Desulfurization of 2-thiouracil nucleosides: Conformational studies of 4-pyrimidinone nucleosides

Article abstract of DOI:10.1016/j.bmc.2011.02.008

4-Pyrimidinone ribofuranoside (H²o⁴U) and 4-pyrimidinone 2′-deoxyribofuranoside (dH²o⁴U) were synthesized by the oxidative desulfurization of parent 2-thiouracil nucleosides with m-chloroperbenzoic acid. The cry

Full text of DOI:10.1016/j.bmc.2011.02.008

Bioorganic & Medicinal Chemistry 19 (2011) 2443–2449
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Bioorganic & Medicinal Chemistry
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Desulfurization of 2-thiouracil nucleosides: Conformational studies
of 4-pyrimidinone nucleosides
a
a
a
b
´
Karina Kraszewska , Iwona Kaczynska , Stefan Jankowski , Janina Karolak-Wojciechowska ,
Elzbieta Sochacka a,
⇑
a Institute of Organic Chemistry, Technical University of Lodz, Zeromskiego 116, 90-924 Lodz, Poland
b Institute of General and Ecological Chemistry, Technical University of Lodz, Zeromskiego 116, 90-924 Lodz, Poland
a r t i c l e i n f o
a b s t r a c t
4-Pyrimidinone ribofuranoside (H2o4U) and 4-pyrimidinone 20-deoxyribofuranoside (dH2o4U) were
synthesized by the oxidative desulfurization of parent 2–thiouracil nucleosides with m-chloroperbenzoic
acid. The crystal structures of H2o4U and dH2o4U and their conformations in solution were determined
and compared with corresponding 2-thiouracil and uracil nucleosides. The absence of a large 2-thiocar-
bonyl/2-carbonyl group in the nucleobase moiety results in C20-endo puckering of the ribofuranose ring (S
conformer) in the crystal structure of H2o4U, which is not typical of RNA nucleosides. Interestingly, the
hydrogen bonding network in the crystals of dH2o4U stabilizes the sugar moiety conformation in the
C30-endo form (N conformer), rarely found in DNA nucleosides. In aqueous solution, dH2o4U reveals a
similar population of the C20-endo conformation (65%) to that of 20-deoxy-2-thiouridine (62%), while
the 62% population of the S conformer for H2o4U is significantly different from that of the parent 2-
thiouridine, for which the N conformer is dominant (71%). Such a difference may be of biological impor-
tance, as the desulfurization process of natural tRNA 2-thiouridines may occur under conditions of
oxidative stress in the cell and may influence the decoding process.
Article history:
Received 2 November 2010
Accepted 3 February 2011
Keywords:
Base modified nucleosides
4-Pyrimidinone nucleoside
20-Deoxy-2-thiouridine
2-Thiouridine
Nucleoside conformation
X-ray
NMR
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
a synthetic biological system.11,12 In this context, the stability of
the 2-thiocarbonyl functionality of nucleoside units during the
Sulfur modification of pyrimidine nucleosides by the replace-
ment of the natural carbonyl group with thiocarbonyl is of great
importance for their biological functions1–8 and for practical appli-
cations.9–13 It has been recognized that the 2-thiocarbonyl group of
2-thiouracil nucleosides strongly influences their conformation
properties and plays a key role in the modulation of base pair rec-
ognition.1,2 These properties are significant for the decoding ability
of natural 2-thiouracil ribonucleosides located in the anticodon
wobble positions of many tRNAs.3,4 2-Thiouridines preferentially
adopt a rigid C30-endo sugar ring conformation,5,6 and therefore
the modified s2U-A base pair in RNA duplexes is more stabilized
than an unmodified one.7,8 Furthermore, due to steric hindrance
and the weaker H-bonding ability of sulfur relative to oxygen,
2-thiouracil ribonucleosides make the U–G wobble base pairs less
stable than base pair containing uridine.7,8 The specific hybridiza-
tion properties of 2-thiouracil ribonucleosides led to their practical
use in the antisense strategy and SNPs analysis,9,10 while 20-deoxy
analogs of 2-thiouracil nucleosides found successful application as
useful tools for enhancing base pairing and replication efficiency in
synthesis of modified oligonucleotide tools as well as their pro-
cessing in cells is of great importance.
However, recently it has been recognized that under different
oxidation conditions 2-thiouracil nucleosides easily undergo
oxidative desulfurization to corresponding 4-pyrimidinone nucleo-
sides and oxidation to natural uridines (Fig. 1). Oxidative desulfur-
ization of the 2-thiopyrimidine moiety has been observed under
treatment with aqueous iodine,14 m-chloroperbenzoic acid,15 dim-
ethyldioxirane16 and trans-2-phenylsulfonyl-3-phenyloxaziridine
(PSO).17 The reactivity of the 2-thiocarbonyl function towards oxi-
dizing agents was found to be the main cause of the low efficiency
of 2-thionucleoside incorporation into the oligonucleotide chain
via the standard phosphoramidite method, as desulfurization oc-
curred at the iodine oxidation step.18–21 Loss of the sulfur atom
from natural thiomodified nucleosides was also reported in the
studies on tRNA probing with different oxidizing agents,22–24 indi-
cating the probability of this process occurring under the condi-
tions of oxidative stress in the cells.
The loss of 2-thiocarbonyl/2-carbonyl function may signifi-
cantly influence the conformational preferences of nucleosides
and, as a consequence, be important for their biological roles. Addi-
tionally, it is worth noting that 4-pyrimidinone nucleosides 3 and 4
are ‘truncated’ nucleoside analogs lacking both the N3-amide
⇑
Corresponding author. Tel.: +48 42 6313155, fax: +48 42 6365530.
