Conformational and electronic properties of the two cis (5S,6R) and (5R,6S) diastereoisomers of 5,6-dihydroxy-5,6-dihydrothymidine: X-ray and theoretical studies

Article abstract of DOI:10.1021/tx9501344

The structure of (+)-cis-(5S,6R)-5,6-dihydroxy-5,6-dihydrothymidine was obtained using X-ray crystallography [space group P2₁ with a = 10.130(3) A, b = 6.434(9) A, c = 11.02(5)A, and β = 112.646(2)°]. The comparison of the two cis diastereoisomers of thymidine glycol (I, II) showed several structural and conformational differences. The solid state structures appear to be in agreement with the results of ¹H NMR studies which were carried out in aqueous solution. Conformational and electronic properties of the ground state of the molecules I and II were obtained using ab initio LSD-DFT theory. Only slight differences between the crystal structure and the optimized geometry are observed for each of the two oxidized nucleosides. On the other hand, molecules I and II exhibit significant differences in their electronic properties. In particular, the dipole moment of (5S,6R)-thymidine glycol (I) is twice smaller than that of the (5R,6S) diastereoisomer (II). It is noteworthy that these differences in the electronic properties between the two compounds may be related to changes in the rotameric population around the C4'-C5' bond. The repartition of the electrostatic potential is different in the two compounds. These observations lead to a better understanding of the structural changes when the above lesions are included within a DNA molecule.

Full text of DOI:10.1021/tx9501344

298
Chem. Res. Toxicol. 1996, 9, 298-305
Con for m a tion a l a n d Electr on ic P r op er ties of th e Tw o Cis
(5S,6R) a n d (5R,6S) Dia ster eoisom er s of
5,6-Dih yd r oxy-5,6-d ih yd r oth ym id in e: X-r a y a n d
Th eor etica l Stu d ies
Franck J olibois,† Lucienne Voituriez,† Andre´ Grand,‡ and J ean Cadet*,†
De´partement de Recherche Fondamentale sur la Matie`re Condense´e/ CEA/ Grenoble, SESAM/ LAN
and SESAM/ CC, 17, Rue des Martyrs, F-38054 Grenoble Cedex 09, France
Received J uly 21, 1995X
The structure of (+)-cis-(5S,6R)-5,6-dihydroxy-5,6-dihydrothymidine was obtained using X-ray
crystallography [space group P21 with a ) 10.130(3) Å, b ) 6.434(9) Å, c ) 11.02(5)Å, and â )
112.646(2)°]. The comparison of the two cis diastereoisomers of thymidine glycol (I, II) showed
several structural and conformational differences. The solid state structures appear to be in
1
agreement with the results of H NMR studies which were carried out in aqueous solution.
Conformational and electronic properties of the ground state of the molecules I and II were
obtained using ab initio LSD-DFT theory. Only slight differences between the crystal structure
and the optimized geometry are observed for each of the two oxidized nucleosides. On the
other hand, molecules I and II exhibit significant differences in their electronic properties. In
particular, the dipole moment of (5S,6R)-thymidine glycol (I) is twice smaller than that of the
(5R,6S) diastereoisomer (II). It is noteworthy that these differences in the electronic properties
between the two compounds may be related to changes in the rotameric population around
the C4′-C5′ bond. The repartition of the electrostatic potential is different in the two
compounds. These observations lead to a better understanding of the structural changes when
the above lesions are included within a DNA molecule.
effective premutagenic lesion. It should be added that
the presence of thymine glycol in pSV2 plasmids did not
affect the transformation frequency in humans cells (25).
Evidence was provided that the “thymine glycol” is a
substrate for the N-glycosylase activity of E. coli endo-
nuclease III (26-29). In addition, 5,6-dihydroxy-5,6-
dihydrothymine was also found to be removed from
oxidized DNA through an excision repair process medi-
ated by the E. coli UV ABC nuclease complex (30, 31).
Efforts were also made to determine the chemical and
conformational features of 5,6-dihydroxy-5,6-dihydrothym-
ine and related nucleoside derivatives. The absolute
configuration of the four cis and trans diastereoisomers
of 5,6-dihydroxy-5,6-dihydrothymidine which may be
produced by the reaction of hydroxyl radicals (5) and
through the photosensitized formation of a pyrimidine
radical cation (32) was assigned (33, 34). The main
change in the conformational properties of these four
In tr od u ction
Much attention has been devoted in the two past
decades to the determination of the mechanisms of
radical oxidation of nucleic acids associated with exposure
to ionizing radiation (1-5). One of the major radiation-
induced nucleobase lesions identified so far within naked
(6-10) and cellular DNA (11, 12) is 5,6-dihydroxy-5,6-
dihydrothymine. It is noteworthy that the so-called
“thymine glycol” has been shown to be generated in cells
under other oxidizing conditions including near-UV ir-
radiation (12) treatment with hydrogen peroxide (13) and
various carcinogens (14, 15). Efforts have also been made
to gain insights into the biological role of 5,6-hydroxy-
5,6-dihydrothymine (16). In this respect, the latter
oxidized base does not appear to be a premutagenic lesion
(17) when assessed in single-stranded DNA by using the
transfection assay. It was also found that the coding
properties of the diol were retained (18-21) despite the
fact that the 5,6-ethylenic bond of the pyrimidine ring is
saturated and the pyrimidine ring is no longer planar.
Thymine glycol was found to be efficiently bypassed by
the Escherichia coli Klenow fragment (19, 22). Similar
observation was made in the same sequence context
when single- and double-stranded genomes containing a
thymine glycol residue were replicated in E. coli strains
(23). However, since 5,6-dihydroxy-5,6-dihydrothymine
was found to form an efficient hydrogen bond with
adenine (24), it was concluded that this important class
of oxidative DNA damage cannot be considered as an
1
thymidine glycols, which was inferred from a detailed H
NMR study in aqueous solutions, deals with the shift in
the dynamic equilibrium between the two puckered sugar
2
2
conformers E (C2′-endo) T 3E (C3′-endo) toward the E
form. This is more pronounced for the (6S) diastereoi-
somers (35). The presence of the thymine glycol in a
duplex DNA was shown to induce significant distortion
in the vicinity of the damage (36).
