Enzymatic synthesis of 2-deoxy-β-d-ribonucleosides of 8-azapurines and 8-aza-7-deazapurines

Article abstract of DOI:10.1055/s-0031-1290679

The enzymatic synthesis of 8-azapurine and 8-aza-7-deazapurine 2-deoxyribonucleosides has been studied. Two methods have been used: (i) transglycosylation employing 2-deoxyguanosine, 2-deoxycytidine, 2-deoxyuridine, and 2-deoxythymidine as 2-deoxy-d-ribofuranose donors and recombinant E. coli purine nucleoside phosphorylase (PNP) as biocatalyst, and (ii) one-pot synthesis from 2-deoxy-d-ribose and nucleobases employing recombinant E. coli ribokinase (RK), phosphopentomutase (PPM) and PNP as biocatalysts. Good substrate activity was observed for all bases studied except 2-amino-8-aza-6-chloro-7-deazapurine, which afforded the desired N⁹-nucleoside in moderate yield due to very low solubility of the base and partial replacement of C6-chloro atom of the base and formed nucleoside with a hydroxy group. The participation of Ser90 O^γ of E. coli PNP in the binding of 8-aza-7-deazapurines in the catalytic center of PNP followed by the formation of a productive complex and glycosidic bond is suggested. Georg Thieme Verlag Stuttgart · New York.

Full text of DOI:10.1055/s-0031-1290679

LETTER
▌1541
letter
Enzymatic Synthesis of 2′-Deoxy-β-D-ribonucleosides of 8-Azapurines and
8-Aza-7-deazapurines
2′-Deoxy-β-D-ribonucleosides of 8-Azapurines and 8-Aza-7-deazapurines
Vladimir A. Stepchenko,a Frank Seela,b Roman S. Esipov,c Anatoly I. Miroshnikov,c Yuri A. Sokolov,a
Igor A. Mikhailopulo*a
a
Institute of Bioorganic Chemistry, National Academy of Sciences, Acad. Kuprevicha 5/2, 220141 Minsk, Belarus
b
Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstr. 11, 48149 Münster, Germany
c
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 GSP,
Moscow B-437, Russian Federation
Fax +375(17)2678761; E-mail: imikhailopulo@gmail.com
Received: 30.01.2012; Accepted after revision: 04.04.2012
deaza analogues.10 The reaction proceeds regio- and ste-
Abstract: The enzymatic synthesis of 8-azapurine and 8-aza-7-
reospecifically, affording 8-aza-2′-deoxyadenosine and 8-
deazapurine 2′-deoxyribonucleosides has been studied. Two meth-
aza-2′-deoxyguanosine. Furthermore, it was found that
ods have been used: (i) transglycosylation employing 2′-deoxy-
guanosine, 2′-deoxycytidine, 2′-deoxyuridine, and 2′-deoxythymidine
the presence of nitrogen-7 of purines and their isosteric
as 2-deoxy-D-ribofuranose donors and recombinant E. coli purine analogues is a prerequisite for the reaction.10 However,
there are several exceptions; 5-aza-7-deazaguanine11 (1),
nucleoside phosphorylase (PNP) as biocatalyst, and (ii) one-pot
synthesis from 2-deoxy-D-ribose and nucleobases employing re-
5-aza-7-deazaisoguanine12 (2) and N-(1,3,4-thiadiazol-2-
combinant E. coli ribokinase (RK), phosphopentomutase (PPM)
yl)-cyanamide13 (3) are substrates for bacterial purine nu-
and PNP as biocatalysts. Good substrate activity was observed for
cleoside phosphorylases (Figure 1).
all bases studied except 2-amino-8-aza-6-chloro-7-deazapurine,
which afforded the desired N9-nucleoside in moderate yield due to
O
N
NH2
H
N
very low solubility of the base and partial replacement of C6-chloro
atom of the base and formed nucleoside with a hydroxy group. The
participation of Ser90 Oγ of E. coli PNP in the binding of 8-aza-7-
deazapurines in the catalytic center of PNP followed by the forma-
tion of a productive complex and glycosidic bond is suggested.
