Monomers for oligonucleotide synthesis with linkers carrying reactive residues: I. The synthesis of deoxynucleoside derivatives with methoxyoxalamide groups in heterocyclic bases

Article abstract of DOI:10.1023/B:RUBI.0000030129.13466.18

A number of monomers for the standard phosphoamidite oligodeoxynucleotide synthesis that carry reactive methoxyoxalamide groups attached to the thymidine, 2′-deoxycytidine, and 2′-deoxyadenosine heterocyclic bases were prepared.

Full text of DOI:10.1023/B:RUBI.0000030129.13466.18

Russian Journal of Bioorganic Chemistry, Vol. 30, No. 3, 2004, pp. 224–233. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 3, 2004, pp. 254–263.
Original Russian Text Copyright © 2004 by Abramova, Vasil’eva, Ivanova, Shishkin, Sil’nikov.
Monomers for Oligonucleotide Synthesis
with Linkers Carrying Reactive Residues:
I. The Synthesis of Deoxynucleoside Derivatives
with Methoxyoxalamide Groups in Heterocyclic Bases
1
T. V. Abramova, S. V. Vasil’eva, 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, June 16, 2003
Abstract—A number of monomers for the standard phosphoamidite oligodeoxynucleotide synthesis that carry
reactive methoxyoxalamide groups attached to the thymidine, 2'-deoxycytidine, and 2'-deoxyadenosine hetero-
cyclic bases were prepared.
Key words: modified nucleosides, linkers carrying reactive groups, oligonucleotide synthesis
1
INTRODUCTION
nylalanine tRNA by an oligodeoxyribonucleotide con-
taining four catalytically active imidazole residues at its
5'-terminus [5]. These residues were introduced after
the completion of the solid-phase synthesis of the
addressing oligodeoxyribonucleotide carrying the reac-
tive methoxyoxalylamide groups at its 5'-end.
The synthesis of modified oligodeoxyribonucle-
otides carrying various fluorescent groups, residues of
intercalating residues, cationic lipids, etc. is currently
2
an almost routine procedure. Many modified oligonu-
cleotides are commercially available. Some companies,
e.g., Glen Research, purchase a variety of reagents for
the synthesis of modified oligonucleotides [1]. How-
ever, solution of the problems of molecular biology
associated with the directed effect on biopolymers
steadily requires new nucleotide derivatives. In view of
this, it is expedient to develop a maximally general
method for their modification. One of the approaches to
the solution of this problem is the precursor strategy
based on the intercalation of active precursor groups
into an oligonucleotide. It consists in the introduction
of linkers carrying chemically active groups that are
subsequently converted into the desired derivatives [2].
This approach is rather universal and also enables the
use of methods of combinatorial chemistry for the syn-
thesis of oligonucleotide derivatives.
One might expect that the introduction of catalyti-
cally active groups into the middle of the addressing
oligonucleotide chain would enhance the cleavage effi-
ciency of target due to a decreased binding of the
reagent to hydrolysis products and would also enable
varying the selectivity of artificial RNase [6]. In this
case, the use of linkers containing reactive groups is
promising since they enable the introduction of a set of
catalytically active groups into the artificial RNase con-
struct, with the synthesis of oligonucleotide addressing
moiety of the construct remaining easy. The phospho-
amidite reagents on the basis of nucleosides with prop-
erly modified heterocyclic bases are convenient to use
for the automated oligonucleotide synthesis when
inserting the linkers with terminal chemically active
groups into various positions of oligodeoxyribonucle-
otide. The possibility of modifying various positions of
purine and pyrimidine bases would allow a thorough
study of the interaction of modified oligonucleotides
with complementary nucleic acids, since the insertion
sites of reactive groups would affect their position in
both the small and large grooves of the complementary
duplex [7].
In recent years, we have been engaged in the synthe-
sis of artificial ribonucleases [3, 4]. The directed highly
specific cleavage of ribonucleic acids by imidazole res-
idues is possible when the synthetic ribonuclease con-
tains an addressing part, an oligodeoxyribonucleotide
complementary to a particular fragment of the ribonu-
cleic acid. A striking example of successful use of link-
ers carrying reactive groups for the synthesis of highly
specific artificial ribonucleases is the cleavage of phe-
This study is devoted to the synthesis of monomers
for the phosphoamidite oligonucleotide synthesis that
contain reactive methoxyoxalylamide groups attached
to the heterocyclic bases of thymidine, 2'-deoxycyti-
dine, and 2'-deoxyadenosine. A particular emphasis
1
Corresponding author; phone (3822) 33-3762; e-mail: abram-
ova@niboch.nsc.ru
2
Abbreviations: TBD, 1,5,7-triazabicyclo[4,4,0]dec-5-ene; TEA,
triethylamine; Tetr, tetrazole; and Tri, 1,2,4-triazole.
1068-1620/04/3003-0224 © 2004 MAIK “Nauka /Interperiodica”

MONOMERS FOR OLIGONUCLEOTIDE SYNTHESIS…I.
225
O
O
O
HN
N
CH3
O
O
H
O
N
O
O
HN
O
N
HN
O
NH2
N
H
O
N
O
O
RO
HO
CH3
O
O
i
N
H
O
O
O
O
N
H3C
N
H
3
OR'
OH
(I)
(III) R = R' = H
(II)
ii
(IV) R = (MeO)2Tr, R' = H
(V) R = (MeO)2Tr
iii
R' = P[O(CH2)2CN]N(iPr)2
Reagents: (i) (II)/TEA; (ii) (MeO)2TrCl; (iii) NC(CH2)2OP[N(iPr)2]2/Tetr-HN(iPr)2.
