In the first part of this study, frictional and normal forces in aqueous solutions were measured between a glass particle and oligopeptide films grafted from a glass plate. Homopeptide molecules consisting of 11 monomers of different amino acids were each "grafted from" an oxidized silicon wafer using microwave-assisted solid phase peptide synthesis. Oligopeptides increased the magnitude of friction compared to a bare hydrophilic silicon wafer. Friction was a strong function of the nature of the monomer unit and was lower for hydrophilic films. There was a strong adhesion and therefore friction between surfaces of opposite charges. Changes in adhesion and friction depended on the hydrophobicity and electrostatic forces: hydrophobic films and oppositely charged films produced high friction, whereas hydrophilic and like-charges produced low friction. Friction was lower in phosphate buffered saline than in pure water due to the screening of the double layer attraction for oppositely charged surfaces and additional lubrication by hydrated salt ions. We also investigated antimicrobial action of poly (allyl amine) (PA) when covalently bonded to glass. Glass surfaces were prepared by a two-step procedure where the glass was first functionalized with epoxide groups using 3-glycidoxypropyltrimethoxy silane (GOPTS) and then exposed to PA to bind via reaction of a fraction of its amine groups. Antibacterial properties of these coatings were evaluated by spraying aqueous suspensions of bacteria on the functionalized glass slides, incubating them under agar, and counting the number of surviving cell colonies. The PA film displayed strong anti-microbial activity against both Gram-positive and Gram-negative bacteria. Films that were prepared by allowing the PA to self assemble onto the solid via electrostatic interactions were ineffective antimicrobials. Such films had an insufficient positive charge and did not extend far from the solid. Thus we found that antimicrobial activity was correlated with a combination of the ability of the polymer chain to extend into solution and a positive surface potential. / Ph. D.

No description available.

<p>Pathogenic bacteria are evolving to drug resistant strains at alarming rates. The threat posed by drug resistant bacterial infections emphasize the need to establish new antimicrobial agents. Of immediate concern regarding the dangers of antibiotic resistance is the existence of intracellular bacteria, which find refuge from bactericidal devices by hiding within mammalian cells. Unfortunately, many therapeutics, such as vancomycin, do not possess membrane penetrating abilities to achieve efficacious eradication of bacteria at the subcellular level, allowing infections to persist. In an effort to target pathogens that thrive within mammalian cells, features of cell penetrating peptides (CPPs) and antimicrobial peptides (AMPs) were combined to develop a dual action antimicrobial CPP, cationic amphiphilic polyproline helices (CAPHs). CAPHs have proven to be an effective antimicrobial agent to combat an array of both Gram negative and Gram positive bacteria. </p> <p> </p> <p>Herein, to improve CAPHs activity, we have demonstrated how the incorporation of strategic modifications has resulted in increased cell uptake, alternative subcellular locations for CAPHs, and advanced antimicrobial potency. By simultaneously extending the helical length of CAPHs while incorporating different hydrophobic groups in place of the original isobutyl moiety that compose CAPHs we have created a <b>FL-P17-5R </b>series of peptides with five carbon aliphatic motifs: <b>Fl-P17-5B</b>, <b>Fl-P17-5C</b> and <b>Fl-P17-5L. </b>Through these modifications the peptides proved to be 2 to 5-fold more efficient in accumulating in macrophage cells than parent peptide Fl-P14LRR and where able to clear intracellular pathogenic bacteria, such as <i>Listeria</i>, from infected macrophages by 26 to 54%. </p> <p> </p> <p>In addition to making the <b>Fl-P17-5R</b> series of CAPHs to potentiate CAPHs activity, modifications to the cationic moiety of CAPHs were explored. By incorporating a new cationic monomer into the CAPHs sequence, a guanylated amino proline (GAP) residue, we produced <b>Fl-P14GAP</b>, a CAPHs peptide with an organized cationic charge display. This modification resulted in a 5-fold increase in cell uptake and a 2 to 16-fold decrease in minimum inhibitory concentration (MIC) values against strains of enteric and ESKAPE pathogens in comparison to Fl-P14LRR. <b>Fl-P14GAP</b> also executed superior clearance of intracellular pathogenic bacteria that resulted in the complete eradication of a drug resistant strain of <i>A. baumannii</i> from infected macrophage cells. Overall, our efforts with the <b>Fl-P17-5R</b> series of CAPHs and <b>Fl-P14GAP</b> have strengthened the therapeutic potential of CAPHs in the hopes of addressing the need for novel antibiotics with the propensity to eradicate intracellular pathogens.</p>

No description available.

