Recent biotechnological developments in the use of peroxidases

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Recent biotechnological developments in the use of peroxidases Stefano Colonna, Nicoletta Gaggero, Carlo Richelmi and Piero Pasta Peroxidases are ubiquitous oxidoreductases that use hydrogen peroxide or alkyl peroxides as oxidants. Advances have recently been made in using them to prepare, under mild and controlled conditions, chiral organic molecules that are valuable for the chemoenzymatic synthesis of a wide range of useful compounds. Horseradish peroxidase can be converted into a peroxygenative enzyme by molecular engineering. Chloroperoxidase, the most versatile peroxidase, behaves like a ‘true’ monooxygenase in sulfoxidations with molecular oxygen and an external reductant, with substantial increases in enantioselectivity and enzyme stability.

eroxidases are oxidoreductases that act with hydrogen peroxide or alkyl hydroperoxides as acceptors. They are found ubiquitously in animals, plants and microorganisms, and efficiently catalyse the oxidation of a variety of substrates1,2. Our understanding of the structure–function relationships and catalytic mechanisms of peroxidases is largely based on work with horseradish peroxidase (HRP). In classical peroxidases, ferric protoporphyrin IX is the prosthetic group and imidazole the fifth iron ligand. The characteristic activity of peroxidases is oneelectron oxidation, which normally proceeds through the mechanism depicted in Fig. 1. Compound I is reduced back to the ferric resting state either by a two sequential one-electron transfer processes from peroxidase to substrates or by two-electron oxidation process associated with the ferryl oxygen transfer to substrates3. The radicals produced in the reaction generally evolve nonenzymatically to nonradical products by pathways characteristic of each substrate (coupling, dismutation etc.). Of the two electrons required for peroxide reduction, one comes from Fe(III), while the other (in HRP) comes from the porphyrin, producing a porphyrin radical cation. One-electron reduction of compound I gives compound II, in which the FeIV5O species remains intact and porphyrin is reduced. Generally, the reaction of compound II with the substrate is at least 10–20 times slower than that of compound I and, under most steady-state conditions, is rate limiting. Chloroperoxidase (CPO), isolated from the marine fungus Caldariomyces fumago, is unique among the peroxidases because it contains a cysteinic thiolate as the fifth axial ligand of the heme instead of the imidazole ligand. For this reason, many of its spectroscopic3 and chemical properties are similar to those of cytochrome

P

S. Colonna ([email protected]), N. Gaggero and C. Richelmi are at Centro CNR and Istituto di Chimica Organica, Facoltà di Farmacia, Università degli Studi di Milano, via Venezian 21, I-20133 Milano, Italy. P. Pasta is at the Istituto di Biocatalisi e Riconoscimento Molecolare CNR, via Mario Bianco 9, I-20131 Milano, Italy. TIBTECH APRIL 1999 (VOL 17)

P-450. CPO is unusually versatile: it catalyses not only the reactions typical of peroxidases but also those of catalases and monooxygenases, and it is also almost unique in catalysing halogenation reactions (except fluorination) in the presence of halide ions and H2O2 (Fig. 1). Peroxidase-catalysed reactions (Fig. 2) can be grouped into four categories. (1) Oxidative dehydrogenation 2 SH 1 H2O2 → 2 S• 1 2 H2O (2) Oxidative halogenation SH 1 H2O2 1 H(1) 1 X(2) → SX 1 2 H2O X = Cl, Br, I (3) H2O2 dismutation 2 H2O2 → 2 H2O 1 O2 (4) Oxygen-transfer reaction SH 1 H2O2 → SOH 1 H2O Oxidative dehydrogenation HRP is the peroxidase with the broadest specificity for oxidative dehydrogenation, being able to produce a great variety of useful compounds. This commercially important enzyme occurs as a large family of isoenzymes. The crystal structure of HRP-C, the major isoenzyme, was recently resolved to 2.15 Å resolution, and this has enabled the prediction of the enzyme region that binds aromatic substrates4 (which has a greater affinity for phenols than for anilines). An important aspect of peroxidase catalysis is the mode of binding of the substrate to the active site. Unlike heme monooxygenases, which are believed to insert the ferryl oxygen into the substrate, classical peroxidases oxidize the substrate by electron transfer to the heme centre and appear to bind the substrate near the heme edge5,6. In the peroxidase-catalysed oxidation of phenols, phenoxide radicals are produced, which dimerize in a non-enzymatic step to form two coupling products: o,o9-biphenyl and the Pummerer ketone; on prolonging the reaction times, a complex mixture of oligomeric oxidative products results. The dimeric products formed in the initial phase have been separated and characterized on a preparative scale; the biphenyl is, in a few instances7,8, the major oxidative

