Oligonucleotide analogues containing a C3′-NH-C(O)-CH 2-C5′ amide internucleotide bond

Article abstract of DOI:10.1134/S1068162010020093

A dinucleotide containing a C3′-NH-C(O)-CH₂-C5′ amide internucleotide bond was synthesized by the interaction of 3′-deoxy-3′-amino-5′-O-(tert-butyldimethylsilyl)thymidine with 3′-O-benzyl-2′-O-tert-butyldimethylsilyl-5′-deoxy-5′- carboxymethylr

Full text of DOI:10.1134/S1068162010020093

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2010, Vol. 36, No. 2, pp. 199–206. © Pleiades Publishing, Ltd., 2010.
Original Russian Text © A.M. Varizhuk, S.V. Kochetkova, N.A. Kolganova, E.N. Timofeev, V.L. Florent’ev, 2010, published in Bioorganicheskaya Khimiya, 2010, Vol. 36, No. 2,
pp. 215–222.
Oligonucleotide AnaloguesContaining a C3'–NH–C(O)–CH2–C5'
Amide Internucleotide Bond
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, 119991 Russia
Received March 24, 2009; in final form, May 18, 2009
A dinucleotide containing a C3'–NH–C(O)–CH2–C5' amide internucleotide bond was synthesized by the
interaction of 3'ꢀdeoxyꢀ3'ꢀaminoꢀ5'ꢀ
butyldimethylsilylꢀ5'ꢀdeoxyꢀ5'ꢀcarboxymethylribosylthymine, which was obtained from 2'ꢀ
benzylꢀ5'ꢀdeoxyꢀ5'ꢀethoxycarbonylmethylribosylthymine through the methanolysis of the acetyl group folꢀ
lowed by silylation of liberated hydroxyl and ester saponification. After standard manipulation with protectꢀ
ing groups, the dinucleotide was converted into 3'ꢀ (2ꢀcyanoethylꢀN,Nꢀdiisopropylphosphoramidite),
O
ꢀ(tertꢀbutyldimethylsilyl)thymidine with 3'ꢀ
O
ꢀbenzylꢀ2'ꢀOꢀtertꢀ
O
ꢀacetylꢀ3'ꢀ
Oꢀ
O
which was used for the synthesis of modified oligonucleotides on an automated synthesizer. The melting
curves of the duplexes formed by modified and complementary natural oligonucleotides were registered, and
the melting temperatures and thermodynamic parameters of the duplex formation were calculated. The
introduction of a single modified bond into the oligonucleotide led to an insignificant decrease in the melting
temperature of these duplexes as compared to unmodified ones.
Key words: oligonucleotides, analogues, amide internucleotide bond, hybridization, thermal stability
DOI: 10.1134/S1068162010020093
1
INTRODUCTION
nucleic acids (PNAs) [11, 12], oligonucleotides conꢀ
taining morpholino cycle instead of furanose [13, 14],
bicyclic sugar (LNA) [15, 16], and an amide internuꢀ
cleotide bond [17, 18].
Therapy with synthetic oligonucleotide analogues
(antisense therapy) is one of the most prospective methods
for the treatment of viral and oncological diseases [1–3].2
The concept of the method is the possibility of the inhibiꢀ
tion of the translation of a target gene through the specific
binding of a synthetic oligonucleotide to the correspondꢀ
ing region of mRNA. An important criterion for choosing
antisense oligonucleotides is the stability of their duplexes
with complementary natural deoxyriboꢀ or ribooligonuꢀ
cleotides [4]. The melting temperature of the modified
duplexes is usually used as a quantitative measure of their
stability. This temperature should be comparable with or
higher than that of the corresponding natural duplexes.
In our previous paper [19], we described the synꢀ
thesis and properties of oligonucleotides containing
the C3'–CH2–C(O)–NH–C5' amide internucleotide
bond (structure (A) in Scheme 1). The present work is
devoted to the synthesis and the study of the physicoꢀ
chemical and hybridization properties of oligonucleꢀ
otide analogues containing a reverse sequence of
atoms in the internucleotide bond (structure (
B) in
Scheme 1).
Over the last 15 years, a vast majority of oligonucleꢀ
otide analogues have been synthesized with modified
heterocyclic bases, carbohydrate residues, or internuꢀ
cleotide bonds (see reviews [5–7]). Few of them, howꢀ
ever, meet the stability criterion. The most prospective
are oligonucleotides in which there are a phosphate
internucleotide bond replaced by a phosphorothioate
O
O
Thy
Thy
O
O
OH
NH
O
O
Thy
Thy
NH
one [8, 9], 2'ꢀ
O
ꢀalkyloligonucleotides [10], peptide
O
O
1
Corresponding author; phone: +7 (499) 135ꢀ2182, +7 (499)
135ꢀ6591; fax: +7 (499) 135ꢀ1405; eꢀmail: flor@imb.ac.ru
O
OH
2
O
Abbreviations: Bn, benzyl; DIPEA, diisopropylethylamine;
TBDMSꢀCl, tertꢀbutyldimethylsilyl chloride; TEAA, triethylamꢀ
monium acetate; (MeO) Tr, dimethoxytrityl; TFA, trifluoroaceꢀ
(
A)
(B)
2
tic acid; THF, tetrahydrofuran; TBAF, tetrabutylammonium fluꢀ
oride; cT, 5'ꢀdeoxyꢀ5'ꢀcarboxymethylꢀ5'ꢀribosylthymine; Ta,
3'ꢀaminoꢀ3'ꢀdeoxythymidine.
