A new and efficient synthetic method for 15N3-labeled cytosine nucleosides: Dimroth rearrangement of cytidine N3-oxides

Article abstract of DOI:10.1021/jo0486241

The treatment of ¹⁵N⁴-labeled cytidine N ₃-oxide and ¹⁵N⁴-labeled 2′-deoxycytidine N₃-oxide, prepared from the appropriate unprotected uridines in three reaction steps, with benzyl bromide in the presence of excess lithium methoxide allowed the smooth occurrence of their Dimroth rearrangement even under mild conditions leading to the corresponding ¹⁵N ₃-labeled uridine 4-O-benzyloximes which can easily undergo the reductive N-O bond cleavage to give the desirable ¹⁵N ₃-labeled cytosine nucleosides in high total yields.

Full text of DOI:10.1021/jo0486241

and subsequent Dimroth rearrangement6 occurring in the
presence of 15NH4Cl-KOH-Et3N7 at room temperature.
Their method for the preparation of the 15N3-labeled
cytidine, however, required a long time for the completion
of the rearrangement (e.g., one week), the introduction
of an exocyclic amino group8 to the 15N3-labeled uridine
derivative obtained after the intramolecular rearrange-
ment, and also the O-deprotection in the sugar moiety
as the final step. In this paper, we describe an alternative
and efficient synthetic method for the 15N3-labeled cyti-
dine and the first synthesis of 15N3-labeled 2′-deoxycyti-
dine using the appropriate 15N4-labeled cytosine nucle-
A New and Efficient Synthetic Method for
15N3-Labeled Cytosine Nucleosides:
Dimroth Rearrangement of Cytidine
N3-Oxides
Magoichi Sako* and Hiroyoshi Kawada
Gifu Pharmaceutical University, 5-6-1, Mitahora-higashi,
Gifu 502-8585, Japan
sako@gifu-pu.ac.jp
3
Received August 9, 2004
osides as the starting material.
The oxidations of 15N4-labeled cytidine3,4 and 15N4-
labeled 2′-deoxycytidine3 with excess m-chloroperbenzoic
acid proceeded smoothly even at ambient temperature
to give the corresponding cytidine N3-oxides (15N4-labeled
1a,b) in almost quantitative yields.9 Analogous results
were obtained with use of magnesium monoperoxyph-
thalate as an oxidant. The treatment of the 15N4-labeled
cytidine N3-oxide (15N4-labeled 1a) with a slight excess
of benzyl bromide in the presence of excess lithium
methoxide at 37 °C for 1 day followed by chromatographic
separation with a short column allowed the isolation of
the desired Dimroth-rearrangement product, the 15N3-
labeled uridine 4-O-benzyloxime (15N3-labeled 2a), in 95%
yield. The structure of the labeled 2a was confirmed
Abstract: The treatment of 15N4-labeled cytidine N3-oxide
and 15N4-labeled 2′-deoxycytidine N3-oxide, prepared from
the appropriate unprotected uridines in three reaction steps,
with benzyl bromide in the presence of excess lithium
methoxide allowed the smooth occurrence of their Dimroth
rearrangement even under mild conditions leading to the
corresponding 15N3-labeled uridine 4-O-benzyloximes which
can easily undergo the reductive N-O bond cleavage to give
the desirable 15N3-labeled cytosine nucleosides in high total
yields.