Two photon absorption (TPA) is the near simultaneous absorption of two photons of light to achieve an electronically excited state. It has led to huge advances in microscopy and microfabrication due to its quadratic dependence on the local light intensity. This thesis describes the design, synthesis and application of dyes with strong TPA properties, and as such is divided into three chapters. The first introduces the theory and measurement of TPA as well as structure-property relationships known to maximise the efficiency of TPA. The subsequent chapters present explorations of the application of these dyes in biological applications; namely two-photon uncaging and two-photon photodynamic therapy. A recurring theme in my research is the discussion and evaluation of strategies for improving the compatibility of organic macromolecules with biological systems. Uncaging is the use of photolysis to achieve a rapid increase in the local concentration of a physiologically active species via a photoremovable protecting groups. Photoremovable protecting groups are covalently attached to the physiologically active species, thus rendering it inactive. At the desired time and location, a light dose releases the molecule in its active form. There are many compounds known to uncage following photoexcitation, but there are few examples of caging groups which exhibit both strong two-photon absorption properties and highly efficient uncaging. Chapter 2 discusses the rational design of such groups through the development of a new mechanism for uncaging, in which a photoinduced electron transfer (PeT) between a two-photon-excited electron donor and an electron acceptor/release group drives the uncaging event. Photodynamic therapy (PDT) is a treatment for neoplastic disorders such as cancer in which localised cell death is induced through photoexcitation of a sensitiser. Following light absorption, the photosensitiser enters a relatively long-lived excited state which reacts with cellular oxygen to produce its highly cytotoxic singlet form. The main challenges of the field are to achieve deep penetration of light into tissue and to reduce coincident damage to unaffected tissue by light scattering during irradiation. In 2008, the Anderson group reported the development of PDT photosensitisers with highly efficient two-photon absorption as well as high singlet oxygen quantum yields. Chapter 3 discusses strategies for improving the pharmacokinetics and defining the sub-cellular localisation of these photosensitisers.

Conformationally constrained ligands and their flexible analogues were prepared as inhibitors of the Grb2 SH2 domain in order to study the structural and energetic effects of ligand preorganization in protein-ligand interactions. The compounds were prepared by using trans-cyclopropane-containing amino acid mimics, macrocyclization, or [alpha,alpha]-disubstituted amino acid residues. All trans-cyclopropane containing peptides were more potent than their corresponding succinate containing analogues due to an enthalpic advantage. Surprisingly, the binding of constrained peptides to the domain was entropically disfavored relative to their flexible controls. Effects of proton transfer and desolvation as being the source of the unprecedented entropic penalty for the constrained ligands relative to their respective controls were precluded, and X-ray crystallographic studies revealed that the binding conformations for the respective cyclopropane and succinate containing ligands were similar. This led us to believe that differential changes in protein dynamics may occur upon binding of the constrained and flexible ligands, which could contribute to the observed binding energetics. Two 23-membered macrocyclic ligands were slightly more potent than their corresponding linear controls. The amino acids used to link the N- and C-termini of the linear peptides to form the macrocycles were found to affect the energetics of binding. In one case, the 23-membered macrocycle was more potent than its control due to an entropic advantage, whereas the other 23-membered macrocycle was more potent than its control because it benefited from an enthalpic advantage. [alpha,alpha]-Disubstituted and [alpha]-monosubstituted residues that varied in hydrophobic character were incorporated into Grb2 SH2 domain binding tripeptides, and binding became more favorable as nonpolar surface area increased only for the set of tripeptides possessing cyclic [alpha,alpha]-disubstituted residues. The increase in affinity was due to an increasing enthalplic term, whereas the entropy of binding became less favorable. A total synthesis of (±)-lycopladine A was achieved in five steps from known compounds. The tricyclic core of the natural product was prepared utilizing a novel two-step sequence comprising a conjugate addition of a metalated picoline derivative followed by an intramolecular enolate arylation. It was demonstrated that the natural product existed in a solvent dependent equilibrium with its isomeric lactol. / text