Scheme 1. Dinucleotides containing an internucleotide
amide bond.
199

200
VARIZHUK et al.
RESULTS AND DISCUSSION
3'ꢀAmidite dinucleotide ( ) for the synthesis of oliꢀ
gonucleotide analogues was prepared according to
Scheme 2. Previously prepared 2'ꢀ ꢀacetylꢀ3'ꢀ ꢀbenꢀ
zylꢀ5'ꢀdeoxyꢀ5'ꢀethoxycarbonylmethylribosylthymine (
a 93% yield of nucleotide (II) containing a deproꢀ
tected 2'ꢀhydroxy group. Compound (II) was conꢀ
verted into a 2'ꢀ ꢀTBDMS derivative (III) by the
interaction with tertꢀbutyldimethylsilyl chloride,
which led to free acid (IV) in a yield of 91% after alkaꢀ
B
O
O
O
I)
[20] was subjected to methanolysis, which resulted in line hydrolysis and the subsequent acidification.
O
N
O
N
O
N
H3C
O
H3C
O
H3C
O
NH
NH
NH
EtO
EtO
EtO
O
O
O
5'
6'
O
O
O
a
b
4'
1'
3'
2'
OAc
OTBDMS
OH
BnO
(
BnO
(
BnO
III)
I)
II
)
(
c
O
N
O
N
O
H3C
H3C
H3C
O
NH
NH
NH
HO
O
O
N
O
TBDMSO
TBDMSO
O
O
O
d
OTBDMS
N3
NH2
VI
BnO
IV
(
V)
(
)
(
)
O
N
O
H3C
H3C
NH
NH
e
O
O
N
R1O
(MeO)2TrO
5'
O
O
4'
1'
O
N
O
N
2'H3C
H3C
3'
NH
NH
O
HN
HN
O
6'
i
(X)
O
5'
O
O
O
4'
1'
3'
OR2
2'
OTBDMS
OTBDMS
O
NCCH2CH2O P
1
2
(
(
(
(
VII): R = TBDMS, R = Bn
N(iPr)2
XI
f
g
1
2
VIII): R = H, R = Bn
(
)
1
2
IX): R = R = H
h
1
2
X
): R = (MeO) Tr, R = H
2
Scheme 2. (a) K CO , MeOH; (b) TBDMSꢀCl, imidazole, acetonitrile; (c) KOH, ethanol/water; (d) HCOONH , Pd/C,
2
3
4
MeOH; (e) carbonyldiimidazole, CH Cl ; (f) 90% TFA; (g) H , Pd/C, THF; (h) (MeO) TrCl, pyridine; and
2
2
2
2
(i) NCCH CH OP(NiPr )Cl, DIPEA, CH Cl .
2
2
2 2 2
3'ꢀAminoꢀ3'ꢀdeoxyꢀ5'ꢀ
was synthesized by the reduction of the corresponding
azide ( ) with ammonium formate in the presence of
10% Pd/C.
O
ꢀTBDMSꢀthymidine (VI
)
with amine (VI) in the presence of carbonyldiimidaꢀ
zole. The primary TBDMS protecting group was
V
selectively removed with trifluoroacetic acid at
An attempt to remove the benzyl group in dinucleotide
VIII) by hydrogenation in ethanol over 20% Pd(OH)2
led to a mixture of 3'ꢀ and 2'ꢀ ꢀTBDMS isomers in a
0°C.
(
Completely protected dinucleotide (VII) was preꢀ
pared in a yield of 81% by the coupling of acid (IV
)
O
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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2010

OLIGONUCLEOTIDE ANALOGUES
201
CH3O Ta (DMTr)
H5'b cT
H5'a cT
2.0
2.5
3.0
3.5
4.0
4.5
5.0
H2'a Ta; H6'a, H6'b cT
H2'a Ta
H5'b Ta
H5'a Ta
H3', H4' cT
H4' Ta
H2' cT
H3' Ta
3'ꢀOH cT
H1' cT
6.0
8.5
H1' Ta
3'ꢀNH Ta
6.0
5.0
4.5
4.0
f 2, m.d.
3.5
3.0
2.5
2.0
Fig. 1. Fragment of the COSY DQF spectrum showing the correlation between protons in the carbohydrate cycles of dinucleotide (
cT is the 5 ꢀdeoxyꢀ5 ꢀcarboxinethylribosylthymine residue and Ta is the 3 ꢀaminoꢀ3 ꢀdeoxythymidine residue. The attribution of
signals to cT protons is designated by the dotted line, to Ta protons, by the points.
X).