NMR spectroscopic studies of DNA and RNA oligo-
nucleotides regiospecifically labeled with 15N-enriched
nitrogen atom(s) provide valuable information regarding
the dynamic structural features of nucleic acids and their
interactions with xenobiotics or proteins in the solution
states.1 Along this line, we have documented the conve-
nient and efficient syntheses of 15N6-labeled adenine
nucleosides2 and 15N4-labeled cytosine nucleosides3 using
the silylation-benzylamination of the appropriate un-
protected nucleosides, inosines and uridines, followed by
the oxidative N-debenzylation with ammonium persul-
fate. Also, Ariza et al.4 recently reported the first
synthesis of the specifically 15N3-labeled cytidine,5 which
involves the N3-nitration of 2′,3′,5′-O-protected uridine
based on its spectral data, i.e., the observation of a
15
molecular ion peak m/z 351.1328 [calcd for C16H20N2
-
NO6 (MH+) 351.1323] in its HR-FABMS spectrum, the
appearance of a singlet signal at δ 4.93 (2H) assignable
to the O-benzylmethylene protons and a doublet signal
with a large coupling constant (J ) 95 Hz) at δ 9.99 (1H)
assignable to the 15N3H-proton in the 1H NMR spectrum,
and the observation of the UV spectral change [272 and
224 nm for the starting N-oxide (cf. 1a); 279, 249, and
208 nm for the uridine 4-O-benzyloxime 2a]. Acid hy-
drolysis of the 15N3-labeled 2a under mild conditions
leading to the 15N3-labeled uridine7 {HR-FABMS m/z
246.0753 [calcd for C9H13N15NO6 (MH+) 246.0744]} pro-
vided further evidence supporting the structure of the
labeled 2a. In a sharp contrast to these results, the
heating of the N-oxide 1a in the absence of benzyl
bromide caused the complete recovery of the starting
N-oxide even in the presence of excess lithium methoxide,
and the heating of 1a in a mixed solvent of phosphate
buffers (pH 7.5-8.5) with methanol containing benzyl
bromide resulted in the formation of the N3-O-benzylated
product, N3-O-benzyl-4-iminouridine (3), as the major
product, together with a small amount of N3-O-benzylu-
ridine (4).10 When the N3-O-benzylated product 3 was
(1) (a) Kawashima, E.; Kamaike, K. Mini-Rev. Org. Chem. 2004, 1,
309-332 and references therein. (b) Wenter, P.; Pitsch, S. Helv. Chim.
Acta 2003, 86, 3955-3974. (c) Shallop, A. J.; Gaffney, B. L.; Jones, R.
A. J. Org. Chem. 2003, 68, 8657-8661. (d) Lagoja, I. M.; Pochet, S.;
Boudou, V.; Little, R.; Lescrinier, E.; Rozenski, J.; Herdewijn, P. J.
Org. Chem. 2003, 68, 1867-1871. (e) Abdi, F. A.; Mundt, M.; Doggett,
N.; Bradbury, E. M.; Chen, X. Genome Res. 2002, 12, 1135-1141. (f)
Milecki, J. J. Labeled Compd. Radiopharm. 2002, 45, 307-337. (g)
Cromsigt, J.; Schleucher, J.; Gustafsson, T.; Kihlberg, J.; Wijmenga,
S. Nucleic Acids Res. 2002, 30, 1639-1645. (h) Lagoja, I. M.; Herdewijn,
P. Synthesis 2002, 301-314 and references therein. (i) Laxer, A.; Major,
D. T.; Gottlieb, H. E.; Fisher, B. J. Org. Chem. 2001, 66, 5463-5481.
(j) Dunger, A.; Limbach, H.-H.; Weisz, K. J. Am. Chem. Soc. 2000, 122,
10109-10114. (k) Sako, M. J. Synth. Org. Chem. Jpn. 1996, 54, 68-
75 and references therein.
(2) Sako, M.; Ishikura, H.; Hirota, K.; Maki, Y. Nucleosides Nucle-
otides 1994, 13, 1239-1246.
(3) Sako, M.; Kihara, T.; Kawada, H.; Hirota, K. J. Org. Chem. 1999,
64, 9722-9723.
(4) Ariza, X.; Vilarrasa, J. J. Org. Chem. 2000, 65, 2827-2829.
(5) The 15N1,15N3-doubly or 15N1,15N3,15N4-triply labeled cytidines
were prepared by total syntheses starting from 15N-labeled urea: Niu,
C.-H. Anal. Biochem. 1984, 139, 404-407. Amantea, A.; Henz, M.;
Strazewski, P. Helv. Chim. Acta 1996, 79, 244-254.
(6) Pertinent reviews for the Dimroth rearrangement, see: (a) El
Ashry, E. S. H.; El Kilany, Y.; Rashed, N.; Assafir, H. Adv. Heterocycl.
Chem. 1999, 75, 79-167. (b) Fujii, T.; Itaya, T. Heterocycles 1998, 48,
359-390.
