This thesis entitled “Investigating the Intercarbonyl X···C′ (X = O/S/N) Interactions in Short Peptides and Peptidomimetics. Evidence of Charge→π* Interactions. Synthesis and Characterization of Thioimidate Isostere Containing Peptidomimetics” is divided into two chapters. First chapter is further subdivided into four sections where investigation of the nature of intercarbonyl X···C′ (X = O/S/N) interactions in short peptides and peptidomimetics has been described. The second chapter also has been subdivided into three parts where the syntheses and characterization of thioimidate (1,3-thiazine) and imidate (1,3-oxazine) isostere containing peptidomimetics have been discussed. Chapter 1: Section A: Revisiting the earlier models for the intercarbonyl O···C′ interactions The proximity between carbonyls is ubiquitous in crystals. Here we review the key reports that have assigned an n→π* nature to interactions between carbonyl oxygen (O) atoms and adjacent carbonyl carbon (C′) atoms (O···C′). Based on earlier hypotheses (by Burgi-Dunitz) that suggest that “the minimum energy trajectory of a nitrogen nucleophile adding to the C′ of carbonyl is at N···C′ distances of ≤ 3.2 Å and along N···C′=O angles of 109±10o”, the optimum trajectory for addition of an O to an adjacent C′ has also been assigned to be the same (O···C′ distance ≤ 3.2 Å and O···C′=O angle is 109±10o). Additionally, all O and C′ atoms within these boundary conditions in crystal structures were assigned a status of interacting and those outside of the same as non-interacting. Based on quantum mechanical models for electronic orbitals that contain the valence electrons of such proximal O and C′ atoms – derived through NBO (Natural Bond Order) calculations (on crystal structures) – it has been proposed that the filled non-bonding lone pair orbital of the O (donor) overlaps with the empty π* orbital of the carbonyl C′ (acceptor), in these O···C′ interactions. Hence, these have been termed as n→π* interactions. Using DFT (Density Functional Theory) calculations energies for these interactions have been predicted to range from 0.5 to 5.0 kcal mol-1, which are similar to those for other strong non-covalent interactions such as H-bonding, weak cation-π, etc. This n→π* interaction model is assumed to prevail between adjacent carbonyls (Oi-1···C′i) at Xaa-Pro dipeptide motifs and to be exclusively responsible for the changes in equilibrium constant values (Kc/t) for the trans to cis isomerisation reaction at Xaa-Pro peptide bond in chosen analogue molecules. Based on this assumption, these Kc/t values have been used as direct experimental equivalents for the energies of these n→π* interactions. Simultaneous to such review of literature, this chapter highlights several anomalies in this n→π* model for the intercarbonyl O···C′ interactions. We discuss the alternate models that also exist for the O···C′ proximities and show that several features – such as improved pyramidalization at the acceptor carbonyl; decrease in Kc/t values at Xaa-Pro peptide bonds; and small changes in 13C NMR chemical shift values for the acceptor carbonyls; etc. – that accompany the shortening of O···C′ distance, can be explained without invoking the n→π* interaction model. Moreover, we discuss key observations such as the presence of near-symmetric antiparallel short contacts between carbonyl groups (C=O) in crystal structures, which cannot be explained by the quantum mechanical n→π* model for the O···C′ interactions. Chapter 1: Section B: Spectroscopic and kinetic investigations into the nature of X···C′ (X = O/S) interactions in N-acyl homoserine lactones (AHLs) In this section the key interactions involving the adjacent carbonyls in model N-acyl homoserine lactones (AHLs) (which are signalling molecules in quorum sensing) in solution, their electronic nature and their influence on solvolysis of the lactone ring have been investigated. Earlier, in the crystal structures of two sterically encumbered synthetic AHL analogues N-trimethyl acetyl homoserine lactone and N-tribromoacetyl homosrine lactone the presence of an n→π* orbital overlap type interaction between Oacyl and C′lact had been suggested. Based primarily on this, the operation of similar OacylC′lact interaction was proposed in all AHLs in their solution conformations as well. More intriguingly, the interaction was hypothesized to decrease the rates of lactone hydrolysis, rendering AHLs with longer biological half-life. This is contrary to physical organic understanding of nucleophilic catalysis of addition to carbonyls. Here we synthesize a variety of AHLs and analyze their NMR and FT-IR data in solution. The spectral data reveal that the role of the N-acyl group in AHLs is to withdraw eˉs from lactone C=O inductively and to improve electronic shielding at C′lact. Lack of appreciable changes in C=O stretching frequencies of lactone and 13C NMR chemical shift values of C′lact indicate the absence of electronic perturbation of the π* of the lactone. Similar non-variance of spectral bands with improvement in nucleophilicity of the N-acyl group indicates the absence of any evidence for n→π* nature for the O···C′ interactions (between the lone pair of eˉs from Oacyl to π* at C′lact). Further the spectroscopic data indicate that any change in charge at the acyl O is felt by C′lact and this weak interaction releases energy in the order of ≤ 0.05 kcal mol-1. The combined influence of the electron withdrawing N-acyl group and the weak Oacyl···C′lact interaction in AHLs is that, increasing the charge at Oacyl increases the rate of solvolysis