E-mail address: elzbieta.sochacka@p.lodz.pl (E. Sochacka).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.008

2444
K. Kraszewska et al. / Bioorg. Med. Chem. 19 (2011) 2443–2449
O
N
O
N
O
N
NH
NH
[ O ]
S
N
O
O
O
O
+
HO
HO
HO
HO
OH(H)
HO
OH(H)
HO
OH(H)
2-thiouridine (s2U, 1)
2’-deoxy-2-thiouridine (ds2U, 2)
4-pyrimidinone ribofuranoside (H2o4U, 3)
4-pyrimidinone 2’-deoxyribofuranoside (dH2o4U, 4)
uridine (U, 5)
2’-deoxyuridine (dU, 6)
Figure 1. Oxidative desulfurization and oxidation of 2-thiouridine and 20-deoxy-2-thiouridine. (The abbreviations H2o4U for 3 means that the hydrogen atom is at C-2 and
the carbonyl function at C-4 of the pyrimidine ring in the structure of 4-pyrimidinone ribofuranoside; dH2o4U denotes the modified nucleoside in 20-deoxy series).
hydrogen and the thiocarbonyl/carbonyl function at C-2 position in
the heterobase moiety characteristic of 2-thiouridines or natural
uridines and thus they offer entirely different possibilities for base
pairing within RNA and DNA duplex complexes.
4-pyrimidinone nucleosides 9 (yield 81%) and 10 (yield 83%),
respectively. Removal of the sugar protecting groups of 9 and 10
by transestrification with 0.1 M NaOMe in MeOH, following
work-up with Dowex (in pyridinium salt form), quantitatively gave
title nucleosides 3 and 4. The purity and structures of H2o4U (3)
and dH2o4U (4) were fully confirmed by chromatography (TLC,
HPLC) and spectral data (UV, MS and NMR).
In the present work, we describe the synthesis and conforma-
tional studies of 4-pyrimidinone nucleosides 3 (H2o4U) and 4
(dH2o4U) in the solid state and in aqueous solution. The crystal
structures of 3 and 4 are compared with the structures of corre-
sponding 2-thiouracil nucleosides: 2-thiouridine (1, s2U),25
20-deoxy-2-thiouridine (2, ds2U) and uracil nucleosides: uridine
(5, U),26 20-deoxyuridine (6, dU),27 respectively. Since the crystallo-
graphic data of ds2U have not been published yet, its crystal struc-
ture is also resolved and reported. The conformational study of 3
(H2o4U) and 4 (dH2o4U) in solution was performed by means of
high-resolution NMR methods. Detailed conformational analysis
of H2o4U and dH2o4U, and a comparison of their conformational
properties with the related nucleosides (s2U,28 U,28,29 ds2U,30
dU31) make it possible to determine how the loss of the 2-thiocar-
bonyl functionality influences the sugar/base conformation of
pyrimidine nucleosides in solution and to gain some insight into
the consequences of the desulfurization process on the hybridiza-
tion properties of double helical sulfur-modified DNA and RNA.
Conformational analysis of nucleosides 3 and 4 was carried out
based on parameters defined by Altona and Sundaralingam.33
Three parameters describe the primary features of the conforma-
tion of a pyrimidine nucleoside: the glycosidic bond torsion angle
v
(O40–C10–N1–C2) describes the orientation of the base relative to
the furanose ring; the C40–C50 torsion angle
c determines the orien-
tation of the 50-hydroxyl group relative to the furanose ring (angle
O50–C50–C40–C30); and the furanose ring puckering is specified by
the pseudorotational phase angle P. Two major furanose ring
conformations are strongly preferred for the nucleosides: C30-endo
(North, N-type conformation) and C20-endo (South, S-type
conformation).33,34
2.2. X-ray structure analysis
The ORTHEP drawings of the 4-pyrimidinone ribofuranoside
H2o4U (3) and 4-pyrimidinone 20-deoxyribofuranoside dH2o4U
(4) are presented in Figure 2 in projections perpendicular to the
plane passing through atoms C10, C40 and O40, showing conforma-
tional details of the molecules. Selected geometrical parameters
indispensable for conformation discussion are listed in Table 1
for ribonucleosides 1, 3, 5 and in Table 2 for 20-deoxy analogs 2,
4, 6. The remaining crystallographic data are given in Supplemen-
tary data. The corresponding ORTHEP for one of four independent
molecules of ds2U (2) structure, is also given in Figure 2, while geo-
metrical details are included in Table 2.
2. Results and discussion
2.1. Synthesis
4-Pyrimidinone ribonucleoside (H2o4U, 3) and 4-pyrimidinone
20-deoxyribofuranoside (dH2o4U, 4) were synthesized according
to the reactions presented in Scheme 1.
Sugar protected 2-thionucleosides 7 and 8 were obtained by the
silyl method of N-glycoside bond formation from the silylated
derivative of 2-thiouracil and 1-O-acetyl-2,3,5-tri-O-benzoyl-ribo-
furanose32 or 2-deoxy-3,5-di-O-toluyl-ribofuranosyl chloride.14
Desulfurization of 7 or 8 by the treatment with 0.1 M m-chloroper-
benzoic acid in dichloromethane/pyridine15 for 3 h at rt afforded
The geometry of the 4-pyrimidinone ring, a heterobase moiety
in both studied nucleosides, ribonucleoside H2o4U (3) and 20-deox-
yribonucleoside dH2o4U (4), is not remarkably changed in
O
O
N
O
N
NH
N
N
N
S
i
ii
O
O
O
(TolO) BzO
(TolO) BzO
(TolO) BzO
(TolO) BzO
HO
OBz (H)
OBz (H)
HO
OH (H)
perBz-s2U (7)
perBz-H2o4U (9)
H2o4U (3)
perTo l - d s 2U (8)
perTo l - d H 2o4U (10)
dH2o4U (4)
Scheme 1. Synthetic route for the preparation of H2o4U (3) and dH2o4U (4). Reagents and conditions: (i) 0.1 M m-chloroperbenzoic acid in CH2Cl2/pyridine 9:1, rt, 3 h;
(ii) 0.1 M NaOMe/MeOH, rt, 30 min.

