2-Thiozebularine: Base modified nucleoside fully constrained in C3'-endo conformation in solution

Article abstract of DOI:10.1135/cccc2011081

2-Thiopyrimidinone ribofuranoside (2-thiozebularine, s²zeb) was synthesized by the adaptation of silyl method of N-glycosidic bond formation and using thionation of protected 2-oxonucleoside derivative (zebularine, zeb) with Lawesson reagent. T

Full text of DOI:10.1135/cccc2011081

2-Thiozebularine: Synthesis, Conformation and Cytotoxity
1103
2-THIOZEBULARINE: BASE MODIFIED NUCLEOSIDE FULLY
CONSTRAINED IN C3′-endo CONFORMATION IN SOLUTION
a1
a2
b1
Katarzyna EBENRYTER , Stefan JANKOWSKI , Janina KAROLAK-WOJCIECHOWSKA ,
Andrzej FRUZIŃSKIb2, Julia KAŹMIERCZAK-BARANSKA , Barbara NAWROT and
c1
c2
a3,
Elzbieta SOCHACKA
*
a
Institute of Organic Chemistry, Technical University of Lodz, Zeromskiego 116, 90-924 Lodz,
Poland; e-mail: k.ebenryter@gmail.com, stefan.jankowski@p.lodz.pl,
elzbieta.sochacka@p.lodz.pl
Institute of General and Ecological Chemistry, Technical University of Lodz, Zeromskiego 116,
90-924 Lodz, Poland; e-mail: janina.karolak-wojciechowska@p.lodz.pl,
andrzej.fruzinski@p.lodz.pl
Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies of the
Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland;
e-mail: julia@bio.cbmm.lodz.pl, bnawrot@bio.cbmm.lodz.pl
1
2
3
b
c
1
2
1
2
Received April 19, 2011
Accepted May 19, 2011
Published online August 24, 2011
This article is dedicated to Professor Antonín Holý on the occasion of his 75th birthday in recognition
of his outstanding contribution to the area of nucleic acids chemistry.
2-Thiopyrimidinone ribofuranoside (2-thiozebularine, s2zeb) was synthesized by the adapta-
tion of silyl method of N-glycosidic bond formation and using thionation of protected
2-oxonucleoside derivative (zebularine, zeb) with Lawesson reagent. The X-ray crystal struc-
ture of s2zeb and NMR determined conformations of s2zeb and zeb in solution were com-
pared with structures of 2-thiouridine (s2U) and uridine (U). In the solid state s2zeb
molecule adopts conformation typical for ribonucleosides: C3′-endo C2′-exo twist type of
ribofuranose pucker, anti of N-glycosidic bond and trans around C4′–C5′ bond. In aqueous
solution, however, almost 100% population of s2zeb exhibits C3′-endo ribofuranose pucker.
The population of N-conformer of s2zeb is about 20% higher than for zeb (analogously to
pair of s2U and U nucleosides) indicating similar influence of steric effect of bulky sulfur
atom on stabilization of N-type ribose conformation. Interestingly, the absence of
4-carbonyl function in zeb and s2zeb raises the population of C3′-endo conformation by
about 30% in comparison to U and s2U as a result of significant anomeric effect. Additive
action of both effects makes the 2-thiozebularine almost fully constrained in C3′-endo con-
formation in aqueous solution. Cytotoxic properties of s2zeb are less pronounced in compar-
ison to zebularine, with IC50 > 100 mM for HeLa and K562 cancer cells and for HUVEC
non-cancerous cells.
Keywords: 2-Thiozebularine; Zebularine; Modified nucleosides; Conformation of nucleo-
sides; X-ray; NMR; Cytotoxity of nucleosides.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 9, pp. 1103–1119
© 2011 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2011081

1104
Ebenryter et al.:
2-Thiozebularine (s2zeb) is one of the 2-pyrimidinone nucleoside analogues
in which carbonyl group at C-2 position of the heterobase moiety was re-
placed by thiocarbonyl function. First synthesis of 2-thiozebularine was de-
scribed by Wightman and Holý in 1973 1. The family of 2-pyrimidinone
nucleosides plays an important role among biologically active analogues of
natural pyrimidine nucleosides and was intensively investigated. Parent
1-β-D-ribofuranosylpyrimidin-2-one (zebularine, zeb) exhibits an antibacte-
rial activity connected with its in vivo transformation into 1-(2′-deoxy-
β-D-ribofuranosyl)pyrimidin-2-one 5′-phosphate, a strong inhibitor of
thymidylate synthetase2,3. Zebularine is also a strong cytidine deaminase
inhibitor4,5 and effectively inhibits DNA methylation6,7
.
Besides of the wide spectrum of biological activity, 2-pyrimidinone nu-
cleosides are important tools as model compounds in structural studies in-
volving RNA, and nucleic acid-binding proteins. This is mainly because
2-pyrimidinone nucleosides lacking N3-amide hydrogen and carbonyl func-
tion at C4 position in heterobase moiety, offer entirely different possibili-
ties for base pairing within RNA duplexes8,9
.
As the sulfur modification of pyrimidine nucleosides by the replacement
of the C2-carbonyl group with C2-thiocarbonyl is known to have a great
impact on their structural features, we are interested in 2-thiozebularine in
the context of our structural studies on modified nucleosides with non-
natural bases10.