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pound I is a strong oxidant and can react with chloride ions to produce HOCl, by analogy with CPO (Fig. 1). The lack of stereospecificity in the halogenation reactions catalysed by CPO is in agreement with the participation of an ‘enzyme-free’ halogenating species, the details still being a matter of debate. A remarkable exception is the regioselective bromohydration of certain saccharide glycals with CPO, HBr and H2O2 to give the corresponding 2-deoxy-2-bromo saccharides, useful as bioactive saccharides16.

O FeIV

AH

A• + OH

2

e

2

A• + H

S Cys Compound II H2O2

e

1

2

AH

H2O O FeIV

S Cys O2 + H2O 2 AX + OH

H2O2

X O

2

FeIII

AH

S Cys

H2O2 dismutation Some peroxidases catalyse the decomposition of hydrogen peroxide to water and oxygen. CPO and, to a lesser extent, HRP in fact have a catalase-type activity17 (Fig. 1). In the absence of synthetically useful applications, this dismutase activity must be minimized.

1 •

S Cys Compound I

X

2

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FeIII

Figure 1 Catalytic cycle of chloroperoxidase (CPO). AH represents the substrate, such as guaiacol (peroxidative pathway) or monochlorodimedone (halogenation pathway), and the oval represents the heme. Compounds I and II represent the ferryl intermediates; X represents Cl, Br or I involved in the halogenation pathway.

product of p-cresol in single-turnover experiments rather than the Pummerer ketone7. Also, the peroxidasecatalysed polymerization of anilines occurs under mild conditions and produces polymeric amines9. Of particular interest are the N- and O-dealkylation reactions catalysed by peroxidases via electron transfer10,11, because these reactions require drastic conditions in preparative organic chemistry. Among their biotechnological applications is their extensive use in the removal of toxic organic substances from industrial waste12. Oxidative halogenation Several heme-containing peroxidases are known to use hydrogen peroxide and halide ions to halogenate a carbon atom of a substrate (AH) that has an ‘activated’ benzylic/allylic C–H bond13. These enzymatic halogenation reactions do not show the typical features of enzymatic reactions, such as reversibility, narrow substrate range and high product selectivity. A variety of alkenes can be converted to a,b halohydrins by CPO, which also catalyses the halogenation of a range of aromatic compounds, including anilines, phenols and heterocycles. Oxidative halogenation can be promoted not only by heme peroxidases but also by vanadium and non-heme peroxidases. In the case of vanadium and heme peroxidases, the active species is probably hypochlorous acid14,15. In particular, CPO catalyses the chlorination reaction involved in the biosynthesis of caldariomycin. Myeloperoxidase from activated leukocytes is a heme peroxidase that differs from HRP in that its com-

