Synthesis and properties of oligodeoxyribonucleotides containing 2′-O-(2,3-dihydroxypropyl)- and 2′-O-(2-oxoethyl)arabinouridine residues

Article abstract of DOI:10.1007/s11172-005-0243-2

2′-O-(2,3-Dihydroxypropyl)arabinouridine-containing oligodeoxyribonucleotides were synthesized starting from a new modified nucleoside, viz., 2′-O-(2,3-dihydroxypropyl)arabinouridine, and the corresponding 3′-phosphoramidite. Oxidation of these oligodeoxy

Full text of DOI:10.1007/s11172-005-0243-2

238
Russian Chemical Bulletin, International Edition, Vol. 54, No. 1, pp. 238—246, January, 2005
Synthesis and properties of oligodeoxyribonucleotides
containing 2´ꢀOꢀ(2,3ꢀdihydroxypropyl)ꢀ and
2´ꢀOꢀ(2ꢀoxoethyl)arabinouridine residues*
T. S. Zatsepin,a Yu. M. Ivanova,a D. A. Stetsenko,b M. J. Gait,b and T. S. Oretskayaa
ꢀ
aDepartment of Chemistry, M. V. Lomonosov Moscow State University,
1 Leninskie Gory, 119992 Moscow, Russian Federation.
Fax: +7 (095) 939 3148. Eꢀmail: oretskaya@belozersky.msu.ru
bMedical Research Council, Laboratory of Molecular Biology,
Hills Road, Cambridge CB2 2QH, UK
2´ꢀOꢀ(2,3ꢀDihydroxypropyl)arabinouridineꢀcontaining oligodeoxyribonucleotides were
synthesized starting from a new modified nucleoside, viz., 2´ꢀOꢀ(2,3ꢀdihydroxypropyl)arabinoꢀ
uridine, and the corresponding 3´ꢀphosphoramidite. Oxidation of these oligodeoxyriboꢀ
nucleotides with sodium periodate afforded oligonucleotides containing 2´ꢀOꢀ(2ꢀoxoꢀ
ethyl)arabinouridine residues. Subsequent modification of the aldehydeꢀcontaining oligonucleꢀ
otides involved the reactions with 9ꢀhydrazinoacridine and Nꢀaminooxyacetyl peptide and
reductive amination by 4ꢀ(1ꢀpyrenyl)butyrohydrazide and biotin hydrazide. Thermal stabilities
of duplexes of modified oligodeoxyribonucleotides with complementary oligodeoxyriboꢀ
nucleotides are slightly lower than those of natural duplexes. Duplexes with complementary
oligoribonucleotides are substantially destabilized.
Key words: modified oligonucleotides, duplexes.
Oligonucleotides including modified oligonucleotides
interacting with particular regions of nucleic acids in
find wide application in various fields of molecular biolꢀ
ogy. They are used as primers for DNA sequencing and
amplification, for the determination of DNA primary
structures by hybridization, and also as potential theraꢀ
peutics.2,3 In particular, modified oligonucleotides are
employed for the preparation of conjugates with various
compounds, such as peptides, oligosaccharides, and fluoꢀ
rescent dyes for studying the behavior of nucleic acids
in vitro or in vivo. Affinity modification of DNAꢀbinding
proteins is used to study the structures and functioning of
the active sites of the latter.4,5
Conjugation of fluorophores or electrochemically acꢀ
tive compounds to oligonucleotides enables the use of
such derivatives in the design of DNA detection systems
by hybridization.6—8 Marker groups substantially increase
sensitivity of DNA detection. Many conjugates of oligoꢀ
nucleotides with peptides, carbohydrates, and lipophilic
compounds more easily penetrate cell membranes than
native nucleic acids, and this fact can be used in antisense
and antigene biotechnology.9—11
nucleic acid—protein complexes. In studies of multiꢀ
protein complexes formed during the replication, translaꢀ
tion, etc., one can determine a protein component interꢀ
acting with a region of nucleic acid recognition12 and
examine cell extracts with the aim of revealing proteins
that selectively interact with particular nucleotide seꢀ
quences. This approach is also applicable in the SELEX
technology for the design of nucleic acid aptamers that
irreversibly bind to proteins.13,14 In recent years, considꢀ
erable progress has been achieved in crystallization of
covalent nucleic acid—protein complexes and elucidaꢀ
tion of their structures by Xꢀray diffraction.15
Earlier, we have demonstrated that 2´ꢀOꢀ(2ꢀoxoꢀ
ethyl)uridineꢀcontaining DNA duplexes can successfully
be used for the affinity modification of DNAꢀbinding
proteins, such as transcription factor NFꢀκB16 and meꢀ
thyl transferase SsoII.17 The reactive aldehyde group can
selectively react with the adjacent εꢀamino group of lysine
in proteins under conditions of reductive amination.
Modifications of a ribose residue at position 2 generꢀ
ally cause only small distortions in the oligonucleotide
structure and allow the preparation of nucleosides conꢀ
taining carbohydrate residues in both the C(2´)ꢀendo (2T3)
and C(2´)ꢀexo conformations (3T2) and direction of the
substituent into either the minor or major groove of
Covalent attachment of proteins to nucleic acids is the
method of choice for solving a series of problems. First
and foremost, it allows identification of the amino acids
* For a preliminary communication, see Ref. 1.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 233—241, January, 2005.
1066ꢀ5285/05/5401ꢀ0238 © 2005 Springer Science+Business Media, Inc.

