Molecular design of a new class of spin-labeled ribonucleosides with N-tert-butylaminoxyl radicals

Article abstract of DOI:10.1021/jo015532s

We designed a new type of spin-labeled nucleosides with an N-tert-butylaminoxyl radical which is introduced to the nucleobase directly. Purine and pyrimidine ribonucleosides containing the aminoxyl radical such as 1a-d, 2, 3, and 4 were synthesized to investigate the stability and behavior of the N-tert-butylaminoxyl radical on a nucleobase. Lithiation of tri-O-silylated 6-chloropurine ribonucleoside (5) followed by reaction with 2-methyl-2-nitrosopropane (MNP) gave the key compound 6a, which was further converted to 6b-d. Oxidation of the obtained 6a-d and their triols (7a-d) with Ag₂O led to formation of the corresponding stable spin-labeled nucleosides (8a-d and 1a-d), which were confirmed by EPR spectroscopy. Similarly, the precursors of spin-labeled pyrimidines (13, 20, and 23) were synthesized by site-selective lithiation of tri-O-protected pyrimidine derivatives (9, 18, and 21) followed by the reaction with MNP and deprotection. An EPR study showed that the aminoxyl radicals (2, 3, and 4) were stable and that their hyperfine structures were dependent on the position of the radical. Electron densities of pyrimidine also affected hyperfine structures.

Full text of DOI:10.1021/jo015532s

J . Org. Chem. 2001, 66, 3513-3520
3513
Molecu la r Design of a New Cla ss of Sp in -La beled Ribon u cleosid es
w ith N-ter t-Bu tyla m in oxyl Ra d ica ls
Mariko Aso,* Takeshi Ikeno, Kouji Norihisa, Masakazu Tanaka, Noboru Koga, and
Hiroshi Suemune*,†
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, J apan
suemune@phar.kyushu-u.ac.jp
Received J anuary 19, 2001
We designed a new type of spin-labeled nucleosides with an N-tert-butylaminoxyl radical which is
introduced to the nucleobase directly. Purine and pyrimidine ribonucleosides containing the
aminoxyl radical such as 1a -d , 2, 3, and 4 were synthesized to investigate the stability and behavior
of the N-tert-butylaminoxyl radical on a nucleobase. Lithiation of tri-O-silylated 6-chloropurine
ribonucleoside (5) followed by reaction with 2-methyl-2-nitrosopropane (MNP) gave the key
compound 6a , which was further converted to 6b-d . Oxidation of the obtained 6a -d and their
triols (7a -d ) with Ag2O led to formation of the corresponding stable spin-labeled nucleosides (8a -d
and 1a -d ), which were confirmed by EPR spectroscopy. Similarly, the precursors of spin-labeled
pyrimidines (13, 20, and 23) were synthesized by site-selective lithiation of tri-O-protected
pyrimidine derivatives (9, 18, and 21) followed by the reaction with MNP and deprotection. An
EPR study showed that the aminoxyl radicals (2, 3, and 4) were stable and that their hyperfine
structures were dependent on the position of the radical. Electron densities of pyrimidine also
affected hyperfine structures.
In tr od u ction
Spin-labeled nucleic acids have been used to study
structures and dynamics of nucleic acids by electron
paramagnetic resonance (EPR) spectroscopy. In the
previous studies of spin-labeled nucleosides,1 only 2,2,6,6-
tetramethylpiperidine-1-oxyl (TEMPO) and related com-
pounds were used as a spin source, which were connected
to the nucleobase with the “linker” part.2 We planned the
synthesis of a new class of spin-labeled nucleosides such
as purine derivatives (1a -d ), a 6-substituted uridine
derivative (2), a 6-substituted cytidine derivative (3), and
a 5-substituted uridine derivative (4) in which a spin
source, an N-tert-butylaminoxyl radical, is directly at-
tached to the purine3 and pyrimidine nucleus (Figure 1).
In these compounds, the unpaired electron of the
aminoxyl radical can interact with electrons of the
nucleobase. It is of interest to study the behaviors of
aminoxyl radicals upon changes of electron density
distribution in the nucleobase because those changes also
might be caused by formation and breakdown of hydro-
gen bonding between complementary base pairs. Fur-
F igu r e 1. Spin-labeled ribonucleosides with N-tert-butyl-
aminoxyl radicals.
thermore, direct introduction of a spin probe into the
nucleobase decreases the complexity of EPR spectra as
flexible linker confers the motion of aminoxyl radical
independent of the DNA. Thus, it might be advantageous
to use spin-labeled oligonucleotides containing 2′-deoxy
and 2′-O-methyl derivatives of 1b, 2, 3, and 4 to study
structures of nucleic acids unless steric bulkiness of the
spin source disrupts the structure of the nucleic acids.
We anticipated that electronic properties of 1b, 2, 3, and
4 were shared with 2′-deoxy and 2′-O-methyl analogues,
which can be synthesized by conversion of the corre-
sponding ribonucleosides by known procedures4,5 or by
procedures similar to those for spin-labeled ribonucleo-
† Phone and fax: +81-92-642-6603.
(1) (a) Duh, J .-L.; Bobst, A. M. Helv. Chim. Acta 1991, 74, 739 and
references therein. (b) Zhdanov, R. I.; Porotikova, V. A.; Rozantsev, E.
G. Synthesis 1979, 267. (c) Gnewuch, T.; Sosnovsky, G. Chem. Rev.
1986, 86, 203.
(2) Spaltensen, A.; Robinson, B. H.; Hopkins, P. B. Biochemistry
1989, 28, 9484-9495.
(3) Preliminary communication of this research: Aso, M.; Norihisa,
K.; Tanaka, M.; Koga, N.; Suemune, H. J . Chem. Soc., Perkin Trans.
2 2000, 1637.
(4) Roelen, H. C. P. F.; Saris, C. P.; Brugghe, H. F.; van den Eist,
H.; Westra, J . G.; van der Marel, G. A.; van Boom, J . H. Nucleic Acids
Res. 1991, 19, 4361-4369.
(5) Shibahara, S.; Mukai, S.; Nishimura, T.; Inoue, H.; Ohtsuka, E.;
Morisawa, H. Nucleic Acids Res. 1987, 59, 4403-4415.
10.1021/jo015532s CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/24/2001

3514 J . Org. Chem., Vol. 66, No. 10, 2001
Aso et al.
Sch em e 1
Sch em e 2
tion of 6a might cause an anisotropic deshielding of C2′-H
by the nitrogen atom at the 3-position.10 Direct synthesis
of the 6-amino derivative (6b) by lithiation of the tri-O-
silylated adenosine derivative followed by reaction with
MNP was not successful, but 6b was synthesized by a
substitution reaction of the chloride group of 6a . Heating
6a in ammoniacal methanol in a sealed tube (60 °C, 4
days) gave 6b in 82% yield. The N-tert-butylhydroxyl-
amino function at the 8-position was not changed under
these conditions. The 6-alkoxy derivatives, 6c and 6d ,
were also formed with ease by treatment of 6a with
sodium alkoxides at room temperature in 70% and 76%
sides. Thus, we synthesized 1a -d , 2, 3, and 4 and studied
their stabilities and electromagnetic behaviors by using
EPR. The N-tert-butylaminoxyl radical, the choice of spin
source in this study, can be obtained by oxidation of the
corresponding hydroxylamino group as used for construc-
tion of stable high-spin poly(N-tert-butylaminoxyl radi-
cal)s containing the m-phenylene framework as organic
super-high-spin molecules.6 We are also interested in
biological activities of ribonucleosides with an N-tert-
butylhydroxylamino group and N-tert-butylaminoxyl radi-
cal as N-tert-butylhydroxylamine7 and aminoxyl radical8
are reported to be potent antioxidants.
1
yields, respectively. In the H NMR spectra, the C2′-H
of 6b-d were also observed at lower fields, similar to
that of 6a (6b, 5.35 ppm; 6c, 5.19 ppm; 6d , 5.21 ppm).
The final step of the synthesis was desilylation of 6a -d .