S
N
N
N
N
N
C
N
N
N
N
NH2
N
O
H
H
1
2
3
Key words: azapurine, nucleosides, enzyme catalysis, glycosyl-
ation, phosphorylases
Figure 1 Some unusual substrates of E. coli purine nucleoside phos-
phorylase in synthetic reactions
We applied the enzyme-catalyzed glycosylation to di-
verse range of heterocyclic bases belonging to the classes
of 8-azapurines and 8-aza-7-deazapurines. The work pre-
sented here reports on the use of various donors of the 2′-
deoxy-D-ribofuranosyl residue and recombinant E. coli
PNP14 as a biocatalyst (transglycosylation reaction;
Scheme 1, path A) and enzymatic cascade transformation
of 2-deoxy-D-ribose into the nucleosides (Scheme 1,
path B). This cascade reaction uses recombinant E. coli
ribokinase (RK),15 phosphopentomutase (PPM),16 and
PNP14 (one-pot synthesis16). We studied the enzymatic
synthesis of N9-2′-deoxy-β-D-ribonucleosides of a num-
ber of 8-azapurines (4 and 5) and 8-aza-7-deazapurines
(6–9) (Figure 2).
Nucleosides with 8-azapurines and 8-aza-7-deazapurines
(purine numbering throughout) are applicable as drugs1
and tools in chemistry, chemical biology, and molecular
diagnostics.2–5 Chemical syntheses of these nucleoside
shape mimics suffer from the formation of mixtures of
isomers, which makes it necessary to conduct time-con-
suming separation procedures. As a result, the desired nu-
cleosides are obtained in moderate or low yields (for
example see refs.4,6 and those in the Supporting Informa-
tion).
Nucleoside phosphorylases (NP), in particular E. coli NP,
are very efficient biocatalysts in glycosylation reactions.7
Doskocil and Holy8 have shown that 8-azaguanine is a
good ribosyl acceptor in the enzymatic reaction catalyzed
by E. coli purine nucleoside phosphorylase (PNP; product
of deoD gene; EC 2.4.2.1;7,9). Later, Votruba et al. studied
alginate gel entrapped cells of an auxotrophic thymine-
dependent strain of E. coli as a biocatalyst for the transfer
of the 2′-deoxy-D-ribofuranosyl moiety of 2′-deoxyuri-
dine to purine and pyrimidine bases including aza and
First, the substrate specificity of bases 4–8 was tested in
the transglycosylation reaction performed under standard
conditions using 2′-deoxyguanosine (10) as a glycosyl do-
nor (Scheme 1, path A).17 All the bases are satisfactory
and the respective nucleosides were obtained in good
yields. The structure of these nucleosides (15–19) was
confirmed by 1H, 13C NMR and UV spectroscopic analy-
sis and compared to previously published data (see the
Supporting Information). We did not observe the forma-
tion of regioisomeric nucleosides.
SYNLETT 2012, 23, 1541–1545
Advanced online publication: 29.05.2012
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6
DOI: 10.1055/s-0031-1290679; Art ID: ST-2012-B0090-L
© Georg Thieme Verlag Stuttgart · New York

1542
V. A. Stepchenko et al.
LETTER
enabling nucleophilic attack of a nitrogen atom of the sp2
hybridized imino -C=N9- tautomer on the electrophilic
center of the co-substrate. An analogous mode of binding
can be assumed with high probability for natural purine
bases as well as for 8-azapurines 4 and 5, leading to the
exclusive formation of N9-glycosides. However, the for-
mation of nucleosides 17–19 was rather unexpected in the
light of the earlier data pointing to the crucial importance
of nitrogen-7 of purines and their isosteric analogues in
the synthetic reaction catalyzed by E. coli PNP.7–10
X
N
X
N
N
N
N
N
N
N
N
Y
Y
H
H
4, X = NH2, Y = H
5, X = OH, Y = NH2
6, X = NH2, Y = H
7, X = OMe, Y = NH2
8, X = OH, Y = H
9, X = Cl, Y = NH2
Figure 2 The structures of 8-aza- and 8-aza-7-deazapurine bases
studied in the synthesis of 2′-deoxy-β-D-ribonucleosides catalyzed by
recombinant E. coli PNP
The mechanism of the synthetic reaction catalyzed by E.
