Adenosine Deaminase in Nucleoside Synthesis. A Review

ADENOSINE DEAMINASE IN NUCLEOSIDE SYNTHESIS. A REVIEW

Mukta GUPTA1 and Vasu NAIR2,*Department of Pharmaceutical and Biomedical Sciences and The Center for Drug Discovery,The University of Georgia, Athens, GA 30602, U.S.A.; e-mail: 1 [email protected],2 [email protected]

Received March 14, 2006Accepted April 27, 2006

Dedicated to Professor Antonín Holý on the occasion of his 70th birthday in recognition of hisoutstanding contributions to the area of nucleic acid chemistry.

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7692. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771

2.1. Occurrence and Biomedical Significance of Adenosine Deaminase . . . . . . . 7712.2. Structure and Biocatalytic Properties of Adenosine Deaminase . . . . . . . . 7712.3. Selected Examples of the Use of Adenosine Deaminase in Nucleoside Synthesis . 774

3. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7854. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785

Adenosine deaminase (ADA) is an enzyme in the purine salvage pathway that catalyzes thedeamination of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively.This deamination is an important factor in limiting the usefulness of adenosine analoguesin chemotherapy. However, the biocatalysis by ADA is also a useful transformation in enzy-matic synthesis. In this review, examples from both the principal investigator’s laboratoryand from the literature, which depict the synthetic usefulness of this enzyme in de-amination, dehalogenation, demethoxylation reactions and in diastereoisomeric resolution,are presented. It is not the intent of this review to comprehensively list all of the bio-transformations induced by adenosine deaminase, but rather to present representative exam-ples to highlight the powerful synthetic utility of this enzyme. A review with 72 references.Keywords: Adenosine deaminase; Nucleosides; Enzyme catalysis; Deamination; Hydrolysis;Dechlorination; Adenosine; Inosine; Nucleoside analogues; Antiviral activity.

1. INTRODUCTION

The applications of enzyme catalysis has immense potential in biotechnol-ogy, drug discovery and molecular medicine. The concept of using enzymesfor producing therapeutic drugs has been around for well over a century.Some of the earliest studies were performed in 1835 by the Swedish chem-

Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 6, pp. 769–787

Adenosine Deaminase 769

© 2006 Institute of Organic Chemistry and Biochemistrydoi:10.1135/cccc20060769

ist, Jon Jakob Berzelius, who termed their chemical action catalytic. En-zymes have the distinct advantage of binding their targets with highaffinity and specificity. They can also function as catalysts in organic trans-formations.

enzyme + organic substrate enzyme–substrate complex enzyme + product

The increasing concerns pertaining to safe environmental scientific prac-tices have encouraged scientists to focus more on environmentally benigncatalysts. The introduction of new chemoenzymatic methodologies is im-portant for the development of key target molecules of biomedical andtherapeutic importance. For this reason, the use of enzymes in the prepa-ration of novel molecules has received a steadily increasing amount ofattention. These novel catalysts, not only promote reactions with highspecificity under very mild conditions of temperature, pH and pressure, butthey also facilitate transformations that are difficult to achieve using moretraditional synthetic approaches. Many of the reagents and chemical cata-lysts that have been used in the past several decades contain transition met-als and heavy metals. Their handling and disposal pose a variety ofproblems. In addition, these metals may have environmental longevity.Replacing them with environmentally acceptable catalysts is desirable.Enzymes are environmentally friendly resources that function best underaqueous conditions, which decrease the necessity for using toxic organicsolvents. Another reason for considering enzymes as catalysts is their utilityand greater ease in enantiospecific or diastereospecific synthesis.

