Efficient desulfurization of 2-thiopyrimidine nucleosides to the corresponding 4-pyrimidinone analogues using trans-2-(phenylsulfonyl)-3- phenyloxaziridine

Article abstract of DOI:10.1016/j.tetlet.2004.07.052

A brief treatment of 2-thiopyrimidine nucleosides (s²U*) with trans-2-phenylsulfonyl-3-phenyloxaziridine (PSO) results in efficient substrate desulfurization leading to the corresponding 4-pyrimidinone analogues (H²U*). The key transformation proceeds through oxidation of the 2-thiocarbonyl group to a sulfur oxyacid derivative and subsequent elimination of sulfur dioxide. 4-Pyrimidinone 1-β-D-riboside (H ²U) has been transformed into the respective phosphoramidite, a ready-to-use monomer for the introduction of a modified nucleoside into an oligonucleotide chain. Moreover, the effective desulfurization of the 2-thiouridine nucleotide could be achieved directly at the oligonucleotide level, by treatment of the TdA(s²U)dGdC oligonucleotide with PSO, as verified by MALDI-TOF mass spectrometry.

Full text of DOI:10.1016/j.tetlet.2004.07.052

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 6729–6731
Efficient desulfurization of 2-thiopyrimidine nucleosides to
the corresponding 4-pyrimidinone analogues using
trans-2-(phenylsulfonyl)-3-phenyloxaziridine
*
_
Elzbieta Sochacka and Iwona Fra˛tczak
_
Institute of Organic Chemistry, Technical University of Lodz, Zeromskiego 116, 90-924 Łodz, Poland
´ ´
´ ´
Received 31 May 2004; revised 7July 2004; accepted 13 July 2004
Available online 29 July 2004
Abstract—A brief treatment of 2-thiopyrimidine nucleosides (s2U*) with trans-2-phenylsulfonyl-3-phenyloxaziridine (PSO) results in
efficient substrate desulfurization leading to the corresponding 4-pyrimidinone analogues (H2U*). The key transformation proceeds through
oxidation of the 2-thiocarbonyl group to a sulfur oxyacid derivative and subsequent elimination of sulfur dioxide. 4-Pyrimidinone
1-b-D-riboside (H2U) has been transformed into the respective phosphoramidite, a ready-to-use monomer for the introduction of a
modified nucleoside into an oligonucleotide chain. Moreover, the effective desulfurization of the 2-thiouridine nucleotide could be
achieved directly at the oligonucleotide level, by treatment of the TdA(s2U)dGdC oligonucleotide with PSO, as verified by MALDI-
TOF mass spectrometry.
Ó 2004 Elsevier Ltd. All rights reserved.
The synthesis of modified nucleosides and their incorpo-
ration into oligonucleotide sequences is an important
strategy for the elucidation of structure–function rela-
tionships of nucleic acids.1–3 A variety of nucleoside
derivatives have been prepared through deletion or by
changing the nature of the functional groups present
on the heterocyclic bases.2–4 One simple base modifica-
tion among uridine nucleotides that has dramatic effect
on nucleoside conformation is the replacement of oxy-
gen at C-2 with sulfur.5 2-Thiopyrimidine nucleosides
are known to adopt preferentially a rigid C30-endo sugar
ring conformation,6,7 so in RNA duplexes, a modified
s2U-A base pair is more stabilized than the unmodified
one.8–10 Furthermore, due to steric hindrance and the
weaker H-bonding ability of sulfur relative to oxygen,
2-thiouridine destabilizes the U-G wobble base pair
compared to uridine.8–10 4-Pyrimidinone nucleosides
are a class of nucleoside analogues lacking both the
N3-amide hydrogen and the 2-carbonyl function,11–14
and thus they do not form the conventional wobble
U-G base pair. An incorporation of a 2-thiouridine
and a 4-pyrimidinone-1-b-D-riboside, instead of uridine,
in the site specific position within the oligonucleotide
chain could provide useful models for the study of bio-
logically important U-G wobble interactions.15,16
In the present report we describe a very efficient trans-
formation of 2-thiopyrimidine nucleosides into the
corresponding 4-pyrimidinone analogues by a brief
treatment with trans-2-phenylsulfonyl-3-phenyloxa-
ziridine (PSO).17 Previously it had been reported that
the desulfurization of 2-thiopyrimidine nucleosides pro-
ceeds in moderate yield under reductive conditions by
dipotassium diazenedicarboxylate treatment11 or Ra-
ney-nickel reduction.12 Oxidative desulfurization of the
2-thiopyrimidine moiety has been observed on treatment
with hydrogen peroxide,11 aqueous iodine,13 m-chlorop-
erbenzoic acid/pyridine13 or dimethyldioxirane.14 Oxa-
ziridine-type oxidizing reagents (2-(phenylsulfonyl)-3-
(3-nitrophenyl)oxaziridine and 10-camphorsulfonyl
oxaziridine) were applied recently in the oxidation step
of oligonucleotide synthesis in H-phosphonate18 and
phosphoramidite19 approaches.
