Isopeptide bonds in chemotactic tripeptides. Synthesis and activity of lysine-containing fMLF analogs.

In order to explore the properties of chemotactic N-formylpeptides containing isopeptide bonds within their backbones, a group of lysine-containing analogs of the prototypical chemotactic tripeptide N-formylmethionyl-leucyl-phenylalanine (fMLF) was synthesized. The new analogs were designed by adding to the HCO-Met or Boc-Met residue a dipeptide fragment made up of Lys and Phe residues joined through Lys N alpha or N epsilon bonds, in all possible combinations. Thus, the following six pairs of tripeptides were synthesized and examined for their bioactivity: RCO-Met-Lys(Z)-Phe-OMe (2a, b), RCO-Met-Lys(Z-Phe)-OMe (3a, b), Z-Lys(RCO-Met)-Phe-OMe (4a, b), Z-Phe-Lys(RCO-Met)-OMe (5a, b), RCO-Met-Phe-Lys(Z)-OMe (6a, b) and Z-Lys(RCO-Met-Phe)-OMe (7a, b), with R=OC(CH3)(3 )and R=H for compounds a and b, respectively. All the new models were characterized fully and their activity (chemotaxis, superoxide anion production and lysozyme release) on human neutrophils determined as agonists (compounds b) and antagonists (compounds a). All N-formyl derivatives 2b-7b are less potent than fMLF-OMe as chemoattractants, but compound 7b exhibits selective activity as superoxide anion producer. Derivatives 2a-7a do not show antagonistic activity towards fMLF induced chemotaxis and O(2)(-) production, however, all these compounds except 4a antagonize lysozyme release by 60%.

Pseudopeptides containing the 2-hydrazonoacyl fragment: analogs of the chemotactic agent HCO-Met-Leu-Phe-OMe.

In order to further examine the properties of pseudopeptides containing the 2-hydrazonoacyl fragment, two new series of analogs of the prototypical chemotactic N-formyl-tripeptide HCO-Met-Leu-Phe-OMe were designed and synthesized. The first group contains the new fragment as the N-terminal residue and is represented by the N-aryl derivatives p-Cl-C6H4-NH-N=C(R)-CO-Leu-Phe-OMe (2 and 3) and by the corresponding N-aroyl analogs p-CH3-C6H4-CO-NH-N=C(R)-CO-Leu-Phe-OMe (4). The second group contains the new fragment in place of the central Leu residue and is represented by compounds HCO-Xaa-NH-N=C(R)-CO-Phe-OMe (7a and 7b) where Xaa is Nle and Met, respectively. The conformational and biochemical properties of the new products were examined.

Synthesis and activity on human neutrophil functions of fMLF-OMe analogs containing alkyl spacers at the central position.

We report here the synthesis and activity of new analogs of the N-formyl and N-tert-butyloxycarbonyl (Boc) derivatives of the tripeptide Met-Leu-Phe-OMe containing an achiral omega-amino acid residue replacing the hydrophobic central leucine. The tripeptides HCO-Met-NH-(CH2)n-CO-Phe-OMe and Boc-Met-NH-(CH2)n-CO-Phe-OMe (n = 3-5) containing the central homomorphic residue of 5-aminopentanoic acid (delta-aminovaleric acid; delta-Ava; n = 4) and the two non-homomorphic residues of 4-aminobutanoic acid (gamma-aminobutyric acid; gamma-Abu; n = 3) and 6-aminohexanoic acid (epsilon-aminocaproic acid; epsilon-Aca; n = 5) have been examined. The activity as agonists and antagonists in chemotaxis, lysozyme release, and superoxide anion production of the new analogs has been determined. The N-Boc derivatives 2a and 2b, incorporating the gamma-Abu and the delta-Ava residues, show good and selective antagonist activity on superoxide anion production.

Bioactive fMLF-OMe analogs containing a N-terminal oximic or formylhydrazonic moiety.

In order to obtain chemotactic peptides with selective bioactivity, a new type of structural modification was introduced at the N-terminal position of HCO-Nle-Leu-Phe-OMe. Two groups of analogs have been synthesized both containing a N-terminal residue of the X=C(R)-CO-type replacing the native HCO-NH-CH(R)-CO-. In particular, the A group of pseudopeptides (2a-d) possesses a N-terminal oximic fragment (X=HO-N) and the B group (3a-d) a formylhydrazone fragment (X=HCO-NH-N). These new ligands have been examined for their capacity to induce chemotaxis and other cellular responses such as superoxide anion production and lysozyme release; although significantly active as chemoattractants they have been found to be practically devoid of secretagog activity, thus exhibiting selective behavior. The adopted chemical modification seems extensible in designing a new class of pseudopeptides (hydrazonopeptides) structurally related to both hydrazinopeptides and peptides containing alpha,beta-unsaturated residues.

