Journal of Biotechnology, 23 (1992) 193-210

193

© 1992ElsevierSciencePublishers B.V. All rights reserved0168-1656/92/$05.00

BIOTEC 00727

The enzymatic synthesis of antiviral agents J ane R. H a n r a h a n and David W. H u t c h i n s o n Chemistry Department, Warwick University, Coventry, UK

(Received 19 August 1991;revisionaccepted 12 October 1991)

Summary The majority of potential antiviral agents which are currently undergoing clinical trials are inhibitors of the replication of nucleic acids.. The most common class of these inhibitors are nucleoside analogues and the elucidation of synthetic routes to these compounds has been of interest for many years as many are anticancer agents. One synthetic development has been the application of biotransformations to nucleoside syntheses. This topic has been reviewed recently (Shirae et al., 1991) but this review is not widely available. In the present review, the application of biotechnology to the synthesis of antiviral agents including those which are not nucleoside analogues will be discussed. Enzymatic syntheses of nucleosides can be simpler and quicker than syntheses carried out by chemical methods. The most useful enzymes are those found in catabolic pathways. Nucleoside phosphorylases and N-deoxyribosyltransferases have both been widely used for nucleoside synthesis catalysing the transfer of sugar residues from a donor nucleoside to a heterocyclic base. Enzymatic methods have also been applied to the resolution of racemic mixtures and adenosine deaminase is a convenient catalyst for the hydrolysis of amino groups on purines and purine analogues. Regioselective deprotection of nucleoside esters has been achieved with lipases and these enzymes have also been applied to the synthesis of esters of sugar-like alkaloids. The latter have potential as inhibitors of the replication of HIV. Esterases have also been used in combined chemical and enzymatic syntheses of organophosphorus antiviral agents.

Correspondence to: D.W. Hutchinson,ChemistryDept., Warwick University,CoventryCV4 7AL, U.K.

194

Adenosine deaminase; Antiviral; Castanospermine; 1-Deoxynojirimycin; N-Deoxyribosyltransferase; Enzymatic synthesis; Esterase; Lipase; Nucleosides; Nucleoside phosphorylases; Phosphinothricin

Introduction

A considerable number of antibacterial drugs have been developed over the past fifty years as there are many enzymatic reactions unique to bacterially-infected cells which can be targets for therapeutic agents. On the other hand, very few drugs are licensed for clinical use as antiviral agents at the present time. A major reason for this discrepancy is that there are very few differences between virally-encoded enzymes and those of the host and hence many compounds tested as antiviral agents have been extremely toxic towards the host. Most of the licensed antiviral drugs are analogues of naturally occurring nucleosides which act by interfering with the synthesis of nucleic acids in infected cells. In this way the replication of the virus is inhibited. These nucleoside analogues are usually activated by phosphorylation to their 5'-triphosphates before inhibiting nucleic acid synthesis and this activation can be carried out either by host cell or virally-induced enzymes. If activation is by host cell enzymes, the nucleoside analogue can be highly toxic as is the case with the two drugs which have been licensed as anti-HIV agents 3'-azido-2',3'-dideoxythymidine (AZT) (1) and 2',3'-dideoxyinosine (ddI) (2) (Fig. 1). On the other hand, the anti-herpetic drug 9-(2-hydroxyethoxymethyl)guanine (Acyclovir) (3) exhibits very low toxicity to the host as the initial activation of this compound is its phosphorylation catalysed

0 0

I..L.J N3 (1)

(2)

O

~o/~/o.J (3)

Fig. 1. Examples of antiviral nucleoside analogues: (1) 3'-azido-2',3'-dideoxythymidine(AZT); (2) 2',3'-dideoxyinosine(ddI); (3) 9-(2-hydroxyethoxymethyl)guanine(acyclovir).

