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Indiana University-Purdue University Indianapolis (IUPUI) / Metallopeptides of the general form M(II)∙Gly1-Gly2-His induce DNA strand scission via minor groove interactions. This peptide system can serve as a nucleic acid-targeted cleavage agent – either as an appendage to other DNA binding agents, or as a stand alone complex. In an effort to further our knowledge of DNA recognition and cleavage, a novel series of phenyl-benzimidazole modified Gly-Gly-His-derived metallopeptides was synthesized via solid phase methods and investigated. The new systems allow the formation of additional contacts to the DNA minor groove through the incorporation of a DNA binding phenyl-benzimidazole moiety, thus strengthening the overall binding interaction and further stabilizing the metal complex-DNA association. In addition, how Lys side chains and an amidinium group influence the efficiency of DNA cleavage was also studied. DNA cleavage studies suggested that the phenyl-benzimidazole-modified Gly-Gly-His metallopeptides possess enhanced DNA cleavage abilities. In particular, when amidines are placed on the benzimidazole moieties, these moieties appeared to play an important role in increasing the DNA cleavage activity of the metal complex, most likely through an enhanced electrostatic attraction to the DNA.

Thesis (PhD)--Stellenbosch University, 1998. / 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.

Thesis (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: The implication of biologically active peptides from different organisms on one another in complex ecological communities is largely unknown at this stage. The elucidation of the nature of this influence may have practical implications in terms of organism resistance and the conservation of an optimal agricultural environment. This study was aimed to elucidate the effect of antimicrobial peptides from different co-habitational organisms, on each other, both in terms of bioactivity and interaction. The two peptides investigated were gramicidin S, a decapeptide from Bacillus brevis, and surfactin, a heptalipopeptide from Bacillus subtilis. Preliminary studies were also done on iturin A and synthetic analogues of iturin A and iturin C, both octalipopeptides from Bacillus subtilis. Analytical antimicrobial assay systems were used to study the effect of surfactin on the antibiotic action of gramicidin S towards three different target cells namely, a Gram-positive bacterium (Micrococcus luteus), a Gram-negative bacterium, (Escherichia coli) and a fungus (Penicillium corylophilium). The investigation of the antifungal activity was hampered by the insensitivity and subjectivity of the majority of antifungal assays and necessitated the development of two new testing methodologies. The investigation showed that surf actin had an antagonistic effect on the antimicrobial activity of gramicidin S against all three of the target cells. This antagonism is dose-dependent at concentrations lower than required for surfactin to exert biological activity. Electrospray mass spectrometry (ESMS) showed the formation of surfactin-gramicidin S complexes in 1:1 and 2: 1 ratios with enhanced complex formation in an apolar environment. Dissociation experiments indicated that the peptide complexes were slightly less stable than the peptides alone. The presence of NaCI up to 80 mM had little effect on the stability of preformed complexes. Incubating surfactin with NaCI and CaCh before titration with gramicidin S also did not affect complex formation. Furthermore, results from the pre-incubation studies with CaCh indicated that surfactin-gramicidin S complexes might be formed through the displacement of the metal ion. The mechanism of this displacement is unlikely to be direct competition but rather the result of conformational' changes induced by peptide-peptide interaction/interactions. A likely point of interaction the p-tums in the peptide ring. Linear iturin A2 and iturin C analogues were synthesised (8-Beta and 8-Betac) with solid phase peptide synthesis and purified using self-assembly and high performance liquid chromatography. The products of the syntheses wete analysed by ESMS and found to be correct. The products, together with commercially obtained iturin A, were used in biological assays and it was found that iturin A antagonises the antibiotic activity of gramicidin S but the linear analogues had no effect. Complex formation between iturin A and gramicidin S was observed using ESMS but no complexes were detected for the analogues, which reinforces the hypothesis that antagonism is related to the formation of inactive complexes. In general, the formation of peptide-gramicidin S complexes may indicate that a defence mechanism may be present in which toxic peptides of the competitor organism are inactivated by peptides from co-habiting organisms. / AFRIKAANSE OPSOMMING: Die invloed wat biologiese aktiewe verbindings van verskillende mikro-organismes in komplekse ekologiese omgewings op mekaar het, is onbekend. Die ontrafeling van die rol mag verskeie vrae ten opsigte van weerstandbiedenheid en ontwrigting van ekologiese landbou-omgewings beantwoord. Die doel van hierdie studie was om die invloed wat antimikrobiese peptiede, afkomstig van verskillende ko-habiterende organismes, op mekaar het te ondersoek- beide in terme van biologiese aktiwiteit en interaksie. Die twee peptiede wat ondersoek is, was gramisidien S, 'n dekapeptied geproduseer deur Bacillus brevis en surfaktien, 'n heptalipopeptied, geproduseer deur Bacillus subtilis. Voorlopige ondersoeke is ook uitgevoer op iturin A, en sintetiese iturin A en iturin C analoë, beide oktalipopeptiede van B. subtilis. Analitiese antimikrobiese toetsstelsels is gebruik om die effek van surfaktien op die biologiese aktiwiteit van gramisidien S te bepaal. Drie teikenselle is gebruik nl. 'n Grampositiewe bakterium (Micrococcus luteus), 'n Gram-negatiewe bakterium (Escherichia coli) en 'n fungus (Penicillium corylophilium) Die gebrek aan sensitiwiteit van bestaande antifungiese toetsstelsels het die ontwikkelling van twee nuwe toetstelsels genoodsaak. Die ondersoek het aangetoon dat surfaktien 'n antagonistiese effek op gramisidien S se antimikrobiese werking teen al drie teikenselle het. Die antagonisme is waarneembaar by surfaktien konsentrasies veel laer as wat nodig is vir biologiese aktiwiteit. Elektrosproeimassaspektrometrie (ESMS) van surfaktien en gramisidien S mengsels het aangedui dat komplekse in 'n 1:1 en 2: 1 stoichiometrie voorkom. Die vorming van peptiedkomplekse word ook deur 'n nie-polêre omgewing bevorder. Die stabiliteit van die peptiedkomplekse is ook geëvalueer met dissosiasie eksperimente en daar is gevind dat die komplekse minder stabiel is as die peptiede alleen - dit is 'n aanduiding van kompleksdissosiasie. Die teenwoordigheid van NaCI tot en met 80 mM het 'n minimale invloed op die stabiliteit van voorafgevormde peptiedkomplekse gehad. Inkubasie van surfaktien met NaCl en CaCh voor titrasie met gramisidien S, het ook nie die vorming van peptiedkomplekse beïnvloed nie en die studies het aangetoon dat die komplekse moontlik gevorm word deur die verplasing van die alkaliemetaalioon. Dit is onwaarskynlik dat die meganisme van ioon-verplasing direkte kompetisie is, maar eerder as gevolg van interaksie in een van die p-draaie. Liniêre iturin A2 en liniêre iturin Canaloë (8-Beta en 8-Betac) is gesintetiseer met behulp van soliede fase peptiedsintese en gesuiwer deur middel van "self-assembly" en "high performance liquid chromatography (HPLC)". Volgens die ESMS analise is die korrekte produkte verkry. Die analoë en kommersieel beskikbare iturin A is aan biologiese toetsing onderwerp en daar is gevind dat iturin A, maar nie die analoë nie, die antibiotiese effek van gramisidien S ophef. Die vorming van iturin en gramisidien S komplekse, wat met ESMS waargeneem is, versterk die teorie dat opheffing van aktiwiteit verband hou met die vorming van inaktiewe komplekse. Verder, die analoë het nie komplekse met gramisidien S gevorm me. Dit blyk vanuit hierdie studies dat die vorming van peptied komplekse moontlik deel kan uitmaak van 'n tipe verdedigingsmeganisme waar toksiese peptiede van kompeterende organismes, deur peptiede van ko-habiterende organismes, geïnaktiveer word.

