A New Route to 2′-O-Alkyl-2-thiouridine Derivatives via 4-O-Protection of the Uracil Base and Hybridization Properties of Oligonucleotides Incorporating These Modified Nucleoside Derivatives

Article abstract of DOI:10.1021/jo035246b

Oligonucleotides containing 2-thiouridine (s²U) in place of uridine form stable RNA duplexes with complementary RNAs. Particularly, this modified nucleoside has proved to recognize highly selectively adenosine, the genuine partner, without form

Full text of DOI:10.1021/jo035246b

A New Rou te to 2′-O-Alk yl-2-th iou r id in e Der iva tives via
4-O-P r otection of th e Ur a cil Ba se a n d Hybr id iza tion P r op er ties of
Oligon u cleotid es In cor p or a tin g Th ese Mod ified Nu cleosid e
Der iva tives
Itaru Okamoto,† Koh-ichiroh Shohda,† Kohji Seio,‡,§ and Mitsuo Sekine*,†,§
Department of Life Science and Frontier Collaborative Research Center, Tokyo Institute of Technology,
Nagatsuta, Midori-ku, Yokohama 226-8501, J apan, and CREST, J ST (J apan Science and Technology
Corporation) Nagatsuta, Midori-ku, Yokohama 226-8501, J apan
msekine@bio.titech.ac.jp
Received August 26, 2003
Oligonucleotides containing 2-thiouridine (s2U) in place of uridine form stable RNA duplexes with
complementary RNAs. Particularly, this modified nucleoside has proved to recognize highly
selectively adenosine, the genuine partner, without formation of a mismatched base pair with the
guanosine counterpart. In this paper, we describe new methods for the synthesis of 2-thiouridine
and various 2′-O-alkyl-2-thiouridine derivatives. Oligoribonucleotides having these modified
nucleoside derivatives were synthesized, and their hybridization and structural properties were
1
studied in detail by the H NMR analysis of these modified nucleosides and Tm experiments of
RNA duplexes with their complementary RNA strands.
In tr od u ction
forms a more stable base pair with adenosine than
guanosine and exhibits predominant selectivity for for-
mation of a base pair with adenosine over guanosine.
Furthermore, our recent paper also revealed that the 2′-
O-methyl-2-thiouridine (s2Um) has a similar base recog-
nition ability.5
2-Thiouridine (s2U) and its derivatives have been
discovered from transfer RNAs.1 It has been recognized
that the 2-thiocarbonyl group of s2U plays a key role in
changing its sugar conformation to a rigid C3′-endo form
seen in typical RNA duplexes so that a modified s2U-A
base pair is more stabilized than the unmodified one.2 It
was also reported that s2U derivatives found at the first
letter of anticodon (the wobble position) improve the
capability of base recognition to distinguish adenosine
from guanosine located at the third letter of codon triplets
on mRNAs and, moreover, enforce the codon-anticodon
minihelix structure.3 Actually, Testa and colleagues
reported that the melting temperatures of RNA duplexes
having an s2U-G wobble base pair were considerably
lower than those of RNA duplexes having a matched
s2U-A base pair.4 This result indicated that 2-thiouridine
The exact base pair recognition is due mainly to the
weak hydrogen bonding property of the 2-thiocarbonyl
group toward the 2-amino group of the guanine base. A
G-U wobble base pair requires two hydrogen bonds.6 One
is the hydrogen bond between the 2-carbonyl oxygen of
the uracil base and the 1-amido proton of the guanine
base. The other is the hydrogen bond between the
6-carbonyl oxygen of the guanine base and the 3-imido
proton of the uracil base. Generally, an electrostatic
interaction between two components is a key factor for
the hydrogen bonding.7 Since the sulfur atom of the
2-thiocarbonyl group is bigger than the oxygen of the
carbonyl group so that the polarized minus charge on the
sulfur is dispersed on its surface, the hydrogen bonding
ability of the 2-thiocarbonyl group is considerably weak-
ened.
* To whom correspondence should be addressed. Phone (fax):
+81-45-924-5706.
† Department of Life Science, Tokyo Institute of Technology.
‡ Frontier Collaborative Research Center, Tokyo Institute of Tech-
nology.
§ CREST.
The thermal stability of RNA duplexes containing s2U
is due mainly to the rigid ribose residue, which prefers
C3′-endo conformation. This C3′-endo predominance is
entropically favorable for stable formation of A-type RNA
duplexes, because all the ribose conformations of A-type
(1) (a) Hall, R. H. In The Modified Nucleosides in Nucleic Acids;
Columbia University Press: New York, 1971. (b) Chackalaparampil,
I.; Cherayil, J . D. Biochem. Int. 1981, 2, 121. (c) Edmonds, C. G.; Crain,
P. F.; Hashizume, T.; Gupta, R.; Stetter, K. O.; McCloskey, J . A. J .
Chem. Soc., Chem. Commun. 1987, 909. (d) Kimura-Harada, F.;
Saneyoshi, M.; Nishimura, S. FEBS Lett. 1971, 13, 335.
(2) (a) Yamamoto, Y.; Yokoyama, S.; Miyazawa, T.; Watanabe, K.;
Higuchi, S. FEBS Lett. 1983, 157, 95. (b) Sierzputowska-Gracz, H.;
Sochacka, E.; Malkiewicz, A.; Kuo, K.; Gehrke, C. W.; Agris, P. F. J .
Am. Chem. Soc. 1987, 109, 7171.
(5) Shohda, K.; Okamoto, I.; Wada, T.; Seio, K.; Sekine, M. Bioorg.
Med. Chem. Lett. 2000, 10, 1795.
(6) Sugimoto, N.; Kierzek, R.; Freier, S. M.; Turner, D. H. Biochem-
istry 1986, 25, 5755.
(3) Yokoyama, S.; Watanabe, T.; Murao, K.; Ishikura, H.; Yamai-
zumi, Z.; Nishimura, S.; Miyazawa, T. Proc. Natl. Acad. Sci. U.S.A.
1985, 82, 4905.
(4) Testa, S. M.; Disney, M. D.; Turner, D. H.; Kierzek, R. Biochem-
istry 1999, 38, 16655.
(7) Kawahara, S.; Wada, T.; Kawauchi, S.; Uchimaru,T.; Sekine, M.
J . Phys. Chem. A 1999, 103, 8516.
10.1021/jo035246b CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/25/2003
J . Org. Chem. 2003, 68, 9971-9982
9971

Okamoto et al.
SCHEME 1
TABLE 1. Th ion a tion of 4-O-Ar ylu r id in e Der iva tives
1a -d w ith La w esson ’s Rea gen t
RNA duplexes are C3′-endo.8 In addition, the stacking
effect between the 2-thiocarbonyl group and the 1- or
9-nitrogen atom of 3′-downstream nucleoside bases is also
important for stabilization of RNA duplexes.8 These
properties of oligoribonucleotides containing 2-thiouridine
or its derivatives are favorable for the antisense strategy
and SNPs analysis.
In general, 2-thiouridine has been prepared by glyco-
sylation of a silylated 2-thiouracil base with ribose
derivatives.9 However, this method incurs several prob-
lems: It requires the use of expensive glycosyl donors
and moisture-sensitive reactions. We have also encoun-
tered difficulty in isolating the desired product because
of inseparable emulsion during the extraction. Therefore,
it seems to us that it is difficult to perform a large-scale
synthesis because of these inherent problems.
In this paper, we report a new convenient route to
2-thiouridine or its derivatives via selective conversion
of the 2-carbonyl group to a 2-thiocarbonyl group. More-
over, the synthesis of various 2′-O-alkyl-2-thiouridine
derivatives is described along with the clarified relation-
ship between the 2′-O-modification and the duplex stabil-
ity.
type protecting groups for avoidance of side reactions on
the 4-carbonyl group of uracil and thymine residues in
their oligonucleotide synthesis.12 Such a 4-O-protection
strategy is also available for the 2-thionation of the
2-carbonyl group of the uracil base because Laweeson’s
reagent and phosphorus pentasulfide do not damage
phenyl ether-type functions.
Resu lts a n d Discu ssion
Syn th esis of 2-Th iou r id in e (s2U). Necessity of
P r otectin g Gr ou p s for P r otection of th e 4-Ca r bon yl
Gr ou p of Ur id in e. Lawesson’s reagent or phosphorus
pentasulfide has been generally used as a reagent for the
conversion of the carbonyl group of nucleobases to a
thiocarbonyl group.10 The uracil base residue has 2- and
4-carbonyl groups. In general, the reactivity of the
4-carbonyl group is much higher than that of the 2-car-
bonyl group. Therefore, when the uracil base residue is
allowed to react with thionating reagents without protec-
tion, 4-thiouracil derivatives are formed predominantly.
This method provides a general route to 4-thiouridine and
2,4-dithiouridine but 2-thiouridine derivatives cannot be
obtained.11 Therefore, protection of the 4-carbonyl group
is necessary to selectively thionate the 2-carbonyl group.
Reese and colleagues reported the use of phenyl ether-
To check this possibility, several 4-O-protected deriva-
tives 1a -d were synthesized from 2′,3′,5′-O-tris(tert-
butyldimethylsilyl)uridine according to a modified pro-
cedure of Reese. It was reported that the reactivity and
solubility of Lawesson’s reagent were superior to those
of phosphorus pentasulfide. Therefore, Lawesson’s re-
agent was chosen for the thionation of 4-O-protected
uridine derivatives 1a -d (Scheme 1). The use of the 2,6-
dimethylphenyl derivative 1d gave the most successful
result, as shown in Table 1.
The 2-thionation of the uracil residue of 1d resulted
in a significant shift of the NMR resonance signal of 1′-H
to low magnetic field, as shown in Figure 1.
The low yields of other 4-O-protected derivatives are
due to partial elimination of the protecting group and
formation of 2,4-dithiouridine derivatives. It is likely that
the use of phenyl groups having more electron-withdraw-
ing or smaller substituents tends to cause elimination of
the protecting group. Therefore, sterically hindered phe-
nol groups with electron-donating substituents such as
a methyl group are suitable for the 2-thionation.
(8) Seanger, W. In Principles of Nucleic Acid Structure; Springer-
Verlag: Berlin, Germany, 1981; Chapter 10.
(9) (a) Vorbru¨ggen, H.; Strehlke, P. Chem. Ber. 1973, 106, 3039. (b)
Niedballa, U.; Vorbru¨ggen, H. J . Org. Chem. 1974, 39, 3654.
(10) (a) Cherkasov, R. A.; Kutyrev, G. A.; Pudovik, A. N. Tetrahedron
1985, 41, 2567. (b) Cava, M. P.; Levinson, M. I. Tetrahedron 1985, 41,
5061.
(11) (a) Chien-Hua, N. Anal. Biochem. 1984, 139, 404. (b) Fox, K.;
Praag, V. P.; Wempen, I.; Doerr, I. L.; Cheong, L.; Knoll, J . E.; Eidinoff,
M. L.; Bendich, A.; Brown, G. B. J . Am. Chem. Soc. 1959, 81, 178.
(12) Reese, C. B.; Skone, P. A. J . Chem. Soc., Perkin Tran. 1 1984,
1263.
9972 J . Org. Chem., Vol. 68, No. 26, 2003