Scheme 1.
was placed on the use of the most readily available its 4-triazolyl- or 4-thioderivatives with the correspond-
starting compounds and the choice of synthetic ing amines [12, 13]. The search for the methods of syn-
schemes providing the greatest yields of target com- thesis of 2'-deoxycytidine derivatives using easily
pounds.
available starting compounds forced us to draw our
attention to a simple method of one-step synthesis of
N4-(6-aminohexyl)-2'-deoxycytidine protected at its 5'-
hydroxy group from N4-benzoyl-5'-O-(4,4'-dimethoxy-
RESULTS AND DISCUSSION
2'-Deoxyuridine or its derivatives are usually used trityl)-2'-deoxycytidine (VI) by the transamination
as starting compounds for the synthesis of thymidine reaction [14]. This method allowed us to similarly syn-
thesize N4-(3-aminopropyl)-2'-deoxycytidine protected
at 5'-hydroxy group (VII) (Scheme 2). The yield of the
transamination reaction was relatively low (40%).
However, the availability of starting compound (VI),
which is a precursor of the phosphoamidite reagent for
the standard oligonucleotide synthesis, and the possi-
bility of regeneration of the starting nucleoside from the
major by-product 5'-(4,4'-dimethoxytrityl)-2'-deoxy-
cytidine (VIII) (Scheme 2) made this method of obtain-
ing (VII) rather attractive. At the next stage of the syn-
thesis, we introduced into the modified nucleoside
(VII) a linker with reactive methoxyoxalylamide
groups by analogy with the reaction of modification of
the aminopropyl thymidine derivative (I) (Scheme 1i).
The last stage of the synthesis consisted in the phosphi-
tylation of 3'-hydroxy group of nucleoside (IX) to yield
phosphoamidite (X) suitable for oligonucleotide syn-
thesis.
analogues modified at the heterocyclic base [8, 9].
However, we used thymidine as a starting compound
because of its commercial availability. The synthesis of
thymidine derivatives containing 5-(2-aminoe-
thyl)oxymethyl linker was reported in [10]. In a similar
way, we obtained the thymidine derivative (I) contain-
ing a 5-(3-aminopropyl)oxymethyl residue (Scheme 1).
The derivative with two methoxyoxalylamide groups
was obtained in [11] by the successive treatment of the
amino group of starting compound with dimethyl
oxalate, tris(2-aminoethyl)amine, and again with dime-
thyl oxalate. We reduced the number of stages in the
synthesis of this derivative by synthesizing tris(meth-
oxyoxalylamidoethyl)amine (II). Reaction of the thy-
midine derivative (I) with (II) led to the formation of
nucleoside (III) in 57% yield. After protecting the 5'-
hydroxy group of deoxyribose and the subsequent
phosphitylation of the nucleoside, phosphoamidite (V)
suitable for the standard oligonucleotide synthesis was
obtained (Scheme 1).
The oligodeoxyribonucleotides containing 5-
methyl-2'-deoxycytidine substituting for 2'-deoxycyti-
The introduction of an alkyl substituent into 4- dine are widely used in molecular biology for studying
amino group of 2'-deoxycytidine does not lead to its the complementary interactions of nucleic acids in
disability to form hydrogen bonds during complemen- duplexes and triplexes [15]. To examine the complete-
tary interactions [9]. The 2'-deoxycytidine analogues ness and the specificity of cleavage of complementary
containing a monoalkylated exocyclic amino group can nucleic acids by modified oligonucleotides containing
be synthesized from 2'-deoxyuridine by the reaction of 2'-deoxycytidine derivatives with various positions of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

226
ABRAMOVA et al.
O
HN
HN
N
NH2
NH2
N
N
N
O
N
O
N
O
(MeO)2TrO
(MeO)2TrO
(MeO)2TrO
O
O
O
i
+
OH
OH
OH
(VI)
(VII)
(VIII)
ii
O
O
HN
N
CH3
O
O
O
H
N
HN
N
H
N
O
CH3
N
H
O
N
O
(MeO)2TrO
O
O
OR
(IX) R = H
(X) R = P[O(CH2)2CN]N(iPr)2
iii
Reagents: (i) íÇD/H2N(CH2)3NH2; (ii) (II)/íÖÄ; (iii) NC(CH2)2OP[N(iPr)2]2/Tetr-HN(iPr)2.
Scheme 2.
reactive groups, we synthesized (XVI) in which active monomer (X) only by the presence of methyl group in
functional groups were attached at the position 5 of the position 5 of its heterocycle. We synthesized (XX) by
heterocyclic base of 2'-cytidine (Scheme 3).
the successive treatment of 5'-O-(4,4'-dimethoxytri-
tyl)thymidine (XVII) with trimethylchlorosilane and
1,2,4-triazole and phosphorus oxychloride. The inter-
mediate triazolide was treated, without isolation, with
1,3-diaminopropane as described in [16]. After treat-
ment with ammonia, N4-(3-aminopropyl)-5'-O-(4,4'-
dimethoxytrityl)-5-methyl-2'-deoxycytidine (XVIII)
was obtained (Scheme 4). A linker with reactive meth-
oxyoxalylamide groups was then introduced by the
reaction with (II), and the modified nucleoside was
phosphitylated to yield the target reagent (XX).