Arabinouridine 2´ꢀaldehyde oligonucleotides
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 1, January, 2005
239
the nucleic acid duplex.18 In 2´ꢀdeoxyꢀ2´ꢀfluoroꢀ and
2´ꢀOꢀalkylribonucleotides, the sugar residue adopts the
stable C(3´)ꢀendo conformation (3T2), the alkyl groups
protruding into the minor groove of the duplex.19—21 On
the contrary, stabilization of the C(2´)ꢀendo conformaꢀ
tion is typical of 2´ꢀSꢀalkylꢀ2´ꢀthioꢀ and 2´ꢀaminoꢀ2´ꢀ
deoxyribonucleotides.22,23 To prepare nucleosides in a reꢀ
quired conformation, one can not only vary the nature of
the 2´ꢀsubstituent but also use the arabino epimer. It has
been demonstrated24,25 that arabinoꢀ and 2´ꢀOꢀalkylꢀ
arabinooligonucleotides adopt the C(2´)ꢀendo conformaꢀ
tion and give Bꢀform duplexes with DNAs. In this case,
2´ꢀsubstituents protrude into the major groove of the DNA
duplex. At the same time, the insertion of a 2´ꢀOꢀmethylꢀ
arabinonucleotide unit into the DNA duplex leads to a
slight decrease in its melting point compared to the
nonmodified duplex.26,27
The aim of the present study was to introduce a
modified unit containing the 2´ꢀaldehyde group into a
DNA duplex. The substituent would be expected to proꢀ
trude into the major groove of the DNA duplex and
cause no noticeable distortions of its local structure. We
chose 2´ꢀOꢀ(2ꢀoxoethyl)arabinouridine as a modified
nucleoside.
When interacting with a DNA duplex, many
DNAꢀbinding proteins recognize a particular nucleotide
sequence and bind to this sequence without unwinding
the double helix. By comparing the results of covalent
attachment of a protein to 2´ꢀOꢀ(2ꢀoxoethyl)uridineꢀ and
2´ꢀOꢀ(2ꢀoxoethyl)arabinouridineꢀcontaining DNA duꢀ
plexes, one can determine whether the reactive lysine
residue is located on the side of the major or minor groove.
Study by NMR spectroscopy demonstrated that sterically
hindered 2´ꢀOꢀalkylarabinonucleotides produce no subꢀ
stantial distortions of the DNA B form.25
We prepared 2´ꢀOꢀ(2ꢀoxoethyl)oligonucleotides using
conventional periodate oxidation of the 1,2ꢀdiol group in
the corresponding precursor.28,29 To retain the C(2´)ꢀendo
conformation (2T3) of the carbohydrate fragment, 2´ꢀOꢀ or
2´ꢀSꢀalkyl derivatives may be used. Since sulfides are oxiꢀ
dized with sodium periodate to give sulfoxides,30 we used
a 2´ꢀOꢀalkylarabino derivative. Synthesis of 2´ꢀmodified
was carried out in the presence of 2ꢀtertꢀbutyliminoꢀ2ꢀ
diethylaminoꢀ1,3ꢀdimethylperhydroꢀ1,3,2ꢀdiazaphosꢀ
phorine (BDDDP).33 Hydroxylation of the double bond
of the allyl group of nucleoside 3 with osmium tetraoxide
in the presence of Nꢀmethylmorpholine Nꢀoxide34 was
carried out under the optimum conditions found in
our earlier investigation.35 Under these conditions, the
5,6ꢀdouble bond of the heterocyclic base remains intact,
which made it possible to prepare the target product 4 in
high yield. The diol group in compound 4 was protected
by acylation with levulinic anhydride in the presence of
DMAP. Levulinic anhydride was generated from levulinic
acid in the presence of N,N´ꢀdicyclohexylcarbodiimide
immediately before the reaction. The levulinoyl protectꢀ
ing group was chosen because it can be selectively reꢀ
moved with hydrazinium acetate. This makes it possible
to subsequently generate the free aldehyde group in a
completely protected oligonucleotide immobilized on a
polymer support and perform solidꢀphase reactions, for
example, in the preparation of combinatorial libraries and
for the coupling with hydrophobic compounds.36 Attempts
to remove the silyl or dinitrophenylsulfenyl protecting
groups in compound 5 by the reaction with Bun NF in
4
THF37 led to removal of the levulinoyl protecting groups
as well. Therefore, we used triethylamine trihydrofluoꢀ
ride as a milder desilylating agent.38 The synthesis of
5´ꢀOꢀ(4,4´ꢀdimethoxytrityl) derivative 7 followed by its
3´ꢀphosphitylation was carried out according to standard
procedures.39 The phosphitylation time was increased
to 2 h, which was associated with steric hindrance of the
3´ꢀhydroxy group in compound 7.
Phosphoramidite derivative 8 was used in the autoꢀ
mated oligonucleotide synthesis. In the step of coupling
of the modified unit, the reaction time was increased to
15 min, and the condensation was carried out twice. Prodꢀ
ucts of the oligonucleotide synthesis were cleaved from
the polymer support and all alkaliꢀlabile protecting groups
were removed with concentrated aqueous ammonia
at 55 °C. The structures of analogs of dodecadeoxyriboꢀ
nucleotide I containing one or two 2´ꢀOꢀ(2,3ꢀdihydroxyꢀ
propyl)arabinouridine residues, viz., dodecadeoxyriboꢀ
nucleotides II—IV, are given in Table 1. Oligodeoxyꢀ
ribonucleotides I—IV are complementary to the region
located in the TAR hairpin structure of HIVꢀ1 mRNA,
which will allow one to study the effect of the attached
molecules (nonradioactive labels, peptide fragments) on
specific binding of oligodeoxyribonucleotides to a target
and on the ability to inhibit Tatꢀdependent transcription
of HIVꢀ1 DNA in vitro.40 5´ꢀDimethoxytrityl derivatives
of modified oligonucleotides I—IV were isolated by reꢀ
versedꢀphase HPLC. The purity of detritylation products
was monitored by ionꢀpair reversedꢀphase HPLC. The
oligonucleotides were characterized by matrixꢀassisted laꢀ
ser desorption ionization timeꢀofꢀflight (MALDI TOF)
mass spectrometry (Table 2).
oligonucleotides involves the preparation of
a
phosphoramidite of a modified nucleoside containing the
protected 1,2ꢀdiol group and automated synthesis folꢀ
lowed by removal of the protecting groups.
Results and Discussion
The synthesis of 1ꢀ[2ꢀOꢀ(2,3ꢀdihydroxypropyl)ꢀβꢀ
Dꢀarabinofuranosyl]uracil derivatives is presented in
Scheme 1. The starting compound 131 was transꢀ
formed into the N(3)ꢀ(2,4ꢀdinitrophenylsulfenyl) derivaꢀ
tive with transient trimethylsilylation of the 2´ꢀOH
group.32 Allylation of N(3)ꢀprotected arabinouridine 2

240
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 1, January, 2005
Zatsepin et al.
