1954
R. E. Bremer et al. / Bioorg. Med. Chem. 8 (2000) 1947±1955
1H), 7.17 (s, 4H), 7.12 (s, 1H), 7.02 (m, 6H), 6.95 (s, 1H),
6.90 (s, 1H), 6.82 (s, 1H), 3.95 (s, 3H), 3.31 (m, 2H), 3.20
(s, 2H), 3.06 (quartet, 2H, J=5.6 Hz), 2.93 (quintet, 2H,
J=4.9 Hz), 2.69 (d, 6H, J=4.5 Hz), 2.2 (br m, 4H), 1.95
(m, 2H), 1.64 (m, 2H); MALDI-TOF-MS (monoiso-
topic) [M+H] 1138.5, 1138.5 calcd for C52H60N21 O+10.
ImDsDsDs-(R)NH2g-DsDsDsDs-b-Dp (700 nmol) was
functionalized with EDTA as described32 (68 nmol, 9.7%
recovery) MALDI-TOF-MS (monoisotopic) [M+ H]
1412.7, 1412.6 calcd for C62H74N23O+17.
Bristol-Myers Squibb and the Ralph M. Parsons Foun-
dation for predoctoral fellowships to R.E.B., the Howard
Hughes Medical Institute for a predoctoral fellowship to
E.E.B., and the National Institutes of Health for a
research service award to J.W.S.
References and Notes
1. Arcamone, F.; Penco, S.; Prezzi, P. G.; Nicolella, V.; Pirelli,
A. Nature 1964, 203, 1064.
2. Zasedatelev, A. S.; Gursky, G. V.; Zimmer, C.; Thrum, H.
Mol. Biol. Rep 1974, 1, 337.
3. Krylov, A. S.; Grokhovsky, S. L.; Zasedatelev, A. S.;
Zhuze, A. L.; Gursky, G. V.; Gottikh, B. P. Nucleic Acids Res.
1979, 6, 289.
4. Van Dyke, M. W.; Hertzberg, R. P.; Dervan, P. B. Proc.
Natl. Acad. Sci. USA 1982, 79, 5470.
5. Van Dyke, M. W.; Dervan, P. B. Nucleic Acids Res. 1983,
11, 5555.
6. Fox, K. R.; Waring, M. J. Nucleic Acids Res. 1984, 12, 9271.
7. Schultz, P. G.; Taylor, J. S.; Dervan, P. B. J. Am. Chem.
Soc. 1982, 104, 6861.
8. Dervan, P. B. Science 1986, 232, 464.
9. Kopka, M. L.; Yoon, C.; Goodsell, D.; Pjura, P.; Dick-
erson, R. E. J. Mol. Biol. 1985, 183, 553.
10. Coll, M.; Frederick, C. A.; Wang, A. H. J.; Rich, A. Proc.
Natl. Acad. Sci. USA 1987, 84, 8385.
11. Klevit, R. E.; Wemmer, D. E.; Reid, B. R. Biochemistry
1986, 25, 3296.
12. Pelton, J. G.; Wemmer, D. E. Biochemistry 1988, 27, 8088.
13. Pelton, J. G.; Wemmer, D. E. Proc. Natl. Acad. Sci. USA
1989, 86, 5723.
14. Chen, X.; Ramakrishnan, B.; Rao, S. T.; Sundaralingam,
M. Nat. Struct. Biol. 1994, 1, 169.
15. Dervan, P. B.; Burli, R. W. Curr. Opin. Chem. Biol. 1999,
3, 688.
16. Wade, W. S.; Mrksich, M.; Dervan, P. B. J. Am. Chem.
Soc. 1992, 114, 8783.
