A facile synthetic method for 15N4-labeled cytosine nucleosides

Full text of DOI:10.1021/jo9911589

9722
J . Org. Chem. 1999, 64, 9722-9723
A F a cile Syn th etic Meth od for 15N4-La beled
Cytosin e Nu cleosid es
Sch em e 1
Magoichi Sako,* Toshiyuki Kihara,
Hiroyoshi Kawada, and Kosaku Hirota
Gifu Pharmaceutical University, 5-6-1, Mitahora-higashi,
Gifu 502-8585, J apan
Received J uly 20, 1999
Recent advances in heteronuclear NMR spectroscopic
techniques on isotopically labeled DNA and RNA oligo-
nucleotides have made it possible to obtain valuable
information regarding the dynamic structural features
of nucleic acids and their interactions with proteins or
xenobiotics in the solution state.1 Along this line, a
variety of methods for the regioselective 15N-labeling of
purine nucleosides has been developed.2 The preparative
methods for regioselectively 15N-labeled pyrimidine nu-
cleosides, however, are still unsatisfactory to use.3
The exocyclic amino group in a cytosine moiety is a
good candidate for the 15N-labeling of nucleic acids,
because this functional group plays very important roles
to form hydrogen bonds with suitable acceptors in the
nucleic acids, proteins, or xenobiotics. The synthetic
methods for the 15N4-labeled cytosine nucleosides previ-
ously reported involve the introduction of a good leaving
group such as SMe, SO3H, and tetrazolyl groups into the
C4-position of the uridine derivatives and subsequent
nucleophilic displacement with 15N-enriched ammonia or
phthalimide (followed by alkaline hydrolysis).4 These
methods, however, required many steps from commer-
cially available starting materials because of the neces-
sity of protecting the primary and secondary alcohols in
the sugar moiety during the reactions for the preparation
of the desired 15N4-labeled cytosine nucleosides.
derivatives followed by oxidative N-debenzylation with
ammonium persulfate under thermal conditions. We
report herein the successful application of this synthetic
method which provides a simple method for the prepara-
tion of 15N4-labeled cytosine nucleosides starting from the
appropriate uridine derivatives.
Treatment of 2′-deoxyuridine with 15N-enriched benz-
ylamine in hexamethyldisilazane (HMDS) containing
excess R-picoline and a catalytic amount of chlorotri-
methylsilane at 140 °C for 2 days6a afforded 15N4-labeled
N4-benzyl-2′-deoxycytidine (1a ) in 74% yield, together
with small amounts of 15N-labeled N4-benzylcytosine and
15N2,15N4-labeled N2,N4-dibenzylaminopyrimidine. The
structure of the labeled compound (1a ) was confirmed
based on its spectral data, i.e., the observation of a
molecular ion peak m/z 318.1335 [calcd for C16H1915N1-
14N2O4 (M+) 318.1345] in its HRMS spectrum and the
appearance of broad double triplet signals with a large
15N-1H coupling constant (J ) 93 Hz) at δ 8.14 (1H) and
a doublet signal at δ 4.51 (2H, J ) 6 Hz) assignable to
the 15N-labeled benzylamino group in the 1H NMR
spectrum. Under analogous conditions,6b the thermal
reaction of uridine with 15N-enriched benzylamine in
HMDS afforded 15N4-labeled N4-benzylcytidine (1b) in
84% yield (Scheme 1).
In a previous paper,5 we have documented a convenient
synthesis of 15N6-labeled adenosine and 2′-deoxyadenos-
ine involving the silylation-benzylamination of inosine
Heating a solution of the N4-benzyl-2′-deoxycytidine
