Rozprawy doktorskie na temat „Covalent Interactions”

Non-covalent interactions taking place in solution are essential in chemical and biological systems. The solvent environment plays an important role in determining the geometry and stability of interactions. This thesis examines aromatic stacking interactions, alkyl-alkyl interactions, edge-to-face aromatic interactions, halogen bonds and hydrogen…hydrogen interactions in solution. Chapter 1 briefly introduces the different classes of non-covalent interactions, in addition to the state-of-the-art models and methods for investigating these weak interactions. The chapter finishes with a focus on dispersion interaction in alkanes and arenes. Chapter 2 investigates dispersion interactions between stacked aromatics in solution using a new class of complexes and thermodynamic double mutant cycles (DMCs). In extended aromatics, dispersion was detected as providing a small but significant contribution to the overall stacking free energies. Chapter 3 concerns the experimental measurement of alkyl-alkyl dispersion interactions in a wide range of solvents using Wilcox torsion balances. The contribution of dispersion interactions to alkyl-alkyl association was shown to be very small, with DMC, QSPR method and Hunter's solvation model. Chapter 4 studies edge-to-face aromatic interactions in series of solvents. In the open system, edge-to-face aromatic interactions were found to be sensitive to the solvent environment. The solvent effects were complicated and cannot be rationalised by a single parameter. Further analysis is needed. Chapter 5 describes a preliminary approach to investigate organic halogen…π interactions in solution using supramolecular complexes and torsion balances. Chapter 6 is a preliminary investigation of the ability of hydrogen atoms to act as H bond acceptors in silane compounds. Computations and 1H NMR demonstrated a weak interaction between silane and perfluoro-tert-butanol.

Les polyphénols naturels forment des complexes non-covalents dans lesquels le π-stacking et les liaisons hydrogène jouent un rôle clé dans la stabilisation. Les calculs DFT incluant la dispersion (DFT-D), la description des processus d'agrégation non-covalente de produits naturels devient fiable. Dans ce travail, les méthodes DFT-D sont appliquées à i) la compréhension de la biosynthèse stéréo- et régio-sélective des oligostilbenoïdes, ii) la prédiction de l'agrégation des antioxydants naturels au sein de la membrane bicouche lipidique, qui pourrait rationaliser la synergie de la vitamine E, la vitamine C et polyphénols dans leur action antioxydante, et iii) la modulation des propriétés optiques de dérivés de chalcones
Natural polyphenols form non-covalent complexes in which π-stacking and H-bonding play a key stabilizing role. The dispersion-corrected DFT calculations have paved the way towards reliable description of aggregation processes of natural products. In this work, these methods are applied at i) understanding of stereo- and regio-selective oligostilbenoids biosynthesis; ii) predicting natural antioxidant aggregation within lipid bilayer membrane, which may allow rationalizing the synergism of vitamin E, vitamin C and polyphenols in their antioxidant action; and iii) modulating optical properties of chalcone derivatives

Lubricant formulations are highly complex mixtures, containing a multitude of additives, each geared towards improving the efficiency of often highly specialised processes. The study of lubrication, or tribology, is a huge area of research, but is often overlooked by chemists in favour of pharmaceutical or agrochemical research. This thesis lays the foundation for the study and further understanding of additive-additive interactions in a lubricant formulation. Chapter one presents a concise introduction to modern lubricant formulations by providing a historical background and examining current understanding, while focusing not on the more widely publicised engineering aspects, but on the chemical properties and mechanisms of lubricant formulations. In order to dissect out additive-additive interactions from additive-solvent interactions, a study of the biggest single component in any lubricant formulation, the base oil, is performed in Chapter two. By using a series of molecular torsion balances which have been shown to be heavily influenced by solvent effects, it is revealed that the complex multitude of available commercial base oils can be substituted with a single common lab solvent. In Chapter three, a computational reparameterisation and experimental examination of previous semi-empirical models for the prediction of hydrogen-bond energies in solution using electrostatic surface potentials was performed. Surprisingly, it was found that a simple DFT/B3LYP/6-31G* method provides a better prediction of H-bond energies in solution than more complex models or functional-group corrected methods. Chapter four focuses on the fundamental understanding of entropy and conformational flexibility of compounds in solution. Using a simple flexible experimental system, which is capable for forming an internal hydrogen bond in solution, the impact of intramolecular hydrogen bonding on the stability of an intermolecular process was studied using both experimental and computational (DFT/molecular dynamics) methods. A distinct cut-off point was observed experimentally where the intramolecular process is completely out-competed by the intermolecular interaction. A computational model which reproduces the experimental trends has yet to be found.

ESI-MS screening methods directly detect ligand-target non covalent complexes in the gas phase and allow inference of affinity (and specificity) of the ligand-target interaction in solution [1, 2]. The identity of different complexes can be directly assessed as the mass of each molecule works as intrinsic label. Biopolymers can be screened either as a single component or a mixture of different targets; in this way it is possible to determine the selectivity of a new chemical entity for different targets. On the other hand, using ESI-MS it is also possible to identify, within a mixture, components that selectively bind the active site of a certain biopolymer and could be profitably used to screen libraries of known compounds. The aim of this PhD project was the study by ESI mass spectrometry of different noncovalent complexes formed with biopolymers identified as possible therapeutic targets (Mismatch Repair Mechanism and Hsp90). The noncovalent interaction between biological targets and possible ligands were studied in order to identify potential inhibitors. The binding affinities determined by ESI-MS were compared to existing data obtained by solution phase methods. A novel MS-based method was implemented for testing different biopolymer, of therapeutic interest, against a library of fragments (molecular weight 100-300 Da) constituted of about 2,000 compounds. Particularly this approach was used for the identification of new molecules able to recognize TG mismatched base pairs in DNA that are responsible for most of the common mutations leading to formation of tumors in humans. TG mismatches are particularly abundant in cells lacking mismatch repair mechanism (MMR). MMR is involved in the correction of DNA polymerase errors that escape proofreading activity. MMR deficiency increases 50-1000-fold spontaneous mutation rates (microsatellite instability MSI) and in addition, MMR deficiency can lead to resistance to several chemotherapeutic agents (DNA damaging agent) [3]. An ESI-MS method was used to study the complexes formed between different DNA duplexes and minor groove binders and intercalator compounds. A hairpin DNA sequence (CTGGsm) bearing a single T:G mismatch and a matched hairpin DNA sequence (CCGG) were prepared and used to set up the method. Two DNA sequences were also synthesized: a self complementary G:C rich DNA sequence (HFM) and a polyAT DNA duplex (A5TG). The association constants (KAs) were directly determined from the MS spectrum and the amount of bound ligand was used to determine the selectivity of a binder among the different DNA sequences. Minor groove binders confirmed their selectivity toward AT rich DNA duplex (A5TG) whereas a tris imidazole lexitropsin derivative proved to be selective for the TG mismatched DNA hairpin (CTGGsm) in agreement with NMR, SPR, and ITC studies. A medium throughput screening (MTS) method was setup for the screening of the fragment library (about 1,000 cps) and the procedure was validated studying the interactions between the two different DNA sequences (HFM and CTGGsm) and minor groove binders. The screening was performed on the fragment library and hit compounds were afterwards tested against HFM and CCGG in order to identify selective ligands. Among the different hits identified, a slight selectivity (1.3) for the single mismatched sequence was highlighted for the fragment FBA-05-094. Twenty close analogues of FBA-05-094 were subsequently tested against CTGGsm and CCGG DNA with the aim to find more selective and higher affinity ligands. The screening of the fragment library on CTGGsm was repeated using ligand mixtures (5 and 10 components) in order to improve the assay throughput. A good agreement between the results obtained by screening the compounds as single component and in mixture was found. We investigated also the binding of small molecules towards two enzymes (PKA and Hsp90) in order to verify the possibility to apply this procedure to protein targets. Hsp90 (heat shock protein 90) is a molecular chaperone and is one of the most abundant proteins expressed in cell [4]. Targeting Hsp90 with drugs has shown promising effects in clinical trials. Inhibition of the Hsp90 ATPase machinery by natural, semi- and totally synthetic inhibitors has shown promising results in clinic. The interactions between Hsp90 and different known ligands that bind either at N or C terminal binding domains were studied and the dissociation constants for this set of ligands (KD from 0.0007 uM to 103 uM) were previously determined by fluorescence polarization (FP). We found a good agreement between these values and the percentage of bound protein determined by mass spectrometry. A screening of the fragment library against Hsp90 was performed and the resulted hit compounds were compared to those obtained by using NMR FAXS technique[5]. A good correlation was found between the data obtained by MS and NMR screenings. Competition experiments using a known ATP competitor were performed and these studies highlighted the presence of a few ATP competitor ligands in agreement with X-ray experiments. [1] Ganem B, Li Y.-T., Henion JD. Detection of noncovalent Receptor-Ligand complexes by Mass Spectrometry. Journal of American Society for Mass Spectrometry, 113, 6294-6296 (1991) [2] Hofstadler SA, Sannes-Lowery KA. Applications of ESI-MS in drug discovery: interrogation of noncovalent complexes. Nature Reviews Drug Discovery, 5(7), 585-595 (2006) [3] Stojic L., Brun R., Jiricny J., Mismatch repair and DNA damage signalling. DNA Repair, 3, 1091-1101 (2004). [4] Chaudhury S., Welch T.R., Blagg B.S., Hsp90 as a Target for Drug Development. ChemMedChem, 1331-1340 (2006). [5] Dalvit C., Fagerness P.E., Hadden D.T.A., Sarver R.W., Stockman B.J. Fluorine-NMR Experiments for High-Throughput Screening: Theoretical Aspects, Practical Considerations, and Range of Applicability. Journal of American Society for Mass Spectrometry, 125, 7696-7703 (2003)