coli PNP nucleoside phosphorylases has not attracted the
attention of researchers thus far, and many important de-
tails are not clear (see, for example the discussion in the
literature7b,c). Thus, the mode of binding of the substrate
or inhibitor in the catalytic center of the enzyme might
shed light on the mechanism of the enzyme’s functioning,
and provide clues that could help to explain some unusual
observations such as the good substrate activity of 8-aza-
7-deazapurines. Detailed analysis of the mechanism of the
phosphorolysis of purine nucleosides by E. coli PNP
showed that the base binding site is formed mainly by
Asp204 and, to some extent, by Phe159.23–26 It is notewor-
thy that replacement of D-aspartic acid residue 204 by al-
anine resulted in complete loss of the catalytic activity of
E. coli PNP (see note on page 1266 of the paper by Ealick
et al.26a and the recent paper by Luic et al.26b). Taking into
account that it is an equilibrium reaction, one can expect
that the same amino acid residues make the main contri-
bution to binding of the substrate in the synthetic reaction.
Asp204 interacts with nitrogen-7 and the substituent at C6
of the purine base, giving rise, in all likelihood, to the op-
timal base orientation and to enhancement of the nucleo-
philic properties of nitrogen-9, i.e., activation of the
substrate (Figure 3). It is, therefore, surprising that re-
placement of nitrogen-7 with a CH group did not abolish
the substrate activity of bases 6–8, which suggests a rather
efficient contribution of the Asp204–C6 substituent inter-
action to correct binding and activation. To establish the
role of such interaction, we investigated the substrate
properties of base 9, which has no groups that could imi-
tate an interaction of natural purine substrates with
Asp204. It was surprising to find that base 9 still retains
The regiospecific 2′-deoxy-D-ribosylation of 8-azaade-
nine (4) and 8-azaguanine (5) catalyzed by recombinant
E. coli PNP is in line with published data.8,9b,10 In contrast,
glycosylation of anions of 8-azapurines with 2-deoxy-3,5-
di-O-(4-toluoyl)-α-D-erythropentofuranosyl
chloride
gave a very complex mixture of regioisomers and their
α,β-anomers.4,18,19 There are a great number of publica-
tions devoted to the tautomerizm of 8-azapurines and the
chemical reactivity of nitrogen atoms of nucleobase an-
ions that are involved in the formation of the glycosyl
bond (reviewed in refs.20,21). The nucleobase anions are
one of the activated species in the chemical glycosylation
of 8-azapurinrs existing in a mixture of different tauto-
mers; the other method of activation of purine bases in-
volves the preparation of trimethylsilyl derivatives, -C–
N9(7)(H)- → -C–N9(7)(TMS)-, leading to the enhancement
of nucleophilicity of nitrogen atoms of the sp2 hybridized
imino -C=N7(9)- tautomers.21 An interplay of the equilibri-
um of tautomeric forms of anions or imino tautomers, on
the one hand, and nucleophilicity of the activated nitrogen
atoms, on the other, are responsible for the formation of
regioisomeric nucleosides. In the case of the E. coli PNP
catalyzed coupling of base and pentofuranose, activation
of natural purines involves fixing a specific sp2 hybridized
imino -C=N9- tautomer of base in the catalytic site.7b,22
From a chemical viewpoint, the formation of the glycosyl
bond results from nucleophilic attack of a nitrogen atom
of the base on the electrophilic C1-carbon atom of 2-de-
oxy-D-ribofuranose 1-phosphate (14). The regioselectivi-
ty of the enzymatic glycosylation is governed by the
binding mode of base in the catalytic center of the enzyme
O
P
HO
HO
B
HO
B
O
O
O
PNP, Pi
– BH
PNP, base
– Pi
OH
O
A
O
HO
HO
HO
14
10, B = guanin-9-yl
11, B = cytosin-1-yl
12, B = uracil-1-yl
13, B = thymin-1-yl
15, B = 8-azaadenin-9-yl
16, B = 8-azaguanin-9-yl
17, B = 8-aza-7-deazaadenin-9-yl
18, B = 8-aza-7-deaza-O6-methylguanin-9-yl
19, B = 8-aza-7-deazahypoxanthin-9-yl
20, B = 2-amino-8-aza-6-chloro-7-deazapurin-9-yl.
