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9 Enzyme chemistry By J. S. PARRATT,* M. C. CRIPPS S. J. FAULCONBRIDGE K. E. HOLT C. L. RIPPE� S. P. SAVAGE and S. J. C. TAYLOR Chiroscience Ltd. 283 Cambridge Science Park Milton Road Cambridge UK CB4 4WE 1 Introduction Biocatalysis is becoming an increasingly useful synthetic tool aiding organic chemists in the preparation of ever more challenging compounds. The principal benefits brought by these enzymatic methodologies include the stereo- regio- and chemoselective transformation of molecules typically under mild reaction conditions with respect to pH and temperature. Designer biocatalysts such as the abzyme are also attracting more interest as they are becoming better characterised and understood. This review follows closely in the footsteps of previous Annual Reports1,2 and has for clarity divided the various forms of enzyme-catalysed reactions.Subsequently biotransformations of both academic as well as commercial interest are discussed along with a variety of interesting practical and synthetic examples. It is hoped that this work adequately illustrates the healthy science that is enzyme chemistry. 2 Hydrolysis and condensation reactions Hydrolysis of complex acids and alcohols (1RS,7RS)-2-Oxotricyclo[2.2.1.0]heptane-7-carboxylic acid (])-2 is a useful intermediate in the synthesis of prostaglandins. Current preparative procedures rely on five successive crystallisations using (S)-1-phenylethylamine and (^)-2 giving at most 12% yield after 4 days. This year however two groups of researchers have investigated the possibility of a biocatalytic resolution and both groups reported the kinetic resolution of the methyl ester (^)-1 using Candida antartica lipase.The methyl ester (])-1 of correct stereochemistry was isolated in 41% yield ([98%ee) (Scheme 1).3,4 Kinetic resolution of amino acid esters catalysed by lipases and proteases has also been studied in some detail this year.5,6 An interesting variation on this theme is the biocatalytic hydrolysis of Schi§ bases derived from racemic amino acid esters and aromatic aldehydes. Such reactions were carried out in 19 1 acetonitrile–water systems where the amino acid precipitated as it was formed. The addition of an organic base 1,4-diazabicyclo[2.2.2]octane (DABCO) to the reaction resulted in racemisation of the D-ester giving up to 88% yield of the L-amino acid.7 Royal Society of Chemistry–Annual Reports–Book B 291 MeO2C O MeO2C O CO2H O i + (±)-1 (+)-1 (-)-2 Scheme 1 Reagents i Candida antartica lipase pH 7 bu§er 50 °C Ph OAc 3 Ph OH (+)-4 i Scheme 2 Reagents i Pseudomonas cepacia lipase pH 7 bu§er 37 °C 5mol% PdCl 2 (MeCN) 2 Scheme 3 Reagents i Porcine pancreatic lipase pH 6.9 bu§er 37 °C Enantiomerically pure 1-phenylethylamine and substituted analogues are popular agents for the classical resolution of chiral carboxylic acids.Aside from enzymecatalysed transamination and lipase-catalysed acylation there are few other suitable methods for the preparation of enantiomerically pure amines. Chapman et al. however have demonstrated a worthy method for the biocatalytic resolution of 1- phenylethylamine and analogues by Candida antartica lipase-mediated hydrolysis of their octyl oxalamic esters.Excellent enantiomeric ratios (E[100) were reported.8 The resolution of secondary alcohols using lipases forms the bulk of the literature on ester hydrolysis. This methodology has found many applications within the pharmaceutical industry giving access to calcium channel blockers,9 antibiotics10,11 and anti-virals.12,13 The preparation of insecticides14,15 and antiferroelectric liquid crystals16 have also enjoyed success using this technology. A useful dynamic resolution of the 2-phenylcyclohexen-2-yl acetate 3 was reported whereby enzymatic hydrolysis using Pseudomonas cepacia lipase was coupled with PdII catalysed in situ racemisation of the substrate. The alcohol (])-4 was isolated in 81% yield and 96%ee (Scheme 2).17 The biocatalytic hydrolysis of prochiral diesters to give optically active monoalcohols is a valuable asymmetric methodology several good examples