of lactone. Analysis of the conformation of the lactone ring in the LuxR receptor-bound and unbound crystal structure forms reveals the flattening of the puckered ring in the LuxR bound state – facilitated by several interactions with the receptor. Conserved interactions between LuxR and AHLs lock the N-acyl carbonyl motif such that they are orthogonal to the lactone carbonyl and intramolecular interaction between Oacyl and C′lact is precluded. We propose the design of flat cyclic analogues of γ-butyrolactone bearing electron withdrawing side chains as potential molecules for taking advantage of bacterial quorum sensing in environmental applications and biotechnology. Chapter 1: Section C: Spectroscopic investigation into the nature of intercarbonyl X•••C′ (X = O/S/N) interactions: Carbamyl-cisPro model systems In this section we investigate the nature of intercarbonyl X···C′ interactions in carbamyl-Pro model systems using spectroscopic methods like FT-IR and 1D NMR. Further we derive the enthalpic and entropic contributions towards the free energy for trans to cis isomerization (Kc/t) at these model carbamyl-Pro systems. Our results reveal that changes in Kc/t values cannot always be used as proof for the presence or absence of electronic interactions, and hence to unambiguously suggest the nature of these interactions. Cis/trans isomerism exists at Xaa-Pro amide and carbamate motifs, and it was proposed that in acyl-Pro systems the O···C′ interactions are responsible for the stability of either cis or trans depending upon their direction of operation (Forward direction: O of Xaa is the donor of electrons to π* at C′ of Pro; Reverse direction: O of Pro is the donor of electrons to π* at C′ of Xaa). Investigation of the carbamyl-Pro systems can shed further light on this hypothesis. Hence we undertook the first spectroscopic and Van’t Hoff analysis of homologous carbamyl-Pro model systems. The Kc/t of the homologous series surprisingly increased with increase in the bulk at R (R varies from Me to tBu). The spectroscopic data revealed the presence of charge→σ* interactions at carbamyl groups. This interaction locks the carbamyl motif in the s-transoid conformation, along the C′-O σ-bond. Such conformational lock is observed to be greater in carbamyl groups where R has at least one Cα-H bond. Interestingly, we observe the absence of X···C′ electronic interactions that may selectively stabilize the cisPro conformer in these molecules. Van’t Hoff analyses on the other hand showed that as the number of Me substituents in R increases (R = Me to iPr), there is a favorable increase in entropy ( So) associated with the transPro to cisPro conformational isomerism. As a result, the population of the cisPro conformer improves significantly as the steric bulk at R increases. We note that the enthalpy of cisPro is however relatively small and remains unfavourable as R-bulk increases (Me to iPr). These data reveal the influence of electrostatic interactions between charged groups, on the change in entropy associated with cis/trans isomerism at carbamayl-Pro motifs. This not only opposes the n→π* model, but also provides an example for the important point that changes in Kc/t can/should not be taken as direct evidences of any single electronic interaction. Importantly, this study provides another example where electronic interactions between charged, polarized carbonyl motif rather than nonbonding lone pair eˉs of carbonyl motifs influence cis/trans isomerism at Xaa-Pro systems. Chapter 1: Section D: Investigation of the stereoelectronic nature of the X···C′ (X = O/S) contacts In this section we provide experimental evidence for the existence of inverse correlation between the charge on the O nucleophile and the O···C′ distances. We show that O and C′ atoms (of adjacent carbonyls), which are separated at distances > 3.20 Å in carefully chosen analogues, come together to σ-bonding distances when the charge on O is increased to -1. Additionally, the influence of backbone steric factors on these charge→π* interactions is investigated. A partial covalent nature was proposed for the O···C′ interactions. Our study showed that the shortest intercarbonyl O···C′ distances between the O of 1°, 2° and 3° amide carbonyls and proximal C′ in molecules found in the Cambridge Structural Database (CSD) (v5.36, November 2014) show an inverse linear correlation with the partial negative charge (δ‾) on the amide carbonyl O rendered by natural amide carbonyl polarization. These data suggest the interaction of charge on the nucleophilic O with π* of the acceptor carbonyl. Further on increasing the charge on nucleophilic carbonyl O to -1 in the model compound, we achieved the formation of σ-bond through non-native (natively disallowed) Oi‾¹→C′i-1 interaction. Here we provide the first experimental evidences that suggest the interaction between charge of O and π* at adjacent C′ (the charge→π* interaction) and the latent covalent nature of the O···C′ interactions. This charge→π* model explains the origins of variations in O···C′ distances (3.20 Å–1.43 Å) in proteins and complexes that occur to suit biological functions; and the mutual interactions between antiparallel carbonyls. Further the effect of 3 key steric factors – namely the allowed τ (N-Cα-C′) angle, entropy and allowed (ϕ,ψ) angles – on the non-native Oi→C′i-1 interactions were investigated in the model compounds. Our kinetic data revealed that, the allowed τ angles have the greatest influence on charge→π* interaction, followed by entropy. Importantly the allowed (ϕ,ψ) torsional