Monomers for oligonucleotide synthesis with linkers carrying reactive residues: II. The synthesis of phosphoamidites on the basis of uridine and cytosine and containing a linker with methoxyoxalamide groups in position 2′

Article abstract of DOI:10.1023/B:RUBI.0000030130.99454.ae

A convenient preparative synthesis of 2′-amino-2′-deoxyuridine was developed. Starting from 2′-amino-2′-deoxyuridine and 2′-amino-2′-deoxycytidine, monomers for the phosphoamidite oligonucleotide synthesis were obtained that carry a linker with methoxyoxalamide groups in position 2′.

Full text of DOI:10.1023/B:RUBI.0000030130.99454.ae

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 3, 2004, pp. 234–241. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 3, 2004, pp. 264–272.
Original Russian Text Copyright © 2004 by Vasil’eva, Abramova, Ivanova, Shishkin, Sil’nikov.
Monomers for Oligonucleotide Synthesis
with Linkers Carrying Reactive Residues:
II.1 The Synthesis of Phosphoamidites on the Basis of Uridine
and Cytosine and Containing a Linker
with Methoxyoxalamide Groups in Position 2'
2
S. V. Vasil’eva, T. V. Abramova, T. M. Ivanova, G. V. Shishkin, and V. N. Sil’nikov
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences,
pr. akademika Lavrent’eva 8, Novosibirsk, 630090 Russia
Received December 20, 2002; in final form, March 3, 2003
Abstract—A convenient preparative synthesis of 2'-amino-2'-deoxyuridine was developed. Starting from
2'-amino-2'-deoxyuridine and 2'-amino-2'-deoxycytidine, monomers for the phosphoamidite oligonucleotide
synthesis were obtained that carry a linker with methoxyoxalamide groups in position 2'.
Key words: 2'-amino-2'-deoxynucleosides, modified nucleosides, methoxyoxalamide groups
21
INTRODUCTION
gates based on monomers containing reactive methoxy-
oxalamide groups in position 5' of thymidine has been
reported in 2000 [17]. Phosphoamidites of 2'-modified
nucleosides make possible the synthesis of series of oli-
gonucleotides containing modified units both at the
ends and in the middle of oligomeric chain.
The goal of this study was the synthesis of mono-
mers for the synthesis of phosphoamidite oligonucle-
otides containing reactive methoxyoxalamide groups in
position 2'.
Modified oligonucleotides containing active groups
are widely used for the solution of many problems of
molecular biology and bioorganic chemistry and also
for therapeutic and diagnostic purposes. The oligonu-
cleotides modified at their carbohydrate moiety are of
particular interest [2, 3]. It has been stressed in litera-
ture that the substituent in position 2' of carbohydrate
moiety plays a unique role in nucleic acids. It directly
affects the conformation of sugar–phosphate backbone
and, thereby, retards the NA hydrolysis by nucleases [4,
5]. It was previously shown that the use of DNA probes
containing 2'-modified pyrimidine nucleosides enables
a regiospecific hybridase cleavage of RNA by RNase H
[6–10]. Nucleosides with an amino group in the ribose
ring are efficient antibacterial and antitumor prepara-
tions and also can act as synthetic inhibitors of a num-
ber of enzymes [11–14]. At the same time, the presence
of 2'-amino group allows an easy functionalization of
nucleotide unit. In particular, the amino groups in this
position can be modified by fluorescein isothiocyanate
(FITC) and carboxylic and dicarboxylic anhydrides
[15].
RESULTS AND DISCUSSION
We chose 2'-amino-2'-deoxyuridine as a starting
compound for the introduction of methoxyoxalamide
groups in position 2' of pyrimidine nucleotides. Several
variants of large-scale laboratory synthesis of this com-
pound have been described; however, a poor reproduc-
ibility [18, 19] and the use of hardly available com-
pounds in the syntheses [20] prompted us to seek for a
simpler and more efficient method. Therefore, we mod-
ified the synthetic method described in [21] (Scheme 1).