PPM
O
P
HO
O
O
RK, ATP
O
B
2-deoxy-D-ribose
recombinant E. coli enzymes:
PNP - purine nucleoside phosphorylase
RK - ribokinase
OH
HO
PPM - phosphopentomutase
21
Scheme 1 Synthesis of nucleosides 15–20 by the transglycosylation reaction (path A) and cascade one-pot transformation of 2-deoxy-D-ribose
into nucleosides (path B)
Synlett 2012, 23, 1541–1545
© Georg Thieme Verlag Stuttgart · New York

LETTER
2′-Deoxy-β-D-ribonucleosides of 8-Azapurines and 8-Aza-7-deazapurines
1543
moderate substrate activity, despite the absence of any in- lowed by activation of the nitrogen-9 in productive com-
teractions with Asp204 and the extremely low solubility.
plex 27 (Figure 4). The contribution of Phe159 of E. coli
PNP to both processes — binding and activation —
appears to be analogous to that of the natural bases. Thus,
in the case of 8-aza-7-deaza purine analogues, the Ser90
residue of the catalytic site of E. coli PNP takes the place
of Asp204 in the case of natural purine substrates.
binding
activation
Asp204
Asp204
O
H
X
O
O
O
H
The spatial tautomeric structures of base 9 in complex
with Ser90-Oγ have been analyzed by the restricted Har-
tree–Fock (RHF) ab initio method using basis set of 6-
31** FIREFLY QC package,29 which is partially based on
the GAMESS (US)30 source code. The files of MOPAC
format containing Z-matrix of internal coordinates ob-
tained by the PM3 geometry optimization31 was used as
starting approximation for the ab initio calculations. The
following main dimensions were obtained for the respec-
tive structures 26: Ser90-OγH···N8 (0.97 and 1.86 Å);
Ser90-Oγ···H–N9 (2.39 and 0.99 Å); =N8–N9(H)- (1.39
Å); 27: Ser90-Oγ···H–N8 (2.37 and 0.99 Å); Ser90-
OγH···N9 (0.96 and 1.83 Å); -N8(H)–N9= (1.37 Å); these
data are in fair agreement with strong hydrogen bonding
of the base, enabling correct binding followed by activa-
tion.
H
X
N
H
N
N
N
N
N
H
N
N
R
R
22
23
X = NH, O; R = H, NH2
binding
Asp204
O
Asp204
O
O
O
H
H
Me
O
O
N
H
N
NH
N
N
N
N
NH2
N
NH2
H
In the next series of experiments, we studied the one-pot
synthesis of nucleosides 15–19 with 2-deoxy-D-ribose
and heterocyclic bases 4–8 in the presence of recombinant
RK, PPM and PNP (Scheme 1, Path B).7b,16 Under the re-
action conditions employed, the formation of 8-aza-2′-de-
oxyadenosine (15) and 8-aza-2′-deoxyguanosine (16)
proceeded slowly, affording the nucleosides in moderate
yields. In contrast, 8-aza-7-deazapurines 6–8 showed sat-
isfactory substrate activity and the respective nucleosides
17–19 were formed in yields of more than 50% after
20 hours (Scheme 1; see also the Supporting Informa-
tion).
24
25
Figure 3 Schematic presentation of binding adenine, hypoxanthine
and guanine (22), followed by activation (23), and a possible binding
of 2-amino-8-aza-6-methoxy-7-deazapurine (7) and 5-aza-7-deaza-
guanine (1); the most populated tautomers are show for bases in struc-
tures 24 and 25
It appears clear that binding of 2-amino-8-aza-6-me-
thoxy-7-deazapurine (7) in the catalytic center of PNP
cannot lead to activation of the substrate. Indeed, analysis
of the tautomeric structures of base 7 by ab initio calcula-
tions using a Polack–Ribiere conjugate gradient (Hyper-
Chem 8.01) showed a strong preference for the sp3-hy-
bridized amino -C-N9(H)- tautomer.27,28 Remarkably, sim-
ilar analyses using a semi-empirical method PM3 (in
water box) for three possible tautomeric structures of 5-
aza-7-deazaguanine (1) revealed a strong preference for
the sp2 hybridized imino -C=N9- tautomer (Figure 3).7b
This electronic structure is in harmony with satisfactory
substrate activity of isosteric guanine analogue 1, imply-
ing the important role of Asp204 and Phe159 residues in
correct binding at the catalytic center of E. coli PNP.
