectively and for the corresponding -xylase derivatives the enzymes from A. oryzae lamb small intestine or Saccharomyces fragalis were required. A regioselective transglycosylation from p-NO--galactopyranoside to 1- deoxynorjirimycin was carried out using a-galactosidase from green coee beans. The major product was 6-O--galactopyranosyl-1-deoxynorjirimycin. Waldmann has reported the development of the tetrabenzylglucosyloxycarbonyl (BGloc) protecting group as an enzymatically removable urethane protecting function for peptide synthesis.BGloc amino acids are synthesized by converting amino acid allyl esters into the respective isocyanates followed by treatment with 2,3,4,6-tetra-O-benzylglucose and C-terminal allyl ester cleavage. The terminal urethane can be selectively cleaved by 55 Annu. Rep. Prog. Chem. Sect. B 1999 95 39¡X58Scheme 25 hydrogenation followed by hydrolysis with - and -glucosidase under mild conditions. 7 Carbon¡Vcarbon bond formation Kyoto Research Laboratories have scaled up an enzymatic process for the production of N-acetylneuraminic acid (Neu5Ac). A recombinant E. coli strain was used to overexpressN-acetylneuraminate lyase and -acyl-glucosamine epimerase. The two 56 Annu.Rep. Prog. Chem. Sect. B 1999 95 39¡X58enzymes were used simultaneously to convert 27 kg of N-acetylglucosamine (GlcNAc) and pyruvate into 29 Kg of Neu5Ac (77% conversion). 2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase catalyses the reaction between 3-phosphoglyceraldehyde (3-PG) and pyruvate. -Glyceraldehyde is accepted as a substrate at 1% the rate of the phosphorylated substrate. Given the synthetic potential of 4-substituted-4-hydroxy-2-ketobutyrates Toone et al. investigated the variability of this component and the possibility that phosphorylated 3-PG analogues may serve as substrates. Using enzymes from E. coli and Z. mobilis there appeared to be no systematic universal variation in reaction rate in response to phosphorylation. Michaelis constants for the 3-PG analogues varied only modestly when compared with changes in turnover rates (k) showing that although the analogues were binding both the C-2 hydroxy and the C-3 phosphate are required for productive catalysis.Oikawa et al. have used a crude preparation of an enzyme from the fungus Alternaria solani to catalyse the two step conversion of prosolanapyrone II into ()- solanapyrones A and D (Scheme 25). The rst step is an oxidation catalysed by an oxidase activity evidenced by the requirement for molecular oxygen and the HO liberated. This gives a system containing an activated dienophile for the second step which is the rst example of an enzyme-catalysed Diels¡XAlder reaction. The E,Zisomer of prosolanapyrone II was oxidized but did not undergo cyclization. From partial purication and preliminary characterisation the authors propose that a single bifunctional enzyme is responsible for the two steps based on its chromatographic behaviour.References 1 S.M. 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