Novel epimerization of aromatic C-nucleosides with electron-withdrawing substituents with trifluoroacetic acid-benzenesulfonic acid using mild conditions

Article abstract of DOI:10.1016/S0040-4039(03)00848-7

Trifluoroacetic acid and benzenesulfonic acid in dichloromethane at ambient temperature have been found to be efficient co-catalysts for the epimerization of C-nucleosides with electron-withdrawing substituents. This approach provides a convenient route to the β anomer of the nucleosides with high yields in the range 54-78%. Synthesis of the 3′-phosphoramidite of 2,4-difluorophenyl C-nucleoside suitable for solid-phase DNA synthesis is described.

Full text of DOI:10.1016/S0040-4039(03)00848-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 4051–4055
Novel epimerization of aromatic C-nucleosides with
electron-withdrawing substituents with trifluoroacetic acid
–benzenesulfonic acid using mild conditionsꢀ
Yu Lin Jiang and James T. Stivers*
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street,
Baltimore, MD 21205-2185, USA
Received 5 March 2003; revised 31 March 2003; accepted 1 April 2003
Abstract—Trifluoroacetic acid and benzenesulfonic acid in dichloromethane at ambient temperature have been found to be
efficient co-catalysts for the epimerization of C-nucleosides with electron-withdrawing substituents. This approach provides a
convenient route to the b anomer of the nucleosides with high yields in the range 54–78%. Synthesis of the 3%-phosphoramidite
of 2,4-difluorophenyl C-nucleoside suitable for solid-phase DNA synthesis is described. © 2003 Elsevier Science Ltd. All rights
reserved.
Chemical approaches have proven to be powerful tools
for understanding the basis of specificity and catalysis
of DNA polymerases and DNA repair glycosylases.1,2
One of the first examples was the use of non-polar
nucleoside analogues to probe the basis of nucleotide
recognition by DNA polymerases.1,3 These analogues
lack the hydrogen bond donor–acceptor groups of nor-
mal DNA molecules, yet mimic their shape, or even the
shape of the entire base pair.1,2 Thus, Kool and col-
leagues have shown that DNA polymerases will specifi-
cally incorporate the non-polar dTTP analogue,
2,4-difluorotoluenyl nucleotide triphosphate (dFTP)
opposite to adenine on the template strand, showing
that high specificity can occur in the absence of hydro-
gen bonding.3 They also found that polymerases can
compounds potential anticancer, antiviral and diagnos-
tic imaging agents.
We are actively using chemical approaches to under-
stand the recognition and catalytic mechanism of the
DNA repair enzyme uracil DNA glycosylase (UDG),
which removes unwanted deoxyuridine residues from
DNA.2,6–11 To better understand the role of base shape
and hydrogen bonding in uracil recognition by UDG,
we wished to incorporate a 2,4-difluorophenyl C-
nucleoside uracil analogue in DNA.12 Although the
efficient synthesis of the difluorotoluenyl C-nucleoside
phosphoramidite has been described and is commer-
cially available, the synthesis of the difluorophenyl
derivative has never been reported.13,14 We initially
attempted synthesis of the difluorophenyl C-nucleoside
phosphoramidite according to Kool’s method, but the
yield was very low in the epimerization step (<20%).
Although we reported previously that trifluoroacetic
acid is an effective catalyst in dichloromethane for the
epimerization of a variety of C-nucleosides with elec-
tron-donating substituents,15 we found that TFA alone
is not an efficient catalyst for the epimerization of
C-nucleosides that contain electron-withdrawing sub-
stituents, requiring the development of new synthetic
methods for these compounds.
specifically incorporate
a pyrenyl (Y) nucleotide
triphosphates (dYTP) opposite to a DNA site that
lacks a base, confirming that steric complimentarity is
an important component of high fidelity DNA replica-
tion.1,4 Some interesting results from Wiebe and co-
workers5
suggest
that
similar
difluorophenyl
C-nucleoside derivatives could also be very useful bio-
pharmaceuticals. The most remarkable properties of
C-nucleosides are their cell permeability, their ability to
bind to proteins with high affinities, and their stability
towards metabolic deglycosylation, which make these
In this work, we report that TFA and benzenesulfonic
acid are efficient, convenient and economical co-cata-
lysts for epimerization of a variety of C-nucleosides at
ambient temperature in dichloromethane (Table 1),
while benzenesulfonic acid by itself is oxidative and
Keywords: epimerization; C-nucleosides; trifluoroacetic acid; benzene-
sulfonic acid; dichloromethane.
