Oligonucleotide analogues containing internucleotide C3′-CH 2-C(O)-NH-C5′ bonds

Article abstract of DOI:10.1134/S106816200902006X

A dinucleoside bearing an amide internucleotide C3′-CH ₂-C(O)-NH-C5′ bond was synthesized by the interaction of 3′-deoxy-3′-carboxylmethylribothymidine-2′,3′-lactone obtained by hydrolysis of 2′-O-acetyl-5′-O-benzoyl-3′-deoxy- 3′-ethoxycarboxyl

Full text of DOI:10.1134/S106816200902006X

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 2, pp. 185–192. © Pleiades Publishing, Ltd., 2009.
Original Russian Text © S.V. Kochetkova, A.M. Varizhuk, N.A. Kolganova, E.N. Timofeev, V.L. Florent’ev, 2009, published in Bioorganicheskaya Khimiya, 2009, Vol. 35, No. 2,
pp. 202–209.
Oligonucleotide Analogues Containing Internucleotide
C3'-CH2-C(O)-NH-C5' Bonds
S. V. Kochetkova, A. M. Varizhuk, N. A. Kolganova, E. N. Timofeev, and V. L. Florent’ev*1
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 119991 Russia
Received May 28, 2008; in final form, July 14, 2008
Abstract—A dinucleoside bearing an amide internucleotide C3'-CH -C(O)-NH-C5' bond was synthesized by the
2
interaction of 3'-deoxy-3'-carboxylmethylribothymidine-2',3'-lactone obtained by hydrolysis of 2'-O-acetyl-5'-O-
benzoyl-3'-deoxy-3'-ethoxycarboxylmethylribothymidine with 5'-deoxy-5'-amino-3'-O-(tert-butyldimethylsilyl)thy-
midine. After standard manipulations with protective groups, the dinucleoside was converted into 3'-O-(2-cyanoet-
hyl-N,N'-diisopropylphosphoroamidite), which was used for the synthesis of modified oligonucleotides on an auto-
matic synthesizer. Duplex melting curves formed by modified and complementary natural oligonucleotides were
measured and the melting temperatures and thermodynamic parameters of duplex formation were calculated. The
introduction of one modified bond into oligonucleotides caused only an insignificant decrease in the duplex melting
temperatures compared with the nonmodified ones.
Key words: amide internucleotide bond, analogues, hybridization, oligonucleotides, thermal stability
DOI: 10.1134/S106816200902006X
INTRODUCTION
oligonucleotides whose stability is practically the same
as that of natural duplexes.
Therapy with synthetic oligonucleotide analogues
(antisense therapy) is one of the promising methods in
the treatment of viral and oncologic diseases [1, 2].2
The method is based on the inhibition of gene transla-
tion due to specific binding of a synthetic oligonucle-
otide to the mRNA corresponding site. The basic mech-
anism of action of synthetic oligonucleotides is an
attack of the resulting DNA–RNA hybrid with intracel-
lular endonuclease RNase H, which breaks a ribo-chain
of the hybrid complex and inhibits translation [3, 4].
High hybridization specificity provides selective inhi-
bition of the expression of genes involved in pathogen-
esis.
We describe in this work the synthesis and study of
the hybridization properties of oligonucleotide ana-
logues, in which a dinucleotide residue is replaced by a
dinucleoside residue containing an amide internucleo-
side bond (structure (D) in Scheme 1).
O
B
O
R1
O
NH
B
During last 15 years, a large number of base-, carbo-
hydrate- or internucleotide bond-modified oligonucle-
otide analogues were synthesized (see reviews [5–7]).
In recent years, researchers paid significant attention to
oligonucleotide analogues, in which a phosphate inter-
nucleotide bond was replaced by an amide bond. Oligo-
nucleotides in which one or several dinucleotides are
replaced by a dinucleoside residue with a C3'–CH2–
C(O)–NH–C5' bond [8–12] (structures (Ä) and (B) in
Scheme 1) are of special interest, because these oligo-
nucleotides form duplexes with complementary natural
O
O
R2
(A): R1 = H, R2 = H
(B): R1 = OH, R2 = OH
(D): R1 = OH, R2 = H, B = Thy
Scheme 1. Dinucleosides with internucleotide amide bon.ds.
RESULTS AND DISCUSSION
1
Corresponding author; phone: +7 (499) 135-2182, +7 (499) 135-
Earlier, we published an improved method of synthesis
of 2'-O-acetyl-5'-é-benzoyl-3'-deoxy-3'-ethoxycarboxyl-
methylnucleosides [13]. The use of these compounds in
oligonucleotide synthesis requires the preparation of prop-
erly protected dinucleosides (D) (Scheme 1). At first
6591; fax: +7 (499) 135-1405; e-mail: flor@imb.ac.ru
2
Abbreviations: TBAF, tetra-n-butylammonium fluoride; TEAA,
triethylammonium acetate; TBDMS, tert-butyldimethylsilyl; aT,
5'-amino-5'-deoxythymidine; cT, 3'-deoxy-3'-carboxymethylri-
bothymidine.
