Claudia Galoppini

Racemization Studies of Fmoc-Ser(tBu)-OH During Stepwise Continuous-Flow Solid-Phase Peptide Synthesis

We observed unexpectedly high levels of racemization of Fmoc-Ser(tBu)-OH during automated stepwise solid-phase peptide synthesis under standard continuous-flow conditions. We set up a model assay, based on the solid-phase assembly of the tripeptide H-Gly-Ser-Phe-NH2, in order to test the effect of the coupling conditions on serine racemization. Protocols based on collidine as the tertiary base added to the coupling reagent enabled incorporation of serine with less than 1 % racemization. We describe unexpectedly high levels of racemization of Fmoc-Ser(tBu)-OH during automated stepwise solid-phase peptide synthesis under standard continuous-flow conditions. We set up a model assay, based on the solid-phase assembly of the tripeptide H-Gly-Ser-Phe-NH2, in order to test the effect of the coupling conditions on serine racemization. Protocols based on collidine as the tertiary base added to the coupling reagent enabled incorporation of serin with less than 1% racemization.

Synthesis and biological activity of new bradykinin pseudopeptide B1 receptor agonists containing alkylic spacers

Abstract The tetrapeptide in the central portion of bradykinin and desArg10-kallidin was replaced by alkyl spacers of various lengths (-NH(CH2)nCO-, n=5–11). When tested as agonists at the kinins receptors, these pseudopeptides showed significant activity only at the B1 receptor. In particular, the introduction of a dodecanoic spacer in the central portion of desArg10-kallidin reduces only tenfold the agonist activity at the B1 receptor.

Toward the rational development of peptidomimetic analogs of the C-terminal endothelin hexapeptide: development of a theoretical model

In an early report on the structure-activity relationship of endothelin (ET) peptides, it was reported that the C-terminal hexapeptide ET(16-21), His-Leu-Asp-Ile-Ile-Trp, is the minimum ET fragment which maintains biological activity in some, but not all the tissues responding to ETs. Subsequently, other authors described a series of analogs of this peptide, in which the His 16 residue was replaced by non-natural amino acids, characterized by bulky aromatic side chains. Among them, two well-characterized non-selective ETA/ETB antagonists were PD 142893 and PD 145065; interest in these potent ET antagonists was, however, reduced by their peptidic structure which was likely to lead to undesirable properties such as poor bioavailability and short duration of action. On the basis of these premises, our previous studies led to the development of a peptidomimetic ligand of ET receptors (compound 3), based on the replacement of the His 16 residue of ET(16-21) with an (E)-N-(benzyloxy)iminoacyl moiety; compound 3 proved to possess a certain affinity for ET receptors, albeit lower than that shown by PD 142893 and PD 145065. We report here on ETA/ETB binding affinity of compounds 4-12, designed as a new series of ET(16-21) analogs. Compounds 4 and 5 were practically devoid of any affinity; derivatives 6-12 exhibited appreciable affinity indices for ETB receptors higher than that shown by 3, even if still lower than that obtained for PD 145065. This paper also describes the development of a pharmacophoric model able to explain the ET receptor binding properties of our hexapeptide analogs compared with those of PD 142893 and PD 145065 and IRL2500, recently reported as a potent ETB selective endothelin antagonist.

Conformational studies on a synthetic C-terminal fragment of the α subunit of GS proteins

It has recently been reported that synthetic peptides corresponding to the C-terminal sequence of G alpha, can be used to study the molecular mechanisms of interaction between this protein and G protein coupled receptors (Hamm et al., Science, 1988, Vol. 241, pp. 832-835). A conformational analysis on a 11 amino acids peptide from the G alpha(S) C-terminus, G alpha(S)(384-394) (H-QRMHLRQYELL-OH), was performed by nmr spectroscopy and molecular modeling methods. Two-dimensional nmr spectra, recorded in hexafluoroacetone/water, a mixture with structure stabilizing properties, showed an unusually high number of nuclear Overhauser effects, forming significative pattern to the drawing of a secondary structure. Conformations consistent with experimental NOE distances were obtained through molecular dynamics and energy minimization methods. These calculations yielded two stable conformers corresponding to an alpha-turn and a type III beta-turn involving the last five C-terminal residues. Interestingly, the alpha-turn conformation was found to overlap with good agreement the crystallographic structure of the same fragment in the G alpha(S) protein.

Structure-activity analysis of C-terminal endothelin analogues

Several synthetic endothelin (ET) analogues of the C-terminal ET hexapeptide (ET16-21) were analyzed by radio-receptor competition binding assays and biologic activity using both ETA and ETB receptor subtypes. In addition, we produced a hybridoma monoclonal antibody, anti-ET15-21, that appeared to crossreact with the entire ET molecule and was able to neutralize its biologic activity. Antibody binding was measured with competition enzyme-linked immunosorbent assays and a surface plasmon resonance-based biosensor (BIA technology). The ET16-21 moiety was modified with systematic replacement of each residue by alanine (Ala-scan). Whereas the C-terminal residues (Asp18, Ile20, and particularly Trp21) were very important for both receptor binding and immunologic activity, Ala substitution in positions 16, 17, and 19 hardly affected such activities. Analysis of another series of synthetic ET16-21 analogues with the His16 residue replaced by a non-amino-acidic block confirmed that the last two C-terminal residues are essential for receptor and antibody binding, whereas the central region of this hexapeptide is much more tolerant to modification. However, a critical steric conformation of the active hexapeptide is necessary.

A structure-activity study on the bradykinin B1 antagonist desArg10-HOE 140: The alanine scan

It has recently been shown that the biological activity of the second generation kinin B1 receptor selective antagonist, desArg10 HOE 140, can be improved by specific amino acid substitutions. Starting from this observation, we undertook a systematic structure-activity relationship study of this antagonist, based on the alanine-scan technique, in order to obtain useful information for the rational design of more analogues. Our data indicate that the sequence of desArg10 HOE 140 does not tolerate the replacement by Ala of most of its residues, with the exception of Ser in position 7 and, to a lesser extent, D-Arg in position 1 and Hyp in position 4. The most critical residues appear to be Pro in position 3 and the C-terminal dipeptide DTic-Oic; Ala replacement at these positions resultes in a total loss of activity. Moreover, replacement by Ala of Gly in position 5 reverts the activity of desArg10 HOE 140 to that of an agonist.

Synthesis and structure–activity relationship studies of new endothelin pseudopeptide analogues containing alkyl spacers

We replaced the Asp18-Ile19 dipeptide of the C-terminal ET analogue Ph-Ph-CH2-O-N=CH-CO-Phe-Asp-Ile-Ile-Trp-OH by alkyl spacers of various lengths to investigate the role of the aminoacidic central portion of the molecule and to define the N-terminal and C-terminal pharmacophoric regions of this analogue. The side-chains of the central dipeptide have been shown to be irrelevant for the binding of the molecule to the receptor, but the distance between the two postulated sites of interaction of the ligand with the ETB receptor appears to be fundamental.