(7) Ariza, X.; Bou, V.; Vilarrasa, J. J. Am. Chem. Soc. 1995, 117,
3665-3673.
(8) Reese, C. B.; Ubasawa, A. Nucleic Acids Res. Symp. Ser. 1980,
7, 5-21.
(9) For the preparation of cytidine N3-oxides, see: (a) Delia, T. J.;
Olsen, M. J.; Bosworth Brown, G. J. Org. Chem. 1965, 30, 2766-2768.
(b) Townsend, L. B.; Panzica, R. P.; Robins, R. K. J. Med. Chem. 1971,
14, 259-260. (c) Ohigashi, H.; Kaji, M.; Sakaki, M.; Koshimizu, K.
Phytochemistry 1989, 28, 1365-1368. (d) Itahara, T. Chem. Lett. 1991,
1591-1594. (e) Rhie, S. Y.; Ryu, E. K. Heterocycles 1995, 41, 323-
328.
10.1021/jo0486241 CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/15/2004
8148
J. Org. Chem. 2004, 69, 8148-8150

SCHEME 1. Dimroth Rearrangement of Cytidine
labeled 2′-deoxycytidine {HR-FABMS: m/z 229.0949 [cal-
cd for C9H14N215NO4 (MH+) 229.0955]} as the desired
compound.
N-Oxides 1a,ba
Thus, the syntheses of the 15N3-labeled cytosine nu-
cleosides were accomplished by using the N3-oxidation
(cf. 1) of the 15N4-labeled cytidines, their Dimroth rear-
rangement in the presence of benzyl halides, and subse-
quent reductive cleavage of the N-O bond in the 15N3-
labeled uridine 4-O-benzyloximes (cf. 2).
Characteristics of the present synthetic method are (1)
the use of the 15N4-labeled cytidine or 15N4-labeled 2′-
deoxycytidine, easily prepared from commercially avail-
able unprotected uridine or 2′-deoxyuridine, as the
starting material, (2) the involvement of a novel type of
Dimroth rearrangement in the cytidine N3-oxides smoothly
proceeding even under mild conditions, and (3) the
reactions effectively proceeding even without protection
of the primary and secondary alcohols in the sugar
moiety. The present synthetic method, in principle, is
applicable for the preparation of 15N3-labeled cytidine
derivatives having the protected hydroxyl groups in the
sugar moiety, which are starting materials for the
syntheses of DNA and RNA oligonucleotides, and the
preparation of the 15N3-labeled cytosine nucleosides with
a modified sugar moiety.13
a Reagents and conditions: (a) BnBr, excess MeOLi, MeOH, 37
°C, 1 day; (b) BnBr, 0.1 M phosphate buffer (pH 7.5-8.5)-MeOH
(4/1), 37 °C; (c) excess MeOLi, MeOH, 37 °C, overnight.
stirred in methanol containing lithium methoxide at 37
°C overnight, the almost quantitative conversion to the
uridine 4-O-benzyloxime 2a was observed. These results
strongly suggest that the presence of alkyl halides and a
strong base in the media are a prerequisite for the
occurrence of the present rearrangement and that the
N3-O-benzyl-4-iminouridine 3 is a key intermediate for
this reaction.