185

186
KOCHETKOVA et al.
O
O
H3C
H3C
NH
NH
O
N
H3C
OH
N
O
BzO
O
N
O
NH
O
O
O
a
R1O
5'
O
O
N
O
O
OAc
4'
3'
1'
H3C
2'
OR2
NH
OEt
6'
c
5'
NH
O
(I)
(II)
O
O
4'
3'
1'
2'
O
O
OR3
H3C
H3C
NH
NH
(V): R1 = H, R2 = H, R3 = TBDMS
(VI): R1 = (MeO)2Tr, R2 = H, R3 = TBDMS
(VII): R1 = (MeO)2Tr, R2 = Ac, R3 = TBDMS
d
e
H2N
N3
N
O
N
O
b
O
O
OTBDMS
OTBDMS
(III)
(VI)
O
f
H3C
NH
O
N
H3C
(MeO)2TrO
N
O
O
N
NH
O
H3C
NH
(MeO)2TrO
O
O
N
OAc
NH
O
H3C
O
NH
O
g
O
O
OAc
NH
O
N(Pr2i)2
O
O
P
OCH2CH2CN
OH
(VIII)
(IX)
Scheme 2. a) 1 – KOH, H O/EtOH, 2 – AcOH. 70°C; b)
HCOONH , Pd/C, MeOH; c) 2-hydroxypyridine, DMF, 70°ë 30 h;
2
4
i
d) (MeO) TrCl, Py; e) Ac O, Py; f) TBAF, THF; g)NCCH CH OP(NPr ) , tetrazole.
2
2
2
2
2 2
glance, the simplest method of dinucleoside synthesis is tone. Saponification of the lactone and interaction with a
large excess of tert-butyldimethylsilyl chloride for 3 days
resulted in two major products: target protected 3'-deoxy-
3'-carboxylmethylribothymidine and its bissilyl deriva-
tive. This mixture was hydrolyzed with an acetone–water–
triethylamine mixture (3 days) to give 5'-é-(MeO)2Tr–2'-
O-TBDMS-3'-deoxy-3'-carboxylmethylribothymidine.
The above procedure is time consuming and is of little use
for the large-scale synthesis of dinucleosides.
the following sequence: alkaline hydrolysis, successive
protection of a 5'-hydroxyl with a dimethoxytrityl and 2'-
hydroxyl with a tert-butyldimethylsilyl group, and final
coupling with 5'-amino-5'-deoxynucleoside. However,
this route proved to be ineffective in the case of 3'-deoxy-
3'-ethoxycarboxylmethylnucleosides. The reason being
that ester hydrolysis was accompanied by 2',3'-lactone
closure. It was shown in [14] that hydrolysis of 2',5'-di-O-
acetyl-3'-deoxy-3'-ethoxycarboxylmethylribothymidine
We used 2',3'-lactone as a key compound for the
followed by tritylation only resulted in protected 2',3'-lac- synthesis of dinucleoside (Scheme 2). Lactone (II) was
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 2 2009

OLIGONUCLEOTIDE ANALOGUES
187
5'-OH cT
H1' aT
H2'b aT
H2'a aT; H6'b cT
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
H3', H6'a cT
H5'a, H5'b aT
H5'b cT
H5'a cT; H4' ‡T
H4' cT
H2' cT
H3' aT
f1 (ppm)
5'-OH cT
H1', 2'-OH cT
6.0
8.0
H1' aT
5'-NH aT
8.0 6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
f2 (ppm)
Fig. 1. A COSY DQF spectral fragment demonstrating correlations among protons of carbohydrate cycles of dinucleoside (V)
(DMSO-d solution). cT, a 3'-deoxy-3'-carboxymethylribothymidine residue; aT, 5'-amino-5'-deoxythymidine residue. The assign-
6
ment of resonances to the cT residue is shown with a dotted line, and to the aT residue, with dots.
obtained in a yield of 84% by alkaline hydrolysis of
nucleoside (I) followed by short heating of the interme-
diate acid in a weak acidic medium. Peterson et al.,
showed that the lactone interaction with amines
occurred with great difficulty and good results could
only be achieved in the presence of a bifunctional cata-
lyst, 2-hydroxypyridine [15]. Indeed, the heating of lac-
tone (II) with a fivefold excess of 5'-amino-5'-deoxy-3'-
O-butyldimethylsilylthymidine (IV) and a twofold
excess of 2-hydroxypyridine in dimethylformamide led
to 86% dinucleoside (V). Further routine procedures of
tritylation, acetylation, and the removal of silyl protec-
tive groups resulted in dinucleoside (VIII) with a total
yield of 78% relative to starting dinucleoside (V). Phos-
phoroamidite (IX) was obtained as was earlier
described in [16].
