Synthesis of 5′-deoxy-5′-ethoxycarbonylmethyl nucleosides, the precursors of oligonucleotides with the amide internucleoside bond C3′-NH-C(O)-CH2-C5′

Article abstract of DOI:10.1134/S1068162009050082

A method for the synthesis of 5′-deoxy-5′-ethoxycarbonylmethyl nucleosides has been developed. 3-O-benzyloxymethyl-1,2-O-isopropylidene- α-D-allofuranose was oxidized by sodium periodate to form a 5′-aldo derivative, which was converted by the reaction wi

Full text of DOI:10.1134/S1068162009050082

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 5, pp. 585–591. © Pleiades Publishing, Ltd., 2009.
Original Russian Text © A.M. Varizhuk, S.V. Kochetkova, N.A. Kolganova, E.N. Timofeev, V.L. Florent’ev, 2009, published in Bioorganicheskaya Khimiya, 2009, Vol. 35, No. 5,
pp. 650–656.
Synthesis of 5'-Deoxy-5'-Ethoxycarbonylmethyl Nucleosides,
the Precursors of Oligonucleotides with the Amide
Internucleoside Bond C3'-NH-C(O)-CH2-C5'
A. M. Varizhuk, S. V. Kochetkova, N. A. Kolganova, E. N. Timofeev, and V. L. Florent'ev1
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 117984 Russia
Received February 6, 2009; in final form, February 18, 2009
Abstract—A method for the synthesis of 5'-deoxy-5'-ethoxycarbonylmethyl nucleosides has been developed.
3-O-benzyloxymethyl-1,2-O-isopropylidene-α-D-allofuranose was oxidized by sodium periodate to form a 5'-
aldo derivative, which was converted by the reaction with triethylphosphonoacetate in the presence of sodium
hydride into a 5-deoxy-5-ethoxycarbonylmethylene derivative. The hydration of the unsaturated compound
gave 5-deoxy-5-ethoxycarbonylmethyl-1,2-O-isopropylidene-α-D-ribofuranose. After the benzylation of 3-
hydroxyl, the removal of the isopropylidene group by heating with acetic acid, and the subsequent acetylation,
1,2-di-O-acetyl-3-O-benzyl-5-deoxy-5-ethoxycarbonylmethyl-D-ribofuranose was obtained, which reacted
with persilylated nucleic acid bases to form 5'-deoxy-5'-ethoxycarbonylmethyl nucleosides.
Key words: nucleoside analogues, synthesis
DOI: 10.1134/S1068162009050082
INTRODUCTION
siRNA and miRNA in the therapy of human diseases
appears to be very promising [11–13].
Therapy with synthetic oligonucleotide analogues
(antisense therapy) is a promising method of the treat-
ment of viral and oncological diseases [1, 2].2 The idea
of the method consists of the suppression of gene trans-
lation through the specific binding of a synthetic oligo-
nucleotide to the corresponding site of mRNA. The
main mechanism of action of synthetic deoxyoligonu-
cleotides is the attack on the resulting DNA–RNA
hybrid of the intracellular endonuclease of RNase H,
which destroys the ribo chain of the hybrid duplex,
thereby preventing the translation [3, 4]. Therefore, the
efforts of investigators have been directed at the synthe-
sis of modified deoxyoligonucleotides. In the last
15 years, a great number of oligonucleotide analogues
modified at the nucleic acid base, carbohydrate residue,
and internucleotide bond have been synthesized (see
reviews [5–7]).
Previously, we have synthesized 3'-deoxy-3'-car-
boxymethyl ribonucleosides, the precursors of oligonu-
cleotides in which the phosphate bond is substituted for
by the C3'–CH2–CO–NH–C5' bond [14]. The present
study is devoted to the synthesis of the precursors of
oligonucleotides with a reverse sequence of atoms in
the internucleoside bond C3'–NH–CO–CH2–C5',
namely, 5'-deoxy-5'-ethoxycarbonylmethyl ribonucleo-
sides.
