Thesis advisor: Jianmin Gao / Thesis advisor: James P. Morken / This thesis includes two projects: "Novel perfluorinated aromatic amino acids: synthesis and applications" and "Thioflavin T dimers as novel amyloid ligands". I) Novel perfluorinated aromatic amino acids: synthesis and applications. Fluorinated amino acids serve as powerful tools in protein chemistry. Using the commercially available Boc-protected pentafluorophenylalanine, we synthesized a series of para-substituted tetrafluorophenylalanines via the regioselective SNAr reaction. These novel unnatural amino acids display useful and unique properties that can be applied to biological systems, including distinct 19F NMR signatures, pH-dependent amphiphilicity, lipid-binding selectivities, and halogen bonding capabilities. II) Thioflavin T dimers as novel amyloid ligands. Fluorescent molecules that specifically target amyloid structures are highly desirable for Alzheimer's disease research. We have designed a dimeric Thioflavin T that, through a reduced entropic penalty, has an improved binding affinity to Aβ amyloid by up to 70 fold. More importantly, the specificity and the "light-up" feature upon amyloid binding have not been sacrificed. Encouraged by the successful dimer design, we are further investigating the potential of amyloid-templated reactions to tailor-make ligands for amyloids. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

No / Beta-2-microglobulin (2m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (~1 m), straight morphology are produced by incubation of the protein at pH=<3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of 2m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states.

O presente trabalho tem como objetivo avaliar se o complexo Európio- Clorotetraciclina (EuCTc) ou o corante Tioflavina T (ThT) podem atuar como biossensores precisos e eficientes de colesterol numa fração específica, através de procedimentos simples. Para isso estudaram-se as propriedades ópticas do complexo EuCTc e ThT na presença da Lipoproteína de Baixa Densidade (LDL) em seu estado nativo e em seu estado oxidado. O primeiro estudo realizado verificou a melhor razão molar entre o Európio e a Clorotetraciclina, em seguida verificou-se a influência da diálise da LDL na emissão do complexo EuCTc, para obtenção de um protocolo para quantificação da concentração da LDL. Foram traçadas as curvas de calibração da emissão do complexo EuCTc com várias concentrações da LDL nativa, LDL oxidada com íons de Cobre e LDL oxidada com íons de Ferro. Em seguida obteve-se o tempo de vida do íon Európio no complexo EuCTc na presença de diferentes concentrações de LDL nativa e LDL oxidada por íons de Cobre. Na segunda parte do trabalho estudou-se a emissão do corante Tioflavina T na presença da LDL nativa e LDL oxidada. Na terceira etapa as propriedades ópticas dos biossensores EuCTc e ThT foram investigadas na presença do plasma sanguíneo e foram comparadas às emissões do complexo EuCTc e o corante ThT com a LDL ultracentrifugada, para verificar a possibilidade de quantificar a LDL diretamente no plasma sanguíneo. Na última etapa do trabalho desenvolveu-se uma nova metodologia para oxidar a partícula de LDL a partir da irradiação com laser de pulsos ultracurtos, a fim de produzir uma oxidação branda da partícula de LDL e controlada. / The present study aims at assessing the complex Europium-Clorotetracycline (EuCTc) or the dye Thioflavin T (ThT) can act as biosensors accurate and efficient in a specific fraction of cholesterol through simple procedures.For this purpose, it was studied the optical properties of Europium- Chlortetracycline (EuCTc) complex and the thioflavin T (ThT) dye in the presence of low-density lipoprotein (LDL) in native state and in oxidized state in vitro. First study realized it was verified the influence of dialysis in the emission of complex EuCTc in the presence of LDL, thereby producing a protocol for use of the complex to obtain the concentration of LDL. It was obtained the calibration curves of the complex with various concentrations of native LDL, the oxidized LDL with copper ions and oxidized LDL with iron ions. It was also obtained from the calibration curve of the emission of the complex in the presence of calcium interferent ion with the concentration found in blood plasma with the oxidized LDL with copper ions. It was obtained the lifetime of the europium ion in the complex in the presence of different concentrations of Native LDL and oxidized LDL by copper ions. In the second part of the work it was studied the emission of the dye thioflavin T in the presence of native LDL and oxidized LDL. In the third part the optical properties of biosensors EuCTc and ThT were investigated in the presence of blood plasma and compared to the emission of the complex EuCTc and the dye ThT with LDL ultracentrifugated to verify the possibility of quantifying directly LDL in blood plasma.In the final part of this work it has developed a new methodology for the LDL particles oxidation from the irradiation of ultrashort laser pulses in order to produce mild and controlled oxidation of the LDL particle.

