2'-C-branched ribonucleosides: Synthesis of the phosphoramidite derivatives of 2'-C-β-methylcytidine and their incorporation into oligonucleotides

Article abstract of DOI:10.1021/jo981329u

We describe a strategy for the incorporation of a 2'-C-branched ribonucleoside, 2'-C-β-methylcytidine, into oligonucleotides via solid- phase synthesis using phosphoramidite derivatives. 4-N-Benzoyl-2'-C-β- methylcytidine (2b) was synthesized by coupling persilylated 4-N- benzoylcytosine with 1,2,3,5-tetra-O-benzoyl-2-C-β-methyl-α-(and β)-D- ribofuranose (1) in the presence of SnCl₄ in acetonitrile, followed by selective deprotection with NaOH in pyridine/methanol. The 3'- and 5'- hydroxyl groups were blocked as a cyclic di-tert-butylsilanediyl ether 3 by treatment with di-tert-butyldichlorosilane/AgNO₃ in DMF. The 2'-hydroxyl group was then protected as a tert-butyldimethylsilyl ether 4a by treatment with tert-butylmagnesium chloride followed by addition of tert- butyldimethylsilyl trifluoromethanesulfonate in THF. As an alternative to 2'- silyl protection, the corresponding 2'O-tetrahydropyranyl ether 4b was prepared by treatment of 3 with 4,5-dihydro-2H-pyran in the presence of a catalytic amount of 10-camphorsulfonic acid in methylene chloride. The di- tert-butylsilanediyl groups of 4a and 4b were removed by treatment with pyridinium poly(hydrogen fluoride) to afford 5a and 5b, respectively. Protection of the 5'-hydroxyl group as a dimethoxytrityl ether and phosphitylation of the 3'-hydroxyl group by the standard procedure gave the phosphoramidite derivatives 7a and 7b. Both 7a and 7b could be used to incorporate 2'-C-β-methylcytidine into oligonucleotides efficiently via standard solid-phase synthesis, but the tetrahydropyranyl group of 7b was more readily removed from oligonucleotides than the tert-butyldimethylsilyl group of 7a. Oligonucleotides containing 2'-C-β-methylcytidine undergo base- catalyzed degradation analogous to natural RNA.

Full text of DOI:10.1021/jo981329u

J . Org. Chem. 1999, 64, 747-754
747
2′-C-Br a n ch ed Ribon u cleosid es: Syn th esis of th e P h osp h or a m id ite
Der iva tives of 2′-C-â-Meth ylcytid in e a n d Th eir In cor p or a tion in to
Oligon u cleotid es
Xiao-Qing Tang, Xiangmin Liao, and J oseph A. Piccirilli*
Howard Hughes Medical Institute, Departments of Biochemistry & Molecular Biology and Chemistry,
The University of Chicago, 5841 South Maryland Avenue, MC 1028, Chicago, Illinois 60637
Received J uly 8, 1998
We describe a strategy for the incorporation of a 2′-C-branched ribonucleoside, 2′-C-â-methylcytidine,
into oligonucleotides via solid-phase synthesis using phosphoramidite derivatives. 4-N-Benzoyl-
2′-C-â-methylcytidine (2b) was synthesized by coupling persilylated 4-N-benzoylcytosine with 1,2,3,5-
tetra-O-benzoyl-2-C-â-methyl-R-(and â)-D-ribofuranose (1) in the presence of SnCl4 in acetonitrile,
followed by selective deprotection with NaOH in pyridine/methanol. The 3′- and 5′-hydroxyl groups
were blocked as a cyclic di-tert-butylsilanediyl ether 3 by treatment with di-tert-butyldichlorosilane/
AgNO3 in DMF. The 2′-hydroxyl group was then protected as a tert-butyldimethylsilyl ether 4a by
treatment with tert-butylmagnesium chloride followed by addition of tert-butyldimethylsilyl
trifluoromethanesulfonate in THF. As an alternative to 2′-silyl protection, the corresponding 2′-
O-tetrahydropyranyl ether 4b was prepared by treatment of 3 with 4,5-dihydro-2H-pyran in the
presence of a catalytic amount of 10-camphorsulfonic acid in methylene chloride. The di-tert-
butylsilanediyl groups of 4a and 4b were removed by treatment with pyridinium poly(hydrogen
fluoride) to afford 5a and 5b, respectively. Protection of the 5′-hydroxyl group as a dimethoxytrityl
ether and phosphitylation of the 3′-hydroxyl group by the standard procedure gave the phosphoram-
idite derivatives 7a and 7b. Both 7a and 7b could be used to incorporate 2′-C-â-methylcytidine
into oligonucleotides efficiently via standard solid-phase synthesis, but the tetrahydropyranyl group
of 7b was more readily removed from oligonucleotides than the tert-butyldimethylsilyl group of
7a . Oligonucleotides containing 2′-C-â-methylcytidine undergo base-catalyzed degradation analogous
to natural RNA.
Ch a r t 1
In tr od u ction
Nucleoside analogues that contain a 2′-C-branched
ribose sugar have been synthetic targets1 over the past
decade because of their potential as chemotherapeutic
agents.1f-l,2 For example, 2′-deoxy-2′-â-C-methylcytidine
exhibits potent cytotoxicity toward several leukemic cell
lines.1h,i,2a Such analogues might also be used to regulate
gene expression by antisense or ribozyme cleavage ap-
proaches because modification of the 2′-position increases
the nuclease resistance of oligonucleotides,3 thereby
increasing their stability in biological fluids. For ribozyme
therapeutics, 2′-C-branched ribonucleosides are of par-
ticular interest because specific 2′-hydroxyl groups in
ribozymes are essential for catalytic activity.4
Some attention has been given to 2′-C-branched ribo-
nucleosides as biochemical probes.3a,5 For example, phos-
phorylated forms of 2′-C-methyluridine have been studied
as substrates for ribonucleotide reductase,5c RNA poly-
merase,6 and nucleases.3a 2′-Branched ribonucleosides
also have potential as tools for exploring the structure
and function of RNA and ribozymes. Our interest arises
from the possibility that a series of nucleoside analogues
(Chart 1) in which the 2′-alkyl substituent is methyl,
monofluoromethyl, difluoromethyl, or trifluromethyl might
allow systematic variation of the pKa of the 2′-hydroxyl
group within the same structural context. For those
enzymes and ribozymes that catalyze RNA strand scis-
sion by activating the 2′-hydroxyl group for attack at the
adjacent 3′-phosphate diester (such as ribonuclease A7
or the hammerhead and hairpin ribozymes8), this series
of nucleosides could reveal a linear free energy relation-
(1) (a) Walton, E.; J enkins, S. R.; Nutt, R. F.; Zimmerman, M.; Holly,
F. W. J . Am. Chem. Soc. 1966, 88, 4524. (b) J enkins, S. R.; Arison, B.;
Walton, E. J . Org. Chem. 1968, 33, 2490. (c) Walton, E.; J enkins, S.
R.; Nutt, R. F.; Holly, F. W. J . Am. Chem. Soc. 1969, 91, 306. (d)
Walton, E.; J enkins, S. R.; Nutt, R. F.; Holly, F. W.; Nemes, M. J . Med.
Chem. 1969, 12, 306. (e) Beigelman, L. N.; Ermolinski, B. S.; Gurskaya,
G. V.; Tsapkina, E. N.; Karpeisky, M. Y.; Mikhailov, S. N. Carbohyd.
Res. 1987, 166, 219. (f) Matsuda, A.; Takenuki, K.; Itoh, H.; Sasaki,
T.; Ueda, T. Chem. Pharm. Bull. 1987, 35, 3967. (g) Matsuda, A.; Itoh,
H.; Takenuki, K.; Sasaki, T.; Ueda, T. Chem. Pharm. Bull. 1988, 36,
945. (h) Ueda, T.; Matsuda, A.; Yoshimura, Y.; Yakenuki, K. Nucleo-
sides Nucleotides 1989, 8, 743. (i) Matsuda, A.; Takenuki, K.; Sasaki,
T.; Ueda, T. J . Med. Chem. 1991, 34, 234. (j) Matsuda, A.; Nakajima,
Y.; Azuma, A.; Tanaka, M.; Sasaki, T. J . Med. Chem. 1991, 34, 2937.