Scheme 1
i. 1) Me3SiCl, pyridine, 2) DnbsCl, pyridine, 3) aqueous NH3, pyridine; ii. H2C=CHCH2Br, BDDDP, MeCN; iii. Aqueous acetone;
iv. (MeCOCH2CH2CO)2O, DMAP, pyridine—CH2Cl2; v. Et3N(HF)3, THF; vi. DMTrCl, pyridine; vii. Pri2NP(Cl)OCH2CH2CN,
EtNPri2, CH2Cl2.
Investigation of the effect of the modified units on the
stability of duplexes with complementary DNA and RNA
fragments (see Table 1) revealed a slight decrease in the
melting point for complementary DNA and a more subꢀ
stantial decrease in the melting point for the duplex with
RNA. Recall that we used a uridine derivative, whereas a
single substitution of 2´ꢀdeoxyuridine for thymidine in
DNA leads to a decrease in the melting temperature of
the duplex by 0.5 °C.41 Our results are in good agreement
with the data obtained earlier26,27 for the modified
oligonucleotide containing the 1ꢀ(2ꢀOꢀmethylꢀβꢀDꢀ
arabinofuranosyl)thymine residues where the melting temꢀ

Arabinouridine 2´ꢀaldehyde oligonucleotides
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 1, January, 2005
241
Table 1. Thermal stability of duplexes formed by 2´ꢀmodified oligonucleotides with
complementary DNA and RNA
Oligonucleotidea
Tm 0.5/°С
TGAGCCTGGGAG r(UGAGCCUGGGAG)
CTCCCAGGCTCA (I)
CTCCCAGGCUCA (II)
CUCCCAGGCTCA (III)
CUCCCAGGCUCA (IV)
56.0
58.6
52.9b (53.1)c
50.3b (55.2)c
49.1b (47.0)c
49.5b (48.7)c
42.3b (49.5)c
40.9b (41.0)c
a
U is a modified unit.
b The melting point of the duplex in which U is 2´ꢀOꢀ(2,3ꢀdihydroxypropyl)arabinoꢀ
uridine.
c The melting point of the duplex in which U is 2´ꢀOꢀ(2ꢀoxoethyl)arabinouridine.
Table 2. Yields and massꢀspectrometric data (MALDIꢀTOF) for 2´ꢀmodified oligonucleotides and related conjugates
Conjugated moiety
Yield (%)
MS, m/z,
for [M + H]+
found
calculated
II´
III´
IV´
II´
III´
IV´
ОН*
—
—
—
3643.4
3645.1
3853.7
3853.9
3897.3
3893.0
3802.6
3800.9
4528.3
4531.0
3643.4
3641.3
3853.7
3855.1
—
3719.4
3718.3
4140.0
4142.1
4228.1
4223.5
4036.8
4038.1
5490.3
5489.2
Biotin hydrazide
74
82
84
65
79
—
85
—
69
74
62
50
4ꢀ(Pyrenꢀ1ꢀyl)butyrohydrazide
9ꢀHydrazinoacridine
H2NOCH2COꢀDPGYIGSRꢀNH2
3802.6
3803.0
—
* OH are oligonucleotides containing 2´ꢀOꢀ(2,3ꢀdihydroxypropyl)arabinouridine.
perature of the duplexes with complementary DNA and
RNA fragments decreased by 3.0 and 4.5 °C, respectively.
Earlier, it has been demonstrated24 that 2´ꢀOꢀmethylꢀ
arabinonucleosides adopt the C(2´)ꢀendo conformation
and thus their introduction into oligodeoxyribonucleotides
causes almost no changes in the local structure, and the
resulting duplexes with DNA have the classical B form.
On the contrary, hybrid DNAꢀRNA duplexes are strucꢀ
turally similar to the A form due to conformational labilꢀ
ity of DNA.42 The bulky 2´ꢀOꢀalkyl substituent in arabinoꢀ
uridine rather rigidly fixes a local conformation of the
oligonucleotide and, correspondingly, hinders the conꢀ
formational transition of the modified duplex (B → A). It
should also be taken into account that the 2,3ꢀdihydroxyꢀ
propyl group can hinder the formation of a duplex with
RNA due to both steric effects and partial displacement of
water molecules associated with the modified oligonucꢀ
leotide.
periodate in a weakly acidic medium (Scheme 2).
Periodate oxidation afforded modified aldehydeꢀcontainꢀ
ing oligonucleotides II´—IV´.
Then we synthesized conjugates of oligonucleotides
II´—IV´ with 9ꢀhydrazinoacridine (9), 4ꢀ(pyrenꢀ1ꢀ
yl)butyrohydrazide (10a) (fluorescent labels), biotin hyꢀ
drazide (10b) (marker group), and Nꢀaminooxyacetyl pepꢀ
tide (11) (see Scheme 2). Peptide 11 is an Nꢀmodified
fragment of the B1 domain of laminin (926—933)43 and
ensures binding to several types of receptors on the cell
surface.
The reactions were carried out in 50% aqueous DMSO
at pH 4.5—5.0. The results of HPLC analysis of the conꢀ
jugate of oligonucleotide II´ with 4ꢀ(pyrenꢀ1ꢀyl)butyroꢀ
hydrazide (10a) (Fig. 1, a) and the results of massꢀspecꢀ
trometric analysis confirmed the formation of the conjuꢀ
gate II´•10a (Fig. 1, b). It should be noted that the conꢀ
jugates of the modified oligonucleotides with aromatic
hydrazine (II´•9—IV´•9) and Nꢀaminooxy peptide
(II´•11—IV´•11), unlike conjugates with hydrazides, are
stable under the reaction conditions and under condiꢀ
The 1,2ꢀdiol group at position 2´ of a carbohydrate
fragment of oligonucleotides II—IV was oxidized to the
aldehyde group with a ~1000ꢀfold excess of sodium

242
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 1, January, 2005
Zatsepin et al.