angles for residues, that govern protein folding pathways, have little influence on the O···C′ electronic interactions. Chapter 2: Section A: Design and synthesis of novel 1,3-Thiazine containing peptidomimetics This section describes the first synthesis of peptidomimetics containing the 1,3-thiazine isostere (thioimidate isostere for the peptide bond), at the C-terminus and also at the middle of the peptide. The synthesis of the 6-membered heterocycles – 1,3-oxazine (Oxa) – have earlier been reported. Oxa motifs constrain preceding amino acid backbones into natively disallowed conformations. Here we present the first synthesis of peptidomimetics containing the 1,3-thiazine (Thi) (the thioimidate analogue of Oxa) motif, by the treatment of N-(3-hydroxypropyl)thioamides with MsCl/Et3N, which leads to intramolecular S-alkylation / cyclization. When placed at the C-terminus of acyl-Pro motifs the Thi group selectively improves the stability of the rare s-cis conformation of the acyl-Pro peptide bond. Further this method has been used to synthesize peptidomimetics in which an endogenous peptide bond is replaced with the Thi isostere. These Thi analogues are shown to be stable to standard conditions of peptide coupling and N- and C- terminus protection, deprotection and can be extended selectively at their N- or C- termini. Chapter 2: Section B: Epimerization in 1,3-Thiazine containing peptidomimetics The epimerization in 1,3-thiazine containing peptidomimetics and its mechanism has been described in this section. Further the aggregation behaviour of these thiazines, in solution and crystal structures, has been studied. It has been well-documented that epimerization (Racemization) occurs at the chiral centers at the C(2) exo methine of 1,3-thiazolines and 1,3-thiazoles. Similar epimerizations in 1,3-thiazines have however not been explored. Here we report our observation of epimerization in chiral aminoacid (non Pro) containing 1,3-thiazine peptidomimetics. Our studies revealed that, the epimerization happens at C2 positions of chiral (non-Pro) amino acids-derived 1,3-thiazine containing peptiomimetics. And NH of chiral (non-Pro) amino acid fused to Thi ring at C2 position is necessary for the epimerization. Further we investigated the Boc-Xaa*-Thi analogues in solution, which showed two resonances for the carbamate N-H (HN) and the H of Xaa*, irrespective of the side chain in Xaa, in CDCl3 a weakly polar solvent. The integral ratios of the major : minor peak increased with increase in concentration for Boc-Val*-Thi, indicating the formation of H-bonded aggregates. Even in the polar aprotic (DMSO-d6) and polar protic (D2O) solvents the two sets of resonances were observed for Boc-Val*-Thi in 1H NMR. But when the thioimidate N is protonated (N of Thi is no longer a H-bond acceptor), showed only a single set of resonances. Formation of intermolecular H-bonds involving N of Thi in solution is thus evident in the aggregates. This is further suggested by the crystal structures obtained for the peptide mimetics Boc-Val*-Thi, Boc-Leu*-Thi and Boc-Phe*-Thi in which the racemic pair, instead of one enantiomer of it, are present in the unit cell and are locked in a pair of intermolecular 10 membered H-bonding interactions between NThi and HLeu* similar to an antiparallel β-sheet. A mechanism for racemization is proposed, where this strong H-bond assists enamination/racemization process. Chapter 2: Section C: Influence of a disallowed conformation of Aib on the structure of a 310-helical fold In this section, the effect of the presence of a disallowed conformation of Aib at the C-terminus of a 310-helical peptide, on the structure and fold of the rest of the peptide body has been studied in solution. We constrain the C-terminal Aib in the Aib-rich octapeptide (N-tert-butoxycarbonyl-Leu1-Aib2-Ala3-Leu4-Aib5-Ala6-Phe7-Aib8-CO2Me (1), which adopts a complete 310-helical conformation throughout the peptide body in the crystal structure and in solution) in one of its disallowed conformations using a method earlier developed in our group. This involves the synthetic modification of the C-terminal ester (Aib8-CO2Me) in 1 to an Oxa (Aib*8-Oxa) in 2 and the study of its effect on the peptide body. Analyses of the solution FT-IR, CD, ¹H, 2D (TOCSY, HSQC, HMBC and ROESY) and solvent polarity dependent NMR data reveal that 2 adopts a 310-helical conformation similar to that of 1. The C-terminal CO2Me → Oxa (E → O) modified Aib*8-Oxa motif is constrained in a unique conformation where the two Cβ atoms of Aib*8 are staggered with respect to the Aib*8 C=O and are both interacting with the two Hβ of Phe7. Here the Aib* backbone is constrained by a 5-membered ring NOxa∙∙∙HAib* H-bond, in a C5i structure. Solvent polarity dependent ¹H NMR data indicate the formation and persistence of C5i H-bond at the Aib*8-Oxa motif in 2. Analyses of the ROESY, solvent polarity dependent ¹H NMR and CD spectra reveal that four crucial changes in ROESY cross peaks occur at the Phe7-Aib*8 motif of 2, compared to that in 1. From these spectroscopic data it has been confirmed that there is no change in the structure of 2 from Leu1 to Ala6. Whatever the crucial changes happened are at Phe7-Aib*8 motif of 2. Hence our study showed that the significant structural consequences of this disallowed conformation of Aib* are primarily observed to occur in the residue in its immediate vicinity, rather than in the whole peptide body. Presence of a disallowed fold at a residue need not result in disruption of the structure, or the overall fold, in the rest of the peptide body.