The conditions for desilylation such as those with TBAF
and HF did not effect formation of the corresponding
triols (7a -d ). Finally, 6a -d were converted to 7a -d in
87-98% yields by heating with ammonium fluoride in
MeOH (Scheme 2).
F or m a tion of N-ter t-Bu tyla m in oxyl Ra d ica l a t
th e C-8 P osition . Oxidation of 6a -d and 7a -d with
Ag2O proceeded easily to afford the corresponding ami-
noxyl radicals (8a -d and 1a -d ) (Scheme 2). For ex-
ample, the toluene solution of 7a gradually turned red
in color after addition of Ag2O and TLC analysis of the
reaction mixture showed formation of less polar, red
aminoxyl radical. Attempts to isolate the radical 1a were
unsuccessful, but formation of the aminoxyl radical was
confirmed by EPR spectroscopy. The EPR spectrum of
1a in a toluene solution at room temperature showed a
triplet with a hyperfine coupling constant of aN ) 9.70 G
due to interaction between an unpaired electron and
nitrogen nucleus of the aminoxyl radical (g value ) 2.006,
Figure 2a). The EPR spectrum of 1a could be well
simulated11 by assuming hyperfine coupling constants (aN
) 9.70, 2.45, 1.06, 0.39, and 0.35 and aH ) 1.00 G) to
indicate that the unpaired electron must be delocalized
Syn th esis of 8-(N-ter t-Bu tylh yd r oxyla m in o) P u -
r in e Der iva tives. Miyasaka et al. reported that treat-
ment of 6-chloropurine derivatives with LDA resulted in
exclusive lithiation at the 8-position and subsequent
reaction with electrophiles afforded 8-substituted purine
nucleosides.9 Introduction of an N-tert-butylhydroxyl-
amino group, the precursor of the aminoxyl radical, into
the purine nucleus at the 8-position was carried out
according to their procedure; tri-O-silylated 6-chloropu-
rine ribonucleoside (5), prepared from inosine, was lithi-
ated with 6 equiv of LDA in dry ether at -78 °C and
subsequent treatment with 2-methyl-2-nitrosopropane
(MNP) at -20 °C gave the desired 6a in 56% yield
(Scheme 1). Purification of 6a by silica gel chromatog-
raphy led to its decomposition, but 6a was purified by
using alumina. The position of the N-tert-butylhydroxyl-
1
amino group was determined by the H NMR spectrum.
The C2′-H of 6a was observed at lower field (5.19 ppm)
than that of 5 (4.59 ppm). The syn-glycosidic conforma-
(6) Fujita, J .; Tanaka, M.; Suemune, H.; Koga, N.; Matsuda, K.;
Iwamura, H. J . Am. Chem. Soc. 1996, 118, 9347 and references therein.
(7) Atmna, H.; Martinez, A. P.; Ames, B. N. J . Biol. Chem. 2000,
275, 6741-6748.
(8) Krishna, M. C.; Russo, A.; Mitchell, J . B.; Goldstein, S.; Dafni,
H.; Samuni, A. J . Biol. Chem. 1996, 271, 26026-26031.
(9) Kato, K.; Hayakawa, H.; Tanaka, H.; Kuramoto, H.; Shindoh,
S.; Shuto, S.; Miyasaka, T. J . Org. Chem. 1997, 62, 6833.
(10) Pless, P.; Dudycz, L.; Stolarski, R.; Shugar, D. Z. Naturforsch.
1978, 33c, 902-907.
(11) Duling, D. R. J . Magn. Reson., Ser. B 1994, 104, 105-110.

New Class of Spin-Labeled Ribonucleosides
J . Org. Chem., Vol. 66, No. 10, 2001 3515
Sch em e 3
F igu r e 2. (a) EPR spectrum of 1a . (b) Simulated spectrum
of 1a (aN ) 9.70, 2.45, 1.06, 0.39, 0.35 and aH ) 1.00 G). (c)
EPR spectrum of 1b. (d) Simulated spectrum of 1b (aN ) 10.50,
2.68, 1.16, 0.40, 0.40 G).
into the purine ring (Figure 2b). The intensity of the
signal decreased gradually, but the triplet due to the
aminoxyl radical persisted for several days. The half-time
period was estimated to be about 4 days at room tem-
perature, which showed that 1a was quite stable. The
EPR spectrum of 1b in toluene also showed a triplet with
a hyperfine coupling constant of aN ) 10.5 G (g value )
2.005, Figure 2c) but the shape of each signal of the
triplet was somewhat different from that of 1a probably
due to the difference of spin density distribution in the
purine ring. The EPR spectrum of 1b was well simulated
by assuming hyperfine coupling constants aN ) 10.5,
2.68, 1.16, 0.40, and 0.40 G (Figure 2d). EPR spectra of
the radicals (1c,d and 8a -d ) in toluene were also
measured to show spectra similar to those of 1a and 1b
(g value ) 2.005-2.006, Supporting Information).
Sch em e 4
Syn th esis of N-ter t-Bu tylh yd r oxyla m in o P yr im i-
d in e Der iva tives. Lithiation reactions of pyrimidine
compounds were also studied by Miyasaka extensively,
and selective synthesis of 5- and/or 6-substituted pyri-
midine derivatives are effected by using the appropriate
base and protective group of the ribose moiety.12 Lithia-
tion of 9 with LDA followed by reaction with MNP gave
10 in 48% yield. X-ray analysis of 10 showed that the
N-tert-butylhydroxylamino group was introduced into the
6-position of uracil (Scheme 3). Attempted deprotection
of MOM and isopropylidene groups with 30% aqueous
trifluoroacetic acid led to decomposition of 10. However,
treatment of O-acetylated 1113 under the same conditions
gave the triol 12, which was further converted to 13.
Introduction of the N-tert-butylhydroxylamino group
into the 6-position of the 4-ethoxy pyrimidine derivative
was carried out successfully under similar conditions
(Scheme 4). Treatment of the tri-O-silylated derivative
1414 with LDA and subsequent reaction with MNP gave
6-substituted 15 in 59% yield. The H NMR spectrum of
15 did not show the signal of the C-6 proton and the C-5
proton was observed at 5.96 ppm. Desilylation of 15 with
TBAF was accompanied by removal of the N-tert-butyl-
1
(12) (a) Tanaka, H.; Hayakawa, H.; Miyasaka, T. Chem. Pharm.
Bull. 1981, 29, 3565-3572. (b) Hayakawa, H.; Tanaka, H.; Obi, K.;
Itoh, M.; Miyasaka, T. Tetrahedron Lett. 1987, 28, 87-90. (c) Tanaka,
H.; Hayakawa, H.; Iijima, S.; Haraguchi, K.; Miyasaka, T. Tetrahedron
1985, 41, 861-866.
(13) 1H NMR spectra of 11 and 12 showed the presence of two
isomers in a ratio of 2:1. Those isomers might be rotational isomers,
and hydrolysis of the acetyl group of 12 afforded 13 as a single isomer
in 80% yield.
(14) Ogilvie, K. K.; Beaucage, S. L.; Schifman, A. L.; Theriault, N.
Y.; Sadana, K. L. Can. J . Chem. 1978, 56, 2768-2780.

3516 J . Org. Chem., Vol. 66, No. 10, 2001
Aso et al.
Sch em e 5
Sch em e 6
12.9, 3.53 G; 24, aN ) 12.7, 2.52 G, Figures 4b and 4c).
In contrast, 5-substituted 4 showed six peaks with
hyperfine coupling constants of aN ) 13.6, aH )1.75 G
(g value ) 2.005, Figure 4d). The intensity of the signal
decreased gradually but persisted for several weeks.