In the present work we describe the results of our conformational studies
of 2-thiozebularine (s2zeb, 1, Fig. 1) in the solid state and in aqueous solu-
tion. The resolved crystal structure of 1 is compared to the published struc-
tures of zebularine, (zeb, 2)11, 2-thiouridine (s2U, 3)12 and uridine (U, 4)13.
Conformational studies in aqueous solution were performed for 1 and also
for not so far precisely determined conformation of 2 by means of high-
FIG. 1
Structures of nucleosides
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2-Thiozebularine: Synthesis, Conformation and Cytotoxity
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resolution NMR methods and the results were compared with the known
data for s2U 14 and U 15. Moreover, cytotoxic properties of s2zeb and zeb as
well as their 2′-deoxyribo analogues were determined by MTT assay in HeLa
and K542 cancer cells and in HUVEC non-cancerous cells.
EXPERIMENTAL
Materials
All reagents were commercially available. In particular, CH3CN was distilled from P2O5;
MeOH from Mg and benzene from Na. SnCl4 was distilled from P2O5 under reduced pres-
sure. All other reagents were carefully dried before use. Silylation of heterobase and the reac-
tion of N-glycoside bond formation were performed under anhydrous conditions.
For column chromatography Merck silica gel 60 (230–400 mesh) was used. TLC was per-
formed on analytical silica plates (Kieselgel 60 F254/0.2 mm thickness).
NMR spectra were obtained on Bruker DPX 250 MHz and Bruker Advance II Plus 700 MHz.
1H and 13C NMR chemical shifts are given in ppm (δ-scale), coupling constants (J) in Hz.
Mass spectra were obtained on Finnigan MAT 95 spectrometer. UV-data were obtained on
HITACHI UV Vis U-2800A.
2-Trimethylsilylmercaptopyrimidine (5)
To a suspension of 2-mercaptopyrimidine (6.2 g, 55 mmol) in anhydrous benzene (550 ml),
trimethylsilyl chloride (9.6 ml, 68.8 mmol) and triethylamine (9.6 ml, 68.8 mmol) dissolved
in 12 ml of anhydrous benzene were added dropwise and the mixture was stirred at room
temperature for 7 days. After this time the resulting precipitate (Et3N·HCl) was filtered off
with careful exclusion of moisture. The filtrate was evaporated to a small volume (15 ml)
and the residue was distilled under reduced pressure (0.1 mm Hg, 105 °C) to afford silylated
2-mercaptopyrimidine 5 (7 g, 38 mmol; 69% yield).
2′,3′,5′-Tri-O-benzoyl-1-(β-D-ribofuranosyl)-1,2-dihydropyrimidine-2-thione (7) and
2S-(2′,3′,5′-Tri-O-benzoyl-β-D-ribofuranosyl)-2-thiopyrimidine (8)
Trimethylsilyl derivative 5 (1.84 g, 10 mmol) in anhydrous acetonitrile (25 ml) was added to
the solution of 1-O-acetyl-2′,3′,5′-tri-O-benzoyl-β-D-ribofuranoside (6; 3.53 g, 7 mmol) in an-
hydrous acetonitrile (50 ml). Then a solution of SnCl4 (1.3 ml, 11 mmol) in 80 ml of aceto-
nitrile was dropped with stirring under exclusion of moisture. After 40 min at room
temperature, when the reaction was judged to be complete by TLC (chloroform–methanol
98:2), the mixture was concentrated to 2/3 of its volume, diluted with CH2Cl2 (100 ml) and
neutralized by addition of saturated aqueous solution of NaHCO3 (100 ml). After filtration
through a layer of Celite to remove the tin salts and repeated washings of the Celite with
CH2Cl2, the layers were separated and the aqueous phase was extracted with 2 × 100 ml of
CH2Cl2. The combined organic phase was washed with water (50 ml) and dried over MgSO4.
The final solution was filtered off, evaporated in vacuo and the residue was purified by silica
gel column chromatography (hexane/ethyl acetate gradient from 2:1 to 1:2 v/v) to give
N-glycoside 7 (3.19 g, 5.74 mmol; isolated yield 82%) and S-glycoside 8 (0.39 g, 0.70 mmol;
isolated yield 10%).
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Ebenryter et al.:
N-Glycoside 7: RF 0.11 CHCl3–MeOH 98:2; 0.51 CH3COOC2H5. 1H NMR (250 MHz,
CDCl3): 8.46 dd, 1 H, 3J(5,4) = 4.0, 4J(6,4) = 2.3 (H4); 8.42 dd, 1 H, 3J(6,5) = 6.7, 4J(6,4) = 2.3
(H6); 7.82–8.09 m, 6 H (Ar-H); 7.26–7.68 m, 9 H (Ar-H); 7.03 d, 1 H, 3J(1′,2′) = 2.0 (H1′);
6.46 dd, 1 H, 3J(5,4) = 4.0, 3J(5,6) = 6.7 (H5); 6.01 dd, 1 H, 3J(2′,1′) = 2.0, 3J(2′,3′) = 5.0 (H2′);
5.77 dd, 1 H, 3J(3′,2′) = 5.0, 3J(3′,4′) = 7.8 (H3′); 4.87–4.97 m, 2 H (H4′,H5′); 4.65–4.72 m, 1
H (H5′′).