Le travail rapporté dans ce manuscrit s'attache à comprendre l'influence des groupements silylés en position C7 sur l'équilibre de valence entre cycloheptatriène (CHT) et norcaradiène (NCD) et sa réactivité afin de développer de nouvelles approches synthétiques. Nous avons ainsi synthétisé 3 nouveaux cycloheptatriènes silylés et réalisé des réactions de cycloaddition [3+2] et [4+2] visant à les désymétriser. Bien qu'une rationalisation complète n'ait pu être établie, ces travaux ont mis en évidence les réactivités complexes des CHT/NCD silylés. Selon les substituants portés par le silicium, des structures cycliques à 7 chainons ou bicycliques à 6 chainons sont rapidement accessibles. De plus, les groupements silylés permettent aussi une bonne discrimination diastéréofaciale. L'utilisation de composés issus de la désymétrisation de cyloheptatriènes silylés pour la synthèse de mimes de sucres constitue un aspect majeur de nos travaux. La réaction de dihydroxylation appliquée aux CHT silylés a conduit à l’obtention de diols qui ont pu être fonctionnalisés pour conduire à trois aminoheptitols originaux. Les endoperoxides issus des réactions de photo-oxygénation ont été désymétrisés par réaction de Kornblum-DeLaMare asymétrique et une étude des différentes fonctionnalisations possibles, notamment par voie radicalaire, a également été réalisée. / This work sets out to understand the influence of the silyl group at the C7 position on the valence equilibrium and reactivity of the cycloheptatriene (CHT) - norcaradiène (NCD) system, and to use this knowledge to develop new synthetic methodologies. Three new cycloheptatrienes have been synthesized and we have undertaken [3+2] and [4+2] cycloaddition reactions with the aim of desymetrizing them. Although a complete rationale could not be established, this study has highlighted the complex reactivities of silylated CHTs/NCDs. Depending on the nature of the silyl group, 7-membered rings or bicyclic 6-membered rings can be rapidly obtained, and the silyl groups also allow diastereofacial discrimination. The application of the desymetrization reaction of silylated cycloheptatrienes to the synthesis of sugar mimics constitutes a major aspect of this work. Dihydroxylation of silylated CHTs has afforded diols which could be functionalized into three novel aminoheptitols. Endoperoxides from photooxygenation have been desymmetrized by an asymmetric Kornblum-DeLaMare reaction, and a study of possible further functionalization (notably by radical processes) has also been carried out.

The extracellular matrix (ECM) provides mechanical and biochemical support to tissues and cells. It is crucial for cell attachment, differentiation, and migration, as well as for ailment-associated processes such as angiogenesis, metastases and cancer development. An approach to study these phenomena is through emulation of the ECM by synthetic gels constructed of natural polymers, such as collagen and fibronectin, or simple but tunable materials such as poly(ethylene glycol) (PEG) crosslinked with short peptide sequences susceptible to digestion by metalloproteases and cell-binding domains. Our lab uses PEG gels to study cell behavior in three dimensions (3D). Although this system fosters cell attachment and crosslinking peptides mentioned, the regenerative process of the ECM has not been mimicked yet in 3D synthetic gels. In an attempt to build in this functionality to PEG-based gels, I performed phage display to identify short oligopeptides that bind either collagen or fibronectin to assess them as potential nucleation points for crosslinking elements in order to emulate the in vivo reconstitution process. A phage display is a library of random oligopeptides expressed on a M13 bacteriophage that allows identification of a phenotype and a genotype with a single screening step. This inexpensive strategy could yield a short oligopeptide with high specificity. I identified the conditions under which phage display is compatible with our targets, and I isolated and identified five peptide candidates for fibronectin binding and two for collagen. Future work includes assessing whether these candidates could facilitate the formation of cell-created crosslinking in 3D synthetic hydrogels.

Human Epidermal Growth Factor Receptor 2 (HER2) is a cell surface receptor tyrosine kinase and plays a role in the signal pathways leading to cell proliferation and differentiation. Overexpression of HER2 is found in various cancers including breast, ovarian, gastric, colon, and non-small-cell lung cancers, which makes it an attractive target for cancer therapy. Specific antibodies, peptides and small molecules are developed by scientists to bind with HER2 as therapeutical agents, dimerization inhibitors and biological makers. Among these molecules, antibodies showed excellent binding affinity and specificity toward HER2. However, uses of antibodies are limited by their high cost of production, long development time, limited ability to penetrate tumor tissue and immunogenicity. Many of these limitations are due to the high molecular weight of antibodies. Compared to antibodies, peptides and small molecule that selectively recognize HER2 have advantages in solubility, permeability and immunogenicity. So far, the design of all peptides and small molecules for binding with HER2 either utilize phage display technique or rely on computational screen of large library of millions of small molecules. These approaches all suffer from the drawbacks of tedious, labor intensive, and time consuming as well as uncertainty of outcome. In this study, it was hypothesized that a novel approach based on molecular interactions of HER2-Pertuzumab complex and Knob-Socket model can be developed to design antibody mimics for targeting HER2. All designed antibody mimics were simulated and docked with HER2 using Molecular Operating Environment (MOE) software to estimate binding energy and analyze the detail interaction map. A series of mimics were then synthesized and characterized. HER2 positive breast cancer cells MDA-MB-361 and ZR-75-1 were used in confocal microscopic and flow cytometric studies to evaluate the binding specificity of all antibody mimics to HER2 in vitro, while human embryonic kidney cell (HEK293) was used as control. After incubation with antibody mimics, high fluorescence intensities were observed on MDA-MB-361 and ZR-75-1 cells, while only background fluorescence were observed on HEK293 cells. Surface plasma resonance (SPR) studies showed that all antibody mimics bind to HER2 protein with KD value in range of 55.4 nM- 525.5 nM. Western blot technique was used to evaluate inhibition capability of antibody mimics on phosphorylation of HER2 downstream signaling Akt and MAPK pathways that were crucial for cell differentiation and survival. When treated with antibody mimics at 10µM for 24 h, more than 85% phosphorylation of Akt pathway was inhibited while phosphorylation of MAPK pathway was not affected. This finding proved that antibody mimics could bind to HER2 extracellular domain and selectively inhibit the dimerization between HER2 and HER3 to block phosphorylation of Akt pathway in a similar way as Pertuzumab. In addition, in vivo studies on tumor bearing nude mice were carried out to investigate the distribution and binding specificity of antibody mimics towards HER2 positive tumor after injecting through vein tail. Signal intensity ratio (SIR) of tumor to muscle revealed about 10-fold increase in tumor retention of HER2-PEP11 compared to the Cy7.5 carboxylic acid and Cy7.5-HER2-PEP22, which confirmed excellent in vivo binding specificity of antibody mimic HER2-PEP11 to HER2 positive tumor. In conclusion, this study demonstrated that a rational design of antibody mimics with both binding specificity and affinity towards HER2 based on the molecular interaction between Pertuzumab and HER2 and Knob-Socket model is feasible.