K. Kraszewska et al. / Bioorg. Med. Chem. 19 (2011) 2443–2449
2445
Figure 2. ORTHEP drawing of H2o4U (3), dH2o4U (4) and ds2U (2) with respect to the plane passing through atoms C10, C40 and O40.
Table 1
Selected torsion angles and conformational parameters for H2o4U (3) in comparison
Table 3
Hydrogen bond geometry in H2o4U (3) and dH2o4U (4)
with s2U (1)25 and U (5)26
D–HꢁꢁA
(sym. code)
DꢁꢁꢁA (Å)
D–HꢁꢁA (°)
H2o4U (3)
s2U (1)25
U (5)26
H2o4U (3)
O20–H20OꢁꢁꢁO4
O50–H50OꢁꢁꢁO4
C30–H3ꢁꢁꢁN3
ꢀ1/2+x,3/2ꢀy,1ꢀz
5/2ꢀx,1ꢀy,ꢀ1/2+z
3/2ꢀx,1ꢀy,ꢀ1/2+z
2.745 (2) 169
2.818 (2) 163
3.490 (2) 168
2.856 (1) 156
3.132 (2) 127
3.220 (2) 119
A
B
(a) Torsion angels [°]
O30–H30OꢁꢁꢁO50 ꢀ1+x,y,z
v
= O40–C10–N1–C2
ꢀ142.1 (1)
ꢀ26.8 (1)
41.2 (1)
ꢀ38.9 (1)
24.6 (1)
1.3 (1)
ꢀ163.0
6.0
ꢀ161.7
10.5
ꢀ155.7
C5–H5ꢁꢁꢁO20
1ꢀx,y,z
1ꢀx,y,z
m
m
m
m
m
c
0 = C40–O40–C10–C20
1 = O40–C10–C20–C30
2 = C10–C20–C30–C40
3 = C20–C30–C40–O40
4 = C30–C40–O40–C10
= O50–C50–C40–C30
3.4
ꢀ27.9
40.4
C6–H6ꢁꢁꢁO20
ꢀ27.0
ꢀ31.4
dH2o4U (4) O30–H30ꢁꢁꢁO4
O50–H50ꢁꢁꢁN3
(3/2ꢀx,2ꢀy,1/2+z)
(1/2+x,5/2ꢀy,1ꢀz)
2.786 (1) 174
2.860 (1) 175
36.0
39.5
ꢀ34.0
18.0
ꢀ169.0
ꢀ34.6
15.3
45.9
ꢀ39.5
C6–H6ꢁꢁꢁO50
(ꢀ1/2+x,3/2ꢀy,1ꢀz) 3.053 (2) 133
22.8
C30–H30ꢁꢁꢁO50
(ꢀ1/2+x,3/2ꢀy,1ꢀz) 3.340 (2) 124
57.6 (1)
39.6
C2–H2ꢁꢁꢁO30
(x,1+y,z)
3.281 (2) 152
(b) Conformational parameters
P [°]
wm [°]
Sugar moiety
C40–C50 bond
C10–N1 bond
160.6 (S)
42.6
C20-endo
gauche (+)
anti
9.7 (N)
36.5
3.8 (N)
39.6
C30-endo
gauche (+)
anti
13.9 (N)
41.6
C30-endo
gauche (+)
anti
C30-endo
trans
(N-type) form of their ribose puckering, characteristic for nucleo-
tide units in double helical RNA, as it was present in the crystal
structures of reference s2U and U nucleosides.25,26 Nevertheless,
modification of the H2o4U heterobase does not influence the con-
formation around the C40–C50 ribose bond and the nucleoside
adopts a typical gauche(+) arrangement.34 The orientation of the
heterocyclic base relative to the sugar moiety in H2o4U molecules,
anti
comparison with those of 2-thiouracil or uracil residues in s2U,25
ds2U and U,26 dU27 structures (for details see Supplementary data).
However, the absence of the bulky group at C-2 position and
hydrogen at N-3 position in H2o4U and dH2o4U heterobases reveals
a significant impact on the conformation of the modified nucleo-
sides treated as a whole (Tables 1 and 2), as well as on hydrogen
bond pattern in their crystals (Table 3).
(v
= ꢀ142.0°) is in the typical anti range similar to s2U and U struc-
tures, although the small hydrogen atom at C-2 position of H2o4U
heterobase instead of the bulky 2-thiocarbonyl group of s2U or the
carbonyl of U does not sterically favor this arrangement around the
N-glycosidic bond. The preference of the anti conformer of 3 is
caused by hydrogen bonding network in the crystal state.
