9032 J. Am. Chem. Soc., Vol. 121, No. 39, 1999
Aso et al.
MeOH gave 12c as a colorless solid: yield 117 mg (98%) silica gel
TLC Rf 0.07 (10:1 CHCl3-MeOH); H NMR (CD3OD) δ 1.12-1.25
CDCl3 layer; δ 4.82 (s, 1H), 4.89 (s, 1H), 6.55 (d, 1H, J ) 6 Hz), and
6.94 (d, 1H, J ) 6 Hz).
1
(m, 6H), 1.29 and 1.31 (each s, 3H), 2.19 (m, 1H), 2.60-2.91 (m,
7H), 3.11 (m, 1H), 3.16 and 3.17 (each s, 6H), 3.66 (m, 2H), 4.00 (m,
2H), 4.10-4.50 (m, 8H), 4.97-5.10 (m, 1H), 5.28 (m, 1H), 6.08 (m,
1H), 6.33 (m, 1H), 7.35-7.56 (m, 4H), 7.88-7.91 (m, 2H), 8.06 (br s,
1H), and 8.29-8.34 (m, 1H); mass spectrum (FAB) m/z 1019.3059
(M + H)+ (C41H53N10O17P2 requires 1019.3065.
3′-(2,5-Dimethoxy-2,5-dihydrofurfuryl) 2′-Deoxycytidylyl-(3′f5′)-
2′-deoxy-3′-guanylate (5a).9g A mixture of 201 mg (0.19 mmol) of
12a and 5 mL of NH4OH was heated at 55 °C in a pressure bottle for
16 h. The solvent was concentrated under diminished pressure and the
residue was coevaporated twice with portions of EtOH. The residue
was purified on a 5-g silica gel colum; elution with 17:1:2 and then
with 7:1:2 i-PrOH-NH4OH-H2O gave 5a as a colorless solid: yield
139 mg (88%); silica gel TLC Rf 0.09 (17:1:2 i-PrOH-NH4OH-H2O);
1H NMR (D2O) δ 1.54 (m, 1H), 2.23 (dd, 1H, J ) 14, 5 Hz), 2.50-
2.65 (m, 1H), 2.70-2.90 (m, 1H), 3.03, 3.09 (each s, 3H), 3.31, 3.32,
and 3.38 (each s, 3H), 3.52 (br s, 2H), 3.74 (dd, 1H, J ) 11, 6 Hz),
3.85-4.00 (m, 4H), 4.27 (m, 1H), 4.53 (m, 1H), 4.95 (m, 1H), 5.51
(br s, 1H), 5.87 (d, 1H, J ) 8 Hz), 6.20-6.98 (m, 4H), 7.50 (d, 1H, J
) 8 Hz), and 7.96 (s, 1H); mass spectrum (FAB), m/z 779.1799 (M +
H)+ (C26H37N8O16P2 requires 779.1803).
3′-(2,5-Dimethoxy-2,3,4,5-tetrahydrofurfuryl) 2′-Deoxycytidylyl-
(3′f5′)-2′-deoxy-3′-guanylate (5b). A mixture of 63 mg (59 µmol)
of 12b and 5 mL of NH4OH was heated at 55 °C in a pressure bottle
for 16 h. The solvent was concentrated under diminished pressure and
coevaporated twice with portions of EtOH. The residue was purified
on a 5-g silica gel column; elution with17:1:2 and then with 7:1:2
i-PrOH-NH4OH-H2O gave 5b as a colorless solid: yield 36 mg
(78%); silica gel TLC Rf 0.10 (17:1:2 i-PrOH-NH4OH-H2O); 1H NMR
(D2O) δ 1.51-1.61 (m, 1H), 1.84-2.15 (m, 4H), 2.24 (dd, 1H, J )
14, 6 Hz), 2.57-2.62 (m, 1H), 2.80 (m, 1H), 3.18, 3.24, and 3.30 (each
s, 6H), 3.52 (m, 2H), 3.71-3.77 (m, 1H), 3.85-3.95 (m, 1H), 3.95-
4.05 (m, 3H), 4.28 (m, 1H), 4.48 (m, 1H), 4.88-5.17 (m, 2H), 5.85
(d, 1H, J ) 7 Hz), 5.98 (d, 1H, J ) 6 Hz), 6.14 (t, 1H, J ) 7 Hz), 7.47
(d, 1H, J ) 7 Hz), and 7.95 (s, 1H); mass spectrum (FAB), m/z
781.1971 (M + H)+ (C26H39N8O16P2 requires 781.1959).