No description available.

La première partie de ce manuscrit est consacrée à l'inhibition des glycosidases. Ces enzymes sont impliquées dans de très nombreux processus biologiques et, par voie de conséquence, dans de nombreuses maladies (diabète, cancer, maldadies lysosomales etc…). Outre l'intérêt de développer de nouveaux médicaments, les glycosides hydrolases font aussi figure de cibles de choix pour étudier les itinéraires conformationnels au cours de l'hydrolyse de la liaison glycosidique et en particulier au niveau de l'état de transition. La mise en parallèle de l'importance de l'orientation des liaisons C2-O2 et C3-O3 dans la stéréosélectivité des réactions de b-mannopyranosylation dirigées par les groupements 4,6-O-benzylidène, avec le rôle des interactions avec la liaison C3-O3 dans la catalyse enzymatique pour les b-mannosidases, nous a conduits à nous intéresser au développement de nouveaux glycoimidazoles, potentiellement inhibiteurs de ces enzymes. Nous nous sommes attachés à développer ce type de molécules car ils sont considérés, à l'heure actuelle, comme les meilleurs mimes de l'état de transition. La catalyse par les b-mannosidases passerait par un état de transition de type B2,5. Ce chemin conformationnel semble inhabituel mais serait le plus approprié au développement du caractère de double liaison entre le carbone anomérique et l'oxygène endocyclique ainsi qu'à la charge positive lorsque le système est proche de l'état de transition. De plus, il semblerait que la conformation B2,5 soit aussi privilégiée pour les a-mannosidases, ce qui renforce l'intérêt thérapeutique de synthétiser de nouveaux inhibiteurs de glycosidases. La stratégie de synthèse s'appuie sur des travaux de Vasella. Elle repose sur la construction du squelette bicyclique des tétrahydroimidazopyridines, via une réaction de cyclisation intramoléculaire d'oxoéthylamidines intermédiaires, qui permet d'obtenir de nouveaux glycoimidazoles en une quinzaine d'étapes. Dans la seconde partie, sont exposés les travaux concernant la préparation et l'utilisation en synthèse de glutaconaldéhydes oxydés en position 2 et de N-acylaminopentadiènals. Après avoir rappelé l'importance des glutaconaldéhydes et des aminopentadiènals dans les hypothèses de biogenèse des alcaloïdes marins de la famille des manzamines, nous avons vu que ces espèces pouvaient être considérés comme des intermédiaires possibles pour la synthèse d'alcaloïdes marins de la famille des pyrrole-2-aminoimidazoles (P-2-AI). La nouvelle méthode de préparation des sels de glutaconaldéhydes nous a permis de synthétiser un glutaconaldéhyde oxydé en position 2 et de commencer à étudier la réactivité des 2-alkoxy-N-acylaminopentadiènals. Le dernier chapitre de cette partie traite d'une méthode de préparation de N-acyl-5-aminopenta-2,4-diènals via l'ouverture de furfurylamines N-acylées développée en parallèle avec les travaux précédemment cités. / The first part of this manuscript is dedicated to glycosidase inhibition. Because enzyme-catalyzed carbohydrate hydrolysis is a biologically widespread process, glycosidase inhibitors have many potential applications as therapeutic agents. Glycosidase inhibitors are extremely useful probes to understand vital functions of glycosidases in living systems. They are also interesting tools to study mechanisms involved in the hydrolysis by different glycosidase families and to understand conformational pathways more particularly close to the transition state of the reaction. In parallel with the importance of the orientation of C2-O2 and C3-O3 bonds for the stereoselectivity of 4,6-O-benzylidene-directed b-mannopyranosylation, the role of interactions with C3-O3 bond during enzymatic hydrolysis for b-mannosidases prompted us to develop new glycoimidazoles, potential glycosidases inhibitors. Nowadays, glycoimidazoles are considered as the best transition state mimics; that is why we were interested in these molecules. Enzyme inhibition through mimicry of the transition state is indeed a major area for the design of new therapeutic agents. The enzymatic catalysis for b-mannosidases appears to use an unusual B2,5 conformational pathway for a better accommodation of the double bond character and the positive charge on the pyranose ring close to the transition state. There is now strong support for a similar pathway for a-mannosidases. Golgi a-mannosidases are involved in cancer metastasis and this strengthens the interest of developing new glycosidases inhibitors. Our strategy leaned on Vasella's work with the construction of a tetrahydroimidazopyridine bicyclic skeleton, via an intramolecular oxoethylamidine cyclization. It allowed us to prepare new glycoimidazoles in about fifteen steps. These compounds are modified in position 3 compared to those reported in the literature. They are expected to adopt a B2,5 conformation which would allow us to get better transition state mimics than those actually known for -mannosidases. In the second part, we discuss the preparation and use of oxidized glutaconaldehydes and N-acylaminopentadienals in organic synthesis. After reminding of the importance of glutaconaldehydes and aminopentadienals in the biosynthetic scenario of manzamine alkaloids, we will see that these species are possible intermediates in the synthesis of pyrrole-2-aminoimidazoles (P-2-AI), a marine alkaloids family. A practical method to access glutaconaldehydes allowed us to prepare these species oxidized in position 2 and to begin a study of the reactivity of 2-alkoxy-N-acylaminopentadienals. Finally, in the last chapter, a new access to N-acyl-5-aminopenta-2,4-dienals through base-induced ring-opening of N-acylated furfurylamines is reported.

