Enzymatic synthesis of novel purine nucleosides bearing a chiral benzoxazine fragment

Article abstract of DOI:10.1111/cbdd.13458

A series of ribo- and deoxyribonucleosides bearing 2-aminopurine as a nucleobase with 7,8-difluoro- 3,4-dihydro-3-methyl-2H-[1,4]benzoxazine (conjugated directly or through an aminohexanoyl spacer) was synthesized using an enzymatic transglycosylation reaction. Nucleosides 3-6 were resistant to deamination under action of adenosine deaminase (ADA) Escherichia coli and ADA from calf intestine. The antiviral activity of the modified nucleosides was evaluated against herpes simplex virus type 1 (HSV-1, strain L2). It has been shown that at sub-toxic concentrations, nucleoside (S)-4-[2-amino-9-(β-D-ribofuranosyl)-purin-6-yl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine exhibit significant antiviral activity (SI?>?32) on the model of HSV-1 in vitro, including an acyclovir-resistant virus strain (HSV-1, strain L2/R).

Full text of DOI:10.1111/cbdd.13458

DR IRINA D. KONSTANTINOVA (Orcid ID : 0000-0001-5563-6549)
Article type : Research Article
Corresponding author mail id: kid1968@yandex.ru
Enzymatic Synthesis of Novel Purine Nucleosides Bearing a Chiral
Benzoxazine Fragment
a
b
b
b
Barbara Z. Eletskaya , Dmitry A. Gruzdev , Victor P. Krasnov , Galina L. Levit , Maria A.
a
a
a
a
Kostromina , Alexander S. Paramonov , Alexei L. Kayushin , Inessa S. Muzyka , Tatyana I.
a
a
c
c
Muravyova , Roman S. Esipov , Valeria L. Аndronova , Georgiy A. Galegov , Valery N.
b,d
a
a
Charushin , Anatoly I. Miroshnikov and Irina D. Konstantinova*
a
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of
Sciences, Miklukho-Maklaya 16/10, 117997 GSP, Moscow B-437, Russian Federation
b
Postovsky Institute of Organic Synthesis, Russian Academy of Sciences (Ural Branch),
S.Kovalevskaya/Academicheskaya St. 22/20, 620137, Ekaterinburg, Russian Federation
c
Ivanovsky Institute of Virology (Gamaleya Research Center of Epidemiology and
Microbiology, Ministry of Healthcare of the Russian Federation), Gamaleya Str.,18, 123098,
Moscow, Russian Federation
d
Ural Federal University named after the first President of Russia B.N. Yeltsin, Mira St. 19,
6
20002, Ekaterinburg, Russian Federation
Running Head: Biocatalisys, Benzoxazine, Purine nucleosides
Keywords: Biocatalisys, Benzoxazine, Purine nucleosides.
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
1
0.1111/cbdd.13458
This article is protected by copyright. All rights reserved.

Abstract:
A series of ribo- and deoxyribonucleosides bearing 2-aminopurine as a nucleobase with 7,8-
difluoro- 3,4-dihydro-3-methyl-2H-[1,4]benzoxazine (conjugated directly or through an
aminohexanoyl spacer) was synthesized using an enzymatic transglycosylation reaction.
Nucleosides 3-6 were resistant to deamination under action of adenosine deaminase (ADA)
E. coli and ADA from calf intestine. The antiviral activity of the modified nucleosides was
evaluated against herpes simplex virus type 1 (HSV-1, strain L2). It has been shown that at
sub-toxic concentrations, nucleoside (S)-4-[2-amino-9-(β-D-ribofuranosyl)-purin-6-yl]-7,8-
difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine exhibit significant antiviral activity
(SI>32) on the model of HSV-1 in vitro, including an acyclovir-resistant virus strain (HSV-1,
strain L2/R).
1
. INTRODUCTION
The permanent interest in obtaining new modified nucleosides is caused by their high
efficiency in treatment of some hematological (fludarabine, cytarabine, cladribine,
clofarabine, nelarabine [1, 2]) and viral (ribavirin, azidothymidine, vidarabine) human
diseases [3, 4].
However, in oncology, the use of drugs with a high systemic toxicity is motivated by their
high efficiency (up to 80% of remission in the treatment of hematological diseases [5-7]) in
treatment of viral diseases [8]; drugs of this class are applied only in case of immediate threat
to human life (for example, ribavirin in the complex therapy of hepatitis С [9, 10]).
Among the huge number of publications related to modified nucleosides and bases, a
particular attention is paid to purine nucleosides with various substituents at the C6 position
of purine base [11-15].
This article is protected by copyright. All rights reserved.

It is known that in the process of metabolism the corresponding enzymes convert nucleosides
into active nucleotides [16]. However, the intracellular enzyme adenosine deaminase (ADA)
often interferes with this process. This enzyme destroys modified nucleosides; as a result,
they cannot produce active nucleoside forms [17]. Thus, the application of vidarabine (Figure
1
) in the treatment of herpes simplex virus types I and II infection is restricted by a fast
degradation of this substance under the action of ADA to 9-β-D-arabinofuranosyl-
hypoxanthine, which has a low antiviral activity [10].
One of the approaches to improvement of stability of nucleosides to ADA action is an
introduction of a chlorine (fluorine) atom into the second position of the heterocyclic base
(fludarabine, clofarabine, and cladribine (Figure 1)) [18-20].
However, those nucleosides have a high systemic toxicity and are used only in
oncohematology. Another possible way to improve the stability of nucleosides to ADA in
vivo is introduction of substituents at the C6-position of a base [11, 13, 14, 21-24]. The only
exception is nelarabine (9-β-D-arabinofuranosyl-6-O-methylguanine, Figure 1), used for the
treatment of T-cell acute lymphoblastic leukemia and lymphoblastic lymphoma. The
introduction of the 6-O-methyl group into 9-β-D-arabinofuranosylguanine (AraG) improved
its solubility in water and bioavailability. Meanwhile, nelarabine is a substrate for human
ADA, which converts the 6-methoxy group into 6-keto group with the formation of active
AraG molecule. [25]
The most effective chemical approach to obtain compounds resistant to ADA action is a
cross-coupling reaction with a protected 6-chloropurine with riboside followed by removal of
3
4
protective groups [14, 26]. In this way, purine ribosides (R = H, NH ) with alkyl (R =
2
isoamyl-, n-propyl, isopropyl, allyl, etc.), aryl, acyclic, and heterocyclic substituents (Figure
This article is protected by copyright. All rights reserved.