Amyloid fibrils results from a type of ordered polypeptide aggregation that is associated with ailments such as Alzheimer's disease (AD). Annually, millions of people in the United States alone develop and die from AD. Therefore, it is necessary to understand not only the process of amyloid formation, but also the causes of this specific type of aggregation. This study used ABeta(25-35) since it is a fragment of the Alzheimer?s peptide that behaves like the full length peptide found in patients with AD. To study the process of amyloid formation, several methods were used so that a more complete picture of the stepped aggregation process could be realized. Several oligomeric species were detected and described many of which could not have been observed without using the complete battery of methods utilized here. The oligomeric species detected included a novel 'rolled sheet' that appeared to be the immediate precursor of amyloid fibrils, and two supermolecular species that appear after amyloid fibrils were formed. In determining the causes of amyloid formation, two significant discoveries were made. First, by partial sequence randomization, truncation, and Ala scanning mutagenesis, the critical amyloidogenic region of ABeta(25-35) was found to be residues 30-35. This critical core region is important because it is thought to be the region that initiates amyloid formation, therefore knowing the residues involved in the region is a useful tool for developing methods of fibril formation prevention. Second, by inserting all naturally occurring amino acids into position 34 of ABeta(25-35), three distinct classes of variants were observed and the effect of several physiochemical properties on amyloidosis were examined. Hydrophobicity, solubility, and ?-strand propensity were found to affect aggregation to the greatest extent. Also within these two studies, our results suggest that early oligomers are the cytotoxic species as opposed to amyloid fibrils or other larger macromolecular assemblies.

O presente trabalho tem como objetivo avaliar se o complexo Európio- Clorotetraciclina (EuCTc) ou o corante Tioflavina T (ThT) podem atuar como biossensores precisos e eficientes de colesterol numa fração específica, através de procedimentos simples. Para isso estudaram-se as propriedades ópticas do complexo EuCTc e ThT na presença da Lipoproteína de Baixa Densidade (LDL) em seu estado nativo e em seu estado oxidado. O primeiro estudo realizado verificou a melhor razão molar entre o Európio e a Clorotetraciclina, em seguida verificou-se a influência da diálise da LDL na emissão do complexo EuCTc, para obtenção de um protocolo para quantificação da concentração da LDL. Foram traçadas as curvas de calibração da emissão do complexo EuCTc com várias concentrações da LDL nativa, LDL oxidada com íons de Cobre e LDL oxidada com íons de Ferro. Em seguida obteve-se o tempo de vida do íon Európio no complexo EuCTc na presença de diferentes concentrações de LDL nativa e LDL oxidada por íons de Cobre. Na segunda parte do trabalho estudou-se a emissão do corante Tioflavina T na presença da LDL nativa e LDL oxidada. Na terceira etapa as propriedades ópticas dos biossensores EuCTc e ThT foram investigadas na presença do plasma sanguíneo e foram comparadas às emissões do complexo EuCTc e o corante ThT com a LDL ultracentrifugada, para verificar a possibilidade de quantificar a LDL diretamente no plasma sanguíneo. Na última etapa do trabalho desenvolveu-se uma nova metodologia para oxidar a partícula de LDL a partir da irradiação com laser de pulsos ultracurtos, a fim de produzir uma oxidação branda da partícula de LDL e controlada. / The present study aims at assessing the complex Europium-Clorotetracycline (EuCTc) or the dye Thioflavin T (ThT) can act as biosensors accurate and efficient in a specific fraction of cholesterol through simple procedures.For this purpose, it was studied the optical properties of Europium- Chlortetracycline (EuCTc) complex and the thioflavin T (ThT) dye in the presence of low-density lipoprotein (LDL) in native state and in oxidized state in vitro. First study realized it was verified the influence of dialysis in the emission of complex EuCTc in the presence of LDL, thereby producing a protocol for use of the complex to obtain the concentration of LDL. It was obtained the calibration curves of the complex with various concentrations of native LDL, the oxidized LDL with copper ions and oxidized LDL with iron ions. It was also obtained from the calibration curve of the emission of the complex in the presence of calcium interferent ion with the concentration found in blood plasma with the oxidized LDL with copper ions. It was obtained the lifetime of the europium ion in the complex in the presence of different concentrations of Native LDL and oxidized LDL by copper ions. In the second part of the work it was studied the emission of the dye thioflavin T in the presence of native LDL and oxidized LDL. In the third part the optical properties of biosensors EuCTc and ThT were investigated in the presence of blood plasma and compared to the emission of the complex EuCTc and the dye ThT with LDL ultracentrifugated to verify the possibility of quantifying directly LDL in blood plasma.In the final part of this work it has developed a new methodology for the LDL particles oxidation from the irradiation of ultrashort laser pulses in order to produce mild and controlled oxidation of the LDL particle.

