106

Natural products are ideal training compounds for enzymaticcatalysis. New transformations have become possible on apreparative scale thanks to molecular biology, which has mademany new enzymes available. Additionally, new syntheticpathways have been developed to regenerate expensivecofactors in situ and to improve enzyme selectivity.

AddressesIstituto di Biocatalisi e Riconoscimento Molecolare, CNR, Via MarioBianco 9, 20131 Milano, Italy; e-mail: Rivas@ico.mi.cnr.it

Current Opinion in Chemical Biology 2001, 5:106–111

1367-5931/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.

IntroductionThe peculiar properties of enzymes, especially their highselectivity and their ability to work under mild reactionconditions, appear to be particularly suitable for supportingthe synthesis and the modification of natural products. Infact, despite the wide use of chemical approaches, seriousproblems are sometimes encountered in the manipulationof these sensitive molecules. Even in cases where a simplechemical procedure exists for a given biotransformation(e.g. alcohol oxidation), the chemoselectivity and regiose-lectivity of the biocatalytic approach may eliminate theprotection/deprotection steps needed in the syntheticchemical route.

This review covers the literature published on this subjectin the past two years. It is a limited time period, but thetopic is impressively wide, as the world of natural com-pounds is not limited to some fancy metabolites isolatedfrom exotic plants or marine organisms but also includescarbohydrates, peptides and, strictly talking, even proteins.Because it is impossible to cover exhaustively a subject thatshould move, for example, from the enzyme-mediated syn-thesis of new oligosaccharides and polysaccharides to theelaboration of proteins by site-directed mutagenesis or bysite-directed modification [1••], this paper presents someselected examples that will give an overview of the poten-tial of biocatalysis in this area.

Enzymatic modification of natural productsSugars and glycosidesOligosaccharides and glycoconjugates have been recognizedas mediators of important biological processes and their ther-apeutic potential is one of the ‘hot’ subjects of biochemistry[2]. The presence of several stereogenic carbons as well asthe similar chemical reactivity of the numerous hydroxygroups of carbohydrate molecules still make the transforma-tion of these natural compounds a challenging target. Theapplication of enzymatic catalysis in this area has followedthe four classical main pathways, detailed below.

Specific aldolasesSpecific enzymes, namely aldolases, have been employed forthe synthesis of sugars and sugar derivatives from simplerprecursors [3•,4–6]. For example, pure D-xylulose-5-phos-phate, a valuable substrate required for enzymatic assays, hasbeen prepared in gram quantities by a multi-enzymatic one-pot procedure starting from the readily available precursorshydroxypyruvate and fructose 1,6-bisphosphate [3•].

Specific transferases and glycosidasesSpecific sugar transferases and sugar hydrolases (glycosidas-es) have been used to transform suitable glycosides intovaluable disaccharides and oligosaccharides as well as intotheir corresponding conjugates with other hydroxylatedcompounds. New and significant examples related to theuse of glycosyltransferases have been reported by Palcic andco-workers [7•] and by Wong and co-workers [8] notable for,respectively, the synthesis of analogues of blood grouptrisaccharide antigens, and a glycopeptide carrying the sialylLewis X moiety. New examples of glucosylation and glu-coronidation of macrolide antibiotics [9•] and alkaloids[10,11] have also been reported (see also Update).

Glycosidases are hydrolytic enzymes that, under suitablereaction conditions, can also be used for the synthesis ofthe glycosidic bond. Major drawbacks of this methodology,when applied to the preparation of oligosaccharides, arethe low yields and the generally poor regioselectivity in theglycosylation of the sugar acceptors. Efforts have focusedon ways to overcome these limitations, and two successfulapproaches have been proposed. The first one simplyrelies on the differing behavior of enzymes isolated fromvarious natural sources [12,13,14•,15•]. For instance,α-galactosidases able to produce terminal α-D-Galp-(1→3)-D-Galp or α-D-Galp-(1→4)-D-Galp (where Galp isgalactopyranosyl) have been isolated from Penicillium mul-ticolor [14•] and from Aspergillus niger [15•], respectively.