Aquesta tesi va dirigida específicament al desenvolupament de poliuretans (PU)s funcionalitzats en la cadena lateral (FPU)s, sintetitzats a partir de diols funcionals que provenen d’àcids grassos i dos diisocianats diferents: el diisocianat d’isoforona (IPDI) i el diisocianat d’hexametilé (HDI). Aquests nous FPUs presenten una amina terciària i grups alquil, al•lil, propargil o la combinació d'aquests en la cadena lateral. Posteriorment els FPUs es modifiquen mitjançant dos mecanismes de post-polimerització basats en enllaços covalents o en enllaços no covalents.En el primer cas, es duen a terme una sèrie de reaccions fotoiniciades d’acoplament tiol-é/í entre el grup al.lil i propargil que presenten els FPUs (formats a partir de IPDI), i tioglicerol. Els hidroxi-PUs obtinguts, exhibeixen una millora del seu caràcter hidrofílic. Alternativament, els FPUs que contenen només una amina terciaria com a grup funcional situat a la cadena lateral del PU, es barregen amb diferents àcids carboxílics per donar una reacció àcid base. Els supramoleculars PUs resultants (SPU)s es caracteritzen per espectroscòpia per a verificar la presència d'enllaços iònics d'hidrogen que uneixen les cadenes de PU mitjançant interaccions fisiques. A més, es demostra la correlació existent entre l'estructura química i les propietats tèrmiques i mecàniques dels materials sintetitzats. Aquests materials presenten prometedores propietats adaptatives. Per exemple, ressalten les bones propietats de regeneració i reciclatge/remodelació, degudes al caràcter reversible de les interaccions físiques. Addicionalment, aquests elastòmers posseeixen una inherent capacitat d’autoreparació, que en termes pràctics es podria veure com una millomillora de la seva sostenibilitat. Finalment, es sintetitzen xarxes de PU que tenen un doble caràcter estructural mitjançant enllaços iònics d'hidrogen dinàmics i entrecreuaments covalents. La variació de la densitat d’entrecreuament covalent introduït per a cada una d’aquestes xarxes, produeixun ajustament sistemàtic de les propietats mecàniques i de la sensibilitat del material a la calor. Aquesta preparació demostra una via simple i eficaç per a la fabricació de poliuretans multifuncionals i l’estratègia seguida també es pot estendre a l'exploració d'altres tipus d'interaccions covalents i no covalents en polímers
Esta tesis está dirigida específicamente al desarrollo de poliuretanos (PU)s funcionalizados en la cadena lateral (FPU)s, sintetizados a partir de dioles funcionales que provienen de ácidos grasos y dos diisocianats diferentes; el diisocianato de isoforona (IPDI) y el diisocianato de hexametileno (HDI). Estos nuevos FPUs presentan una amina terciaria y grupos alquilo, alilo, propargilo o la combinación de éstos en posiciones de cadena lateral. Posteriormente los FPUs se modifican mediante dos mecanismos de post-polimerización basados en enlaces covalentes o en enlaces no covalentes.En el primer caso, se llevan a cabo una serie de reacciones fotoiniciadas de acoplamiento tiol-eno/ino entre el grupo alilo y propargilo que presentan los FPUs (formados a partir de IPDI), y tioglicerol. Los hidroxi-PUs obtenidos, exhiben una mejora de su carácter hidrófilo. Alternativamente, los FPUs que contienen sólo una amina terciaria como grupo funcional situado en la cadena lateral del PU, se mezclan con diferentes ácidos carboxílicos mediante una reacción de ácido base. Los PUs supramoleculares resultantes (SPU)s se caracterizan por espectroscopia para verificar la presencia de enlaces iónicos de hidrógeno que unen las cadenas de PU formando interacciones físicas. Además, se demuestra la correlación existente entre la estructura química y las propiedades térmicas y mecánicas de los materiales sintetizados. Estos materiales presentan prometedoras propiedades adaptativas. Por ejemplo, resaltan las buenas propiedades de regeneración y reciclaje/remodelación, debidas al carácter reversible de las interacciones físicas. Adicionalmente, estos elastómeros poseen una inherente capacidad de autorautorreparación, que en términos prácticos se podría ver como una mejora de su sostenibilidad. Finalmente, se sintetizan redes de PU que tienen un doble carácter estructural mediante enlaces iónicos de hidrógeno dinámicos y entrecruzamientos covalentes. La variación de la densidad de entrecruzamiento covalente introducido para cada una de estas redes produce un ajuste sistemático de las propiedades mecánicas y la sensibilidad del material al calor. Esta preparación demuestra una vía simple y eficaz para la fabricación de poliuretanos multifuncionales.
This Thesis is addressed to the development of side-chain functionalized polyurethanes (FPU)s, with enhanced properties, made from fatty acid-based functional diols and two different diisocyanates; isophorone diisocyanate (IPDI) and hexamethylene diisocyanate (HDI). The novel FPUs present tertiary amine and alkyl, allyl, propargyl moieties or the combination of these, as side-chain positions groups. The FPUs were further modified via two post-polymerization mechanisms based on covalent or non-covalent bonds. In the first case, photoinitiated thiol-ene/yne coupling reaction between allyl, propargyl-functionalized PUs (based on IPDI) and thioglycerol was carried out. Obtained hydroxyl-PUs exhibit different thermal and mechanical properties in comparison with precursor PUs. Moreover, the incorporation of hydroxyl groups leads to PUs with enhanced hydrophilicity. Alternatively, the FPU (based on IPDI) containing only tertiary amine pendant group was mixed with different carboxylic acids in an acid-base reaction. Supramolecular ionic PUs were characterized by spectroscopic tools to verify the presence of ionic hydrogen bond as ionic interaction. Correlation between structure and thermal and mechanical properties was demonstrated. Samples show rapid thermal reversibility and recyclability thanks to the reversible bonds. In addition, elastomeric supramolecular PUs networks were prepared from HDI and aminodiol. The resulting materials exhibit some promising adaptive material properties such as effective energy dissipation upon deformation through unzipping the ionic hydrogen bonding network, combined with good shape-regeneration property and recycling/reshaping capability arising from their recoverable nature. More importantly, the resulting biobased elastomers possess the inherent self-healing ability, which can be seen as an upgrade of their sustainability.A novel thermo-reversible network is constructed by the thiol-ene functionalized polyurethane via dynamic ionic hydrogen bonds and covalent cross-links. By varying the covalent cross-linking density, the mechanical properties and the stimuli-responsive behaviour can be systematically tuned. This synthesis demonstrates a simple and effective pathway to fabricate multifunctional polyurethanes with desired functions.

Non-covalent interactions underpin the whole of chemistry and biology, but their study is extremely difficult in complicated biological systems. This thesis presents the application of synthetic molecular balances for gaining fundamental insights into the physicochemical phenomena that govern molecular recognition processes. Chapter 1 reviews the use of small synthetic molecules that exist in two conformational states via slow rotation of a bond, in the quantification of non-covalent interactions. Chapter 2 presents a new molecular torsion balance, based on a slowly rotating tertiary formyl amide for the study of non-covalent interactions. The incorporation of a fluorine atom in one of the rings allows the quantification of solvent effects in a wide range of solvents. Intramolecular electrostatic interactions and intermolecular solvation effects (but not solvophobic effects) are shown to be important in determining the position of the conformational equilibria. Correlations with calculated molecular properties show that solvent effects are fully dissected, revealing the idealistic behavior of the system in the gas phase. Chapter 3 discusses through-space substituent effects on the properties of aromatic rings. Electronic communication between both electron-rich and electron-deficient substituents with the electron density of an adjacent aromatic ring is predicted by molecular electrostatic potential calculations. The effect is confirmed to occur experimentally and is quantified using synthetic molecular balances. Chapter 4 describes the work done towards the investigation of solvent bridging interactions in molecular torsion balances. No experimental evidence of bridging interactions was observed. This might be attributed to the entropic penalty associated with this binding mode, or the non-ideal geometry of the potential bridging sites. Chapter 5 outlines a steric blocking effect observed in certain balances with bulky substituents in chloroform and dichloromethane. Chapter 6 presents synthetic procedures and compound characterisation including a thorough analysis of NMR data obtained in this study.

In this thesis, the design and synthesis of novel C60 fullerene compounds with different functional groups is reported. Chapter 1 introduces the background of the project with a general introduction into the covalent and non-covalent types of bonding interactions, and the chemistry and reactions of C60 fullerenes. Chapter 2 focuses on co-crystallisation of pristine C60 with selected aromatic compounds. Single crystals have been obtained and characterised by X-ray diffraction. Chapter 3 is concerned with the synthesis and characterisation of novel fullerene compounds by using Prato cycloaddition reactions, such novel compounds are further characterised in the solid state by single crystal X-ray diffraction. Hirshfeld surfaces are used to investigate the non-covalent interaction of the obtained crystal structures. Chapter 4 describes the synthesis and characterisation of novel fullerene compounds by using Bingel cycloaddition reactions. Crystallisation experiments have been attempted and theoretical studies using the semi-empirical method, parameterized model number 3, have been employed to investigate the charge transfer between the ligand and the C60 molecule. The novel molecules in this work have been characterised by infrared and NMR spectroscopies, and mass spectrometry. Single crystal X-ray diffraction was used when appropriate.

Non-covalent interactions in solution are subject to modulation by surrounding solvent molecules. This thesis presents two experimental molecular balances that have been used to quantify solvent effects on non-covalent interactions, including electrostatic and dispersion interactions. The first chapter introduces literature where non-covalent interactions have been studied in a range of solvents, particularly those where the effects of aqueous or fluorous solvents have been investigated. These solvents are of particular interest as they both invoke solvophobic effects on organic molecules, but have differing chemical and physical properties. The second chapter describes the adaptation of the Wilcox molecular torsion balance to study interactions between organic and fluorinated carbon chains in a range of solvents. Solvent cohesion was found to be the principle force driving both the alkyl and fluorous chains together in aqueous solvents, where no contribution to the interaction energy arising from dispersion forces could be detected. In fluorous and polar organic solvents evidence was found for weak favourable dispersion interactions between the alkyl chains. In contrast dispersion forces between the chains were found to be disrupted by competitive van der Waals interactions with surrounding solvent molecules in apolar organic solvents. Association of the fluorous chains was found to be solely driven by solvent cohesion. The final chapter describes the design and synthesis of a novel synthetic molecular-balance framework and describes its application to simultaneously measure solvent and substituent effects on the position of conformational equilibria. Despite the simplicity of the model system, surprisingly complicated behaviour emerged from the interplay of conformational, intramolecular and solvent effects. Nonetheless, a large data set of experimental equilibrium constants was analysed using a simple solvent model, which was able to account for both the intuitive and more unusual patterns observed. A means of dissecting electrostatic and solvent effects to reveal pseudo gas-phase behaviour has resulted from the analysis of experimental data obtained in many solvents.