The use of enzymes in organic synthesis is now widely accepted and willcontinue to gain momentum as more synthetic research utilizes enzymes asalternative and more desirable sources for specific biocatalysis. This is par-ticularly so for the field of nucleoside chemistry. The majority of potentialantiviral agents which are in clinical use or undergoing clinical trials arenucleoside analogues. The chemical synthesis of novel nucleosides forchemotherapeutic screening usually involves multistage processes whichcan be time-consuming and inefficient1. The application of enzymaticmethods for the synthesis and modification of antiviral nucleosides showsgreat promise because of the simplicity and high specificity of enzymaticreactions. Some of the more useful enzymes for nucleosides are those foundin nucleoside catabolic pathways. Nucleoside phosphorylases andN-deoxyribosyltransferases have both been used for nucleoside synthesis,catalysing the transfer of sugar residues from a donor nucleoside to aheterocyclic base. Enzymatic methods have also been applied to the resolu-


770 Gupta, Nair:

tion of racemic mixtures2. One enzyme that has received considerable inter-est in bioorganic synthesis is adenosine deaminase. This review articlefocuses on representative examples of synthetic biotransformations in-duced by adenosine deaminase.

2. RESULTS AND DISCUSSION

2.1. Occurrence and Biomedical Significance of Adenosine Deaminase

Adenosine deaminase or adenosine aminohydrolase (ADA, EC 3.5.4.4) is anenzyme that catalyzes the hydrolytic deamination of adenosine, its deriva-tives and its analogues. The enzyme is present in almost all human tissues,the highest levels being in the lymphoid system3. Studies have also shownthat ADA is not only a cytosolic enzyme but also an ecto-enzyme4. In hu-mans and animals, adenosine deaminase shows two kinetically distinctisozymes, ADA1 and ADA2 5. These are distributed differently and havedifferent molecular weights. ADA1 can exist as a 41 kDa monomer or asa 280 kDa dimer coupled to a binding protein, while ADA2 is a 110 kDaprotein. The isoenzymes have closely related active centers but with somedifferences.

Adenosine deaminase has been the subject of considerable medical inter-est in recent years, mainly because in humans a congenital defect in the en-zyme causes severe combined immunodeficiency disease6,7. In 1972, Giblettet al. hypothesized that since ADA in red blood cells is similar to that pro-duced by normal lymphocytes, its deficiency in inherited immune diseaseis due to impaired lymphocyte function8. Severe combined immunodefi-ciency disease is characterized by deficiencies in both B- and T-cell medi-ated immunity and, with the onset early in life, of severe infections. ADA isimplicated in a variety of diseases including acquired immune deficiencysyndrome, anemia, various lymphomas and leukemias9. Green and Chan10

suggested that the immune deficiency disease associated with a deficiencyof ADA may be the result of pyrimidine starvation. The role of ADA ranges,amongst others, to modulating neuron membrane permeability and thuscellular excitability and to detoxification of adenosine11–13.

2.2. Structure and Biocatalytic Properties of Adenosine Deaminase

ADA participates in purine metabolism, where it degrades either adenosine or2′-deoxyadenosine producing inosine or 2′-deoxyinosine, respectively (Fig. 1).



adenosine + H2O inosine + NH3

Cleavage of inosine (or its deoxy counterpart) leads to hypoxanthine,which can be either transformed into uric acid by xanthine oxidase or sal-vaged into mononucleotides by the action of hypoxanthine-guanine phos-phoribosyltransferase (HGPRT).

Structurally, it is a glycoprotein containing galactosamine and glucos-amine and has a predominance of acid amino acids14. This enzyme is also awell studied model for catalytic functions through binding of inhibitors in-cluding ground- and transition-state analogues15. In 1971, Zielke andSuelter suggested that no cofactors were required for activity16. However, in1991 Wilson et al.17, using X-ray crystallographic data, showed that thezinc cofactor is a key element, not only in the catalytic function of ADA,but also in correlating many of its mutational effects. The mechanism ofaction of ADA is of the addition-elimination type with a direct addition ofwater to the C-6 position of the purine ring followed by elimination ofammonia (Fig. 2)17.


