G. M. Grotenbreg et al. / Bioorg. Med. Chem. 11 (2003) 2835–2841
2841
[M+H]+, 1445.9 [M+Na]+ 1H NMR (DMSO-d6): d
References and Notes
8.94 (d, 1H, NH dPhe, J=2.6 Hz), 8.55 (d, 1H, NH Orn,
J=8.8 Hz), 8.36 (d, 1H, NH Leu, J=8.6 Hz), 7.26 (bs,
6H, Harom dPhe, NH Val), 6.77 (m, 1H, dNH Orn), 4.70
(m, 1H, Ha Orn), 4.54 (m, 2H, Ha Leu, Amp), 4.39 (m,
2H, Ha dPhe, Val), 4.00 (m, 1H, Hg Amp), 3.82 (m, 1H,
Hd Amp), 2.99 (m, 1H, Hb dPhe), 2.88 (m, 3H, Hd Orn,
Hb dPhe), 2.48 (m, 1H, Hd Amp), 2.30 (m, 1H, Hb Amp),
2.01 (m, 1H, Hb Val), 1.67 (m, 1H, Hb Orn), 1.59 (m, 1H,
Hb Amp), 1.43–1.15 (m, 15H, 3 Â CH3 Boc, 1 Â Hb Orn,
2 Â Hg Orn, 2 Â Hb Leu, 1 Â Hg Leu), 0.89–0.77 (m,
12H, 3 Â Hd Leu, 3 Â Hg Val).
1. Gause, G. F.; Brazhnikova, M. G. Nature 1944, 154, 703.
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cock, R. E. W.; Hodges, R. S. Int. J. Peptide Protein Res.
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J. Am. Chem. Soc. 2002, 124, 1203 and references cited therein.
9. Ovchinnikov, Y. A.; Ivanov, V. T. In The Proteins; Neurath,
H., Hill, R. Eds.; Academic: New York, 1979; Vol. 5, p 391.
10. (a) Jelokhani-Niaraki, M.; Kondejewski, L. H.; Farmer,
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chem. J. 2000, 349, 747. (b) Kondejewski, L. H.; Lee, D. L.;
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therein.
11. Gibbs, A. C.; Kondejewski, L. H.; Gronwald, W.; Nip,
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D
cyclo( Phe-2S,4S-Azp-Val-Orn(Boc)-Leu)2 (17). Start-
ing from 5 (51 mg, 35 mmol) peptide 17 was obtained as
described for 16, as a white amorphous solid (42 mg,
30 mmol, 86%). MS (ESI): m/z=1424.2 [M+H]+,
1445.9 [M+Na]+ 1H NMR (DMSO-d6): d 8.81 (d, 1H,
d
NH Phe, J=2.8 Hz), 8.57 (d, 1H, NH Orn, J=8.9 Hz),
8.34 (d, 1H, NH Leu, J=8.9 Hz), 7.27 (bs, 5H, Harom
dPhe), 7.18 (d, 1H, NH Val, J=8.9 Hz), 6.80 (m, 1H, dNH
Orn), 4.69 (m, 1H, Ha Orn), 4.55 (m, 3H, Ha Leu, Ha Val,
Ha Amp), 4.35 (m, 1H, Ha dPhe), 4.11 (m, 1H, Hg Amp),
3.41 (d, 1H, 1 Â Hd Amp, J=11.5 Hz), 2.92 (m, 4H, 2 Â
Hd Orn, 2 Â Hb dPhe), 2.73 (m, 1H, 1 Â Hd Amp), 2.20 (d,
1H, 1 Â Hb Amp, J=13.3 Hz), 1.96 (m, 1H, Hb Val), 1.71
(m, 2H, 1 Â Hb Amp, 1 Â Hb Orn), 1.48–1.16 (m, 15H, 3 Â
CH3 Boc, 1 Â Hb Orn, 2 Â Hg Orn, 2 Â Hb Leu, 1 Â Hg
Leu), 0.87–0.80 (m, 12H, 3 Â Hd Leu, 3 Â Hg Val).
Biological activity
The following bacterial strains were used: Staphylo-
coccus aureus (ATCC 29213), S. epidermidis (ATCC
12228), E. faecalis (ATCC 29212), E. coli (ATCC
25922), P. aeruginosa (ATCC 27853) and Bacillus cereus
(ATCC 11778). Bacteria were stored at ꢀ70 ꢁC and
grown at 35 ꢁC on Columbia Agar with sheep blood
(Oxoid, Wesel, Germany) overnight and diluted in 0.9%
NaCl. Large plates (25 wells of 3 mL) as well as micro-
titre plates (96 wells of 100 mL) were filled with Mueller
Hinton II Agar (Becton Dickinson, Cockeysvill, USA)
containing serial 2-fold dilutions of peptides 1–11. To
the wells was added 3 mL of bacteria, to give a final
inoculum of 104 colony forming units (CFU) per well.
The plates were incubated overnight at 35 ꢁC and the
MIC was determined as the lowest concentration inhi-
biting bacterial growth.
14. Aimoto, S. Bull. Chem. Soc. Jpn. 1988, 61, 2220.
15. Florsheimer, A.; Riniker, B. In Peptides, 1990. Giralt, E.,
¨
Andreu, D., Eds. ESCOM: 1991; p 131.
16. Castro, B.; Dormoy, J. R.; Evin, G.; Selve, C. Tetrahedron
Lett. 1975, 16, 1219.
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18. Gangamani, B. P.; Kumar, V. A.; Ganesh, K. N. Tetra-
hedron 1991, 34, 2787.
19. Klein, L. L.; Li, L. P.; Chen, H. J.; Curty, C. B.; DeGoey,
D. A.; Grampovnik, D. J.; Leone, C. L.; Thomas, S. A.;
Yeung, C. M.; Funk, K. W.; Kishore, V.; Lundell, E. O.;
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Lartey, P. A.; Plattner, J. J. Bioorg. Med. Chem. 2000, 8, 1677.
20. Reduction of azides 16 and 17 by Pd-catalysed hydro-
genolysis resulted in undesirable loss of Boc-protecting groups
under several reaction conditions.
21. Ramachandran, G. N.; Chandrasekaran, R.; Kopple,
K. D. Biopolymers 1971, 10, 2113.
22. Wuthrich, K. NMR of Proteins and Nucleic Acids; John
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Acknowledgements
23. Wishart, D. S.; Sykes, B. D.; Richards, F. M. Biochem-
istry 1992, 31, 1647.
This work was financially supported by the Council for
Chemical Sciences of the Netherlands Organization for
Scientific Research (CW-NWO), the Netherlands Tech-
nology Foundation (STW) and DSM Research. We
thank Nico Meeuwenoord and Hans van der Elst for
their technical assistance and are grateful to Kees Erke-
lens and Fons Lefeber for their help with recording the
NMR spectra.
24. It was previously reported that the chemical shifts of these
residues did not significantly alter when using methanol
instead of water as solvent system. Therefore, to enhance
solubility, CD3OH was employed. Krauss, E. M.; Chan, S. I.
J. Am. Chem. Soc. 1982, 104, 6953.
25. Stanger, H. E.; Syud, F. A.; Espinosa, J. F.; Giriat, I.;
Muir, T.; Gellman, S. H. Proc. Natl. Acad. Sci. U.S.A. 2001,
98, 12015.