High resolution mass spectrometric strategies in drug discovery for the investigation of covalent and non covalent interactions Dott. Danilo De Maddis PhD tutor: Prof. Giancarlo Aldini The research work here described was focused on the set-up and application of analytical methods for studying compounds effective as inhibitors of the advanced glycation end-products (AGEs) and advanced lipoxidation end-products (ALEs) as well as as antagonists of the receptors of AGEs (RAGE). AGEs and ALEs represent a quite complex and heterogeneous class of compounds that are formed by different mechanisms, by heterogeneous precursors and can be formed either exogenously or endogenously. AGEs represent a class of covalently modified proteins generated by oxidative and non-oxidative pathways, involving sugars or their degradation products. The term ALEs includes a variety of covalent adducts which are generated by the non-enzymatic reaction of reactive carbonyl species (RCS), produced by lipid peroxidation and lipid metabolism, with the nucleophilic residues of macromolecules, especially proteins. AGEs and ALEs share some common properties, for example, both consist of non-enzymatic, covalently modified proteins and oxidative stress is often (although not always) involved in the mechanism of their formation. Moreover some AGEs and ALEs have the same structure, since they arise from common precursors, as in the case of carboxymethyllysine (CML) which is generated by glyoxal, which in turn is formed by both lipid and sugar oxidative degradation pathways [1]. Besides being considered as reliable biomarkers of oxidative damage, as well as predictors and prognostic factors, more recently, AGEs and ALEs have also been recognized as important pathogenetic factors of some oxidative based diseases, as supported by the following facts: 1) a strict correlation between the amount of AGEs/ALEs in tissues and fluids and disease states has been found, in both animal and human subjects; 2) a substantial amount of literature is now available reporting the molecular and cellular pathogenic mechanisms for the AGEs/ALEs involvement in the onset and progression of different oxidative-based diseases including diabetes [2], chronic renal failure [3], cardiovascular diseases [4] and neurological disorders [5]. The AGEs/ALEs damaging effect is mediated by different mechanisms, including the dysfunction of the proteins undergoing the oxidative modification, protein polymerization, signal transduction, immunoresponse and RAGE activation. Some of the biological effects are due to the loss of function of the target proteins undergoing the covalent modification, such as in the case of extracellular matrix proteins that lose their elastic and mechanical functions when modified as AGEs/ALEs and in particular, when cross-links are involved [6]. Other examples of a direct damaging effect of AGEs/ALEs can be ascribed to the covalent modification of enzymes and receptors that lose their activity due to the covalent modification involving the catalytic or binding site, or following a conformational change of the protein structure. Moreover AGEs and ALEs can be immunogenic. Hence AGEs/ALEs are now considered as promising drug targets and a substantial effort is dedicated to delve the molecular strategies aimed at preventing, reducing or removing these protein oxidation products. The different molecular approaches thus far reported can be grouped by considering at which level of the damaging AGEs/ALEs cascade they are effective and in particular if they act by inhibiting the AGEs/ALEs formation, accelerating their catabolism or blocking their biological effects. The first level of action, the inhibition of AGEs/ALEs formation, also consists of different approaches, which target the different inducers (ROS, metal ions) and intermediate products (mainly reactive carbonyl species, RCS) involved in the AGEs/ALEs formation. Antioxidants, radical scavengers, metal-ion chelators and reactive carbonyl compounds quenchers (RCS sequestering agents) represent the most promising approaches so far reported for inhibiting AGEs/ALEs formation. In some cases, as found both for natural or synthetic compounds, the inhibition of AGEs/ALEs formation does not proceed through a single specific mechanism but implicates multiple mechanisms, involving at least two of the following ones: antioxidant, radical-scavenging, metal ion chelation and RCS trapping. The second level of intervention consists of accelerating the catabolism of already formed AGEs/ALEs and this can be achieved by potentiating the endogenous proteolytic system or by using xenobiotics able to catalytically degrade AGEs/ALEs. The third level of intervention, which is also the most innovative, consists of blocking the biological response of AGEs/ALEs by inhibiting the activation of the RAGE receptors through different classes of antagonists. It should be noted that such an approach permits the blocking of the damaging effect induced not only by endogenously formed AGEs/ALEs but also by exogenously derived AGEs/ALEs. In view of a drug discovery program aim to search bioactive compounds, the first step of the research program was to set up analytical methods based on high resolution MS strategies able to test the efficacy and selectivity of compounds effective as sequestering agents of RCS and also acting as RAGE antagonists. The methods so far reported in the literature to test RCS sequestering compounds consist to measure the consumption of the aldehydes when incubated with the test compounds by a direct spectrophotometric analysis and depending on the aldehyde, the derivatization reaction should also be considered. The main limitations of such approach is that it cannot be applied to mixtures or extracts, it cannot be applied to a high throughput screening and in several cases the experimental conditions such as the acid condition of the derivatization process can dissociate the adducts between the aldehydes and the sequestering agents. Therefore, based on the limits above reported, the initial aim of this research project was focused to set-up and then apply suitable analytical methods able to screen novel RCS sequestering agents. An HPLC method was firstly set-up in order to measure the consumption of RCS (reactivity) and of endogenous carbonyls such as pyridoxal (selectivity) when incubated in presence of the tested compounds. The method was optimized in order to use a mobile phase buffered at pH 7.4 in order to avoid the acid catalyzed degradation of the formed adducts. Reactivity towards RCS was evaluated by testing the ability of the tested compound to quench 4-hydroxy-nonenal (HNE) chosen as a model of alfa,beta-unsaturated aldehydes, glyoxal (GO) and methylglyoxal (MGO) as di-carbonyl derivatives. Selectivity was tested by measuring the consumption of pyridoxal as an endogenous aldehyde. The method was firstly validated by testing the reactivity and selectivity of known RCS scavenger compounds such as edaravone (EDA), hydralazine (HY), aminoguanidine (AG), and pyridoxamine (PYR). Even though they are very effective as RCS detoxifying agents, their usage is limited due to their lack of selectivity because they react with physiological aldehydes such as pyridoxal and by a promiscuous activity: ED is a neuroprotective compound, HY an antihypertensive drug and AG a NOS inhibitor. The HPLC method was then applied to study the reactivity and selectivity of carnosine (CAR) and derivatives such as anserine, n-acetyl carnosine and of carnosine analogues designed to be stable to carnosinase, a specific metal-ion dependent homodimeric dipeptidase (carnosinase, CN1 EC.3.4.13.20),. The screening permitted to identify carnosinol, a carnosine peptidomimetic characterized by the replacement of the carboxyl group with a hydroxyl group, as a selective and reactive RCS sequestering agent, characterized by a suitable PK profile since resistant to serum carnosinase and recognized by HPEPT1 (patent application: WO2011080139). Beside evaluating the reactivity and the selectivity, the approach was then implemented by an ESI-MS approach aimed to fully characterize the covalent adducts between RCS and the sequestering agents. The investigation of the mechanisms of carbonyl quenching have so ensued a broad understanding of the biochemical mechanisms in vitro as well as in vivo that occur between cytotoxic RCS and the "sweepers" detoxifying agents. The method was then applied to test the RCS sequestering activity of proteinogenic histidine-containing dipeptides with the C-terminus capped by a methyl ester or by a primary amido group so as to study dipeptides which should be still recognized by peptide transporters and resistant to proteolysis. Moreover, the study considered diastereoisomeric pairs of dipeptides produced by alternating the absolute configuration of the histidine residue thus revealing the effect of configuration on the quenching activity (7). The second step of the work was to set-up a rapid and accurate method for testing the ability of RCS sequestering agents to inhibit protein carbonylation. To set-up the method, HNE, which is one of the most abundant and reactive lipid-derived RCS, was used as RCS, and ubiquitin [8] as a model of protein substrate. Going into details, ubiquitin was selected as a protein target since it is commercially available at high purity and at a reasonable cost and because its molecular weight is suitable for intact protein analysis using a high resolution mass spectrometer such as the Orbitrap. Despite lacking cysteine residues, ubiquitin exposes a set of reactive lysines and a highly reactive histidine, which make this protein a suitable benchmark to test protein carbonylation and its inhibition. The method was firstly validated by using known RCS sequestering agents and the results well correlate with those obtained by HPLC analysis. The method was found suitable to test mixtures or extracts and to be used for HTS applications. AGEs and most probably ALEs are ligands and activators of the receptor RAGE whose activation is involved in a NF-kB pathway, leading to NADPH oxidase activation, oxidative stress and a condition of inflammatory and pro-fibrotic response. Since the damaging AGEs-RAGE axis has been associated to the onset and/or progression of many chronic and degenerative oxidative based disease and in many metabolic disorders, it has been recently hypothesized that the AGE-RAGE axis represents a promising drug target. One approach to counteract the AGEs-RAGE axis is represented by the design of RAGE antagonists. It should be considered that a drug design approach aimed to screen RAGE antagonists firstly requires a reliable analytical method able to test the binding affinity of small molecules and the set-up of such a method represents the aim of the present research program that is based on the following steps:  RAGE expression as recombinant protein in E. coli strain ORIGAMI B (DE3) with recombinant plasmid pET-15b VC1;  Application of the automated loop injection High Resolution Mass Spectrometry (MS) method, based on the High Resolution Mass Spectrometry method developed to study the Ubiquitin-RCS interaction, to inquire in vitro non-covalent binding relationship between low molecular weight AGEs and recombinant RAGE. The receptor for advanced glycation end products (RAGE) is a type I transmembrane glycoprotein of the mmunoglobulin superfamily of cell surface receptors. RAGE extracellular portion is involved in ligand binding and contains one “V” –type followed by two “C”-type immunoglobulin-like domains (V-C1-C2 structure). V-C1 domains form an integrated unit whereas C2 domain is attached to V-C1 by a flexible linker but is a fully independent unit. As a general strategy, we decided to express the V-C1 portion of the extracellular domain of human RAGE (sRAGE) as protein target since it is water soluble and involved in the molecular recognition and engagement of the AGEs ligands. Therefore, V, V-C1 and s-RAGE expression was focused on the recombinant protein expression carried out by the protein studies obtained through the E. coli expression system. Despite a greater facility to handling the microorganism, E. coli in contrast to eukaryotic expression system, is not capable to perform post-translational modifications, including glycosylation, which are instead present in the human RAGE. Regardless for the lack of this peculiarity, numerous studies reported in literature, indicate that, post-translational modifications have not significantly effect on the RAGE properties regarding for instance the bind activity toward certain ligands and so that, the bacterial E. coli expression system represent one of the most used host for the heterologous proteins expression. A great advantage by using E.coli is its peculiarity of secreting the protein of interest in the medium that it can be easily purified in one-step. This advantage have avoided the use of tags, such as the Poly-His, that could affect the property of sRAGE during high throughput screening of libraries of compounds and making unnecessary the removal step by specific protease treatments. The high resolution mass spectrometric (ESI-MS) approach in top-down and bottom-up approach was then used in order to verify the identity of the protein. The multi-charged ESI-MS spectrum of the recombinant protein corresponding deconvoluted MS spectrum showing the MW of 24580 Da which is consistent with the theoretical one. GSHMAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEGIFRCQAMNRNGKETKSNYRVRVYQIPGKPEIVDSASELTAGVPNKVGTCVSEGSYPAGTLSWHLDGKPLVPNEKGVSVKEQTRRHPETGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPV-PLEEVQLVVE. The primary sequence was then identified by a bottom-up approach consisting to enzymatically digest the protein, separate the peptides by reversed phase capillary column. Eluted peptides were then sequenced by MS/MS analyses. The primary structure of the protein corresponding to the predicted sequence on the bases of the designed nucleotide sequence of sRAGE. After having characterized the protein, a MS approach to study the non covalent binding of RAGE with ligands was set-up. The ligand-binding properties of RAGE was studied by a native MS method that is suitable to study the non covalent interactions between ligands and recombinant V-C1. The advantages of native MS over other methods is that it does not require labeled target or ligands, it is characterized by high sensitivity, low sample consumption, fully automatation and that ligands can be screened as mixture. The experiments consist in maintaining the concentration of VC1 constant and increasing the ligand concentration. The ligands used are well known low molecular weight sRAGE ligands such as carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) derived peptide [9]. Unmodified peptides were also used as controls. Validation of the method was obtained by comparing the Kd values obtained by native MS analysis in respect to the Kd values determined from fluorescence titration experiments. The native MS method was found accurate and suitable to test libraries in order to understand the structure requirements for RAGE recognition as well as for searching antagonists. References [1] Baynes JW . Chemical modification of proteins by lipids in diabetes. Clin Chem Lab Med 2003 ; 41 : 1159 – 1165. [2] Yamagishi S , Maeda S , Matsui T , Ueda S , Fukami K , Okuda S . Role of advanced glycation end products (AGEs) and oxidative stress in vascular complications in diabetes. Biochim Biophys Acta 2012 ; 1820 : 663 – 671. [3] Iacobini C , Menini S , Ricci C , Scipioni A , Sansoni V , Mazzitelli G , et al . Advanced lipoxidation end-products mediate lipid-induced glomerular injury: role of receptormediated mechanisms . J Pathol 2009 ; 218 : 360 – 369. [4] Del Turco S , Basta G . An update on advanced glycation endproducts and atherosclerosis. Biofactors 2012 ; 38 : 266 – 274. [5] Li JL , Liu DN , Sun L , Lu Y , Zhang Z . Advanced glycation end products and neurodegenerative diseases: Mechanisms and perspective. J Neurol Sci 2012 ; 317 : 1 – 5. [6] Ciulla MM , Paliotti R , Carini M , Aldini G . Fibrosis, enzymatic and non-enzymatic cross-links in hypertensive heart disease. Cardiovasc Hematol Disord Drug Targets 2011. [7] Vistoli G, De Maddis D, Straniero V, Pedretti A, Pallavicini M, Valoti E, Carini M, Testa B, Aldini G. Exploring the space of histidine containing dipeptides in search of novel efficient RCS sequestering agents. Eur J Med Chem. 2013 Aug;66:153-60 [8] Liang X, Chen Y, Zhuang J, Zhang M, Xiong W, Guo H, Jiang F, Hu P, Guo D, and Shi W, Advanced oxidation protein products as prognostic biomarkers for recovery from acute kidney injury after coronary artery bypass grafting. Biomarkers 17 (2012) 507-12. [9] Xue J, Rai V, Singer D, Chabierski S, Xie J, Reverdatto S, Burz DS, Schmidt AM, Hoffmann R, Shekhtman A. Advanced glycation end product recognition by the receptor for AGEs. Cell Press. 2011 May 11;19(5):722-32.