We began the synthesis of (XVI) from the conver-
sion of 5',3'-diacetyl-5-(3-trifluoroacetamidopro-
pyl)oxymethyl-2'-deoxyuridine (XI), an intermediate
in the synthesis of (I), into a 2'-deoxycytidine derivative
by the reaction with 1,2,4-triazole and phosphorus oxy-
chloride as described in [16]. After treatment with
ammonia, 5-(3-aminopropyl)oxymethyl-2'-deoxycyti-
dine (XII) was obtained. The reaction of this nucleo-
side with (II) led to (XIV); in this case, the exocyclic
amino group did not react. At the next stage of the syn-
thesis, 4-amino group of the 2'-deoxycytidine moiety
was blocked by benzoic anhydride as described in [17]
to form (XVI). 5'-Hydroxy group of the modified
nucleoside was protected, and the reaction product
(XV) was phosphitylated to yield phosphoamidite
(XVI), suitable for the oligonucleotide synthesis.
We have an interest in studying the cleavage of
nucleic acids at pyrimidine nucleotides and, therefore,
synthesized a purine nucleoside containing methoxy-
oxalylamide groups. 2'-Deoxyadenosine served as a
starting compound for the modification. The key com-
pound in the synthesis of 2'-deoxyadenosine derivatives
Phosphoamidite (XX) was synthesized (Scheme 4) modified at position 6 is 9-(2'-deoxy-β-D-ribofurano-
to determine the effect of 5'-methyl group in the 2'- syl)-6-chloropurine [18–20], which is obtained either
deoxycytidine analogue on the efficiency of comple- by the chlorination of 2'-deoxyinosine [21] or by the
mentary interactions of nucleic acids. It differs from deamination of 2'-deoxyadenosine by the action of
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

MONOMERS FOR OLIGONUCLEOTIDE SYNTHESIS…I.
227
O
N
NH2
H
HN
O
N
CF3
N
O
NH2
O
O
O
N
AcO
HO
O
O
i, ii
OAc
OH
(XII)
(XI)
O
O
O
HN
CH3
NHR''
N
O
O
O
H
N
O
N
N
N
H
O
O
iii
RO
CH3
O
N
H
O
OR'
(XIII) R = R' = R'' = H
(XIV) R = R' = H, R'' = Bz
(XV) R = (MeO)2Tr, R' = H, R'' = Bz
(XVI) R = (MeO)2Tr, R' = P[O(CH2)2CN](iPr)2, R'' = Bz
iv
v
vi
Reagents: (i) Tri/POCl3; (ii) NH3; (iii) (II)/íÖÄ; (iv) (C6H5CO)2O; (v) (MeO)2TrCl;
(vi) NC(CH2)2OP[N(iPr)2]2/Tetr-HN(iPr)2.
Scheme 3.
amyl nitrite in the presence of CCl4 [22]. A drawback of
these synthetic methods is a high sensitivity of 2'-
deoxyadenosine derivatives to apurinization under
acidic conditions, which reduces the reaction yield.
EXPERIMENTAL
The following reagents were used: thymidine and
2'-deoxyadenosine (NPO Vostok; Omutninsk, Russia);
N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycyti-
dine, 5'-O-(4,4'-dimethoxytrityl)thymidine (Group of
oligonucleotide synthesis, Novosibirsk Institute of
Bioorganic Chemistry, Russia); 1,2,4-triazole and 4,4'-
dimethoxytrityl chloride (OKhP, Novosibirsk Institute
of Organic Chemistry, Russia); trimethylchlorosilane
(Fluka, Switzerland); phosphoryl chloride (Merck,
Germany); and tris(2-aminoethyl)amine, N,N,N',N'-tet-
raisopropyl-(2-cyano)ethyl phosphodiamidite, and
dimethyl oxalate (Aldrich, USA). 5-Bromomethyl-
5',3'-diacetyl-2'-deoxyuridine was synthesized as
described in [10], 3-N-trifluoroacetylamidopropanol as
described in [25], and 1,2-bis[(dimethylamino)methyl-
ene]hydrazine as described in [26]. Other reagents and
solvents were Russian preparations of analytical and
reagent grade. TLC was carried out on precoated Kie-
selgel 60 F254 plates (Merck, Germany) in 9 : 1 methyl-
ene chloride–methanol mixture unless otherwise indi-
It was proposed to synthesize 6-modified 2'-deoxy-
adenosine derivatives through the formation of inter-
mediate triazolides [23, 24]. We synthesized N6-(3-ami-
nopropyl)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenos-
ine (XXIII) by analogy with the method described in
[24] (Scheme 5). After the introduction of a linker with
methoxyoxalylamide groups by the reaction with (II)
and phosphitylation of nucleoside (XXIV), the target
phosphoamidite (XXV) was obtained.
Thus, we obtained synthons for oligodeoxyribonu-
cleotide synthesis (V), (X), (XVI), (XX), and (XXV),
which allow the introduction of reactive methoxyoxal-
amido groups into any position of oligodeoxyribonu-
cleotide sequence. The synthesis of all the modified
nucleosides requires no expensive starting compounds,
is readily reproduced, and leads to target products in
relatively high yields.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

228
ABRAMOVA et al.
O
N
HN
NH2
CH3
CH3
N
N
O
O
N
(MeO)2Tr
O
(MeO)2TrO
i, ii, iii, iv
O
O
OH
(XVII)
OH
(XVIII)
O
O
HN
N
CH3
O
O
O
H
HN
N
N
CH3
H
N
O
CH3
N
H
O
N
O
v
(MeO)2TrO
O
O
OR
(XIX) R = H
(XX) R = P[O(CH2)2CN]N(iPr)2
vi
Reagents: (i) Me3SiCl; (ii) Tri/POCl3; (iii) H2N(CH2)3NH2; (iv) ammonia; (v) (II)/íÖÄ;
(vi) NC(CH2)2OP[N(iPr)2]2/Tetr-HN(iPr)2.
