– CF3-PEPTAIBOLS

TTo gain insight into the behaviour of Harzianin HK-VI (HZ) and Alamethicin F30/3 (ALM) towards membranes, a structural analysis was considered using solid-state 19F-NMR spectroscopy of these two membrane-embedded peptaibols. For these highly sensitive NMR measurements, specific tailor-made trifluoromethyl group labelled amino acids have been designed and incorporated into the peptides. The different steps of our investigations have been:
1) Optimization of the synthesis of various enantiomerically pure trifluoromethyl group containing ?-amino acids designed to fit into the Aib-type peptide backbone.
2) Incorporation of these fluorinated?amino acids into different positions of the two peptaibols, which is challenging per se due to the low reactivity of the nitrogen atom of the amino acid because of the strong electron withdrawing effect of the fluorine atoms.
3) Verification of the conformational and biological intactness of all wild-type and selectively labelled peptide analogues, using oriented circular dichroism and antimicrobial activity tests, respectively.
4) Solid-state 19F-NMR structure analysis of HZ and ALM in lipid membranes, to resolve the backbone conformation, molecular alignment, oligomerization state and dynamics.

Wild type and fluorinated peptaibols have been synthesized by solid phase peptide synthesis. Considering the difficult peptide coupling reaction at the N-terminal position of the fluorinated amino acids, their incorporation was achieved by mean of tripeptides or dipeptides previously prepared in solution phase. Although the Harzianin peptide shows no antibacterial and a weak antifungus activity, the circular dichroism measurements show that this peptaibol can adopt an unexpected helical structure consistent with a membrane thickness. The solid state 19F NMR and the synchrotron oriented circular dichroism show that the orientation of the peptide is depending on the lipid membrane. The skills acquired in this programme resulted in other international partnership with Italian and Swiss groups in the area of peptaibols and fluorinated peptides biological applications.

A better understanding of the action mechanism of the antimicrobial peptides will allow the design of new antimicrobial peptides which could give an answer to the resistance phenomenon.

The synthesis of fluorinated peptides and preliminary biophysical results has been reported at several international and national symposiums on organic and fluorine chemistry (International Symposium on Fluorine Chemistry, French fluorine network symposium, young investigator meetings) and peptide chemistry (International Conference for Antimicrobial Research, European Peptide Society Symposium, French peptide network).
Two workshops gathering the participants and invited speakers have been organized in Germany and in France. Publications in peer-review journals are in progress.