772 Gupta, Nair:

HOO

OHOH

O

OHOH

HONH3

(H) (H)

ADAHN

N N

N

O

N

N N

N

NH2

PNP

IMP

HGPRT

H2O

(d)ATP

(d)ADP

purinesalvage

(deoxy)adenosine (deoxy)inosine

methylation reactions(S)-adenosylmethionine

(d)AMP

cAMPnucleic acids

hypoxanthine

FIG. 1Pathway of purine metabolism illustrating the site of action of adenosine deaminase14

Knowledge of the three-dimensional structure of adenosine deaminasecomplexed with transition-state analogues has led to considerable new mo-lecular understanding of the catalytic mechanism of the enzyme. Adeno-sine deaminase is a single-chain α/β protein with 352 amino acids. ADAcontains a parallel α/β barrel motif, with 8 central β-strands and 8 peri-pheral α-helices (also called a TIM barrel). The oblong-shaped deep activesite is lined by the COOH-terminal segments and connecting loops of theβ-barrel strands. The active site also contains a zinc atom, which partici-pates directly in the deamination mechanism17. There are several hydrogenbonds between the substrate and the enzyme that stabilize the binding ofsubstrate in the transition state (Fig. 3). The zinc ion is located deep within



O

HO OH

OH

HN

O H

HO

O

HO

Zn

OO H

N

N N

N

NH2

O

NNH

Asp 295

His 238

Glu 217

Gly 184

Asp 296_

FIG. 2Mechanism of hydrolytic deamination of adenosine by adenosine deaminase

FIG. 3Ribbon diagram of the crystal structure of adenosine deaminase17. ADA has a penta-coordinated zinc metal ion in the deep end of the active site pocket. Hydrophobic residues(part of α-helix) are closely located at the opening of the active site, apparently acting as a lid

the substrate binding cleft and is coordinated in a tetrahedral geometry tonitrogens of His-15, His-17 and His-214 and the oxygen of Asp-295. A watermolecule, which shares the ligand coordination site on zinc with Asp-295,is involved in replacing the amine group at the C-6 position of adenosinethrough a stereospecific addition-elimination mechanism17–20. The aminoacid sequence comparisons of human, mouse, and bovine adenosinedeaminases are known21–24. The primary amino-acid sequence of ADA ishighly conserved across species.

ADA is commercially available in purified form. The enzymatic prepa-ration from calf intestinal mucosa is the most frequently used enzyme innucleoside chemistry. ADA catalyzes the hydrolytic deamination ofadenosine and deoxyadenosine to inosine and deoxyinosine, respectively(Scheme 1). The conversion of adenosine to inosine catalyzed by ADA canbe monitored by UV spectroscopy by following the change in λmax from260 nm (ε 15 000) to 248 nm (ε 12 200). ADA exhibits broad substrate ac-tivity. It finds many synthetic applications in the transformation of purinenucleosides that are modified in the base or the ribose moiety25–29.

2.3. Selected Examples of the Use of Adenosine Deaminase inNucleoside Synthesis

An early report of the potential of ADA in synthetic work was that byMinato and Nakanishi30 in 1967 that stated ADA from takadiastase cata-lyzed the hydrolytic dechlorination of 6-chloropurine riboside to giveinosine. This activity was competitively inhibited by adenine and 6-chloro-purine. Several enzyme preparations had the same ratio of deaminase activ-ity to dechlorinase activity. They concluded that a single enzyme might beinvolved in both the deamination and the dechlorination reactions.


