Notes
J . Org. Chem., Vol. 64, No. 26, 1999 9723
lowed by a single-electron oxidation of the resulting 15N4-
labeled N4-benzylcytidine derivatives (1) with a generated
sulfate anion radical in the pH 7.0 buffer solution.
Advantages of the present synthetic method are (1) the
use of 15N-enriched benzylamine easily prepared from the
relatively inexpensive 15N-enriched benzamide as a source
of 15N-labeling, (2) the deprotection of the N4-benzyl group
smoothly proceeded even under mild conditions, and (3)
the reactions proceeding effectively even without protec-
tion of the hydroxyl groups in the sugar moiety. The
present synthetic method is, in principle, applicable to
the preparation of 15N4-labeled cytidine derivatives hav-
ing the protected hydroxyl groups in the sugar moiety,
which are starting materials for the syntheses of DNA
and RNA oligonucleotides.
µL, 3.84 mmol) containing ammonium sulfate (13 mg, 0.10 mmol)
was heated at 140 °C under an argon atmosphere for 1 day. The
after-treatment was similar to the case of 1a followed by
chromatographic separation that allowed the isolation of the
desired 15N-labeled cytidine derivative (1b) (267 mg, 84%),
together with 15N2,15N4-labeled N2,N4-dibenzylaminopyrimidine
(36 mg, 13%) and 15N4-labeled N4-benzylcytosine (2 mg, 1%).
F or 15N4-la beled N4-ben zylcytid in e (1b): mass m/z 334
(M+), 303, 261, 242, 231, 202 (B), 107, 91; HRMS m/z 334.1300
[calcd for C16H1915N114N2O5 (M+) 334.1295]; IR (KBr) 1649, 1570
1
cm-1; H NMR (DMSO-d6) δ 3.60 (2H, m), 3.84 (1H, br s), 3.96
(2H, br s), 4.51 (2H, d, J ) 6 Hz), 4.99 (1H, d, J ) 5 Hz), 5.05
(1H, b), 5.30 (1H, d, J ) 5 Hz), 5.79 (1H, br s), 5.84 (1H, d, J )
7 Hz), 7.3-7.4 (5H, m), 7.87 (1H, d, J ) 7 Hz), 8.02 (1H, br dt,
J ) 93 and 6 Hz).
P r ep a r a tion of 15N4-La beled 2′-Deoxycytid in e (2a ). A
solution of 15N4-benzyl-2′-deoxycytidine (1a ) (170 mg, 0.53 mmol)
and ammonium persulfate (Wako, 98% purity) (130 mg, 0.56
mmol) in 0.5 mol phosphate buffer (pH 7.0)-acetonitrile (1/2)
(3 mL) was heated at 80 °C for 30 min. After removal of the
solvent under reduced pressure, the resulting residue was
subjected to a silica gel column eluting with chloroform-
methanol (5/1) to isolate the desired compound (2a ) (96 mg, 79%),
15N4-labeled cytosine (2 mg, 4%), and 15N4-labeled N4-benzyl-
cytosine (3 mg, 3%), together with the starting (1a ) (20 mg, 12%).
Exp er im en ta l Section
1H NMR spectra were obtained at 400 MHz. Column chro-
matography was performed on silica gel (Cica Merck No. 9385-
5B; silica gel 60). 15N-Enriched benzylamine was prepared by
the LiAlH4 reduction of 15N-enriched benzamide (99 atom % 15N,
Isotec Inc.), according to a previously reported procedure.8 Unless
otherwise noted, materials obtained from commercial suppliers
were used without further purification.
1
The H NMR spectrum of the first fraction showed the presence
of a small amount of benzaldehyde.
F or 15N4-la beled 2′-d eoxycytid in e (2a ): mass m/z 228 (M+),
154, 139, 112; HRMS m/z 228.0883 [calcd for C9H1315N1-
14N2O4 (M+) 228.0877]; 1H NMR (DMSO-d6) δ 1.95 and 2.11 (each
1H, each m), 3.57 (2H, m), 3.78 (1H, m), 4.22 (1H, m), 5.00 (1H,
br), 5.22 (1H, br d, J ) 4 Hz), 5.75 (1H, br d, J ) 7 Hz), 6.17
(1H, dd, J ) 6 and 7 Hz), 7.08 and 7.19 (each 1H, each br d, J
) 89 Hz), 7.81 (1H, d, J ) 7 Hz).
P r ep a r a tion of 15N4-La beled N4-Ben zyl-2′-d eoxycytid in e
(1a ). A mixture of 2′-deoxyuridine (Aldrich, 99+% purity) (218
mg, 0.95 mmol) and 15N-enriched benzylamine (205 mg, 1.90
mmol) in HMDS (Aldrich, 99.9% purity) (606 µL, 2.87 mmol)
containing R-picoline (250 µL, 2.53 mmol) and chlorotrimethyl-
silane (12 µL, 0.1 mmol) was heated at 140 °C under an argon
atmosphere for 2 days. After treatment with 10% aqueous
methanol at 60 °C overnight, the resulting mixture was evapo-
rated to dryness and subjected to a silica gel column eluting with
chloroform-methanol (10/1) to isolate the desired 15N4-labeled
N4-benzyl-2′-deoxycytidine (1a ) (223 mg, 74%), 15N4-labeled N4-
benzylcytosine (14 mg, 7%), and 15N2,15N4-labeled N2,N4-di-
benzylaminopyrimidine (11 mg, 4%).