Uridine (I) was converted into 2,2'-O-anhydro-1-(β-D-
arabinofuranosyl)uracil (II) in good yield using the
procedure described in [18]. The interaction of (II) with
sodium azide in the presence of dibenzo-18-crown-6 in
dimethylacetamide led to 2'-azido-2'-deoxyuridine
(III) in 78% yield as indicated by HPLC. Despite the
high content of nucleoside (III) in the reaction mixture,
we were unable to find conditions for isolating the tar-
get compound without the use of chromatographic sep-
aration. Note that the increase in the amount of crown
ether does not increase the yield of target product. The
The nucleosides with amino groups in ribose ring
are useful synthons for obtaining phosphoamidites con-
taining active groups. The use of such monomers can
help synthesize series of oligonucleotide conjugates on
the basis of one precursor; a similar approach was real-
ized in [16]. The synthesis of oligonucleotide conju-
1
For Part I, see [1].
2
Corresponding author; phone +7 (3832) 33-3762; e-mail:
vasilieva_2001@ngs.ru
1068-1620/04/3003-0234 © 2004 MAIK “Nauka /Interperiodica”

MONOMERS FOR OLIGONUCLEOTIDE SYNTHESIS…II.
235
O
N
O
N
O
HN
HN
HN
O
O
O
N
HO
O
HO
HO
O
O
(PhO)2CO/NaHCO3
DMF
NaN3/dibenzo-18-crown-6
CH3CONMe2
OH N3
OH OH
OH
(I)
(II)
(III)
O
O
N
HN
HN
O
N
O
HO
HO
O
O
CF3COOC2H/Et3N
MeOH
H2/Pd(C)
MeOH
OH NH2
OHHN
O
(IV)
CF3
(V)
O
O
HN
HN
O
N
O
N
(MeO)2Tr O
(MeO)2Tr O
O
O
(MeO)2TrCl
NH3
pyridine
dioxane
OHHN
CF3
OH NH2
(VII)
O
(VI)
Scheme 1. Synthesis of 2'-amino-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyuridine.
optimum temperature for the reaction is 130–135°ë. of nucleoside (VII) in 25% yield from uridine without
The temperature of 140°ë and above leads to a consid- the use of chromatographic separations of reaction
erable gum formation and a decreased yield. At the mixtures on silica gel.
same time, a lower temperature (125°ë) induces a
There is only a short report on the synthesis of 5'-O-
sharp fall of the reaction rate.
(4,4'-dimethoxytrityl)-2'-methoxyoxalylamido-2'-deoxy-
The introduction of dimethoxytrityl protective
group for 5'-hydroxyl at this step seems very attractive
uridine (VIII) in literature [22]. We describe here a mod-
ified procedure for the synthesis of this compound,
at first sight. However, this leads to the formation of
which is similar to that described in [17]. It was
complex reaction mixtures requiring chromatographic
obtained by the reaction of nucleoside (VII) with dime-
separation. Another way, which involved the use of a
thyl oxalate (Scheme 2). The phosphitylation of (VIII)
temporary protection of 2'-amino group, turned out to
by the standard procedure [1] led to phosphoamidite
be more rational. Crude nucleoside (III) was catalyti-
(IX).
cally hydrogenated, and the resulting 2'-amino-2'-deoxy-
uridine (IV) was purified on a KRS-2p ion-exchange
2'-[N,N-Bis(2-methoxyoxalylamidoethyl)aminoet-
column. Then, 2'-amino group was protected by triflu- hyl]amidooxalamido-5'-O-(4,4'-dimethoxytrityl)-2'-
oroacetyl group, nucleoside (V) was tritylated at the 5'- deoxyuridine (XI) can be obtained either from nucleo-
hydroxyl group, and the trifluoroacetyl was removed to side (VII) by the successive elongation of the linker
give the target 2'-amino-5'-O-(4,4'-dimethoxytrityl)-2'- chain (Scheme 2, pathway A) or by treatment with the
deoxyuridine (VII). This pathway enables the synthesis preliminarily synthesized tris(carbomethoxycarbam-
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VASIL’EVA et al.
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MONOMERS FOR OLIGONUCLEOTIDE SYNTHESIS…II.
237
oylethyl)amine [1] (Scheme 2, pathway B). The second and had reagent, analytical, or special purity grades.
pathway appeared to be preferable due to a smaller They were purified, if necessary, by standard proce-
number of the synthetic stages. However, the yield of dures [25]. Preparative HPLC was carried out using a
the product by this reaction was very low under various Millikhrom 4 chromatograph equipped with a column
conditions. At the same time, successive reactions of (2 × 50 mm) packed with Lichrosorb RP-18, 10 µm
nucleoside (VII) with dimethyl oxalate, tris(2-aminoet- (Merck, Germany) and eluted with a linear gradient of
hyl)amine, and dimethyl oxalate again without the iso- acetonitrile concentration in water (0–40%) at a rate of
lation of intermediate (X) allowed us to obtain nucleo- 100 µl/min. For column chromatography, Porasil Silica
side (XI) in a good yield. Note that the introduction of 125A (55–105 µm; Millipore, United States) was used.