(k) Yoshimura, Y.; Saitoh, K.; Ashida, N.; Sakata, S.; Mutsuda, A.
Bioorg. Med. Chem. Lett. 1994, 4, 721. (l) Yoshimura, Y.; Satoh, H.;
Sakata, S.; Ashida, N.; Miyazaki, S.; Matsuda, A. Nucleosides Nucle-
otides 1995, 14, 427. (m) Yoshimura, Y.; Iino, T.; Mutsuda, A.
Tetrahedron Lett. 1991, 32, 6003. (n) Wolf, J .; J arrrige, J . M.; Florent,
J . C.; Grierson, D. S.; Monneret, C. Synthesis 1992, 773. (o) Iino, T.;
Yoshimura, Y.; Mutsuda, A. Tetrahedron 1994, 50, 10397. (p) Wolfe,
M. S.; Harry-O’Kuru, R. E. Tetrahedron Lett. 1995, 36, 7611. (q) Harry-
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* To whom correspondence should be addressed. Tel: (773) 702-
9312. Fax: (773) 702-0271. E-mail: jpicciri@midway.uchicago.edu.
10.1021/jo981329u CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/09/1999

748 J . Org. Chem., Vol. 64, No. 3, 1999
Tang et al.
Sch em e 1a
a
Conditions: (a) persilylated 4-N-benzoylcytosine, SnCl4; (b) NaOH; (c) t-Bu2SiCl2/AgNO3/Et3N; (d) for 4a , (i) t-BuMgCl; (ii) t-BuMe2SiOTf;
for 4b, 4,5-dihydro-2H-pyran/10-camphorsulfonic acid, CH2Cl2; (e) HF-pyridine; (f) for 6a , DMTrCl/AgNO3/pyridine; for 6b, DMTrCl; (g)
i-Pr2NP(OCH2CH2CN)Cl/1-methylimidazole/i-Pr2NEt.
ship between the catalytic rate and the pKa of the
nucleophile, thereby providing information about the
degree of bond-making between the phosphorus and the
2′-OH nucleophile in the transition state.
To employ these nucleoside analogues as ribozyme
therapeutics, antisense drugs, or probes for ribozyme
mechanism, we must incorporate them into DNA and
RNA oligonucleotides. Schmit et al. derivatized 2′-deoxy-
2′-C-branched nucleosides as phosphoramidites and in-
corporated them into DNA by solid-phase synthesis,9 but
there is no method reported for the site specific incorpo-
ration of the corresponding ribonucleosides (such as those
shown in Chart 1) into oligonucleotides. For phosphoram-
idite derivatives of the natural nucleosides, the 2′-
hydroxyl group is commonly protected as a tert-butyldi-
methylsilyl (TBDMS) ether.10 Because the tertiary 2′-
hydroxyl group of 2′-C-branched ribonucleosides is hin-
dered and therefore more difficult to protect selectively,
obtaining suitable phosphoramidite derivatives is more
challenging. Herein, we report a solution to this problem
and strategies for the incorporation of 2′-C-â-methyl-
cytidine into oligonucleotides via phosphoramidite chem-
istry. We also show that, analogous to natural RNA,
oligonucleotides containing 2′-C-â-methylcytidine de-
grade under basic conditions by a reaction in which the
tertiary 2′-hydroxyl group attacks the adjacent 3′-phos-
phodiester.
(2) (a) Takenuki, K.; Matsuda, A.; Ueda, T.; Sasaki, T.; Fujii, A.;
Yamagami, K. J . Med. Chem. 1988, 31, 1063. (b) Matsuda, A.;
Takenuki, K.; Tanaka, M.; Sasaki, T.; Ueda, T. J . Med. Chem. 1991,
34, 812. (c) Yamagami, K.; Fujii, A.; Arita, M.; Okumoto, T.; Sakata,
S.; Matsuda, A.; Ueda, T. Cancer Res. 1991, 51, 2319. (d) Awano, H.;
Shuto, S.; Baba, M.; Kira, T.; Shigeta, S.; Mastuda, A. Bioorg. Med.
Chem. Lett. 1994, 4, 367. (e) Cory, A. H.; Samano, V.; Robins, M. J .;
Cory, J . P. Biochem. Pharmacol. 1994, 47, 365.
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Padyukova, N, Sh.; Yakovlev, G. I.; Karpeisky, M. Ya. Carbohydr. Res.
1983, 124, 75. (b) Beigelman, L.; Karpeisky, A.; Matulic-Adamic, J .;
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Yang J . H.; Perreault, J . P.; Labuda, D.; Usman, N.; Cedergren, R.
Biochemistry 1990, 29, 11156. (c) Olsen, D. B.; Benseler, F.; Aurup,
H.; Pieken, W. A.; Eckstein, F. Biochemistry 1991, 30, 9735. (d)
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M.; Berzal-Herranz, A.; Keller, C. F.; Burke, J . M. J . Biol. Chem. 1993,
268, 19458. (h) Strobel, S. A.; Cech, T. R. Science 1995, 267, 675. (i)
Narlikar G. J .; Khosla M.; Usman, N.; Herschlag D. Biochemistry 1997,
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(5) (a) Baker, C. H.; Banzon, J .; Bollinger, J . M.; Stubbe, J .; Samano,
V.; Robins, M. J .; Lippert, B.; J arvi, E.; Resvick, R. J . Med. Chem. 1991,
34, 1879. (b) Mikhailov, S. N.; Oivanen, M.; Oksman, P.; Lonnberg, H.
J . Org. Chem. 1992, 57, 4122. (c) Ong, S. P.; McFarlan, S. C.;
Hogenkamp, H. P. C. Biochemistry 1993, 32, 11397. (d) van der Donk,
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8381.
Resu lts a n d Discu ssion
We prepared 1,2,3,5-tetra-O-benzoyl-2-C-â-methyl-R-
(and â)-D-ribofuranose (1) (Scheme 1) according to the
literature1p-q from commercially available R-D-ribofura-
(6) (a) Savochkina, L. P.; Sviriyeva, T. V.; Beigelman, L. N.;
Padyukova, N. Sh.; Kuznetsov, D. A.; Lysov, Yu. P.; Mikhailov, S. N.;
Beabealashvilli, R. Sh. Mol. Biol. (USSR) 1989, 23, 1700. (b) Mikhailov,
S. N.; Padyukova, N. Sh.; Lysov, Yu. P.; Savochkina, L. P.; Chidgea-
vadze, Z. G.; Beabealashvilli, R. Sh. Nucleosides Nucleotides 1991, 10,
339.
(7) Gerlt, J . A. In Nucleases; Linn, S. M., Lloyd, R. S., Roberts, R.
J ., Eds; Cold Spring Harbor Laboratory Press: Plainview, NY, 1993;
pp 7-8, 16-18.
(8) Been, M. D. In Nucleases; Linn S. M., Lloyd, R. S., Roberts, R.
J . Eds; Cold Spring Harbor Laboratory Press: Plainview, NY, 1993;
pp 425-428.
(9) (a) Schmit, C.; Bevierre, M. O.; MesMaeker, A. D.; Altman, K.
H. Bioorg. Med. Chem. Lett. 1994, 4, 1969. (b) Schmit, C. Synlett 1994,
238, 241.
(10) (a) Ogilvie, K. K.; Beaucage, S. L.; Schifman, A. L.; Theriault,
N. Y.; Sadana, K. L. Can. J . Chem. 1978, 56, 2768. (b) Ogilvie, K. K.;
Schifman, A. L.; Penney, C. L. Can. J . Chem. 1979, 57, 2230. (c)
Hakimelahi, G. H.; Proba, Z. A.; Ogilvie, K. K. Tetrahedron Lett. 1981,
22, 4775. (d) Hakimelahi, G. H.; Proba, Z. A.; Ogilvie, K. K. Can. J .
Chem. 1982, 60, 1106. (e) Usman, N.; Pon, R. T.; Ogilvie, K. K.
Tetrahedron Lett. 1985, 26, 4567. (f) Usman, N.; Ogilvie, K. K.; J iang,
M. Y.; Cedergren, R. J . J . Am. Chem. Soc. 1987, 109, 7845.

2′-C-Branched Ribonucleosides
J . Org. Chem., Vol. 64, No. 3, 1999 749
nose 1,3,5-tribenzoate. Even with the steric influence of
the â-C2 methyl group, the reaction of 1 with persilylated
nucleobases under Vorbruggen conditions still affords
â-nucleosides exclusively.1p-q The reaction of 1 with
DMSCl/AgNO3/pyridine10c,d in THF, the starting material
was recovered unchanged. Consequently, we tested vari-
ous bases as possible activators of the 2′-hydroxyl group.