Scheme 4.
cated. For column chromatography, Porasil Silica absorption at 254 nm. The product was eluted with
125A, 55–100 µm (Waters, United States) was used. 1.2 M ammonia. The ammonia solution of (I) was
The compositions of eluents for column chromatogra- evaporated, and the residue was coevaporated with ace-
phy are given in volume percent. NMR spectra were tonitrile. After drying in a vacuum, 5-(3-aminopro-
recorded on an AM-400 spectrometer (Bruker, Ger- pyl)oxymethyl-2'-deoxyuridine (I) was obtained; yield
many); chemical shifts are given in ppm, and spin–spin 0.28 g (0.89 mmol, 72%); Rf 0.45 (99 : 1 methanol–ace-
tic acid); 1H NMR (D2O): 7.93 (1 H, s, H6), 6.37 (1 H,
quasi-triplet, J 6.5, H1'), 4.53 (1 H, m, H3'), 4.34 (2 H,
s, CH2O), 4.11 (1 H, m, H4'), 3.96 (1 H, dd, 2J 12.3, J
coupling constants in Hz. Tetramethylsilane was used
1
as an internal standard in H NMR spectra, and 85%
phosphoric acid as an external standard in 31P NMR
spectra.
'
'
3.1, H5a ), 3.86 (1 H, dd, J 4.5, H5b ), 3.74 (2 H, t, J 5.9,
OCH2), 3.15 (2 H, t, J 6.8, CH2NH2), 2.52–2.37 (2 H,
m, H2'), 2.00 (2 H, m, CH2CH2CH2).
5-(3-Aminopropyl)oxymethyl-2'-deoxyuridine (I).
5-Bromomethyl-5',3'-diacetyl-2'-deoxyuridine (0.5 g,
1.23 mmol) was dissolved in dry DMF (5 ml), and 3-N-
trifluoroacetylamidopropanol (2 g, 11.7 mmol) was
added. The reaction mixture was stirred for 16 h at
room temperature and evaporated. The resulting oil was
treated with concentrate ammonia (20 ml), and the mix-
ture was kept for 3 h at 45°ë. When the unblocking was
over, the solution was evaporated. The residue was dis-
solved in water (40 ml) and applied onto a column
Tris(2-methoxyoxalylamidoethyl)amine
(II).
Tris(2-aminoethyl)amine (1.8 ml, 12 mmol) was dis-
solved in dry methanol (6 ml), and dimethyl oxalate
(4.45 g, 40 mmol) in 20 ml of dry methanol was added
to the stirred solution dropwise for 2 h. Stirring was
continued for additional 2 h, and then the reaction mix-
ture was refluxed for 10 min and evaporated. The resi-
packed with Dowex 50Wx2 (50 ml, H+-form). The col- due was dissolved in methylene chloride (10 ml) and
umn was washed with water until the absence of UV passed through a column of silica gel. After the elution
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

MONOMERS FOR OLIGONUCLEOTIDE SYNTHESIS…I.
229
N N
N
NH2
N
HN
N
NH2
N
N
N
N
N
N
N
N
HO
HO
(MeO)2Tr O
iii, iv
i, ii
N
N
O
O
O
OH
(XXI)
OH
OH
(XXII)
(XXIII)
O
O
HN
CH3
CH3
O
O
H
HN
N
N
N
H
N
N
O
O
v
(MeO)2Tr
O
N
O
N
N
O
H
O
OR
(XXIV) R = H
(XXV) R = P[O(CH2)2CN]N(iPr)2
vi
Reagents: (i) Me3SiCl/[(CH3)2NCH=N]2; (ii) MeOH; (iii) (MeO)2TrCl; (iv) H2N(CH2)3NH2; (v) (II)/íÖÄ;
(vi) NC(CH2)2OP[N(iPr)2]2/Tetr-HN(iPr)2.
Scheme 5.
with 5% methanol in methylene chloride, the target ane, if the next stage of the synthesis was the phosphi-
fractions were evaporated, and the residue was tritu-
rated with ether. The crystalline product was dried in a
vacuum to yield 3.9 g of tris(2-methoxyoxalylamidoet-
hyl)amine (9.8 mmol, 80%); Rf 0.55; mp. 95–97°C;
tylation. In another case, the chromatographic fractions
were evaporated, and the residual foam product was
dried.
5-{3-[N,N-Di(2-methoxyoxalylamidoethyl)amino-
ethyl]amidooxalylamidopropyl}oxymethyl-2'-deoxy-
uridine (III) was obtained from (I) by the above-
described procedure. The final concentration of metha-
nol in the eluting solution was 25%. (III): yield 57%; Rf
1H NMR (CDCl3): 7.46 (3 H, br t, J 5.6, NH), 3.88
(9 H, s, OCH3), 3.39 (6 H, quasi-quartet, CH2CH2NH),
2.69 (6 H, t, J 5.8, CH2CH2N ).
