Cyclic dipeptides have limited conformational freedom due to their diketopiperazine backbone and their small size. They are relatively simple to synthesise, making them ideal subjects for investigation into their biological effects. Cyclic dipeptides have also been known for their multitude of biological activities, including antimicrobial, anticancer and haematological properties. In this study the cyclic dipeptides, cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro), were synthesised from their corresponding linear precursors using a modified phenol-induced cyclisation procedure. The phenol induced cyclisation procedure resulted in good yields and purity of the cyclic dipeptides. Quantitative analysis and evaluation of the physiochemical properties of the cyclic dipeptides was achieved using high-performance liquid chromatography, scanning electron microscopy, thermal analysis and X-ray powder diffraction. Structural elucidation of the cyclic dipeptides was done by means of infrared spectroscopy, mass spectroscopy, nuclear magnetic resonance spectroscopy and molecular modelling. The study‟s aim was to determine the biological activity of cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro) with respect to their anticancer, antimicrobial, haematological and ant-diabetic studies. Anticancer studies revealed that cyclo(Phe-4Cl-Pro) and cyclo(D-Phe-4Cl-Pro) inhibited the growth of HeLa (cervical cancer), HT-29 (colon cancer) and MCF-7 (breast cancer) cancer cell lines. Both cyclic dipeptides also inhibited the growth of certain selected Gram-positive, Gram-negative and fungal microorganisms in the antimicrobial study. Although the inhibition of growth in the anticancer and antimicrobial studies was statistically significant, the clinical relevance is questionable, since the inhibition produced by both cyclic dipeptides was very limited compared to other pre-existing anticancer and antimicrobial agents. Both cyclic dipeptides caused a significant shortening of the APTT and PT clotting times and an increase in the fibrin and D-Dimer formation. Cyclo(D-Phe-4Cl-Pro) at a screening concentration of 12.5 mM and 3.125 mM, showed significant anti-platelet activity. Both cyclic dipeptides failed to produce any inhibition of the α-Glucosidase enzyme and very limited inhibition of the α-Amylase enzyme.

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This thesis targets cyclic peptide supramolecular structures for biomaterial applications. The introduction gives a brief insight into supramolecular interactions, peptides, and their application in biomaterials. These supramolecular interactions range from the weak forces of electrostatics and van der Waals interactions, to hydrogen bonding and metal-coordination. The application of peptides and supramolecular interactions has become a highly studied area of chemistry, which has quickly gotten attention in the area of biomaterials. The use of peptides in biomaterials seems obvious since in vivo rejection of this material might be limited. Nature can be used as a blue print to direct the path for hydrogen bonding motifs and metal-coordinating interactions and can be applied potentially towards supramolecular biomaterials. Finally, the introduction reviews the use of cyclic peptides and accounts for the synthetic design of the cyclic octapeptide to be used throughout the thesis work. The second chapter of the thesis provides the details by which the synthetic scheme for creating the linear peptides of interest and ultimately the cyclic peptides is described in detail. Many synthetic challenges were met and overcome during this thesis work; the most notable was overcoming purification challenges and poor amino acid coupling reactions that resulted in low yields. This thesis focuses primarily on the di-substituted pyridylalanine cyclic octapeptide, however much of the initial work on the mono-substituted cyclic octapepide was carried out in tandem allowing for comparison of the two peptides necessary for future work.