The structures of all synthesized compounds were
confirmed by 1H NMR spectra. In the case of dinucleo-
sides (V)–(VIII), the resonances were assigned using
COSY DQF spectra (as an example, a two-dimensional
spectrum of dinucleoside (V) is given in Fig. 1).
The two-dimensional spectrum (Fig. 1) provided
unambiguous identification of the resonances of termi-
nal 5' and 3'dinucleoside residues. Since the spectrum
was registered in DMSOd6, it contained resonances of
the changing protons, namely, those of 2' and
5'-hydroxy groups, with the T residue (identified by dis-
appearance of proton resonances of hydroxyl groups
when 2H2O was added to the sample). In the 1D spec-
trum the 2'-OH resonance was partially overlapped
with the H1' resonance; therefore they gave a united
peak with H2', which interacted in turn with H3', whose
resonance was overlapped with that of the solvent. The
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 2 2009

188
KOCHETKOVA et al.
Table 1. Mass spectra of the synthesized oligonucleotides
Mass spectra
Code
Sequence, 5'
3'
found, m/z
Modified oligonucleotides
calculated [M – H]–
M1
M2
M3
M4
M5
M6
M7
G-D-TT-TT-TT-TT-G
G-TT-TT-TT-TT-D-G
G-TT-TT-D-TT-TT-G
G-AC-AC-D-CT-AA-G
G-AC-AA-D-AT-CA-G
G-AC-AG-D-GT-CA-G
G-AA-GT-D-GA-CA-G
3614.9
3614.6
3615.8
3606.7
3629.3
3662.5
3683.5
3614.45
3614.45
3614.45
3607.47
3629.50
3661.49
3685.52
Natural oligonucleotides
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
G-TT-TT-TT-TT-TT-G
C-AA-AA-AA-AA-AA-C
G-AC-AC-TT-CT-AA-G
G-AC-AA-TT-AT-CA-G
G-AC-AG-TT-GT-CA-G
G-AA-GT-TT-GA-CA-G
C-TT-AG-AA-GT-GT-C
C-TG-AT-AA-TT-GT-C
C-TG-AC-AA-CT-GT-C
C-TG-TC-AA-AC-TT-C
3637.8
3647.3
3628.9
3653.2
3683.1
3709.2
3660.7
3622.5
3606.4
3679.7
6337.38
3647.46
3628.4
3652.42
3684.42
3708.54
3659.41
3633.4
3604.37
3579.36
H3' proton formed cross-peaks with H4' and H6'a and lated using a model of two states (Table 2). When cal-
H6'b. Proton H4' interacted with H5'a and H5'b. Addi-
tional information was obtained from cross peaks
between 5'-OH, H5'a and H5'b. In the case of an aT pro-
ton, H1' formed cross peaks with H2'a and H2'b, which
interacted with H3'. With the above data, it was easy to
assign the H4' resonance and resonances of H5'a and
H5'b, whose signals in the 1D spectrum were com-
pletely overlapped with the signal of residual water. In
addition, the cross peak between H5'a and H5'b and 5'-
NH was observed.
All the dinucleosides were characterized by MALDI
mass spectra.
Phosphoramidite (IX) was used for the synthesis of
oligonucleotide analogues (D-modified dinucleoside
(Scheme 1)) (Table 1). In addition, complementary nat-
ural oligonucleotides were also prepared (Table 1).
The synthesized oligonucleotides were character-
ized by mass spectra (Table 1).
Profiles of the thermal dissociation of natural and
modified duplexes were measured (Fig. 2‡). Duplex
experimental melting temperatures were determined by
the derivative maximum after the differentiation of
experimental melting curves (Fig. 2b).
culating, temperature dependences of the duplex and
individual chains were taken into account. Since melt-
ing temperatures of short duplexes depended on the
concentration, experimental melting temperatures were
recalculated to a standard chain concentration of 10–5 M
(the next to last row in Table 2).
As is seen in Table 2, one modification caused a
decrease in the duplex melting temperatures by 0.4–
1.3°ë, depending on the sequence if compared with that
of natural duplexes. The data on the thermal stability of
duplexes formed by antisense and complementary nat-
ural oligonucleotides were summarized in [17]. Of the
analogues with amide internucleotide bonds, oligonu-
cleotides containing modified dinucleoside (A)
(Scheme 1) or internucleotide C3'−CH2–NH–C(O)–C5'
bonds formed the most stable duplexes. In the first case,
δTm was –0.1 – +0.9, and in the second, –0.8 – +0.4°C.