Scheme 2
R1, R2 are deoxyribonucleotides
Irel•10–3
3893.08
A260
20
a
b
15
10
5
0.02
0.01
0
5
10
15
20
25 t/min
2
3
4
5
6
m/z•10–3
Fig. 1. (a) HPLC analysis of the reaction mixture after the reaction of oligonucleotide II´ with 4ꢀ(pyrenꢀ1ꢀyl)butyrohydrazide
(II´•10a) and (b) the MALDI TOF mass spectrum of conjugate II´•10a.

Arabinouridine 2´ꢀaldehyde oligonucleotides
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 1, January, 2005
243
acetophenone (40 mg mL–1 in methanol) and ammonium
hydrocitrate (80 mg mL–1 in water) (1 : 1, v/v) was used for
oligonucleotides. Elemental analysis was carried out on a
Thermosinigan CHNSꢀAnalyzer/EA1112 instrument. The abꢀ
sorbance and UV spectra were recorded on a Varian Cary 50
singleꢀbeam spectrophotometer (USA) in quartz cells with a
1 cm optical path length.
tions of ionꢀpair reversedꢀphase HPLC and do not reꢀ
quire reduction. This fact agrees well with the literature
data.44 The conjugates of compounds 9—11 were isolated
by reversedꢀphase HPLC and analyzed by ionꢀpair reꢀ
versedꢀphase HPLC and MALDI TOF mass spectromꢀ
etry (see Table 2).
To summarize, we synthesized a new modified
arabinouridine derivative containing the 1,2ꢀdiol group at
position 2´ of the ribose, 1ꢀ[2ꢀOꢀ(2,3ꢀdihydroxypropyl)ꢀ
βꢀDꢀarabinofuranosyl]uracil, and its 3´ꢀphosphoramidite.
Two new types of modified oligonucleotides containꢀ
ing the 1,2ꢀdiol or aldehyde group at position 2´ of
the arabinouridine residue were synthesized. The reactivꢀ
ity of the aldehyde group in the arabinonucleoside is
virtually identical to that of the aldehyde group in the
ribonucleoside.45 Duplexes based on modified oligoꢀ
nucleotides can be used for affinity modification of
DNAꢀbinding proteins because the introduction of
2´ꢀOꢀ(2ꢀoxoethyl)arabinouridine into the oligomer chain
causes no essential changes in local structures of nucleic
acids.
1ꢀ[3,5ꢀOꢀ(1,1,3,3ꢀTetraisopropyldisiloxaneꢀ1,3ꢀdiyl)ꢀβꢀDꢀ
arabinofuranosyl]ꢀ3ꢀ(2,4ꢀdinitrophenylsulfenyl)uracil (2). Comꢀ
pound 1 (2.85 g, 5.9 mmol), which was prepared according to a
known procedure,30 was dried by coevaporation with pyridine
(3×10 mL) and dissolved in anhydrous pyridine (90 mL). Then
Me3SiCl (17.7 mmol, 7.2 mL) was added with stirring at
room temperature under argon. After 30 min (TLC control;
CHCl3—EtOH, 9 : 1, Rf 0.8), 2,4ꢀdinitrobenzenesulfenyl chloꢀ
ride (DnbsCl) (9.4 mmol, 2.2 g) was added to the reaction
mixture. After 2 h (TLC control; CHCl3—EtOH, 95 : 5, Rf 0.5),
a concentrated ammonia solution (8.8 mL) and water (12.3 mL)
were added. The completeness of detrimethylsilylation was moniꢀ
tored by TLC (CHCl3—EtOH, 9 : 1, Rf 0.5). After 30 min, the
reaction mixture was concentrated and the residue (oil) was
dissolved in EtOAc (100 mL). The solution was washed with
a saturated aqueous NaHCO3 solution (50 mL) and water
(2×50 mL) and dried with anhydrous Na2SO4. The solvent was
removed, the residue was dissolved in a minimum volume of
chloroform, and hexane (100 mL) was added. The precipitate of
bis(2,4ꢀdinitrophenyl) disulfide that formed was filtered off. The
target nucleoside was isolated from the filtrate by column chroꢀ
matography (gradient of ethyl acetate in benzene 0→20%). The
yield of compound 2 was 2.68 g (67%).
Experimental
We used the following reagents: 3´ꢀ(2ꢀcyanoethylꢀN,Nꢀdiisoꢀ
propyl)phosphoramidites of 5´ꢀOꢀdimethoxytritylꢀ2´ꢀdeoxyꢀ
ribonucleosides, 5ꢀSꢀethylthiotetrazole (Glen Research, USA),
1ꢀ(βꢀDꢀarabinofuranosyl)uracil (Sigma, USA), βꢀcyanoethyl
N,Nꢀdiisopropylphosphoramidochloridite, 1,3ꢀdichloroꢀ1,1,3,3ꢀ
tetraisopropyldisiloxane, 2,4ꢀdinitrobenzenesulfenyl chloride,
triethylamine, chlorotrimethylsilane, 4ꢀ(N,Nꢀdimethylꢀ
amino)pyridine, 2ꢀtertꢀbutyliminoꢀ2ꢀdiethylaminoꢀ1,3ꢀdiꢀ
methylperhydroꢀ1,3,2ꢀdiazaphosphorine (BDDDP), allyl broꢀ
mide, DMSO (Fluka, USA), Nꢀmethylmorpholine Nꢀoxide, osꢀ
mium tetraoxide, triethylamine trihydrofluoride, tetrabutylꢀ
ammonium fluoride, 4,4´ꢀdimethoxytrityl chloride, N,Nꢀdiisoꢀ
propylethylamine, levulinic acid, N,N´ꢀdicyclohexylcarboꢀ
diimide, sodium periodate, 9ꢀhydrazinoacridine, sodium cyanoꢀ
borohydride, lithium perchlorate (Aldrich, USA), 4ꢀ(1ꢀpyrꢀ
enyl)butyrohydrazide, biotin hydrazide (Pierce, USA), sodium
hydrocarbonate, sodium carbonate, sodium sulfate, sodium chloꢀ
ride, sodium acetate, 30% aqueous ammonia, ethanol, acetic
acid, dichloromethane, pyridine, tetrahydrofuran, acetone
(Reakhim, Russia), and acetonitrile (Cryochrom, Russia). The
peptide NꢀaminooxyacetylꢀDPGYIGSRꢀamide was synthesized
according to a known procedure.45 Thinꢀlayer chromatography
was carried out on Kieselgel 60 F254 plates (Merck, Germany).