During our evaluation of 2-thiouridine stability under
different oxidizing conditions, used for automated oligo-
nucleotide synthesis, we discovered that treatment of a
2-thiopyrimidine nucleoside with PSO led to complete
loss of sulfur giving the 4-pyrimidinone analogue
quantitatively.20
Keywords: 2-Thiopyrimidine nucleosides; Oxidative desulfurization;
4-Pyrimidinone 1-b-D-ribosides; Oxaziridine.
*
Corresponding author. Tel.: +48-42-631-3141; fax: +48-42-636-
5530; e-mail: ejsochac@p.lodz.pl
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.07.052

6730
E. Sochacka, I. Fra˛tczak / Tetrahedron Letters 45 (2004) 6729–6731
the 4-pyrimidinone nucleosides. To confirm this, 20,30,50-
O
O
O
O
Ph S
O
R
O-tribenzoyl-2-thiouridine in anhydrous methylene
chloride or acetonitrile was treated with 2equiv of
PSO under a stream of argon. The emerging argon
was analyzed for sulfur oxides. Using known proce-
dures,23 we showed that sulfur dioxide was the only gas-
eous reaction product. Moreover, the solid reaction
residue, separated on a silica gel column, gave in quan-
titative yield the tribenzoyl derivative of the correspond-
ing 4-pyrimidinone ribonucleoside together with the
sulfonimine PhSO2N = CHPh.
R
N
CH
Ph
HN
N
N
S
H
N
HO
HO
O
O
pyridine; r.t.; 0.5h
OH
HO
OH
HO
s2U*
H2U*
1. a: R = H
2. a: R = H
b: R = OCH3
b: R = OCH3
c: R = CH2NHCH3
c: R = CH2NHCH3
d: R = CH2COOCH3
d: R = CH2COOCH3
Scheme 1.
PSO-assisted desulfurization was also used for trans-
forming 50-O-(dimethoxytrityl)-20-O-(tert-butyldimethyl-
silyl)-2-thiouridine 324,25 into 4, which allowed us to
prepare the phosphoramidite 5, ready to use as a mono-
mer for the introduction of a 4-pyrimidinone nucleoside
into an oligonucleotide chain (Scheme 2). Desulfuriza-
tion of 3 was performed by its brief treatment (30min)
with two molar equivalents of PSO in anhydrous methyl-
ene chloride (after silica gel column chromatography
the isolated yield of 4 was 77%). Pyrimidinone deriva-
tive 4 was then easily converted into the respective phos-
phor-amidite 5 by the reaction with 2-cyanoethyl
diisopropylchlorophosphoramidite.26 The structure of
phosphoramidite 5 was confirmed by 31P NMR and
HR mass spectrometry.27
Thus, 2-thiouridine 1a and its 5-substituted analogues
1b–d, commonly found at the wobble position of the an-
ticodon loop of tRNA,16 were studied in the desulfuriza-
tion process (Scheme 1). Treatment of 2-thiouridine21
with an excess of PSO solution in pyridine (minimum
2equiv) for 30min at room temperature afforded 4-
pyrimidinone 2a in 81% isolated yield.22 Modified 2-
thiouridines 1b–d underwent similar desulfurization in
quantitative yields. The courses of the reactions were
1
monitored by H NMR spectroscopy. A significant up-
field shift of the resonance signal of 10-H was observed
during the 1 ! 2 transformation (from 7.2–7.0ppm for
1b–d to 5.9–5.7ppm for 2b–d).