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Oxygen-transfer reactions The enantioselective introduction of an oxygen atom into organic substrates is the most interesting process catalysed by peroxidases, because these enzymes allow the selective oxidation of organic molecules in mild, controlled conditions. The prevalent feature of these applications is the use of low-cost and environmentally compatible oxidants (e.g. hydrogen peroxide, dioxygen). The development of enantioselective enzymatic processes that allow the efficient oxidation of organic substrates is an area of great scientific and industrial interest, because there are few reliable chemical examples on a large scale (using transition-metal complexes as catalysts and dioxygen or hydroperoxides as oxidants)18,19. Moreover, as a result of environmental concerns, catalytic oxidations with low-environmentalimpact solvents, as achieved by the use of peroxidases, is one of the goals of future research. This interest is stimulated by their possible use in the manufacture of natural products, fine chemicals, drugs and agrochemicals through industrially attractive processes. In these biotransformations, peroxidases behave as monooxygenases. Such oxygenase-type reactions can be grouped as follows: (1) heteroatom oxidation (Soxidation and N-oxidation); (2) epoxidation; and (3) carbon–hydrogen-bond oxidation (benzylic/allylic oxidation, alcohol oxidation and indole oxidation). Heteroatom oxidation Sulfur oxidation Enantiomerically pure sulfoxides are important chiral synthons in asymmetric synthesis, for enantioselective carbon–carbon-bond formation, in particular20. The sulfoxide functional group is involved in different biological activities and optically pure sulfoxides are of great pharmaceutical interest21. Several heme peroxidases, such as horseradish peroxidase4,22,23, cytochrome-c peroxidase24, microperoxidase25 and lactoperoxidase26, catalyse the enantioselective sulfoxidation of alkyl aryl sulfides. In general, their turnover numbers and enantioselectivities [measured as enantiomeric excess (ee)] are rather low. However, the substantial enantioselectivity of the HRP-catalysed oxidation of some aryl methyl sulfides with hydrogen peroxide was described by Colonna and co-workers22. The enantioselectivity of the HRP oxidation of alkyl aryl sulfides can be increased considerably by TIBTECH APRIL 1999 (VOL 17)

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molecular engineering27, the ee of aryl methyl sulfoxides being in all cases greater than 94%. The conversion of HRP into a nearly stereospecific sulfoxidation catalyst, owing to the replacement of Phe-41 with leucine, is very significant because it represents the first step in the conversion of HRP into a versatile oxygenative catalyst. Indeed, the replacement of the Phe-41 by smaller amino acids such as leucine improves access to the ferryl oxygen, which is responsible for the monooxygenase activity. This has recently been confirmed by the fact that leucine-modified HRP catalyses the epoxidation of styrene and bmethylstyrenes, a reaction not catalysed by native HRP27,28. A peroxidase-related soybean oxidoreductase is also capable of performing enantioselective sulfoxidation of methyl p-tolyl sulfide to the (S) sulfoxide at 90% ee29. CPO is the heme peroxidase of choice for sulfoxidation reactions owing to its high enantioselectivity and versatility. Indeed, in contrast to HRP, it catalyses the oxidation of a large variety of alkyl aryl, dialkyl and heterocyclic sulfides with high chemical conversion and ee (Table 1). By taking into account the unavoidable minor spontaneous sulfoxidation by H2O2 or other oxidants, the enantioselectivity is, in many instances, almost absolute. Electronic and, particularly, steric factors dramatically affect the outcome of the reaction30–34. Excellent yields and very high ees (97–100%) have been obtained in the sulfoxidation of a number of sulfides structurally related to methyl p-tolyl sulfide. Binding experiments show that these substrates fit the active-site topology of CPO6. The reactions are likely to proceed either through a direct oxygen transfer from compound I to the substrate or through a P-450-like mechanism, involving an initial electron transfer from the substrate to compound I with the formation of a substrate radical species and tightly coupled compound II, followed by rapid oxygen transfer from compound II to sulfur within the same cage of solvent, as indicated by kinetic, spectroscopic and 18O-labeling studies, together with observed high enantioselectivity. In all cases examined, chloroperoxidase preferentially forms the sulfoxide with the (R) absolute configuration. The best results were obtained with H2O2 either in aqueous buffer solutions or in a tert-butyl-alcohol– water mixture32. When racemic hydroperoxides were used as oxidants, optically active alcohols and alkyl hydroperoxides were obtained (up to 88% ee)16. Nitrogen oxidation Aryl amines can be oxidized to the corresponding nitroso compounds with CPO and H2O2 as oxidant35. Quantitative incorporation of 18O into the nitroso function was found when H218O2 was the oxidant substrate. The N-oxidation occurs via oxygen transfer from compound I. Many amidoximes are biologically active and interest in the oxidation of these compounds is increased by the possible formation of nitric oxide, an important biological mediator. The oxidation of arylamidoximes generally affords a mixture of compounds, whereas their oxidation by H2O2 in the presence of HRP yields the corresponding O-arylimidoyl arylamidoximes in 30–70% yields36. TIBTECH APRIL 1999 (VOL 17)