1
) were synthesized [27, 28]. All compounds were studied as antagonists of adenosine
receptors [27, 28]; some of them possessed an antiproliferative activity [14, 23].
The alternative approach to the synthesis of modified nucleosides was developed by Abdalla
E.A. Hassan et al. New analogues of purine ribosides containing alkyl- and cycloalkyl-
substituents at the C6-position were obtained, and their substrate specificity to purine
nucleoside phosphorylase E. coli (PNP E. coli) was investigated [29]. It has been shown that
PNP E. coli has a high tolerance to the steric and hydrophobic environment at the C6-position
of ribosides synthesized. This allows using the C6-substituted bases and their ribosides as
substrates for transglycosylation under the action of PNP E. coli to obtain new nucleosides.
The new compounds can be obtained with both natural and modified carbohydrate residues
[
30, 31].
An advantage of enzymatic approach to the synthesis of modified nucleosides is a stereo- and
regioselectivity of the process (9-β-D-nucleosides only) [32-34].
Recently we have synthesized a number of 6-substituted derivatives of purine and 2-
aminopurine containing amino acids [35-37] and heterocyclic amines [38, 39] fragments.
Among them, compounds with high antimycobacterial were found [37]. To obtain
enantiomerically pure heterocyclic amines (the precursors of bases 1 and 2), the method of
kinetic resolution of racemates was used [40-42]. Four bases of the series (conjugates of 2-
aminopurine with the enantiomers of 7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine
[
[
(R)-1, 2 and (S)-1,2], (Figure 2)) were used in the enzymatic synthesis of β-D-arabinosides
39]. Unfortunately, bases 1 and 2, as well as their arabinosides did not show a high level of
antiherpetic activity in in vitro experiments on the HSV type-1 model. However, they did not
show a high cytotoxicity in Vero E6 cells (unpublished data).
This article is protected by copyright. All rights reserved.

Among the modified nucleosides, namely ribosides exhibit a higher anti-herpes activity in
comparison with 2-deoxyribosides and arabinosides. [43].
In addition, C6-substituted purine bases are usually poorly soluble in water. Ribosylation is
often used to improve the solubility of bases. That is why we decided to synthesize ribosides
and deoxyribosides of 2-aminopurine with the enantiomers of 7,8-difluoro-3,4-dihydro-3-
methyl-2H- [1,4] benzoxazine and investigate their substrate properties with respect to ADA
and antiherpes virus activity.
2
. METHODS AND MATERIALS
The conjugates of 2-aminopurine with the enantiomers of 7,8-difluoro-3,4-dihydro-3-methyl-
H-[1,4]benzoxazine attached directly to the 6-position [(R)-1, (S)-1] and through an
aminohexanoyl spacer [(R)-2, (S)-2] were prepared as described earlier [44].
2
2
.1. General methods.
Unless otherwise noted, materials were obtained from commercial suppliers and used
without any purification. The method used to produce recombinant E. coli phosphorylases
was described earlier [45]. The method used to produce recombinant E. coli adenosine
deaminase was described in Supporting Information. Bases (R)-1 and (S)-1 [39], (R)-2 and
(S)-2 [43] were obtained according to the known procedures.
Column chromatography was performed on Silica gel 60 (Fluka, Buchs, Switzerland).
High performance liquid chromatography (HPLC) was performed on a Waters system
Waters 1525, Waters 2489, Breeze 2) using a Nova Pak C18 column, 4.6 × 150 mm, eluent
А - 0.1% TFA–H O, eluent В - 70% MeCN in 0.1% TFA–H O, flow rate 0.5 mL/min,
(
2
2
detection at 254 nm. Gradient: 0  100 B over 20 min. NMR spectra were recorded on
This article is protected by copyright. All rights reserved.

Bruker Avance II 700 spectrometers (Bruker BioSpin, Rheinstetten, Germany) in DMSO-d6
°
at 30 C. Chemical shifts in ppm (δ) were measured relative to the residual solvent signals as
internal standards (2.50). Coupling constants (J) were measured in Hz. Liquid
chromatography mass spectrometry was performed on an Agilent 6210 TOF LC/MS system
(Agilent Technologies, USA) in positive ion mode (ESI+).
The reaction conditions of nucleosides 3–6 synthesis (a ratio of reagents, PNP
concentration, duration of reaction, and conversion of base into nucleoside) are reported in
Table 1. Physicochemical properties of nucleosides are shown in Table 2.
2
.2. Enzymatic reactions of nucleosides synthesis
Bases 1 or 2 and natural nucleosides (Urd, dUrd, dIno) were dissolved in water at 40–50

C and pH was adjusted up to 7.0. The enzymes were added and the reaction mixture was
incubated at 50 C. Reactions were monitored by HPLC. When conversion reached the
maximal value, reaction was stopped by heating to 70 C. The products 3–6 were isolated by
reversed-phase column chromatography (silica gel C , Fluka), column (20 × 100 mm, elution
18
conditions see in Table 1). Yields, physicochemical and spectral properties of nucleosides are
given in Table 2. The NMR spectra and HPLC data are presented in Supporting Information.
2
.3. Preparation and determination of enzymatic activity of adenine
deaminase from E. coli (EcADA)
Preparation of adenine deaminase from E. coli (EcADA) and determination of enzymatic
activity are presented in Supporting Information. Experimental data for the substrate
specificity of adenosine deaminase E.coli (EcADA) and ADA from calf intestine (CiADA)
with respect to nucleosides 3–6, adenosine (Ado), 2’-deoxyadenosine (2'-dAdo) and 9-(β-D-
arabinofuranosyl)adenine (AraA), as well as previously synthesized arabinosides (R)-1-Ara
(R)-2-Ara and (S)-2-Ara, [39], given in Table 3.
This article is protected by copyright. All rights reserved.

2
.4. Antiviral Activity and Cytotoxicity
African green monkey kidney cells (Vero E6) which were used for evaluating the activity of
hetorocyclic bases 1, (R)-2 and nucleosides 4 – 6 against herpes simplex virus type 1 (HSV-1)
and their cytotoxicity were kindly provided by Dr. Butenko A.M. (D. I. Ivanovsky Institute of
5
Virology). Vero E6 cells were seeded in 96-well tissue plates at a density of 4 × 10 cell/ml in
1
00 µl growth medium (Dulbecco's modified Eagle's medium [DMEM] containing 5%
newborn calf serum, 100 IU/ml benzylpenicillin, and 2 mM L-glutamine) and incubated in a
humid atmosphere with 5% CO at 37 C until a confluent monolayers formed.
2
HSV-1 strain L was originally obtained from the State Collection of Viruses of the D.I.
2
Ivanovski Institute of Virology of the N.F. Gamaleya Research Center of Epidemiology and
Microbiology, Ministry of Healthcare of the Russian Federation [46].
ACV-resistant HSV-1 strain L /R was obtained by serial passages of etalon strain HSV/L in
2
2
the presence of increasing concentrations of ACV.
The following compounds purchased from Sigma-Aldrich, (USA) were used as reference
drugs: 9-[(2-hydroxyethoxy)methyl]guanine (Acyclovir, CAS Number: 59277-89-3), sodium
phosphonoformate tribasic hexahydrate (Foscarnet, CAS Number: 34156-56-4) and 1-β-D-
Ribofuranosyl-1,2,4-triazole-3-carboxamide (Ribavirin, CAS Number: 36791-04-5).
Investigation of antiherpes activity of the compounds in vitro was carried out in accordance
with the internationally accepted cytopathic effect (CPE) inhibition assay [47-49] In 96-well
.
plastic plates (Linbro, Flow Laboratories, UK) with the generated cell monolayers, the two-
fold serial dilutions of the compounds were prepared in the support medium (Eagle medium
and medium 199, 1: 1). Infectious material was then introduced with final dilution providing a
This article is protected by copyright. All rights reserved.