of which were reported this year.18–20 This technique has also been applied to the monoselective hydrolysis of chemically-equivalent diesters e.g.the diacetate of butynediol 5 was converted exclusively into the monoacetate 6 with an impressive 95% yield (Scheme 3).21 Transesterifcation of complex acids and alcohols As in previous years the most studied area of biotransformations proves to be lipase-catalysed transesterifications. The regio- and stereo-selective properties of such enzyme-catalysed reactions can often be essential for the direct enantiomeric resolution of novel pharmaceuticals or in the formation of enantiomerically pure syn- 292 J. S. Parratt et al.OH SBut OAc SBut OH SBut OH SBut OAc SBut OAc SBut OH SBut OH SBut i 25% yield >99% ee 50% yield >99% ee 24% yield >99% ee Scheme 4 Reagents i Lipase PS-30 (50% w/w) vinyl acetate hexane 37 °C 8 days thons.22,23 Examples include the synthesis of both enantiomers of the non-steroidal anti-inflammatory Suprofen by Mertoli et al. employing the commercially available enzymes Candida antarctica lipase and Mucor miehei lipase.24 The resolution of synthetic intermediates such as terminal diepoxides used in two-directional chain synthesis has been explored by Hoye and Tan.25 Transesterification of bis(b-hydroxyalkyl) thioethers catalysed by Amano-PS lipase with vinyl acetate as the acyl donor yields the corresponding diol monoacetate and diacetates with extremely high optical purity in all cases ([99%ee Scheme 4).The corresponding enantiomerically pure a,x-diepoxides are subsequently synthesised by S-alkylation and base promoted intramolecular displacement. Lipase from Candida antarctica has played a key role in the synthesis of stable highly unsaturated glycerides. Previous research has shown that glycerols of polyunsaturated fatty acids can be just as unstable as the free fatty acids and therefore can only be handled in an inert atmosphere. However glycerols of stable unsaturated fatty acids such as the unnatural carotenoic acids studied by Partali et al. have been synthesised using the aforementioned enzyme.26 Biotransformations have proved extremely useful in the original syntheses of many novel compounds. Enantiomerically pure biindolizines were prepared on a preparative scale by a transesterification catalysed by Candida antartica lipase.27 (])-Crooksidine an indole alkaloid from the perennial shrub Haplophyton crooksii was synthesised in high optical purity by a Lipase-PS mediated esterification of the racemic 1-benzyloxycarbonyl-1,2,5,6-tetrahydropyridin-5-ol precursor.28 The successful bioresolution of hydroxy groups in steroids has previously been well reported.However it should be noted that in all cases the hydroxy group was located in the steroid ring. 293 Enzyme chemistry OAc OH O OH 20 R,S 20 S 20 R 20( S):20( R) = 2:1 70% yield 20( S):20( R) = 4:1 i + Scheme 5 Reagents i Pseudomonas cepecia lipase vinyl acetate chloroform 68 h S OH Fe S OAc Fe S OH Fe 48% ee 90% ee i Scheme 6 Reagents i Novozyme 435 diisopropyl ether vinyl acetate 32% conversion 55 min Ferraboschi et al.have recently reported that successful acylation of the steroid side-chain hydroxy groups can also be achieved using Pseudomonas cepecia lipase as illustrated in Scheme 5.29 Enzyme-mediated reactions have also had a part to play in organometallic chemistry. Chromium(0) complexes of cyclic trienes are useful intermediates for the formation of stereochemically rich polycyclic ring systems. Many of these cycloadducts were previously synthesised by auxilliary-control methods however Rigby and Sugathapala have reported the synthesis of these compounds with good optical purity utilising Lipase PS-30 with isopropenyl acetate as the acyl donor.30 Lambusta et al. demonstrated the enzymatic resolution of compounds such as 2-hydroxymethyl-1- methylferrocene which are particularly interesting compounds in asymmetric synthesis since they can be used in homogeneous catalysis.31 Utilizing Candida antarctica lipase-catalysed transesterificati the 1R enantiomer of the ferrocenyl derivative was furnished with good stereopurity (Scheme 