Gas-phase tautomers of protonated 1-methylcytosine
1419
ethyl 2-cyano-3-oxopentanoate (III). GC–MS showed essen-
tially one peak, mass spectrum (m/z, % relative intensity):
169 (47), 141(39), 123(44), 112(92), 95(52), 68(69), 57(100).
Compound III (17.6 g) was dissolved in 50 ml of 90%
DMSO and 10% water and the mixture was heated under
1-Methyl-6-ethyl-5,6-dihydrocytosine (II)
Compound VI (0.55 g, 3.5 mmol) was dissolved in a mini-
mum volume of hot dioxane (5 ml), one crystal of sodium
amide (20 mg) was added to the solution, and the mixture
°
was maintained at 70 C overnight under stirring. Prod-
°
argon at 120 C until the evolution of CO2 ceased. The
uct II that crystallized upon cooling was collected and the
°
°
mixture was cooled to 0 C, saturated NaCl solution (250 ml)
supernatant was placed at 4 C to yield a second crop of
was added, and the product was extracted with 3 ð 50 ml
of CH2Cl2. The dichloromethane extract was dried over
anhydrous Na2SO4, the solvent was evaporated, and the
residue was distilled in vacuo (0.2 torr) to give 8.6 g (85%)
of 3-oxopentanenitrile (IV). IR (neat, cmꢀ1)ꢁmax: 1630 (C O),
2247 (CN). Mass spectrum (m/z, % relative intensity): 68 (16),
57(48), 42(9), 41(15), 40(22), 39(9), 29(100), 27(42).
crystalline II. Total yield: 0.41 g (73%). Mass spectrum (m/z,
% relative intensity): 155(11), 126(100), 85(15), 83(43), 42(28).
1
°
H-NMR(DMSO-d6, 25 C): 0.82 (t, 3H, CH3CH2-), 1.28, 1.53
(m, 2H, CH3-CH2-), 2.34 (d, 1H, J D 15 Hz, Heq-5), 2.52 (dd,
1H, J D 15 and 7.5 Hz, Hax-5), 2.82 (br s, 3H, N-CH3), 3.23 (m,
1H, H-6), 7.21(br s, 1H), 7.43(br s, 1H).
1-Methyl-6-ethyl-[6-2H1]-5,6-dihydrocytosine (IIa)
IIa was prepared analogously from VIa. Mass spec-
trum (m/z, % relative intensity): 156.1121 (C7H12N3OD
requires 156.1118, M, 15%), 155 (3), 127(100), 126(24), 86(12),
3-(Methylamino)pentanenitrile (V)
A solution of IV (8.5 g, 87.5 mmol) in 30 ml methanol was
mixed with 50 ml of 2M methylamine solution in methanol
and the mixture was stirred at room temperature for
15 min. Then a solution of sodium cyanoborohydride (2.2 g,
35 mmol) in 25 ml of methanol was added dropwise over
30 min under stirring. After another 30 min, the reaction
mixture was additioned with solid KOH (15 g), and the
volume was reduced to 50 ml on a rotary evaporator. Water
(10 ml) and saturated brine (25 ml) were added, the organic
layer was separated, and the aqueous layer was extracted
with 2 ð 50 ml of ether. The combined organic layers were
extracted with 3 ð 20 ml of 20% HCl. The HCl extract was
1
°
84(29), 43(19), 42(11). H-NMR(DMSO-d6, 25 C): 0.82 (t,
3H, CH3CH2-), 1.28, 1.53 (m, 2H, CH3-CH2-), 2.33 (d, 1H,
J D 15 Hz, Heq-5), 2.53 (d, 1H, J D 15 Hz, Hax-5), 2.82 (br s,
3H, N-CH3).
1-Methyl-6-ethyl-[5,N-2H2]-5,6-dihydrocytosine (IIb)
Compound II (260 mg) was dissolved in a 1 : 1 mixture of
D2O and CD3OD (10 ml) that contained K2CO3 to adjust to
pH 9. The mixture was kept in the dark at room temperature
for 3 days. Then it was acidified with CD3COOD, the solvents
were reduced on a rotary evaporator, the product was
extracted with acetonitrile, and the solvent was evaporated.