On the basis of these facts, the formation of the uridine
4-O-benzyloxime 2a in this reaction can be reasonably
explained by considering the transient formation of the
intermediate carbamate (cf. A) by the initial attack of a
methoxide ion on the C2-carbonyl group in the N3-O-
benzyated 4-iminouridine 3 as illustrated in Scheme 1.11
Experimental Section
4-Amino-1-(3,4-dihydroxy-5-hydroxymethyltetrahydro-
furan-2-yl)-1H-pyrimidin-2-one 3-Oxide (Cytidine N3-
Oxide) (1a). To a suspension of cytidine (Aldrich, 99% purity;
491.4 mg, 2.0 mmol) in dry methanol (30 mL) was added
m-chloroperoxybenzoic acid (Aldrich, 77% purity; 1.345 g, 6.0
mmol), and the mixture was stirred at ambient temperature
overnight under an argon atmosphere. TLC analysis of the
reaction mixture with use of chloroform-methanol-acetic acid
(16/6/3) as the developing solvent showed almost the complete
consumption of the starting cytidine and the formation of a more
polar compound as the sole product. After removal of the solvent
under reduced pressure, the resulting residue was triturated
with acetone, collected by filtration, and then washed well with
acetone to obtain the desired N3-oxide (1a) 9 (447.8 mg, 86%) as
a colorless powder in an almost pure state: mp 263-269 °C dec,
from methanol; FABMS m/z 260 (MH+), 244, and 128; UV
Hydrogenation of the 15N3-labeled uridine 4-O-benzy-
loxime (15N3-labeled 2a) in the presence of a PtO2 catalyst
proceeded quickly to afford the corresponding 15N3-labeled
cytidine4 in an almost quantitative yield.12 Structural
proof for this product rests upon the observation of a
molecular ion peak m/z 245.0911 [calcd for C9H14N215NO5
(MH+) 245.0904] in its HR-FABMS spectrum and the
(MeOH) 277 and 225 nm; IR (KBr) 3370, 1692, and 1653 cm-1
;
1H NMR δ 3.57 and 3.68 (each 1H, each br d, J ) 12 Hz), 3.87
(1H, m), 3.94 (1H, m), 3.99 (1H, m), 5.09 (1H, d, J ) 5 Hz), 5.14
(1H, br t), 5.51 (1H, br s), 5.81 (1H, d, J ) 4 Hz), 6.07 (1H, d, J
) 8 Hz), 7.75 and 8.25 (each 1H, each br), and 7.79 (1H, d, J )
8 Hz) ppm; HR-FABMS m/z 260.0884 [calcd for C9H14N3O6
(MH+) 260.0883].
1
Analogous results (the isolated yield of 1a: 85% in the 2.0
mmol scale experiment) were obtained by the employment of
magnesium monoperoxyphthalate hexahydrate (Ardrich, 80%
purity) in place of m-chloroperoxybenzoic acid as the oxidant,
though the column chromatographic purification with chloroform-
methanol-acetic acid (16/6/3) as an eluting solvent was required
to separate the phthalate concurrently formed in this case.
4-Amino-1-(4-hydroxy-5-hydroxymethyltetrahydrofuran-
2-yl)-1H-pyrimidin-2-one 3-Oxide (2′-Deoxycytidine N3-
observation of H NMR and UV spectral data identical
with those of the nonlabeled cytidine. Analogous results
were obtained when using the 15N4-labeled 2′-deoxycyti-
dine as the starting material that produced the 15N3-
(10) No drastic changes were observed even after prolonged reaction
times (e.g., 2 days).
(11) An analogous mechanism has been proposed for the conversion
of acetic acid 3-acetoxy-5-(2,8-dimethyl-5-oxo[1,2,4]triazolo[1,5-c]pyri-
midin-6-yl)tetrahydrofuran-2-ylmethyl ester to 6-(4-hydroxy-5-hy-
droxymethyltetrahydrofuran-2-yl)-3,8-dimethyl-6H-[1,2,4]triazolo[4,3-
c]pyrimidin-5-one in the presence of a methoxide ion. Cf.: Loakes, D.;
Brown, D. M.; Salisbury, S. A. J. Chem. Soc., Perkin Trans. 1 1999,
1333-1337. Loakes, D.; Brown, D. M. Recent Res. Dev. Org., Bioorg.
Chem. 2001, 4, 89-95.
Oxide) (1b). In a manner similar to that of the cytidine N3-
oxide 1a, the desired N3-oxide 1b
9d
(206.3 mg, 85%) was
(13) Matsuda, A.; Sasaki, T. Cancer Sci. 2004, 95, 105-111. Miura,
S.; Izuta, S. Curr. Drug Targets 2004, 5, 191-195. Estlin, E. J.; Yule,
S. M.; Lowis, S. P. Cancer Treat. Rev. 2001, 27, 339-350. Azuma, A.;
Matsuda, A.; Sasaki, T.; Fukushima, M. Nucleosides Nucleotides 2001,
20, 609-619. Holy, A.; Guenter, J.; Dvorakova, H.; Masojidkova, M.;
Andrei, G.; Snoeck, R.; Balzarini, J.; De Clercq, E. J. Med. Chem. 1999,
42, 2064-2086.