O uso de peptídeos na terapêutica apresenta inúmeras limitações, desde características físico-químicas até perfil farmacocinético inadequado para absorção oral. Entretanto, novas estratégias sintéticas para redução de custos e aumento da estabilidade química e metabólica de peptídeos, associada a vias alternativas de administração foram desenvolvidas e permitiram o acesso a essas moléculas no mercado farmacêutico. Diante disso, peptídeos naturais bioativos, subexplorados do ponto de vista químico medicinal, são considerados bons protótipos para o desenvolvimento de biblioteca de análogos não-naturais, gerando informações relevantes para a compreensão da relação entre a estrutura química e a atividade biológica destes compostos. No presente estudo, os peptídeos cíclicos rufomicina B (tuberculostático), chaiyaphumine A (anti-malárico), ciclozantoxilano A e desotamide B (antibiótico) foram estudados do ponto de vista sintético sob diferentes metodologias. Para tal, os aminoácidos Fmoc-N-Metil-L-Leucina e Fmoc-L-crotil-glicina foram sintetizados com rendimentos satisfatórios para síntese do antibiótico rufomicina B. O aminoácido Fenil-acetil-L-treonina foi obtido em bons rendimentos e a síntese de chaiyaphumine A e derivado foi testada em fase sólida, em solução e empregando combinação dos dois métodos. O peptídeo desotamide B e ciclozantoxilano A foram sintetizados, mesmo que em baixo rendimentos, em fase sólida com ciclização solução. Ainda, a síntese de desotamide B também foi possível através da metodologia \"safety-catch\". Por fim, realizamos análise sistemática das propriedades físico-químicas e estruturais dos peptídeos no mercado e em fase clínica e comparamos os resultados obtidos com os alvos desse estudo / The use of peptides in therapy presents several limitations, from physicochemical characteristics to inadequate pharmacokinetic profile for oral absorption. However, new synthetic strategies for cost reduction and increase of the chemical and metabolic stability of peptides associated with alternative routes of administration were developed and allowed access to these molecules in the pharmaceutical market. Therefore, natural bioactive peptides, is an underexploited class from the medicinal chemist\'s point of view, are considered good prototypes for the development of similar series, generating information relevant to understanding the relationship between chemical structure and biological activity of these compounds. In the present study, cyclic peptides rufomycin B (antituberculosis activity), chaiyaphumine A (anti-malarial), cyclozanthoxylane A and desotamide were studied from the synthetic point of view, through different metodologies. In this sense, the amino acids Fmoc-N-Methyl-L-Leu and Fmoc-L-crotyl-Gly were synthetized in good yields for the total synthesis of rufomycin B. The amino acid Phenyl-acetyl-L-threonine was obtained in high yield and the synthetic studies on the chaiyaphumine A and analogue has been performed in solution and solid phase peptide synthesis and also through the combination of the 2 methods. The peptides desotamide B and cyclozanthoxylane A were synthetized, in low yields, in solid phase and in solution cyclization. Desotamide B could also be synthetized through safety-catch solid phase method. At end, we performed a systematic review of the physical chemical properties of all market and on clinical trial peptide drugs and we compared the results with the target molecules in this study.

<p> This study addresses the issue of how to convert peptides into drug-like non-peptides while retaining the biological activity at peptide receptors. Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Ang II) was used as a model peptide.</p><p> Small bioactive peptides are in most cases conformationally flexible molecules. Rigidified peptide analogues or peptidomimetic scaffolds can be introduced into the peptide, to enforce a particular backbone conformation, and thereby locate the side-chains at defined positions in space. The conformationally constrained analogues are of considerable value in determining biologically active conformation(s) of the studied peptide. The strategy applied in this thesis includes identification of non-pharmacophoric amino acid residues, rigidification, conformational analysis and incorporation of turn mimicking scaffolds in </p><p>Ang II. Several side-chain cyclized (disulfide and methylendithioether) Ang II analogues have been synthesized. The binding studies of the rigidified analogues demonstrated that the compounds designed for the AT₁-receptor had affinity for both receptor subtypes, while the compounds designed for the AT₂-receptor displayed high selectivity only for this receptor subtype. Conformational evaluation revealed that several of the cyclized Ang II analogues most probably adopt a <i>γ</i>-turn like conformation around Tyr-4 while interacting with the </p><p>Ang II receptor. Based on this hypothesis, three different <i>γ</i>-turn mimetics replacing amino acid residues 3-5 were designed, synthesized and incorporated into Ang II. One of the synthesized pseudopeptides, incorporating an azepine-containing <i>γ</i>-turn mimetic, exerted high binding affinity and agonistic activity. These results strongly support the theory that Ang II adopts a <i>γ</i>-turn like conformation when activating the AT₁ receptor. The other Ang II analogues, incorporating bicyclic and aromatic <i>γ</i>-turn mimetics, did not display any binding to the AT₁ receptor.</p>