In a second approach, the more reactive sugar primary OHgroups have been selectively acylated by lipases or proteas-es in organic solvents [16•] so that glycosylation could occurselectively on one of the secondary OH groups [17••,18•].As shown in Figure 1, the trisaccharide iso-globotriose wasprepared from lactose in just two enzymatic steps followedby standard deacetylation [17••]. Additionally, glycosidasescan be used for the synthesis of alkyl glycosides in biphasicsystems, and the search for more suitable reaction condi-tions is still an important topic [19–21].

Lipases and proteasesHydrolytic enzymes such as lipases and proteases havefound interesting applications in the regioselective acyla-tion and deacylation of carbohydrates. The unusualexploitation of these enzymes (lipids a nd proteins are the

Biocatalytic modification of natural productsSergio Riva

Biocatalytic modification of natural products Riva 107

natural substrates of lipases and proteases, respectively) inorganic solvents constitutes an interesting approach to theselective protection of the different sugar hydroxylgroups [22••,23].

Recent reports range from the optimization of reactionconditions [24,25] to new applications, such as a support tothe chemo-enzymatic synthesis of ganglioside analogs [26],the preparation of specific sugar esters (i.e. esterification ofascorbic acid with a carotenoid, bixin, to protect the latter

compound from air oxidation [27]), and the synthesis ofsugar-based monomers suitable for radical polymerizationto get biodegradable macromolecules [28,29].

Since the first report on this subject was published [23], ithas been clear that enzymatic acylation of natural glyco-sides usually occurs on the sugar moiety of thesemolecules. A significant recent example is given by thelipase-catalyzed acylation of digitonin (11, Figure 2), a com-plex monodesmosidic saponin possessing haemolytic

Figure 1

OHOO

OHOH

OH

O

OHOH

OH OH

OHOO

OHOH

OH

O

OHOH

OR OH

OHOO

OHOH

OH

O

OOH

OR OH

OH

O

OHOH

OH

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OHO

OHOH

OH

NO2

R-OCH2CF3

Protease N; DMF

Lactose

CF3CH2OH

OH

NO2

α-Galactosidase

MeONa/MeOH

DeacetylationIso-globotriose (R = H)

Current Opinion in Chemical Biology

R = CH3CO ; CH3CH2CO ; CH3CH2CH2CO

Enzymatic synthesis of the trisaccharide iso-globotriose from lactose [17•• ]. DMF, N,N-dimethylformamide.

Figure 2

1 R = H R′ = H 2 R = Ac R′ = Ac 3 R = Ac R′ = H4 R = H R′ = Ac5 R = H R′ = Lauryl

O

OO

ROO

O

O

O

O

OOH

OH

O

O

OH

OR′O

OH

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HOHO HO

Current Opinion in Chemical Biology

HO

HO

HOHO

HO

HO

Lipase-catalyzed regioselective acylation of digitonin (1, [30•• ]). Bold arrows indicate acylated OH groups.

108 Biocatalysis and biotransformation

activity, which has been isolated from the seeds of Digitalispurpurea L [30••]. Depending on the acylating agent used,only one of the four primary OH groups and one of theeleven secondary OH groups of the pentasaccharidic moi-ety of digitonin could be esterified by action of the lipaseB from Candida antarctica (Figure 2).

Other papers report the regioselective acylation of flavonoidglucosides [31] and of nucleosides [32•,33•,34]. For instance,Santaniello and co-workers [32•] reported an interestingexample of complementary regioselective acylation of inosineand 2′-deoxyinosine: by choosing the appropriate enzyme, itwas possible to acetylate a secondary hydroxyl without touch-ing the more reactive primary hydroxyl. The papers describedin [33•] and [34] further exemplify the preparation of sugar-based polymers by a chemo-enzymatic approach.

Finally, concerning sugar esterification, the seminal workof Wong and co-workers [35••] in the selective sulfation ofcarbohydrates and glycopeptides by action of a recombi-nant Nod factor sulfotransferase, has to be acknowledged.

Enzymes for the selective oxidation or reduction of carbohydratesC-2 oxidation of the 1→6 disaccharides gentiobiose, isomal-tose and melibiose has been catalyzed, on a preparative scale,by a pyranose 2-oxidase isolated from the fungus Trametesmulticolor [36]. In another example, raw enzymatic extractsfrom engineered Escherichia coli strains have been used forthe chemo-enzymatic synthesis of the 3,6-dideoxy sugardiphosphate nucleoside TDP-β-L-ascarylose [37]. Dordick’sefforts in the chemo-enzymatic synthesis of new polymershave been further exemplified in a paper describing the oxi-dation of galactose or galactosamine catalyzed by a galactoseoxidase, followed by their polymerization by chemicalreductive amination [38•].