'
'
'
'
1 : 0.7 ratio according to the NMR spectrum. It was dinucleotides (VII)–(X), the attribution of signals to
shown [21] that the migration of the TBDMS group is the nucleotide protons was performed by COSY DQF
reduced in dry aprotic solvents. Indeed, while hydroꢀ spectra. As an example, the twoꢀdimensional specꢀ
genating in absolute tetrahydrofuran over 10% Pd/C trum of dinucleotide (X) is presented in Fig. 1.
for 5 h, only a 2'ꢀ
obtained. The subsequent dimethoxytritylation led to
dinucleotide ( ), the interaction of which with 2ꢀcyaꢀ
noethylꢀ ꢀdiisopropylchlorophosphoramidite in
the presence of diisopropylethylamine resulted in
phosphoroamidite (XI).
OꢀTBDMS derivative (IX) was
This spectrum allows for the unambiguous identifiꢀ
cation of the proton signals in both nucleotide resiꢀ
X
dues. Since the spectrum was registered in DMSOꢀd6
,
N,N
it revealed the signals of exchanging protons, namely,
hydroxyl groups, which were identified by disappearꢀ
ance while adding D2O to the sample. The proton of
The structure of all of the synthesized compounds the secondary hydroxyl group (doublet) gives a cross
was confirmed by the 1H NMR spectra. In the case of peak with the 3'ꢀproton of the cT residue (solid line in
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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202
VARIZHUK et al.
Table 1. Synthesized oligonucleotides and their mass spectra (B is modified dinucleotide, see Scheme 1)
Mass spectra
Code
Sequence, 5'
3'
Obtained
,
m
/
z
Calculated for [M
–H]–
Modified oligonucleotides
M1
M2
M3
M4
Gꢀ
B
ꢀTTꢀTTꢀTTꢀTTꢀG
3614.9
3614.7
3614.2
3568.4
3614.45
3614.45
3614.45
3568.59
GꢀTTꢀTTꢀTTꢀTTꢀ
GꢀTTꢀTTꢀ ꢀTTꢀTTꢀG
Gꢀ ꢀTTꢀ ꢀTTꢀ ꢀG
BꢀG
B
B
B
B
Natural oligonucleotides
S1
S2
GꢀTTꢀTTꢀTTꢀTTꢀTTꢀG
CꢀAAꢀAAꢀAAꢀAAꢀAAꢀC
3637.8
3646.9
6337.38
3647.46
Table 2. Thermodynamic parameters of duplex formation from single strands
Δ
H
°
,
Δ
S
°
,
T
, °C
6
m
Duplex
c
×
10 , M
Tm
,
°
C
0.5
Δ
T
, °C**
cal mol–1 K–1
normalized
*
m
kcal/mol
S1·S2
M1·S2
M2·S2
M3·S2
M4·S2
10.37
4.86
–90.2 0.7
–88.1 1.7
–89.2 1.0
–91.4 1.5
–86.4 1.2
–266.1 2.3
–260.6 5.5
–263.2 3.2
–271.5 4.7
–258.7 3.9
36.2
33.7
34.9
34.5
31.1
36.2
35.4
35.7
34.5
31.0
–0.8
–0.5
–1.7
–5.2
6.79
10.18
10.30
–5
Notes: * Calculated for a 10 M concentration.
** Difference between T of modified and natural duplexes.
m
Fig. 1), which unambiguously points to the presence and fully masked by the signal of the CH3 groups in the
dimethoxytrityl group of another nucleotide. In the
2D spectrum, the cross peaks with H2' and with H5'a
and H5'b in the cT residue allow for the identification
of the H3' and H4' signals, respectively.
of a free 3'ꢀhydroxyl, i.e., 2'ꢀOꢀTBDMS derivative.
Moreover, the 2D spectrum allows for the identificaꢀ
tion of overlapping signals. The signals of H3' and H4'
in the cT residue in the 1D spectrum are overlapped
All of the dinucleotides were characterized by
MALDI mass spectra (see the Experimental section).
1.00
0.75
0.50
0.25
0
Phosphoramidite (XI) was used for the synthesis of
oligonucleotide analogues. In addition, complemenꢀ
tary natural oligonucleotides were prepared (Table 1).
The structure of the synthesized oligonucleotides
was confirmed by mass spectra (Table 1). The thermal
dissociation profiles of the natural and modified
duplexes were registered (Fig. 2).
The obtained curves allowed for the calculation of
the thermodynamic parameters of the duplex formaꢀ
tion from single strands using the twoꢀstate model
(Table 2). The temperature dependence of the adsorpꢀ
tion of a duplex (Ad) and single strands (Ass) was taken
into account. Since the melting temperature of short
duplexes depends on the concentration, the experiꢀ
mental melting temperatures were normalized to the
standard 10–5 M concentration of the strands (second
to last column in Table 2).
10
20
30
Temperature,
40
50
60
°
C
Fig. 2. Experimental melting curves for synthesized
duplexes (left to right): M4·S2 M1·S2 M3·S2 M2·S2,
S1·S2. For convenience, hyperchromicity was normalꢀ
,
,
,
As seen from Table 2, a single modification caused
only an insignificant decrease in the duplex melting
ized to 1.
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OLIGONUCLEOTIDE ANALOGUES
203
temperature as compared to that of the corresponding many). TLC was fulfilled on Kieselgel 60 F254 plates
natural complex.
