Synthesis of 4-thiopseudoisocytidine and 4-thiopseudouridine as components of triplex-forming oligonucleotides

Article abstract of DOI:10.1246/cl.2009.174

In this paper, we report convenient methods for the synthesis of 4-thiopseudoisocytidine (s⁴ψiC) and 4-thiopseudouridine (s ⁴ψ). ¹HNMR spectral analysis of these modified nucleosides showed that both s⁴ψ and s

Full text of DOI:10.1246/cl.2009.174

174
Chemistry Letters Vol.38, No.2 (2009)
Synthesis of 4-Thiopseudoisocytidine and 4-Thiopseudouridine
as Components of Triplex-forming Oligonucleotides
Itaru Okamoto, Shiqi Cao, Hiroto Tanaka, Kohji Seio, and Mitsuo Sekineꢀ
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501
(Received November 26, 2008; CL-081117; E-mail: msekine@bio.titech.ac.jp)
In this paper, we report convenient methods for the synthesis
of 4-thiopseudoisocytidine (s4 iC) and 4-thiopseudouridine
In the synthesis of 4-thiouridine (s4U), it was reported that
the thiolation of the pyrimidine ring at position 4 could be
achieved by the reaction of 4-(2,4,6-triisopropylbenzenesulfo-
nyl)pyrimidinone nucleoside derivatives with 3-sulfanylpropio-
nitrile.17–19 In addition, many reactions with pyrimidine rings
substituted with leaving groups at position 4 were reported.
Therefore, such types of substitution reactions might also pro-
duce 4-substituted ꢀ derivatives. Townsend et al. reported 2,4-
dichloro-5-(2,3,5-tri-O-acetyl-ꢀ-D-ribofuranosyl)pyrimidine as
a ꢀ derivative that has chloro groups at positions 2 and 4 on
the pyrimidine ring.20 Considering the reactivity of compound,
we expected that the substitution reaction might occur predom-
inantly at position 4.21 According to Townsend’s procedure
(Scheme 1), pseudouridine 3 was converted to 20,30,50-tri-O-ace-
tylpseudouridine (4) in 92% yield. Compound 4 was further
treated with excess POCl3 to give 2,4-dichloropseudouridine
derivative 5 in 91% yield.
1
(s4ꢀ). H NMR spectral analysis of these modified nucleosides
showed that both s4ꢀ and s4 iC prefer C30-endo ribose pucker-
ing. These conformational properties are favorable for the stabi-
lization of triplex formation.
Using the antigene strategy, a large number of modified nu-
cleosides have been synthesized to enhance the thermal stability
of DNA triplexes formed by hybridization of the third DNA
strands with DNA duplexes.1–5 These studies showed that the
use of homopyrimidine–oligodeoxynucleotides containing cyto-
sine or 5-methylcytosine bases as triplex-forming oligodeoxynu-
cleotides (TFOs) under weakly acidic conditions resulted in sig-
nificant stabilization of the resulting parallel triplex structures.
This was due to the formation of protonated cytosine or 5-
methylcytosine bases that could bind to guanine bases at the
Hoogsteen base-pairing site.6–9 However, those acidic condi-
tions limit the sequences of TFOs; therefore, antigene therapy
using this strategy is not generally applicable. To overcome this
limitation, several modified nucleosides have been developed
to mimic the structure of the 3-N-protonated cytosine base.10–15
20-O-Methylpseudoisocytidine ( iCm) is known to form a
triplet base pair with a G–C base pair under neutral conditions.
However, TFOs containing iCm could not stabilize the triplex
structure sufficiently at neutral pH.10,11
On the other hand, we have recently reported that TFOs con-
taining 20-O-methyl-2-thiouridine (s2Um) or 2-thiothymidine
(s2T) formed quite stable parallel triplex structures.16 Enhance-
ment of the thermal stability of these parallel triplexes can be ex-
plained by means of the strong stacking interaction of the 2-thio-
carbonyl group with the 50-upstream or 30-downstream bases. In
particular, it was found that a consecutive alignment of s2Um or
s2T in TFOs resulted in a more effective increase in the binding
ability toward DNA duplexes.16
After that, as expected, the reaction of compound 5 with
2-(trimethylsilyl)ethanethiol in N,N-dimethylacetamide in the
presence of triethylamine formed only the 4-thiolated compound
6 in a high yield of 84%. The structure of this product was deter-
1
mined from the correlation between the H signal of the 2-(tri-
methylsilyl)ethyl group and the 13C signal of 4C on the pyrimi-
dine ring, obtained by HMBC spectrum analysis. The chloro
group of compound 6 was converted to an amino group by the
reaction with concd NH3 to form compound 7 in 44% yield.
Treatment of 7 with Bu4NF formed s4 iC (1) in 62% yield.22
It was expected that a consecutive pile of 4-thiopseudoiso-
cytidine (1: s4 iC) in combination with s2Um or s2T might
cause an increase in the thermal stability of the parallel triplex
structures. In this paper, we report convenient methods for the
synthesis of 1 and 4-thiopseudouridine (2: s4ꢀ), which can be
derived from a synthetic intermediate of the former. Chemical
structures of these modified nucleosides were shown in Figure 1.
Scheme 1. Reagents and conditions: (i) Ac2O (10 equiv), pyri-
dine, rt; (ii) N,N-diethylaniline hydrochloride (1.0 equiv), POCl3
(20 equiv), reflux; (iii) 2-(trimethylsilyl)ethanethiol (1.2 equiv),
triethylamine (1.2 equiv), DMA, rt; (iv) concd NH3, dioxane,
ꢁ
ꢁ
.
100 C; (v) TBAF (3.0 equiv), THF, 50 C; (vi) LiOH H2O
(5.0 equiv), DMA, 60 ꢁC; and (vii) TBAF (1.5 equiv), THF,
60 ꢁC.
Figure 1. Chemical structures of 4-thiopseudoisocytidine and
4-thiopseudouridine.
Copyright Ó 2009 The Chemical Society of Japan

