Several methods have been used for the characterisation of minerals in powders and in rocks. Particular emphasis has been directed towards the use of chemical probes to elucidate the surface mineralogy in order to provide detection profiles suitable for both qualitative and quantitative chemometric analysis. The first probe evaluated was cyclohexylamine (CHA). Ni[2+]- and Al[2+]-exchanged montmorillonite (SWy-2 -Wyoming, USA), were exposed to cyclohexylamine vapour (CHA). The samples were characterised by X-ray diffraction (XRD), thermogravimetric analysis (TGA) and evolved gas analysis (EGA), which included TGA, real time gas phase FTIR (RT-GP-FTIR), real time mass spectrometry (RT-MS) and organic trap module-gas chromatography-mass spectrometry (OTM-GC-MS). CHA decomposed along different routes via interrelated mechanisms that were cation dependent. Over Al[3+]-SWy-2 hydrodenitrogenation via the Hofmann degradation was the predominant route whereas ring dehydrogenation to aniline either directly from CHA or possibly via cyclohexylimine was significant over Ni[2+]-SWy-2. Very little unmodified cyclohexylamine was detected in the evolved gases which means that the desorption of CHA cannot be used as a quantitative measure of the acidity of cation-exchanged clays. Nonetheless, as a qualitative probe, CHA has the potential to distinguish the difference between Ni[2+] and Al[3+]-SWy-2.The second probe evaluated was 3-cyanopyridine (3CYP). TGA has shown that temperature programmed desorption (TPD) of 3CYP is capable of distinguishing between the Ni[2+]- and Al[3+] exchanged forms of SWy-2. 3CYP was found to be unsuitable for a variety of reasons highlighted by its ability to condense within the EGA system and the long incubation times necessary to produce detectable thermal events. The most suitable probe used so far is Dimethylformamide (DMF), being able to intercalate quickly and showing no evidence of modification. Principal component analysis (PCA) and partial least squares (PLS) modelling techniques were applied to the EGA DMF detection profiles of four clay mineral groups. Each group was successfully characterised and provided valuable information about intra and inter group relationships. The use of RT-MS detection profiles for ions m/z =18 and 73 (DMF parent ion) were shown to have the most potential for more sensitive quantitative applications. Application of quantitative PLS modelling to mixed mineral standards has shown that it is possible to estimate the clay mineral constituents in both powders and rocks. The use of the ion m/z = 73 provided an R[2] value of 0.98. Use of ion m/z =18 (H[2]O) has perhaps shown most potential, due to its ability to produce training sets with excellent correlation coefficients of 0.95 for SWy-2, 0.97 for KGa-2 and 0.95 for CCa-1. However, because SWy-2 and 1-Mt (illite) demonstrate overlapping detection of DMF (where uptake of DMF by 1-Mt is comparatively small), it is impossible to directly separate their relative contributions at the concentration levels under study. The use of mass spectrometry has shown that it is possible to detect to levels of 1% with considerable scope for detecting smaller levels. Environmental scanning electron microscope (ESEM) and energy dispersive X-ray analysis (EDX) of cation exchanged SWy-2 and a sandstone rock exposed to 2-bromopyridine (2BPY) have shown the potential of using the bromine functional group as a tag to locate the presence of swelling minerals in rocks by EDX.

Thesis advisor: Jianmin Gao / Thesis advisor: Eranthie Weerapana / Cysteine residues are known to perform an array of functional roles in proteins, including nucleophilic and redox catalysis, regulation, metal binding, and structural stabilization, on proteins across diverse functional classes. These functional cysteine residues often display hyperreactivity, and electrophilic chemical probes can be utilized to modify reactive cysteines and modulate their protein functions. A particular focus was placed on three peptide-based cysteine-reactive chemical probes (NJP2, NJP14. and NJP15) and their particular biological applications. NJP2 was discovered to be an apoptotic cell-selective inhibitor of glutathione S-transferase omega 1 and shows additional utility as an imaging agent of apoptosis. NJP14 aided in the development of a chemical-proteomic platform to detect Zn2+-cysteine complexes. This platform identified both known and unknown Zn2+-cysteine complexes across diverse protein classes and should serve as a valuable complement to existing methods to characterize functional Zn2+-cysteine complexes. Finally, NJP15 was part of a panel of site-selective cysteine-reactive inhibitors of protein disulfide isomerase A1 (PDIA1). These inhibitors show promise in clarifying the unique and redundant properties of PDIA1's dual active-sites, as well as interrogating the protein's role in cancer. Together, these case studies illustrate the potential of cysteine-reactive chemical probes to modulate protein activities, interrogate biological systems, and aid in the development of powerful therapeutic drugs. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

The primary objective of this DPhil research project was to develop selective and cell-active inhibitors of the histone lysine demethylase KDM2A, which could potentially lead to the discovery of a novel chemical probe. Chapter one of this thesis introduces the role of histone lysine demethylases (KDMs) in the epigenetic regulation of gene expression and discusses the value of chemical probes as tools to study these enzymes. Chapter two describes the synthesis of a library of indoline-based KDM2A inhibitors using a modular synthetic approach to explore key structure-activity relationships and a chiral counterion-mediated strategy to synthesize lead candidates enantioselectively. Chapter three discusses investigations into the cellular activity of lead compounds and explores strategies to address limitations associated with cytotoxicity and promiscuity. Chapter four describes the application of a variety of experimental techniques to identify the mode of target inhibition. Finally, chapter five focuses on the development of an enantioselective C-acylation reaction to access spirocyclic fragments asymmetrically.