774 Gupta, Nair:

N

N

N

N

NH2

O

OHHO

HOO

OHHO

HO

NH

NN

N

O

adenosine deaminase

λmax 260 nm λmax 248 nm

SCHEME 1The ADA-catalyzed conversion of adenosine to inosine

Nair and Wiechert31 demonstrated that nucleosides in which the adeninering has been moved from the 1′-position to the 5′-position are resistant todegradation by the enzyme, ADA. They investigated minimum structuralrequirements for substrate binding and significant substrate activity andconcluded that the adenine ring, the tetrahydrofuran moiety bearing a5′-CH2OH and the β-D stereochemistry were all required. Dramatic changesin substrate activity occur when the 5′-CH2OH is altered. They examined aclass of nucleosides called “reversed” nucleosides where the purine ring hasbeen moved from the 1′-position to the 5′-position. These compounds wereremarkably stable compared to adenosine toward hydrolytic deaminationby ADA. The results were consistent with other observations of the impor-tance of 5′-CH2OH in ADA substrate activity32.

Oxetanocin, a natural nucleoside with a surrogate carbohydrate moietyisolated from bacterial sources, exhibits anti-HIV activity33. The chemo-enzymatic synthesis of a hypoxanthine analogue of oxetanocin by Shimadaand co-workers used ADA 34. Further, this hypoxanthine compound wasoxygenated by actinomycetes Norcardia interforma to yield the xanthine an-alogue of oxetanocin, which was converted chemically into the correspond-ing 2,6-diaminopurine derivative. Treatment of the latter with adenosinedeaminase produced the guanine analogue of oxetanocin (Scheme 2).



N

NN

N

NH2

NH

NN

N

ONH

NH

N

N

O

O

N

NN

N

NH2

NH2

NH

NN

N

O

NH2

ADA

ADA

OHO

OH

OHO

OH

OHO

OH

OHO

OH

OHO

OH

oxetanocin

Nocardiainterforma

3 chemicalsteps

guanine analogue

hypoxanthine analogue

actinomycetes

SCHEME 2Use of ADA in the synthesis of analogues of oxetanocin

In 1986, Montgomery and co-workers35 reported the synthesis of9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)guanine (Scheme 3) from1,3-di-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-D-arabinofuranose and 2,6-di-chloropurine in six steps using an enzymic deamination by ADA in the laststep. The target compound was stable to cleavage by purine nucleosidephosphorylase. It was found to be cytotoxic in two cell lines, including aT-cell line. Incubation of the compound with L1210 cells resulted in inhibi-tion of DNA synthesis as evidenced from reduced incorporation of labeledthymidine into DNA.

Seela and Kaiser36 reported the conversion of 2′ ,3′-dideoxy-8-aza-7-deazaadenosine into allopurinol 2′,3′-dideoxyribofuranoside with ADAin phosphate buffer (Scheme 4).

In 1988, Farina and Benigni37 prepared dideoxynucleosides in high opti-cal purity from L-glutamic acid. The condensation reactions between acti-vated 2,3-dideoxypentofuranoses and silylated purines afforded diastereo-isomeric dideoxynucleosides (β/α mixtures) which are difficult to separate.However, in the case of the synthesis of the anti-HIV active compound,dideoxyinosine (ddI), the diastereoisomeric mixture of dideoxyadenosine(ddA) and its diastereoisomer could be easily separated through the actionof ADA, which quantitatively deaminates the β-anomer, leaving behind


776 Gupta, Nair:

NH2

NH

N

N

O

N

O

HO

FHO

N

N

N

NH2

NH2N

O

HO

FHO

ADA

SCHEME 3Structure of a cytotoxic compound synthesized with ADA in the final step

NH

NNN

O

OHO

NNN

NH2

OHO

N ADA

phosphate buffer

SCHEME 4Use of ADA in the synthesis of a derivative of allopurinol

unreacted α-isomer (Scheme 5). Dideoxynucleosides are of considerable in-terest as reagents for DNA sequencing and some dideoxynucleosides are po-tent inhibitors of HIV replication through their triphosphates.

In 1989, Nair and Buenger38 synthesized novel and stable congeners ofthe antiretroviral compound, 2′,3′-dideoxyadenosine (ddA), through metal-mediated cross-coupling reactions and free radical conversions as the keysteps. As expected, these compounds were intrinsically more stable thanddA with respect to hydrolytic deamination by ADA.