RESULTS AND DISCUSSION
The starting compound for the synthesis was 1,2 :
5,6-di-O-isopropylidene-α-D-allofuranose (I) [15] (see
the scheme). After the protection of 3-hydroxyl by the
benzyloxymethyl group [compound (II)], the 5,6-iso-
propylidene group was selectively removed by heating
in 50% acetic acid to 50°ë. The resulting diol (III) was
oxidized by sodium periodate to aldehyde (IV). Alde-
hyde (IV) was converted into the unsaturated com-
pound (V) by the Wadsworth–Horner–Emmons reac-
tion [16]. The resulting olefin was hydrated at atmo-
spheric pressure. In this case, simultaneously with the
hydration of the double bond, the benzyloxymethyl
protection [compound (VI)] is removed.
With the discovery of RNA interference, the interest
in chemically modified ribooligonucleotides greatly
increased (see reviews [8–10]). RNA interference is a
natural mechanism of regulation of gene expression in
which short (20–30 nt) RNAs (siRNA, short interfering
RNAs; miRNA, microRNA) play a key role. In view of
this, the idea to use chemically modified analogues of
1
Corresponding author; phone: (499) 135-21-82, (499) 135-65-91;
fax: (499) 135-14-05; e-mail: flor@imb.ac.ru
2
Abbreviations: Bn, benzyl; BSA, N-O-bis(trimethylsilyl)aceta-
mide; DIPEA, diisopropylethylamine; TBDMS, tert-butyldime-
thylsilyl; Tf, trifluoromethanesulfonate.
585

586
VARIZHUK et al.
H3C
H3C
H3C
H3C
HO
HO
O
O
O
O
O
a
b
O
O
O
O
O
CH3
BnOCH2O
EtOOC
O
CH3
CH3
BnOCH2O
HO
(I)
O
O
CH3
CH3
CH3
(II)
(III)
O
EtOOC
O
c
d
e
O
O
O
CH3
BnOCH2O
EtOOC
O
O
O
CH3
CH3
CH3
BnOCH2O
EtOOC
HO
O
O
(IV)
(V)
CH3
(VI)
CH3
EtOOC
B
O
i
j
O
O
f
OAc
O
CH3
BnO
OAc
BnO
BnO
OAc
O
(VII)
(VIII)
(IX‡–e)
CH3
B = Thy (a); Cyt (b); Ade (c);
6-N-BzAde (d); 2-N-AcGua (e)
(a) BnOCH Cl, DIPEA, methylene chloride; (b) 50% AcOH, 50°C; (c) NaIO ; (d) (EtO) P(O)CH COOEt, NAH, THF; (e) H ,
2
4
2
2
2
Pd(OH) /C, ethanol; (f) BnBr, NaH, THF; (i) 80% AcOH, boiling, then Ac O, pyridine; (j) nucleic acid bases, BSA, Me Si-Otf,
2
2
3
CH CN.
3
The choice of a new protecting group for 3-hydroxyl D2O to the sample), but also to determine their position
in the molecule. The spectrum of aldehyde (IV) showed
a characteristic signal of H5 at 9.676 ppm. The double
bond in compound (V) made itself evident in the
appearance of two signals of olefin protons at 6.960
(H5) and 6.156 ppm (H6), both having the form of a
doublet of doublets. This is related to both the interac-
tion of protons with each other (J5, 6 15.71 Hz) and with
H4 (J4, 5 5.19, 4J4, 6 1.46). Based on the spin–spin cou-
pling constants, it can be assumed that the double bond
has an E configuration. The hydration of the double
bond in (V) leads to the disappearance of olefin proton
signals and the appearance of signals of H5a and H5b
(1.899 and 1.637 ppm) and 6-CH2 (2.365 ppm) in the
expectedly high field of the spectrum. In the spectrum
of diacetate (VIII), the signals of two methyls of the
isopropylidene group are absent, and two singlets of the
acetyl groups (at 1.568 and 1.337 ppm) appear. Inter-
estingly, the diastereotropism of methylene protons of
the OCH2CH3 group manifests itself only in the spec-
trum of diacetate. The signal of the CH2 group in the
was dictated by the following requirements: first, it
should be stable under acidic conditions (the next stage
of synthesis) and, second, because the resulting nucle-
osides has to be used in the oligonucleotide synthesis,
the protecting group should enable one to clearly dis-
criminate between 2- and 3-hydroxyl groups of sugar.
The benzyl group best satisfies these conditions. After
the benzylation of (VI), the completely protected deriv-
ative (VII) was subjected to acid hydrolysis (boiling in
80% acetic acid) followed by acetylation. The resulting
diacetate (VIII) was used for the synthesis of nucleo-
sides analogues (IX‡)–(IXe) by the standard method
[17] with some modifications.
