Biscarbamate analogues of the chemotactic tripeptide fMLF-OMe.

Based on the sequence of the prototypical chemotactic tripeptide HCO-Met-Leu-Phe-OH (fMLF) and by taking into account the versatility shown by its N-terminal carbamate analogues, the new biscarbamates MeOCO-Met-Leu-gPhe-COOMe (2) and Boc-Met-Leu-gPhe-COOMe (4) were synthesized. These two new ligands are characterized by the presence of a gem-diamino residue (gPhe) replacing the C-terminal Phe and a carbamate functionality positioned at both the ends of the molecule. The activity of the two new compounds has been determined on human neutrophils and compared to that shown by the corresponding N-terminal monocarbamates MeOCO-Met-Leu-Phe-OMe (1) and Boc-Met-Leu-Phe-OMe (3).

Studies on fMLP-receptor interaction and signal transduction pathway by means of fMLP-OMe selective analogues.

For-Thp-Leu-Ain-OMe ([Thp(1), Ain(3)] fMLP-OMe) (2), for-Met-delta(z)Leu-Phe-OMe ([delta(z)Leu(2)] fMLP-OMe) (3), for-Thp-Leu-Phe-OMe ([Thp(1)] fMLP-OMe) (4), and for-Met-Leu-Ain-OMe ([Ain(3)] fMLP-OMe) (5) are for-Met-Leu-Phe-OMe (fMLP-OMe) (1) analogues which discriminate between different responses of human neutrophils. Peptides 3 and 5, similar to fMLP-OMe, enhance neutrophil cyclic AMP (cAMP) as well as calcium levels, while analogues 2 and 4, which evoke only chemotaxis, do not alter the concentration of these intracellular messengers. When we tested the peptides' ability to displace [3H]-fMLP from its binding sites, the following order of potency was observed: analogue 1 > 3 > 5 > 2 > 4. A particularly low activity at the receptor level characterized analogues 2 and 4. Their low effectiveness was not improved by the addition of cytochalasin B, by different incubation temperatures, or by the absence of endogenous guanine nucleotides, conditions known to influence fMLP receptor fate and functionality. We speculate that, in certain conditions, the fMLP receptor may undergo conformational changes that impede the binding of pure chemoattractants.

The role of (Z)-2,3-didehydrophenylalanine and phenylalanine C-terminal residues in determining the chemotactic activity of formylpeptides.

Several formylpeptides, analogs of the chemotactic agent HCO-Met-Leu-Phe-OMe, having the HCO-Xaa-Leu-delta ZPhe-OMe and HCO-Xaa-Leu-delta ZPhe-Phe-OMe structures (delta ZPhe = (Z)-2,3-didehydrophenylalanine), have been synthesized. The biological activity of these ligands has been determined on human neutrophils and compared to that of the corresponding HCO-Xaa-Leu-Phe-OMe derivatives not containing the unsaturated residue. The replacement of the C-terminal Phe with delta ZPhe causes, in all the examined tripeptides, the loss of any biological activity. On the other hand, the introduction into the delta ZPhe containing models of an additional C-terminal Phe residue leads to the formyltetrapeptides HCO-Xaa-Leu-delta ZPhe-Phe-OMe which show a biological activity very similar to that exhibited by the corresponding HCO-Xaa-Leu-Phe-OMe analogues.

Chemotactic peptide analogues. Synthesis and chemotactic activity of N-formyl-Met-Leu-Phe analogues containing (S)-phenylalaninol derivatives.

The synthesis and the biological activity towards human neutrophils of some N-formyl-Met-Leu-Phe-OMe analogues containing (S)-phenylalaninol (Pheol) or its derivatives in place of the native phenylalanine are reported. While the analogue containing Pheol (4) was found to be devoid of significant biological activity, its esters 3 and 5, although inactive as chemoattractants, are able to strongly stimulate superoxide production and are active with a lower efficacy in the lysozyme release.

For-Met-Lys-Phe-For-Met-Lys-Phe-: a new cyclic analogue of the chemotactic formylpeptides.

As a continuation of the studies on chemotactic N-formylpeptides, we report here the synthesis and activity of a new cyclic analogue of the prototypical ligand For-Met-Leu-Phe-OMe. The new compound For-Met-Lys-Phe-For-Met-Lys-Phe- (4) contains a 20-membered cyclic moiety made up of a dimeric -Lys-Phe- sequence in which For-Met is attached to each Lys alpha-NH2 and hence remains outside the ring. The conformation in the crystal of the cyclic precursor of 4, namely Boc-Lys-Phe-Boc-Lys-Phe- (2) and the activity of the structurally related linear analogue For-Met-Lys(Z)-Phe-OBzl (6), have also been examined. The new analogues 4 and 6 are active as chemoattractants, secretagogues, and superoxide anion generating agents, when tested on human neutrophils. The structure-activity relationship is discussed and related to that of a previously studied cyclic model.