ꢀ This work was supported by NIH grant RO1 GM56834 (J.T.S.).
* Corresponding author. Tel.: 410-502-2758; fax: 410-955-3023;
e-mail: jstivers@jhmi.edu
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)00848-7

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Y. L. Jiang, J. T. Sti6ers / Tetrahedron Letters 44 (2003) 4051–4055
a
Table 1. Epimerization of C-nucleosides with trifluoroacetic acid–benzenesulfonic acid in CH2Cl2
causes substrate decomposition and dehydration.13,14 It
was found that 2 was converted to 3 in the presence of
the combination catalysts TFA–benzenesulfonic acid
(5:1 molar ratio)16 at 40°C for 40 h to afford b isomer
3 (58%) and a isomer 2 (37%). The a isomer 2 was
retreated using identical conditions to give more b-iso-
mer 3 (22%). Thus, the combined yield of the b isomer
was 80% after two epimerizations. The total synthesis
of 2,4-difluorophenyl C-nucleoside phosphoramidite is
shown in Scheme 1.17 The results for the epimerization
of difluorophenyl and other aromatic C-nucleosides are
listed in Table 1.
19F NMR measurements suggest the formation of car-
bocation intermediate during the epimerization reaction
(Fig. 1). The chemical shifts of 1-bromo-2,4-
difluorobenzene in CDCl3 show only a small up-field
shift (0.09 ppm) when the co-catalysts were added,
indicating that these catalysts do not alter the electronic
structure of the aromatic ring. However, upon addition

Y. L. Jiang, J. T. Sti6ers / Tetrahedron Letters 44 (2003) 4051–4055
4053
indicating that the oxygen in the sugar ring is proto-
nated with the possible formation of a benzylic cation
(Scheme 2).18,19 In contrast with previous proce-
dures,13,14 no dehydration, carbonization or decomposi-
tion is observed, and the solvent CH2Cl2 is easily
removed. The yields of products are about fourfold
better than when benzenesulfonic acid alone is utilized
with toluene as the solvent at 110°C for 8 h.13 Although
an alternative epimerization method using BF3 in
nitromethane has been reported, this approach requires
high temperature.20
Scheme 1. Total synthesis of the difluorophenyl C-nucleoside
phosphoramidite. Reagents and conditions: (a) Cd(Fp)2/THF,
rt; (b) TFA–benzenesulfonic acid (5:1), CH2Cl2, 40°C, 48 h;
(c) NaOMe/MeOH, 23°C; (d) 4,4%-dimethoxytrityl chloride,
DMAP, pyridine, CH2Cl2, 23°C; (e) N,N%-diisopropyl-2-O-
cyanoethyl phosphonamidic chloride, DIPEA, CH2Cl2, 5 min,
23°C.
In conclusion, we provide an efficient new method for
the epimerization of difluorophenyl and other C-
nucleosides with electron-withdrawing substituents
using the co-catalysts TFA-benzenesulfonic acid (5:1
molar ratio) in dichloromethane. This method should
find wide application in the synthesis of such com-
pounds, which may be incorporated into DNA via
phosphoramidite chemistry for structure–activity stud-
ies in biological systems.
of the co-catalysts, the 19F NMR chemical shifts of 2
showed 0.48 and 1.15 ppm down-field shifts for the
fluorine atoms at the 2 and 4 positions, respectively,
Figure 1. 19F NMR spectra of 15 mM 1-bromo-2,4-difluorobenzene and a isomer 2 in the absence and presence of 4.1% (v/v)
combination catalyst in CDCl3 at rt (370 MHz 19F frequency). TFA was used as an internal reference for the fluorine chemical
shifts. The asterisk symbols * and ** are the 4-F and 2-F atoms of the b isomers 3, which were assigned from an authentic sample
of this isomer.