Bovint β-laktoglobulinprotein (bLG) är ett litet globulärt protein med 162 aminosyrarester, som vanligtvis finns i mjölkvassle. Under sura förhällanden dissocierar dessa dimera proteiner och bildar amyloidliknande fibriller. Studien av β- laktoglobulinfibriller kan vara ett värdefullt verktyg för att förstå strukturen och dynamiken hos patogena amyloidproteiner och relaterade sjukdomar (t.ex. Alzheimers). Det är dessutom viktigt att förstå den korrekta formationen och elucideringen av dessa protein-nanofibriller (PNF) och deras sammansättningutgör också en grund för vidare design av nya biobaserade material. Således är framställningen av en signifikant homogen morfologi av nano-fibriller från bLG för proteinstrukturstudier huvudsyftet med detta projekt. Studien omfattar också användning av rekombinant β-laktoglobulin renat från Escherichia coli Origami (DE3) - celler. Omfattningen av bildandet av dessa PNF kan påverkas genom att variera experimentets olika förhållanden. Huvudsyftet med denna avhandling är att modifiera reaktionsparametrarna såsom inkubationstid, temperatur, koncentrationer, såningsanalyser och även hitta nya för att maximera homogeniteten hos den beredda PNF. En detaljerad analys av alla effekter av olika förhållanden på reaktionsprocessen och vilken provtyp eller beredningsprocess som leder till ökad mängd fibriller är huvudresultatet av denna avhandling. Detta kan i sin tur fungera som en bas för framtida modeller eller proteiner som kan användas för att få en bättre förståelse för amyloidrelaterade patologier eller andra associerade applikationer, till exempel i livsmedelsindustrin eller design av nya material. I slutet av studien visade det sig att den högsta mängden fibriller bildades för de prover som inkuberades vid 70 ℃, förvarades i 48 timmar, vid 300 rpm, med 10 % fröprov som sonikerades två gånger med ett intervall på 60 minuter . / Bovine β-lactoglobulin protein (bLG) is a 162 residue small globular protein, usually found in the whey component of milk. These dimeric proteins under acidic conditions and high temperatures dissociates and form amyloid-like fibrils. The study of bLG fibrils can be a valuable tool for understanding the structure and dynamics of pathogenic amyloid proteins and related diseases (e.g., Alzheimer’s). Also, proper formation and elucidation of these protein nano-fibrils (PNF’s) and their assembly provides a foundation for further design of new bio-based materials. Thus, producing a significant homogenous morphology of the nano-fibrils from bLG for protein structure study is the main objective of this project. The study involves also the use of recombinant β- lactoglobulin purified from Escherichia coli Origami (DE3) cells. The extent of formation of these PNF’s can be influenced by varying the different conditions of the experiment. The main aim of this thesis is to modify the reaction parameters such as the incubation time, temperature, concentrations, seeding assays and also find new ones so as to maximize the homogeneity of the prepared PNF. A detailed analysis of all the effects of different conditions on the reaction process and which sample type or preparation process leads to increase in the amount of fibrils is the main outcome of this thesis. This can in turn serve as a base for future models or proteins that can be used to gain a better understanding of amyloid-related pathologies or any other associated applications such as in food industries or design of new materials. In the end of the study, it was found that highest amount of fibrils were formed for those samples incubated at 70 ℃, kept for 48 hours, at 300 rpm, with the 10 % seed sample that was sonicated twice at an interval of 60 minutes.