195

by virally coded enzymes. Acyclovir is not phosphorylated by host cell enzymes and hence is not activated in uninfected cells. In recent years, much effort has been expended on the synthesis of nucleoside analogues. The chemical syntheses of nucleoside analogues are often multistage processes which can be difficult and time consuming (Moffatt, 1979). For example, if a naturally-occurring nucleoside is to be modified chemically, protecting groups are usually required on the heterocyclic base a n d / o r the sugar residue to allow modification of specific groups in the starting material. These protecting groups must be added before the relevant synthetic step and removed at a later stage and this can lower the overall yield of the synthetic reaction. If the alternative synthetic route of direct fusion of a heterocyclic base to an activated sugar residue is carried out chemically to produce the nucleoside analogue, mixtures of isomers of the analogue can be formed. The target compound must then be separated from this mixture, again resulting in poor yields. The unparalleled specificity of enzymatic reactions can be used to great advantage in the synthesis of complex biological molecules (Davies et al., 1989). Thus, reactions can take place at one reactive centre among fnany of similar reactivity without the need for protecting groups. The high catalytic efficiency of enzymatic reactions allows reactions to be carried out on analogues of natural substrates for the enzymes and the use of non-aqueous as well as aqueous solvents. The use of organic solvents instead of water as reaction media allows degrading enzymes such as peptidases or lipases to be used to catalyse synthetic reactions. Under these conditions, substrate specificity and the stereospecificity of enzyme-catalysed reactions can differ significantly from those observed in aqueous solution allowing a new range of products to be prepared. The enzymatic synthesis of nucleoside analogues offers advantages over chemical methods as protecting groups are not usually required and highly stereospecific reactions can occur. For example, enzymatic glycosyl transfer reactions usually lead exclusively to the "natural" /3-anomer of the nucleoside and only one site in the heterocyclic base is glycosylated. Enzyme-catalysed reactions are usually so efficient that analogues of naturally occurring substrates can often be employed in synthetic reactions with little diminution in yield. While the rate of enzymatic reactions with substrate analogues may not be as high as those with natural substrates, the syntheses still proceed at acceptable rates. This review will consider the application of enzyme catalysed reactions to the synthesis of three classes of antiviral agents (a) nucleoside analogues (b) sugar-like alkaloids and (c) organophosphorus compounds.

Nucleoside analogues Two main classes of enzyme have been used to synthesize nucleoside analogues by transferring glycosyl residues from a donor to an acceptor base: (a) nucleoside phosphorylases and (b) N-deoxyribosyltransferases.

196

Nucleoside phosphorylases Nucleoside phosphorylases catalyse the reversible phosphorolysis of ribo- or deoxyribonucleosides affording ribose- or deoxyribose-l-phosphate with release of base. Addition of an acceptor base to reaction (i) can result in the formation of a new nucleoside (reaction (ii)) (Bzowska et al., 1990).

Reactions catalysed by nucleoside phosphorylases (i) dRib-Base(1) + Pi = dRib-l-phosphate + Base(l) (ii) dRib-l-phosphate + Base(2) = dRib-Base(2) + Pi Where: dRib = 2-deoxy-D-ribose, Base = heterocyclic base e.g. purine or pyrimidine. Both pyrimidine and purine nucleoside phosphorylases are known. Nucleoside phosphorylases can be obtained from bacterial or mammalian sources and they can differ in substrate specificity. For example, the major difference between mammalian and bacterial purine nucleoside phosphorylases is that the mammalian enzyme will not accept adenosine as a natural substrate but will carry out the phosphorolysis of inosine and guanosine. The enzyme from Escherichia coli on the other hand will accept all three purine nucleosides as substrates. Coupled phosphorylase systems have been used on a synthetic scale to prepare a variety of base-modified nucleoside analogues. For example, using either uridine or thymidine phosphorylase from E. coli together with purine nucleoside phosphorylase from the same source, ribo- and deoxyribonucleosides of imidazo[4,5c]pyridines (3-deazapurines) have been prepared (Krenitsky et al., 1986) including 3-deazaadenosine which has antiviral properties and which will inhibit the replication of a number of viruses including Rous sarcoma virus (Bodner et al., 1981). Nucleoside syntheses by coupled enzymatic reactions rely on the establishment of equilibria and the success of a synthetic reaction depends inter alia on the relative strengths of binding of donor and acceptor bases to the enzymes, the nucleophilicity of the acceptor base etc. One attractive modification of these coupled reactions involves the use of a salt of 7-methylguanosine (4) as a glycosyt donor (Hennen and Wong, 1989). The production of ribose-l-phosphate in the first stage of the phosphorolysis reaction is essentially irreversible as 7-methylguanine does not function as a nucleophile in the reverse reaction. Using 1,2,4-triazole-3-carboxamide (TCA) as an acceptor, the broad spectrum antiviral agent ribavirin has been produced from a salt of 7-methylguanosine in high yield (Fig. 2). 3-Deazaadenosine has also been prepared in this manner. Crude preparations of nucleoside phosphorylases or whole cells have been widely used for the preparation of nucleoside analogues. While the enzymes carrying out the synthetic reaction often have not been identified, the synthetic reaction usually requires the presence of orthophosphate and it is reasonable to assume that nucleoside phosphorylases are involved. The earliest recorded synthesis of this kind was the preparation in low yield"of the 2'-deoxynucleoside derived from 5-trifluoromethyluracil with the aid of ruptured E. coli B cells (Heidelberger et al., 1964). Ribavirin has been synthesised from inosine and TCA by a partially