SteroidsSuccessful bioconversions of steroids are among the mile-stones of biocatalysis [39]. Recent papers exploit the catalyticpower of different enzymes, moving from the hydroxylationactivity of different microbial strains [40] to the regiospecificoxidoreductions of several bile acids catalyzed by a new3α-hydroxysteroid dehydrogenase isolated from Pseudomonas

Figure 3

NH

NH

Cl

O

OH

NH

NH

O

O

HO

O

OH

NH

O

ONH

HOOC

ONHMe

O

NH2

OH

OH

O

Cl

Cl

OHAcHN

OH

OH

ClCl

MeOOC

MeOOC

MeOOC

NHAc NHAc

O

OH

Cl

Cl

OH

NHAc

MeOOC

COOMe+

ClOH

MeOOC

AcHN

OH

O

OH Cl

OH

NHAc

MeOOC NHAc

COOMe

O

Cl

AcHN

OH

HRP

HRP

(Vancomycin aglycone)

Current Opinion in Chemical BiologyHO

Synthesis of the bis-diaryl ether fragment of vancomycin aglycone catalyzed by horseradish peroxidase (HRP) [51•• ].

Biocatalytic modification of natural products Riva 109

paucimobilis [41]. In a third report, the lipase fromPseudomonas cepacia has been used for the stereoselectiveacetylation of the primary hydroxyl group of a steroidsidechain [42].

β-Lactam antibioticsPenicillin-acylase-catalyzed synthesis of 6-aminopenicillamicacid (6-APA) is one of the very few examples of bioconver-sion that has been able to displace an established chemicalprocedure on an industrial scale. Moreover, the cognate bi-enzymatic process used to produce 7-aminocephalosporanicacid (7-ACA) from cephalosporin C is on its way to repeat-ing the same success.

Exploitation of penicillin acylase for the preparation ofsemisynthetic β-lactam antibiotics is still an active researcharea [43,44]. Less is known on the properties of glutarylacylase, one of the two biocatalysts involved in the pro-duction of 7-ACA, an enzyme that has been the subject ofa very recent publication [45•].

Amino acids and peptidesSeveral papers have been recently published on theenzymatic modification of these basic natural com-pounds. For instance, hydrolases have been used for thekinetic resolution of racemic aspartic acid diesters [46]and to support the synthesis of labile glycopeptides[47•], an area in which the Waldmann and Wong groupsare particularly active.

Non-natural amino acids have been prepared by catalysis oflyases and transferases. Specifically, phenylalanine ammonialyase has been used for the synthesis of several fluoro- andchloro-phenylalanines [48•], whereas a serine hydroxymethyl-transferase has been able to catalyze the condensationbetween glycine and 4-pentenaldehyde affording an enan-tiopure derivative of L-threonine [49]. Sih and co-workers[50,51••,52•] have been investigating the use of oxidativeenzymes (horseradish peroxidase and chloroperoxidase) ontyrosine-containing peptides to support the synthesis of natur-al macrocyclic peptides. For example, as described in Figure 3,a bis-diaryl fragment of the vancomycin aglycon has beenobtained, albeit in quite low overall yields, by two consecutiveoxidative couplings catalyzed by horseradish peroxidase[51••]. In another paper [52•], chlorination of vancomycin andof some of its derivatives by action of a chloroperoxidase fromCaldariomyces fumago has been described.

Conclusions: will combinatorial biocatalysisbe the next step?A few years ago, it was suggested that, analogously to com-binatorial chemistry, isolated enzymes and whole cellscould be used in iterative reactions to generate libraries ofnatural-compound derivatives [53]. Indeed, combinatorialbiocatalysis proved to be a powerful addition to theexpanding array of combinatorial methods for the genera-tion and optimization of lead compounds in drug discoveryand development, as has been exemplified with the anti-cancer diterpenoid paclitaxel [54].