In the crystal structure of H2o4U (3) the sugar residue adopts
C20-endo (S-type) conformation (phase angle of pseudorotation
P = 160.6°, pseudorotation amplitude 42.6°), which is not often ob-
served in the structure of RNA nucleosides.34 Usually the pyrimi-
dine ribonucleosides in the solid state are fixed in the C30-endo
Analogous conformational parameters derived from crystallo-
graphic studies of dH2o4U (4) (Fig. 2 and Table 2) show that mod-
ified 20-deoxynucleoside 4 crystallizes in the C30-endo, gauche(+),
anti conformation. The anti orientation around the N-glycosidic
Table 2
Selected torsion angles and conformational parameters for dH2o4U (4) in comparison with ds2U (2) and dU (6)27
dH2o4U (4)
ds2U (2)
dU (6)27
A
B
C
D
A
B
(a) Torsion angles [°]
v
ꢀ99.3 (1)
ꢀ4.1 (1)
ꢀ17.4 (1)
30.7 (1)
ꢀ33.7 (1)
24.1 (1)
57.9 (1)
ꢀ157.3 (7)
4.0 (1)
ꢀ150.0 (7)
ꢀ0.1 (7)
ꢀ17.5 (8)
27.5 (8)
ꢀ155.3 (7)
1.9 (8)
ꢀ146.6 (7)
ꢀ0.5 (8)
ꢀ21.6 (8)
34.0 (8)
ꢀ154.0
ꢀ16.8
33.9
ꢀ155.0
ꢀ13.0
33.1
m0
m1
m2
m3
m4
c
ꢀ26.4 (8)
37.6 (8)
ꢀ36.6 (8)
20.6 (8)
54.1 (9)
ꢀ24.3 (8)
34.8 (8)
ꢀ34.4 (8)
21.3 (8)
60.8 (9)
ꢀ37.5
ꢀ35.3
ꢀ29.1 (8)
ꢀ33.5 (8)
28.8
28.8
18.4 (7)
22.7 (8)
ꢀ7.6
ꢀ10.9
192.0
56.1 (9)
49.3 (9)
186.0
(b) Conformational parameters
P [°]
24.9 (N)
34.5
C30-endo
gauche (+)
anti
12.6 (N)
35.8
C30-endo
gauche (+)
anti
19.2 (N)
35.1
C30-endo
gauche (+)
anti
15.1 (N)
39.3
C30-endo
gauche (+)
anti
18.8 (N)
34.4
C30-endo
gauche (+)
anti
173.5 (S)
37.7
C20-endo
gauche (ꢀ)
anti
177.7 (S)
35.3
C20-endo
gauche (ꢀ)
anti
wm [°]
Sugar
C40–C50
C10–N1

2446
K. Kraszewska et al. / Bioorg. Med. Chem. 19 (2011) 2443–2449
bond and gauche(+) around C40–C50 of dH2o4U (4) is the same as in
the structure of ribonucleoside H2o4U (3), as well as in ds2U (2)
and dU (6) reference 20-deoxynucleosides. Unexpectedly, the sugar
moiety of dH2o4U (4) adopts the C30-endo conformation, which is
rarely found in 20-deoxynucleosides in the solid state.34 The C30-
endo folding of 20-deoxyribose residue was also observed in the re-
solved ds2U structure (Fig. 2, Table 2). In the case of ds2U structure
it is mainly caused by the steric interactions of the bulky 2-tiocar-
bonyl group in heterobase residue with sugar moiety, while the
non-typical folding of the 20-deoxyribose moiety in dH2o4U is
maintained by the specific hydrogen bond system in the crystal.
It is important to underline that the absence of an N-3 hydrogen
donor and a 2-tiocarbonyl/2-carbonyl acceptor function in the
heterobase of the modified nucleosides 3 and 4 has a crucial
impact on molecular packing in their crystals and causes a differ-
ent hydrogen bond system as compared to the related pyrimidine
C20-endo (S conformer) or C30-endo (N conformer). The percentage
3
0
0
of S and N conformers can be estimated based on JH1 –H2 and
3
0
0
JH3 –H4 coupling constants applying the equations:
%
C20-
C30-endo = 100 ꢀ %
0
0
0
0
0
0
endo = 100 JH1 –H2 /(JH1 –H2 + JH3 –H4
) and %
C20-endo.35,36 The experimental JH–H coupling constants and the
calculated populations of C20-endo and C30-endo conformers are
listed in Table 4 and Table 5, respectively.
3
Complete pseudorotation analysis of H2o4U (3), dH2o4U (4) was
performed using the PSEUROT software (version 6.2).39 In this
program, minimization of the differences between the experimen-
tal and calculated couplings is accomplished by non-linear
Newton–Raphson minimization, while the quality of the fit is ex-
pressed by root–mean–square (rms) differences. The input of five
coupling constants: J(H10, H20), J(H10, H200), J(H20, H30), J(H200, H30)
and J(H30, H40) made it possible to define the ratio of two conform-
ers with rms <0.01 for 20-deoxynucleoside dH2o4U. The S con-
nucleosides s2U,25
U
26 and ds2U, dU.27 The architecture of crystals
former is more populated (60%) with PS = 135.3°,
while the minor conformer (40%) is characterized by
PN = 340.8°, m(N) = 30.3°. PSEUROT calculations performed for
wm(S) = 33.6°,
of H2o4U and dH2o4U nucleosides (both in the P212121 space
group) is based on loops of helical and columnar molecular
arrangements forming infinitive three dimensional structures.
Comprehensive descriptions of the observed hydrogen bonding
interactions for both structures are summarized in Table 3. The
corresponding illustrations are placed in Supplementary data (Figs.
S8 and S9 for H2o4U and dH2o4U, respectively). A description of
molecular packing for the ds2U structure is also enclosed
(Fig. S10, Supplementary data). As in the molecules of H2o4U (3)
and dH2o4U (4) there is no strong H-bond donor in the heterobase,
the primary molecular bonding in the crystals is based on the
strong hydrogen bonds of O20-HꢁꢁꢁO4 in H2o4U and O30-HꢁꢁꢁO4 in
dH2o4U leading to the main motif connecting the heterobase with
the sugar moiety (‘base-to-sugar’ motif), whereas the molecules in
the crystals of reference nucleosides s2U,25 U,26 dU27 and ds2U are
mainly joined by strong N3-HꢁꢁꢁO4 bonds, providing a ‘base-to-
base’ dominated pattern. Thus, the different molecular packing of
modified nucleosides H2o4U and dH2o4U as compared to unmodi-
fied pyrimidine nucleosides resulted in the observed atypical
C20-endo folding of the sugar residue in the structure of H2o4U
and C30-endo folding for 20-deoxynucleoside dH2o4U in their crys-
tal structures.