197

~H3~J~ NH OH3 O X- +NI ~ J ~ NH ¢/ I + H2P04(~ I~N/,~NH2 Nucleoside == HO'~ Phos~orylase + .o

o.

(4)

~ OPO3H2 HO OH

~"~ OPO3H2 t~N'N HO OH H TCA

Phospholylase

/~NsN HO"~O,~ + H2PO4-

==

HO OH dbavirin

Fig. 2. Enzymaticsynthesisof ribavirinfroma salt of 7-methylguanosine(4). purified purine nucleoside phosphorylase from Enterobacter aerogenes. In this case, the ribose-l-phosphate produced in the first step of the reaction was isolated by ion exchange chromatography before being used in the next synthetic step. The microbiological synthesis of ribavirin from TCA has also attracted much attention. Wet cell pastes of a number of organisms such as Bacillus brevis and Arthrobacter oxidans are capable of synthesising ribavirin from ribose-l-phosphate and TCA (Utagawa et al., 1986) while Brevibacterium acetylicum or Enterobacter aerogenes cells are capable of utilising TCA and guanosine for the synthesis of virazole (Shirae et al., 1988a). Erwinia carotovora cells can use orotidine as the ribosyl donor in this synthetic reaction (Shirae et al., 1988b). Whole cells of various bacteria including Enterobacter aerogenes, E. coli, Erwinia herbicola and Aeromonas salmonicida are capable of transferring the arabinofuranosyl residue from arabinofuranosyl uracil to adenine (Utagawa et al., 1985a). With Enterobacter aerogenes cells, 9-fl-D-arabinofuranosyladenosine (araA) formed and the product precipitated from solution. The synthesis of arabinofuranosyl guanine (araG) with whole cells of E. coli has also been reported (Zinchenko et al., 1990). Whole cells of Serratia marcescens carry out the synthesis of 5-trifluorothymidine from 5-trifluorouracil and thymidine (Hayashi et al., 1989). The anti-herpetic drug (E)-5-(2bromovinyl)-2'-deoxyuridine (BVDU) has been prepared from 2'-deoxy-guanosine or thymidine as glycosyl donor and (E)-5-(2-bromovinyl)-uracil as acceptor in the presence of a suspension of glutaraldehyde-treated E. coli BM-11 cells in potassium phosphate solution (Kalinichenko et al., 1989). Two procedures for the production of 5-methyluridine, a useful intermediate in the synthesis of AZT, have been reported. In the first method 5-methyluridine is synthesised from guanosine

198

and thymidine in the presence of resting cells of Erwinia carotovora (Ishii et aI., 1989). Immobilised thermostable purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus can utilise thymine and inosine for the production of 5-methyluridine in a continuous enzyme reactor (Hori et al., 1991). While the purified nucleoside phosphorylases appear to have a wide tolerance of different structures for the acceptor base, they do not appear to accept many sugar modified nucleosides as glycosyl donors. One exception to this is preparation of 2',3'-dideoxynucleosides of 6-substituted purines (Koszalka et al., 1988). These compounds which are potential anti-HIV agents have been prepared from the purine and 2',3'-dideoxythymidine in the presence of thymidine phosphorylase and purine nucleoside phosphorylase absorbed on to ion-exchange cellulose. The anti-HIV drugs 2',3'-dideoxyadenosine and -inosine have been prepared from 2',3'-dideoxyuridine and the corresponding purine by resting cells of E. coli (Shirae et al., 1989). Inorganic phosphate is an essential component of the reaction mixture in this case. The only other sugar-modified nucleosides which have been synthesised in this manner are 2'-amino-2'-deoxynucleosides of purines. Among the 26 genera of bacteria which have been examined, Erwinia herbicola, Enterobacter aerogenes and E. coli were found to be the most effective (Utagawa et al., 1985b).