A New Route to 2′-O-Alkyl-2-thiouridine Derivatives
F IGURE 1. 1H NMR spectra of compounds 1d and 2d .
1,3-diyl (TIPDS) group. Subsequently, the base moiety
of the resulting product 4 was protected with the 2,6-
dimethylphenyl group under conditions similar to those
described by Reese via transient protection of the 2′-
hydroxyl group with a trimethylsilyl group. However, the
yield of the desired product 5 was significantly low. The
reason for the low yield is explained on the assumption
that the base moiety was also masked by the trimethyl-
silyl group so that the base modification was impossible
under these conditions. Accordingly, protection of the
4-carbonyl group was performed by use of phase-transfer
conditions.13 These conditions gave the desired product
5 in good yield. The 2′-O-methylation of 6 with CH3I-
NaH in DMF gave the 2′-O-methylated product 6. The
2-thionation of 6 was successively done by using Lawes-
son’s reagent. However, separation of the desired product
7 from byproducts derived from Lawesson’s reagent was
very difficult. Therefore, phosphorus pentasulfide was
used as an alternative thionating reagent that allows
much easier workup. Thus, the product 7 was synthe-
sized in good yield (77%). Finally, both the TIPDS and
2,6-dimethylphenyl groups of 7 were removed in the
usual manner. Thus, the desired product 3b could be
successfully obtained in an overall yield of 38% from
uridine (Scheme 2).
F IGURE 2. Uridine derivatives 3a -e protected with various
aryl groups.
Next, to demonstrate the utility of our method, we
synthesized various 2′-O-alkylated-2-thiouridine deriva-
tives (Figure 2).
Syn th esis of 2′-O-Alk yl-2-th iou r id in e Der iva tives
(3b-d ). We previously reported the synthesis of 2′-O-
methyl-2-thiouridine 3b via selective 2′-O-methylation of
2-thiouridine.5 In this method, we used 2-thiouridine 3a
as the starting material. Our new method gave 3b via
3′,5′-O-(1,1,3,3-tetraisopropylsiloxane-1,3-diyl)uridine (4)
more easily from commercially available uridine as the
starting material. The synthetic intermediate 5 can be
used as the common precursor for the synthesis of various
2′-O-alkylated 2-thiouridine derivatives. We thought the
2′-alkylation of intermediate 5 was better than that of
the 4-O-(2,6-dimethylphenyl)-2′,3′-O-(1,1,3,3-tetraisopro-
pyldisilane-1,3-diyl)-2-thiouridine intermediate, since the
synthesis of this compound requires an additional two
reactions for introduction and removal of a protecting
group at the 2′-position to avoid side reactions during the
2-thionation. The 2-thiocarbonyl group of the 2-thoiuracil
base moiety might be more reactive to electrophiles such
as alkyl halides than the 2-carbonyl group of the uracil
base moiety. Therefore, in this study, we selected the
intermediate 5 as the common precursor and the 2′-O-
alkylation reactions were carried out before the 2-thion-
ation.
Syn th esis of 2′-O-Allyl-2-th iou r idin e (3c: s2Uallyl).
Sproat and colleagues reported the synthesis of 2′-O-
allylated nucleosides.14 2′-O-Allyl nucleosides are potent
antisense molecules because of their high nuclease
resistance. Moreover, the allyl intermediate 9 could be
converted into other 2′-O-alkyl compounds.15 Therefore,
we synthesized 2′-O-allyl-2-thiouridine. The fully pro-
tected uridine derivative 9 was synthesized by treatment
of 5 with allyl ethyl carbonate in the presence of a tris-
(13) Sekine, M. J . Org. Chem. 1989, 54, 2321.
Syn th esis of 2′-O-Meth yl-2-th iou r idin e (3b: s2Um ).
First, the 3′- and 5′-hydroxyl groups of uridine were
protected by use of the 1,1,3,3-tetraisopropyldisiloxane-
(14) (a) Sproat, B. S.; Iribarren, A.; Beijer, B.; Pieles, U.; Lamond,
A. I. Nucleosides Nucleotides 1991, 10, 25. (b) Sproat B. S.; Iribarren,
A. M.; Garcia, R. G.; Beijer, B. Nucleic Acids Res. 1991, 19, 733.
(15) Maglott, E.; Glick, G. D. Nucleic Acids Res. 1998, 26, 1301.
J . Org. Chem, Vol. 68, No. 26, 2003 9973

Okamoto et al.
SCHEME 2. Syn th esis of s2Um a
a
Reagents and conditions: (i) (a) TPSCl, Bu4NBr in CH2Cl2/Na2CO3 aq, (b) 2,6-dimethylphenol, Et3N, 1,4-diazabicyclo-2,2,2-octane in
CH3CN, 92%; (ii) NaH, CH3I in DMF, 84%; (iii) P2S5, K2CO3 in toluene, 77%; (iv) syn-o-nitrobenzaldoxime, TMG in CH3CN, 70%; (v)
Bu4NF in THF, 99%.
SCHEME 3. Syn th esis of s2Ua llya
a
Reagents and conditions: (i) allyl ethyl carbonate, PPh3, tris(dibenzylideneacetone)-dipalladium(0) in THF, 97%; (ii) Lawesson’s reagent
in toluene, 77%; (iii) AcOH, Bu4NF in THF, 100%; (iv) syn-o-nitrobenzaldoxime, TMG in CH3CN, 77%.
(dibenzylideneacetone)-dipalladium(0) complex.16 The
2-thionation of the uridine derivative 9 was carried out
by using Lawesson’s reagent to give the 2′-O-allyl product
10 in good yield. The usual deprotection of 10 gave the
desired product 3c in an overall yield of 53% from the
4-O-protected uridine derivative 5 via intermediate 11,
as shown in Scheme 3.
Syn th esis of 2′-O-Hyd r oxyeth yl-2-th iou r id in e
(3d : s2Uh eth yl). Next, we synthesized the 2′-O-hy-
droxyethyl-2-thiouridine. Oxidation of the 2′-O-allyl prod-
uct 9 with OsO4 in the presence of N-methylmorpholine
N-oxide gave the diol 12, which, in turn, was allowed to
react with NaIO4. The resulting aldehyde 13 was reduced
by treatment with NaBH4 to afford the saturated alcohol
14. Treatment of 14 with Ac2O in pyridine gave the
acetate 15. A similar 2-thionation of 15 gave the product
16 in good yield. Thus, the desired product 3d could be
(16) (a) Lakhmiri, R.; Lhoste, P.; Sinou, D. Tetrahedron Lett. 1989,
30, 4669. (b) Kumar, V.; Gopalakrishnan, V.; Ganesh, K. N. Bull. Chem.
Soc. J pn. 1992, 65, 1665.
9974 J . Org. Chem., Vol. 68, No. 26, 2003

A New Route to 2′-O-Alkyl-2-thiouridine Derivatives
SCHEME 4. Syn th esis of s2Uh eth yla
a
Reagents and conditions: (i) OsO4, NMO in acetone/water, 100%; (ii) NaIO4 in dioxane/water, 98%; (iii) NaBH4 in MeOH, 86%; (iv)
Ac2O in pyridine, 91%; (v) Lawesson’s reagent in toluene, 86%; (vi) syn-o-nitrobenzaldoxime, TMG in CH3CN, 76%; (vii) AcOH, Bu4NF in
THF, 99%; (viii) conc NH3, 99%.
successfully synthesized after deprotection in an overall
yield of 44% from uridine derivative 5 via intermediates
17 and 18 (Scheme 4).
uridine derivative 21. The successive 2-thionation of 21
was achieved in the same manner as described for
compound 10. Finally, both the TBDMS and 2,6-dimeth-
ylphenyl groups were removed from 22 via a two-step
procedure in the usual manner. Thus, the desired product
3e could be synthesized in an overall yield of 48% from
2′-O-(methoxyethyl)uridine 19 (Scheme 5).
1H NMR An a lysis of 2′-O-Alk yl-2-th iou r id in es. To
study the conformational properties of 2′-O-alkyl-2-
thiouridine derivatives, the 1H NMR spectra of these
nucleosides were measured in sodium phosphate buffer
(pH 7.0). The ratios of the C3′-endo and C2′-endo con-
formers of these nucleosides were calculated by using the
coupling constants J 1′2′ and J 3′4′. These results are shown
in Table 2.
The % N (C3′-endo) values of 3d and 3e were calculated
to be 76% and 72%, respectively, while those of s2U and
s2Um reported previously were 70% each.2b,5 These
results indicated that the gauche effect of the 2′-O-
hydroxyethyl group and 2′-O-methoxyethyl group leads
to predominance of the C3′-endo conformer of 3d and 3e.
On the other hand, the 2′-O-allylation of s2U slightly
reduced the % N value.
Syn th esis of 2′-O-Meth oxyeth yl-2-th iou r id in e
(3e: s2Um oe). It was reported that oligoribonucleotides
containing 2′-O-methoxyethylated nucleosides form stable
RNA duplexes and have nuclease resistance.17 Therefore,
one can expect that more favorable antisense oligonucle-
otides could be created by a combination of the 2′-O-
methoxyethyl group and the 2-thiouracil residue. Our
interest was focused on this possibility.
Reese and colleagues reported a convenient method for
the synthesis of 2′-O-(methoxyethyl)uridine 19 via a ring-
opening substitution of 2,2′-anhydrouridine with metal
methoxyethoxides.17 Therefore, 2′-O-methoxyethyl-2-
thiouridine was synthesized by a series of reactions from
19 as follows: Silylation of 2′-O-(methoxyethyl)uridine
19 followed by 4-O-protection of the intermediate 20 with
the 2,4-dimethylphenyl group gave the fully protected
(17) (a) Martin, P. Helv. Chim. Acta 1995, 78, 486. (b) Altman, K.-
H.; Dean, N. M.; Fabbro, D.; Freier, S. M.; Geiger, T.; Ha¨ner, R.;
Hu¨sken, D.; Martin, P.; Monia, B. P.; Mu¨ller, M.; Natt, F.; Nicklin, P.;
Phillips, J .; Pieles, U.; Sasmor, H.; Moser, H. E. Chimia 1996, 50, 168.
J . Org. Chem, Vol. 68, No. 26, 2003 9975