A general procedure for the introduction of a
linker carrying two methoxyoxalylamide groups. A
solution of nucleoside modified with the aminopropyl
linker (1 mmol) and of triethylamine (3 mmol) in meth-
anol (4 ml) was added in portions for 4 h to a solution
of (II) (2.5 mmol, 1.01 g) in dry methanol (10 ml). The
reaction mixture was stirred for 3 h and evaporated. The
target products were purified by chromatography on sil-
ica gel, eluting the product by a gradient of methanol
(from 0%) in methylene chloride (the final concentra-
tion of methanol in the eluting solution depended on a
particular compound; see below). The fractions with
target product were evaporated, and it was precipitated
from methylene chloride by a tenfold volume of hex-
1
0.37 (4 : 1 methylene chloride–methanol); H NMR
(C5D5N): 13.27 (1 H, br s, H3), 9.29 J 5.8,
CH2NHC(O)), 9.25 J 4.8, CH2NHC(O)), 9.14 J 5.5,
CH2NHC(O)), 8.48 (1 H, s, H6), 6.98 (1 H, quasi-trip-
let, J 6.5, H1'), 5.01 (1 H, m, H3'), 4.45 (1 H, m, H4'),
4.41 (1 H, d, J 11.9, CH‡HbO), 4.31 (1 H, d, J 11.9,
2
'
CHaHbO), 4.22 (1 H, dd, J 12.0, J 3.0, H5a ), 4.14 (1 H,
'
dd, J 3.0, H5b ), 3.74 (6 H, Ò, OCH3), 3.62–3.48 (10 H,
m, CH2CH2CH2NH, OCH2CH2, and CH2CH2N ),
2.78 (6 H, m, CH2CH2N ), 2.69 (2 H, m, H2'), 1.91
(2 H, m, CH2CH2CH2).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

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ABRAMOVA et al.
N4-{3-[N,N-Di-(2-methoxyoxalylamidoethyl)ami-
noethyl]amidooxalylamidopropyl}-5'-O-(4,4'-dime-
thoxytrityl)-2'-deoxycytidine (IX) was obtained from
compound (VII) according to the above-described pro-
cedure. The final concentration of methanol in the elut-
A general procedure for the introduction of the
4,4'-dimethoxytrityl protecting group. The corre-
sponding nucleoside (1 mmol) was dissolved in dry
pyridine (5 ml), and 4,4'-dimethoxytrityl chloride
(1.15 mmol) was added at stirring. After the end of
reaction (2–3 h), the reaction mixture was evaporated,
and the residue was dissolved in methylene chloride
and applied onto a silica gel column. The elution with a
gradient of methanol (0%–12%) in methylene chloride
supplemented with 1% pyridine led to the target prod-
uct fractions. They were evaporated, and the product
was precipitated from the solution in methylene chlo-
ride by a tenfold volume of hexane.
1
ing solution was 12%. Yield of (IX) 55%; Rf 0.18; H
NMR (CDCl3): 8.23 (1 H, t, J 6.0, CH2NHC(O)), 8.10
(3 H, m, CH2NHC(O)), 7.75 (1 H, d, J 6.8, H6), 7.45–
7.18 [9 H, m, (MeO)2Tr], 6.81 [4 H, d, J 8.6,
(MeO)2Tr], 6.32 (1 H, quasi-triplet, J 5.8, H1'), 5.71 (1
H, br t, NHCH2), 5.39 (1 H, d, H5), 4.49 (1 H, m, H3'),
4.05 (1 H, m, H4'), 3.78 (6 H, s, OCH3), 3.77 (6 H, s,
OCH3), 3.60–3.24 (12 H, m, H5, NHCH2CH2CH2,
CH2CH2CH2NH and CH2CH2N ), 2.72–2.52 (6 H, m,
5-{3-[N,N-Di-(2-methoxyoxalylamidoethyl)amino-
ethyl]amidooxalylamidopropyl}oxymethyl-5'-O-(4,4'-
dimethoxytrityl)-2'-deoxyuridine (IV) was obtained
from (III) as described above. The product was precip-
itated from a solution in a 4 : 1 methylene chloride–
pyridine mixture by a tenfold volume of hexane, and
the precipitate was washed with hexane to give (IV);
'
'
CH2CH2N ), 2.35 (1 H, m, H2a ), 2.21 (1 H, m, H2b ),
1.79 (2 H, m, CH2CH2CH2).
5-(3-Aminopropyl)oxymethyl-2'-deoxycytidine
(XII). 3-N-Trifluoroacetylamidopropanol (4 g, 23.4 mmol)
was added to a solution of 5-bromomethyl-3',5'-
diacetyl-2'-deoxyuridine (1 g, 2.46 mmol) in DMF
(10 ml). The reaction mixture was stirred for 16 h at
room temperature and evaporated. The oily residue was
dissolved in methylene chloride (50 ml) and washed
with water (3 × 25 ml). The organic layer was dried
with Na2SO4, evaporated, dried by coevaporation with
anhydrous acetonitrile, and dissolved in 6 ml of anhy-
drous acetonitrile. Separately, phosphoryl chloride
(0.95 ml, 10 mmol) and triethylamine (7 ml, 50 mmol)
1
yield 65%; Rf 0.21; H NMR (C5D5N): 13.30 (1 H, s,
H3), 9.24 (2 H, t, J 4.8, CH2NHC(O)), 9.19 (1 H, t,
J 5.8, CH2NHC(O)), 9.12 (1 H, t, J 5.6, CH2NHC(O)),
8.04 (1 H, s, H6), 7.76–7.08 [9 H, m, (MeO)2Tr], 6.98
(4 H, m, (MeO)2Tr), 6.92 (1 H, quasi-triplet, J 6.5, H1'),
4.92 (1 H, m, H3'), 4.50 (1 H, m, H4'), 4.21 (1 H, d, J
11.2, CHaHbO), 4.36 (1 H, d, J 11.2, CHaHbO), 3.74 (6
H, s, OCH3), 3.70 (6 H, s, OCH3), 3.66 (2 H, m, H5'),
3.54 (10 H, m, ëç2CH2CH2NH, OCH2CH2, and
CH2CH2N ), 2.78 (6 H, m, CH2CH2N ), 2.67 (2 H, m, were successively added to a suspension of 1,2,4-triaz-
ole (3.45 g, 50 mmol) in dry acetonitrile (60 ml) under
cooling with ice and stirring. Stirring was continued for
30 min, and then the nucleoside solution in acetonitrile
was added to the reaction mixture. Cooling was termi-
nated, and the reaction mixture was stirred for 1.5 h at
room temperature and evaporated. The residue was dis-
solved in methylene chloride (100 ml), washed with 5%
NaHCO3 (50 ml) and water (50 ml). The organic layer
was dried with Na2SO4 and evaporated, the residue was
dissolved in 20 ml of dioxane, and concentrate ammo-
nia (20 ml) was added. The reaction mixture was kept
for 16 h at room temperature and evaporated. The resi-
due was dried by coevaporation with acetonitrile and
triturated with ether to give (XII); yield 0.47 g
(1.5 mmol, 60%); Rf 0.26 (99 : 1 methanol–acetic acid);
H2'), 1.85 (2 H, m, CH2CH2CH2).