Electron density of the amino group of the pyrimidine
ring also affected hyperfine couplings. The EPR spectrum
of cytidine derivative 25 showed nine peaks, whereas that
of 26, the amino group of which was benzoylated, showed
six peaks (Figure 5).
hydroxylamino group, and the triol 16 was obtained in
low yield. However, heating 15 with ammonium fluoride
in methanol gave 16 in 75% yield. Unfortunately, conver-
sion of 15 to N-tert-butylhydroxylamino cytidine deriva-
tive 17 by heating in ammoniacal methanol15 proceeded
at only 6% (isolated yield, 33% conversion yield). Next,
the synthesis of 6-substituted cytidine derivative 19 from
1816 was carried out. Lithiation of 18 with LDA and the
following reaction with MNP gave 19 in 48% yield
(Scheme 5). Heating 19 in ammoniacal methanol (65 °C,
6 h) afforded 17 in 98% yield. Desilylation of 17 also
proceeded with ammonium fluoride to give the radical
precursor 20 in 52% yield.
Introduction of the N-tert-butylhydroxylamino group
into the 5-position of the uracil moiety was studied with
21.12c Lithiation of 21 with sec-BuLi and subsequent
reaction with MNP gave 22 albeit in low yield (19%
isolated yield, 45% conversion yield, Scheme 6). In the
1H NMR spectrum of 22, the signal of the C-5 proton was
not observed and the C-6 proton was observed at 7.61
ppm. For desilylation, treatment of 22 with TBAF gave
23 in 90% yield, whereas 23 was obtained in low yield
(ca. 20%) by heating 22 with ammonium fluoride.
EP R Mea su r em en t of Sp in -La beled P yr im id in e
Der iva tives. Oxidation of pyrimidine derivatives con-
taining the N-tert-butylhydroxylamino group (13, 20, 16,
and 23) with Ag2O proceeded easily to afford the corre-
sponding aminoxyl radicals (2, 3, 24, and 4) (Figure 3).
Interestingly, hyperfine couplings of EPR spectra were
dependent on the position of the N-tert-butylaminoxyl
group. The EPR spectrum of 2 in CH2Cl2 containing 1%
MeOH showed nine peaks with hyperfine coupling con-
stants of aN ) 12.6, 2.55 G (g value ) 2.005, Figure 4a).
F igu r e 3. Pyrimidine ribonucleosides with N-tert-butylhy-
droxylamino groups and N-tert-butylaminoxyl radicals.
Discu ssion
Syn th esis a n d Con for m a tion of Ribon u cleosid es
w ith N-ter t-Bu tyh yd r oxyla m in o Gr ou p . As precur-
sors of 1a -d , 2, 3, and 4, purine ribonucleosides (6a -d )
and pyrimidine ribonucleosides (13, 20, and 23) were
synthesized by site-selective lithiation and the following
reaction with 2-methyl-2-nitrosopropane and deprotec-
tion. The conformational properties of the modified
6-Substituted 3 and 24 showed similar spectra (3, aN
)
(15) Matsuda, A.; Itoh, H.; Takenuki, K.; Sasaki, T.; Ueda, T. Chem.
Pharm Bull 1988, 36(3), 945-953.
(16) Ogilvie, K. K.; Schifman, A. L.; Penny, C. L. Can. J . Chem. 1979,
56, 2230-2238.

New Class of Spin-Labeled Ribonucleosides
J . Org. Chem., Vol. 66, No. 10, 2001 3517
ppm (C2′-H of 7b; 4.89 ppm, C2′-H of adenosine; 4.61
ppm), which was smaller than that of 8-bromoadenosine
relative to adenosine (0.48 ppm; C2′-H of 8-bromoad-
enosine; 5.09 ppm). It is likely that the syn population of
7b is smaller than that of 8-bromoadenosine. For pyri-
midine ribonucleosides, the chemical shift of C2′-H of
6-methyluridine is shifted downfield (4.81 ppm in D2O)
compared to that of uridine (4.32 ppm) to indicate the
preferred syn conformation of 6-methyluridine in solu-
tion.12a We compared the chemical shift of C2′-H of 13
(4.70 ppm) and that of uridine. Compared to the chemical
shift change of C2′-H of 6-methyluridine relative to
uridine (0.49 ppm), that of 13 was smaller (0.38 ppm).
The chemical shift data of 7b and 13 indicate that steric
bulkiness of an N-tert-butylhydroxylamino group intro-
duced into the 8-position of purine and the 6-position of
pyrimidine is smaller than that of bromide and methyl
groups. In contrast, the C2′-H of 5-substituted 23 was
observed at 4.32 ppm in D2O which is comparable to that
of uridine and 23 might exist in an anti conformation
similar to 5-methyluridine. Introduction of N-tert-butyl-
hydroxylamino group into the 5-position of pyrimidine
might minimize structural disruption of the oligonucle-
otide duplex.
F igu r e 5. EPR spectra of (a) 25 (aN ) 12.8, 1.99 G) and (b)
26 (aN ) 14.3, aN ) 2.73 G).
EP R Stu d y of Sp in -La beled P u r in e a n d P yr im i-
d in e Ribon u cleosid es. An EPR study of spin-labeled
purine and pyrimidine ribonucleosides indicated that
aminoxyl radicals attached directly to nucleobases were
quite stable. In EPR spectra of 1a and 1b, the largest
hyperfine coupling constants were 9.70 and 10.50 G
(Figures 2a and 2c). These values are reasonable for an
N-tert-butylaminoxyl radical on an sp2 hybridized car-
bon.19 The EPR spectra of 1a -1d could be well simulated
with five hyperfine coupling constants of nitrogens (for
1a , aN ) 9.70, 2.45, 1.06, 0.39, and 0.35 and aH ) 1.00
G). This indicated that the unpaired electron of the
aminoxyl radical might be delocalized into the purine ring
(Figures 2b and 2d). In spin-labeled pyrimidine ribo-
nucleosides, 6-substituted 2, 3, and 24 showed nine peaks
with two hyperfine coupling constants of nitrogens (for
2, aN ) 12.6, 2.55 G). In contrast, 5-substituted 4 showed
six peaks with hyperfine coupling constants of aN ) 12.7,
aH ) 3.53 G (Figure 4). It is likely that hyperfine
couplings of spin-labeled pyrimidines are more influenced
by the position of the radical than the structure of
pyrimidines. In addition to its electronic effect, the steric
influence of an N-tert-butylaminoxyl radical on the
conformation including the dihedral angles between N-O
bonds and the pyrimidine ring planes must be important
because spin density distribution is dependent on inter-
action of the unpaired radical electron and electrons of
pyrimidines. Influences of substituents of the purine and
pyrimidine rings on hyperfine couplings were also ob-
served. The EPR spectra of 6-substitued 26 with the
benzoylated amino group a showed sharp contrast to that
of 25, probably due to the difference of electron densities
of the pyrimidine ring (Figure 5). These data prompted
us to study behaviors of 2′-O-methyl and 2′-deoxy ana-
logues of 1b, 2, 3, and 4 in oligonucleotides, and their
synthetic study is in progress.
F igu r e 4. EPR spectra of (a) 2 (aN ) 12.6, 2.55 G), (b) 3 (aN
) 12.9, 3.53 G), (c) 24 (aN ) 12.7, 2.52 G), and (d) 4 (aN
)
13.6, aH ) 1.75 G). The spectra were obtained from solutions
in CH2Cl2 containing 1% MeOH.
nucleosides are associated with their biological behaviors
and conformations of oligonucleotides and polynucleotides
containing them. It is known that introduction of a bulky
substituent into the 8-position of purine and the 6-posi-
tion of pyrimidine causes a shift of the syn/anti equilib-
rium to syn due to interaction of substituent and ribose.
In 1H NMR spectra, characteristic changes in chemical
shifts of C1′-H, C2′-H, and C3′-H relative to those of
unmodified nucleosides are observed. Especially, the
chemical shifts of C2′-H are shifted downfield signifi-
cantly when nucleosides have a high population of the
syn form. These changes result from the anisotropic
influence of the N(3) atom and the ring currents of the
purine moiety or of the 2-keto group of the pyrimidine
in the conformation syn. Therefore, the syn/anti confor-
mational preference was determined by comparison of
1
chemical shifts of H NMR spectra in D2O and DMSO-
10,17,18
d6.
