Efficient epimerization of pyrene and other aromatic C-nucleosides with trifluoroacetic acid in dichloromethane

Article abstract of DOI:10.1016/S0040-4039(02)02481-4

Trifluoroacetic acid has been found to be an efficient and non-oxidative catalyst for the epimerization of pyrene and other C-nucleosides in dichloromethane at ambient temperature. Aspects of the epimerization mechanism are also elucidated.

Full text of DOI:10.1016/S0040-4039(02)02481-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 85–88
Efficient epimerization of pyrene and other aromatic
C-nucleosides with trifluoroacetic acid in dichloromethaneꢀ
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 18 September 2002; revised 30 October 2002; accepted 5 November 2002
Abstract—Trifluoroacetic acid has been found to be an efficient and non-oxidative catalyst for the epimerization of pyrene and
other C-nucleosides in dichloromethane at ambient temperature. Aspects of the epimerization mechanism are also elucidated.
© 2002 Elsevier Science Ltd. All rights reserved.
Recently, non-polar nucleoside analogs have attracted
much interest as probes of protein-nucleic acid recogni-
tion, because they lack the hydrogen bond donor–
acceptor groups of the natural DNA bases, yet mimic
the shape of natural bases or even base pairs. Impor-
tantly, the pioneering work of Kool and colleagues has
shown that DNA polymerase will specifically incorpo-
rate a pyrene (Y) nucleoside triphosphate (dYTP)
opposite to DNA sites that lack bases, confirming that
steric complimentarity is an important component of
high fidelity DNA replication.1,2 In our laboratory,
pyrene has been found to be a very useful tool in the
studies of DNA base flipping by uracil DNA glycosyl-
ase (UDG), and the rational engineering of a DNA
glycosylase that is specific for an unnatural C:Y base
pair.4 In general, placement of a pyrene nucleotide
opposite to a damaged base in duplex DNA forces the
base into an extrahelical conformation and enhances
enzymatic damage recognition. The flipping of DNA
bases and chemical rescues of base flipping mutations
of enzymes by pyrene have also been reported.3–6
We report here that trifluoroacetic acid is a very effec-
tive catalyst for the epimerization of a variety of C-
nucleosides in dichloromethane solvent. Trifluoroacetic
acid has been found to be a catalyst for the isomeriza-
tion of indolo[2,3-a]quinolizidine derivatives,9 3-indolyl
sulfides to 2-indolyl sulfides10,11 and a-aryloxymethyl-
cinnamic acids.12 Most importantly, trifluoroacetic acid
is non-oxidative but it is also a strong acid. Typically,
to a solution of a-isomer (0.180 g, 0.32 mmol) in 6 ml
dichloromethane, were added trifluoroacetic acid (60
ml). The mixture was stirred for 18 h at room tempera-
ture. The reaction was quenched by adding onefold
excess of triethylamine (200 ml). The solvents were
removed in vacuo and the residue was purified on silica
gel (acetone/CH2Cl2/hexanes, 3:9:88) to give b-isomer
Although pyrene nucleoside phosphoramidite and
related C-nucleosides can be prepared as described
previously (Scheme 1), the yield is very low in the
epimerization step. The reaction requires high tempera-
ture and the oxidative catalysts benzenesulfonic acid
and concentrated sulfuric acid, causing dehydration
and carbonization side reactions.7,8
Scheme 1. Published synthesis of pyrene nucleoside phospho-
ramidite and the yield at each step.7,8 (a) CdY2/THF/reflux;
(b) benzenesulfonic acid–H2SO4–H2O–toluene, reflux, 6 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 23°C.
Keywords: epimerization; pyrene nucleoside; C-nucleosides; trifluoro-
acetic acid and 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 © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02481-4