It was recently reported that t-BuMgCl could be used for
oxygen-selective phosphorylation20 and alkylation21 of
nucleoside derivatives and that potassium hydride (KH)/
18-crown-6 could be used for the successful protection of
highly hindered hydroxyl groups with TBDMSCl or
TBDMSOTf.22 We found that both t-BuMgCl and KH
were effective for the silylation of the 2′-hydroxyl group
of 3, but t-BuMgCl afforded a higher yield of the desired
2′-O-TBDMS product 4a than KH did. Compound 3 was
treated with t-BuMgCl for 0.5 h followed by overnight
incubation with TBDMSOTf in THF. After workup,
purification by silica gel column chromatography afforded
4a in 64% yield. Selective removal of the di-tert-butylsil-
anediyl group from 4a with PPHF in THF at 0 °C for 5
min gave 5a in 82% yield. During protection of the 5′-
hydroxyl group of 5a by overnight treatment with
dimethoxytrityl chloride (DMTrCl) in pyridine,23 some
migration of the TBDMS group from the 2′-hydroxyl to
the 3′-hydroxyl occurred. To circumvent this problem, we
used AgNO3 to accelerate the tritylation reaction.10d One
hour in the presence of DMTrCl and AgNO3 was suf-
ficient to convert 5a to 6a . Under these conditions, no
migration of the 2′-O-TBDMS group to the 3′-oxygen was
observed.
11
persilylated 4-N-benzoylcytosine in the presence of SnCl4
in acetonitrile at room temperature afforded 2′,3′,5′-tri-
O-benzoyl-4-N-benzoyl-2′-C-â-methylcytidine 2a in 86%
yield; reactions carried out at elevated temperature (83
°C) gave much lower yields. Selective deprotection of 2a
with NaOH in pyridine and methanol gave 4-N-benzoyl-
2′-C-â-methylcytidine 2b in 94% yield. Sodium methoxide
or ammonia in methanol removed all four benzoyl groups
from 2a .
Standard procedures for preparation of nucleoside
synthons used in solid-phase synthesis of oligonucleotides
include protection of the amino groups on the nucleobases
as amides, protection of the 5′- and 2′-hydroxyl groups
as dimethoxytrityl (DMTr) and TBDMS ethers, respec-
tively, and phosphitylation of the 3′-oxygen to the â-cya-
noethyl N,N-diisopropylphosphoramidite.10 Although pro-
tection of the 5′-hydroxyl group of 2b as a DMTr ether
proceeded well, attempts to protect the 2′-hydroxyl group
with tert-butyldimethylsilyl chloride (TBDMSCl) either
in the presence of imidazole in pyridine or AgNO3 in THF
gave exclusively the 3′-O-TBDMS ether.12 We tried to
obtain the 2′-isomer by exposing the 3′-isomer to isomer-
ization conditions,10a but treatment with imidazole in
DMF at room temperature for 1 week afforded only 8%
of the 2′-isomer.14 These results suggested that if a
TBDMS group was to be regioselectively installed at the
tertiary 2′-hydroxyl group of 2′-C-â-methylcytidine, the
secondary 3′-hydroxyl group would have to be protected
first.
Di-tert-butyldichlorosilane appeared to be a useful
reagent for this purpose because it simultaneously pro-
tects the 3′- and 5′-hydroxyl groups, and the resulting
cyclic di-tert-butylsilanediyl ether can be cleanly and
chemoselectively removed within minutes by exposure to
pyridinium poly(hydrogen fluoride) (PPHF)16 or tributyl-
ammonium fluoride17 without affecting a TBDMS ether
moiety. Thus, treatment of 2b with di-tert-butyldichlo-
rosilane in the presence of AgNO318 in DMF provided the
corresponding 3′,5′-O-(di-tert-butylsilanediyl) ether 3 in
95% yield. However, our initial attempts to protect the
tertiary 2′-hydroxyl group of 3 as a TBDMS ether were
unsuccessful. Even after heating 3 in the presence of
either tert-butyldimethylsilyl trifluoromethylsulfonate
(TBDMSOTf)/2,6-lutidine in methylene chloride19 or TB-
We found that this procedure for silylation of the
tertiary 2′-hydroxyl group of 2′-C-methylcytidine using
t-BuMgCl/TBDMSOTf was not effective for silylation of
the 2′-hydroxyl group of 2′-C-â-trifluoromethyl nucleoside
derivatives (Chart 1) because they decomposed under the
reaction conditions.24 We later found that the removal of
the TBDMS group from oligonucleotides occurs much
more slowly from 2′-C-â-methylcytidine residues than
from cytidine residues (see below). These observations
led us to examine whether other 2′-hydroxyl protecting
groups could be used for the solid-phase synthesis of
oligonucleotides containing 2′-C-â-methylcytidine. The
1-(2-fluorophenyl)-4-methoxypiperidin-4-yl group25 seemed
to be a good alternative for two reasons: it can be
introduced with mild acid, conditions under which 2′-C-
â-trifluoromethyl nucleosides should be stable, and nu-
cleoside phosphoramidites that possess this 2′-protecting
group have recently become commercially available.
Unfortunately, the reaction of 3 with 1-(2-fluorophenyl)-
4-methoxy-1,2,5,6-tetrahydropyridine under various con-
ditions failed to give any desired product.
We next examined the THP group for the protection
of the 2′-hydroxyl of 2′-C-â-methylcytidine. This group
has been used successfully for solid-phase synthesis of
oligoribonucleotides via the phosphoramidite approach,26
and the conditions necessary for its removal are relatively
mild (0.01 N HCl, pH 2; ca. 3-4 h at 20 °C) and do not
promote significant migration of the 3′-phosphoryl group
to the adjacent 2′-oxygen.27 Reaction of 3 with excess 4,5-
(11) (a) Niedballa, U.; Vorbruggen, H. J . Org. Chem. 1974, 39, 3654,
3664, 3668, 3672. (b) Niedballa, U.; Vorbruggen, H. J . Org. Chem. 1976,
41, 2084. (c) Vorbruggen, H.; Hofle, G. Chem. Ber. 1981, 114, 1256.
(12) Tang, X. Q.; Piccirilli, J . A. Unpublished results. Similarly,
Mikhailov et al. reported that treatment of 5′-O-benzyl-3′-C-methyl-
uridine with excess acetic anhydride or tosyl chloride yielded only the
2′-O derivative.13
(13) Mikhailov, S. N.; Fomitcheva, M. V. Bioorg. Khim. 1990, 16,
192.
(14) Analogously, Reese and Trentham reported that the benzoyl
group of 2′-O-benzoyl-3′-C-methyluridine showed no evidence of isomer-
ization to the neighboring oxygen after storage in pyridine at room
temperature for a half-year, whereas those of 2′- and 3′-O-benzoyl-
uridine do isomerize under these conditions.15
(20) Uchiyama, M.; Aso, Y.; Noyori, R.; Hayakawa, Y. J . Org. Chem.
1993, 58, 373.
(21) Wada, T.; Tobe, M.; Nagayama, T.; Furusawa, K.; Sekine, M.
Tetrahedron Lett. 1995, 36, 1683.
(22) Braish, T. F.; Fuches, P. L. Synth. Commun. 1986, 16, 111.
(23) (a) Shaller, H.; Weimann, G.; Lerch, B.; Khorana, H. G. J . Am.
Chem. Soc. 1963, 85, 3821. (b) Agarwal, K. L.; Yamazaki, A.; Cashion,
P. J .; Khorana, H. G. Angew. Chem., Int. Ed. Engl. 1972, 11, 451.
(24) Tang, X. Q.; Piccirilli, J . A. Unpublished results.
(25) Rao, M. V.; Reese, C. B.; Schehlmann, V.; Yu, P. S. J . Chem.
Soc., Perkin Trans. 1 1993, 43 and references therein.
(15) Reese, C. B.; Trentham, D. R. Tetrahedron Lett. 1965, 2467.
