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KHANDAZHINSKAYA et al.
4.69 (2 H, d, J 6.8, CH2 of Fmoc), 4.34 (1 H, t, H9 of and H8 of Fmoc), 7.53 and 7.45 (4 H, 2 t, J 7.5, H2, H3,
H6, and H7 of Fmoc), 4.21 (2 H, d, J 6.8, CH2 of
Fmoc), 4.04 (1 H, t, H9 of Fmoc), 3.86 (2 H, m, CH2O),
3.25 (2 H, m, CH2CH2OP), 2.85 (2 H, m, CH2NHCO2),
2.19 [2 H, m, CH2CO], and 1.53–1.10 [6 H, m,
(CH2)3CH2N]; 31P NMR (D2O): –10.24 (1 P, d, J 20.3,
Pγ), –10.64 (1 P, d, J 19.3, Pα), and –22.67 (1 P, dd, Pβ).
The substrate activity of the synthesized com-
pounds. The labeled oligonucleotide was obtained by
the procedure described earlier [3]. The reaction
mixture (10 µl) containing 0.02 µM [5'-32P]-labeled
14-membered oligodeoxynucleotide, 0.2 U of TDT,
100 mM sodium cacodylate (pH 7.2), 2 mM CoCl2,
0.1 mM dithiothreitol, and the substrates at various
concentrations was incubated for 10 min at 37°C, ter-
minated with formamide (5 µl) containing 0.5 mM
EDTA and 0.1% Bromophenol Blue and Xylene
Cyanole. The products were separated in 15% denatur-
ing polyacrylamide gel. The gels were exposed to a
Kodak RX roentgen film. For the quantitative determi-
nation, the autoradiograph was scanned on a Molecular
Dynamics 300A Computing densitometer.
Fmoc), 3.89 (2 H, m, CH2O), 3.03 (2 H, m, CH2N), and
1.63–1.19 [8 H, m, (CH2)4CH2N]; 31P NMR (D2O):
−10.15 (1 P, d, J 19.3, Pγ), –10.24 (1 P, d, J 20.3, Pα),
and −22.67 (1 P, dd, Pβ).
N-[(9-Fluorenylmethoxycarbonyl)aminohexan-
oyl]-2-aminoethyl triphosphate (II). A solution of N-
9-fluorenylmethoxycarbonyl chloride (400 mg,
1.6 mmol) in dioxane (800 µl) was added to a solution
of 6-aminohexanoic acid (260 mg, 2 mmol) in aqueous
Na2CO3 (860 mg, 8 mmol, 8 ml), and the mixture was
kept for 18 h at room temperature. The reaction mixture
was diluted with water to 100 ml and extracted with
ether (3 × 20 ml). The aqueous fraction (pH 10) was
adjusted to pH 5 with 1 M HCl, the precipitate was fil-
tered, washed with water, and dried to give 721 mg
(93%) of N-(9-fluorenylmethoxycarbonyl)aminohex-
anoic acid; 1H NMR (CDCl3): 7.75 and 7.58 (4 H, 2 d,
J 7.5, H1, H4, H5, and H8 of Fmoc), 7.53 and 7.45
(4 H, 2 t, J 7.5, H2, H3, H6, and H7 of Fmoc), 4.87
(1 H, br. s, NH), 4.39 (2 H, d, J 6.5, CH2 of Fmoc), 4.21
(1 H, t, H9 of Fmoc), 3.19 (2 H, m, CH2N), 2.35 (2 H,
m, CH2CO), and 1.64–1.36 [6 H, m, (CH2)3CH2N].
2-Aminoethyl phosphate (52 mg, 0.3 mmol) was
refluxed in N,O-bis(trimethylsilyl)acetamide (3 ml) for
10 h. The reagent was evaporated, and the residue was
dissolved in a mixture of hexamethyldisilazane (1 ml)
and DMF (1 ml). A solution obtained by the addition of
thionyl chloride (220 µl, 3 mmol) to a solution of N-(9-
fluorenylmethoxycarbonyl)aminohexanoic acid in
dichloromethane (5 ml) was stirred for 3 h at room tem-
perature, evaporated, and added to a solution of the
silylated 2-aminoethyl phosphate. The reaction mixture
was stirred for 12 h at room temperature, and evapo-
rated. After the dissolution of residue in DMF (5 ml),
CDI (324 mg, 2 mmol) was added, the mixture was
stirred for 5 h, and 0.23 mM bis(tributylammonium)
pyrophosphate (3.5 ml, 0.8 mmol) in DMF was added.
After 3-h stirring, the mixture was applied onto a
Dowex 50 column (2 × 4 cm) and eluted with 50%
methanol (50 ml). The solvents were evaporated, and
the residue was chromatographed on a LiChroprep RP-
18 column (2 × 18 cm) in a gradient of methanol (0
20%) in water to give 18 mg (14%) of (Id); Rf 0.46; 1H
NMR (D2O): 7.67 and 7.47 (4 H, 2 d, J 7.5, H1, H4, H5,
ACKNOWLEDGMENTS
The work was supported by the Russian Foundation
for Basic Research, project no. 04-04-49621. The
authors are grateful to E.A. Shirokova and Yu.S. Skob-
lov for the assistance in the preparation of the manu-
script.
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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 4 2005