the subsequent methyl oxalate groups was more easy The columns were eluted with a gradient of methanol
than that of the first group, which was probably facili- (0–20%) in chloroform. TLC was carried out on Kiesel-
tated by decreased steric hindrances. The standard gel 60 F254 Alufolien (Merck, Germany) using the fol-
phosphitylation of (XI) led to phosphoamidite (XII).
lowing solvent systems: (A) 9 : 1 dichloromethane–
methanol, (B) 9 : 1 : 0.1 dichloromethane–methanol–
triethylamine, (C) 4 : 1 ethyl acetate–methanol, (D) 9 :
0.8 : 0.1 dichloromethane–methanol–triethylamine, (E)
8.7 : 1.2 : 0.1 dichloromethane–methanol–triethyl-
amine, (F) 8 : 2 : 0.1 dichloromethane–methanol–tri-
ethylamine, and (G) 8 : 6 : 3 : 3 tert-butyl alcohol–
methylethylketone–formic acid–water. 1ç and 31P
NMR spectra were recorded on a Bruker WP-200-SY
and Bruker AM-400 spectrometers (Germany) in
CDCl3, CD2O, or deuteroacetone. Tetramethylsilane
An attempt to synthesize 2'-amino-5'-O-(4,4'-
dimethoxytrityl)-2'-deoxycytidine (XVIII) by the
method that was used for obtaining nucleoside (IV)
starting from N4-benzoyl-5'-O-(4,4'-dimethoxytri-
tyl)cytidine [9] failed. On the other hand, uridine can be
easily transformed into cytidine [18, 19, 23]. We
obtained (XVIII) using 2'-azido-2'-deoxyuridine (III)
as a starting compound (Scheme 3). Uridine was con-
verted into cytidine through the triazolide derivative
(XV) as described in [19]. The starting nucleoside (III)
was tritylated to form 2'-azido-5'-O-(4,4'-dimethoxytri-
tyl)-2-deoxyuridine (XIII). The subsequent reaction
with trimethylchlorosilane led to 2'-azido-5'-O-(4,4'-
dimethoxytrityl)-3'-O-trimethylsilyl-2'-deoxyuridine
(XIV), which was converted into the triazolide deriva-
tive (XV) by treatment with a mixture of POCl3 and
1,2,4-triazole. The treatment of (XV) with ammonia
yielded 2'-azido-5'-O-(4,4'-dimethoxytrityl)-2'-deoxy-
cytidine (XVI), and the azide group in this compound
was reduced by catalytic hydrogenation. The reaction
of dimethyl oxalate with amino group of the resulting
nucleoside (XVII) proceeds much quicker and under
milder conditions than the similar reaction with 2'-
amino-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyuridine
(VII) and does not affect the exocyclic amino group of
the base. The following protection of this amino group
by benzoic anhydride [24] leads to (XIX) in a good
yield. The standard phosphitylation of this nucleoside
yields monomer (XX).
1
was used as an internal standard for H NMR spectra,
and 85% phosphoric acid in D2O for 31P NMR spectra.
Chemical shifts (δ, ppm) and spin–spin coupling con-
stants (J, Hz) are given. Melting points were measured
on a Koffler plate (VEB Analytic, Germany).
2,2'-O-Anhydro-1-(b-D-arabinofuranosyl)uracil
(II) was obtained from uridine (I) (40 g, 0.16 mol) in
95% yield as described in [18].
2'-Amino-2'-deoxyuridine (IV). A suspension of
nucleoside (II) (10 g, 44 mmol), sodium azide (5.75 g,
88 mmol), and dibenzo-18-crown-6 (3.2 g, 8.8 mmol)
in dimethylacetamide (200 ml) was heated at 130–
135°ë under stirring in argon atmosphere and were kept
at this temperature for 36 h when analyzing the mixture
by HPLC. The reaction mixture was cooled to room
temperature, and the precipitate was filtered and
washed with ethyl alcohol. The filtrate and alcohol
washings were evaporated in a vacuum. Water (200 ml)
was added to the residue, and the precipitate was fil-
tered off and washed with water. The resulting aqueous
solution was passed through a column (4 × 18 cm) of
Thus, we obtained monomers (IX), (XII), and (XX)
with reactive methoxyoxalamide groups in position 2',
which can be used for the phosphoamidite oligonucle- KRS-2p ion-exchange resin (ç+ form), and the column
otide synthesis.