The present thesis is concerned with the quantification of weak non-covalent interactions between a simple π system and functional groups through dynamic NMR studies of conformational equilibria of specifically designed molecular balance systems. The work begins with an introduction which provides a formal review of the current literature on various aspects of non-covalent π-interactions, starting with an examination of the physical properties of non-covalent arene interactions, including arene-arene interactions, the behaviour of aromatic rings as hydrogen bond acceptors and the interactions between aromatic rings and cationic species. The use of molecular balance systems for studying these weak interactions will then be discussed together with some other important methods used. The introduction concludes with an account of olefinic !-interactions and methods for their study as described in the current chemical literature. The second section presents and discusses the results obtained in the present study and begins with the description of the synthetic routes employed to prepare two series of molecular balance systems, both of which feature a common bicyclo[3.2.2]nona-6,8-diene framework and a flexible three carbon atom bridge, whose central carbon atom can possess two different functional groups. The relative abundance of each of the two possible conformers in solution provides a measure of the relative interaction energies. Dynamic NMR studies in a range of solvents were employed to study each derivative. The results from these studies were then compared in order to gain insight into the relative strengths of both olefinic and aromatic π-interactions. The thesis concludes with a formal description of the experimental procedures used in this study and appendices detailing the data obtained from dynamic NMR studies and crystal structure data.

This thesis describes an investigation of the weak non-covalent interactions between the 7r-system of an aromatic ring and various functional groups. The first section comprises a review of the current literature on the through space aromatic ring interactions based on theoretical, biological and experimental chemistry and especially focuses on the -interactions involving a functional group. The second section describes the synthesis of a series of novel 9,10-propanoanthracenes, with a common framework, consisting of a rigid structure containing two independent aromatic systems and a flexible bridge. The central carbon atom of the bridge can be substituted with two different functional groups, which are held in close proximity to the centre of one of the aromatic rings. This gives rise to two possible conformers, whose population relies on the difference in the strength of the interactions between the aromatic ring and each substituent of the bridge, relative to the solvent being employed. The conformational equilibria of each derivative were studied by NMR spectroscopy using variable temperature techniques and solvents. The presumed equilibria under those two variables were compared in order to give some insight into the nature of the n interactions involved. The thesis concludes with a formal account of the experimental procedures used, the compounds prepared and an appendix detailing theoretical data obtained by computational chemistry and crystal structures data.

Phosphonium ions readily compare to ammonium ions in regards to their aggregate characteristics, thermal stability, and antibacterial activity. Ionic aggregation in phosphonium-based polymers provides thermoreversible crosslinks, ideal for reversible self-assembly, self-healing, and smart response. In polymers, these ionic functionalities aggregate, providing improved moduli, and altering the size and structure of ionic aggregates regulates polymer melt processability. This dissertation highlights phosphonium-based chemistry for the synthesis of novel step-growth ionomers and structure-property relationships in ionic polymers. The synthesis of phosphonium endcapping reagents for melt polyester reactions afforded a thermally stable ionic functionality that controlled molecular weight. Weak association was present with phosphonium ions at low ion concentrations below 7.7 mole %. The use of novel ionic bisacetoacetate monomers in the formation of networks from Michael addition reactions led to the synthesis of ionic networks with increased and broadened glass transitions and improved tensile stresses at break and strains at break compared to those in the non-ionic networks. The first electrospun fibers from Michael addition crosslinking reactions are reported, and equilibrium ionic liquid uptake experimental results indicated that ionic functional networks absorb close to three times the amount of ionic liquid as non-ionic, poly(ethylene glycol)-based films. Chain-extending polyurethanes with a phosphonium diol and subsequently varying the hard segment content led to changes in ionic aggregation, crystallinity, and thermal transitions in the polymers. Additionally, novel phosphonium-based methacrylate monomers incorporated into diblock copolymers with styrene exhibited microphase separation. Overall, the inclusion of phosphonium ions pendant to or in the main chain of various types of polymers led to changes in morphology, improved tensile properties, enhanced moduli, broadened transitions, changes in crystalline melting points, changes in solubility, and appearance of ionic aggregation.
Ph. D.

Acid and base-induced unfolding of myoglobin in solution is monitored by electrospray ionization mass spectrometry. It is shown that acid-induced Mb unfolding causes the appearance of different charge state distributions in positive and negative ion modes compared to base-induced unfolding, suggesting different protein conformations in solution. Collision cross sections of both apomyoglobin and holomyoglobin ions are measured at various orifice-skimmer voltage differences (AVOS). The results show that at low AVOS, apomyglobin ions have greater collision cross sections than holomyoglobin ions, indicating that heme, when attached to the globin, helps maintain a more compact myoglobin structure. Coulomb effects in binding of heme in gas phase holomyoglobin ions are studied. Positive and negative ions are formed from solutions of Fe⁺² (ferromyoglobin) and Fe⁺³ (ferrimyoglobin). The energy that must be added to the resulting holomyoglobin ions to cause heme loss is measured with a triple quadrupole MS/MS system. With negative ions, neutral heme is lost, regardless of the charge state of Fe in solution. It is likely that the Fe⁺³ is reduced to Fe⁺² in the negative electrospray process. With positive ions, predominantly neutral heme loss is observed for ions formed from ferromyoglobin in solution, and positive heme loss for ions formed from ferrimyoglobin in solution. The energies required to induce neutral heme loss are similar for positive and negative ions. The energies required to induce charged heme loss from positive holomyoglobin ions are significantly less. Coulomb repulsion between the charged heme and charged protein appears to lower the barrier for heme loss. These results are consistent with a simple model of a long range Coulomb repulsion and a short range attraction between the heme and protein.
Science, Faculty of
Chemistry, Department of
Graduate

