Desulfuration of 2-thiouridine with hydrogen peroxide in the physiological pH range 6.6-7.6 is pH-dependent and results in two distinct products

Article abstract of DOI:10.1016/j.bmcl.2013.08.114

The 2-thiomodified nucleosides, located at first position of tRNAs anticodon, may constitute a primary target for oxidative attack under conditions of oxidative stress. Desulfuration of 2-thiouridine (S2U) was investigated in the ¹H NMR scale in the presence of 100 mM H₂O₂ and phosphate buffer in the physiological pH range, from pH 6.6 to 7.6. The obtained data demonstrate an intriguing result that within one unit of the pH range uridine is the major product of the S2U desulfuration in the pH 7.6, while the 4-pyrimidinone nucleoside (H2U) is dominant in pH 6.6. The possible desulfuration pathway and the biological importance of the transformation of S2U either to U or H2U are discussed in the context of the tRNA oxidative damage.

Full text of DOI:10.1016/j.bmcl.2013.08.114

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5803–5805
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Desulfuration of 2-thiouridine with hydrogen peroxide
in the physiological pH range 6.6–7.6 is pH-dependent and results
in two distinct products
a
a
b
b,
⇑
Elzbieta Sochacka , Paulina Bartos , Karina Kraszewska , Barbara Nawrot
a
Institute of Organic Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland
Centre of Molecular and Macromolecular Studies of the Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The 2-thiomodified nucleosides, located at first position of tRNAs anticodon, may constitute a primary
target for oxidative attack under conditions of oxidative stress. Desulfuration of 2-thiouridine (S2U)
Received 30 July 2013
Revised 29 August 2013
Accepted 30 August 2013
Available online 8 September 2013
was investigated in the 1H NMR scale in the presence of 100 mM H
O and phosphate buffer in the phys-
2 2
iological pH range, from pH 6.6 to 7.6. The obtained data demonstrate an intriguing result that within one
unit of the pH range uridine is the major product of the S2U desulfuration in the pH 7.6, while the 4-pyri-
midinone nucleoside (H2U) is dominant in pH 6.6. The possible desulfuration pathway and the biological
importance of the transformation of S2U either to U or H2U are discussed in the context of the tRNA oxi-
dative damage.
Keywords:
2
-Thiouridine
tRNA
Ó 2013 Elsevier Ltd. All rights reserved.
Modified nucleoside
Oxidative stress
Desulfuration
The 5-substituted 2-thiouridines (x5S2U) are hypermodified
nucleosides located at the first position of the anticodon (the 34
demonstrated.7,8 Besides, other mechanisms of tRNA damage
may occur which alter the tRNA structure and cellular function.9
In this context, we turned our attention to x5S2U nucleosides,
present in the anticodon region of several tRNAs, as these units
contain a thiocarbonyl function and are susceptible to the attack
of reactive oxygen species (ROS). Moreover, the ROS-sensitive units
may be potential damage sites for the tRNA molecule. Early in vitro
studies have suggested that the S2U-containing tRNAs treated with
hydrogen peroxide (mimics of the ROS) under in vitro conditions
undergo desulfuration and are transformed predominantly to uri-
Lys3
position, wobble) in transfer RNAs specific for lysine (tRNA
),
Glu
Gln 1
glutamic acid (tRNA ) and glutamine (tRNA ). Their structural
features play fundamental role in precise codon reading and effi-
ciency of the translation process.2 Substitution of the oxygen
atom at C2 of uridine with a sulfur atom shifts the puckering equi-
,3
0
librium of the ribose ring towards the C3 -endo form, present in the
4
,5
A-type RNA and enhances the base stacking in RNA helix. In this
conformation, 2-thiouridines recognize predominantly adenosine
as a third letter of the codon by the classical Watson–Crick base
pairing, while the wobble base pairing (with a G residue) is re-
stricted due to the less preferred formation of hydrogen bond be-
tween sulfur acceptor of S2U and N1H donor of G.2
1
0–12
dine-containing tRNAs.