Figure 4

O

OOH

O

OH

OH

OH

O

OOH

O

OH

OH

OH

Cl

O

O

O

OOH

O

OH

OH

Cl

O

OH

OOH

O

OOH

O

OH

OH

α-Galactosidase

α-GalactosidaseX

Chloroperoxidase

Chloroperoxidase

Current Opinion in Chemical Biology

MeO

HO

MeO

HO

HO HO

MeO

HO

HO HOOH

OH

MeO

HO

Orthogonality issues in iterative biocatalysis: an example of the significance of reaction order on the production of analogs of bergenin.

A recent interesting paper [55••] describes the applicationof this methodology to the synthesis of new derivatives ofthe flavonol bergenin and addresses some issues related tothe iterative use of biocatalysts. As shown in Figure 4, oneof these combinatorial methods is the so-called ‘orthogo-nality’: the very same substrate selectivity of enzymesmakes the correct choice of the reaction sequence of para-mount importance in order to enrich the number ofsubstrate analogues.

Despite some problems, it is reasonable to forecast thatthe development of molecular biology, which will givenew enzymes with improved catalytic performances, aswell as the so-called ‘medium engineering’, which allowsthe optimization of reaction conditions, will strengthenthe biocatalytic approach to the modification of naturalcompounds even further.

UpdateRecent papers have reported impressive advances in theenzymatic synthesis of oligosaccharides, moving from thelarge-scale production of activated sugar nucleotides and fromthe optimized expression and overproduction of bacterial gly-cosyltransferases [56] to the precise enzymatic synthesis oflabeled oligosaccharides on an intact glycoprotein [57].

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest••of outstanding interest

1. Jones JB, DeSantis G: Toward understanding and tailoring the•• specificity of synthetically useful enzymes. Acc Chem Res 1999,

32:99-107.A recent review on the ways to affect enzyme selectivity by chemical modifi-cation and site-directed mutagenesis.

2. Koeller KM, Wong CH: Emerging themes in medicinalglycoscience. Nat Biotechnol 2000, 18:835-841.

3. Zimmermann FT, Schneider A, Schörken U, Sprenger GA,• Fessner WD: Efficient multi-enzymatic synthesis of D-xylulose

5-phosphate. Tetrahedron Asymmetry 1999, 10:1643-1646.A classical application of a recombinant transketolase for the synthesis of asugar derivative.

4. Schoevaart R, van Rantwijk F, Sheldon RA: Carbohydrates fromglycerol: an enzymatic four-step, one-pot synthesis. ChemCommun 1999:2465-2466.

5. Guanti G, Banfi L, Zannetti MT: Phosphonic derivatives ofcarbohydrates: chemoenzymatic synthesis. Tetrahedron Lett 2000,41:3181-3185.

6. Zannetti MT, Walter C, Knorst M, Fessner WD: Fructose1,6-bisphosphate aldolase from Staphylococcus carnosus:overexpression, structure prediction, stereoselectivity, andapplication in the synthesis of bicyclic sugars. Chem Eur J 1999,5:1882-1890.

7. Seto NOL, Compston CA, Szpacenko A, Palcic MM: Enzymatic• synthesis of blood group A and B trisaccharide analogues.

Carbohydr Res 2000, 324:161-169.Two glycosyltransferases have been cloned and overexpressed and theirsubstrate specificity has been investigated.

8. Koeller KM, Smith MEB, Huang RF, Wong CH: Chemoenzymaticsynthesis of a PSGL-1 N-terminal glycopeptide containingtyrosine sulfate and αα-O-linked sialyl Lewis X. J Am Chem Soc2000, 112:4241-4242.

9. Quiros LM, Carbajo RJ, Brana AF, Salas JA: Glycosylation of• macrolide antibiotics. J Biol Chem 2000, 275:11713-11720.A macrolide glycosyltransferase has been cloned and used to glucosylatethe antibiotic oleandomycin.

10. Jenkins GN, Stachulski AV, Scheinmann F, Turner NJ: The enzymaticglucoronidation of 3-O-protected morphine. A new route to7,8-dihydromorphine-6-glucoronide. Tetrahedron Asymmetry 2000,11:413-416.

11. Stevenson DE, Kren V, Halada P, Sedmera P: Enzymatic preparationof lysergol ββ-D-glucoronide. Collect Czech Chem Commun 2000,65:117-121.

12. Zeng X, Yoshino R, Murata T, Aijsaka K, Usui T: Regioselectivesynthesis of p-nitrophenyl glycosides of ββ-D-galactopyranosyl-disaccharides by transglycosylation with ββ-D-galactosides.Carbohydr Res 2000, 325:120-131.