We compared the chemical shifts of C2′-H of 7b
and those of adenosine and 8-bromoadenosine in DMSO-
d6 to estimate conformation of 7b in solution because
preferred conformation of adenosine is anti and that of
8-bromoadenosine is predominantly syn.17 The chemical
shift change of C2′-H of 7b relative to adenosine was 0.28
Exp er im en ta l Section
Melting points were taken on a Yanagimoto melting point
apparatus and are not corrected. 1H NMR spectra were
obtained on a J EOL GX-270 (270 MHz) spectrometer. Chemi-
(17) J ordan, F.; Niv, H. Biochim. Biophys. Acta 1977, 476, 265-
271. They reported that the changes of chemical shifts in DMSO-d6
showed the same trends in D2O.
(18) Schweizer, M. P.; Bantam, E. B.; Witkowski, J . T.; Robins, R.
K. J . Am. Chem. Soc. 1973, 95, 3770-3778.
(19) Buettner, G. R. Free Rad. Biol. Med. 1987, 3, 259-303.

3518 J . Org. Chem., Vol. 66, No. 10, 2001
Aso et al.
cal shifts are reported as δ values with respect to tetrameth-
ylsilane (TMS) or 2,2-dimethyl-2-silapentane 5-sulfonate (DSS)
as an internal standard or are referenced to a residual proton
at 2.49 ppm for DMSO-d6 or at 3.30 ppm for CD3OD. 13C NMR
spectra were taken on a J EOL GX-270 (68 MHz) or a Varian
UNITY-plus (125 MHz) spectrometer. IR spectra were taken
on a J ASCO IR A-100 infrared spectrophotometer. High-
resolution mass spectra (HRMS) were determined on an SX-
102 or a J MS-700T spectrometer. EPR spectra were recorded
on a Bruker ESP 300 X-band (9.4 GHz) spectrometer equipped
with a Hewlett-Packard 5350B microwave frequency counter.
For thin-layer chromatography, precoated TLC plates (Merck,
Kieselgel 60 F254) were used. Column chromatography was
carried out on silica gel 70-230 mesh (Merck, Kieselgel 60)
or on alumina.
1H), 4.14 (s, 3H), 4.05-3.96 (m, 2H), 3.73 (dd, J ) 13.4, 6.1
Hz, 1H), 1.39 (s, 9H), 0.94 (s, 9H), 0.83 (s, 9H), 0.76 (s, 9H),
0.15 (s, 3H), 0.13 (s, 3H), 0.02 (s, 3H), 0.00 (s, 3H), -0.12 (s,
3H), -0.35 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 160.5 (s),
154.7 (s), 151.4 (s), 151.3 (d), 119.5 (s), 88.6 (d), 84.0 (d), 73.1
(d), 71.4 (d), 61.9 (t), 60.2 (s), 54.2 (q), 26.2 (q), 26.0 (q), 25.9
(q), 25.8 (q), 18.5 (s), 18.1 (s), 17.9 (s), -4.2 (q), -4.5 (q), -4.6
(q), -4.9 (q), -5.3 (q), -5.6 (q). Anal. Calcd for C33H65N5O6-
Si3: C, 55.66; H, 9.21; N, 9.84. Found: C, 55.89; H, 9.13; N,
9.69.
6-E t h oxy-8-(N-ter t-b u t ylh yd r oxyla m in o)-9-(2,3,5-t r is-
O-TBDMS-â-D-r ibofu r a n osyl)p u r in e (6d ). To a solution of
6a (40 mg, 0.06 mmol) in 2 mL of dry EtOH was added NaOEt
(11 mg, 0.17 mmol) under Ar, and the mixture was stirred at
room temperature. After 4 h, EtOH was removed under
reduced pressure and the residue was diluted with AcOEt and
washed with saturated aqueous ammonium chloride solution
and brine. The organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified
on alumina. Elution with hexane/AcOEt (10:1) gave 6d (31 mg,
76%) as a colorless oil: IR (CHCl3) 3400, 2960, 2940, 2850,
1600 cm-1; 1H NMR (270 MHz, CDCl3) δ 8.43 (s, 1H), 6.37 (s,
1H), 6.18 (d, J ) 5.0 Hz, 1H), 5.21 (t, J ) 5.0 Hz, 1H), 4.66 (q,
J ) 7.0 Hz, 2H), 4.59 (t, J ) 4.6 Hz, 1H), 4.06-3.97 (m, 2H),
3.79-3.72 (m, 1H), 1.50 (t, J ) 7.0 Hz, 3H), 1.41 (s, 9H), 0.96
(s, 9H), 0.85 (s, 9H), 0.79 (s, 9H), 0.17 (s, 3H), 0.15 (s, 3H),
0.04 (s, 3H), 0.02 (s, 3H), -0.09 (s, 3H), -0.32 (s, 3H); 13C NMR
(125 MHz, CDCl3) δ 160.4 (s), 154.7 (s), 151.5 (s), 151.4 (d),
119.7 (s), 88.6 (d), 84.1 (d), 73.0 (d), 71.7 (d), 63.1 (t), 62.1 (t),
60.3 (s), 26.2 (q), 26.0 (q), 26.0 (q), 25.8 (q), 18.4 (s), 18.1 (s),
17.9 (s), 14.6 (q), -4.2 (q), -4.4 (q), -4.6 (q), -4.9 (q), -5.3
(q), -5.6 (q); HRMS (FAB) m/z calcd for C34H68N5O6Si3 ([M +
H]+) 726.4477, found 726.4489.
6-Ch lor o-8-(N-ter t-b u t ylh yd r oxyla m in o)-9-(2,3,5-t r is-
O-TBDMS-â-D-r ibofu r a n osyl)p u r in e (6a ). To a dry ether
solution containing LDA (8.20 mmol) was added dropwise a
solution of 6-chloro-9-(2,3,5-tris-O-TBDMS-â-D-ribofuranosyl)-
purine (860 mg, 1.37 mmol) in 10 mL of dry ether at -78 °C
under an Ar atmosphere, and the temperature was maintained
for 1.5 h. A solution of 2-methyl-2-nitrosopropane (MNP) (360
mg, 4.1 mmol) in 5 mL of dry ether was added to the reaction
mixture, and the temperature was raised to -40 °C. A solution
of MNP (360 mg, 4.1 mmol) in 5 mL of dry ether was added
again, and the temperature was raised to -20 °C. The reaction
mixture was quenched by adding saturated aqueous am-
monium chloride solution, and the organic layer was extracted
with ether, washed with brine, and dried over Na2SO4. The
solvent was evaporated under reduced pressure, and the
residue was purified on alumina. Elution with hexane/EtOAc
(10:1) gave 6a (550 mg, 56%) as a colorless solid: mp 144 °C;
IR (CHCl3) 3400, 2900, 1590, 1250, 1160, 1130, 1070, 830 cm-1
;
1H NMR (270 MHz, CDCl3) δ 8.62 (s, 1H), 6.68 (brs, 1H, D2O
exchanged), 6.21 (d, J ) 5.4 Hz, 1H), 5.19 (t, J ) 5.1 Hz, 1H),
4.53 (t, J ) 4.3 Hz, 1H), 4.08-3.72 (m, 2H), 3.68 (m, 1H), 1.46
(s, 9H), 0.96 (s, 9H), 0.86 (s, 9H), 0.78 (s, 9H), 0.17 (s, 3H),
0.16 (s, 3H), 0.06 (s, 3H), 0.04 (s, 3H), -0.10 (s, 3H), -0.34 (s,
3H); 13C NMR (125 MHz, CDCl3) δ 157.1 (s), 151.5 (s), 151.0
(d), 149.6 (s), 123.0 (s), 88.8 (d), 84.4 (d), 73.2 (d), 71.4 (d), 61.9
(t), 60.5 (s), 26.1 (q), 25.91 (q), 25.90 (q), 25.7 (q), 18.5 (s), 18.1
(s), 17.9 (s), -4.2 (q), -4.5 (q), -4.6 (q), -5.0 (q), -5.3 (q), -5.6
(q). Anal. Calcd for C32H62ClN5O5Si3: C, 53.64; H, 8.72; N, 9.77.