Okamoto et al.
SCHEME 5. Syn th esis of s2Um oea
a
Reagents and conditions: (i) TBDMSCl, imidazole in DMF, 86%; (ii) (a) TPSCl, Bu4NBr in CH2Cl2/Na2CO3 aq, (b) 2,6-dimethylphenol,
Et3N, 1,4-diazabicyclo-2,2,2-octane in CH3CN, 89%; (iii) Lawesson’s reagent in toluene; (iv) AcOH, Bu4NF in THF, 78% (2 steps); (v)
syn-o-nitrobenzaldoxime, TMG in CH3CN, 80%.
SCHEME 6. Syn th esis of P h osp h or a m id itesa
a
Reagents and conditions: (i) DMTrCl in pyridine; (ii) 2-cyanoethoxy-bis(diisopropylamino)-phosphine, diisopropylethylammonium
1H-tetrazolide in CH2Cl2.
TABLE 2. Cou p lin g Con sta n ts (Hz) a n d % N
Ua Umb s2Ua s2Umc s2Uallyl s2Uhethyl s2Umoe
sides, we synthesized the phosphoramidite building block
25c-e.
2′-O-Alkyl-2-thiouridine phosphoramidite building block
25c-e was synthesized in the usual manner (Scheme 6).
The 2′-O-methyl-2-thiouridne phosphoramidite 25b was
prepared by the method reported previously.5 Oligoribo-
nucleotides containing 2′-O-alkyl-2-thiouridine were syn-
thesized with use of commercially available PAC-
protected phosphoramidites and standard coupling condi-
tions except for the use of tert-butyl hydroperoxide
oxidation on a commercial DNA synthesizer.21 Release,
deprotection, and desilylation of each oligonucleotide
were achieved with aqueous ammonia-EtOH and 1 M
tetrabutylammonium fluoride in THF. The fully depro-
tected RNA was purified by anion-exchange HPLC. The
J 1′2′
J 3′4′
4.6 4.0
5.2 5.9
9.8 9.9
2.5
6.0
8.5
70
3.1
7.0
10.1
70
3.4
6.7
10.1
67
2.4
7.6
10.0
76
2.9
7.3
10.2
72
J 1′2′ + J 3′4′
% Nd
53
60
(C3′-endo)
a
b
These coupling constants were reported in ref 2b. These
coupling constants were reported in ref 19. c These coupling
constants were reported in ref 5. These values were calculated
d
according to the equation % N (C3′-endo) ) {J 1′H-2′H (Hz)/(J 1′H-2′H
(Hz) + J 3′H-4′H (Hz)} × 100.20
The C3′-endo predominance is a favorable property for
stable RNA duplex formation, because all ribose pucker-
ing modes of RNA duplexes are fixed in the C3′-endo
conformation and pre-fixation of ribose conformation in
this manner is entropically favorable. Next, we examined
the stability of RNA duplexes containing modified nu-
cleosides.
Syn th esis of 2′-O-Alk yl-2-th iou r id in e Con ta in in g
Oligor ibon u cleotid es. To evaluate the duplex stability
of oligoribonucleotides containing these modified nucleo-
(18) Legorburu, U.; Reese, C. B.; Song, Q. Tetrahedron 1999, 55,
5635.
(19) Davies, D. B. Prog. NMR Spectrosc. 1978, 12, 135.
(20) Davies, D. B.; Danyluk, S. S. Biochemistry 1974, 13, 4417.
(21) (a) Kumar, R. K.; Davis, D. R. J . Org. Chem. 1995, 60, 7726.
(b) Kumar, R. K.; Davis, D. R. Nucleosides Nucleotides 1997, 16, 1469.
(c) Kumar, R. K.; Davis, D. R. Nucleic Acids Res. 1997, 25, 1272.
9976 J . Org. Chem., Vol. 68, No. 26, 2003

A New Route to 2′-O-Alkyl-2-thiouridine Derivatives
TABLE 3. Meltin g Tem p er a tu r e An a lysis of RNA-RNA
Du p lex Con ta in in g 2-Th iou r id in e Der iva tives
other 2′-O-alkylated thiouridine derivatives are now
under way in this lab. In conclusion, oligoribonucleotides
incorporating 2-thiouridine derivatives would provide a
potential tool for the design of new antisense molecules.
U*
Ua
36.7 43.6
+6.9 +5.5
s2Ua s2Uma s2Uallyl s2Uhethyl s2Umoe
Exp er im en ta l Section
Tm (°C)
42.2
40.1
40.6
41.4
∆Tm (°C)b
+3.4
+3.9
+4.7
4-O-(2-Nit r op h en yl)-2′,3′,5′-O-t r is(ter t-b u t yld im et h yl-
silyl)u r id in e (1a ). 2′,3′,5′-O-Tris(tert-butyldimethylsilyl)uri-
dine (2.00 g, 3.41 mmol) was dissolved in dry CH2Cl2 (70 mL),
and Et3N (542 µL, 3.75 mmol), 2,4,6-triisopropylbenzensulfonyl
chloride (820 mg, 3.75 mmol), and 4-(dimethylamino)pyridine
(104 mg, 0.85 mmol) were added. After being stirred at room
temperature for 18 h, the mixture was diluted with CHCl3.
The CHCl3 solution was washed once with saturated NaHCO3
(aqueous) and twice with brine. The combined organic extracts
were dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was rendered anhydrous by repeated coevapora-
tion with dry pyridine. The residue was further coevaporated
with dry toluene and finally dissolved in dry CH3CN (35 mL).
A solution of 2-nitrophenol (474 mg, 3.41 mmol), Et3N (3.45
mL, 25 mmol), and 1,4-diazabicyclo[2,2,2]octane (38 mg, 0.34
mmol) in dry CH3CN (35 mL) was added. After being stirred
at room temperature for 30 min, the mixture was concentrated
in vacuo. The residue was dissolved in CHCl3. The CHCl3
solution was washed once with saturated NaHCO3 (aqueous)
and twice with brine. The combined organic extracts were
dried over Na2SO4, filtered, and concentrated in vacuo. The
residue was purified by column chromatography with hex-
anes-Et2O (hexanes-ethyl acetate in the case of 1b-d ) to
afford 1a as a foam (1.54 g, 64% yield): 1H NMR (270 MHz,
CDCl3) δ 0.02-0.20 (18H, m), 0.87-0.97 (27H, m), 3.77-4.15
(5H, m), 5.64 (1H, s), 6.12 (1H, d, J ) 7.26 Hz), 7.22-8.12
(4H, m), 8.62 (1H, d, J ) 7.26 Hz); 13C NMR (67.8 MHz, CDCl3)
δ -5.4, -5.1, -4.9, -4.0, -3.8, 18.2, 18.7, 26.0, 26.0, 26.2, 60.5,
68.6, 76.0, 82.8, 91.4, 94.0, 125.6, 125.9, 126.5, 134.7, 141.6,
145.0, 145.3, 154.8, 170.6. HRMS (ESI) m/z (M + H) calcd for
a
b
These values were reported in ref 5. ∆Tm ) (Tm value of a
modified duplex) - (Tm value of a wild-type duplex).
oligoribonucleotides obtained were characterized by MAL-
DI-TOF mass spectrometry.
Stu d y of RNA Du p lex Sta bility. The hybridization
properties of the modified oligoribonucleotides toward
their complementary strands were evaluated by melting
temperature (Tm) experiment. These results are shown
in Table 3.
These data showed that all 2′-O-alkyl-2-thiouridine
derivatives enhanced the hybridization affinity for the
complementary RNA strand. The RNA oligomer contain-
ing 2-thiouridine 3a formed the most stable RNA duplex.
The RNA oligomer containing 2′-O-methyl-2-thiuridine
3b formed the second most stable RNA duplex. The order
of the stability of the modified RNA oligomers tested was
2′-hydroxyl > 2′-O-methyl> 2′-O-methoxyethyl > 2′-O-
hydroxyethyl > 2′-O-allyl.
Although the C3′-endo predominance of 3d and 3e was
much larger than that of 3a and 3b, the stability of RNA
duplexes was slightly decreased. It can be assumed that
increase of the hydrophobicity of the alkyl group results
in disturbance of the hydrogen bonding network around
the 2′-position so that RNA duplexes are significantly
destabilized. Despite the loss of the hydrogen bonding
network, the RNA oligomers containing these modified
nucleosides were still capable of formation of more stable
RNA-RNA duplexes than the unmodified RNA oligomer.
These results indicated that the 2-thiocarbonyl group
potentially affects stabilization of RNA duplexes.
C
33H58N3O8Si3 708.3536, foun: 708.3580.
4-O-P h en yl-2′,3′,5′-O-t r is(ter t-b u t yld im et h ylsilyl)u r i-
d in e (1b): 1H NMR (270 MHz, CDCl3) δ 0.03-0.23 (18H, m),
0.86-0.95 (27H, m), 3.77-4.15 (5H, m), 5.65 (1H, s), 6.00 (1H,
d, J ) 7.25 Hz), 7.11-7.37 (5H, m), 8.55 (1H, d, J ) 7.25 Hz);
13C NMR (67.8 MHz, CDCl3) δ -5.5, -5.1, -5.1, -5.0, -4.0,
-3.9, 18.2, 18.7, 25.9, 26.0, 26.2, 60.4, 68.5, 76.0, 82.6, 91.3,
94.4, 121.7, 125.6, 129.4, 144.4, 151.5, 155.2, 171.2. HRMS
(ESI) m/z (M + H) calcd for C33H59N2O6Si3 663.3678, found
663.3679.
Con clu sion
4-O-(2,6-Dich lor op h n e yl)-2′,3′,5′-O-t r is(ter t-b u t yld i-
m eth ylsilyl)u r id in e (1c): 1H NMR (270 MHz, CDCl3) δ
0.05-0.19 (18H, m), 0.87-0.98 (27H, m), 3.77-4.15 (5H, m),
5.69 (1H, s), 6.13 (1H, d, J ) 7.25 Hz), 7.07-7.32 (3H, m),
8.58 (1H, d, J ) 7.25 Hz,); 13C NMR (67.8 MHz, CDCl3) δ -5.4,
-5.1, -5.0, -4.9, -4.1, -3.9, 18.2, 18.2, 18.7, 26.0, 26.2, 60.6,
68.8, 76.1, 82.9, 91.3, 91.31, 93.6, 127.0, 128.6, 128.9, 144.7,
We have developed a new method for the synthesis of
2-thiouridine derivatives 3b-e via 4-O-protection. 2′-O-
Methyl, 2′-O-allyl, 2′-O-hydroxyethyl, and 2′-O-methoxy-
ethyl 2-thiouridine derivatives were successfully synthe-
sized with our method. The thermal stabilities of
oligoribonucleotides containing 2′-O-alkyl-2-thiouridine
derivatives were also studied. The melting temperature
analysis of oligoribonucleotides containing 2-thiouridine
derivatives suggested that the 2-thiocarbonyl group is an
essential factor for the stabilization of RNA-RNA du-
plexes. Even if unfavorable hydrophobic substituents are
introduced into the 2′-hydroxyl position, the 2-thionation
can compensate the destabilization arising from this
substitution effect. Although we did not analyze the base
pair selectivity of oligoribonucleotides having 2′-O-alkyl-
2-thiouridine derivatives in this study, our previous
paper5 on melting temperature studies of RNA duplexes
containing s2Um strongly indicated that the 2′-O-methy-
lation of 2-thiouridine does not affect the original base
recognition ability of 2-thiouridine that can form precisely
a W-C base pair only with the adenine base. Further
more detailed studies of the base recognition ability of
145.2, 154.9, 169.4. HRMS (ESI) m/z (M + H) calcd for C33H57
Cl2N2O6Si3 731.2901, found 731.2917.
-
4-O-(2,6-Dim et h ylp h n eyl)-2′,3′,5′-O-t r is(ter t-b u t yld i-
m eth ylsilyl)u r id in e (1d ): 1H NMR (270 MHz, CDCl3) δ
0.06-0.21 (18H, m), 0.89-0.98 (27H, m), 2.13 (6H, s), 3.79-
4.16 (5H, m), 5.72 (1H, s), 6.03 (1H, d, J ) 7.26 Hz), 7.02 (3H,
s), 8.62 (1H, d, J ) 7.26 Hz); 13C NMR (67.8 MHz, CDCl3) δ
-5.4, -5.1, -5.0, -4.9, -4.1, -3.9, 16.6, 16.6, 18.1, 18.2, 18.7,
25.9, 26.2, 60.7, 68.7, 76.1, 82.9, 91.1, 91.1, 93.7, 125.6, 128.5,
130.1, 144.5, 149.0, 155.4, 170.4. HRMS (ESI) m/z (M + H)
calcd for C35H63N2O6Si3 691.3994, found 691.3952.
Gen er a l P r oced u r e for t h e 2-Th ion a t ion of 4-O-
P r otected Ur id in e Der iva tives (2a -d ). An appropriate 4-O-
protected uridine derivative 1a -d (1 mmol) was dissolved in
anhydrous toluene (10 mL). Lawesson’s reagent (607 mg, 1.5
mmol) was added. After being stirred under reflux for 2 h, the
mixture was cooled to room temperature. The insoluble
materials were filtered off, and the filtrate was concentrated
J . Org. Chem, Vol. 68, No. 26, 2003 9977