6).Although enzyme-mediated resolutions to obtain chiral building blocks have been well reported this year many studies have also been carried out on the optimisation of biotransformation conditions in most cases to increase optical purity. The enhanced enantioselectivity of Lipase-PS in the bioresolution of allyl alcohols has been studied by Takagi et al.32 The addition of 5 mol% of thiacrown ether to the substrate in propan-2-ol with 1.5 equiv. of vinyl acetate not only increased the stereoselectivity of the process but also had a remarkable e§ect on the reaction rate.A high proportion of the optimisation studies performed have been centred around the e§ect of the acyl donor.33,34 The enantiomer ratio of three secondary alcohols by resolution with Canida antarctica lipase was found to be dependent on the alkyl part 294 J. S. Parratt et al. of the acyl donor. 2-Chloroethyl butanoate 2,2,2-trichloroethyl butanoate vinyl butanoate and butanoic anhydride were studied by Ho§ et al.35 It was reported that the highest E value was observed with 2-chloroethyl butanoate. Interestingly butanoic anhydride and vinyl butanoate caused the lipase to be less specific. One of the most popular acyl donors at present is vinyl acetate largely due to its high reactivity its ability to make the reaction irreversible and a§ord products in high chemical and optical purity in short reaction times.However its use is often limited as the acetaldehyde liberated from the donor often inactivates enzymes. A novel variation on this 1-ethoxyvinyl acetate has been studied by Kita et al.36 This has the major advantage of liberating ethyl acetate as a by-product which is significantly less harmful to biocatalysts. Another area of intense interest is the optimisation of enzyme-catalysis using microwave irradiation to increase the reaction rate.37,38 Candida antarctica lipase catalysed esterifications of methyl a-glucopyranosides under focussed microwave radiation were compared with those using classical methods of heating. After 5 h complete conversions were obtained under the microwave heated conditions whereas a conversion of only 55% was obtained under classical heating conditions.It was concluded that improvements could be a result of more expeditious water removal under irradiation and possibly some specicific non-thermal e§ects of microwaves. Amide hydrolysis and condensation The use of enzymes to remove phenylacetamido protecting groups from chemically sensitive phosphopeptides has been further described by Waldmann et al.39 Penicillin G acylase e¶ciently hydrolyses the phenylacetamido group at pH6.5 and room temperature without attacking peptide bonds C-terminal esters or phosphates. Furthermore this methodology has been shown to work e§ectively across a broad spectrum of structurally diverse phosphopeptides making this an extremely useful synthetic tool in the preparation of biologically active peptide conjugates.In a similar vein a native phthalyl amidase enzyme isolated from Xanthobacter agilis was reported as being able to deprotect a variety of phthalimido substrates once having been partially hydrolysed into their corresponding mono-acids (pH 8.0 bu§er solution).40 Although only a few phthalimido substrates have been reported to date its potential as a mild deprotection catalyst is clear. The preparation of biologically active peptides is currently enjoying a period of resurgence with intensive research programmes being carried out both in academia as well as industry. Subsequently many advances have been made in the application of enzyme processes to furnish such peptides for the pharmaceutical food and flavours industries and is the subject of a recent review.41 The cross-linked enzyme crystal (CLEC) has proven to be a useful technology when applied to amide bond formations.Such biocatalysts are extremely stable to exposure with organic solvents and aqueous –organic mixtures with a recent example CLEC-subtilisin being used to e¶ciently catalyse the synthesis of a range of optically active alkylamides. The enzyme was able to accept a broad range of substrates which possessed