Improved synthesis of oligonucleotides containing 2-thiouridine derivatives by use of diluted iodine solution

Article abstract of DOI:10.1016/j.tetlet.2005.11.044

It is known that the 2-thiocarbonyl group of 2-thiouridine (s²U) derivatives reacts easily with various oxidizing agents used in oligonucleotide synthesis to give a complex mixture. In this letter, we report an improved method for the synthesis of oligonucleotides containing s²U derivatives. It turned out that the 2-thiocarbonyl group of oligonucleotides containing s²U derivatives was stable in a 0.02 M solution of iodine in pyridine-THF-H₂O. These conditions were successfully applied to the synthesis of oligonucleotides containing s²U derivatives on an automated DNA/RNA synthesizer. Moreover, no undesirable side reactions were detected so that these modified oligonucleotides could be obtained in markedly improved yields.

Full text of DOI:10.1016/j.tetlet.2005.11.044

Tetrahedron
Letters
Tetrahedron Letters 47 (2006) 583–585
Improved synthesis of oligonucleotides containing
2-thiouridine derivatives by use of diluted iodine solution
Itaru Okamoto,a,c Kohji Seiob,c and Mitsuo Sekinea,c,
*
aDepartment of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
bFrontier Collaborative Research Institute, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
cCREST, JST (Japan Science and Technology Agency), 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
Received 13 October 2005; revised 2 November 2005; accepted 4 November 2005
Available online 29 November 2005
Abstract—It is known that the 2-thiocarbonyl group of 2-thiouridine (s2U) derivatives reacts easily with various oxidizing agents
used in oligonucleotide synthesis to give a complex mixture. In this letter, we report an improved method for the synthesis of oligo-
nucleotides containing s2U derivatives. It turned out that the 2-thiocarbonyl group of oligonucleotides containing s2U derivatives
was stable in a 0.02 M solution of iodine in pyridine–THF–H2O. These conditions were successfully applied to the synthesis of oligo-
nucleotides containing s2U derivatives on an automated DNA/RNA synthesizer. Moreover, no undesirable side reactions were
detected so that these modified oligonucleotides could be obtained in markedly improved yields.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
It is known that various oxidizing agents react with the
2-thiocarbonyl group of 2-thiouridine derivatives.11–15
In oligonucleotides synthesis, an oxidation step was
required for the phosphoramidite and H-phosphonate
methods. In earlier papers, 2-thiothymidine (s2T)
was incorporated into DNA by the H-phosphonate
and phosphoramidite method but no side reactions at
the oxidation step using iodine were reported.16,17
However, Kuimelis and co-workers reported that, in
incorporation of s2T into oligonucleotides via the phos-
phoramidite method, side reactions occurred at the
iodine oxidation step.18 In order to avoid these side reac-
tions at the iodine oxidation step, they used the toluoyl
group for protection of the base moiety.19
2-Thiouridine (s2U) and its derivatives have been discov-
ered from tRNAs.1–4 It is known that the thionation of
the uracil ring of the 2-carbonyl group leads to the C30-
endo conformation because of steric repulsion between
the 2-thiocarbonyl group and the 20-hydroxy group.5,6
Thus, oligonucleotides containing its derivatives can
form stable duplexes with the complementary RNAs.7–10
Moreover, s2U and its derivatives proved to have an
exact base-pair recognition ability.9,10 In a U–G wobble
base pair, a hydrogen bond between the 2-carbonyl group
of the uracil base and the amino group of the guanine
base is formed. Since the ability of the sulfur atom as a
donor of the hydrogen bonding is lower than that of
the carbonyl oxygen atom, a 2-thiouracil-guanine wobble
base pair is destabilized so that the base recognition abil-
ity of s2U toward A significantly increases. Actually, our
melting temperature analysis of oligoribonucleotides
having s2U and s2Um revealed predominant base recog-
nition ability of the 2-thiouridine derivatives over the
uracil base.10 These properties of s2U derivatives are
favorable for antisense strategy as well as SNPs analysis.
In the synthesis of RNA containing s2U, it was reported
that the 2-thiocarbonyl group of the 2-thiouracil base
also reacted with iodine. Davis et al. proposed the use
of a solution of tert-butyl hydroperoxide in CH3CN as
the oxidizing agent in place of a solution of iodine in
pyridine–H2O to avoid this problem.20 Recently, Socha-
cka reported the reactivity of the 2-thiocarbonyl moiety
of s2U-loaded LCAA-CPG toward various oxidizing
agents, which were prescribed for the usual solid-phase
synthesis of oligonucleotides.14 They claimed that the
2-thiocarbonyl moiety underwent side reactions with
tert-butyl hydroperoxide in CH3CN and reported that
the best result was obtained by the use of carbon
*
Corresponding author. Tel.: +81 45 924 5706; fax: +81 45 924
5772; e-mail: msekine@bio.titech.ac.jp
0040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.11.044

