Synthesis and conformational analysis of D-2′-deoxy-2′, 2′-difluoro-4′dihydro-4′-thionucleosides

Article abstract of DOI:10.1039/b914679b

An efficient synthesis of D-2′-deoxy-2′,2′-difluoro- 4′-dihydro-4′-thionucleosides is described. The conformations of D-2′-deoxy-2′,2′-difluoro-4′-dihydro-4'-thiouridine were studied by X-ray crystallography, NMR spectroscopy and molecular modeling in an attempt to explore the roles of the two gem-difluorine atoms in the puckering preferences of the thiosugar ring. No matter which conformation (south or north) the thiosugar adopts, there is always one fluorine in a pseudoaxial position, with the other in a pseudoequitorial position and thus the strong antiperiplanar (ap) effects from C-H and C-C σ-bonds to σ*C-F are equal to each other in these two conformers. Therefore, the other weak effects, such as dipole-dipole interactions and electrostatic attractions, become more important for determining the overall conformation of the sugar ring. Based on the results of NMR spectroscopy, high-level quantum computations and molecular dynamic simulations were performed to study the preferred pucker of the thiosugar ring in solution. Our results showed that the strong antiperiplanar preference of C-H and C-C σ-bonds to σ*C-F and σ*C-O seemed to be responsible for the favored S-conformation in solution, and the weak electrostatic attractions between ^δ-C2-Fβ ^δ- and ^δ+H6-C6^δ- may stabilize the preferred structure further, and keep the base moiety in a high anft'-rotamer population in solution. In contrast, the packing forces (hydrogen bond OH ... O=C, dipole-dipole interaction C-F ... C=O) in the solid state compensated the energetic disadvantage of the relatively less stable N-conformation, and drove the thiouridine to crystallize in the N-conformation. These results, along with the earlier empirical rules regarding proton chemical shifts in carbohydrates and nucleosides, were used to propose a method based on proton chemical shifts for the analysis of the N ? S equilibrium of the fluorinated sugar ring. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b914679b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and conformational analysis of
D-2¢-deoxy-2¢,2¢-difluoro-4¢-dihydro-4¢-thionucleosides†
Feng Zheng,a Lin Fu,b Renxiao Wangb and Feng-Ling Qing*a
Received 6th August 2009, Accepted 29th September 2009
First published as an Advance Article on the web 2nd November 2009
DOI: 10.1039/b914679b
An efficient synthesis of D-2¢-deoxy-2¢,2¢-difluoro-4¢-dihydro-4¢-thionucleosides is described. The
conformations of D-2¢-deoxy-2¢,2¢-difluoro-4¢-dihydro-4¢-thiouridine were studied by X-ray
crystallography, NMR spectroscopy and molecular modeling in an attempt to explore the roles of the
two gem-difluorine atoms in the puckering preferences of the thiosugar ring. No matter which
conformation (south or north) the thiosugar adopts, there is always one fluorine in a pseudoaxial
position, with the other in a pseudoequitorial position and thus the strong antiperiplanar (ap) effects
from C–H and C–C s-bonds to s*C–F are equal to each other in these two conformers. Therefore, the
other weak effects, such as dipole–dipole interactions and electrostatic attractions, become more
important for determining the overall conformation of the sugar ring. Based on the results of NMR
spectroscopy, high-level quantum computations and molecular dynamic simulations were performed to
study the preferred pucker of the thiosugar ring in solution. Our results showed that the strong
antiperiplanar preference of C–H and C–C s-bonds to s*C–F and s*C–O seemed to be responsible for
the favored S-conformation in solution, and the weak electrostatic attractions between d+C2–Fbd- and
d+H6–C6d- may stabilize the preferred structure further, and keep the base moiety in a high anti-rotamer
=
population in solution. In contrast, the packing forces (hydrogen bond OH ◊ ◊ ◊ O C, dipole–dipole
=
interaction C–F ◊ ◊ ◊ C O) in the solid state compensated the energetic disadvantage of the relatively less
stable N-conformation, and drove the thiouridine to crystallize in the N-conformation. These results,
along with the earlier empirical rules regarding proton chemical shifts in carbohydrates and
nucleosides, were used to propose a method based on proton chemical shifts for the analysis of the N ꢀ
S equilibrium of the fluorinated sugar ring.
4¢-thionucleosides,6 such as 1 and 2, and truncated nucleosides
Introduction
without 4¢-hydroxymethyl,7,8 such as 3 and 4, possess potent
antiviral and antitumor activities. In view of the above findings,
we designed and synthesized our target molecules 5a–c based on
bioisosteric rationale (Fig. 1).