N
w
the modified ribonucleoside H2o4U, based on three coupling con-
stants J(H10,H20), J(H20,H30), and J(H30,H40), lead to a varied family
of conformations within the range of P = 135°–155° for the S con-
former and P = 330°–350° for the N conformer. Puckering ampli-
tudes for both conformers were fixed and equal to 37°. For
20-deoxyribonucleosides, dH2o4U (4), ds2U (2) and dU (6), the con-
formations of the deoxyribose ring (the population of C20-endo/C30-
endo) are nearly the same (Table 5). However, the 2-thiolation in
ribonucleosides significantly affects the conformational character-
istics of the ribose moiety. The population of the C30-endo confor-
mation of 2-thiouridine (1) is much higher than that of the
canonical uridine (5).5,40,41 The rigidity of the C30-endo form of
s2U is caused by the large steric effect of the 2-thiocarbonyl group
of the heterobase and the 20-hydroxyl group of the ribose ring. This
inherent conformational rigidity of the 2-thiopyrimidine ribonu-
cleosides strongly contributes to the stability of s2U-modified
polyribonucleotides7 and influences the decoding process by 2-
thiomodified tRNAs.40,41 Recently, hybrid DFT and MP2 calcula-
tions showed that it is distant electrostatic effects rather than
the hydrogen bond between 20-OH and the 2-tiocarbonyl function
that enhance the selectivity of the C30-endo conformation of ribose
2.3. NMR conformational analysis
1D and 2D NMR techniques were used to determine the solu-
tion conformations of H2o4U (3) and dH2o4U (4). Nucleoside con-
formation in solution is characterized by the dominant sugar
pucker and preferred glycosidic bond arrangement.35–38
Table 5
3
Population of sugar S and N conformers (%) calculated on the basis of JH–H coupling
constants
Nucleoside
XS
XN
Nucleoside
XS
XN
H2o4U (3)
s2U35 (1)
U35 (5)
62
29
47
38
71
53
dH2o4U (4)
ds2U (2)
dU (6)
65
62
64
35
38
36
2.3.1. Conformation of the sugar moiety
It was assumed that the sugar ring of a nucleoside in solution
exists as an equilibrium mixture of the two puckered forms
Table 4
Proton–proton vicinal (n = 3) and geminal (n = 2) coupling constants (Hz) for H2o4U (3) and dH2o4U (4) (25 °C in D2O) in comparison with data for s2U (1), U (5) and ds2U (2), dU
(6)
nJ (H,H)
H2o4U (3)
s2U (1)28
U (5)29
dH2o4U (4)
ds2U (2)31
dU (6)30
2–6
5–6
2.6
7.7
5.7
—
—
5.4
—
3.5
3.2
4.1
12.7
—
—
2.5
7.8
6.8
6.4
14.4
6.5
3.9
3.7
3.6
4.9
—
—
6.6
6.3
—
6.6
4.9
4.1
4.0
5.0
—
—
6.7
6.6
14.3
6.8
4.1
3.7
3.5
5.1
a
a
8.3
2.5
—
—
4.0
—
6.0
1.6
3.0
13.5
8.1
4.8
—
—
5.2
—
5.4
2.9
4.4
12.7
10–20
10–200
20–200
20–30
200–30
30–40
40–50
40–500
50–500
a
a
12.6
—
12.5
a
Not reported in cited publications.

K. Kraszewska et al. / Bioorg. Med. Chem. 19 (2011) 2443–2449
2447
in the 2-thiouridine molecule.42 The desulfurization of s2U (1)
Table 8
leading to H2o4U (3) dramatically changes the conformation of
the sugar residue. In solution, the dominating conformer of 1 is
C30-endo (71%), while the conformer C20-endo (62%) dominates in
the case of 3, similarly as in the desulfurized DNA nucleoside
dH2o4U (65%).
Population (%) of gauche(+), trans and gauche(ꢀ) conformers at 25 °C in D2O
3
3
0
0
0
00
Nucleoside
JH4 –H5 [Hz]
JH4 –H5 [Hz] gauche(+) trans gauche(ꢀ)
H2o4U (3)
s2U (1)28
U (5)29
3.2
1.6
3.1
4.2
3.0
4.4
4.8
5.0
5.1
62
94
61
55
52
49
29
5
32
33
37
39
9
1
7
12
11
12
dH2o4U (4) 3.5
ds2U (2)31
dU (6)30
4.0
3.5
2.3.2. Conformation around the glycosidic bond
The syn/anti conformation around the N-glycosidic bond was
probed by means of vicinal carbon-proton couplings and NOE ef-
fects. Proton-coupled 13C spectra reveal carbon-proton couplings
that indicate the syn/anti conformation according to the following
the dominating anti conformation around the N-glycosidic bond of
the desulfurized nucleosides H2o4U and dH2o4U.
relationships JC6–H1 > JC2–H1 = anti; JC2–H1 > JC6–H1 = syn.37,38,43,44
0
0
0
0
For example, the magnitudes of the corresponding
J values
0
0
0
JC6–H1 = 3.6 Hz and JC2–H1 = 2.4 Hz for uridine, and JC6–H1 = 3.6 Hz
2.3.3. Conformation around the C40–C50 bond
and JC2–H1 = 2.1 Hz for 20-deoxyuridine, clearly confirm the prefer-
0
The local conformation around the C40–C50 bond was examined
by analysis with three staggered forms: gauche(+), trans, and
gauche(ꢀ), using coupling constants H50 and H500 protons with
H40.37,46 The absolute assignment of the H50 and H500 proton in
1H NMR spectra was based on the deshielding effect of the phos-
phate group on H50 and H500 in 30-monophosphates of uridines.47
It was shown that the H50 and H500 spectral region shows a similar
ence of anti conformation for both nucleosides in solution.44 The
population of the anti conformer can be estimated from the equa-
tion% anti = [10-(JC2–H1 + JC6–H1 )/6.4]. In the case of H2o4U and
44
0
0
dH2o4U, JC6–H1 and JC2–H1 are similar (3.3–3.5 Hz), and the popula-
tions of syn and anti conformers are practically equal (Table 6).