S-Glycoside 8: RF 0.47 CHCl3–MeOH 98:2; 0.64 CH3COOC2H5. 1H NMR (250 MHz, CDCl3):
8.56 d, 2 H, 3J(6,5) = 3J(4,5) = 4.9 (H4, H6); 7.30–8.13 m, 15 H (Ar-H); 7.03 t, 1 H, 3J(5,6) =
3J(5,4) = 4.9 (H5); 6.51 d, 1 H, 3J(1′,2′) = 3.8 (H1′); 6.04 dd, 1 H, 3J(2′,3′) = 5.0, 3J(2′,1′) = 3.8
(H2′); 5.96 dd, 1 H, 3J(3′,2′) = 5.0, 3J(3′,4′) = 5.9 (H-3′); 4.80 m, 1 H (H4′); 4.71 dd, 1 H,
2J(5′,5′′) = 12.1, 3J(5′,4′) = 4.0 (H5′); 4.60 dd, 1 H, 2J(5′′,5′) = 12.1, 3J(5′′,4′) = 4.1 (H5′′).
1-(β-D-Ribofuranosyl)-1,2-dihydropyrimidine-2-thione (s2zeb, 1)
The benzoylated derivative 7 (800 mg, 1.44 mmol) was suspended in anhydrous MeOH
(57 ml), slightly warmed and then the solution was allowed to cool down to the room tem-
perature. Afterwards 1 M solution of MeONa in MeOH was added (1.4 ml). The mixture was
stirred at room temperature for 30 min. After this time TLC analysis (15% 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 vacuo and coevaporated with toluene. The oily residue was dissolved in water
(15 ml) and washed with diethyl ether (3 × 10 ml). The aqueous layer was frozen and
lyophilized to give N-glycoside 1 (0.25 g, 1.02 mmol; yield 71%). 1H NMR (700 MHz, D2O):
8.82 dd, 1 H, 3J(6,5) = 6.8, 4J(6,4) = 2.2 (H6); 8.49 dd, 1 H, 3J(5,4) = 4.4, 4J(6,4) = 2.2 (H4);
7.09 dd, 1 H, 3J(5,4) = 4.4, 3J(5,6) = 6.8 (H5); 6.38 s, 1 H (H1′); 4.37 d, 1 H, 3J(2′,3′) = 4.9
(H2′); 4.22 ddd, 1 H, 3J(4′,5′) = 2.4, 3J(4′,5′) = 3.5, 3J(4′,3′) = 9.4 (H4′); 4.10 dd, 1 H, 3J(3′,2′) =
4.9, 3J(3′,4′) = 9.4 (H3′); 4.05 dd, 1 H, 3J(4′,5′) = 2.4, 2J(5′,5′′) = 13.2 (H5′); 3.85 dd, 1 H,
3J(4′,5′) = 3.5, 2J(5′,5′′) = 13.2 (H5′′). 13C NMR (175 MHz, D2O): 179.76 (C2), 160.24 (C4),
145.69 (C6), 111.50 (C5), 95.93 (C1′), 83.55 (C4′), 74.54 (C2′), 67.26 (C3′), 59.23 (C5′).
MS-FAB (m/z, %): 245.1 (100) [M + H]+, calculated for C9H12O4N2S: 244. UV-Spectrum
(H2O): λmax 281 nm (ε 15739), 216 nm (ε 6630), 351 nm (ε 2662).
2S-(β-D-Ribofuranosyl)-2-thiopyrimidine (9)
The benzoylated derivative 8 (100 mg, 0.18 mmol) was dissolved in saturated methanolic
ammonia (10 ml) and the mixture was stirred at room temperature for 48 h. After this time
the reaction was judged to be complete by TLC (20% MeOH in CHCl3) and the mixture was
evaporated in vacuo. The residue was dissolved in water (10 ml) and washed with diethyl
ether (3 × 10 ml) to remove the methyl benzoate and benzamide. The crude product was pu-
rified by silica gel column chromatography (CHCl3/MeOH gradient from 100 to 90%). The
fraction containing product was evaporated, dissolved in water, frozen and lyophilized to
give S-glycoside 9 (36 mg, 0.15 mmol; 82%). 1H NMR (250 MHz, D2O): 8.55 d, 2 H, 3J(4,5) =
3J(6,5) = 4.9 (H4, H6); 7.23 t, 1 H, 3J(5,6) = 3J(5,4) = 4.9 (H5); 5.89 d, 1 H, 3J(1′,2′) = 5.0
(H1′); 4.20–4.31 m, 2 H, 3J(3′,4′) = 9.4, 3J(2′,1′) = 5.0 (H3′, H2′); 4.02–4.08 m, 1 H, 3J(4′,5′) =
3.7, 3J(4′,5′′) = 4.9, 3J(4′,3′) = 9.4 (H4′); 3.70 dd, 1 H, 2J(5′,5′′) = 12.5, 3J(5′,4′) = 3.7 (H5′);
3.61 dd, 1 H, 2J(5′,5′′) = 12.5, 3J(5′′,4′) = 4.9 (H5′′). 13C NMR (62 MHz, D2O): 168.91 (C2),
158.15 (C6, C4), 118.32 (C5), 86.29 (C1′), 85.05 (C4′), 74.43 (C2′), 70.74 (C3′), 61.44 (C5′).
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2-Thiozebularine: Synthesis, Conformation and Cytotoxity
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MS-CI (m/z, %): 245.1 (100) [M + H]+, calculated for C9H12O4N2S : 244. UV-Spectrum (H2O):
λmax 241.5 nm (ε 10049).