(12) On the other hand, the Pd/C reduction of the uridine 4-O-
benzyloxime 2a caused the concurrent occurrence of O-debenzylation
leading to the corresponding unsubstituted uridine 4-oxime (22%, by
TLC densitometry).
J. Org. Chem, Vol. 69, No. 23, 2004 8149

prepared by starting from 2′-deoxycytidine monohydrate (Ald-
rich, 99+% purity; 245.2 mg, 1.0 mmol): FABMS m/z 244 (MH+),
228, and 128; UV (H2O) 271 and 223 nm; IR (KBr) 3389, 1703,
and 1657 cm-1; 1H NMR δ 2.02 and 2.22 (each 1H, each m), 3.54
and 3.59 (each 1H, each br d, J ) 12 Hz), 3.83 (1H, dd, J ) 7
and 3 Hz), 4.21 (1H, d, J ) 2 Hz), 5.03 (1H, br), 5.27 (1H, br),
6.08 (1H, d, J ) 8 Hz), 6.13 (1H, t, J ) 6 Hz), 7.76 (1H, d, J )
8 Hz), 6.90 and 8.30 (each 1H, each br) ppm; HR-FABMS m/z
244.0925 [calcd for C9H14N3O5 (MH+) 244.0934].
Reduction of 15N3-Labeled Uridine 4-O-Benzyloxime
15N3-Labeled 2a) or 15N3-Labeled 2′-Deoxyuridine 4-O-
(
Benzyloxime (15N3-Labeled 2b). To a suspension of the 15N-
labeled uridine 4-oxime (15N3-labeled 2a; 25.0 mg, 0.07 mmol)
in dry methanol (1.0 mL) was added platinum(IV) oxide (PtO2,
Aldrich) (2.5 mg), and the mixture was stirred vigorously at 60
°C for 1 h under a hydrogen atmosphere. TLC analyses of the
reaction mixture with chloroform-methanol-acetic acid (16/6/
3) and chloroform-methanol (10/1) as the developing solvents
showed the complete conversion of the starting 2a to a more
polar compound. After removal of the solvent under reduced
pressure, the resulting residue was subjected to a silica gel short
column by eluting with chloroform-methanol (5/1 to 3/1) to
isolate the desired 15N3-labeled cytidine (triturated with acetone,
15.7 mg, 92%):4 UV (MeOH) 273 and 224 (sh) nm; 1H NMR,
identical with the data for the unlabeled cytidine; HR-FABMS
m/z 245.0911 [calcd for C9H14N215NO5 (MH+) 245.0904].
1-(3,4-Dihydroxy-5-hydroxymethyltetrahydrofuran-2-
yl)-1H-pyrimidin-2,4-dione 4-O-Benzyloxime (Uridine 4-O-
Benzyloxime) (2a). To a suspension of the N3-oxide 1a (77.7
mg, 0.30 mmol) and lithium hydride (Aldrich, 95% purity; 12.6
mg, 1.5 mmol) in dry methanol (5 mL) was added benzyl bromide
(Tokyo Kasei, >98% purity; 40.0 µL, 0.33 mmol), and the mixture
was stirred at 37 °C for 1 day under an argon atmosphere. TLC
analyses of the reaction mixtures with chloroform-methanol-
acetic acid (16/6/3) and chloroform-methanol (10/1) as the
developing solvents showed complete consumption of the starting
N-oxide 1a for the almost quantitative conversion to a less polar
compound. After being neutralized with 1 N HCl solution and
subsequent removal of the solvent under reduced pressure, the
resulting residue was subjected to a short silica gel column by
eluting with chloroform-methanol (20/1) to isolate the desired
oxime 2a (triturated with diethyl ether, 99.5 mg, 95%) as a
colorless amorphous powder: mp 123-125 °C (from methanol);
FABMS m/z 350 (MH+), 260, 218, 217, and 91; UV (MeOH) 279,
249, and 208 nm; IR (KBr) 3409 and 1691 cm-1; 1H NMR δ 3.34-
3.63 (2H, m), 3.76 (1H, m), 3.90 (1H, m), 3.95 (1H, m), 4.93 (2H,
s), 4.98 (1H, t, J ) 5 Hz), 5.01 (1H, d, J ) 5 Hz), 5.23 (1H, d, J
) 6 Hz), 5.54 (1H, br d, J ) 8 Hz), 5.72 (1H, d, J ) 6 Hz), 7.12
(1H, d, J ) 8 Hz), 7.24-7.38 (5H, m), and 9.99 (1H, br s); HR-
FABMS m/z 350.1361 [calcd for C16H20N3O6 (MH+) 350.1352].