For 15N4-la beled cytosin e: mass m/z 112 (M+); HR-MS m/z
112.0398 [calcd for C4H515N114N2O (M+) 112.0403]; IR (KBr) 1656
cm-1 1H NMR (DMSO-d6) δ 5.59 (1H, d, J ) 7 Hz), 6.89 and
;
7.12 (each 1H, each br d, J ) 90 Hz), 7.33 (1H, d, J ) 7 Hz).
P r ep a r a tion of 15N4-la beled cytid in e (2b). The labeled
cytidine (2b) was prepared by a procedure similar to the case of
2a . The column chromatographic separation of the reaction
mixture obtained from 1b (49 mg, 0.15 mmol) allowed the
isolation of 2b (19 mg, 53%), 15N4-labeled cytosine (2.6 mg, 16%),
and 15N4-labeled N4-benzylcytosine (1.8 mg, 6%), together with
F or 15N4-la beled N4-ben zyl-2′-d eoxycytid in e (1a ): mass
m/z 318 (M+), 287, 244, 229, 202 (base peak), 107, 91; HRMS
m/z 318.1335 [calcd for C16H1915N114N2O4 (M+) 318.1345]; 1H
NMR (DMSO-d6) δ 1.96 and 2.13 (each 1H, each m), 3.57 (2H,
m), 3.78 (1H, m), 4.22 (1H, br), 4.51 (2H, d, J ) 6 Hz), 4.96 (1H,
t, J ) 5 Hz), 5.19 (1H, d, J ) 4 Hz), 5.84 (1H, d, J ) 7 Hz), 6.18
(1H, dd, J ) 6 and 7 Hz), 7.26-7.38 (5H, m), 7.80 (1H, d, J ) 7
Hz), 8.14 (1H, br dt, J ) 93 and 6 Hz).
1
the starting 1b (10 mg, 20%). The H NMR spectrum of the first
fraction showed the presence of a small amount of benzaldehyde.
F or 15N4-la beled cytid in e (2b): mass m/z 245 (M+), 207,
185 (B); HRMS m/z 245.0911 [calcd for C9H1415N114N2O5 (M+)
1
245.0904]; H NMR (DMSO-d6) δ 3.56 and 3.67 (each 1H, each
F or 15N4-la beled N4-ben zylcytosin e: mass m/z 202 (M+),
107, 91; HRMS m/z 202.0864 [calcd for C11H1115N114N2O (M+)
d, J ) 10 Hz), 3.84 (1H, m), 3.96 (2H, br s), 5.07, 5.11, and 5.30
(each 1H, each br), 5.76 (1H, dd, J ) 6 and 7 Hz), 5.78 (1H, d,
J ) 8 Hz), 7.20 and 7.35 (each 1H, each br d, J ) 95 Hz), 7.89
(1H, d, J ) 8 Hz).
202.0873]; IR (KBr) 1641, 1606, 1512 cm-1
.
F or 15N2,15N4-la beled N2,N4-d iben zyla m in op yr im id in e:
mass m/z 292 (M+), 201, 186, 107, 91; HRMS m/z 292.1477 [calcd
for C18H1815N214N2 (M+) 292.1472]; 1H NMR (CDCl3) δ 4.49 (2H,
d, J ) 6 Hz), 4.58 (2H, d, J ) 5 Hz), 5.04 (1H, br d, J ) 90 Hz),
5.30 (1H, br d, J ) 90 Hz), 5.71 (1H, d, J ) 6 Hz), 7.2-7.3 (10H,
m), 7.81 (1H, d, J ) 6 Hz).
Under the conditions employed above, the deglycosylation of
cytidine to cytosine proceeded more smoothly (in 56% yield)
compared with the case (in 14% yield) of 2′-deoxycytidine. Thus,
the prolonged reaction time for the oxidative debenzylation of
the 15N-labeled cytidine (1b) caused a decrease in the isolated
yield of the labeled cytidine (2b).
P r ep a r a tion of 15N4-La beled N4-Ben zylcytid in e (1b). A
mixture of uridine (Aldrich, 99% purity) (234 mg, 0.95 mmol)
and 15N-labeled benzylamine (205 mg, 1.90 mmol) in HMDS (810
Su p p or tin g In for m a tion Ava ila ble: 1H NMR and MS
data for 1a,b and 2a,b. This material is available free of charge
(8) (a) Nystrom, R. F.; Brown, W. G. J . Am. Chem. Soc. 1948, 70,
3738-3740. (b) Brown, H. C.; Heim, P. J . Org. Chem. 1973, 38, 912-
916. (c) Horneman, U. Carbohydr. Res. 1973, 28, 171-174.
J O9911589