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I. Okamoto et al. / Tetrahedron Letters 47 (2006) 583–585
tetrachloride–N-methylmorpholine in pyridine–water–
acetonitrile. However, in our hands, this oxidation
system did not give the desired oligonucleotides at
all. Therefore, we reexamined suitable oxidizing agents
for the synthesis of oligonucleotides containing s2U
derivatives.
derivative 3. Our result indicated that prolonged reac-
tion time resulted in damage of the 2-thiocarbonyl
group. Since the conditions used by Davis et al. required
a relatively long reaction time (each oxidation required
12 min), it was likely that the desired oligonucleotides
could not be obtained in good yields.
Contrary to this result, only the desulfurization product
2 was formed with a 0.1 M solution of iodine in pyri-
dine–H2O. To our surprise, however, it was found that
the use of a one-fifth diluted solution of iodine, that is,
a 0.02 M solution of iodine in pyridine–H2O,22 resulted
in virtually no formation of 2 even after 24 h. Under this
diluted condition, it was confirmed that compound 1
remained unchanged. These results are summarized in
Table 1.
2. Results and discussion
Oligonucleotides containing s2U derivatives were syn-
thesized in our previous studies.10,21 However, the yields
of modified oligonucleotides were quite low (ca. 5–20%)
when DavisÕ oxidizing agent was used. In order to
improve the yield of oligonucleotides containing s2U
derivatives, several conditions for the oxidation step
were examined.
Based on these unexpected but satisfactory results, the
diluted iodine solution was applied to the synthesis of
oligonucleotides containing s2U derivatives on an auto-
mated DNA/RNA synthesizer. In this study, we used 20-
O-methyl-2-thiouridine (s2Um) instead of s2U because
the 20-O-methylated nucleoside was easily handled.
The s2Um 30-phosphoramidite derivative was synthe-
sized according to our previous paper.10,21
First, we checked the stability of the 2-thiocarbonyl
groups toward tert-butyl hydroperoxide and iodine in
solution. The s2U derivative 1 was prepared by use of
VorbruggenÕs method. When compound 1 was treated
¨
with a 0.1 M solution of tert-butyl hydroperoxide in
CH3CN, the 2-thiocarbonyl group of 1 was completely
converted to a carbonyl group in 12 h to give the uridine
Table 1. Stability of 2-thiouridine derivatives under conditions using tert-butyl hydroperoxide or iodine
O
O
O
N
NH
N
NH
oxidizing agent
BzO
BzO
BzO
N
S
N
H
O
O
O
O
BzO
OBz
BzO
OBz
BzO
OBz
3
1
2
Conditions
Equiva
Time (h)
Recovery of 1 (%)
Product (%)b
2
3
0.1 M tert-Butyl hydroperoxide in CH3CN
0.1 M Iodine in pyridine–H2O (9:1, v/v)
0.02 M Iodine in pyridine–H2O (9:1, v/v)
10
10
2
12
1
24
ndc
53
70
nd
nd
86
nd
a Equivalent of an oxidizing agent for compound 1.
b Isolated yield.
c The abbreviation of ÔndÕ refers to not detected.
Figure 1. RP-HPLC Profiles of 50-dTTTTTTTs2UmT-30: (a) tert-BuOOH–CH3CN 60 s, (b) tert-BuOOH–CH3CN 15 s, (c) I2–pyridine–H2O–THF
15 s.

I. Okamoto et al. / Tetrahedron Letters 47 (2006) 583–585
Table 2. Yields and MALDI-TOF mass analysis of oligonucleotides containing s2Um
585
Oligonucleotidea
Calcd [MꢀH]ꢀ
Found
Yield (%)
dTTTTTTTXT
2705.4
3941.6
4005.6
4069.5
2705.8
3941.9
4004.8
4071.1
53
31
30
19
dCGTTTTXTTTTGC
dCGTTTXXXTTTGC
dCGTTXXXXXTTGC
a = s2Um.
When tert-butyl hydroperoxide in CH3CN was used
as the oxidizing agent, many undesired products were
observed as expected (Fig. 1a). Moreover, when the
time for the oxidation was reduced at every oxidation
step, little desired oligonucleotides could be formed
because of incomplete oxidation (Fig. 1b). On the
other hand, with the use of a 0.02 M solution of
iodine in pyridine–H2O–THF22 as the oxidizing agent,
it was found that the synthesis of oligonucleotides
containing s2Um could be carried out in good yields
(ca. 20–50%) without any detectable side reaction
products (Fig. 1c and Table 2). In the synthesis of
the oligonucleotide derivative containing five modified
bases, the main reason of the low yield (19%) is due
to the low coupling efficiency of the s2Um phosphor-
amidite unit because of the steric hindrance of the
20-O-methyl group.
References and notes
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In conclusion, it turned out that the 2-thiouracil base is
very sensitive to the concentration of iodine so that there
is a clear-cut difference in reactivity between 0.1 and
0.02 M solutions of iodine in pyridine–THF–water. It
was unclear why such pronounced change of its reactiv-
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iodine. It should be noted that our finding is of great
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Acknowledgments
This work was supported in part by a grant of the Gen-
ome Network Project from the Ministry of Education,
Culture, Sports, Science and Technology, Japan. This
work was also supported by CREST of JST (Japan
Science and Technology) and partially supported by
COE21 project.
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Chem. 2003, 68, 9971–9982.
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