Nucleoside 5′-Diphosphate Imidazolide
J . Org. Chem., Vol. 64, No. 16, 1999 5837
(0. 5 mL), and triethylamine (2 mL) with stirring. The sodium
salt of ImppA was obtained in 82% yield (48 mg, 1180 ODU
at 260 nm) as white precipitates which were collected by a
glass filter, washed with dry acetone and then with dry ether,
and dried in a desiccator. The ImppA is stable under dry
conditions at low temperature and can be stored in a refriger-
ated desiccator. Analytical HPLC: tR ) 24.2 min (system B).
λmax ) 260 nm. 1H NMR δ (D2O): 8.39 (s, 1H), 8.26 (s, 1H),
7.94 (s, 1H), 7.30 (s, 1H), 6.98 (s, 1H), 6.10 (d, 1H, J 1′,2′ ) 5.9
Hz), 4.71 (dd,1H), 4.40 (dd,1H), 4.33 (m, 1H), 4.09 (m, 2H).
Syn t h esis of 7-Met h ylgu a n osin e 5′-Dip h osp h a t e
(p p 7m G). The triethylammonium salt of 7-methylguanosine
5′-monophosphate16 (15120 ODU at 260 nm, 1.5 mmol) was
dried by coevaporation with DMF twice. N,N′-Carbonyldiimi-
dazole (2.4 g, 14.7 mmol) and dry DMF (10 mL) were added to
the residue, and the solution was stirred for 1 h at 40 °C. After
an excess amount of N,N′-carbonyldiimidazole was degraded
by addition of a small amount of dry methanol to the reaction
mixture, tris(tri-n-butylammonium)phosphate (10 mmol) in
dry DMF (20 mL) was added and the solution was stirred for
3 d at room temperature. The completion of the reaction was
checked by HPLC. The reaction mixture was poured into a
solution containing dry acetone (300 mL), ether (150 mL),
NaClO4-saturated acetone (6 mL), and triethylamine (2 mL)
to precipitate 7-methylguanosine 5′-diphosphate as a sodium
salt. The precipitates were collected with a glass filter, washed
with acetone and ether, and dried. The resulting precipitates
were dissolved in water, passed through a column on activated
carbon, washed with water to remove the phosphate, and
eluted with a solution of pyridine:ethanol:water (1:20:20). The
eluate was evaporated to dryness and applied to an anion-
exchange column (3 × 50 cm) on QAE-Sephadex (bicarbonate
form) with a linear gradient elution of triethylammonium
bicarbonate buffer (0-0.5 M). Appropriate fractions were
collected and lyophilized several times to remove triethyl-
ammonium bicarbonate. 7-Methylguanosine 5′-diphosphate
triethylammonium was obtained in 57% yield, 8450 ODU at
In this paper we report a synthesis and the hydrolytic
stability of nucleoside 5′-diphosphate imidazolide and an
application of 7-methyylguanosine 5′-diphosphate imi-
dazolide to the nonenzymatic synthesis of the capped
oligoribonucleotides from 5′-phosphorylated oligoribo-
nucleotides in aqueous solution.
Ma ter ia ls a n d Meth od s
Ma t er ia ls. Adenosine and guanosine 5′-monophosphates
(pA and pG) were purchased from Seikagaku Kogyo. Adenosine
5′-diphosphate was from Boeringer. The 5′-monophosphate of
hexariboadenylate (pAAAAAA) was prepared by partial diges-
tion of poly(A) with nuclease SW as described previously.15 The
chemically synthesized 5′-monophosphate of RNA 11mer
(pACACUUGCUUU) was purchased from Genset. Nuclease
P1 was from Seikagaku Kogyo, and Venom phosphodiesterase
and alkaline phosphatase were from Worthington Biochemi-
cals. 7-Methylguanosine 5′-monophosphate (p7mG) was pre-
pared from pG and methyl iodide by slight modification of the
published procedure.16 Adenosine 5′-phosphorimidazolide
(ImpA)11c and 7-methylguanosine 5′-phosphorimidazolide
(Imp7mG)10 were prepared from pA and p7mG, respectively,
as described previously. All other chemicals were of reagent
grade.
HP LC. HPLC on an ODS-silica gel column (4 mm × 250
mm) was done with a linear gradient elution from 2.4 to 12
(system A), 24 (system B), or 40% (system C) methanol in 0. 1
M triethylammonium acetate at pH 7.0 in 30 min at a flow
rate of 1.0 mL/min. HPLC on an RPC-5 column (4 mm × 250
mm) was carried out with a linear gradient elution from 0 to
0.08 M NaClO4 in 10 mM Tris-acetate (pH 7.5) containing 1.0
mM EDTA in 30 min at a flow rate of 1.0 mL/min. The eluate
was monitored by UV absorption at 260 nm and by fluores-
cence with excitation at 279 nm and emission at 393 nm.