Relevant structural and conformational information
regarding both the pyrimidine moiety and the furanose
ring of the (-)-cis-(5R,6S)-“thymidine glycol” was inferred
from a X-ray crystallographic investigation (37). In
addition, it should be added that the X-ray structure of
the cis isomer of 5,6-dihydrothymine was also resolved
(38). Theoretical study of the structures of the four cis
and trans enantiomers of 5,6-dihydroxy-5,6-dihydrothym-
ine was carried out using Hartree-Fock ab initio quan-
* To whom correspondence should be addressed. Phone: (33)-76-
88-49-87; Fax: (33)-76-88-50-90; E-Mail: cadet@drfmc.ceng.cea.fr.
† SESAM/LAN.
‡ SESAM/CC.
X Abstract published in Advance ACS Abstracts, December 15, 1995.
0893-228x/96/2709-0298$12.00/0 © 1996 American Chemical Society
+
+

5,6-Diastereoisomers of Thymidine Glycols
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 299
Ta ble 1. Cr ysta llogr a p h ic Da ta a n d Refin em en t
P a r a m eter s of I
tum chemical models (39). However, no theoretical
investigations were made on the related nucleoside
derivatives.
formula
C10H16N2O7
space group
a (Å)
P21
10.130(3)
The aim of the present work is to provide structural
and conformational information on the (+)-cis-(5S,6R)-
“thymidine glycol” obtained by X-ray crystallographic
diffraction and to compare the structure of the two cis
diastereoisomers in the solid state. In addition, a theo-
retical study on both (+)-cis-(5S,6R) and (-)-cis-(5R,6S)
diastereoisomers (I, II) was carried out using available
crystallographic data. Emphasis was placed on the
determination of conformational features of both the
pyrimidine ring and the sugar moiety. This also allowed
access to the electronic properties. The whole geometries
of nucleosides were optimized with the local spin density
(LSD) quantum chemical method. Electronic properties
(partial charges, dipole moments, electrostatic potentials)
were determined at the same level of calculation. We
also determined dipole moments by using the ab initio
Hartree-Fock theory.
b (Å)
6.434(9)
c (Å)
11.020(5)
â (deg)
112.646(2)
661.93
2
1.35
cell volume (Å3)
Z (molecules/cell)
density F (g‚cm-3
radiation
diffractometer
scan range
)
Mo KR (Ge monochromatized)
ENRAF-NONIUS CAD4
2° < 2θ < 60°
scan mode
ω scan (scan rate ) 1deg‚min-1
(h, +k, +l
)
data collected
no. of data collected
no. of parameters for mean
square
1309
180 using 759 reflections
R and Rw (%)
4.5 and 3.9
Exp er im en ta l P r oced u r es
Ch em ica ls. Thymidine, which was obtained from Sigma (St.
Louis, MO), was used without further purification.
The (+)-cis-(5S,6R) and (-)-cis-(5R,6S) diastereoisomers of
5,6-dihydroxy-5,6-dihydrothymidine were prepared by the spe-
cific conversion of the (+)-trans-(5R,6R)- and (-)-trans-(5S,6S)-
5-bromo-6-hydroxy-5,6-dihydrothymidine under slightly alkaline
solutions. The (+)-trans-(5R,6R) and (-)-trans-(5S,6S) diaste-
reoisomers of 5-bromo-6-hydroxy-5,6-dihydrothymidine were
synthesized according to an adaptation of Baudish and Davidson
procedure (40). Typically, 200 µL of bromine was added to 20
mL of water containing 2 g of thymidine (41, 42). The solution
was kept in an ice bath for 15 min. Then, the excess of bromine
was removed by bubbling nitrogen gas for 1 h. Subsequently,
the aqueous solution was neutralized by adding 2.5 mL of 5 M
sodium acetate. The solution was injected directly on a HPLC
Prep LC/500 (Waters Associates, Milford, MA) apparatus, and
the two thymidine bromohydrins were separated on a prepara-
tive (50 × 5 cm i.d.) octadecylsilyl silica gel column. The eluent
was a mixture of water-methanol (80:20) at a flow rate of 100
mL/min. Evaporation to dryness of the fastest eluting HPLC
fraction (k′ ) 1.60) gave 605 mg of the (5S,6S)-bromohydrin.
The second HPLC eluting fractions (k′ ) 2.64) were combined
and evaporated to dryness, yielding 1.450 g of the (5R,6R)-
bromohydrin. Each of the two bromohydrins was transferred
in a round flask. Then, 350 mL of water containing 350 mg of
NH4HCO3 (for 1 g of bromhydrin) was added, and the resulting
solution was heated for 3 h at 100 °C. The solutions were then
evaporated and deposited on the above preparative column. The
separation was achieved using water as the isocratic eluent at
a flow rate of 100 mL/min. The (+)-(5R,6R)-bromohydrin leads
to the formation of the trans-(-)- and cis-(+)-thymidine glycol.
Evaporation to dryness in vacuum of the HPLC fraction (k′ )
3.61) yields 620 mg of (+)-cis-(5S,6R)-5,6-dihydroxy-5,6-dihy-
drothymidine. The conversion of (-)-(5S,6S)-bromohydrin gives
rise to the trans-(+)- and cis-(-)-thymidine glycol. Evaporation
to dryness under reduced pressure of the HPLC fraction (k′ )
3.45) gives 605 mg of (-)-cis-(5R,6S)-5,6-dihydroxy-5,6-dihy-
drothymidine. The four products were characterized by exten-
sive spectroscopic measurements including FAB-mass spec-
trometry, U.V, circular dichroism, homonuclear 1H NMR 1-D
and 2-D COSY, 2-D NOESY, and heteronuclear 1H-13C analy-
sis.
F igu r e 1. General structure of (+)-cis-(5S,6R)-5,6-dihydroxy-
5,6-dihydrothymidine (I).
least-squares refinement of the setting angles for 25 reflections
(2θ >20°). The data collection was performed on the same
crystal, and intensities of 1309 independent reflections were
collected in an ω scan mode (scan rate ) 1 deg‚min-1). They
were corrected for Lorentz and polarization factors but not for
absorption. The stability of the crystal was monitored by
measuring the intensities of three controlled reflections after
every 100 measurements and 3600 s of exposure time. No
significant trend in the intensities was observed during the data
acquisition. Crystal data, together with details of the diffraction
experiment and subsequent calculations, are listed in Table 1.