The focus of this thesis is the question of how non-covalent interactions affect chemical systems' electronic and structural properties. Non-covalent interactions can exhibit a range of binding strengths, from strong electrostatically-bound salt bridges or multiple hydrogen bonds to weak dispersion-bound complexes such as rare gas dimers or the benzene dimer. To determine the interaction energies (IE) of non-covalent interactions one generally takes the supermolecular approach as described by the equation \begin{equation} E_{IE} = E_{AB} - E_{A} - E_{B}, \end{equation} where subscripts A and B refer to two monomers and AB indicates the dimer. This interaction energy is the difference in energy between two monomers interacting at a single configuration compared to the completely non-interacting monomers at infinite separation. In this framework, positive interaction energies are repulsive or unfavorable while negative interaction energies signify a favorable interaction. We use prototype systems to understand systems with complex interactions such as π-π stacking in curved aromatic systems, three-body dispersion contributions to lattice energies and transition metal catalysts affect on transition state barrier heights. The current "gold standard" of computational chemistry is coupled-cluster theory with iterative single and double excitation and perturbative triple excitations [CCSD(T)].\cite{Lee:1995:47} Using CCSD(T) with large basis sets usually yields results that are in good agreement with experimental data.\cite{Shibasaki:2006:4397} CCSD(T) being very computational expensive forces us to use methods of a lower overall quality, but also much more tractable for some interesting problems. We must use the available CCSD(T) or experimental data available to benchmark lower quality methods in order to ensure that the low quality methods are providing and accurate description of the problem of interest. To investigate the effect of curvature on the nature of π-π interactions, we studied concave-convex dimers of corannulene and coronene in nested configurations. By imposing artificial curvature/planarity we were able to learn about the fundamental physics of π-π stacking in curved systems. To investigate these effects, it was necessary to benchmark low level methods for the interaction of large aromatic hydrocarbons. With the coronene and corannulene dimers being 60 and 72 atoms, respectively, they are outside the limits of tractability for a large number of computations at the level of CCSD(T). Therefore we must determine the most efficient and accurate method of describing the physics of these systems with a few benchmark computations. Using a few benchmark computations published by Janowski et al. (Ref. \cite{Janowski:2011:155}) we were able to benchmark four functionals (B3LYP, B97, M05-2X and M06-2X) as well as four dispersion corrections (-D2, -D3, -D3(BJ), and -XDM) and we found that B3LYP-D3(BJ) performed best. Using B3LYP-D3(BJ) we found that both corannulene and coronene exhibit stronger interaction energies as more curvature is introduced, except at unnaturally close intermolecular distances or high degrees of curvature. Using symmetry adapted perturbation theory (SAPT)\cite{Jeziorski:1994:1887, Szalewicz:2012:254}, we were able to determine that this stronger interaction comes from stabilizing dispersion, induction and charge penetration interactions with smaller destabilizing interactions from exchange interactions. For accurate computations on lattice energies one needs to go beyond two-body effects to three-body effects if the cluster expansion is being used. Three-body dispersion is normally a smaller fraction of the lattice energy of a crystal when compared to three-body induction. We investigated the three-body contribution using the counterpoise corrected formula of Hankins \textit{et al.}.\cite{Hankins:1970:4544} \begin{equation} \Delta ^{3} E^{ABC}_{ABC} = E^{ABC}_{ABC} - \sum_{i} E^{ABC}_{i} - \sum_{ij} \Delta ^{2} E^{ABC}_{ij}, \end{equation} where the superscript ABC represents the trimer basis and the E(subscript i) denotes the energy of each monomer, where {\em i} counts over the individual molecule of the trimer. The last term is defined as \begin{equation} \Delta ^{2} E^{ABC}_{ij} = E^{ABC}_{ij} - E^{ABC}_{i} - E^{ABC}_{j}, \end{equation} where the energies of all dimers and monomers are determined in the trimer basis. Using these formulae we investigated the three-body contribution to the lattice energy of crystalline benzene with CCSD(T). By using CCSD(T) computations we resolved a debate in the literature about the magnitude of the non-additive three-body dispersion contribution to the lattice energy of the benzene crystal. Based on CCSD(T) computations, we report a three-body dispersion contribution of 0.89 kcal mol⁻¹, or 7.2\% of the total lattice energy. This estimate is smaller than many previous computational estimates\cite{Tkatchenko:2012:236402,Grimme:2010:154104,Wen:2011:3733,vonlilienfeld:2010:234109} of the three-body dispersion contribution, which fell between 0.92 and 1.67 kcal mol⁻¹. The benchmark data we provide confirm that three-body dispersion effects cannot be neglected in accurate computations of the lattice energy of benzene. Although this study focused on benzene, three-body dispersion effects may also contribute substantially to the lattice energy of other aromatic hydrocarbon materials. Finally, density functional theory (DFT) was applied to the rate-limiting step of the hydrolytic kinetic resolution (HKR) of terminal epoxides to resolve questions surrounding the mechanism. We find that the catalytic mechanism is cooperative because the barrier height reduction for the bimetallic reaction is greater than the sum of the barrier height reductions for the two monometallic reactions. We were also able to compute barrier heights for multiple counter-ions which react at different rates. Based on experimental reaction profiles, we saw a good correlation between our barrier heights for chloride, acetate, and tosylate with the peak reaction rates reported. We also saw that hydroxide, which is inactive experimentally is inactve because when hydroxide is the only counter-ion present in the system it has a barrier height that is 11-14 kJ mol⁻¹ higher than the other three counter-ions which are extremely active.

Conventional studies of ionomers have focused on ionomers bearing monovalent carboxylate or sulfonate pendant ions. There are relatively fewer studies on ionomers containing multivalent pendant ions, such as divalent phosphonate. In this dissertation, poly(ethylene terephthalate) (PET) and polystyrene ionomers with divalent phosphonate pendant ions have been synthesized, and the influence of divalent phosphonate pendant ions on the structure-morphology-property relationship has been compared to the ionomers with monovalent sulfonate pendant ions. The phosphonate groups generated a stronger physically crosslinked network in phosphonated ionomers as compared to sulfonated analogues. Higher plateau modulus, longer relaxation time, and significantly higher zero-shear viscosity were noted for phosphonated ionomers by a dynamic melt rheology study. Compared to the ionic aggregates generated from sulfonate groups, larger ionic aggregates with associated phosphonate groups have been observed. Furthermore, phosphonated ionomers displayed significantly higher glass transition temperatures than sulfonated ionomers. Ionomers have proven to be attractive, interfacially active compatibilizers for a number of polymer blend systems because of specific interactions that may develop between the ionic groups and complementary functional groups on other polar polymers within the blends. The successful compatibilization of polyester/polyamide blends (prepared by solution mixing and melt blending methods) using phosphonated PET ionomers as a minor-component compatibilizer has been demonstrated. The phase-separated polyamide domain dimension decreased with increasing mol % phosphonated monomers and this decrease was attributed to the specific interactions between the ionic phosphonate groups on the polyester ionomer and the amide linkages of polyamide. More importantly, the divalent phosphonate pendant ions are more effective at compatibilizing polyester/polyamide blends in comparison to the monovalent sulfonate pendant ions. Phosphonated PET ionomer-compatibilized polyester/polyamide blends required 6 times fewer ionic monomers to achieve domain dimension < 1 μm as compared to sulfonated PET-containing blends. Deep eutectic solvents (DES) have been reported to be the next generation solvents due to the superior biocompatibility, biodegradability, and sustainability as compared to ionic liquids. Two types of deep eutectic solvents, choline chloride : malic acid (ChCl:MA) and L-arginine : levulinic acid (Arg:LA), have been demonstrated as effective plasticizers for poly(vinyl alcohol) (PVOH) films. The plasticization effects on the properties of PVOH films were evidenced by lower crystallizability and improved film ductility. In addition, ChCl:MA deep eutectic solvent was more effective in plasticizing PVOH as compared to propylene glycol, one of the most widely studied alcohol-type plasticizers. From an applied perspective, DES-plasticized PVOH film is a promising candidate in the packaging market of heath-related products.
Doctor of Philosophy

Non-covalent interactions including nucleobase hydrogen bonding and ionic aggregation were studied in block and end-functional polymers synthesized via living polymerization techniques such as nitroxide mediated polymerization and anionic polymerization. The influence of non-covalent association on the structure/property relationships of these materials were studied in terms of physical properties (tensile, DMA, rheology) as well as morphological studies (AFM, SAXS). Hydrogen bonding, a dynamic interaction with intermediate enthalpies (10-40 kJ/mol) was introduced through complementary heterocyclic DNA nucleobases such as adenine, thymine and uracil. Hydrogen bonding uracil end-functionalized polystyrenes and poly(alkyl acrylate)s were synthesized via nitroxide mediated polymerization from novel uracil-functionalized alkoxyamine unimolecular initiators. Terminal functionalization of poly(alkyl acrylate)s with hydrogen bonding groups increased the melt viscosity, and as expected, the viscosity approached that of nonfunctional analogs as temperature increased. A novel difunctional alkoxyamine, DEPN2, was synthesized and utilized as an efficient initiator in nitroxide-mediated controlled radical polymerization of triblock copolymers. Complementary hydrogen bonding triblock copolymers containing adenine (A) and thymine (T) nucleobase-functionalized outer blocks were synthesized. Hydrogen bonding interactions were observed for blends of the complementary nucleobase-functionalized block copolymers in terms of increased specific viscosity as well as higher scaling exponents for viscosity with solution concentration. Multiple hydrogen bonding interactions were utilized to attach nucleobase-functional quaternary phosphonium ionic guests to complementary adenine-functionalized triblock copolymers. Ionic interactions, which possess stronger interaction energies than hydrogen bonds (~150 kJ/mol) were studied in the context of sulfonated poly(styrene-b-ethylene-co-propylene-b-styrene) block copolymers. Strong ionic interactions resulted in the development of a microphase separated physical network and greater extents for the rubbery plateau in DMA analysis compared to sulfonic acid groups, which exhibit weak hydrogen bonnding interactions. In contrast to the physical networks consisting of sulfonated or hydrogen bonding block copolymers, covalent networks were synthesized using carbon-Michael addition chemistry of acetoacetate functionalized telechelic oligomers to diacrylate Michael acceptors. The thermomechanical properties of the networks based on poly(propylene glycol) oligomers were analyzed with respect to the molecular weight between crosslink points (Mc) and the critical molecular weight for entanglement (Me).
Ph. D.