Transformation of 2′,3′,5′-Tri-O-benzoylzebularine to
2′,3′,5′-Tri-O-benzoyl-2-thiozebularine (7) with Lawesson Reagent
To a solution of 0.25 g (0.46 mmol) 2′,3′,5′-tri-O-benzoyl-1-(β-D-ribofuranosyl)-1,2-dihydro-
pyrimidine-2-one in anhydrous dioxane (11.5 ml) Lawesson reagent (0.25 g, 0.51 mmol) was
added and the mixture was stirred at boiling point for 1.5 h. After this time the reaction was
judged to be complete by TLC (2% MeOH in CHCl3) and 4.7 ml of water was added to the
reaction mixture. After 30 min the mixture was evaporated and the residue was dissolved in
10 ml of CHCl3 and then washed with 2 × 20 ml of 5% aqueous solution of NaHCO3 and
2 × 20 ml of water. The combined organic phase was dried over MgSO4. The final solution
was filtered off, evaporated in vacuo and the residue was purified by silica gel column chro-
matography (CH2Cl2/(CH3)2CO gradient from 100 to 95%) to give 89 mg of N-glycoside 7
(0.16 mmol; yield 35%).
X-ray Structure Analysis
Crystal data for 2-thiozebularine: C9H12N2O4S, M = 244.28, monoclinic, space group P21, a =
4.9452(1) Å, b = 7.6077(2) Å, c = 13.7185(4) Å, β = 92.783(4)°, V = 515.50(2) Å3, Z = 2, Dx
=
1.574 g cm–3, T = 293 K, µ = 0.315 mm–1, λ = 0.71073 Å, data/parameters = 1779/147; flack
x = 0.06(5), final R1 = 0.0181.
Crystals of 2-thiozebularine were obtained by slow evaporation from their methanolic so-
lutions. The measurements of the crystals were performed on a SMART diffractometer with
graphite-monochromated MoKα radiation (λ = 0.71073 Å) at room temperature. The struc-
tures were solved by direct method and refine with SHELXTL 16. E-maps provided 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
idealized locations and refined using a riding model, with C–H = 0.93 Å and Uiso(H) = 1.2
Ueq(C).
CCDC 818090 contains the supplementary crystallographic data for this paper. These data
can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the
Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge, CB2 1EZ, UK; fax:
+44 1223 336033; or deposit@ccdc.cam.ac.uk).
NMR Studies of Modified Nucleoside
The one- and two-dimensional NMR experiments were recorded 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 pro-
cessed by means of TopSpin 2.1 software (Bruker BioSpin). In the case of overlapping signals
in 1H 1D-NMR spectra the DAISY (Bruker BioSpin) deconvolution procedure was applied.
1H-13C vicinal coupling constants were derived from the coupled 13C NMR spectra and veri-
fied by J-HMBC experiments17. NOESY spectra were recorded with 0.5, 1 and 2 s mixing-
times.
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Ebenryter et al.:
Cells and MTT Cytotoxicity Assay
The cytotoxicity experiments were carried out with two cancer cell lines HeLa (human cer-
vix carcinoma) and K562 (human chronic myelogenous leukemia).
The HeLa and K562 cells were cultured in RPMI 1640 medium supplemented with antibi-
otics and 10% fetal calf serum in a 5% CO2–95% air atmosphere. 7 × 103 cells were seeded
on each well on 96-well plate (Nunc). 24 h later cells were exposed to the test compounds
for another 24 or 48 h. Stock solutions (100 mM) of test compounds were freshly prepared
in water (distilled, MiliQ). The final concentrations of the compounds tested in the cell cul-
tures were 1, 1 × 10–2, 1 × 10–4 and 1 × 10–6 mM. The values of IC50 (the concentration of
test compound required to reduce the cell survival fraction to 50% of the control) were cal-
culated from dose-response curves and used as a measure of cellular sensitivity to a given
treatment.
The cytotoxicity of all compounds was determined by the MTT (3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO) assay as described pre-
viously18,19. Briefly, after 24 or 48 h of incubation with drugs, cells were treated with the
MTT reagent and incubation was continued for 2 h. MTT-formazan crystals were dissolved
in 20% SDS and 50% DMF at pH 4.7 and absorbance was read at 570 and 650 nm on an
microplate reader FLUOstar Omega (BMG LABTECH). As a control (100% viability), cells
grown in the presence of vehicle (1% DMSO) only were used.
RESULT AND DISCUSSION
Chemical Synthesis
The general methodology of nucleosides synthesis by N-glycosidic bond
formation was successfully applied for 2-thiopyrimidinone nucleoside.
Using this methodology, 2-thiozebularine was obtained for the first time by
Wightman and Holý, in the reaction of metal salts of 2-mercapto-
pyrimidine and 2,3,5-tri-O-benzoyl ribofuranosyl chloride1 to give a mix-
ture of S- and N-glycosides. Further efficient conversion of S-glycoside to
the N-isomer on treatment with tin tetrachloride raised the yield of desired
per-benzoylated 2-thiozebularine to 68%. Final removal of benzoyl groups
from N-riboside sugar moiety by alkaline methanolysis afforded the
deprotected 2-thionucleoside in good overall yield.
