A new odorless procedure for the synthesis of 2'-deoxy-6-thioguanosine and its incorporation into oligodeoxynucleotides

Article abstract of DOI:10.1080/15257770903155576

6-S-[2-[(2-ethylhexyl)oxycarbonyl]ethyl)}-3',5'-O-bis(tert- butyldimethylsilyl)-2'-deoxy-6-thiogua nosine (2) was synthesized in high yield from the corresponding 6-O-mesitylenesulfonyl derivative by the reaction with 2-ethylhexyl 3-mercapto-propionate. T

Full text of DOI:10.1080/15257770903155576

Nucleosides, Nucleotides and Nucleic Acids, 28:752–760, 2009
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770903155576
A NEW ODORLESS PROCEDURE FOR THE SYNTHESIS
OF 2ꢀ-DEOXY-6-THIOGUANOSINE AND ITS INCORPORATION INTO
OLIGODEOXYNUCLEOTIDES
Kazumitsu Onizuka, Yosuke Taniguchi, and Shigeki Sasaki
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan
and CREST, Japan Science and Technology Agency, Saitama, Japan
6-S-{2-[(2-ethylhexyl)oxycarbonyl]ethyl)}-3ꢁ,5ꢁ-O-bis(tert-butyldimethylsilyl)-2ꢁ-deoxy-6-thiogua
2
nosine (2) was synthesized in high yield from the corresponding 6-O-mesitylenesulfonyl derivative by
the reaction with 2-ethylhexyl 3-mercapto-propionate. The phosphoramidite precursor derived from
2 was successfully applied to an automated DNA synthesizer to produce 2ꢁ-deoxy-6-thioguanosine
containing ODN. The results showed that 2-ethylhexyl 3-mercaptopropionate is useful as an odor
less reagent and also as an S-protecting group of 2ꢁ-deoxy-6-thioguanosine.
Keywords
2ꢁ-Deoxy-6-thioguanosine;
odorless
synthesis;
2-Ethylhexyl
3-mercaptopropionate; phosphoroamidite; DNA synthesis
INTRODUCTION
Oligonucleotides containing a thio-substituted base, such as 6-
thioguanine (6-thio-dG, 6-thio-G) and 4-thiouracil (4-thio-U), have been
widely used for various purposes in biology.[1–7] Their photo-induced cross-
linking ability has been used to study the three-dimensional interaction
of RNA-RNA or RNA-proteins at the atomic level.[1–4] The high nucle-
ophilicity of the sulfur atom of 6-thioguanosine was used as a modification
site of oligode-oxynucleotides (ODN).[5–7] Recently, we developed the
functionality-transfer reaction for the site-specific modification of DNA
and RNA by utilizing the ODN containing 2ꢁ-deoxy-6-thioguanosine. The
site-specific NO modification of the target cytosine was demonstrated by
Received 3 March 2009; accepted 17 June 2009.
This work was supported by a Grant-in-Aid for Scientific Research (A) from the Japan Society for
the Promotion of Science (JSPS), and CREST from the Japan Science and Technology Agency. We thank
Research Fellowship of the Japan Society for the Promotion of Science (JSPS) for Young Scientists (K.O.).
Address correspondence to Shigeki Sasaki, Graduate School of Pharmaceutical Sciences, Kyushu
University, 3-1-1 Maidashi, Higashi-ku, Fukuoka812-8582, Japan. E-mail: sasaki@phar.kyushu-u.ac.jp
752

A New Odorless Procedure for 2ꢁ-Deoxy-6-thioguanosine
753
the NO transfer reaction from S-nitroso-6-thioguanosine in the ODN.[8,9]
The nitrosylated amino group of cytosine caused deamination to form
uracil, and its potential application as an artificial tool for RNA editing is
now under investigation. This strategy was expanded to the functionality-
transfer reaction of the relatively large organic entity to the amino group
of cytosine base, and site-specific covalent modification of RNA has been
achieved.[10,11]
The excellent synthesis of 2ꢁ-deoxy-6-thioguanosine, the phospho-
ramidite derivative and its incorporation into the ODN have already
been reported.[12–18] One approach utilized the preformed 2ꢁ-deoxy-6-
thioguanosine to protect the 6-thiocarbonyl group with the cyanoethyl
group[12][13] or the 2,4-dinitorophenyl group.[17,18] These protecting
groups are useful, because they can be removed under mild alkaline
conditions. For the other approach, the 6-carbonyl group was transformed
into a labile leaving group such as a halide or arylsulfonyloxy group for
displacement with 2-cyanoethanethiol.[14–16] In both approaches, odor-
ous agents, such as 2-cyanoethanethiol or hydrogen sulfide, are used.