However, our recent, more detailed studies,13 carried out on the
S2U-containing model anticodon-stem loop RNA, have clearly
–4
2 2
shown that in aqueous 100 mM H O the S2U unit is transformed
Recently, some attention has been paid to the response of the
cellular RNA, in particular tRNA, to oxidative stress.6 The well-
structured tRNA molecule is especially susceptible to stress-in-
duced modifications occurring within the anticodon loop region.
Numerous studies have demonstrated that under oxidative stress
conditions tRNA is cleaved by codon-specific nucleases to yield
the 5 - and 3 -halves of tRNA molecules. Such tRNA fragments
have been identified intracellularly, and their multiple functions,
including interfering with cell metabolism, have been already
predominantly to 4-pyrimidinone riboside (H2U, Fig. 1), instead of
uridine, which was the commonly accepted major dethiolation
1
1,12
product.
A 4-pyrimidinone nucleoside is a uridine analog de-
prived of the N3 amide hydrogen and the carbonyl function at
0
the C2 position (Fig. 1). H2U adopts preferentially the C2 -endo
conformation, characteristic for the B-DNA type units rather than
for the parent RNA units (as for S2U, which predominantly exists
0
0
7
0
14,15
in the C3 -endo conformation).
ent possibilities for base pairing.
Thus, H2U offers entirely differ-
Our studies have shown that hybridization affinity of the H2U-
RNA to the Watson–Crick complement (A opposite to H2U), was
much smaller than the affinity of S2U-RNA or U-RNA to the com-
plementary RNA. In contrast, stability of duplexes with wobble
⇑
Corresponding author. Tel.: +48 42 681 6970; fax: +48 42 681 5483.
E-mail address: bnawrot@cbmm.lodz.pl (B. Nawrot).
0
960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.114

5
804
E. Sochacka et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5803–5805
O
O
O
NH
N
N
N
S
O)
N
N
S
O
O
O
HO
HO
HO
(
HO
OH
HO
OH
HO
OH
S2U (U)
H2U
geS2U
Figure 1. The donor/acceptor pattern of S2U (U), H2U and geS2U units. Acceptors are marked in red, and donors are marked in blue.
base pairs S2U-G and H2U-G was almost unchanged, as compared
1
3
to the classical U-G bp. This interesting observation became even
more intriguing in the light of the recent discovery by Liu and oth-
ers, who identified 5-substituted-2-thiouridines, in which the sul-
fur atom in the parent nucleosides is functionalized with a geranyl
1
6
residue (geS2U, Fig. 1). The studies have shown that such gerany-
Lys
lated tRNA preferably recognizes AAG codons over AAA codons,
thus the geS2U-G base pair seems to be more stable than the
geS2U-A base pair. We realized that in geS2U the double bond-pat-
tern in the base moiety is similar to that in H2U. Although the con-
version of S2U-containing tRNA to corresponding H2U derivatives
in the natural systems has not been yet identified, this possibility
cannot be excluded.
Therefore, it was tempting to search for the parameters promot-
ing transformation of 2-thiouridine into H2U. For this purpose, 2-
thiouridine was subjected to a desulfuration reaction under
in vitro oxidative stress conditions, that is, in the presence of
1
2 2
00 mM H O in 200 mM phosphate buffer (Scheme 1). The reac-
1
tion was carried out at a H NMR scale using a 10 mM S2U solution.
The reaction was monitored by the H NMR spectroscopy. The
diagnostic range of H NMR spectra used for quantification of the
1
1
products were the chemical shifts of heterobase protons H5 and
0
H6 as well as the H-1 anomeric proton of the sugar moiety. The
reference spectra for the S2U substrate and for the U and H2U
products are shown in Fig. S1.
Figure 2. The time course of the desulfuration reaction of 2-thiouridine carried out
2 2
in the presence of 100 mM H O in a 200 mM phosphate buffer pH 6.6 (upper panel)
and pH 7.6 (lower panel).