Ternary systems comprising DNA/RNA, proteins and one (or more) metal ion are generating increased interest due to its biological relevance. The knowledge gained from the study of these systems could provide important clues regarding the precise mechanism for transcription factors, repair proteins and metal complexes with anti-tumoral/anti-viral activities.The interactions occurring among the components of these ternary systems can be broadly grouped into covalent and non-covalent. The first kind of interactions can lead to the irreversible transformation of the components in the system, while the second is thought to be reversible leading to transient states and fluxionality. Both kinds of interaction are generally present in living systems, complementing the function of each other.Monofunetional Platinum-nucleobase complexes (MPNs) are synthesized via substitution of a chloride ligand by a nucleobase in platinum complexes with trans geometry. MPNs are particularly interesting for the study of ternary systems since they mimic the first step in the formation of a platinum-DNA adduct and their interaction with aminoacids/proteins provide a good first approach for more complex systems.The presence of the nucleobase as a ligand, significantly modifies the biological activity of these complexes by reducing its cytotoxicity and generating a promising anti-viral activity, especially against HIV-1 virus. The specific role of the nucleobase ligand on these complexes as a non-covalent motif, important for protein recognition, was explored in models involving tryptophan/N-acetyl tryptophan and a small protein domain called zinc finger, containing also a tryptophan residue.The coordination of the nucleobase to a metal ion such as Pt(II) or Pd(II) was found to increase its π-stacking interaction towards aromatic residues in proteins, specifically tryptophan. The enhancing effect was found to depend on the nature of the metal ion, nature of nucleobase and size/complexity of the protein model. Furthermore, DFT studies revealed an important change in the energy for the lowest unoccupied molecular orbital (LUMO) in the coordinated nucleobases, which could place this orbital in an favored position to interact with the highest occupied molecular orbital (HOMO) in the tryptophan residue. Results from calculations showed a good correlation with experimental evidence and could indicate an important role for the frontier molecular orbitals (HOMO/LUMO) of the species involved in the π-stacking interaction.This study was extended to a zinc finger domain from an essential protein in HIV-1 virus, i.e. nucleocapsid protein NCp7. Findings showed that the nucleobase ligand in addition to modulate hydrolysis and reaction rates for MPNs can also be responsible for an initial non-covalent recognition towards a specific protein. This initial recognition has been proposed as the first stage in a two-step mechanism of action for these platinum complexes that ultimately can lead to zinc ejection from the zinc finger domain in the viral NCp7. The significance of the data presented show that is possible to modulate the ligand coordination sphere in metal complexes to can result in great differences in terms of biological effects.The novel chemistry derived from DNA adducts with platinum complexes with a trans geometry was also explored in silico. The molecular dynamics of two free DNA 20-mer is compared with the corresponding metallated-adducts, namely monofunctional, 1,2-bifunctional interstrand and 1,3-bifunctional intrastrand. The differences in terms of structure and energy are compared for these systems, in general the monofunctional adduct exhibited the most interesting feature in terms of structural change in the DNA double strand causing the destacking of the metallated nucleobase. Bifunctional adducts exhibited loss of Watson-crick bonds and localized change in sugar puckering. These results showed that important differences can be found for platinated DNA even at short simulation times < 1 ns.

Gas phase molecular clusters present an ideal medium for observing factors that drive chemical reactions without outside interferences from excessive solvent molecules. Introducing an ion into the cluster promotes ion-molecule interactions that may manifest in a variety of non-covalent or even covalent binding motifs and are of significant importance in many fields including atmospheric and astronomical sciences. For instance, in outer space, molecules are subject to ionizing radiation where ion-molecule reactions become increasingly competitive to molecule-molecule interactions. To elucidate individual ion-molecule interaction information, mass spectrometry was used in conjunction with appropriate theoretical calculations. Three main categories of experiment were conducted in this dissertation. The first of which were thermochemical equilibrium measurements where an ion was introduced to an ion mobility drift cell wherein thermalizing collisions occur with helium buffer gas facilitating a reversible reaction with a neutral molecule allowing the standard changes in enthalpy and entropy to be determined. The second type of experiment was an ion mobility experiment where an ionized homo- or hetero-cluster was injected into the drift cell at specific conditions allowing the reduced mobility and collisional cross-section to be evaluated. Thirdly, kinetics measurements were taken following injection of an ion into the drift cell were an irreversible reaction ensued with the neutral species hindering equilibrium, but prompting rate constant assessment. Previous research has laid the groundwork for this dissertation as the results and discussion contained herein will build upon existing data while maintaining originality. For example, past work has given support for ion-molecule reactions involving precursor species such as acetylene and hydrogen cyanide to form more complex organics, perhaps leading to biologically relevant species. The chemical systems studied for this research are either ionized substituted benzenes like phenylacetylene and benzonitrile or polycyclic aromatic nitrogen-containing hydrocarbons like quinoline and quinoxaline interacting with a variety of neutral species. Hydrogen bonding and its many sub-sections are of the utmost importance to the kinds of reactions studied here. Past work has shown the tendency of organic radical cations to form conventional and unconventional ionic hydrogen bonds with gas phase solvents. Other non-covalent modes of interaction have also been detected in addition to the formation of covalently bound species. Gas phase reactions studied here will explore, via mass-selected ion mobility, reversible and irreversible reactions leading to binding enthalpy and entropy and rate constant determination, respectively, in addition to collisional cross-section determination.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Dec. 18, 2007). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry." Discipline: Chemistry; Department/School: Chemistry. On t.p., [beta] is the Greek letter.

The majority of drugs are small organic molecules, so-called ligands, that influence biochemical processes by interacting with proteins. The understanding of how and why they interact and form complexes is therefore a key component for elucidating the mechanism of action of drugs. The research presented in this thesis is based on studies of acetylcholinesterase (AChE). AChE is an essential enzyme with the important function of terminating neurotransmission at cholinergic synapses. AChE is also the target of a range of biologically active molecules including drugs, pesticides, and poisons. Due to the molecular and the functional characteristics of the enzyme, it offers both challenges and possibilities for investigating protein-ligand interactions. In the thesis, complexes between AChE and drug-like ligands have been studied in detail by a combination of experimental techniques and theoretical methods. The studies provided insight into the non-covalent interactions formed between AChE and ligands, where non-classical CH∙∙∙Y hydrogen bonds (Y = O or arene) were found to be common and important. The non-classical hydrogen bonds were characterized by density functional theory calculations that revealed features that may provide unexplored possibilities in for example structure-based design. Moreover, the study of two enantiomeric inhibitors of AChE provided important insight into the structural basis of enthalpy-entropy compensation. As part of the research, available computational methods have been evaluated and new approaches have been developed. This resulted in a methodology that allowed detailed analysis of the AChE-ligand complexes. Moreover, the methodology also proved to be a useful tool in the refinement of X-ray crystallographic data. This was demonstrated by the determination of a prereaction conformation of the complex between the nerve-agent antidote HI-6 and AChE inhibited by the nerve agent sarin. The structure of the ternary complex constitutes an important contribution of relevance for the design of new and improved drugs for treatment of nerve-agent poisoning. The research presented in the thesis has contributed to the knowledge of AChE and also has implications for drug discovery and the understanding of biochemical processes in general.

Photophysical properties of an array of various polyaromatic hydrocarbons were benchmarked with B3LYP, M06 and B97D methods coupled with Pople and CEP-31G(d) basis sets. Results from the benchmark show the importance of diffuse basis sets when modeling the electronic properties of highly conjugated systems and provide qualitative reliable accuracy with certain levels of theory. B97D and M06 are applied to modeling pyrene adducts governed by non-covalent interactions in both gaseous and condensed states to reproduce experimental spectra. DFT calculations with both B97D and M06 functionals show qualitatively and quantitatively that pyrene dimer is a stronger π–base as compared to its monomer. Binding energies coupled with MEP, PCA and Qzz results show that the difference in π-basicity of the monomer and dimer impacts the supramolecular chemistry involved in adducts formed with super π-acidic silver cyclometallic trimer (CTC). Non-covalent interactions between coinage metal CTCs and ammonia/phosphine substrates is reported. Interactions between these substrates and the facial plane of the π-rich gold CTC reveal a novel interaction, where the typical Lewis acid/base roles are reversed for the substrates. Adducts formed through this type of interaction define typical Lewis bases like ammonia and phosphine as Lewis acids, wherein the partially positive hydrogens coordinate to the metallo-aromatic center through dipole-quadrupole interactions. Interactions of ammonia at the side positions is shown to heavily impact the Lewis basicity of the CTC facial plane leading to similar interactions exhibited by the ammonia-gold CTC adducts. Structural and electronic properties of the adducts modeled are examined.

This thesis reports the development of several novel chemical sensing systems, self-assembled aggregates, and membrane transporters in which noncovalent and/or dynamic covalent interactions operate. Perylenebisimide dyes functionalized with boronic acid groups were designed as effective chirality sensors for [alpha]-hydroxy carboxylates. Binding of chiral ?-hydroxy carboxylate guests via boronate ester linkage leads to formation of optically active helical stacks of perylenebisimide dyes in water, giving diagnostic induced circular dichroism signals in the perylene absorption region. A boronic acid-functionalized pyrene fluorophore forms excimer-emissive stacks upon cooperative binding of fluoride ion and catechol to the boron centre, allowing sensitive sensing of fluoride at ppm levels in aqueous solution which was unprecedented for boronic acids. The stabilization of boron-fluoride adduct in the aggregate and increase of Lewis acidity via catechol binding were proposed responsible for the unprecedented affinity, as supported by control experiments. A dynamic covalent amphiphile comprised of 4-formylphenylboronic acid and octylamine forms vesicular aggregates selectively with glucose which can bind two boronic acids thus forming “Gemini-type” amphiphiles. The aggregates feature stabilization of imine bond and boronate ester linkage, with the two dynamic covalent bonds working in synergy promoting the formation of each other despite the spatial separation. The system allows selective sensing of glucose against the interference of fructose, for the first time without resorting to any synthesis. A dynamic covalent approach was employed to transmembrane transport of amino acids by the formation of a three-component assembly. A mixture of a squaramide and a lipophilic and electrophilic aldehyde is shown to synergistically transport glycine across phospholipid vesicle membranes. The transport is proposed to occur via a hydrogen-bonded anionic glycine hemiaminal/imine, with control experiments supporting the role of hemiaminal/imine in the observed facilitated glycine transport Finally, the issue of electrogenic/electroneutral transport mechanisms and potential proton or hydroxide transport for synthetic anionophores were examined. It is shown that depending on acidity, many synthetic anionophores can facilitate electrogenic proton or hydroxide transport. However, two newly-developed small molecules are shown to promote chloride transport without significant proton/hydroxide transport (pH gradient dissipation) at low concentrations, essentially mimicking the electrogenic cationophore valinomycin. The chloride > proton/hydroxide selective anionophores feature encapsulation of chloride ion via weak hydrogen or halogen bonds.