The unique role of the substituent (hydrogen or hydroxyl) on the
C-2¢ atom in nucleoside acids as the distinguishing feature between
DNA and RNA prompted the investigation of the biological prop-
erties of nucleosides containing substituents other than hydrogen
or hydroxyl at this position. Accordingly, it was interesting to
study the biological properties of nucleosides containing fluorine,
which could mimic both hydrogen and hydroxyl to some extent,
at the C2¢ position. Furthermore, the fluorine atoms at the 2¢-
position increase the hydrolytic stability of the nucleoside due
to destabilization of the transition state (positive charge at the
anomeric center) during hydrolytic cleavage of the nucleosidic
bond. So far, a number of 2¢-fluorinated nucleosides1–3 have been
synthesized and biologically evaluated, such as 2¢-deoxy-2¢,2¢-
difluorocytidine (Gemcitabine), which has beenapproved as a drug
for solid tumor treatment.4,5 Recently, it has been reported that
aKey Laboratory of Organofluorine Chemistry, Shanghai Institute of Organic
Chemistry, Chinese Academy of Sciences, 354 Fenglin Lu, Shanghai, 200032,
China. E-mail: flq@mail.sioc.ac.cn; Tel: 02154925185
bState Key Laboratory of Bioorganic and Natural Products, Shanghai
Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin
Lu, Shanghai, 200032, China
Fig. 1 Rationale for the design of the target compounds 5a–c.
The conformations of the gem-difluoromethylene-containing
thiouridine 5a were studied using X-ray crystallography, NMR
spectroscopy and ab initio calculations. The conformation of the
thiosugar ring was defined by the concept of pseudorotation9
† Electronic supplementary information (ESI) available: Scans of NMR
spectra, computational methods and results of MD simulations. CCDC
reference number 740876. For crystallographic data in CIF or other
electronic format see DOI: 10.1039/b914679b
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(two pseudorotation parameters: the phase angle P and the
maximum puckering amplitude umax), and the conformation
of the base was defined by the glycosyl rotamer angle c.10
Altona and Sundralingham defined pseudorotational parameters
and correlated them with highly populated solid-state nucle-
oside/nucleotide conformations.9,11 Altona et al.,12 Chattopad-
hyaya et al.,13 Marquez et al.,14 and others have refined and
added additional parametrization to the fundamental Altona–
Sundralingham approach11 for analysis of the predominant con-
former populations of the nucleosides. For the sugar-fluorinated
nucleosides, in 1998, a new complicated seven-parameter Karplus
relationship based on JHF coupling constants was derived15
3
and later modified slightly by Mikhailopulo, et al.16 The JHF
coupling constants have been widely used in the conformational
analysis of fluorinated sugars and nucleosides, while chemical
shifts of protons are almost ignored in the conformational or
structure analysis. For the simple fluorinated nucleoside molecules,
conversion of the conformation from N to S, and vice versa, would
result in considerable changes in the chemical shifts of the protons,
especially for those experiencing interactions with the fluorine
atom in various ways, since the reoriented highly electronegative
fluorine atom could profoundly alter chemical environments, as
well as the stereoelectronic environments of the protons. This
prompted us to propose a method dependent on the chemical shifts
of the protons to analyze the N ꢀ S equilibrium of compound
Scheme 1 Synthesis of compounds 5a–c. Reagents and conditions: (a) i.
O3, CH2Cl2, -78 ◦C, ii. NaBH4; (b) i. TFA–H2O–THF (1 : 1 : 1), ii. NaIO4,
acetone, iii. NaBH4; (c) i. MsCl, pyridine, ii. Na2S.·9H2O, DMF, 90 ◦C;
(d) i. m-CPBA, CH2Cl2, -78 ◦C, ii. silylated base, TMSOTf, DCE; (e) BCl3,
CH2Cl2, -70 ◦C; (f) NH3, CH3OH
5a. In addition, we wish to gain more insight into the weak but
17
=
meaningful C–F ◊ ◊ ◊ C O and C–F ◊ ◊ ◊ H–C interactions, which
may influence the overall conformation of the nucleoside.
Fig. 2 Packing patterns of (A) 5a, which exhibits important intermolecu-
=
lar hydrogen bonding and F ◊ ◊ ◊ C O interaction; and the crystal structures
of (B) 5a.
Results and discussion
Synthesis
˚
=
intermolecular hydrogen bond (C3¢–OH ◊ ◊ ◊ O C4, ~2.002 A).
The CF2 and uracil base units form intermolecular weak C2¢–
Fb¢ ◊ ◊ ◊ C2 O dipole–dipole interaction, leading to an extended
gem-Difluorinated synthon 6 was easily prepared according to our
reported methodology.18 Compound 6 was converted to alcohol
7 by oxidation with O3 followed by reduction with NaBH4. Diol
8 was prepared in 86% overall yield by the following steps: (1)
acidic hydrolysis of the isopropylidene group with TFA–THF–
H2O and (2) oxidative scission of the resulting diol with sodium
periodate and subsequent reduction of the resultant aldehyde
with NaBH4. Mesylation of 8 followed by treatment with sodium
sulfide in DMF at 90 ◦C for 30 min resulted in a ring closure
to give thiofuranose 9 in 84% yield. Finally, oxidation of 9 with
m-CPBA followed by condensation with silylated base using the
Pummerer reaction afforded our desired protected nucleosides
regioselectively. Subsequent removal of the protecting groups
provided the target thionucleosides 5a–c (Scheme 1).