The structure was solved by direct methods using the
MULTAN program (43). An E-map based on 190 phased
reflections with E >1.6 revealed the positions of all non-
hydrogen atoms. The structure was refined by a least-squares
method based on 759 reflections with |Fo| > 2.6σ(Fo), using the
XFLSN program (44). The positions of all hydrogen atoms were
determined from a Fourier difference map and were fixed for
refinement. Non-hydrogen atoms were assigned with anisotro-
pic temperature factors whereas the hydrogens were assumed
to have isotropic thermal motions (B ) 5 Å2). The final
refinement of 180 variables reached values R ) 0.045 and Rw
) 0.039 respectively (with R defined by ∑(|Fo| - |Fc|)/∑|Fo| and
2
Rw by (∑ω(|Fo| - |Fc|)2/∑ω|Fo| )1/2).
Structural and conformational data, which were obtained
from the X-ray structure, are reported in Table S1 (non-
hydrogen atom coordinates), Table S2 (hydrogen atom coordi-
nates), Table S3 (bond lengths), Table S4 (bond angles), Table
S5 (torsional angles), Table 2 (Cremer-Pople parameters for
the pyrimidine ring (45)), Table 3 (hydrogen bonds), and Table
4 (pucker parameters for the sugar ring). Tables S1-S5 are
available in the supporting information. The general structure
of the (+)-cis-(5S,6R) diastereoisomer of 5,6-dihydroxy-5,6-
dihydrothymidine (I) is shown in Figure 1, and a view of the
packing is given in Figure 2.
X-r a y Str u ctu r e Deter m in a tion . (+)-cis-(5S,6R)-5,6-Di-
hydroxy-5,6-dihydrothymidine (I) was crystallized by evapora-
tion from a concentrated water solution. The monoclinic P21
(Z ) 2) space group with the following cell dimensions: a )
10.130(3) Å, b ) 6.434(9) Å, c ) 11.02(5)Å, and â ) 112.646(2)°
were determined on a single crystal (approximately 0.2 × 0.2
× 0.25 mm) using an automatic ENRAF-NONIUS CAD4 dif-
fractometer with Mo KR (Ge monochromatized) radiation, by
Th eor etica l Ca lcu la tion s. The theoretical study of the
conformational and electronic properties of both (+)-cis-(5S,6R)
+
+

300 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
J olibois et al.
orbitals. All properties were calculated on a CRAY 94 super-
computer with 4 CPU process (CEA-CEN, Grenoble).
Resu lts a n d Discu ssion
(a ) Cr ysta l Str u ctu r e of (+)-cis-(5S,6R)-5,6-Dih y-
d r oxy-5,6-d ih yd r oth ym id in e. (1) P yr im id in e Rin g.
Bond lengths and bond angles are given in Tables S3 and
S4, respectively. The pyrimidine ring exhibits a half-
chair conformation geometry as already described for 5,6-
saturated 2,4-dioxopyrimidine derivatives (37, 38, 58).
The best four-atom mean plane is defined by N1, C2, N3,
and C4 atoms. The average displacement of the related
atoms from the mean plane is 0.02 Å. On the other hand,
the C5 and C6 atoms are displaced 0.40 Å to the right
and 0.39 Å to the left, respectively (see Figure 3). When
the base is viewed in its anti conformation with respect
to the N-glycosyl bond, the left represents displacement
toward O4′ and the right toward C2′. On the other hand,
for the (5R,6S) diastereoisomer (II), the C5 and C6 atoms
are displaced to the left and the right, respectively (37),
as expected from the enantiomeric relationship existing
between the two pyrimidine bases of I and II. The
5-methyl and the 6-hydroxyl groups of both I and II adopt
a pseudoaxial orientation, whereas the 5-hydroxyl group
is pseudoequatorial. Similar structural features were
also inferred from the X-ray crystallographic structure
(38) and the ab initio calculations (39) of cis-thymine
glycol. It should be added that the substituents on the
C5-C6 carbons of I are orientated in the range of
classically-staggered saturation system ((60°,180°) (see
Table S5).
F igu r e 2. View of the packing of (+)-cis-(5S,6R)-5,6-dihydroxy-
5,6-dihydrothymidine (I).
and (-)-cis-(5R,6S) diastereoisomers of 5,6-dihydroxy-5,6-dihy-
drothymidine (I, II) was carried out using crystallographic data
(37). The approach involved a quantum chemistry ab initio
method: the all-electron local spin version of density functional
theory (LSD-DFT) (46, 47).
Cremer-Pople data for I and II are reported in Table
2. I appears in the “Southern” CP hemisphere S(CP) (90°
< θ < 180°), whereas II is in the “Northern” CP
hemisphere N(CP) (0° < θ < 90°). I shows the most
pronounced extent of puckering (QI ) 0.52 Å, QII ) 0.48
Å) with a φI value ) 89°, which is different from that of
II (φII ) 272°). This is in agreement with the predictions
of Hruska et al. (37).
Gaussian type orbital approach implemented in the Dgauss
program (48, 49) of UNICHEM 2.3 package (50) was used to
solve LSD-DFT equations. An orbital basis set was required
to describe molecular orbitals as well as an auxilliary one for
the electronic distribution. The auxilliary basis set was also
utilized to describe the exchange-correlation potential and
energy. The double zeta valence plus polarization (DZVP) (621/
41/1) orbitals basis set and the A1 auxilliary (7/3/3; 7/3/3) basis
set (51, 52) were used for this purpose. Nonlocal Becke Perdew
(53-55) corrections to exchange and correlation energy were
included after the SCF process, in a perturbation mode. This
approach is communly used for such studies. As preliminary
attempts, several calculations were carried out using different
methods (DFT, Hartree-Fock, semiempirical) and several basis
sets for the DFT method (DZVP, DZVP2, TZVP). It appears
that the DFT method with DZVP plus A1 basis sets is the more
appropriate for the present study.
(2) N-Glycosyl Bon d . The N1-C1′ bond length
(Table S3) and the bond angles involving N1-C1′ (Table
S4) are similar to those of II. By considering ø(O4′-C1′-
N1-C2) and ø′(O4′-C1′-N1-C6) torsional angles (Table
S5), I adopts an anti conformation about the N-glycosyl
bond. However, we may note slight differences in the
values of ø (-96.9° and -111.6° for I and II, respectively)
and ø′ (62.1° and 67.6° for I and II, respectively). These
are related to the differences in the pucker of the
pyrimidine bases (the base of I is in the S(CP) hemi-
sphere, whereas the base of II is in the N(CP) hemi-
sphere). The latter data clearly indicate that the H6
atom of I, which adopts a pseudoequatorial orientation,
is located on the right side of the pyrimidine ring.
