Ines Torrini

Effect of cyclic AMP level reduction on human neutrophil responses to formylated peptides

The increase in human neutrophil cyclic adenosine monophosphate (cAMP) levels evoked by formylated peptides is significantly reduced in the presence of MDL 12330A, SQ 22536, GDPssS and clonidine, which inhibit the adenylyl cyclase system by acting at different sites in this enzyme complex. A similar effect is exerted by adenosine deaminase and dipyridamole, which alter the extracellular adenosine concentration. Neutrophil preincubation with adenylyl cyclase inhibitors or dipyridamole reduces chemotaxis and superoxide anion production triggered by peptides; adenosine deaminase, on the contrary, has no effect on neutrophil responses. Our results seem to indicate that: (1) the peptide-induced increase in neutrophil cAMP is due mainly to an action on the adenylyl cyclase system; (2) an enhancement of this cyclic nucleotide, even slight and necessarily transient, is required for chemotaxis and O2 production induced in neutrophils by formylated peptides; and (3) cAMP does not represent the crucial second messenger for adenosine in the modulation of neutrophil responses.

γ-Turn conformation induced by α,α-disubstituted amino acids with a cyclic six-membered side chain

Abstract 4-Aminotetrahydrothiopyran-4-carboxylic acid (Thp) is an unusual achiral cyclic α,α-disubstituted amino acid mimicking the natural Met residue. The conformational energy map computed for Ac-Thp-NHMe shows that the γ-turn is the lowest minimum. 1 H-NMR and IR studies performed on For-Thp-Leu-OMe ( 2a ), a short peptide unable to give a 4…>1 H-bond, indicate that the γ-turn is adopted in CDCl 3 solution, whereas is not retained in (CD 3 ) 2 SO. An analogous conformational behaviour in solution has been observed for the strictly related For-Ac 6 c-Leu-OMe ( 2b ), containing 1-aminocyclohexane carboxylic acid (Ac 6 c).

Two For-Met–Leu–Phe-OMe analogues trigger selective neutrophil responses: A differential effect on cytosolic free Ca2+

For-Thp-Leu-Ain-OMe and for-Met-delta(z)Leu-Phe-OMe are two conformationally restricted fMLP-OMe analogues able to discriminate between different biological responses of human neutrophils. In this paper, we demonstrate that the former peptide, which evokes only chemotaxis, does not alter human neutrophil Ca2+ levels. In contrast, for-Met-delta(z)Leu-Phe-OMe, which induces superoxide anion release and degranulation but not chemotaxis, significantly increases the cation concentration. The chelation of Ca2+ in both extracellular and intracellular media abolishes O2- production triggered by for-Met-delta(z)Leu-Phe-OMe, while the same procedure does not affect neutrophil chemotaxis towards for-Thp-Leu-Ain-OMe. We therefore suggest that chemotaxis, unlike superoxide anion release, is independent of Ca2+ enhancement in human neutrophils.

Retrosulfonamido peptide analogues. Synthesis and crystal conformation of Boc-Pro-Leu-Ψ(NH-SO2)-Gly-NH2

Abstract Boc-Pro-Leu-Ψ(NH-SO 2 )-Gly-NH 2 ( 3 ) has been synthesized as the first example of a pseudopeptide incorporating the NH-SO 2 junction. The crystal structure of 3 indicates that the Ψ(NH-SO 2 ) induces a cisoidal (gauche − ) conformation which induces a backbone folding and prevents the H-bonded β-turn found in the parent peptide.

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, conformation, and biological activity of two fMLP‐OMe analogues containing the new 2‐[2′‐(methylthio) ethyl] methionine residue

The new Cα‐tetrasubstituted α‐amino acid residue 2‐[2′‐(methylthio) ethyl]methionine (Dmt) has been introduced into the reference chemotactic tripeptide HCO‐Met‐Leu‐Phe‐OMe (fMLP‐OMe) in place of the leucine or methionine, respectively. The biological activity of the new analogues [Dmt2] fMLP‐OMe (2) and [Dmt1] fMLP‐OMe (3) has been determined; whereas 2 is active toward human neutrophils, stimulating directed migration, superoxide anion generation, and lysozyme release, 3 results practically inactive in all tested assays. A conformational analysis on 2 and 3 has been performed in solution by using ir absorption and 1H‐nmr. The conformation of 2 was also examined in the crystal by x‐ray diffraction methods. Both 2 and 3 adopt fully extended conformation in correspondence with the Dmt residue. Biological and conformational results are discussed and compared with related previously studied models.

Selective acylations of aminophenols and hydroxyalkylphenols with 1-acetyl-v-triazolo[4,5-b]pyridine

Abstract The title triazolide serves as a convenient reagent for higly chemoselective acetylations of aminophenols and hydroxyalkylphenols.

Modified chemotactic peptides: Synthesis, conformation, and activity of HCO‐Thp‐Ac6c‐Phe‐OMe

HCO-Thp-Ac6c-Phe-OMe (3) has been synthesized as a new analogue of the prototypical chemotactic agent HCO-Met-Leu-Phe-OMe (fMLP-OMe). Compound 3 contains 4-aminotetra-hydrothiopyran-4-carboxylic acid (Thp) and 1-aminocyclohexane-1-carboxylic acid (Ac6c) as achiral, conformationally restricted mimics of Met and Leu, respectively. In the crystal, the formyltripeptide adopts an helical conformation at the Thp and Ac6c residues, of the type alpha R and alpha L, respectively, whereas the C-terminal phenylalanine is quasi-extended. A system of two consecutive gamma-turns, centered at the first two residues, better explains the nmr data as compared with an alternative beta-turn structure. The conformation of the new analogue 3 is compared with those of two related peptides containing Thp as N-terminal residue. The biological activity of 3 has been determined on human neutrophils and compared to that of the previously studied model [Ac6c2] fMLP-OMe. While the above analogue is highly active in the superoxide anion production, the new tripeptide 3 is practically unable to elicit any of the tested biological activities.

For-Met-ΔzLeu-Phe-OMe: A new active analog of chemotactic N-formyltripeptides with β-turn crystal conformation

Abstract The title Δ z Leu containing N-formyltripeptide 2 , has been synthesized and found active in the superoxide production whereas inactive in the stimulation of human neutrophil migration. The X-ray crystallographic analysis reveals that 2 adopts a β-turn conformation stabilized by an intramolecular H-bond.

Water induced β-turn modification in a chemotactic tetrapeptide. Synthesis, crystal conformation, and activity of HCO-Met-Leu-ΔZPhe-Phe-OMe

In order to study the influence of the conformation on the activity and bioselectivity, the new tetrapeptide ligand of the chemotactic formylpeptide receptors HCO-Met-Leu-Δ Z Phe-Phe-OMe ( 2 ) has been studied. Compound 2 has been designed so as to induce a preferential β-turn conformation with the N -formyl group located outside the backbone loop. The crystallographic analysis reveals that 2 adopts only in part the expected conformation due to the presence of a water molecule isnide the turn. Details on the H-bonding network stabilizing the “open-turn” are given. The tetrapeptide 2 is active towards human neutrophils, stimulating directed migration, superoxide anion generation and lysozyme release. The influence of the backbone conformation on the bioselectivity is discussed.