=
˚
layer packing. The F ◊ ◊ ◊ C distance is 3.042 A, which is about
˚
˚
0.13 A shorter than the sum of the van der Waals radii (3.17 A).
Similar dipole–dipole interactions have been observed between
aromatic fluorine compounds and their target proteins.19,20 Herein,
aliphatic C–F is also found to be involved in such interactions. In
5a (Fig. 2B), the thiosugar ring adopts a northern conformation
with a pseudorotational phase angle of P = 9.4◦ and a maximum
puckering amplitude of umax = 45.3◦, which is in contrast to its
nonfluorinated parent 4¢-thiothymidine21 that adopts a southern
conformation in the solid state. Such a big difference may be due
to the gauche relationships22,23 between OH at C3¢ and both of the
two gem fluorines at C2¢ in 5a. The base is anti to the thiosugar
ring, with a glycosyl rotamer angle of c = -143.0◦. The torsion
angle of H1¢–C1¢–C2¢–Fb¢ was the ideal 90◦.
Solid-state: X-ray crystallography
The solid state structure of 5a is shown in Fig. 2.‡ All of the struc-
ture shown represents the absolute configuration for the molecule.
The structure relates to a monoclinic space group P21, and between
the neighboring molecules within the same packing layer is a
Molecular modeling: quantum calculations and MD simulations
Using the experimentally determined crystal structure as a starting
point, the structures of compound 5a were minimized by the
CHARMm24 force field to convergence. Then, each conformation
was further minimized at the B3LYP/6-311++G(2d,2p) level
with the Gaussian 03 program.25 Frequency analysis was also
performed to confirm that these minimized structures were true
minima on the potential energy surface. In order to determine
the most stable conformation in methanol, the solvation free
‡ Crystal data for 5a: C8H8F2N2O3S, M = 250.22, monoclinic, a =
˚
6.1849(8), b = 18.082(2), c = 8.9343(11) A, a = 90, b = 100.122(2),
◦
3
˚
g = 90 , V = 983.6(2) A , T = 293 K, space group P21, Z = 4, 5594
reflections collected, 2129 unique (Rint = 0.0244), R1 = 0.0433 [I > 2d(I)],
wR2 = 0.1110.
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Table 1 Comparison of structure parameters for compound 5a as determined by X-ray crystallography and quantum calculations
u0
u1
u2
u3
u4
P
umax
c
X-Ray
ab intio
44.75
-47.52
-38.55
34.62
19.26
-11.67
5.26
-14.49
-29.67
37.66
9.43
177.71
45.36
47.56
-142.97
-125.38
Table 2 Comparison of the computed proton chemical shifts of the N- and S-conformers with the experimental data at 300 K
H6
H5
H1¢
H3¢
H4a¢
H4b¢
N-Conformer
S-Conformer
Experimental data
7.78
7.92
8.01
5.80
5.80
5.78
6.82
7.31
6.51
4.50
4.42
4.39
3.08
2.98
2.86
3.13
3.55
3.45
energy of each conformation was calculated at the B3LYP/6-
311++G(2d,2p) level in couple with the SCIPCM solvation
model. The lowest energy conformation identified after such
conformational sampling was observed to adopt a typical S-
conformation with P = 177.7◦, umax = 47.6◦ and c = -125.4◦,
whereas in the X-ray structure, the thiosugar ring pucker was
basically in the pure N-conformation (P = 9.4◦, Table 1).
effect arising from anisotropy in the magnetic susceptibility of
nearby atoms or portions of a molecule, and the electrostatic
interaction between hydrogen and strongly electronegative atoms
or groups nearby.28 The pyrimidine base adopts a similar anti
conformation with c = -143.0◦ for solid conformer N, and c =
-125.4◦ for solution conformer S (Table 1), whichindicates thatthe
effect of the base moiety on the chemical-shift of H4b¢ and H4a¢ is
alike in these two conformers. Then, we focused on the anisotropy
effects of the sugar ring to rationalize the different chemical shifts
of H4b¢ and H4a¢ between the N- and S-conformers using the
earlier empirical rules, which were concluded from extensive stud-
ies on 1H NMR of carbohydrates and their derivates.26,29–34 In the
S-conformer, H4b¢ was strongly deshielded by the axial hydroxyl
at C3¢, which is at an opposing position to it,32 and was further
deshielded by the spatially close axial fluorine atom because of the
through space electrostatic interaction d+C2¢–Fb¢d- ◊ ◊ ◊ d+H4b¢–C4¢d-.