(16) (a) Trost, B. M.; Caldwell, C. G. Tetrahedron Lett. 1981, 22,
4999. (b) Sekine, M.; Limura, S.; Furusawa, K. J . Org. Chem. 1993,
58, 3204.
(17) Furusawa, K. Chem. Lett. 1989, 509.
(18) Furusawa, K.; Ueno, K.; Katsura, T. Chem. Lett. 1990, 97.
(19) Corey, E. J .; Cho, H.; Rucker, C.; Hua, D. H. Tetrahedron Lett.
1981, 22, 3455.

750 J . Org. Chem., Vol. 64, No. 3, 1999
Tang et al.
Sch em e 2a
a
Conditions: (a) [γ-32P]-ATP, T4 polynucleotide kinase; *P indicates 32P; (b) TBAF or 0.01 N HCl (pH 2.0); (c) 0.1 N NaOH.
F igu r e 1. Deprotection of 5′-radiolabeled oligonucleotides with TBAF. TBAF/THF (25 °C) removes the 2′-O-TBDMS group from
*pdUC2′OTBDMSU (9c) (lanes 1-5) much more rapidly than from *pdUC2′Me/OTBDMSU (9a ) (lanes 6-10).28
dihydro-2H-pyran in the presence of a catalytic amount
of 10-camphorsulfonic acid in methylene chloride gave
4b in 95% yield (p-toluenesulfonic acid as catalyst gave
4b in 50% yield) as a mixture of two diastereomers, which
could not be separated by silica gel chromatography.
However, after desilyation of 4b by treatment with
PPHF, the two diastereomers of 5b were distinguishable
by TLC and were separated by silica gel chromatography
to afford equal quantities of each diastereomer in a
combined yield of 92%. This strategy for 2′-protection may
also be effective for the 2′-(monofluoromethyl)- and the
2′-(trifluoromethyl)ribonucleosides shown in Chart 1.24
showed four peaks at 152.9, 152.2, 150.3, and 150.0 ppm
due to the presence of the chiral THP group.
To determine the suitability of both the 2′-O-TBDMS
(7a ) and 2′-O-THP (7b) ribonucleoside phosphoramidites
as building blocks for the incorporation of 2′-C-â-meth-
ylcytidine into oligonucleotides, we synthesized three
trinucleotides (Scheme 2), 2′-deoxyuridylyl(3′-5′)2′-C-â-
methyl-2′-O-(tert-butyldimethylsilyl)cytidylyl(3′-5′)uri-
dine (dUC2′Me/OTBDMSU, 8a ), 2′-deoxyuridylyl(3′-5′)2′-C-
â-methyl-2′-O-(tetrahydopyranyl)cytidylyl(3′-5′)uridine
(dUC2′Me/OTHPU, 8b), and the control trinucleotide 2′-
deoxyuridylyl(3′-5′)2′-O-(tert-butyldimethylsilyl)cyti-
dylyl(3′-5′)uridine (dUC2′OTBDMSU, 8c). During solid-
phase synthesis using standard protocols, 7a and 7b
coupled as efficiently as commercial phosphoramidites,
according to the release of the DMTr cation. The tri-
nucleotides 8a -c were 5′-32P-labeled with [γ-32P]-ATP
and polynucleotide kinase (PNK) and purified by poly-
acrylamide gel electrophoresis (PAGE) to give the corre-
Overnight treatment of the diastereomeric mixture of
5b with DMTrCl in pyridine afforded 6b in 91% yield.
Under the standard conditions,10f both 6a and 6b were
phosphitylated with 2-cyanoethyl N,N-diisopropylchlo-
rophosphoramidite to give the corresponding phosphoram-
idites 7a and 7b, which were characterized by HRMS and
31P NMR. The 31P NMR spectrum of 7a showed two peaks
at 151.0 and 150.9 ppm, and as expected, that of 7b
sponding 5′-radiolabeled trinucleotides *pdUC2′Me/OTBDMS
U
(9a ), *pdUC2′Me/OTHPU (9b), and *pdUC2′OTBDMSU (9c),
respectively (*p indicates 32P phosphate). Deprotection
of the 2′-O-TBDMS group with tetrabutylammonium
fluoride (TBAF) was much slower for 2′-C-â-methylcyti-
dine than for normal cytidine. For example, after treat-
ment of *pdUC2′Me/OTBDMSU (9a ) with TBAF in THF for
24 h, none of the desilylated trinucleotide could be
(26) (a) Seliger, H.; Zeh, D.; Azuru, G.; Chattopadhyaya, J . B. Chem.
Scr. 1983, 22, 95. (b) Caruthers, M. H.; Dellinger, D.; Prosser, K.;
Brone, A. D.; Dubendorff, J . W.; Kierzek, R.; Rosendahl, M. Chem. Scr.
1986, 26, 25. (c) Kierzek, R.; Caruthers, M. H.; Longfellow, C. E.;
Swinton, D.; Turner, D. H.; Freier, S. M. Biochemistry 1986, 25, 7840.
(d) Tanimura, H.; Fukazawa, T.; Sekine, M.; Hata, T.; Efcavitch, J .
W.; Zon, G. Tetrahedron Lett. 1988, 29, 577. (e) Koizumi, M.; Iwai, S.;
Ohtsuka, E. FEBS Lett. 1988, 228, 228. (f) Koizumi, M.; Iwai, S.;
Ohtsuka, E. FEBS Lett. 1988, 239, 285. (g) Koizumi, M.; Hayase, Y.;
Iwai, S.; Kamiya, H.; Inoue, H.; Ohtsuka, E. Nucleic Acids Res. 1989,
17, 7059. (h) Tanimura, H.; Maeda, M.; Fukazawa, T.; Sekine, M.;
Hata, T. Nucleic Acids Res. 1989, 17, 8135. (i) Tanimura, H.; Imada,
T. Chem. Lett. 1990, 1715. (j) Tanimura, H.; Imada, T. Chem. Lett.
1990, 2081.
detected (Figure 1, lanes 6-10),28 whereas *pdUC2′OTBDMS
U
(9c) was almost completely desilylated to 10c during this
time (Figure 1, lanes 1-5). Sekine et al. recently dem-
onstrated that the 2′-O-TBDMS group can be efficiently
removed from oligonucleotides under acidic conditions29
(10% acetic acid or 8% formic acid or 0.01 N HCl). We
(27) (a) Griffin, B. E.; J arman, M.; Reese, C. B. Tetrahedron 1968,
24, 639. (b) Griffin, B. E.; Reese, C. B. Tetrahedron Lett. 1964, 2925.

2′-C-Branched Ribonucleosides
J . Org. Chem., Vol. 64, No. 3, 1999 751
F igu r e 2. Deprotection of 5′-radiolabled oligonucleotides with acid. Acidic conditions (0.01 N HCl, pH 2; 30 °C) remove the
2′-O-TBDMS group from *pdUC2′OTBDMSU (9c) in less than 6 h (lanes 1-5) and the 2′-O-THP group from *pdUC2′Me/OTHPU (9b) in
less than 1 h (lanes 13-17); however, even after 6 h, acidic conditions do not remove the 2′-O-TBDMS group from *pdUC2′Me/OTBDMSU
(9a ).
also found that 0.01 N HCl readily desilylated 9c to 10c
within 6 h (Figure 2, lanes 1-5) but failed to desilylate
any of *pdUC2′Me/OTBDMSU (9a ) within this time (Figure
2, lanes 7-11). Longer exposure to these conditions (120
h) resulted in decomposition of 9a . Results obtained using
10% acetic acid or 8% formic acid were similar (data not
shown). In contrast, the THP group of trinucleotide
dUC2′Me/OTHPU (8b) was readily removed with 0.01 N HCl
(pH 2) at 30 °C to give the corresponding free trinucle-
otide *pdUC2′MeU (10a ) within 1 h (Figure 2, lanes 14-
17), even less time than required for desilyation of
F igu r e 3. Cleavage of 5′-radiolabeled oligonucleotides with
*pdUC2′OTBDMSU (9c) to 10c. Thus, the 2′-O-THP phos-
phoramidite appears to be a better choice for synthesizing
oligonucleotides containing 2′-C-â-methylcytidine. Ad-
ditionally, it is compatible with the commercial 2′-O-
TBDMS phosphoramidites because both the THP and the
TBDMS groups are easily removed from synthetic oligo-
nucleotides with 0.01 N HCl.