Scheme 2. Proposed mechanism for the epimerization of aromatic C-nucleosides containing fluorine in presence of the
combination catalyst TFA–benzenesulfonic acid in CH2Cl2.

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Y. L. Jiang, J. T. Sti6ers / Tetrahedron Letters 44 (2003) 4051–4055
was added 0.5 ml of the combination catalyst. The
References
homogenous mixture was sealed in a sample vial and
shaken for 40 h at 40°C, and then washed using 15 ml 5%
NaHCO3. The solvents were removed in vacuo and the
residue was purified on silica gel (ethyl acetate/hexanes,
4:96) to give 3 (0.105 g) and 2 (0.068 g), the latter was
treated again to give more 3 (0.040 g). The overall yield
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1
was 80%. H NMR (CDCl3, ppm) l 7.99 (d, 2H, J=8.4
Hz); 7.91 (d, 2H, J=8.4 Hz); 7.50 (m, 1H); 7.29 (d, 2H,
J=7.6 Hz); 7.22 (d, 2H, J=8.4 Hz); 6.81 (m, 2H); 5.61
(m, 1H); 5.47 (dd, 1H, J=5.2 Hz); 4.67 (m, 2H); 4.54 (m,
1H); 2.62 (m, 1H); 2.42 (s, 3H); 2.40 (s, 3H); 2.16 (m,
1H). 19F NMR (CDCl3, ppm) l −39.41 (2-F); −35.70
(4-F). HRMS (MALDI-FTMS) calcd for C27H24F2O5Na
(M+Na) 489.148, found 489.146.
4: To a solution of 3 (0.90 g, 1.8 mmol) in 5 ml CH2Cl2,
were added 20 ml MeOH and 0.25 ml NaOMe (in
MeOH, 25%). The mixture was stirred at room tempera-
ture for 1 h. The solvent was removed in vacuo and the
residue was purified on silica gel using EtAc then acetone
10. Krokan, H. E.; Nilsen, H.; Skorpen, F.; Otterlei, M.;
Slupphaug, G. FEBS Lett. 2000, 476, 73.
1
to give 4 (0.40 g, 95%). 4 H NMR (CDCl3, ppm) l 7.43
11. Slupphaug, G.; Mol, C. D.; Kavli, B.; Arvai, A. S.;
Krokan, H. E.; Tainer, J. A. Nature 1996, 384, 87.
12. Jiang, Y. L.; McDowell, L.; Studelska, D.; Song, F.;
Schaefer, J.; Stivers, J. T., manuscript in preparation.
13. Ren, R. X.-F.; Chaudhuri, N. C.; Paris, P. L.; Rumney,
S., IV; Kool, E. T. J. Am. Chem. Soc. 1996, 118, 7671.
14. Chaudhuri, N. C.; Ren, R. X. F.; Kool, E. T. Synlett
1997, 341.
15. Jiang, Y. L.; Stivers, J. T. Tetrahedron Lett 2003, 44, 85.
16. Combination catalyst. Trifluoroacetic acid (0.50 ml, 0.74g,
6.4 mmol) was added to benzenesulfonic acid (0.22g, 1.4
mmol). The mixture was vortexed and centrifuged to give
a clear solution (0.58 ml). The residue was found to be
about 0.06 g.
(m, 1H); 6.83 (m, 1H); 6.79 (m, 1H); 5.34 (dd, 1H, J=5.6
Hz); 4.48 (m, 1H); 4.03 (m, 1H); 3.85 (m, 1H); 3.78 (m,
1H); 2.32 (m, 0.5H); 2.30 (m, 0.5H); 2.05 (m, 1H). HRMS
(MALDI-FTMS) calcd for C11H12F2O3Na (M+Na)
253.065, found 253.065.
