The Chemoenzymatic Synthesis of 2-Chloro- and 2-Fluorocordycepins

Article abstract of DOI:10.1055/s-0036-1590804

Two approaches to the chemoenzymatic synthesis of 2-fluorocordycepin and 2-chlorocordycepin were studied: (i) the use of 3′-deoxyadenosine (cordycepin) and 3′-deoxyinosine (3′dIno) as donors of 3-deoxy- d -ribofuranose in the transglycosylation of 2-fluoro- (^2F Ade) and 2-chloroadenine (^2Cl Ade) catalyzed by the recombinant E. coli purine nucleoside phosphorylase (PNP), and (ii) the use of 2-fluoroadenosine and 3′-deoxyinosine as substrates of the cross-glycosylation and PNP as a biocatalyst. An efficient method for 3′-deoxyinosine synthesis starting from inosine was developed. However, the very poor solubility of ^2Cl Ade and ^2F Ade is the limiting factor of the first approach. The second approach enables this problem to be overcome and it appears to be advantageous over the former approach from the viewpoint of practical synthesis of the title nucleosides. The 3-deoxy-α- d -ribofuranose-1-phosphate intermediary formed in the 3′dIno phosphorolysis by PNP was found to be the weak and marginal substrate of E. coli thymidine (TP) and uridine (UP) phosphorylases, respectively. Finally, one-pot cascade transformation of 3-deoxy- d -ribose in cordycepin in the presence of adenine and E. coli ribokinase, phosphopentomutase, and PNP was tested and cordycepin formation in ca. 3.4% yield was proved.

Full text of DOI:10.1055/s-0036-1590804

SYNTHESIS
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© Georg Thieme Verlag Stuttgart · New York
2017, 49, A–H
paper
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en
A. O. Denisova et al.
Paper
Synthesis
The Chemoenzymatic Synthesis of 2-Chloro- and 2-Fluorocordy-
cepins
Alexandra O. Denisovaa
Yulia A. Tokunovaa
Ilja V. Fateeva
Alexandra A. Breslava
Vladimir N. Leonova
Elena V. Dorofeevaa
Olga I. Lutoninaa
Inessa S. Muzykaa
Roman S. Esipova
Alexey L. Kayushina
Irina D. Konstantinovaa
Anatoly I. Miroshnikova
Vladimir A. Stepchenkob
Igor A. Mikhailopulo*b
a Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry,
Russian Academy of Sciences, Miklukho-Maklaya 16/10,
Moscow B-437, 117997 GSP-7, Russian Federation
b Institute of Bioorganic Chemistry, National Academy of
Sciences, Acad. Kuprevicha 5/2, 220141 Minsk, Belarus
imikhailopulo@gmail.com
Received: 04.04.2017
Accepted after revision: 25.05.2017
Published online: 20.07.2017
since then cordycepin and its analogues have attracted the
continuous attention of researchers. There is extensive lit-
erature on the biological properties of cordycepin and re-
search results before 1979 are summarized in the compre-
hensive review by R. J. Suhadolnik.4 A large number of pub-
lications devoted to the chemical synthesis of cordycepin
and related 3′-deoxyribonucleosides have been reviewed;5
reports relevant to this publication are also available.6–17 In
biological fluids, cordycepin undergoes deamination by
ubiquitous adenosine deaminase (ADA) to give 3′-deoxy-
inosine (8), which is devoid of biological activity.4 For this
reason, the study of the biological activity of cordycepin is
carried out with the simultaneous application of an ADA in-
hibitor18 or using 2-fluoro(chloro) derivatives of cordycepin
[3′d2F(Cl)Ado], which are resistant to the action of a variety
of adenosine deaminases from diverse sources.14,19 The 2-
halogenated derivatives of cordycepin are therefore of con-
siderable interest as potential therapeutic agents, in partic-
ular, against human African trypanosomiasis (HAT).14 Nota-
bly, 2-fluorocordycepin (3′d2FAdo) was shown to be a very
poor substrate19b for ADA from calf intestine, whereas 2-
chloro-2′-deoxycordycepin displayed weak inhibitory activ-
ity for the same ADA (Ki = 2.09 × 10–5 M)19d and these prop-
erties of 3′d2F(Cl)Ado were suggested to be the reason for the
enhanced cytotoxicity of the 2-halogenated derivatives of
cordycepin and related nucleosides.9,20,21
DOI: 10.1055/s-0036-1590804; Art ID: ss-2017-t0220-op
Abstract Two approaches to the chemoenzymatic synthesis of 2-fluo-
rocordycepin and 2-chlorocordycepin were studied: (i) the use of 3′-de-
oxyadenosine (cordycepin) and 3′-deoxyinosine (3′dIno) as donors of 3-
deoxy-D-ribofuranose in the transglycosylation of 2-fluoro- (2FAde) and
2-chloroadenine (2ClAde) catalyzed by the recombinant E. coli purine nu-
cleoside phosphorylase (PNP), and (ii) the use of 2-fluoroadenosine and
3′-deoxyinosine as substrates of the cross-glycosylation and PNP as a
biocatalyst. An efficient method for 3′-deoxyinosine synthesis starting
from inosine was developed. However, the very poor solubility of 2ClAde
and 2FAde is the limiting factor of the first approach. The second ap-
proach enables this problem to be overcome and it appears to be ad-
vantageous over the former approach from the viewpoint of practical
synthesis of the title nucleosides. The 3-deoxy-α-D-ribofuranose-1-
phosphate intermediary formed in the 3′dIno phosphorolysis by PNP
was found to be the weak and marginal substrate of E. coli thymidine
(TP) and uridine (UP) phosphorylases, respectively. Finally, one-pot cas-
cade transformation of 3-deoxy-D-ribose in cordycepin in the presence
of adenine and E. coli ribokinase, phosphopentomutase, and PNP was
tested and cordycepin formation in ca. 3.4% yield was proved.