These deshielded effects on H4b¢ in the S-conformer far outweigh
the deshielded effects from the C2¢(exo)–C3¢(endo) bond and the
relatively weak electrostatic interaction between d+C3¢–O3¢d- and
d+H4b¢–C4¢d- on H4b¢ in the N-conformer. Therefore, the chemical
shift of H4b¢ in the S-conformer is at a correspondingly lower
field than that in N-conformer. A similar analysis for H4a¢ was
performed. The deshielding effect of the C2¢(endo)–C3¢(exo) bond,
and electrostatic interaction between d+C3¢–O3¢d- and d+H4a¢–
C4¢d- on H4a¢ in the S-conformer is estimated to be weaker than
that in the N-conformer, arising from the electrostatic interaction
between d+C3¢–O3¢d- and d+H4a¢–C4¢d-, d+C2¢–Fa¢d- and d+H4a¢–
C4¢d-, and thus H4a¢ in the S-conformer is shifted upfield from
its counterpart in the N-conformer. Thus, the differences in the
chemical shifts (Dd) between H4b¢ and H4a¢ of the S-conformer
(Dd = 0.59 ppm) are much larger than that in the N-conformer
(Dd = 0.05 ppm).35
For nucleosides, the main factors which affect the chemical shift
signals of the protons are the conformation of the sugar moiety
and that of the nucleobase.26 Therefore, to further confirm the
1
predominant conformation of compound 5a in solution, the H
NMR spectra of the N- and S-conformers were predicted using
the GIAO/DFT method at the B3LYP/6-311++G(2d,2p) level.
The experimental data basically match with the chemical shifts
predicted for the S ring pucker (Table 2).
The hydrogen atoms H6, H4a¢ and H4b¢ on compound 5a
should be paid special attention since they reveal some impor-
tant structural information. In the low-energy S-conformation
deduced by quantum calculations, the distance between Fb¢ and
˚
˚
H6 is 2.47 A, about 0.20 A shorter than the sum of the van der
˚
Waals radii (2.67 A) of the two relevant atoms. The chemical
shift of H6 on the S-conformer was shifted downfield from its
counterpart on the N-conformer by 0.20 ppm. It indicates that
a through-space electrostatic attraction between d+C2¢–d-Fb¢ and
d+H6–d-C6 exists in the S-conformer. A qualitative inspection of
1
the experimental 13C NMR and H NMR also points to such
interaction between d+C2¢–d-Fb¢ and d+H6–d-C6: the C6 signal in
13C NMR spectra splits into a doublet (J = ~4.2 Hz), which
presumably results from through-space (ts) coupling27 with Fb¢,
and a similar doublet (J = ~1.8 Hz) for H6 signal is also observed.
The chemical shifts of H4b¢ and H4a¢ in chemically similar
environments are largely dependent on the “neighbor anisotropy”
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Table 3 The main contributors to the preferred conformation in solid and in solution respectively
Conformer N
Conformer S
ap s to s*a
sC3¢–H3¢ to s*C2¢–Fa¢
sC3¢–C4¢ to s*C2¢–Fb¢
sC1¢–C2¢ to s*C3¢–O3¢
sC1¢–H1¢ to s*C2¢–Fb¢
sC4¢–H4b¢ to s*C3¢–O3¢
sC3¢–C4¢ to s*C2¢–Fa¢
Electrostatic attractionb
Packing forcesc
d+C2¢–Fa¢d- ◊ ◊ ◊ d+H4a¢–C4¢d-
d+C2¢–Fb¢d- ◊ ◊ ◊ d+H6–C6d-
d+C2¢–Fb¢d- ◊ ◊ ◊ d+H4b¢–C4¢d-
=
OH ◊ ◊ ◊ O
C
O
=
C–F ◊ ◊ ◊ C
a Potential antiperiplanar effects depicted by arrows. b Through space electrostatic attraction depicted by dashed line. c Intermolecular packing forces
depicted by yellow hashed line in Fig. 2A.
Table 4 The 1H NMR chemical shifts (ppm) of compound 5a at six
Many of the conformational adjustments imparted by substi-
tution of electronegative (or other) atoms into the carbohydrate
different temperaturesa
ring have been rationalized based on arguments relating to the
T/K
H6
H5
H1¢
H3¢
H4b¢
H4a¢
anomeric15 and gauche effects.22,36 Recent reports that studied
similar difluorinated structures have invoked an alternative theory
by Brunck and Weinhold,37 which suggests that, not only do vicinal
electronegative atoms prefer a gauche arrangement, but the origins
of this effect may be more due to an antiperiplanar (ap) s to s*
stabilization when the donating bond is the least polar one and
the acceptor orbital is at the most polarized bond. For compound
5a, in solution, three strong ap effects (sC1¢–H1¢ to s*C2¢–Fb¢,
sC3¢–C4¢ to s*C2¢–Fa¢ and sC4¢–H4b¢ to s*C3¢–O3¢) appear
to be responsible for keeping the ring pucker in the preferred
213
233
253
273
293
313
8.153
8.116
8.081
8.048
8.014
7.977
5.800
5.794
5.789
5.785
5.778
5.771
6.511
6.509
6.508
6.506
6.503
6.496
4.413
4.404
4.402
4.396
4.391
4.386
3.472
3.465
3.458
3.451
3.442
3.436
2.850
2.851
2.853
2.854
2.858
2.860
a Data were referenced to CD3OD at 3.31 ppm.
solid state, the packing forces (Table 3) are able to overcome the
energetic disadvantage and make compound 5a crystallize as a
type N-conformer.