5: To 4 (0.200 g, 0.87 mmol) in 4 ml pyridine, were added
4-(dimethylamino)pyridine (20 mg, 0.16 mmol) and 4,4%-
dimethoxytrityl chloride (0.52 g, 1.53 mmol). If the reac-
tion did not go to completion, more 4,4%-dimethoxytrityl
chloride was needed. The reaction mixture was stirred at
rt for 1 h, and quenched by adding 20 ml 5% NaHCO3.
The solvent was removed in vacuo and the residue was
purified on silica gel (Et3NH3/EtAc/hexanes, 0.5:24.5:75)
to give 5 (0.335 g, 77%). 5 1H NMR (CDCl3, ppm) l 7.43
(m, 1H); 7.28 (m, 5H); 7.18 (d, 4H, J=10.4 Hz); 6.84 (d,
4H, J=8.8 Hz); 6.84 (m, 1H); 6.74 (m, 1H); 5.36 (m, 1H);
4.42 (m, 1H); 3.84 (m, 1H); 3.79 (s, 6H); 3.76 (m, 1H);
3.35 (m, 0.5H); 3.28 (m, 0.5H); 2.34 (m, 1H); 2.01 (m,
1H). Mass (ESI) calcd for C32H30F2O5Na (M+Na) 555,
found 555.
17. Total synthesis of 2,4-difluorophenyl C-nucleoside
phosphoramidite:
2 and 3. To dry THF (10 ml) in a two-necked round-bot-
tomed flask equipped with a condenser, magnesium turn-
ings (0.288 g, 12 mmol), 1-bromo-2,4-difluorobenzene
(1.35 ml, 12 mmol) and two crystals of iodine were added
consecutively. The mixture was stirred and heated slightly
to achieve slow reflux for 0.5 h using magnetic stirrer.
Dry cadmium chloride was then added to the above
warm solution in one portion. The mixture was heated
for a few minutes until most of the CdCl2 was dissolved.
To the reaction mixture, a suspension of 1%-a-chloro-3%,5%-
di-O-toluyl-2%-deoxyribose (3.0 g, 7.8 mmol) in 10 ml
THF suspension were added dropwise at rt over 10 min.
The mixture was stirred at 40°C for 2 h, and then rt
overnight. The solvent was removed in vacuo and the
residue was purified on silica gel (EtAc/hexanes, 4:96) to
give 2 (1.82 g, 76%) and 3 (0.24 g, 11%). 2 1H NMR
(CDCl3, ppm) l 7.97 (d, 2H, J=8.0 Hz); 7.66 (d, 2H,
J=8.0 Hz); l 7.56 (m, 1H); 7.26 (d, 2H, J=7.2 Hz); 7.18
(d, 2H, J=7.6 Hz); 6.89 (m, 1H); 6.80 (m, 1H); 5.60 (dd,
1H, J=3.2 Hz); 5.56 (m, 1H); 4.72 (m, 1H); 4.56 (m, 2H);
2.44 (m, 1H); 2.42 (s, 3H); 2.39 (s, 3H); 2.30 (m, 0.5H);
2.27 (m, 0.5H). 19F NMR (CDCl3, ppm) l −38.43 (2-F);
−36.33 (4-F). HRMS (MALDI-FTMS) calcd for
C27H24F2O5Na (M+Na) 489.148, found 489.146.
3: In addition, 3 can be prepared by epimerization of 2.