Found: C, 53.57; H, 8.70; N, 9.81.
6-Ch lor o-8-(N-ter t-b u t ylh yd r oxyla m in o)-9-(â-D-r ib o-
fu r a n osyl)p u r in e (7a ). To a solution of 6a (100 mg, 0.14
mmol) in 5 mL of MeOH was added ammonium fluoride (52
mg, 1.40 mmol), and the mixture was heated at 65 °C. After
10 h, MeOH was removed under reduced pressure and the
residue was purified on Florisil. Elution with CHCl3/MeOH
(5:1) gave 7a (45 mg, 87%) as a colorless solid: mp 105-107
°C; IR (KBr) 3300, 2900, 1580, 1320 cm-1; 1H NMR (270 MHz,
DMSO-d6) δ 9.63 (s, 1H, D2O exchanged), 8.64 (s, 1H), 6.11
(d, J ) 5.6 Hz, 1H), 5.21 (m, 2H, D2O exchanged), 5.00 (m,
2H, 1H was exchanged with D2O), 4.32 (dd, J ) 9.6, 5.0 Hz,
6-Am in o-8-(N-ter t-bu tylh yd r oxyla m in o)-9-(2,3,5-tr is-O-
TBDMS-â-D-r ib ofu r a n osyl)p u r in e (6b ). Ammonia was
bubbled through a solution of 6a (400 mg, 0.56 mmol) in MeOH
(40 mL) at -40 °C, and the resultant ammoniacal solution was
heated at 60 °C in a sealed tube for 4 days. The solvent was
evaporated under reduced pressure, and the residue was
purified on alumina. Elution with hexane/AcOEt (10:1) gave
6b (320 mg, 82%) as a colorless solid: mp 140-142 °C; IR
1H), 3.90 (m, 1H), 3.70 (m, 1H), 3.56 (m, 1H), 1.38 (s, 9H); 13
C
NMR (125 MHz, DMSO-d6) δ 157.3 (s), 151.5 (s), 150.3 (d),
146.8 (s), 129.5 (s), 89.1 (d), 85.2 (d), 71.3 (d), 70.2 (d), 61.7 (t),
59.8 (s), 25.8 (q); HRMS (FAB) m/z calcd for C14H21N5O5Cl ([M
+ H]+) 374.1231, found 374.1245.
6-Am in o-8-(N-ter t-bu tylh ydr oxylam in o)-9-(â-D-r ibofu r a-
n osyl)p u r in e (7b). To a solution of 6b (96 mg, 0.14 mmol) in
5 mL of MeOH was added ammonium fluoride (51 mg, 1.38
mmol), and the mixture was heated at 65 °C. After 10 h, MeOH
was removed under reduced pressure and the residue was
purified on Florisil. Elution with CHCl3/MeOH (5:1) gave 7b
(46 mg, 94%) as a colorless solid: mp 187-189 °C; IR (KBr)
3350, 2980, 2940, 1640, 1600, 1360, 1330, 1300 cm-1; 1H NMR
(270 MHz, DMSO-d6) δ 8.89 (s, 1H, D2O exchanged), 8.06 (s,
1H), 7.20 (brs, 2H, D2O exchanged), 6.08 (d, J ) 6.6 Hz, 1H),
5.87-5.83 (m, 1H, D2O exchanged), 5.20 (d, J ) 4.0 Hz, 1H,
D2O exchanged), 5.06 (d, J ) 7.3 Hz, 1H, D2O exchanged), 4.89
(dd, J ) 12.5, 6.9 Hz, 1H), 4.20 (m, 1H), 3.94 (m, 1H), 3.66
(dm, J ) 12.4 Hz, 1H), 3.58-3.49 (m, 1H), 1.29 (s, 9H); 13C
NMR (125 MHz, DMSO-d6) δ 155.5 (s), 152.1 (s), 151.5 (d),
147.8 (s), 116.7 (s), 87.8 (d), 85.8 (d), 71.8 (d), 70.8 (d), 62.1 (t),
59.0 (s), 26.0 (q); HRMS (FAB) m/z calcd for C14H23N6O5 ([M
+ H]+) 355.1730, found 355.1761.
6-Meth oxy-8-(N-ter t-bu tylh yd r oxyla m in o)-9-(â-D-r ibo-
fu r a n osyl)p u r in e (7c). To a solution of 6c (80 mg, 0.11 mmol)
in 5 mL of MeOH was added ammonium fluoride (42 mg, 1.13
mmol), and the mixture was heated at 65 °C. After 10 h, MeOH
was removed under reduced pressure and the residue was
purified on Florisil. Elution with CHCl3/MeOH (5:1) gave 7c
(40 mg, 98%) as a colorless solid: mp 115-117 °C; IR (KBr)
3350, 2990, 2870, 1600, 1580, 1480, 1350, 1320 cm-1; 1H NMR
(CHCl3) 3410, 2900, 1620, 1240, 1150, 1120, 1070, 830 cm-1
;
1H NMR (270 MHz, CDCl3) δ 8.24 (s, 1H), 6.98 (brs, 1H), 6.14
(d, J ) 4.9 Hz, 1H), 5.64 (brs, 2H), 5.29 (t, J ) 5.0 Hz, 1H),
4.55 (t, J ) 4.0 Hz, 1H), 4.08-3.97 (m, 2H), 3.71 (m, 1H), 1.36
(s, 9H), 0.93 (s, 9H), 0.85 (s, 9H), 0.77 (s, 9H), 0.15 (s, 3H),
0.13 (s, 3H), 0.08 (s, 3H), 0.02 (s, 3H), -0.10 (s, 3H), -0.30 (s,
3H); HRMS (FAB) m/z calcd for C32H65ClN6O5Si3 ([M + H]+)
697.4324, found 697.4312. Anal. Calcd for C32H64ClN6O5Si3:
C, 55.13; H, 9.25; N, 12.05. Found: C, 55.17; H, 9.22; N, 12.00.
6-Meth oxy-8-(N-ter t-bu tylh yd r oxyla m in o)-9-(2,3,5-tr is-
O-TBDMS-â-D-r ibofu r a n osyl)p u r in e (6c). To a solution of
6a (65 mg, 0.09 mmol) in 3 mL of dry MeOH was added
NaOMe (15 mg, 0.28 mmol) under an Ar atmosphere, and the
mixture was stirred at room temperature. After 4 h, MeOH
was removed under reduced pressure and the residue was
diluted with AcOEt and washed with saturated aqueous
ammonium chloride and brine. The organic layer was dried
over Na2SO4 and concentrated under reduced pressure. The
residue was purified on alumina. Elution with hexane/AcOEt
(10:1) gave 6c (40 mg, 70%) as a colorless solid: mp 106-108
1
°C; IR (CHCl3) 3400, 2960, 2940, 2850, 1600, 1210 cm-1; H
NMR (270 MHz, CDCl3) δ 8.43 (s, 1H), 6.44 (brs, 1H), 6.17 (d,
J ) 5.0 Hz, 1H), 5.19 (t, J ) 5.0 Hz, 1H), 4.55 (t, J ) 4.6 Hz,

New Class of Spin-Labeled Ribonucleosides
J . Org. Chem., Vol. 66, No. 10, 2001 3519
(270 MHz, CDCl3) δ 8.29 (s, 1H), 7.93 (brs, 1H, D2O ex-
changed), 6.63 (brs, 1H, D2O exchanged), 6.31 (d, J ) 7.9 Hz,
1H), 5.15 (dd, J ) 7.6, 5.0 Hz, 1H), 4.46 (d, J ) 4.6 Hz, 1H),
4.28 (s, 1H), 4.15 (s, 3H), 3.93 (d, J ) 11.9 Hz, 1H), 3.75-3.70
(m, 1H), 1.30 (s, 9H); 13C NMR (68 MHz, CDCl3) δ 160.1 (s),
154.4 (s), 151.0 (d), 149.5 (s), 119.1 (s), 89.6 (d), 87.0 (d), 77.2
(d), 72.9 (d), 63.3 (t), 61.3 (s), 54.5 (q), 25.9 (q); HRMS (FAB)
m/z calcd for C15H24N5O6 ([M + H]+) 370.1726, found 370.1691.