Key words 2-chlorocordycepin, 2-fluorocordycepin, 3′-deoxyinosine,
2-fluoroadenosine, E. coli nucleoside phosphorylases
Cordycepin (3′-deoxyadenosinе, 3′dAdo, 1) is the first
representative of a large family of nucleoside antibiotics; it
was originally isolated from Cordyceps militaris in 19511,2
and 13 years later from Aspergillus nidulans.3 Its high cyto-
static activity was discovered shortly after its isolation and
Chemical synthesis of 3′-deoxyribonucleosides has de-
veloped within the two traditional approaches, namely: (i)
the modification of the carbohydrate moiety or heterocyclic
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–H

B
A. O. Denisova et al.
Paper
Synthesis
NH2
O
NH2
N
N
N
NH
N
HO
HO
N
O
HO
N
O
HO
2NH2Ade
Pi
N
X
O
O
O
O
O
O-P-OH
O–
–Uracil
– Pi
OH
3dRib-1P
OH
OH
OH
3'd2NH2Ado (X = NH2; 64%)
3'dCyd
3'dUrd
3'd2FAdo [X = F (43%)]
plus
Schiemann
Reaction
Biocatalysts:
the intact E. coli cells BM-11 & BMT-4D/1A
60 mM K-phosphate buffer (pH 7.0), 52 °C, 26 h
3'diGuo [X = OH ( 7%)]
Scheme 1 Chemoenzymatic synthesis of 2-amino-3′-deoxyadenosine (2-aminocordycepin, 3′d2NH2Ado) and its transformation into 2-fluorocordy-
cepin (3′d2FAdo) and 3′-deoxyisoguanosine (3′diGuo)22
base of readily available natural ribonucleosides,5a,b,7–9,12,14–17
and (ii) the convergent synthesis of nucleosides by the con-
densation of heterocyclic base with suitably protected gly-
cosylated reagent.5c,6,10,11,13 Both approaches suffer from
well-known shortcomings, which are inherent in the chem-
istry of nucleosides, such as a multistage character, the
need for the introduction of the base and sugar protective
groups followed by their removal after the glycoside bond
formation, the absence of regioselectivity of the glycoside
bond formation, and finally purification of the desired com-
pounds by column chromatography resulting in the low
yields of the desired end products.
Previously, we have developed the chemoenzymatic
synthesis of 2-fluorocordycepin (3′d2FAdo) starting from
2,6-diaminopurine (2NH2Ade) as an acceptor and 3′-deoxy-
cytidine (3′dCyd) as a donor of 3-deoxy-D-ribofuranose us-
ing the selected intact E. coli BM-11 and BMT-4D/1A cells as
a biocatalyst (Scheme 1).22 Note that 3′dCyd is not a sub-
strate of E. coli thymidine (TP) and uridine (UP) phosphory-
lases and is deaminated to 3′-deoxyuridine (3′dUrd) by the
powerful cytidine deaminase of E. coli BM-11 cells, which is
the precursor of the interim 3-deoxy-D-ribofuranose-1-
phosphate (3dRib-1P).
(3′dCyd) and 3′-deoxyuridine24 (3′dUrd) from commercially
available cytidine and uridine is an essential drawback of
this approach.
In the present work, we have studied the other ap-
proaches to the synthesis of 2-fluorocordycepin (3) and
2-chlorocordycepin (9) using the recombinant E. coli PNP25
as the sole biocatalyst.
Analysis of the various variants of chemoenzymatic
synthesis of 2-halogenated derivatives of cordycepin (see
Supporting Information, Table 1) led us to the conclusion
that the use of the relevant purine 3′-deoxyriboside as a do-
nor of 3-deoxy-D-ribofuranose and the recombinant E. coli
PNP as a sole biocatalyst is fairly reasonable. As a verifica-
tion of this approach, we primarily tested the transglyco-
sylation reaction of 2-fluoroadenine by using cordycepin
(3′dAdo, 1) as a donor of the carbohydrate moiety. It was
found that incubation of cordycepin and 2-fluoroadenine
(2FAde) in 50 mM phosphate buffer (pH 7.0) in the presence
of PNP (2000 units per mmol of 2FAde) at 40 °C for 120
hours (monitoring the reaction progress by HPLC) gave, af-
ter workup and chromatography, 2-fluorocordycepin (3) in
62% yield (Scheme 2). It was thus shown that cordycepin is
a satisfactory donor of the carbohydrate moiety in the
transglycosylation of 2-fluoroadenine. Notably, the fairly ef-
ficient two-step chemical synthesis of 3′dAdo from adeno-
sine (73% overall yield) was recently described.7c
2-Amino-3′-deoxyadenosine (3′d2NH2Ado) is valuable
starting compound for the synthesis of a number of C2 and
C6 substituted purine 3′-deoxyribosides. However, the rath-
er laborious chemical synthesis of 3′-deoxycytidine23
Commercially available inosine (4) was further selected
as the starting nucleoside for the synthesis of 3′-deoxy-
inosine (8) aiming at its utilization as a 3-deoxy-D-ribofura-
NH2
NH2
N
N
N
N
O
HO
O
HO
N
HO
N
2FAde
O
N
PNP/Pi
N
F
O
P
OH
O
O
Ade
+
–Pi
OH
OH
OH
1
2
3 (62%)
Scheme 2 Reagents and conditions: 3′-deoxyadenosine (3′dAdo)/2-fluoroadenine (2FAde) (mol. ratio, 1.11:1.0), 50 mM phosphate buffer (pH 7.0), E.
coli PNP (2000 units), 40 °C, 120 h, 62%.