In order to further substantiate this observation, 1D 1H NOE
experiments were performed45 (Table 7). Irradiation of the H2 pro-
ton in H2o4U (3) gave a NOE of 19.1% for H10 and 2.7% for H20 and
H30. Such a relationship of NOEs is observed when the anti con-
former is dominating. To confirm this, the H6 proton was irradi-
ated and almost equal NOEs of 9.8% at H10 and 8.8% at H20 and
H30 were observed. Additionally, the signal of H5 was enhanced
by 16.1%. The irradiation of H10 gave a NOE of 17.3% at H2 and of
3.2% at H6. These results indicate that the H6 proton is situated
closer to sugar H20 and H30 protons than H2. The opposite relation-
ship applies to distances between H2, H6 and H10.
0
0
characteristic spectral pattern: d(H50) > d(H500) and J4 –5 <J4 –5 in a
number of other nucleosides and nucleotides.35 Therefore, it ap-
pears reasonable to assume that for all these nucleosides where
this pattern is observed the more shielded proton is assigned as
H500.
0
0
0
00
The specific assignments of H50 and H500 methylene protons in
the 1H NMR spectra of H2o4U and dH2o4U and measurements of
0
0
0
00
the J4 –5 and J4 –5 proton–proton coupling constants, enabled the
unequivocal determination of the population of three exocyclic
C40–C50 rotamers gauche(+), trans and gauche(ꢀ). The results are gi-
ven in Table 8. All of the presented nucleosides exhibit preferen-
tially gauche(+) conformation. An appreciably higher population
of the gauche(+) rotamer in s2U (94%) than in the other listed
nucleosides (50–60%) is due to the dominant C30-endo, anti confor-
mation of 2-thioribonucleoside.5,40
Analogous experiments were performed for dH2o4U (4), and
again NOEs were consistent with the dominating anti conformer.
Irradiation of H2 led to NOEs of 16.0% at H10 and 2.0% at H20 and
H30, an analogous experiment with H6 led to NOEs of 15.4% at
H5, 7.3% at H10 and 6.0% at H20 and H30. The experiment with H10
produced uneven enhancement of H2 (16.7%) and H6 (1.4%) sig-
nals. Moreover, in the last experiment the assignments of the
2.50 ppm signal as the H200 proton was confirmed by a NOE of 6.0%.
Although measurements of vicinal proton–carbon coupling
constants suggested almost equal populations of syn and anti
conformers, the results of 1D NOE experiments are consistent with
3. Conclusions
The modified nucleosides H2o4U (3) and dH2o4U (4) were
synthesized in good overall yields by desulfurization of appropriate
2-thiouracil nucleosides with m-chloroperbenzoic acid. X-ray crys-
tallography and NMR spectroscopy made it possible to determine
the conformational parameters of H2o4U and dH2o4U nucleosides,
Table 6
and compare them to reference ribonucleosides s2U,
U and
Population of anti and syn conformers of H2o4U and dH2o4U calculated on the basis of
3
20-deoxynucleosides ds2U, dU. In the DNA nucleoside series, desul-
furization of 20-deoxy-2-thiouridine does not remarkably influence
the sugar moiety conformation in solution. The dH2o4U nucleoside
exhibits a slightly higher proportion of C20-endo sugar pucker than
ds2U or dU. However, in the solid state dH2o4U adopts C30-endo
0
JC–H1 coupling constants (Hz) in D2O at 25 °C (in%)
3
3
0
0
Nucleoside
JH1 –C2
JH1 –C6
anti
syn
H2o4U (3)
3.4
3.3
3.5
3.4
48
51
52
49
dH2o4U (4)
Table 7
Results of 1D NOE experiments for H2o4U (3) and dH2o4U (4) (25 °C in D2O at 700 MHz)
Compound
Irradiated proton
H2
NOE (%)
Compound
Irradiated proton
H2
NOE (%)
H2o4U
H10 (19.1)
H20 + H30 (2.7)
H10 (9.8)
H20 + H30 (8.8)
H5 (16.1)
dH2o4U
H10 (16.0)
H20 + H30 (2.0)
H10 (7.3)
H6
H6
H20 + H30 (6.0)
H5 (15.4)
H2 (16.7)
H6 (1.4)
H10
H2 (17.3)
H6 (3.2)
H10
H200 (6.0)
H40 (2.3)

2448
K. Kraszewska et al. / Bioorg. Med. Chem. 19 (2011) 2443–2449
conformation, rarely found in the crystals of 20-deoxynucleosides,
resulting from a specific hydrogen bond network. By contrast, in
the RNA nucleoside series the conformational characteristics of
the ribofuranose ring are dramatically affected by desulfurization,
and the 4-pyrimidinone nucleoside 3 predominantly takes the
C20-endo form (S conformer) in aqueous solution. This ribose
pucker is also fixed for H2o4U molecules in the crystal state. The
observed difference between the s2U and H2o4U ribose folding
(71% of N conformer for s2U, 62% of S conformer for H2o4U) may
have important biological consequences as desulfurization of nat-
ural 2-thiouridines may occur under conditions of oxidative stress
in the cell.
Additionally, the absence of a 2-thiocarbonyl/2-carbonyl accep-
tor and a N3-H donor in the structure of H2o4U and dH2o4U may
play a significant role in modifying hydrogen bonding specificity
with regard to the pair with purine nucleosides. The development
of an efficient synthesis of modified nucleosides allows for the gen-
eration of phosphoramidites or nucleotide triphosphates, followed
by incorporation into oligonucleotides for further biophysical stud-
ies of modified sequences. It remains to be determined how these
modified nucleosides behave within the context of the oligonu-
cleotide. We are currently assessing the conformational impact of
H2o4U (3) and dH2o4U (4) within the context of model DNA and
RNA oligonucleotides.
4H, Ar), 7.56 (dd, 1H, J = 2.5 Hz, 7.9 Hz, H6), 7.91 (m, 4H, Ar), 8.61
(d, 1H, J = 2.5 Hz, H2).