15N3-Labeled Uridine 4-O-Benzyloxime (15N3-Labeled
2a). FABMS m/z 351 (MH+), 261, 219, 218, and 91; 1H NMR,
identical with the above data described for the unlabeled oxime
2a, except δ 9.99 (1H, d, J ) 95 Hz) ppm; HR-FABMS m/z
351.1328 [calcd for C16H20N215NO6 (MH+) 351.1323].
In a similar manner, the PtO2 reduction of the 15N3-labeled
2′-deoxyuridine 4-O-benzyloxime (15N3-labeled 2b; 0.1 mmol) was
accomplished to obtain the desired 15N3-labeled 2′-deoxycytidine
in 95% yield after silica gel column chromatographic purification
with chloroform-methanol (5/1 to 3/1): UV (MeOH) 273 and
232 (sh) nm; 1H NMR, identical with the data for the unlabeled
15
2′-deoxycytidine; HR-FABMS m/z 229.0949 [calcd for C9H14N2
-
NO4 (MH+) 229.0955].
Reactions of Cytidine N3-Oxide (1a) with Benzyl Bro-
mide in a Mixed Solvent of Phosphate Buffers (pH 7.5 and
8.5) with Methanol. A solution of cytidine N3-oxide (1a; 26.0
mg, 0.1 mmol) in 0.1 mol phosphate buffer (pH 7.5 or 8.5)-
methanol (4/1) (2.5 mL) containing benzyl bromide (36 µL, 0.3
mmol) was stirred at 37 °C overnight. TLC analyses of the
reaction mixtures with chloroform-methanol-acetic acid (16/
6/3) and chloroform-methanol (10/1) as the developing solvents
showed the complete consumption of the starting N-oxide 1a and
the formation of a less polar product as the major product,
together with a small amount of more less-polar product. The
product ratios in these reactions were almost equal. After
removal of the solvent under reduced pressure, the resulting
residue was subjected to a silica gel column by eluting with
chloroform-methanol (10/1 to 3/1) to isolate 3-benzyloxy-1-(3,4-
dihydroxy-5-hydroxymethyltetrahydrofuran-2-yl)-4-imino-3,4-di-
hydro-1H-pyrimidin-2-one (3) (triturated with diethyl ether, 31.5
mg, 90%) {IR (KBr) 1729 and 1660 cm-1; UV (MeOH) 281, 229,
and 206 nm; 1H NMR δ 3.59 and 3.72 (each 1H, each br dd, J )
12 and 5 Hz), 3.93 (1H, br s), 3.96 (1H, m), 4.08 (1H, m), 5.14
(1H, d, J ) 6 Hz), 5.18 (2H, s), 5.21 (1H, t, J ) 5 Hz), 5.59 (1H,
d, J ) 5 Hz), 5.71 (1H, d, J ) 3 Hz), 6.10 (1H, d, J ) 8 Hz), 7.42
(3H, m), 7.59 (2H, m), 8.14 (1H, d, J ) 8 Hz), and 9.56 (1H, br);
HR-FABMS m/z 350.1365 [calcd for C16H20N3O6 (MH+) 350.1352]}
as a colorless amorphous powder and 3-benzyloxy-1-(3,4-dihy-
droxy-5-hydroxymethyltetrahydro- furan-2-yl)-1H-pyrimidine-
2,4-dione (4) (2.0 mg, 6%) {IR (KBr) 1719 and 1674 cm-1; UV
(MeOH) 263 and 205 nm; 1H NMR δ 3.56 and 3.66 (each 1H,
each br dd, J ) 12 and 4 Hz), 3.87 (1H, m), 3.97 (1H, dd, J ) 10
and 5 Hz), 4.06 (2H, m), 5.02 (2H, s), 5.09 (1H, d, J ) 5 Hz),
5.13 (1H, t, J ) 5 Hz), 5.45 (1H, d, J ) 5 Hz), 5.77 (1H, d, J )
5 Hz), 5.83 (1H, d, J ) 8 Hz), 7.39 (3H, m), 7.53 (2H, m), and
7.96 (1H, d, J ) 8 Hz); HR-FABMS m/z 351.1200 [calcd for
C16H19N2O7 (MH+) 351.1192]} as an oily product.