was then washed with water. All water fractions were
combined and evaporated. The resulting crude 2'-azido-
2'-deoxyuridine (III) was obtained as a yellowish
brown foam. An analytical sample was purified by col-
umn chromatography on a Preparative C18 reversed
phase column (Waters). The elution with a methanol
gradient (0 to 20%) in water and evaporation of the tar-
get fractions gave the product as a white foam; Rf 0.66
(B); UV [MeOH, λmax, nm, (ε)]: 260 (10 179); IR (KBr,
EXPERIMENTAL
We used uridine, diphenyl carbonate, dimethyl
oxalate, 2-cyanoethyl N,N,N',N'-tetraisopropylphos-
phoroamidite, tris(2-aminoethyl)amine, and palladium
on active carbon from Aldrich (United States); trimeth-
ylsilylchloride from Fluka (Switzerland); 4,4'-
dimethoxytrityl chloride and 1,2,4-triazole from Chem-
IMPEX (United States); phosphoryl chloride from
Merck (Germany); and sodium azide from (Germany).
ν, cm–1): 2114 (N3); 1H NMR (200 MHz, D2O): 8.0 (1
H, d, J 8, H6), 6.11 (1 H, d, J 4, H1'), 6.12 (1 H, d, J 8,
H5), 4.55 (1 H, t, J 5, H3'), 4.47 (1 H, dd, J1',2' 4, J2',3' 5,
Solvents and other chemical reagents were from Russia H2'), 4.24 (1 H, m, H4'), and 4.03 (2 H, m, H5').
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

238
VASIL’EVA et al.
O
N
O
O
N
NH
NH
NH
O
N
O
O
HO
(MeO)2Tr
O
(MeO)2Tr O
O
O
O
Me3SiCl
Et3N
(MeO)2TrCl
pyridine
OH N3
OH N3
O
N3
SiMe3
(XIV)
(III)
(XIII)
N
N
N
NH2
N
N
N
O
N
O
(MeO)2Tr
O
O
(MeO)2Tr
O
O
NH3 (ammonia)
POCl3/ 1,2,4-triazole
Et3N
dioxane
O
N3
OH N3
SiMe3
(XV)
NH2
(XVI)
NH2
N
N
O
N
O
N
O
CH3
O
O
(MeO)2Tr
O
(MeO)2Tr O
H3C
O
O
O
H2/Pd(C)
MeOH
Et3N/MeOH
O
OH NH2
(XVII)
OHHN
CH3
O
O
O
(XVIII)
O
HN
N
HN
N
N
N
P
N
O
CN
O
N
O
(MeO)2Tr O
(MeO)2Tr O
O
O
(C6H5CO)2O
CH3CONMe2
O
O
OHHN
CH3
HN
CH3
O
O
O
O
O
CN
P
(XIX)
O
N
(XX)
Scheme 3. Synthesis of a phosphoamidite reagent based on cytidine, with a methoxyoxalylamide group in position 2'.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

MONOMERS FOR OLIGONUCLEOTIDE SYNTHESIS…II.
239
A suspension of 5% Pd/C catalyst (1 g) in methanol reaction mixture was kept for 10 h at 50°ë and left
(5 ml) was saturated with hydrogen at 1.5 atm pressure overnight at room temperature. The reaction mixture
under stirring, and a solution of (III) in methanol was evaporated to a volume of 3–4 ml and precipitated
(95 ml) was added. The mixture was kept overnight at with hexane. The precipitate was chromatographed on
1.5 atm pressure of hydrogen, the catalyst was filtered a silica gel column eluted with a gradient of methanol
off and washed several times with methanol. The meth- (0–10%) in chloroform with 1% of triethylamine. The
anol fractions were combined, fivefold diluted with fractions containing the product were evaporated, and
water, and passed through a column of KRS-2p resin the residue was dissolved in chloroform (2 ml) and pre-
(H+ form, diameter 3 cm, height 7 cm). The column was
washed with 20% aqueous methanol (50 ml). The prod-
cipitated with 20 ml of hexane. After drying the precip-
itate in a vacuum, the yield of a white powder of (VIII)
1
uct was eluted from the resin with 25% ammonia was 304 mg (52%); Rf 0.71 (A); H NMR (400 MHz,
(20 ml) and washed with 20% aqueous methanol
(15 ml). The evaporation of fractions containing the tar-
get product (V) gave a foam; yield 4.8 g (44.8%); Rf
0.44 (G); mp 197–198°C (ethanol); UV spectrum cor-
responds to that reported in [21]; 1H NMR (400 MHz,
D2O): 7.94 (1 H, d, J 8, H6), 6.01 (1 H, d, J 8, H1'), 6.02
(2 H, d, J 8, H5), 4.32 (1 H, dd, J3',4' 3, J3',2' 6, H3'), 4.27
(1 H, dd, J4',3' 3, J4',5' 5, H4'), 3.96 (2 H, m, H5'), 3.68 (1
H, dd, J2',1' 8, J2',3' 6, H2').