Un très grand nombre de travaux de recherche fait état de l’intérêt des oligonucléotides en tant qu’agents thérapeutiques. Les modes d’actions envisageables sont très variés (thérapie antisens, antigène, interférence ARN, etc.). Cependant, les propriétés pharmacocinétiques et pharmacodynamiques des oligonucléotides naturels ne permettent pas leurs utilisations in vivo. Leurs propriétés peuvent être améliorées par des modifications chimiques. Notre travail consiste à synthétiser une nouvelle génération d’oligoribonucléotides modifiés : les oligomères de nucléosides aminooxy acides. Dans ces oligomères, la liaison phosphodiester de l’ARN est remplacée par une liaison N-oxyamide -CONHO-. Cette liaison est stable vis-à-vis des hydrolyses chimiques et enzymatiques et est facilement engagée dans des liaisons hydrogène. La préparation de différents nucléosides aminooxy esters protégés à partir de l’uridine ou du D-(+)-glucose est présentée. Par ailleurs, les N-oxy PNA constituent une autre famille d’oligonucléotides modifiés présentant une liaison N-oxyamide. L’analyse structurale des monomères et des dimères de N-oxy PNA est détaillée.De plus, un projet en collaboration avec le LBPA s’intéresse à une méthode de détermination de la structure secondaire des ARN. Dans ce but, nous avons conçu, synthétisé et étudié des sondes chimiques ciblant le ribose des nucléotides non appariés d’ARN. L’emploi de catalyseurs nucléophiles comme la DMAP permet d’augmenter la réactivité des sondes. / A large number of researches report the interest of oligonucleotides as therapeutic agents. The modes of actions are very varied (antisense therapy, antigen therapy, RNA interference, etc.). However, the pharmacokinetic and pharmacodynamic properties of natural oligonucleotides do not allow their in vivo uses. Their properties can be improved by chemical modifications. Our work consists to synthesize a new generation of modified oligoribonucleotides: the oligomers of aminooxy acids nucleosides. In such oligomers, the phosphodiester bond of the RNA is replaced with a N-oxyamide bond -CONHO-. This linkage is stable to chemical and enzymatic hydrolysis and is easily engaged in hydrogen bondings. The preparation of different protected aminooxy esters nucleosides starting from uridine or D-(+)-glucose is presented. Furthermore, N-oxy PNA constitute another family of modified oligonucleotides having a N-oxyamide bond. Structural analysis of the monomers and the dimers of N-oxy PNA is detailed.In addition, a project in collaboration with the LBPA focuses on a method for determining the secondary structure of RNA. To this end, we designed, synthesized and studied chemical probes targeting ribose of unpaired nucleotides. The use of nucleophilic catalysts such as DMAP increases the reactivity of the probes.

<div>Protein N-terminal methyltransferases (NTMTs) are a family of enzymes that methylate the α-N-terminus of a variety of protein substrates. Both NTMT1 and NTMT2 recognize a unique N-terminal X-P-K/R motif (X represents any amino acid other than D/E) to install 1-3 methyl group(s) on the substrates. NTMT1 plays important roles in mitosis regulation, chromatin interactions, and DNA damage repair. Another member NTMT2 shares ~50% sequence similarity and the same substrate recognition motif although NTMT2 was initially characterized as a mono-methyltransferase. To understand the molecular mechanism of NTMT2, we obtained the first co-crystal structure of NTMT2 in complex with its peptide substrate. After an extensive investigation of substrate recognition and methylated products of NTMT1/2, we found out that NTMT2 can fully methylate G/P-PKRIA peptides despite a predominant mono-methyltransferase. Moreover, we identified a gatekeeper N89 in NTMT2 that controls the substrate entry and the product specificity of NTMT2.</div><div>To elucidate the biological functions of NTMT1/2-catalyzed N-terminal methylation, we applied two different strategies to discover cell-potent inhibitors. Guided by the co-crystal structures of NTMT1 in complex with previously reported inhibitors, we designed and synthesized a series of new peptidomimetic inhibitors. By introducing more hydrophobic groups, the most cell-potent peptidomimetic inhibitor GD562 (IC50 = 0.93 ± 0.04 µM) exhibited over 2-fold increased inhibition on cellular N-terminal methylation levels with an IC50 value of ~50 µM compared to previously reported peptidomimetic inhibitor DC541. Meanwhile, we also discovered the first potent small molecule inhibitor Genz-682452 (IC50 = 0.5 ± 0.04 µM) after screening ~58,000 compounds. Subsequent structural modifications led to the discovery of GD433 (IC50 = 27 ± 0.5 nM) with a 20-fold increased potency compared to the initial hit Genz-682452. Inhibition mechanism indicated both inhibitors bind to peptide-binding pocket and co-crystal structures of both Genz-682452 and GD433 with NTMT1 confirmed their binding modes. Furthermore, GD433 shows over 7-fold selectivity over other major 40 protein methyltransferases and DNA methyltransferase and exhibits improved selectivity for NTMT1 over glucosylceramide synthase (GCS). GD433 significantly decreases the cellular N-terminal methylation level of NTMT1 substrates RCC1 and SET at 10 nM in both HEK293 and HCT116 cells, providing a valuable probe for cell-based studies in the future.<br></div><p><br></p>