Recently, Mitsui Chemicals, Inc. reported an enzymatic
method for the production of nucleosides.32 Surprisingly
they found that recombinant E. coli PNP is able to cata-
lyze the reaction of cytosine and 2-deoxy-D-ribofuranose
1-phosphate in Tris·HCl buffer (100 mM, pH 8.0; 50 °C,
20 h) giving rise to the formation of 2′-deoxycytidine (11;
dC) in 57.5% yield (HPLC). We have confirmed this find-
ing and found that dC is phosphorolyzed under very mild
conditions (nucleoside 0.05 mmol; 80 mM K,Na-phos-
phate buffer, pH 7.0; 27 units14 PNP; 14–15°С), resulting
in more than 60% product yield (Figure 5). Furthermore,
2′-deoxyuridine (12; dU) and, to a lesser extent, 2′-de-
oxythymidine (13; dT) are also cleaved. However, a pla-
teau is reached in the phosphorolysis of dT implying two
competing reactions: phosphorolysis and glycosylation. It
is noteworthy that Pugmire and Ealick performed detailed
structural analysis of NP and defined two distinct families
of the enzymes: NP-I and NP-II.33 The first family in-
cludes homotrimeric and hexameric enzymes from both
prokaryotes and eukaryotes that accept purine (inosine,
guanosine and adenosine) as well as pyrimidine (uridine)
nucleosides as substrates. The second family encompass-
es bacterial pyrimidine phosphorylases and eukaryotic
Analysis of the crystal structure of the ternary complex of
hexameric E. coli PNP with formycins A and B showed
that Ser90 is involved in binding of the bases.23,24 More-
over, crystallographic data for adenine binding to the ac-
tive site of E. coli PNP clearly showed the close proximity
of Ser90 Oγ to carbon-8 of the base.25,26a These data, to-
gether with the aforementioned considerations, suggest a
possible explanation for the good substrate properties of
bases 6–8 and moderate activity of base 9; it is apparent
that Ser90 Oγ is hydrogen bonded to nitrogen-8 (purine
numbering) of the base in 26, giving rise to the correct
base orientation in the catalytic site of E. coli PNP, fol-
© Georg Thieme Verlag Stuttgart · New York
Synlett 2012, 23, 1541–1545

1544
V. A. Stepchenko et al.
LETTER
binding
productive complex
product formation
Asp204
Asp204
Asp204
O
OH
O
OH
O
OH
Cl
N
Cl
N
Cl
N
H
H
N
H
N
H
N
N
N
H
O
N
H
N
N
Oγ
Ser90
HO
N
O
Oγ
Oγ
NH2
NH2
NH2
Ser90
HO
Ser90
H
O
H
O
O
O
HO
P
HO
P
HO
O
O
O
26
27
28
Figure 4 Schematic presentation of binding 2-amino-8-aza-6-chloro-7-deazapurine (9), and the formation of productive complex 27 and
nucleoside 28
thymidine phosphorylases. The activity of E. coli PNP tioned nucleosides through intermediate formation of 2-
against dU, dC and dT deserves detailed structural analy- deoxy-D-ribofuranose 5-phosphate (21), 2-deoxy-α-D-ri-
sis. It should be stressed that the corresponding ribonucle- bofuranose 1-phosphate (14), and finally nucleosides cat-
osides showed no substrate activity with E. coli PNP alyzed by recombinant E. coli RK, PPM and PNP,
under the aforementioned reaction conditions.
respectively.7c,16 The methods employed in the present
work for the synthesis of 2′-deoxy-β-D-ribonucleosides
are complementary to each other. We tested both methods
to assess, first of all, the feasibility of the latter for the syn-
thesis of base-modified nucleosides. It is clear that the
choice of method depends on the availability of the en-
zymes and the starting pentose or its donor.
The participation of Ser90 Oγ of E. coli PNP in the binding
of 8-aza-7-deazapurines in the catalytic center of PNP fol-
lowed by the formation of productive complex and the
glycosidic bond is suggested.
The reaction conditions in both types of enzymatic reac-
tions have not been optimized. Despite this, the heterocy-
clic bases 4–8 showed satisfactory substrate properties
and the desired N9-2′-deoxy-β-D-ribofuranosyl nucleo-
sides have been prepared in good yields.
Figure 5 Phosphorolytic cleavage of 2′-deoxycytidine, 2′-deoxyuri-
dine, and 2′-deoxythymidine with recombinant E. coli PNP (HPLC
analysis of the formation of the corresponding bases).