(3) Su ga r Moiety. The respective bond lengths and
bond angles regarding the sugar moiety of I and II are
similar. The furanose ring of I adopts a C2′-endo
conformation with a phase angle of pseudorotation P )
Ground state geometries were obtained using the gradient
optimization technique (56) and were used for the determination
of electronic properties. The electronic features determined are
the following:
•Mulliken charge population analysis (57);
•Dipole moment, according to the equation:
µm
)
AZAµm(A) - fρ(r)µm(r) dr
∑
where µm is defined with regard to the center of mass A as:
µm ) µmx,my,mz ) (x - Ax)mx(y - Ay)my(z - Az)mz
•Electrostatic potential as defined by:
2
169.9° (S type pucker T3 (59)) and a maximum amplitude
of pucker τm ) 37.4° (Table 4). The S (C2′-endo) type of
pucker of I is more pronounced than that of I (P ) 151.2°,
τm ) 36.5°).
The staggered orientation of the exocyclic hydroxym-
ethyl group about the C4′-C5′ bond of thymidine glycol
I is trans with γ ) -71.8° (60). Comparison between the
crystallographic data of I and II shows important differ-
ences in the C4′-C5′ bond geometry since II exhibits a
gauche+ conformation. Interestingly, the solid state
Vesp(r) )
AZA/|RA - r| - fρ(r)/|r - r′|dr′
∑
The determination of dipole moment and electrostatic poten-
tial was based on the electronic density obtained from molecular
+
+

5,6-Diastereoisomers of Thymidine Glycols
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 301
F igu r e 3. Schematic view of the base of the two diastereoisomers I and II. The horizontal axis gives the deviation in Å to the left
(positive) and to the right (negative) of the best four-atom mean plane, N1-C2-N3-C4.
Ta ble 2. Cr em er -P op le P u ck er in g P a r a m eter s for
(b) Th eor etica l Con for m a tion a l P r op er ties.
A
P yr im id in e Ba ses
geometry optimization of the two cis (5S,6R) and (5R,6S)
diastereoisomers of 5,6-dihydroxy-5,6-dihydrothymidine
(I, II) was carried out using the local spin density theory
with the X-ray coordinates as starting data. It should
be noted that the optimized geometries of the molecular
structures were obtained in empty space at 0 K. The
geometry optimization was carried out on the whole
structure, without any constraints. It has to be added
that the calculations were based on the gradient optimi-
zation technique, using Cartesian coordinate representa-
tion.
moleculea
Q (Å)
θ (deg)
φ (deg)
1
2
3
4
0.52
0.46
0.48
0.50
124
129
62
89
96
272
268
59
a
(1) (5S,6R) crystal structure; (2) (5S,6R) optimized geometry;
(3) (5R,6S) crystal structure; (4) (5R,6S) optimized geometry.
conformational features of I are in agreement with the
preferential trans conformation of the 5-(hydroxymethyl)
group in aqueous solution as inferred from a 1H NMR
study (35).
(1) P yr im id in e Rin g. The respective bond lengths
and bond angles of the crystals and optimized structures
are similar (Tables S3 and S4). Comparison between
dihedral angles (Table S5) shows that the conformational
features are identical for all geometries. These include
a pseudo-half-chair conformation for the pyrimidine ring,
a pseudoaxial orientation for the 5-methyl and 6-hydroxyl
groups, and a pseudoequatorial conformation for the
5-hydroxyl group. Considering the Cremer-Pople data
(Table 2), geometries of I are in the “Southern” CP
hemisphere with a more pronounced puckered conforma-
(4) Hyd r ogen Bon d s. Hydrogen bond data are listed
in Table 3. Molecular packing is stabilized by three weak
(distance D-A >2.8 Å) and one strong (distance D-A
<2.8 Å) hydrogen bonds in the following network: N3-
H...O5-H...O4′-H...O6-H...O5. Three other strong hy-
drogen bonds are involved with a water solvent mol-
ecule: These include O3′-H...O10, O10-H1...O4′, and
O10-H2...O3′. Finally, an intermolecular close contact
is noted between H6 and O2 (2.24 Å). The O5, O4′, and
O6 donor atoms also serve as acceptors, but this is not
the case for the O3′ and N3 atoms. Because of molecular
configuration differences, hydrogen bonds are not identi-
cal for the two compounds I and II. The O2 atom, which
participates in a weak bond in the (5R,6S) diastereoiso-
mer II (37), is involved in a close contact in the (5S,6R)
diastereoisomer I. We also established that the H6 atom
of I is included in an intermolecular close contact with
O2, whereas the related atom of II participates in an
intramolecular close contact with O5′. It should be added
that, in both cases, O4 does not participate in molecular
packing.
tion for the crystal structure (QI ) 0.52 Å and QI-LSD
)
0.46 Å). It should be noted that I represents the (5S,6R)
crystal structure and I-LSD, the (5S,6R) calculated
geometry. We used the same notation for the (5R,6S)
diastereoisomer II, where II-LSD represents the calcu-
lated geometry. In contrast, geometries of II are in the
“Northern” CP hemisphere with a more pronounced
puckering for the optimized structure (QII ) 0.48 Å and
QII-LSD ) 0.50 Å). The best contiguous four-atom plane
is defined by N1, C2, N3, and C4 as for the X-ray
structures. Displacement of C5 to the right is higher for
Ta ble 3. Hyd r ogen Bon d s a n d Close Con ta ct for (5S,6R) Str u ctu r e
D-H...A
acceptor A
D-H (Å)
H...A (Å)
D...A (Å)
D-H...A (deg)
O3′-H...O10
O10-H...O4′
O10-H...O3′
N3-H...O5
O5-H...O4′
O4′-H...O6
O6-H...O5
C6-H...O2
x, y, z
1.05
1.00
0.94
1.01
0.91
0.88
0.95
1.11
1.67
1.93
2.40
2.16
2.39
1.96
2.44
2.24
2.68
2.84
2.77
3.08
3.14
2.71
3.31
3.19
159.8
149.9
102.9
150.8
140.3
140.9
151.4
140.3
-x, -1/2 + y, 2 - z
1
-x, /2 + y, 2 - z
x, -1 + y, z
1 + x, y, z
1
-x, /2 + y, 1 - z
1 - x, -1/2 + y, 1 - z
x, 1 + y, z
+
+

302 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
J olibois et al.
