muramidase and formylmethionyl-leucyl-phenylalanine-methyl-ester

In the present study, we report synthesis and biological evaluation of the N-Boc-protected tripeptides 4a-l and N-For protected tripeptides 5a-l as new For-Met-Leu-Phe-OMe (fMLF-OMe) analogues. All the new ligands are characterized by the C-terminal Phe residue variously substituted at position 4 of the aromatic ring. The agonism of 5a-l and the antagonism of 4a-l (chemotaxis, superoxide anion production, lysozyme release as well as receptor binding affinity) have been examined on human neutrophils. No synthesized compounds has higher activity than the standard fMLF-OMe tripeptide to stimulate chemotaxis, although compounds 5a and 5c with -CH(3) and -C(CH(3))(3), respectively, in position 4 on the aromatic ring, are better than the standard tripeptide to stimulate the production of superoxide anion, in higher concentration. Compounds 4f and 4i, containing -F and -I in position 4, respectively, on the aromatic ring of phenylalanine, exhibit significant chemotactic antagonism. The influence of the different substitution at the position 4 on the aromatic ring of phenylalanine is discussed.

Topics: Amino Acid Sequence; Anions; Chemotaxis; Humans; Molecular Conformation; Muramidase; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oligopeptides; Phenylalanine; Reference Values; Stereoisomerism; Superoxides

Two analogs of the prototypical peptide for-Met-Leu-Phe-OMe (fMLP-OMe), for-Gln-Tyr-Phe-OMe (1) and for-Gln-Tyr-Tyr-OMe (2), carrying unusual hydrophilic residues, were synthesized in order to investigate whether they provoked specific biological responses, as well as intracellular calcium mobilization, in human neutrophils. Whereas neither compound stimulates chemotaxis, both are able to elicit lysosomal enzyme production. However compound 1 is able to trigger copious superoxide anion production while compound 2 only elicits minor superoxide anion production. In binding experiments on formylpeptide receptors, the newly synthesized compounds for-Gln-Tyr-Phe-OMe (1) and for-Gln-Tyr-Tyr-OMe (2) showed affinity values in the micromolar range. These derivatives demonstrate inability to find a positive contribute from single substitutions. A very important result of this research is the evidence of the ability of the formyl group alone to trigger the primary target of the human neutrophil activity, i.e. killing mechanisms, by activating the specific receptor conformation.

Topics: Binding, Competitive; Chemotaxis, Leukocyte; Cytotoxicity, Immunologic; Humans; In Vitro Techniques; Muramidase; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Radioligand Assay; Structure-Activity Relationship; Superoxides

The alpha/beta3-mixed tripeptides R-CO-beta3-HMet-Leu-Phe-OMe (1a,b), R-CO-Met-beta3-HLeu-Phe-OMe (2a,b) and R-CO-Met-Leu-beta3-HPhe-OMe (3a,b) (a, R = tert-butyloxy-; b, R = H-), analogues of the potent chemoattractant For-Met-Leu-Phe-OMe, have been synthesized by classical solution methods and fully characterized. The activities of the new analogues as chemoattractants, superoxide anion producers and lysozyme releasers have been determined on human neutrophils. Whereas all of the three N-formyl derivatives are significantly less active than the parent tripeptide as chemoattractants, compound 1b has been found to be highly active as a superoxide anion producer and 3b as a lysozyme releaser. The results show that the replacement of the native Leu residue at the central position is, in each of the examined cases, the least favourable modification. The three N-Boc derivatives are, as expected, devoid of activity as agonists, but they are all good inhibitors of chemotaxis. Information on the solution conformation has been obtained by examining the involvement of the NH groups in intramolecular H-bonds using 1H NMR. The conformation of the N-Boc analogue 1a has also been determined in the crystal state by x-ray diffraction analysis. The molecule is extended at the beta3-HMet residue (phi1 = -87 degrees; theta1 = 172 degrees; psi1 = 126 degrees) and no intramolecular H-bond is present.

Topics: Amino Acid Sequence; Chemotaxis, Leukocyte; Crystallization; Humans; Molecular Conformation; Molecular Sequence Data; Muramidase; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oligopeptides; Structure-Activity Relationship; Superoxides; X-Ray Diffraction

for-Met-Ser(Bzl)-Phe-OMe, for-Met-Cys(Bzl)-Phe-OMe, for-Met-Tyr(Bzl)-Phe-OMe and for-Met-Lys(Z)-Phe-OMe were synthesized to investigate the importance of a bulky protecting group on the side-chain of a hydrophilic residue at position 2 on the biological activities of human neutrophils. Our results indicate that these compounds do not trigger a good chemotactic response, which, in any case, is not improved with respect to that induced by the analogs with the unprotected residues. Instead, both superoxide anion production and, particularly, lysozyme release are more efficient.

Topics: Chemotaxis, Leukocyte; Chromatography, Thin Layer; Humans; Muramidase; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Nuclear Magnetic Resonance, Biomolecular; Oligopeptides; Structure-Activity Relationship; Superoxides

The peptides for-Met-Ser-Phe-OMe 1, for-Met-Cys-Phe-OMe 2, for-Met-Lys-Phe-OMe 3, and for-Met-Tyr-Phe-OMe 4 were synthesized in order to investigate the importance of a hydrophilic side-chain on the residue at position 2 on biological activities of human neutrophils. Our results seem to highlight that this type of substitution does not facilitate good chemotaxis, although it elicits both superoxide anion production and particularly lysozyme release, in some cases even more potent than the parent fMLP-OMe, if the hydrophilicity is associated with steric hindrance.

Topics: Amino Acid Substitution; Chemotaxis; Humans; In Vitro Techniques; Muramidase; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oligopeptides; Superoxides

Two analogs of chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine were examined for their capacity to activate several functions of human neutrophils. The C-terminus methyl ester derivative of the chemotactic peptide was found to possess strong biological activity and was able to induce levels of chemotaxis, enzyme secretion, and superoxide generation comparable to those observed with the same concentrations of N-formyl-L-methionyl-L-leucyl-L-phenylalanine. The analog containing a tert-butyloxycarbonyl group at the N-terminus, as well as the C-terminal methyl ester, was completely devoid of activity towards neutrophils. From these results, it appears that the free carboxyl group is not necessary for biological function. In contrast, the substituent at the N-terminus may play a critical role in the induction of the cellular response.

Topics: Chemotaxis, Leukocyte; Humans; In Vitro Techniques; Kinetics; Muramidase; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Structure-Activity Relationship