656
P. K. Mandal et al. / Bioorg. Med. Chem. Lett. 17 (2007) 654–656
rescence polarization assay.19 Peptide 10a, containing
Ser(CONH2), exhibited an IC50 of 379 nM, which was
approximately 3-fold less active than peptide 3. Interest-
ingly, peptide 10b was 6-fold less active.
5. Yu, H.; Jove, R. Nat. Rev. Cancer 2004, 4, 97.
6. Stahl, N.; Farruggella, T. J.; Boulton, T. G.; Zhong, Z.;
Darnell, J. E.; Yancopoulos, G. D. Science 1995, 267,
1349.
7. Gerhartz, C.; Heesel, B.; Sasse, J.; Hemmann, U.; Landg-
raf, C.; Schneider-Mergener, J.; Horn, F.; Heinrich, P. C.;
Graeve, L. J. Biol. Chem. 1996, 271, 12991.
8. Wiederkehr-Adam, M.; Ernst, P.; Muller, K.; Bieck, E.;
Gombert, F. O.; Ottl, J.; Graff, P.; Grossmuller, F.; Heim,
M. H. J. Biol. Chem. 2003, 278, 16117.
9. Skinner, C. G.; McCord, T. J.; Ravel, J. M.; Shive, W.
J. Am. Chem. Soc. 1956, 78, 412.
10. DeWald, H. A.; Craft, M. K.; Nicolaides, E. D. J. Med.
Chem. 1963, 6, 741.
11. Donaldson, V. H.; Ratnoff, O. D. Proc. Soc. Exp. Biol.
Med. 1967, 125, 145.
12. Miller, K.E. PCT Int. Appl. 2003 WO 2003022261.
13. Cho, C. Y.; Moran, E. J.; Cherry, S. R.; Stephans,
J. C.; Fodor, S. P. A.; Adams, C. L.; Sundaram,
A.; Jacobs, J. W.; Schultz, P. G. Science 1993, 261,
1303.
14. Moran, E. J.; Wilson, T. E.; Cho, C. Y.; Cherry, S. R.;
Schultz, P. G. Biopolymers (Peptide Science) 1995, 37,
213.
Previously, we showed that replacing the side-chain
amide protons of glutamine with one or two methyl
groups severely impaired binding of phosphopeptides
to Stat3.19 Furthermore, replacing Gln with isosteric
methionine sulfoxide caused a 20-fold reduction in
affinity. Taken together with the observation that
Gln at pY+3 is a specificity determinant for binding
to Stat3, these results suggest that the side-chain
amide protons form important hydrogen bonds with
groups on the protein. It is unclear whether the loss
in affinity of 10a is due to reduced hydrogen bonding,
slightly altered geometry of the OC(O)NH2 group rel-
ative to the CH2C(O)NH2 of 3, increased energy of
de-solvation of the carbamate group, or other factors.
However, the 6-fold loss in affinity of 10b suggests
that the side chain of Gln resides in a tight pocket
or cleft, and that the b-methyl group of Thr(CONH2)
results in steric clash with this site.
15. Paikoff, S. J.; Wilson, T. E.; Cho, C. Y.; Schultz, P. G.
Tetrahedron Lett. 1996, 37, 5653.
16. Alsina, J.; Rabanal, F.; Chiva, C.; Giralt, E.; Albericio, F.
Tetrahedron 1998, 54, 10125.
17. Fernandez-Forner, D.; Huerta, J. M.; Ferrer, M.; Casals,
G. R.; Ryder, H.; Giralt, E.; Albericio, F. Tetrahedron
Lett. 2002, 43, 3543.
18. Ren, Z.; Cabell, L. A.; Schaefer, T. S.; McMurray, J. S.
Biorg. Med. Chem. Lett. 2003, 13, 633.
19. Coleman, D. R., IV; Ren, R.; Mandal, P. K.; Cameron, A.
G.; Dyer, G. A.; Muranjan, S.; Chen, X.; McMurray, J. S.
J. Med. Chem. 2005, 48, 6661.
In summary, we have synthesized peptides containing
Ser(CONH2) and Thr(CONH2) using solid-phase tech-
niques. As glutamine analogues, these amino acids re-
duce affinity of a peptide for the SH2 domain of Stat3,
which requires Gln at the pY+3 residue. There are cur-
rently no X-ray crystallographic or NMR structures of
Stat3 complexed with Y(p)XXQ peptides that would re-
veal phosphopeptide–protein interactions. Thus, the re-
duced activity of the threonine analogue provides insight
into the binding pocket for Gln.
20. 3 ESI-MS calcd, 671.27; found, 671.35. Analytical C18
HPLC TR (a) 18.02 min 0–40% ACN/30 min (both
solvents with 0.1% TFA) (b) 15.28 min 0–40% ACN in
0.01 M NH4OAc.
21. Compound 7a white solid mp 167–169 ꢁC. 1H NMR
(300 MHz) CHCl3d 3.6–3.67 (m, 2H), 4.19–4.24 (m, 2H),
4.34–4.45 (m, 2H), 4.54 (s, 2H), 5.24 (br s, 1H), 7.25–7.42
(m, 11H), 7.58 (d, J = 7.5 Hz, 2H), 7.76 (d, J = 7.5 Hz,
2H), 8.22 (d, J = 7.5 Hz, 2H). ESI-MS calcd, 581.18;
found, 582.30. Compound 7b white solid mp 193–195 ꢁC.
1H NMR (300 MHz) CHCl3d 1.37 (d, J = 6.6 Hz, 3H),
4.22 (t, J = 6.3 Hz, 1H), 4.42–4.6 (m, 5H), 5.48 (m, 1H),
5.61 (d, J = 8.1 Hz, 1H), 6.48 (br s, 1H), 7.28–7.45 (m,
12H), 7.59 (d, J = 7.2 Hz, 2H), 7.78 (d, J = 7.5 Hz, 2H),
8.26 (d, J = 9.3 Hz, 2H). ESI-MS calcd, 595.20; found,
596.40.
Acknowledgments
We are grateful to the National Cancer Institute
(CA096652) and the MDACC University Cancer Fund
for support of this work. Funding as an Odyssey Fellow
(Z.R.) was supported by the Odyssey Program and the
Cockrell Foundation Award for Scientific Achievement
at UTMDACC. We also acknowledge the NCI Cancer
Center Support Grant CA016672 for the support of
our NMR facility and the Peptide Synthesis Core (Dr.
Martin Campbell) that provided mass spectrometry.
22. Szardenings, A. K.; Gordeev, M. F.; Patel, D. V.
Tetrahedron Lett. 1996, 37, 3635.
References and notes
23. Compound 10a ESI-MS calcd, 673.25; found, 673.32.
Analytical C18 HPLC TR (a) 15.58 min, 0–40% ACN/
30 min (both solvents with 0.1% TFA) (b) 13.85 min, 0–
40% ACN in 0.01 M NH4OAc. Compound 10b ESI-MS
calcd, 687.27; found, 687.28. Analytical C18 HPLC TR (a)
19.07 min, 0–40% ACN/30 min (both solvents containing
0.1% TFA) (b) 13.85 min, 0–40% ACN in 0.01 M
NH4OAc.
1. Bowman, T.; Garcia, R.; Turkson, J.; Jove, R. Oncogene
2000, 19, 2474.
2. Buettner, R.; Mora, L. B.; Jove, R. Clin. Cancer Res. 2002,
8, 945.
3. Bromberg, J. J. Clin. Invest. 2002, 109, 1139.
4. Darnell, J. E., Jr. Nat. Rev. Cancer 2002, 2, 740.