Ta ble 5. Dip ole Mom en ts (in D) of th e Tw o
Ta ble 4. P u ck er P a r a m eter s for Su ga r F r a gm en tsa
Dia ster eoisom er s I a n d II Ca lcu la ted w ith Differ en ts
Meth od s a n d Ba sis Sets
1
2
3
4
P (deg)
τm (deg)
169.9
37.4
159.4
37.8
151.2
36.5
171.8
35.4
(5S,6R)
(5R,6S)
DFT
3.56
4.15
4.13
4.09
7.12
7.79
7.67
7.71
a
(1) (5S,6R) crystal structure; (2) (5S,6R) optimized geometry;
Hartree-Fock 3-21G
Hartree-Fock 3-21G**
Hartree-Fock 6-21G
(3) (5R,6S) crystal structure; (4) (5R,6S) optimized geometry.
the crystal structure of I (crystal: 0.40 Å, optimized: 0.28
Å). On the other hand, displacement of C6 to the left is
more pronounced for the calculated geometry (crystal:
0.39 Å, optimized: 0.43 Å). It should be added that the
displacement of C5 to the left for II is more pronounced
in the theoretical geometry (crystal: 0.28 Å, optimized:
0.36 Å). In addition, displacement of C6 to the right is
more important in the crystal structure (crystal: 0.42 Å,
optimized: 0.38 Å).
the inversion of the C6 and C5 configurations induces
significant changes in the electronic properties.
(1) P oin t Ch a r ges a n d Dip ole Mom en ts. Point
charges are given in Table S6 (available in the supporting
information) and dipole moments are reported in Table
5 and Figure 4. Dipole moments µ are different in both
value and direction. Surprisingly, the value of µI (dipole
moment of I) is twice smaller than the value of µII (dipole
moment of II). Other calculations of the dipole moment
were carried out using the ab initio Hartree-Fock theory
program GAUSSIAN 92 (61) with several basis sets (see
Table 5). In each case, the values of µI are approxima-
tively twice smaller than those of µII. Therefore, it may
be concluded that the observed difference in the values
does not depend on either the method used or a basis set
effect. In addition, the directions of the dipole moments
are also different. Qualitatively, µI and µII directions are
along the straight lines defined by (C1′,C2′) and (N3,-
C5′), respectively.
(2) N-Glycosyl Bon d . The respective N1-C1′ bond
lengths and N1-C1′-X bond angles are similar (Tables
S3 and S4) for the crystal structures and optimized
geometries. The values of ø(O4′-C1′-N1-C2) torsion
angles are indicative of an anti conformation about the
N-glycosyl bond for the two cis-thymidine glycols (I, II),
both in the solid state and from theoretical calculations.
However, according to the displacement of C6 toward O′4
in the (5S,6R) diastereoisomer, ø′(O4′-C1′-N1-C6) is
higher in the crystal structure than in the optimized
geometry (ø′crystal: 62.1°, ø′optimized: 49.8°). For the (5R,6S)
diastereoisomer II, ø′ exhibits a higher value in the
crystal structure (ø′crystal: 67.6°, ø′optimized: 66.9°), and the
displacement of C6 toward C2′ is more important in the
crystal.
Partial charges are similar for the related atoms, with
the exception of C6, H6, and O5′ atoms. Partial charge
on O5′ is higher for I, -0.497 C, than for II, -0.544 C.
Point charge on H6 is higher for I, 0.328 C, than for II,
0.227 C.
(3) Su ga r Moiety. All bond lengths and bond angles
of the crystal and optimized structures of I and II are
similar. The pucker parameters for the furanose ring of
the four experimental and theoretical geometries are
reported in Table 4. In all cases, the furanose ring adopts
a C2′-endo conformation (151.2° e P e 171.8°). The data
for the crystal structure of II and the optimized geom-
At the present time, we can rationalize these differ-
ences in term of change in the electronic density. The
calculation of these properties is directly connected to the
electronic distribution. Differences in conformationnal
features and, especially, the existence of an intramolecu-
lar close contact between O5′ and H6 in (5R,6S) diaste-
reoisomer II induce important modifications in the
electronic density. Effectively, the latter close contact
is likely to lead to an electronic delocalization from H6
to O5′. This large delocalization provides an explanation
for the observed differences in dipole moment and point
charges between the two diastereoisomers. The close
contact also leads to a decrease in the partial charge on
C6 (for II: -0.258 C, for I: -0.126 C).
(2) Electr osta tic P oten tia l. The electrostatic poten-
tial at a point r is defined as an electrostatic interaction
energy of a probe charge of value +1 au with all nuclei
and electrons of the concerned molecular system. Ac-
cording to this definition, regions of negative electrostatic
potential are favorable to electrophilic attack including
hydrogen bonding.
Negative electrostatic potentials at -24 kcal/mol are
observed for both compounds I and II as shown in Figure
5. Negative regions are localized around all the oxygen
atoms with the exception of the O5′ atom for II, which is
involved in close contact with H6. We can observe a
decrease of the electrostatic potentials in the vicinity of
the O4 and O5 atoms for I and near O3′ and O4′ for II.
Each of the thymidine glycols I and II exhibits a region
of high reactivity delocalized around a few different
atoms. For the glycol I, the negative well is localized
around O2, O6, and O4′, whereas the reactivity region
is localized around O2, O4, O6, and O5 atoms for the
glycol II.
2
etries of I are indicative of a T1 S type pucker. The two
2
others structures display a T3 S type pucker. It should
be noted that the S (C2′-endo) type of pucker for the
(5R,6S) optimized structure exhibits the highest value
of pseudorotation angle (P ) 171.8°), whereas the (5R,6S)
crystal structure shows the smallest value (P ) 151.2°).
Comparison of the two (5S,6R) geometries shows that the
crystal structure (P ) 169.9°) has a stronger S (C2′-endo)
type of pucker than the optimized geometry (P ) 159.4°).