Table 1. Oxidation of sulfides by CPO and H2O230–32 Sulfide

Buffer

R1

R2

Conversion (%)

Enantiomeric excess(%)

C6H5 C6H5 C6H5 p-CH3–C6H4 p-OCH3–C6H4 m-OCH3–C6H4 o-OCH3–C6H4 p-NO2–C6H4 p-Cl–C6H4 m-Cl–C6H4 p-Br–C6H4 m-Br–C6H4 p-Cl–C6H4

CH3 CH2–CH2 n-C3H7 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH2–CH3

100 83 3 83 53 37 3 19 78 59 15 11 33

99 99 27 99 99 99 99 99 99 99 99 99 99

CH3

100

99

CH3

100

99

S N S

Epoxidation Asymmetric epoxidation is of fundamental importance not only from the synthetic point of view but also in biological systems. Optically active epoxides are very useful chiral synthons because they can give bifunctional compounds through stereospecific ring opening37. Native HRP usually does not catalyse the epoxidation reaction, whereas various mutants (F41L, F41T, F41A, H42V) lead to optically active styrene-oxide derivatives28,38. The synthetic importance of this reaction is limited by the formation of large amounts of rearranged aldehydes as byproducts. Similar results are obtained in the epoxidation of styrenes catalysed by cytochrome-c peroxidase24 (up to 32% ee). By contrast, the CPO-catalysed epoxidation recently discovered by Colonna and co-workers39 and Hager and co-workers40 proceeds with high chemical and optical yields (Table 2). With styrene derivatives, tertbutyl–OOH rather than H2O2 is the oxidant of choice in view of the higher chemical yield obtained, whereas the ee was practically the same (66–67%) with both oxidizing agents39. The R epoxides were formed preferentially and all the data support the view of oxygen delivery from the ferryl oxygen directly to the substrates. Excellent enantioselectivity is observed in the CPO-catalysed epoxidation of short-chain cis alkenes with a chain length of nine or fewer carbon atoms, except for monosubstituted olefins, which often function as reversible suicide inhibitors of the enzyme40–42. Trans olefins are highly unreactive substrates, and terminal alkenes lead to heme alkylation and subsequent enzyme deactivation43. The epoxidation reaction can be optimized by using branched 1-alkenes44. An important application of CPO as an enantioselective epoxidation catalyst is the efficient synthesis of (R)-(2)-mevalonolactone42. This example represents the first multistep synthesis based on the enantioselective

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Table 2. Chloroperoxidase-catalysed epoxidation of olefins41,44 Olefin

Product, yield and ee

Byproducts and yield

Turnover number

O 2100 Yield 100%, ee 95% 50%

O

840 OH OH Yield 40%, ee 95% O 63

OHC 35% Yield 3% O

34 Yield 2%, ee 10% O 1700 Yield 23%, ee 95% CHO

O

COOH

840

24% Yield 40%, ee 49%

epoxidation catalysed by CPO. The effect of chain length on the enantioselective CPO-catalysed formation of v-bromo-2-methyl-1-alkene epoxides has been recently reported45. Especially in large-scale reactions, the use of tert-butyl–OOH appears to be more effective than H2O2 because CPO is relatively sensitive to H2O2, losing activity rapidly in the presence of excess reagent. Benzylic/allylic hydroxylation The selective hydroxylation of hydrocarbons with chemical reagents is a demanding task, but not when CPO is used as a catalyst. The first example was the oxidation of cyclohexene to cyclohex-2-en-1-ol46. More recently, CPO-catalysed benzylic hydroxylation was studied by Ortiz de Montellano and co-workers47, and Zaks and Dodds41. Remarkably, the oxidation of ethylbenzene results in the formation of the (R)-2phenethyl alcohol (97% ee), whereas propylbenzene gives the alcohol of opposite stereochemistry41. The oxygen atom in the benzylic alcohol is derived from H2O2, which is consistent with a mechanism involving direct oxygen transfer from CPO. The selective hydroxylation of aromatic compounds is very difficult in preparative organic chemistry