multiplicity of infection (MOI) 0.1 PFU/cell; incubation was performed at 37 °C. Two wells
were used for each concentration of the tested compound and three independent experiments
were assessed. Three controls were used. 1. Toxicity control. Uninfected cells were passaged
in the presence of the drug in the same concentration range as in the experiment. 2. Virus
control. Cells were infected under conditions described above, but the support medium did
not contain the drug. 3. Cells control. Uninfected cell cultures were incubated in the support
medium, which contained no drug.
After 48 h, when 95-100% CPE was developed in virus control, an antiviral activity was
assessed by determining of ID and ID for compounds concentrations that inhibit the
5
0
95
development of virus-induced CPE as compared with control by 50% and almost completely
respectively (Table 4).
Cytotoxic properties of the compounds in vitro were evaluated after 72 h incubation of cells
in the presence of a known concentration of a compound. A maximal tolerable concentration
(MTC) of compound that does not cause destructive changes of the cellular monolayer in
comparison with controls was determined by state of a cell monolayer with the light
microscope. Cytotoxicity was determined quantitatively also by trypan blue exclusion method
and expressed as CD , the concentration of the drug, at which 50% of cells die [50-53].
5
0
3
. RESULTS AND DISCUSSION
.1. Biosynthesis of nucleosides
The purpose of the present research was to synthesize nucleosides with natural sugar residues
ribose and 2-deoxyribose) by the transglycosylation reaction (Scheme 1) and to study their
3
(
cytotoxicity and anti-herpes virus activity.
This article is protected by copyright. All rights reserved.

As in the case of the synthesis of arabinosides [39], bases 1 and 2 proved to be good
substrates for PNP E. coli in the synthesis of 2’-deoxyribosides (3 and 4) and ribosides (5 and
6
). The degree of conversion of bases to nucleosides was rather high; the data are presented in
Figures 3 and 4.
We should note the differences in reaction rates for heterocyclic bases (S)-1 and (R)-1 having
a benzoxazine substituent attached directly to the purine base.
In the case of the (S)-configuration of the benzoxazine substituent, the rate of the ribosylation
reaction of (S)-1 was lower than that for (R)-isomer. Earlier we found that (S)-1-arabinoside
cannot be obtained using transglycosylation reaction [39].
Influences of the configuration of the chiral center of hexanoyl-containing bases 2 on the
substrate-specific properties of PNP were not revealed.
The dependence of the enzymatic synthesis efficiency on various ribose donors (Ado, Ino,
and Urd) was studied. As shown in Figure 5, the conversion of base (R)-2 to riboside (R)-6
does not depend on type of a ribose donor.
It is preferable to synthesize ribosides from uridine using two enzymes: PNP and uridine
phosphorylase (UP) (6 units of PNP and 4 units of UP per 1 mL of the reaction mixture); a
base–Urd ratio is 1:3.
Similar data was obtained for the synthesis of 2'-deoxyribosides: 2'-deoxyinosine (dIno) and
2
'-deoxyuridine (dUrd) can be used. In the synthesis of 2'-deoxyribosides from dIno,
equilibrium of the reaction was achieved in a short time (1 to 2 h), then the amount of the
This article is protected by copyright. All rights reserved.

desirable product in the reaction mixture was decreased. We believe that this is due to the
instability of 2-deoxyribose-1-alpha-phosphate under the reaction conditions [54, 55]. We
tried to influence this phenomenon by decreasing the amount of PNP (from 5 to 0.5 units) and
increasing a base–dIno ratio from 1:1.5 to 1:3 (Figure 6).
From the results obtained it is obvious that the optimal conditions are the following: a
threefold excess of dIno to base (S)-2 and 0.5–1 units of PNP per 1 mL of the reaction
mixture. These conditions were used in the synthesis of the 2'-deoxyribosides of the bases 1
and 2. It is very important to control the conversion of bases to the nucleoside until the
conversion of the base reaches the plateau.
General experimental data for the enzymatic synthesis of β-D-ribo- and β-D-2'-
deoxyribonucleosides are shown in Table 1.
Compounds (S)-2, (R)-2, 4, and 6 are slightly soluble in water. Therefore, the reaction mixture
was applied to a chromatographic column without concentrating. Compounds 4 and 6
containing the hexanoyl spacer were eluted in a gradient of methanol in water (7–70%). For
nucleosides 3 and 5 without spacer, a gradient 0–50% was used. The fractions containing the
desired products were combined, the solvent was evaporated, and the residue was dried in
1
13
vacuo over P O . For all nucleosides synthesized, the H- and C-NMR data (including
2
5
1
1
1
13
[
H, H] and [ H, C] 2D: COSY, HSQC, HMBC) were obtained. The NMR spectra and
HPLC data are presented in Supporting Information.
3
.2. Adenosine deaminase assays
It is known that the action of intracellular adenosine deaminase can lead to inactivation of
modified nucleosides and accordingly affects their activity.
This article is protected by copyright. All rights reserved.

We tested the substrate specificity of adenosine deaminase E. coli (EcADA) and ADA from
calf intestine (CiADA) toward new C6-modified deoxyribosides 3 and 4, ribosides 5 and 6
and earlier synthesized arabinosides [39]. CiADA was chosen because this enzyme isolated
from calf intestine is similar to human ADA and exhibits almost identical Km and kcat for
adenosine and analogues [56]. Adenosine (Ado), 2'-deoxyadenosine (2'-dAdo), adenine-β-D-
arabinofuranoside (AraA) were used as controls under the same conditions. No reaction
products (according to HPLC and mass spectrometry data) were detected in the reaction
mixtures containing modified nucleosides after 1 and 24 h (Table 3). These results show that
nucleosides 3–6 are not substrates for ADA (E. coli and calf intestinal). The introduction of
benzoxazine substituents in the C6-position protects modified nucleosides from undesirable
action of ADA.
3
.3. Antiviral assays
The antiviral activity of the modified nucleosides was evaluated against herpes virus type 1
HSV-1, strain L ). Cytotoxic properties of compounds 1, (R)-2, 4–6 against Vero E6 cells
(
2
were determined by incubation of a cell culture in the presence of nucleosides and
nucleobases at the range of investigated concentrations up to 1000 g/mL during 72 h (Table
4
). A minimum of three experimental replicates were performed.
As follows from the presented results, nucleoside (S)-6 can be attributed to low-toxic
compound: the CD values characterizing the level of toxicity for the Vero E6 cell culture are
5
0
close to or exceed a concentration of 1000 μg/mL.
Other nucleosides exhibited a moderate cytotoxicity (CD > 300 μg/mL). Their cytotoxicity
5
0
turned out to be lower than the cytotoxicity of bases 1 and (R)-2, which are close in structure,
but do not contain a sugar residue.
This article is protected by copyright. All rights reserved.