N-Deoxyribosyltransferases Some organisms, notably the Lactobacilli, which do not contain high levels of purine nucleoside phosphorylase, can contain appreciable quantities of N-deoxyribosyltransferases (Chawdhri et al., 1991). Two types of N-deoxyribosyltransferases have been identified: type I which catalyses the transfer of 2-deoxyribose residues exclusively between purine bases and type II which catalyses the transfer of 2-deoxyribose residues between two purines, pyrimidines and purines or between two pyrimidines (Holguin and Cardinaud, 1975).

Reactions catalysed by N-deoxyribosyltransferases Type I (catalyses transfer between purines only) dRib-Pur(1) + Pur(2) = dRib-Pur(2) + Pur(1) Type II (catalyses transfer between purines and pyrimidines) (a) dRib-Pur(1) + Pur(2) = dRib-Pur(2) + Pur(1) (b) dRib-Pur + Pyr = dRib-Pyr + Pur (c) dRib-Pyr(1) + Pyr(2) = dRib-Pyr(2) + Pyr(1) Where: dRib = 2-deoxy-D-ribose, Pur = purine, Pyr = pyrimidine. The two transferase types, which have different molecular weights and thermal stabilities (Betbeder et al., 1991), offer a route for the synthesis of antiviral nucleoside analogues which is an alternative to nucleoside phosphorylases. For synthetic work, the two transferases from either Lactobacillus helveticus or L. leichmannii are usually not separated but are used with the minimum of purifica-

199

tion. As with the nucleoside phosphorylases, the glycosyl transfer reaction is highly stereospecific and only the/3-anomer of the nucleoside is formed (Betbeder et al., 1989). With the crude N-deoxyribosyltransferases, thymidine or 2'-deoxycytidine are the most effective glycosyl donors and considerable structural variation in the acceptor bases can be tolerated. For example, the 2'-deoxynucleoside of 2-chloroadenine which has antileukemic and immunosuppressive activity has been synthesised with a transferase preparation from L. leichmannii (Carson et al., 1984). This preparative reaction is well suited to the preparation of radioactively labelled compounds and crude N-deoxyribosyltransferases from L. helveticus have been used to synthesise 2-[~4C]-2'-deoxyribofuranosyl-5-trifluoromethyluracil (Heidelberger et al., 1964). The synthesis of 2',3'-dideoxynucleosides can also be carried out with the N-deoxyribosyltransferases. The crude enzymes from L. helveticus were reported to catalyse the synthesis of 2',3'-dideoxynucleosides natural bases (Carson and Wasson, 1988) and 2-halo-adenines (Haertle et al., 1988). The latter only showed anti-HIV activity in cells expressing 2'-deoxycytidine kinase activity. The transferases from L. leichmannii can catalyse the transfer of 2,3-dideoxyribose from 2',3'-dideoxycytidine to N(6)-substituted adenines (Betbeder et al., 1990). These compounds had appreciable anti-HIV activity in several cell lines but were not as toxic to cells as AZT. The synthesis of several pyrazolo[3,4-d]pyrimidine and triazolo[4,5-d]pyrimidine derivatives from 2',3'-dideoxycytidine and the corresponding bases catalysed by purified N-deoxyribosyltransferase II from L. leichmannii has been reported recently (Fischer et al., 1990).