(1a ) and a slight excess of ammonium persulfate in a
mixed solvent of 0.5 mol phosphate buffer (pH 7.0) with
acetonitrile at 80 °C for 30 min followed by chromato-
graphic separation allowed the isolation of 15N4-labeled
2′-deoxycytidine (2a )4b in 79% yield, together with 15N4-
labeled N4-benzylcytosine, 15N4-labeled cytosine,7 and
benzaldehyde. Structural proof for the product (2a ) rests
upon the observation of a molecular ion peak: m/z
228.0883 [calcd for C9H1315N114N2O4 (M+) 228.0876] in its
HRMS spectrum and the observation of two broad
doublet signals with large coupling constants (each J )
89 Hz) at δ 7.08 and 7.19 (each 1H) assignable to the
15N-labeled primary amino group in its 1H NMR spec-
trum. Analogous results were obtained for the oxidation
of the N4-benzylcytidine (1b) with ammonium persulfate
that produced 15N4-labeled cytidine (2b).
(1) (a) Moore, P. B. Acc. Chem. Res. 1995, 28, 251-256. (b) Lynch,
S. R.; Tinoco, I., J r. Nucleic Acids Res. 1998, 26, 980-987. (c) Zhang,
X.; Gaffney, B. L.; J ones, R. A. J . Am. Chem. Soc. 1998, 120, 6625-
6626. (d) Leitner, D.; Schroeder, W.; Weisz, K. J . Am. Chem. Soc. 1998,
120, 7123-7124. (e) Dingley, A. J .; Grzesiek, S. J . Am. Chem. Soc.
1998, 120, 8293-8297. (f) Pervushin, K. V.; Ono, A.; Fernandez, C.;
Szyperski, T.; Kainosho, M.; Wuthrich, K. Proc. Natl. Acad. Sci., USA,
1998, 95, 14147-14151. (g) Wuthrich, K. Nat. Struct. Biol. 1998, 492-
495. (h) Masse, J . E.; Allain, F. H.-T.; Yen, Y.-M.; J ohnson, R. C.;
Feigon, J . J . Am. Chem. Soc. 1999, 121, 3547-3548.
(2) (a) Grenner, G.; Schmidt, H.-L. Chem. Ber. 1977, 110, 373-375.
(b) Leonard, N. J .; Henderson, T. R. J . Am. Chem. Soc. 1975, 97, 4990-
4999. (c) Kieper, I.; Schmidt, T.; Fera, B.; Ruterjans, H. Nucleosides
Nucleotides, 1988, 7, 821-825. (d) De Napoli, L.; Messere, A.; Mon-
tesarchio, D.; Piccialli, G. J . Org. Chem. 1995, 60, 2251-2253. (e) Orji,
C. C.; Kelly, J .; Ashburn, D. A.; Silks, L. A., III J . Chem. Soc., Perkin
Trans. 1 1996, 595-597. (f) Zhao, H.; Pagano, A. R.; Wang, W.; Shallop,
A.; Gaffney, B. L.; J ones, R. A. J . Org. Chem. 1997, 62, 7832-7835
and references therein.
Thus, the syntheses of the desired 15N4-labeled cytosine
nucleosides (2) were accomplished using the silylation-
benzylamination of appropriate uridine derivatives fol-
(3) (a) Sako, M. J . Synth. Org. Chem., J pn. 1996, 54, 54-61 and
references therein. (b) Amantea, A.; Henz, M.; Strazewski, P. Helv.
Chimica. Acta 1996, 79, 244-254.
(4) (a) Niu, C.-H. Anal. Biochem. 1984, 139, 404-407. (b) Kupfer-
schmitt, G.; Schmidt, J .; Schmidt, T.; Fera, B.; Buck, F.; Ruterjans,
H. Nucleic Acids Res. 1987, 15, 6225-6241. (c) Kamaike, K.; Taka-
hashi, M.; Utsugi, K.; Tomizuka, K.; Ishido, Y. Tetrahedron Lett. 1995,
36, 91-94.
(5) Sako, M.; Ishikura, H.; Hirota, K.; Maki, Y. Nucleosides Nucle-
otides, 1994, 13, 1239-1246.
(6) (a) Vorbruggen, H.: Krolikiewicz. K.; Niedballa, U. Liebigs Ann.
Chem. 1975, 988-1002. (b) Vorbruggen, H.: Krolikiewicz. K. Liebigs
Ann. Chem. 1976, 745-761. (c) Vorbruggen, H. Acc. Chem. Res. 1995,
28, 509-520.
(7) Wempen, I.; Brown, G. B.; Ueda, T.; Fox, J . J . Biochemistry 1965,
4, 54-57.
10.1021/jo9911589 CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/04/1999

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
via the Internet at http://pubs.acs.org.
(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