An aliquot of the deuterated intermediate (170 mg) was
dissolved in 3 ml of a 4 : 1 methanol–water mixture and the
solution was stirred at room temperature for 1 h. The solvents
were removed and the solid product was characterized by
°
cooled to 0 C, made basic by adding solid NaOH, and
extracted with 3 ð 20 ml of ether. The ether extract was
washed with saturated brine, dried over K2CO3, ether was
distilled off in vacuo, and the crude product was purified
°
by vacuum distillation (150 C/0.2 torr) to give 0.40 g (4%)
of V. IR (neat, cmꢀ1): 2261 (CN), 3447 (NH); GC–MS gave
essentially one peak (>95%). Mass spectrum (m/z, % relative
intensity): 83(78), 73(15), 72(100), 57(28), 56(15), 44(12), 42(74),
41(15), 30(31), 28(32).
1
°
H-NMR (DMSO-d6, 25 C): 0.84 (t, 3H, CH3CH2-), 1.42, 1.58
(m, 2H, CH3-CH2-), 2.52 (d, 1H), 2.89 (br s, 3H, N-CH3), 3.38
(dd, 1H, J D 4.8 and 8.1 Hz, H-6). Mass spectrum (m/z, %
relative intensity):158(7), 157 (17), 156(23), 155(14), 129(22),
128(61), 127(88), 126(57), 86(14), 85(27), 84(41), 83(31), 42(100).
This showed that the product was a mixture of mainly D1
and D2 species that, rather unexpectedly, had an axial proton
and an equatorial deuteron at C-5.
[3-2H1]-3-(Methylamino)pentanenitrile (Va)
Va was prepared analogously from IV using sodium
cyanoborodeuteride. Mass spectrum (m/z, % relative inten-
sity): 84(75), 83(16), 73(100), 72(31), 58(25), 57(24), 43(72),
42(65), 30(32), 29 (18), 28(17).
Methods
1H-NMR spectra were measured on a Bruker Avance 300
N-(1-Cyano-2-butyl)-N-methylurea (VI)
Nitrile V (0.39 g. 3.5 mmol) was stirred with nitrourea (0.56 g,
5.3 mmol) in 10 ml of 4 : 1 water/methanol at 50 C for
1 h. The solvent was removed on a rotary evaporator to
give a solid residue (0.49 g, 90%). Mass spectrum (m/z, %
relative intensity): 126(3), 115(46), 83(54), 72(100), 57(12),
56(13), 44(23), 42(46), 30(12). IR(cmꢀ1): 1653 (C O), 2250
(CN), 3448 (NH).
°
spectrometer at 300.13 MHz in DMSO-d6 at 25 C. GC–MS
°
was performed on an HP 5971A instrument equipped with
a silicone elastomer DB5 GC capillary column. Electron
and chemical ionization mass spectra were measured on
a JEOL HX-110 double-focusing mass spectrometer of an
EB geometry (electrostatic sector E precedes magnet B).
Samples were introduced from a heated solid probe at
°
°
120–150 C. The ion source temperature was 200 C. EI
ionization conditions were as follows: emission current,
100 µA; electron energy, 70 eV; acceleration voltage, 10 kV.
Chemical ionization (CI) mass spectra were obtained with
N-([2-2H1]-1-Cyano-2-butyl)-N-methylurea (VIa)
VIa was prepared analogously from Va. Compound VIa gave
essentially a single peak on GC–MS (>95%): Mass spectrum
(m/z, % relative intensity): 127(3), 116(46), 115(10), 84(47),
83(12), 73(100), 72(22), 58(12), 57(14), 56(11), 44(29), 43(36),
42(28), 31(5), 30(15).
C
C
NH3/NH4 or ND3/ND4 as the ionization reagents.
Collisionally activated dissociation (CAD) spectra were
obtained with air as a collision gas, which was admitted
Copyright 2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 1417–1428