La maladie d'Alzheimer est caractérisée par la présence de dégénérescences neurofibrillaires et l'accumulation de plaques amyloïdes dans le cerveau. Ces plaques contiennent principalement un peptide nommé amyloïde-β (Aβ) sous forme agrégée. Le processus d'agrégation des peptides Aβ en plaques amyloïdes représente une étape clé dans l'apparition de la pathologie, la coordination du cuivre, et également du zinc, favorisant la formation d'espèces agrégées impliquées dans la neurotoxicité. Notre objectif consiste à concevoir des complexes bifonctionnels avec d'une part un analogue de la Thioflavine T (ThT) et d'autre part un complexe de Ru(II), ce travail de thèse s'articule donc selon ces deux axes. I- Nous nous sommes d'abord intéressés à l'interaction entre le peptide Aβ et la Thioflavine T (ThT), fluorophore classiquement utilisé pour étudier l'agrégation du peptide Aβ. Cette interaction a été étudiée principalement par spectroscopie RMN. Les résultats obtenus ont permis d'identifier le site d'interaction de la ThT au peptide Aβ. Par la suite, les effets de la ThT et du Zn(II) sur l'agrégation du peptide Aβ ont été évalués en combinant la RMN et la spectroscopie de fluorescence. A partir des données obtenues, nous avons montré que la ThT et le Zn(II) ne sont pas inertes sur la cinétique d'agrégation du peptide Aβ. Les résultats ont également révélé des différences importantes concernant les informations apportées par la fluorescence et la RMN. II- La coordination du cuivre et du zinc implique principalement les noyaux imidazoles des résidus histidines. Afin d'empêcher la coordination de ces ions métalliques aux peptides Aβ, une stratégie thérapeutique innovante consiste en l'utilisation de complexes platinoïdes comportant des sites labiles et capables de se lier aux résidus histidines du Aβ. En raison de la toxicité des complexes de Pt(II), nous avons envisagé la synthèse de complexes de Ru(II), principalement basés sur le motif fac-Ru(CO)32+. Différents complexes avec des ligands de type glycinate, hydroxyquinolinate et éthylenediamine ont été synthétisés. L'étude de leur interaction avec le peptide Aβ a été réalisée par différentes techniques spectroscopiques (RMN, RPE, fluorescence, spectrométrie de masse). Les résultats obtenus ont montré, en particulier, que les complexes sont capables d'inhiber l'agrégation du peptide Aβ induite par le zinc. / The Alzheimer's disease is characterized by the presence of neurofibrillary tangles and amyloid plaques in the brain. These plaques are formed by aggregated amyloid-β (Aβ) peptide. The Aβ aggregation represents a key event in the appearance of the pathology, copper and zinc coordination favoring the formation of aggregated species involved in the neurotoxicity. Our objective consists in designing bifonctional complexes with, on one hand, a Thioflavine T (ThT) analog and, on the other hand, a Ru(II) complex : this thesis is thus centered around these two axes. I- In this context, we first investigated the interaction between Aβ and ThT, which is a classical dye commonly used to study the aggregation process. This interaction was mainly studied by NMR spectroscopy. Our first results allowed us to identify the interaction site of the ThT with the Aβ peptide. Then, the ThT and Zn(II) effects on the aggregation process were assessed by NMR and fluorescence spectroscopy. From the obtained data, we showed that ThT and Zn (II) are involved in the aggregation kinetic. The results also revealed important differences concerning the information brought by fluorescence and NMR. II- Copper and zinc coordination mainly implies imidazole ring of the histidine residues. In order to prevent the coordination of these metallic ions to Aβ, an innovative therapeutic strategy consists of the use of platinoid complexes containing labile sites which are able to bind the Aβ histidine residues. Because of Pt(II) complexes toxicity, we envisaged the synthesis of Ru(II) complexes, mainly based on fac-Ru(CO)32+ motive. Different complexes with glycinate, hydroxyquinolinate or ethylenediamine ligand were synthesized. The study of their interaction with the Aβ peptide was realized by various spectroscopy techniques (RMN, RPE, fluorescence, mass spectrometry and demonstrated that the complexes are able to prevent the Aβ aggregation induced by zinc.