Supramolecular, or non-covalent, interactions remain a hallmark of biological systems, dictating biologic activity from the structure of DNA to protein folding and cell-substrate interactions. Harnessing the power of supramolecular interactions commonly experienced in biological systems provides numerous functionalities for modifying synthetic materials. Hydrogen bonding, ionic interactions, and metal-ligand interactions highlight the supramolecular interactions examined in this work. Their broad utility in the fields of nanoparticle formulations, polymer chemistry, and additive manufacturing facilitated the generation of numerous biological materials. Metal-ligand interactions facilitated carbon nanohorn functionalization with quantum dots through the zinc-sulfur interaction. The incorporation of platinum-based chemotherapeutic cisplatin generated a theranostic nanohorn capable of real-time imaging and drug delivery concurrent with photothermal therapies. These nanoparticles remain non-toxic without chemotherapy, providing patient-specific. Furthermore, metal-ligand interactions proved vital to retaining quantum dots on nanoparticle surfaces for up to three days, both limiting their toxicity and enhancing their imaging potential. Controlled release of biologics remain highly sought-after, as they remain widely regarded as next-generation therapeutics for a number of diseases. Geometry-controlled release afforded by additive manufacturing advances next-generation drug delivery solutions. Poly(ether ester) ionomers composed of sulfonated isophthalate and poly(ethylene glycol) provided polymers well suited for low-temperature material extrusion additive manufacturing. Ionic interactions featured in the development of these ionomers and proved vital to their ultimate success to print from filament. Contrary to ionic interactions, hydrogen bonding ureas coupled poly(ethylene glycol) segments and provided superior mechanical properties compared to ionic interactions. Furthermore, the urea bond linking together poly(ethylene glycol) chains proved fully degradable over the course of one month in solution with urease. The strength of these supramolecular interactions demanded further examination in the photopolymerization of monofunctional monomers to create free-standing films. Furthermore, the incorporation of both hydrogen bonding acrylamides and ionic groups provided faster polymerization times and higher moduli films upon light irradiation. Vat photopolymerization additive manufacturing generated 3-dimensional parts from monofunctional monomers. These soluble parts created from additive manufacturing provide future scaffolds for controlled release applications. Controlled release, whether a biologic or chemotherapeutic, remains a vital portion of the biomedical sciences and supramolecular interactions provides the future of materials for these applications.
Ph. D.

Non-covalent interactions are fundamental to molecular recognition processes that underpin the structure and function of chemical and biological systems. Their study is often difficult due to the interplay of multiple interactions and solvent effects common in complex systems. Herein, chapter one provides some general background on the area before presenting a literature review of key, contemporary developments on the use of folding molecules for the quantification of non-covalent interactions. Chapter two investigates the magnitude and extent of energetic cooperativity in H-bond chains. Utilising supramolecular complexes and synthetic molecular torsion balances, direct measurements of energetic cooperativity are presented in an experimental system in which the geometry and number of H-bonds in a chain were systematically controlled. Strikingly, it was found that adding a second H-bond donor to form a chain can almost double the strength of the terminal H-bond, while further extension had very little effect. Computations provide insights into this strong, short-range cooperative effect in a range of H-bonding contexts. Chapters three and four build on the concepts and molecular models discussed in chapter two. Chapter three discusses the effects of interplay and competition between strong H-bond acceptors such as formyl groups and the weaker organofluorine H-bond acceptor. There has been some debate in recent literature about the latter’s ability to accept H-bonds, the work presented shows that although organofluorine is a weak H-bond acceptor, it can have a significant modulating effect on stronger interactions when in direct competition. Chapter four investigates deuterium isotope effects on conformational equilibria governed by non-covalent interactions. The results show that any deuterium isotope effect which exists is less than the margins of experimental error. Finally, chapter five discusses a molecular torsion balance designed to investigate halogen∙∙∙arene interactions. The interaction energies were investigated in a range of solvents and mixtures in order to dissect out the dispersive and solvophobic components of folding. Overall, these interactions were found to be weak. Nonetheless, a model was used to dissect trends in solvophobic and electronic contributions to the binding using multiple linear regression based upon the cohesive energy density and polarisabilities of the solvents.

It is widely accepted that the sharing of electrons constitutes a bond. Conversely, molecular interactions that do not involve electron transfer, such as van der Waals forces and electrostatics are defined as "non-bonding" or "non-covalent" interactions. More recently computational and experimental observations have shown situations where the division between "bonding" and "non-bonding" interactions is blurred. One such class of interactions are known as σ-hole interactions. Chapter 1 provides a literature review of investigations into the nature of σ-hole interactions, highlighting the individual contributing factors. Chapter 2 provides a detailed analysis into the nature of chalcogen-bonding interactions. Synthetic molecular balances are employed for experimental measurements of conformational free energies in different solvents, facilitating a detailed examination of the energetics and associated solvent and substituent effects on chalcogen-bonding interactions. The chalcogen-bonding interactions examined were found to have surprisingly little solvent dependence. The independence of the conformational free energies on solvent polarity, polarisability and H-bond characteristics showed that electrostatic, solvophobic or dispersion forces were not dominant factors in accounting for the experimentally observed trends. A molecular orbital analysis provided a quantitative relationship between the experimental free energies and the molecular orbital energies, which was consistent with chalcogen-bonding interactions being dominated by an n→σ* orbital delocalisation. Chapters 3 and 4 both use the molecular orbital modelling approach established in Chapter 2 to investigate the potential partial covalency in H-bonding and carbonyl···carbonyl interactions. H-bonding is generally considered to be an electrostatically dominated interaction. However, computational results have suggested a partial covalent character in H-bonding. The molecular orbital analysis revealed an n→σ* electron delocalisation in all H-bonding systems evaluated. However, no quantitative correlation could be found with experimental free energies. Similarly, the nature of carbonyl···carbonyl interactions has been subject to debate, with electrostatic or an n→π* electron delocalisation having been proposed as the dominant factors. The molecular orbital analysis employed here showed that n→π* delocalisation was exceptionally geometry dependent. Studies of literature systems reveal that n→π* delocalisation contributes to overall stability of a range of systems, with a quantitative link between molecular orbital energy and conformational free energies.

Les interactions non-covalentes (NCI pour Non-Covalent Interactions) stabilisant les complexes non-covalents biologiques (NCX pour Non-Covalent compleXes) régissent la majorité des processus cellulaires indispensables au développement et au bon fonctionnement de tout organisme vivant. Toutes les fonctions de l'ADN, tels que son conditionnement, sa réplication et la régulation de son expression, sont permises par la formation et la dissociation de NCI avec des protéines. La compréhension des bases de ces processus cellulaires de l'ADN au niveau moléculaire est un sujet d'actualité et d'une importance fondamentale. Des informations essentielles peuvent être obtenues par spectrométrie de masse (MS pour Mass Spectrometry) qui joue un rôle de plus en plus important dans ce domaine. Malgré la technologie avancée déjà mise en ¿uvre, le développement de nouveaux concepts d'ionisation et d'activation implémentent perpétuellement la MS. Les travaux de thèse exposés à travers ce manuscrit présente l'étude de la stabilité des NCI maintenant les NCX biologiques par la comparaison des voies de fragmentations observées en mode positif et en mode négatif mais aussi par l'application de certains concepts récents de la MS comme : (i) l'utilisation d'agents de " superchargement " et, (ii) le développement et l'utilisation d'une source V-EASI (pour Venturi Easy Ambiant Sonic-spray Ionization) permettant l'aspiration libre de la solution et la désorption/ionisation des analytes par la seule vélocité du gaz de nébulisation.

My research interests during my doctoral years have been focused on high resolution rotational studies of molecules and weakly bound molecular complexes. Information on the molecular structure, internal motions and intermolecular interactions that can be obtained by applying suitable theoretical models to the analysis of these unusually complex spectra allows the determination and understanding of the driving forces involved in formation of the molecular complex. In this way, many types of non-covalent interactions have been characterized, from pure van der Waals interactions in complexes of rare gases to moderate-strength and weak hydrogen bonds (HBs) and to the most recent halogen bonds, pnicogen4 or chalcogen bonds. In this thesis, we first introduce the theory of rotational spectroscopy, including that of the asymmetrical rotor, the effects of centrifugal distortion, nuclear quadrupole coupling effects end those of internal motions In the second part, we introduce the experimental apparatuses that were used and related theoretical knowledge. In the third part, chloropentafluorobenzene (C6F5Cl) and bromopentafluorobenzene (C6F5Br) are chosen as case studies to investigate the effect of perfluorination on the molecular structure and electronic properties.In the fourth and fifth parts, we discuss the 1:1 complexes of acrolein-methanol and acrolein-ethanol. In chapter six to eight I report the results on the microwave detection and analysis of the 1:1 complexes of dimethyl sulfoxide (DMSO) with water, methanol and ethanol, respectively, in the gas phase.