Selective enzymatic modification of sugar or base residues

Sugar modification

The selective protection and deprotection of hydroxyl groups of nucleosides is of importance both in the synthesis of oligonucleotides and in the preparation of pro-drugs of antiviral compounds. Selective protection of hydroxyl groups of nucleosides has recently been investigated. Acylation of 2'-deoxy-5-fluorouridine and 2'-deoxy-5-trifluoromethyluridine in the presence of Amano PS (a lipase from Pseudomonas sp.) was reported to afford preferentially the 3'-O-acyl derivatives in high yields (Nozaki et al., 1990). While the selective deprotection of mono- and oligosaccharides has been extensively studied (Kloosterman et al., 1989), little work appears to have been carried out on nucleosides. Enzymatically removable protecting groups for nucleosides and nucleotides have been proposed for oligonucleotide synthesis, (Taunton-Rigby, 1973) and the regioselective deacylation of dicarboxylate esters of 2'-deoxynucleosides has been observed (Uemara et al., 1989). However, the only case of the synthesis of a sugar-modified nucleoside with antiviral activity is the preparation of 2'-O-acyl esters of araA by the action of a cell paste of Bacillus subtilis on the 2',3',5'-tri-O-acyl esters of araA (Baker et al., 1979).

200 Gua ATP

, HSV-1thyrnidinekinase HO racemic

B. H203PO

Ctotatus atrox

racemic

(5)

HO~l~Gua HO (+)

Gua

H203PO" ~ HO (-)

Gua

~, H O ' ~ HO (.)

Fig. 3. Enzymaticresolution of racemic 2'-ara-fluoroguanosine (5). Although the 2'-O-acyl esters were totally resistant to adenosine deaminase, they showed little inhibitory activity against genital herpes. One interesting application of enzymes to the synthesis of antiviral nucleosides is in the resolution of racemic carbocyclic nucleosides. For example, racemic 2'-ara-fluoroguanosine (5) was found to be a potent antiherpetic agent but it was not known which was the more active antipode. The chemically synthesised racemic nucleoside analogue was phosphorylated by ATP and thymidine kinase which had been isolated from Herpes Simplex Virus-1 (Fig. 3). Hydrolysis of the racemic mixture of monophosphates obtained in this manner with a 5'-nucleotidase from Crotalus atrox, venom gave a dextrorotatory nucleoside analogue which was assumed to correspond to naturally occurring guanosine. Hydrolysis of the remaining monophosphate with alkaline phosphatase gave the laevorotatory isomer. The dextrorotatory isomer of (5) was approximately twice as potent as the laevo-isomer as an anti-herpes agent (Borthwick et al., 1988). Application of this method to the resolution of racemic aristeromycin gave the dextro- and laevorotatory antipodes. Only the laevorotatory antipode of aristeromycin showed antiviral activity (Secrist et al., 1987).

Base modification The most common enzymatic modification of base residues in the preparation of antiviral compounds is the deamination of 6-aminopurines with adenosine deaminase. This enzyme which is of widespread occurrence in animal tissue, is commercially available and is a convenient catalyst for carrying out this deamination reaction under mild conditions. The synthesis of the hypoxanthine analogue (6) of oxetanocin (7) was an early example of the use of adenosine deaminase to prepare an antiviral agent (Fig. 4) (Shimada et al., 1987). Compound (6) could be oxygenated by the actinomycete Nocardia interforma to yield the xanthine analogue (8) which was converted chemically into the corresponding 2,6-diaminopurine derivative. Treatment of the latter with adenosine deaminase pro-

201

•

NH2

O adenosinecleami.el=

HOg'OH

~ I~NJ

HO~OH (6)

(7)

II=

Nocat~a interfomla

i io N ~ ;

H

threechemicalsteps

"N.~U~.N~ O

(8) NH2

I I adenosinedeaminase

IB

O

N~

NH

~N/~NH2

H O ~ O H

HOv~OH

(9) Fig. 4. Synthesis of guanine analogue of oxetanocin (9).

~~ NH2

N

N . . ~ N°

N

•H O ~

H

?"5°C

Racemic (11)

(-)-carbovir (lo) + O

NH2

(÷)-earl~vir (10)

(+)-(11)

Fig. 5. Synthesis of the optical antipodes of 2',3'-didehydro-2',3'-dideoxyguanosine(carbovir) (11).

