Design, synthesis and biomedical applications of Azabicycloalkanone Amino Acid Peptidomimetics

Peptidomimetics are promising structures that replicate peptide function and conformation. They offer the potential to improve molecular-recognition, to enhance transport across biological membranes, and to resist metabolism. Among peptidomimetic classes, azabicyclo[X.Y.0]alkanone amino acids are particularly attractive, because of their capacity to rigidify backbone and side-chain geometry to replicate specific secondary structures such as turn conformations. This thesis reveals an effective means for synthesizing azabicycloalkanone amino acids having different ring sizes. Strategies for adding ring substituents to the bicyclic ring system have been developed to mimic side-chain function. Finally, a set of indolizidin-2-one amino acid (I2aa) analogs has been made based on a lead allosteric modulator of the prostaglandin-F2α (PGF2α) receptor (FP) with the goal to address the unmet biomedical need of preterm birth. A study of transannular cyclization of unsaturated lactams has provided access to a series of constrained bicyclic peptide surrogates using a common synthetic strategy featuring preparation of the cyclic precursor by coupling of enantiomerically pure ω-unsaturated amino acids followed by ring closing metathesis. Transannular iodoamidation and iodoacetoxylation of different unsaturated lactams have been accomplished where regioselectivity and diastereoselectivity was contingent on ring size, olefin position, solvent, amine protection and the use of a hypervalent iodine(III) additive. Transannular iodolactamization of different 8-, 9- and 10-member unsaturated lactams gave access to fused 5,5-, 5,6-, 6,4-, 6,5-, 6,6- and 7,5-bicycles. Although 8- and certain 9-membered lactams were resistant to iodoamidation using iodine in different solvents, transannular cyclization was achieved by adding (diacetoxyiodo)benzene to the reaction mixture. X-ray crystallographic and spectroscopic analyses of 8-, 9-, and 10-member macrocycles, and 5,5-, 5,6-, 6,5- and 7,5-bicycles have demonstrated the potential of these constrained dipeptides to mimic the central residues of ideal type I, II’, II and VI β-turn geometries. Different methods were investigated to introduce functionality onto these ring systems with the purpose of replicating side-chain presentation at such turn regions. For example, copper-catalyzed SN2' reaction of the zincate from N-(Boc)iodo-alanine onto (Z)-1,4-dichlorobut-2-ene has provided methyl 2-N-(Boc)amino-4-(chloromethyl)hexenoate. Intra- and intermolecular displacement of the chloride from this ω-unsaturated amino acid building block has given effective entry to enantiomerically pure 4-vinylproline (Vyp) and 4-vinylornithine (Von) derivatives. Side-chain diversity has also been added by C-H bond activation and allylic oxidation of dehydro-indolizidin-2-one residues. In the pursuit of agents to prolong labor and prevent preterm birth (so-called tocolytics), diversity-oriented methods were developed to prepare a series of analogues of the I2aa FP modulator PDC113.824. A series of ten different I2aa analogs was synthesized to study structure-activity relationships for FP modulation in a myometrial contraction assay. The addition of substituents was achieved on both the five and six membered rings. Certain I2aa analogues exhibited activity and efficacy in inhibiting myometrial contractions likely by way of FP modulation. An effective, atom-economical means for producing lactam and bicyclic peptidomimetics of different ring sizes has been developed using a common synthetic strategy. A wealth of X-ray structural data of the different peptidomimetics has been obtained indicating potential for turn mimicry. Mechanistic insights into the transannular cyclization to make the bicycles may have general utility for the organic chemistry community engaged in alkaloid natural product synthesis. The applications of specific azabicycloalkanone amino acids in medicinal chemistry was demonstrated by the synthesis of FP modulators possessing ring-substituted I2aa residues with the objective of developing therapeutics to delay labour and inhibit preterm birth. This thesis has thus addressed important issues in organic reactivity for the synthesis of heterocycles, in peptide science with respect to mimicry of secondary structure, and in medicinal chemistry towards treatment of the unmet medical need of preterm birth.Les molécules peptidomimétiques représentent des structures prometteuses ayant la capacité de mimer les fonctions et conformations des peptides. Elles offrent de nombreux avantages potentiels comme une meilleure reconnaissance moléculaire, un transport accru de part et d’autre des membranes biologiques et une résistance à la dégradation enzymatique. Parmi