sodium hydroxide. In 0.1 N NaOH (25 °C), *pdUCU (10c)
(lanes 1-5) and *pdUC2′MeU (10a ) (lanes 6-10) undergo base-
catalyzed degradation at similar rates.30
photransesterification reaction. When *pdUC2′MeU (10a )
was treated with 0.1 N NaOH, a faster migrating species
formed, presumably 11a containing a 2′,3′-cyclic phos-
phate (Figure 3, lanes 6-10).31 This cleavage product
forms at a rate similar to the product 11c from base-
promoted cleavage of the trinucleotide *pdUCU (10c)
(Figure 3, lanes 1-5) and has similar electrophoretic
mobility. These results indicate that 2′-C-â-methylcyti-
dine residues in oligonucleotides undergo base-catalyzed
degradation as efficiently as natural RNA even though
the 2′-nucleophile is bonded to a more hindered, tertiary
carbon center. Thus, ribozymes that cleave RNA by this
pathway might be active toward oligonucleotide sub-
strates containing a 2′-C-branched substituent at the
cleavage site, raising the possibility that 2′-C-branched
ribonucleosides could be used to probe the mechanism
of these ribozymes. The results also imply a cis relation-
ship between the 2′-hydroxyl and 3′-hydroxyl groups of
the 2′-C-â-methylcytidine residue, confirming that the
methyl group of 1 was installed with the correct stereo-
chemistry.
RNA undergoes base-catalyzed degradation by a path-
way in which the 2′-oxygen attacks the adjacent 3′-
phosphodiester to give products containing 2′,3′-cyclic
phosphate and 5′-hydroxyl termini.30 We examined
whether the tertiary hydroxyl of 2′-C-â-methylcytidine
could participate in an analogous base-catalyzed phos-
(28) We also synthesized both a 2′-C-â-methylcytidine containing
dinucleotide C2′MeOTBDMS
U using 7a and a control dinucleotide
C
2′OTBDMSU. Following purification by reversed-phase HPLC,
C2′Me/OTBDMSU gave the expected molecular ion in the mass spectrum
(M- ) 676 m/z), indicating that 7a had been correctly installed into
the dinucleotide. Removal of the TBDMS group from each dinucleotide
was monitored by reversed-phase HPLC. As observed for the trinucle-
otides (9a and 9c), TBAF/THF removed the TBDMS group more readily
from the cytidine residue than from the 2′-C-â-methylcytidine residue;
C
C
2′OTBDMSU was completely deprotected in less than 4 h, whereas
2′Me/OTBDMSU was only about 70% deprotected after 27 h. Following
purification by reversed-phase HPLC, C2′Me
U gave the expected
molecular ion in the mass spectrum (M- ) 562 m/z), confirming that
7a can be used to incorporate 2′-C-â-methylcytidine into oligonucle-
otides via solid-phase synthesis. The slower desilyation of the trinucle-
otides (9a and 9c) compared to the dinucleotides may be due to the
presence of the 5′-monophosphate group, which could limit their
solubility in TBAF/THF. We also synthesized two 17 residue RNA
oligonucleotides containing at position 9 either cytidine or 2′-C-â-
methylcytidine. Deprotection was monitored by labeling the 5′-end of
the oligonucleotide with 32P phosphate followed by PAGE. After 3 days
in TBAF/THF, about 70% of the 2′-C-â-methylcytidine residue was
deprotected. Thus, although TBAF/THF removes the 2′-O-TBDMS
group from 2′-C-â-methylcytidine slowly, the desired oligonucleotide
can be obtained using the phosphoramidite 7a .
In conclusion, we have developed a strategy for the
synthesis of phosphoramidite derivatives of 2′-C-â-me-
thylcytidine and their incorporation into oligonucleotides
via solid-phase synthesis. Either the TBDMS or the THP
group can be used to protect the tertiary 2′-hydroxyl
group of 2′-C-â-methylcytidine. The corresponding phos-
phoramidites couple efficiently during solid-phase oligo-
(29) Kawahara, S.; Wada, T.; Sekine, M. J . Am. Chem. Soc. 1996,
118, 9461.
(30) (a) Kirby, A. J .; Marriott, R. E. J . Am. Chem. Soc. 1995, 117,
833. (b) Breslow, R. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1208. (c)
Eftink, M.; Biltonen, R. L. Biochemistry 1983, 22, 5134. (d) Abrash,
H. I.; Cheung, C. S.; Davis, J . D. Biochemistry 1967, 6, 1298.
(31) If the products of base-promoted cleavage are electrophoresed
further in the gel, they resolve into a doublet, suggesting that the 2′,3′-
cyclic phosphate at least partially hydrolyzes to a mixture of the 2′-
and 3′-monophosphates. Mikhailov et al. reported that under basic
conditions 2′-C-â-methyluridine 2′,3′-cyclic phosphate hydrolyzes nearly
as fast as uridine 2′,3′-cyclic phosphate.5b

752 J . Org. Chem., Vol. 64, No. 3, 1999
Tang et al.
50WX8-200 resin. The resin was filtered and washed with H2O/
pyridine (4:1, 400 mL). The filtrate was evaporated to dryness,
and the residue was purified by silica gel chromatography
eluting with 10% methanol in chloroform to give 2b (2.38 g,
nucleotide synthesis using standard conditions and pro-
tocols. Although the THP group adds another chiral
center into the phosphoramidite, it is more advantageous
than the commonly used TBDMS group because it is
more easily introduced and more readily removed from
synthetic oligonucleotides. The conditions required for
removal of the THP group (0.01 N HCl; 30 °C) also
remove the TBDMS group of commercial phosphoramid-
ites, precluding the need for an additional deprotection
step. This work represents the first synthesis of oligo-
nucleotides containing a 2′-C-branched ribonucleoside
and expands the repertoire of modified nucleic acids for
use as antisense molecules or biochemical probes. Our
approach should be generally applicable to the incorpora-
tion of other 2′-C-branched ribonucleosides (such as those
in Chart 1) into oligonucleotides.
1
94%) as a white solid: mp 154-155 °C; H NMR (500 MHz)
(CDCl3) δ 8.60 (d, J ) 5.00 Hz, 1H), 8.01 (d, J ) 7.50 Hz, 2H),
7.63 (t, J ) 7.50 Hz, 1H), 7.52 (t, J ) 7.50 Hz, 2H), 7.34 (d, J
) 5.00 Hz, 1H), 5.98 (s, 1H), 5.20 (b, 1H), 5.18 (b, 2H), 3.86-
3.88 (m, 2H), 3.67-3.74 (m, 2H), 1.01 (s, 1H); 13C NMR (125
MHz) (CDCl3) δ 167.4, 162.8, 154.7, 145.0, 133.2, 132.8, 129.3,
128.5, 96.0, 91.9, 82.3, 78.5, 71.4, 58.8, 19.8; HRMS (FAB) calcd
for C17H20N3O6 [MH]+ 362.1352, found 362.1344.
3′,5′-O-(Di-ter t-bu tylsila n ed iyl)-2′-C-â-m eth yl-4-N-ben -
zoylcytid in e (3). To a solution of 2b (1.22 g, 3.38 mmol) and
AgNO3 (1.27 g, 7.50 mmol) in DMF (30 mL) at 0 °C was added
dropwise di-tert-butyldichlorosilane (0.79 mL, 3.75 mmol) with
vigorous stirring. The mixture was warmed to room temper-
ature and subsequently stirred for 15 min. Triethylamine (1.05
mL, 7.50 mmol) was added, and the mixture was stirred for
an additional 5 min. The solvent was evaporated under
reduced pressure, and the residue was partitioned between
methylene chloride and H2O. The organic layer was separated,
washed with brine, and dried over Na2SO4. After removal of
the solvent under reduced pressure, the residue was purified
by silica gel chromatography eluting with 5% methanol in
chloroform to give 3 (1.61 g, 95%) as a white solid: 1H NMR
(500 MHz) (CDCl3) δ 8.86 (b, 1H), 7.91 (d, J ) 7.50 Hz, 3H),
7.69 (d, J ) 6.02 Hz, 1H), 7.62 (t, J ) 7.50 Hz, 1H), 7.52 (t, J
) 7.50 Hz, 2H), 6.23 (s, 1H), 4.55 (dd, J ) 5.02 and 8.50 Hz,
1H), 4.15 (m, 1H), 4.06 (t, J ) 9.75 Hz, 1H), 3.56 (d, J ) 9.02
Hz, 1H), 2.67 (b, 1H), 1.23 (s, 3H), 1.08 (s, 9H), 1.05 (s, 9H);
13C NMR (125 MHz) (CDCl3) δ 162.5, 143.3, 133.2, 133.0, 129.0,
127.6, 96.8, 93.4, 80.6, 77.7, 74.3, 67.5, 27.3, 27.1, 22.8, 20.4;
HRMS (FAB) calcd for C25H36N3O6Si [MH]+ 502.2373, found
502.2365.