(Merck, Germany) in systems of ethanol–methylene
chloride in ratios of 1 : 30 (A), 1 : 19 (B), 1 : 9 (C), and
1 : 9 + 0.1% TEA (D).
Thus, the prepared oligonucleotides meet the staꢀ
bility criterion applicable to antisense oligonucleꢀ
otides.
3'ꢀ
bosylthymine (II). К2CO3 (0.17 g, 1.2 mmol) was
added to the solution of 2'ꢀ ꢀacetylꢀ3'ꢀ ꢀbenzylꢀ5'ꢀ
deoxyꢀ5'ꢀethoxycarbonylmethylꢀ5ꢀribosylthymine (
OꢀBenzylꢀ5'ꢀdeoxyꢀ5'ꢀethoxycarbonylmethylriꢀ
O
O
EXPERIMENTAL
I)
We used pyridine, chloroform, methylene chloride,
dimethylformamide, ethyl acetate, acetonitrile, tetꢀ
rahydrofuran (purity grade, Khimmed, Russia), abs.
methanol (Panreac, Spain), ammonium formiate, triꢀ
ethylamine, acetanhydride (Fluka, Switzerland),
[20] (1.4 g, 3.0 mmol) in abs. methanol (50 ml), and
the mixture was stirred for 4 h. Acetic acid (1.5 ml) was
then added with stirring for 5 min, and the solvent was
evaporated. The residue was divided between water (50
ml) and chloroform (50 ml). The water layer was
dimethoxytrityl chloride, 2ꢀcyanoethylꢀN,Nꢀdiisoꢀ
extracted with chloroform (
chloroform extracts were washed with a saturated
NaHCO3 solution (3 50 ml) and water, dried with
2
×
20 ml). The combined
propylchlorophosphoramidite, and DEAEꢀToyopearl
650M (Aldrich, United States). 3'ꢀDeoxyꢀ3'ꢀazidoꢀ
thymidine was kindly provided by the AZT Associaꢀ
tion (Russia). Dimethylformamide, methylene chloꢀ
ride, and acetonitrile were dried by reflux over phosꢀ
phorus pentoxide; pyridine, over calcium hydride; and
tetrahydrofuran, over lithium alumohydride.
×
anhydrous Na2SO4, and evaporated. The residue was
separated by chromatography on a silicaꢀgel column
using a methylene chloride–2% methanol mixture as
an eluent. Fractions containing the product were
combined and evaporated. Product (II) was obtained
The NMR spectra ( , ppm; SSCC, Hz) of comꢀ
δ
in a solid foam form in a yield of 1.18 g (93%),
(А). 1H NMR (CDCl3): 8.774 (1 H, bs, H3), 7.29–7.39
(5H, m, Ph), 7.047 (1 H, bd, 1.14, H6), 5.601 (1 H,
d, 1',2' 4.23, H1'), 4.639 (2 H bs, PhCH2O), 4.256 (1 H,
dd, J1',2' 4.23, J2',3' 5.77, H2'), 4.111 (2 H, qv, 7.14,
OCH2CH3), 4.012 (1 H, m, H4'), 3.144 (1 H, dd, J2',3'
5.77, 3',4' 3.75, H3'), 2.434 (1 H, m, H6'a), 2.367 (1 H,
dd, J5',6'b 7.22, J6'a,6'b 16.59, H6'b), 2.011 (1 H, m,
H5'a), 1.927 (1 H, m, H 5'b), 1.913 (3 H, d, 1.14, 5ꢀ
CH3), 1.232 (3 H, t, 7.14, OCH2CH3).
Rf 0.27
pounds in DMSOꢀd6 (if not specially mentioned) were
registered on a Bruker AMXIIIꢀ400 spectrometer with
a working frequency of 400 MHz. Spectra were proꢀ
cessed by the MestReNova 5.3.0 program, (Mestrelab
Research SL).
The duplexes’ melting curves were recorded on a
UV 160ꢀA spectrophotometer (Shimadzu, Japan)
supplied with a thermostatic device. The absorption of
a duplex ( A260) was registered in 0.5°C intervals. The
calculation of the thermodynamic parameters was
performed by the nonlinear regression method in the
DataFit 9.0.059 program (Oakdale Engineering,
United States).
J
J
J
J
2
J
J
3'ꢀ ꢀBenzylꢀ2'ꢀOꢀtertꢀbutyldimethylsilylꢀ5'ꢀdeoxyꢀ5'ꢀ
O
ethoxycarbonylmethylribosylthymine (III). A mixture
of nucleotide (II) (1.08 g, 2.58 mmol) and imidazole
(0.7 g, 10.3 mmol) was evaporated with abs. acetoniꢀ
MALDI analysis of modified and natural oligonuꢀ
cleotides was carried out on an MSꢀ30 mass spectromꢀ
eter (Kratos, Japan) in the linear mode using negativeꢀ
ion registration.
trile (
3 5 ml) and dissolved in abs. acetonitrile (5 ml).
×
After the addition of TBDMSꢀCl (0.97 g, 6.45 mmol),
the reaction mixture was kept overnight under stirring.
