Cyclic peptides possess desirable characteristics as potential pharmaceutical scaffolds. The cyanobactin family of cyclic peptide natural products boast diverse structures and bioactivity. Exemplars are the patellamides, which have attracted attention due to their ability to reverse the effects of multi-drug resistance in human leukemia cells. In addition to their macrocyclic architecture patellamides contain azol(in)e heterocycles and d-amino acids. This structural complexity makes them challenging targets for chemical synthesis. Understanding their biosynthesis will enable the development of a biotechnological ‘toolkit' for the synthesis of new pharmaceutical compounds. Patellamides are ribosomally-synthesised and post-translationally modified peptides (RiPPs) and much of their biosynthesis has been elucidated, however there are still elements of their biosynthesis that are not yet fully understood. PatA and PatG contain C-terminal domains of unknown function (DUFs). The crystal structure of PatG-DUF has been solved and subsequent to biochemical and biophysical investigation PatG-DUF was found not to constitute an essential part of the biotechnological ‘toolkit' and can be excluded from in vitro enzyme-based synthesis of cyanobactin-like cyclic peptides. The cyanobactin heterocyclases are able to introduce heterocycles into a peptide backbone, seemingly irrespective of the neighbouring residues; however a molecular rational governing substrate recognition is unknown. Additionally the mechanism of heterocyclisaton is disputed. Analysis of crystal structures of LynD in complex with cofactor and substrate (solved by Dr Jesko Koehnke) enabled the active site and substrate recognition site to be located. A new mechanism for heterocyclisation has been proposed. Guided by the substrate recognition observed in complex structures a constituently active heterocyclase (AcLynD) has been engineered, which is able to process short, leaderless peptide substrates. Epimerisation in cyanobactin biosynthesis is believed to be spontaneous, but its precise timing is uncertain. NMR analysis of selectively labelled peptide substrates processed by the modifying enzymes, identified epimerisation to be spontaneous on the macrocycle, regardless of whether the neighbouring heterocycles have been oxidised. A one-pot in vitro synthesis of cyanobactins has been developed, and employed to create a number of patellamide D analogues to ascertain structural-activity relationships.

Le travail présenté dans ce mémoire est consacré à la synthèse de cycles pseudopeptidiques construits à partir d'aza-β³-aminoacides, et à la détermination des conformations adoptées par ces cycles. Le travail réalisé a permis de montrer que les cycles obtenus à 8, 16 et 24 liaisons adoptent des conformations privilégiées dans lesquelles la configuration relative des atomes d'azote chiraux est fixée en dépit du phénomène d'inversion pyramidale associé à la structure électronique de cet élément chimique, en réponse à des contraintes structurelles qui varient selon la taille du macrocycle. Ces cycles existent alors sous la seule forme de deux invertomères en équilibre. La constante de vitesse de cet équilibre, qui est indiscernable de la barrière d'inversion pyramidale des atomes d'azote, est maintenue à des valeurs étonnamment faibles par les contraintes conformationnelles. L'étude de ces macrocycles originaux dans le domaine de la chiralité a permis d'apporter en particulier des résultats nouveaux concernant l'influence de l'encombrement stérique des chaînes latérales sur la vitesse d'inversion pyramidale des atomes d'azote, mais aussi sur le transfert de chiralité d'éléments d'asymétrie exocycliques vers la séquence chirale du squelette, et enfin de montrer également l'intérêt des nouveaux cycles à 8 chaînons à travers l'étude de leur conformation. / The work depicted here is devoted to the synthesis of pseudopeptides built from aza-β³-aminoacid units, and to their conformational analysis. The results show that the cycles with 8, 16, and 24 bonds each adopt a ground conformation where the relative configuration of the chiral nitrogen atom is fixed in response to specific structural constraints, and despite the nitrogen pyramidal inversion phenomenon. The cycles just undergo equilibrium between two invertomeric forms, and the energetic barrier associated with the macrocycle inversion reveals surprisingly slow considering the size of the compounds. The influence of steric crowding of the side chains on the inversion rate has been carefully studied, but also the transfer of chirality from exocylic elements towards chirotopic nitrogen atoms inside the backbone. A specific chapter is devoted to the 8-membered rings, that reveal the interest of these newly described compounds in the domain of nitrogen chirality.