Okamoto et al.
under reduced pressure. The residue was dissolved in CHCl3
and washed twice with saturated NaHCO3 (aqueous). The
combined organic extracts were dried over Na2SO4, filtered,
and concentrated in vacuo. The residue was purified by
chromatography with hexanes-Et2O to give the compound
2a -d as listed in Table 1.
4-O-(2-Nit r op h en yl)-2′,3′,5′-O-t r is(ter t-b u t yld im et h yl-
silyl)-2-th iou r id in e (2a ): 1H NMR (270 MHz, CDCl3) δ 0.03-
0.18 (18H, m), 0.87-0.98 (27H, m), 3.82-4.35 (5H, m), 6.21
(1H, s), 6.40 (1H, d, J ) 7.58 Hz), 7.32-7.43 (2H, m), 7.63-
7.68 (1H, m), 8.09 (1H, m), 8.98 (1H, d, J ) 7.58 Hz); 13C NMR
(67.8 MHz, CDCl3) δ -5.4, -4.9, -4.9, -4.6, -3.7, -2.9, 18.3,
18.4, 18.8, 26.1, 26.3, 26.3, 60.2, 68.4, 76.0, 82.7, 95.1, 98.8,
125.4, 126.0, 126.7, 144.7, 164.8, 180.9. HRMS (ESI) m/z (M
+ H) calcd for C33H58N3O7SSi3 724.3303, found 724.3379.
144.5, 149.1, 155.3, 171.0. Anal. Calcd for C29H46N2O7Si2: C,
58.95; H, 7.85; N, 4.74. Found: C, 58.98; H, 7.74; N, 4.83.
2′-O-Met h yl-4-O-(2,6-d im et h ylp h en yl)-3′,5′-O-(1,1,3,3-
tetr a isop r op yld isiloxa n e-1,3-d iyl)u r id in e (6). Compound
5 (591 mg, 1 mmol) was rendered anhydrous by repeated
coevaporation with dry pyridine. The residue was further
coevaporated with dry toluene and finally dissolved in dry
DMF (10 mL). CH3I (311 µL, 5 mmol) and NaH (80 mg, 2
mmol) were added, and the mixture was stirred at room
temperature for 10 min. The mixture was quenched by
addition of acetic acid and diluted with ethyl acetate. The ethyl
acetate solution was washed once with saturated NaHCO3
(aqueous) and twice with brine. The combined organic extracts
were dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by chromatography with hexanes-
ethyl acetate to afford 6 as a white foam (507 mg, 84% yield):
1H NMR (270 MHz, CDCl3) δ 0.99-1.12 (28H, m), 2.12 (6H,
s), 3.69 (3H, s), 3.83-4.30 (5H, m), 5.80 (1H, s), 6.03 (1H, d, J
) 7.26 Hz), 7.05 (3H, s), 8.30 (1H, d, J ) 7.26 Hz); 13C NMR
(67.8 MHz, CDCl3) δ 12.3, 12.9, 13.0, 13.4, 16.4, 16.8, 16.9,
17.0, 17.3, 17.4, 17.5, 59.1, 59.5, 67.8, 81.7, 83.2, 89.4, 93.6,
125.9, 128.7, 130.2, 144.1, 149.2, 155.3, 171.0. Anal. Calcd for
4-O-P h en yl-2′,3′,5′-O-tr is(ter t-bu tyldim eth ylsilyl)-2-th io-
1
u r id in e (2b): H NMR (270 MHz, CDCl3) δ 0.03-0.23 (18H,
m), 0.92-1.01 (27H, m), 3.86-4.43 (5H, m), 6.29 (1H, s), 6.31
(1H, d, J ) 7.58 Hz), 7.19-7.45 (5H, m), 8.96 (1H, d, J ) 7.58
Hz); 13C NMR (67.8 MHz, CDCl3) δ -5.5, -5.0, -5.0, -4.7,
-3.7, -2.9, 18.2, 18.4, 18.7, 26.1, 26.1, 26.2, 60.1, 68.3, 75.9,
82.6, 95.0, 99.0, 121.4, 125.9, 129.5, 145.9, 151.5, 165.4, 181.1.
HRMS (ESI) m/z (M + H) calcd for C33H59N2O5SSi3 679.3452,
found 679.3387.
C
30H48N2O7Si2: C, 59.57; H, 8.00; N, 4.63. Found: C, 59.64;
H, 8.10; N, 4.84.
2′-O-Met h yl-4-O-(2,6-d im et h ylp h en yl)-3′,5′-O-(1,1,3,3-
tetr a isop r op yld isiloxa n e-1,3-d iyl)-2-th iou r id in e (7). Com-
pound 6 (302 mg, 0.5 mmol) was rendered anhydrous by
repeated coevaporation with dry pyridine. The residue was
further coevaporated with dry toluene and finally dissolved
in dry toluene (10 mL). Phosphorus pentasulfide (144 mg, 0.65
mmol) and K2CO3 (415 mg, 3 mmol) were added. The resulting
mixture was refluxed for 3 h and then cooled to room
temperature. The insoluble materials were filtered. The filtrate
was concentrated in vacuo, and the residue was dissolved in
CHCl3. The CHCl3 solution was washed once with saturated
NaHCO3 (aqueous) and twice with brine. The combined organic
extracts were dried over Na2SO4, filtered, and concentrated
in vacuo. The residue was purified by chromatography with
hexanes-ethyl acetate to afford 6 as a pale yellow foam (239
mg, 77% yield): 1H NMR (270 MHz, CDCl3) δ 1.00-1.11 (28H,
m), 2.13 (6H, s), 3.77 (3H, s), 3.96-4.30 (5H, m), 6.21 (1H, d,
J ) 7.26 Hz), 6.34 (1H, s), 7.07 (3H, s), 8.54 (1H, d, J ) 7.26
Hz); 13C NMR (67.8 MHz, CDCl3) δ 12.4, 12.8, 13.0, 13.4, 16.4,
16.8, 16.9, 17.0, 17.2, 17.4, 59.4, 60.3, 68.1, 82.2, 82.7, 93.8,
97.7, 126.2, 128.9, 130.1, 145.5, 149.0, 165.5, 181.1. Anal. Calcd
for C30H48N2O6SSi2: C, 58.03; H, 7.79; N, 4.51; S, 5.16.
Found: C, 57.94; H, 7.97; N, 4.61; S, 4.97.
4-O-(2,6-Dich lor op h n e yl)-2′,3′,5′-O-t r is(ter t-b u t yld i-
m eth ylsilyl)-2-th iou r id in e (2c): 1H NMR (270 MHz, CDCl3)
δ 0.05-0.19 (18H, m), 0.82-0.98 (27H, m), 3.82-4.38 (5H, m),
6.26 (1H, s), 6.40 (1H, d, J ) 7.26 Hz), 7.11-7.36 (3H, m),
8.94 (1H, d, J ) 7.26 Hz); 13C NMR (67.8 MHz, CDCl3) δ -5.4,
-4.9, -4.6, -3.7, -2.9, 18.3, 18.5, 18.8, 26.0, 26.1, 26.2, 26.3,
60.3, 68.6, 76.1, 77.5, 82.8, 95.0, 98.1, 127.2, 128.8, 128.8, 144.6,
146.6, 163.7, 181.2. HRMS (ESI) m/z (M + H) calcd for C33H57
Cl2N2O5SSi3 747.2673, found 747.2776.
-
4-O-(2,6-Dim et h ylp h en yl)-2′,3′,5′-O-t r is(ter t-b u t yld i-
m eth ylsilyl)-2-th iou r id in e (2d ): 1H NMR (270 MHz, CDCl3)
δ 0.03-0.17 (18H, m), 0.82-1.07 (27H, m), 2.12 (6H, s), 3.81-
4.31 (5H, m), 6.17 (1H, d, J ) 7.59 Hz), 6.29 (1H, s), 7.05 (3H,
s), 8.87 (1H, d, J ) 7.59 Hz); 13C NMR (67.8 MHz, CDCl3) δ
-5.5, -5.1, -5.0, -4.7, -3.8, -3.0, 16.6, 16.7, 18.2, 18.4, 18.6,
26.0, 26.1, 60.3, 68.5, 76.0, 82.7, 94.8, 94.9, 97.5, 126.0, 128.7,
130.0, 146.1, 148.9, 164.9, 181.5. HRMS (ESI) m/z (M + H)
calcd for C35H63N2O5SSi3 707.3765, found 707.3782.
4-O -(2,6-D im e t h y lp h e n y l)-3′,5′-O -(1,1,3,3-t e t r a is o -
p r op yld isiloxa n e-1,3-d iyl)u r id in e (5). To a solution of 3′,5′-
O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)uridine 4 (4.86 g,
10 mmol) in CH2Cl2 (200 mL) were added Na2CO3 (0.2 M
solution 400 mL), tetrabutylammnium bromide (1.29 g, 4
mmol), and 2,4,6-triisopropylbenzenesulfonyl chloride (3.94 g,
13 mmol). The resulting two-phase solution was stirred
vigorously at room temperature for 15 h. The organic phase
was collected, and the aqueous phase was washed twice with
CH2Cl2. The combined organic extracts were dried over Na2-
SO4, filtered, and concentrated in vacuo. The residue was
rendered anhydrous by repeated coevaporation with dry
pyridine. The residue was further coevaporated with dry
toluene and finally dissolved in dry CH3CN (100 mL). A
solution of 2,6-dimethylphenol (1.2 g, 10 mmol), Et3N (4.1 mL,
30 mmol), and 1,4-diazabicyclo[2,2,2]octane (112 mg, 1 mmol)
in dry CH3CN (100 mL) was added. After being stirred at room
temperature for 30 min, the mixture was concentrated in
vacuo. The residue was dissolved in CHCl3. The CHCl3 solution
was washed once with saturated NaHCO3 (aqueous) and twice
with brine. The combined organic extracts were dried over Na2-
SO4, filtered, and concentrated in vacuo. The residue was
purified by chromatography with hexanes-ethyl acetate to
afford 5 as a white foam (5.4 g, 92% yield): 1H NMR (270 MHz,
CDCl3) δ 0.97-1.07 (28H, m), 2.09 (6H, s), 3.31 (1H, br s),
3.95-4.35 (5H, m), 5.71 (1H, s), 6.02 (1H, d, J ) 7.26 Hz),
7.01 (3H, s), 8.10 (1H, d, J ) 7.26 Hz); 13C NMR (67.8 MHz,
CDCl3) δ 12.4, 12.5, 12.7, 12.9, 13.3, 16.4, 16.8, 16.8, 16.9, 17.2,
17.3, 17.4, 60.3, 68.8, 74.8, 82.0, 92.2, 94.0, 125.9, 128.7, 130.1,
2′-O-Met h yl-3′,5′-O-(1,1,3,3-t et r a isop r op yld isiloxa n e-
1,3-d iyl)-2-th iou r id in e (8). Compound 7 (700 mg, 1.13 mmol)
was rendered anhydrous by repeated coevaporation with dry
pyridine. The residue was further coevaporated with dry
toluene and finally dissolved in dry CH3CN (5 mL). A solution
of 1,1,3,3-tetramethylguanidine (428 µL, 3.4 mmol) and syn-
o-nitrobenzaldoxime (565 mg, 3.4 mmol) in CH3CN (5 mL) was
added. After being stirred at room temperature for 3 h, the
mixture was concentrated in vacuo, and the residue was
dissolved in CHCl3. The CHCl3 solution was washed once with
saturated NaHCO3 (aqueous) and twice with brine. The
combined organic extracts were dried over Na2SO4, filtered,
and concentrated in vacuo. The residue was purified by
chromatography with hexanes-Et2O to afford 8 as a white
foam (426 mg, 70% yield): 1H NMR (270 MHz, CDCl3) δ 0.93-
1.11 (28H, m), 3.72 (3H, s), 3.88-4.30 (5H, m), 6.00 (1H, d, J
) 8.25 Hz), 6.25 (1H, s), 8.10 (1H, d, J ) 8.25 Hz), 10.82 (1H,
br s); 13C NMR (67.8 MHz, CDCl3) δ 12.4, 12.8, 13.2, 13.4, 17.0,
17.2, 17.5, 59.3, 60.2, 68.5, 82.1, 83.3, 92.6, 92.7, 106.1, 106.2,
140.4, 160.5, 174.6. HRMS (ESI) m/z (M + H) calcd for
C
22H41N2O6SSi2 517.2224, found 275.0701.
2′-O-Meth yl-2-th iou r id in e (3b). To a solution of compound
8 (168 mg, 0.33 mmol) in THF (3 mL) was added tetrabutyl-
ammonium fluoride (172 mg, 0.66 mmol). After being stirred
at room temperature for 15 min, the mixture was diluted with
9978 J . Org. Chem., Vol. 68, No. 26, 2003