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5%

because it is laborious, time consuming and inefficient. Klibanov and co-workers have found that HRP can catalyse the hydroxylation of some aromatic compounds by molecular oxygen in the presence of dihydroxyfumaric acid as hydrogen donor. Three important drugs have been produced using this enzymatic hydroxylation with a chemical yield of up to 70%: L-3,4-dihydroxyphenylalanine (L-DOPA), D-(2)-3,4dihydroxyphenylglycine and L-epinephrine48. Alcohol oxidation In contrast to other peroxidases, which are generally restricted to the oxidation of phenols via the classical free-radical intermediates, CPO is able to transform allylic, propargylic or benzylic alcohols to the corresponding aldehydes with H2O2 as oxidant49. However reactive aldehydes such as 5-hydroxymethyl-furfural are further oxidized to the corresponding acids via direct oxygen transfer from the ferryl species50. Indole oxidation Substituted oxindoles have interesting biological properties. The chemical methods available for their production mostly require multistep procedures. Direct oxidation of indole and indoles with substituents at the TIBTECH APRIL 1999 (VOL 17)

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Cl

OCH3

Cl

O

OH

O

n H3C CH3 Dichlorodimedone

Oligomeric oxidative products

OCH3 OCH3 a

Cl H

b

O

O

Cl2 1 H3C CH3

Peroxidases 2H2O 1 O2

H2O

O

p-tol-S-CH3

+ H2O2

c

d

H3C

H3CO

p-tol

S CH3

N

a N H

H3C

N

N CH3 Ellipticine quinone imine

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O

CH3

Figure 2 Examples of reactions catalysed by peroxidases: (a) oxidative dehydrogenation; (b) oxidative halogenation; (c) H2O2 dismutation; (d) oxygentransfer reactions.

4, 5 or 6 position with H2O2 and CPO gives the corresponding oxindoles in nearly quantitative yields51. Conclusions The small-scale applications of peroxidases in the area of synthetic organic chemistry, especially when regioand enantioselective oxidations are sought, are both numerous and appealing. The highly enantioselective sulfoxidations and epoxidations of a variety of substrates to yield useful chiral synthons are particularly important. Compared with cytochrome P-450 and other monooxygenases, peroxidases are economically more attractive as synthetic catalysts because they use H2O2 or other peroxides as oxidants instead of the more expensive molecular oxygen and NAD(P)H. In the latter case, preparative reactions with isolated enzymes can be carried out only if effective coenzyme-regeneration systems are employed. Of the various peroxidases, CPO is the best suited to catalysing stereoselective reactions because, even though its active site resembles that of classical peroxidases, the TIBTECH APRIL 1999 (VOL 17)

substrates have access to the heme iron and ferryl oxygen, thus favoring the stereoselective transfer of the oxygen atom. With other peroxidases, such as HRP and lactoperoxidase, direct oxygen transfer is hampered by the fact that the substrates cannot approach the reactive oxo groups in the distal pocket of the enzyme. However, conversion of HRP into a versatile and enantioselective oxygenative catalyst has been shown to be possible by means of molecular engineering27. In spite of the remarkable synthetic possibilities of peroxidases, commercial processes based on these enzymes have not yet been developed. One reason is the fact that peroxides inactivate heme enzymes, the most versatile peroxidases, by oxidation of the porphyrin ring. Enzyme stability can be notably improved by maintaining a low peroxide concentration, through stepwise or continuous addition of the oxidant31,51. Increases in the resistance of HRP to H2O2 have also been sought through genetic manipulation but, so far, no satisfactory amelioration has been obtained52.

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Fe31 1 H2O2 (Fe=O)31 1 S Fe31 1 H2O2 (Fe5O)31 1 S (Fe5O)21 • S1•

K1 K2 K1 K3 K4

(Fe5O)31 1 H2O

(1)

Fe31 1 SO

(2)

(Fe5O)31 1 H2O

(1)

(Fe5O)21 • S1•

(3)

Fe31 1 SO

(4)

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Figure 3 Proposed mechanisms for oxygen transfer to substrates in chloroperoxidase-catalysed reactions: direct oxygen transfer (1,2) or the rebound mechanism (1,3,4).