1-n-Butyl-5-methyleneazacyclopent-3-ene (15) by Treatment of
14a with n-BuNH2. To 485 mg (1.76 mmol) of 13a was added 20 mL
of 0.1 N HCl solution. The reaction mixture was stirred at room
temperature for 1 h, then treated with 20 mL of 0.2 N n-BuNH2 and
stirred at room temperature for 30 min. The reaction mixture was
extracted with CHCl3, and the CHCl3 layer was washed with brine,
dried (Na2SO4), and concentrated under diminished pressure. The
residue was purified on a 5-g silica gel column; elution with n-hexane
and then with 1:10 AcOEt-n-hexane gave 15 as a colorless liquid:
yield 40 mg (19%); silica gel TLC Rf 0.28 (1:8 AcOEt-n-hexane); IR
(neat) 1700, 1630, 1400, 820, and 810 cm-1; 1H NMR (CDCl3) δ 0.93
(t, 3H, J ) 7 Hz), 1.35 (m, 2H), 1.56 (m, 2H), 3.60 (t, 2H, J ) 7 Hz),
4.82 (d, 1H, J ) 1 Hz), 4.89 (d, 1H, J ) 1 Hz), 6.18 (dd, 1H, J ) 6,
1 Hz), and 6.94 (d, 1H, J ) 6 Hz); 13C NMR (CDCl3) δ 13.71, 20.05,
30.71, 38.77, 96.23, 125.04, 136.94, 145.60, and 170.38; mass spectrum
(FAB), m/z 152.1061 (M + H)+ (C9H14NO requires 152.1075).
1-n-Butyl-2-oxo-5-hydroxy-5-methylazacyclopent-3-ene (18). To
a solution of 2.1 g (15.8 mmol) of 2-oxo-5-chloro-5-methyloxacyclo-
pent-3-ene (17)10 in 10 mL of pentane was added 1.57 mL (1.16 g,
15.8 mmol) of n-BuNH2 at 0 °C; the reaction mixture was maintained
at 0 °C for 5 days. The reaction mixture was treated with 5 mL of H2O
and the organic layer was extracted with CH2Cl2. The CH2Cl2 layer
was washed with brine, dried (Na2SO4), and concentrated under
diminished pressure. The residue was purified on a 5-g silica gel
column; elution with 1:2 and then with 1:1 AcOEt-n-hexane gave 18
as a colorless solid: yield 481 mg (32% based on consumed 17); silica
gel TLC Rf 0.1 (1:1 AcOEt-n-hexane); mp 65-69 °C dec; IR (CHCl3)
3400 and 1695 cm-1; 1H NMR (CDCl3) δ 0.94 (t, 3H, J ) 7 Hz), 1.35
(m, 2H), 1.54 (s, 3H), 1.42-1.72 (m, 2H), 2.71 (s, 1H, ex D2O), 3.23
(ddd, 1H, J ) 14, 10, 6 Hz), 3.39 (ddd, 1H, J ) 14, 10, 6 Hz), 6.02 (d,
1H, J ) 6 Hz), and 6.89 (d, 1H, J ) 6 Hz); mass spectrum (FAB), m/z
170.1180 (M + H)+ (C9H16NO2 requires 170.1181). Lactam 18
gradually decomposed when stored at room temperature.
Lactam 15 from 18. To a solution of 108 mg (0.64 mmol) of 18 in
5 mL of dry pyridine was added 0.07 mL (114 mg, 0.96 mmol) of
SOCl2 at -40 °C. After being maintained at - 40 °C for 1 h, the
reaction mixture was poured into 5 mL of H2O and extracted with CH2-
Cl2. The organic layer was washed successively with H2O and brine,
then dried (Na2SO4) and concentrated under diminished pressure. The
residue was purified on a 5-g silica gel column; elution with ether gave
15 as a colorless liquid: yield 8.2 mg (11% based on consumed 18);
UV (MeOH) λmax 255 nm (log ꢀ 3.94) and 294 nm (log ꢀ 3.69); IR
(neat) 1700, 1630, 1400, 820, and 810 cm-1; 1H NMR (CDCl3) δ 0.93
(t, 3H, J ) 7 Hz), 1.35 (m, 2H), 1.56 (m, 2H), 3.60 (t, 2H, J ) 7 Hz),
4.82 (d, 1H, J ) 1 Hz), 4.89 (d, 1H, J ) 1 Hz), 6.18 (dd, 1H, J ) 6,
1 Hz), and 6.94 (d, 1H, J ) 6 Hz); 13C NMR (CDCl3) δ 13.71, 20.05,
30.71, 38.77, 96.23, 125.04, 136.93, 145.60, and 170.38; mass spectrum
(FAB), m/z 152.1061 (M + H)+ (C9H13NO requires 152.1075).
Lactam 15 by Treatment of 14a with n-BuNH2 (pH 5.3 and 7).