N4-(3-Aminopropyl)-5'-O-(4,4'-dimethoxytrityl)-
2'-deoxycytidine (VII). TBD (1.3 g, 9.4 mmol) and
1,3-diaminopropane (3.5 ml, 42 mmol) were added to a
solution of (VI) (2 g, 3.16 mmol) in DMF (10 ml). The
reaction mixture was heated at 60°ë for 16 h and evap-
orated. The residue was dissolved in methylene chlo-
ride (100 ml) and washed with 1 M NaOH (50 ml) and
water (5 × 50 ml). The organic layer was dried with
anhydrous Na2SO4 and evaporated. The residue was
dissolved in methylene chloride and applied onto a sil-
ica gel column. The product was eluted in a gradient of
methanol concentration in methylene chloride (0
20%) supplemented with 10% triethylamine. The frac-
tions with target product were evaporated, the product
(VII) was precipitated from the solution in methylene
chloride with a tenfold volume of hexane; yield 0.74 g
(1.26 mmol, 40%); Rf 0.13 (7 : 2 : 1 methylene chlo-
1H NMR (D2O): 8.04 (1 H, s, H6), 6.33 (1 H, t, J 6.5,
H1'), 4.51 (1 H, m, H3'), 4.48 (2 H, s, CH2O), 4.17 (1 H,
m, H4'), 3.95 (1 H, dd, 2J 12.5, J 3.0, H5'), 3.84 (1 H,
dd, J 4.1, H5'), 3.70 (2 H, t, J 5.8, OCH2CH2), 3.18
ride–methanol–triethylamine); 1H NMR (CDCl3): 7.56
'
(1 H, d, J 7.8, H6), 7.42–7.03 [9 H, m, (MeO)2Tr], 6.73 (2 H, t, J 7.2, CH2CH2NH2), 2.55 (1 H, m, H2a ), 2.40
[4 H, d, J 8.6, (MeO)2Tr], 6.15 (1 H, quasi-triplet, J 6.0,
'
(1 H, m, H2b ), and 2.09 (2 H, m, CH2CH2CH2).
H1'), 5.62 (1 H, d, H5), 4.38 (1 H, m, H3'), 4.00 (1 H,
m, H4'), 3.66 (6 H, s, OCH3), 3.38 (2 H, quasi-triplet, J
5.5, NHCH2CH2), 3.29 (2 H, m, H5'), 2.95 (2 H, t, J 5.0,
5-{3-[N,N-Bis(2-methoxyoxalylamidoethyl)amino-
ethyl]amidooxalylamidopropyl}oxymethyl-2'-deoxy-
cytidine (XIII) was obtained from (XII) by the above-
described procedure, using 35% methanol as the final
eluting solvent; yield 58%; Rf 0.18 (4 : 1 methylene
'
CH2CH2NH2), 2.38 (1 H, m, H2a ), 2.05–1.78 (3 H, m,
'
H2b , CH2CH2CH2).
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MONOMERS FOR OLIGONUCLEOTIDE SYNTHESIS…I.
231
1
triethylamine (5.15 ml, 36.8 mmol) were successively
added to an ice-cooled suspension of 1,2,4-triazole
(2.54 g, 36.8 mmol) in dry acetonitrile (50 ml). The sus-
pension was stirred for 30 min, treated with the nucleo-
side solution in acetonitrile, and then stirred for 1.5 h at
room temperature, and evaporated. The residue was
dissolved in methylene chloride (100 ml); the solution
was washed with 5% NaHCO3 (50 ml) and water
(50 ml), dried with Na2SO4, and evaporated. The resi-
due was dissolved in acetonitrile (20 ml), and 1,3-
diaminopropane (1.54 ml, 18.4 mmol) was added. After
1 h, 10 ml of concentrate ammonia was added to the
reaction mixture, and it was kept overnight at room
temperature and evaporated. The residue was dissolved
in methylene chloride (100 ml), washed with 0.1 M
NaOH (30 ml) and water (5 × 50 ml), dried with
Na2SO4, and evaporated. The residue was dissolved in
methylene chloride (5 ml), and the product (XVIII)
was precipitated with hexane; yield: 0.9 g (1.5 mmol,
81%); Rf 0.14 (7 : 2 : 1 methylene chloride–methanol–
chloride–methanol); H NMR ((CD3)2SO): 8.64 (3 H,
br t, CH2NHC(O)), 8.45 (1 H, br t, CH2NHC(O)), 7.69
(1 H, s, H6), 6.14 (1 H, quasi-triplet, J 6.4, H1'), 4.22 (1
H, m, H3'), 4.16 (2 H, s, CH2O), 4.11 (1 H, m, H4'),
3.79 (6 H, s, OCH3), 3.62 (4 H, m, H5', OCH2CH2),
3.41–3.11 (8 H, m, CH2CH2CH2NH and CH2CH2N ),
2.60 (6 H, m, CH2CH2N ), 2.22–1.88 (2 H, m, H2'),
and 1.72 (2 H, m, CH2CH2CH2).