a variety of N-protecting and ester groups.42 Elsewhere biocatalytic amide condensation reactions have been improved through the use of activated ester groups as the acyl donor instead of the more usual methyl ester. Such an approach was found to increase greatly the e¶ciency of non-protein amino acid insertion into peptides using the protease a-chymotryp- 295 Enzyme chemistry O HO HO NHAc O pNP OH O HO HO NHAc OH OH O HO HO AcHN OH O O HO NHAc OH OH + i 7 8 9 O HO HO O pNP HO OH O HO HO O O HO AcHN OH O HO NHAc OH OH HO OH ii 10 11 Scheme 7 Reagents i N-acetylhexosaminidase; ii b-mannosidase; pNP\p-nitrophenyl sin.43 Candida antarctica lipase has been widely reported with respect to its ability to catalyse amidation reactions.Recent reports describe the resolution of (^)-transcyclohexane- 1,2-diamine,44 (^)-ethyl 2-methyloctanoate45 and the enantioselective aminolysis of prochiral 3-hydroxyglutarate.46 Glycosidation reactions Crout and co-workers have been amongst the most prolific researchers in the area of enzymatic glycosidation chemistry. Recent work has centred around the use of b- galactosidase derived from Bacillus circulans to synthesise a variety of interesting oligosaccharides.47,48 However the same research group has also reported an elegant application for the b-mannosidase and b-N-acetylhexosaminidase enzymes from Aspergillus oryzae.In a two step process the donor 7 is coupled with the acceptor 8 to form the disaccharide 9 using the part-purified b-N-acetylhexosaminidase. Subsequently the trisaccharide 11 is exclusively formed by the transfer of a b-mannosyl unit from 10 to the disaccharide 9 using the b-mannosidase activity (Scheme 7).49 Ohrlein and co-workers have turned their attention to the use of cloned a(2-3)sialyltransferase (from rat liver)50 and fucosyltransferase VI.51 In both cases the catalysts have demon- 296 J. S. Parratt et al.strated a wide substrate tolerance with non-natural di- and tri-saccharides being readily sialylated or fucosylated accordingly. Epoxide and nitrile hydrolysis Epoxide hydrolases are becoming increasingly popular as biocatalysts for the synthesis of enantiomerically pure epoxides and diols especially from microbial sources such as fungi and bacteria. These allow almost unlimited production of the catalyst. Enzymes from mammalian sources are utilised on an analytical scale but their applications on a preparative scale are impeded by the limited supply of the enzyme.52 It has been shown that the biocatalysed hydrolysis of racemic epoxides can be complementary to chemical approaches where the product obtained shows a low ee value. The epoxide of dihydronaphthalene and epoxyvindene cannot be obtained in an optically pure form via chemical methods.Epoxyvindene is a key intermediate in the synthesis of an orally active HIV protease inhibitor which is one of a group of compounds that are in advanced trials for the treatment of AIDS. The ee values obtained using a fungal epoxide hydrolase mediated hydrolysis are much higher than those from a chemical asymmetric method i.e. Sharpless dihydroxylation process. The drawback of using a biocatalysed hydrolysis is the intrinsic yield limitation (to 50%) of such resolution processes. Therefore the combined use of Jacobsen–Katsuki asymmetric epoxidation and the biocatalytic approach constitutes an excellent compromise to obtain the previously mentioned epoxides in high yields and approaching 100%ee.53 The mechanism employed by epoxide hydrolases has recently been studied and consequently has provided information on the stereochemical outcome of kinetic resolutions of racemic epoxides.Knowledge of the regio- and stereo-chemical course of microsomal epoxide hydrolase (mEH)-catalysed hydrolysis is useful in understanding the biological e§ect and small scale kinetic resolution of epoxides as well as in the preparation of chiral vicinal diols.54 The enantioconvergent transformation of racemic cis-dialkyl substituted epoxides to the (R,R)-threo-diols has been demonstrated both using a microsomal epoxide hydrolase and Nocardia EH1-epoxide hydrolase. In the former case the