The desulfurization procedure shown here represents a
significant improvement over current methods.11–13
High yields, mild reaction conditions, the stability of
common nucleosides and oligonucleotides to the action
of oxaziridine-type oxidizing agents18,19 encouraged us
to apply this method to the post-synthetic modification
of oligonucleotides containing 2-thiopyrimidine nucleo-
sides. The preliminary experiment was performed on the
model pentamer TdA(s2U)dGdC. The reaction sub-
strate and products were analyzed by MALDI TOF
mass spectrometry (Fig. 1).
Unexpectedly, the same quantitative transformation of
s2U to H2U was observed when the PSO-assisted oxida-
tion was carried out in the presence of oxygen or nitro-
gen nucleophiles (methanol, water, n-propylamine), so it
was possible to perform efficient desulfurization of 2-thio-
pyrimidine nucleosides in aqueous media. An aqueous
solution of the 2-thiouridine was treated with 3equiv
of PSO dissolved in acetonitrile (30min, 25°C) and,
after washing with ethyl acetate, was concentrated in va-
cuo. The crude reaction product was purified by silica
gel column chromatography in chloroform/methanol
solution and pure derivative 2a was isolated in 79% yield.
The peak at m/z 1495 corresponding to s2U-containing
oligonucleotide was shifted to m/z 1463 after PSO treat-
ment. This result indicated an efficient loss of sulfur
atom during oxidation, resulting in the formation of
an oligonucleotide with a modified H2U unit.
It is noteworthy that common DNA and RNA nucleo-
sides were not affected by PSO under these reaction
conditions.
We suggest that the PSO-assisted desulfurization of 2-
thiouridines proceeds via the initial formation of an sul-
fur oxyacid14 followed by subsequent decomposition to
Further work on optimization of the desulfurization
protocol for 2-thiouridine-containing oligonucleotides,
also bound to the solid support is in progress.
O
O
O
HN
N
N
i
ii
N
S
N
H
N
H
DMTO
DMTO
DMTO
O
O
O
OTBDMS
O
HO
OTBDMS
OTBDMS
HO
O P
N
3
5
4
N
Scheme 2. Reagents and conditions: (i) PSO 2equiv/CH2Cl2; rt; 30min. (ii) DIPEA/CH2Cl2; 2-cyanoethyl diisopropylchlorophosphoramidite; argon;
rt; 30min.

E. Sochacka, I. Fra˛tczak / Tetrahedron Letters 45 (2004) 6729–6731
6731
Figure 1. MALDI TOF mass spectra of (a) TdA(s2U)dGdC in 2,4,6-trihydroxyacetophenone as a matrix; (b) reaction mixture after TdA(s2U)dGdC
(0.2OD in 10lL of water) treatment with 0.1M PSO solution in acetonitrile (10lL).
13. Kuimelis, R. G.; Nambiar, K. P. Tetrahedron Lett. 1993,
34, 3813–3816.
Acknowledgements
14. Saladino, R.; Mincione, E.; Cearsini, C.; Mezzetti, M.
Tetrahedron 1996, 52, 6759–6780.
15. Masquida, B.; Westhof, E. RNA 2000, 6, 9–15.
16. Takai, K.; Yokoyama, S. Nucleic Acids Res. 2003, 31,
6383–6391.
This work was supported by the State Committee for
Scientific Research (project PBZ-KBN-059/T09/07to
E.S.).
17. Davis, F. A.; Sheppard, A. C. Tetrahedron 1989, 45,
5703–5742.
References and notes
18. Wada, T.; Sato, Y.; Honda, F.; Kawahara, S.; Sekine, M.
J. Am. Chem. Soc. 1997, 119, 12710–12721.
19. Gianolio, D. A.; McLaughlin, L. W. Nucleos. Nucleot.
1999, 18, 1751–1769.