N4-Benzoyl-5-{3-[N,N-bis(2-methoxyoxalylamido-
ethyl)aminoethyl]amidooxalylamidopropyl}oxyme-
thyl-2'-deoxycytidine (XIV). Benzoic anhydride
(0.181 g, 0.8 mmol) was added to a solution of 0.6 g
(0.87 mmol) of (XIII) in DMF (4 ml). The reaction
mixture was kept for 16 h at room temperature and
treated with additional benzoic anhydride (18 mg).
After 1 h at 45°ë, the mixture was evaporated. The res-
idue was triturated with ether (3 × 5 ml) and dried in a
vacuum to give (XIV); yield 0.4 g (0.5 mmol, 58%); Rf
1
0.45 (4 : 1 methylene chloride–methanol); H NMR
1
(C5D5N): 9.35–9.07 (4 H, m, CH2NHC(O)), 8.53 (2 H,
m, Bz), 8.47 (1 H, s H6), 7.50 (3 H, m, Bz), 6.85 (1 H,
quasi-triplet, J 6.1, H1'), 5.02 (1 H, m, H3'), 4.60 (1 H,
d, J 12.0, CHaHbO), 4.48 (1 H, d, J 12.0, CHaHbO), 4.46
triethylamine); H NMR (CDCl3): 7.58 (1 H, s, H6),
7.42–7.11 [9 H, m, (MeO)2Tr], 6.80 [4 H, d, J 8.0,
(MeO)2Tr], 6.43 (1 H, quasi-triplet, J 6.1, H1'), 4.50 (1
H, m, H3'), 4.04 (1 H, m, H4'), 3.77 (6 H, s, OCH3),
3.72 (1 H, m, NHCH2), 3.62 (2 H, m, NHCH2CH2),
(1 H, m, H4'), 4.26 (1 H, dd, 2J 12.0, J 3.0, H5'), 4.09
(1 H, dd, J 2.9, H5'), 3.78 (8 H, m, OCH3, OCH2CH2),
3.68–3.42 (8 H, m, CH2CH2CH2NH and ëH2CH2N ),
2.88–2.55 (8 H, m, CH2CH2N and H2'), and 1.96
(2 H, m, CH2CH2CH2).
2
3.44 (1 H, dd, J 10, J 3.0, H5'), 3.30 (1 H, dd, J 2.9,
H5), 2.87 (2 H, t, J 5.9, CH2CH2NH2), 2.54 (1 H, m,
'
'
H2a ), 2.20 (1 H, m, H2b ), 1.70 (2 H, m, CH2CH2CH2),
and 1.46 (3 H, s, CH3).
N4-Benzoyl-5-{3-[N,N-bis(2-methoxyoxalylamido-
ethyl)aminoethyl]amidooxalylamidopropyl}oxyme-
thyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine (XV)
was obtained from (XIV) by the above-described pro-
cedure, using 10% methanol as the final eluting solvent;
yield 0.27 g (0.27 mmol, 62%); Rf 0.28; H NMR
(CDCl3): 8.20 (2 H, d, J 6.9, Bz), 7.91 (2 H, m, H6,
N4-{3-[N,N-Bis(2-methoxyoxalylamidoethyl)ami-
noethyl]amidooxalylamidopropyl}-5'-O-(4,4'-dime-
thoxytrityl)-5-methyl-2'-deoxycytidine (XIX) was
obtained from (XVIII) by the above-described proce-
dure, using 12% final concentration of methanol in the
eluting solution; yield 41%; Rf 0.18; 1H NMR (CDCl3):
8.05 (1 H, t, J 6.1, CH2NHC(O)), 7.70(3 H, m,
CH2NHC(O)), 7.61 (1 H, s, H6), 7.52–7.15 [9 H, m,
(MeO)2Tr), 6.80 [4 H, d, J 8.2, (MeO)2Tr), 6.43 (1 H,
quasi-triplet, J 6.4, H1'), 5.94 (1 H, br t, J 6.1,
NHCH2CH2CH2), 4.50 (1 H, m, H3'), 4.04 (1 H, m,
H4'), 3.82 (6 H, s, OCH3), 3.77 (6 H, s, OCH3), 3.58–
3.27 (12 H, m, H5, NHCH2CH2CH2, CH2CH2CH2NH,
and CH2CH2N ), 2.75–2.60 (6 H, m, CH2CH2N ),
1
CH2NHCO), 7.68 (2 H, quasi-triplet,
J
6.5,
CH2NHC(O)), 7.58–7.12 (13 H, m, MeO)2Tr,
CH2NHC(O) and Bz), 6.80 (4 H, d, (MeO)2Tr), 6.35
(1H, quasi-triplet, J 6.4, H1'), 4.55 (1 H, m, H3'), 4.13
(2 H, s, CH2O), 4.05 (1 H, m, H4'), 3.81 (6 H, s, OCH3),
3.76 (8 H, m, OCH3, OCH2CH2), 3.60–3.13 (10 H, m,
H5, CH2CH2CH2NH and CH2CH2N ), 2.79–2.36 (8 H,
m, CH2CH2N and H2'), 1.68 (2 H, m, CH2CH2CH2).