epoxides used included racemic 9,10-epoxystearic acid racemic 5,6-epoxyhexadecane racemic cis 11,12-epoxyhexadecan-1-ol and meso cis-9,10-epoxyoctadecane.55 In the latter case racemic cis-2,3-epoxyheptane was specified.56 The enantioconvergent pathway involved nucleophilic attack at the (S) configuration carbon resulting in production of the respective (R,R)-threo-diol in a[90%ee.Brennan et al. recently identified two cobalt-containing nitrile hydratases from R. Rhodochrous J1. By comparison of the K-edge X-ray absorption spectrum for Co3` with that of Fe3` found in the nitrile hydratase from Rhodococcus sp. R312 it was deduced that the cobalt is either five or six coordinate in the nitrile hydratase. The use of Fourier filtered EXAFS revealed the coordination sphere of the low spin cobalt complex to be S 2 N 2 O 2 . NO coordination was found not to exist in this particular hydratase however charge-transfer transitions support coordination of sulfur in the low-spin six-coordinated cobalt complex.It was concluded that the nitrile hydratase from R. Rhodochrous J1 is the first example of a native non-corrin cobalt enzyme exhibiting biological Co–S coordination.57 Turner and co-workers have reported the application of enzymatic nitrile hydrolysis 297 Enzyme chemistry O OTBS I O CO2H OH CN CN OBn CO2H CN OBn CN OTBS 14 65% yield 88% ee 15 13 12 i ii iii 86% yield 88% ee Scheme 8 Reagents i Brevibacterium R312; ii TBAF THF; iii Rhodococcus sp. SP 361 phosphate bu§er for the synthesis of the protected lactone 12 which is a precursor to the mevinic acids which are e§ective antihypercholestemic agents. The formal synthesis of the silyl protected lactone 12 was successfully completed in 12 steps starting from the readily prepared 3-benzyloxyglutaronitrile 13 (Scheme 8).The nitrile hydratase from Rhodococcus sp. and Brevibacterium sp. catalysed the hydrolysis of the pro-S nitrile group of protected 3-hydroxyglutaronitrile. This produced the 3-(S)-acid 14 in 65% yield with an 88%ee. A second non-steroselective application of the two step nitrile hydratase –amidase biotransformation catalysed by the immobilised cell preparation SP361 was used for hydrolysis of the nitrile group in 15 producing the corresponding acid in 86% yield.58 3 Reduction reactions Once again a number of reductions of carbonyl groups using bakers’ yeast (Saccharomyces cerevisiae) have been reported which lead to useful synthons.59–62 This area has been widened by a study into the relative diastereoselective reduction of chiral organometallic or organic deuterioaldehydes which demonstrates the importance of both planar chirality and aldehyde configuration in the diastereoselection process.63 Facial diastereoselectivity of spiro[4.5]decane-1,4-dione derivatives ranged from excellent to non-existent when using bakers’ yeast to mediate reduction.Whereas better results from sodium borohydride reduction indicated influence from stereoelectronic phenomena from which the yeast-mediated reductions could not benefit significantly. 64 Hitherto unprecedented the reduction of vinylic nitro functions was used to prepare 298 J. S. Parratt et al. N O R R Ph O2 N CN Ph NH2 16 17 i R = Ph R = Me R = H 75% yield 80% yield 75% yield Scheme 9 Reagents i bakers’ yeast 5-aminoisoxazoles 17 from substituted (Z)-3-nitropropenenitriles 16 in moderate to good yields as outlined in Scheme 9.65 The prevention of over-reduction improvement of regioselectivity and enantioselectivity in the bakers’ yeast mediated reduction of a-diketones was achieved by the composite e§ect of using an enzyme inhibitor and thermal pre-treatment.Subsequently the reduction reaction proceeded without any formation of the associated diol and in high ee.66 Nakamura and co-workers expanded on their initial work which showed that acetone powder from G. Candidum IFO4597 (APG4) catalysed the reduction of ketones yielding alcohols with improved optical purity by investigating trifluorinated analogues which reacted via a completely di§erent stereochemical course. For example the reduction of 1,1,1-trifluorodecan-2-one yielded the corresponding (S)- alcohol