The supramolecular chemistry of novel synthetic biomacromolecular assemblies

Naidoo, Venthan B. (2004-04)

Dissertation (PhD)--Stellenbosch University, 2004

Thesis

ENGLISH ABSTRACT: Over the past decade peptide bola-amphiphiles have been the subject of much attention because of their role as potential models of functionalised membranes and as new generation surfactants. In the quest for new surfactants a peptidomimetic-based approach was used to design a library of novel 'hybrid' bola-amphiphilic peptide surfactants derived from sapecin B and a model symmetrical oligo-glycine bola-amphiphile. The library was divided into different series, each one purpose-built; first, to investigate hierarchal supramolecular architecture and, second, to investigate potential antimicrobial activity. The bola-amphiphiles were synthesised using Fmoc-polyamide based solid phase peptide synthesis and purified via high performance liquid chromatography. The peptide hybrids were characterised using electro spray mass spectrometry, nuclear magnetic resonance, different modes of electron microscopy, Fourier-transform infrared spectroscopy and, in some cases, further studies were done using circular dichroism and bioactivity tests. The model bola-amphiphile suberamide(GGh was synthesised using peptide fragment condensation based on solid phase peptide synthesis. The synthesis is bi-directional (N~C and C~N) and versatile, making it possible to synthesis new dicarboxylic oligopeptide bola-amphiphiles and other analogous compounds. The product, suberarnide(GG)2, was purified using its inherent ability to self-assemble in an acidic solution. Novel asymmetrical bola-amphiphiles composed of dipeptide head groups linked via an aliphatic (I)-amino acid, serving as a hydrocarbon spacer, were also synthesized. Two small libraries of bola-amphiphiles were established - the first involved variation in to-amino acid length and the other variation in the C-terminal amino acid. The bolaamphiphiles were self-assembled in either 0.1% trif1uoroacetic acid or 0.1% triethylamine. Electron microscopy revealed the formation of a variety of higher order supramolecular architectures based on ~-sheet self-assembly. FT-IR spectrometry indicated that interlayer and intralayer hydrogen bond networks, together with strong selfassociation, promoted by the hydrophobic effect and, in certain instances, electrostatic interactions, are responsible for the variety of supramolecular architectures. Variations in the higher order structures can be attributed to amino acid composition, specifically length of m-amino acid, nature of the C-terminal amino acid and the optimised solvent conditions used for the self-assembly process. A third library of novel 'hybrid' bola-amphiphilic peptide surfactants, in which a cationic tripeptide motif from antimicrobial peptides was combined in a hybrid molecule containing a oi-amino acid residue, was established. These bola-amphiphiles displayed potent antimicrobial activity against both Gram-positive and Gram-negative bacteria; the analogues were as active or more active than the leader peptides yet, remarkably, displayed little or no appreciable haemolytic activity. These organopeptide bolaamphiphiles thus demonstrated selective toxicity towards bacteria. The hydrophobicity imparted by the co-amino acid has contrasting effects on haemolysis and antimicrobial activity of the peptide analogues. The other unique feature of these peptides and their analogues is the fact they self-assembled into complex supramolecular architectures, composed primarily of ~-sheets. Their self-assembly is primarily governed by hydrophobic interactions together with inter and intralayer hydrogen bonding. Electron microscopy clearly revealed higher order structures for both peptides and analogues. The generation of higher order supramolecular architecture is dependent on optimisation of ~- sheet self-assembly whereas antimicrobial activity is dependent on the balance between net positive charge and optimum hydrophobicity of the peptide hybrids. This study has demonstrated that it is possible to design hybrid peptide surfactants capable of producing higher order supramolecular architecture and improving the antimicrobial activity whilst reducing the haemolytic effect. The study and design of these versatile 'purpose-built' bio-inspired surfactants heralds a novel approach, one that shows tremendous potential.