Finally, the transglycosylation reaction of 8-azaadenine
(4) and 8-aza-7-deazaadenine (6) with 2′-deoxycytidine
(dC), 2′-deoxyuridine (dU) and 2′-deoxythymidine (dT)
as sugar donors, and recombinant E. coli PNP as biocata-
lyst, was studied.34 In the case of dC/base combination of
substrates, the reaction proceeded smoothly and the nucle-
osides 15 and 17 were formed in 10 and 60% yields,
respectively, after 72 hours. Under these reaction condi-
tions, the formation of nucleosides was not observed in
the case of dU/base and dT/base substrate combinations.
The use of dU and dT as glycosyl donors in enzymatic
synthesis of nucleosides is under study.
Acknowledgment
Financial support by the International Science and Technology
Centre (project #B-1640) is gratefully acknowledged. I.A.M. is de-
eply thankful to the Alexander von Humboldt-Stiftung (Bonn –
Bad-Godesberg, Germany) for partial financial support of this stu-
dy. The authors thank Drs I.D. Konstantinova, K.V. Antonov, and
I.V. Fateev (Shemyakin-Ovchinnikov Institute of Bioorganic Che-
mistry, Russian Academy of Sciences) for repeating some experi-
ments and for performing analyses on the reaction mixtures.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.SnuIpofoiprmntgirStanoIuifpog
r
t
iornat
In conclusion, efficient enzymatic methods for the prepa-
ration of N9-2′-deoxy-β-D-ribonucleosides of 8-azapu-
rines and 8-aza-7-deazapurines are described. One of the
approaches involves pentofuranose transfer from 2′-deoxy-
guanosine or 2′-deoxycytidine to heterocyclic bases cata-
lyzed by recombinant E. coli PNP.7 The uses the one-pot
transformation of 2-deoxy-D-ribose into the aforemen-
References and Notes
(1) Elion, G. B. Cancer Res. 1969, 29, 2448.
(2) Seela, F.; Pujari, S. Bioconjugate Chem. 2010, 21, 1629.
(3) Jiang, D.; Seela, F. J. Am. Chem. Soc. 2010, 132, 4016.
(4) Seela, F.; Jiang, D.; Xu, K. Org. Biomol. Chem. 2009, 7,
3463; and references cited therein.
Synlett 2012, 23, 1541–1545
© Georg Thieme Verlag Stuttgart · New York

LETTER
2′-Deoxy-β-D-ribonucleosides of 8-Azapurines and 8-Aza-7-deazapurines
1545
(5) (a) Pokharel, S.; Jayalath, P.; Maydanovych, O.; Goodman,
R. A.; Wang, S. C.; Tantillo, D. J.; Beal, P. A. J. Am. Chem.
Soc. 2009, 131, 11882. (b) Veliz, E. A.; Easterwood, L. M.;
Beal, P. A. J. Am. Chem. Soc. 2003, 125, 10867.
(c) Wierzchowski, J.; Ogiela, M.; Iwanska, B.; Shugar, D.
Anal. Chim. Acta 2002, 472, 63.
(6) (a) Elliot, R. D.; Montgomery, J. A. J. Med. Chem. 1976, 19,
1186. (b) Kazimierczuk, Z.; Cottam, H. B.; Revankar, G. R.;
Robins, R. K. J. Am. Chem. Soc. 1984, 106, 6379; and
references therein.
(7) For recent reviews, see: (a) Mikhailopulo, I. A. Curr. Org.
Chem. 2007, 11, 317. (b) Mikhailopulo, I. A.; Miroshnikov,
A. I. Acta Naturae 2010, 2, 36. (c) Mikhailopulo, I. A.;
Miroshnikov, A. I. Mendeleev Commun. 2011, 21, 57.
(8) Doskocil, J.; Holy, A. Collect. Czech. Chem. Commun.
1977, 42, 370; and works cited therein.
(9) (a) Bzowska, A.; Kulikowska, E.; Shugar, D. Pharmacol.
Ther. 2000, 88, 349. (b) Wierzchowski, J.; Wielgus-
Kutrowska, B.; Shugar, D. Biochim. Biophys. Acta 1996,
1290, 9.
(10) Votruba, I.; Holy, A.; Dvorakova, H.; Gunter, J.; Hockova,
D.; Hrebabecky, H.; Cihlar, T.; Masojidkova, M. Collect.