As already mentioned, the crystal structure of I has a
stronger S (C2′-endo) puckered geometry than the crystal
of II. Interestingly, a similar trend was noted for the
corresponding optimized geometries. In addition, the
differences in the puckering features of the two optimized
structures are similar to that observed for the crystal
structures.
Conformation about C4′-C5′ is trans for both experi-
mental and theoretical geometries of I, whereas a gauche+
orientation is noted for the two geometries of II. The
differences between the crystal and optimized structures
are close to 10°.
(c) Electr on ic P r op er ties. From the results of
geometry optimization, a study of the electronic proper-
ties was carried out in order to gain insight into molec-
ular reactivity features. UNICHEM2.3 package allows
the determination of some electronic properties based on
the use of the LSD-DFT method. These include dipole
moments, point charges, and electrostatic potential. As
a first general observation, it is important to note that
As for the two other properties, the modifications of
electrostatic potential can be rationalized in terms of
+
+

5,6-Diastereoisomers of Thymidine Glycols
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 303
F igu r e 4. Representation of the dipole moment of the two diastereoisomers I and II.
F igu r e 5. Volumetric representation of the electrostatic potential at -24 kcal/mol of the two diastereoisomers I and II.
electronic density delocalization. The observed decrease
in the negative electrostatic potential region near O4 of
I is likely to be associated with a reduction in reactivity
around the latter atom including hydrogen bonding. This
is reflected by molecular packing (see before) in which
O4 is not implicated in any hydrogen bond. It should be
noted that the atom is directly implicated in a hydrogen
bond with adenine in the DNA double strand.
in the electronic density repartition which consequently
may give rise to differences in the electronic properties.
The presence of I and II within DNA may cause severe
structural distortions of the double strand (36, 39). Yet,
we can envisage a difference of behavior between the two
cis-thymidine glycols. The differences in the electrostatic
interactions within DNA are expected to be related to
modifications in dipole moments and electrostatic poten-
tials. It should be remembered that the dipole moment
of the (5R,6S) diastereoisomer is twice that of the (5S,6R)
diastereoisomer. Considering the repartition of the
negative electrostatic potentials (Figure 5), again it is
likely that the extend of the distortion of the DNA helix
will depend on the stereoconfiguration of the thymidine
glycol.
Con clu sion
Both reported experimental and theoretical results
show that the two cis diastereoisomers of thymidine
glycol exhibit differences in the conformational and
electronic properties. The structures of the two com-
1
pounds are in agreement with the results of the H NMR
analysis in aqueous solution. The general conformation
of the pyrimidine ring is similar to that was found for
the cis-thymine glycol (38, 39). We may note that the
presence of the sugar has little effect on the base
conformation.
Ack n ow led gm en t. This research was partly sup-
ported by a grant from the French Ministry of Science
and Research. We thank Prof. Robert Subra and Prof.
Vincenzo Barone for fruitful discussions.
Important differences were observed in the electro-
static potential, the partial charges, and the dipole
moment of the two compounds. The principal cause of
these changes may be explained in term of an intramo-
lecular close contact between H6 and O5′ atoms in the
thymidine glycol II which does not exist in the other
diastereoisomer I. This is due to differences in the
rotameric population of the C4′-C5′ bond which is trans
in the diastereoisomer I and gauche+ in the diastereoi-
somer II. This close contact may lead to modifications
Su p p or tin g In for m a tion Ava ila ble: Final atomic crystal-
lographic coordinates of both non-hydrogen (Table S1) and
hydrogen (Table S2) atoms of the (+)-cis-(5S,6R)-5,6-dihydroxy-
5,6-dihydrothymidine, bond lengths (Table S3), bond angles
(Table S4), torsion angles (Table S5), and the partial charges
of the two cis diastereoisomers of the 5,6-dihydroxy-5,6-dihy-
drothymidine (Table S6) (9 pages). This material is contained
in many libraries on microfiche, immediately follows this article
in the microfilm version of the journal, can be ordered from the
ACS, and can be downloaded from the Internet; see any current
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304 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
J olibois et al.
esis and molecular modeling studies. Proc. Natl. Acad. Sci. U.S.A..
86, 7677-7681.
(24) Clark, J . M., and Beardsley, G. P. (1987) Functional effects of
cis-thymine glycol lesions on DNA syntheses in vitro. Biochemistry
26, 5398-5403.
masthead page for ordering information and Internet access
instructions.
Refer en ces
(25) Spivak, G., Leadon, S. A., Vos, J . M., Meade, S., Hanawalt, P. C.,
and Ganesan, A. K. (1988) Enhanced transforming activity of
pSV2 plasmids in human cells depends on the type of damage
introduced into the plasmid. Mutat. Res. 193, 97-108.
(26) Demple, B., and Linn, S. (1980) DNA-N-glycosylases and UV
repair. Nature 287, 203-208.
(27) Breimer, L. H., and Lindahl, T. (1985) Thymine lesions produced
by ionizing radiation in double-stranded DNA. Biochemistry 24,
4018-4022.
(1) Te´oule, R., and Cadet, J . (1978) Radiation-induced degradation
of the base component in DNA and related substances. Final
products. In Effects of Ionizing Radiation on DNA (Hu¨tterman,
J ., Ko¨hnlein, W., Te´oule, R., and Bertinchamps, A. J ., Eds.) pp
171-203, Springer, Berlin.
(2) Te´oule, R. (1987) Radiation-induced DNA damage and its repair.
Int. J . Radiat. Biol. 51, 573-589.
(3) von Sonntag, C. (1987) The Chemical Basis of Radiation Biology,
Taylor and Francis.
(28) Dizdaroglu, M., Laval, J ., and Boiteux, S. (1993) Substrate
specificity of the Escherichia coli Endonuclease III: Excision of
thymine and cytosine-derived lesions in DNA produced by radia-
tion-generated free radicals. Biochemistry 32, 12105-12111.
(29) Cunningham, R. P., Ahern, H., Xing, D., Thayer, M. M., and
Tainer, J . A. (1994) Structure and function of Escherichia coli
Endonuclease III. Ann. N.Y. Acad. Sci. 726, 215-222.
(30) Lin, J . J ., and Sancar, A. (1989) A new mechanism for repairing
oxidative damage to DNA: (A)BC exonuclease removes AP sites
and thymine glycols from DNA. Biochemistry 28, 7979-7984.