Vince and Brownell39 utilized ADA for the enzymatic resolution ofcarbovir, a nucleoside analogue with potent anti-HIV activity. Racemiccis-[4-(2,6-diamino-9H-purin-9-yl)cyclopent-2-en-1-yl]methanol was usedas a precursor of carbovir and the deamination of this racemic precursorwith adenosine deaminase at 25 °C gave laevorotatory carbovir in good yield(Scheme 6).



N

NN

N

NH2

OHO

NH

N

N

O

N

OHO

N

NN

N

NH2

OHO

ADA+

SCHEME 5Structures of two anti-HIV dideoxynucleosides and the synthesis of ddI from ddA using ADA

N

N

NH2

NH2N

HO

N

N

N

O

NH2N

HO

NH

NHN

O

NH2N

HON

N

N

N

NH2

NH2N

HOADA, 37 oC

(±)

+

(+) precursor

(-) carbovircarbovir precursor

(+) carbovir

ADA, 25 oC

SCHEME 6Optical isomer resolution in the synthesis of the anti-HIV active compound, (–)-carbovir

Nair and Sells40,41 reported a convenient enzymatic synthesis of 2′,3′-di-deoxyguanosine (ddG) in high yields by utilizing the hydrolase activity ofADA. Dideoxyguanosine has been shown to have in vitro anti-HIV activity(ED50 = 0.1, 1.0, 7.6 µM in H-9, MT-2 and MT-4 cells, respectively)42. Itsbioactive form, the corresponding triphosphate, has been shown to be aninhibitor of HIV reverse transcriptase in cell free systems (Km = 16.5 µM, Ki =0.5 µM)43. The immediate precursor in the synthesis of ddG was 2-amino-6-chloro-purine 2′ ,3′-dideoxyriboside, which is readily prepared fromguanosine. In the key enzyme-mediated step, rapid and complete conver-sion to ddG was observed and the transformation was easily monitored byfollowing the change in absorbance from 307 to 252 nm (Scheme 7).Robins et al.44 synthesized sugar-modified guanine nucleosides using ADAin the key final step of the synthesis. Ciuffreda et al.45, reported the ADA-catalyzed deamination of 2′-deoxyadenosine to 2′-deoxyinosine on a pre-parative scale.

In 1991, Nair et al.46 synthesized 2′,3′-dideoxy-3-isoadenosine, a uniquestructural isomer of the anti-HIV compound, 2′ ,3′-dideoxyadenosine(Scheme 8). 2′,3′-Dideoxy-3-isoadenosine (λmax 270 nm) is stable at ambienttemperature in organic solvents. However, it slowly isomerizes to ddA(λmax 260 nm) in aqueous solution at room temperature. 2′,3′-Dideoxy-3-isoadenosine is a substrate for mammalian adenosine deaminase, but it is


778 Gupta, Nair:

N

N

Cl

NH2N

O

OHHO

HO

N

N

N NH

O

NH2N

OHO

Cl

NH2N

OHO

N

N

N

N

O

NH2

NH

NN

O

OHHO

HO

ADA phosphate buffer

pH 7.4

SCHEME 7Efficient ADA-based synthesis of anti-HIV active ddG from guanosine

only slowly deaminated by this enzyme (<10% of the activity of adenosine)to give the corresponding dideoxyisoinosine (261 nm), which slowly rear-ranges to ddI (248 nm).

Farina et al.47 patented the preparation of dideoxyinosine via enzymicdeamination of dideoxyadenosine with adenosine deaminase. An anomeric(α/β) mixture of dideoxyadenosine prepared by chemical synthesis was in-cubated with ADA in water at 30 °C for 2.5 h. The product was separatedand purified by crystallization.