Exclusive formation of 2-mercaptopyrimidine N-riboside was reported
by Niedballa and Vorbruggen using the silyl method of nucleoside synthe-
sis20. Reaction of the silylated 2-mercaptopyrimidine with 1-O-acetyl-2,3,5-
tri-O-benzoyl-β-D-ribofuranose in the presence of SnCl4 afforded per-
benzoylated 2-thiozebularine in very good yield, but the sugar deprotected
nucleoside was not characterized20.
For our conformational study 2-thiozebularine (s2zeb, 1) was synthesized
using the silyl method, according to Scheme 1.
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2-Thiozebularine: Synthesis, Conformation and Cytotoxity
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Condensation of silyl derivative of 2-mercaptopyrimidine 5 with
1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose in acetonitrile, catalyzed
with SnCl4, gave the protected N-glycoside 7 and S-glycoside 8 in 82 and
10% of isolated yield, respectively.
It is worth to note that deprotection of benzoyl groups of N-glycoside 7
with standard methanolic ammonia treatment led to nucleoside decompo-
sition while the S-isomer 8 was stable under these conditions. Removal of
the sugar protecting groups of 7 by transestrification with 0.1 M NaOMe in
MeOH, following work-up with Dowex (in pyridinium salt form), quantita-
tively gave the title nucleoside 1. The purity and structural identity of s2zeb
and its S-isomer 9 were fully confirmed by TLC chromatography and spec-
tral data (UV, MS and NMR).
Additionally, the new approach for the synthesis of s2zeb using transfor-
mation of zebularine derivative with Lawesson thionation reagent21 was
elaborated. The starting 2′,3′,5′-tri-O-benzoyl-zebularine obtained according
Vorbruggen procedure20 was treated with 1.1 molar excess of Lawesson re-
agent in dioxane at 101 °C for 1.5 h. Purification of the product by column
chromatography afforded per-benzoylated 2-thiozebularine in 35% yield.
SCHEME 1
Synthetic route for the preparation of 2-thiozebularine 1. Reaction conditions are as follows:
(i) SnCl4, acetonitrile, r.t., 40 min; (ii) 0.1 M NaOMe/MeOH, r.t., 30 min; (iii) NH3/MeOH, r.t.,
48 h
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1110
Structural Analysis
Ebenryter et al.:
Conformational analysis of modified nucleosides was carried out based on
parameters defined by Altona and Sundaralingam22,23. Three parameters de-
scribe the primary features of the conformation of a pyrimidine nucleoside:
the glycosidic bond torsion angle χ (O4′–C1′–N1–C2) describes the orienta-
tion of the base relative to the furanose ring; the C4′–C5′ torsion angle γ
determines the orientation of the 5′-hydroxyl group relative to the furanose
ring (angle O5′–C5′–C4′–C3′); and the furanose ring puckering is specified
by the pseudorotational phase angle P. Two major furanose ring conforma-
tions are strongly preferred for the nucleosides: C3′-endo (North, N-type
conformation) and C2′-endo (South, S-type conformation)22,23
.
X-ray Structure Analysis
2-Thiozebularine was crystallized from methanol solution to give yellow
needles. The detailed structure of 1 was established by X-ray crystallogra-
phy and required data were deposited at the CCDC database. Figure 2a pres-
ents the ORTEP drawings of 2-thiozebularine molecule in projections
perpendicular to the plane passing through atoms C1′, C4′ and O4′, show-
ing conformational details of the molecule. Selected geometrical parameters
of s2zeb and related nucleosides: zeb, s2U and U, indispensable for confor-
mations comparison and discussion are listed in Table I.
a
b
FIG. 2
The ORTEP drawings of 2-thiozebularine (a); hydrogen bonds pattern in the crystal of s2zeb
down a-axis (b)
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2-Thiozebularine: Synthesis, Conformation and Cytotoxity
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The geometry of the 2-thiopyrimidinone heterobase moiety of s2zeb is
not remarkably changed in comparison to those of 2-pyrimidinone,
2-thiouracil or uracil residues in zeb 11, s2U 12, and U 13 structures. However,
the presence of the bulky sulfur atom at C-2 position of heterobases influ-
ences the conformation of the modified nucleoside treated as a whole
(Fig. 2a, Table I), as well as the hydrogen bonds pattern in the crystals
(Fig. 2b, Table II).