A New Route to 2′-O-Alkyl-2-thiouridine Derivatives
CHCl3. The CHCl3 solution was washed with H2O. The
aqueous phase was collected and concentrated in vacuo. The
residue was purified by column chromatography on C-18 gel
and eluted with water-acetonitrile. The pure product 3b was
180.8. HRMS (ESI) m/z (M + H) calcd for C20H25N2O5S
405.1484, found 405.1473.
2′-O-Allyl-2-th iou r id in e (3c). Compound 11 (1.27 g, 3
mmol) was rendered anhydrous by repeated coevaporation
with dry pyridine. The residue was further coevaporated with
dry toluene and finally dissolved in dry CH3CN (20 mL). A
solution of 1,1,3,3-tetramethylguanidine (392 µL, 3.1 mmol)
and syn-o-nitrobenzaldoxime (518 mg, 3.1 mmol) in CH3CN
(20 mL) was added. After being stirred at room temperature
for 3 h, the mixture was diluted with Et2O. The Et2O solution
was washed twice with H2O, and the combined aqueous
extracts were concentrated in vacuo. The residue was purified
by chromatography with CHCl3-MeOH to afford 3c as a white
powder (690 mg, 77% yield): 1H NMR (400 MHz, D2O) δ 3.61
(1H, s), 3.82-3.98 (2H, m), 4.14-4.25 (2H, m), 4.29-4.33 (1H,
m), 6.17 (1H, d, J ) 8.25 Hz), 6.75 (1H, d, J ) 3.0 Hz), 8.10
(1H, d, J ) 8.08 Hz); 13C NMR (67.8 MHz, DMSO) δ 60.4, 68.7,
71.8, 82.0, 85.4, 91.8, 107.3, 117.7, 135.3, 141.8, 160.8, 176.3.
HRMS (ESI) m/z (M + H) calcd for C12H17N2O5S 301.0858,
found 301.0901.
2′-O-(2,3-Dih yd r oxyp r op yl)-4-O-(2,6-d im eth ylp h en yl)-
3′,5′-O-(1,1,3,3-t e t r a isop r op yld isiloxa n e -1,3-d iyl)u r i-
d in e (12). Compound 9 (2.67 g, 4.23 mmol) was dissolved in
a mixture of acetone-H2O (40 mL, 6:1, v/v). N-Methylmor-
pholine N-oxide (1.09 g, 9.31 mmol) and OsO4 (21 mg, 0.085
mmol) were added. After the mixture was stirred in the dark
at room temperature for 20 h, saturated sodium bisulfate (1
mL) was added. The resulting osmium salts were filtered off.
The filtrate was diluted with Et2O. The ethereal solution was
washed twice with saturated NaHCO3 (aqueous). The com-
bined organic extracts were dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by chroma-
tography with CHCl3-MeOH to afford 12 as a white foam (2.81
g, 100% yield): 1H NMR (270 MHz, CDCl3) δ 0.92-1.11 (28H,
m), 2.12 (6H, s), 3.49-4.30 (9H, m), 5.76 (1H, s), 6.06 (1H, d,
J ) 7.58 Hz), 7.04 (3H, s), 8.22 (1H, d, J ) 7.58 Hz); 13C NMR
(67.8 MHz, CDCl3) δ 12.6, 12.7, 12.9, 12.9, 13.0, 13.5, 16.5,
16.9, 17.0, 17.1, 17.3, 17.3, 17.4, 17.5, 59.3, 63.5, 63.9, 67.8,
67.9, 70.3, 70.3, 73.2, 74.2, 81.8, 81.8, 82.8, 83.1, 89.9, 90.2,
94.0, 125.9, 128.6, 130.0, 143.5, 149.0, 155.3, 171.0. HRMS
(ESI) m/z (M + H) calcd for C32H53N2O9Si2 665.3289, found
665.3234.
2′-O-(2-Oxoe t h yl)-4-O-(2,6-d im e t h ylp h e n yl)-3′,5′-O-
(1,1,3,3-tetr a isop r op yld isiloxa n e-1,3-d iyl)u r id in e (13). To
a solution of compound 12 (3.7 g, 5.56 mmol) in 1,4-dioxane:
H2O (60 mL, 3:1) was added NaIO4 (1.43 g, 6.67 mmol). After
being stirred in the dark at room temperature overnight, the
mixture was diluted with Et2O. The ethereal solution was
washed twice with saturated NaHCO3. The combined organic
extracts were dried over Na2SO4, filtered, and concentrated
in vacuo. The residue was purified by chromatography with
CHCl3-MeOH to afford 13 as a white foam (3.44 g, 98%
yield): 1H NMR (270 MHz, CDCl3) δ 0.86-1.11 (28H, m), 2.12
(6H, s), 3.95-4.58 (7H, m), 5.78 (1H, s), 6.05 (1H, d, J ) 7.58
Hz), 7.04 (3H, s), 8.25 (1H, d, J ) 7.25 Hz), 9.78 (1H, s); 13C
NMR (67.8 MHz, CDCl3) δ 12.4, 13.0, 13.5, 16.5, 16.5, 16.9,
16.9, 17.0, 17.0, 17.1, 17.4, 17.5, 17.5, 59.4, 68.0, 76.2, 81.8,
82.9, 89.8, 93.9, 125.9, 128.6, 130.0, 130.0, 143.7, 149.0, 155.2,
171.0, 200.4. HRMS (ESI) m/z (M + H) calcd for C31H49N2O8-
Si2 633.3027, found 633.3018.
2′-O-H yd r oxyet h yl-4-O-(2,6-d im et h ylp h en yl)-3′,5′-O-
(1,1,3,3-tetr a isop r op yld isiloxa n e-1,3-d iyl)u r id in e (14). To
a solution of compound 13 (1.88 g, 2.97 mmol) in CH3OH (30
mL) was added NaBH4 (34 mg, 0.89 mmol). After being stirred
at room temperature overnight, the mixture was diluted with
Et2O and washed once with saturated NaHCO3 and twice with
brine. The combined organic extracts were dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was purified
by chromatography with hexanes-ethyl acetate to afford 14
as a white foam (1.62 g, 86% yield):1H NMR (270 MHz, CDCl3)
δ 0.86-1.11 (28H, m), 2.12 (6H, s), 3.69-4.31 (9H, m), 5.77
(1H, s), 6.05 (1H, d, J ) 7.26 Hz), 7.04 (3H, s), 8.24 (1H, d, J
1
obtained as a white solid (90 mg, 99%): H NMR (400 MHz,
D2O) δ 3.61 (3H, s), 3.82-4.01 (2H, m), 4.06-4.17 (2H, m),
4.29-4.33 (1H, m), 6.17 (1H, d, J ) 8.25 Hz), 6.75 (1H, d, J )
3.0 Hz), 8.10 (1H, d, J ) 8.08 Hz); 13C NMR (67.8 MHz, DMSO)
δ 58.4, 59.4, 67.8, 83.5, 84.6, 90.8, 106.5, 140.6, 159.3, 175.6.
HRMS (ESI) m/z (M + H) calcd for C10H15N2O5S 275.0701,
found 275.0691.
2′-O-Allyl-4-O-(2,6-d im eth ylp h en yl)-3′,5′-O-(1,1,3,3-tet-
r a isop r op yld isiloxa n e-1,3-d iyl)u r id in e (9). Compound 5
(590 mg, 1 mmol) was rendered anhydrous by repeated
coevaporation with dry pyridine. The residue was further
coevaporated with dry toluene and finally dissolved in dry THF
(10 mL). Triphenylphosphine (52 mg, 0.2 mmol) and tris-
(dibenzylideneacetone)-dipalladium(0) (18 mg, 0.02 mmol)
were added. Fianlly, allyl ethyl carbonate (263 µL, 2 mmol)
was added dropwise with stirring under argon, and the
reaction mixture was refluxed for 30 min. The reaction mixture
was cooled to room temperature and concentrated in vacuo.
The residue was purified by column chromatography on silica
gel with hexanes-ethyl acetate. The pure product 9 was
1
obtained as a white foam (614 mg, 97%): H NMR (270 MHz,
CDCl3) δ 0.91-1.12 (28H, m), 2.12 (6H, s), 3.95-4.01 (2H, m),
4.12-4.45 (5H, m), 5.14-5.44 (2H, m), 5.78 (1H, s), 5.88-5.98
(1H, m), 6.00 (1H, d, J ) 7.58 Hz), 7.04 (3H, s), 8.10 (1H, d, J
) 7.58 Hz); 13C NMR (67.8 MHz, CDCl3) δ 12.5, 12.9, 13.0,
13.4, 16.4, 16.8, 16.9, 17.0, 17.3, 17.3, 17.4, 17.5, 59.5, 67.6,
71.0, 80.6, 81.7, 89.9, 93.4, 116.9, 125.7, 128.5, 130.0, 134.3,
143.8, 149.0, 155.1, 170.7. HRMS (ESI) m/z (M + H) calcd for
C
32H51N2O7Si3 631.3235, found 631.3329.
2′-O-Allyl-4-O-(2,6-d im eth ylp h en yl)-3′,5′-O-(1,1,3,3-tet-
r a isop r op yld isiloxa n e-1,3-d iyl)-2-th iou r id in e (10). Com-
pound 9 (2.47 g, 3.97 mmol) was rendered anhydrous by
repeated coevaporation with dry pyridine. The residue was
further coevaporated with dry toluene and finally dissolved
in dry toluene (40 mL). Lawesson’s reagent (2.37 g, 5.87 mmol)
was added, and the mixture was refluxed for 1 h. The mixture
was cooled to room temperature. The insoluble materials were
filtered, and the materials were washed twice with cooled
EtOH. The organic phase was concentrated in vacuo. The
residue was dissolved in hexane and washed three times with
brine. The combined organic extracts were dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was purified
by chromatography with hexanes-ethyl acetate to afford 10
as a pale yellow foam (1.95 g, 77% yield): 1H NMR (270 MHz,
CDCl3) δ 0.91-1.12 (28H, m), 2.12 (6H, s), 3.95-4.01 (2H, m),
4.12-4.45 (5H, m), 5.14-5.44 (2H, m), 5.78 (1H, s), 5.88-5.98
(1H, m), 6.00 (1H, d, J ) 7.58 Hz), 7.04 (3H, s), 8.10 (1H, d, J
) 7.58 Hz); 13C NMR (67.8 MHz, CDCl3) δ 12.6, 12.8, 13.1,
13.4, 16.5, 16.8, 16.9, 17.0, 17.0, 17.2, 17.3, 17.4, 17.5, 59.4,
67.8, 72.3, 80.4, 82.2, 94.0, 97.4, 117.0, 126.0, 128.8, 130.0,
134.7, 145.4, 148.8, 165.3. HRMS (ESI) m/z (M + H) calcd for
C
32H51N2O6SSi3 647.3006, found 647.3004.
2′-O-Allyl-4-O-(2,6-d im eth ylp h en yl)-2-th iou r id in e (11).
To a solution of compound 10 (1.95 g, 3.01 mmol) in THF were
added tetrabutylammonium fluoride (1.97 g, 7.53 mmol) and
acetic acid (431 µL, 7.53 mmol). After being stirred at room
temperature for 10 min, the mixture was diluted with CHCl3.
The CHCl3 solution was washed twice with saturated NaHCO3.
The combined organic extracts were dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was purified
by chromatography with hexanes-ethyl acetate to afford 11
as a pale yellow foam (808 mg, 100% yield):1H NMR (270 MHz,
CDCl3) δ 2.12 (6H, s), 3.90-4.24 (5H, m), 4.47-4.83 (2H, dd),
5.20-5.39 (2H, m), 5.88-6.00 (1H, m), 6.24 (1H, d, J ) 7.58
Hz), 6.63 (1H, s), 7.06 (3H, s), 8.99 (1H, d, J ) 7.58 Hz); 13C
NMR (67.8 MHz, CDCl3) δ 16.6, 59.0, 66.6, 72.4, 81.2, 84.1,
93.1, 98.0, 118.1, 126.2, 128.9, 130.1, 133.8, 146.7, 148.8, 165.4,
J . Org. Chem, Vol. 68, No. 26, 2003 9979