AFRIKAANSE OPSOMMING: Die afgelope dekade het bola-amfifiliese peptiede baie aandag geniet weens hulle rolle as potensiële modelle van gefunksionaliseerde membrane en as 'n nuwe generasie surfaktante. In die soeke na nuwe surfaktante is 'n peptiedornimetiese benadering gevolg om 'n biblioteek van nuwe "hibried" bola-amfifiliese peptiedsurfaktante van sapesien B en 'n simmetriese oligoglisien bola-amfifil af te lei. Die biblioteek is in verskillende reekse onderverdeel. Elke reeks is doelmatig vervaardig om ondersoek in te stel na twee aspekte, nl. die rangorde van die supramolekulêre strukture en die potensiële antirnikrobiese aktiwiteit. Fmoc-poliamied gebaseerde soliedefase-peptied-sin-tese is aangewend vir die sintese van die bola-amfifile en hulle is met behulp van hoë doeltreffendheid vloeistofchromatografie gesuiwer. Die peptiedhibriede is gekarakteriseer met behulp van elekrosproei massaspektrometrie, kern-magnetiese resonansie, verskillende modusse elektronrnikroskopie, Fourier-transform infrarooispektrometrie en, in sommige gevalle is verdere studies met sirkulêre dichroïsme en bioaktiwiteitstoetsing uitgevoer. Die bola-amfifilsuberamiedtflfij--model is met behulp van peptiedfragment-konden-sasie gesintetiseer gegrond op soliedefase-peptiedsintese. Dit sintese vind in twee rigtings plaas (N~C en C~N) en is veelsydig aangesien dit die sintese van sowel nuwe dikar-boksielbola- amfifile as ander analoë verbindings moontlik maak. Die produk, suber-arnied(GG)2, is gesuiwer met behulp van die verbinding se inherente vermoë tot self-montering in suur oplossings. Nuwe assimetriese bola-amfifile, saamgestel uit dipeptiedkopgroepe, gekoppel via 'n alifatiese ro-aminosuur, wat as koolwaterstofspasieerder dien, is ook gesintetiseer. Twee klein bola-amfifilbiblioteke is saamgestel - die een het variasies in die ro-aminosuur se lengte omvat en die ander een variasies in die C-terrninale aminosuur. Selfmontering van die bola-amfifile het plaasgevind in of 0,1 % trifluorasynsuur Of 0,1 % trietielamien. Elektronrnikroskopie het die bestaan van 'n verskeidenheid hoërorde supramolekulêre strukture, gegrond op p-plaatselfmontering, aangetoon. Uit FT-IR-spektrometrie blyk dit dat inter - en intralaag waterstofbinbdingsnetwerke en sterk selfassosiasie, 19. word bevorder deur die hidrofobiese effek en, in sekere gevalle, elektrostatiese interaksies, is verantwoordelik vir die verskeidenheid supramolekulêre strukture. Variasies in die hoërorde strukture kan toegeskryf word aan aminosuursamestelling, in besonder die lengte van die ro-aminosuur, die aard van die C-terminale aminosuur en die geoptimiseerde oplosmiddelkondisies wat gebruik is vir die selfmonteringsproses. 'n Derde biblioteek nuwe "hibried" bola-amfifiliese peptiedsurfaktante, waarin 'n kationiese tripeptiedmotief uit antimikrobiale peptiede gekombineer is met 'n m-aminosuurresidu, is geskep. Sommige van hierdie bola-amfifile het 'n kragtige antimikrobiese aktiwiteit teenoor sowel Gram-positiewe as Gram-negatiewe bakterieë gertoon. Die analoë strukture was aktief, of selfs meer aktief as die voorste peptiede maar het, verbasend genoeg, nie 'n beduidende hemolitiese aktiwiteit vertoon nie. Hierdie organopeptied bola-amfifil het dus 'n selektiewe toksisiteit teenoor bakterieë vertoon. Die hidrofo-bisiteit, as gevolg van die ui-aminosuur, het 'n resiproke effek op hemolise en die antimikrobiese aktiwiteit van die peptiedanaloë. Die ander uitstaande kenmerk van die peptiede en hulle analoë is die vermoë om te selfmonteer en komplekse supramolekulêre strukture, bestaande hoofsaaklik uit ~-plate, te vorm. Hierdie selfmontering word in hoofsaak beheer deur hidrofobiese interaksies asook inter - en intralaagwaterstofbinding. Elektronmikroskopie het duidelik hoërorde strukture getoon by sowel dié peptiede as hulle analoë. Die ontwikkeling van hoërorde supramolekulêre struktuurvorms is afhanklik van die optimalisering van die ~-plaatselfmontering. Daarteenoor is die antimikro-biese aktiwiteit afhanklik van die balans tussen die netto positiewe lading en die opti-male hidrofobisiteit van die peptiedhibriede. Hierdie studie het getoon dat dit moontlik is om hibriedsurfaktante te ontwerp wat hoërorde supramolekulêre strukture te produseer en om die antimikrobiese aktiwiteit te verbeter terwyl die hemolitiese effek verminder word. Die studie en ontwerp van hier-die veeldoelige, "doelmatig-gesintetiseerde" biogeïnspireerde surfakante stel 'n unieke benadering daar, wat oor groot potensiaal beskik.

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