The structures of all compounds synthesized were
1
confirmed by H NMR spectra. The spectra of com-
pounds (I), (III), and (VI), which contain hydroxy
groups, were measured in DMSO-d6, which made it
possible not only to show the presence of free hydrox-
yls by the occurrence of their signals (signals were
identified from their disappearance after the addition of spectra of preceding compounds is a quartet, whereas
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 5 2009

SYNTHESIS OF 5'-DEOXY-5'-ETHOXYCARBONYLMETHYL NUCLEOSIDES
587
H6
H1′
H2′
CH3CH2O
1.2
5-CH3
H5′b
H5′a
2.0
2.2
2.4
2′-CH3CO2
H6′a, H6′b
H3′, H4′
CH3CH2O
4.0
4.2
4.4
4.6
PhCH2 Hb
PhCH2 Ha
H2′
5.4
5.6
H1′
H6
7.0
5.6
4.6
4.2
f2 (ppm.)
2.4
2.0
A fragment of the COSY-DQF spectrum of 2'-O-acetyl-3'-O-benzyl-5'-deoxy-5'-ethoxycarbonylmethyl-5-ribosylthymine (IXa),
which illustrates the interaction of protons of the carbohydrate cycle (dashed line), thymine (dotted line), ethoxygroup (dot-and-
dash line), and the methylene group of benzene (two dots-and-dash line).
in the spectrum of diacetate, it is a complex multiplet. the signal of methyl of the 2'-acetyl group, and the H5'b
signal overlaps with the signal of 5-ëç3.
In the spectra of nucleosides, the characteristic features
of the spectrum of diacetate are retained, and the sig-
nals of the nucleic acid base appear (see the figure).
Thus, we developed a convenient method for the
synthesis of all four 5'-deoxy-5'-ethoxycarbonylmethyl
ribonucleosides.
The 2D spectrum of compound (IXa) (see the fig-
ure) enables one to unambiguously assign the signals to
the corresponding protons of the molecule and identify
signals that overlap in the 1D spectra. Thus, the peak at
4 ppm contains two signals, H3' (is identified from the
cross peak with H2') and H4' (cross peaks with H5'a
EXPERIMENTAL
The following preparations were used: benzene,
chloroform, methylene chloride, acetonitrile, ethyl ace-
and H5'b). It is seen that the H5'a signal overlaps with tate, tetrahydrofuran, acetic acid, and pyridine (all of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 5 2009

588
VARIZHUK et al.
2
extra purity grade) (Khimmed, Russia); acetic anhy- (1 H, d, Ja,b 11.55 Ha OCH2O), 4.662 (1 H, t, J 4.01,
dride of extra purity grade (Reakhim, Russia); cytosine, H2), 4.572 (1 H, d, 2Ja,b 11.5, Hb OCH2O), 4.221 1 H, dt,
thymine, adenine, and guanine (Fluka, Switzerland); J4,5 3.62, J5,6 6.75, H5), 3.992 (1 H, dd, J3,4 8.89, J4,5 3.62
and triethylphosphonoacetate, benzyloxymethylchlo- H4), 3.958 (1 H, dd, J5,6a 6.78, J6a,6b 8.08, H6a), 3.912
ride, benzyl bromide, BSA, trimethylsilyl trifluo- (1 H, dd, J2,3 4.12, J3,4 8.89, H3), 3.805 (1 H, dd, J5,6b
romethanesulfonate, sodium hydride, sodium perio- 6.73, J6a,6b 8.08, H6b), 1.463 (3 H, s, CH3), 1.324 (3 H, Ò,
date, and DIPEA (Aldrich, United States). Benzene, CH3), 1.263 (3 H, Ò, CH3), 1.259 (3 H, s, CH3).
acetonitrile, and methylene chloride were dried by dis-
3-é-Benzyloxymethyl-1,2-O-isopropylidene-a-D-
tillation over phosphorus pentoxide; tetrahydrofurane
allofuranose (III). Water (30 ml) was added to a solu-
was dried over lithium alumohydride; and pyridine,
tion of compound (II) (12.7 g, 33.4 mmol) in acetic
over calcium hydride. 2-N-Acetylguanine was obtained
acid (30 ml) and the suspension was heated with stir-
as described in [18].
ring to 50°ë until a homogeneous solution formed
NMR spectra (δ, ppm, spin–spin coupling constant,
Hz) were recorded on a Bruker AMXIII-400 spectrom-
eter with a working frequency of 400 MHz. The spectra
were processed using the program MestReNova, ver-
sion 5.3.0 (Mestrelab Research SL). For compounds
containing hydroxy groups (spectrum in DMSO-d6),
the multiplicity of signals of nonexchanging protons
that interact with the proton of the hydroxy group was
determined after the addition of 2H2O to the sample.