We now describe a new practical synthesis of the 3ꢁ-O-phosphoramidite
derivative of 6-S-protected 2ꢁ-deoxy-6-thioguanosine by using 2-ethylhexyl
3-mercaptopropionate[19,20] as an odorless and inexpensive reagent and its
incorporation into the oligodeoxynucleotides.
RESULTS AND DISCUSSION
The synthetic route for 2ꢁ-deoxy-6-thioguanosine phosphoramidite
is shown in Scheme 1. 2ꢁ-Deoxyguanosine 1 protected with tert-butyl-
dimethylsilyl (TBDMS) groups was reacted with 2-mesitylenesulfonyl chlo-
ride to activate the 6-position of guanine, and the formed intermediate
was treated with 2-ethylhexyl 3-mercaptopropionate in the presence of
N -methylpyrrolidine. This one-flask, two-step procedure gave 2 in a good
yield (95%) after purification using a silica gel column. As the reaction
and workup can be easily performed without any smell, this procedure
can be applied to a large scale synthesis. Although racemic 2-ethylhexyl
3-mercaptopropionate was used, two diasteroisomers of 2 did not produce
any trouble either in purification by column chromatography or in structure
determination by NMR. The 2-amino group of 2 was protected with phe-
noxyacetyl chloride, and the TBDMS groups were deprotected with TBAF
to give the corresponding diol product 3 in 71% yield for two steps. The
5ꢁ-hydroxyl group of 3 was selectively protected with 4,4ꢁ-dimethoxytrityl
(DMTr) chloride in pyridine (94%), and the phoshporamidite precur-
sor 4 was synthesized by a standard procedure with 2-cyanoethyl-N ,N -
diisopropylchlorophosphoramidite in 77% yield after silica gel column
chromatography followed by precipitation in hexane at −78◦C. The amidite
precursor 4 was applied to an automated DNA synthesizer for incorporation
into the ODN 5ꢁ-d(CTTTGsTTCTCCTTTCT).

754
K. Onizuka et al.
O
O
O
N
S
N
N
N
N
N
NH
NH2
N
a, b
c, d
NH2
HO
TBSO
O
O
OTBS
2
OH
1
O
O
O
O
S
N
S
N
N
N
N
N
N
N
e, f
NHPac
NHPac
DMTrO
HO
O
O
O
OH
P OCH2CH2CN
3
4
N
SCHEME 1 Synthesis of 6-S-protected 2ꢁ-deoxy-6-thioguanosine by using 2-ethylhexyl 3-
mercaptopropionate. (a) TBDMSCl, imidazole, DMF, room temperature, 94%; (b) 2-mesitylenesulfonyl
chloride, Et3N, DMAP, CH2Cl2, 0◦C to room temperature, then N -methylpyrrolidine, 2-ethylhexyl
3-mercaptopropionate, 0◦C to room temperature, 95%; (c) phenoxyacethyl chloride, 1-HOBt, pyridine,
CH3CN, room temperature, 84%; (d) TBAF, THF, room temperature 85%; (e) DMTrCl, pyridine, room
temperature, 94%; (f) amidite reagent, DIPEA, CH2Cl2, 0◦C, 77%.
When the synthesized ODN5 was cleaved from the CPG resin by
the treatment with a 1M NaOH and 0.01M NaSH solution, the 2-
ethylhexylpropionate group was not deprotected but was hydrolyzed to give
ODN6 after HPLC purification and subsequent DMTr deprotection with
5% AcOH (Scheme 2). On the other hand, the prior treatment of ODN5

A New Odorless Procedure for 2ꢁ-Deoxy-6-thioguanosine
755
OH
O
O
O
S
N
S
N
N
N
N
N
N
DMTrO
DNA
NH2
N
NHPac
DNA
O
O
DNA
DNA
1) 1M NaOH aq,
0.01M NaSH aq
2) HPLC purification
3) 5% AcOH
6
5
CPG
1) 1M DBU in CH3CN
2) 1M NaOH aq,
0.01M NaSH aq
3) HPLC purification
4) 5% AcOH
S
N
NH
N
N
NH2
DNA
O
DNA
7
SCHEME 2 Conditions for deprotection of 2-ethylhexylpropionate and cleavage from the CPG resin.