Cyclic peptides have potential as scaffolds for novel pharmaceuticals, however their chemical synthesis can be challenging and as such natural sources are often explored. Several species of cyanobacteria produce a family of cyclic peptides, the cyanobactins, through the ribosomal synthesis of precursor peptides and post-translational tailoring. The patellamides, a member of the cyanobactin family, are cyclic octapeptides containing D-stereo centres and heterocyclised amino acids. A single gene cluster, patA - patG, contains the genes for the expression of the precursor peptide and the enzymes responsible for post-translational modifications including a heterocyclase, protease, macrocyclase and oxidase. Biochemical and structural analysis on the patellamide and related cyanobactin pathways has been carried out. The crystal structure of PatF, a proposed prenyl transferase, has been determined, highlighting that it is likely evolutionary inactive due to changes to key residues when compared to active homologues. This is in agreement with the knowledge that no naturally prenylated patellamides have been discovered to date. The crystal structure of the macrocyclase domain of PatG has been determined in complex with a substrate analogue peptide. The structure, together with biochemical analysis has allowed a mechanism of macrocyclisation to be proposed, confirming the requirement of a specific substrate conformation to enable macrocyclisation. Using isolated enzymes from the patellamide and related pathways, a small scale library of macrocycles made up of diverse sequences has been created in vitro and characterised by mass spectrometry and in certain cases NMR. In order to further enhance diversity, macrocycles containing unnatural amino acids have been created using three approaches; SeCys derived precursor peptides, intein-mediated peptide ligation and pEVOL amber codon technology. Finally, two oxidase enzymes from cyanobactin pathways have been purified, characterised and confirmed active for thiazoline oxidation. Native X-ray datasets on crystals of the oxidase CyaGox have been collected and phasing trials are on-going.

The chromosome passenger complex (CPC) is an essential mitotic regulator with key roles in mitotic processes such as chromosome condensation, spindle dynamics, chromosome bi-orientation, the spindle checkpoint and cytokinesis. The Aurora B kinase is the CPC’s catalytic subunit. Its targeting and activation are dependent on interactions with the other components of the complex: inner centromere protein (INCENP), survivin and borealin/Dasra B. INCENP serves both as a scaffolding subunit for the CPC as a whole and as an activator of Aurora B via its highly conserved INbox domain. Aurora B is a putative anti‐cancer target; several inhibitors of the kinase are currently in clinical trials. All these are ATP-analogues targeting the kinase active site. The protein-­protein interaction between Aurora B and the INCENP INbox is also essential for CPC function. Earlier studies have demonstrated that INCENP INbox mutants unable to bind and/or activate Aurora B cannot rescue lethality in the absence of endogenous INCENP. The first goal of this study was to test the in vivo effects of disrupting the interaction between endogenous wild type INCENP and Aurora B. For this, a cell-based CPC function assay was developed in HeLa cells. Using this assay, I show that expression of soluble INbox in HeLa cells produces a significant increase in multinucleated and micronucleated cells: both effects consistent with Aurora B loss of function. Expression of soluble INbox bearing the mutations W845G and/or F881A does not elicit this effect suggesting that those mutants cannot bind to Aurora B and occlude INCENP binding. The result concerning the F881A mutant contrasts with earlier reports that equivalent mutants could bind, but not activate, Aurora B. Expression of an INbox mutant lacking the C-­terminal TSS motif reported to be involved in Aurora B activation but not binding has effects similar to those of the wild type INbox. Using the INbox/Aurora B interaction as a model, a secondary goal of this study was to develop and evaluate a novel approach to identify small peptides capable of dissociating intracellular protein‐protein interactions. For this, a library of small (5-­9 residues long) circular peptides (CPs) mimicking the INbox was generated using the split intein circular ligation of proteins and peptides (SICLOPPS) methodology and assayed using the cell-­based CPC function assay. Over two successive rounds of screening, a small number of CPs were identified that caused a significant increase in rates of multinucleated and micronucleated cells. Although statistically significant, these increases were very modest. Furthermore, due to high heterogeneity in SICLOPPS processing efficiencies, it was not practicable to compare the effects of different peptides side-­by-side by transfection. The level of variation in processing efficiency – thus, CP production – was unexpectedly high and puts into question the functional complexity of more commonly used combinatorial cyclic peptide libraries derived using current SICLOPPS methodology. The results of this study are divided into three sections. The first is a methods section concerning the testing of SICLOPPS in HeLa cells and the development of a cell­‐based CPC function assay. In the second, the effects of expressing soluble INbox and mutants thereof in HeLa cells are presented. The final results section presents the results of the feasibility study of the rationally-­designed genetically encoded library approach.