Dans le contexte de la chimiothérapie, la réparation de l’ADN réduit les dommages induits par les agents alkylants de l’ADN dont le témozolomide (TMZ), conduisant à la chimiorésistance. Une des voies principales de réparation de l’ADN est la voie par excision de base (BER) au sein de laquelle une enzyme clée, APE1 (endonucléase AP 1), clive les sites abasiques générés suite aux traitements par les agents alkylants et initie la réparation de la coupure simple-brin. Ce mécanisme représente une source majeure de chimiorésistance dans certains cancers. Plusieurs études ont ainsi validé la voie BER et plus particulièrement APE1 comme une cible importante dans le but d’améliorer l’efficacité des agents anticancéreux; pour ces raisons, de nombreux inhibiteurs d’APE1 ont été développés. Cependant, à la place d’une inhibition directe de l’enzyme, une stratégie alternative consiste à cibler le substrat de cette dernière : les sites abasiques. Les composés macrocycliques de type naphtalénophane ont montré la capacité à se lier fortement et sélectivement aux sites abasiques. Ce processus interfère avec la reconnaissance de ces derniers par APE1 et conduit in vitro à deux effets : l’inhibition du clivage enzymatique d’APE1 et le clivage du site AP par les macrocycles par un mécanisme différent de celui d'APE1, de type β-élimination. Ainsi, une nouvelle série de naphtalénophanes fonctionnalisés, composée de neuf nouveaux dérivés, a été synthétisée et étudiée. La plupart des macrocycles démontre la capacité à se lier fortement et sélectivement aux sites abasiques de l’ADN ainsi qu’à inhiber l’activité d’APE1 in vitro, avec des constantes d’inhibition s'étalant de 39 nM à 25 µM. De plus, l’activité d’inhibition d’APE1 par les ligands, caractérisée par les valeurs de Kı, a pu être corrélée avec leur affinité et leur sélectivité pour les sites abasiques. La structure moléculaire des macrocycles montre une forte influence sur l’activité de clivage de ces derniers pouvant conduire à une abolition ou à une très haute activité de clivage des sites abasiques. De façon intéressante, la formation d’un adduit covalent ADN – ligand avec un des macrocycles a été caractérisée. Enfin, l’activité biologique des naphtalénophanes sur la lignée cellulaire de glioblastome T98G résistante au TMZ a été étudiée. La plupart des ligands affiche une cytotoxicité élevée, avec des GI₅₀ de l’ordre du micromolaire. De plus, un remarquable effet synergique lors du traitement des cellules avec le TMZ et le MMS en combinaison avec un ligand (2,7-BisNP-O4Me) a été démontré. Ce macrocycle augmente également le nombre de sites abasiques et le nombre de coupures double-brins après un co-traitement cellulaire avec les agents alkylants suggérant ainsi l'inhibition d'APE1 attendue. Ces résultats mettent ainsi en évidence le fort intérêt thérapeutique de ce composé. / In the context of chemotherapy, DNA repair reduces the DNA damage induced by DNA-alkylating drugs such as temozolomide, leading to chemoresistance. One of the most important pathways of DNA repair is Base Excision Repair (BER), where a key enzyme, APE1 (AP endonuclease 1), cleaves abasic sites generated following treatment with DNA-alkylating drugs and initiates the repair of the single-strand break. The DNA repair activity of APE1 was identified as the major source of chemoresistance in certain cancers. Several studies validated the BER pathway and, particularly, the APE1 enzyme as important drug targets for improvement the efficacy of anti-cancer drugs; for this reason, several APE1 inhibitors have been developed. However, instead of direct inhibition of the enzyme, an alternative strategy can rely on targeting its substrate: the AP sites in DNA. Macrocyclic compounds, namely naphthalenophanes, show a strong and selective binding to abasic sites in the DNA. This process interferes with the recognition of the latter by APE1 and leads in vitro to two effects: inhibition of the APE1-induced DNA cleavage and macrocycle-induced DNA cleavage by a mechanism different from that of APE1, namely β-elimination. Herein, a novel serie of functionalized naphthalenophanes, composed of nine novel derivatives, has been synthesized and studied. Most ligands demonstrate a strong and selective binding to AP-sites in DNA and an inhibition of APE1 activity in vitro, with inhibitory constants from 39 nM to 25 µM. Moreover, the inhibitory activity of ligands, as characterized by Kı values, could be directly related to their affinity and selectivity to AP-sites. The molecular design of macrocycles has a crucial influence on their intrinsic AP-site cleavage activity leading either to total abolition, or to an exceptionally high AP-site cleavage activity. Interestingly, an unprecedented formation of a covalent DNA-ligand adduct with one of the ligands have been characterized. Finally, the biological activity of naphthalenophanes was assessed in the TMZ-resistant glioblastoma cell line T98G. Most compounds are highly active, with GI₅₀ values in sub-micromolar or low-micromolar range. In addition, a remarkable synergic effect upon co-treatment of TMZ or MMS with one ligand (2,7-BisNP-O4Me) was demonstrated. This ligand was found to increase the number of AP-sites and the number of double-strands break in DNA upon co-treatment with TMZ and MMS suggesting APE1 inhibition as excepted. These observations highlight the hight therapeutic interest of this compound.

The time-dependent visible spectra and the crystal structure of [Fe2(C20H24N8O2)(CH3CN)4]·PF6 (diketo-dimer) were studied. The spectra showed that the most significant chemistry occurred during the initial 1.5 hours of the synthetic reaction. The starting materials 343 nm peak shifted to a lower energy, at 360 nm, and a new shoulder appeared at 490 nm. This change suggests the formation of a new intermediate whose spectrum has an exceptional resemblance to the starting materials mixed valent species, [Fe2(TIED)(Cl)4]+1 (TIED = tetraiminethylene dimacrocycles). Two isosbestic points were found at 538 and 371 nm. The diketo-dimer's crystals appear to have individual colors, a physical characteristic called pleochroism. Pleochroism is a topic in the study of optical crystallography which is discussed and applied to the diketo-dimer. The extinction angle was estimated to be 14°, a value consistent for triclinic crystals. X-ray crystallography found that the diketo-dimer is triclinic, and has a space group of P-1. A noteworthy feature is the bond length, 1.406 Å, between the two linking bridgehead carbons. This bond length matches the value for partial double bonds of aromatic compounds. This argues for a delocalized electron circulating within the macrocycle. The NMR spectra of a diiron macrocycle, [Fe2(TIED)(CH3CN)4]4+, were examined. Temperature dependent, pH dependent, D+ substitution, selectively decoupled, and COSY 1H NMR experiments were performed. Two sets of structural equilibria were found. One set is temperature dependent, and the other is pH dependent. Of particular interest are the peaks centered at 9.7 ppm and assigned to the imine carbon protons H2. Its resonance indicates an imine proton in an extensively conjugated aromatic environment with an electron deficient metal.

No description available.