(0.5 h, control TLC). The solution was evaporated and
the residue was evaporated several times with water and
then with absolute benzene. Chromatography on a sil-
ica gel column (ethanol–chloroform 2 : 49) yielded
9.1 g (80%) of crystalline substance (III); mp 62–63°ë
(from a cyclohexane–ethyl acetate mixture 30 : 1), Rf
0.34 (Ä). 1ç NMR (DMSO-d6): 7.39–7.26 (5 H, m, Ph),
5.697 (1 H, d, J1,2 3.64, H1), 4.822 (1 H, d, J 4.79 5-OH),
4.793 (1 H, d, 2Ja,b 6.76, Ha PhCH2O), 4.733 (1 H, d, 2Ja,b
2
6.76, Hb PhCH2O), 4.683 (1 H, d, Ja,b 10.89, Ha
Substances were purified by flash chromatography
on silica gel [19]. Silica gel 60, 40–63 µm (Merck, Ger-
many) was used. TLC was carried out on Silica gel 60
F254 plates (Merck, Germany) in systems of ethanol–
chloroform 1 : 49 (system A); isopropanol–hexane 1 :
19 (system B); isopropanol–hexane 3 : 47 (system C);
isopropanol–hexane 1 : 49 (system D); methanol–chlo-
roform 1 : 49 (system E); and methanol–chloroform 1 :
19 (system F).
OCH2O), 4.624 (1 H, t, J 4.07, H2), 4.537 (1 H, d, 2Ja,b
10.89, Hb OCH2O), 4.498 (1 H, d, J 5.61, 5-OH), 4.091
(1 H, dd, J2,3 4.44, J3,4 8.81, H3), 4.007 (1 H, dd, J3,4 8.81,
J4,5 2.58, H4), 3.700 (1 H, m, H5), 3.467 (1 H, dd, J5,6a
6.02, J6a,6b 11.92, H6a), 3.363 (1 H, dd, J5,6b 7.48, J6a,6b
11.92, H6b), 1.457 (3 H, Ò, CH3), 1.257 (3 H, s, CH3).
3-é-Benzyloxymethyl-1,2-é-isopropylidene-a-
D-ribo-pentadialdo-1,4-furanose (IV). Sodium perio-
date (8.56 g, 40 mmol) and water (5 ml) were added to
a solution of derivative (III) (9.1 g, 26.7 mmol) in ethyl
acetate (50 ml). The mixture was stirred at room tem-
perature overnight (control TLC in a system of isopro-
panol–hexane 1 : 19), filtered, and the residue on the fil-
ter was washed with ethyl acetate (2 × 20 ml). The fil-
trate was washed with water, dried over anhydrous
Na2SO4, and evaporated. The yield of product (IV) as a
1,2,5,6-Di-O-isopropylidene-a-D-allofuranose (I)
was obtained from 1,2,5,6-di-O-isopropylidene-α-D-
1
glucofuranose as described in [15]. H NMR (DMSO-
d6): 5.657 (1 H, d, J1,2 3.63, H1), 5.046 (1 H, d, J 7.13, 3-
OH), 4.456 (1 H, t, J 4.21, H2), 4.228 (1 H, dt, J4,5 7.15,
J5,6 2.75, H5), 3.929 (1 H, dd, J3,4 9.06, J4,5 7.15, H4),
3.828 (2 H, m, H6a, H6b), 3.743 (1 H, dd, J2,3 4.62, J3,4
9.06, H3), 1.446 (3 H, Ò, CH3), 1.323 (3 H, s, CH3), 1.275
(3 H, Ò, CH3), 1.267 (3 H, s, CH3).