In the crystal structure of s2zeb the sugar residue adopts C3′-endo/C2′-exo
twist type N-conformation (3 T), with phase angle of pseudorotation P =
2
6.7° and pseudorotation amplitude 40.4°. It is worth to note that in the
structure of s2zeb the sugar twist conformation is less symmetrical (more
TABLE I
Selected torsion angles and conformational parameters from X-ray crystal structure of
2-thiozebularine (s2zeb) in comparison to zebularine (zeb)11, 2-thiouridine (s2U)12 and
uridine (U)13
U13
s2zeb
zeb11
s2U12
A
B
Torsion angels, °
χ
–168.3
8.2
–178.5
12.6
–163.0
6.0
–161.7
10.5
–155.7
3.4
ν0
ν1
ν2
ν3
ν4
γ
–30.1
39.4
–31.9
38.3
–27.0
36.0
–31.4
39.5
–27.9
40.4
–35.5
17.3
–31.8
12.1
–34.0
18.0
–34.6
15.3
–39.5
22.8
–169.8
52.3
–169.0
45.9
39.6
Conformational parameters
P, °
6.7 (N)
40.4
–0.3 (N)
38.3
9.7 (N)
36.5
3.8 (N)
39.6
13.9 (N)
41.6
ψ
m, °
C3′-endo
C2′-exo
C3′-endo
C2′-exo
C3′-endo
C2′-exo
C3′-endo
C2′-exo
Sugar moiety
C3′-endo
C4′–C5′ bond
C1′–N1 bond
trans
anti
gauche (+)
trans
anti
gauche (+)
gauche (+)
anti
anti
anti
χ = O4′–C1′–N1–C2, ν0 = C4′–O4′–C1′–C2′, ν1 = O4′–C1′–C2′–C3′, ν2 = C1′–C2′–C3′–C4′,
ν3 = C2′–C3′–C4′–O4′, ν4 = C3′–C4′–O4′–C1′, γ = O5′–C5′–C4′–C3′
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 9, pp. 1103–1119

1112
Ebenryter et al.:
C3′-endo) than in zebularine molecule, for which nearly ideal twist confor-
mation occurs. For s2zeb, the C3′ atom is displaced by 0.44 Å from the
plane C1′–O1′–C4′ on the same side as C5′, while by 0.31 Å for zeb, whereas
C2′ is at a distance of 0.21 Å from this plane on the opposite side for s2zeb
and 0.32 Å for zeb.
The orientation of the heterocyclic base relative to the sugar moiety in
s2zeb molecules (χ = –168.3°) is in the typical anti range, similar to zeb, s2U
and U structures (Table I).
The conformation around the C4′–C5′ ribose bond of s2zeb is in trans ar-
rangement, observed less frequently than gauche(+) in pyrimidine nucleo-
sides having N-type sugar ring pucker23. The preference of 1 to exist as the
C4′–C5′ trans conformer comes from the intermolecular hydrogen bonding
between 5′O-H sugar group and 2-thiocarbonyl function of another mole-
cule (Table II), similarly to hydrogen bonds pattern in the crystals of s2U
molecules12.
It is important to underline that the absence of N-3 hydrogen donor and
4-carbonyl acceptor functions in the heterobase influences the molecular
packing of s2zeb in crystals. The observed net of intermolecular hydrogen
bonds varies from those ones observed for pyrimidine nucleosides: s2U 12
,
and U 13. Comprehensive description of the hydrogen bonds in s2zeb crys-
tal structure is summarized in Table II. The architecture of crystals of s2zeb
(in the P21 space group) is based on helical molecular arrangements by only
the “sugar-to-sugar” junctions, with the strongest O2′–H2′···O3′ hydrogen
bonds (Fig. 2b). Two remaining H-bonds, one strong (O3′–H3′···O5′) and
one weak (5′O –H5′···S2), are responsible for joining helices present in the
crystal (Table II). It is interesting that the identified three hydrogen bonds
are managed to form 11-membered rings with graph-set notation of
[R3 (11)] 24, the same as in the structure of s2U.
3
TABLE II
Hydrogen bond geometry in s2zeb structure
D–H···A
Sym. code
D–H, Å
H···A, Å
D···A, Å
D–H–A, °
O2′–H2′···O3′
O3′–H3′···O5′
O5′–H5′···S2
2–x, 1/2+y, 1–z
1–x, 1/2+y, 1–z
–1+x, y, z
0.94
0.92
0.93
1.76
1.90
2.42
2.697(1)
2.784(2)
3.296(1)
171
162
157
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 9, pp. 1103–1119

2-Thiozebularine: Synthesis, Conformation and Cytotoxity
1113
NMR Conformational Analysis
1D and 2D NMR techniques were used to determine the solution conforma-
tions of 2-thiozebularine (s2zeb) and zebularine (zeb). In general, nucleo-
side conformation in solution is characterized by the dominant sugar
pucker and preferred glycosidic bond arrangement25–28
.
Conformation of the Sugar Moiety
It was assumed that the sugar ring of nucleoside in solution exists as an
equilibrium mixture of the two puckered forms: C2′-endo (S conformer) or
C3′-endo (N conformer). The percentage of S and N conformers can be esti-
3
3
mated based on the values JH1′-H2′ and JH3′-H4′ coupling constants accord-
ing to the following equations: % C2′-endo = 100 JH1′-H2′/(JH1′-H2′ + JH3′-H4′
)
and % C3′-endo = 100 – % C2′-endo 25,26. The experimental JH-H coupling
constants and the calculated populations of C2′-endo and C3′-endo con-
formers of s2zeb and zeb in comparison to s2U and U are listed in Table III.