Other modifications of amide, carbamide, and carbam-
ate internucleotide bonds decreased duplex ím by more
than 2°ë. To summarize, stability of the duplexes
formed by the synthesized with complementary natural
oligonucleotides was only insignificantly lower than
that of the corresponding natural duplexes and compa-
rable to the stability of the above-described most stable
Based on the curves, thermodynamic parameters of
duplex formation from individual chains were calcu- modified duplexes.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 2 2009

OLIGONUCLEOTIDE ANALOGUES
189
EXPERIMENTAL
10
20
30
40
50
60
70
Pyridine, chloroform, methylene chloride, dime-
thylformamide, diethyl ether, ethyl acetate, acetonitrile,
tetrahydrofurane, and acetic acid of a chemically pure
grade were from Khimmed, Russia; ammonium for-
mate, triethylamine, and acetic anhydride were from
Fluka, Switzerland; dimethoxytrityl chloride, tert-
butyldimethylsilyl chloride, imidazole, tetrazole, and
2-hydroxypyridine, from Aldrich, United States; anhy-
drous methanol, from Pancreac, Spain. Dimethylfor-
mamide, methylene chloride, and acetonitrile were
dried by distillation over phosphorus pentachloride;
pyridine, over calcium hydride; and tetrahydrofurane,
over lithium aluminium hydride. 2-Cyanoethyl-
N,N,N',N'-tetraisopropylphosphoroamidite was pre-
pared as described in [18].
NMR spectra (δ, ppm, J, Hz) of DMSO-d6 solutions
(if not stated otherwise) were registered on a Bruker
AMXIII-400 spectrometer with a working frequency of
400 MHz. The spectra were treated using the MestReN-
ova program, version 5.2.3 (Mestrelab Research SL).
The multiplet centers are shown.
1.0
3
(b)
2
4
1
0.5
1.0
(a)
0.8
0.6
0.4
0.2
0
4
2
3
1
10
20
30
40
50
60
70
Temperature, °C
Duplex melting curves were registered on a UV
160-A spectrophotometer (Shimadzu, Japan) supplied
with a thermostatic device. Duplex absorption A260 was
registered at intervals of 0.5°ë.
Fig. 2. a, Experimental melting curves of natural duplexes:
1, S5·S9, 3, S1·S2; and modified duplexes 2, M6·S9, 4,
M2·S2. b, a derivative obtained by differentiation of exper-
imental curves. For the convenience of comparison, hyper-
chromicity and the derivative were normalized to 1.
MALDI analysis of modified and natural oligonu-
cleotides was performed on a MS-30 mass-spectrome-
ter (Kratos, Japan) in a linear mode and registration of
negative peaks.
Oligonucleotides were synthesized on an automatic
ASM-102U DNA synthesizer (Biosun, Russia) using
by reverse-phase HPLC on columns (25 × 4 mm2) with
Hypersil C18 in 0.05 M TEAA buffer (pH 7.8). DMTr-
protected oligomers were isolated in a gradient of ace-
tonitrile (10–50%) for 30 min. Exhaustively deblocked
the standard protocol. Oligonucleotides were purified oligonucleotides were additionally purified in a gradi-
Table 2. Thermodynamic parameters of duplex formation from single chains
∆S°,
Tm, °C
Tm, °C
Duplex
c × 106, M
∆H°, kJ/mol
∆Tm, °C**
J/mol–1 ä–1
0.5
scaled*
S1·S2
7.46
9.96
9.42
1.12
8.82
4.84
8.76
9.04
7.53
8.62
7.70
5.53
–368.6 5.4
–294.3 5.4
–275.9 12.7
–289.7 7.9
–344.5 3.7
–275.9 5.0
–364.7 3.4
–265.9 12.6
–338.7 5.0
–292.7 5.9
–357.4 5.4
–962.1 17.2
–851.6 29.7
–791.7 42.2
–838.2 32.2
–987.7 12.9
–737.3 16.3
–1053.0 10.9
–740.6 40.6
–965.1 15.5
–822.3 18.1
–1016.6 18.5
–681.2 22.2
33.6
33.9
33.7
33.7
41.3
38.2
41.0
40.2
42.2
41.4
44.4
42.0
34.3
33.9
33.9
33.4
41.6
40.3
41.3
40.5
42.9
41.8
45.0
44.0
M1·S2
M2·S2
M3·S2
S3·S7
–0.4
–0.4
–0.9
M4·S7
S4·S8
–1.3
–0.8
–1.1
–1.0
M5·S8
S5·S9
M6·S9
S6·S10
M7·S10
–249.9 7.1
–5
Notes: * Recalculated to a concentration of 10 M.
** Differences between T of modified and natural duplexes.
m
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 2 2009

190
KOCHETKOVA et al.
ent of acetonitrile (10–25%) for 30 min in the above H2'b), 1.911 (3 H, d, J 1.24, 5-CH3), 0.861 (9 H, s,
buffer.