Column chromatography was performed on silica gel 33—70
(BDH, UK). The 1H, 13C, and 31P NMR spectra were recorded
on a Bruker DRXꢀ500 instrument in CDCl3 and DMSOꢀd6,
using residual CHCl3 and DMSO, respectively, as the internal
standards. The 31P NMR spectra were measured with H3PO4 as
the external standard. The MALDI TOF mass spectra were obꢀ
tained on a Voyager DE instrument (PerSeptive Biosystems,
USA). Solutions of 2,4,6ꢀtrihydroxyacetophenone (10 mg mL–1
in methanol) or 2,5ꢀdihydroxybenzoic acid (10 mg mL–1 in
methanol) were used as matrices in massꢀspectrometric analyꢀ
sis. A freshly prepared mixture of solutions of 2,6ꢀdihydroxyꢀ
Found (%): C, 47.71; H, 5.37; N, 8.21; S, 4.81.
C27H40N4O11SSi2. Calculated (%): C, 47.35; H, 5.89; N, 8.18;
1
S, 4.68. H NMR (CDCl3), δ: 8.78 (s, 1 H, HAr(3)); 8.26 (m,
1 H, Ar); 7.89 (d, 1 H, H(6), J5,6 = 6.3 Hz); 7.79 (m, 1 H, Ar);
5.97 (m, 1 H, H(1´)); 5.67 (d, 1 H, H(5)); 4.78—4.50 (m, 2 H,
H(3´), H(4´)); 4.01 (br.d, 2 H, H(5´)); 3.55 (m, 1 H, H(2´));
0.8—1.0 (m, 28 H, Pri). MS: found 682.0; calculated for
[M + H]+ 685.9.
1ꢀ[3,5ꢀOꢀ(1,1,3,3ꢀTetraisopropyldisiloxaneꢀ1,3ꢀdiyl)ꢀ2ꢀOꢀ
allylꢀβꢀDꢀarabinofuranosyl]ꢀ3ꢀ(2,4ꢀdinitrophenylsulfenyl)uracil
(3). Compound 2 (2.68 g, 3.9 mmol) was dried by coevaporation
with anhydrous toluene (3×10 mL) and the residue was disꢀ
solved in anhydrous MeCN (40 mL). 2ꢀtertꢀButyliminoꢀ2ꢀ
diethylaminoꢀ1,3ꢀdimethylperhydroꢀ1,3,2ꢀdiazaphosphorine
(1.6 mL, 5.2 mmol) and allyl bromide (0.6 mL, 6.5 mmol) were
added with stirring. The reaction was carried out at ~20 °C
for 2 h. The completeness of the reaction was monitored by TLC
(CHCl3—EtOH, 97 : 3, Rf 0.71). The solvent was removed, the
residue (oil) was dissolved in benzene, and the target compound
was isolated by column chromatography (gradient of ethyl acꢀ
etate in benzene 0→15%). The yield of compound 3 was
2.3 g (81%).
Found (%): C, 49.87; H, 5.76; N, 7.81; S, 4.36.
C30H44N4O11SSi2. Calculated (%): C, 49.71; H, 6.12; N, 7.73;
1
S, 4.42. H NMR (CDCl3), δ: 8.82 (s, 1 H, HAr(3)); 8.23 (d,
1 H, HAr(5), JHAr(5),HAr(6) = 2.3 Hz); 7.75 (d, 1 H, H(6), J5,6
=
8.0 Hz); 7.55 (d, 1 H, HAr(6)); 6.17 (d, 1 H, H(5)); 6.05 (d, 1 H,
H(1´), J1´2´ = 3.0 Hz); 5.81 (m, 3 H, —CH2—CH=CH2); 5.20
(dd, 1 H, H(3´), J2´,3´ = 3.5 Hz, J3´,4´ = 0.5 Hz); 4.55 (d, 2 H,
H(5´), J4´,5´ = 3.6 Hz); 4.35 (dd, 2 H, —CH2—CH=CH2, J1´´ ,2´´
=
4.9 Hz, J1´´,1´´ = 12.1 Hz); 4.15 (m, 1 H, H(4´)); 3.75 (dd, 1 H,
a
b

244
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 1, January, 2005
Zatsepin et al.
H(2´)); 1.00—1.13 (m, 28 H, Pri). MS: found 721.1; calculated
for [M + H]+ 725.9.
1.68 mmol) in anhydrous THF (10 mL) at ~20 °C, and
the reaction mixture was stirred for 3 h (TLC control;
CHCl3—EtOH, 9 : 1, Rf 0.1). Column chromatography (gradiꢀ
ent of ethanol in chloroform 0→15%) afforded compound 6 in a
yield of 0.73 g (85%).
Found (%): C, 51.27; H, 5.96; N, 5.44. C22H30N2O12. Calꢀ
culated (%): C, 51.36; H, 5.88; N, 5.45. 1H NMR (DMSOꢀd6),
δ: 10.61 (s, 1 H, H(3)); 8.11 (d, 1 H, H(6), J5,6 = 8.1 Hz);
6.12 (m, 1 H, H(1´)); 5.89 (d, 1 H, H(5)); 4.45 (m, 1 H,
1ꢀ[3,5ꢀOꢀ(1,1,3,3ꢀTetraisopropyldisiloxaneꢀ1,3ꢀdiyl)ꢀ2ꢀOꢀ
(2,3ꢀdihydroxypropyl)ꢀβꢀDꢀarabinofuranosyl]ꢀ3ꢀ(2,4ꢀdinitroꢀ
phenylsulfenyl)uracil (4). Compound 3 (2.3 g, 3.19 mmol) was
dissolved in acetone (35 mL). A solution of Nꢀmethylmorpholine
Nꢀoxide (0.56 g, 4.8 mmol) in water (7 mL) and a solution of
osmium tetraoxide (4 mg) in acetone (1 mL) were added with
stirring to the reaction mixture at room temperature. The reacꢀ
tion was monitored by TLC (CHCl3—EtOH, 95 : 5, Rf 0.2).
After 3 days, a saturated Na2S2O3 solution (1 mL) was added.