Water (2 ml) was then added, and the mixture was
stirred additionally for 30 min followed by evaporaꢀ
tion. The residue was dissolved in CH2Cl2 (50 ml),
Oligonucleotides were synthesized on an autoꢀ
mated 3400 DNA Synthesizer (Applied Biosystems,
United States). The introduction of natural nucleꢀ
otides was carried out according to the standard protoꢀ
col; when a modified dinucleotide was used, the couꢀ
pling time was increased by a factor of three. The silyl
protecting group was removed by 1 M TBAF in THF
followed by desalting on a column with 0.5 ml of
DEAE Toyopearl using 0.1 M TEAA, pH 7, as an eluꢀ
ent. The oligonucleotide was eluted by 1 M TEAA.
The purification of oligonucleotides was performed by
reverseꢀphase HPLC on Hipersil C18 columns (25 cm ×
washed with 10% KHSO4 (2
saturated NaHCO3 (2 20 ml), dried with anhydrous
Na2SO4, and evaporated. The residue was chromatoꢀ
graphed on a silicaꢀgel column (3.5 20 cm) with a
×
20 ml) and then with
×
×
methylene chloride–1% methanol mixture. Fractions
containing the product were combined and evapoꢀ
rated. Product (III) was obtained in a solid foam form
1
in a yield of 1.33 g (97%), Rf 0.68 (А). H NMR
(CDCl3): 8.396 (1 H, bs, H3), 7.26–7.37 (5 H, m, Ph),
7.126 (1 H, bd, J 1.11, H6), 5.677 (1 H, d, J1',2' 3.53,
mm2) in a 0.05 M TEAA buffer. (MeO)2TrꢀProtected
2
4
H1'), 4.704 (1H, d, Ja,b 11.74, Ha PhCH2O), 4.470
(1 H, d, 2Ja,b 11.74, Hb PhCH2O), 4.378 (1 H, t,
H2'), 4.129 (1 H, m, H4'), 4.108 (2 H, qv,
J
J 7.14,
4.30
,
oligomers were isolated in an acetonitrile gradient
(10–50%). Fully deprotected oligonucleotides were
additionally purified in a gradient from 0 to 25% in the
same buffer.
OCH2CH3), 3.559 (1 H, dd, J2',3' 5.11, J3' 4' 5.75,H3'),
2.450 (2 H, m, H6'a, H6'b), 2.036 (1 H, m, H5'a),
For the preparative isolation, we used flash chroꢀ 1.949 (1 H, m, H5'b), 1.936 (3 H, d,
J 1.11, 5ꢀCH3),
matography on Kieselgel 60, 40–63 m (Merck, Gerꢀ 1.226 (3 H, t, 7.14, OCH2CH3), 0.885 (9 H, s,
µ
J
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
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204
VARIZHUK et al.
(CH3)3CSi), 0.088 (3 H, s, CH3Si), 0.083 (3 H, s, H3), 7.475 (1 H, bd, J 1.18, H6), 6.238 (1 H, t, J 6.15,
2
CH3Si).
H1'), 3.910 (1 H, dd, J4',5'a 3.12, J5'a,5'b 11.39, H5'a),
2
3.824 (1 H, dd, J4',5'b 2.80, J5'a,5'b 11.39, H5'b), 3.725
(1 H, m, H4'), 3.616 (1 H, m, H3'), 2.184 (2 H, m,
H2'a, H2'b), 1.901 (3 H, d, J 1.06, 5ꢀCH3), 0.915 (9 H,
s, (CH3)3CSi), 0.104 (3 H, s, CH3Si), 0.099 (3 H, s,
CH3Si).
3'ꢀ ꢀBenzylꢀ2'ꢀOꢀtertꢀbutyldimethylsilylꢀ5'ꢀdeoxyꢀ
O
5'ꢀcarboxymethylribosylthymine (IV). A solution of 4 N
KOH (1.6 ml) was added to compound (III) (1.3 mg,
2.4 mmol) in ethanol (4.2 ml). After stirring the mixꢀ
ture for 1 h, water (20 ml) and 1 M KHSO4 (7 ml,
pH 2) were added followed by extraction with CH2Cl2
3'ꢀ
O
ꢀBenzylꢀ2'ꢀ
Oꢀtertꢀbutyldimethylsilylꢀ5'ꢀdeoxyꢀ
ꢀ(5 tertꢀbutyldimethylsilylꢀ3'ꢀdeoxythymiꢀ
(4
×
20 ml). The extracts were washed with water, dried
5'ꢀ{[
N
'ꢀOꢀ
with anhydrous Na2SO4, and evaporated. Acid (IV
)
dineꢀ3'ꢀyl)carboxamido]methyl}ribosylthymine (VII).