ImPyPyPy-(R)EDTAꢀ-PyPyPyPy-ꢁ-Dp (1-E). ImPyPy
Py-(R)NH2g-PyPyPyPy-b-PAM-resin was synthesized in
a stepwise fashion by manual solid-phase methods from
Boc-b-PAM-resin (600 mg, 0.75 mmol/g).30 A sample of
polyamide resin (350mg, 0.44mmol/g) was cleaved with
dimethylaminopropylamine (2 mL, 55ꢁC, 18h). The crude
polyamide was diluted to 8 mL with 0.1% (w/v) TFA then
puri®ed by reverse phase HPLC. ImPyPyPy-(R)NH2g-
PyPyPyPy-b-Dp was recovered upon lyophilization of
the appropriate fractions as a white powder (25.3 mg,
20.5 mmol, 13.3% recovery); UV (H2O) lmax 312
1
(66,000); H NMR (DMSO-d6): d 10.40 (s, 1H), 9.93 (s,
1H), 9.92 (s, 1H), 9.89 (s, 2H), 9.87 (s, 2H), 9.75 (br s,
1H), 8.05 (m, 1H), 7.97 (m, 1H), 7.84 (t, 1H, J=5.4 Hz),
7.35 (s, 1H), 7.24 (d, 1H, J=0.9 Hz), 7.20 (s, 2H), 7.16
(s, 1H), 7.14 (s, 2H), 7.12 (m, 2H), 7.01 (s, 2H), 6.99 (s,
2H), 6.92 (s, 1H), 6.83 (s, 1H), 6.78 (s, 1H), 3.94 (s, 3H),
3.80 (s, 12H), 3.75 (m, 9H), 3.23 (m, 5H), 3.00 (m, 2H),
2.23 (m, 2H), 2.16 (m, 2H), 2.04 (m, 2H), 2.03 (s, 6H),
1.42 (m, 2H); MALDI-TOF-MS (monoisotopic) [M+H]
1236.7, 1236.6 calc'd for C59H74N21O+10. ImPyPyPy-
(R)NH2g-PyPyPyPy-b-Dp (6.4 mg, 5.2 mmol) was func-
tionalized with EDTA as described32 (3.5 mg, 2.3 mmol,
43% recovery) MALDI-TOF-MS (monoisotopic) [M+
H] 1510.9, 1510.7 calcd for C69H88N23O+17.
17. White, S.; Szewczyk, J. W.; Turner, J. M.; Baird, E. E.;
Dervan, P. B. Nature 1998, 391, 468.
18. White, S.; Baird, E. E.; Dervan, P. B. Chem. Biol. 1997, 4,
569.
19. Mrksich, M.; Wade, W. S.; Dwyer, T. J.; Geierstanger, B.
H.; Wemmer, D. E.; Dervan, P. B. Proc. Natl. Acad. Sci. USA
1992, 89, 7586.
20. Kielkopf, C. L.; Baird, E. E.; Dervan, P. D.; Rees, D. C.
Nat. Struct. Biol. 1998, 5, 104.
21. White, S.; Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc.
1997, 119, 8756.
22. Pilch, D. S.; Poklar, N.; Baird, E. E.; Dervan, P. B.; Bres-
lauer, K. J. Biochemistry 1999, 38, 2143.
23. Pelton, J. G.; Wemmer, D. E. J. Am. Chem. Soc. 1990,
112, 1393.
24. Kielkopf, C. L.; White, S.; Szewczyk, J. W.; Turner, J. M.;
Baird, E. E.; Dervan, P. B.; Rees, D. C. Science 1998, 282, 111.
25. Urbach, A. R.; Szewczyk, J. W.; White, S.; Turner, J. M.;
Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1999, 121, 11621.
26. Mrksich, M.; Parks, M. E.; Dervan, P. B. J. Am. Chem.
Soc. 1994, 116, 7983.
MPE footprinting and anity cleavage
All reactions were carried out in a ®nal volume of 400 mL.
A polyamide stock solution (or water for reference lanes)
was added to an assay buer where the ®nal solution
conditions were as follows: 20 mM HEPES (pH 7.3),
200 mM NaCl, 50 mg/mL glycogen and 20 kcpm 30- or
50-end-labeled EcoRI/PvuII fragment of pSES4. The
reactions were equilibrated for 16h at 22 ꢁC. For MPE
footprinting, MPEꢀ Fe(II) was added to a ®nal concentra-
tion of 0.5 mM and equilibrated for 5 min.4 For anity
cleavage experiments, Fe(NH4)2(SO4)2 was added to a
®nal concentration of 0.5 mM and equilibrated for
20min.8,39 Cleavage was initiated by the addition of DTT
to a ®nal concentration of 5 mM and allowed to proceed
for 30min. Reactions were terminated with ethanol (1 mL)
and 10mL of a precipitation buer (2.8mg/mL glycogen,
140 mM bp calf thymus DNA). The labeled DNA was
precipitated and analyzed on a 8% denaturing poly-
acrylamide gel as described for DNase I footprinting.41,42
27. Parks, M. E.; Baird, E. E.; Dervan, P. B. J. Am. Chem.
Soc. 1996, 118, 6147.
28. Pilch, D. S.; Poklar, N.; Gelfand, C. A.; Law, S. M.; Bres-
lauer, K. J.; Baird, E. E.; Dervan, P. B. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 8306.
Acknowledgements
29. deClairac, R. P. L.; Geierstanger, B. H.; Mrksich, M.; Der-
van, P. B.; Wemmer, D. E. J. Am. Chem. Soc. 1997, 119, 7909.
30. Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 118,
6141.
We are grateful to the National Institutes of Health for
research support, the National Science Foundation,