(CH3)3CSi), 0.058 (6 H, s, (CH3)2Si).
For column chromatography, a Kieselgel 60
5'-Deoxy-5'-amino-3'-O-(tert-butyldimethylsi-
(Merck, Germany) was used. TLC was performed on lyl)thymidine (IV). A mixture of 10% Pd/C (0.42 g)
Kieselgel 60 F254 plates (Merck, Germany) in 1 : 49 eth- and a solution of azide (III) (4.2 g, 11 mmol) in anhy-
anol–chloroform (system A); 1 : 19 ethanol–chloro- drous methanol (50 ml) was stirred at room tempera-
form (system B); 1 : 9 ethanol–chloroform (system C); ture. In approximately 10 min, a gas was liberated and
and 1 : 19 ethanol–chloroform + 0.5% triethylamine the mixture was heated. After 2 h stirring, the mixture
(system D).
was filtered through Cellit, the Cellit was washed with
anhydrous methanol (2 × 15 ml) on the filter, and the fil-
trate was evaporated. The residue was mixed with satu-
rated NaHCO3 (50 ml) and extracted with chloroform
(3 × 30 ml). Chloroform extracts were washed with
water, dried with anhydrous Na2SO4, and evaporated to
give amine (IV) (3.82 g, 98%) as a thick oil; Rf 0.28 (B).
1H NMR (CDCl3): 7.325 (1 H, br. d, J 1.26, H6), 6.176
(1 H, t, J 6.6, H1'), 4.316 (1 H, m, H3'), 3.811 (1 H, m,
3'-Deoxy-3'-carboxymethylribothymidine 2',3'-
lactone (II). A solution of potassium hydroxide (1.51g,
27 mmol) in water (5 ml) was added under stirring to a
solution of 2'-O-acetyl-5'-O-benzoyl-3'-deoxy-3'-
ethoxycarbonylmethylribothymidine (1.28 g, 2.7 mmol)
(I) in ethanol (20 ml) [13]. The mixture was kept under
stirring at room temperature overnight. Concentrated
HCl (2.23 ml) and then water (20 ml) were added, the
mixture was evaporated to half a volume, and the solu-
tion was extracted with ether (3 × 20 ml). Acetic acid
(5 ml) was added to the aqueous layer and heated at 70–
80°ë for 1 h. The mixture was evaporated to dryness;
the residue was evaporated with ethanol (4 × 10 ml) and
dissolved in absolute ethanol (10 ml). Silica gel (6 g)
2
H4'), 3.051 (1 H, dd, J4',5'a 3.75, J5'a,5'b 13.45, H5'a),
2
2.881 (1 H, dd, J4',5'b 5.76, J5'a,5'b 13.45, H5'b), 2.258
(1 H, m, H2'a), 2.175 (1 H, m, H2'b), 1.916 (3 H, d,
J 1.26, 5-CH3), 0.881 (9 H, s, (CH3)3CSi), 0.073 (6 H, s,
(CH3)2Si).
3'-Deoxy-3'-{[N-(3'-O-tert-butyldimethylsilyl)-
was added to the solution and the mixture was evapo- 5'-deoxythymidine-5'-yl)carboxamido]methyl}-
rated to dryness. The residue was loaded onto the col- ribothymidine(V). A solution of lactone (II) (0.56 g,
umn (3.5 × 20 cm) with silica gel and chromatographed 2 mmol), amine (IV) (3.56 g, 10 mmol), and 2-hydrox-
with elution with system C. The fractions containing ypyridine (0.38 g, 4 mmol) in anhydrous DMF (20 ml)
lactone were evaporated to dryness to give 0.64 g (84%) was heated at 70°ë for 30 h. The solvent was removed
of lactone (II) as thick oil. Rf 0.25 (C). 1H NMR: 11.366 in vacuum, and the residue was dissolved in chloroform
(1 H, br. s, H3), 7.623 (1 H, br d, J 1.16, H6), 5.913 (100 ml). The solution was washed with 10% KHSO4
(1 H, d, J1' ,2' 1.63, H1'), 5.104 (1 H, dd, J1' ,2' 1.63, J2', 3' (3 × 30 ml), saturated with NaHCO3 (2 × 30 ml) and
7.12, H2'), 5.043 (1 H, t, J 5.04, 5'-OH), 3.854 (1 H, m, water, and dried with anhydrous Na2SO4. The solvent
2
H4'), 3.678 (1 H, dd, J4' ,5'a 3.54, J5'a ,5'b 12.08, H5'a), was evaporated, and the residue was dissolved in chlo-
2
3.597 (1 H, dd, J4' ,5'b 4.39, J5'a,5'b 12.08, H5'b), 3.144 roform and loaded on a silica gel column (20 × 4 cm).