New fMLF-OMe analogues containing constrained mimics of phenylalanine residue.

In the context of a research program aimed at elucidating the HCO-Met-Leu-Phe-OMe (fMLF-OMe) structural features which control interactions with the receptor in correspondence with the C-terminal residue, four different analogues of the native ligand have been synthesized and evaluated. Compounds 1-3 possess the general formula HCO-Met-Leu-Xaa-OMe with Xaa = N-benzylglycine, N-benzylphenylalanine, and alpha,alpha-dibenzylglycine, respectively. In the analogue 4 the constraint at the C-terminus has been obtained by incorporating a 2-oxopiperazine ring, made up of two phenylalanine residues, to replace the native C-terminal Phe residue. The consequences of the chemical modifications on the activity of the new analogues are discussed.

Modified chemotactic peptides: synthesis and activity of an azaTic-containing fMLP-OMe analogue.

The synthesis and the biological activity of a pseudopeptide analogue of the chemotactic N-formyltripeptide fMLP-OMe, containing the azaTic (3,4-dihydro-2(1H)-phthalazinecarboxylic acid) residue replacing the native phenylalanine, is described. Whereas pseudopeptides containing linear alpha-azaamino acids are currently studied, data on the new group of analogues containing cyclic alpha-aza residues capable of limiting the rotameric distribution of the side chains (topological control) are just emerging in the literature. At our best knowledge, the here described [azaTic3]fMLP-OMe represents the first example of the introduction of this new type of alpha-aza residue into a natural bioactive peptide.

Synthesis, conformation, and biological activity of two fMLP-OMe analogues containing the new 2-[2'-(methylthio)ethyl]methionine residue.

The new C alpha-tetrasubstituted alpha-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.

Chemotactic peptide analogues. Centrally constrained chemotactic N-formyltripeptides: synthesis, conformation, and activity of two new analogues.

The role exercised by the central residue of the chemotactic N-formyltripeptide HCO-Met-Leu-Phe-OMe (fMLP-OMe) in controlling both the backbone conformation and the biochemical activity is the subject of recent interest. Here, two new centrally constrained fMLP-OMe analogues, namely HCO-Met-azaPro-Phe-OMe (4) and HCO-Met-(gamma-lactam)-Phe-OMe (6) have been synthesized and their CDCI3 solution conformation and activity have been studied. The azapeptide 4 adopts beta-folded conformation with the azaPro residue at the i+2 position and an intramolecular H-bond involving the formylic oxygen and the Phe NH. The gamma-lactam tripeptide 6 prefers a semi-extended backbone conformation. When tested on human neutrophils both the new models were found practically devoid of biological activity. The role exerted by the NH groups as well as by the conformational preferences is discussed.

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.

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.

Modified chemotactic peptides: synthesis and biological activity of HCO-Met-delta ZLeu-delta ZPhe-OMe.

For-Met-delta ZLeu-delta ZPhe-OMe (3) has been synthesized as a new analogue of the prototypical chemotactic agent For-Met-Leu-Phe-OMe (fMLP-OMe). Compound 3 is characterized by presence of two consecutive alpha,beta-didehydro amino acid residues [delta ZLeu = (Z)-alpha,beta-didehydroleucine; delta ZPhe = (Z)-alpha,beta- didehydrophenylalanine] located at the central and C-terminal position, respectively. When tested on human neutrophils the N-formyltripeptide 3, although less active than the parent, is able to induce chemotaxis, superoxide anion production and lysozyme release. The activity of 3 has been compared to that of related fMLP-OMe analogues containing a single delta ZPhe residue located at the C-terminal position.

Modified chemotactic peptides: synthesis, crystal conformation, and activity of For-Hse(Me)-Leu-Phe-OMe.

