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In order to probe the role of peptide backbone conformation on the biological activity of chemotactic peptides through conformationally constrained peptides, we synthesized the following three analogs of N-formyl-Met-Leu-Phe-OH (fMLF) containing dehydrophenylalanine (delta ZPhe) and dehydroleucine (delta ZLeu): formyl-Met-delta ZPhe-Phe-OCH3 (1), formyl-Met-delta ZLeu-Phe-OCH3 (2) and formyl-Met-delta ZPhe-delta ZPhe-OCH3 (3) and studied their conformational behavior in solution by 1H NMR and IR spectroscopy. The conformation of (1) was also examined by x-ray diffraction methods. Biological activity of these analogs was assessed for their ability to induce the release of beta-glucosaminidase from rabbit neutrophils. In addition, the chemotactic activity of analog (2) was also determined. We found that, in the solid state, (1) favors a type II beta-turn structure, stabilized by a 4-->1 intramolecular hydrogen bond. A similar structure was reported recently for (2) also. 1H NMR studies in solution suggest that the Phe NH is solvent shielded in both (1) and (2) and that a major population of peptide molecule exists in an intramolecular hydrogen bond stabilized type II beta-turn conformation. None of the NH groups in (3) and another analog, formyl-Met-Phe-Phe-OCH3 (4), appear solvent shielded, favoring an extended structure for these analogs. Analogs (2) and (4) are highly active indicating that both extended and beta-turn backbone conformations may be compatible with high activity and that the phenylalanine ring in the middle position is well accepted. Highly reduced activities of (1) and (3) suggest that delta ZPhe residue in position 2, irrespective of the preferred peptide backbone conformation, is not acceptable for high bioactivity. These results suggest that an induced fit mechanism may possibly be the most relevant one, but the nature and the topography of the side chains, particularly the middle residue, may be crucial for appropriate receptor ligand interactions.