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A. O. Denisova et al.
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Synthesis
O
O
N
O
N
N
N
N
N
NH
NH
NH
HO
N
HO
N
AcO
N
O
O
O
a
b
d
X
HO
OH
O
O
OAc
6, X = Br
MeO
Me
c
4
5
7, X = H
NH2
N
N
N
HO
N
Cl
O
O
N
OH
O
e
N
9
NH
HO
N
O
f
H3C
NH
O
OH
HO
N
O
8
OH
10
Scheme 3 Reagents and conditions: (a) DMF, MeC(OMe)3, CHCl2CO2H (cat.), 60 °C, 1 h, 97%; (b) AcBr, MeCN, 0 → 10 °C, 8 h, 80%; (с) benzoyl peroxide,
Bu3SnH, toluene, reflux 3 h, 88%; (d) 25% NH4OH/MeOH (2:1), 63%; (e) mol ratio 8/2ClAde (2:1), potassium phosphate buffer (2 mM, pH 7.0), E. coli PNP
(1320 units), 50 °C, 8 d; 40%; (f) mol ratio 8/Thy (1:1), potassium phosphate buffer (2 mM, pH 7.0), E. coli PNP (0.78 units), E. coli ТP (2.25 units), 50 °C,
3 d, 10%.
nose donor in the synthesis of 2-halogenated derivatives 9
and 3 of cordycepin (Scheme 3). Inosine (4) was trans-
formed into 2′,5′-di-O-acetyl-3′-deoxyinosine (7) essential-
ly as described by Takamatsu et al. (54% overall yield)17 with
some modifications. Treatment of inosine (4) with trimeth-
yl orthoacetate in DMF in the presence of catalytic amounts
of dichloroacetic acid gave the derivative 5, condensation of
which with acetyl bromide afforded, after workup and silica
gel column chromatography, the xylo-bromide 6 in 78%
combined yield (cf.8,17). The radical-mediated hydrodehalo-
genation of 6 with tributyltin hydride in the presence of
dibenzoyl peroxide in toluene under reflux gave the diace-
tate 7 (88%) after silica gel column chromatography. Con-
ventional deacetylation followed by column chromatogra-
phy on Separon gave the desired 3′-deoxyinosine (8) in 63%
yield (Scheme 3).
reaction is strongly displaced to the formation of the de-
sired product, 2-chloro-3′-deoxyadenosine (9), clearly
pointing to its much lower substrate activity for PNP vs that
of 3′-deoxyinosine (8).
The efficiency of 3′-deoxyinosine (8) as a universal do-
nor of 3-deoxy-D-ribofuranose in transglycosylation reac-
tions was tested in the synthesis of 2-chloro-3′-deoxyade-
nosine (9, 3′d2ClAdo) and 1-(3-deoxy-β-D-ribofurano-
syl)thymine (10, 3′dRib-Thy) under standard reactions
conditions (Figure 1). It was found that the PNP-catalyzed
phosphorolysis of 3′-deoxyinosine (8) reached equilibrium
at the nucleoside/hypoxanthine ratio of ca. 8:2. In the pres-
ence of 2-chloroadenine, the flow of the transglycosylation
Figure 1 The E. coli PNP catalyzed phosphorolysis of 3′-deoxyinosine
(8; 3′dIno) [progress of the formation of hypoxanthine (Hyp)] as well as
3′dIno as a 3-deoxy-D-ribofuranose donor in the transglycosylation of (i)
2-chloroadenine (2ClAde) [(progress of the formation of 2-chloro-3′-de-
oxyadenosine (9, 3′d2ClAdo), and (ii) thymine [progress of the forma-
tion of 1-(3-deoxy-β-D-ribofuranosyl)thymine (10, 3′dRib-Thy)]
catalyzed by E. coli PNP and TP vs. PNP and UP. Reagents and conditions:
in all cases total volume = 0.5 mL, 2 mМ 3′dIno, 2 mM KH2PO4 (pH 7.0),
50 °C, PNP (0.78 units), 2ClAde 0.5 mМ (30 units PNP per 1 μmol base);
thymine 2 mМ, and TP (2.25 units) or UP (1.35 units).
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–H

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A. O. Denisova et al.
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Synthesis
In contrast to the 3′d2ClAdo synthesis, conversion of thy-
mine in 3′dRib-Thy (10) catalyzed by E. coli TP is stopped at
the ca. 10% concentration of the latter and was not ob-
served at all in the presence of E. coli UP (Figure 1). It ap-
pears that the intermediary phosphate 2 is a very poor sub-
strate for E. coli TP and practically devoid such activity for
E. coli UP.
On the other hand, apparently both enzymes of the in-
tact E. coli cells BM-11 and BMT-4D/1A catalyze the phos-
phorolysis of the interim 3′-deoxyuridine enabling the fair-
ly efficient synthesis of 2-amino-3′-deoxyadenosine
(3′d2NH2Ado, Scheme 1). On the whole, these results are res-
onate with our recently published data on the substrate
specificity of E. coli PNP, on the one hand, and E. coli TP and
UP, on the other.26 Indeed, UP and TP show more stringent
requirements for the structure and stereochemistry of α-D-
pentofuranose-1-phosphates than PNP.