Okamoto et al.
) 7.25 Hz); 13C NMR (67.8 MHz, CDCl3) δ 12.3, 13.0, 13.1,
13.6, 16.5, 16.8, 17.0, 17.0, 17.1, 17.4, 17.5, 17.5, 51.8, 59.5,
67.3, 67.7, 81.6, 82.2, 89.4, 93.7, 125.8, 128.6, 130.0, 143.9,
149.0, 155.2, 170.4, 170.9. HRMS (ESI) m/z (M + H) calcd for
room temperature for 15 min, the mixture was diluted with
CHCl3. The CHCl3 solution was washed once with pyridine-
H2O (1:1, v/v). The organic phase were collected and concen-
trated in vacuo. The residue was dissolved in ethyl acetate.
The desire product 19 was precipitated from the ethyl acetate
solution by addition of hexanes-Et2O (100 mL, 1:1, v/v). Then
precipitates were collected and dried to give the pure product
18 as a white powder (529 mg, 99%): 1H NMR (270 MHz,
CDCl3) δ 2.07 (3H, s), 3.94-4.37 (9H, m), 5.98 (1H, d, J ) 8.24
Hz), 6.45 (1H, s), 8.40 (1H, d, J ) 8.24 Hz); 13C NMR (67.8
MHz, DMSO) δ 21.6, 60.1, 64.0, 68.6, 69.4, 83.2, 85.2, 92.0,
107.2, 141.3, 160.6, 171.0, 176.5. HRMS (ESI) m/z (M + H)
calcd for C13H19N2O7S 347.0913, found 347.0859.
2′-O-Hyd r oxyeth yl-2-th iou r id in e (3d ). Compound 18 (52
mg, 0.15 mmol) was dissolved in CH3OH:28% aqueous NH3 (2
mL, 3:1, v/v). After being stirred at room temperature for 24
h, the mixture was diluted with Et2O and washed repeatedly
with H2O. The aqueous phase was collected and concentrated
in vacuo. The residue was purified by column chromatography
on C-18 gel with water-acetonitrile to afford 3d as a white
powder (45 mg, 99% yield): 1H NMR (400 MHz, D2O) δ 3.76-
3.88 (4H, m), 3.98-4.08 (2H, m), 4.16-4.22 (2H, m), 4.27-
4.30 (1H, dd), 6.16 (1H, d, J ) 8.09 Hz), 6.75 (1H, d, J ) 2.44
Hz), 8.12 (1H, d, J ) 8.08 Hz); 13C NMR (67.8 MHz, D2O) δ
59.8, 60.8, 68.2, 72.7, 82.3, 84.0, 92.0, 106.8, 141.8, 162.8, 175.9.
HRMS (ESI) m/z (M + H) calcd for C11H16N2O6S 305.0807,
found 305.0807.
C
31H51N2O8Si2 635.3184, found 635.3239.
2′-O-Acet oxylet h yl-4-O-(2,6-d im et h ylp h en yl)-3′,5′-O-
(1,1,3,3-t et r a isop r op yld isiloxa n e-1,3-d iyl)u r id in e (15).
Compound 14 (1.62 g, 2.55 mmol) was rendered anhydrous
by repeated coevaporation with dry pyridine and finally
dissolved in dry pyridine (25 mL). Ac2O (264 µL, 2.81 mmol)
was added. After being stirred at room temperature overnight,
the resulting mixture was concentrated in vacuo. The residue
was dissolved in Et2O and washed once with brine and twice
with saturated NaHCO3. The combined organic extracts were
dried over Na2SO4, filtered, and concentrated in vacuo. The
residue was purified by chromatography with hexanes-ethyl
acetate to afford 15 as a white foam (1.57 g, 91% yield): 1H
NMR (270 MHz, CDCl3) δ 0.80-1.12 (28H, m), 2.01 (3H, s),
2.09 (6H, s), 3.94-4.30 (9H, m), 5.73 (1H, s), 5.99 (1H, d, J )
7.58 Hz), 7.01 (3H, s), 8.24 (1H, d, J ) 7.25 Hz); 13C NMR
(67.8 MHz, CDCl3) δ 12.5, 12.9, 13.0, 13.4, 16.4, 16.9, 17.0,
17.0, 17.1, 17.3, 17.32, 17.4, 17.5, 20.9, 59.4, 63.4, 67.7, 69.0,
81.6, 81.9, 89.8, 93.5, 125.7, 128.5, 130.0, 143.8, 149.0, 155.1,
170.75, 170.8. HRMS (ESI) m/z (M + H) calcd for C33H53N2O9-
Si2 677.3289, found 677.3263.
2′-O-Acet oxylet h yl-4-O-(2,6-d im et h ylp h en yl)-3′,5′-O-
(1,1,3,3-t e t r a isop r op yld isiloxa n e -1,3-d iyl)-2-t h iou r i-
d in e (16). Compound 15 (1.57 g, 2.32 mmol) was rendered
anhydrous by repeated coevaporation with dry pyridine. The
residue was further coevaporated with dry toluene and finally
dissolved in dry toluene (50 mL). Lawesson’s reagent (1.41 g,
3.48 mmol) was added, and the mixture was refluxed for 1 h.
The mixture was cooled to room temperature. The insoluble
materials were filtered, and the filtration was twice washed
with cooled EtOH. The organic phase was concentrated in
vacuo. The residue was dissolved in hexane and washed three
times with brine. The combined organic extracts were dried
over Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by chromatography with hexane-CHCl3 to afford
16 as a pale yellow foam (1.39 g, 86% yield): 1H NMR (270
MHz, CDCl3) δ 0.91-1.12 (28H, m), 2.12 (6H, s), 3.95-4.01
(2H, m), 4.12-4.45 (5H, m), 5.14-5.44 (2H, m), 5.78 (1H, s),
5.88-5.98 (1H, m), 6.00 (1H, d, J ) 7.58 Hz), 7.04 (3H, s),
8.10 (1H, d, J ) 7.58 Hz); 13C NMR (67.8 MHz, CDCl3) δ 12.6,
12.8, 13.1, 13.4, 16.5, 16.8, 16.9, 16.96, 17.0, 17.2, 17.3, 17.4,
17.5, 59.4, 67.8, 72.3, 80.4, 82.2, 94.0, 97.4, 117.0, 126.0, 128.8,
130.0, 134.7, 145.4, 148.8, 165.3. HRMS (ESI) m/z (M + H)
calcd for C33H53N2O8SSi2 693.3061, found 693.3028
2′-O-Meth oxyeth yl-3′,5′-O-bis(ter t-bu tyld im eth ylsilyl)-
u r id in e (20). 2′-O-Methoxyethyluridine 19 was prepared by
Reese’s method.17 To a solution of compound 19 (1.51 g, 5
mmol) in dry DMF were added imidazole (851 mg, 12.5 mmol)
and tert-butyldimethylsilyl chloride (1.72 g, 11 mmol). After
being stirred at room temperature for 12 h, the reaction
mixture was diluted with ethyl acetate. The ethyl acetate
solution was washed once with brine and twice with saturated
NaHCO3. The combined organic extracts were dried over Na2-
SO4, filtered, and concentrated in vacuo. The residue was
purified by chromatography with hexanes-ethyl acetate to
afford 20 as a white solid (2.27 g, 86% yield): 1H NMR (270
MHz, CDCl3) δ 0.01-0.12 (12H, m), 0.90-0.95 (18H, m), 3.35
(3H, s), 3.55-4.24 (9H, m), 5.67 (1H, d, J ) 8.24 Hz), 5.94
(1H, d, J ) 1.98 Hz), 8.04 (1H, d, J ) 8.24 Hz), 9.69 (1H, br s);
13C NMR (67.8 MHz, CDCl3) δ -5.6, -5.4, -5.0, -4.6, 18.1,
18.4, 25.7, 25.9, 58.9, 60.7, 68.5, 69.7, 71.9, 83.1, 83.7, 87.7,
101.7, 140.0, 150.0, 163.5. HRMS (ESI) m/z (M + H) calcd for
C
24H47N2O7Si2 531.2922, found 531.2887.
2′-O-Met h oxyet h yl-4-O-(2,6-d im et h ylp h en yl)-3′,5′-O-
bis(ter t-bu tyld im eth ylsilyl)u r id in e (21). To a solution of
compound 20 (10.2 g, 19.2 mmol) in CH2Cl2 (200 mL) were
added Na2CO3 (0.2 M solution 400 mL), tetrabutylammnium
bromide (2.48 g, 7.68 mmol), and 2,4,6-triisopropylbenzene-
sulfonyl chloride (7.56 g, 24.96 mmol). The resulting two-phase
solution was stirred vigorously at room temperature overnight.
The organic phase was collected, and the aqueous phase was
washed twice with CH2Cl2. The combined organic extracts
were dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was rendered anhydrous by repeated coevapora-
tion with dry pyridine. The residue was further coevaporated
with dry toluene and finally dissolved in dry CH3CN (100 mL).
A solution of 2,6-dimethylphenol (2.55 g, 19.2 mmol), Et3N (8
mL, 57.6 mmol), and 1,4-diazabicyclo[2,2,2]octane (646 mg,
5.76 mmol) in dry CH3CN (100 mL) was added. After being
stirred at room temperature for 3 h, the mixture was concen-
trated in vacuo. The residue was dissolved in CHCl3. The
CHCl3 solution was washed once with saturated NaHCO3 and
twice with brine. The combined organic extracts were dried
over Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by chromatography with hexanes-ethyl acetate
to afford 21 as a white foam (10.8 g, 89% yield): 1H NMR (270
MHz, CDCl3) δ 0.04-0.14 (12H, m), 0.83-0.99 (18H, m), 2.11
(6H, s), 3.33 (3H, s), 3.54-3.58 (2H, m), 3.76-3.81 (3H, m),
4.06-4.18 (4H, m), 5.88 (1H, s), 6.00 (1H, d, J ) 7.25 Hz),
2′-O-Acet oxylet h yl-3′,5′-O-(1,1,3,3-t et r a isop r op yld isi-
loxa n e-1,3-d iyl)-2-th iou r id in e (17). Compound 16 (1.39 g,
2 mmol) was rendered anhydrous by repeated coevaporation
with dry pyridine. The residue was further coevaporated with
dry toluene and finally dissolved in dry CH3CN (20 mL). The
solution of 1,1,3,3-tetramethylguanidine (754 µL, 6 mmol) and
syn-o-nitrobenzealdoxime (997 mg, 6 mmol) in CH3CN (20 mL)
was added. After being stirred at room temperature for 5 h,
the mixture was diluted with Et2O. The Et2O solution was
washed twice with H2O, and the combined aqueous extracts
were concentrated in vacuo. The residue was purified by
chromatography with CHCl3-MeOH to afford 17 as a white
powder (900 mg, 76% yield): 1H NMR (270 MHz, CDCl3) δ
0.76-1.03 (28H, m), 2.01 (3H, s), 3.88-4.24 (9H, m), 5.88 (1H,
d, J ) 8.24 Hz), 6.16 (1H, s), 8.00 (1H, d, J ) 8.24 Hz), 10.28
(1H, s); 13C NMR (67.8 MHz, CDCl3) δ 12.7, 12.9, 13.18, 13.2,
13.5, 16.9, 17.0, 17.1, 17.14, 17.2, 17.3, 17.4, 17.5, 17.6, 59.3,
63.7, 68.6, 70.1, 82.2, 82.3, 92.8, 92.9, 106.1, 140.1, 159.9, 170.9,
174.4. HRMS (ESI) m/z (M + H) calcd for C25H45N2O8SSi2
589.2435, found 589.2420.
2′-O-Acet oxylet h yl-2-t h iou r id in e (18). Compound 17
(900 mg, 1.53 mmol) was dissolved in THF (15 mL) and
tetrabutylammonium fluoride (1.0 g, 3.83 mmol) and acetic
acid (219 µL, 3.83 mmol) were added. After being stirred at
9980 J . Org. Chem., Vol. 68, No. 26, 2003