The presence of the sulfur atom of the methionine side chain exerts significant effects at different levels on biochemical behavior of chemotactic N-formylpeptides. In order to acquire more information on this point, the synthesis, the conformation in the crystal, and the activity of For-Hse(Me)-Leu-Phe-OMe (2)--an oxygen analogue of For-Met-Leu-Phe-OMe (fMLP-OMe) containing the O-methyl-L-homoserine in place of the native methionine at position 1--is reported. The new analogue 2 adopts a conformation that is extended at the first two residues and folded at the C-terminal phenylalanine. This conformation is different from that of the parent fMLP-OMe and strikingly similar to that adopted by fMLP-OBu(t). The side-chain spatial orientation of 2 corresponds to that adopted by fMLP-OH when cocrystallized with an immunoglobulin possessing binding properties similar to those of neutrophil receptors. When tested on human neutrophils the formylpeptide 2 is more active than the parent in the stimulation of directed mobility and maintains both the granule enzyme release activity and the superoxide anion production.

Synthesis, conformation, and activity of HCO-Met-delta Z Leu-Phe-OMe, an active analogue of chemotactic N-formyltripeptides.

In order to induce a beta-turn conformation into the chemotactic linear tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), the new analogue N-formyl-L-methionyl-delta Z leucyl-L-phenylalanine methyl ester [delta Z Leu]2fMLP-OMe (1) has been synthesized. The conformational and biochemical consequences of this chemical modification have been determined. Analogue 1 has been synthesized by using N-carboxy-(Z)-alpha,beta-didehydroleucine anhydride as key compound to introduce the unsaturated residue at the central position of the tripeptide 1. The x-ray analysis shows that 1 adopts in the crystal a type II beta-turn conformation in which the new residue occupies the (i + 2) position, and an intramolecular H bond is formed between the formylic oxygen and the Phe NH. 1H-nmr analysis based on nuclear Overhauser effect measurements suggests that the same folded conformation is preferred in CDCl3 solution; this finding is also supported by molecular dynamics simulation. The biological activity of 1 has been determined on human neutrophils (polymorphonuclear leukocytes) and compared to that shown by fMLP-OMe. Chemotactic activity, granule enzyme release, and superoxide anion production have been determined. Analogue 1 is practically inactive as chemoattractant, highly active in the superoxide generation, and similar to the parent in the lysozyme release. The conformational restriction imposed on the backbone by the presence of the unsaturated residue is discussed in relation with the observed bioselectivity.

New chemotactic peptide analogs with high biological activity for human neutrophils.

As a part of a research programme aimed at studying structure activity relationships in the field of chemotactic peptides, modified analogs of the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) were examined for their capacity to activate several functions of human neutrophils. 4-Aminotetrahydrothiopyran-4carboxylic acid (Thp) and 2-aminoindane-2-carboxylic acid (Ain) were chosen as achiral, conformationally restricted amino acids suitable for mimicking the external Met and Phe residues of FMLP-OMe. The replacement of both produces a high locomotion activity, greater than the parent peptide; in contrast, the two Thp-containing analogs induce neither superoxide production nor lysozyme release. From these results we can hypothesize two different signal transduction systems: one which provides for movement, the other for superoxide generation and granule enzyme release.

Synthesis and properties of chemotactic peptide analogs. II. HCO-Met-Leu-Phe-OMe analogs containing cyclic alpha,alpha-disubstituted amino acids as Met and Phe mimicking residues.

As a part of a research program aimed at studying structure activity relationship in the field of chemotactic peptides, modified analogs of the potent chemoattractant HCO-Met-Leu-Phe-OH (fMLP) of the general formula HCO-Xaa-Leu-Yaa-OMe are examined. 4-Aminotetrahydrothiopyran-4-carboxylic acid (Thp) and 2-aminoindane-2-carboxylic acid (Ain) have been chosen as achiral, conformationally restricted amino acids suitable to mimick the external Met and Phe residues of fMLP-OMe. Studies on a first model, namely [Ain3]fMLP-OMe 1, have already been reported (12). Here the two remaining analogs [Thp1, Ain3] 2 and [Thp1] 3 have been synthesized. The conformation in the crystal of the disubstituted analog 2 has been determined and compared with those adopted by the parent fMLP-OMe and by previously studied models. The backbone conformation of 2 is characterized by helical folding centred at each of the three residues with the central Leu presenting helical handedness opposite to those of the two adjacent achiral residues. This conformation presents strong similarities with that adopted in the crystal by fMLP-OMe and resembles the conformation of fMLP bound to immunoglobulin (Bence-Jones dimer). The conformationally restricted analogs 2 and 3 are more active than the parent in the stimulation of directed mobility of human neutrophils but are practically inactive in the superoxide production. Crystals of 2 are orthorhombic, s.g. P2(1)2(1)2(1), with a = 21.934 (8), b = 10.856 (2), c = 10.380 (2) A. The structure has been refined to R = 0.071 for 2301 independent reflections with I greater than 1.5 sigma.