S orientation. In addition, the small but meaningful through
d-
space electrostatic attractions (d+C2¢–Fb¢d- ◊ ◊ ◊ d+H6–C6 , ~2.46 A;
˚
and d+C2¢–Fb¢d- ◊ ◊ ◊ d+H4b¢–C4 , ~2.84 A) contribute additional
d-
˚
The conformation of the uracil base is both anti in the N-
and S-sugar puckers, which is confirmed by the crystal structure,
quantum computation and the experimental 1H NMR, 13C NMR
and NOESY spectra. The electrostatic attractions between Fb¢
and H6 in the S-conformer may be the main factor for keeping
the base moiety in a high anti rotamer population in solution.10
Similar interactions have been observed in the C2¢-up or C3¢-up
fluorinated nucleosides.40–42
stabilization to the preferred structure (Table 3). The anomeric and
ap effects arising from sulfur seem to be a weak contributor to the
structure, since sulfur is much less electronegative in comparison
to oxygen and fluorine. The 1H NMR chemical shifts of compound
5a were measured in CD3OD at six different temperatures from
213 to 313 K in 20 K increments (Table 4). The chemical shift
changes in compound 5a in CD3OD showed liner dependence as
a function of temperature,38 which is typical for systems where
intermolecular interactions are not significant.39
Conformational equilibrium of the thiosugar ring in compound 5a
For the N-conformer, the ap effects from C–H and C–C s-
bonds to s*C–F are equal to that of the S-conformer (Table 3).
However, the hyperconjugation coming from the more efficient
C–H bond in the S-conformer rather than the C–CF2 bond in the
N-conformer overlap with the s*C–O orbital, together with the
additional electrostatic attraction d+C2¢–Fb¢d- ◊ ◊ ◊ d+H6–C6d- lead to
an energy-favored S conformation in methanol solution. In the
The temperature-dependent chemical shifts reflect time-averaged
values of several rapidly interconverting conformers on the NMR
time scale in solution. The observed chemical shifts depend not
only on the geometries but also on the mole fraction of conformer
N (XN) and conformer S (1 - XN) present in dynamic equilibrium,
and can be writtenas d = XNdN + (1 - XN)dS. Taking the error
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estimation into consideration,43 the chemical shift values of H4b¢
(Table 4) are selected, which are influenced most by an intercon-
version from the N-conformer to the S-conformer, and XN = 0.22
is obtained at 300 K. This is only a rough estimate since there is
an extremely small structural database available for compounds
containing a gem-CF2 group in nucleoside templates. Our MD
simulations (by using the AMBER 9 program44) of the dynamic
equilibrium between the N- and S-conformers suggest that the
N ꢀ S equilibrium in methanol is driven toward the S-conformer
preferentially with a ratio of S/N = 10 : 1 at 300 K (Fig. 3), which
is in good agreement with experimental observation.
equilibrium is driven by enthalpy towards S-type conformer for
compound 5a, and the negative entropy (DS◦ = -0.56 J K-1)
suggests a more organized S-conformer, which is consistent with
the low-energy conformer from high-level ab initio calculations, in
which an additional strong intramolecular d+C2¢–Fb¢d- ◊ ◊ ◊ d+H6–C6d-
attractive interaction was shown.
Finally, the NMR data of compounds 5b and 5c were compared
to that of compound 5a. The signals of the hydrogen atoms of
the sugar ring in the upfield area are almost the same to that
of compound 5a (Fig. 5), including chemical shifts and coupling
matters, despite the different bases they had, which indicated that
the predominant conformation which compound 5b and 5c adopt
in solution, was type S. The doublet of C6 signals and doublet
of doublets of H6 signals were also observed in the spectra of
compound 5b and 5c, just like that of compound 5a, which
suggested that the d+C2¢–Fb¢d- ◊ ◊ ◊ d+H6–C6d- interaction may also
exist in compound 5b and 5c in methanol solution.
Fig. 3 MD simulations of the dynamic equilibrium between two con-
formers in methanol. The structure on the left is the N-conformer; while
the one on the right is the S-conformer. The X axis is the RMSD values
computed using the N-conformer as reference, while the Y axis is the
RMSD values computed using the S-conformer as reference. Each circle
represents a conformation sampled by MD simulations. Conformations
close to the S-conformer are apparently more populated. The S/N ratio
is computed to be 10 : 1.
To further evaluate the variation of the population ratios
for compound 5a in a more quantitative way, van’t Hoff plots
were employed. A van’t Hoff analysis using populations of N
and S pseudorotamers based on the chemical shifts of H4b¢ at
six different temperatures (Table 4) enabled calculation of the
enthalpy and entropy contributions that drive N ꢀ S equilibrium
in methanol (Fig. 4). The van’t Hoff analysis shows that the N ꢀ S
Fig. 5 Partial 1H NMR spectra of compounds 5a, 5b and 5c in CD3OD
at 300 K as referenced to CD3OD at 3.31 ppm.
Conclusions
We have designed and efficiently synthesized the new gem-CF2-
containing thionucleosides. The conformational analysis of com-
pound 5a using X-ray crystallography, NMR spectroscopy and ab
initio calculations shows that compound 5a exists in the solid state
as a type-N thiosugar pucker with anti conformation of the base,
whereas from the NMR data and the high level calculations, it
follows that the predominant conformer in methanol solution is
type-S with anti conformation of the base. The strong ap effects
from C–H and C–C s-bonds to s*C–F and s*C–O are responsible
for the favored S conformation in solution. Beside ap effects,
the weak electrostatic attraction betweend+C2¢–Fb¢d- and d+H6–
C6d- may also contribute additional stabilization to the preferred
structure and hold the base in a high anti rotamer population
in solution. However, in the solid state, the packing forces
Fig.