A New Route to 2′-O-Alkyl-2-thiouridine Derivatives
7.00 (3H, s), 8.48 (1H, d, J ) 7.25 Hz); 13C NMR (67.8 MHz,
CDCl3) δ -5.4, -5.3, -4.9, -4.5, 16.6, 18.1, 18.5, 25.7, 26.1,
58.8, 60.2, 67.7, 69.7, 71.7, 77.5, 82.8, 83.0, 89.3, 93.7, 125.6,
128.5, 130.0, 144.3, 149.0, 155.2, 170.5. HRMS (ESI) m/z (M
+ H) calcd for C32H55N2O7Si2 635.3548, found 635.3458.
134.7, 136.0, 140.9, 144.1, 149.5, 158.5, 158.6, 159.6, 174.7.
HRMS (ESI) m/z (M + Na) calcd for C33H34N2NaO7S 625.1985,
found 625.1902.
2′-O-Acet oxylet h yl-5′-O-(4,4′-d im et h oxyt r it yl)-2-t h io-
u r id in e (24d ): 1H NMR (270 MHz, CDCl3) δ 2.08 (3H, s), 3.78
(6H, s), 3.92-4.54 (9H, m), 5.36 (1H, d, J ) 8.24 Hz), 6.46
(1H, s), 6.80-6.85 (4H, m), 7.24-7.37 (9H, m), 8.37 (1H, d, J
) 8.24 Hz), 9.76 (1H, br s); 13C NMR (67.8 MHz, CDCl3) δ 21.0,
55.2, 60.2, 63.2, 67.8, 70.4, 76.5, 83.0, 83.2, 87.1, 91.8, 91.8,
106.6, 106.6, 113.0, 113.2, 127.1, 127.6, 127.7, 127.9, 128.0,
129.0, 130.0, 130.1, 134.7, 135.0, 140.8, 144.1, 158.5, 158.5,
160.0, 170.9, 174.5. HRMS (ESI) m/z (M + Na) calcd for
2′-O-Met h oxyet h yl-4-O-(2,6-d im et h ylp h en yl)-3′,5′-O-
bis(ter t-bu tyld im eth ylsilyl)-2-th iou r id in e (22). Compound
21 (8.25 g, 13 mmol) was rendered anhydrous by repeated
coevaporation with dry pyridine. The residue was further
coevaporated with dry toluene and finally dissolved in dry
toluene (200 mL). Lawesson’s reagent (7.89 g, 19.5 mmol) was
added, and the mixture was refluxed for 2 h. The mixture was
cooled to room temperature. The insoluble materials were
filtered, and the filtrate was twice washed with cooled EtOH.
The organic phase was concentrated in vacuo. The residue was
dissolved in hexane and washed three times with brine. The
combined organic extracts were dried over Na2SO4, filtered,
and concentrated in vacuo. The crude product was used in the
next reaction without further purification.
2′-O-Met h oxyet h yl-4-O-(2,6-d im et h ylp h en yl)-2-t h io-
u r id in e (23). To a solution of the crude compound 22 in THF
(140 mL) were added tetrabutylammonium fluoride (7.32 g,
28 mmol) and acetic acid (1.6 mL, 28 mmol). After being stirred
at room temperature for 20 h, the mixture was diluted with
CHCl3. The CHCl3 solution was washed once with H2O. The
aqueous phase was collected and concentrated in vacuo. The
residue was purified by chromatography with CHCl3-MeOH
to afford 23 as a white powder (4.64 g, 78% yield): 1H NMR
(270 MHz, CDCl3) δ 2.03 (6H, s), 3.32 (3H, s), 3.46-3.59 (2H,
m), 3.88-4.36 (7H, m), 6.19 (1H, d, J ) 8.25 Hz), 6.43 (1H, s),
6.99 (3H, s), 8.74 (1H, d, J ) 8.25 Hz); 13C NMR (67.8 MHz,
CDCl3) δ 16.6, 58.8, 59.4, 67.1, 71.4, 71.6, 82.9, 84.0, 93.9, 98.1,
126.1, 128.8, 130.1, 145.9, 148.9, 165.3, 180.8. (two-step yield).
HRMS (ESI) m/z (M + H) calcd for C20H27N2O6S 423.1590,
found 423.1563.
C
34H36N2NaO9S 671.2040, found 671.1981.
5′-O-(4,4′-Dim eth oxytr ityl)-2′-O-m eth oxyeth yl -2-th io-
u r id in e (24e): 1H NMR (270 MHz, CDCl3) δ 3.36 (3H, s), 3.77
(6H, s), 3.50-3.62 (4H, m), 3.84-4.51 (5H, m), 5.39 (1H, d, J
) 8.24 Hz), 6.47 (1H, s), 6.80-6.84 (4H, m), 7.21-7.37 (9H,
m), 8.33 (1H, d, J ) 8.24 Hz), 10.56 (1H, br s); 13C NMR (67.8
MHz, CDCl3) δ 55.2, 58.9, 60.5, 68.3, 71.3, 71.6, 83.3, 87.0,
113.2, 127.1, 127.9, 128.0, 129.0, 130.0, 130.1, 134.8, 135.1,
140.8, 144.2, 158.5, 158.5, 159.9, 174.6. HRMS (ESI) m/z (M
+ Na) calcd for C33H36N2NaO8S 643.2090, found 643.2095.
P h osp h ityla tion of 3′-Hyd r oxyl Gr ou p (25c-e). Com-
pound 24c-e (0.5 mmol) was rendered anhydrous by repeated
coevaporation with dry pyridine. The residue was further
coevaporated with dry toluene and finally dissolved in dry CH2-
Cl2 (5 mL). To the solution were added diisopropylammonium
tetrazolide (0.3 mmol) and 2-cyanoethoxy[bis(diisopropyl-
amino)]phosphine (0.6 mmol) and the solution was stirred at
room temperature for 6 h. The mixture was partitioned
between CH2Cl2 and saturated NaHCO3. The organic layer was
collected, dried over Na2SO4, filtered, and concentrated in
vacuo. The concentrated solution was poured into hexanes-
Et2O. The resulting precipitate was dissolved in CH2Cl2 and
concentrated in vacuo, then the product 25c-e was obtained
as a white foam.
2′-O-Met h oxyet h yl-2-t h iou r id in e (3e). Compound 23
(4.07 g, 9.63 mmol) was rendered anhydrous by repeated
coevaporation with dry pyridine. The residue was further
coevaporated with dry toluene and finally dissolved in dry CH3-
CN (90 mL). The solution of 1,1,3,3-tetramethylguanidine (3.63
mL, 28.9 mmol) and syn-o-nitrobenzaldoxime (4.8 g, 28.9
mmol) in CH3CN (90 mL) was added. After being stirred at
room temperature for 3 h, the mixture was diluted with Et2O.
The Et2O solution was washed twice with H2O, and the
combined aqueous extracts were concentrated in vacuo. The
residue was purified by chromatography with CHCl3-MeOH
to afford 3e as a white powder (2.47 g, 80% yield): 1H NMR
(400 MHz, D2O) δ 3.05 (3H, s), 3.67-3.69 (2H, dd), 3.83-4.29
(7H, m), 6.17 (1H, d, J ) 8.24 Hz), 6.75 (1H, d, J ) 2.90 Hz),
8.12 (1H, d, J ) 8.24 Hz); 13C NMR (67.8 MHz, DMSO) δ 58.1,
59.4, 67.9, 69.8, 71.2, 82.3, 84.5, 91.1, 106.4, 140.7, 159.4, 175.7.
HRMS (ESI) m/z (M + H) calcd for C12H18N2O6S 319.0964,
found 319.0984.
2′-O-Allyl-5′-O-(4,4′-dim eth oxytr ityl)-2-th iou r idin e (24c).
2′-O-Allyl-2-thiouridine 3c (636.7 mg, 2 mmol) was rendered
anhydrous by repeated coevaporation with dry pyridine and
finally dissolved in dry pyridine (20 mL). 4,4′-Dimethoxytrityl
chloride was added. After being stirred at room temperature
for 90 min, the mixture was concentrated under reduced
pressure. The resulting oily residue was dissolved in CHCl3.
The CHCl3 solution was washed twice with brine and twice
with saturated NaHCO3 (aqueous). The combined organic
extracts were dried over Na2SO4, filtered, concentrated in
vacuo. The residue was purified by column chromatography
with hexanes-ethyl acetate containing 0.5% pyridine to afford
24c as a white foam (1.16 g, 94% yield): 1H NMR (270 MHz,
CDCl3) δ 3.58 (2H, s), 3.78 (6H, s), 4.02-4.06 (2H, m), 4.38-
4.63 (3H, m), 5.22-5.40 (3H, m), 5.84-5.98 (1H, m), 6.57 (1H,
s), 6.80-6.86 (4H, m), 7.24-7.67 (9H, m), 8.35 (1H, d, J ) 8.24
Hz), 10.42 (1H, br s); 13C NMR (67.8 MHz, CDCl3) δ 55.3, 55.3,
60.5, 68.0, 72.2, 81.3, 83.4, 87.2, 91.8, 91.9, 106.7, 106.7, 113.2,
118.5, 123.7, 127.1, 128.0, 128.0, 129.9, 130.0, 130.1, 133.4,
2′-O-Allyl-5′-O-(4,4′-dim eth oxytr ityl)-3′-O-(2-cyan oeth yl-
N,N-d iisop r op ylp h osp h or a m id ite)-2-th iou r id in e (25c):
1H NMR (270 MHz, CDCl3) δ 1.02-1.22 (14H, m), 2.40-2.61
(2H, m), 3.42-3.93 (13H, m), 4.14-4.64 (4H, m), 5.18-5.36
(3H, m), 5.86-6.02 (1H, m), 6.53-6.50 (1H, m), 6.80-6.85 (4H,
m), 7.21-7.37 (9H, m), 8.34 (1H, m); 13C NMR (67.8 MHz,
CDCl3) δ 15.4, 20.5, 20.6, 24.6, 24.7, 24.7, 24.8, 43.3, 43.3, 43.5,
43.5, 55.3, 55.3, 58.3, 60.3, 65.8, 72.2, 72.2, 77.2, 87.2, 87.3,
92.7, 92.8, 100.5, 106.6, 113.2, 116.9, 117.1, 127.2, 127.9, 128.3,
130.2, 134.3, 134.4, 134.8, 134.9, 135.0, 135.1, 140.8, 140.9,
144.2, 158.7, 158.7, 159.3, 174.8; 31P NMR (109.25 MHz,
CDCl3) δ 150.46, 151.21. HRMS (ESI) m/z (M + Na) calcd for
C
42H51N4NaO8P: 825.3063, found 825.3144.
2′-O-Acetoxyleth yl-5′-O-(4,4′-d im eth oxytr ityl)-3′-O-(2-
cya n oeth yl-N,N-d iisop r op ylp h osp h or a m id ite)-2-th iou r i-
d in e (25d ): 1H NMR (270 MHz, CDCl3) δ 0.82-1.23 (14H, m),
2.02 (3H, s), 2.40-2.60 (2H, m), 3.51-4.24 (19H, m), 4.42-
4.61 (1H, m), 5.32-5.35 (1H, m), 6.44-6.47 (1H, m), 6.77-
6.83 (4H, m), 7.19-7.36 (9H, m), 8.35 (1H, m); 13C NMR (67.8
MHz, CDCl3) δ 14.2, 20.4, 20.5, 20.5, 21.0, 21.0, 22.7, 24.5,
24.6, 24.7, 24.8, 24.8, 25.4, 31.6, 34.7, 43.3, 43.4, 55.3, 55.3,
57.9, 58.2, 59.9, 63.6, 63.6, 69.5, 77.1, 81.9, 87.1, 87.2, 92.9,
106.6, 113.2, 113.2, 117.3, 127.2, 127.9, 128.3, 130.2, 134.7,
134.8, 140.7, 140.8, 144.0, 144.1, 158.7, 158.7, 158.7, 159.4,
170.7, 170.7, 174.8; 31P NMR (109.25 MHz, CDCl3) δ 150.25,
151.42. HRMS (ESI) m/z (M + Na) calcd for C43H53N4NaO10
PS 871.3118, found 871.3163.
-
5′-O-(4,4′-Dim eth oxytr ityl)-3′-O-(2-cya n oeth yl-N,N-d i-
isop r op ylp h osp h or a m id it e)-2′-O-m et h oxyet h yl-2-t h io-
u r id in e (25e): 1H NMR (270 MHz, CDCl3) δ 1.01-1.28 (14H,
m), 2.40-2.65 (2H, m), 3.34 (3H, s), 3.44-4.60 (18H, m), 5.30-
5.35 (1H, m), 6.51-6.57 (1H, m), 6.79-6.84 (4H, m), 7.13-
7.39 (9H, m), 8.35-8.38 (1H, m); 13C NMR (67.8 MHz, CDCl3)
δ 20.1, 20.2, 20.3, 20.4, 23.0, 23.0, 23.0, 23.1, 24.6, 24.7, 24.7,
24.8, 43.2, 43.2, 43.4, 43.4, 45.3, 45.4, 55.3, 55.3, 57.9, 58.1,
58.2, 59.1, 71.0, 72.0, 72.2, 76.5, 77.5, 87.1, 87.2, 92.7, 92.8,
J . Org. Chem, Vol. 68, No. 26, 2003 9981