Compounds 4 and 6 exhibit a minimal antiviral activity only at a sub-toxic concentration
Selectivity index, SI = CD /ID , 1 ÷ 3). Riboside (S)-5 exhibits a significant selective
(
5
0
50
activity against HSV-1 (SI > 32) in vitro, including an acyclovir-resistant virus strain (HSV-1,
strain L /R, SI = 23). Compounds (S)-6 and (R)-6 show not high but significant antiviral
2
activity on the HSV-1 model in vitro, including the HSV-1 strain L /R.
2
The observed antiviral effect of the compounds is equally exposed in both used viral models,
that is, it does not depend on the sensitivity of HSV-1 to ACV.
4
. CONCLUSION
Thus, we obtained a series of modified ribo- and deoxyribonucleosides, conjugates of 2-
aminopurine and chiral 7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine attached
directly to the 6-position and through 6-aminohexanoyl spacer. Nucleosides 3-6 were resistant
to deamination under action of adenosine deaminase (ADA) E. coli and ADA from calf
intestine. The antiviral activity of the modified nucleosides was evaluated against herpes virus
type 1 (HSV-1, strain L ). It has been shown that at sub-toxic concentrations, modified
2
ribosides containing (R)- and (S)-enantiomers of 7,8-difluoro-3,4-dihydro-3-methyl-2H-
[
1,4]benzoxazine exhibit significant antiviral activity on the model of HSV-1 in vitro,
including an acyclovir-resistant virus strain (HSV-1, strain L /R).
2
ACKNOWLEDGEMENTS
This work was financially supported by the Russian Science Foundation (grant no. 14-13-
0
1077). The authors would like to thank Dr. Tamara A. Balashova for assistance in analysis
of NMR spectra. NMR experiments were partially carried out using the equipment provided
by the IBCH сore facility (CKP IBCH, supported by the Russian Ministry of Education and
Science, grant no. RFMEFI62117X0018).
This article is protected by copyright. All rights reserved.

CONFLICT OF INTEREST
The authors declare no conflict of interest.
Figure legends
Figure 1. С2- and С6-substituted purine nucleosides
Figure 2. Conjugates of 2-aminopurine with the enantiomers of 7,8-fluoro-3,4-dihydro-3-
methyl-3,4-dihydro-2H-[1,4]benzoxazine for transglycosilation
Scheme 1. Reaction of transglycosylation.
Figure 3. Conversion of bases 1 and 2 to 2’-deoxyribosides 3 and 4. Reaction conditions:
base (2 mM) and sugar moiety donor dIno (6 mM) in 1.0 mL of the buffer (pH 7.0; 5 mM
KH PO ), 5 units of PNP E. coli, 50 C, 4 h.
2
4
Fig. 4. Conversion of bases 1 and 2 to ribosides 5 and 6.
Reaction conditions: base (2 mM) and sugar moiety donor Urd (6 mM) in 1.0 mL of the
buffer (pH 7.0; 5 mM KH PO ), 5 units of PNP E. coli and 5 units of UP E. coli, 50 C, 4 h.
2
4
Fig. 5. Conversion of (R)-2 into riboside (R)-6.
Reaction conditions: 1.0 ml of the buffer (pH 7.0; 5 mM KH PO ), 50 C, 4 h, base (S)-2 (2
o
2
4
mM) and sugar moiety donor: Urd (6 mM), 6 units of E. coli PNP and 4 units of UP; Ino (6
mM), 5 units of E. coli PNP; Ado (6 mM), 5 units of E. coli PNP.
Fig. 6. Conversion of base (S)-2 to 2-deoxyriboside (S)-4.
Reaction conditions: base (S)-2 (2 mM) and sugar moiety donor dIno (3, 4, 6 mM) in 1.0 mL
of the buffer (pH 7.0; 5 mM KH PO ), 0.5, 1, 2, 5 units of E. coli PNP, 50 C, 4 h.
2
4
Supplementary data
Supplementary data associated with this article can be found in the online version
This article is protected by copyright. All rights reserved.

References
1
2
3
. Robak P, Robak T. Older and new purine nucleoside analogs for patients with acute
leukemias. Cancer Treat. Rev. 39(8), 851-861 (2013).
. Parker WB. Enzymology of purine and pyrimidine antimetabolites used in the treatment of
cancer. Chem. Rev. 109(7), 2880-2893 (2009).
. Jordheim LP, Durantel D, Zoulim F, Dumontet C. Advances in the development of
nucleoside and nucleotide analogues for cancer and viral diseases. Nat. Rev. Drug
Discovery 12(6), 447-464 (2013).
4
. Schwartz P, Novack J, Shipman CJr, Drach JC. Metabolism of arabinosyladenine in herpes
simplex virus-infected and uninfected cells correlation with inhibition of dna synthesis
and role in antiviral selectivity. Biochem. Pharmacol. 33(15), 2431-2438 (1984).
. Reilly KM, Kisor DF. Profile of nelarabine: use in the treatment of T-cell acute
lymphoblastic leukemia. OncoTargets Ther. 2, 219-228 (2009).
5
6
. Ravandi F, O'Brien S, Jorgensen J, Pierce S, Faderl S, Ferrajoli A, Koller C, Phallagundla
P, York S, Brandt M, Luthra R, Burger J, Thomas D, Keating M, Kantarjian H. Phase
2
1
study of cladribine followed by rituximab in patients with hairy cell leukemia. Blood
18 (14), 3818-3823 (2011).
7
. Thompson PA, Tam CS, O’Brien SM, Wierda WG, Stingo F, Plunkett W, Smith SC,
Kantarjian HM, Freireich EJ, Keating MJ. Fludarabine, cyclophosphamide, and
rituximab treatment achieves long-term disease-free survival in IGHV-mutated
chronic lymphocytic leukemia. Blood 127 (3), 303-309 (2016).
8
9
1
. Daifuku R. Stealth nucleosides: mode of action and potential use in the treatment of viral
diseases. BioDrugs 17, 169-177.
. De Clercq E. Milestones in the discovery of antiviral agents: nucleosides and nucleotides.
Acta Pharm. Sin. 2(6), 535-548 (2012).
0. Gentry BG, Shen W, Breitenbach JM, Hilfinger J, Drach JC.. Vidarabine Prodrugs as
This article is protected by copyright. All rights reserved.