1-(4-Hydroxy-5-hydroxymethyltetrahydrofuran-2-yl)-
1H-pyrimidin-2,4-dione 4-O-Benzyloxime (2′-Deoxyuri-
dine 4-O-Benzyloxime) (2b). In a manner similar to that of
the uridine 4-O-benzyloxime 2a, the desired oxime 2b (301 mg,
90%) was obtained by starting from the 2′-deoxycyctidine N3-
oxide (1b) (243 mg, 1.0 mmol): mp 119-122 °C; FABMS m/z
334 (MH+), 218, 217, and 91; UV (MeOH) 279, 240, and 204 nm;
1
IR (KBr)′ 3352, 1724, and 1674 cm-1; H NMR δ 1.95 and 2.45
(each 1H, each m), 3.50 (2H, br s), 3.70 (1H, m), 4.18 (1H, br s),
4.89 (1H, br s), 4.93 (2H, s), 5.18 (1H, br s), 5.53 (1H, br d, J )
8 Hz), 6.13 (1H, t, J ) 7 Hz), 7.11 (1H, d. J ) 8 Hz), 7.24-7.38
(5H, m), and 9.96 (1H, br s); HR-FABMS m/z 334.1398 [calcd
for C16H20N3O5 (MH+) 334.1403].
15N3-Labeled 2′-Deoxyuridine 4-O-Benzyloxime (15N3-
Labeled 2b). FABMS m/z 335 (MH+), 219, 218, and 91; 1H
NMR, identical with the above data described for the 2′-
deoxyuridine 4-O-benzyloxime 2b except δ 9.96 (1H, d, J ) 94
Hz) ppm; HR-FABMS m/z 335.1385 [calcd for C16H20N215NO5
(MH+) 335.1373].
Independent Syntheses of the Uridine 4-O-Benzy-
loximes (2a,b).14To a suspension of cytidine (102.2 mg, 0.42
mmol) or 2′-deoxycytidine (monohydrate, 100.0 mg, 0.42 mmol)
in methanol (5 mL) containing sodium methoxide (2.0 mmol)
was added O-benzylhydroxylamine HCl (Aldrich, 99% purity;
319.2 mg, 2.0 mmol), and the mixture was then stirred at
ambient temperature for 3 days. After removal of the solvent
under reduced pressure, the resulting residue was subjected to
a silica gel column by eluting with chloroform-methanol (5/1)
to isolate the desired oximes 2a,b in 21% and 10% yields,
respectively. The structures of the products 2a,b were confirmed
by comparison with the 1H NMR spectra of the authentic
compounds described above.
Supporting Information Available: Experimental pro-
cedures for the acid-hydrolysis of the 15N3-labeled 2a leading
to the 15N3-labeled uridine and for the chemical conversion of
1
the N3-O-benzylated 4-iminouridine 3 to 2a, and IR, UV, H
NMR, and/or MS spectra for the compounds 1a,b, 2a,b, 3, and
4, 15N3-labeled cytidine, 15N3-labeled 2′-deoxycytidine, and 15N3-
labeled uridine. This material is available free of charge via
the Internet at http://pubs.acs.org.
(14) Budovskii, E. I.; Simukova, N. A.; Shibaeva, R. P.; Kochetkiv,
N. K. Biokhimiya 1965, 30, 902-908.
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