13. Murata T, Morimoto S, Zeng X, Watanabe S, Usui T: Enzymaticsynthesis of αα-L-fucosyl-N-acetyllactosamines and 3′′-O-αα-L-fucosyllactose utilizing αα-L-fucosidases. Carbohydr Res 2000,320:192-199.

14. Singh S, Scigelova M, Crout DHG: Glycosidase-catalyzed• synthesis of αα-galactosyl epitopes important in

xenotransplantation and toxin binding using the αα-galactosidasefrom Penicilium multicolor. Chem Commun 1999:2065-2066.

Highly regioselective galactosyl transfer to give products containing the α-D-Galp-(1→3)-D-Galp epitope.

15. Scigelova M, Crout DHG: Purification of αα-galactosidase from• Aspergillus niger for application in the synthesis of complex

oligosaccharides. J Mol Cat B: Enzymatic 2000, 8:175-181.An α-galactosidase capable of catalyzing the α-galactosyl transfer to theC-4 hydroxyl of pyranosidic sugar acceptors is described.

16. Riva S, Roda G: Sugar transformations using enzymes in non• aqueous media. In Methods in Non-aqueous Enzymology. Edited by

Gupta MN. Basel: Birkäuser Verlag; 2000:146-159.A recent review on the enzymatic modification of sugars and sugar derivatives.

17. Weignerova L, Sedmera P, Unkova Z, Halada P, Kren V, Casali M,•• Riva S: Enzymatic synthesis of iso-globotriose from partially

protected lactose. Tetrahedron Lett 1999, 40:9297-9299.The title trisaccharide was prepared in just three steps, exploiting, insequence, the action of two enzymes (a protease and a glycosidase) and aquantitative alkaline hydrolysis.

18. MacManus DA, Vulfson EN: Regioselectivity of enzymatic• glycosylation of 6-O-acyl glycosides in supersaturated solutions.

Biotechnol Bioeng 2000, 69:585-590.The same bi-enzymatic approach has been used for the synthesis of a seriesof disaccharides.

19. Mori T, Fukusho S, Kojima J, Okahata Y: Enzymatic syntheses ofglycolipids catalyzed by a lipid-coated glycoside hydrolase in theorganic-aqueous two phase system. Polym J 1999, 31:1105-1108.

20. Kobayashi T, Adachi S, Nakanishi K, Matsuno R: Synthesis of alkylglycosides through ββ-glucosidase-catalyzed condensation in anaqueous-organic biphasic system and estimation of theequilibrium constants for their formation. J Mol Cat B: Enzymatic2000, 11:13-21.

21. Huneke FU, Bailey D, Nucci R, Cowan D: Sulfolobus solfataricusββ-glycosidase-catalyzed synthesis of sugar-alcohol conjugates inthe presence of organic solvents. Biocatal Biotrans 2000,18:291-299.

22. Carrea G, Riva S: Properties and synthetic applications of•• enzymes in organic solvents. Angew Chem Int Ed Engl 2000,

39:2227-2254.A complete overview of what enzymes can do in organic solvents

23. Riva S, Chopineau J, Kieboom APG, Kilbanov AM: Protease-catalyzed regioselective esterification of sugars and relatedcompounds in anhydrous dimethylformamide. J Am Chem Soc1988, 110:584-589.

24. Cao L, Bornscheuer UT, Schmid RD: Lipase-catalyzed solid-phasesynthesis of sugar esters. Influence of immobilization onproductivity and stability of the enzyme. J Mol Cat B: Enzymatic1999, 6:279-285.

25. Heo JH, Kim SY, Kim HS, Yoo KP: Enzymatic preparation of acarbohydrate ester of medium-chain fatty acid in supercriticalcarbon dioxide. Biotechnol Lett 2000, 22:995-998.

110 Biocatalysis and biotransformation

26. Zhang X, Kamiya T, Otsubo N, Ishida H, Kiso M: Chemoenzymaticsynthesis of ganglioside GM4 analogs as potentialimmunosuppressive agents. J Carbohydr Chem 1999, 18:225-239.

27. Humeau C, Rovel B, Girardin M: Enzymatic esterification of bixin byL-ascorbic acid. Biotechnol Lett 2000, 22:165-168.