20. Sochacka, E. Nucleos. Nucleot. Nucleic Acids 2001, 20,
1871–1879.
21. Vorbruggen, H. Chem. Ber. 1981, 114, 1279–1286.
1. Zimmermann, R. A.; Gait, M. J.; Moore, M. J. In
Modification and Editing of RNA; Grosjean, H., Benne, R.,
Eds. ; ASM: Washinghton, DC, 1998; Chapter IV, pp 59–
84.
2. Earnshaw, D. J.; Gait, M. J. Biopolymers 1998, 48, 39–
55.
3. Verma, S.; Eckstein, F. Annu. Rev. Biochem. 1998, 67,
99–134.
4. Grasby, J. A.; Gait, M. J. Biochimie 1994, 76, 1223–
1234.
5. Davis, D. R. In Modification and Editing of RNA;
Grosjean, H., Benne, R., Eds.; ASM: Washinghton, DC,
1998; Chapter V, pp 85–102.
6. Yamamoto, Y.; Yokoyama, S.; Miyazawa, T.; Watanabe,
K.; Higuchi, S. FEBS Lett. 1983, 157, 95–99.
7. Sierzputowska-Gracz, H.; Sochacka, E.; Malkiewicz, A.;
Kuo, K.; Gehrke, C.; Agris, P. F. J. Am. Chem. Soc. 1987,
109, 7171–7177.
3
22. 1H NMR (250MHz, D2O, dppm) 3.75 (dd, 1H, J5 -4
=
=
00
0
0
4.0Hz, J5 -5 = 12.8Hz, H500), 3.85 (dd, 1H, J5 -4
3
3
00
0
0
3
3.1Hz, J5 -5 = 12.8Hz), 4.15–4.25 (m, 2H, H30, H40),
00
0
3
4.28–4.36 (m 1H, H20), 5.55 (d, 1H, J1 -2 = 5.7Hz, H1 0),
6.43 (d, 1H, J5-6 = 7.7Hz, H5), 8.03 (dd, 1H, J6-2 =
0
0
3
4
3
4
2.3Hz, J6-5 = 7.7Hz, H6), 8.60 (d, 1H, J2-6 = 2.3Hz,
H2); 31C NMR (D2O, d ppm) 60.5, 69.8, 72.8, 74.7, 85.8,
94.6, 112.2, 140.4, 151.9, 173.4; FAB MS: positive ions––
m/z = 229, [M+H]+; negative ions––m/z = 227, [MꢀH]ꢀ.
23. Karchmer, J. H. The Analytical Chemistry of Sulfur and its
Compounds; Wiley-Interscience: New York, 1970; pp 163–
167.
8. Kumar, R. K.; Davis, D. R. Nucleic Acids Res. 1997, 25,
1272–1280.
9. Testa, S. M.; Disney, M. D.; Turner, D. H.; Kierzek, R.
Biochemistry 1997, 38, 16655–16662.
10. Shohda, K.; Okamoto, I.; Wada, S.; Seio, K.; Sekine, M.
Bioorg. Med. Chem. Lett. 2000, 10, 1795–1798.
11. Ogihara, T.; Mitsunobu, O. Chemistry Lett. 1982,
1621–1624.
12. Rajur, S. B.; McLaughlin, L. W. Tetrahedron Lett. 1992,
33, 6081–6084.
24. Agris, P. F.; Malkiewicz, A.; Kraszewski, A.; Everett, K.;
Nawrot, B.; Sochacka, E.; Jankowska, J.; Guenther, R.
Biochimie 1995, 77, 1272–1280.
25. Kumar, R. K.; Davis, D. R. Nucleic Acids Res. 1997, 25,
6383–6391.
26. Dahma, M. J.; Ogilvie, K. K. Methods Mol. Biol. 1993, 20,
81–115.
27. 31P NMR (benzene, d ppm); 151.8; 148,6; FAB HRMS:
calcd for C45H61N4O8SiPNa 867.3897, found m/z =
867.3901 [M+Na]+.