Another disadvantage is that the oxidation of some substrates by peroxidases is in competition with their spontaneous oxidation by peroxides, which reduces the purity of the products when enantioselectivity is the target. In this case, too, background oxidation can be drastically reduced by keeping the peroxide concentration low throughout the reaction period. Recently, it has been found with CPO that spontaneous oxidation can be suppressed and enzyme stability increased by using O2 in the presence of either dihydroxyfumaric acid or ascorbic acid as a hydrogen donor as in the conversion of methyl phenyl sulfide to (R)-methyl phenyl sulfoxide53. A third drawback, intrinsic to most enzymatic systems, that limits the scaling-up of peroxidase-catalysed reactions is the low water solubility of most of the substrates of synthetic interest. The solution to this general problem could come by designing strategies that enhance enzymatic activity in organic solvents54 or by the use of hydrophobic matrices that act as a reservoir for both substrates and products. In conclusion, peroxidases, especially chloroperoxidase, represent a versatile tool for performing oxygen-transfer reactions to a large series of structurally very different substrates. Furthermore, the capacity of CPO to undergo reactions with oxygen in the presence of reducing agents raises the possibility that it may perform such oxygentransfer reactions by behaving as a true monooxygenase. References 1 van Deurzen, M. P. J., van Rantwijk, F. and Sheldon, R. A. (1997) Tetrahedron 53, 13183–13220 2 Everse, J., Everse, K. E. and Grisham, M. B. (1991) Peroxidases in Chemistry and Biology, CRC Press 3 Dawson, J. H. (1988) Science 240, 433–439 4 Gaihede, M., Schuller, D. J., Henriksen, A., Smith, A. T. and Poulos, T. L. (1997) Nat. Struct. Biol. 4, 1032–1038 5 Ortiz de Montellano, P. R. (1987) Acc. Chem. Res. 20, 289–294 6 Casella, L. et al. (1992) Biochemistry 31, 9451–9459 7 Hewson, W. D. and Dunford, H. B. (1976) J. Biol. Chem. 251, 6043–6052 8 Pietikäinen, P. and Adlercreutz, P. (1990) Appl. Microbiol. Biotechnol. 33, 455 9 Zemel, H. and Quinn, J. F. (1993) Chem. Eng. News 71, 36–37 10 Kedderis, G. L. and Hollenberg, P. F. (1984) J. Biol. Chem. 259, 3663–3668 11 Meunier, G. and Meunier, B. (1985) J. Am. Chem. Soc. 107, 2558–2560 12 Klibanov, A. M., Tu, T-M. and Scott, K. P. (1983) Science 221, 259–261 13 Neidleman, S. L. and Geigert, J. (1986) Biohalogenation: Principles, Basic Roles and Applications, Ellis Horwood 14 Franssen, M. C. R. and van der Plas, H. C. (1992) Adv. Appl.