1
dense oil was 8.1 g (98.4%); Rf 0.41 (B). H NMR
(CDCl3): 9.676 (1 H, d, J4,5 1.82, H5), 7.38–7.26 (5 H, m,
3-é-Benzyloxymethyl-1,2,5,6-di-O-isopropylidene-
a-D-allofuranose (II). DIPEA (7.24 g; 9.59 ml,
0.056 mol) and then benzyloxymethyl chloride (7.52 g,
6.59 ml, 0.048 mol) were added with stirring to a solu-
tion of 10.4 g (0.04 mol) of diisopropylidene allose (I)
in 40 ml of absolute methylene chloride. The mixture
was refluxed for 5 h and left overnight under stirring.
Water (40 ml) was added and the mixture was stirred
for 30 min. The organic layer was separated and the
Ph), 5.842 (1 H, d, J1,2 3.31, H1), 4.891 (1 H, d, 2Ja,b 7.08,
2
Ha PhCH2O), 4.850 (1 H, d, Ja,b 7.08, Hb PhCH2O),
2
4.713 (1 H, d, Ja,b 11.57, Ha OCH2O), 4.664 (1 H,
2
pseudo s, t, J 3.71, H2), 4.598 (1 H, d, Ja,b 11.57, Hb
OCH2O), 4.442 (1 H, dd, J3,4 9.44, J4,5 1.82, H4), 4.066
(1 H, dd, J2,3 4.32, J3,4 9.44, H3), 1.601 (3 H, Ò, CH3),
1.353 (3 H, s, CH3).
3-é-Benzyloxymethyl-5-deoxy-1,2-O-isopropy-
aqueous layer was extracted by methylene chloride. lidene-5-ethoxycarbonylmethylene-a-D-ribofura-
The combined organic extracts were washed with nose (V). A 60% suspension of sodium hydride (1.26 g,
water, a 10% KHSO4 solution, and water, dried over 31.6 mmol) in mineral oil and dry THF (15 ml) were
anhydrous Na2SO4, and evaporated. Purification on a placed in a retort equipped with a magnetic stirrer, a
column of silica gel and elution with isopropanol : hex- dropping funnel, and a thermometer. The mixture was
ane 2 : 49 yielded 13.3 g (87.4%) of the crystalline sub- cooled in an ice bath, and triethylphosphonoacetate
stance (II); yield; Rf 0.8 (A), mp 46–47°ë (from cyclo- (7.08 g, 6.27 ml, 31.6 mmol) in dry THF (21 ml) was
hexane). 1H NMR (DMSO-d6): 7.39–7.26 (5 H, m, Ph), added dropwise with stirring so that the temperature
5.715 (1 H, d, J1,2 3.61, H1) 4.818 (1 H, d, 2Ja,b 6.78, Ha was no higher than 5°ë. The mixture was stirred for
2
PhCH2O), 4.797 (1 H, d, Ja,b 6.78, Hb PhCH2O), 4.687 another 20 min at 0–5°ë. Then, a solution of aldehyde
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 5 2009

SYNTHESIS OF 5'-DEOXY-5'-ETHOXYCARBONYLMETHYL NUCLEOSIDES
589
(IV) (8.1 g, 26.3 mmol) in dry THF (50 ml) was added J4,5a 4.01, J4,5b 8.38, H4), 3.413 (1 H, dd, J2,3 4.36, J3,4
and care was taken that the temperature was no higher 8.90, H3), 2.394 (2 H, m, H6a, H6b), 2.054 (1 H, m,
than 5°ë. The temperature was brought to room tem- H5a), 1.767 (1 H, m, H5b), 1.568 (3 H, s, CH3), 1.337
perature and the mixture was stirred for 90 min. During (3 H, s, CH3), 1.222 (3 H, t, J 7.14, OCH2CH3).
stirring, a resin-like sediment of sodium diethylphos-
3-O-Benzyl-5-deoxy-1,2-di-O-acetyl-5-ethoxy-
phate was formed. The mixture was diluted with water
carbonylmethyl-D-ribofuranose
(VIII).
Water
(100 ml) and extracted with ethyl acetate. Organic
extracts were washed with a 10% sodium bicarbonate
solution and water, dried over anhydrous Na2SO4, evap-
orated, and the residue was chromatographed on a silica
gel column. Elution with isopropanol–hexane 3 : 97
yielded 8.2 g (82.4%) of (V) as a dense oil; Rf 0.38 (B).
