In this thesis I examined the potential of Bio-Oligomers such as peptoids, peptides and aptamers, as therapeutic and diagnostic entities. Therapeutic Bio-Oligomers; A series of peptoid analogs have been designed and synthesised using solid phase synthesis. These peptoids have been subjected to biological evaluation to determine structure-activity relationships that define their antimicrobial activity. In total 13 peptoids were synthesised. Out of 13 different peptoids, only one peptoid called Tosyl-Octyl-Peptoid (TOP) demonstrated significant broad-spectrum bactericidal activity. TOP kills bacteria under non-dividing and dividing conditions. The Minimum Inhibitory Concentrations (MIC) values of TOP for S. epidermidis, E. coli and Klebsiella were 20 μM, whereas Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA) were 40 μM. The highest MIC values were observed for Pseudomonas aeruginosa (PAO1) at 80 μM. The selectivity ratio (SR) or Therapeutic index (TI) was calculated, by dividing the 10% haemolysis activity (5 mM) by the median of the MIC (50 μM) yielding a TI for TOP as 100. This TI is well above previously reported peptidomimetics TI of around 20. TOP demonstrates selective bacterial killing in co-culture systems and intracellular bacterial killing activity. Diagnostic Bio-Oligomers; In the second part of my thesis, I investigated aptamer and peptide-based molecular probes to detect MRSA. As well as screening aptamers and peptide probes against whole MRSA, I over-expressed and purified PBP2A protein. This purified protein was used as a target for aptamer and peptide probes to detect MRSA. Two different aptamer libraries were initially screened for utility. In-vitro conditions for SELEX were optimised. Biopanning with a phage derived peptides was also performed. Target sequences for both methods were identified and chemically synthesised. Evaluation of fluorescently labelled sequences with flow cytometry and confocal imaging showed no specificity for MRSA detection with either method. The Bio-Oligomers and the in-vitro selection methodology require further refinement to improve diagnostic utility.

Efficient delivery into cells is essential for many applications. However, cellular access of “cell-impermeable” molecules, such as drugs, sensors, proteins and oligonucleotides, can often be severely limited due to the plasma membrane which protects cells from unregulated influx of hydrophilic materials. In order to solve this issue, several physical techniques and bio-chemical products are today available. One of them is called peptoids (N-alkylglycines). These compounds are peptidomimetics which are resistant to enzymatic degradation, non-immunogenic and are readily prepared by an Fmoc chemical approach. Peptoids based on the "TAT"-peptide (RKKRRQRRR) offer rapid cellular uptake/delivery and low cytotoxicity. In this thesis, based on previous works using fluorescein-cationic peptoids, various fluorescent N-substitued glycines (lysine-like) were prepared by the monomer method followed by solid-phase synthesis. Their cellular uptakes in vitro into several cell lines (such as HeLa, B16F10, HEK293T and primary immune cells) were examined via flow cytometry and microscopy. The cellular delivery of small molecules mediated by the 9mer polymer achieved an efficient and rapid penetration. These results open up a vast number of applications for delivery of macromolecules using nonalysine-like peptoid. In order to demonstrate this ability, the nonalysinelike carrier was used to deliver various biopolymer molecules such as peptides, GFP protein and DNA (in collaboration with Dr. Stefano Caserta). In addition, thanks to the non-cytotoxicity of this cellular transpoter (MTT assays); experiments were carried out in vivo in mice using peptoids labelled near-infrared dyes. The first results have shown that the peptoid is not toxic for the mouse and does not block cell movements. These results allowed the use of 9mer-peptoid as a cellular tracking agent. Based on the development on antimicrobial peptides, the polylysine-like peptoid was also tested as an antibiotic. Recent experiments carried out in collaboration with Dr. Kevin Dhaliwal have revealed a new antimicrobial property of the peptoids. In vitro and in vivo studies have been carried out using both gram positive and negative bacteria. These results present a promising alternative to conventional antibiotics and antimicrobial peptides (AMPs).

In recent studies it was reported that the D-amino acid containing peptide HYD1 was used in the treatment of necrotic cell death in multiple myeloma cell lines and showed promising biological activity and in vivo activity. It was meaningful to explore strategies for increasing the therapeutic efficacy of HYD1, a linear peptide. These efforts led to the development of MT1-101 (cyclized peptidomimetics), a lead compound that showed increased in vitro activity and in vivo activity. MTI-101 was found to bind the cell adhesion molecule CD44 and induce programmed necrosis in myeloma cell lines. It was important to improve on the binding efficiency of the MTI-101 to this target and explore more cost effective ways to synthesize this peptide. This lead to developing Cyclic beta-hairpin-like peptoid scaffolds, which introduced diverse families of random peptoid-body libraries that will be screened to find small stable scaffolds that compete with and can replace antibodies as cell-surface targeting reagents. The synthesis of peptoids on solid-support can be more cost effective and a large library can be developed using a diverse library of primary amines. This initiated this thesis project to develop a generalized scheme for the synthesis of TRP-like primary amine peptoid side chains used in the cyclic beta-hairpin - Like scaffolds.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23920号 / 工博第5007号 / 新制||工||1781(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 秋吉 一成, 教授 大内 誠, 教授 大塚 浩二 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

No description available.