in[99%ee while the reduction of decan-2-one proceeds on the opposite face of the carbonyl yielding the corresponding (S)-alcohol in[99%ee.67,68 Both enantiomers of d-hydroxymethyl valerolactone and e-hydroxymethyl caprolactone were obtained in good yields and high enantiomeric purity using bakers’ yeast and Rhizopus arrhizus catalysed reduction of ethyl 2-oxocyclopentane- or 2- oxocyclohexane-carboxylates respectively.69 Similar results were reported for 2-carboxyethyl- 1-tetralactone when various microorganisms were used; a simple predictive model was constructed from the consistent stereochemical features observed.70 Elsewhere fungi yeasts and bacteria screens for reduction of a,a,a-trifluoro-a@-phenylsulfenyl ketones provided five organisms which gave either or both the anti and syn isomers with satisfactory results.71 The stereoselective reduction of acylsilanes with free and immobilised (calciumalginate matrix) resting cells of Trigonopsis variabilis to the a-hydroxysilanes was reported in high chemical and stereochemical yields.Storage of the immobilised cells at 4 °C for 2 h did not visibly a§ect their activity and they could be recycled for three biotransformations before any drop in activity was observed. This drop was however stabilised after 15–16 cycles thus providing a useful route to compounds with a stereogenic silicon centre.72 Comparison of the enantioselectivity of yeast-mediated reduction of model prochiral ketones under high hydrostatic pressure and high pressure showed in some cases increased yields and inversion of enantioselectivity compared with control reactions carried out at atmospheric pressure.However a more detailed knowledge of the protein structure of the enzyme will be necessary in order to understand fully the promising results reported.73 Elsewhere the isolation and characterisation of a reductase enzyme which is respon- 299 Enzyme chemistry OOH OOH OH OOH OOH R2 R1 R1 R1 R1 R1 R1 R2 R2 R2 R2 R2 OH OH OH OH OH OH OH ( R R) ( R S) ( S S) ( S R) ( R* R*) ( R* S*) + + i R1 = Me Et Pri Bu t R2 = H Scheme 10 Reagents i horseradish peroxidase guaiacol sible for the reduction of an enone carbon–carbon double bond has been reported.74 Also of interest is the horse radish peroxidase enzyme which was shown to synthesise optically active a,b-unsaturated hydroxy functionalised hydroperoxides where no other method is presently available.This methodology is illustrated in Scheme 10.75 4 Oxidation reactions Optically pure sulfoxides are of great pharmaceutical interest. However the use of such sulfoxides has been hampered by di¶culties in their preparation. This year sees a large number of publications on the subject illustrating that an enzymatic approach can be used to solve the problem. Colonna et al. reviewed the application of bacterial cyclohexanone monooxygenase (CMO) to the enantioselective oxidation of organic sulfur compounds to sulfoxides.76 More specifically they have also examined the asymmetric oxidation of 2-substituted dithianes dithiolanes and oxathiolanes catalysed by CMO.77 Most flavin monooxygenases investigated so far utiliseNADPH as a reductant.This is less easily regenerated than NADHand therefore costly and not easily scaled-up. This could be overcome by using whole cell catalysts. To this end Kelly et al. have been investigating the whole cell oxidation of aryl alkyl sulfides (18 Scheme 11) and found it to be only slightly less enantioselective than the isolated 300 J. S. Parratt et al. SR¢ R S R R¢ O S R R¢ O + ( R) ( S) R HF Me Me Me Me Me Me Et Pr R¢ 100% ( R) 40% ( R) 34% ( S) 80% ( S) 58% ( S) Acinetobacter sp. NCIMB 9871 (WC) 18 •• •• i Scheme 11 S S S S O i A. calcoaceticus 76% yield 98% ee ( R) Pseudomonas sp. 85% yield 57% ee ( S) 19 Scheme 12 Reagents i Acinetobacter calcoaceticus NCIMB 9871 or Pseudomonas sp. NCIMB 9872 enzyme transformation.78 Elsewhere the preparative scale enantioselective oxidation of 1,3-dithiane to the corresponding mono sulfoxide a useful chiral auxilary has been described.79 Here two bacteria Acinetobacter calcaceticus NCIMB 9871 and Pseudomonas sp.NCIMB 9872 were used to