1682
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 9, September, 2006
Vasil´eva et al.
target product was no longer detected in the organic phase. The
combined organic layer was dried with anhydrous Na2SO4. Yield
0.45 g (52%), foam. The spectroscopic characteristics of the
product corresponded to published data.20
(d, 1 H, H(1´), J = 8 Hz); 6.85 (m, 4 H, (Ar)); 7.65—7.19 (m,
12 H, 3 H (COC6H5), 9 H (Ar)); 8.12 (d, 2 H, COC6H5,
J = 8 Hz); 8.42 (s, 1 H, H(2)); 8.54 (s, 1 H, H(8)).
MS (MALDIꢀTOF), m/z 759.26 [M + H]. C41H38N6O9. Calcuꢀ
lated: M = 758.27.
9ꢀ[2ꢀDeoxyꢀ2ꢀmethoxalylaminoꢀ5ꢀOꢀ(4,4´ꢀdimethoxytrityl)ꢀ
3ꢀ(2ꢀcyanoethoxyꢀN,Nꢀdiisopropylaminophosphino)ꢀβꢀDꢀriboꢀ
furanosyl]ꢀN6ꢀbenzoyladenine (11) was prepared by a standard
procedure from nucleoside 10 (50 mg, 0.065 mmol). The reacꢀ
tion time was 3 h. Yield 47 mg (75%). Rf 0.62 (CHCl3—MeOH,
9 : 1). 31P NMR (acetoneꢀd6), δ: 152.53; 152.41.
MS (MALDIꢀTOF), m/z: 959.3724 [M + H]. C50H55N8O10P.
Calculated: M = 958.3778.
9ꢀ[2ꢀ(NꢀBenzyloxycarbonyl)aminoꢀ2ꢀdeoxyꢀβꢀDꢀfuranosyl]ꢀ
N6ꢀbenzoyladenine (8) was prepared by a known procedure20
from compound 7 (134 mg, 0.356 mmol) with 1,2ꢀdichloroethane
as the solvent. After completion of transglycosylation, the reacꢀ
tion mixture was concentrated, and the residue was dissolved in
a 1 M solution of tetrabutylammonium fluoride in dioxane and
stirred for 1 h at room temperature. The reaction mixture was
concentrated, and the residue was dissolved in CHCl3 and
chromatographed on silica gel. The product was eluted with a
MeOH—CHCl3 gradient mixture (0→10%). The target fracꢀ
tions with Rf 0.75 (EtOAc—MeOH, 8 : 2) were concentrated.
The resulting compound 8 was used in the next step without
additional purification
Synthesis of oligonucleotide conjugates
After completion of the solidꢀphase synthesis, the resin with
attached oligonucleotides ONꢀ3, ONAꢀ4, ONꢀ5, and ONAꢀ6
was transferred into tubes, treated with 200 µL of ethylenediꢀ
amine, and kept for 2 h at 30 °C. Complete cleavage of the
oligonucleotides from the resin and removal of the protecting
groups were performed by adding concentrated aqueous ammoꢀ
nia (0.5 mL) followed by stirring of the reaction mixture for 24 h
at 30 °C. After preliminary purification of the reaction mixtures
on a PolyꢀPakTM cartridge according to manufacturer´s recomꢀ
mendations, the solutions of oligonucleotides were concentrated
to 100 µL and treated with a 8% solution of LiClO4 in acetone
(1 mL). The precipitated lithium salts of oligonucleotides conꢀ
taining an amine linker group in the 11th residue were dissolved
in the minimum volume of water (20—50 µL). A 1/10 volume of
a 1 M solution of NaHCO3 and a saturated solution of fluoresꢀ
cein isothiocyanate (FITC) were added (in five portions over a
period of 12 h at 30 °C, 20ꢀfold molar excess of FITC in DMF).
The oligonucleotide conjugates were purified by gel filtration on
Sephadex Gꢀ10 (a 2×50 mm column), elution with 10 mM
TrisꢀHCl, 1 mM EDTA, pH 7.4. The purified conjugates were
reprecipitated with a 2% solution of LiClO4 in acetone.