28. Park OJ, Kim DY, Dordick JS: Enzyme-catalyzed synthesis of sugar-containing monomers and linear polymers. Biotechnol Bioeng2000, 70:208-216.

29. Kitagawa M, Chalermisrachai P, Fan H, Tokiwa Y: Chemoenzymaticsynthesis of biodegradable polymers containing glucobiosebranches. Macromol Symp 1999, 144:247-256.

30. Danieli B, Luisetti, M, Steurer S, Michelitsch A, Likussar W, Riva S,•• Reiner J, Schubert-Zvilavecz M: Application of lipase-catalyzed

regioselective esterification in the preparation of digitoninderivatives. J Nat Prod 1999, 62:670-673.

An interesting example of the regioselective acylation of a natural glycosidepossessing a complex oligosaccharide substituent.

31. Ishihara K, Nakajima N, Itoh T, Yamaguchi H, Nakamura K, Furuya T,Hamada H: A chemoenzymatic synthesis of aromatic carboxylicacid vinyl esters. J Mol Cat B: Enzymatic 1999, 7:307-310.

32. Ciuffreda P, Casati S, Santaniello E: Lipase-catalyzed protection of• the hydroxy groups of the nucleosides inosine and 2′′-

deoxyinosine: a new chemoenzymatic synthesis of the antiviraldrug 2′′,3′′-dideoxyinosine. Bioorg Med Chem Lett 1999,9:1577-1582.

Different regioselective acylation outcomes are obtained by changing enzyme.

33. Wang Q, Linhardt RJ, Dordick JS: Affinity chromatography using• enzymatically synthesized nucleotide-containing DNA binding

polymers. Biotechnol Tech 1999, 13:463-467.An interesting example of new polymers obtained using a chemo-enzymatic protocol.

34. Kitagawa M, Fan H, Raku T, Kurane R, Tokiwa Y: Preparation of vinylthymidine ester catalyzed by protease and its chemicalpolymerization. Biotechnol Lett 2000, 22:883-886.

35. Burkart MD, Izumi M, Chapman E, Lin CH, Wong CH: Regeneration•• of PAPS for the enzymatic synthesis of sulfated oligosaccharides.

J Org Chem 2000, 65:5565-5574.An efficient sulfotransferase-catalyzed reaction, coupled with a PAPS regen-eration system based on a recombinant aryl sulfotransferase, was used forthe synthesis of various oligosaccharide sulfates that were further glycosy-lated using glycosyltransferases.

36. Volc J, Leitner C, Sedmera P, Halada P, Haltrich D: Enzymaticformation of dicarbonyl sugars: C-2 oxidatoin of 1→→6disaccharides gentiobiose, isolmaltose and melibiose bypyranose 2-oxidase from Trametes multicolor. J Carbohydr Chem1999, 18:999-1007.

37. Klaffke W, Chambon S: Chemo-enzymatic synthesis of TDP-ββ-L-ascarylose. Tetrahedron Asymmetry 2000, 11:639-644.

38. Liu XC, Dordick JS: Sugar-containing polyamines prepared using• galactose oxidase coupled with chemical reduction. J Am Chem

Soc 1999, 121:466-467.Selective galactose oxidation gives new sugar-based monomers.

39. Riva S: Enzymatic modification of steroids. In Applied Bsiocatalysis.Edited by Blanch HW, Clark DS. New York: Marcel Dekker;1991:179-220.

40. Holland HL, Lakshmaiah G: Microbial hydroxylation of2-oxatestosterone. J Mol Cat B: Enzymatic 1999, 6:83-88.

41. Bianchini E, Chinaglia N, Dean M, Giovanini PP, Medici A, Pedrini P,Poli S: Regiospecific oxidoreductions catalyzed by a newPseudomonas paucimobilis hydroxysteroid dehydrogenase.Tetrahedron 1999, 55:1391-1398.

42. Ferraboschi P, Pecora F, Reza-Elahi S, Santaniello E:Chemoenzymatic syntheses of (25R)- and (25S)-25-hydroxy-27-nor-cholesterol, a steroid bearing a secondary hydroxyl group inthe side chain. Tetrahedron Asymmetry 1999, 10:2497-2500.

43. van Langen LM, de Vroom E, van Rantwijik F, Sheldon R: Enzymaticsynthesis of ββ-lactam antibiotics using penicillin-G acylase infrozen media. FEBS Lett 1999, 456:89-92.