Optimization of the synthesis of 2-chloro-3′-deoxyaden-
osine (9, 3′d2ClAdo) was performed by testing the influence
of the ratio of substrates, pH of the phosphate buffer, PNP
quantity, and the reaction temperature on the yield of de-
sired nucleosides (data not shown).
It was found that the optimal conversion of the base
into nucleoside proceeds at the 3′-deoxyinosine (8)/base
mol ratio of 1.5:1.0, 2 mM phosphate buffer (pH 7.0), and at
a reaction temperature of 50 °C; moreover, in the presence
of 4000–4500 units of PNP per 1 mmol of base, the reaction
continued 8 days resulting in the formation of the desired
nucleoside in 65–70% yield according to HPLC and 3′d2ClAdo
was isolated by silica gel 100 C18-RP chromatography in 40%
yield.
The study of the aforementioned reactions revealed that
the use of 2-fluoro- and 2-chloroadenines as substrates in
an enzymatic synthesis of their nucleosides is a bottleneck
in this approach owing to the very low solubility of both
bases in the water reaction mixtures.27 One of the possible
solutions to this problem consists of the use of nucleosides
of poorly soluble heterocycles as heterocyclic base donors
as it was successfully realized in the synthesis of 9-(β-D-
arabinofuranosyl)guanine (ara-G)28 and 9-(β-D-arabinofu-
ranosyl)-2-fluoroadenine29 (Fludarabine). With this aim in
view, the cross-glycosylation between 2-fluoroadenosine
(11) as a 2-fluoroadenine donor and 3′-dIno (8) as a source
of 3-deoxy-D-ribofuranose-1-phosphate (3dRib-1P) in the
presence of PNP was studied (Scheme 4). The efficient syn-
thesis of 2-fluoroadenosine (11) from commercially avail-
able guanosine was published earlier.29
Optimization of two important parameters of the cross-
glycosylation, viz., the ratio of substrates and the PNP quan-
tity, was performed (see Supporting information) and
based on these data, 3′-deoxy-2-fluoroadenosine (3) was
prepared in 58% yield. The structures of all compounds syn-
thesized in this work was proved by the integrity of spectral
methods and the data are given in the Supporting Informa-
tion.
Finally, the cascade successive transformation of 3-de-
oxy-D-ribose into 3-deoxy-D-ribofuranose-5-phosphate
[catalyzed by E. coli ribokinase (RK) in the presence of ATP],
3-deoxy-α-D-ribofuranose-1-phosphate (2) [catalyzed by E.
coli phosphopentomutase (PPM)], and eventually cordy-
cepin (catalyzed by PNP in the presence of adenine)26,30–34
was tested (Scheme 5). The synthesis of adenosine under
similar reaction conditions was performed for comparison
reasons.
Cordycepin was used as a source of 3-deoxy-D-ribose af-
ter heating its water solution (HCl, pH 1.0) at 90 °C for 30
minutes monitoring the formation of adenine; the reaction
mixture was neutralized with 5 M KOH to pH 7.0 and this
solution was used in the cascade synthesis. Under analo-
gous reaction conditions (1.76 units of PPM) the formation
of adenosine reached equilibrium at the 1:2.2 ratio of ade-
nine and its riboside after 60 minutes, whereas the forma-
tion of cordycepin proceeded very slowly attaining 1.6%
(HPLC; LC/MS) after 48 hours. Preliminary tests of the im-
pact of the quantity of RK, PPM, and PNP on the cascade
synthesis pointed to the most decisive role of PPM and en-
hancement its content in the mixture until 3.96 units re-
sulted in acceleration of cordycepin formation attaining
3.4% in 24 hours. Thus, the cascade synthesis of cordycepin
tested in this study as well as other purine 3′-deoxy-β-D-
ribofuranosides is, in principle, possible, but needs thor-
ough optimization to reach a preparative level and this
work is in progress.
In conclusion, the diverse approaches to the synthesis of
2-fluorocordycepin (3) and 2-chlorocordycepin (9) were
studied. First, it was found that 3′-deoxyadenosine (cordy-
cepin, 1) and 3′-deoxyinosine (8) are satisfactory donors of
3-deoxy-D-ribofuranose in the E. coli PNP catalyzed the
NH2
NH2
O
N
N
N
N
N
N
N
N
N
NH
HO
HO
HO
N
F
N
F
O
O
O
+
Inosine
+
OH
HO
OH
OH
3 (58%)
11
8
Scheme 4 The cross-glycosylation of 2-fluoroadenosine (11) and 3′-dIno (8). Reagents and conditions: (a) mol ratio 11/8 (1.0:1.5), potassium phos-
phate buffer (2 mM; pH 7.0), E. coli PNP (1720 units), 52 °C, 12 d, 58%.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–H