4
van’t Hoff plots according to the relation: ln(XS/XN)
=
-DH◦/RT + DS◦/R. The values of DH◦ and DS◦ were calculated from
slopes and intercepts of the van’t Hoff plots. DH◦ = -2.78 kJ mol-1 and
DS◦ = -0.56 J K-1 were found.
=
(hydrogen bond C3¢–OH ◊ ◊ ◊ O C4 and dipole–dipole interaction
=
C2¢–Fb¢ ◊ ◊ ◊ C2 O) overcome the energetic disadvantage of the
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relatively less stable N-conformer, and drive compound 5a to
crystallize in type-N-conformer. The relatively larger differences
in the chemical shifts (Dd) between H4b¢ and H4a¢ of the S-
conformer (Dd = 0.59 ppm) than that of the N-conformer (Dd =
0.05 ppm) could be roughly and readily rationalized using the
earliest empirical rules, which were concluded from extensive
organic layer was washed with brine (20 mL). After removal of
the solvent in vacuo, the residue was dissolved in acetone (10 mL),
followed by treatment with a solution of NaIO4 (3.49 g, 16.3 mmol)
in water (10 mL) at 0 ◦C with stirring. After stirring for 1.5 h,
the reaction was quenched with ethylene glycol (0.6 mL). The
mixture was extracted with CH2Cl2 (10 mL ¥ 3) and the solvent
was removed in vacuo. The crude product was dissolved in CH3OH
(8 mL) and then NaBH4 (680 mg, 17.9 mmol) was added at 0 ◦C.
The reaction mixture was then warmed to room temperature
and stirred for 30 min. Then the reaction was quenched with
water (10 mL). The resultant mixture was extracted with Et2O
(15 mL ¥ 3). The combined organic layers were washed with brine
(15 mL), dried over anhydrous Na2SO4, filtered, and the solvent
removed in vacuo. The residue was purified by silica gel column
chromatography (petroleum ether : ethyl acetate = 2 : 1) to give
2.96 g (86% yield for three steps) of compound 8 as a clear oil:
[a]D25 = 17.4 deg cm3 g-1 cm-1 (c 1.05, CHCl3); 1H NMR (400 MHz,
CDCl3) d 7.41–7.38 (m, 5H), 4.79 (d, J = 11.4 Hz, 1H), 4.67 (d,
1
studies on H NMR of carbohydrates and their derivates. The
conformational equilibrium between conformation N and S in
methanol has been studied by MD simulations as well as chemical
shift-based analyses, both of which reveal the similar preference
of the N ꢀ S interconversion towards the S-conformer. Taking
all the above into consideration, the conformational analysis for
sugar pucker of the fluorinated nucleoside based on chemical-
shifts sounds feasible since the chemical shifts of the protons,
especially for those involved in ap effects and interaction with
strong electronegative atoms (F, O), could be obviously shifted
by an conversion of the conformation from N to S in fluorinated
nucleosides. Moreover, the signals for the protons in NMR spectra
are well dispersed due to the profound effects of the highly
electronegative fluorine atom on the chemical properties as well as
on the stereoelectronic properties of the modified nucleosides and
could be easily assigned.
J = 11.4 Hz, 1H), 3.93 (m, 1H), 3.84–3.72 (m, 4H), 2.90 (s, 2H); 13
C
NMR (100.7 MHz, CDCl3) d 137.1, 128.5, 128.1, 128.0, 121.4 (dd,
J = 249.4, 247.1 Hz), 78.4 (dd, J = 28.3, 25.7 Hz), 73.7, 60.8 (dd,
J = 32.6, 28.5 Hz), 59.6 (dd, J = 6.2, 2.7 Hz); 19F NMR (282 MHz,
CDCl3) d -111.1 (ddd, J = 267.9, 21.7, 12.1 Hz, 1F), -115.7
(dm, J = 267.5 Hz, 1F); IR (KBr)max 3383, 2945, 1456, 1101, 914,
742 cm-1; MS (ESI) m/z 250.2 (M+ + H2O), 255.2 (M+ + Na); Anal.
Calcd for C11H14O3F2: C, 56.89; H, 6.08. Found: C, 56.70; H, 6.15.
The chiral HPLC analytical data: Chiralpak AD column, detected
at l = 214 nm, eluent: n-hexane : i-PrOH (80 : 20), 0.7 mL min-1,
tR (minor) = 7.58 min, tR (major) = 6.94 min, 88% ee.