The time course of the desulfuration reaction of S2U was ana-
lyzed at two different pH conditions within physiological range,
namely 6.6 and 7.6. The results are shown in Figure 2 (upper and
lower panels, respectively). The reaction carried out at pH 6.6 re-
quired ca. 320 min to complete and resulted in the formation of
4
-pyrimidinone nucleoside as a major product (93%), while uridine
1
was a minor product (7%), as determined by integration of H NMR
resonances (Fig. 2, upper panel and Fig. S2). In contrast, the S2U
desulfuration in phosphate buffer pH 7.6 was complete after
ꢀ
50 min and uridine was a major product (82%), while H2U was
present in 18% only (Fig. 2, lower panel and Fig. S3). Upon kinetic
analysis, the S2U desulfuration at 25 °C was determined as a pseu-
do-first order reaction (see Fig. S4). The rate constant values
ꢂ3
ꢂ1
ꢂ3
ꢂ1
k = 8.94 ꢁ 10 min and k = 62.40 ꢁ 10 min were found for
the reactions carried out at pH 6.6 and pH 7.6, respectively.
The ratios of the S2U desulfuration products were determined
within a pH range 6.6–7.6, changing pH with 0.2 unit steps. The
reaction mixtures were analyzed by 1H NMR. The corresponding
proton spectra, collected after 320 min, are shown in Figure 3.
0
The respective H6, H5 and H1 signals from H2U are marked by
Figure 3. The pH dependent desulfuration reaction of 2-thiouridine (10 mM)
carried out in the presence of 100 mM H in 200 mM phosphate buffer at a pH of
.6, 6.8, 7.0, 7.2, 7.4 and 7.6. 1H NMR spectra were collected after 320 min of
reaction time. The respective H6, H5 and H1 of H2U nucleoside are marked by red
lines while the same signals of uridine are marked by green lines.
O
N
O
O
2 2
O
6
NH
N
NH
0
S
N
H
+
N
O
O
O
O
HO
H
2
O
2
HO
HO
2
00 mM buffer
red lines, while the same signals belonging to uridine are marked
by green lines. Thus, the change of pH by one unit dramatically
altered both the reaction pathway and the ratio of the products.
This intriguing result indicates that oxidative desulfuration of
HO
OH
HO
OH
HO
OH
Scheme 1. The desulfuration of 2-thiouridine with hydrogen peroxide in 200 mM
phosphate buffer at physiological pH range (6.6–7.6).

E. Sochacka et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5803–5805
5805
2
-thiouridine is strongly dependent on the pH of the reaction solu-
hydrogen bond pattern than uridine and 2-thiouridine, incompat-
ible with the classical Watson–Crick hydrogen bonding. Addition-
ally, this nucleoside adopts the C2 -endo B-type sugar ring
puckering. Both these features are crucial in respect to the wobble
nucleoside interactions with complementary unit of the third posi-
tion of the codon.
In the context of the possible biological significance of the ob-
tained results, further studies are designed to get a proof of the
concept on the desulfuration of the S2U in the tRNA molecules
in vitro and at the cellular level.
tion. The oxidative desulfuration reaction leading to the H2U prod-
uct is preferred in lower pH (6.6) and its rate is much slower in
comparison to the S2U desulfuration reaction carried out in higher
pH. One may conclude that the rate of the formation of U is pH-
dependent and increases remarkably when the pH is elevated,
while the formation of H2U nucleoside is less sensitive to pH
0
change and its yield is limited by the presence of the substrate S2U.
By analogy to desulfuration of cyclic thioureas,1
7–21
a possible
pathway of 2-thiouridine desulfuration may involve an initial oxi-
dation of the thiol-tautomeric form of S2U by a nucleophilic attack
of the SH function on the oxygen atom of the hydrogen peroxide
molecule (Scheme 2). The resulting sulfenic acid intermediate
Acknowledgments
(
n = 1) is then oxidized to yield a sulfinic acid product (n = 2), in
This research was financially supported by The National Science
Centre in Poland, Project Number UMO-2011/03/B/ST5/02669 to
B.N. and E.S. for years 2012–2015.