Potential Inhibitors of Adenosine Deaminase. Antiviral Res. 78(2), A56 (2008).
1. Fleysher MH, Hakala MT, Bloch A, Hall RH. Synthesis and biological activity of some
N6-alkyladenosines. J. Med. Chem. 11(4), 717-720 (1968).
1
1
2. Ashton TD, Aumann KM, Baker SP, Schiesser CH, Scammells PJ. Structure–activity
relationships of adenosines with heterocyclic N6-substituents. Bioorg. Med. Chem.
Lett. 17(4), 6779-6784 (2007).
1
1
1
1
3. Hocek M, Silhar P, Shih I, Mabery E, Mackman R. Cytostatic and antiviral 6-arylpurine
ribonucleosides. Part 7: Synthesis and evaluation of 6-substituted purine L-
ribonucleosides. Bioorg. Med. Chem. Lett. 16(20), 5290-5293 (2006).
4. Drenichev MS, Oslovsky VE, Mikhailov SN. Cytokinin Nucleosides - Natural
Compounds with a Unique Spectrum of Biological Activities. Curr. Top. Med. Chem.
1
6 (23), 2562-2576 (2016).
5. Voller J, Zatloukal M, Lenobel R, Dolezal K, Bereš T, Kryštof V, Spichal L, Niemann P,
Dzubak P, Hajduch M, Strnad M. Anticancer activity of natural cytokinins: A
structure–activity relationship study. Phytochemistry 71(11-12), 1350-1359 (2010).
6. Sampath D, Ashutosh RA, Plunkett W. Mechanisms of apoptosis induction by nucleoside
analogs. Oncogene 22(56), 9063-9074 (2003).
1
1
7. Galmarini CM, Mackey JR, Dumontet C. Nucleoside analogues: mechanisms of drug
resistance and reversal strategies. Leukemia 15(16), 875-890 (2001).
8. Plunkett W, Huang P, Searcy CE, Gandhi V. Gemcitabine: Preclinical pharmacology and
mechanisms of action. Semin. Oncol. 5(10), 3-15 (1996).
1
9. Bonate PL, Arthaud L, Cantrell WR, Stephenson K, Secrist JA, Weitman S. Discovery
and development of clofarabine: a nucleoside analogue for treating cancer. Nat. Rev.
Drug Discovery 5(10), 855-863 (2006).
2
2
0. Liliemark J. The clinical pharmacokinetics of cladribine. Clin Pharmacokinet. 32(2), 120-
1
31 (1997).
1. Link A, Heidler P, Kaiser M, Brun R. Synthesis of a series of N 6-substituted adenosines
This article is protected by copyright. All rights reserved.

with activity against trypanosomatid parasites. Eur. J. Med. Chem. 44(9), 3665-3671
2009).
(
2
2. Silhar P, Hocek M, Pohl R, Votruba I, Shih I, Mabery E, Mackman R. Synthesis,
cytostatic and anti-HCV activity of 6-(N-substituted aminomethyl)-, 6-(O-substituted
hydroxymethyl)- and 6-(S-substituted sulfanylmethyl)purine nucleosides. Bioorg.
Med. Chem. 16(5), 2329-2366 (2008).
2
2
3. Too K, Brown DM, Bongard E, Yardley V, Vivas L, Loakesa D, Anti-malarial activity of
N6-modified purine analogues. Bioorg. Med. Chem. 15(16), 5551-5562 (2007).
4. Ottria R, Casati S. Manzocchi A, Baldoli E, Mariotti M, Maier JA, Ciuffreda P. Synthesis
and evaluation of in vitro anticancer activity of some novel isopentenyladenosine
derivatives. Bioorg. Med. Chem. 18(12), 4249-4254 (2010)
2
2
5. Curbo S, Karlsson A. 9-β-D-Arabinofuranosylguanine. From: Cancer Drug Discovery and
Development: Deoxynucleoside Analogs in Cancer Therapy. Ed. G.J.Peters, p. 215-
2
24 (2006).
6. Hocek M, Holy A, Votruba I, Dvorakova H. Synthesis and Cytostatic Activity of
Substituted 6-Phenylpurine Bases and Nucleosides: Application of the Suzuki-
Miyaura Cross-Coupling Reactions of 6-Chloropurine Derivatives with Phenylboronic
Acids. J. Med. Chem. 43(9), 1817-1825 (2000).
2
2
2
7. Van Galen PJM, Ijzerman AP, Soudijn W. Adenosine derivatives with N6-alkyl, -
alkylamine or -alkyladenosine substituents as probes for the A1-receptor. FEBS Lett.
2
3(1), 197-201 (1987).
8. Gao ZG, Blaustein JB, Gross AS, Melman N, Jacobson KA. N6-Substituted adenosine
derivatives: selectivity, efficacy, and species differences at A3 adenosine receptors.
Biochem Pharmacol. 65(10), 1675-84 (2003).
9. Hassan AEA, Abou-Elkhair RAI, Riordan JM, Allan PW, Parker WB, Khare R, Waud
WR, Montgomery JA, Secrist JA 3rd. Synthesis and evaluation of the substrate
This article is protected by copyright. All rights reserved.

activity of C-6 substituted purine ribosides with E. coli purine nucleoside
phosphorylase: Palladium mediated cross-coupling of organozinc halides with 6-
chloropurine nucleosides. Eur. J. Med. Chem. 47(1), 167-174 (2012).
3
3
3
0. Mikhailopulo IA, Miroshnikov AI. New trends in nucleoside biotechnology. Acta Naturae
2
(2), 36-58 (2010).
1. Mikhailopulo IA, Miroshnikov AI. Biologically important nucleosides: modern trends in
biotechnology and application. Mendeleev Commun. 21(2), 57-68 (2011).
2. Konstantinova ID, Antonov KV. Fateev IV, Miroshnikov AI, Stepchenko VA,
Baranovsky AV, Mikhailopulo IA. Chemo-enzymatic synthesis of b-D-
arabinofuranosyl purine nucleosides. Synthesis 10, 1555-1560 (2011).
3
3. Barai VN, Zinchenko AI, Eroshevskaya LA, Kalinichenko EN, Kulak TI, Mikhailopulo
IA. A universal biocatalyst for the preparation of base- and sugar-modified
nucleosides via an enzymatic transglycosylation. Helv. Chim. Acta. 85(7), 1901-1908
(2002).
3
3
4. Bzowska A, Kulikowska E, Shugar D. Purine nucleoside phosphorylases: properties,
functions, and clinical aspects. Pharmacol. Ther. 88(3), 349-425 (2000).
5. Vigorov AYu, Krasnov VP, Gruzdev DA, Men’shikova AA, Demin AM, Levit GL,
Charushin VN. Novel synthetic routes to N-(2-amino-9H-purin-6-yl)-substituted
amino acids. Mendeleev Commun. 24(1), 35-36 (2014).
3
3
6. Krasnov VP, Vigorov AYu, Gruzdev DA, Levit GL, Demin AM, Nizova IA, Tumashov
AA, Sadretdinova LSh, Gorbunov EB, Charushin VN. Synthesis of enantiomers of n-
(2-aminopurin-6-yl)amino acids. Russ. Chem. Bull. 64(9), 2106-2113 (2015).
7. Krasnov VP, Vigorov AYu, Musiyak VV, Nizova IA, Gruzdev DA, Matveeva TV, Levit
GL, Kravchenko MA, Skornyakov SN, Bekker OB, Danilenko VN, Charushin VN.
Synthesis and antimycobacterial activity of N-(2-aminopurin-6-yl) and N-(purin-6-yl)
amino acids and dipeptides. Bioorg. Med. Chem. Lett. 26(11), 2645-2648 (2016).
This article is protected by copyright. All rights reserved.