Resonance signal of H1′ proton for s2zeb was observed as a single line
with 1.85 Hz half-width. Assuming similar puckering for s2zeb and zeb
sugar rings and the same sum of values of J(H1′,H2′) and J(H3′,H4′) cou-
pling constants are equal to 10 Hz 25,26, the coupling constant J(H1′,H2′) for
3
TABLE III
Proton-proton vicinal (n = 3) and geminal (n = 2) coupling constants for s2-zeb and zeb
(25 °C in D2O) in comparison to data for s2U and U. Population of sugar S and N conform-
3
ers is calculated on the basis of JH-H coupling constants
nJ (H,H), Hz
s2zeb
zeb
s2U14
U15
4-5
4.4
6.8
4.5
–
–
8.1
5-6
6.7
2.7
8.3
–
4-6
2.2
–
1′-2′
0a
2.0
2.5
4.0
6.0
1.6
3.0
13.5
71/29
4.8
2′-3′
4.9
5.0
5.2
3′-4′
9.5
8.0
5.4
4′-5′
2.2
2.6
2.9
4′-5′′
3.6
4.3
4.4
5′-5′′
13.2
100/0
13.0
80/20
12.7
53/47
Population N/S, %
a
Signal for H1′ proton was observed as a singlet.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 9, pp. 1103–1119

1114
Ebenryter et al.:
s2zeb was estimated at 0.5 Hz. A lack of splitting of H1′ line was confirmed
by simulation of the line shape for different coupling constants by means
of DAISY software. Splitting of the 1.85 Hz half-width line was observed
when coupling constant was higher than 1 Hz. On the basis of J(H1′,H2′) =
0.5 Hz the population of C3′-endo conformer was estimated as at least 95%.
Complete pseudorotation analysis of s2zeb and zeb was performed using
the PSEUROT software (version 6.2)29. In this program, minimization of
the differences between the experimental and calculated values of cou-
plings is accomplished by non-linear Newton–Raphson minimization,
while the quality of the fit is expressed by root-mean-square (rms) differ-
ences. Calculations for s2zeb, based on three coupling constants J(H1′,H2′),
J(H2′,H3′) and J(H3′,H4′) were performed by means of the PSEUROT auto-
mated procedure which led to 2800 results. For further analysis were taken
only data with rms below 0.55 and puckering amplitude 30–40°. For dif-
ferent values of J(H1′,H2′) (0.0, 0.5 and 1.0 Hz) the dominating was
N conformer (99%) and the best results were achieved for J(H1′,H2′) equal
to 1 Hz. The lowest rms 0.17 corresponds to N conformer with the angle of
pseudorotation PN = 31° and puckering amplitude ΨmN = 41°, when for the
minor S conformer PS = 169° and ΨmS = 31°. Analogous PSEUROT analysis
of zebularine led to 89% population of N conformer (PN = 29°, ΨmN = 36°,
PS = 172°, ΨmS = 38°, rms 0.00).
Our results evidently confirm that heterobase modification strongly af-
fects conformational characteristics of the ribose moiety (Table III). Struc-
ture of pyrimidine nucleobase influences the pentafuranose conformation
mainly through the steric effects and steroelectronic interactions within
O4′–C1′–N1 fragment (anomeric effect)30. The replacement of C2 oxygen
atom in U by more sterically demanding sulfur atom causes an increase of
N conformer population of s2U by about 20% (Table III). This feature is at-
tributed to steric effects between the 2-tiocarbonyl group of nucleobase and
2′-hydroxyl group of the ribose ring31–33, although recent hybrid DFT and
MP2 calculations showed that the distant electrostatic effects between
2′-OH and the 2-tiocarbonyl function may enhance the selectivity of the
C3′-endo conformation of ribose in the 2-thiouridine molecule34. The same
extent of increase of the C3′-endo population was observed when
2-carbonyl function of zeb was replaced by bulky 2-tiocarbonyl group in
s2zeb (Table III; 80% of N conformer for zeb, 100% for s2zeb). Interestingly,
an absence of the carbonyl oxygen at C4 in zeb and s2zeb raises the popula-
tion of the C3′-endo conformers by about 30% in comparison to U and s2U,
respectively (Table III). This feature can be attributed to the presence of the
strong anomeric effect caused by electron-deficient 2-pyrimidinone or
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 9, pp. 1103–1119

2-Thiozebularine: Synthesis, Conformation and Cytotoxity
1115
2-thiopyrimidinone heterobase. In the case of s2zeb, strong steric and
stereoelectronic effects are additive and therefore the N conformer popula-
tion increases to almost 100%. It is an unique case where the structure of
nucleobase forces the ribose conformation to almost exclusive C3′-endo
pucker, without any sugar modification.
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 effects. Proton-coupled
13C spectra reveal carbon-proton couplings that indicate the syn/anti con-
formation according to the following relationships JC6-H1′ > JC2-H1′ = anti;
JC2-H1′ > JC6-H1′ = syn 27,28,35,36
.
For example, the magnitudes of the corresponding J values JC6-H1′ = 3.6 Hz
and JC2-H1′ = 2.4 Hz for uridine clearly confirm the preference of anti confor-
mation for this nucleoside in solution36.
In the case of 2-thiozebularine and zebularine the measured smaller val-
ues of JC2-H1′ than JC6-H1 (1.8 vs 2.1 Hz for s2zeb and 1.1 vs 2.6 Hz for zeb)
evidently indicate domination of the anti conformation around their
N-glycoside bond. The population of the anti conformer can be estimated
from the equation % anti = [10 – (JC2-H1′ + JC6-H1′)/6.4] 31 and for s2zeb and
zeb nucleosides the contents of anti conformers are similar and equal to 95
and 98%, respectively.
To confirm domination of anti conformation around N-glycosidic bond
of s2zeb and zeb the NOE measurements were performed. Results of 1D
NOE experiments were inconclusive because resonance of heterobase H6
and H4 protons were to close for selective irradiation of diagnostic H6 one.