(a) pH 7.0: A mixture of 72 mg (0.28 mmol) of 13a and 1 mL of 0.1
M HCl was stirred at room temperature for 1 h; the reaction mixture
was then neutralized with 0.1 M NaOH. To this was added 94 µL of
n-BuNH2 in 1 mL of H2O, adjusted to pH 7.0 with HOAc. The reaction
mixture was stirred at room temperature for 2.5 h. The reaction mixture
was extracted with four portions of ether, and the ether layer was dried
(Na2SO4) and concentrated under diminished pressure at 0 °C. The
residue was purified on a 5-g silica gel column; elution with n-hexane
and then with 5:1 pentane-ether gave 15 as a colorless liquid: yield
3′-(2,2-Dimethoxypropionyl) 2′-Deoxycytidylyl-(3′f5′)-2′-deoxy-
3′-guanylate (5c). A mixture of 75 mg (74 µmol) of 12c and 10 mL
of NH4OH was heated at 55 °C in a pressure bottle for 16 h. The solvent
was concentrated under diminished pressure and coevaporated twice
with portions of EtOH. The residue was purified on a 5-g silica gel
column; elution with 17:1:2 and then with 7:1:2 i-PrOH-NH4OH-
H2O gave 5c as a colorless solid: yield 47 mg (86%); silica gel TLC
1
Rf 0.10 (17:1:2 i-PrOH-NH4OH-H2O); H NMR (D2O) δ 1.28 (s,
3H), 1.53 (m, 1H), 2.22 (ddd, 1H, J ) 14, 6, 2 Hz), 2.56 (ddd, 1H, J
) 14, 6, 3 Hz), 2.79 (m, 1H), 3.14 (s, 3H), 3.15 (s, 3H), 3.51 (m, 2H),
3.72 (m, 2H), 3.90-4.00 (m, 3H), 4.27 (m, 1H), 4.50 (m, 1H), 4.92
(m, 1H), 5.83 (d, 1H, J ) 8 Hz), 5.97 (dd, 1H, J ) 8, 6 Hz), 6.13 (t,
1H, J ) 6 Hz), 7.45 (d, 1H, J ) 8 Hz), and 7.93 (s, 1H); mass spectrum
(FAB), m/z 737.1707 (M - H)- (C24H35N8O15P2 requires 737.1697).
1H NMR Study of the Formation of 4a, 14a, and 14c. Dinucleotide
5a (1.5 mg, 1.84 µmol) was dissolved in 0.5 mL of D2O in an NMR
1
tube and a H NMR spectrum was taken. To this solution was added
3 drops of (COCl)2 and the reaction mixture was maintained at room
temperature. After 1 h, the formation of 4a was confirmed by the
disappearance of the signals corresponding to the methoxyl groups of
5a (3.03-3.09, 3.31-3.38, multiplets due to the presence of diaster-
eomers). Hydrolyses of 13a and 13c were carried out similarly. 14a:
1H NMR (D2O in the presence of (COCl)2) δ 3.31 and 3.34 (each d,
3H, J ) 11 Hz), 3.55-3.70 (m, 2H), 5.60 and 5.81 (each s, 1H), and
5.86 (m, 2H). 14c: 1H NMR (D2O in the presence of (COCl)2) δ 1.96
(s, 3H), 3.39 (d, 3H, J ) 11 Hz), and 4.39 (d, 2H, J ) 8 Hz).
1H NMR Study of the Formation of CpGp from 4a in the
Presence of n-BuNH2. To the acidic solution of 4a obtained above
was added 32 equiv (5.8 µL, 58.8 µmol) of n-BuNH2. The reaction
mixture was shaken for 5 min and then extracted with 0.5 mL of CDCl3.
The CDCl3 layer was separated and dried (Na2SO4), and 1H NMR
spectra of the D2O and CDCl3 layers were taken. The appearance of
CpGp was observed in the D2O layer; lactam 15 was observed in the
1
35 mg (82%); H NMR (CDCl3) δ 0.93 (t, 3H, J ) 7 Hz), 1.35 (m,
2H), 1.56 (m, 2H), 3.60 (t, 2H, J ) 7 Hz), 4.82 (d, 1H, J ) 1 Hz),
4.89 (d, 1H, J ) 1 Hz), 6.18 (dd, 1H, J ) 6, 1 Hz), and 6.94 (d, 1H,
J ) 6 Hz). (b) pH 5.3: A mixture of 100 mg (0.40 mmol) of 13a and
1 mL of 0.1 M HCl was stirred at room temperature for 1 h; the reaction
mixture was then neutralized with 0.1 M NaOH. To this was added
0.12 mL of n-BuNH2 in 1 mL of H2O, adjusted to pH 5.3 with HOAc.
The reaction was carried out in analogy with that at pH 7.0 to give 15
as a colorless liquid: yield 43 mg (73%).
Lactam 19 by treatment of 14a with BnNH2. (a) pH 12: A mixture
of 125 mg (0.49 mmol) of 13a and 1 mL of 0.1 M HCl was stirred at