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2. O’Donnell, M. J.; McLaughlin, L. W. In Bioorganic Chemistry; Nucleic Acids; Hecht,
S. M., Ed.; Oxford: NewYork, 1996; pp 216–243.
3. Liu, L.; Li, Y.; Liotta, D.; Lutz, S. Nucleic Acids Res. 2009, 37, 4472.
4. Jameson, D. M.; Eccleston, J. F.. In Methods in Enzymology; Brand, L., Johnson, M.
L., Eds.; Academic: San Diego, 1997; Vol. 278, pp 363–390.
5. Rist, M. J.; Marino, J. P. Curr. Org. Chem. 2002, 6, 775.
6. Kool, E. T. Acc. Chem. Res. 2002, 35, 936.
7. Berry, D. A.; Jung, K. Y.; Wise, D. S.; Sercel, A. D.; Pearson, W. H.; Mackie, H.;
Randolph, J. B.; Somers, R. L. Tetrahedron Lett. 2004, 45, 2457.
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the substrate. Similar binding effects, albeit this time in favor of the
substrate, could explain the three-fold gain in activity for 4, the
fluorescent version of d4T. Unexpectedly, pyrrolo analog 5 shows
a 40-fold drop in activity relative to the non-fluorescent ddC, a
trend opposite to the slight gain observed with its 20-deoxyribosoyl
analog 2. Conformational differences in the sugar moiety are unli-
kely to account for the differences as 3 possesses the same 20,30-
dideoxyribosyl portion, yet shows 10-fold higher activity. Future
studies exploring the conformational preferences of furano and
pyrrolo-pyrimidines in regard to their syn-anti orientation might
assist in rationalizing their difference in performance.
In summary, the application of fluorescent nucleoside analogs
as molecular probes provides a new tool for studying the uptake
and metabolism of antiviral prodrugs as demonstrated in our lab-
oratory and by other research groups.3,17,18 Herein, we have shown
that nucleoside analogs with furano and pyrrolo modification are
synthetically readily accessible and can serve as substrates for
the type-I 20-deoxyribo-nucleoside kinase from Drosophila melano-
gaster. In bacterial and mammalian cell cultures, these fluorescent
analogs have shown no unusual cytotoxicity, hence making them
suitable reporters to study cellular uptake and phosphorylation,
as well as to evaluate large combinatorial libraries of kinases for
variants with substrate specificity for the corresponding prodrugs
by fluorescence activated cell sorting (FACS).
9. Robins, M. J.; Vinayak, R. S.; Wood, S. G. Tetrahedron Lett. 1990, 31, 3731.
10. Compound 4: UV (H2O) kmax 331 nm; IR (cmꢀ1) 3440 (br), 3195, 1670, 1642; 1
H
NMR (400 MHz, DMSO-d6) d 2.29 (s, 3H), 3.61 (m, 2H), 4.88 (s, 1H), 5.05 (t, 1H,
J = 5.6 Hz), 6.01 (d, 1H, J = 6.0 Hz), 6.38 (m, 2H), 6.95 (s, 1H), 8.53 (s, 1H); 13C
NMR (100 MHz, DMSO-d6) d 14.27, 62.78, 88.88, 92.61, 100.95, 107.50, 127.22,
135.24, 138.19, 154.98, 155.60, 172.18; HRMS (FAB) m/z 271.0684, calcd for
C12H12O4N2Na 271.0689 (M+Na).
11. Woo, J. S.; Meyer, R. B.; Gamper, H. B. Nucleic Acids Res. 1996, 24, 2470.
12. Compound 5: UV (H2O) kmax 335 nm; IR (cmꢀ1) 3350 (br), 1670, 1560, 1090; 1
H
NMR (400 MHz, CDCl3/CD3OD) d 1.91–1.93 (m, 2H), 2.18 (m, 1H), 2.41(s, 3H),
2.54 (m, 1H) 3.82 (dd, 1H, J = 4.0, 12 Hz), 4.08 (dd, 1H, J = 2.8, 12.4 Hz), 4.26 (m,
1H), 4.80 (d, 1H, J = 1.6 Hz), 6.22 (dd, 1H, J = 2.4, 6.8 Hz), 8.44 (s, 1H); 13C NMR
(100 MHz, CDCl3) d 13.5, 24.2, 33.9, 62.5, 83.2, 88.8, 97.9, 110.6, 134.9, 138.6,
155.5, 158.6; HRMS (FAB) m/z 250.1183, calcd for C12H16O3N3 250.1186 (M+H).
13. McGuigan, C.; Carangio, A.; Snoeck, R.; Andrei, G.; De Clercq, E.; Balzarini, J.
Nucleosides Nucleotides Nucleic Acids 2004, 23, 1.
14. Czernecki, S.; Valery, J. M. Synthesis 1991, 239.
15. Compound 6 was crystallized from ethyl acetate. UV (H2O) kmax 331 nm; IR
(cmꢀ1) 3469 (br), 2839, 1667, 1638, 1573, 1482; 1H NMR (400 MHz, CDCl3) d
2.27 (d, 3H, J = 7.2 Hz), 2.30–2.36 (m, 1H), 2.57–2.64 (m, 1H), 3.29 (br, 1H),
3.71–3.75 (m, 1H), 3.90–3.95 (m, 2H), 4.18 (dd, 1H, J = 6.8, 13.2 Hz), 6.10–6.14
(m, 1H), 8.65 (s, 1H); 13C NMR (100 MHz, CDCl3) d 14.1, 39.2 58.6, 60.5, 85.7,
88.0, 100.1, 108.4, 136.2, 155.2, 156.2, 171.9; HRMS (FAB) m/z 292.1036, calcd
for C12H14O4N5 292.1039 (M+H).
Acknowledgements
16. Compound 7: UV (H2O) kmax 335 nm; IR (cmꢀ1) 3350 (br), 1670, 1560, 1090; 1
H
NMR (600 MHz, DMSO-d6) d 2.20 (m, 1H), 2.33 (s, 3H), 2.73 (m, 1H), 3.66 (m,
2H), 4.33 (dt, 1H, J = 25.8 Hz), 5.32 (dd, 1H, J = 56.4 Hz), 6.22 (q, 1H), 6.44 (s,
1H), 8.60 (s, 1H). 13C NMR (150 MHz, DMSO-d6) d 13.6, 60.6, 86.2, 87.6, 95.4,
100.4, 106.8, 136.5, 154.3, 171.56. 19F NMR (376 MHz, DMSO-d6) d ꢀ174.9;
HRMS (FAB) m/z 269.0932, calcd for C12H14O4N2F1 269.0932 (M+H).
This work was supported in part by the National Institutes of
Health (GM69958), as well as by a grant to the Emory Center for
AIDS Research (AI050409) from the National Institutes of Health
and by institutional funding from the Emory University Health Sci-
ence Center.
17. Patching, S. G.; Baldwin, S. A.; Baldwin, A. D.; Young, J. D.; Gallagher, M. P.;
Henderson, P. J.; Herbert, R. B. Org. Biomol. Chem. 2005, 462.
18. Zhang, J.; Sun, X. J.; Smith, K. M.; Visser, F.; Carpenter, P.; Barron, G.; Peng, Y. S.;
Robins, M. J.; Baldwin, S. A.; Young, J. D. Biochemistry 2006, 1087.
References and notes
1. Hermanson, G. T. Bioconjugate Tech.; Academic: San Diego, 2008.