6-E t h oxy-8-(N-ter t-b u t ylh yd r oxyla m in o)-9-(â-D-r ib o-
fu r a n osyl)p u r in e (7d ). To a solution of 6d (110 mg, 0.15
mmol) in 5 mL of MeOH was added ammonium fluoride (56
mg, 1.51 mmol), and the mixture was heated at 65 °C. After
12 h, MeOH was removed under reduced pressure and the
residue was purified on Florisil. Elution with CHCl3/MeOH
(5:1) gave 7d (52 mg, 90%) as a colorless solid: mp 102-104
°C; IR (KBr) 3300, 2940, 1600, 1450, 1320 cm-1; 1H NMR (270
MHz, CDCl3) δ 8.26 (s, 1H), 7.97 (brs, 1H, D2O exchanged),
6.56 (brd, J ) 11.6 Hz, 1H, D2O exchanged), 6.32 (d, J ) 7.9
Hz, 1H), 6.07 (brs, 1H, D2O exchanged), 5.24-5.20 (m, 1H),
4.67-4.46 (m, 3H), 4.29 (s, 1H), 4.07 (brs, 1H, D2O exchanged),
3.92 (d, J ) 12.6 Hz, 1H), 3.76 (t, J ) 12.6 Hz, 1H), 1.52 (t, J
) 7.1 Hz, 3H), 1.28 (s, 9H); 13C NMR (68 MHz, CDCl3) δ 159.8
(s), 154.2 (s), 151.1 (d), 149.5 (s), 119.0 (s), 89.6 (d), 87.0 (d),
73.0 (d), 72.8 (d), 63.9 (t), 63.4 (t), 61.5 (s), 25.9 (q), 14.3 (q);
HRMS (FAB) m/z calcd for C16H25N5O6 ([M + H]+) 384.1883,
found 384.1861.
5′-Meth oxym eth yl-6-(N-ter t-bu tylh yd r oxyla m in o)-2′,3′-
O-isop r op ylid en eu r id in e (10). To a soluiton of LDA in THF
(6 mL), prepared from n-BuLi (6.2 mL, 9.68 mmol) in hexane
and diisopropylamine (1.36 mL, 9.68 mmol), was added a
solution of 5′-methoxymethyl-2′,3′-O-isopropylideneuridine (9)12a
(530 mg, 1.61 mmol) in 11 mL of THF at -78 °C, and the
mixture was stirred for 1 h at -70 °C. A solution of MNP (842
mg, 9.68 mmol) in 7.8 mL of THF was added dropwise, and
the mixture was stirred for 3 h. A saturated NH4Cl solution
was added to the reaction mixture, and the organic layer was
extracted with AcOEt, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by silica gel
chromatography (AcOEt) to give 10 (263 mg, 48%) as colorless
needles: mp 185 °C (recrystallized from hexane), 1H NMR (270
MHz, CDCl3) δ 8.95 (br, 1H, D2O exchanged), 6.77 (br, 1H,
D2O exchanged), 6.59 (s, 1H), 5.78 (s, 1H), 5.09 (dd, J ) 6.3,
1.0 Hz, 1H), 4.80 (t, J ) 5.6 Hz, 1H), 4.73 (d, J ) 6.6 Hz, 1H),
4.65 (d, J ) 6.6 Hz, 1H), 4.32-4.29 (m, 1H), 3.87 (dd, J ) 8.9,
3.6 Hz, 1H), 3.75 (t, J ) 8.9 Hz, 1H), 3.38 (s, 3H), 1.55 (s, 3H),
1.33 (s, 3H), 1.24 (s, 9H); HRMS (FAB) m/z calcd for C18H30O8N3
([M + H]+) 416.2033, found 416.2053.
5′-Meth oxym eth yl-6-(N-ter t-bu tyla cetoxyla m in o)-2′,3′-
O-isop r op ylid en eu r id in e (11). To a solution of 10 (163 mg,
0.39 mmol) in CH2Cl2 (7 mL) were added pyridine (0.05 mL,
0.59 mmol), acetic anhydride (0.05 mL, 0.59 mmol), and DMAP
(4.8 mg, 0.04 mmol), and the mixture was stirred at room
temperature for 3 h. A saturated aqueous NaHCO3 solution
was added to the reaction mixture, and the organic layer was
extracted with CH2Cl2, washed with brine, and dried over Na2-
SO4. After concentration, the residue was purified by silica gel
chromatography (AcOEt) to give 11 (169 mg, 94%) as a
colorless solid: 1H NMR (270 MHz, CDCl3) δ (major) 9.78 (bs,
1H), 6.97 (s, 1H), 5.73 (s, 1H), 5.14 (d, J ) 6.6 Hz, 1H), 4.92
(m, 1H), 4.65 (dd, J ) 7.8, 6.8 Hz, 2H), 4.31-4.24 (m, 1H),
3.76-3.73 (m, 2H), 3.35 (s, 3H), 2.08 (s, 3H), 1.56 (s, 3H), 1.34
(s, 3H), 1.27 (s, 9H), (minor) 9.59 (bs, 1H), 6.97 (s, 1H), 5.75
(s, 1H), 5.03 (d, J ) 5.9 Hz, 1H), 4.92 (m, 1H), 4.71 (s, 2H),
4.31-4.24 (m, 1H), 3.76-3.73 (m, 2H), 3.35 (s, 3H), 2.08 (s,
3H),1.56 (s, 3H), 1.36 (s, 3H), 1.30 (s, 9H); HRMS (FAB) m/z
calcd for C20H32O9N3 ([M + H]+) 458.2139, found 458.2172.
6-N-ter t-Bu tyla cetoxyla m in ou r id in e (12). A mixture of
11 (21.4 mg, 0.05 mmol) and 30% aqueous trifluoroacetic acid
(3 mL) was stirred at room temperature for 5 h. The reaction
mixture was concentrated under reduced pressure, and the
residue was purified by silica gel chromatography (CHCl3:
MeOH ) 10:1) to give 12 (11.8 mg, 68%) as a colorless oil: 1H
NMR (270 MHz, CD3OD) δ (major) 7.73 (s, 1H), 6.57 (s, 1H),
5.53 (5.59) (s, 1H), 4.33 (4.24) (s, 1H), 3.73-3.48 (m, 2H), 3.15-
3.13 (m, 2H), 1.94 (s, 3H), 1.14 (s, 9H), (minor) 7.73 (s, 1H),
6.57 (s, 1H), 5.59 (s, 1H), 4.24 (s, 1H), 3.73-3.48 (m, 2H), 3.15-
3.13 (m, 2H), 1.94 (s, 3H), 1.14 (s, 9H); HRMS (FAB) m/z calcd
for C13H22O7N3 ([M + H]+) 374.1563, found 374.1554.
6-N-ter t-Bu tylh yd r oxyla m in ou r id in e (13). A mixture of
12 (11.8 mg, 0.03 mmol) and a saturated methanolic ammonia
solution (2 mL) was stirred at room temperature for 30 min.
The reaction mixture was concentrated under reduced pres-
sure, and the residue was purified by silica gel chromatography
(CHCl3:MeOH ) 4:1) to give 13 (8.4 mg, 80%) as a colorless
oil: 1H NMR (270 MHz, DMSO-d6) δ 9.09 (s, 1H, D2O
exchanged), 6.18 (d, J ) 2.0 Hz, 1H), 5.51 (s, 1H), 4.99 (d, J )
7.3 Hz, 1H, D2O exchanged), 4.92 (d, J ) 5.0 Hz, 1H, D2O
exchanged), 4.63 (m, 1H, D2O exchanged), 4.36 (m, 1H), 4.18
(m, 1H), 3.58 (m, 2H), 1.15 (s, 9H), (D2O) δ 6.45 (d, J ) 3.1
Hz, 1H), 5.94 (s, 1H), 4.70 (dd, J ) 6.7, 3.1 Hz, 1H), 4.42 (t, J
) 6.7 Hz, 1H), 3.89-3.86 (m, 2H), 3.72 (dd, J ) 12.8, 7.0 Hz,
1H), 1.25 (s, 9H); HRMS (FAB): m/z calcd for C13H22O7N3 ([M
+ H]+) 332.1458, found 332.1459.