Czech. Chem. Commun. 1994, 59, 2303.
(11) Rosemeyer, H.; Seela, F. J. Org. Chem. 1987, 52, 5136.
(12) Voegel, J. J.; Altorfer, M. M.; Benner, S. A. Helv. Chim.
Acta 1993, 76, 2061.
(13) Hayden, F. G.; Tunkel, A. R.; Treanor, J. J.; Betts, R. F.;
Allerheiligen, S.; Harris, J. Antimicrob. Agents Chemother.
1994, 38, 1178.
Conditions B: Total volume: 2 mL; 2 mM ATP, 50 mM
KCl, 3 mM MnCl2, 20 mM Tris×HCl (pH 7.5); 1.3 mM 2-
deoxy-D-ribose, 1 mM heterocyclic base; units of enzymes:
RK15 (9), PPM16 (4), and PNP14 (14); 40 °C. Yields by HPLC
of nucleoside and reaction time: 15 (8 %; 15 h), 16 (23%;
25 h), 17 (52%; 25 h), 18 (50%; 20 h) and 19 (60%; 10 h).
(18) Kazimierczuk, Z.; Binding, U.; Seela, F. Helv. Chim. Acta
1989, 72, 1527.
(19) Seela, F.; Lampe, S. Helv. Chim. Acta 1993, 76, 2388.
(20) (a) Shcherbakova, I.; Elguero, J.; Katritzky, A. R. Adv.
Heterocycl. Chem. 2000, 77, 51. (b) Elguero, J.; Katritzky,
A. R.; Denisko, O. V. Adv. Heterocycl. Chem. 2000, 76, 1.
(21) Vorbrüggen, H.; Ruh-Pohlenz, C. Org. React. (N. Y.) 2000,
55, 1.
(22) (a) Schramm, V. L. Arch. Biochem. Biophys. 2005, 433, 13.
(b) Taylor Ringia, E. A.; Schramm, V. L. Curr. Top. Med.
Chem. (Sharjah, United Arab Emirates) 2005, 5, 1237.
(23) Koellner, G.; Luic, M.; Shugar, D.; Saenger, W.; Bzowska,
A. J. Mol. Biol. 1998, 280, 153.
(24) Koellner, G.; Bzowska, A.; Wielgus-Kutrowska, B.; Luic,
M.; Steiner, T.; Saenger, W.; Stepinski, J. J. Mol. Biol. 2002,
315, 351.
(25) Bennett, E. M.; Li, C.; Allan, P. W.; Parker, W. B.; Ealick,
S. E. J. Biol. Chem. 2003, 278, 47110.
(26) (a) Zhang, Y.; Parker, W. B.; Sorscher, E. J.; Ealick, S. E.
Curr. Top. Med. Chem. (Sharjah, United Arab Emirates)
2005, 5, 1259. (b) Mikleusevic, G.; Stefanic, Z.; Narczyk,
M.; Wielgus-Kutrowska, B.; Bzowska, A.; Luic, M.
Biochimie 2011, 93, 1610.
(14) Esipov, R. S.; Gurevich, A. I.; Chuvikovsky, D. V.;
Chupova, L. A.; Muravyova, T. I.; Miroshnikov, A. I.
Protein Expression Purif. 2002, 24, 56.
(15) Chuvikovsky, D. V.; Esipov, R. S.; Skoblov, Y. S.;
Chupova, L. A.; Muravyova, T. I.; Miroshnikov, A. I.;
Lapinjoki, S.; Mikhailopulo, I. A. Bioorg. Med. Chem. 2006,
14, 6327.
(16) Miroshnikov, A. I.; Esipov, R. S.; Konstantinova, I. D.;
Fateev, I. V.; Mikhailopulo, I. A. The Open Conf. Proc. J.
2010, 1, 98.
(27) Among the 8-aza-7-deazapurines studied in the present
work, the chemical and the physical properties of allopurinol
(8) (the neutral molecule as well as cation and anion) have
been an object of numerous studies (see for example refs.
20a and 28). Notably: (1) the neutral molecule is present as
a mixture of tautomeric forms with predominant population
of the keto =N8-N9(H)-tautomer28d (purine numbering);
(2) the anion of allopurinol is also present as a mixture of
tautomeric forms and yields upon methylation N9,N1-, N8,N1-,
and N8,N3-dimethyl derivatives 28a
.