Thus the NOESY experiments were performed. The cross-peaks were inte-
grated and interproton distances were calculated from the relation ηA/ηB =
r6 /r6 27,37, where ηA and ηB are NOEs measured for two pairs of protons A
B
A
and B, and rA and rB are distances for these two pairs of protons. As refer-
ence the NOE for H6 and H5 protons was used as the distance between
these protons is known and equal to 2.45 Å. For both nucleosides the rela-
tionship H6-H1′ > H6-H3′ was observed (3.2 > 2.7 Å and 3.2 > 2.6 Å for
s2zeb and zeb, respectively), characteristic for anti conformer domination.
Conformation Around the C4′–C5′ Bond
The local conformation around the C4′–C5′ bond was examined by analysis
with three staggered forms: gauche(+), trans, and gauche(–), using coupling
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 9, pp. 1103–1119

1116
Ebenryter et al.:
constants H5′ and H5′′ protons with H4′ 27,38. The stereospecific assignment
1
of the H5′ and H5′′ proton in H NMR spectra was based on the deshielding
effect of the phosphate group on H5′ and H5′′ in 3′-monophosphates of
uridines39. It was shown that the H5′ and H5′′ spectral region shows a simi-
lar characteristic spectral pattern: δ(H5′) > δ(H5′′) and J4′-5′ < J4′-5′′ in a num-
ber of other nucleosides and nucleotides25. Therefore, it appears reasonable
to assume that for all these nucleosides where this pattern is observed the
more shielded proton is assigned as H5′′. The stereospecific assignments of
1
H5′ and H5′′ methylene protons in the H NMR spectra of s2zeb and zeb
and measurements of the J4′-5′ and J4′-5′′ proton-proton coupling constants
enabled the determination of the population of three exocyclic C4′–C5′
rotamers gauche(+), trans and gauche(–). The results are given in Table IV. All
of analyzed nucleosides exhibit preferentially gauche(+) conformation with
the higher population of this rotamer for 2-thionucleosides.
TABLE IV
Population (in %) of gauche(+), trans and gauche(–) conformers at 25 °C in D2O of s2zeb and
zeb in comparison with s2U and U
Nucleoside
3JH4′-H5′, Hz
3JH4′-H5′′, Hz
gauche(+)
trans
gauche(–)
s2-zeb (1)
zeb (2)
2.2
2.6
1.6
3.1
3.6
4.3
3.0
4.4
74
68
94
61
26
31
5
0
1
1
7
s2U (3)28
U (4)29
32
Cytotoxicity of Zebularine and 2-Thiozebularine and
Their 2′-Deoxyanalogues
A series of compounds: zebularine 2, 2-thiozebularine 1, 2′-deoxyzebularine 10
and 2-thio-2′-deoxyzebularine 11 was screened for cytotoxic properties
against K562 (leukemia), HeLa (human cervix carcinoma) and HUVEC (nor-
mal) cells. The viability of cells was determined in the MTT assay at four
different compound concentrations: 1, 1 × 10–2, 1 × 10–4 and 1 × 10–6 mM.
Compounds 1, 10 and 11 were not toxic toward cells used in the study
both after 24 and 48 h incubation time (IC50 > 1 mM). However,
zebularine 2 showed limited toxicity toward K562 leukemia cancer cells
only. Cytotoxicity of 2 seems to be time-dependent, as its concentration
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 9, pp. 1103–1119

2-Thiozebularine: Synthesis, Conformation and Cytotoxity
1117
needed to induce cell death after 48 h (100 µM) is six-fold lower comparing
to IC50 at 24 h (600 µM).
CONCLUSIONS
The s2zeb nucleoside was synthesized in good overall yield by coupling of
silyl derivative of 2-mercaptopyrimidine with 1-O-acetyl-2,3,5-tri-O-benzoyl-
β-D-ribofuranose in acetonitrile, catalyzed with SnCl4, and removal of the
sugar protecting groups with NaOMe in MeOH, or alternatively, by the
thionation of appropriate zebularine derivative with Lawesson reagent.
X-ray analysis revealed that in solid state s2zeb molecules adopt C3′-endo/
C2′-exo twist puckering of ribofuranose ring and anti of N-glycosidic bond
conformation, generally consistent with those of zeb, s2U and U crystal
structures. NMR spectroscopy analysis of s2zeb and zeb conformations in
solution and their comparison to reference s2U and U compounds evidently
confirms that the sugar pucker can be steered by heterobase structure (from
53% of N population in uridine to almost 100% in 2-thiozebularine). The
replacement of oxygen atom at C2 either of zeb or U by more sterrically de-
manding sulfur atom, leads to 20% increase of C3′-endo conformer popula-
tion, as in s2zeb and s2U. The lack of carbonyl oxygen at position 4, as in
s2zeb or zeb, gives the 30% increase of the C3′-endo conformer population
due to the electron-withdrawing effect of modified heterobase on sugar
moiety. In the case of s2zeb both effects operate simultaneously and, there-
fore, the population of N conformer increases to almost 100%. 2-Thio-
zebularine is unique example of pyrimidine ribonucleoside which in
aqueous solution is fully constrained in the C3′-endo conformation and this
feature is exerted exclusively through nucleobase steric and stereoelectronic
interactions with ribose ring. The cytotoxicity studies indicate that replace-
ment of oxygen by sulfur at position 2 of the nucleobase of zebularine,
which is known anticancer agent, completely abolishes cytotoxic properties
of the resulting s2zeb (IC50 > 100 mM).
This work was supported by grant DzS I-18/15/2011 (Technical University of Lodz).
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