(31) Kow, Y. W., Wallace, S. S., and Van Houten, B. (1990) UvvABC
nuclease complex repairs thymine glycol, an oxidative DNA base
damage. Mutat. Res. 235, 147-156.
(32) Decarroz, C., Wagner, J . R., van Lier, J . E., Murali Krishna, C.,
Riesz, P., and Cadet, J . (1986) Sensitized photo-oxidation of
thymidine by 2-methyl-1,4-naphthoquinone. Characterization of
the stable photoproducts. Int. J . Radiat. Biol. 50, 491-505.
(33) Cadet, J ., Ulrich, J ., and Te´oule, R. (1975) Isomerization and new
specific synthesis of thymine glycol. Tetrahedron 31, 2057-2061.
(34) Cadet, J ., Ducolomb, R., and Te´oule, R. (1977) Pre´paration,
isome´risation et configuration absolue des “hydrates” de thymine
(Preparation, isomerization, and absolute configuration of thy-
midine “hydrates”). Tetrahedron 33, 1603-1607.
(35) Cadet, J ., Ducolomb, R., and Hruska, F. E. (1979) Proton magnetic
resonance studies of 5,6-saturated thymidine derivatives produced
by ionizing radiation. Conformational analysis of 6-hydroxylated
diastereoisomers. Biochim. Biophys. Acta 563, 206-215.
(36) Kao, J . I., Goljer, I., Phan, T. A., and Bolton, P. H. (1993)
Characterizations of the effects of a thymine glycol residue on
the structure, dynamics, and stability of duplex DNA by NMR.
J . Biol. Chem. 268, 17787-17793.
(37) Hruska, F. E., Sebastian, R., Grand, A., Voituriez, L., and Cadet,
J . (1987) Characterization of a γ-radiation-induced decomposition
product of thymidine. Crystal and molecular structure of the (-)
cis (5R,6S) thymidine glycol. Can. J . Chem. 65, 2618-2623.
(38) Flippen, J . L. (1973) The crystal and molecular structures of
reaction products from γ-irradiation of thyline and cytosine: cis-
thymine glycol, C5H8N2O4, and trans-1-carbamoyl-imidazoli-
done-4,5-diol, C4H7N3O4. Acta Crystallogr. B29, 1756-1762.
(39) Miaskiewicz, K., Miller, J ., and Osman, R. (1993) Ab initio
theoretical study of the structures of thymine glycol and dihy-
drothymine. Int. J . Radiat. Biol. 63, 677-686.
(40) Baudisch, O., and Davidson, D. (1925) The mechanism of oxidation
of thymine. 4,5-Dihydroxyhydrothymine (thymine glycol). J . Biol.
Chem. 64, 233-239.
(41) Cadet, J ., and Te´oule, R. (1973) Isome´risation des 5-bromo-5,6-
dihydro-6-hydroxythymidines. Pre´paration des formes anome`res
de la thymidine et de la 1-(2-de´soxy-D-e´rythro-pentofuranosyl)-
thymine (Isomerization of 5-bromo-5,6-dihydro-6-hydroxy-
thymidines. Preparation of anomeric forms of thymidine and 1-(2-
deoxy-D-erythro-pentofuranosyl)thymine). Carbohydr. Res. 29,
345-361.
(42) Lustig, M. J , Cadet, J ., Boorstein, J ., and Teebor, G. W. (1992)
Synthesis of the diastereoisomers of thymidine glycol, determi-
nation of concentrations and rates of interconversion of their cis-
trans epimers at equilibrium and demonstration of differential
alkali lability within DNA. Nucleic Acids Res. 20, 4839-4845.
(43) Declercq, J . P., Germain, G., Main, P., and Woolfson, M. M. (1973)
On the application of phase relationship to complex structures.
V. Finding the solution. Acta Crystallogr. A29, 231-234.
(44) Busing, W. R., Martin, K. O., and Levy, H. A. (1971) ORFLS3,
Report ORNL 59-4-37. Oak Ridge National Laboratory, Oak
Ridge, TN.
(45) Cremer, D., and Pople, J . A. (1975) A general definition of ring
puckering coordinates. J . Am. Chem. Soc. 97, 1354-1358.
(46) Khon, W., and Sham, L. J . (1965) Self-consistent equations
including exchange and correlation effects. Phys. Rev. A140,
1133-1138.
(4) Teebor, G. W., Boorstein, R. J ., and Cadet, J . (1988) The
repairability of oxidative free radical mediated damage to DNA.
Int. J . Radiat. Biol. 54, 131-150.
(5) Cadet, J . (1994) DNA damage caused by oxidation, deamination,
ultraviolet radiation and photoexcited psoralens. In DNA ad-
ducts: Identifications and biological significance (Hemminki, K.,
Dipple, A., Shuker, D. E. G., Kadlubar, F. F., Segerba¨ck, D., and
Bartsch, H., Eds.) IARC Scientific Publications 125, pp 245-276,
International Agency for Cancer Research, Lyon.
(6) Te´oule, R., Bert, C., and Bonicel, A. (1977) Thymine fragment
damage retained in the DNA polynucleotide chain after gamma
irradiation in aerated solution. Radiat. Res. 72, 190-200.
(7) Frenkel, K., Goldstein, M., and Teebor, G. W. (1981) Identification
of the cis-thymine glycol moiety in chemically oxidized and γ
irradiated deoxyribonucleic acid by high-pressure liquid chroma-
tography analysis. Biochemistry 24, 7566-7571.
(8) Hubbard, K., Huang, H., Laspia, M. F., Erlanger, B. F., and
Wallace, S. S. (1989) Immunochemical quantitation of thymine
glycol in oxidized and X-irradiated DNA. Radiat. Res. 118, 257-
268.
(9) Dizdaroglu, M. (1985) Application of capillary gas chromatogra-
phy-mass spectrometry to chemical characterization of radiation-
induced base damage of DNA: implications for assessing DNA
repair processes. Anal. Biochem. 144, 593-603.
(10) Teebor, G. W., Cummings, A, Frenkel, K., Shaw, A., Voituriez
L., and Cadet J . (1987) Quantitative measurement of the dias-
tereoisomers of cis thymidine glycol in γ-irradiated DNA. Free.
Radical Res. Commun. 2, 303-309.