Plaques of amyloid β peptide (Aβ) are a hallmark trait of Alzheimer’s disease (AD). However, the precise role of Aβ aggregates is not well understood. Recent studies have identified that naturally occurring N-terminal truncation and pyroglutamylation of Aβ significantly increases its neurotoxicity by an unknown mechanism. Content of pyroglutamylated Aβ (pE-Aβ) in AD brains has been shown to reach up to 50% of total Aβ. Modified pE-Aβ co-aggregates with Aβ by a seeding mechanism and forms structurally distinct and highly toxic oligomers. We studied structural transitions of the full-length Aβ1-42, its pyroglutamylated form AβpE3-42, their 9:1 (Aβ1-42/AβpE3-42) and 1:1 molar combinations. Transmission electron microscopy was used to directly visualize the fibrils of the samples in a buffer mimicking physiological environment. Atomic force microscopy measurements were done to determine rate of second nucleation events in fibrils. Thioflavin-T fluorescence indicated that low ionic strength suppressed the aggregation of AβpE3-42 but promoted that of Aβ1-42, suggesting different paths of fibrillogenesis of unmodified Aβ and pE- Aβ. Interestingly, AβpE3-42 at only 10% significantly facilitated the fibrillization of Aβ1-42 at near-physiological ionic strength but had little effect at low salt. Circular dichroism and Fourier transform infrared (FTIR) spectroscopy were used to characterize the structural transitions during fibrillogenesis. In aqueous buffer, both unmodified Aβ and pE-Aβ peptides adopted parallel intermolecular β-structure. Interestingly, AβpE3-42 contained lower β-sheet content than 13C-Aβ1-42, while retaining significantly larger fractions of α-helical and turn structures. Structural details of Aβ and pE-Aβ combinations were unveiled by isotope-edited FTIR spectroscopy, using 13C-labeled Aβ1-42 and unlabeled AβpE3-42. When exposed to environmental humidity, AβpE3-42 not only maintained an increased fraction of α-helix but also was able to reverse 13C-Aβ1-42 β-sheet structure. These data provide a novel structural mechanism for pE-Aβ hypertoxicity; pE-Aβ undergoes faster nucleation due to its increased hydrophobicity, thus promoting formation of smaller, hypertoxic oligomers of partial α-helical structure.

The nucleic acid determination in biological samples has been essential in the last decades for many analytical processes, such as polymerase chain reaction (PCR), genotyping, clinical diagnosis, genetically modified organism determination in food ingredients, fetal DNA maternal blood test, forensic applications and others. Absorbance measurements in the UV-vis region are frequently employed for nucleic acid quantifications. Nevertheless, the results are often approximated and measurement accuracy is limited. Thus, this work presents a low-cost and simple method for DNA and/or RNA determination employing Thioflavin T (TT) by molecular fluorescence. The reaction mechanism consists of the interaction of TT and DNA or RNA. This mechanism is based on the off-on concept in which the free TT in solution shows low fluorescence but when interacts with the nucleic acid there is a rotation restriction in the TT molecule, becoming rigid, which allows the alignment of π orbitals and consequently increases its fluorescence intensity. After the addition of DNA, TT showed a redshift (bathochromic effect) in the molecular absorption spectra, indicating that there is interaction with the macromolecule. Experimental conditions were optimized applying salmon test DNA as experimental model for nucleic acids (stDNA) in pH = 5, MES buffer solution (10 mM) and TT concentration of 5 μM. For the ionic strength studies, it was observed that increasing NaCl concentration (0 – 150 mM) the TT-DNA interaction process is disfavored, indicating that the interaction mechanism occurs through electrostatic attractions. Moreover, binding mode assays with competitors (ethidium bromide and berenil) were performed, showing that the second type of interaction occurs preferentially by groove. In those conditions, the probe was able to respond immediately to different nucleic acids, with the signal stable for at least 120 min. The proposed method presents a 0.1 – 1.5 mg L-1 linear range in stDNA, with a LOD of 0.012 mg L-1 relative standard deviation lower than 3.7 %. Applying RNA as a standard, the method shows linear range of 0.25 – 3.0 mg L-1, LOD of 0.073 mg L-1 and RSD lower than 3.2 %. Fe(III) ion and albumin and lysozyme proteins were the main interference species to the method. At last, the concentration of nucleic acids in a DNA sample extracted from blood plasma was determined (stDNA – 57 mg L-1 and ctDNA – 48,5 mg L-1 standards), moreover, it was performed a recovery assay applying ctDNA and stDNA in saliva samples whose recoveries in the range of 67 – 89%. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A determinação de ácidos nucleicos em amostras biológicas tem sido imprescindível nas últimas décadas para muitos processos analíticos, como reação de polimerase em cadeia (PCR), genotipagem, diagnóstico clínico, determinação de organismos geneticamente modificados em ingredientes alimentares, testes para determinação de DNA fetal em sangue materno, aplicações forenses, entre outros. Medidas de absorvância no UV-vis são comumente usadas para quantificação de ácidos nucleicos. Entretanto, os resultados obtidos são aproximados e a exatidão das medidas é limitada. Dessa forma, este trabalho apresenta um método simples e rápido empregando a Tioflavina T (TT) como sonda sensível e seletiva para determinação de DNA e/ou RNA através de fluorescência molecular. O mecanismo da reação consiste na interação da TT com DNA ou RNA. Este mecanismo se baseia no conceito off-on em que a tioflavina livre em solução apresenta baixa fluorescência, mas ao interagir com o ácido nucleico, ocorre restrição de rotação na molécula de TT, tornando-a planar e possibilitando o alinhamento dos orbitais π, desta forma, provocando aumento na intensidade de fluorescência. Após a adição de DNA, a TT mostrou um desvio para o vermelho (efeito batocrômico) nos espectros de absorção molecular, indicando que ocorre interação com a macromolécula. As condições experimentais foram otimizadas empregando DNA de salmão como modelo experimental de ácido nucleico (stDNA) em pH = 5, tampão MES (10 mM) e a concentração de TT em 5 μM. No estudo da força iônica observou-se que o aumento da concentração de NaCl (0 - 150 mM) desfavorece o processo de interação TT-DNA, indicando que a interação se dá por meio de atrações eletrostáticas. Além disso, realizou-se o estudo do modo de ligação com os competidores como brometo de etídio e berenil evidenciando que um segundo tipo de interação ocorre preferencialmente via groove. Nestas condições, a sonda respondeu a diferentes ácidos nucleicos (DNA e RNA) de forma imediata, sendo o sinal estável pelo menos até 120 min. O método proposto apresentou faixa linear de concentração de 0,1-1,5 mg L-1 em stDNA com limite de detecção (LOD) de 0,012 mg L-1 e desvio padrão relativo (RSD) menor que 3,7%. Quando se utilizou RNA como padrão a faixa linear foi de 0,25-3,0 mg L-1 com LOD de 0,073 mg L-1 e RSD menor que 3,2 %. Os íons Fe(III) e as proteínas albumina e lisozima foram os principais interferentes do método. Por fim, determinou-se a concentração de ácidos nucleicos em uma amostra de DNA extraído de plasma sanguíneo (padrão stDNA – 57 mg L-1 e ctDNA – 48,5 mg L-1), além disso, também foi realizado o ensaio de recuperação empregando ctDNA e stDNA em amostras de saliva, no qual obteve-se recuperações na faixa de 67-89%.

<p>Therapeutic peptides have become a clinically and commercially important drug class providing novel treatment options in variety of disease areas. Today, more than 80 peptide drugs are marketed worldwide and hundreds more are in development. However, the development of peptide drugs can be hindered by their tendency to self-associate to form fibrils, an impurity that can affect potency and increase the potential for adverse immune responses in patients. Fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. From a pharmaceutical development perspective, early detection of instabilities can inform the development of mitigation strategies to minimize the risk of product failure and avoid costly delays in clinical development. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and formulations.</p> <p>The objective of this dissertation was to develop structurally modified fibrillation-resistant peptides based on a mechanistic understanding of the fibrillation process. The therapeutic peptides studied were human calcitonin (hCT), a glucagon/GLP-1 analog, and human insulin B-chain (INSB). Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) and other biophysical methods were used to provide mechanistic understanding of the intermolecular interactions and structural transitions during peptide fibrillation. Coupled with proteolytic digestion, pulsed HDX-MS of fibrillating peptides enabled identification of the residues involved in the early interactions leading to fibrillation based on their differential deuterium exchange rates. The high-resolution residue level information was used to make site-specific modifications to hCT, with phosphorylation in the central region resulting in complete inhibition of fibrillation for the phospho-Thr-13 hCT analog under the stress conditions employed. Reversible ‘prodrug’ modifications such as phosphorylation can aid the rational design of fibrillation-resistant therapeutic peptides. Furthermore, the effects of structural modifications on peptide fibrillation were evaluated by reducing the Cys1-Cys7 disulfide bond in hCT, and by C-terminal amidation or substitution with a helix-stabilizing residue (α-aminoisobutyric acid, Aib) in the glucagon/GLP-1 analog peptide. Finally, studies of insulin B-chain probed fibrillation mechanisms of this therapeutically important peptide, contributing to our understanding of the mechanisms of insulin fibrillation with the broad goal of developing fibrillation-resistant, rapid-acting, monomeric insulin analogs. Overall, the results demonstrate that small structural changes can have significant effects on peptide fibrillation, that pulsed HDX-MS can be used to probe these effects, and that an understanding of these effects can inform the rational development of fibrillation-resistant peptide drugs. </p>