[ES] Toda esta disertación ha utilizado la química computacional como herramienta fundamental para el análisis científico. Por ello, en el Capítulo 2 se explican los modelos y métodos teóricos sobre este tema. La primera parte del capítulo se centra en los fundamentos de la química cuántica y, en concreto, se explica con detalle la Teoría del Funcional de la Densidad la cual constituye la base de los métodos computacionales aplicados. En esta sección, las nociones básicas del método Hartree-Fock sirven de prólogo a la DFT. El Capítulo 3 presenta los primeros resultados de este trabajo correspondientes a la reacción de metanol a olefinas catalizada por diferentes zeolitas con cavidades de poro pequeño. Esta reacción es un proceso industrial relevante que produce olefinas de cadena corta como eteno (C2=), propeno (C3=) y buteno (C4=) a escala industrial a partir de la biomasa. El sistema catalítico comprende tanto la estructura inorgánica de la zeolita que contiene los sitios ácidos Brønsted como las especies orgánicas confinadas, que forman la "hydrocarbon pool" y producen olefinas ligeras mediante pasos sucesivos de metilación y craqueo. Hemos centrado nuestros esfuerzos en comprender la naturaleza de la "hydrocarbon pool", una molécula de benceno polimetilada, y sus mecanismos de reacción para poder discernir entre ellos e identificar los catalizadores adecuados para mejorar la producción de propeno o eteno en función de la topología de cada cavidad zeolitica. Hemos podido identificar el grado de metilación de la "hydrocarbon pool" como el factor clave para potenciar el mecanismo de la ruta "paring", donde el propeno es el producto mayoritario, o el mecanismo de la ruta "side-chain", siendo el eteno el producto predominante. Este hallazgo nos permite establecer una relación entre la estabilización de los dos intermedios clave y la selectividad experimental observada con un alto grado de correlación. En el Capitulo 4 presentamos una nueva herramienta para el estudio de reacciones competitivas catalizadas por zeolitas. Utilizando un cribado computacional rápido con "force fields" para los intermedios clave de la reacción y un detallado estudio mecanístico usando la teoría del funcional de la densidad somos capaces de reconocer y cuantificar sutiles diferencias en la estabilización de intermedios y estados de transición dentro de huecos microporosos similares, aproximándonos así al nivel de reconocimiento molecular de las enzimas. Con estas herramientas somos capaces de seleccionar como catalizador una zeolita que obstaculice el mecanismo "alkyl-transfer" reduciendo la producción de eteno no deseado y potenciando al mismo tiempo el mecanismo "diaryl-mediated pathway". También somos capaces de obstaculizar la desproporción de dietilbenceno, una ruta no deseada del mecanismo "diaryl-mediated pathway" que conduce a la producción de trietilbenceno, mientras que se favorece la transalquilación de dietilbenceno aumentando el rendimiento obtenido de etilbenceno. en la primera sección del Capítulo 5, estudiamos la afinidad energética de cationes alquilamonio comercialmente disponibles con ligeras diferencias en sus grupos alquilo, TEA, MTEA y DMDEA, para la síntesis de CHA y sus efectos sobre la calidad del material obtenido. Evaluamos las energías de interacción entre la zeolita y el catión de diferentes combinaciones de agentes directores y cationes Na+ con métodos DFT periódicos pudiendo distinguir pequeños efectos de estabilización causados por ligeras diferencias estructurales entre moléculas que repercuten en la estructura final sintetizada. Durante la segunda sección del Capítulo 5, identificamos las características estructurales de diferentes agentes directores de estructura para la síntesis de AEI que mejoran las probabilidades de dispersión del Al en posiciones tetraédricas distintas de T1 obteniendo un catalizador AEI diferente de los sintetizados clásicamente. / [CA] Tota aquesta dissertació utilitza la química computacional com eina fonamental per a l'anàlisi científica. Per això, en el Capítol 2 s'expliquen els models i mètodes teòrics sobre aquest tema. La primera part del capítol es centra en els fonaments de la química quàntica i, en concret, s'explica amb detall la Teoria del Funcional de la Densitat la qual constitueix la base dels mètodes computacionals aplicats. En aquesta secció, les nocions bàsiques del mètode Hartree-Fock serveixen de pròleg a la DFT. El Capítol 3 presenta els primers resultats d'aquest treball corresponents a la reacció de metanol a olefines catalitzada per diferents zeolites amb cavitats de porus petit. Aquesta reacció és un procés industrial rellevant que produeix olefines de cadena curta com etè (C2=), propè (C3=) i butè (C4=) a escala industrial a partir de la biomassa. El sistema catalític comprèn tant l'estructura inorgànica de la zeolita que conté els llocs àcids Brønsted com les espècies orgàniques confinades, que formen la "hydrocarbon pool" i produeixen olefines lleugeres mitjançant passos successius de metilació i craqueig. Hem centrat els nostres esforços en comprendre la naturalesa de la "hydrocarbon pool", una molècula de benzè polimetilada, i els seus mecanismes de reacció per a poder discernir entre ells i identificar els catalitzadors adequats per millorar la producció de propè o etè en funció de la topologia de cada cavitat zeolitica. Hem pogut identificar el grau de metilació de la "hydrocarbon pool" com el factor clau per a potenciar el mecanisme de la ruta "paring", on el propè és el producte majoritari, o el mecanisme de la ruta "side-chain", sent l'etè el producte predominant. Al Capítol 4 presentem una nova eina per a l'estudi de reaccions competitives catalitzades per zeolites. Utilitzant un cribratge computacional ràpid amb "force fields" per als intermedis clau de la reacció i un detallat estudi mecanístic amb la teoria del funcional de la densitat som capaços de reconèixer