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

Rautenbach, Marina (1998-12)

Thesis (PhD)--Stellenbosch University, 1998.

Thesis

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.

AFRIKAANSE OPSOMMING: Iturin A, ’n antifungiese lipopeptied, word deur Bacillus subtilis geproduseer. Hierdie sikliese peptied bestaan uit sewe D- en L-aminosuurresidue (L-Asn2-D-Tyr3-D-Asn4-L-Gln5-L-Pro6- D-Asn7-L-Ser8) en ’n beta-aminovetsuurresidu. Agt analoë van iturin A2 is gesintetiseer en m.b.v. hoë doeltreffendheid chromatografie (HPLC) gesuiwer. Die hoë chemiese suiwerheid van die sintetiese produkte is deur elektrosproei-ionisasie massaspektrometrie (ESI-MS), aminosuuranalise en HPLC bevestig. Die gesuiwerde peptiede is gebruik om die invloed van primêre struktuur op konformasie, hidrofobisiteit, interaksie met alkalimetaal-ione en bioaktiwiteit te ondersoek. Twee lae-energie in vacuo strukture van die lineêre iturin A2 analoog (8-Beta), een met ‘n verwronge W-ruggraatstruktuur en een met ’n gedraaide S-ruggraatstruktuur, is deur HyperChem®4.5 voorspel. Kernmagnetiese resonansspektrometrie het die bestaan van twee interomskakelende konformasies bevestig, moontlik ’n W<->S ewewig. Die voorspelde S-struktuur van 8-Beta bevat twee draaie wat neig na beta-draaie. Dieselfde twee peptiedeenhede, beta-aminotetradekanoiël-L-Asn2-D-Tyr3-D-Asn4 en L-Gln5-L-Pro6-D-Asn7-L-Ser8, neem elk ’n tipe II beta-draai konformasie in die natuurlike iturin A aan. ESI-MS fragmentasiepatrone van die 8-Beta natruimaddukte het aangedui dat die natriumione met die meeste van die amiedbinding-suurstowwe in die voorspelde draaie interaksie het. Die lineêre peptiede het met een of twee alkalimetaal-ione geassosieer, terwyl die sikliese analoë slegs met een ioon geassosieer het. Al die lipopeptiede se alkalimetaal-ioon selektiwiteitsvolgorde was Na+>K+>Rb+, wat aandui dat daar ’n grootte limiet is in die interaksieholtes. Iturin A het moontlik ’n direkte interaksie met alkalimetaal-ione en dit word voorgestel dat hierdie ione deur die karbonielsuurstowwe in enige een van die twee beta-draaie van natuurlike iturin A gechelateer word. Daar is gevind dat hoe meer hidrofobies die iturin A2 analoog is, hoe beter is die interaksie daarvan met lipiedmembrane en oktadekanoïelsilaanmatrikse (HPLC retensie), behalwe as dit ’n groot tendens het om in oplossing te aggregeer. Aggregasie in die membraan is deel van iturin A se meganisme van aksie. Daar word voorgestel dat die aggregate in oplossing nie die aktiewe vorm van iturin A is nie, omdat die lipopeptiedpreparate wat in oplossing selfaggregeer antibakteriële aktiwiteit verloor. Sirkulêre dichroïsme (CD) spektra van die peptiede in liposome het die moontlikheid van tipe II beta-draaie in al die oktalipopeptiede uitgewys. Alhoewel daar merkbare verskille tussen die totale sikliese en lineêre strukture in die membrane voorgekom het, was die diastereomere, wat verskil t.o.v. konfigurasie van beta-aminotetradekanoësuurresidu (beta-NC14), se strukture baie dieselfde. Die moontlikheid van selfverpakking van sintetiese iturin A2 in antiparallele beta-plate is ook deur CD aangedui. Hemolitiese aktiwiteit van die iturin A2 analoë het afgehang van siklisering, die insluiting van L-Asn2 en die beta-NC14-konfigurasie. Hemolise is moontlik stereoselektief want sintetiese iturin A2 en sy lineêre analoog was die aktiefste. Groei-inhibisie van Micrococcus luteus het hoofsaaklik afgehang van hidrofobiese interaksie en nie van siklisering of die konfigurasie van die beta-NC14-residu nie, dus verskil hierdie aktiwiteit in meganisme van aksie van diè van hemolise. Die lise van M. luteus protoplaste daarenteen neem af met verkorting van peptiedketting: 8-Beta>7-Beta>6-Beta. Die aktiwiteit teen Botrytis cinerea het hoofsaaklik afgehang van siklisering. Die hidrofobiese eenheid, gevorm deur die invariante Tyr-residu en die beta-NC14-residu, is ’n moontlike sleutel tot die antifungiese aktiwiteit. Hierdie eenheid is afwesig in die voorspelde S-struktuur van 8-Beta en mag ook in die sikliese 8-Beta en korter analoë beïnvloed word deur die konfigurasie van die beta-NC14-residu, wat lei tot goeie algemene bioaktiwiteit van slegs die sintetiese iturin A2.

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