(11) Hariharan, P. V., and Cerutti, P. A. (1972) Formation and repair
of γ-ray induced thymine damage in Micrococcus radiodurans.
J . Mol. Biol. 66, 65-81.
(12) Hariharan, P. V., and Cerutti, P. A. (1976) Excision of ultraviolet
and gamma ray products of the 5,6-dihydroxydihydrothymine type
by nuclear preparations of xeroderma pigmentosum cells. Bio-
chim. Biophys. Acta 447, 375-378.
(13) Kaneko, M., Leadon, S. A., and Ito, K. (1988) Relationship between
the induction of mitotic gene conversion and the formation of
thymine glycols in yeast S. cerivisiae treated with hydrogen
peroxyde. Mutat. Res. 207, 17-22.
(14) Kaneko, M., and Leadon, S. A. (1986) Production of thymine
glycols in DNA by N-hydroxy-2-naphthylamine as detected by a
monoclonal antibody. Cancer Res. 46, 71-73.
(15) Leadon, S. A. (1990) Production and repair of DNA damage in
mammalian cells. Health Phys. 59, 15-22.
(16) Cathchart, R., Schwiers, E., Saul, R. L., and Ames, B. N. (1984)
Thymine glycol and thymidine glycol in human and rat urine: A
possible assay for oxidative DNA damage. Proc. Natl. Acad. Sci.
U.S.A.. 81, 5633-5637.
(17) Hayes, R. C., Petrullo, L. A., Huang, H., Wallace, S. S., and
Leclerc, J . E. (1988) Oxidative damage in DNA: lack of mutage-
nicity by thymine glycol lesions. J . Mol. Biol. 201, 239-246.
(18) Ide, H., Kow, Y. W., and Wallace, S. S. (1985) Thymine gycol and
urea residues in M13 DNA constitute replicative blocks in vitro.
Nucleic Acids Res. 13, 8035-8052.
(19) Hayes, R. C., and Leclerc, J . E. (1986) Sequence dependence for
bypass of thymine glycols in DNA by DNA-polymerase I. Nucleic
Acids Res. 14, 1045-1061.
(20) Clark, J . M., and Beardsley, G. P. (1986) Thymine glycol lesions
terminate chain elongation by DNA-polymerase I in vitro. Nucleic
Acids Res. 14, 737-749.
(21) Ide, H., and Wallace, S. S. (1988) Dihydrothymine and thymidine
glycol triphosphates as substrates for DNA polymerases: dif-
ferential recognition of thymine C5-C6 bond saturation and
sequence specificity of incorporation. Nucleic Acids Res. 16,
11339-11354.
(22) Evans, J ., Maccabee, M., Hatahet, Z., Courcelle, J ., Bockrath, R.,
Ide, H., and Wallace, S. S. (1993) Thymine ring saturation and
fragmentation products: lesion bypass, misinsertion and implica-
tions for mutagenesis. Mutat. Res. 299, 147-156.
(23) Basu, A. K., Loechler, E. L., Leadon, S. A., and Essigmann, J . M.
(1989) Genetic effetcs of thymidine glycol: Site-specific mutagen-
(47) Vosko, S. H., Wilk, L., and Nusair, M. (1980) Accurate spin-
dependent electron liquid correlation energies for local spin
density calulation: a critical analysis. Can. J . Phys. 58, 1200-
1211.
+
+

5,6-Diastereoisomers of Thymidine Glycols
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 305
(48) Sambe, H., and Felton, R. H. (1975) A new computational
approach of the Slater’s XR equation. J . Chem. Phys. 62, 1122-
1126.
(49) Dunlap, B. I., Connolly, J . W. D., and Sabin, J . R. (1979) On some
approximations in applications of XR theory. J . Chem. Phys. 71,
3396-3402.
(56) Schlegel, H. B. (1987) Ab initio Methods in Quantum Chemistry
(Lawley, K. P., Ed.) p 249, Wiley.
(57) Mulliken, R. S. (1955) Electronic population analysis on LCAO-
MO molecular wave functions. J . Chem. Phys. 23, 1833-1840.
(58) Grand, A., and Cadet, J . (1978) Crystal and molecular structure
of (-)-(5S)-5-hydroxy-5,6-dihydrothymidine. Acta Crystallogr.
B34, 1524-1528.
(50) UNICHEM2.3 (1994) Cray Research Inc., 2360 Pilot Knob Rd.,
Mendota Heights, MN 55120.
(51) Andzelm, J ., Radzio, E., and Salahub, D. R. (1985) Compact basis
sets for LCAO-LSD calculations. Part I. Method and bases for
scandium to zinc. J . Comput. Chem. 6, 520-532.
(52) Godbout, N., Salahub, D. R., Andzelm, J ., and Wimmer, E. (1992)
Optimization of gaussian-type basis sets for local spin density
functionnal calculations. Part I. Boron through neon, optimization
technique and validation. Can. J . Chem. 70, 560-571.
(53) Becke, A. D. (1988) Density functionnal exchange-energy ap-
proximation with correct asymptotic behaviour. Phys. Rev. A38,
3098-3100.
(54) Becke, A. D. (1988) A multicenter numerical integration scheme
for polyatomic molecules. J . Chem. Phys. 88, 2547-2552.
(55) Perdew, J . P. (1986) Density functionnal approximation for the
correlation energy of the inhomogeneous electron gas. Phys. Rev.
B33, 8822-8824.
(59) Altona, C., and Sundaraligam, M. (1972) Conformational analysis
of the sugar ring in nucleosides and nucleotides. A new description
using the concept of pseudorotation. J . Am. Chem. Soc. 94, 8205-
8212.
(60) Saenger, W. (1983) Principles of nucleic acid structure, Chapters
2 and 4, Springer Verlag, New York.
(61) Frisch, M. J ., Trucks, G. W., Head-Gordon, M., Gill, P. M. W.,
Wong, M. W., Foresman, J . B., J ohnson, B. G., Schlegel, H. B.,
Robb, M. A., Replogle, E. S., Gomperts, R., Andres, J . L.,
Raghavachari, K., Binkley, J . S., Gonzales, C., Martin, R. L., Fox,
D. J ., DeFrees, D. J ., Baker, J ., Stewart, J . J . P., and Pople, J . A.
(1992) GAUSSIAN92. Gaussian Inc., Pittsburgh, PA.
TX9501344
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