The synthesis of optically pure (–)-adenallene and (+)-adenallene were re-ported using ADA 48. Racemic (±)-adenallene was subjected to deaminationwith ADA and monitored by HPLC using a Chiralcel CA-1 column to give(–)-adenallene and the deaminated product, (+)-hypoxallene. The latter wasthen converted by known methodologies to optically pure (+)-adenallene.The R-enantiomer inhibited the replication and cytopathic effect of HIV-1in ATH8 cell culture with an IC50 of 5.8 µM, whereas the S-enantiomer wasinactive (IC50 > 200 µM). Kinetics of deamination of (R)- and (S)-adenallenecatalyzed by adenosine deaminase gave the following parameters: Km valuesof S-form and R-form were 0.41 and 0.52 mM with Vmax being 530 and18.5 µmol/min, respectively.

In an anti-HIV drug discovery effort, Nair and coworkers49–51 reportedthe synthesis of (S,S)-isodideoxyadenosine (IsoddA), a compound whichwas found to have potent anti-HIV activity (Scheme 9). The synthetic ap-



NH2

N

NN

N

OHO

N

NH2

N

NN

OHO

NH

NN

N

O

OHO

N

NNH

N

O

HOO

ADApH 7.4

isomerization

isomerization

SCHEME 8Synthesis of dideoxy-3-isoinosine by ADA-catalyzed hydrolytic deamination of dideoxy-3-isoadenosine

proaches involved either direct coupling with inversion at the 2-position ofa preformed dideoxygenated sugar using the base moiety as nucleophile orconstruction of the base moiety onto a stereochemically defined aminosugar precursor. (S,S)-IsoddA possessed extremely high stability with respectto glycosidic bond cleavage. Unlike most adenine-based nucleosides, thiscompound was very resistant to deamination by ADA (Vmax/Km = 0.0008%of adenosine).

Diastereoselective deamination52 was used to differentiate between (6′R)-and (6′S)-6′-C-methylneplanocin A. The 6′R-isomer has potent antiviralactivity. Katagiri et al.53 studied the effect of pressure on the ADA-catalyzeddeamination of cyclaradine, a carbocyclic analogue of ara-A having anti-HSV activity. The rate of deamination of (+)-cyclaradine increased with in-creasing pressure, but (–)-cyclaradine was not deaminated even under highpressures.

In 2000, Ciuffreda et al.54 confirmed the crucial role of the 5′-hydroxygroup for ADA substrate activity. They prepared all the acetates of adeno-sine, 2′-deoxyadenosine and 3′-deoxyadenosine by lipase-catalyzed reac-tions (Fig. 4). Only the acetates with free 5′-hydroxy group weredeaminated by adenosine deaminase. The importance of the 5′-hydroxygroup for significant ADA activity had been reported previously by Nair andWiechert31 and by Bloch and Robins32. This observation was further sup-ported by X-ray studies of the complex formed by ADA with the inhibitor,2′-deoxycoformicin55.

Easterwood et al.56 reported the synthesis and analysis of substrateanalogues designed to provide insight into the mechanistic relationshipbetween the RNA-editing ADARs and the well-understood nucleoside-modifying enzyme, adenosine deaminase (ADA). ADARs are adenosinedeaminases that act on RNA and are responsible for RNA-editing that occurin eukaryotic mRNAs and can lead to codon changes in the mRNA. Under-standing of the mechanism of the ADAR-catalyzed reaction is limited.