with 1M DBU in CH3CN was effective for the β-elimination to remove the
2-ethylhexylpropionate group from ODN5 on the CPG resin. Subsequent
cleavage of the ODN from the CPG resin by the treatment with a 1M NaOH
and 0.01M NaSH solution produced the DMTr-protected ODN7. An alkaline
0.01M NaSH solution was used to protect against air oxidation and sulfur
loss. The HPLC analysis of the crude products indicated that both the
DNA synthesis and the deprotection of the 2-ethylhexylpropionate group
efficiently proceeded (Figure 1A). Finally, the terminal 5ꢁ-DMTr group was
deprotected with 5% AcOH to give the desired ODN7 containing 2ꢁ-deoxy-
6-thioguanosine as a single peak in ca 60% isolated yield (Figure 1B). There

756
K. Onizuka et al.
FIGURE 1 HPLC Chart and MALD-TOF/MS of the synthesized DNA. A) HPLC chart of the crude
sample of DMTr-protected ODN7. B) HPLC of ODN7 purified by HPLC after the treatment of 5%
AcOH. C) MALDI-TOF/MS of ODN7 calcd. 4767.79 ([M-H]−), found 4767.12.
was no peak with a lower molecular weight than the calculated one in
the MALDI-TOF/MS spectra of the ODN7 (Figure 1C), confirming that
desulfurization did not take place during the ODN synthesis and isolation
procedure.
CONCLUSION
In conclusion, we have established the efficient and odorless
synthesis of 6-S-{2-[(2-ethylhexyl)oxycarbonyl]ethyl)}-3ꢁ,5ꢁ-O-bis(tert-butyldi
methylsilyl)-2ꢁ-deoxy-6-thioguanosine (2) from the corresponding 6-
O-mesitylenesulfonyl derivative by the reaction with 2-ethylhexyl-3-
mercaptopropionate; an odorless and inexpensive reagent. The phospho-
ramidite precursor was also derived from 2 by the conventional method,
and was successfully applied to an automated DNA synthesizer to produce
2ꢁ-deoxy-6-thioguanosine containing ODN. The described procedure is
expected to be useful as a standard protocol for the synthesis of ODN
incorporating 2ꢁ-deoxy-6-thioguanosine.
EXPERIMENTAL
6-S-{2-[(2-ethylhexyl)oxycarbonyl]ethyl)}-3ꢁ,5ꢁ-O-bis(tert-
butyldimethylsilyl)-2ꢁ-deoxy-6-thioguanosine(2)
3ꢁ,5ꢁ-O-Bis(tert-butyldimethylsilyl)-2ꢁ-deoxy-6-guanosine (1 g, 2.0 mmol)
was dried by azeotropic evaporation with dry CH3CN, and dissolved in
dry CH2Cl2 (60 mL). To the suspension were added dry Et3N (1.1 ml,
7.9 mmol), 2-mesitylenesulfonyl chloride (530 mg, 2.4 mmol), and DMAP
(12.3 mg, 0.010 mmol) at 0◦C under an argon atmosphere. After be-
ing stirred for 12 hours at room temperature, the reaction mixture was

A New Odorless Procedure for 2ꢁ-Deoxy-6-thioguanosine
757
cooled to 0◦C. N -methylpyrrolidine (2.1 mL, 20 mmol) and 2-ethylhexyl
3-mercaptopropionate (4.6 mL, 20 mmol) were added to the mixture,
and then the mixture was stirred at room temperature for 5.5 hours.