Experimental section
(R)-3-(benzyloxy)-3-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-
difluoropropan-1-ol (7)
A solution of compound 6 (5.12 g, 17.2 mmol) in CH2Cl2
(80 mL) was ozonized at -78 ◦C for 1 h. Then, a suspension
of NaBH4 (1.27 g, 33.4 mmol) in C2H5OH (15 mL) was added
to the reaction mixture. The reaction mixture was warmed to
ambient temperature and was stirred for 30 min. Then the reaction
was quenched with water (50 mL), and the organic layer was
separated. The resultant aqueous layer was extracted with EtOAc
(25 mL ¥ 3). The combined organic layers were washed with brine
(50 mL), dried over anhydrous Na2SO4, filtered, and the solvent
was removed in vacuo. The residue was purified by silica gel column
chromatography (petroleum ether : ethyl acetate = 7 : 1) to give
4.52 g (87% yield) of compound 7 as a clear oil: [a]2D6 = 13.7
deg cm3 g-1 cm-1 (c 1.81, CHCl3); 1H NMR (400 MHz, CDCl3), d
7.37–7.31 (m, 5H), 4.81 (d, J = 11.2 Hz, 1H), 4.77 (d, J = 11.2 Hz,
1H); 4.42 (m, 1H), 4.11 (m, 1H), 4.09–4.05 (m, 2H); 3.77 (m, 2H),
3.05 (br, 1H), 1.43 (s, 3H), 1.36 (s, 3H); 13C NMR (100.7 MHz,
CDCl3) d 137.3, 128.4, 128.1, 128.0, 121.6 (t, J = 247.6 Hz), 108.8,
76.5 (dd, J = 27.1, 23.4 Hz), 75.7, 74.4, 65.1(d, J = 4.4 Hz), 61.8
(dd, J = 32.0, 28.8 Hz), 26.2; 25.0 19F NMR (282 MHz, CDCl3) d
-114.8 (dm, J = 258.6, 1F), -116.7 (ddd, J = 262.8, 29.3 Hz, 13.0,
1F); IR (KBr)max 3443, 2989, 1456, 1374,1136, 1029, 699 cm-1; MS
(ESI) m/z 303.2 (M+ + H); Anal. Calcd for C15H20O4F2: C, 59.59;
H, 6.67. Found: C, 59.64; H, 6.75.
(S)-4-(benzyloxy)-3,3-difluorotetrahydrothiophene (9)
To compound 8 (2.77 g, 11.9 mmol) in anhydrous CH2Cl2 (40 mL)
and pyridine (8 mL) was added MsCl (3.70 mL, 47.6 mmol)
◦
slowly at 0 C. The reaction mixture was then warmed to room
temperature and stirred overnight. The reaction was quenched
with water (30 mL) and the organic layer was separated. The
resultant aqueous layer was extracted with Et2O (15 mL ¥ 3). The
combined organic layers were washed with 1 N HCl (20 mL ¥
3), saturated NaHCO3 solution (30 mL), water (30 mL) and
brine (30 mL), dried over anhydrous Na2SO4, filtered, and the
solvent was removed in vacuo. The residue was dissolved in DMF
(60 mL) and Na2S·9H2O (4.19 g, 17.4 mmol) was added. Then
the reaction mixture was heated to 90 ◦C. After stirring for
30 min, the reaction mixture was cooled to room temperature and
water (70 mL) was added. The resulting mixture was extracted
with diethyl ether (30 mL ¥ 3). The combined organic layers
were washed with water (50 mL) and brine (50 mL), dried over
anhydrous Na2SO4, filtered, and the solvent was removed in vacuo.
The residue was purified by silica gel column chromatography
(petroleum ether : ethyl acetate = 40 : 1) to give 2.31 g (84% yield
for two steps) of compound 9 as a light yellow oil: [a]2D8 = -16.3
1
deg cm3 g-1 cm-1 (c 1.95, CHCl3); H NMR (300 MHz, CDCl3)
(R)-3-(benzyloxy)-2,2-difluorobutane-1,4-diol (8)
d 7.38–7.28 (m, 5H), 4.77 (d, J = 12.0 Hz, 1H), 4.66 (d, J =
12.0 Hz, 1H), 4.04 (m, 1H), 3.26 (m, 1H), 3.11 (m, 1H), 2.98
(m, 1H), 2.87 (m, 1H); 13C NMR (100.7 MHz, CDCl3) d 137.1,
128.5, 128.1, 128.0 (dd, J = 260.5, 250.8 Hz), 127.8, 79.0 (dd, J =
31.1, 21.4 Hz), 72.6, 32.5 (t, J = 26.7 Hz), 30.5 (t, J = 3.2 Hz);
19F NMR (282 MHz, CDCl3) d -104.4 (dm, J = 231.2 Hz, 1F),
Compound
7
(4.48 g, 14.8 mmol) was dissolved in
CF3COOH/H2O–THF (1 : 1 : 1, 9 mL). After the reaction mixture
was stirred for 2 h, the mixture was diluted with EtOAc (10 mL)
and the reaction was quenched with solid NaHCO3. The resulting
mixture was extracted with EtOAc (10 mL ¥ 3). The combined
168 | Org. Biomol. Chem., 2010, 8, 163–170
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-115.0 (dm, J = 232.1 Hz, 1F); IR (KBr)max 3033, 2952, 1455, 1261,
1120, 697 cm-1; MS (ESI) m/z 231.2 (M+ + H), 248.2 (M++ H2O);
HRMS Calcd for C11H12OF2S+ (M+): 230.0577, Found: 230.0580.