4-Eth oxy-1-(2′,3′,5′-tr is-O-ter t-bu tyld im eth ylsilyl-â-D-r i-
bofr an osyl)-2(1H)-pyr im idin e (14). To a solution of 4-ethoxy-
1-(â-D-ribofranosyl)pyrimidine15 (1.12 g, 4.10 mmol) in dry
DMF (22 mL) were added TBDMSCl (3.09 g, 20.5 mmol) and
imidazole (1.40 g, 20.5 mmol), and the mixture was stirred at
room temperature for 10 h. The reaction mixture was diluted
with H2O, and the organic layer was extracted with AcOEt,
dried over Na2SO4, and concentrated under reduced pressure.
The residue was purified by silica gel chromatography (hexane:
AcOEt ) 10:1) to give 14 (2.47 g, 98%) as a colorless solid: 1H
NMR (270 MHz, CDCl3) δ 8.35 (d, J ) 7.3 Hz, 1H), 5.72 (d, J
) 7.6 Hz, 1H), 5.68 (s, 1H), 4.40-4.35 (m, 2H), 4.10-3.98 (m,
4H), 3.74 (d, J ) 11.9 Hz, 1H), 1.31 (t, J ) 7.1 Hz, 3H), 0.91
(s, 3H), 0.87 (s, 9H), 0.84 (s, 9H), 0.09 (s, 3H), 0.07 (s, 3H),
0.04 (s, 3H), 0.03 (s, 3H), -0.04 (s, 3H), -0.06 (s, 3H); HRMS
(FAB) m/z calcd for C31H65O6N4Si3 ([M + H]+) 615.3681, found
615.3662.
4-Eth oxy-6-(N-ter t-bu tylh yd r oxyla m in o)-1-(2′,3′,5′-tr is-
O-ter t-bu tyld im eth ylsilyl)-2(1H)-p yr im id in e (15). To a
solution of LDA in THF (14 mL), prepared from n-BuLi (10.3
mL, 16.1 mmol) in hexane and diisopropylamine (2.26 mL, 16.1
mmol), was added a solution of 14 (2.47 g, 4.02 mmol) in 27
mL of THF at -78 °C under an Ar atmosphere, and the
mixture was stirred for 1 h at -70 °C. A solution of MNP (1.75
g, 20.1 mmol) in 18 mL of THF was added dropwise, and the
mixture was stirred for 3 h. A saturated NH4Cl solution was
added to the reaction mixture, and the organic layer was
extracted with AcOEt, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by silica gel
chromatography (AcOEt:hexane ) 1:30) to give 15 (1.49 g,
53%) as a colorless oil: 1H NMR (270 MHz, CDCl3) δ 6.39 (d,
J ) 6.9 Hz, 1H), 6.35 (br, 1H), 5.96 (s, 1H), 5.28 (m, 1H), 4.45-
4.35 (m, 2H), 4.29-4.26 (m, 1H), 4.00-3.90 (m, 2H), 3.89-
3.69 (m, 1H), 1.34 (t, J ) 7.3 Hz, 3H), 1.27 (s, 9H), 0.94 (s,
9H), 0.89 (s, 9H), 0.84 (s, 9H), 0.13 (s, 3H), 0.10 (s, 3H), 0.06
(s, 3H), 0.05 (s, 3H), 0.02 (s, 3H), -0.06 (s, 3H); HRMS (FAB)
m/z calcd for C33H68O7N3Si3 ([M + H]+) 702.4365, found
702.4338.
4-E t h oxy-6-(N-ter t-b u t ylh yd r oxyla m in e)-1-(â-D-r ib o-
fr a n osyl)p yr im id in e (16). To a solution of 15 (210 mg, 0.3
mmol) in 15 mL of dry MeOH was added ammonium fluoride
(110.9 mg, 3.0 mmol) under an Ar atmosphere, and the mixture
was heated at reflux for 30 min. After cooling, the reaction
mixture was concentrated under reduced pressure and the
residue was purified by silica gel chromatography (CHCl3:
MeOH ) 7:1) to give 16 (178 mg, 75%) as a colorless solid: 1H
NMR (270 MHz, CD3OD) δ 6.49 (d, J ) 2.6 Hz, 1H), 6.08 (s,
1H), 4.61 (dd, J ) 6.6, 3.0 Hz, 1H), 4.46 (t, J ) 6.4 Hz, 1H),
4.39 (q, J ) 6.9 Hz, 2H), 3.90-3.82 (m, 2H), 3.79-3.65 (m,
1H), 1.34 (t, J ) 6.9 Hz, 2H), 1.24 (s, 9H); HRMS (FAB) m/z
calcd for C15H26O7N3 ([M + H]+) 360.1771, found 360.1820.
6-(N -t er t -B u t y lh y d r o x y la m in o )-2′,3′,5′-t r is -O -t er t -
bu tyld im eth ylsilylcytid in e (17). Ammonia was bubbled into
a solution of 15 (168 mg, 0.24 mmol) in 4 mL of MeOH at 0 °C
for 15 min, and the mixture was heated at 100 °C in a sealed
tube. After 48 h, the mixture was cooled to room temperature
and concentrated under reduced pressure. The residue was

3520 J . Org. Chem., Vol. 66, No. 10, 2001
Aso et al.
purified by silica gel chromatography (AcOEt) to give 17 (10
mg, 6% isolated yield; 33% conversion yield) as a colorless
solid: 1H NMR (270 MHz, CDCl3) δ 6.51 (br, 1H), 6.37 (d, J )
6.9 Hz, 1H), 5.80 (s, 1H), 5.32-5.26 (m, 2H), 4.25 (d, J ) 3.3
Hz, 1H), 3.98-3.86 (m, 2H), 3.74-3.72 (m, 1H), 1.26 (s, 9H),
0.93 (s, 9H), 0.89 (s, 9H), 0.85 (s, 9H), 0.13 (s, 3H), 0.10 (s,
3H), 0.08 (s, 3H), 0.05 (s, 3H), 0.03 (s, 3H), -0.04 (s, 3H);
HRMS (FAB) m/z calcd for C31H65O6N4Si3 ([M + H]+) 673.4242,
found 673.4219.
of 2112c (433 mg, 0.74 mmol) in 3 mL of THF was added
dropwise at -70 °C. After 2 h, a solution of MNP (385 mg,
4.43 mmol) in 2 mL of THF was added to the reaction mixture
and stirred for 2.5 h. The reaction was quenched by addition
of a saturated aqueous NH4Cl solution, and the organic layer
was extracted with AcOEt, dried over Na2SO4, and concen-
trated under reduced pressure. The residue was purified by
silica gel chromatography (AcOEt:hexane ) 1:1) to give 22 (95
mg, 19% yield, 45% isolated yield) as a colorless oil: 1H NMR
(270 MHz, CDCl3) δ 8.95 (brs, 1H, D2O exchanged), 7.61 (s,
1H), 7.02 (br, 1H, D2O exchanged), 5.97 (d, J ) 7.3 Hz, 1H),
4.17-4.12 (m, 1H), 4.06-4.01 (m, 2H), 3.80 (dd, J ) 8.6, 3.0
Hz, 1H), 3.73 (dd, J ) 8.6, 3.0 Hz, 1H), 1.18 (s, 9H), 0.97 (s,
9H), 0.93 (s, 9H), 0.89 (s, 9H), 0.15 (s, 3H), 0.12 (s, 3H), 0.09
(s, 3H), 0.06 (s, 3H), 0.02 (s, 3H), -0.02 (s, 3H); HRMS (FAB)
m/z calcd for C31H63O7N3Na ([M + Na]+) 696.3872, found
696.3892.