The reaction mixture was diluted with CH2Cl2 (40 mL) and washed with
1M KH2PO4 (70 mL × 3). The combined organic layers were dried over
Na2SO4, then evaporated. The crude product was purified by silica gel
chromatography (hexane/ethyl acetate = 4:1) to give 2 (1.34 g, 95%) as a
colorless oil. 1H NMR (400 MHz, CDCl3): δ: 7.93 (1H, s), 6.28 (1H, t, J = 6.7
Hz), 5.13 (2H, brs), 4.57–4.54 (1H, m), 4.05–3.98 (2H, m), 3.97–3.95 (1H,
m), 3.79 (1H, dd, J = 11.2, 4.2 Hz), 3.73 (1H, dd, J = 11.2, 3.4 Hz), 3.56 (2H,
t, J = 7.0 Hz), 2.81 (2H, t, J = 7.0 Hz), 2.62 (1H, ddd, J = 13.1, 6.6, 6.1 Hz),
2.23 (1H, ddd, J = 13.1, 6.1, 3.7 Hz), 1.57–1.52 (1H, m), 1.33 (2H, quint, J
= 7.3 Hz), 1.30–1.20 (6H, m), 0.89 (9H, s), 0.88 (9H, s), 0.86 (6H, t, J = 7.3
Hz), 0.09 (6H, s), 0.03 (6H, s); IR (neat): 3321, 3193, 1734, 1591, 1564, 1255,
836 cm−1; HR-ESIMS calcd for C33H61N5O5SSi2 (M+H)+: m/z 696.4005,
found: 696.4006.
6-S-{2-[(2-ethylhexyl)oxycarbonyl]ethyl)}-2-N-phenylacetyl-3ꢁ,5ꢁ-
O-bis(tert-butyldimethylsilyl)-2ꢁ-deoxy-6-thioguanosine
Compound 2 (1.18 g, 1.7 mmol) was dried by azeotropic evaporation
with dry CH3CN, and dissolved in dry CH3CN (5 mL) and dry pyridine
(5 mL). To the solution was added 1-hydroxybenzotriazol (573 mg, 4.2
mmol) in the presence of molecular sieves 4A at room temperature. After
being stirred for 1.5 hours, phenoxyacetyl chloride (1.2 mL, 8.7 mmol)
was added to the reaction mixture. The reaction mixture was stirred for
an additional 8.5 hours, diluted with ethyl acetate (50 mL), and filtered
through Celite. The filtrate was washed with brine (50 mL × 2), dried
over Na2SO4, then evaporated. The crude product was purified by silica gel
chromatography (hexane/ethyl acetate = 5:1) to give the title compound
(1.18 g, 84%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3): δ: 8.84 (1H,
brs), 8.16 (1H, s), 7.33 (2H, t, J = 7.6 Hz), 7.04 (1H, t, J = 7.6 Hz), 7.01
(2H, d, J = 7.9 Hz), 6.40 (1H, t, J = 6.4 Hz), 4.73 (2H, s), 4.61–4.58 (1H,
m), 4.03–3.98 (3H, m), 3.84 (1H, dd, J = 11.3, 4.3 Hz), 3.75 (1H, dd, J =
11.3, 3.4 Hz), 3.60 (2H, t, J = 7.0 Hz), 2.88 (2H, t, J = 7.0 Hz), 2.63 (1H,
ddd, J = 12.9, 6.6, 6.1 Hz), 2.41 (1H, ddd, J = 12.9, 6.1, 3.7 Hz), 1.57–1.54
(1H, m), 1.35–1.25 (8H, m), 0.89 (9H, s), 0.88 (9H, s), 0.85 (6H, t, J =
7.3 Hz), 0.09 (6H, s), 0.06 (6H, s); IR (neat): 3408, 1730, 1576, 1495, 1380,
1216, 837; ESI-MS: m/z 830.5 (M+H)+
6-S-{2-[(2-ethylhexyl)oxycarbonyl]ethyl)}-2-N-phenylacetyl-2ꢁ-
deoxy-6-thioguanosine(3)
To a solution of the above compound (993 mg, 1.2 mmol) in dry
THF (10 mL) was added TBAF (946 mg, 3.0 mmol) at room temperature.