3
3
8. Gruzdev DA, Musiyak VV, Chulakov EN, Levit GL, Krasnov VP. Synthesis of purine
and 2-aminopurine conjugates bearing the fragments of heterocyclic amines at position
6
. Chem. Heterocycl. Compd. 51(8), 738-744 (2015).
9. Eletskaya BZ, Konstantinova ID, Paramonov AS, Esipov RS, Gruzdev DA, Vigorov
AYu, Levit GL, Miroshnikov AI, Krasnov VP, Charushin VN. Chemoenzymatic
arabinosylation of 2-aminopurines bearing the chiral fragment of 7,8-difluoro-3,4-
dihydro-3-methyl-2H-[1,4]benzoxazines. Mendeleev Commun. 26(1), 6-8 (2016).
0. Krasnov VP, Levit GL, Bukrina IM, Andreeva IN, Sadretdinova LSh, Korolyova MA,
Kodess MI, Charushin VN, Chupakhin ON. Kinetic resolution of (±)-2,3-dihydro-3-
methyl-4H-1,4-benzoxazine, (±)-2-methyl-1,2,3,4-tetrahydroquinoline and (±)-2-
methylindoline using N-tosyl-(S)-prolyl chloride. Tetrahedron: Asymmetry 14(14),
4
1
985-1988 (2003).
4
1. Gruzdev DA, Chulakov EN, Levit GL, Ezhikova MA, Kodess MI, Krasnov VP. A
comparative study on the acylative kinetic resolution of racemic fluorinated and non-
fluorinated 2-methyl-1,2,3,4-tetrahydroquinolines and 3,4-dihydro-3-methyl-2H-
[
1,4]benzoxazines. Tetrahedron: Asymmetry 24(19), 1240-1246 (2013).
4
4
2. Gruzdev DA, Vakarov SA, Levit GL, Krasnov VP. N-Tosyl-(S)-Prolyl Chloride in
Kinetic Resolution of Racemic Heterocyclic Amines. Chem. Heterocycl. Compd.
4
9(12), 1795-1807 (2014).
3. Kharitonova MI, Denisova AO, Аndronova VL, Kayushin AL, Konstantinova ID,
Kotovskaya SK, Galegov GA, Charushin VN, Miroshnikov AI. New modified 2-
aminobenzimidazole nucleosides: synthesis and evaluation of their activity against
herpes simplex virus type 1. Bioorg.Med. Chem. Lett. 27, 2484-2487 (2017).
4. Krasnov VP, Gruzdev DA, Chulakov EN, Vigorov AYu, Musiyak VV, Matveeva TV,
Tumashov AA, Levit GL, Charushin VN. Synthesis of novel purin-6-yl conjugates
with heterocyclic amines linked via 6-aminohexanoyl fragment. Mendeleev Commun.
4
This article is protected by copyright. All rights reserved.

2
5(6), 412-414 (2015).
4
5. Esipov RS, Gurevich AI, Chuvikovsky DV, Chupova LA, Muravyova TI, Miroshnikov
AI. Overexpression of Escherichia coli genes encoding nucleoside phosphorylases in
the pET/Bl21(DE3) system yields active recombinant enzymes. Protein Expression
Purif. 24(1), 56-60 (2002).
4
4. Gus’kova AA, Skoblov MYu, Korovina AN, Yasko MV, Karpenko IL, Kukhanova MK,
Andronova VL, Galegov GA, Skoblov YuS. Antiherpetic properties of acyclovir 5’-
hydrogenphosphonate and the mutation analysis of herpes virus resistant strains Chem.
Biol. Drug Des. 74(4), 382-389. (2009)
4
4
5. De Clercq E, Descamps J, Verheist G, Walker RT, Jones AS, Torrence PF. Comparative
Efficacy of Antiherpes Drugs against Different Strains of Herpes Simplex Virus. J.
Infect. Dis. 141(5), 563-573 (1980).
6. Fedorov II, Kazmina EM, Gurskaya GV, Jasko MV, Zavodnic VE, Balzarini J, De Clercq
E, Faraj A, Sommadossi JP, Imbach JL, Gosselin G. Novel 3‘-C/N-substituted 2‘,3‘-β-
d-dideoxynucleosides as potential chemotherapeutic agents. 1. Thymidine derivatives:ꢀ
synthesis, structure, and broad spectrum antiviral properties. J. Med. Chem. 40(4),
4
86-494 (1997).
4
7. Petrera E, Níttolo AG, Alché LE. Antiviral action of synthetic stigmasterol derivatives on
herpes. Simplex virus replication in nervous cells in vitro. BioMed Res. Int.
ArticleID947560 (2014).
4
4
8. Holy A, De Clercq E, Votryba I. Phosphonylmethyl ethers of nucleosides and their acyclic
analogues.ACS Symposium Series, 401, 50-71 (1989).
9. Selvam P, Lakra DR, Pannecouque C, De Clercq E. Synthesis, antiviral and cytotoxicity
studies of novel N-substituted indophenazine derivatives. Indian J. Pharm. Sci. 74(3),
2
75-278 (2012).
5
0. Galegov GA, Shobukhov VM, Leont'eva NA, Yas'ko MV. Synthesis and antiherpetic
activity of acyclovir phosphoesters. Russ. J. Bioorg. Chem. 23(1), 906-909 (1997).
This article is protected by copyright. All rights reserved.

5
5
1. Andronova VL, Skorobogatyi MV, Manasova EV, Berlin YuA, Korshun VA, Galegov
GA. Antiviral activity of some 2′-deoxyuridine 5-arylethynyl derivatives. Russ. J.
Bioorg. Chem. 29(3), 290-295 (2003).
2. Nixon AE, Hunter JL, Bonifacio G, Eccleston J, Webb MR. Purine nucleoside
phosphorylase: its use in a spectroscopic assay for inorganic phosphate and for
removing inorganic phosphate with the aid of phosphodeoxyribomutase. Anal.
Biochem. 265(2), 299-307 (1998).
5
5
3. Koellner G, Bzowska A, Wielgus-Kutrowska B, Luic M, Steiner T, Saenger W, Stepinski
J. Open and closed conformation of the E. coli purine nucleoside phosphorylase active
center and implications for the catalytic mechanism. J. Mol. Biol. 315(3), 351-371
(2002).
4. Spector T, Jones TE, Beacham LM III. Conversion of 2,6-diamino-9-(2-
hydroxyethoxymethyl)purine to acyclovir as catalyzed by adenosine deaminase.
Biochem. Pharmacol. 32(17), 2505-2511 (1983).
This article is protected by copyright. All rights reserved.