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Microbiol. 37, 41–98 15 Zacks, A., Yabannavar, A. V., Dodds, D. R., Anderson Evans, C., Das, P. R. and Malchow, R. (1996) J. Org. Chem. 61, 8692–8695 16 Fu, H., Kondo, H., Ichikawa, Y., Look, G. C. and Wong, C-H. (1992) J. Org. Chem. 57, 7265–7270 17 Thomas, J. A., Morris, D. R. and Hager, L. P. (1970) J. Biol. Chem. 245, 3129–3134 18 Sheldon, R. A. and Kochi, J. K. (1981) Metal Catalysed Oxidation of Organic Compounds, Academic Press 19 Strukul, G. (1992) Catalytic Oxidations with Hydrogen Peroxide as Oxidant, Kluwer 20 Walker, A. J. (1992) Tetrahedron Asymmetry 3, 961–998 21 Carreño, M. C. (1995) Chem. Rev. 95, 1717–1760 22 Colonna, S., Gaggero, N., Carrea, G. and Pasta, P. (1992) J. Chem. Soc., Chem. Commun. 357–358 23 Harris, R. Z., Newmyer, S. L. and Ortiz de Montellano, P. R. (1993) J. Biol. Chem. 268, 1637–1645 24 Miller, V. P., De Pillis, G. D., Ferrer, J. C., Mauk, G. and Ortiz de Montellano, P. R. (1992) J. Biol. Chem. 267, 8936–8942 25 Colonna, S., Gaggero, N., Carrea, G. and Pasta, P. (1994) Tetrahedron Lett. 35, 9103–9104 26 Colonna, S., Gaggero, N., Richelmi, C., Carrea, G. and Pasta, P. (1995) Gazzetta Chim. Ital. 125, 479–482 27 Ozaki, S. and Ortiz de Montellano, P. R. (1995) J. Am. Chem. Soc. 117, 7056–7064 28 Newmyer, S. L. and Ortiz de Montellano, P. R. (1995) J. Biol. Chem. 270, 19430–19438 29 Blée, E. and Schuber, F. (1989) Biochemistry 28, 4962–4967 30 Colonna, S. et al. (1990) Biochemistry 29, 10465–10468 31 Colonna, S., Gaggero, N., Casella, L., Carrea, G. and Pasta, P. (1992) Tetrahedron Asymmetry 3, 95–106 32 van Deurzen, M. P. J., Remkes, I. J., van Rantwijk, F. and Sheldon, R. A. (1997) J. Mol. Catal. A Chem. 117, 329–337 33 Colonna, S., Gaggero, N., Carrea, G. and Pasta, P. (1997) J. Chem. Soc., Chem. Commun. 439–440 34 Allenmark, S. G. and Andersson, M. A. (1996) Tetrahedron Asymmetry 7, 1089–1094 35 Doerge, D. R. and Corbett, M. D. (1991) Chem. Res. Toxicol. 4, 556–560 36 Boucher, J-L., Vadon, S., Tomas, A., Viossat, B. and Mansuy, D. (1996) Tetrahedron Lett. 37, 3113–3116 37 Gorzynski Smith, J. (1984) Synthesis 8, 629–656 38 Ozaki, S. and Ortiz de Montellano, P. R. (1994) J. Am. Chem. Soc. 116, 4487–4488 39 Colonna, S., Gaggero, N., Casella, L., Carrea, G. and Pasta, P. (1993) Tetrahedron Asymmetry 4, 1325–1330 40 Allain, E. J., Hager, L. P., Deng, L. and Jacobsen, E. N. (1993) J. Am. Chem. Soc. 115, 4415–4416 41 Zaks, A. and Dodds, D. R. (1995) J. Am. Chem. Soc. 117, 10419–10424 42 Lakner, F. J. and Hager, L. P. (1996) J. Org. Chem. 61, 3923–3925 43 Dexter, A. F. and Hager, L. P. (1995) J. Am. Chem. Soc. 117, 817–818 44 Dexter, A. F., Lakner, F. J., Campbell, R. A. and Hager, L. P. (1995) J. Am. Chem. Soc. 117, 6412–6413 45 Lakner, F. J., Cain, K. P. and Hager, L. P. (1997) J. Am. Chem. Soc. 119, 443–444 46 McCarthy, M. B. and White, R. E. (1983) J. Biol. Chem. 258, 9153–9158 47 Miller, V. P., Tschirret-Guth, R. A. and Ortiz de Montellano, P. R. (1995) Arch. Biochem. Biophys. 319, 333–340 48 Klibanov, A. M., Berman, Z. and Alberti, B. N. (1981) J. Am. Chem. Soc. 103, 6263–6264 49 Geigert, J., Dalietos, D. J., Neidleman, S. L., Lee, T. D. and Wadsworth, J. (1983) Biochem. Biophys. Res. Commun. 114, 1104–1108 50 van Deurzen, M. P. J., van Rantwijk, F. and Sheldon, R. A. (1997) J. Carbohydr. Chem. 16, 299–309 51 van Deurzen, M. P. J., van Rantwijk, F. and Sheldon, R. A. (1996) J. Mol. Catal. B Enzym. 2, 33–42 52 Hiner, A. N. P., Hernández-Ruíz, J., Arnao, M. B., Garcia-Canovas, F. and Acosta, M. (1996) Biotechnol. Bioeng. 50, 655–662 53 Pasta, P., Carrea, G., Monzani, E., Gaggero, N. and Colonna, S. Biotechnol. Bioeng. (in press) 54 Klibanov, A. M. (1997) Trends Biotechnol. 15, 97–101

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