L'identification de nouveaux agents anti-infectieux, actifs contre les pathogènes et les micro-organismes multi-résistants, reste un enjeu majeur pour la science. Parmi les molécules développées pour combattre ces infections, les peptides thérapeutiques apparaissent comme un champ prometteur de recherche. Ils se synthétisent rapidement, grâce à la synthèse sur support solide automatisée, et leur structure modulable facilite la découverte et l'amélioration d'activités biologiques. Le principal inconvénient des peptides est leur manque de résistance face à la dégradation protéolytique, et donc leur rapide élimination du corps humain. L'introduction d'aminoacides modifiés, comme les aza-bêta³-aminoacides, au sein de la séquence peptidique, permet de renforcer la biodisponibilité de ces peptides, et peut conduire à une augmentation de l’activité biologique et/ou de la sélectivité. Les aza-bêta3-aminoacides sont des analogues aza des bêta³-aminoacides, où le carbone portant la chaîne latérale est remplacé par un atome d'azote chiral à configuration non-fixée. Introduit au sein d'une séquence peptidique, ces monomères donnent accès à des pseudopeptides dont la biodisponibilité est augmentée, et l’activité et/ou la sélectivité peuvent être améliorée. Ce mémoire de thèse présente la synthèse et la fonctionnalisation d’aza-bêta³-aminoacides, à chaînes latérales protéinogènes ou non, en vue de leur insertion en synthèse peptidique sur support solide. Deux séries de pseudopeptides cycliques de novo ont été développées. La première série cible les vésicules d’endocytose résultant d’une infection adénovirale, et la seconde série mime la séquence RGD, ligand des intégrines alpha-nu-bêta₃, qui est une cible d’intérêt contre la néo-angiogénèse tumorale. / Discovering new anti-pathogenic agent, which are effective against new or multi-drug resistant microorganisms, is still a major challenge for science. Among all the drugs, which are currently developed to fight these infections, therapeutic peptides arise as a promising research field. Their synthesis is fast, due to automated solid phase synthesis, and their adjustable structure makes the discovery and the enhancement of biological activities easier. The main drawback of peptides is their lack of resistance against proteolytic degradation, and therefore their quick elimination from the human body. Modification of peptide sequence, by introduction of aminoacids analogues, such as aza-bêta3-aminoacids, reinforces the peptide bioavailability, and can lead to an increase of the biological activity, and/or of the selectivity. Aza-bêta3-amino acids are aza analogues of bêta3-amino acids, where the side chain is carried by a chiral nitrogen atom, with a non-fixed configuration. Their introduction in a peptide sequence affords pseudopeptides, with a better bioavailability, and with an activity/selectivity which could be increased. This report describes the synthesis of aza-bêta3-aminoacids, with proteinogenic side chains or not, in order to insert them in solid phase peptide synthesis. Two sets of cyclic pseudopeptides de novo have been developed. The first one targets endocytosis vesicles, resulting from an adenoviral infection, and the second one copies the RGD sequence, ligand of alpha-nu- bêta₃ integrins, which is one the main targets against the tumorous neo-angiogenesis.