The reaction mixture was diluted with EtOAc and washed with a
saturated NaHCO3 solution (50 mL) and water (2×10 mL). The
organic phase was dried with anhydrous Na2SO4 and concenꢀ
trated in vacuo. The target product was isolated by column chroꢀ
matography (gradient of ethanol in chloroform 0→15%). The
yield of compound 4 was 2.1 g (90%).
—CH2—CH(OLev)—CH2OLev); 4.38 (d, 1 H, H(2´), J1´,2´
=
4.1 Hz); 4.37 (m, 2 H, CH2—CH(OLev)—CH2OLev); 4.05 (m,
1 H, H(3´)); 3.84 (m, 1 H, H(4´)); 3.76 (m, 2 H, H(5´)); 2.78
(m, 2 H, CH2—CH(OLev)—CH2OLev); 2.65—2.48 (m, 8 H,
CH2—CH(OLev)—CH2OLev); 2.18 (s, 6 H, CO—CH3). MS:
found 513.9; calculated for [M + H]+ 514.5.
1ꢀ[2ꢀOꢀ(2,3ꢀDilevulinoyloxypropyl)ꢀ5ꢀOꢀ(4,4´ꢀdimethoxyꢀ
trityl)ꢀβꢀDꢀarabinofuranosyl]uracil (7). Compound 6 (0.73 g,
1.42 mmol) was dried by coevaporation in vacuo with anhydrous
pyridine (3×10 mL) and dissolved in anhydrous pyridine (25 mL).
4,4´ꢀDimethoxytrityl chloride (1.7 mmol, 0.63 g) was added
with stirring at ~20 °C (TLC control; CHCl3—EtOH, 95 : 5,
Rf 0.4). After 2 h, excess dimethoxytrityl chloride was quenched
by the addition of MeOH (5 mL) and the reaction mixture was
concentrated. The residue (oil) was dissolved in CHCl3 (100 mL)
and washed with a saturated NaHCO3 solution (2×50 mL) and
water (2×50 mL). Standard workꢀup and column chromatograꢀ
phy (gradient of ethanol in chloroform 0→10%) afforded comꢀ
pound 7 in a yield of 1.1 g (95%).
Found (%): C, 47.77; H, 5.66; N, 7.44; S, 4.26.
C30H46N4O13SSi2. Calculated (%): C, 47.48; H, 6.11; N, 7.38;
1
S, 4.22. H NMR (CDCl3), δ: 8.78 (s, 1 H, HAr(3)); 8.20 (d,
1 H, HAr(5), JHAr(5),HAr(6) = 2.0 Hz); 7.70 (d, 1 H, H(6), J5,6
=
6.9 Hz); 7.31 (d, 1 H, HAr(6)); 6.10 (m, 1 H, H(1´)); 5.71
(d, 1 H, H(5)); 4.35 (m, 1 H, H(3´)); 4.13 (m, 2 H, H(5´)); 4.00
(m, 1 H, H(4´)); 3.73 (d, 1 H, H(2´), J1´,2´ = 3.0 Hz); 3.51
(br.d, 2 H, CH2—CH(OH)—CH2OH); 3.31 (m, 3 H,
CH2—CH(OH)—CH2OH); 3.08 (m, 2 H, OH); 1.21—0.95 (m,
28 H, Pri). MS: found 756.2; calculated for [M + H]+ 760.0.
1ꢀ[3,5ꢀOꢀ(1,1,3,3ꢀTetraisopropyldisiloxaneꢀ1,3ꢀdiyl)ꢀ2ꢀOꢀ
(2,3ꢀdilevulinoyloxypropyl)ꢀβꢀDꢀarabinofuranosyl]ꢀ3ꢀ(2,4ꢀdiꢀ
nitrophenylsulfenyl)uracil (5). Levulinic acid (0.5 mL, 5.5 mmol)
and N,N´ꢀdicyclohexylcarbodiimide (0.5 g, 2.8 mmol) in dioxꢀ
ane (30 mL) were vigorously stirred for 2 h. N,N´ꢀDicycloꢀ
hexylurea that precipitated was filtered off and the resulting
solution of levulinic anhydride was added to a solution of comꢀ
pound 4 (2.1 g, 2.8 mmol) in anhydrous pyridine (20 mL). Then
a catalytic amount (5 mg) of 4ꢀ(N,Nꢀdimethylamino)pyridine
was added. After 18 h (the reaction was monitored by TLC;
CHCl3—EtOH, 95 : 5, Rf 0.3), the reaction mixture was conꢀ
centrated in vacuo, and the residue (oil) was dissolved in EtOAc
(50 mL). The solution was washed with a saturated NaHCO3
solution (50 mL) and water (2×50 mL) and dried with anhyꢀ
drous Na2SO4. Target product 5 was isolated by column chroꢀ
matography (gradient of ethanol in chloroform 0→10%) in a
yield of 1.6 g (60%).
Found (%): C, 63.17; H, 5.96; N, 3.44. C43H48N2O14. Calꢀ
1
culated (%): C, 63.23; H, 5.92; N, 3.43. H NMR (CDCl3), δ:
10.21 (s, 1 H, H(3)); 7.68 (d, 1 H, H(6), J5,6 = 7.8 Hz);
7.45—6.88 (m, 13 H, DMTr); 6.19 (d, 1 H, H(1´), J1´,2´
=
2.8 Hz); 5.56 (d, 1 H, H(5)); 5.27 (s, 1 H, C(3´)—OH); 4.45
(m, 1 H, CH2—CH(OLev)—CH2OLev); 4.37 (m, 2 H,
CH2—CH(OLev)—CH2OLev); 4.25 (dd, 1 H, H(3´), J2´,3´
3.2 Hz, J3´,4´ = 0.2 Hz); 4.03 (dd, 1 H, H(2´)); 3.80 (s, 6 H,
OCH3); 3.54 (m, 1 H, H(4´)); 3.45 (d, 2 H, Ha(5´), JH(4´),H (5´)
=
=
a
2.4 Hz); 3.45 (d, 2 H, Hb(5´), JH(4´),H (5´) = 10.4 Hz); 2.78
(m, 2 H, CH2—CH(OLev)—CH2OLev)b; 2.65—2.48 (m, 8 H,
CH2—CH(OLev)—CH2OLev); 2.18 (s, 6 H, CO—CH3). MS:
found 816.2; calculated for [M + H]+ 817.8.