N4-(3-Aminopropyl)-5'-O-(4,4'-dimethoxytrityl)-
5-methyl-2'-deoxycytidine (XVIII). Trimethylchlo-
rosilane (0.47 ml, 3.7 mmol) was added to a stirred
'
'
2.52 (1 H, m, H2a), 2.22 (1 H, m, H2b), 1.84 (2 H, m,
CH2CH2CH2), and 1.54 (3 H, s, CH3).
N6-(1,2,4-Triazole-4-yl)-2'-deoxyadenosine (XXII).
Trimethylchlorosilane (0.5 ml, 4 mmol) and 1,2-
bis[(dimethylamino)methylene]hydrazine (1.11 g,
7.8 mmol) were added to a solution of 2'-deoxyadenos-
ine (XXI) (0.5 g, 2 mmol) in dry pyridine (5 ml). The
reaction mixture was refluxed for 5 h and kept for 16 h
at room temperature. An additional quantity of trimeth-
solution
of
5'-O-(4,4'-dimethoxytrityl)thymidine
(XVII) (1 g, 1.84 mmol) in dry pyridine (5 ml). After
30 min, the reaction mixture was evaporated, the oil
was dissolved in methylene chloride (100 ml), and the
solution was washed with 5% NaHCO3 (50 ml) and
water (50 ml). The organic layer was dried with
Na2SO4, evaporated, coevaporated with toluene to
remove pyridine (3 × 10 ml), dissolved in dry acetoni- ylchlorosilane (0.5 ml) was added to the reaction mix-
trile (5 ml), and mixed with triethylamine (0.5 ml). Sep- ture, and after 20 min the solution was diluted with
arately, phosphoryl chloride (0.69 ml, 7.36 mmol) and methylene chloride (20 ml) and extracted with 1 N HCl
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004

232
ABRAMOVA et al.
(3 × 20 ml). The organic layer was dried with Na2SO4, monitored by TLC. After 2 h, an additional quantity of
phosphitylating reagent (2.2 ml, 6.9 mmol) was added.
After 3 to 7 h, the reaction mixture was evaporated, the
residue was treated with hexane (50 ml), and the mix-
ture was kept overnight. Hexane was then decanted,
and the residue was dissolved in methylene chloride
(20 ml) and chromatographed on silica gel. Elution
with a gradient of methanol in methylene chloride (0 to
10%) gave the target fractions; the product was precip-
itated from methylene chloride with hexane; 31P NMR
(CD3CN): 149.37 and 149.10 for (V); 149.01 for (X);
149.21 for (XVI); 148.88 for (XX); and 149.06 and
148.92 for (XXV).
evaporated, and then three times coevaporated with tol-
uene to remove pyridine. The residue was dissolved in
methanol (10 ml) and refluxed for 6 h. After recrystal-
lization from methanol, (XXII) was obtained; yield
0.38 g (1.25 mmol, 62%); Rf 0.14; 1H NMR (D2O): 9.60
(2 H, s, H3'', 5'', 8.88 (1 H, s, H8), 8.76 (1 H, s, H2),
6.64 (1 H, quasi-triplet, J 6.6, H1'), 4.18 (1 H, m, H4'),
'
3.81 (2 H, m, H5'), 2.93 (1 H, m, H2a ), 2.69 (1 H, m,
'
H2b ).
N6-(3-Aminopropyl)-5'-O-(4,4'-dimethoxytrityl)-
2'-deoxyadenosine (XXIII). Dimethoxytrityl protect-
ing group was introduced into (XXII) by the above-
described procedure. The chromatographic fractions
with the target product were evaporated, the residue
was dissolved in pyridine (10 ml), and 1,3-diaminopro-
pane (0.85 ml, 10 mmol) was added. The reaction mix-
ture was heated for 8 h at 70°ë and evaporated, and the
residue was dissolved in methylene chloride (50 ml)
and washed with water (3 × 25 ml). The organic layer
was dried with Na2SO4 and evaporated. The product
(XXIII) was precipitated from its solution in methylene
chloride with a tenfold volume of hexane; yield 0.56 g
(0.91 mmol, 73%); Rf 0.11 (7 : 2 : 1, methylene chlo-
ride–methanol–tryethylamine); 1H NMR (CDCl3): 8.28
(1 H, s, H8), 7.85 (1 H, s, H2), 7.73–7.09 [9 H, m,
(MeO)2Tr), 6.78 [4 H, d, J 8.9, (MeO)2Tr), 6.39 (1 H,
quasi-triplet, J 6.5, H1'), 6.31 (1 H, br s, NHCH2), 4.67
(1 H, m, H3'), 4.10 (1 H, m, H4'), 3.76 (8 H, m, OCH3,
NHCH2CH2), 3.39 (2 H, m, H5'), 2.85 (2 H, t, J 6.5,
ACKNOWLEDGMENTS
The study was supported by the Russian Foundation
for Basic Research (projects nos. 02-04-48664a and 01-
03-32439a).
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CH2CH2NH2), 2.78 (1 H, m, H2a ), 2.52 (1 H, m, H2b ),
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N6-{3-[N,N-Bis(2-methoxyoxalylamidoethyl)ami-
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 30 No. 3 2004