CDCl3): 8.10 (1 H, d, J 8, 2'-NH), 7.64 (1 H, d, J 8, H6),
7.38–7.20 (9 H, m, Ar), 6.81 (4 H, m, Ar), 6.21 (1 H, d,
J 8, H1'), 5.40 (1 H, d, J 8, H5), 4.7 (1 H, m, H2'), 4.49
(1 H, dd, J2',3' 5, J3',4' 2, H3'), 4.2 (1 H, dd, J3',4' 2, J4',5' 3,
H4'), 3.83 (3 H, s, CO2CH3), 3.76 (6 H, s, Ar, OCH3),
3.42 (2 H, d, J 3, H5').
A general procedure for obtaining phosphoamid-
ite reagents for oligonucleotide synthesis. 2-Cyanoet-
hyl-N,N,N',N'-tetraisopropylphosphoroamidite (4.5 ml,
14 mmol and, after 2, h 2.2 ml, 6.9 mmol) was added
portionwise under stirring to a solution of the protected
nucleoside (10 mmol) and diisopropylammonium tetra-
zolide (900 mg, 4.47 mmol) in dichloromethane
(50 ml). After 3–7 h (TLC monitoring), the reaction
mixture was evaporated to dryness, covered with hex-
ane, and left overnight. Then hexane was decanted, and
the residue was chromatographed on a silica gel col-
umn. The elution with a gradient of methanol (0–10%)
in dichloromethane and the evaporation of target frac-
tions gave the product, which was precipitated from
dichloromethane by hexane and dried in a vacuum.
2'-Amino-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyuri-
dine (VII). Ethyl trifluoroacetate (8.42 ml, 70.8 mmol)
was added to a mixture of nucleoside (IV) (8.6 g,
35.4 mmol) and triethylamine (9.85 ml, 70.8 mmol) in
dry methanol (90 ml) under stirring. The reaction mix-
ture was kept for 2 h, evaporated to dryness and then
two times coevaporated with dry pyridine. A solution of
4,4'-dimethoxytrityl chloride (12.0 g, 35.4 mmol) in
pyridine (70 ml) was dropwise added under stirring to
a solution of the nucleoside in pyridine (20 ml) on an
ice bath. The mixture was kept for 1 h at this tempera-
ture, heated to room temperature, left overnight, and
evaporated. The residue was dissolved in dichlo-
romethane (50 ml), and the solution was washed with
water (2 × 50 ml) and 5% sodium bicarbonate (50 ml),
and evaporated. The residue was dissolved in dioxane
(70 ml), 25% ammonia (140 ml) was added under stir-
ring, and the mixture was left overnight and evaporated.
The residue was dissolved in dichloromethane (50 ml),
and washed with water (2 × 50 ml) and 5% sodium
bicarbonate (50 ml). The organic layer was dried with
anhydrous sodium sulfate, evaporated to a volume of
10 ml, and precipitated with hexane (100 ml). The
reprecipitation was repeated twice. The product (VII)
was obtained as a yellow powder; yield 13 g (67.3%);
Rf 0.33 (B); UV spectrum corresponds to that reported
5'-O-(4,4'-Dimethoxytrityl)-2'-methoxyoxalyl-
amido-3'-(2-cyanoethyl-N,N-diisopropylamino)phos-
phinyl-2'-deoxyuridine (IX) was obtained from (VIII)
(137 mg, 0.22 mmol) by the standard procedure. The
reaction proceeded for 3.5 h and gave 116 mg (64%) of
a white powder; Rf 0.76 (D); 31P NMR: (400 MHz,
CDCl3): 151.4, 151.3.
