The synthesis and characterisation of analogues of the antimicrobial peptide iturin A₂

[摘要] ENGLISH ABSTRACT: Iturin A, an antifungal lipopeptide, is produced by Bacillus subtilis. This cyclic peptide consists of seven D- and L-amino acid residues (L-Asn2-D-Tyr3-D-Asn4-L-Gln5-L-Pro6-DAsn7- L-Ser8) and a beta-amino fatty acid residue. Eight analogues of iturin A2 were synthesised and purified by high performance chromatography (HPLC). Electrospray ionisation mass spectrometry (ESI-MS), amino acid analysis and HPLC confirmed high chemical purity of the synthetic products. The influence of primary structure on conformation, hydrophobicity, interaction with alkali metal ions and bioactivity was investigated using the purified peptides. Two low energy in vacuo structures of a linear iturin A2 analogue (8-Beta), one with a distorted W-backbone structure and one with a twisted S-backbone structure, were predicted with HyperChem®4.5. Nuclear magnetic resonance spectrometry confirmed the existence of two slow interconverting conformations, possibly a W<->S equilibrium. The predicted S-structure of 8-Beta includes two turns that approximate beta-turns. In natural iturin A, the same two peptide moieties, beta-aminotetradecanoyl-L-Asn2-D-Tyr3-D-Asn4 and L-Gln5-L-Pro6-DAsn7- L-Ser8, each adopt a type II beta-turn conformation. ESI-MS fragmentation patterns of sodiated 8-Beta indicated that the sodium interacts with the majority of the amide bond oxygens in the predicted turns. The linear peptides associated with either one or two alkali metal ions, while the cyclic analogues associated only with one ion. The alkali metal ion selectivity sequence of all the lipopeptides was Na+>K+>Rb+, indicating a size limitation in interaction cavities. Iturin A possibly has a direct interaction with alkali metal ions and it is proposed that these ions are chelated by the carbonyl oxygens in either one of the two beta-turns of natural iturin A. It was found that the more hydrophobic the iturin A2 analogue, the better it interacted with lipid membranes and octadecanoylsilane matrices (HPLC retention), except if it had a high tendency to aggregate in solution. Aggregation in the membrane is part of iturin A's mechanism of action. It is proposed that solution-phase aggregates are not the active form of iturin A as the lipopeptide preparations, which self-aggregated in solution, lost their antibacterial activity. Circular dichroism (CD) spectra of the peptides in liposomes revealed the possibility of type II b-turns in all the octalipopeptides. There is, however, a marked difference between the overall cyclic and linear structures in membranes, although diastereomers, differing in configuration of b-aminotetradecanoic acid (b-NC14) residue, had similar structures. The possibility of self-assembly of synthetic iturin A2 in antiparallel beta-sheets was also indicated by CD. Haemolytic activity of the iturin A2 analogues depended on cyclisation, inclusion of L-Asn2 and b-NC14 configuration. This activity is possibly stereoselective as synthetic iturin A2 and its linear analogue were the most haemolytic. Growth inhibition of Micrococcus luteus mainly depended on hydrophobic interaction and not on cyclisation or configuration of the beta-NC14 residue, therefore this activity differs in mechanism of action from that of haemolysis. Lysis of M. luteus protoplasts, however, decreased with decrease in peptide length: 8-Beta>7-Beta>6- Beta. The activity against Botrytis cinerea depended mainly on cyclisation. The hydrophobic hub, formed by the invariant Tyr residue and the beta-NC14 residue, is a possible key to antifungal activity. This hub is absent in the predicted S-structure of 8-Beta and may be influenced in cyclic 8-Beta and shorter analogues by the configuration of the beta-NC14 residue, resulting in good overall bioactivity of only the synthetic iturin A2.

[发布机构] Stellenbosch University