To a solution of 2 (0.180 g, 0.37 mmol) in 2 ml CH2Cl2,
6: To 5 (0.320 g, 0.58 mmol) in 4 ml CH2Cl2 and 0.44 ml
N,N-diisopropylethylamine, was added 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (0.22 ml, 0.96
mmol) in a dropwise fashion. The reaction mixture was
stirred at rt for 5 min, and quenched by adding 5 ml 5%
NaHCO3. The solvent was removed in vacuo and the
residue was purified on silica gel (Et3NH3/EtAc/hexanes,
2:25:73) to give 6 (0.39 g, 90%). 6 1H NMR (CDCl3,
ppm) l 7.21–7.54 (m, 12H); 6.81 (m, 4H); 5.35 (m, 1H);
4.52 (m, 1H); 4.21 (m, 1H); 3.83 (m, 1H); 3.79 (6H, s);
3.69 (m, 1H); 3.60 (m, 2H); 3.29 (m, 2H); 2.60 (m, 1H);
2.46 (m, 2H); 1.95 (m, 1H); 1.16 (m, 12H). 31P NMR
(CDCl3, ppm) l 149.36 (s) and 148.90 (s). Mass (ESI)
calcd for C41H47F2N2O6P (M+H, M+Na and M+Cl,)
733, 755 and 767, found 733, 755 and 767, respectively.
18. Frenking, G.; Fau, S.; Marchand, C. M.; Grutamacher,
H. J. Am. Chem. Soc. 1997, 119, 6648.
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Clercq, E.; Knaus, E. E. Nucleosides Nucleotides Nucleic
Acids 2001, 20, 11.

Y. L. Jiang, J. T. Sti6ers / Tetrahedron Letters 44 (2003) 4051–4055
4055
1
21. 9a: 1H NMR (CDCl3, ppm) l 7.97 (d, 2H, J=8.0 Hz);
7.81 (d, 2H, J=8.4 Hz); 7.41 (m, 2H); 7.25 (d, 2H, J=8.0
Hz); 7.20 (d, 2H, J=8.4 Hz); 7.04 (m, 2H); 5.59 (m, 1H);
5.34 (m, 1H); 4.69 (m, 1H); 4.57 (m, 2H); 2.93 (m, 1H);
2.41 (s, 3H); 2.40 (s, 3H); 2.26 (m, 1H). 19F NMR
(CDCl3, ppm) l −38.34. HRMS (MALDI-FTMS) calcd
for C27H25FO5Na (M+Na) 471.158, found 471.156.
23. 12a: H NMR (CDCl3, ppm) l 7.98 (d, 2H, J=8.0 Hz);
7.79 (d, 2H, J=8.4 Hz); 7.62 (d, 1H, J=7.2 Hz); 7.12–
7.25 (m, 7H); 5.62 (m, 1H); 5.51 (t, 1H, J=6.8 Hz); 4.77
(m, 1H); 4.59 (m, 2H); 3.00 (m, 1H); 2.41 (s, 3H); 2.39 (s,
3H); 2.39 (s, 3H); 2.30 (s, 3H), 2.15 (m, 1H). HRMS
(MALDI-FTMS) calcd for C28H28O5Na (M+Na)
467.183, found 467.182.
1
22. 9b: 1H NMR (CDCl3, ppm) l 7.99 (d, 2H, J=8.4 Hz);
7.94 (d, 2H, J=8.0 Hz); 7.38 (m, 2H); 7.29 (d, 2H, J=7.6
Hz); 7.24 (d, 2H, J=8.0 Hz); 7.00 (m, 2H); 5.61 (d, 1H,
J=6.0 Hz); 5.23 (m, 1H); 4.65 (m, 2H); 4.53 (m, 1H);
2.54 (m, 1H); 2.44 (s, 3H); 2.41 (s, 3H); 2.19 (m, 1H). 19F
NMR (CDCl3, ppm) l −37.38. HRMS (MALDI-FTMS)
calcd for C27H25FO5Na (M+Na) 471.158, found 471.156.
24. 12b: H NMR (CDCl3, ppm) l 7.99 (d, 2H, J=8.4 Hz);
7.95 (d, 2H, J=8.0 Hz); 7.58 (m, 1H); 7.12–7.28 (m, 7H);
5.61 (d, 1H, J=4.4 Hz); 5.44 (m, 1H); 4.68 (m, 2H);
4.53 (m, 1H); 2.59 (m, 1H); 2.44 (s, 3H); 2.39 (s, 3H); 2.39
(s, 3H); 2.35 (s, 3H), 2.15 (m, 1H). HRMS (MALDI-
FTMS) calcd for C28H28O5Na (M+Na) 467.183, found
467.182.