An enzymatic transglycosylation of purine bases

Article abstract of DOI:10.1080/15257770701506343

An enzymatic transglycosylation of purine heterocyclic bases employing readily available natural nucleosides or sugar-modified nucleosides as donors of the pentofuranose fragment and recombinant nucleoside phosphorylases as biocatalysts has been investigated. An efficient enzymatic method is suggested for the synthesis of purine nucleosides containing diverse substituents at the C6 and C2 carbon atoms. The glycosylation of N6-benzoyladenine and N2-acetylguanine and its O6-derivatives is not accompanied by deacylation of bases. Copyright Taylor & Francis Group, LLC.

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An Enzymatic Transglycosylation of
Purine Bases
Jarkko Roivainen a , Tatiana Elizarova a , Seppo Lapinjoki a , Igor A.
Mikhailopulo a , Roman S. Esipov b & Anatoly I. Miroshnikov b
a Department of Pharmaceutical Chemistry, University of Kuopio,
Kuopio, Finland
b Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian
Academy of Sciences, Moscow, Russia
Published online: 10 Dec 2007.
To cite this article: Jarkko Roivainen , Tatiana Elizarova , Seppo Lapinjoki , Igor A. Mikhailopulo ,
Roman S. Esipov & Anatoly I. Miroshnikov (2007) An Enzymatic Transglycosylation of Purine Bases,
Nucleosides, Nucleotides and Nucleic Acids, 26:8-9, 905-909, DOI: 10.1080/15257770701506343
To link to this article: http://dx.doi.org/10.1080/15257770701506343
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Nucleosides, Nucleotides, and Nucleic Acids, 26:905–909, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701506343
AN ENZYMATIC TRANSGLYCOSYLATION OF PURINE BASES
Jarkko Roivainen, Tatiana Elizarova, Seppo Lapinjoki,
and Igor A. Mikhailopulo
Department of Pharmaceutical Chemistry, University of
2
Kuopio, Kuopio, Finland
Roman S. Esipov and Anatoly I. Miroshnikov
Shemyakin-Ovchinnikov
2
Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia
An enzymatic transglycosylation of purine heterocyclic bases employing readily available natural
2
nucleosides or sugar-modified nucleosides as donors of the pentofuranose fragment and recombinant
nucleoside phosphorylases as biocatalysts has been investigated. An efficient enzymatic method is
suggested for the synthesis of purine nucleosides containing diverse substituents at the C6 and C2
carbon atoms. The glycosylation of N6-benzoyladenine and N2-acetylguanine and its O6-derivatives
is not accompanied by deacylation of bases.
Keywords Base- and sugar-modified purine nucleosides; enzymatic synthesis; recombi-
nant nucleoside phosphorylases
INTRODUCTION
It was shown that the reaction of an enzymatic transglycosylation offers
obvious advantages over chemical methods.[1] Two approaches are success-
fully employed for the pentofuranosyl-transfer reactions catalyzed by nucle-
oside phosphorylases: one of these is based on the use of the whole bacterial
cells displaying the uridine phosphorylase (UP) and/or thymidine phos-
phorylase (TP) and purine nucleoside phosphorylase (PNP) activities as a
biocatalyst, the other makes use of the purified enzymes or cloned enzymes
and overexpressed in bacteria. On the whole, both approaches mutually
complement one another allowing to find out the most efficient way toward
the desired nucleoside.
In the present communication, we have investigated an enzymatic trans-
glycosylation of purine bases employing readily available pyrimidine or
purine nucleosides as donors of the carbohydrate moiety, purine bases as
The study at the University of Kuopio was supported by the Technology Development Center of
Finland (TEKES, Finland).
Address correspondence to I. A. Mikhailopulo, Department of Pharmaceutical Chemistry, Univer-
sity of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. E-mail: Igor.Mikhailopulo@uku.fi
905

906
J. Roivainen et al.
SCHEME 1 Structure of donors of a pentofuranose residue and purine heterocyclic bases investigated
as acceptors in the reaction of an enzymatic transglycosylation.
acceptors and the purified recombinant thymidine (TP) and/or purine
(PNP) nucleoside phosphorylases[2] as biocatalysts.
RESULTS AND DISCUSSION
Structure of donors and acceptor used in this study are shown
in Scheme 1. The ribosylation, 2-deoxyribosylation and 3-amino-2,3-
dideoxyribosylation of a number of purine heterocyclic bases was investi-
gated employing commercially available guanosine (1), 2ꢁ-deoxyguanosine
(2) and thymidine (3) as well as previously described 3ꢁ-amino-2ꢁ,3ꢁ-
dideoxyguanosine (4)[3] as donors of the corresponding sugar moieties and
either PNP or TP, or both enzymes simultaneously as biocatalysts.
SCHEME 2 Structure of nucleosides 14-21.