4.1.3. General procedure for sugar deprotection
Compound 9 or 10 (2.9 mmol) was suspended in 0.1 M solution
of MeONa in MeOH. The mixture was stirred at room temperature
for 30 min. After this time TLC analysis (20% MeOH in CHCl3)
showed that the starting material was completely consumed. The
mixture was worked up with Dowex (pyridine salt form) and after
filtered off the resin, the remaining solution was evaporated in va-
cuo and coevaporated with toluene. The oily residue was dissolved
in water (15 ml) and washed with diethyl ether (3 ꢂ 10 ml). The
aqueous layer was frozen and lyophilized to give
respectively.
3
or 4,
4.1.3.1. 1-(b-
D
-Ribofuranosyl)-4-pyrimidinone (3).
Yield =
92%, TLC Rf: 0.23 (CH3Cl/CH3OH, 80:20), 0.42 (isopropanol/aq 25%
NH3/H2O, 7:1:2), 0.50 (n-butanol/ethanol/H2O, 40:11:19); 1H
NMR (D2O) d 3.75 (dd, 1H, J = 4.1 Hz, 12.7 Hz, H500), 3.82 (dd, 1H,
J = 3.2 Hz, 12.7 Hz, H50), 4.19 (ddd, 1H, J = 3.2 Hz, 3.5 Hz, 4.1 Hz,
H40), 4.24 (dd, 1H, J = 3.5 Hz, 5.4 Hz, H30), 4.32 (dd, 1H, J = 5.4 Hz,
5.7 Hz, H20), 5.58 (d, 1H, J = 5.7 Hz, H10), 6.43 (d, 1H, J = 7.7 Hz,
H5), 8.07 (dd, 1H, J = 2.6 Hz, 7.7 Hz, H6), 8.58 (d, 1H, J = 2.6 Hz,
13
H2); C NMR (D2O) d 60.9 (C50), 70.1 (C30), 75.0 (C40), 86.1 (C20),
94.8 (C10), 112.5 (C5), 140.7 (C6), 152.2 (C2), 173.8 (C4); FAB MS
m/z 229 [M+H]+; 227 [MꢀH]ꢀ; HRMS calculated for C9H13O5N2
([M+H]+) 229.0835, found m/z 229.0813; UV (H2O) kmax 243 nm;
4. Experimental
kmin 210 nm;
e .
= 7200 Mꢀ1cmꢀ1
4.1. Synthesis of modified nucleosides
4.1.3.2.
(4).
1-(b-
Yield = 91%, TLC Rf: 0.23 (CH3Cl/CH3OH, 80:20), 0.45 (iso-
D
-20-Deoxyribofuranosyl)-4-pyrimidinone
4.1.1. General methods
All reagents were commercially available. CH2Cl2 was predried
with K2CO3 and distilled from K2CO3/P2O5; pyridine was stirred
with NaOH, refluxed over ninhydrin for several hours, distilled
once from ninhydrin and once from CaH2; MeOH was distilled from
Mg; toluene was distilled from Na. Silica gel column chromatogra-
phy was performed using Merck silica gel 60 (230–400 mesh). TLC
was performed on analytical silica plates (Kieselgel 60 F254/0.2 mm
thickness). NMR spectra were obtained on Bruker Avance 250 1H
and 13C NMR chemical shifts are in ppm, and J values are in Hz.
Mass spectra were obtained on Finnigan MAT 95 spectrometer.
propanol/aq 25% NH3/H2O, 7:1:2); 1H NMR (D2O) d 2.44 (ddd, 1H,
J = 6.5 Hz, 6.8 Hz, 14.4 Hz, H20), 2.50 (ddd, 1H, J = 3.9 Hz, 6.4 Hz,
14.4 Hz, H200), 3.64 (dd, 1H, J = 4.9 Hz, 12.6, H500), 3.72 (dd, 1H,
J = 3.6 Hz, 12.6, H50), 4.00 (ddd, 1H, J = 3.6 Hz, 3.7 Hz, 4.9 Hz, H40),
4.40 (ddd, 1H, J = 3.7 Hz, 3.9 Hz, 6.5 Hz, H30), 5.93 (dd, 1H,
J = 6.4 Hz, 6.8 Hz, H10), 6.33 (d, 1H, J = 7.8 Hz, H5), 7.95 (dd, 1H,
J = 2.5 Hz, 7.8 Hz, H6), 8.49 (d, 1H, J = 2.5 Hz, H2), 13C NMR (D2O)
d 38.28 (C20), 59.22 (C50), 68.59 (C30), 85.77 (C40), 90.04 (C10),
110.45 (C5), 139.24 (C6), 150.49 (C2), 171.85 (C4); FAB MS m/z
213 [M+H]+; HRMS calculated for C9H13O4N2 ([M+H]+) 213.0834,
found
e
m/z
213.0815;
.
UV
(H2O)
kmax = 241 nm,
4.1.2. General procedure for oxidative desulfurization
= 16,200 Mꢀ1 cmꢀ1
Nucleoside 7 or 8 (3 mmol) was dissolved in the mixture of
anhydrous CH2Cl2 (25 mL) and anhydrous pyridine (5 ml) and then
0.2 M mCPBA in CH2Cl2 (30 ml) was added. The solution was stir-
red at room temperature and after 3 h reaction was judged to be
complete by TLC (2% MeOH in CHCl3). The mixture was washed
with 10% Na2SO3 (50 ml), 5% NaHCO3 (50 ml), dried over MgSO4
and evaporated. The crude product was coevaporated with toluene
(3 ꢂ 10 ml) and purified by silica gel column chromatography
(0–5% MeOH in CHCl3) to give 9 or 10, respectively.
4.2. X-ray structure analysis
Crystal data for dH2o4U (4): C9H12N2O4, M = 212.21, orthorom-
0
0
bic, space group P212121, a = 7.6490(2) ÅA, b = 8.1401(2) ÅA, c =
0
0
15.2382(4) ÅA, V = 948.79(4) ÅA3, Z = 4, Dx = 1.486 g cmꢀ3, T = 293 K,
0
l
= 0.118 mmꢀ1, k = 0.71073 ÅA, data/parameters = 1669/139; Flack
x = 0.0(9), final R1 = 0.0226.