Chemistry Letters Vol.38, No.2 (2009)
175
Table 1. Conformational analysis of s4ꢀ and s4 iC in D2O
ed in part by a grant of the Genome Network Project from the
Ministry of Education, Culture, Sports, Science and Technology,
Japan and by the COE21 project.
ꢀ
s4ꢀ
s4 iC
%N (C30-endo)a
50%
5.4 Hz
5.4 Hz
78%
2.2 Hz
7.8 Hz
66%
3.9 Hz
7.1 Hz
0
J1 H2 H
0
References and Notes
0
J3 H4 H
0
´ `
C. Helene, J.-J. Toulme, Biochim. Biophys. Acta 1990, 1049, 99.
1
2
a%N values of nucleosides were determined by following
equation: %N (C30-endo) = J3 H4 H=ðJ1 H2 H þ J3 H4 HÞ ꢂ 100.
D. Praseuth, A. L. Guieysse, C. Helene, Biochim. Biophys. Acta
1999, 1489, 181.
´ `
0
0
0
0
0
0
3
4
5
K. R. Fox, Curr. Med. Chem. 2000, 7, 17.
S. Buchini, C. J. Leumann, Curr. Opin. Chem. Biol. 2003, 7, 717.
R. V. Guntaka, B. R. Varma, K. T. Weber, Int. J. Biochem. Cell
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C. Des los Santos, M. Rosen, D. Patel, Biochemistry 1989, 28,
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7
8
P. Rajagopal, J. Feigon, Biochemistry 1989, 28, 7859.
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9
J. Robles, A. Grandas, E. Pedroso, F. J. Luque, R. Eritja, M.
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Figure 2. UV spectra of s4 iC and s4ꢀ in H2O.
10 A. Ono, P. O. P. Ts’o, L. S. Kan, J. Am. Chem. Soc. 1991, 113,
4032.
On the other hand, hydrolysis of compound 6 with LiOH afford-
ed 4-(2-trimethylsilyl)ethyl-4-thiopseudouridine (8) in 36%
yield. The low yields of the above two reactions of compound
6 forming compounds 7 and 8 were due to side reactions of
position 4, since it is known that pyrimidine derivatives having
alkylthio or sulfanyl groups at position 4 or 2 react easily with
nucleophilic reagents.23–26 The TBAF-mediated deprotection
of compound 8 formed s4ꢀ in 67% yield.27
11 A. Ono, P. O. P. Ts’o, L. S. Kan, J. Org. Chem. 1992, 57, 3225.
12 G. Xiang, W. Soussou, L. W. McLaughlin, J. Am. Chem. Soc. 1994,
116, 11155.
13 R. Berressem, J. W. Engels, Nucleic Acids Res. 1995, 23, 3465.
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15 G. Xiang, R. Bogacki, L. W. McLaughlin, Nucleic Acids Res. 1996,
24, 1963.
To clarify the sugar conformations of s4ꢀ and s4 iC,
1H NMR spectral analysis was performed. As shown in
Table 1, it was found that s4ꢀ and s4 iC showed C30-endo
ribose puckering forms (%N; s4ꢀ: 78%; s4 iC: 66%) more pre-
dominantly than ꢀ. It was reported that s2U derivatives prefer
C30-endo ribose puckering.28–30 This conformational predomi-
nance is known to be caused by steric repulsion between the
2-thiocarbonyl group of s2U and the 20-hydroxy group.28 The
C30-endo predominance observed could be explained by the
same type of steric repulsion.
It is known that s4U exhibited a unique UV absorption spec-