prepare both enantiomers of 1,3-dithiane 1-oxide 19 (Scheme 12). Allenmark and Andersson have been investigating the chloroperoxidase (CPO) catalysed oxidation of a series of rigid aromatic bicyclic sulfides.80 This approach allows (R)-sulfoxides 20–22 to be obtained with higher enantiomeric excess than previously reported showing an increased applicability of CPO (Table 1). Site-directed mutagenesis is a technique which may be used to alter the characteristics of an enzyme. Myoglobin (Mb) can catalyse peroxygenation of a variety of substrates including olefin epoxidation and thioether sulfoxidation but turnover numbers are low.Thus Ozaki et al. have performed site-directed mutagenesis studies on Mb successfully giving mutants with greatly increased rates and enantioselectivity. 81 Baeyer–Villiger oxidations also feature significantly in the literature of 1996. Ottolina et al. update us on their e§orts to find an active-site model for one of the most important enzymes cyclohexanone monooxygenase from Acinetobacter NCIMB 9871.82 Elsewhere with the aim of extending the scope of these biotransformations Alphand et al. has shown that a variety of a-substituted cyclohexanones may be transformed into optically active caprolactones using whole cell cultures of Acinetobacter TD63 or the MO2 enzyme purified from Pseudomonas putida NCIMB 10007.83 The use of whole cells a§ords a very straightforward single step procedure allowing for the preparative scale synthesis of these useful chirons.The catalytic 301 Enzyme chemistry Table 1 Yield (%) Ee (%) S S S O O O O •• •• 20 99.5 99 21 10 96 22 9 95 X R¢ R X R¢ R OH OH XH R¢ R OH O R X R' R X OH OH R¢ R XH O OH R¢ i i cis/trans cis/trans E.g. R¢ = H R = Me X = S yield 11% ee 48% cis/trans 60:40 E.g. R¢ = H R = Me X = S yield 79% ee >98% cis/trans 78:22 Scheme 13 Reagents i oxygen P. putida UV4 repertoire of baker’s yeast has successfully been expanded to include enantioselective Baeyer–Villiger oxidations.84 This catalyst was created by inserting the Acinetobacter sp.CMOgene into a yeast expression vector creating a ‘designer yeast’ that performed whole cell Baeyer–Villiger reactions on a 1 mmol scale in high yields and with high enantioselectivity.The regio- and stereo-selective introduction of oxygen into an unactivated carbon –hydrogen bond is another important application of oxidative enzymes. In a set of 302 J. S. Parratt et al. H H H H R OH R [O] i 23 R = Me OH Br N3 I Scheme 14 Reagents i P. putida UV4 O 2 OH R1 OH R1 OH O R1 O O O R1 R2 i i ii R 1= H Me Cl Major product Scheme 15 Reagents i tyrosinase CHCl 3 O 2 ; ii R2CHCH 2 (R2\OEt Ph) three publications Griengl and co-workers investigate the microbial oxidation of 2-cycloalkylbenzoxazoles.85–87 Elsewhere the substrate selectivity of the a-oxidation of carboxylic acids by crude homogenate of young pea leaves was investigated.88 In contrast to previous reports this work showed that not only long chain fatty acids but a broad variety of saturated acids and even oleic acid with an unsaturated C18 chain are recognised as substrates giving the corresponding (R)-2-hydroxy acid products.There have been many reports in recent years of the hydroxylation of a variety of arenes with strains of Pseudomonas putida. This year Hudlicky et al. report the synthesis of new chiral synthons from the biooxidation of 1- and 2-bromonaphthalenes using whole cells of P. putida NCIB 9816-11.89 In addition a new range of heterocyclic ring cis/trans-dihydrodiol derivatives have been obtained from the metabolism of mono- and bi-cyclic heteroarenes (Scheme 13).90 Meanwhile Boyd et al. report the P. putida UV4 catalysed synthesis of enantiopure benzylic alcohols 23 containing two stereogenic centres in a cis relationship from the stereoselective monohydroxylation of achiral 2-substituted indans (Scheme 14).91 Domino reactions are sequential bond forming or breaking processes during which the subsequent transformations occur at the functionalities generated in the preceding step.With this in mind Muller and Waldmann have shown that simple phenols can be converted in high yields to highly