(17) Standard reaction conditions A: K,Na-phosphate buffer
(10 mM, pH 7.0); donor/acceptor ratio was 1.5:1.0 (mol);
1000 units of E. coli PNP14 was used per 1 mmol of base (ca.
0.10–0.15 mmol of base in 10–15 mL buffer was taken in the
reaction); 50 °C, 48–50 h. The reaction mixture was filtered,
silica gel (ca. 2 mL) was added to the filtrate and the mixture
was evaporated to dryness and co-evaporated with EtOH.
The silica gel with adsorbed product was put on the top of a
silica gel column (ca. 50 mL) and product was eluted with
CHCl3–MeOH. HPLC data [Kromasil C18, 4.6 × 250 mm, 5
μm; elution with H2O–MeOH–TFA, 90:10:0.1; flow rate 1
mL/min]: Rt (base/2′-deoxyriboside) = 4.7/6.8 (4/15),
7.8/13.6 (5/16), 4.2/8.4 (6/17), 10.7/17.0 (7/18), 5.6/12.2
(8/19), 6.1/14.3 (9/20) min. Yields for individual isolated
products 15–19: 66, 60, 77, 76 and 68%, respectively.
Synthesis of nucleoside 20: To a solution of 2′-deoxy-
guanosine (95 mg, 0.356 mmol) in K,Na-phosphate buffer
(18 mM; pH 7.5) was added PNP (10 mg, 270 units) and the
reaction mixture was stirred at 50 °C for 1 h. A solution of 9
(40 mg, 0.237 mmol) in DMSO (2 mL) was added dropwise
over 1 h and the mixture was heated at 50 °C for 72 h
(progress of the reaction monitored by HPLC). The mixture
was cooled to r.t., the guanine formed and the remaining
base 9 was filtered off, the filtrate was evaporated, and the
desired product 20 was isolated by silica gel column
chromatography (MeOH–CHCl3, 1:10) to give 20 (17 mg,
25%) as an amorphous product. UV (H2O): λmax = 305 nm,
λmin = 228 nm.
(28) (a) Bergmann, F.; Frank, A.; Neiman, Z. J. Chem. Soc.,
Perkin Trans. 1 1979, 2795. (b) Bundgaard, H.; Falch, E. Int.
J. Pharm. 1985, 24, 307. (c) Shukla, M. K.; Mishra, P. C.
Spectrochim. Acta, Part A 1996, 52, 1547. (d) Costas, M. E.;
Acevedo-Chavez, R. J. Comput. Chem. 1999, 20, 200.
(e) Villegas-Ortega, R.; Costas, M. E.; Acevedo-Chavez, R.
THEOCHEM 2000, 504, 105.
(29) Granovsky A. A.; Firefly version 7.1.G, URL:
http://classic.chem.msu.su/gran/firefly/index.html.
(30) Schmidt, M. W.; Baldridge, K. K.; Boatz, J. A.; Elbert, S. T.;
Gordon, M. S.; Jensen, J. H.; Koseki, S.; Matsunaga, M.;
Nguyen, K. A.; Su, S.; Windus, T. L.; Dupuis, M.;
Montgomery, J. A. J. Comput. Chem. 1993, 14, 1347.
(31) Stewart, J. J. P. J. Comput. Chem. 1989, 10, 209.
(32) Araki, T.; Ikeda, I.; Matoishi, K.; Abe, R.; Oikawa, T.;
Matsuba, Y.; Ishibashi, H.; Nagahara, K.; Fukuiri, Y.
Method for producing cytosine nucleoside compounds;
US Patent 6858721, 2005.
(33) Pugmire, M. J.; Ealick, S. E. Biochem. J. 2002, 361, 1.
(34) The following reaction conditions employed in these
experiments: the ratio of donor to acceptor was 1.1:1.0
(mol), 10 mM K,Na-phosphate buffer (pH 7.0) containing
10% (vol) DMSO; 1000 units14 PNP per 1.0 mmol base,
reaction temperature was 50 °C. The progress of the
nucleoside formation was followed by HPLC column
HYPERSIL 150 × 4.6 mm, 5 μm; eluent: H2O; 1 mL/min;
tR = 4.1 (4), 6.0 (15), 2.0 (6), 5.9 (17) min.
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Synlett 2012, 23, 1541–1545

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