which, at lower pH, the C–S bond scission occurs, followed by elim-
ination of a molecule of sulfur dioxide.1
type intermediate is stabilized by migration of the proton
exchangeable in D O) from the N3 function to the carbene centre
resulting in the formation of H2U (an ‘oxidative reduction’ prod-
8,19
The released carbene-
(
2
Supplementary data
1
9
1
uct). In contrast, at higher pH, the S2U to H2U pathway is sup-
pressed, while an alternative mechanism, involving
nucleophilic attack of a water molecule on the C2 atom of the sul-
Supplementary data (the H NMR spectra of the reference S2U,
1
a
U and H2U nucleosides (Fig. S1), the H NMR data for analysis of
the kinetics of the desulfuration of S2U in pH 6.6 (Fig. S2) and
fur-oxidized intermediate (n = 2 or 3) is prevailing, and leads to the
7
.6 (Fig. S3) and the plot of dependence of ln c = f(t) for the reaction
18
formation of uridine as a main reaction product. In each of the
tested reaction mixtures, independently of the pH used, we con-
firmed the presence of sulfur trioxide by precipitation of barium
sulfate. The detected SO
of sulfur dioxide by excess hydrogen peroxide,
carried out in pH 6.6 (Fig. S4a) and 7.6 (Fig. S4b) as well as the data
on the ratio of products in the reaction mixtures of the S2U desulf-
uration carried out in phosphate buffer of the pH range from 6.6 to
1
9
3
may be produced either by oxidation
7
.6 (Table S1)) associated with this article can be found, in the on-
18,22
or by direct re-
line version, at http://dx.doi.org/10.1016/j.bmcl.2013.08.114.
lease from the corresponding sulfonate intermediate (n = 3). Be-
cause the latter process requires strong basic conditions (for the
References and notes
release of SO
3
from the considerably stable sulfonate deriva-
tives ), the elimination of SO from sulfinic acid intermediate is
Further studies on the mechanism of the
hydrogen peroxide or oxone promoted oxidative desulfuration of
2
3
1. http://rna-mdb.cas.albany.edu.
2
1
8,19
2. (a) Agris, P. F.; Vendeix, F. A. P.; Graham, W. D. J. Mol. Biol. 2007, 366, 1; (b)
Yokoyama, S.; Nishimura, S. TRNA: Structure, Biosynthesis and Function; ASM:
Washington, DC, 1995. p 207; (c) Jackman, J. E.; Alfonzo, J. D. WIREs RNA 2013,
4, 5. http://dx.doi.org/10.1002/wrna.1144.
more plausible.
2
-thiouridine are in progress.
3.
(a) Curran, J. F. Modification and Editing of RNA; ASM Press: Washington, DC,
998. p 493; (b) Watanabe, K. Bull. Chem. Soc. Jpn. 2007, 80, 1253.
The pH dependence of the above reaction pathway may be impor-
1
tant, because in cells with pathological metabolism (e.g., in tumour or
neurodegenerated cells), pH may be lower in comparison to the
4.
(a) Sierzputowska-Gracz, H.; Sochacka, E.; Malkiewicz, A.; Kuo, K.; Gehrke, Ch.
W.; Agris, P. F. J. Am. Chem. Soc. 1987, 109, 7171; (b) Yokoyama, S.; Yamaizumi,
Z.; Nishimura, S.; Miyazawa, T. Nucleic Acids Res. 1979, 6, 2611; (c) Yamamoto,
Y.; Yokoyama, S.; Miyazawa, T.; Watanabe, K.; Higuchi, S. FEBS Lett. 1983, 157,
24
healthy cells. Therefore, when S2U-tRNA is subjected to ROS, the
H2U lesions may be more frequent than the uridine lesions.
While the transformation S2U ? U may be considered not par-
ticularly dramatic, since both nucleosides exhibit similar hydrogen
95.
5. Zhang, R.; Erikson, L. A. Phys. Chem. Chem. Phys. 2010, 12, 3690.
6
.
(a) Wurtmann, E.; Wolin, S. L. Crit. Rev. Biochem. Mol. Biol. 2009, 44, 34; (b)
Hofer, T.; Badouard, C.; Bajak, E.; Ravanat, J.-L.; Mattsson, A.; Cotgreave, I. A.