Enzymatic Transglycosylation
907
Recently, ribosides of N 6-benzyladenine (5) and kinetin (6) attract
much attention as regulators that play an important role in the differen-
tiation and development of human normal and malignant cells and might
be clinically useful for treating some cancers.[4,5] Base halogenated deriva-
tives of N 6-benzyladenine as well as sugar modified derivatives of cytokinines
represent very important family of agonists and antagonists for adenosine
receptors and are under intense all-round investigation.[6]
Biocatalytic ribosylation of N 6-benzyladenine (5) and kinetin (6) has
been described using the whole E. coli cells as a biocatalyst and inosine as a
donor of the ribofuranose residue.[7,8] In the present study, we investigated
the ribosylation, 2-deoxyribosylation and 3-amino-2,3-dideoxyribosylation of
both N 6-substituted purine bases using the respective nucleosides 1, 2, or
3 as donors of the sugar moiety and either PNP or TP, or both enzymes
simultaneously as biocatalysts.[9] It was found that both N 6-benzyladenine
(5) and kinetin (6) are good acceptors of the pentofuranose moieties stud-
ied and the desired nucleosides 14–18 have been obtained in 40–88% iso-
lated yields (Scheme 2). Spatial arrangements of N 6-benzyladenine (5) and
N 6-benzoyladenine (7) are similar. Analysis of their electronic structures
(Hypercube, Inc., 2002; release 7.1; ab initio calculations, 6-31G∗) also dis-
played a close similarity of both heterocycles especially the charges of N 7-
(−0.264 e and −0.267 e, respectively) and N 9-nitrogens (-0.323 e and -0.322
e, respectively) that are involved in binding into the active center and cou-
pling with a pentofuranose (see below). Indeed, N 6-benzoyladenine (7) was
found to be an excellent substrate of PNP, and 2ꢁ-deoxyribosides 19 [donor
2ꢁ-deoxyguanosine (2)] and 20 (donor compound 4) have been isolated
from the reaction mixtures in 85 and 82% yields. Treatment of the latter
with FMOC-OSU/DMF gave the crystalline FMOC-derivative (80%) 21 with
SCHEME 3 An enzymatic trans-2-deoxyribosylation of N 2-acetylguanine (8).

908
J. Roivainen et al.
FIGURE 1 Computer-generated (ab initio, 6-31G*) electronic structures of isomeric N 2-acetyl-N 7(H)
-and -N 9(H)-guanine.
an orthogonal protection of two amino functions that is very useful for the
possible further applications of this compound (Scheme 2).
A number of N 2,O6-substituted guanine derivatives 8–13 was in-
vestigated as substrates in the transglycosylation reaction using 2ꢁ-
deoxyguanosine (2) or thymidine (3) as donors of the carbohydrate moi-
ety. It was found that N 2-acetylguanine (8) is very good substrate for PNP
and its 2-deoxy-D-ribosylation employing thymidine (3) as the pentofura-
nose donor and TP and PNP as biocatalysts gave rather interesting results
(Scheme 3).
It was found that the N 7-isomer 22 predominantly forms at the begin-
ning of the reaction along with the N 9-isomer 23. The primarily formed N 7-
isomer 22 is rearranged into the latter on prolonged heating of the reaction
mixture that clearly points to the higher thermodynamic stability of the N 9-
isomer 18. Indeed, the ab initio calculations showed that the N 9-isomer 18 is
thermodynamically favored over the N 7-isomer 17 by -3.36 kcal/mol. Inter-
estingly, the ab initio calculations of isomeric N 7(H) and N 9(H) isomers of
N 2-acetylguanine (8) manifested their thermodynamic similarity (Figure 1).
The use of thymidine as a donor (the ratio of 3/8 was 1.5:1.0, mol; stan-
dard reaction conditions) and TP and PNP as biocatalysts gave rise after
48 h to a ca. 1:6 mixture of N 7 -and N 9-isomers, from which the individual
nucleosides 22 and 23 have been isolated in 9 and 55% yield, respectively.
Under similar reaction conditions, the use 2ꢁ-deoxyguanosine as a donor
and PNP as a biocatalyst led to a similar mixture, chromatographic separa-
tion of which gave individual nucleosides 22 (11%) and 23 (74%).
It was found that heterocyclic bases carrying out the bulky isobutyryl and
palmitoyl substituents at the N 2-nitrogen, 11 and 12, or diphenylcarbamoyl
group at the O6-oxygen 13 are not substrates for recombinant PNP as well
as PNP within the whole E. coli cells. The formation of the corresponding
nucleosides was not detected in the reaction mixtures.

Enzymatic Transglycosylation
909
On the contrary, N 2-acetyl-O6-methylguanine (9) and N 2-acetyl-O6-
benzylguanine (10) have been found to be the good substrates for PNP and
the respective N 9-2ꢁ-deoxy-β-D-ribofuranosyl nucleosides have been isolated
in 95 and 50%, respectively. In both reactions, the formation of the N 7-
isomers was not established by TLC and HPLC.
CONCLUSIONS
An efficient enzymatic method is suggested for the synthesis of purine
nucleosides containing diverse substituents at C6 and C2 carbon atoms.
N 2-Acetylguanine and N 2-acetyl-O6-methyl- and -benzylguanine have been
found to be good substrates of PNP. Initially, the former is predominantly
glycosylated at the N 7-nitrogen and the N 7-glycoside formed is rearranged
into thermodynamically more stable N 9-glycoside that is in accord with the
ab initio calculations [ꢀE(N 9 − N 7) = −3.36 kcal/mol]. Steric control of
the bulkiness of the N 2- and O6-guanine derivatives realized by PNP is out-
lined. These data allow predicting the substrate properties of new purine
analogues, which can be employed for an enzymatic transglycosylation aim-
ing at the preparation of new biologically active compounds.
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9. Standard reaction conditions: the donor-acceptor ratio was 1.5–3:1 (mol); 5 mM K-phosphate buffer
(pH 7.0); heterogeneous reaction mixture was gentle stirred at 50 ◦C monitoring the progress of
reaction by TLC and HPLC; after the reaction reached constant equilibrium, SiO2 was added and
the mixture was evaporated to dryness, residue co-evaporated twice with abs EtOH, and SiO2 with
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