Carbonyldiimidazole (0.42 g, 2.6 mmol) was added to
a solution of acid (VI) (1.1 g, 2.2 mmol) in abs. CH2Cl2
(10 ml), and the reaction mixture was stirred for 1 h
followed by the addition of amine (VI) (0.86 g,
2.4 mmol) in abs CH2Cl2 (5 ml). After a night, the mixꢀ
ture was diluted by CH2Cl2 (50 ml) and water (50 ml),
divided, and the water layer was extracted with CH2Cl2
was obtained in a solid foam form in a yield of 1.1 g
(93%), Rf 0.33 (D). 1H NMR (CDCl3): 9.311 (1 H, bs,
H3), 7.26–7.39 (5 H, m, Ph), 7.148 (1 H, bd,
H6), 5.631 (1 H, d,
1',2' 3.13, H1'), 4.699 (1 H, d, 2Ja,b
11.69, Ha PhCH2O), 4.455 (1 H, d, Ja,b 11.69, Hb
PhCH2O), 4.410 (1 H, t, 3.85, H2'), 4.153 (1 H, m,
H4'), 3.557 (1 H, dd, 2',3' 5.01, 3',4' 5.99, H3'), 2.507
(2 H, m, H6'a, H6'b), 2.045 (1 H, m, H5'a), 1.961
(1 H, dd, 5'a,5'b 16.44 H5'b), 1.961 (1 H, dd,
4',5'b 9.11, 2
H5'b) 1.908 (3 H, d, 1.16, 5ꢀCH3), 0.887 (9 H, s,
(CH3)3CSi), 0.097 (3 H, s, CH3Si), 0.087 (3 H, s,
CH3Si).
3'ꢀAzidoꢀ3'ꢀdeoxyꢀ5'ꢀ
midine (V). A mixture of 3'ꢀdeoxyꢀ3'ꢀazidothymidine
(1.07 g, 4 mmol) and imidazole (90.41 g, 6 mmol) was
evaporated with abs. acetonitrile (
solved in abs. acetonitrile (10 ml) followed by the addiꢀ
tion of TBDMSꢀCl (0.75 g, 5 mmol) under stirring.
The mixture was kept overnight at room temperature.
After the addition of water (10 ml), the mixture was
evaporated and divided between water (50 ml) and
ethyl acetate (50 ml). The water layer was extracted
with ethyl acetate (20 ml); the combined organic layꢀ
J 1.16,
J
2
J
J
J
(2
ous KHSO4 (2
(2 20 ml), and water; dried with anhydrous Na2SO4
×
20 ml). The extracts were washed with 10% aqueꢀ
J
J
×
20 ml); saturated aqueous NaHCO3
J
×
;
and evaporated. The residue was chromatographed on
a silicaꢀgel column with a methylene chloride–3%
ethanol mixture. Dinucleotide (VII) was obtained in
solid foam form in a yield of 1.5 g (81%), Rf 0.64(B).
1H NMR (here and after, cT is the residue of 5'ꢀdeoxyꢀ
5'ꢀcarboxymethylribosylthymine and Ta is the residue
of 3'ꢀaminoꢀ3'ꢀdeoxythymidine): 11.329 (1 H, bs, H3
O
ꢀ(tertꢀbutyldimethylsilyl)thyꢀ
3
×
5 ml) and disꢀ
cT), 11.288 (1 H, bs, H3 Ta), 8.286 (1 H, d,
3'ꢀNH Ta), 7.485 (1 H, bd, 1.16, H6 cT), 7.476 (1 H,
bd, 1.09, H6 Ta), 7.26–7.39 (5 H, m, Ph cT), 6.181
(1 H, t,
J 7.32,
J
J
J
6.64, H1' Ta), 5.787 (1 H, d, J1',2' 6.06, H1'
cT), 4.654 (1 H, d, 2Ja,b 11.84, Ha PhCH2O cT), 4.582
2
(1 H, d, Ja,b 11.84, Hb PhCH2O cT), 4.482 (1 H, t,
5.52, H2' cT), 4.297 (1 H, m, H3' Ta), 3.965 (1 H, m,
H4' cT), 3.69–3.84 (4 H, m, H3' cT; H4', H5'a,b Ta),
2.218 (2 H, m, H6'a,b cT), 2.117 (2 H, m, H2'a,b Ta),
1.931 (2 H, m, H5'a,b cT), 1.816 (3 H, d,
J
ers were washed with 10% aqueous KHSO4 (2
saturated aqueous NaHCO3 (2 20 ml), and water;
dried with anhydrous Na2SO4; and evaporated. Azide
) was obtained in crystal form in a yield of 1.4 g
(92%), Rf 0.64 (B), tm 89–90 (from an ethyl aceꢀ
tate–hexane mixture, 2 : 1). 1H NMR (CDCl3): 8.383
(1 H, bs, H3), 7.412 (1 H, bd, 1.06, H6), 6.202 (1 H,
t, 6.52, H1'), 4.226 (1 H, m, H4'), 3.960 (1 H, m,
×
20 ml),
×
J1.16, 5ꢀCH3
(
V
cT), 1.782 (3 H, d, 1.09, 5ꢀCH3 Ta), 0.871 (9 H, s,
J
°
C
(CH3)3CSi), 0.814 (9 H, s, (CH3)3CSi), 0.059 (6 H, s,
(CH3)2Si), 0.038 (3 H, s, CH3Si), –0.028 (3 H, s,
CH3Si). Mass spectrum: m/z 841.6. Calculated 841.14
J
J
[M
–H]– (C41H62N5O10Si2).