Binding to protein surfaces or shallow grooves with synthetic molecules poses a unique challenge, since this inherently requires large areas to facilitate interactions. Peptoids have been shown to interact with proteins, and combinatorial libraries of peptoids have been proven to be effective in discovering new ligands for protein binding. Unfortunately, most peptoids are flexible and lack the surface area required to compete with larger protein interactions. To combat these problems, we have created spiroligomers that have a rigid backbone, exhibit functionality comparable to proteins, and are resistant to proteases. To facilitate the rapid installment of spiroligomers into peptoid subunits, we required a more streamlined approach for functionalization of spiroligomers. To this end we applied a single-pot alkylation method, with which we installed over 25 unique functional groups onto different spiroligomer hydantoins. These spiroligomer hydantoins are spirocycles that possesses two stereocenters, of which we have complete control, as well as a protected proline amino acid. These new proline amino acids (enhanced prolines) have been incorporated into peptides via Fmoc-SPPS. Finally, we have functionalized these enhanced proline residues with another functional group and a protected primary amine, which allow for their use in peptoid synthesis. We developed methods to tether multiple spiroligomers together utilizing a peptoid backbone, as well as being able to incorporate spiroligomers into peptoid macrocycles. These spiroligomer-peptoid hybrids are large, diverse, and preorganized structures that have a large potential interacting surface area for binding to protein surfaces or shallow grooves. / Chemistry