9ꢀ[2ꢀ(NꢀBenzyloxycarbonyl)aminoꢀ2ꢀdeoxyꢀ5ꢀOꢀ(4,4´ꢀdiꢀ
methoxytrityl)ꢀβꢀDꢀribofuranosyl]ꢀN6ꢀbenzoyladenine (9). Comꢀ
pound 8 obtained in the preceding step was concentrated with
dry pyridine (15 mL) to 1 mL. 4ꢀDimethylaminopyridine (5 mg)
and 4,4´ꢀdimethoxytrityl chloride (DMT2Cl) (117 mg,
0.35 mmol) were added and the mixture was stirred for 16 h. The
reaction mixture was concentrated, dissolved in CHCl3 conꢀ
taining 0.1% pyridine, and washed with water, a 5% solution of
NaHCO3, and again water. The organic layer was dried with
Na2SO4 and concentrated to 5 mL, and hexane (50 mL) was
added. The product was chromatographed on a column with
silica gel using chloroform containing 0.1% pyridine as the eluꢀ
ent. The target fractions with Rf 0.38 (CHCl3—MeOH, 9 : 1)
were concentrated and precipitated with hexane. Yield 70 mg
(25%) based on 1ꢀ(2ꢀaminoꢀ2ꢀdeoxyꢀβꢀDꢀribofuranosyl)uraꢀ
cil (6). 1H NMR (CD3OD), δ: 3.5 (m, 2 H, H(5´)); 3.75 (s, 6 H,
OMe(Ar)); 4.28 (m, 1 H, H(4´)); 4.50 (m, 1 H, H(3´)); 5.00 (s,
2 H, C(O)OCH2C6H5); 5.3 (dd, 1 H, H(2´), J2´,1´ = 2 Hz,
J
2´,3´ = 7 Hz); 6.25 (d, 1 H, H(1´), J = 4 Hz); 6.84 (m, 4 H,
(Ar)); 7.72—7.26 (m, 17 H, 3 H (COC6H5), 9 H (Ar), 5 H
(C(O)OCH2C6H5)); 8.11 (d, 2 H, COC6H5, J = 4 Hz); 8.51 (s,
1 H, H(2)); 8.62 (s, 1 H, H(8)).
This work was supported by the Russian Foundation
for Basic Research (Projects No. 05ꢀ04ꢀ48985 and No. 04ꢀ
04ꢀ48566) and the Russian Academy of Sciences (Fundaꢀ
mental Research Program of the RAS "Biosphere Origin
and Evolution").
9ꢀ[2ꢀAminoꢀ2ꢀdeoxyꢀ5ꢀOꢀ(4,4´ꢀdimethoxytrityl)ꢀβꢀDꢀriboꢀ
furanosyl]ꢀN6ꢀbenzoyladenine (5). Compound 9 (403 mg,
0.5 mmol) was hydrogenated under atmospheric pressure in the
presence of 5% Pd/C catalyst (50 mg) and 30% NH3(aq.)
(0.5 mL) in dioxane (20 mL) for 12 h. The catalyst was filtered
off and washed with dioxane (3×10), and the combined dioxane
solution was concentrated. The yield of the product formed as a
foam was 290 mg (87%). Rf 0.19 (CHCl3—MeOH, 9 : 1).
1H NMR (CD3OD), δ: 3.5 (m, 2 H, H(5´)); 3.76 (s, 6 H, OMe);
4.34 (m, 1 H, H(4´)); 4.5 (m, 1 H, H(3´)); 4.62 (m, 1 H, H(2´));
6.20 (d, 1 H, H(1´), J = 4 Hz); 6.84 (m, 4 H, (Ar)); 7.72—7.20
(m, 12 H, 3 H (COC6H5), 9 H (Ar)); 8.11 (d, 2 H, COC6H5,
J = 4 Hz); 8.51 (s, 1 H, H(2)); 8.62 (s, 1 H, H(8)).
MS (MALDIꢀTOF), m/z: 673.23 [M + H]; 694.93 [M + Na].
C38H36N6O6. Calculated: M = 672.7290.
9ꢀ[2ꢀDeoxyꢀ2ꢀmethoxalylaminoꢀ5ꢀOꢀ(4,4´ꢀdimethoxytrityl)ꢀ
βꢀDꢀribofuranosyl]ꢀN6ꢀbenzoyladenine (10) was synthesized from
nucleoside 5 (124 mg, 0.184 mmol) by a procedure described for
compound 3. A white powder, yield 60 mg (44%). Rf 0.47
(CHCl3–MeOH, 9 : 1). 1H NMR (acetoneꢀd6), δ: 3.50 (m, 2 H,
H(5´)); 3.76 (s, 3 H, OMe); 3.79 (s, 6 H, OMe(Ar)); 4.36 (m,
1 H, H(4´)); 4.73 (m, 1 H, H(3´)); 5.55 (m, 1 H, H(2´)); 6.37
References
1. A. F. Nasonov and G. A. Korshunova, Usp. Khim., 1999, 68,
532 [Russ. Chem. Rev., 1999, 68, 532 (Engl. Transl.)].
2. M.T. Tierney and M. W. Grinstaff, Org. Lett., 2000, 2, 3413.
3. H. Rosemeyer, N. Ramzaeva, E. M. Becker, E. Feiling, and
F. Seela, Bioconjug. Chem., 2002, 13, 1274.
4. T. S. Zatsepin, E. A. Romanova, and T. S. Oretskaya, Usp.
Khim., 2004, 73, 757 [Russ. Chem. Rev., 2004, 73, 701 (Engl.
Transl.)].
5. M. Manoharan, Biochim. Biophys. Acta, 1999, 1489, 117.
6. H. Ozaki, S. Momiyama, K. Yokotsuka, and H. Sawai, Tetraꢀ
hedron Lett., 2001, 42, 677.
7. K. Yamana, T. Mitsui, H. Hayashi, and H. Nakano, Tetraꢀ
hedron Lett., 1997, 38, 5815.