202 NH2

(3 NH2 aOen°sinedeaminase~

~N~NH

2

F (12)

Racemic

Fig. 6. Synthesis of ,8-1-(2-amino-6-oxo- 1H,9H-purin-9-yl)-2-deoxy-2,2-difluororibose(12).

duced the guanine analogue of oxetanocin (9) which inhibited the replication of Herpes Simplex Virus-2. Adenosine deaminase has also been used to prepare the optical antipodes of 2',3'-didehydro-2',3'-dideoxyguanosine (carbovir) (10), a nucleoside analogue with potent anti-HIV activity (Vince and Brownell, 1990). Racemic cis-[3-(2,6)-diamino-9H-purin-9-yl]cyclopentenyl carbinol (11)was used as a precursor of carbovir in this experiment. Deamination of this racemic precursor with adenosine deaminase at 25°C produced laevorotatory carbovir in good yield (Fig. 5). Hydrolysis of the filtrate from the first hydrolytic reaction with additional adenosine deaminase at 37°C gave dextrorotatory carbovir. The laevorotatory isomer of carbovir was a highly active inhibitor of the replication of HIV-1 while the dextrorotatory isomer had little activity. This methodology was first applied to the resolution of racemic carbocyclic nucleoside analogues derived from 6aminopurines but no data on the antiviral activity of the individual isomers was given (Herdewijn et al., 1985). Adenosine deaminase has also been used in the preparation of /3-1-(2-amino-6-oxo-lH,9H-purin-9-yl)-2-deoxy-2,2-difluororibose (12) from a racemic mixture of 1-(2,6-diamino-9H-purin-9-yl)-2-deoxy-2,2-difluororibose (Hertel et al., 1989; Fig. 6). Adenosine deaminase can also deaminate purine analogues e.g. pyrazolopyrimidines and the 2',3'-dideoxynucleoside of allopurinol has been obtained by the action of this enzyme on the chemically synthesised 2',3'-dideoxynucleoside of

+ o

(-)-Carbovir (10)

98 % ee 45%yield (13)

Separation 10steps

"N~ H 2 ~ cO~H+ O ~

NH

(+)-Carbovir(10)

98 %ee 45%yield Fig. 7. Preparation of the optical antipodes of carbovir starting from a common intermediate (13) using 1, Rhodococcus equi NCIB 40213; 2, Pseudomonas solanacearum NCIB 40249.

203 4-amino-lH-pyrazolo[3,4-d]pyrimidine (Seela and Kaiser, 1988). The allopurinol nucleoside analogue did not possess anti-HIV activity. The enzymatic resolution of the racemic lactam 2-azabicyclo[2.2.1]hept-5-en-3one (13), an intermediate in the synthesis of carbocyclic nucleosides e.g. carbovir has recently been described. Two distinct whole cell biocatalysts were used in the resolution of the racemic lactam to produce both enantiomers with high enantiomeric excess (Fig. 7). Thus, both enantiomers of the desired nucleoside were accessible (Taylor et al., 1990).

Sugar-like alkaloids A number of plant alkaloids such as 1-deoxynojirimycin (DNJ) (14) and castanospermine (15) are potent inhibitors of a-glucosidases and are highly biologically active as they prevent the removal of glucose residues from glycoproteins during processing and maturation. These compounds are of interest as antiviral agents and they show great promise in vitro as anti-HIV compounds. When cells infected with HIV are treated with (14) or (15), glucose residues are not hydrolysed from the virion surface glycoprotein gpl60. This processing of gpl60 which gives rise to glycoprotein gpl20 is an essential step in the maturation of the virus (Jones and Jacob, 1991). The inhibitory effect of DNJ may be due to the retardation of this processing reaction. Changes in conformation of gpl20 have been observed when the latter is formed in the presence of DNJ derivatives which may also alter binding of the virus to the host cell and hence inhibit virus replication. The toxicity of the sugar-like alkaloids towards host cells may restrict their clinical use so it is of interest that N-alkylation or O-acylation reduces their toxicity. For example, N-Butyl DNJ (16) (Ratner et al., 1991; Karpas et al., 1988) and 6-O-butyryl castanospermine (17) (Taylor et al., 1991; Fig. 8) are more active anti-HIV agents in cell culture than their parents' DNJ or castanospermine and appear to be less toxic to host cells. Castanospermine contains four secondary hydroxyl groups of similar reactivity and hence the extensive use of protecting groups is required in any chemical synthesis of derivatives acylated at specific positions. While a chemical synthesis of (17) has been devised (Liu et al., 1990) enzymatic methods should offer advantages for the regiospecific acylation of castanospermine and related compounds in view of the highly specific nature of many enzymatic reactions. This has proved to be the case. Subtilisin Carlsberg catalyses the synthesis of a variety of acylated castanospermines (Margolin et al., 1990). For example, treatment of castanospermine in a solution of pyridine with 2,2,2-trichloroethyl butyrate in the presence of subtilisin leads to 1-O-butyryl castanospermine (18) in high yield. Further acylation by 2,2,2-trichloroethyl butyrate of (18) as a suspension in tetrahydrofuran catalysed by subtilisin gives rise to 1,6-O-dibutyryl castanospermine (19). Under these conditions, lipases show a different regioselectivity, for example lipases from porcine pancreas or Chromobacterium viscosum catalyse the acylation by 2,2,2-trichloroethyl butyrate at the 7-position of (18). Subtilisin can be used to catalyse the