2'-[N,N-Bis(2-methoxyoxalylamidoethyl)aminoet-
hyl]-5'-O-(4,4'-dimethoxytrityl)amidooxalylamido-
2'-deoxyuridine (XI). A solution of (VIII) (304 mg,
0.48 mmol) in methanol (7 ml) was added portionwise
(200 µl in each portion) for 1 h under stirring to a solu-
tion of tris(2-aminoethyl)amine (288 µl, 1.92 mmol) in
methanol (2 ml). After 2 h, the reaction mixture was
evaporated, and the residue was dissolved in chloro-
form (30 ml) supplemented with 1% triethylamine. The
solution was washed with water (2 × 30 ml) and a 5 M
sodium chloride (30 ml). The organic layer was dried
with anhydrous sodium sulfate, evaporated, and then
coevaporated with dry acetonitrile (20 ml) and dry
in [18]; 1H NMR (200 MHz, acetone-d6): 7.73 (1 H, d,
J 8, H6), 7.49–7.21 (9 H, m, Ar), 6.80 (4 H, m, Ar), 6.01
(1 H, d, J 4, H1'), 5.40 (1 H, d, J 8, H5), 4.64 (1 H, dd,
J2',3' 7, J3',4' 4, H3'), 4.34 (1 H, dd, J2',1' 4, J2',3' 7, H2');
4.23 (1 H, m, H4'); 3.76 (6 H, s, CH3), and 3.47–3.30
(2 H, m, H5').
5'-O-(4,4'-Dimethoxytrityl)-2'-methoxyoxalyl- methanol (20 ml). The product in the form of a white
amido-2'-deoxyuridine (VIII). A solution of nucleo- foam (367 mg) was dissolved in methanol–triethyl-
side (VII) (536 mg, 0.98 mmol) in a mixture of trieth- amine mixture (3 + 1.8 ml) and dropwise added under
ylamine (750 l, 5.4 mmol) and methanol (10 ml) was stirring to a solution of dimethyl oxalate (579 mg,
dropwise added under stirring to a solution of dimethyl 4.9 mmol) in methanol (2 ml). After 6, the reaction
oxalate (640 mg, 5.4 mmol) in methanol (5 ml). The mixture was evaporated to a volume of 2 ml and precip-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

240
VASIL’EVA et al.
itated with a 1 : 1 hexane–diethyl ester mixture. The sodium chloride (20 ml). The organic phase was evap-
precipitate was chromatographed on a silica gel col- orated and dissolved in dioxane (25 ml). Ammonia
umn, eluted with a gradient of methanol (0–10%) in (25%, 25 ml) was added, and the mixture was stirred for
chloroform supplemented with 1% triethylamine. The 48 h at room temperature. The reaction mixture was
fractions containing the product were evaporated, and evaporated, dissolved in methylene chloride (30 ml)
the residue was dissolved in 2 ml of dichloromethane supplemented with 1% triethylamine, and washed with
and precipitated with 20 ml of ether. The product (XI) water (2 × 20 ml) and a 5 M sodium chloride (20 ml).
was obtained as a white powder; yield 235 mg (54%); The organic layer was dried with anhydrous sodium
Rf 0.45 (Ä); 1H NMR (200 MHz, CDCl3): 8.9 (1 H, s,
H3), 8.4 (1 H, d, J 8, 2'-NH), 7.9 (1 H, t, J 5,
CONHCH2), 7.7 (2 H, t, J 5, −CONHCH2–), 7.65 (1 H,
d, J 8, H6), 7.37–7.23 (9 H, m, Ar), 6.81 (4 H, m, Ar),
6.31 (1 H, d, J 8, H5), 4.58 (2 H, m, H2', H3'), 4.26 (1
H, dd, J3',4' 1, J4',5' 3, H4'), 3.81 (6 H, s, CO2CH3), 3.75
(6 H, s, Ar, OCH3), 3.44 (2 H, d, J4',5' 3, H5'), 3.40 (6 H,
m, CONHCH2), and 2.7 [6 H, m, N(CH2CH2)3].
2'-[N,N-Bis(2-methoxyoxalylamidoethyl)amino-
ethyl]amidooxalylamido-5'-O-(4,4'-dimethoxytri-
tyl)-3'-(2-cyanoethyl-N,N-diisopropylamino)phos-
phinyl-2'-deoxyuridine (XII) was obtained by the
method described above from nucleoside (XI) (235 mg,
0.26 mmol). The reaction time was 7 h. The product
was obtained as a white powder; yield 269 mg (93%);
Rf 0.65 (E); 31P NMR (400 MHz, CDCl3): 151.7, 151.4.