The protein-protein interactions (PPIs) occur when two or more proteins are bound together. Also, this protein-protein interactions (PPIs) cause the various biological processes in the body. Due to this reason, abilities of controlling or inhibiting PPIs can give us promising advantages like (1) better understanding of biological systems, (2) development of new diagnostic approaches for health or disease, and (3) establishment of novel molecular therapeutics. Many proteins adopt the secondary structures, where most of protein-protein interactions take place. -Helices and -sheets are the prevalent secondary conformations, but there are extended secondary structures such as -hairpins, -turns, 310 helix, and so on. As a result, construction of molecules mimicking these protein secondary structures is tractable target for drug design. Moreover, in drug discovery, designing peptidomimetics or non-peptidic mimetics is a popular strategy instead using peptides or truncated peptides because peptides or truncated peptides are prone to proteolysis and degraded in the body. Also, peptidomimetics and non-peptidic mimetics have not only the similar topology as peptides but also resistance to proteolysis. Due to these advantages, in this study, peptidomimetics or non-peptidic mimetics were synthesized and tested for different targets: (1) synthesis of non-peptidic -helical mimetics for p53-MDM2 inhibition, (2) solution-phase synthesis of -hairpin peptide for the inhibition of multiple myeloma cells (MM) adhesion, and (3) synthesis of -hairpin peptoid-peptide hybrids. The synthesis in all three different studies was succeeded, but they still need some improvements. For instance, non-peptidic -helical mimetics, terpyrimidyl derivatives, were synthesized successfully, but they did not show any bioactivity against p53-MDM2. Also, they have a solubility problem. Based on these results, it is necessary to improve the pharmacokinetic properties and bioactivity by changing the substituents on the rings or structures. The -hairpin peptide for the second case already showed good bioactivity against multiple myeloma (MM). For the next level of bio-study, the considerable amount of a -hairpin peptide was demanded. In order to make the substantial -hairpin peptide, the solution phase peptide synthesis was chosen instead of the solid phase peptide synthesis because of the cost-effect. Two methodology were tried for the solution-phase peptide synthesis: (1) segment ligation and (2) continuous synthesis. In the former case, the -hairpin peptide synthesis was successful, but, in the latter case, it is necessary to investigate the appropriate coupling reagents for each step. Peptoid-peptide hybrids has been one of the popular peptidomimetics in the last two decades. Also, mimicking the peptide secondary structure in peptoids has been studied extensively these days. The combination of these two factors was the goal for the third case. Because peptoid-peptide hybrids with a secondary structure can be recognizable by native proteins and resistant to proteolysis. So far, three sets of peptoid-peptide hybrids were synthesize and checked the secondary structure formation by using NMR. However, there was no indication of the secondary structure formation in the three sets of peptoid-peptide hybrids. This result suggests that it is necessary to introduce the more constrained components in peptoid-peptide hybrids. In the above three chapters, it has been tried to find the new drug candidates by synthesizing peptidomimetics or non-peptidic mimetics. Even though the synthesis was successful, some intended results such as the bioactivity or the secondary structure formation were not obtained. However, these results can give us the inspirations to improve properties of peptidomimetics or non-peptidic mimetics for a certain purpose, which leads to earn the intended results and eventually find new drug candidates.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2017-03-27T17:41:33Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TeseKeilaCunha-Química2016.pdf: 14937894 bytes, checksum: cac41daeb22968873c67113d6f511e66 (MD5) / Made available in DSpace on 2017-03-27T17:41:33Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TeseKeilaCunha-Química2016.pdf: 14937894 bytes, checksum: cac41daeb22968873c67113d6f511e66 (MD5) Previous issue date: 2016-10-21 / CNPQ / Materiais bionspirados em peptídeos e proteínas possuem ampla diversidade de aplicações científicas e tecnológicas. Este trabalho se concentrou na descrição de propriedades dinâmicas e conformacionais de dois biomiméticos: peptóides e catecóis. Peptóides são oligômeros de glicinas N-substituídas de interesse biológico devido à resistência a proteases, maior estabilidade térmica, ambiental e facilidade de síntese, em comparação a peptídeos. Todavia, os desafios à aplicação de peptóides são devidos a flexibilidade ocasionada pela perda das ligações de hidrogênio intra-cadeia principal e interconversão cis/trans à temperatura ambiente. A simulação molecular clássica de peptóides tem sido limitada pelos campos de força existentes que não permitem a rotação interna da ligação amida sem o uso de técnicas para melhorar a amostragem. Neste trabalho foi desenvolvido um conjunto de parâmetros para a simulação de peptóides que permitiu descrever corretamente as populações conformacionais cis/trans de unidades monomêricas, em comparação a dados de RMN. As simulações de oligômeros mostraram estruturas bastantes flexíveis, não condizentes com conclusões propostas a partir de espectros de dicroísmo circular, que tem atribuído estruturas helicoidais a estes oligômeros. Os resultados sugerem que as transições eletrônicas favorecidas pelas conformações cis são as principais responsáveis pelos espectros obtidos. Adicionalmente, simulações por dinâmica molecular foram utilizadas para caracterizar a adsorção de moléculas catecóis em superfícies minerais. Proteínas de pés de mexilhões inspiraram nossos colaboradores na síntese de dois tipos de moléculas: iniciadores acrilatos para aplicação em resinas odontológicas e surfactantes zwiteriônicos para transistores orgânicos de efeito de campo (OFETs). Dentre as moléculas sintetizadas, os catecóis apresentaram melhores resultados nas aplicações propostas e forte adesão a superfícies minerais. A modelagem molecular permitiu elucidar como mudanças na estrutura molecular de catecóis acrilatos e da superfície mineral afetaram a adsorção. Os nossos resultados mostraram ainda que a automontagem de monocamadas de catecóis zwiteriônicos é governada pelo balanço entre ligações de hidrogênio, interações entre anéis aromáticos e dessolvatação da superfície mineral. / Peptides and proteins bioinspired materials have wide range of scientific and technological applications. This work has focused on the description of dynamic and conformational properties of two biomimetics: peptoids and catechols. Peptoids are N-substituted glycine oligomers of biological interest due proteases resistance, higher thermal and environmental stability and easier synthesis, when compared to peptides. However, the flexibility caused by the lack of intra-backbone hydrogen bonds and cis/trans interconversion at room temperature represents the main challenge for applications based on structure-function relationship. Classical molecular simulation of peptoids has been limited by the existing force fields, which do not allow amide bond internal rotation without the use of enhanced sampling techniques. In this work, the development of a set of parameters for peptoids simulation allowed the correctly description of cis/trans conformational populations in monomeric units, when compared to NMR data. The oligomers simulations showed flexible structures and that are not consistent with the withdrawn conclusions from circular dichroism spectra that has attributed helical structures to these oligomers. The results suggested that electronic transitions favored by cis conformations are mainly responsible by the obtained spectra. In addition, molecular dynamics simulations were used to characterize the adsorption of catechol molecules on mineral surfaces. Mussel foot proteins inspired our coworkers to synthesize two types of molecules: acrylic primers for dental resins and zwitterionic surfactants for organic field effect transistors (OFETs). Among the synthesized molecules, catechols showed best results for the proposed applications and strongest adhesion on mineral surfaces. Molecular modeling allowed elucidating how changes in molecular structure of acrylic catechols and on mineral surface affected adsorption. Our results also showed that the self-assembling of zwiterionic catechol monolayers is governed by the balance between hydrogen bonds, interactions between aromatic rings and desolvation of the mineral surface.

No description available.

No description available.