204

HOs.

OH

OH HO., ~'~ ~OH

OH

"H ~ I H 2 0 H (14) OH ,o,A,o

00OC3H7

H (15)

°"~ , [~iH2OU n-C4H9 (16) OH

k'.N~OH (17)

C0C3H7 (~8)

OCOC3H7 HO"...f~..,,,OH L N~OGOC3H7 (19) Fig. 8. Alkaloids with anti-HIV activity: 1-deoxynoiirimycin (DNJ) (14); castarlospermine (15); N-butyl DNJ (16); 6-O-butyryl castanospermine (17); 1-O-butyryl castanospermine (18); 1,6-O-dibutyryl castanospermine (19).

selective hydrolysis of the 1-O-butyryl moiety from the 1,6- or 1,7-dibutyrates leaving the 6- or 7-O-butyrate of castanospermine. N-Butyl DNJ (16) is usually prepared by chemical means e.g. the catalytic reduction of a mixture of (14) and n-butanal. However, an enzymatic synthesis of (16) from 1-hydroxyacetone phosphate and 3-(N-butyl)-2-hydroxypropanal in the presence of fructose diphosphate aldolase has been reported briefly (vonder Osten et al., 1989). Several syntheses of 1-deoxynojirimycin and related compounds which combine an aldol condensation with chemical steps have been published (vonder Osten et al., 1989; Pederson et al., 1989; Straub et al., 1990). The most versatile synthetic route (vonder Osten et al., 1989) uses a recombinant bacterial fructose-l,6-diphosphate aldolase to catalyse the reaction between (S)-3-azido-2-hydroxypropanal and dihydroxyacetone phosphate, hydrolytic removal of the phosphoryl residue from the condensation product followed by hydrogenation of the azido-group leads to DNJ. The corresponding manno-derivative, 1-deoxymannonojirimycin (DMJ), is obtained in a similar manner using (R)-3-azido-2-hydroxypropanal in place of the (S)-isomer (Fig. 9). In a short chemical synthesis of DNJ and DMJ involving a combination of chemical and microbiological methodology, glucose (or mannose) was converted into 1-amino-l-deoxy-D-sorbitol by reductive amination. The 1-amino group was then protected and the product oxidised with Gluconobacter oxidans. DNJ (or DMJ) was isolated after removal of the protecting group from the oxidised product (Fig. 10; Kinast and Schedel, 1981). A chemoenzymatic synthesis of castanosper-

205 0

o

H2OaPO v

v

OH

J¢

H

Na OH

FDP ak:lolase

O 1"12OaPO~ ~ . ~

v

OH

y

L

y -A. ,

OH

OH

(a) acid phosphatase (b} H2/ Pd

DNJ or DMJ

Fig. 9. Chemoenzymatic synthesis DNJ or DMJ. CH2OH

CH2OH OH

~

HO " ~ 1

CH2OH HR

~

HO ~ - ] OH

02

OH

NHR

H

OH

OH

CH2OH H~ Pa

H OH DNJ

where R = Cbz

Fig. 10. Chemoenzymatic synthesis of DNJ. O

O

O

CHaO

D~lx~asct/s~.