758
K. Onizuka et al.
After being stirred for 1.5 hours, the reaction mixture was evaporated. The
residue was purified by silica gel chromatography (CHCl3/MeOH = 99:1)
to give 3 (613 mg, 85%) as a pale yellow solid. 1H NMR (400 MHz, CDCl3):
δ: 9.03 (1H, s), 8.15 (1H, s), 7.33 (2H, t, J = 7.9 Hz), 7.04 (1H, t, J = 7.9 Hz),
7.02 (2H, d, J = 7.9 Hz), 6.40 (1H, t, J = 6.7 Hz), 4.95–4.94 (1H, m), 4.67
(2H, s), 4.14–4.13 (1H, m), 4.05–3.98 (2H, m), 3.94 (1H, dd, J = 10.1, 2.4
Hz), 3.85 (1H, dd, J = 10.1, 2.4 Hz), 3.59 (2H, t, J = 7.0 Hz), 3.02–2.92 (1H,
m), 2.86 (2H, t, J = 7.0 Hz), 2.83 (2H, brs), 1.56–1.53 (1H, m), 1.57–1.54
(1H, m), 1.33–1.24 (8H, m), 0.85 (6H, t, J = 7.3 Hz); IR (neat): 3398(br),
1729, 1578, 1495, 1380, 1214; HR-ESIMS calcd for C29H39N5O7S (M+H)+:
m/z 602.2643, found: 602.2678.
6-S-{2-[(2-ethylhexyl)oxycarbonyl]ethyl)}-5ꢁ-O-(4,4ꢁ-
dimethoxytrityl)-2-N-phenylacetyl-2ꢁ-deoxy-6-thioguanosine
Compound 3 (370 mg, 0.62 mmol) was dried by azeotropic evaporation
each with dry CH3CN and dry pyridine, and dissolved in dry pyridine
(6 mL). To the solution was added 4,4ꢁ-dimethoxytrityl chloride (521 mg,
1.54 mmol) at room temperature, and then the mixture was stirred for 1
hour. The reaction mixture was diluted with ethyl acetate (40 mL), washed
water and brine (each 40 mL). The organic phase was dried over Na2SO4,
then evaporated. The residue was purified by silica gel chromatography
(CHCl3/MeOH = 49:1, 0.5% pyridine) to give the title compound (520 mg,
1
94%) as a pale yellow foam. H NMR (400 MHz, CDCl3): δ: 8.92 (1H, s),
8.06 (1H, s), 7.39 (2H, d, J = 7.3 Hz), 7.34 (2H, t, J = 7.9 Hz), 7.28 (4H, d,
J = 8.9 Hz), 7.22 (2H, t, J = 7.3 Hz), 7.16 (1H, t, J = 7.3 Hz), 7.05 (1H, t,
J = 7.9 Hz), 7.00 (2H, d, J = 7.9 Hz), 6.77 (4H, d, J = 8.9 Hz), 6.64 (1H, t,
J = 6.7 Hz), 4.81–4.79 (1H, m), 4.62 (2H, s), 4.24–4.21 (1H, m), 4.07–3.99
(2H, m), 3.74 (6H, s), 3.60 (2H, t, J = 7.0 Hz), 3.43 (1H, dd, J = 10.1, 4.9
Hz), 3.42 (1H, brs), 3.34 (1H, dd, J = 10.1, 4.3 Hz), 2.87 (2H, t, J = 7.0 Hz),
2.72 (1H, ddd, J = 13.4, 6.7, 6.7 Hz), 2.64 (1H, ddd, J = 13.4, 6.1, 3.7 Hz),
1.59–1.54 (1H, m), 1.38–1.26 (8H, m), 0.87 (6H, t, J = 7.3 Hz); IR (neat):
3405, 1727, 1576, 1508, 1380, 1249, 729; HR-ESIMS calcd for C50H57N5O9S
(M+H)+: m/z 904.3950, found: 904.3933.
6-S-{2-[(2-ethylhexyl)oxycarbonyl]ethyl)}-5ꢁ-O-(4,4ꢁ-
dimethoxytrityl)-2-N-phenylacetyl-2ꢁ-deoxy-6-thioguanosine-3ꢁ-O-
(2-cyanoethyl)(N,N-diisopropyl)phosphoramidite(4)
The above compound (308 mg, 0.34 mmol) was dried by azeotropic
evaporation each with dry CH3CN, and dissolved in dry CH2Cl2 (3.5 mL). To
the solution was added diisopropylethylamine (360 µL, 2.1 mmol) at 0◦C.