E
A. O. Denisova et al.
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NH2
N
N
O
P
N
3-Deoxy-D-ribose
vs
HO
O
P
O
HO
HO
N
O
O
O
O
Ade/PNP
PPM
RK/ATP
OH
OH
O
O
D-Ribose
– Pi
X
OH
X
OH
X
OH
3dRib-5P
Rib-5P
3dRib-1P
Rib-1P
Cordycepin (X = H; 3.4%)
Adenosine (X = OH; 68.8%)
Biocatalysts: the recombinant E. coli enzymes -
RK - ribokinase
PPM - phosphopentomutase
PNP - purine nucleoside phosphorylase
Scheme 5 One-pot cascade synthesis of cordycepin and adenosine from 3-deoxy-D-ribose and D-ribose, respectively. Reagents and conditions: reaction
mixtures (1 mL) containing 2 mM 3-deoxy-D-ribose or D-ribose, 3 mM MnCl2, 50 mM KCl, 3 mM ATP, 2 mM adenine, and 2 mM KH2PO4 in 20 mM
Tris·HCl (pH 7.5) in the presence of PNP (2.34 units), RK (7.65 units) and PPM (3.96 units) were stored at 50 °C; the formation of nucleoside was moni-
tored by HPLC; the yield of (a) adenosine was 68.8% after 1 h, and (b) cordycepin was 3.4% after 72 h.
transglycosylation of 2-fluoro- (2FAde) and 2-chloroadenine
2ClAde). An efficient method for the synthesis of 3′-deoxy-
NMR spectra were recorded on Bruker Avance II 700 spectrometers
(Bruker BioSpin, Rheinstetten, Germany) in DMSO-d6 at 30 °C relative
to the residual solvent signals as internal standard (δ = 2.50). Liquid
chromatography mass spectrometry was performed on Agilent 6210
TOF LC/MS system (Agilent Technologies, USA). UV spectra were re-
corded on the Shimadsu Spectrophotometer UV-160 (Japan) in water
(
inosine (8) starting from readily available inosine was de-
veloped. Second, the cross-glycosylation between 2-fluoro-
adenosine (11), donor of 2-fluoroadenine, and 3′-deoxy-
inosine (8), donor of 3-deoxy-D-ribofuranose, catalyzed by
PNP was realized to give the desired 3′-deoxy-2-fluoroade-
nosine (3) in 58% yield. The 3-deoxy-α-D-ribofuranose-1-
phosphate (2) intermediary formed at the 3′dIno phospho-
rolysis by PNP was found to be the weak and marginal sub-
strate of E. coli thymidine (TP) and uridine (UP) phosphory-
lases, respectively. Finally, preliminary experiments with
one-pot cascade transformation of 3-deoxy-D-ribose in
cordycepin in the presence of adenine and E. coli ribokinase
(RK), phosphopentomutase (PPM) and PNP unequivocally
points to cordycepin formation in 3.4% yield (HPLC; LC/MS).
It is possible that the results of this study will allow en-
hancement of cordycepin production and its analogues af-
ter careful optimization of the reaction conditions.
solutions at pH 7; ε is given in M–1 cm–1
.
Further information is given in the Supporting Information includes:
(1) HPLC of the reaction mixture of the synthesis of 1-(3-deoxy-β-D-
ribofuranosyl)thymine (10; 3′dRib-Thy), and MS and UV data for the
corresponding HPLC peak. (2) NMR spectra and HPLC analysis of 9-(3-
deoxy-β-D-erythro-pentofuranosyl)hypoxanthine (8). (3) NMR spec-
tra and HPLC analysis of 6-amino-9-(3-deoxy-β-D-erythro-pentofura-
nosyl)-2-fluoropurine (3). (4) NMR spectra and HPLC analysis of 6-
amino-2-chloro-9-(3-deoxy-β-D-erythro-pentofuranosyl)purine (9).
2′,3′-Di-O-(1-methoxyethylidene)inosine (5)
Trimethyl orthoacetate (11.6 mL, 93.4 mmol) and dichloroacetic acid
(0.09 mL, 1.2 mmol) were added to a suspension of inosine (4, 5 g,
18.7 mmol) in DMF (25 mL) and the mixture was incubated at 60 °C
[TLC monitoring, MeCN/H2O (85:15) and CHCl3/MeOH (7:1)]. After 1
h, the mixture allowed to cool to r.t., i-Pr2NEt (0.5 mL) was added, and
the mixture was concentrated with a rotary evaporator to 10 mL.
Then, this mixture was diluted to 30 mL with 20% MeOH/CHCl3 and
the desired product was precipitated by addition of hexane (1.0 L);
yield 5.87 g (18.1 mmol, 97%); diastereomeric mixture, purity 93%;
HPLC (method I); tR = 10.94 and 11.14 min; ratio 51:49.
1H NMR (700 MHz, DMSO-d6): δ = 12.40 (s, 2 H, NH), 8.30 and 8.29 (2
s, 2 H, H-8), 8.09 and 8.08 (2 s, 2 H, H-2), 6.26 (d, J = 2.8 Hz, 1 H, H-1′)
and 6.14 (d, J = 2.3 Hz, 1 H, H-1′), 5.45 and 5.34 (2 m, 2 H, H-2′), 5.07
and 4.94 (2 m, 2 H, H-3′), 4.30 and 4.21 (2 m, 2 H, H-4′), 3.56 and 3.52
(2 m, 4 H, H-5′), 3.34 and 3.18 (2 s, 6 H, C-OCH3), 1.63 and 1.53 (2 s, 6
H, C-CH3).