Figure 1. С2- and С6-substituted purine nucleosides
Figure 2. Conjugates of 2-aminopurine with the enantiomers of 7,8-fluoro-3,4-dihydro-3-
methyl-3,4-dihydro-2H-[1,4]benzoxazine
This article is protected by copyright. All rights reserved.

Scheme 1. Reaction of transglycosylation.
This article is protected by copyright. All rights reserved.

Figure 3. Conversion of bases 1 and 2 to 2’-deoxyribosides 3 and 4. Reaction conditions:
base (2 mM) and sugar moiety donor dIno (6 mM) in 1.0 mL of the buffer (pH 7.0; 5 mM
KH PO ), 5 units of PNP E. coli, 50 C, 4 h.
2
4
Fig. 4. Conversion of bases 1 and 2 to ribosides 5 and 6.
Reaction conditions: base (2 mM) and sugar moiety donor Urd (6 mM) in 1.0 mL of the
buffer (pH 7.0; 5 mM KH PO ), 5 units of PNP E. coli and 5 units of UP E. coli, 50 C, 4 h.
2
4
This article is protected by copyright. All rights reserved.

Fig. 5. Conversion of (R)-2 into riboside (R)-6.
Reaction conditions: 1.0 ml of the buffer (pH 7.0; 5 mM KH PO ), 50 C, 4 h, base (S)-2 (2
o
2
4
mM) and sugar moiety donor: Urd (6 mM), 6 units of E. coli PNP and 4 units of UP; Ino (6
mM), 5 units of E. coli PNP; Ado (6 mM), 5 units of E. coli PNP.
Fig. 6. Conversion of base (S)-2 to 2-deoxyriboside (S)-4.
Reaction conditions: base (S)-2 (2 mM) and sugar moiety donor dIno (3, 4, 6 mM) in 1.0 mL
of the buffer (pH 7.0; 5 mM KH PO ), 0.5, 1, 2, 5 units of E. coli PNP, 50 C, 4 h.
2
4
This article is protected by copyright. All rights reserved.

Table 1
Experimental data for the enzymatic synthesis of β-D-2’-deoxyribosides 3, 4 and β-D-
a
ribonucleosides 5, 6
Compd, Acceptor, Acceptor, Donor, Donor, Volume,
Ratio PNP, UP,
Time, Conversion Eluent
c
mol wt
mol wt
mg
mol wt mg
mL
A/D
e.u.
(B)
e.u.
(C)
h
of base into (E) ,
b
b
(
mmol)
A)
(mmol) [5 mM
nucleoside
(HPLC
data), %
85.61
%
(
(D)
KН PO ]
2 4
(
4
S)-3
51.2
(S)-1
318.1
25
(78.6)
dIno
252.2
60
(238)
40
40
35
19
47
40
35
35
1:3
55
-
3
3
3
3
2
2
2
2
0-50
0-50
0-50
0-50
7-70
7-70
7-70
7-70
(0.7)
(
4
R)-3
51.2
(R)-1
318.1
25
(78.6)
dIno
252.2
60
(238)
1:3
55
-
93.70
94.17
94.15
61.32
56.12
95.10
90.33
(0.7)
(
5
S)-4
64.2
(S)-2
432.2
30
(70)
dUrd
228.2
48
(210)
1:3
39
122
(0.56) (0.58)
(
5
R)-4
64.2
(R)-2
432.2
16
(37)
dUrd
228.2
26
(114)
1:3.1
1:2.6
1:3.5
1:3.5
1:3.5
10 33
(0.27) (0.30)
39 90
(
4
S)-5
35.2
(S)-1
318.1
30
(94)
Urd
244.2
60
(246)
(0.42) (0.36)
(
4
R)-5
35.2
(R)-1
318.1
25
(78.6)
Urd
244.2
68
(278)
39
90
(0.5)
(0.32)
(
5
S)-6
48.2
(S)-2
432.2
25
(58)
Urd
244.2
50
(205)
39
90
(0.67) (0.44)
(
R)-6
48.2
(R)-2
432.2
25
(58)
Urd
244.2
50
(205)
82
126
5
(1.4)
(0.61)
a
All enzymatic reactions were performed in potassium phosphate buffer (5 mM; pH 7.0) at 50 C under monitoring the reaction
progress by HPLC; the reactions were stopped when base was transformed into nucleoside like in test reactions. Solutions of
recombinant E. сoli UP and PNP in 5 mM potassium phosphate buffer (pH 7.0) with activities 900 and 918 units per mL,
respectively, were used [43]. Products were purified by silica gel C (Merck) column (20 × 100 mm) chromatography.
1
8
b
B - ratio of PNP (enzyme units) [43] per base (1 mmol); C – ratio of UP (enzyme units) [43] per nucleoside [uridine (Urd) or 2′-
deoxyuridine (dUrd)] (1 mmol).
c
Nucleosides were eluted from the C18 column using a water-methanol mixture as an eluent (vol. %).
This article is protected by copyright. All rights reserved.

Table 2
a
Physicochemical properties of nucleosides 3-6
c
d
Compd
HPLC)
Yield (%),
(mg)
Purity (%)
[t (min)]
R
UV
HRMS: m/z calcd
HRMS: m/z found
a)
b
(
(
(
(
λmax (nm)
+
+
S)-3
15
84.09
[10.3]
96.31
[10.1]
98.16
[10.81]
200, 212, 290, 322
209, 290, 322
435.1587 [M+H] ,
319.1113 [base+H]
435.1569 [M+H] ,
+
+
+
+
(
5)
15
5)
60
319.1102 [base+H]
+
+
R)-3
435.1587 [M+H] ,
435.1586 [M+H] ,
+
+
(
319.1113 [base+H]
319.1104 [base+H]
+
+
S)-4
202, 212, 248, 320
548.2432 [M+H] ,
548.2411 [M+H] ,
(25)
432.1954 [base+H] ,
432.1942 [base+H] ,
+
+
1
095.4755 [2M+H]
1095.4752 [2M+H]
+
+
(
(
(
(
(
R)-4
S)-5
R)-5
S)-6
R)-6
a
97
97.52
[10.79]
202, 212, 248, 320
212, 250, 284, 300
212, 250, 284, 302
214, 250, 284, 300
214, 250, 284, 302
548.2432 [M+H] ,
548.2411 [M+H] ,
+
+
(34)
432.1954 [base+H] ,
432.19406 [base+H] ,
+
+
1
095.4755 [2M+H]
1095.47556 [2M+H]
+
+
71
97.80
[10.05]
451.1536 [M+H] ,
451.1520 [M+H] ,
+
+
(34)
319.1113 [base+H] ,
319.1096 [base+H] ,
+
+
9
01.2999 [2M+H]
901.2969 [2M+H]
+
+
50
97.96
[9.86]
451.1536 [M+H] ,
451.1519 [M+H] ,
+
+
(10)
319.1113 [base+H] ,
319.1095 [base+H] ,
+
+
9
01.2999 [2M+H]
901.2966 [2M+H]
+
+
32
99.38
[10.6]
564.2382[M+H] ,
564.2356 [M+H] ,
+
+
(20)
432.1954 [base+H] ,
432.1937 [base+H] ,
+
+
1
127.4680 [2M+H]
1127.4649 [2M+H]
+
+
43
98.15
[10.5]
564.2382[M+H] ,
564.2358 [M+H] ,
+
+
(25)
432.1954 [base+H] ,
127.4680 [2M+H]
432.1938[base+H] ,
1127.4653[2M+H]
+
+
1
All nucleosides were synthesized as white amorphous powders.
b
HPLC was performed on Waters system (Waters 1525, Waters 2487, Breeze 2; USA) with Ascentis® Express C18 column
(2.7 µm, 7.5×3.0 mm) using gradient of eluent В in A. Eluent А: 0.1% ТFA/water, eluent В: 0.1% ТFA/70% MeCN in water.
Gradient: 0 - 100% B, 20 min, flow rate 0.5 mL/min, detection at 254 nm.
c
The UV spectra were recorded on the Agilent 6224, ESI-TOF, LC/MS (USA) in a water–acetonitrile mixture, рН 3.0.
d
The mass spectra were recorded on the Agilent 6224, ESI-TOF, LC/MS (USA) in positive ion mode, electrospray
ionization.
This article is protected by copyright. All rights reserved.