2
H3'), 3.935 (1 H, dd, J4',5'a 2.62, J5'a,5'b 11.12, H5'a),
3.796 (1 H, dd, J4',5'b 2.02, J5'a,5'b 11.12, H5'b) 2.423
2
3'ꢀ
N
OꢀBenzylꢀ2'ꢀOꢀtertꢀbutyldimethylsilylꢀ5'ꢀdeoxyꢀ
(1 H, m, H2'a), 2.217 (1 H, m, H2'b), 1.912 (3 H, d,
1.06, 5ꢀCH3), 0.930 (9 H, s, (CH3)3CSi), 0.123 (6 H, s,
(CH3)2Si).
3'ꢀAminoꢀ3'ꢀdeoxyꢀ5'ꢀ
midine (VI). 10% Pd/C (0.11 g) and then ammonium for 20 min at
formate (1.14 g) were added to a solution of azide ( vigorous stirring in cooled saturated NaHCO3 (150 ml)
(1.14 g, 3 mmol) in abs. methanol (24 ml). The mixꢀ and extracted with CH2Cl2 ( 20 ml). Extracts were
ture was boiled under stirring for 2 h. After cooling, the washed with saturated aqueous NaHCO3 (2 20 ml)
mixture was filtered through celite, washed with methꢀ and water, dried with anhydrous Na2SO4, and evapoꢀ
anol ( 15 ml), and evaporated. The residue was rated. The residue was chromatographed on a silicaꢀ
mixed with saturated NaHCO3 (50 ml) and extracted gel column (3.5 15 cm) with a methylene chloride–
with CH2Cl2 (4 20 ml). The combined extracts were 5% ethanol mixture. The yield of compound ( VIII in
washed with water (20 ml), dried with anhydrous solid foam form was 1 g, (81%).
f 0.63 (В). 1H NMR:
Na2SO4, and evaporated. Amine (VI) was obtained in 11.332 (1 H, bs, H3 cT), 11.242 (1 H, bs, H3 Ta),
crystal form in a yield of 94%, Rf 0.28 (b), Tm 89–90°C 8.281 (1 H, d, 7.26, 3'ꢀNH Ta), 7.754 (1 H, bd, 0.85
(from ethyl acetate). 1H NMR (CDCl3): 8.193 (1 H, bs, H6 cT), 7.483 (1 H, bd,
0.65, H6 Ta), 7.26–7.39
J
5'{[ ꢀ(3'ꢀdeoxythymidineꢀ3'ꢀyl)carboxamido]methyl}
ribosylthymine (VIII). Water (1 ml) was added to the
solution of dinucleotide (VII) (1.4 g, 1.7 mmol) in
O
ꢀ(tertꢀbutyldimethylsilyl)thyꢀ TFA, cooled to
0°C
(9 ml), and the mixture was stirred
0 C. The mixture was then poured under
°
V)
3
×
×
2
×
×
×
(
)
R
J
J
,
J
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
No. 2
2010

OLIGONUCLEOTIDE ANALOGUES
205
(5 H, m, Ph cT), 6.183 (1 H, t,
(1 H, d, 1',2' 6.16, H1' cT), 5.045 (1 H, t,
Ta), 4.656 (1 H, d, 2Ja,b 11.84, Ha PhCH2O cT), 4.588 dd,
J
6.59, H1' Ta), 5.792 t,
5.15, 5'ꢀOH m, H3', H4' cT), 3.734 (6H, s, (CH3)2Tr), 3.273 (1 H,
4',5'a 4.41, 2
5'a,5'b 10.56, H5'a Ta), 3.171 (1 H, dd,
4',5'a 1.99, 2
5'a,5'b 10.56, H5'b Ta), 2.319 (1 H, m, H2'a
5.61, H2' cT), 4.321 (1 H, m, H3' Ta), 3.972 (1 H, m, Ta), 2.08–2.25 (3 H, m, H6'a,b cT; H2'b Ta), 1.908
H4' cT), 3.762 (2 H, m, H3' cT; H4' Ta), 3.632 (1 H, (1 H, m, H5'a cT), 1.823 (1 H, m, H5'b cT). 1.792
m, H5'a Ta), 3.551 (1 H, m, H5'b Ta), 2.13–2.30 (3 H, (3 H, d, 0.99, 5ꢀCH3 cT), 1.445 (3 H, d, 1.1, 5ꢀCH3
m, H6'a,b cT; H2'a TA), 2.073 (1 H, m, H2'b Ta), Ta), 0.824 (9 H, s, (CH3)3CSi), 0.035 (3 H, s, CH3Si),
1.925 (2 H, m, H5'a,b cT), 1.819 (3 H, d, 0.85, 5ꢀCH3 0.001 (3 H s, CH3Si). Mass spectrum: m/z 939.8. Calꢀ
cT), 1.784 (3 H, d, 0.65, 5 CH3 Ta), 0.814 (9 H, s, culated 939.12 [
–H]– (C49H60N5O12Si)
tertꢀButyldimethylsilylꢀ5'ꢀdeoxyꢀ5'ꢀ{[
ꢀdimethoxytrityl)ꢀ3'ꢀdeoxythymidineꢀ3'ꢀyl)carboxaꢀ
mido]methyl}ribosylthymineꢀ3'ꢀ(2ꢀcyanethylꢀ ꢀdiisoꢀ
J 4.99, H2' cT), 3.876 (1H, m, H4' Ta), 3.734 (2 H,
J
J
J
J
2
(1 H, d, Ja,b 11.84, Hb PhCH2O cT), 4.490 (1 H, t,
J
J
J
J
J
J
J
M
.