Biol. Chem. 2005, 386, 333; (c) Chan, C. T.; Dyavaiah, M.; DeMott, M. S.;
Taghizadeh, K.; Dedon, P. C.; Begley, T. J. PLoS Genet. 2010, 6, e1001247.
(a) Thompson, D. M.; Lu, C.; Green, P. J.; Parker, R. RNA 2008, 14, 2095; (b)
Thompson, D. M.; Parker, R. Cell 2009, 138, 215.
(a) Fu, H.; Feng, J.; Liu, Q.; Sun, F.; Tie, Y.; Zhu, J.; Xing, R.; Sun, Z.; Zheng, X. FEBS
Lett. 2009, 583, 437; (b) Lu, J.; Esberg, A.; Huang, B.; Byström, A. S. Nucleic Acids
Res. 2008, 36, 1072; (c) Dhahbi, J. M.; Spindler, S. R.; Atamna, H.; Yamakawa, A.;
Boffelli, D.; Mote, P.; Martin, D. I. BMC Genomics 2013, 14, 298.
0
bond pattern and the C3 -endo sugar ring preference, the formation
of the H2U-type products may be harmful to the damaged tRNA-
containing cells. This is because H2U exerts another type of the
7
8
.
.
O
O
O
NH
NH
C:
N
9. Nawrot, B.; Sochacka, E.; Duechler, M. Cell. Mol. Life Sci. 2011, 68, 4023.
H2U
10. Rao, Y. S.; Cherayil, J. D. Biochem. J. 1974, 143, 285.
1
1
1. Watanabe, K. Biochemistry 1980, 19, 5542.
2. Houssier, C.; Degee, P.; Nicoghosian, K.; Grosjean, H. J. Biomol. Struct. Dynam.
1988, 5, 1259.
N
S
N
N
Ribose
Ribose
Ribose
1
1
1
3. Sochacka, E.; Kraszewska, K.; Sochacki, M.; Sobczak, M.; Janicka, M.; Nawrot, B.
main product
at pH 6.6
Chem. Commun. (Camb) 2011, 4914.
4. Kraszewska, K.; Kaczy n´ ska, I.; Jankowski, S.; Karolak-Wojciechowska, J.;
Sochacka, E. Bioorg. Med. Chem. 2011, 19, 2443.
C-S bond
scission
5. Sochacka, E.; Fratczak, I. Tetrahedron Lett. 2004, 45, 6729.
O
N
O
N
O
16. Dumelin, C. E.; Chen, Y.; Leconte, A. M.; Chen, Y. G.; Liu, D. R. Nat. Chem. Biol.
012, 8, 913.
2
H
2
O
2
NH
17. Karkhanis, D. W.; Field, L. Phosphorus and Sulfur 1985, 22, 49.
18. Grivas, S.; Ronnie, E. Acta Chem. Scand. 1995, 49, 225.
19. Schantl, J. G.; Lagoja, I. M. Heterocycles 1998, 49, 929.
20. Chang, J. H.; Lee, K. W.; Nam, D. H.; Kim, W. S.; Shin, H. Org. Process Res. Dev.
2002, 6, 674.
nucleophilic
attack at C2
U
buffered
solutions
-
N
SH
N
SO
N
O
n
pH 6.6 - 7.6
Ribose
Ribose
main product
at pH 7.6
Ribose
21. Kim, S. S.; Choi, B. S.; Lee, J. H.; Lee, K. K.; Lee, T. H.; Kim, Y. H.; Shin, H. Synlett
2009, 599.
2
2
2
2. Schönherr, H. J.; Wanzlick, H. W. Chem. Ber. 1970, 103, 1037.
3. Ronnie, E.; Grivas, S.; Olsson, K. Acta Chem. Scand. 1994, 48, 823.
4. Harjes, U.; Bensaad, K.; Harris, A. L. Br. J. Cancer 2012, 107, 1207.
Scheme 2. The plausible pathway of desulfuration of 2-thiouridine with hydrogen
peroxide in phosphate buffer at physiological pH range (pH 6.6–7.6) (n = 1–3).