i quantificar subtils diferències en l'estabilització d'intermedis i estats de transició dins de buits microporosos similars, aproximant-nos així al nivell de reconeixement molecular dels enzims. en la primera secció del Capítol 5, estudiem l'afinitat energètica de cations alquilamoni comercialment disponibles amb lleugeres diferències als seus grups alquil, TEA, MTEA i DMDEA, per a la síntesi de CHA i els seus efectes sobre la qualitat del material obtingut. Avaluem les energies d'interacció entre la zeolita i el catió entre diferents combinacions d'agents directors i cations Na+ amb mètodes DFT periòdics podent distingir petits efectes d'estabilització causats per lleugeres diferències estructurals entre molècules que repercuteixen en l'estructura final sintetitzada. Durant la segona secció del Capítol 5, identifiquem les característiques estructurals de diferents agents directors d'estructura per a la síntesi d'AEI que milloren les probabilitats de propagació de l'Al a través de posicions tetrahedriques diferents de T1 obtenint un catalitzador AEI diferent dels sintetitzats clàssicament. / [EN] Computational chemistry has been used as the fundamental tool during the whole work. Therefore, the theoretical models and methods on this subject are explained in Chapter 2. The first part sketches the fundamentals of quantum chemistry and specifically explains the Density Functional Theory that constitutes the basis of the computational methods applied. In this section, basic notions of the Hartree-Fock method serve as prologue for DFT after which more practical aspects are elucidated. Chapter 3 presents the first results of this work corresponding to the methanol to olefins reaction catalysed by different small-pore cage-like zeolites. This reaction is a relevant process that produces short chain olefins such as ethene, propene and butene at industrial scale from biomass. The catalytic system comprises both the zeolite inorganic framework containing the Brønsted acid sites and the confined organic species, that form the hydrocarbon pool and produce light olefins by successive methylation and cracking steps. Our efforts are focused on understanding the nature of the hydrocarbon pool, a polymethylated benzene molecule, and its reaction mechanisms in order to be able to discern between them and identify the proper catalysts to enhance propene or ethene production based on each zeolite cavity topology. We have been able to identify the hydrocarbon pool methylation degree as the key factor to enhance paring route mechanism where propene is the predominant product, or side-chain mechanism, with ethene being the predominant product. This finding enables us to establish a relation between the stabilization of the two key intermediates and the experimental selectivity observed with a high degree of correlation. In Chapter 4 we present a new tool for the study of competing reactions catalyzed by zeolites. Using a fast computational screening with force fields for the key intermediates of the reaction and a detailed density functional theory mechanistic study we are able to recognize and quantify subtle differences in the stabilization of intermediates and transition states within similar microporous voids, thus approaching the level of molecular recognition of enzymes. With these tools we are able to select a zeolite catalyst that hinders alkyl-transfer mechanism reducing the production of non-desired ethene while enhancing the diaryl-mediated pathyways mechanism. Once we discard the non-desired mechanism, we are also able to hinder the diethylbenzene disproportionation, a non-desired route of the diaryl-mediated pathways that leads to triethylbenzene production, while favouring diethylbenzene transalkylation increasing the obtained yield of ethylbenzene. To close this chapter, the theoretical results are compared with experimental selectivities obtained for eight candidate zeolites obtaining a good correlation between theory and experiment. in the first section of Chapter 5, we study the energetic affinity of commercially available alkylammonium cations with slight differences on their alkyl chain groups, as TEA, MTEA and DMDEA, for CHA synthesis and its effects on the quality of the material obtained. We evaluate the host-guest interaction energies of different combinations of OSDAs and Na+ cations with periodic DFT methods being able to distinguish small stabilization effects caused by slight structural differences between molecules that have an impact on the final structure synthesized. On the other hand, we present a new theoretical methodology to address Al positioning prediction in SSZ-39 zeolite with the AEI framework. During the second section of Chapter 5, we identify the structural features of different OSDAs for AEI synthesis that improve the probabilities of spreading Al through different T-site positions other than T1 obtaining an AEI catalyst different from the classically synthesized. / Vull agrair al Instituto de Tecnología Química per la concessió d’un contracte predoctoral, a la Red Española de Supercomputación (RES), al Centre de Càlcul de la Universitat de València, al Flemish Supercomputer Center (VSC) de la Ghent University pels recursos computacionals i el suport tècnic, a la Unió Europea i al Gobierno de España pel finançament d’aquest projecte a traves dels programes ERC-AdG-2014- 671093 (SynCatMatch) “Severo Ochoa” (SEV-2016-0683, MINECO) i dels projectes MAT2017-82288-C2-1-P i PID2020-112590GB-C21 (AEI/FEDER, UE), i al CSIC pel finançament de la estada al CMM a través del projecte i- Link (LINKA20381). / Ferri Vicedo, P. (2023). Ab initio design of efficient zeolite catalysts for methanol and hydrocarbons conversion [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/193493