1H NMR (CDCl3): 7.36–7.26 (5 H, m, Ph), 6.960 (1 H,
dd, J4,5 5.19, J5,6 15.71, H5), 6.156 (1 H, dd, 4J4,6 1.46, J5,6
15.71, H6), 5.782 (1 H, d, J1,2 3.52, H1), 4.902 (1 H, d,
(10 ml) was added to a solution of compound (VII)
(4.8 g, 13.7 mmol) in acetic acid (40 ml) and the solu-
tion was heated under stirring and weak boiling until
the termination of the reaction (0.5 h). The solution was
evaporated and the residue was reevaporated with water
to remove acetic acid and then with absolute pyridine
(4 × 5 ml). The residue was dissolved in absolute pyri-
dine (30 ml), acetic anhydride (5.61 g, 7.17 ml,
55 mmol) was added, and the mixture was stirred over-
night at room temperature. Water (2 ml) was added, and
the solution was kept for 30 min and evaporated. The
residue was dissolved in chloroform (100 ml), washed
with a saturated NaHCO3 solution, and dried over anhy-
drous Na2SO4, after which the solvent was evaporated.
The chromatography on a silica gel column and elution
with isopropanol–hexane 1 : 49 yielded 5 g (92.5%) of
compound (VIII) as a dense oil; Rf 0.48 (D). 1H NMR
(CDCl3): 7.39–7.26 (5 H, m, Ph), 6.088 (1 H, s, H1),
2
2Ja,b 6.99, Ha PhCH2O), 4.820 (1 H, d, Ja,b 6.99, Hb
2
PhCH2O), 4.744 (1 H, d, Ja,b 11.43, Ha OCH2O), 4.646
(1 H, pseudo s, t, J 3.92, H2), 4.626 (1 H, ddd, J4,3 9.26,
2
J4,5 5.19, 4J4,6 1.46, H4), 4.577 (1 H, d, Ja,b 11.43, Hb
OCH2O), 4.182 (2 H, q, J 7.12, OCH2CH3), 3.713 (1 H,
dd, J2,3 4.19, J3,4 9.26, H3), 1.604 (3 H, Ò, CH3), 1.337
(3 H, s, CH3), 1.266 (3 H, t, J 7.12, OCH2CH3).
5-Deoxy-1,2-O-isopropylidene-5-ethoxycarbon-
ylmethyl-a-D-ribofuranose (VI). Pd(OH)2/C (0.8 g)
was added to a solution of unsaturated compound (V)
(8.1 g, 21.4 mmol) in ethanol (40 ml) and the mixture
was hydrated at room temperature and atmospheric
pressure for 24 h. The mixture was filtered through cel-
lite, cellite on the filter was washed with ethanol (3 ×
20 ml), and the solvent was evaporated. The yield of
compound (VI) was 5.5 g (98.7%) as a dense oil;
2
5.279 (1 H, d, J2,3 4.04, H2), 4.604 (1 H, d, Ja,b 11.34,
2
Ha PhCH2O), 4.450 (1 H, d, Ja,b 11.34, Hb PhCH2O),
4.105 (2 H, m, OCH2CH3), 4.068 (1 H, m, H4), 3.937
(1 H, dd, J2,3 4.04, J3,4 7.82, H3), 2.381 (2 H, m, H6a,
H6b), 2.105 (3 H, Ò, 2-CH3CO), 2.056 (3 H, s, 1-CH3CO),
2.018 (1 H, m, H5a), 1.853 (1 H, m, H5b), 1.232 (3 H,
t, J 7.14, OCH2CH3).
1
Rf 0.32 (C). H NMR (DMSO-d6): 5.627 (1 H, d, J1,2
A general method of obtaining the nucleoside
analogues (IXa)–(IXe). BSA was added with stirring
to a suspension of a nucleic acid base in absolute aceto-
nitrile and the mixture was heated to 60°ë until the res-
idue was dissolved (0.5–1 h). A solution of diacetate
(VIII) in absolute acetonitrile followed by trimethylsi-
lyltriflate was added to the solution. The mixture was
heated under weak boiling for 4 h, cooled, and poured
under stirring into a cold saturated NaHCO3 solution
(50 ml). The mixture was stirred for 30 min and
extracted with chloroform. The extracts were washed
with water and dried over anhydrous Na2SO4. The sol-
vent was evaporated and the residue was chromato-
graphed on a column of silica gel.