Okamoto et al.
106.5, 113.1, 113.2, 113.2, 117.3, 127.2, 127.7, 127.7, 127.9,
128.2, 128.3, 129.0, 130.2, 134.7, 134.8, 134.9, 135.0, 140.9,
144.0, 144.1, 158.6, 158.7, 159.3, 174.8, 175.0; 31P NMR (109.25
MHz, CDCl3) δ 150.15, 150.97. HRMS (ESI) m/z (M + Na) calcd
for C42H53N4NaO9PS 843.3269, found 843.3257.
Oligon u cleot id es Syn t h esis. Oligoribonucleotides were
synthesized on an Applied Biosystems 392 oligonucleotide
synthesizer on a 1 µmol scale, using PAC phosphoramidites
(Pac-A, isopropyl-Pac-G, and acetyl-C) from Glen Research. A
0.1 M solution of each modified nucleoside phosphoramidite
was used and the time for coupling was set up to be 10 min.
The procedure for oxidation with 10% tert-butyl hydroperoxide
in acetonitrile was twice repeated by use of the reaction time
of 5 min each. The standard PAC RNA phosphoramidite
chemistry was used for all the procedures prescribed for
deprotection and purification.
Meltin g Tem p er a tu r e An a lysis. A solution of an ap-
propriate oligonucleotide and the complementary strand, both
of which were arranged to be 2 µM, was prepared in 10 mM
phosphate buffer (pH 7.0) containing 150 mM NaCl and 0.1
mM EDTA. The Tm experiments were done on a BECKMAN
DU 650 spectrophotometer. The solution was heated at 60 °C
and then cooled to 5 °C within 55 min. Data points were
collected every time when the temperature was increased by
1 °C, and the solution was equilibrated at the same temper-
ature for 1 min. Each melting curve was calculated by use of
Igor Pro. software (WaveMetrics, Inc.).
1H NMR Sp ectr a An a lysis. An appropriate nucleoside
derivative (84.6A260 nm) was dissolved in phosphate buffer (600
µL) and lyophilized three times with 99.8% D2O and finally
with 99.95% D2O (600 µL). The spectra were recorded at 400
MHz.
Ack n ow led gm en t. This work was supported by a
Grant from “Research for the Future” Program of the
J apan Society for the Promotion of Science (J SPS-
RFTF97I00301) and a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Culture, Sports,
Science and Technology, J apan. This work was also
supported by CREST of J ST (J apan Science and Tech-
nology) and partially supported by the COE21 project.
Su p p or tin g In for m a tion Ava ila ble: General procedure
1
and H, 13C, and 31P NMR spectra of all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
J O035246B
9982 J . Org. Chem., Vol. 68, No. 26, 2003