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Y. L. Jiang, J. T. Sti6ers / Tetrahedron Letters 44 (2003) 85–88
(0.124 g) and a-isomer (0.052 g). The a-isomer was
resubjected to identical conditions to give more b-iso-
mer (0.033 g). The combined yield of b-isomer was 87%
after two epimerization steps.
observed, and the solvent CH2Cl2 was easily removed.
The yields of products were increased by more than
twofold.7,8 Trifluoroacetic acid is also an effective cata-
lyst for the reverse epimerization of the b-isomer to the
a-isomer (Fig. 1). Therefore, the epimerization is
reversible and the equilibrium constant K is 2.3 (ka=
2.3×10−4 s−1 and kb=1.0×10−4 s−1). We note that the
relative rates of epimerization in Table 1, and the
optimal concentrations of TFA that are required for
efficient epimerization, are likely related to the electron
donating ability of the aromatic group, which would
serve stabilize the proposed carbocation intermediate
(see below). We routinely convert these C-nucleosides
The results for epimerization of pyrene and other aro-
matic C-nucleosides are listed in Table 1. Our goal was
to make these C-nucleosides by the most efficient and
economical method for common use. These results
show the effectiveness and convenience of using tri-
fluoroacetic acid in CH2Cl2 at room temperature for this
epimerization. In contrast with previous procedures,7,8
no dehydration, carbonization or decomposition was
a
Table 1. Epimerization of C-nucleosides with trifluoroacetic acid (TFA) in CH2Cl2

Y. L. Jiang, J. T. Sti6ers / Tetrahedron Letters 44 (2003) 85–88
87
did not quench the fluorescence of pyrene itself. The
selective fluorescence quenching of the pyrene
nucleoside isomers may directly or indirectly reflect the
protonation of the oxygen in the sugar ring. This is a
key step in the epimerization process, allowing the
benzylic cation of pyrene to be formed (Scheme 2).8
In conclusion, we have shown an efficient new method
for the epimerization of pyrene and other air-sensitive
C-nucleosides using dichloromethane solvent and trifl-
uoroacetic acid catalyst. This procedure makes these
very useful compounds readily accessible in high yields,
and should find wide application in the studies which
incorporate these analogs into DNA via phospho-
ramidite chemistry.
Figure 1. Reaction curves of the epimerization of pyrene
nucleosides (52 mM) either from a-isomer to b-isomer (lower
panel) or from b-isomer to a-isomer (upper panel) in CH2Cl2
in the presence of 1% trifluoroacetic acid and at room temper-
ature. For each point, 20 mL sample was taken from reaction
mixture and immediately quenched by 40 ml 5% triethylamine
in CH2Cl2. After evaporation to dryness using a N2 stream,
the sample was dissolved in 0.6 ml CDCl3 and analyzed 1H
NMR spectroscopy (400 MHz). The relative amounts of
a-isomer and b-isomer were quantitated by integration of
characteristic NMR peaks over time. The curves are single
exponential fits to the data (kobsd=ka+kb). The equations are
K=kb/ka and kb=kobsd/(1/K+1).13
Acknowledgements
This work was supported by National Institutes of
Health Research Grant RO1GM56834 (J.T.S.).
References
1. Matray, T. J.; Kool, E. T. Nature 1999, 399, 704.
2. Morales, J. C.; Kool, E. T. Nature Struct. Biol. 1998, 5,
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to the corresponding nucleoside phosphoramidites for
use in solid phase DNA synthesis and ultimately bio-
logical studies in our laboratory.3–6
4. Stivers, J. T.; Kwon, K.; Jiang, Y. L. Biophys. J. 2002,
82, 595.
Other aliphatic acids and solvents were also tested for
the epimerization of pyrene C-nucleoside. No reaction
was observed using formic acid or acetic acid, and the
reaction was very slow using the popular DNA synthe-
sis reagent trichloroacetic acid or dichloroacetic acid.
No epimerization was observed using chloroform, tolu-
ene, THF, ethyl acetate, acetone, methanol or ethanol
as the solvent. The poor ability of TCA to catalyze
epimerization is fortuitous given the wide use of this
reagent in solid-phase DNA synthesis.
5. Jiang, Y. L.; Drohat, A. C.; Ichikawa, Y.; Stivers, J. T. J.
Biol. Chem. 2002, 277, 15385.
6. Jiang, Y. L.; Stivers, J. T.; Song, F. Biochemistry 2002,
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S., IV; Kool, E. T. J. Am. Chem. Soc. 1996, 118, 7671.
8. Chaudhuri, N. C.; Ren, R. X. F.; Kool, E. T. Synlett
1997, 341.
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H.; Tolvanen, A. Tetrahedron 1998, 54, 10205.
10. Hamel, P.; Girard, Y.; Atkinson, J. G. J. Org. Chem.
1992, 57, 2694.
The addition of trifluoroacetic acid to CH2Cl2 was
found to dramatically quench the pyrene fluorescence
of the a and b isomers. Surprisingly, trifluoroacetic acid
11. Hamel, P.; Girard, Y.; Atkinson, J. G. J. Chem. Soc.,
Chem. Commun. 1989, 63.
Scheme 2. Proposed pathway for the epimerization of aromatic C-nucleoside by CF3COOH in CH2Cl2.