44. Park CB, Lee SB, Ryu DDY: Penicillin acylase-catalyzed synthesisof cefalozin in water-solvent mixtures: enhancement effect ofethyl acetate and carbon tetrachloride on the synthetic yield.J Mol Cat B: Enzymatic 2000, 9:275-281.

45. Monti D, Carrea, G, Riva S, Baldaro E, Frare G: Characterization of• an industrial biocatalyst: immobilized glutaryl-7-ACA acylase.

Biotechnol Bioeng 2000, 70:239-244.A complete characterization of the performances of an important industrial enzyme.

46. Liljeblad A, Kanerva LT: Enzymatic methods for the preparation ofenantiopure malic and aspartic acid derivatives in organicsolvents. Tetrahedron Asymmetry 1999, 10:4405-4415.

47. Sander J, Waldmann H: Enzymatic protecting group techniques for• glyco- and phosphopeptide chemistry: synthesis of a

glycophosphopeptide from human serum response factor. ChemEur J 2000, 6:1564-1577.

The use of enzyme-labile protecting groups, both at carboxyl and amino ter-minus, in a growing glycopeptide chain is described.

48. Gloge A, Zon J, Kövari A, Poppe L, Retey J: Phenylalanine ammonia• lyase: the use of its broad substrate specificity for mechanistic

investigations and biocatalysis. Synthesis of L-arylalanines. ChemEur J 2000, 6:3386-3389.

The enzymatic synthesis of several fluorinated or chlorinated phenylalaninederivatives is described.

49. Jackson BG, Pedersen SW, Fisher JW, Misner JW, Gardner JP,Staszak MA, Doecke C, Rizzo J, Aikins J, Farkas E et al.:Enantioselective syntheses of 1-carbacephalosporins fromchemoenzymically derived ββ-hydroxy-αα-amino acids: applicationsto the total synthesis of carbacephem antibiotic Loracarbef.Tetrahedron 2000, 56:5667-5677.

50. Guo ZW, Machiya K, Salamonczyc GM, Sih CJ: Total synthesis ofBastadins 2, 3, and 6. J Org Chem 1998, 63:4269-4276.

51. Malnar I, Sih CJ: Synthesis of the bis-diaryl ether fragment of•• vancomycin via enzymatic oxidative phenolic coupling.

Tetrahedron Lett 2000, 41:1907-1911.Vancomycin aglycon is still far away, but enzymes can play their gamein vitro.

52. Malnar I, Sih CJ: Chloroperoxidase-catalyzed chlorination of• didechloroaglucovancomycin and vancomycin. J Mol Cat B:

Enzymatic 2000, 10:545-549.An interesting example of enzyme-mediated halogenation of complex macro-cyclic natural compounds.

53. Michels PC, Khmelnitsky YL, Dordick JS, Clark DS: Combinatorialbiocatalysis: a natural approach to drug discovery. TrendsBiotechnol 1998, 16:210-215.

54. Khmelnitsky, YL, Budde C, Arnold JM, Usyatinsky A, Clark DS,Dordick JS: Synthesis of water-soluble paclitaxel derivatives byenzymatic acylation. J Am Chem Soc 1997, 119:11554-11555.

55. Krstenansky JL, Khmelnitsky Y: Biocatalytic combinatorial synthesis.•• Bioorg Med Chem Lett 1999, 7:2157-2162.New examples of combinatorial biocatalysis and some general remarks onthis methodology are presented.

56. Endo T, Koizumi S: Large-scale production of oligosaccharides usingengineered bacteria. Curr Opin Struct Biol 2000, 10:536-541.

57. Miyazaki T, Sato H, Sakakibara T, Kajihara Y: An approach to theprecise chemoenzymatic synthesis of 13C-labeledsialyloligosaccharide on an intact glycoprotein: a novel one-pot[3–13C]-labeling method for sialic acid analogues by control ofthe reversible aldolase reaction, enzymatic synthesis of [3–13C]-NeuAc-αα-(2→→3)-[U-13C]-Gal-ββ-(1→→4)-GlcNAc-ββ- sequence ontoglycoprotein, and its conformational analysis by developed NMRtechniques. J Am Chem Soc 2000, 122:5678-5694.

Biocatalytic modification of natural products Riva 111