(1 H, m, H3'), 2.847 (1 H, dd, J3' ,6'a 8.61, 2J6'a, 6'b 18.07, The product was eluted with a mixture of chloroform–
H6'a), 2.464 (1 H, dd, J3' ,6'b 1.51, 2J6'a, 6'b 18.07, H6'b), EtOH (gradient from 5 to 15% EtOH) to give two sub-
1.769 (3 H, d, J 1.26, 5-CH3).
stances with Rf 0.38 and 0.66 (C). The compound with
a higher mobility (1.4 g) was a side product, 5'-deoxy-
5'-formamido-3'-O-(tert-butyldimethylsilyl)thymidine,
whereas the fraction with lower mobility contained the
target dinucleoside (V) as solid foam (1.1 g, 86%).
1H NMR: 11.261 (1 H, br. s, H3 cT), 11.233 (1 H, br. s,
H3 aT), 8.102 (1 H, br. t, J 5.57, NH aT), 7.998 (1 H, br.
d, J1.36, H6 cT), 7.437 (1 H, br. d, J1.14, H6 aT), 6.112
(1 H, dd, J1',2'a 8.25, J1',2'b 5.79, H1' aT), 5.654 (1 H, d, J
4.91, 2'-OH cT), 5.637 (1 H, d, J1',2' 1.41, H1' cT), 5.103
(1 H, d, J4.94, 5'-OH cT), 4.312 (1 H, m, H3' aT), 4.137
(1 H, m, H2' cT), 3.848 (1 H, m, H4' cT), 3.756 (1 H,
m, H5'a cT), 3.748 (1 H, m, H4' aT), 3.530 (1 H, m,
H5'b cT), 3.39–3.23 (2 H, m, H5'a,b aT), 2.49–2.38
(2 H, m, H3', H6'a cT), 2.25–2.11 (2 H, m, H6'b cT,
H2'a aT), 2.007 (1 H, m, H2'b aT), 1.799 (3 H, d, J 1.36,
5-CH3 cT), 1.758 (3 H, d, J 1.14, 5-CH3 aT), 0.862 (9 H,
s, (CH3)3CSi), 0.067 (6 H, s, (CH3)2Si). Mass: m/z 637.3.
Calculated 636.8 [M–H]– (C26H42N5O10Si).
5'-Deoxy-5'-azido-3'-O-(tert-butyldimethylsi-
lyl)thymidine (III). A mixture of 5'-deoxy-5'-azido-
thymidine (3.2 g, 12 mmol) [19] and imidazole (1.63 g,
24 mmol) was evaporated with anhydrous acetonitrile
(3 × 10 ml). The residue was dissolved in anhydrous
acetonitrile (25 ml) and tert-butyldimethylsilyl chlo-
ride (2.7 g, 24 mmol) was added under stirring. The
mixture was kept at room temperature overnight, water
(10 ml) was added, evaporated, and water and ethyl
acetate (50 ml of each) were added to the residue. The
aqueous layer was extracted with ethyl acetate (20 ml),
the united organic fractions were washed with 10%
KHSO4 (2 × 10 ml), saturated NaHCO3 (2 × 10 ml), and
water, dried with anhydrous Na2SO4, and evaporated to
give 4.5 g (98%) of azide (III) as crystalline mass,
Rf 0.51 (A), mp 89–90°ë (2 : 1 ethyl acetate–hexane).
1H NMR (CDCl3): 9.433 (1 H, br. s, H3), 7.281 (1 H, br.
d, J 1.24, H6), 6.214 (1 H, t, J 6.56, H1'), 4.325 (1 H, m,
H3'), 3.905 (1 H, m, H4'), 3.661 (1 H, dd, J4',5'a 3.29,
5'-O-(4,4'-Dimethoxytriryl)-3'-deoxy-3'-{[N-(3'-O-
2J5'a,5'b 13.27, H5'a), 3,468 (1 H, dd, J4',5'b 3.62, J5'a,5'b tert-butyldimethylsilyl-5'-deoxythymidine–5'-yl)car-
2
13.27, H5'b), 2.256 (1 H, m, H2'a), 2.149 (1 H, m, boxamido]methyl}-ribothymidine (VI). A solution of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 2 2009

OLIGONUCLEOTIDE ANALOGUES
191
dinucleoside (V) (0.6 g, 0.94 mmol) in anhydrous pyri- 2.026 (3 H, s, CH3CO), 2.001 (1 H, m, H2'b aT), 1.781
dine (10 ml) was evaporated to dryness. The residue
was dissolved in anhydrous pyridine (10 ml) and
(CH3O)2TrCl (0.41 g, 1.2 mmol) was added. The mix-
ture was allowed to stay overnight under stirring at
room temperature; water (1 ml) and triethylamine
(0.2 ml) were added, kept for 30 min, and evaporated.