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

Amino acids are important building blocks for the synthesis of a large number of biologically active compounds and drugs. Amino acids with the pentacyclo-undecane (1) and trishomocubane (2) frameworks fall into the class of conformationally constrained non-natural amino acids. Conformationally constrained amino acids are found in many naturally occurring, biologically active compounds. It was found that incorporating cage structures into drugs induces a range of positive effects: promotes transport across the cell membrane, drugs can be designed to target the central nervous system, increased receptor site specificity, and retards metabolic degradation. In the light of this, it was decided to investigate the incorporation of cage amino acids into peptides. A synthetic route has been established for the efficient synthesis of amino acids 1 and 2, and for their incorporation into peptides. Several chiral macrocyclic crown ethers and related analogues have been shown to be capable of forming complexes enantioselectively with chiral organic ammonium salts. The design and synthesis of host chiral macrocycles which are able to distinguish between the enantiomers of guest organic ammonium salts is of interest in the areas that include synthesis of enzymes, electrodes for specific ions or molecules, drugs targeted for specific sites, and enantiomer separation. A synthetic procedure has been established for the synthesis of cage annulated chiral crown ethers derived from amino acids. The advantage of using cage compounds in crown ethers is due to increased rigidity, increased solubility in non-polar solvents and increased chirality. Various techniques for the determination of enantiomeric recognition have been studied and include NMR spectroscopy, fluorescence emission spectroscopy and computational methods. The cage crown ether 3 represents a typical example of these new cage annulated, chiral crown ethers. / Thesis (M.Sc.)-University of Natal, Durban, 2001.