3.69, H1), 5.040 (1 H, d, J 6.92, 3-OH), 4.429 (1 H,
pseudo s, t, J 4.11, H2), 4.048 (2 H, q, J 7.11,
OCH2CH3), 3.685 (1 H, dt, J3,4 8.61, J4,5a 4.21, J4,5b 8.22,
H4), 3.486 (1 H, dd, J2,3 4.11, J3,4 8.61, H3), 2.365 (2 H,
m, H6a, H6b), 1.899 (1 H, m, H5a), 1.637 (1 H, m,
H5b), 1.421 (3 H, s, CH3), 1.250 (3 H, s, CH3), 1.175
(3 H, t, J 7.11, OCH2CH3).
3-O-Benzyl-5-deoxy-1,2-O-isopropylidene-5-
ethoxycarbonylmethyl-a-D-ribofuranose (VII). A
suspension of NaH (1.12 g, 28 mmol) was added with
stirring and cooling to a solution of compound (VI)
(5.21 g, 20 mmol) in dry THF (20 ml). After the termi-
nation of hydrogen release, the suspension was stirred
for an additional 10 min and benzyl bromide (4.1 g,
2.85 ml, 24 mmol) was added. The mixture was stirred
2'-O-Acetyl-3'-é-benzyl-5'-deoxy-5'-ethoxycar-
at room temperature for 4 h, acetic acid (2 ml) was bonylmethyl-5-methyluridine (IXa) was obtained
added, and extraction with chloroform was carried out. from thymine (0.41 g, 3.3 mol), BSA (1.52 g, 1.83 ml,
Chloroform extracts were washed with a saturated 7.5 mmol) in absolute acetonitrile (10 ml), diacetate
NaHCO3 solution and water, dried over anhydrous (VIII) (1 g, 2.5 mmol) (a solution in 5 ml of absolute
Na2SO4, and chromatographed. The purification of the acetonitrile), and trimethylsilyltriflate (0.72 g, 0.58 ml,
product on a column of silica gel and elution with iso- 3.3 mmol). Elution with methanol–chloroform 1 : 99
propanol–hexane 1 : 49 yielded 4.9 g (69.9%) of com- yielded 1.1 g (95.6%) of nucleoside (IXa) as a solid
1
1
pound (VII) as a dense oil; Rf 0.56 (D). H NMR foam; Rf 0.74 (E). H NMR (CDCl3): 8.488 (1 H, s, br,
(CDCl3): 7.39–7.26 (5 H, m, Ph), 5.687 (1 H, d, J1,2 3.80, H3), 7.39–7.26 (5 H, m, Ph), 7.004 (1 H, br d, J 1.14,
H1), 4.763 (1 H, d 2Ja,b 11.92, Ha PhCH2O), 4.543 (1 H, H6), 5.694 (1 H, d, J1',2' 3.39, H1'), 5.355 (1 H, dd, J1',2'
2
2
t, J 4.05, H2), 4.530 (1 H, d, Ja,b 11.92, Hb PhCH2O), 3.39, J2',3' 4.88, H2'), 4.581 (1 H, d, Ja,b 11.28, Ha
4.095 (2 H, q, J 7.14, OCH2CH3), 4.018 (1 H, dt, J3,4 8.90, PhCH2O), 4.478 (1 H, d, 2Ja,b 11.28, Hb PhCH2O), 4.106
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 5 2009

590
VARIZHUK et al.
(2 H, q, J 7.14, Ha OCH2CH3), 4.00–3.92 (2 H, m, H3', CH3COO), 2.102 (1 H, m, H5'a), 2.007 (1 H, m, H5'b),
H4'), 2.409 (2 H, m, H6'a, H6'b), 2.095 (3 H, s,
2'-CH3COO), 2.059 (1 H, m, H5'a), 1.926 (3 H, s, br,
J 1.14, 5-CH3), 1.914 (1 H, m, H5'b), 1.277 (3 H, t,
J 7.14, OCH2CH3).
1.199 (3 H, t, J 7.13, OCH2CH3).