88
Y. L. Jiang, J. T. Sti6ers / Tetrahedron Letters 44 (2003) 85–88
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13. Sato, Y.; Tateno, G.; Seio, K.; Sekine Eur. J. Org.
J=5.2 Hz); 4.70 (m, 2H); 4.59 (m, 1H); 2.59 (m, 1H);
2.44 (s, 3H); 2.39 (s, 3H), 2.37 (m, 1H). HRMS
(MALDI-FTMS) calcd for C32H30O6Na (M+Na)
533.193, found 533.193.
Chem. 2002, 87.
16. 7a: 1H NMR (CDCl3, ppm) l 8.63 (d, 2H, J=9.2 Hz);
8.45 (s, 1H); 8.03, 7.47, 7.25 (m, 12H); 7.90 (d, 2H,
J=8.0 Hz); 6.80 (m, 1H); 5.88 (m, 1H); 4.89 (m, 1H);
4.69 (m, 1H); 4.50 (m, 1H); 2.81 (m, 1H); 2.45 (s, 3H);
2.40 (s, 3H); 2.35 (m, 1H). HRMS (MALDI-FTMS)
calcd for C35H30O5Na (M+Na) 553.198, found 553.197.
17. 7b: 1H NMR (CDCl3, ppm) l 8.68 (d, 2H, J=9.2 Hz);
8.44 (s, 1H); 8.10, 7.42, 7.25 (m, 14H); 6.66 (dd, 1H,
J=12 Hz, J=6.0 Hz); 5.91 (m, 1H); 4.92 (m, 2H); 4.64
(m, 1H); 2.58 (m, 1H); 2.44 (s, 3H); 2.40 (s, 3H);
2.35 (m, 1H). HRMS (MALDI-FTMS) calcd for
C35H30O5Na (M+Na) 553.198, found 553.197.
14. 5a: 1H NMR (CDCl3, ppm) l 7.99 (d, 2H, J=8.0 Hz);
7.82 (s, 1H); 7.68–7.72 (m, 3H); 7.50 (d, 1H, J=8.0 Hz);
7.26 (d, 2H, J=8.4 Hz); 7.14 (m, 2H); 7.09 (d, 2H,
J=8.0 Hz); 5.65 (m, 1H); 5.50 (m, 1H); 4.76 (m, 1H);
4.60 (m, 2H); 3.93 (s, 3H); 3.00 (m, 1H); 2.41 (s, 3H);
2.40 (m, 1H); 2.35 (s, 3H). HRMS (MALDI-FTMS)
calcd for C32H30O6Na (M+Na) 533.193, found 533.193.
15. 5b: 1H NMR (CDCl3, ppm) l 8.01 (d, 2H, J=8.0 Hz);
7.98 (d, 2H, J=7.6 Hz); 7.79 (s, 1H); 7.71 (d, 1H,
J=8.4 Hz); 7.64 (d, 1H, J=8.8 Hz); 7.48 (d, 1H, J=8.4
Hz); 7.30 (d, 2H, J=7.6 Hz); 7.22 (d, 2H, J=8.4 Hz);
7.11 (m, 2H); 5.66 (m, 1H); 5.40 (dd, 1H, J=10.8 Hz,