Unless otherwise noted, materials were obtained from commercial
suppliers and used without any purification. Inosine was obtained
from Biosynthes Corp. (Penza, Russia). Trimethyl orthoacetate, DMF,
KH2PO4, and AcBr were purchased from Aldrich (St. Louis, Mo, USA).
Dichloroacetic acid, i-Pr2NEt, Bu3SnH, and benzoyl peroxide were pur-
chased from Fluka (Buchs, Switzerland). MeCN was distilled over CaH2
and stored over molecular sieves 4Å.
TLC was performed on silica gel plates (TLC Silica gel 60 F254, Merck,
Darmstadt, Germany). Column chromatography was performed on
Silica gel 60 (Fluka, Buchs, Switzerland), Separon SGX C18 60 (Tessek
Ltd., Prague, Czech Republic), or on Silica gel 100 C18-RP (Fluka, Buchs,
Switzerland). HPLC was performed on the Waters system (Waters
1525, Waters 2489, Breeze 2) using column Nova Pak C18, 4.6 × 150
mm, flow rate 0.5 mL/min, detection at 260 nm. HPLC Method I: A:
20% MeOH/H2O, B: MeOH; gradient I: 0 → 100 B, 15 min. HPLC Meth-
od II: A: 0.1% TFA/H2O, B: 0.1% TFA in MeCN/H2O (70:30); gradient II:
0 → 20 B, 20 min.
13C NMR (176 MHz, DMSO-d6): δ = 156.37 (2 C, C=O), 147.64 and
147.62 (2 C, C4), 145.94 and 145.92 (2 C, C2), 138.78 and 138.59 (2 C,
C8), 125.04 and 124.57 (2 C, Me-O-C-Me), 124.37 and 124.30 (2 C,
C5), 89.25 and 89.22 (2 C, C1′), 86.70 (2 C, C4′), 84.15 and 83.88 (2 C,
C2′), 81.79 and 80.93 (2 C, C3′), 61.38 and 61.16 (2 C, C5′), 49.77 and
49.11 (2 C, OCH3), 23.20 and 21.16 (2 C, C-CH3).
15N NMR (71 MHz, DMSO-d6): δ = 250.0 (N7), 213.97 (N3), 175.32
(N9), 175.06 (N1).
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–H

F
A. O. Denisova et al.
Paper
Synthesis
HRMS (ESI+): m/z [M + H]+ calcd for C13H17N4O6: 325.1148; found:
325.1150; m/z [sugar + H]+ calcd for C8H13O5: 189.0763; found:
189.0766; m/z [base + H]+ calcd for C5H5N4O: 137.0463; found:
137.0466; m/z [M + Na]+ calcd for C13H16N4NaO6: 347.0968; found:
347.0971.
15N NMR (71 MHz, DMSO-d6): δ = 250.04 (N7), 213.34 (N3), 173.89
(N9).
HRMS (ESI+): m/z [M + H]+ calcd for C14H17N4O6: 337.1148; found:
337.1160; m/z [sugar + H]+ calcd for C9H13O5: 201.0763; found:
201.0779; m/z [base + H]+ calcd for C5H5N4O: 137.0463; found:
137.0480; m/z [M + Na]+ calcd for C14H16N4NaO6: 359.0968; found:
359.0987.
9-(2′,5′-Di-O-acetyl-3′-bromo-3′-deoxyxylofuranosyl)hypoxan-
thine (6)
A mixture of AcBr (5.5 mL, 74.4 mmol) in MeCN (5 mL) was added
over 2 h to a suspension of 5 (5 g, 15.4 mmol) in MeCN (20 mL) at 0 °C.
The mixture was kept at 10 °C for 8 h [TLC monitoring, MeCN/H2O
(85:15) and CHCl3/MeOH (7:1)]. The mixture was cooled to 0 °C, sat.
aq Na2CO3 solution (15 mL) was added (low alkaline pH), the mixture
was evaporated till precipitate formation started, CHCl3 (25 mL) was
added, and the layers were separated. The aqueous layer was washed
with CHCl3 (3 × 25 mL), the combined organic layers were washed
with 0.1 M triethylammonium bicarbonate (2 × 25 mL), dried (dry
Na2SO4), and concentrated. The desired product was isolated by col-
umn chromatography (silica gel, 2.5 × 20 cm, gradient MeOH/CHCl3,
0% → 8%, 800 mL, flow rate 6.0 mL/min) as a white foam; yield: 5.09 g
(12.3 mmol, 80%); purity 88%; HPLC (method I); tR = 8.43 min.
3′-Deoxyinosine (8)
25% Aq NH3 (12 mL) was added to a solution of diacetate 7 (0.6 g (1.80
mmol) in MeOH (6 mL) and the mixture was stirred at r.t. After 1 h,
the deacetylation was complete [TLC monitoring CHCl3/MeOH (9:1)],
the mixture was evaporated to dryness and the target product was
isolated by a column chromatography (Separon, 1.8 × 25 cm, gradient
MeOH/H2O 0 → 30%, 0.8 L, flow rate 6 mL/min) as a lyophilized pow-
der; yield: 288 mg (1.14 mmol, 63%); purity 98.6%; HPLC (method I);
tR = 6.95 min.