Crystal data for H2o4U (3): C9H12N2O5, M = 228.21, orthorombic,
0
0
4.1.2.1. 20,30,50-Tri-O-benzoyl-1-(b-
none (9).
D
-ribofuranosyl)-4-pyrimidi-
space group P212121, a = 06.5028(2) ÅA, b = 9.4577(3) ÅA, c =
0
15.7834(4) ÅA, V = 970.70(5) ÅA3, Z = 4, Dx = 1.561 g cmꢀ3, T = 293 K,
Yield = 81%; TLC Rf: 0.26 (CH3Cl/CH3OH, 95:5);
0
0.23 (benzene/EtOAc, 7:3); 1H NMR (CDCl3) d 4.73 (dd, 1H,
J = 2.6 Hz, 12.0 Hz, H500), 4.84 (m, 1H, H40), 4.88 (dd, 1H,
J = 2.5 Hz, 12.0 Hz, H50), 5.67 (pt, 1H, J = 5.8 Hz, H30), 5.83 (m, 2H,
H20, H5), 6.13 (d, 1H, J = 7.9 Hz, H10), 7.36–7.68 (m, 10H, H6, Bz),
7.90–8.12 (m, 6H, Bz), 8.35 (d, 1H, J = 2.6 Hz, H2).
l
= 0.129 mmꢀ1, k = 0.71073 ÅA, data/parameters = 1726/147; Flack
x = 0.2(8), final R1 = 0.0221.
Crystal data for ds2U (2): C9H12N2O4 S, M = 244.28, triclinic,
0
0
0
space group P1, a = 10.377(3) ÅA, b = 11.784(3) ÅA, c = 13.434(4) ÅA,
0
a
= 100.2(1)°, b = 112.6(1)°,
c
= 103.9(1)°, V = 1403.4(7) ÅA30 , Z = 4,
Dx = 1.156 g cmꢀ3, T = 293 K,
l
= 0.231 mmꢀ1, k = 0.71073 ÅA, data/
4.1.2.2. 30,50-Di-O -toluyl-1-(b-
midinone (10).
D
-20-deoxyribofuranosyl)-4-pyri-
Yield = 83%; TLC Rf: 0.23 (CH3Cl/CH3OH,
parameters = 4410/580; Flack x = 0.3 (1), final R1 = 0.0631.
Crystals of H2o4U (3) and dH2o4U (4) as well as ds2U (2) were
obtained by slow evaporation from their aqueous methanolic solu-
tions. The measurements of the crystals were performed on a
SMART diffractometer with graphite-monochromated MoK
tion (k = 0.71073 ÅA) at room temperature. The structures were
95:5); 1H NMR (CDCl3) d 2.43 (d, 6H, J = 2.1 Hz, CH3), 2.50 (m,
1H, H200), 2.77 (ddd, 1H, J = 1.8 Hz, 5.5 Hz, 14.3 Hz, H20), 4.60 (m,
1H, H40), 4.71 (m, 2H, H50 and H500), 5.66 (m, 1H, H30), 5.95 (dd,
1H, J = 5.5 Hz, 8.5 Hz, H10), 6.14 (d, 1H, J = 7.9 Hz, H5), 7.27 (m,
a
radia-
0

K. Kraszewska et al. / Bioorg. Med. Chem. 19 (2011) 2443–2449
2449
solved by direct method and refine with SHELXTL.48 E-maps pro-
vided positions for all non-H-atoms. The full-matrix least-squares
refinement was carried out on F2’s using anisotropic temperature
factors for all non-H-atoms. All C-bound H atoms were placed in
4. Agris, P. F. Nucleic Acids Res. 2004, 32, 223; Agris, P. F.; Vendeix, F. A. P.; Graham,
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16655.
idealized locations and refined using
a riding model, with
CꢀH = 0.93 Å and Uiso(H) = 1.2Ueq(C).
Special comments to structure ds2U (2): The structure was
solved and refined in P1 non-centrosymmetric space group with
four, insignificantly differing, molecules in independent crystallo-
graphic unit. In brought channel shaped by four molecules down
a-axis numbers of disordered solvents (water and ethanol) are
localized. The final refinement of that structure was based on
SQUEEZE procedure from PLATON packet.49
9. Shohda, K.; Okamoto, I.; Wada, S.; Seio, K.; Sekine, M. Bioorg. Med. Chem. Lett.
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Crystallographic data (excluding structural factors) for the
structure reported in this paper have been deposited at the Cam-
bridge Crystallographic Data Center and allocated with the deposi-
tion numbers: CCDC 798540–798542 for compounds 2,3,4 Copies
of the data can be obtained free of charge on application to CCDC,
12 Union Road, Cambridge CB2 1EW, UK (Fax: Int code + (1223)336
033; E-mail: deposit@ccdc.cam.ac.uk).
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4.3. NMR studies of modified nucleosides
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The one- and two-dimensional NMR experiments were re-
corded on 700.2 MHz spectrometer at 25 °C in D2O with DSS as
the internal standards. The samples for the NMR measurements
were prepared by dissolving 6–8 mg of the nucleosides in 0.6 mL
of D2O. Spectra were processed by means of TopSpin 2.1 software
(Bruker BioSpin). In the case of overlapping signals in 1D, 1H NMR
spectra the DAISY (Bruker BioSpin) deconvolution procedure was
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coupled 13C NMR spectra and verified by J-HMBC experiments.50
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Acknowledgements
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This work was supported by grant DzS I-18/15/2010 (Technical
University of Lodz) and by PBZ-MNiSW-07/I/2007 for years
2008–2010.
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Supplementary data
Supplementary data (1H, 13C, 1D NOE NMR spectra for H2o4U
(3), dH2o4U (4) and crystallographic data for compounds 3, 4, 2
(ds2U)) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmc.2011.02.008.
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