m/z 251.0 (M+ + H), HRMS Calcd for C8H9N2O3F2S (M+ + H):
251.0296. Found: 251.0296.
Acknowledgements
1-((2R,4S)-3,3-difluoro-4-hydroxytetrahydrothiophen–2-yl)
The National Natural Science Foundation of China and Shanghai
Municipal Scientific Committee are greatly acknowledged for
funding this work.
uracil (5a)
A solution of m-CPBA (80%, 116 mg, 0.54 mmol) in CH2Cl2
(5 mL) was added dropwise to a solution of compound 9 (124 mg,
0.54 mmol) in CH2Cl2 (5 mL) at -70 ◦C. The reaction mixture was
stirred at -40 ◦C for 40 min. Then, the mixture was quenched with
saturated NaHCO3 solution (10 mL) and the organic layer was
separated. The resultant aqueous layer was extracted with CH2Cl2
(10 mL ¥ 3). The combined organic layers were washed with 10%
aqueous Na2SO3 (10 mL) and brine (15 mL), dried over anhydrous
Na2SO4, filtered, and the solvent was removed in vacuo to give the
sulfoxide, which was used directly in next step without purification.
To a solution of silylated uracil, prepared from refluxing uracil
(182 mg, 1.62 mmol) and ammonium sulfate (catalytic amount) in
HMDS (4 mL), in anhydrous DCE (2 mL) was added a solution
of the sulfoxide in anhydrous DCE (4 mL) followed by addition of
TMSOTf (195 mL, 1.08 mmol) at 0 ◦C, and the mixture was stirred
at the same temperature for 30 min. The mixture was quenched
with saturated aqueous NaHCO3 solution (5 mL), filtered and
poured into CH2Cl2 (10 mL). The organic layers were washed
with brine (15 mL), dried over anhydrous Na2SO4, filtered, and
the solvent was removed in vacuo. The residue was purified by silica
gel column chromatography (petroleum ether : ethyl acetate =
3 : 1) to give 69 mg of the anti isomer of protected uridine and
36 mg of the syn isomer of protected uridine. To a solution of
the anti isomer (69 mg, 0.20 mmol) in anhydrous CH2Cl2 (10 ml)
was added BCl3 (1M in CH2Cl2, 4.0 ml, 4.0 mmol) at -70 ◦C.
After the reaction mixture was stirred for 2 h at -70 ◦C, the
mixture was quenched with MeOH (5 ml), and the solvent was
removed in vacuo. The residue was purified by silica gel column
chromatography (CH2Cl2 : MeOH = 20 : 1) to give compound 5a
(43 mg, 32% yield for three steps) as a white solid: [a]2D6 = -51.2
deg cm3 g-1 cm-1(c 2.15 MeOH); 1H NMR (300 MHz, MeOH-d4)
d 8.01 (dd, J = 8.1, 1.8 Hz, 1H), 6.50 (dd, J = 12.3, 9.6 Hz, 1H),
5.78 (d, J = 8.1 Hz, 1H), 4.39 (m, 1H), 3.45 (m, 1H), 2.86 (m,
1H); 13C NMR (75.5 MHz, MeOH-d4) d 165.6, 152.6, 143.8 (d,
J = 4.2 Hz), 126.4 (dd, J = 263.1, 256.2 Hz), 103.1, 72.9 (dd, J =
30.4, 22.3 Hz), 59.7 (dd, J = 31.1, 18.9 Hz), 32.8 (t, J = 2.5 Hz);
19F NMR (282 MHz, MeOH-d4) d -110.3 (ddd, J = 236.9, 9.0,
3.9 Hz, 1F), -112.9 (ddd, J = 236.3, 20.6, 11.2 Hz, 1F); IR (KBr)
Notes and references
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3221, 3060, 1693, 1455, 1382, 1078 cm-1; MS (ESI) m/z 251.0
max
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The syn isomer of protected uridine was also deprotected using
the similar procedure as described for the anti isomer to give 5a¢
(21mg, 15% yield for three steps) as a white solid: [a]2D6 = 15.2
deg cm3 g-1 cm-1 (c 1.25 MeOH); 1H NMR (300 MHz, MeOH-d4)
d 8.21 (dd, J = 8.1, 1.5 Hz, 1H), 6.35 (dd, J = 14.1, 4.2 Hz, 1H),
5.73 (d, J = 8.1 Hz, 1H), 4.42 (m, 1H), 3.18 (m, 1H), 3.10 (m, 1H);
13C NMR (75.5 MHz, MeOH-d4) d 165.8, 152.8, 144.8, 126.6 (dd,
J = 266.7, 255.3 Hz), 102.2, 73.1 (dd, J = 33.7, 22.1 Hz), 61.4
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max
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experimental data (DdS - DdN = 0.59 ppm).
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by an axial hydroxyl was set at 0.5 ppm. On this basis, for the differences
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values was taken into consideration.
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170 | Org. Biomol. Chem., 2010, 8, 163–170
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The Royal Society of Chemistry 2010
©