1H NMR (700 MHz, DMSO-d6): δ = 12.32 (s, 1 H, NH), 8.32 (s, 1 H, H-
8), 8.05 (s, 1 H, H-2), 5.86 (d, J = 1.2 Hz, 1 H, H-1′), 5.66 (d, J = 3.4 Hz, 1
H, OH-2′), 5.00 (d, J = 5.1 Hz, 1 H, OH-5′), 4.51 (m, 1 H, H-2′), 4.36 (m,
1 H, H-4′), 3.69 and 3.53 (2 m, 2 H, H-5′), 2.21 (ddd, J = 13.1, 9.3, 5.9
Hz, 1 H, H-3a′), 1.90 (ddd, J = 13.1, 5.9, 2.3 Hz, 1 H, H-3b′).
1H and 13C NMR spectra are in agreement with the literature data.17
1H NMR (700 MHz, DMSO-d6): δ = 12.38 (br s, 1 H, NH), 8.25 (s, 1 H,
H-8), 8.09 (s, 1 H, H-2), 6.15 (d, J = 2.9 Hz, 1 H, H-1′), 5.82 (m, 1 H, H-
2′), 4.91 (dd, J = 4.7, 2.4 Hz, 1 H, H-3′), 4.57 (m, 1 H, H-4′), 4.36 (m, 2 H,
H-5′), 2.11 and 2.06 (2 s, 6 H, 2′- and 5′-CO-CH3).
13C NMR (176 MHz, DMSO-d6): δ = 169.94 (5′-CO-Me), 169.20 (2′-CO-
Me), 156.42 (C6), 147.85 (C4), 146.33 (C2), 137.81 (C8), 124.31 (C5),
86.83 (C1′), 81.63 (C2′), 77.94 (C4′), 64.82 (C5′), 49.11 (C3′), 20.52 and
20.40 (2 C, 5′- and 2′-CO-CH3).
13C NMR (176 MHz, DMSO-d6): δ = 157.20 (C=O), 148.12 (C4), 146.30
(C2), 138.50 (C8), 124.70 (C5), 91.15 (C1′), 81.43 (C4′), 75.49 (C2′),
62.70 (C5′), 34.29 (C3′).
15N NMR (71 MHz, DMSO-d6): δ = 249.30 (N7), 214.58 (N3), 178.25
(N9), 174.55 (N1).
HRMS (ESI+): [M + H]+ calcd for C10H13N4O4: 253.0937; found:
253.0949; m/z [base + H]+ calcd for C5H5N4O: 137.0463; found:
137.0491; m/z [M + Na]+ calcd for C10H12N4NaO4: 275.0756; found:
275.0799; m/z [2 M + H]+ calcd for C20H25N8O8: 505.1795; found:
505.1815; m/z [3 M + H]+ calcd for C30H37N12O12: 757.2654; found:
757.2656.
15N NMR (71 MHz, DMSO-d6): δ = 250.77 (N7), 213.78 (N3), 175.75
(N1), 170.75 (N9).
HRMS (ESI+): m/z [M
+
H]+ calcd for C14H16N4O6Br:
417.0233/415.0253, Br81/Br79; found: 417.0217/415.0251, Br81/Br79
;
UV (H2O, pH 7.0): λ (ε): 248 (12,100, max), 224 nm (4,500, min) [Lit.7b
UV (H2O, pH 7.0): λ = 250.5 nm (11,300, max)].
m/z [sugar
+
H]+ calcd for di-Ac-Br-ribose C9H12BrO5:
280.9848/278.9868, Br81/Br79; found: 280.9856/278.9854, Br81/Br79
;
m/z [base + H]+ calcd for C5H5N4O: 137.0463; found: 137.0452.
2-Chloro-9-(3-deoxy-β-D-ribofuranosyl)adenine (9)
2-Chloroadenine (50 mg, 0.295 mmol) was dissolved in H2O (660 mL)
under heating and stirring. The solution was cooled to 50 °C, 3′-de-
oxyinosine (8, 0.1 g, 0.396 mmol) and KH2PO4 (180 mg, 1.323 mmol)
were added, and the pH of the mixture was adjusted to 7.0 (2.0 mM);
PNP (1320 IU) was added and the mixture was incubated at 50 °C for
8 d under stirring. The precipitate was filtered off, the solution was
concentrated and the desired product was isolated by column chro-
matography (silica gel 100 C18-RP, 2.5 × 19 cm, 10% aq EtOH, flow rate
5 mL/min) as a lyophilized powder; yield: 34 mg (0.119 mmol, 40%);
purity 99.3%; HPLC (method II); tR = 11.41 min.
2′,5′-Di-O-acetyl-3′-deoxyinosine (7)
A solution of the bromide 6 (0.9 g, 2.17 mmol) and benzoyl peroxide
(630 mg, 2.60 mmol) in toluene (40 mL) was evaporated to 18 mL and
the flask was filled with argon. Bu3SnH (1.71 mL, 6.51 mmol) was
added and the mixture was heated under reflux for 3 h [HPLC (meth-
od I) monitoring]. The mixture was cooled and concentrated, and the
desired product was isolated by column chromatography (silica gel,
2.5 × 30 cm, gradient MeOH/CHCl3 0% → 8%, 2 L, flow rate 11 mL/min)
as a white foam; yield: 644 mg (1.92 mmol, 88%); purity 84%; HPLC
(method I); tR = 8.10 min.