1ꢀ{2ꢀOꢀ(2,3ꢀDilevulinoyloxypropyl)ꢀ3ꢀOꢀ[(2ꢀcyanoꢀ
ethoxy)(N,Nꢀdiisopropylamino)phosphino]ꢀ5ꢀOꢀ(4,4´ꢀdiꢀ
methoxytrityl)ꢀβꢀDꢀarabinofuranosyl}uracil (8). βꢀCyanoethyl
N,Nꢀdiisopropylphosphoramidochloridite (0.18 mL, 2.7 mmol)
was added dropwise to a solution of compound 7 (1.1 g,
1.35 mmol), which was thoroughly predried, and N,Nꢀdiꢀ
isopropylethylamine (0.27 mL, 2.7 mmol) in anhydrous diꢀ
chloromethane (20 mL) at ~20 °C. After 2 h (TLC control;
CH2Cl2—Et3N, 98 : 2, Rf 0.4), the reaction mixture was diluted
with dichloromethane (15 mL) and washed with a cold satuꢀ
rated NaCl solution (2×25 mL). After standard workꢀup, target
phosphoramidite 8 was isolated by column chromatography (graꢀ
dient of methanol in dichloromethane containing 0.5% of triꢀ
ethylamine 0→3%) in a yield of 0.9 g (70%).
Found (%): C, 50.57; H, 5.76; N, 5.74; S, 3.29.
C40H58N4O17SSi2. Calculated (%): C, 50.30; H, 6.12; N, 5.87;
1
S, 3.36. H NMR (CDCl3), δ: 8.89 (s, 1 H, HAr(3)); 8.28 (d,
1 H, HAr(5), JHAr(5),HAr(6) = 3.0 Hz); 7.70 (d, 1 H, H(6), J5,6
=
7.4 Hz); 7.55 (d, 1 H, HAr(6)); 6.28 (d, 1 H, H(1´)); 6.20 (d,
1 H, H(5)): 5.79 (m, 1 H, CH2—CH(OLev)—CH2OLev);
5.58 (m, 1 H, H(3´)); 4.47 (m, 2 H, H(5´)); 4.11 (m,
2
H, CH2—CH(OLev)—CH2OLev); 3.80 (m,
CH2—CH(OLev)—CH2OLev); 3.55 (m, H, H(4´));
3.39 (d, 1 H, H(2´), J1´,2´ = 3.6 Hz): 2.75 (m, 4 H,
OCO—CH2—CH2—COMe); 2.65 (m, H,
OCO—CH2—CH2—COMe); 2.10 (s, H, CO—CH3);
2
H,
1
4
6
1H NMR (CDCl3), δ: 10.01 (s, 1 H, H(3)); 7.68 (d, 1 H,
H(6), J5,6 = 7.2 Hz); 7.45—7.20, 6.85—6.73 (m, 13 H, DMTr);
6.23 (d, 1 H, H(1´), J1´,2´ = 3.0 Hz); 5.58 (d, 1 H, H(5)); 5.32
(m, 1 H, CH2CH); 4.45—4.28 (m, 2 H, H(2´), CHCHaHb);
4.17—4.12 (m, 2 H, H(4´), CHCHaHb); 3.89—3.79 (m, 2 H,
H(3´), CH2CH); 3.73 (s, 6 H, DMTr); 3.52 (dd, 1 H, Ha(5´),
1.68—0.95 (m, 28 H, Pri). MS: found 952.7; calculated for
[M + H]+ 956.2.
1ꢀ[2ꢀOꢀ(2,3ꢀDilevulinoyloxypropyl)ꢀβꢀDꢀarabinofuranoꢀ
syl]uracil (6). Triethylamine trihydrofluoride (5.3 mmol, 1 mL)
was added with stirring to a solution of compound 5 (1.6 g,

Arabinouridine 2´ꢀaldehyde oligonucleotides
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 1, January, 2005
245
JH(4´),H (5´) = 6.0 Hz, JH (5´),H (5´) = 12.1 Hz); 3.65 (m, 4 H,
ammonium acetate; the gradient of acetonitrile concentration
was 0—40% (80 min).
a
CH(CH3)2, POCH2); 3.4a 7 (dbd, 1 H, Hb(5´), JH(4´),H (5´)
=
10.0 Hz); 2.78 (m, 4 H, CO2CH2); 2.66 (m, 4 H, CH2COb); 2.54
(t, 2 H, CH2CN); 2.15 (s, 6 H, COCH3); 1.10 (m, 12 H,
CH3CH). 13C NMR (CDCl3), δ: 206.63 (2 C, C=O, Lev); 172.41
(2 C, OC=O, Lev); 162.59 (1 C, C(4)); 158.67 (2 C, DMTr);
150.54 (1 C, C(2)); 144.43 (1 C, DMTr); 140.45 (1 C, C(6));
132.28, 130.19, 128.29, 127.96, 127.12, 117.74 (11 C, DMTr);
117.62 (1 C, CN); 113.22, 112.11 (4 C, DMTr); 103.05
(1 C, C(5)); 100.04 (1 C, C(1´)); 87.16 (1 C, C(4´)); 86.47
(1 C, C(2´)); 84.64 (1 C, C(3´)); 83.04 (1 C, DMTr); 68.27
(1 C, C(2´´)); 65.49 (1 C, C(1´´)); 62.27 (1 C, C(5´)); 62.04
(1 C, C(3´)); 59.92 (1 C, POCH2); 55.32 (2 C, DMTr); 42.79
(1 C, PNCH); 37.51 (2 C, CH2COCH3); 29.92 (2 C, CO2CH2);
29.76 (2 C, COCH3); 26.11 (4 C, CH3, Pri); 20.26
(1 C, CH2CN). 31P NMR (CDCl,), δ: 150.40, 151.73. MS:
found 1016.1; calculated for [M + H]+ 1018.1.
Automated oligodeoxyribonucleotide synthesis was carried
out on an Applied Biosystems 394A synthesizer (USA) acꢀ
cording to a standard protocol using commercial reagents
and solvents. Controlled pore glass (CPGꢀ500) containing
an immobilized nucleoside was used as the polymer support.