Table 3
Experimental data for the substrate specificity of adenosine deaminase E.coli (EcADA) and
a
ADA from calf intestine (CiADA)
Substrate
Product
Conversion of substrate to
product (HPLC data), %
b
EcADA
CiADA
Time, h
Time, h
1
n/f
24
1
24
n/f
c
(
(
(
(
(
(
S)-3 or (R)-3
S)-4 or (R)-4
S)-5 or (R)-5
S)-6 or (R)-6
S)-2-Ara- or (R)-2-Ara
R)-1-Ara
-
-
-
-
-
-
n/f
n/f
n/f
n/f
n/f
n/f
n/f
n/f
n/f
n/f
n/f
n/f
n/f
n/f
n/f
n/f
n/f
n/f
n/f
n/f
n/f
n/f
3
0.85 98.65 19.93 99.39
Ado
Inosine
2
9
'-dAdo
-(β-D-arabinofuranosyl)adenine (AraA)
2’-
17.71 98.47 13.08 96.65
13.06 96.57 4.47 51.27
Deoxyinosine
Arabinosyl-
hypoxantin
a
Reaction conditions. The reaction mixtures contained 1 mM modified nucleoside (2'-deoxyribosides 3 or 4, ribosides 5 or 6,
arabinosides (3R)- or (3S)-4-[6-(2-amino-9-(β-D-arabinofuranosyl)purin-6-yl)aminohexanoyl]-7,8-difluoro-3,4-dihydro-3-
methyl-2H-[1,4]benzoxazine, (3R)-4-[2-amino-9-(β-D-arabino-furanosyl)purin-6-yl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-
[
1,4]benzoxazine) [38]); 1 e.u. EcADA or 1 e.u. CiADA in 1.0 mL of potassium phosphate buffer (20 mM, pH 7.0) at 25 C.
Enzymes used: recombinant ADA E. сoli (0.75 mg/mL, 50 e.u./mg) and ADA from calf intestine (Roche) (1 mg/mL, 20
e.u./mg).
b
All enzymatic reactions were performed under monitoring of the reaction progress by HPLC: Waters system (Waters 1525,
Breeze 2; USA) with Ascentis® Express C18 column (2.7 µm, 7.5×3.0 mm) using gradient of eluent В in A. Eluent А: 0.1%
ТFA/water, eluent В: 0.1% ТFA/70% MeCN in water. Method for modified nucleosides: 0% - 100% B, 20 min, flow rate 0.5
mL/min. UV detection at 254 nm.
c
n/f – the product was not found in the reaction mixture.
This article is protected by copyright. All rights reserved.

Table 4
Cytotoxic properties and antiviral activity of bases 1-2 and nucleosides 2-Ara, 4–6 in Vero E6
cell culture on the models of herpes simplex virus 1 (HSV-1 strain L2) and acyclovir-resistant
HSV-1 (HSV-1 strain L2/R) (multiplicity of infection 0.1 PFUa/cell)
b
c
Compd
MTC ,
M)
CD50
(M)
HSV-1 strain L2
HSV-1 strain L2/R
(
SI
d
e
ID50
M)
ID95
(Selectivity
Index,
ID50
(M)
ID95
(M)
SI
(
(M)
CD /ID )
5
0
50
(
S)-4
5
93.75
730.77 ± 50.00
593.75
>593.75
1.2
593.75
>593.75
1.2
(
(
R)-4
S)-5
593.75
593.75
593.75
690.38 ± 36.53
> 1187.5
855.82 ±6.28
296.88
37.11
37.11
593.75
593.75
296.88
2.3
>32
23
296.88
37.11
37.11
593.75
593.75
296.88
2.3
> 32
23
(
(
R)-5
S)-6
1
5
187.5
93.75
1865.62 ± 34.38
1441.38 ± 75.86
593.75
445.32
1187.5
1187.5
3.1
3.2
1187.5
445.32
1187.5
1187.5
1.6
3.2
(
R)-6
(
(
(
S)-1
R)-1
S)-2
148.44
74.22
> 296.88
167.61 ± 9.66
37.11
74.22
148.44
148.44
> 8
2
37.11
74.22
148.44
148.44
> 8
2
2
96.83
96.83
187.5
660.80 ± 5.30
660.20
148.44
593.75
218.75
1.73
1200.50
>296.83
1187.5
593.75
6.93
1
3
660.20
148.44
593.75
-
1200.50
>296.83
1187.5
-
1
3
(
(
(
R)-2
2
1
462.50 ± 11.50
S)-2-Ara
R)-2-Ara
1982.76± 65.52
3.3
>4
3.3
-
>875
>875
nd
Acyclovir
Foscarnet
Ribavirin
a
>
444
>256
>4
> 444
104.15
511.88
> 444
0
>
416.61
nd
104.15
511.88
208.30
2047.50
208.30
2047.50
>4
> 8
>
4095
nd
> 8
PFU – plaque forming unit.
b
MTC - maximal tolerable concentration - highest concentration of a compound that does not cause destructive changes of
cellular monolayer visible with the light microscope.
c
CD50 - 50% cytotoxic concentration of compound required to reduce the viability of Vero E6 cells by 50%.
ID50 - 50% effective concentration of compound achieving 50% protection of Vero E6 cells against the cytopathic effect of
d
HSV-1.
e
ID95 - 95% effective concentration of compound, achieving 95% protection of Vero E6 cells against the cytopathic effect of
HSV-1.
“-“ – not determined
This article is protected by copyright. All rights reserved.