OH

CH30

N..--r

N,Jr

I

I

~

BOC

(20)

(21)

0 OAC CHaO

Lipase

m- CHaO

-

0 O~ "1-

"

CH30

N- ~ t

N.~I

N.~I

I

I BOC

BOC

BOC

I

(22)

Fig. 11. Enzymatic preparation of precursors to castanospermine.

206

mine has also been published (Fig. 11; Bhide et al., 1990). In this cage, compound (20) was reduced by the yeast Dipodascus sp. to the (S)-alcohol (21) which was converted in a multi-stage chemical process into castanospermine. Alternatively, (S)-(21) can be obtained by the lipase-catalysed enantioselective hydrolysis of the racemic acetate (22). The demonstration that glucose is the major precursor of both DNJ and DMJ in the culture media of Streptomyces subrutilus opens the possibility of obtaining semisynthetic DNJ and DMJ analogues following the addition of glucose derivatives to growing populations of this organism (Hardick et al., 1991).

Organophosphorus compounds Streptomyces viridochromogenes can produce a tripeptide y-(hydroxymethyl phosphinyl)-L-a-aminobutyryl-L-alanyl-L-alanine,(Bialaphos) which has herbicidal and fungicidal activity. Hydrolysis of Bialaphos with E. coli protease liberates 4-(hydroxymethylphosphinyl)-L-2-aminobutanoic acid or L-phosphinothricin (23). Phosphinothricin is widely used as a herbicide and a fungicide and has been shown to possess antiviral activity against both DNA and RNA viruses (Fukuyasu et al., 1978). D-, DL- and L-phosphinothricin have been obtained from o-, DL- and L-3-amino2-pyrrolidinone (24) using an enzymatic hydrolysis as a key step in the synthesis (Natchev, 1988a). Acetylation of (24) followed by treatment with ethyl methylphosphinate afforded (25). Acidic hydrolysis of (25) led to racemisation but enzymatic hydrolysis first with a phosphodiesterase and then with acylase and finally glutaminase led to D-, DL- and L-phosphinothricin with little racemisation (Fig. 12). Hydrolysis of racemic diethyl phosphinothricin using a-chymotrypsin gave (23) and the unchanged diethyl ester of D-phosphinothricin and the P-O-monoethyl ester of L-phosphinothricin. Phosphodiesterase I converted the latter into L-(21) in practically quantitative yield. This enzyme also hydrolysed the P-O-monoethyl ester of D-phosphinothricin into D-(23). Fully esterified Bialaphos was prepared chemically

N• H (24)

AcOPO3H2

m,

N~HoCOCH3c2HsOP(O)HCH3 h:tNOC\ /CH3 I. ~:;HCH2CH2P :O CH3CONH/ \OC2H5 H (25)

Enzymatichydrolysis

HOOC .

/CH3 HCH2CH2P~O H2Ni/C \OH X

phosphinothdcin (23) Fig. 12. Enzymatic synthesis of L-phosphinothricin (23).

207

by the active ester method and the C-terminus hydrolysed using alkaline mesintericopeptidase. Phosphodiesterase I was employed in the final stage of the synthesis to remove the O-ethyl group from phosphorus (Natchev 1989, 1988b).

Conclusion

The application of enzymes to the synthesis of antiviral compounds is still in its infancy. The most progress so far has been made in the application of glycosyl transferases to the synthesis of nucleoside analogues. Methodology is now available for the enzymatic synthesis of 2'-deoxy- and 2',3'-dideoxynucleosides of a wide variety of heterocyclic bases. The enzymatic resolution of enantiomers of nucleoside analogues and the selective acylation or hydrolysis of other derivatives are beginning to be studied. The formation of carbon-carbon bonds catalysed by aldolases is another highly stereospecific reaction which is being exploited in the synthesis of alkaloids such as DNJ. These reactions should prove to be valuable adjuncts in the synthesis of complex molecules in view of the high specificity towards substrate which is shown in enzymatic reactions. With the present intense world-wide interest in biotransformations and the rapid increase in the number of enzymes currently being exploited for the synthesis of commercially valuable organic compounds, the application of enzymes to the synthesis of antiviral agents is expected to be an important tool in the hands of the medicinal chemist.

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