The specific loading of the polymer with the first monomer
unit was 25—30 µmol g–1. 3´ꢀPhosphoramidites of natural
2´ꢀdeoxyribonucleosides were used as 0.1 M solutions in anꢀ
hydrous acetonitrile. The concentrations of the 3´ꢀphosphorꢀ
amidite derivative of arabinouridine 8 in acetonitrile was
0.15 mol L–1. The reaction time in the step of coupling of the
modified monomer was increased to 15 min, and condensation
was carried out twice. In this step, the degree of conversion
was approximately 95%, which is slightly lower than that for
3´ꢀphosphoramidites of natural 2´ꢀdeoxyribonucleosides. Oligoꢀ
deoxyribonucleotides were deprotected and liberated from the
polymer support with a concentrated aqueous ammonia soluꢀ
tion at 55 °C for 18 h, the solutions of the 2´ꢀmodified oligoꢀ
nucleotides were concentrated in vacuo, and the target products
were isolated by ionꢀpair reversedꢀphase HPLC. The oligoꢀ
nucleotides were desalted by five consecutive evaporations with
50% aqueous ethanol and treated with 80% aqueous acetic acid
(1 mL) at ~20 °C for 30 min. Then the acetic acid was evapoꢀ
rated, and 50% aqueous ethanol was added and distilled off
three times. The residue was dissolved in water and the resulting
oligodeoxyribonucleotides were analyzed by ionꢀpair reversedꢀ
phase HPLC.46
Synthesis of modified 2´ꢀaldehydeꢀcontaining oligonucleotides
II´—IV´. Modified 2´ꢀOꢀ(2,3ꢀdihydroxypropyl)arabinouridineꢀ
containing oligonucleotides (0.5 OE260) were dissolved in a 0.1 M
acetate buffer, pH 4.6 (10 µL). Then a 5 mM sodium periodate
solution (5 µL) was added, the reaction mixture was kept at
~20 °C for 1 h, and a 2 M acetate buffer (20 µL) was added. The
oligonucleotide material was twice precipitated with ethanol
and washed with ethanol (200 µL).
Synthesis of conjugates of 2´ꢀaldehydeꢀcontaining oligoꢀ
nucleotides II´—IV´ with hydrazides. Modified aldehydeꢀconꢀ
taining oligonucleotides (0.5 OE260) were dissolved in a 0.4 M
acetate buffer, pH 4.6 (10 µL). Then a 20 mM hydrazide soluꢀ
tion in DMSO (1 µL) was added. After 1 h, a 0.5 M NaBH3CN
solution (5 µL) was added at ~20 °C, the reaction mixture was
kept for 2 h, and then a 4 M NaOAc solution (5 µL) and ethanol
(200 µL) were added. The conjugate that precipitated was
reprecipitated with ethanol and washed with ethanol (200 µL).
The target compounds were dissolved in a deionized water
(50 µL) and analyzed by ionꢀpair reversedꢀphase HPLC.
Synthesis of conjugates of 2´ꢀaldehydeꢀcontaining oligoꢀ
nucleotides II´—IV´ with 9ꢀhydrazinoacridine and Nꢀaminooxyꢀ
acetyl peptide. Modified aldehydeꢀcontaining oligonucleotides
(0.5 OE260) were dissolved in a 0.4 M acetate buffer, pH 4.6
(10 µL). Then a 20 mM solution of 9ꢀhydrazinoacridine or
Nꢀaminooxy peptide in DMSO (1 µL) was added, the reaction
mixture was kept at ~20 °C for 1 h, and a 4 M sodium acetate
solution (5 µL) and ethanol (200 µL) were added. The precipiꢀ
tate of the conjugate was reprecipitated with ethanol and washed
with ethanol (200 µL). The target compounds were dissolved in
deionized water (50 µL) and analyzed by ionꢀpair reversedꢀ
phase HPLC.
Study of thermal stability of duplexes. The temperature deꢀ
pendences of UV absorption of nucleic acid duplexes were meaꢀ
sured on a Varian Cary 50 singleꢀbeam spectrophotometer (USA)
at a wavelength of 260 nm, the rate of temperature increase was
0.5 K min–1 in a temperature range of 5—85 °C followed by a
decrease in the temperature with a rate of 0.5 K min–1 in the
same temperature range in temperatureꢀcontrolled Pye Unicam
quartz cells (UK) with an optical path length of 1 cm. Thermal
stability of the duplexes was studied in a phosphate buffer
(100 mM NaCl, 10 mM Na2HPO4, 1 mM EDTA, pH 7.0) at a
duplex concentration of 4 µmol L–1
.
Analysis and isolation of oligonucleotides. After completion
of the reactions, the mixtures of oligonucleotides were analyzed
and the products (after their isolation) were tested for purity
by ionꢀpair reversedꢀphase HPLC on a Waters chromatoꢀ
graph (USA) equipped with a 4×250 mm column packed with
Diasorbꢀ130ꢀC16T (particle size, 7 µm). The conditions of anaꢀ
lytical separation: the column temperature was 45 °C, the flow
rate was 1 mL min–1. The eluent: 48 mM potassium phosphate
buffer, pH 7.0, containing 2 mM tetraꢀnꢀbutylammonium
dihydrophosphate. The gradient of acetonitrile concentration:
5—20.4% (1 min); 20.4—21.6% (1 min); 21.6—23.2% (3 min);
23.2—24.4% (5 min); 24.4—25.6% (10 min). Oligodeoxyriboꢀ
nucleotides and their conjugates were isolated by reversedꢀphase
HPLC on a Tracor chromatograph (Netherlands) equipped with
a 4×250 mm column packed with Diasorbꢀ130ꢀC16T (particle
size, 7 µm). The conditions of separation: the column temperaꢀ
ture was 45 °C, the flow rate was 1 mL min–1. The eluent: 0.1 M
This study was financially supported by the Wellcome
Trust (Grant CRIG 069419) and the Russian Foundation
for Basic Research (Project No. 03ꢀ04ꢀ48957).
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Received October 4, 2004;
in revised form December 20, 2004