The residue was dissolved in ethyl acetate (50 ml),
washed with saturated NaHCO3 (2 × 20 ml), water, and
(3 H, d, J 1.22, 5-CH3 cT), 1.514 (3 H, d, J 1.17, 5-CH3
aT), 0.852 (9 H, s, (CH3)3CSi), 0.05 (6 H, s, (CH3)2Si).
Mass: m/z 981.2. Calculated 981.2 981.2 [M–H]–
(C51H62N5O13Si).
2'-O-Acetyl-5'-O-(4,4'-dimethoxytrityl)-3'-deoxy-
3'-{[N-(5'-deoxythymidine-5'-yl)carboxamido]methyl}-
ribothymidine (VIII). Anhydrous TBAF [20] (0.25 g,
dried with anhydrous Na2SO4. The residue was dis- 0.97 mmol) was added to a solution of dinucleoside
solved in chloroform containing 0.2% triethylamine
and loaded on a silica gel column (20 ×4 cm). The prod-
uct was eluted with a chloroform–EtOH mixture con-
taining 0.2% triethylamine (gradient from 2 to 10%
EtOH) to give compound (VI) as solid foam (0.74 g,
94%); Rf 0.52 (C). 1H NMR: 11.304 (1 H, br. s, H3 cT),
11.272 (1 H, br. s, H3 aT), 8.066 (1 H, br. t, J 5.83, NH
aT), 7.525 (1 H, br. d, J 1.27, H6 cT), 7.46 (1 H, br. d, J
1.18, H6 aT), 7.41–6.86 (13 H, m, Ar-H, (CH3O)2Tr),
6.09 (1 H, dd, J1',2'a 8.05, J1',2'b 6.09, H1' aT), 5.671 (1 H,
d, J 4.96, 2'-OH cT), 5.671 (1 H, d, J1',2' 1.73, H1' cT),
4.289 (1 H, m, H3' aT), 4.221 (1 H, m, H2' cT), 4.003
(1 H, m, H4' cT), 3.73 (6 H, s, CH3, (CH3O)2Tr), 3.702
(1 H, m, H4' aT), 3.14–3.32 (4 H, m, H5'a,b cT, H5'a,b
aT), 2.652 (1 H, m, H3' cT), 2.429 (1 H, dd, J3',6'a 8.01,
2J6'a,6'b 15.16, H6'a cT), 2.166 (1 H, m, H2'a aT), 1.95–
2.04 (2 H, m, H6'b cT, H2'b aT), 1.784 (3 H, d, J 1.27,
5-CH3 cT), 1.366 (3 H, d, J 1.18, 5-CH3 aT), 0.849 (9 H,
s, (CH3)3CSi), 0.047 (3 H, s, CH3Si), 0.043 (3 H, s,
CH3Si). Mass: m/z 939.1. Calculated 939.1 [M – H]–
(C49H60N5O12Si).
(VII) (0.63 g, 0.64 mmol) in absolute THF (10 ml), and
the mixture was stirred for 3 h. The solvent was evapo-
rated, and the residue was dissolved in ethanol (20 ml).
Silicagel (10 g) was added to the solution, ethanol was
evaporated, and the dry residue was loaded on a silica
gel column (20 × 4 cm). The mixture was chromato-
graphed with a chloroform–EtOH mixture containing
0.2% triethylamine (gradient from 5 to 15% EtOH) to
give dinucleoside (VIII) as solid foam (0.51 g, 92%);
Rf 0.24 (C). 1H NMR: 11.283 (1 H, br. s, H3 cT), 11.170
(1 H, br. s, H3 aT), 7.99 (1 H, br. t, J 5.53, NH aT),
7.466 (1 H, br. d, J 1.08, H6 cT), 7.444 (1 H, br.d, J 1.18,
H6 aT), 7.41–6.86 (13 H, m, Ar-H, (CH3O)2Tr), 6.111
(1 H, dd, J1',2'a 7.49, J1',2'b 6.39, H1' aT), 5.734 (1 H, d, J1',2'
3.09, H1' cT), 5.36 (1 H, dd, J1',2' 3.09, J2',3' 6.97, H2' cT),
4.094 (1 H, dd, H3' aT), 3.995 (1 H, m, H4' cT), 3.739
(6 H, s, CH (6 H, c, CH3, (CH3O)2Tr), 3.715 (1 H, m, H4'
aT), 3.14–3.32 (4 H, m, H5'a,b cT, H5'a,b aT), 3.026
(1 H, m, H3'), 2.331 (1 H, dd, J3',6'a 8.42, 2J6'a,6'b 15.19,
H6'a cT), 2.132 (1 H, dd, J3',6'b 6.93, 2J6'a,6'b 15.19, H6'b
cT), 2.04–2.12 (2 H, m, H2'a,b aT), 2.029 (3 H, s,
CH3CO), 1.775 (3 H, d, J 1.08, 5-CH3 cT), 1.552 (3 H,
d, J 1.18, 5-CH3 aT). Mass: m/z 867.3. Calculated 866.9
866.9 [M – H]– (C45H48N5O13).