780 Gupta, Nair:

N

NH2

O

OH

N

N N

OCH2OH

OH

OHOHO

TsO

CH2OBzseveral steps two steps

SCHEME 9Synthesis of the potently anti-HIV active, ADA-resistant compound, (S,S)-IsoddA

Nucleoside analogues of known ADA substrate activity were incorporatedinto an editing substrate and the substrate activities of these analogues withADAR2 were analyzed. When 6-O-methylinosine was located at the editingsite in the RNA substrate, ADAR2 converted it to inosine with a kobs =0.001 ± 0.0001 min–1. This reaction rate was slower than that for adenosine(kobs = 0.060 ± 0.0001 min–1). These data along with those known for ADAsuggest that both ADA and ADAR2 can deaminate groups at the 6-positionother than the amino group consistent with much early reports by Coryand Suhadolnik25,26. However, in contrast to ADA, ADAR2 deaminated7-deazaadenosine in this RNA substrate (kobs = 0.060 ± 0.003 min–1), whichsuggests that N-7 is not necessary for ADAR2 substrate recognition andactivity. Thus, while ADAR2 and ADA share mechanistic similarities, theseenzymes have different substrate binding requirements for significant sub-strate activity.

The synthesis of [2′-deoxy-4′-C-(hydroxymethyl)-2′-fluoro-D-arabino-furanosyl]purine nucleosides using calf intestinal ADA in the final step hasbeen reported57.

In 2001, Nair and his group58–60 reported the ADA-catalyzed hydrolyticdechlorination of 6-chloropurine nucleosides with functionalized substitu-tion at the 2-position. The target hypoxanthine nucleosides were designedas potential inhibitors (as their monophosphates) of the enzyme, inosinemonophosphate dehydrogenase (IMPDH) (Scheme 10). An example is theADA-mediated synthesis of 2-vinylinosine in high yield (Scheme 11)58. Thiscompound had been previously synthesized by Nair and coworkers usingan entirely chemical approach, but in much lower yields61. 2-Vinylinosine



NH

NN

N

O

R2R1

HO

ON

NN

N

NH2

O

OR3R2O

R1ON

O

N

N

N

NH2

R2O

R1O

NH2

OR1O

N

NN

N

OR2

b: R1 = H, R2 = R3 = Acc: R1 = R3 = H, R2 = Acd: R1 = R2 = H, R3 = Ace: R1 = R2 = R3 = Ac

a: R1 = Ac, R2 = H

b: R1 = H, R2 = Ac

c: R1 = R2 = Ac

a: R1 = Ac, R2 = Hb: R1 = H, R2 = Ac

c: R1 = R2 = Ac

a: R1 = Ac, R2 = R3 = H a: R1 = R2 = OAc

b: R1 = OAc, R2 = OH

c: R1 = OAc, R2 = H

d: R1 = H, R2 = OAc

FIG. 4Structures of adenosine and inosine derivatives used to establish the importance of the5′-hydroxy group in ADA substrate activity

shows broad-spectrum RNA antiviral activity62. The monophosphate of thiscompound is a potent inhibitor of inosine monophosphate dehydrogenase(IMPDH) with a Ki of 3.98 µM 58. The antiviral activity of 2-vinylinosinemay be explained by its cellular conversion to the monophosphate throughthe sequential action of PNP and HGPRT followed by inhibition of IMPDHby the cellularly produced 2-vinylinosine 5′-monophosphate.


782 Gupta, Nair:

N

N

N

Cl

RN

O

OHHO

HO

NH

N

N

O

RN

O

OHHO

HO

CH2OH

CH2F

F

ADA, pH 7.4

0.1 M phosphate buffer

R =

R =

R =

R =

R =

R =

SCHEME 10ADA-mediated synthesis of 2-functionalized hypoxanthine nucleosides

N

OP-O

O

O-

NH

N

N

O

OHHO

O

N

OH

N

N

N

Cl

O

HO

HO

NH

N

N

O

N

O

OHHO

HO

ADA

POCl3


pH 7.0, 4 days, 25 oC

triethyl phosphate

SCHEME 11Chemoenzymatic synthesis of IMPDH inhibitor, 2-vinylinosine monophosphate

Stereoselectivity in the ADA deamination of (5′RS)-5′-O-methyl-2′,3′-O-iso-propylidene adenosine involving a new stereogenic center at the 5′-positionhas also been examined to see if there was preference for the R- or S-isomer(Scheme 12)63. There was a clear preference for the S-isomer in the de-amination.