(CH3)3CSi), 0.039 (3 H, s, CH3Si), –0.029 (3 H, s,
2'ꢀ
(4,4
Oꢀ
Nꢀ(5'ꢀOꢀ
CH3Si). Mass spectrum: m/z 727.4. Calculated 726.88
'
[M
–H]– (C35H48N5O10Si).
N
propylphosphoramidite) (XI). DIPEA (0.41 g, 0.54 ml,
3.2 mmol) and then 2ꢀcyanethylꢀN,N'ꢀdiisopropylꢀ
chlorophosphoramidite (0.37 g, 1.58 mmol) were
2'ꢀOꢀtertꢀButyldimethylsilylꢀ5'ꢀdeoxyꢀ5'ꢀ{[Nꢀ(3'ꢀ
deoxythymidineꢀ3'ꢀyl)carboxamido]methyl}ribosylthꢀ
ymine (IX). Dinucleotide (VIII) (0.9 g, 1.2 mmol) in
abs. THF (10 ml) was hydrogenated over Pd/C (10%,
0.18 g) at room temperature and atmospheric pressure
for 5 h. The reaction mixture was filtered through
added to the solution of dinucleotide (
0.79 mmol) in dry CH2Cl2 (5 ml). The mixture was
stirred for 2 h, poured into cold saturated NaHCO3
X) (0.74 g,
,
and stirred for 5 min. CH2Cl2 (50 ml) was added and
the mixture was divided. The water layer was extracted
with CH2Cl2 (20 ml), organic extracts were washed
celite, the residue was washed with THF (
4 10 ml),
×
and the filtrate was evaporated. Compound (IX) was
prepared in solid foam form in a yield of 0.76 g (99%),
Rf 0.47 (В). 1H NMR: 11.290 (1 H, bs, H3 cT), 11.236
with saturated NaHCO3 (2 20 ml) and saturated NaCl
×
(20 ml), dried with anhydrous Na2SO4, and evapoꢀ
rated. The residue was chromatographed on a silicaꢀ
gel column with a methylene chloride–2% methanol
mixture containing 0.2% triethylamine. The yield
of compound (XI) in solid foam was 0.66 g (74%),
Rf 0.46 (А). 31P NMR: 148.464, 146.749. Mass specꢀ
(1 H, bs, H3 Ta), 8.271 (1 H, d,
7.754 (1 H, bd, 0.96, H6 cT), 7.447(1 H, bd,
H6 Ta), 6.181 (1 H, t, 6.52, H1' Ta), 5.725 (1 H, d,
J1',2' 5.11, H1' cT), 5.031 (1 H, t, 4.98, 5'ꢀOH Ta),
4.891 (1 H, d, 5.61, 3'ꢀOH, cT), 4.312 (1 H, m, H3'
Ta), 4.207 (1 H, t, 5.05, H2' cT), 3.749 (3 H, m, H3',
H4' cT; H4' Ta), 3.629 (1 H, m, H5'a Ta), 3.549 (1 H,
m, H5'b Ta), 2.205 (3 H, m, H6'a,b cT; H2'a Ta), 2.075
(1 H, m, H2'b Ta), 1.946 (1 H, m, H5'a cT), 1.836
J
7.26, 3'ꢀNH Ta),
J
J
0.87
,
J
J
J
J
trum: m/z 1139.8
.
Calculated 1139.34 [M
–H]–
(C58H77N7O13PSi)
.
(1 H, m, H5'b cT), 1.803 (3 H, d,
1.781 (3 H, d, 0.87, 5 CH3 Ta), 0.827 (9 H, s,
(CH3)3CSi), 0.039 (3 H, s, CH3Si), 0.004 (3 H, s,
CH3Si). Mass spectrum: m/z 637.5. Calculated 636.75 tion for Basic Research, project no. 08ꢀ04ꢀ01078.
–H]– (C28H42N5O10Si).
2'ꢀ tertꢀButylmethylsilylꢀ5'ꢀdeoxyꢀ5'ꢀ{[
(4,4'ꢀdimethoxytrityl)ꢀ3'ꢀdeoxythymidineꢀ3'ꢀyl)carboxaꢀ
mido]methyl]ribosylthymine (X). Dinucleotide (IX
(0.7 g, 1.1. mmol) was evaporated with abs. pyridine
(2 5 ml) and dissolved in abs. pyridine (5 ml).
J 0.96, 5ꢀCH3 cT),
ACKNOWLEDGMENTS
The work was supported by the Russian Foundaꢀ
J
[
M
Oꢀ
Nꢀ(5'ꢀOꢀ
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2010