functionalised bicyclic 1,2-diketones in a one-pot domino reaction.92 This consists of a hydroxylation (tyrosinase) an oxidation (tyrosinase) and a subsequent Diels–Alder reaction (Scheme 15). (R)-([)-Mevalonolactone 24 is an important intermediate in biosynthetic pathways leading to sterols terpenes carotenoids and other pentanoid compounds. Lakner and Hagar report the first multistep synthesis based on an enantioselective epoxidation moderated by chloroperoxidase (Scheme 16).93 Since chloroperoxidase is readily available and is highly selective its use in the synthesis of di¶cult intermediates should feature in future work.303 Enzyme chemistry O OEt O OEt O O HO O i 24 93% ee Scheme 16 Reagents i CPO ButOOH citrate pH 5.5 5 Carbon–carbon bond forming reactions Stereoselective carbon–carbon bond formation using oxynitrilases continues to develop and as in previous years the oxynitrilases from almond meal and Sorghum bicolor have attracted the most attention. Kiljunen and Kanerva94 compared both enzymes using either free HCN (by di§usion from a diisopropyl ether solution) or HCN generated in situ from acetone cyanohydrin and found that free HCN was generally preferable to maintain good ee.Enantiomeric purity of the (S)-cyanohydrins from Sorghum bicolor was lowered using acetone cyanohydrin possibly due to the high water content needed to decompose the acetone cyanohydrin or from the acetone generated. In a di§erent study Danieli et al.95 investigated the influence of a stereocentre already present in the substrate on the stereoselectivity of almond meal oxynitrilase. Compounds 25 and 26 were chosen as substrates. For 25 with the CHO CHO 25 26 X NH2 HO2C OH 26 X = S O stereocentre close to the aldehyde the enzyme gave a mixture of all four diastereomers but for 3-phenylbutyraldehyde 26 the two main products were the (2R,4S) and (2R,4R) isomers with only traces of the 2S containing isomers. The use of recombinantE.coli transketolase is reported with more detail on scale-up of the biocatalyst production by Hobbs et al. who scaled the fermentation to 1000 l and showed that a clarified cell extract was a suitable biocatalyst.96 Other work by Morris et al. describes improved synthetic methods for the key substrate potassium hydroxypyruvate and a more convenient biotransformation in unbu§ered aqueous media with automatic pH control.97 A novel aldolase was described by Bycroft et al.,98 from Streptomyces amakusaensis which catalysed a reverse aldolase reaction on (2S,3R)-b-hydroxy-a-amino acids such as 3-phenylserine. The enzyme tolerated other aromatic (heterocyclic) side-chains and 304 J. S. Parratt et al. was used to prepare optically pure (2R,3R)-3-(2-thienyl)serine and (2R,3R)-3-(2- furyl)serine from the readily synthesised threo mixtures 26.6 Abzymes Catalytic antibodies are continuing to find useful applications in organic synthesis. Recent reports have further highlighted the ever widening spectrum of reactions that can be successfully catalysed by abzymes. Examples of such reaction types include isomerisation,99,100 2,3-elimination,101 retro-aldol102 and the hydrolysis of tetrasubstituted phosphorus(V) compounds.103 Antibodies have also been shown to exhibit thiol-S-transferase activity whereby thiol nucleophiles are added into a nitro-substituted styrene derivatives.104 Although antibodies have been shown to catalyse a broad number of reactions they have often been unable to accept a wide variety of substrates while retaining their selectivity.However Fujii and co-workers have reported an abzyme which was able to hydrolyse the L-isomers of racemic N-(benzyloxycarbonyl)- amino acid esters exhibiting both a high degree of enantioselectivity as well as a wide substrate spectrum.105 Hence a genuinely useful synthetic catalyst has been achieved from antibody technology making this an increasingly attractive area of research. References 1 S. J. Faulconbridge K. E. Holt J. S. Parratt S. P. Savage and S. J. C. Taylor Ann. Rep. Prog. Chem. 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ISSN:0069-3030    

DOI:10.1039/oc093291

出版商:RSC    

年代:1997

数据来源: RSC