Interestingly, while ADA was unable to convert 5′-substituted derivativessuch as acetate, acetamido, and azido, the enzyme, adenylate deaminase(AMPDA, EC 3.5.4.6), for which the normal substrates are AMP and relatedcompounds, was able to convert all of these as substrates for theirbiotransformation to the corresponding 5′-substituted inosine derivatives(Scheme 13)64,65.

Okuyama and coworkers66 reported the efficient enzymatic synthesis of2′-deoxyguanosine using nucleoside deoxyribosyltransferase-II followed byADA. Nucleoside deoxyribosyltransferase-II (NdRT-II) from Lactobacillushelveticus, catalyzes the transfer of a glycosyl residue from a donor deoxy-



NH2

N

NN

N

O

OO

H3C

OH

NH

NN

N

O

O

OO

H3C

OH

N

NN

N

NH2

O

OO

H3C

OHADA

(S) (R)

1 h +

SCHEME 12Diastereoselectivity in the ADA-catalyzed deamination of 2′,3′-O-isopropylidene 5′-methyl-adenosine

N

O

NH

N

N

O

OO

R

N

NN

N

O

O

NH2

O

R AMPDAa: R = OHb: R = OAcc: R = N3

d: R = NH2

e: R = NHAc

SCHEME 13AMPDA-catalyzed deamination of adenosine derivatives

ribonucleoside to an acceptor base. 6-Substituted purines were found to besubstrates as acceptor bases for NdRT-II, and, utilizing this property of theenzyme, they synthesized 2′-deoxyguanosine (dGuo). This involved trans-glycosylation from thymidine to a 6-substituted purine and then conver-sion of the resulting 2-amino-6-substituted purine 2′-deoxyriboside todGuo with bacterial ADA (Scheme 14).

In addition to the studies of the differences in catalytic activity of ADAand AMPDA referred to above, more recent studies have provided exten-sions of work in this area67–71.

Gupta and Nair72 reported a facile methodology for the chemoenzymaticsynthesis of 2-acetonylinosine. Hydrolytic dechlorination at the final stepusing bovine intestinal adenosine deaminase was achieved in 2 days at25 °C and was monitored by observing the change in UV λmax from 265to 250 nm (Scheme 15). 2-Acetonylinosine exhibits spectacular antiviralactivity (therapeutic index of >1000!) against a RNA virus of the genus,Bunyavirus. The synthetic strategy, which has generality, is a dramatic im-provement on the methodologies currently available for the synthesis of2-functionalized purine nucleosides of therapeutic interest.


784 Gupta, Nair:

O

HO

HON

NH

O

O

H3C N

NN

N

X

NH2H

N

NH

O

O

H3C

H

HO

N

N

N

X

NH2N

O

HO

NH

N

NO

NH2N

O

HO

HO

NdRT-II

ADAH2O

NH4+

(Cl-)

DAP (X = NH2)ACP (X = Cl)

SCHEME 14Enzymatic synthesis of deoxyguanosine from thymidine

3. CONCLUSION

The development of efficient and facile chemoenzymatic routes to generatestructurally diverse functionalized purine nucleoside analogues is of muchsigni?cance in drug discovery and medicinal chemistry. The applications ofADA biocatalysis in nucleoside chemistry have led to many useful examplesof introduction of functional groups, of specific stereochemistry and of op-tical resolution. ADA is a representative example of a number of ecologi-cally benign biocatalysts that will shape the synthetic methodologies of thefuture, particularly in the field of nucleoside chemistry.

This project was supported by grant No. AI056540 from the National Institutes of Health. Itscontents are solely the responsibility of the authors and do not necessarily represent the official viewsof the NIH.

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NH

N

N

O

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HO OH

HOCH3

O

NH

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NH2N

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Adenosine Deaminase in Nucleoside Synthesis. A Review

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