N-Ben zoyl-2′,3′,5′-t r is-O-ter t-b u t yld im et h ylsilylcyt i-
d in e (18). Compound 18 was prepared according to Ogilvie’s
procedure.16 1H NMR (270 MHz, CDCl3) δ 8.71 (br, 1H), 8.60
(d, J ) 7.26 Hz, 1H), 7.91 (d, J ) 7.26 Hz, 2H), 7.47-7.63 (m,
4H), 4.04-4.17 (m, 4H), 3.82 (d, J ) 10.89 Hz, 1H), 0.99 (s,
9H), 0.92 (s, 9H), 0.90 (s, 9H), 0.23 (s, 3H), 0.17 (s, 3H), 0.15
(s, 3H), 0.12 (s, 3H), 0.09 (s, 3H), 0.04 (s, 3H); HRMS(FAB)
m/z calcd for C34H60O6N3Si3([M + H]+) 690.3790, found 690.3792.
6-(N-ter t-Bu tylh yd r oxyla m in o)-N-ben zoyl-2′,3′,5′-tr is-
O-ter t-bu tyld im eth ylsilylcytid in e (19). To a solution of
LDA in THF (5.6 mL), prepared from n-BuLi (hexane solution;
6.2 mL, 9.68 mmol) and diisopropylamine (1.36 mL, 9.68
mmol), was added a solution of 18 (530 mg, 1.61 mmol) in 11
mL of THF at -78 °C under an Ar atmosphere, and the
mixture was stirred for 1 h at -70 °C. A solution of MNP (842
mg, 9.68 mmol) in 8 mL of THF was added dropwise, and the
mixture was stirred for 3 h. A saturated NH4Cl solution was
added to the reaction mixture, and the organic layer was
extracted with AcOEt, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by silica gel
chromatography (AcOEt:hexane ) 1:2) to give 15 (263 mg,
48%) as a colorless solid: 1H NMR (270 MHz, CDCl3) δ 7.92-
7.89 (m, 2H),7.69-7.48 (m, 4H), 6.38 (m, 2H), 5.26 (m, 1H),
4.32 (m, 1H), 3.94 (m, 2H), 3.73 (m, 1H), 1.37 (s, 9H), 0.94 (s,
9H). 0.90 (s, 9H), 0.86 (s, 9H), 0.14 (s, 3H), 0.11 (s, 3H), 0.08
(s, 3H), 0.07 (s, 3H), 0.04 (s, 3H), -0.05 (s, 3H); HRMS(FAB)
m/z calcd for C38H69N4O7Si3 ([M + H]+).
17 fr om 19. Ammonia was bubbled into a solution of 19
(207 mg, 0.26 mmol) in 19 mL of MeOH at 0 °C for 15 min,
and the mixture was heated at 65 °C in a sealed tube. After 6
h, the mixture was cooled to room temperature and concen-
trated under reduced pressure. The residue was purified by
silica gel chromatography (AcOEt) to give 17 (175 mg, 98%).
6-(N-ter t-Bu tylh yd r oxyla m in o)cytid in e (20). To a solu-
tion of 17 (78.3 mg, 0.12 mmol) in dry MeOH (1.9 mL) was
added ammonium fluoride (15 mg, 0.4 mmol), and the mixture
was heated at reflux for 3.5 h. After cooling, the reaction
mixture was concentrated under reduced pressure and the
residue was purified by silica gel chromatography (CHCl3:
MeOH ) 4:1) to give 20 (20 mg, 52%) as a colorless solid: 1H
NMR (270 MHz, DMSO-d6) δ 8.97 (s, 1H, D2O exchanged),
7.27-7.16 (m, 2H, D2O exchanged), 6.23 (d, J ) 2.0 Hz, 1H),
5.79 (s, 1H), 4.37 (m, 1H), 4.30 (m, 1H), 3.61-3.57 (m, 2H,
D2O exchanged), 3.38-3.34 (m, 4H, 1H was exchanged with
D2O), 1.13 (s, 9H), (D2O) δ 6.50 (d, J ) 3.1 Hz, 1H), 6.10 (s,
1H), 4.68 (dd, J ) 6.7, 3.1 Hz, 1H), 4.43 (t, J ) 6.7 Hz, 1H),
3.93-3.84 (m, 2H), 3.72 (dd, J ) 12.2, 6.1 Hz, 1H), 1.22 (s,
9H); HRMS (FAB) m/z calcd for C13H23N4O6 ([M + H]+)
332.1458, found 332.1459.
5-N-ter t-Bu tylh yd r oxyla m in ou r id in e (23). To a solution
of 22 (79 mg, 0.12 mmol) in 5 mL of THF was added TBAF (1
M solution in THF; 0.41 mL, 0.41 mmol), and the mixture was
stirred at room temperature for 30 min. The reaction mixture
was concentrated under reduced pressure, and the residue was
purified by silica gel chromatography (CHCl3:MeOH ) 7:1) to
give 23 (35 mg, 90%) as a colorless oil: 1H NMR (270 MHz,
DMSO-d6) δ 8.27 (s, 1H, D2O exchanged), 7.90 (s, 1H), 5.81
(d, J ) 5.9 Hz, 1H), 5.37 (d, J ) 5.9 Hz, 1H, D2O exchanged),
5.09 (d, J ) 4.6 Hz, 1H, D2O exchanged), 5.03 (t, J ) 4.5 Hz,
1H, D2O exchanged), 4.01 (m, 1H), 3.95 (m, 1H), 3.86 (m, 1H),
3.51 (m, 2H), 1.03 (s, 9H), (D2O) δ 8.06 (s, 1H), 5.95 (d, J )
4.3 Hz, 1H), 4.32 (t, J ) 4.3 Hz, 1H), 4.22 (t, J ) 5.5 Hz, 1H),
4.15 (m, 1H), 3.92 (dd, J ) 12.8, 2.4 Hz, 1H), 3.80 (dd, J )
12.8, 3.4 Hz, 1H), 1.12 (s, 9H); HRMS(FAB) m/z calcd for
C
13H22O7N3 [M + H]+) 332.1458, found 332.1463.
EP R Stu d y of Am in oxyl Ra d ica ls. To toluene or CH2Cl2
containing a 1% MeOH solution of the appropriate hydroxyl-
amine was added Ag2O, and the mixture was left at room
temperature. After 1 h, the supernatant liquid was taken from
the reaction mixture and filtered. The solution containing the
aminoxyl radical was placed in a 5 mm o.d. quartz tubes,
degassed by three freeze-and-thaw cycles, and sealed. EPR
spectra were taken at room temperature.
Sim u la tion of EP R F in e Str u ctu r e. The simulation of
the EPR fine structure was performed by using Winsim
program from P.E.S.T., Public EPR Software Tools, provided
by National Institute of Environmental Health Science, Na-
tional Institutes of Health. The line width of 0.5 G was used
for simulation.
Ack n ow led gm en t. We are grateful to Drs. Ryuichi
Isobe and Keisuke Mimura (Tohwa University) for
HRMS measurement. This work is supported in part
by the Hayashi Memorial Foundation for Female Natu-
ral Scientists.
Su p p or tin g In for m a tion Ava ila ble: EPR spectra and
simulated spectra of 1c, 1d , and 8a -d . 1H NMR charts of 6a -
d , 7a -d , 10, 11, 12, 13, 15, 16, 17, 19, 20, 22, and 23. X-ray
crystallographic data for 10. This material is available free of
charge via the Internet at http://pubs.acs.org.
5-(N -t er t -B u t y lh y d r o x y la m in o )-2′,3′,5′-t r is -O -t er t -
bu tyld im eth ylsilylu r id in e (22). A mixture of sec-BuLi in
THF (1.8 mL, 1.85 mmol) and TMEDA (0.28 mL, 1.85 mmol)
in 6 mL of THF was stirred at -78 °C for 30 min. A solution
J O015532S