2'-O-Acetyl-3'-é-benzoyl-5'-deoxy-5'-ethoxycar-
bonylmethyl-2-N-acetylguanosine
(IXe)
was
obtained from 2-N-acetylguanine (0.184 g, 0.95 mmol)
in absolute acetonitrile (5 ml), BSA (0.67 g, 0.81 ml,
3.3 mmol), diacetate (VIII) (0.34 g, 0.86 mmol) (a solu-
tion in 4 ml of absolute acetonitrile), and trimethylsilyl-
triflate (0.22 g, 0.18 ml, 1 mmol). Elution with metha-
nol–chloroform 1 : 49 yielded 0.33 g (72.6%) of nucle-
2'-O-Acetyl-3'-é-benzyl-5'-deoxy-5'-ethoxycar-
bonylmethylcytidine (IXb) was obtained from
cytosine (0.16 g, 1.42 mmol) in absolute acetonitrile
(6 ml), BSA (1 g, 1.2 ml, 4.9 mmol), diacetate (VIII)
(0.43 g, 1.1 mmol) (a solution in 4 ml of absolute ace-
tonitrile), and trimethylsilyltriflate (0.32 g, 0.26 ml,
1.42 mmol). Elution with methanol–chloroform 1 : 24
yielded 0.44 g (90.6%) of nucleoside (IXb) as a solid
foam; Rf 0.39 (F). 1H NMR (CDCl3): 7.39–7.26 (5 H, m,
Ph), 7.348 (1 H, d, J5,6 7.42, H6), 5.798(1 H, d, J5,6 7.42,
H5), 5.719 (1 H, d J1',2' 2.06, H1'), 5.494 (1 H, dd, J1',2'
1
oside (IXe) as a solid foam; Rf 0.56 (E). H NMR
(CDCl3): 7.662 (1 H, s, H8), 7.39–7.26 (5 H, m, Ph),
5.93 (1 H, d, J1',2' 5.76, H1'), 5.582 (1 H, pseudo s, t,
J 5.36, H2'), 4.542 (2 H, s, br, Ha, Hb PhCH2O), 4.359
(1 H, m, H3'), 4.229 (1 H, m, H4'), 4.126 (2 H, m,
OCH2CH3), 2.424 (2 H, m, H6'a, H6'b), 2.291 (3 H, s,
2-CH3CON), 2.058 (3 H, Ò, 2'-CH3COO), 2.09 (1 H, m,
H5'a), 2.005 (1 H, m, H5'b), 1.251 (3 H, t, J 7.11,
OCH2CH3).
2
2.06, J2',3' 5.29, H2'), 4.589 (1 H, d, Ja,b 11.23, Hb
PhCH2O), 4.411 (1 H, d, 2Ja,b 11.23, Hb PhCH2O), 4.106
(2 H, m, OCH2CH3), 3.995 (1 H, dt, J3',4' 8.17, J4',5'a 4.04,
J4',5'b 8.35, H4'), 3.876 (1 H, dd, J2',3' 5.29, J3',4' 8.17, H3'),
2.438 (2 H, m, H6'a, H6'b), 2.096 (3 H, s, 2'-CH3COO),
2.056 (1 H, m, H5'a), 1.923 (1 H, m, H5'b), 1.222 (3 H,
t, J 7.08, OCH2CH3).
ACKNOWLEDGMENTS
This work was supported by the Russian Foundation
for Basic Research (project no. 08-04-01078).
2'-O-Acetyl-3'-é-benzyl-5'-deoxy-5'-ethoxycar-
bonylmethyladenosine (IXc) was obtained from ade-
nine (0.16 g, 1.2 mmol) in absolute acetonitrile (5 ml),
BSA (0.84 g, 1 ml, 4.1 mmol), diacetate (VIII) (0.36 g,
0.91 mmol) (a solution in 4 ml of absolute acetonitrile),
and trimethylsilyltriflate (0.27 g, 0.22 ml, 1.2 mmol).
Elution with methanol–chloroform 1 : 49 yielded
0.23 g (53.7%) of nucleoside (IXc) as a solid foam;
Rf 0.55 (F). 1H NMR (CDCl3): 8.318 (1 H, s, H2), 8.054
(1 H, s, H8), 7.39–7.26 (5 H, m, Ph), 5.997 (1 H, d, J1',2'
3.05, H1'), 5.854 (1 H, dd, J1',2' 3.05, J2',3' 5.25, H2'),
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