trum with maximum absorbance at 330 nm.31 As shown in
Figure 2, the UV absorption maxima of 4-thiopseudo-nucleo-
sides s4 iC and s4ꢀ were shifted markedly from that of
(260 nm) to 345 and 331 nm, respectively. These spectral
changes were very similar to those from U to s4U. Since the
structure of s4U resembles that of s4ꢀ, these UV spectral
changes also supported the view that the thiolation occurred at
position 4 of the pyrimidine ring.
16 I. Okamoto, K. Seio, M. Sekine, Bioorg. Med. Chem. Lett. 2006, 16,
3334.
17 R. S. Coleman, J. M. Siedlecki, Tetrahedron Lett. 1991, 32, 3033.
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hedron Lett. 1994, 35, 765.
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20 2,4-Dichloro-5-(ꢀ-D-ribofuranosyl)pyrimidines and substituted
derivatives: L. B. Townsend, D. S. Wise, R. A. Earl, G. Belton,
U.S. Pat. Appl. 4092472, 1989.
21 R. G. Shepherd, J. L. Fedrick, Adv. Heterocycl. Chem. 1965, 4, 145.
22 Physical properties of 4-thiopseudoisocytidine (1) are shown in
Supporting Information is available electronically on the CSJ-
Journal Web site, http://www.csj.jp/journals/chem-lett/index.html.
23 J. J. Fox, D. Van Praag, I. Wempen, I. L. Doerr, L. Cheong, J. E.
Knoll, M. L. Eidinoff, A. Bendich, G. B. Brown, J. Am. Chem.
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26 H. C. Van der Plas, B. Zuurdeeg, H. W. van Meeteren, Recl. Trav.
Chim. Pays-Bas 1969, 88, 1156.
In conclusion, we synthesized s4 iC and s4ꢀ successfully.
The 1H NMR studies of these modified nucleosides showed that
both s4 iC and s4ꢀ prefer C30-endo ribose puckering. These
conformational properties are favorable for the stabilization of
both RNA-duplex and parallel triplex formation. Synthesis of
oligonucleotides containing s4 iC and study of their duplex-
and triplex-forming abilities are now in progress.
27 Physical properties of 4-thiopseudouridine (2) are shown in Sup-
porting Information. Supporting Information is available electroni-
cally on the CSJ-Journal Web site, http://www.csj.jp/journals/
chem-lett/index.html.
28 Y. Yamamoto, S. Yokoyama, T. Miyazawa, K. Watanabe, S.
Higuchi, FEBS Lett. 1983, 157, 95.
29 H. Sierzputowska-Gracz, E. Sochacka, A. Malkiewicz, K. Kuo,
C. W. Gehrke, P. F. Agris, J. Am. Chem. Soc. 1987, 109, 7171.
30 P. F. Agris, H. Sierzputowska-Gracz, W. Smith, A. Malkiewicz,
E. Sochacka, B. Nawrot, J. Am. Chem. Soc. 1992, 114, 2652.
31 W. Saenger, Principles of Nucleic Acid Structure, Springer-
Verlag, New York, 1987.
This work was supported by a grant from CREST of JST
(Japan Science and Technology Agency) and a Grant-in-Aid
for Scientific Research from the Ministry of Education, Culture,
Sports, Science and Technology, Japan. This work was support-
Published on the web (Advance View) January 24, 2009; doi:10.1246/cl.2009.174