2'-Azido-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyuri-
dine (XIII) was obtained from 2'-azido-2'-deoxyuri-
dine (III) (1.66 g, 6.2 mmol) as described in [19] with
the addition of 4-dimethylaminopyridine (189 mg,
1.6 mmol) to the reaction mixture; yield 2.94 g (83%);
Rf 0.66 (B); 1H NMR (200 MHz, acetone-d6): 7.83 (1 H,
d, J 8, H6), 7.48–7.26 (9 H, m, Ar), 6.88 (4 H, m, Ar),
5.9 (1 H, d, J 8, H1'), 5.34 (1 H, d, J 8, H5), 4.72 (1 H,
t, J 6, H2'), 4.29 (1 H, dd, J2',3' 6, J3',4' 4, H3'), 4.11 (1 H,
m, H4'), 3.77 (6 H, s CH3), 3.48 (2 H, d, J4',5' 4, H5').
sulfate, evaporated, and the residue was chromato-
graphed on a silica gel column. Elution with a gradient
of methanol (0–20%) in methylene chloride supple-
mented with 1% triethylamine. After the evaporation of
target fractions, the product was obtained as a white
1
foam; yield 451 mg (16%); Rf 0.27 (B); H NMR
(200 MHz, acetone-d6): 7.96 (1 H, d, J 4, H6), 7.49–
7.22 (9 H, m, Ar), 6.91 (4 H, m, Ar), 5.86 (1 H, d, J 2,
H1'), 5.71 (1 H, d, J 4, H5), 4.75 (1 H, dd, J1',2' 2, J2',3' 3,
H2'), 4.27 (1 H, dd, J2',3' 3, J3',4' 2, H3'), 4.10 (1 H, m,
H4'), 3.76 (6 H, s, CH3), 3.48 (2 H, d, J4',5' 2, H 5').
2'-Amino-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycy-
tidine (XVII). A suspension of 5% Pd/C catalyst
(140 mg) in methanol (5 ml) was saturated with hydro-
gen at a pressure of 1.5 atm under stirring. Then a solu-
tion of (XVI) (191 mg, 0.33 mmol) in methanol (5 ml)
was added and kept for 2 h at H2 pressure of 1.5 atm.
The catalyst was filtered off and washed with methanol
(2 × 10 ml). The combined washings were evaporated to
give 160 mg of a white foam; yield 88%; Rf 0.38 (F); 1H
NMR (200 MHz, acetone-d6): 7.72 (1 H, d, J 4, H6),
7.48–7.23 (9 H, m, Ar), 6.88 (4 H, m, Ar), 5.98 (1 H, d,
J 2, H1'), 5.70 (1 H, d, J 4, H5), 4.62 (1 H, m, H2'),
4.13–4.23 (2 H, m, H3', H4'), 3.77 (6 H, s, CH3), 3.30–
3.35 (2 H, m, H5'), 2.80 (2 H, br s, 2'-NH2). The NMR
spectrum corresponds to that reported in [18].
5'-O-(4,4'-Dimethoxytrityl)-2'-methoxyoxalyl-
amido-2'-deoxycytidine (XVIII). A solution of
(XVII) (160 mg, 0.29 mmol) in a mixture of triethy-
lamine (821 µl, 5.9 mmol) and methanol (2 ml) was
dropwise added under stirring to a solution of dimethyl
oxalate (349 mg, 2.96 mmol) in methanol (2 ml). After
3 h, the reaction mixture was evaporated, and the resi-
due was chromatographed on a silica gel column eluted
by a gradient of methanol (0–20%) in methylene chlo-
ride supplemented with 1% triethylamine. Fractions
containing the product were evaporated and precipi-
tated from methylene chloride (2 ml) with hexane
(20 ml). The product was obtained as a white powder;
yield 177 mg (97%); Rf 0.37 (C); 1H NMR (200 MHz,
acetone-d6): 7.98 (1 H, d, J 4, H6), 7.47–7.29 (9 H, m,
Ar), 6.92 (4 H, m, Ar), 6.26 (1 H, d, J 4, H1'), 6.08 (1 H,
d, J 4, H5), 4.99 (1 H, m, H2'), 4.29 (1 H, m, H3'), 4.14
(1 H, m, H4'), 3.77 (3 H, s, CO2CH3), 3.69 (6 H, s,
OCH3), 3.6–3.5 (2 H, m, H5').