Exp er im en ta l Section
All reagents and anhydrous solvents were purchased from
Aldrich; other solvents were from Fisher unless otherwise
noted. Melting points are uncorrected. Mass spectra were
obtained from the Department of Chemistry, University of
California at Riverside. Microanalyses were performed by
Atlantic Microlab, Inc., Norcross, GA. Merck silica gel (9385
grade, 230-400 mesh, 60 Å, Aldrich) was used for column
chromatography. Silica gel on glass with fluorescent indicator
(Sigma) was used for TLC. Reactions and workup procedures
were at room temperature unless otherwise noted.
All nucleotide modifying enzymes were from U. S. Bio-
chemicals. [γ-32P]-Adenosine triphosphate ([γ-32P]ATP) was
obtained from New England Nuclear. PAGE was performed
using 20% polyacrylamide (acrylamide/bis-acrylamide 29:1,
Fisher) with 7.5 M urea (Fisher), 0.1 M Tris (Fisher), 0.1 M
boric acid (Fisher), and 2 mM EDTA (Fisher). Gels were pre-
electrophoresed at constant power (15 W) for 1 h before sample
loading. Gel loading buffer contained 8 M urea (Fisher), 50
mM EDTA (pH 8.0, Fisher), 0.02% bromophenol blue (EM
Science), and 0.02% xylene cyanol FF (Kodak).
2′,3′,5′-Tr i-O-b en zoyl-4-N-b en zoyl-2′-C-â-m et h ylcyt i-
d in e (2a ). A stirred suspension of 4-N-benzoylcytosine (4.42
g, 19.30 mmol) and (NH4)2SO4 (50 mg) in freshly distilled
1,1,1,3,3,3-hexamethyldisilazane (150 mL) was heated at
reflux overnight under argon. During reflux, the mixture
became clear within 1 h. The clear solution was evaporated
under vacuum, and dry toluene (60 mL) was added and
subsequently distilled off. The crude bis(trimethylsilyl) deriva-
tive obtained was dissolved in dry acetonitrile (150 mL), and
1,2,3,5-tetra-O-benzoyl-2-C-methyl-R-(and â)-D-ribofuranose
(1)1p-q (5.83 g, 10.05 mmol) was added. Under an argon
atmosphere, SnCl4 (2.35 mL, 20.0 mmol) was added in one
portion with vigorous stirring and exclusion of moisture. The
resultant homogeneous pale yellow solution was stirred over-
night. When TLC indicated that 1 was completely consumed,
the reaction was quenched carefully by addition of 10%
NaHCO3 (100 mL) and stirred for an additional 15 min. The
mixture was extracted with methylene chloride, and the
organic phase was washed with brine and dried over Na2SO4.
After evaporation of the solvent, the residue was purified by
silica gel chromatography eluting with 50% ethyl acetate in
hexane to give 2a (5.81 g, 86%) as a white solid: 1H NMR (500
MHz) (CDCl3) δ 8.67 (s, 1H), 8.07 (d, J ) 7.54 Hz, 2H), 8.03
(d, J ) 7.55 Hz, 2H), 7.93 (d, J ) 6.43 Hz, 1H), 7.84 (d, J )
5.87 Hz, 2H), 7.80 (d, J ) 7.35 Hz, 2H), 7.35-7.57 (m, 8H),
7.20 (t, J ) 7.28 Hz, 2H), 6.72 (s, 1H), 5.75 (s, 1H), 4.86 (d, J
) 3.93 Hz, 2H), 4.64 (q, J ) 4.29 Hz, 1H), 1.70 (s, 3H).
4-N-Ben zoyl-2-C-â-m eth ylcytid in e (2b). To a solution of
2a (4.74 g, 7.0 mmol) in a mixture of pyridine (65 mL) and
methanol (11.7 mL) was added dropwise ice-cooled aqueous
NaOH (2.0 M, 10.6 mL, 21.2 mmol) at -20 °C with stirring.
The reaction mixture was stirred at -20 °C to -15 °C for an
additional 30 min and then neutralized to pH 7 with Dowex
3′,5′-O-(Di-ter t-bu tylsilan ediyl)-4-N-ben zoyl-2′-C-â-m eth -
yl-2′-O-(ter t-bu tyld im eth ylsilyl)cytid in e (4a ). To a solution
of 3 (251.5 mg, 0.50 mmol) in THF (5 mL) was added a solution
of t-BuMgCl in THF (1.0 M, 1.0 mL, 1.0 mmol; Aldrich) at 0
°C under a nitrogen atmosphere. The solution was allowed to
warm to room temperature and stirred for 30 min. A solution
of TBDMSOTf (230 µL, 1.0 mmol) in THF (2 mL) was added,
and the mixture was stirred overnight. When TLC indicated
that all the starting material had been consumed, the reaction
mixture was carefully quenched with saturated aqueous NH4-
Cl (2 mL) and extracted with methylene chloride. The organic
layer was washed with brine and dried over Na2SO4. After
evaporation of the solvent, the residue was purified by silica
gel chromatography eluting with 3% methanol in chloroform
to give 4a (197.6 mg, 64%) as a white foam: 1H NMR (500
MHz) (CDCl3) δ 9.12 (b, 1H), 7.93 (d, J ) 7.50 Hz, 2H), 7.71
(d, J ) 7.50 Hz, 1H), 7.61 (t, J ) 7.50 Hz, 1H), 7.51 (t, J )
7.50 Hz, 2H), 6.22 (s, 1H), 4.55 (m, 1H), 4.16 (m, 1H), 4.07 (t,
J ) 9.50 Hz, 1H), 3.56 (d, J ) 9.00 Hz, 1H), 2.91 (s, 1H), 1.23
(s, 3H), 1.07 (s, 9H), 1.05 (s, 9H), 0.91 (s, 9H), 0.09 (s, 6H); 13
C
NMR (125 MHz) (CDCl3) δ 160.2, 148.0, 134.7, 130.4, 129.4,
129.1, 96.3, 93.7, 80.1, 77.9, 74.9, 67.1, 27.3, 27.1, 25.6, 22.7,
20.4, -3.6; HRMS (FAB) calcd for
616.3238, found 616.3258.
C
31H50N3O6Si2 [MH]+
3′,5′-O-(Di-ter t-bu tylsilan ediyl)-4-N-ben zoyl-2′-C-â-m eth -
yl-2′-O-(tetr a h yd r op yr a n yl)cytid in e (4b). To a solution of
3 (720.0 mg, 1.44 mmol) and 3,4-dihydro-2H-pyran (1.31 mL,
14.4 mmol) in dry methylene chloride was added 10-camphor-
sulfonic acid (150.0 mg). The reaction mixture was stirred
overnight, diluted with methylene chloride, washed with 5%
NaHCO3, and dried over Na2SO4. After evaporation of the
solvent, the residue was purified by silica gel chromatography
eluting with 3% methanol in chloroform to give 4b (798.7 mg,
95%) as a mixture of two diastereomers: 1H NMR (500 MHz)
(CDCl3) δ 8.81 (b, 1H), 7.91 (d, J ) 7.50 Hz, 2H), 7.68 (d, J )
6.50 Hz, 1H), 7.62 (t, J ) 7.50 Hz, 1H), 7.52 (t, J ) 7.50 Hz,
2H), 6.58 + 6.10 (s, 1H), 5.59 + 5.48 (s, 1H), 4.56 (dd, J )
9.00, 5.00 Hz, 1H), 4.27-4.37 (m, 1H), 4.07-4.13 (m, 1H),
4.01-4.05 (m, 1H), 3.46-3.64 (m, 2H), 1.52-1.92 (m, 6H), 1.31
(s, 3H), 1.07+1.05 (s, 9H), 1.06 + 1.04 (s, 9H); 13C NMR (125

2′-C-Branched Ribonucleosides
J . Org. Chem., Vol. 64, No. 3, 1999 753
MHz) (CDCl3) δ 162.1, 143.1, 133.3, 129.1, 127.5, 94.4, 93.3,
92.2, 92.1, 83.0, 82.4, 81.7, 74.2, 67.8, 62.9, 61.6, 60.7, 31.8,
31.5, 30.7, 27.9, 27.8, 27.3, 26.9, 25.4, 25.3, 22.9, 20.3, 18.8,
18.4, 16.9; HRMS (FAB) calcd for C30H44N3O7Si [MH]+ 586.2949,
found 586.2931.