Modified chemotactic peptides: synthesis, conformation, and biological activity of For-Thp-Leu-delta ZPhe-OMe.

For-Thp-Leu-delta ZPhe-OMe (2), an analogue of the chemotactic tripeptide For-Met-Leu-Phe-OMe, containing 4-aminotetrahydrothiopyran-4-carboxylic acid (Thp) and (Z)-2,3-didehydrophenylalanine (delta ZPhe) as achiral, conformationally restricted mimics of Met and Phe, respectively, has been synthesized. In the crystal the new formyltripeptide adopts a type I beta-turn conformation stabilized by a weak H bond between the formylic oxygen and the delta ZPhe NH. 1H-nmr analysis based on NH solvent accessibility and nuclear Overhauser effect experiments suggests that the beta-turn is not preferred in CDCl3 solution where a gamma-turn, centered at the Thp residue, prevails. The biological activity of 2 has been determined on human neutrophils and compared to that of previously studied analogues. The tripeptide 2 is practically unable to elicit superoxide anion production and lysozyme release, while slight, but not statistically significant activity was induced in chemotaxis. The role of the orientation of the aromatic ring with respect to the backbone adjacent atoms is discussed.

Two new formylated peptides able to activate chemotaxis and respiratory burst selectively as tools for studying human neutrophil responses.

Two new For-Met-Leu-Phe-OH (FMLP) methyl ester analogues, For-Thp-Leu-Ain-OMe [Thp1, Ain3] and For-Met-delta zLeu-Phe-OMe [delta zLeu2], able to activate selectively chemotaxis and superoxide anion (O2-) release, respectively modulate intracellular cyclic AMP (cAMP) levels in different ways. FMLP and [delta zLeu2] enhance human neutrophil cAMP levels per se, and this effect is potentiated by Ro 201724, a non-xanthinic phosphodiesterase (PDE) inhibitor, whereas it is counteracted by 3-isobutyl-1-methyl-xanthine (IBMX), a blocker of both phosphodiesterase and adenosine receptors. In contrast, [Thp1, Ain3] is ineffective. However, no formylated peptides influence cAMP phosphodiesterase activity. Neutrophil preincubation with Ro 201724 or IBMX drastically reduces chemotaxis and superoxide anion (O2-) production triggered by peptides. Our results suggest that: (1) peptide-induced cAMP increase is probably indirect, and due mainly to the action on adenosine-sensitive adenylate cyclase; (2) formylated peptide, endowed solely with chemotactic activity is unable to increase neutrophil cAMP concentration; (3) cAMP elevation may represent a feed-back mechanism to inhibit the physiological responses induced by formylated peptides.

Synthesis and chemotactic activity of the fMLP analog HCO-Hmb-Leu-Phe-OMe.

The new fMLP analog HCO-Hmb-Leu-Phe-OMe (1), containing (S)-2-hydroxy-4-(methylthio)butyric acid (Hmb) in place of L-methionine at the N-terminal position, has been synthesized and fully characterized. The peptide 1 has been designed in order to improve the understanding of the role exerted by the formamido group in the binding interaction with the formylpeptide chemotactic receptors. Chemotaxis, superoxide anion production, and lysozyme release have been measured for both 1 and its deformylated analog Hmb-Leu-Phe-OMe 2. Results indicate that a strong hydrogen bond of the OH....O = C type may complement a weak H-bonding interaction involving the formylic proton as H-bond donor.

Synthesis and properties of chemotactic peptide analogs. I. Crystal structure and molecular conformation of HCO-Met-leu-Ain-OMe.

HCO-Met-Leu-Ain-OMe (2), an analog of the chemotactic peptide HCO-Met-Leu-Phe-OH, containing the conformationally blocked residue of the 2-aminoindane-2-carboxylic acid (Ain) has been synthesized and its crystal and molecular conformation has been determined. Crystals of 2 are monoclinic, space group P2(1), with a = 15.059(7), b = 18.548(7), c = 9.600(4) A; beta = 85.04(3) degrees. The structure has been solved by direct methods and refined to R = 0.069 for 2813 independent reflections with I greater than 2.5 sigma (I). Two independent molecules A and B have been found in the asymmetric unit of the crystal of 2. Their conformation can be described as extended at the Met and Leu residues, but folded at the C-terminal Ain residue. The helical folding is left- and right-handed in the A and B molecule, respectively. The crystal packing is characterized by ribbons of intermolecular hydrogen bonded molecules extended along the c direction. The constrained analog 2 is highly active in the superoxide production, thus indicating that a stabilization of a helical folding at the C-terminal region of chemotactic tripeptides maintains the activity. The orientation of the aromatic ring, with respect to its adjacent backbone atoms, does not seem critical for the activity.