Non-covalent intermolecular interactions play a large role in determining the properties of a given system, from segmented copolymers to interactions of functionalized polymers with non-viral nucleic acids delivery vehicles. The ability to control the intermolecular interactions of a given system allow for tailoring of that system to yield a desired outcome, whether it is a copolymers mechanical properties or the colloidal stability of a pDNA-delivery vector complex. Each chemical system relies on one or more types of intermolecular interaction such as hydrogen bonding, cooperative À-À stacking, electrostatic interactions, van der waals forces, metal-ligand coordination, or hydrophobic/solvophobic effects. The following research describes the tailoring of specific intermolecular interactions aimed at altering the physical properties of segmented copolymers and non-viral gene delivery vectors.
    Amide containing segmented copolymers relies heavily on hydrogen bonding intermolecular interactions for physical crosslinking to impart the necessary microphase separated morphology responsible for a copolymers physical properties. Amide containing hard segments are composed of various chemical structures from crystalline aramids to amorphous alkyl amides with each structure possessing unique intermolecular interactions. Variations to either of the copolymer segments alters the copolymers physical properties allowing for tuning of a copolymers properties for a particular application. The synthetic strategies, structure-property relationships, and physical properties of amide containing segmented copolymers are thoroughly reported in the literature. Each class of segmented copolymer that contain amide hydrogen bonding groups exhibits a wide range of tunable properties desirable for many applications. The segmented copolymers discussed here include poly(ether-block-amide)s, poly(ether ester amide)s, poly(ester amide)s, poly(oxamide)s, PDMS polyamides, and polyamides containing urethane, urea, or imide groups.
    The structure-property relationships (SPR) of poly(oxamide) segmented copolymers is not well understood with only one report currently found in literature. The effects of oxamide spacing in the hard segment and molecular weight of the soft segments in PDMS poly(oxamide) segmented copolymers demonstrated the changes in physical properties associated with minor structural variations. The optically clear PDMS poly(oxamide) copolymers possessed good mechanical properties after bulk polymerization of ethyl oxalate terminated PDMS oligomers with alkyl diamines or varied length. FTIR spectroscopy experiments revealed an ordered hydrogen bonding carbonyl stretching band for each copolymer and as the spacing between oxamide groups increased, the temperature at which the hard segment order was disrupted decreased. The increased spacing between oxamide groups also led to a decrease in the flow temperature observed with dynamic mechanical analysis. Copolymer tensile properties decrease with increased oxamide spacing as well as the hysteresis. The structure-property investigations of PDMS poly(oxamide) segmented copolymers showed that the shortest oxamide spacing resulted in materials with optimal mechanical properties.
    A new class of non-chain extended segmented copolymers that contained both urea and oxamide hydrogen bonding groups in the hard segment were synthesized. PDMS poly(urea oxamide) (PDMS-UOx) copolymers displayed thermoplastic elastomer behavior with enhanced physical properties compared to PDMS polyurea (PDMS-U) controls. Synthesis of a difunctional oxamic hydrazide terminated PDMS oligomer through a two-step end capping procedure with diethyl oxalate and hydrazine proved highly efficient. Solution polymerization of the oxamic hydrazide PDMS oligomers with HMDI afforded the desired PDMS-UOx segmented copolymer, which yielded optically clear, tough elastomeric films. Dynamic mechanical analysis showed a large temperature insensitive rubbery plateau that extended up to 186 ÚC for PDMS-UOx copolymers and demonstrated increased rubbery plateau ranges of up to 120 ÚC when compared to the respective PDMS-U control. The increase in thermomechanical properties with the presence of oxamide groups in the hard segment was due to the increased hydrogen bonding, which resulted in a higher degree of microphase separation. DMA, SAXS, and AFM confirmed better phase separation of the PDMS-UOx copolymers compared to PDMS-U controls and DSC and WAXD verified the amorphous character of PDMS-UOx. Oxamide incorporation showed a profound effect on the physical properties of PDMS-UOx copolymers compared to the controls and demonstrated promise for potential commercial applications.
    Two novel segmented copolymers based on a poly(propylene glycol) (PPG) that contained two or three oxamide groups in the hard segment were synthesized. Synthesis of non-chain extended PPG poly(trioxamide) (PPG-TriOx) and PPG poly(urea oxamide) (PPG-UOx) segmented copolymers utilized the two-step end-capping procedure with diethyl oxalate and hydrazine then subsequent polymerization with oxalyl chloride or HMDI, respectively. The physical properties of the PPG-TriOx and PPG-UOx copolymers were compared to those of PPG poly(urea) (PPG-U) and poly(oxamide) (PPG-Ox) copolymers. FTIR studies suggested the presence of an ordered hydrogen bonded hard segment for PGG-TriOx and PPG-Ox copolymers with PPG-TriOx possessing a lower energy ordered hydrogen bonding structure. PPG-UOx copolymers exhibited a larger rubbery plateau and higher moduli compared to PPG-U copolymers and also a dramatic increase in the tensile properties with the increased hydrogen bonding. The described copolymers provided a good example of the utility of this new step-growth polymerization chemistry for producing segmented copolymers with strong hydrogen bonding capabilities.
    Non-viral nucleic acid delivery has become a hot field in the past 15 years due to increased safety, compared to viral vectors, and ability to synthetically alter the material properties. Altering a synthetic non-viral delivery vector allows for custom tailoring of a delivery vector for various therapeutic applications depending on the target disease. The types of non-viral delivery vectors are diverse, however the lack of understanding of the endocytic mechanisms, endosomal escape, and nucleic acid trafficking is not well understood. This lack of understanding into these complex processes limits the effective design of non-viral nucleic acid delivery vehicles to take advantage of the cellular machinery, as in the case of viral vectors.
    Mechanisms for cellular internalization of polymer-nucleic acid complexes are important for the future design of nucleic acid delivery vehicles. It is well known that the mammalian cell surface is covered with glycosaminoglycans (GAG) that carry a negative charge. In an effort to probe the effect of GAG charge density on the affinity of cationic poly(glcoamidoamine) (PGAA)-pDNA complexes, quartz crystal microbalance was employed to measure the mass of GAGs that associated with a polyplex monolayer. Affinity of six different GAGs that varied in the charge density were measured for polyplexes formed with poly(galactaramidopentaethylenetetramine) (G4) cationic polymers and pDNA. Results showed that the affinity of GAGs for G4 polyplexes was not completely dependent on the electrostatic interactions indicating that other factors contribute to the GAG-polyplex interactions. The results provided some insight into the interactions of polyplexes with cell surface GAGs and the role they play in cellular internalization.
    Two adamantane terminated polymers were investigated to study the non-covalent inclusion complexation with click cluster non-viral nucleic acid delivery vehicles for passive targeting of the click cluster-pDNA complexes (polyplex). Incorporation of adamantyl terminated poly(ethylene glycol) (Ad-PEG) and poly(2-deoxy-2-methacrylamido glucopyranose) (Ad-pMAG) polymers into the polyplex formulation revealed increased colloidal stability under physiological salt concentrations. Ad-pMAG polyplexes resulted in lower cellular uptake for HeLa cells and not two glioblastoma cell lines indicating the pMAG corona imparts some cell line specificity to the polyplexes. Ad-pMAG provided favorable biological properties when incorporated into the polyplexes as well as increased polyplex physical properties.

Ph. D.

Ce manuscrit présente une méthodologie rationnelle de synthèse, caractérisation, détermination de la structure électronique et du comportement dynamique d’espèces bimétalliques électroniquement insaturées de type Cr(0)-M (avec M = Pd(II), Pt(II) ou Rh(I)). Ces nouvelles espèces constituent de rares exemples d’entités électro-insaturées à 14 électrons ayant la spécificité d’être persistantes en solution. Leur cohésion structurale provient essentiellement de la compensation d’un faible caractère de type donneur/accepteur entre les deux métaux par des interactions non-covalentes Coulombiennes. Nous montrons ainsi qu’en tirant profit du caractère ambiphile d’un ligand hétéroditopique capable de chélater un centre métallique par l’établissement d’une liaison covalente et d’une interaction non-covalente, de nouvelles espèces coordinativement insaturées peuvent être obtenues. Nous proposons d’appeler ce nouveau mode de chelation : « Hemichelation »
The present manuscript will present a rational method of synthesis, characterization, determination of the electronic structure and dynamic behaviour of solution-persistent, and formally unsaturated binuclear Cr(0)-M complexes (with M= Pd(II), Pt(II) or Rh(I)). This new class of complexes constitutes rare examples of persistent coordinatively unsaturated 14-electrons complexes, whose cohesion stems essentially from a compensation of insufficient donor/acceptor Cr-M bonding by non-covalent interactions of preponderant attractive Coulombic nature. By taking advantage of the ambiphilic character of a heteroditopic ligand capable of chelating a metal centre through covalent and noncovalent bonds, truly coordination-unsaturated complexes can be synthesized in a manageable form. We propose to name “Hemichelation” the half-covalent/half noncovalent bonding-relationship between the ambiphilic heteroditopic ligand and the electron-unsaturated metallic centre

The Streptomyces avidinii protein streptavidin binds the small molecule biotin (vitamin H / B₇) with extraordinary stability, resulting in the streptavidin-biotin interaction being one of the strongest non-covalent interactions known in nature (K_d ~ 10^-14 M). The stable and rapid biotin-binding, together with high resistance to heat, pH and proteolysis, has given streptavidin huge utility, both in vivo and in vitro. Accordingly, streptavidin has become a widely used tool in many different biotechnological applications. Streptavidin has also been the subject of extensive research efforts to glean insights into this paradigm for a high-affinity interaction, with over 200 mutants of the protein reported to date. Despite the high stability of the streptavidin-biotin interaction, it can and does fail under certain experimental conditions. For example, streptavidin-biotin dissociation is accelerated by an increased temperature or lower pH (conditions often encountered in cellular imaging experiments), and by mechanical stress, such as the shear force arising from fluid flow (encountered when streptavidin is used as a molecular anchor in biosensor chips and arrays). This study details efforts made at increasing further the utility of streptavidin, by increasing the stability of biotin and biotin-conjugate binding. A rational site-directed mutagenesis approach was used to create 27 mutants, with eight of these mutants possessing higher-stability biotin-binding. The most stable biotin-binding mutant was named traptavidin and was extensively characterised. Kinetic characterisation revealed traptavidin had a decreased dissociation rate from biotin and biotin-conjugates when compared to wildtype streptavidin, at both neutral pH and pH 5. Atomic force microscopy and molecular motor displacement assays revealed the traptavidin-biotin interaction possessed higher mechanical stability than the streptavidin-biotin interaction. Cellular imaging experiments revealed the non-specific cell binding properties of streptavidin were unchanged in traptavidin. X-ray crystallography was also used to generate structures of both apo- and biotinbound traptavidin at 1.5 Å resolution. The structures were analysed in detail and compared to the published structures of streptavidin, revealing the characteristics of traptavidin arose from the mutations stabilising a flexible loop over the biotin-binding pocket, as well as reducing the conformational change on biotin-binding to traptavidin. Traptavidin has the potential to replace streptavidin in many of its diverse applications, as well as providing an insight into the nature of ultra-stable noncovalent interactions.