0.0, -2.5, -3.2; HRMS (FAB) calcd for C44H52N3O8Si [MH]+
778.3824, found 778.3515.
4-N-Ben zoyl-5′-O-(d im eth oxytr ityl)-2′-C-â-m eth yl-2′-O-
(tetr a h yd r op yr a n yl)cytid in e (6b). To a solution of the two
diastereomers of 5b (488.9 mg, 1.10 mmol) in dry pyridine
(15.0 mL) at 0 °C was added dropwise a solution of dimethoxy-
trityl chloride (391.4 mg, 1.32 mmol) in pyridine (10.0 mL) over
1 h. The reaction mixture was warmed to room temperature
and stirred overnight until TLC indicated that the reaction
was complete. Methanol (1.0 mL) was added to quench the
reaction, and the mixture was stirred for an additional 15 min
and concentrated under reduced pressure. The residue was
dissolved in methylene chloride, washed with 5% NaHCO3,
water, and dried over Na2SO4. The solvent was evaporated,
and the residue was purified by silica gel chromatography
eluting with 2% methanol in chloroform to give 6b (750.8 mg,
91%) as a slightly yellow foam: 1H NMR (500 MHz) (CDCl3)
δ 8.69 (m, 2H), 7.88 (d, J ) 7.50 Hz, 2H), 7.59 (m, 1H), 7.49
(m, 2H), 7.43 (m, 2H), 7.28-7.35 (m, 7H), 6.89 (d, J ) 9.00
Hz, 4H), 6.81 + 6.21 (s, 1H), 5.34 + 5.04 (m, 1H), 3.96 (m,
1H), 3.83 + 3.84 (s, 6H), 3.69 (m, 1H), 3.59 (m, 1H); 13C NMR
(125 MHz) (CDCl3) δ 166.2, 158.6, 145.1, 144.0, 135.6, 135.3,
133.0, 130.2, 130.1, 129.1, 128.9, 128.3, 128.0, 127.8, 127.7,
127.5, 127.2, 127.1, 95.5, 95.2, 91.2, 88.4, 87.2, 87.1, 85.8, 84.5,
82.1, 81.7, 73.8, 73.0, 64.8, 64.3, 60.7, 60.6, 55.2, 32.0, 31.9,
24.9, 24.8, 20.9, 20.5, 17.0, 15.6; HRMS (FAB) calcd for
4-N-Ben zoyl-2′-C-â-m eth yl-2′-O-(ter t-bu tyld im eth ylsil-
yl)cytid in e (5a ). HF‚pyridine (100 µL, 3.84 mmol; Aldrich)
was carefully diluted with pyridine (0.51 mL) and then added
dropwise to a solution of 4a (582.7 mg, 0.95 mmol) in THF (5
mL) at 0 °C. The mixture was warmed to room temperature,
stirred for 5 min, and then diluted with pyridine (1.5 mL).
Extraction was performed with methylene chloride/H2O. The
organic layer was collected, washed with 5% NaHCO3, and
dried over Na2SO4. After evaporation of the solvent under
reduced pressure, the residue was purified by silica gel
chromatography eluting with 8% methanol in chloroform to
give 5a (369.0 mg, 82%) as a white foam: 1H NMR (500 MHz)
(CDCl3) δ 8.57 (d, J ) 7.50 Hz, 1H), 7.97 (d, J ) 7.50 Hz, 2H),
7.63 (d, J ) 7.50 Hz, 1H), 7.61 (t, J ) 7.50 Hz, 1H), 7.52 (t, J
) 7.50 Hz, 2H), 6.18 (s, 1H), 4.07-4.13 (m, 2H), 3.89-3.94
(m, 2H), 1.23 (s, 3H), 0.97 (s, 9H), 0.16 (s, 3H), 0.14 (s, 3H);
13C NMR (125 MHz) (CDCl3) δ 162.5, 156.1, 145.1, 133.3, 132.8,
129.0, 127.6, 97.0, 92.8, 84.2, 78.7, 73.1, 61.2, 25.9, 20.2, 18.5,
-5.5; HRMS (FAB) calcd for C23H34N3O6Si [MH]+ 476.2217,
found 476.2194.
4-N-Ben zoyl-2′-C-â-m eth yl-2′-O-(tetr a h yd r op yr a n yl)cy-
tid in e (5b). HF‚pyridine (116 µL, 4.45 mmol; Aldrich) was
carefully diluted with pyridine (0.60 mL) and then added
dropwise to a solution of 4b (650.8 mg, 1.11 mmol) in THF (5
mL) at 0 °C. The mixture was warmed to room temperature,
stirred for 5 min, and then diluted with pyridine (2 mL).
Extraction was performed with methylene chloride/H2O, and
the organic layer was collected, washed with 5% NaHCO3, and
dried over Na2SO4. The solvent was evaporated under reduced
pressure. Chromatography on silica gel, eluting with 8%
methanol in chloroform, separated the two diastereomers of
5b (455.4 mg, 92%). The fast isomer: 1H NMR (500 MHz)
(CDCl3) δ 9.01 (s, 1H), 8.65 (d, J ) 7.50 Hz, 1H), 7.87 (d, J )
8.00 Hz, 2H), 7.60 (t, J ) 7.50 Hz, 1H), 7.49 (t, J ) 7.50 Hz,
2H), 6.21 (s, 1H), 5.04 (d, J ) 5.00 Hz, 1H), 4.15 (m, 2H), 4.03
(m, 2H), 3.88-3.94 (m, 2H), 3.54 (m, 1H), 3.27 (b, 1H), 1.88
(m, 2H), 1.60 (m, 2H), 1.54 (m, 2H), 1.25 (s, 3H); 13C NMR
(125 MHz) (CDCl3) δ 162.3, 145.3, 133.1, 129.0, 127.6, 96.9,
95.3, 91.4, 85.9, 82.6, 72.4, 64.8, 59.6, 31.9, 24.9, 20.9, 15.7;
HRMS (FAB) calcd for C22H28N3O7 [MH]+ 446.1927, found
446.1941. The slow isomer: 1H NMR (500 MHz) (CDCl3) δ 9.08
(b, 1H), 8.63 (b, 1H), 7.87 (d, J ) 7.50 Hz, 2H), 7.59 (t, J )
7.02 Hz, 2H), 7.54 (m, 1H), 7.48 (t, J ) 7.50 Hz, 2H), 6.75 (b,
1H), 5.30 (b, 1H), 4.17 (d, J ) 12.00 Hz, 1H), 4.06 (m, 1H),
4.01 (m, 2H), 3.69 (b, 2H), 3.10 (b, 1H), 1.85 (m, 1H), 1.76 (m,
1H), 1.53-1.65 (m, 4H), 1.25 (s, 3H); 13C NMR (125 MHz)
(CDCl3) δ 162.4, 155.7, 145.8, 133.1, 128.9, 127.6, 96.9, 95.7,
84.6, 83.0, 73.1, 64.3, 59.6, 32.0, 27.3, 25.0, 20.6, 16.9.
C
43H44N3O9 [MH]+ 748.3234, found 748.3207.