2'-O-Acetyl–5'-O-(4,4'-dimethoxytrityl)-3'-deoxy-
3'-{[N-(3'-O-tert-butyldimethylsilyl-5'-deoxythymi-
dine-5'-yl)carboxamido]methyl}-ribothymidine (VII).
Dinucleoside (VI) (0.7 g, 0.75 mmol) was evaporated
with anhydrous pyridine (2 × 5 ml). The residue was
dissolved in anhydrous pyridine (8 ml), acetic anhy-
dride (0.54 g, 0.44 ml, 5.25 mmol) was added, and the
mixture was allowed to stay overnight. Water (0.5 ml)
and triethylamine (0.1 ml) were added and the mixture
was stirred for 30 min. The solvent was removed, and
the residue was dissolved in ethyl acetate (50 ml),
washed with saturated NaHCO3 (2 × 20 ml) and water,
and dried with anhydrous Na2SO4. The mixture was
chromatographed on a silica gel column (20 × 3 cm) in
a chloroform–EtOH mixture containing 0.2% triethy-
lamine (gradient from 2 to 7% EtOH) to give dinucleo-
side (VII) as solid foam (0.66 g, 90%); Rf 0.27 (B).
1H NMR: 11.334 (1 H, br. s, H3 cT), 11.249 (1 H, br. s,
H3 aT), 8.082 (1 H, br. t, J 5.95, NH aT), 7.488 (1 H, br.
d, J 1.22, H6 cT), 7.47 (1 H, br. d, J 1.17, H6 aT), 7.41–
6.86 (13 H, m, Ar-H, (CH3O)2Tr), 6.092 (1 H, dd, J1',2'a
7.98, J1',2'b 6.06, H1' aT), 5.737 (1 H, d, J1',2' 3.06, H1' cT),
5.358 (1 H, dd, J1',2' 3.06, J2',3' 6.94, H2' cT), 4.27 (1 H,
dd, H3' aT), 3.997 (1 H, m, H4' cT), 3.732 (6 H, s, CH3,
(CH3O)2Tr), 3.703 (1 H, m, H4' aT), 3.14–3.32 (4 H, m,
H5'a,b cT, H5'a,b aT), 3.011 (1 H, m, H3'), 2.319 (1 H,
dd, J3',6'a 8.75, 2J6'a,6'b 15.14, H6'a cT), 2.182 (1 H, m, H2'a
2'-O-Acetyl-5'-O-(4,4'-dimethoxytrityl)-3'-deoxy-
3'-{[N-(3'-O-(N,N-diisopropylamino-2-cyanoethox-
yphosphinyl)-5'-deoxythymidine-5'-yl)carboxam-
ido]methyl}-ribothymidine (IX). Absolute pyridine
(56 mg, 57 µl, 0.7 mmol), tetrazole (45 mg, 0.65 mmol)
and then 2-cyanoethyl-N,N,N',N'-tetraisopropylphos-
phoroamidite were added under stirring to a solution of
dinucleoside (VIII) (0.47 g, 0.54 mmol) in dry methyl-
ene chloride (2.5 ml). Cold saturated NaHCO3 (20 ml)
and methylene chloride (50 ml) were added to the mix-
ture in 1 h. The organic phase was separated, and the
aqueous phase was extracted with methylene chloride
(2 × 10 ml). United organic fractions were washed with
saturated NaCl (20 ml), dried with anhydrous Na2SO4,
and evaporated. The residue was loaded onto a silica
gel column (15 × 2 cm) in a chloroform–EtOH mixture
(1 : 49) containing 0.2% triethylamine and eluted with
a chloroform–EtOH mixture containing 0.2% triethy-
lamine (a gradient from 2 to 5% EtOH) to give phos-
phoroamidite (IX) (0.46 g, 80%) as solid foam; Rf 0.59
(D). 31P NMR (acetonitrile-dd3): 151.731, 151.465.
Mass: m/z 1067.2. Calculated 1067.1 [M – H]–
aT), 2.113 (1 H, dd, J3',6'b 6.12, 2J6'a,6'b 15.14, H6'b cT), (C54H65N7O14P).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 35 No. 2 2009

192
KOCHETKOVA et al.
11. von Matt, P., Mesmaeker, D.A., Pieles, U., Zurcher, W.,
ACKNOWLEDGMENTS
and Altmann, K.-H., Tetrahedron Lett., 1999, vol. 40,
The work was supported by the Russian Foundation
for Basic Research, project no. 08-04-01078.
pp. 2899–2902.
12. Rozners, E., Katkevica, D., Bizdena, E., and Stromberg, R.,
J. Am. Chem. Soc., 2003, vol. 125, pp. 12125–12136.
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