After being stirred for 25 minutes, N ,N -diisopropylchlorophosphoramidite
(190 µL, 0.85 mmol) was added, and then the mixture was stirred for
an additional 1 hour at the same temperature. The reaction mixture
was quenched with saturated aqueous NaHCO3 (15 mL), extracted with

A New Odorless Procedure for 2ꢁ-Deoxy-6-thioguanosine
759
ethyl acetate (15 ml × 3). The organic phases were dried over Na2SO4,
then evaporated. The residue was purified by silica gel chromatography
(hexane/ethyl acetate = 2:1) to give the purified material, which was
precipitated in hexane at −78◦C. The hexane was removed by decantation,
and the solid material was dried in a vacuum for several hours to give 4
1
(288 mg, 77%) as a pale yellow foam. H NMR (400 MHz, CDCl3): δ: 8.78
(1H, brs), 8.06 (0.5H, s), 8.05 (0.5H, s), 7.39–7.33 (4H, m), 7.29–7.16 (7H,
m), 7.05 (1H, t, J = 7.9 Hz), 7.02 (2H, d, J = 7.9 Hz), 6.78–6.74 (4H, m),
6.43 (1H, t, J = 6.1 Hz), 4.79–4.74 (3H, m), 4.30–4.26 (1H, m), 4.08–4.00
(2H, m), 3.87–3.54 (6H, m), 3.77 (3H, s), 3.76 (3H, s), 3.40–3.31 (2H, m),
2.93–2.89 (2H, m), 2.86–2.68 (2H, m), 2.61 (1H, t, J = 6.4 Hz), 2.45 (1H,
t, J = 6.4 Hz), 1.59–1.55 (1H, m), 1.39–1.26 (8H, m), 1.19–1.10 (12H, m),
0.87 (6H, t, J = 7.3 Hz); 31P NMR (161 MHz, CDCl3): δ: 149.6; IR (neat):
3406, 2252, 1728, 1575, 1508, 1379, 1249, 1178, 1035, 728; HR-ESIMS calcd
for C50H57N5O9S (M+H)+: m/z 1104.5028, found: 1104.5050.
Synthesis of Oligodeoxynucleotides (ODN7)
ODN 7 was synthesized at a 1 µmol scale on a Model 394 DNA/RNA
synthesizer (Applied Biosystems, Foster City, CA, USA) with standard
β-cyanoethyl phosphoramidite chemistry. The 5ꢁ-terminal dimethoxytrityl-
bearing ODNs were deprotected with 1.0 M DBU in dry CH3CN at room
temperature for 5 hours to remove the 2-[(2-ethylhexyl)oxycarbonyl]ethyl
group, and removed from the solid support by treatment with 1 M NaOH
and 0.01 M NaSH (1 mL) for 4 hours. The crude product was purified
by reverse-phase HPLC (Column: nacalai tesque: COSMOSIL 5C18-AR-II,
10 × 250 mm; Solvent: A: 0.1M TEAA Buffer, B: CH3CN, B: 10–40% per
20 minutes, linear gradient; flow rate: 3.0 ml/min; monitored by UV
detector at 254 nm). The dimethoxytrityl group of the purified ODN was
cleaved with 5% AcOH and the mixture was additionally purified by HPLC
(solvent: A: 0.1M TEAA Buffer, B: CH3CN, B: 10–30% per 20 minutes). The
isolated ODN7 was quantified by UV measurement to be 60% isolated yield.
MALDI-TOFMS (m/z) 7: calcd 4767.79, found 4767.12.
REFERENCES
1. Lencastre, A.D.; Hamill, S.; Pyle, A.M. A single active-site region for a group II intron. Nat. Struct.
Mol. Biol. 2005, 12, 626–627.
2. Christian, E.L.; McPheeters, D.S.; Harris, M.E. Identification of individual nucleotides in the
bacterial ribonuclease P ribozyme adjacent to the pre-tRNA cleavage site by short-range photo-cross-
linking. Biochemistry 1998, 37, 17618–17628.
3. Wang, Z.; Rana, T.M. RNA-protein interactions in the Tat-trans-activation response element complex
determined by site-specific photo-cross-linking. Biochemistry 1998, 37, 4235–4243.
4. Nikiforov, T.T.; Connolly, B.A. Oligodeoxynucleotides containing 4-thiothymidine and 6-
thiodeoxyguanosine as affinity labels for the Eco RV restriction endonuclease and modification
methylase. Nucleic Acids Res. 1992, 20, 1209–1214.

760
K. Onizuka et al.
5. Coleman, R.S.; Siedlcki, J.M. Synthesis of a 4-thio-2ꢁ-deoxyuridine-containing oligonucleotide.
Development of the thiocarbonyl group as a linker element. J. Am. Chem. Soc. 1992, 114, 9229–9230.