1H and 13C NMR spectra are in agreement with the literature data.14
1H and 13C NMR spectra are in agreement with the literature data.17
1H NMR (700 MHz, DMSO-d6): δ = 8.38 (s, 1 H, H-8), 7.77 (s, 2 H, NH2),
5.81 (d, J = 1.9 Hz, 1 H, H-1′), 5.66 (br s, 1 H, OH-2′), 4.99 (m, 1 H, OH-
5′), 4.53 (m, 1 H, H-2′), 4.36 (m, 1 H, H-4′), 3.69 and 3.53 (2 m, 2 H, H-
5′), 2.22 (ddd, J = 13.1, 8.9, 5.7 Hz, 1 H, H-3a′), 1.91 (ddd, J = 13.1, 6.1,
2.8 Hz, 1 H, H-3b′).
13C NMR (176 MHz, DMSO-d6): δ = 157.18 (C6), 150.36 (C4), 139.83
(C8), 118.55 (C5), 91.15 (C1′), 81.36 (C4′), 75.17 (C2′), 62.77 (C5′),
34.21 (C3′).
1H NMR (700 MHz, DMSO-d6): δ = 8.16 (s, 1 H, H-8), 8.04 (s, 1 H, H-2),
6.08 (d, J = <1 Hz, 1 H, H-1′), 5.61 (m, J = 6.0, <1 Hz, 1 H, H-2′), 4.50 (m,
1 H, H-4′), 4.27 and 4.11 (2 m, 2 H, H-5′), 2.59 (ddd, J = 14.0, 10.0, 6.03
Hz, 1 H, H-3a′), 2.20 (dd, J = 14.0, 5.5 Hz, 1 H, H-3b′), 2.08 and 1.90 (2
s, 6 H, 2′- and 5′-CO-CH3).
13C NMR (176 MHz, DMSO-d6): δ = 170.06 (5′-CO-Me), 169.77 (2′-CO-
Me), 158.20 (C6), 147.91 (C4), 147.22 (C2), 138.12 (C8), 124.37 (C5),
88.35 (C1′), 77.82 (C4′), 77.37 (C2′), 64.41 (C5′), 32.43 (C3′), 20.65 and
20.43 (2 C, 5′- and 2′-CO-CH3).
15N NMR (71 MHz, DMSO-d6): δ = 241.41 (N7), 220.38 (N3), 174.09
(N9), 86.94 (NH2).
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2017, 49, A–H

G
A. O. Denisova et al.
Paper
Synthesis
HRMS (ESI+): m/z [M
+
H]+ calcd for C10H13N5O3Cl:
Acknowledgment
288.0677/286.0707, 37Cl/35Cl; found: 288.0691/286.0721, 37Cl/35Cl;
m/z [base + H]+ calcd for C5H5N5Cl: 172.0204/170.0233, 37Cl/35Cl;
found: 172.0219/170.0249, 37Cl/35Cl.
The authors would like to thank Dr. T. A. Balashova (Shemiakin and
Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of
Sciences) for assistance in the measurements and analysis of the
NMR-spectra and Dr. Tatyana I. Muravyova for recombinant nucleo-
side phosphorylases for experiments.
UV (H2O, pH 7.0): λ (ε) = 264 (12,100, max), 232 nm (4,700, min) [Lit.9
UV (H2O): λ = 265 nm (max)].
9-(3-Deoxy-β-D-ribofuranosyl)-2-fluoroadenine (3) from 3′-De-
oxyinosine (8) and 2-Fluoroadenosine (11)
Supporting Information
2-Fluoroadenosine29 (11, 90 mg, 0.316 mmol) was dissolved in H2O
(315 mL) under heating and stirring. The solution was cooled to 50 °C,
3′-deoxyinosine (8, 0.12 g, 0.476 mmol) and KH2PO4 (85 mg, 0.625
mmol) were added, and the pH of the mixture was adjusted to 7.0 (2.0
mM); PNP (1720 IU) was added and the mixture was incubated at 52
°C for 12 d under stirring. The desired product was isolated as de-
scribed above as a lyophilized powder; yield: 49 mg (0.182 mmol,
58%); purity 99.8%; HPLC (method II); tR = 13.22 min.
1H NMR (700 MHz, DMSO-d6): δ = 8.38 (s, 1 H, H-8), 7.77 (s, 2 H, NH2),
5.81 (d, J = 1.9 Hz, 1 H, H-1′), 5.66 (br s, 1 H, OH-2′), 4.99 (m, 1 H, OH-
5′), 4.53 (m, 1 H, H-2′), 4.36 (m, 1 H, H-4′), 3.69 and 3.53 (2 m, 2 H, H-
5′), 2.22 (ddd, J = 13.1, 8.9, 5.7 Hz, 1 H, H-3a′), 1.91 (ddd, J = 13.1, 6.1,
2.8 Hz, 1 H, H-3b′).
13C NMR (176 MHz, DMSO-d6): δ = 158.99 (d, J = 203.8 Hz, 1 C, C2),
158.01 (d, J = 20.9 Hz, 1 C, C6), 150.58 (d, J = 20.0 Hz, 1 C, C4), 139.72
(C8), 117.84 (C5), 91.16 (C1′), 81.38 (C4′), 75.21 (C2′), 62.83 (C5′),
34.37 (C3′).
15N NMR (71 MHz, DMSO-d6): δ = 241.87 (N7), 174.54 (N9), 87.57
(NH2).
Supporting information for this article is available online at
https://doi.org/10.1055/s-0036-1590804.
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Funding Information
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Financial support by the International Science and Technology Centre
(project #B-1640) and the Byelorussian Republican Foundation for
Fundamental Research (project No. X13MC-027) is gratefully ac-
knowledged.
n
I
etrnoaitn
a
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Secince
a
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6
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