4-N-Ben zoyl-5′-O-(d im eth oxytr ityl)-2′-C-â-m eth yl-2′-O-
(ter t-bu tyld im eth ylsilyl)cytid in e 3′-N,N-Diisop r op yl(cya -
n oet h yl)p h osp h or a m id it e (7a ). To a solution of N,N-
diisopropylethylamine (185.9 µL, 1.07 mmol), 1-methylimidazole
(21.4 µL, 0.27 mmol), and 2-cyanoethyl N,N-diisopropylchlo-
rophosphoramidite (178.0 µL, 0.80 mmol) in dry methylene
chloride (5.0 mL) at 0 °C was added dropwise a solution of 6a
(257.7 mg, 0.33 mmol) in methylene chloride (5.0 mL) under
an argon atmosphere. The reaction mixture was warmed to
room temperature and stirred until the starting material was
consumed (∼3 h) as indicated by TLC. The reaction mixture
was concentrated under reduced pressure, and the residue was
purified by flash chromatography on silica gel eluting with 4%
acetone in methylene chloride containing 0.2% triethylamine
to give the phosphoramidite derivative 7a (271.3 mg, 84%) as
a white foam: 31P NMR (500 MHz) (CDCl3) δ 151.0, 150.9;
HRMS (FAB) calcd for C53H69N5O9PSi [MH]+ 978.4602, found
978.4614.
4-N-Ben zoyl-5′-O-(d im eth oxytr ityl)-2′-C-â-m eth yl-2′-O-
(tetr a h yd r op yr a n yl)cytid in e 3′-N,N-Diisop r op yl(cya n o-
eth yl)p h osp h or a m id ite (7b). To a solution of 6b (305.8 mg,
0.41 mmol) in dry methylene chloride (15.0 mL) at 0 °C under
an argon atmosphere were added quickly N,N-diisopropyl-
ethylamine (190.9 µL, 1.10 mmol), 2-cyanoethyl N,N-diisopro-
pylchlorophosphoramidite (182.5 µL, 0.82 mmol), and 1-meth-
ylimidazole (22.2 µL, 0.28 mmol). The reaction mixture was
then warmed to room temperature and stirred until TLC
indicated that the reaction was complete (∼3 h). The reaction
mixture was concentrated under reduced pressure, and the
residue was purified by flash chromatography on silica gel
eluting with 4% acetone in methylene chloride containing 0.2%
triethylamine to give the phosphoramidite derivative 7b (348.1
mg, 90%) as a slightly yellow foam: 31P NMR (500 MHz)
(CDCl3) δ 152.9, 152.2, 150.3, 150.0; HRMS (FAB) calcd for
4-N-Ben zoyl-5′-O-(d im eth oxytr ityl)-2′-C-â-m eth yl-2′-O-
(ter t-bu tyld im eth ylsilyl)cytid in e (6a ). To a solution of 5a
(423.4 mg, 0.89 mmol) in dry THF (10.0 mL) under argon were
added anhydrous pyridine (360.5 µL, 4.46 mmol), AgNO3 (151.4
mg, 0.89 mmol), and DMTrCl (138.5 mg, 0.90 mmol) succes-
sively with vigorous stirring and exclusion of moisture. The
resultant pale-yellow suspension was stirred for 1 h and
filtered into a 5% NaHCO3 solution. The filtrate was extracted
with chloroform, washed with 5% NaHCO3 and brine, and
dried over Na2SO4. The solvent was evaporated, and the
residue was purified by flash chromatography on silica gel
eluting with 2% methanol in chloroform to give 6a (450.2 mg,
65%) as a white foam: 1H NMR (500 MHz) (CDCl3) δ 8.75 (d,
7.50 Hz, 2H), 7.89 (d, J ) 6.52 Hz, 2H), 7.60 (t, J ) 7.50 Hz,
1H), 7.50 (t, J ) 7.50 Hz, 2H), 7.26-7.43 (m, H), 6.90 (d, J )
8.50 Hz, 4H), 6.26 (s, 1H), 4.14 (dd, J ) 11.00, 9.50 Hz, 1H),
3.93 (m, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.58-3.67 (m, 2H), 2.29
(d, J ) 12.00 Hz, 1H), 1.27 (s, 3H), 0.90 (s, 9H), 0.41 (s, 3H),
0.26 (s, 2H); 13C NMR (125 MHz) (CDCl3) δ 158.7, 143.9, 135.6,
135.2, 133.0, 130.2, 130.1, 129.0, 128.4, 128.1, 127.5, 127.2,
113.3, 91.3, 87.3, 82.7, 82.6, 73.5, 60.5, 55.2, 25.8, 19.5, 18.2,
C
52H63N5O10P [MH]+ 948.4313, found 948.4342.
In cor p or a tion of 2′-C-â-Meth ylcytid in e in to Oligo-
n u cleotid es by Solid -P h a se Syn th esis. Oligonucleotides
(the trinucleotides 8a -c described in the text and the dinucle-
otides and 17 residue oligonucleotides described in ref 28) were
synthesized on a 1 µmol scale with standard phosphoramidites
(Perseptive Biosystems and Glen Research) using a Millipore
Expidite nucleic acid synthesis system and standard DNA and
RNA protocols. Both phosphoramidites 7a and 7b were coupled
according to the standard RNA synthesis protocol and gave
yields comparable to those of the commercial phosphoramid-
ites. Synthesized oligonucleotides were deprotected at 55 °C
for 18-24 h with concentrated aqueous NH3 and ethanol (2:
1) and subsequently evaporated to dryness. Oligonucleotide

754 J . Org. Chem., Vol. 64, No. 3, 1999
Tang et al.
10c was desilylated by treatment with 1.0 M TBAF/THF,
shaken in the dark for 24 h, neutralized with 0.1 M triethyl-
ammonium acetate (TEAAC, pH 6.9), and purified by elution
through a NAP-10 gel-filtration column (Pharmacia Biotech.).
Overall yields of oligonucleotides containing the 2′-modification
were comparable to those for oligonucleotides lacking the
modification.
R a d iola belin g of Oligon u cleot id es. Oligonucleotides
were 5′-end labeled with [γ-32P]-ATP and PNK. Reactions
included [γ-32P]-ATP (30 µCi), PNK (17 units), and oligonucle-
otide (20 pmol) in a 10 µL volume. After 1 h incubation at 37
°C, the reactions were quenched with gel loading buffer (8 µL),
and the components were separated by PAGE. Products were
visualized by autoradiography, excised from the gel, and eluted
at 4 °C with H2O. The eluent was applied to a Sep-Pac C18
cartridge (Waters), which was then washed with water (2 mL),
50% aqueous acetonitrile (2 mL), and 75% aqueous acetonitrile
(2 mL). Fractions containing oligonucleotide were evaporated
to dryness and brought up into water to a concentration of 20
nM as determined by specific radioactivity.
Depr otection of 5′-Radiolabeled Oligon u cleotides with
Acid s. 5′-Radiolabled oligonucleotides (1 µL) were treated with
10% acetic acid (1 µL), 8% formic acid (1 µL), and 0.01 N HCl
(pH 2.0, 1 µL), respectively, at 30 °C. The reactions were
quenched with 1X TBE (5 µL) and gel loading buffer (8 µL).
The samples were stored at -78 °C before loading on the gel
(Figure 2 shows results from deprotection with 0.01 N HCl).
Clea va ge of 5′-Ra d iola beled Oligon u cleotid es w ith
Sod iu m Hyd r oxid e. 5′-Radiolabled oligonucleotides (1 µL)
were treated with 0.1 N NaOH (1 µL) at room temperature.
The reactions were quenched with 1X TBE (5 µL) and gel
loading buffer (8 µL). The sample was stored at -78 °C before
loading on the gel (see Figure 3).
Ack n ow led gm en ts We thank Larrisa Munishkina
for assistance with oligonucleotide synthesis, Qing Dai,
Michelle Hamm, and J ason Schwans for critical reading
of the manuscript, and Cecilia Cortez for assistance in
preparing the revised manuscript. X.Q.T is a Research
Associate and J .A.P. is an Assistant Investigator of the
Howard Hughes Medical Institute.
Depr otection of 5′-Radiolabeled Oligon u cleotides with
TBAF . 5′-Radiolabeled oligonucleotides (1 µL) were evaporated
to dryness, taken up into a solution of TBAF in THF (1.0 M,
5 µL), shaken in the dark at room temperature, and neutral-
ized with 0.1 M TEAAC (pH 6.9). Most of the THF was
evaporated, and gel loading buffer (8.0 µL) was added. The
sample was stored at -78 °C before loading on the gel (see
Figure 1).
Su p p or tin g In for m a tion Ava ila ble: NMR spectra for
obtained compounds (23 pages). See any current masthead
page for ordering and Internet access information.
J O981329U