Thesis advisor: Marc Snapper / Thesis advisor: Steven Bruner / Peptide natural products have diverse, elaborate scaffolds and are important leads in the development of new drugs. A complete understanding of the natural biosynthetic pathways of these compounds can improve chemical syntheses and boost bioengineering efforts. There are two classes of peptide natural products: ribosomal and nonribosomal peptides. Ribosomally produced and posttranslationally modified peptides (RiPPs) are produced by the ribosome using the 20 canonical amino acids and undergo extensive tailoring to yield the active natural products. Nonribosomal peptides (NRPs) are assembled through an enzyme dependent system and can incorporate over 500 different amino and acyl building blocks to impart complexity. These peptides can also undergo additional tailoring to further modify the core peptide. The microviridins are a class of RiPPs that are modified by two ATP dependent ligases to create a total of three macrocyclic bonds. We have solved the three dimensional protein structures of each of these ligases to establish the mechanism of substrate recognition and cyclization. Vancomycin is a NRP that contains five nonproteinogenic aromatic amino acids that are necessary for biological activity. One of these amino acids is derived from a polyketide pathway and undergoes a four-electron oxidation by a cofactor independent dioxygenase, DpgC. We have solved the structure of this enzyme and have established a radical mechanism. We have investigated this mechanism using synthetic probes and mutagenesis. We have examined O₂ binding using molecular dynamics and mutagenesis. NRPs are synthesized by the multidomain, modular nonribosomal peptide synthetases (NRPSs) in an enzyme templated, ATP-dependent manner. We have synthesized domain specific probes to study the structures and mechanisms of these pathways. Our continued work will provide the insight necessary to manipulate these pathways to provide biologically active compounds. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Honors Project--Smith College, Northampton, Mass., 2010. / Includes bibliographical references (p. 102-105).

Although peptides and proteins are considered as lead compounds for the discovery and development of new therapeutic agents, poor metabolic and physical properties have limited their optimisation as drug candidates (Adessi & Soto, 2002). Research by medicinal chemists however, generated the discovery of structural similarities between some peptides and diketopiperazines and the common occurrence of such compounds in natural products. This discovery initiated the synthesis of diketopiperazines from amino acids in an attempt to bypass the previously mentioned limitations of using peptides as drug candidates (Dinsmore & Beshore, 2002). Diketopiperazines (DKPs) are the simplest form of cyclic dipeptides, and a class of unexplored bioactive peptides that have great potential for the future. The compounds are relatively simple to synthesise and are prevalent in nature (Prasad, 1995). The DKP backbone is rigid and therefore poses conformational constraint on the compounds. This rigidity allows for simple conformational analysis of the compounds and also gives insight into the conformational requirements for interaction with the targets involved in their biological activity. The reduced conformational freedom also increases the receptor specificity and thus the compounds are proposed to have less unfavourable effects (Anteunis, 1978). The aim of the study was to synthesise compounds that would exhibit metabolic stability, receptor specificity and enhanced lipophilicity which would increase the bioavailability of the compounds. This was to be achieved by the introduction of fluorine and chlorine elements into the DKPs. The structure of the DKPs would be altered which in turn would improve the physicochemical properties and the biological activity of the compounds (Naumann, 1999). Cyclo(D-Phe-2Cl-Pro) and cyclo(Phe-4F-Pro) were synthesised using the method of Milne et al. (1992) and by boiling the linear counterparts under reflux in sec-butanol-toluene. The structures of the synthesised DKPs were elucidated using mass spectrometry, nuclear magnetic resonance spectroscopy, infrared spectroscopy and molecular modeling. Qualitative analysis and evaluation of the physicochemical properties of the DKPs were performed using high-performance liquid chromatography, scanning electron microscopy, thermogravimetric analysis, differential scanning calorimetry and x-ray powder diffraction. The study aimed to determine the biological activity of cyclo(D-Phe-2Cl-Pro) and cyclo(Phe-4F-Pro) with respect to their anticancer, antimicrobial, haematological and antidiabetic effects. The anticancer results obtained indicated that the percentage inhibition produced by both DKPs were lower than those proposed by Graz et al. (2000) for proline-containing DKPs where, a greater than 50% inhibition was observed for cyclo(Phe-Pro). Antimicrobial studies revealed that both DKPs demonstrated marginal effects on Gram-positive and Gram-negative organisms but showed significant effects against C. albicans. The haematological studies revealed that cyclo(D-Phe-2Cl-Pro) at a screening concentration of 12.5 mM, significantly decreased the levels of D-dimer (P < 0.0001). The antidiabetics studies showed limited activity of the DKPs in inhibiting the activity of α-glucosidase and α-amylase enzymes.