2'-Azido-5'-O-(4,4'-dimethoxytrityl)-3'-O-trime-
thylsilyl-2'-deoxyuridine (XIV) was obtained from
(XIII) (2.94 g, 5.14 mmol) as described in [18]. The
product was isolated as a foam; yield 3.17 g (96%); Rf
0.72 (Ä); 1H NMR (200 MHz, acetone-d6): 7.85 (1 H, d,
J 5, H6), 7.49–7.25 (9 H, m, Ar), 6.93 (4 H, m, Ar), 5.94
(1 H, d, J 2, H1'), 5.35 (1 H, d, J 5, H5), 4.71 (1 H, t, J
3, H2'), 4.24 (1 H, dd, J2',3' 3, J3',4' 2, H3'), 4.10 (1 H, m,
H4'), 3.79 (6 H, s, CH3, 3.57–3.43 (1 H, dd, J4',5' 2, H5'),
0.10 [9 H, s, (CH3)3Si].
2'-Azido-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycy-
tidine (XVI) was obtained as described in [19]. Phos-
phoryl chloride (1.83 ml, 19.7 mmol) and, after 15 min,
triethylamine (13.7 ml, 98.4 mmol) were added under
stirring to a solution of 1,2,4-triazole (6.79 g,
98.4 mmol) in dry acetonitrile (20 ml) cooled on an ice
bath. The reaction mixture was kept for 30 min, a solu-
tion of (XIV) (3.17 g, 4.92 mmol) in dry acetonitrile
(20 ml) was added dropwise, and the mixture was
heated to room temperature. After 1.5 h, the reaction
N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-meth-
mixture was evaporated. The residue was dissolved in oxyoxalylamido-2'-deoxycytidine (XIX). Benzoic
chloroform (40 ml) supplemented with 1% triethyl- anhydride (63.3 mg, 0.308 mmol) was added to a solu-
amine and washed with water (2 × 20 ml) and a 5 M tion of (XVIII) (177 mg, 0.28 mmol) in DMF (2 ml)
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

MONOMERS FOR OLIGONUCLEOTIDE SYNTHESIS…II.
241
8. Shmidt, S., Kuznetsova, L.G., Romanova, E.A.,
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under stirring. The reaction mixture was left overnight,
evaporated, and the residue was chromatographed on a
silica gel column. Elution with a gradient of methanol
(0–10%) in dichloromethane supplemented with 0.1%
pyridine gave the fractions containing the product,
which were evaporated and precipitated from dichlo-
romethane (2 ml) with hexane (20 ml). The product was
obtained as a white powder; yield 73 mg (33%); Rf 0.71
10. Manoharan, M., Biochim. Biophys. Acta, 1999,
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1
(B); H NMR (200 MHz, acetone-d6): 8.15 (3 H, m,
COC6H5, H6), 7.53–7.33 (13 H, m, Tr, COC6H5, H5),
6.93 (4 H, m, Tr), 6.34 (1 H, d, J 8, H1'), 4.74 (2 H, m,
H2', H4'), 4.31 (1 H, m, H3'), 3.79 (3 H, s, CO2OCH3),
3.77 (6 H, s, OCH3), 3.51 (2 H, m, H5').
11. Suhadolnik, R.J., Nucleosides as Biological Probes,
New York: Wiley Interscience, 1979.
12. Suhadolnik, R.J., Nucleoside Antibiotics, New York:
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oxyoxalylamido-3'-(2-cyanoethyl-N,N-diisopropyl-
amino)phosphinyl-2'-deoxycytidine (XX) was
obtained by the standard method from 73 mg
(0.1 mmol) of nucleoside (XIX). The reaction time was
3 h. Yield of (XX) 52 mg (60%); 31P NMR (400 MHz,
CDCl3): 151.96, 151.61.
14. Verlinde, C.L., Callens, M., van Aerschot, A.,
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ACKNOWLEDGMENTS
The study was supported by the Russian Foundation
for Basic Research (project nos. 01-03-32439, 02-04-
48664), the Foundation for Assistance to National Sci-
ence and, a of BRHE Rec-008 grant.
18. McGee, D.P.C., Settle, A., Vargeese, Ch., and Zhai, Y.,
J. Org. Chem., 1996, vol. 61, pp. 781–785.
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