6. Coleman, R.S.; Kesicki, E.A. Synthesis and postsynthetic modification of oligodeoxynucleotides
containing 4-thio-2ꢁ-deoxyuridine (ds4U). J. Am. Chem. Soc. 1994, 116, 11636–11642.
7. Coleman, R.S.; Pires, R.M. Covalent cross-linking of duplex DNA using 4-thio-2ꢁ-deoxyuridine as a
readily modifiable platform for introduction of reactive functionality into oligonucleotides. Nucleic
Acids Res. 1997, 25, 4771–4777.
8. Ali, M.M.; Alam, M.R.; Kawasaki, T.; Nakayama, S.; Nagatsugi, F.; Sasaki, S. Sequence- and base-
specific delivery of nitric oxide to cytidine and 5-methylcytidine leading to efficient deamination. J.
Am. Chem. Soc. 2004, 126, 8864–8865.
9. Ali, M.M.; Nagatsugi, F.; Nakayama, S.; Alam, M.R.; Kawasaki, T.; Sasaki, S. Design of high efficient
and selective transfer reaction of nitrosyl group to dC and dmC resulting in specific deamination.
Nucleosides, Nucleotides, and Nucleic Acids 2005, 24, 721–724.
10. Onizuka, K.; Taniguchi, Y.; Sasaki, S. Site-specific covalent modification of RNA guided by
functionality-transfer oligodeoxynucleotides. Bioconjugate Chem. 2009, 20, 779–803.
11. Onizuka, K.; Taniguchi, Y.; Sasaki, S. Development of novel thioguanosine analogs with the ability to
specifically modified cytidine. Nucleic Acids Symp. Series 2007, 51, 5–6.
12. Christopherson, M.S.; Broom, A.D. Synthesis of oligonucleotides containing 2ꢁ-deoxy-6-
thioguanosine at a predetermined site. Nucleic Acids Res. 1991, 19, 5719–5724.
13. Adams, C.J.; Farrow, M.A.; Murray, J.B.; Kelly, S.M.; Price, N.C.; Stockley, P.G. Incorporation of 6-
thioinosine into oligoribonucleotides. Tetrahedron Lett. 1995, 36, 5421–5424.
14. Rao, T.S.; Jayaraman, K.; Durland, R.H.; Revankar, G.R. A novel synthesis of S6-cyanoethyl-2ꢁ-
deoxythioguanosine and its incorporation into triple helix forming oligonucleotides. Tetrahedron
Lett. 1992, 33, 7651–7654.
15. Rao, T.S.; Durland, R.H.; Seth, D.M.; Myrick, M.A.; Bodepudi, V.; Revankar, G.R. Incorporation
of 2ꢁ-deoxy-6-thioguanosine into G-rich oligodeoxyribonucleotides inhibits G-tetrad formation and
facilitates triplex formation. Biochemistry 1995, 34, 765–772.
16. Kadokura, M.; Wada, T.; Seio, K.; Sekine, M. Synthesis of 4-thiouridine, 6-thioinosine, and 6-
thioguanosine 3ꢁ,5ꢁ-O-bisphosphates as donor molecules for RNA ligation and their application
to the synthesis of photoactivatable TMG-capped U1 snRNA fragments. J. Org. Chem. 2000, 65,
5104–5113.
17. Wang, Y.; Lattmann, E.; Zheng, Q. Preparation of 6-thioguanosine phosphoramidite for oligoribonu-
cleotide synthesis. Nucleosides, Nucleotides, and Nucleic Acids 2003, 22, 1247–1249.
18. Zheng, Q.; Wang, Y.; Lattmann, E. Synthesis of S6-(2,4-dinitrophenyl)-6-thioguanosine phos-
phoramidite and its incorporation into oligoribonucleotides. Bioorg. Med. Chem. Lett. 2003, 13,
3141–3144.
19. Itoh, T.; Mase, T. A general palladium-catalyzed coupling of aryl bromides/triflates and thiols. Org
Lett. 2004, 6, 4587–4590.
20. Itoh, T.; Mase, T. Practical thiol surrogates and protective groups for arylthiols for Suzuki-Miyaura
conditions. J. Org. Chem. 2006, 71, 2203–2206.