Syn th esis of Ad en osin e 5′-Dip h osp h a te Im id a zolid e
(Im p p A). Adenosine 5′-diphosphate (ppA) was at first purified
by anion-exchange column chromatography on Sephadex A-25
using a linear gradient (0-0.5 M) of triethylammonium
hydrogen carbonate buffer (pH 7.5), as commercially available
ppA contains some amounts of adenosine 5′-monophosphate
(pA). The triethylammonium salt of ppA (1440 ODU at 260
nm, 0.09 mmol) was lyophilized several times by adding small
amounts of water to remove excess triethylammonium bicar-
bonate and coevaporated three times with pyridine and then
twice with dry DMF. Imidazole (63 mg, 0.9 mmol), di-2-pyridyl
disulfide (203 mg, 0.9 mmol), triphenylphosphine (244 mg, 0.9
mmol), and dry DMF (10 mL) containing triethylamine (0.5
mL) and tri-n-octylamine (0.2 mL) were added to the residue,
and the solution was stirred overnight at 24 °C. The completion
of the reaction was checked by HPLC. The reaction mixture
was poured into a solution containing dry acetone (100 mL),
dry ether (50 mL), acetone saturated with sodium perchlorate
260 nm. Analytical HPLC: tR ) 11.9 min (system B). λmax
)
1
258 and 280 nm. H NMR δ (D2O): 9.18 (s, 1H), 6.05 (d, 1H,
J 1′,2′ ) 3.5 Hz)), 4.67 (t, 1H), 4.51 (m, 1H), 4.40 (m, 1H), 4.33,
4.21 (m, 2H), 4.11 (s, 3H).
Syn th esis of 7-Meth ylgu a n osin e 5′-Dip h osp h a te Im i-
d a zolid e (Im p p 7m G). The Impp7mG was prepared from
pp7mG and imidazole by a similar procedure for the synthesis
of ImppA. In brief, the triethylammonium salt of pp7mG (1183
ODU at 260 nm, 0.1 mmol) was condensed with imidazole (78
mg, 12.2 mmol) in dry DMF (15 mL) using triphenylphosphine
(301 mg, 1.2 mmol) and di-2-pyridyl disulfide (253 mg, 1.2
mmol) as a condensing agent at room temperature overnight.
The completion of the reaction was checked by HPLC. Impp7mG
was isolated as a sodium salt by pouring the above reaction
mixture into a solution of dry acetone and ether containing
sodium perchlorate with stirring. The resulting white precipi-
tates were collected by centrifuge, washed with acetone and
ether, and dried in a desiccator. The isolated yield was 71%
(840 ODU at 260 nm, 53 mg). Impp7mG was kept in a
desiccator stored in a freezer. Analytical HPLC: tR ) 16.8 min
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1
(system B). λmax ) 258 and 280 nm. H NMR δ (D2O): 8.68 (s,
1H), 7.94 (s, 1H), 7.32 (s, 1H), 7.03 (s, 1H), 6.04 (d, 1H, J 1′,2′
3.2 Hz), 4.65 (m, 1H), 4.51 (m, 1H), 4.38 (m, 1H), 4.20-4.30
(m, 2H), 4.12 (s, 3H).
)
Hyd r olytic Sta bility of Im p p 7m G a n d Im p p A. The
Impp7mG (25 mM) was dissolved in 0.25M Tris-acetate buffer
(pH 7.5) and kept at 25 °C. An aliquot of the sample solution
was withdrawn at appropriate intervals and analyzed by
HPLC. Hydrolysis of Impp7mG to pp7mG was observed. The
half-life of Impp7mG against hydrolysis was estimated from
the time course of the reaction. Hydrolysis of Imp7mG was
also carried out under the same conditions as that for
Impp7mG to compare the hydrolytic stability of the phosphor-
imidazolide bond of nucleoside 5′-diphosphate and 5′-mono-
phosphate. Hydrolytic stabilities of ImppA and ImpA were also
examined by the same procedure as described above.
Dia d en osin e 5′,5′-Tr ip h osp h a te (Ap p p A) F or m a tion
fr om Im p p A a n d p A b y Diva len t Met a l Ca t a lyst in
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