Die vorliegende Arbeit befasst sich mit der Entwicklung von Biosensoren mit dem Ziel, mit Hilfe der Kopplung molekular geprägter Polymere (MIPs) als neuartiges Rezeptormaterial und dem sensitiven Nachweisprinzip der Fluoreszenz eine neue Qualität des Analytnachweises zu erreichen. Es wurde eine neue Strategie zur Optimierung der Bindungseigenschaften von molekular geprägten Polymeren in wässrigen Lösungsmitteln entwickelt, die die Kopplung aus Design of Experiments und der Optimierung multipler Zielgrößen umfasst. Damit konnten die Polymerbindungseigenschaften für alle vier betrachteten Parameter wesentlich verbessert werden. Mit Hilfe stationärer und zeitaufgelöster Fluoreszenztechniken wurde die Aufklärung der Wechselwirkung zwischen MIP und Analyt auf molekularer Ebene sowie die Charakterisierung einer neuen Nachweisstrategie basierend auf einen Förster-Resonanzenergietransfer-Mechanismus realisiert. Es wurde ferner ein MIP-Sensor für biologische Proben mit mikrofluidischer Probenzuführung aufgebaut und mittels Fluoreszenzspektrometer als konventionelles Nachweisverfahren etabliert. Darauf aufbauend wurde der optische Nachweis miniaturisiert und somit miniaturisierte Lichtquellen und Detektoren sowie eine faser-optische Lichtleitung eingesetzt. Davon ausgehend erfolgte die Optimierung des Messaufbaus hinsichtlich der Sensitivität und Nachweisgrenze des fluoreszierenden Analyten. Schließlich wurden erstmalig fluoreszenzmarkierte MIP-Partikel zur Lokalisation und Quantifizierung auf Zelloberflächen eingesetzt, d.h. diese dienten als Antikörperersatz der Immunfärbung. / This thesis deals with the development of biosensors with the aim to couple molecularly imprinted polymers (MIPs) as new receptor material with the sensitive detection principle of fluorescence in order to improve analyte detection. A new strategy for optimization of binding parameters of molecularly imprinted polymers in aqueous media was developed which is based on the coupling of design of experiments and the optimization of multiple objective parameters. Due to that the polymer binding properties for all four considered parameters could be optimized considerably. With the help of steady state and time-resolved fluorescence techniques the interaction between MIP and analyte could be clarified on a molecular basis. Furthermore the characterization of a new detection strategy based on a Förster resonance energy transfer mechanism was realized. Moreover a MIP sensor with microfluidic sample handling for biological samples was built-up and established with fluorescence spectroscopy as conventional detection method. Based on that, the optical detection was miniaturized with respect to light sources, detectors as well as optical fibers for light guidance. This set-up was optimized concerning sensitivity and limit of detection of the fluorescent analyte. Finally, for the first time fluorescently marked MIP particles were applied for imaging on cell surfaces – meaning that they were used for immunostaining as antibody mimics.

Die vorliegende Arbeit befasst sich mit der Entwicklung von Biosensoren mit dem Ziel, mit Hilfe der Kopplung molekular geprägter Polymere (MIPs) als neuartiges Rezeptormaterial und dem sensitiven Nachweisprinzip der Fluoreszenz eine neue Qualität des Analytnachweises zu erreichen. Es wurde eine neue Strategie zur Optimierung der Bindungseigenschaften von molekular geprägten Polymeren in wässrigen Lösungsmitteln entwickelt, die die Kopplung aus Design of Experiments und der Optimierung multipler Zielgrößen umfasst. Damit konnten die Polymerbindungseigenschaften für alle vier betrachteten Parameter wesentlich verbessert werden. Mit Hilfe stationärer und zeitaufgelöster Fluoreszenztechniken wurde die Aufklärung der Wechselwirkung zwischen MIP und Analyt auf molekularer Ebene sowie die Charakterisierung einer neuen Nachweisstrategie basierend auf einen Förster-Resonanzenergietransfer-Mechanismus realisiert. Es wurde ferner ein MIP-Sensor für biologische Proben mit mikrofluidischer Probenzuführung aufgebaut und mittels Fluoreszenzspektrometer als konventionelles Nachweisverfahren etabliert. Darauf aufbauend wurde der optische Nachweis miniaturisiert und somit miniaturisierte Lichtquellen und Detektoren sowie eine faser-optische Lichtleitung eingesetzt. Davon ausgehend erfolgte die Optimierung des Messaufbaus hinsichtlich der Sensitivität und Nachweisgrenze des fluoreszierenden Analyten. Schließlich wurden erstmalig fluoreszenzmarkierte MIP-Partikel zur Lokalisation und Quantifizierung auf Zelloberflächen eingesetzt, d.h. diese dienten als Antikörperersatz der Immunfärbung. / This thesis deals with the development of biosensors with the aim to couple molecularly imprinted polymers (MIPs) as new receptor material with the sensitive detection principle of fluorescence in order to improve analyte detection. A new strategy for optimization of binding parameters of molecularly imprinted polymers in aqueous media was developed which is based on the coupling of design of experiments and the optimization of multiple objective parameters. Due to that the polymer binding properties for all four considered parameters could be optimized considerably. With the help of steady state and time-resolved fluorescence techniques the interaction between MIP and analyte could be clarified on a molecular basis. Furthermore the characterization of a new detection strategy based on a Förster resonance energy transfer mechanism was realized. Moreover a MIP sensor with microfluidic sample handling for biological samples was built-up and established with fluorescence spectroscopy as conventional detection method. Based on that, the optical detection was miniaturized with respect to light sources, detectors as well as optical fibers for light guidance. This set-up was optimized concerning sensitivity and limit of detection of the fluorescent analyte. Finally, for the first time fluorescently marked MIP particles were applied for imaging on cell surfaces – meaning that they were used for immunostaining as antibody mimics.