This study addresses the issue of how to convert peptides into drug-like non-peptides while retaining the biological activity at peptide receptors. Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Ang II) was used as a model peptide. Small bioactive peptides are in most cases conformationally flexible molecules. Rigidified peptide analogues or peptidomimetic scaffolds can be introduced into the peptide, to enforce a particular backbone conformation, and thereby locate the side-chains at defined positions in space. The conformationally constrained analogues are of considerable value in determining biologically active conformation(s) of the studied peptide. The strategy applied in this thesis includes identification of non-pharmacophoric amino acid residues, rigidification, conformational analysis and incorporation of turn mimicking scaffolds in Ang II. Several side-chain cyclized (disulfide and methylendithioether) Ang II analogues have been synthesized. The binding studies of the rigidified analogues demonstrated that the compounds designed for the AT1-receptor had affinity for both receptor subtypes, while the compounds designed for the AT2-receptor displayed high selectivity only for this receptor subtype. Conformational evaluation revealed that several of the cyclized Ang II analogues most probably adopt a γ-turn like conformation around Tyr-4 while interacting with the Ang II receptor. Based on this hypothesis, three different γ-turn mimetics replacing amino acid residues 3-5 were designed, synthesized and incorporated into Ang II. One of the synthesized pseudopeptides, incorporating an azepine-containing γ-turn mimetic, exerted high binding affinity and agonistic activity. These results strongly support the theory that Ang II adopts a γ-turn like conformation when activating the AT1 receptor. The other Ang II analogues, incorporating bicyclic and aromatic γ-turn mimetics, did not display any binding to the AT1 receptor.

IgM monoclonal gammopathy is a common age-related demyelinating sensory and motor polyneuropathy. It has been shown to be associated with antibodies against myelin-associated glycoproteins (MAG/SGPG). The HNK-1 carbohydrate epitope is a terminal 3-sulfo-glucuronyl residue attached to lactosamine structures and it is shared both in MAG and SGPG (SO4-3-GlcA(β1-3)Gal-(β1-4)GlcNAc(β1-3)Gal-(β1-4)Glcβ(1-1′)Cer). It is mostly expressed in the nervous system and plays an important role in preferential motor reinnervation. Nevertheless, the HNK-1 epitope is difficult to be isolated and synthesized and diagnostic assays used in the clinics are not always reproducible and reliable. Therefore in our study, our goal is to identify a simple synthetic diagnostic tool (peptide or monosaccharide), mimetic of the HNK-1 epitope, able to recognize antibodies in neurogammopathies sera by Surface Plasmon Resonance to be used in earlier stage patients and possibly to monitor disease activity. For this reason, we firstly tried to synthesize this trisaccharide and then we achieved the synthesis of its terminal monosaccharides with different function groups (octyl glucopyranoside, octyl glucuronic acid, octyl 3-O-sulfo-glucuronic acid and 8-amino octyl 3-O-sulfo-glucuronic acid). Then 10 linear and cyclic peptides conformationally and/or structurally mimicking HNK-1 were also synthesized (LSETTI, LSETTl, cyclo(-TTILSE-), cyclo(-TTlLSE-), cyclo(-TKTlLSE-), cyclo(-TETKlLSE-), TYTKlLSE, TY(SO3)TKlLSE, cyclo(-TYTKlLSE-) and cyclo(-TY(SO3)TKlLSE-)). The SPR kinetic binding affinities of all these sugar and peptide mimics were studied with commercial anti HNK-1 antibody using Biacore. Moreover, mimics with highest binding affinities were chosen for antigen-antibody interaction study in IgM gammopathy patients' serum.