les différents types de mimes peptidiques, les azabicyclo[X.Y.0]alkanones présentent un intérêt particulier, notamment grâce à leur tendance à rigidifier les chaines peptidiques et les chaines latérales, ainsi que leur capacité à mimer certains éléments de structure secondaire tels que les repliements. L’objectif de cette thèse est d’introduire des stratégies efficaces de synthèses d’acides aminés de type azabicycloalkanone possédant des cycles de différentes tailles. De plus, différentes stratégies pour introduire des substituants sur les différents cycles ont été développées dans le but de mimer les différentes chaines latérales des acides aminés naturels. Finalement, une série d’analogues d’un modulateur allostérique du récepteur prostaglandin-F2α (FP) contenant des acides aminés indolizidin-2-one (I2aa) a été synthétisée pour tenter d’exploiter cette voie de signalisation pour le traitement des accouchements prématurés. Une étude de cyclisation trans annulaire de lactames insaturées a permis l’obtention d’une série de systèmes bicycliques rigides en utilisant une stratégie commune, la synthèse des précurseurs cycliques par couplage d’acides aminés ω-insaturés énantiopurs suivi par la fermeture du cycle par métathèse croisée. L’iodo amidation et l’iodo acétoxylation trans annulaire de différent lactames insaturés ont été accomplies de manière régio- et diastéréosélective dépendant de la taille du cycle, ii de la position de l’oléfine, du solvant, du groupe protecteur présent sur la portion amine, et de l’utilisation d’additifs d’iode (III) hypervalent. L’iodolactamisation trans annulaire de différents lactames insaturés de 8-, 9- et 10 chaînons ont fourni des bicycles 5,5-, 5,6-, 6,6-, et 7,5. Malgré le fait que certains lactames à 8 et 9 chaînons se sont avérés résistants à l’iodoamidation par l’iode moléculaire dans différents solvants, la cyclisation a été obtenue lors de l’ajout de diacétoxyiodobenzene dans le mélange réactionnel. Les analyses spectroscopiques et de cristallographie par diffraction des rayons X des macrocycles à 8, 9 et 10 chaînons ainsi que des systèmes bicycliques 5,5, 5,6 et 7,5 ont démontré le potentiel de ces composés à mimer le résidu central des tour β de type I, II’, II et VI. Différentes méthodes ont été utilisées pour introduire une variété de fonctionnalités sur les cycles dans le but de mimer les chaines latérales des acides aminés impliqués dans ce type de repliements. Par exemple, une réaction de type SN2’ catalysée au cuivre entre l’organozincate dérivé de la N-(Boc)iodo-alanine et le (Z)-1,4-dichlorobut-2-ène a permis l’obtention de la méthyle 2-N-(Boc)amino-4- (chlormethyl)hexanoate. Le déplacement intra et intermoléculaire du chlore de cet acide aminé ω- insaturé s’est avéré être une voie de synthèse efficace de la 4-vinyle-proline (Vyp) et de la 4- vinyle-ornithine (Von) sous forme énantiopure. De la diversité structurale a aussi été introduite par oxydation allylique et activation des liens C-H de résidus déhydro-indolizidin-2-one. Dans le but de développer des agents pouvant retarder le travail lors de l’accouchement et ainsi aider à prévenir les accouchements prématurés (composé tocolytiques), une stratégie orientée vers la diversité a été employée pour préparer une série d’analogues I2aa du modulateur du récepteur FP PDC113.824. Une série de dix différents analogues I2aa a été synthétisée pour étudier la relation structure-activité pour la modulation du récepteur FP dans un essai de contraction myométrial. L’addition de substituants a été possible à la fois sur les cycles à cinq et six chaînons. iii Certains de ces analogues I2aa ont démontré de l’activité en inhibant les contractions myométriales, Probablement par modulation du récepteur FP. Une méthodologie de synthèse efficace et avec une bonne économie atomique pour l’obtention de lactames et de composés peptidomimétiques bicycliques avec différentes tailles de cycles a été développée. Une grande quantité de données structurales provenant de rayons X ont été obtenus, indiquant le potentiel de ces structures à mimer divers repliements. Certains éléments mécanistiques des cyclisations trans annulaires menant aux différents bicycles pourraient avoir un impact au sein de la communauté de chimie organique impliquée dans la synthèse de produits naturel alcaloïdes. L’application d’acides aminés azabicycloalcanones spécifiques a été démontrée par la synthèse de modulateurs du récepteur FP de type I2aa comportant des substituants sur les différents cycles dans le but de développer des composés tocolytiques pour la prévention des accouchements prématurés. Le contenu de cette thèse a, par conséquent, fait un apport à la chimie organique impliquant la synthèse d’hétérocycles, à la chimie peptidique en présentant de nouveaux mimes de structure secondaire et à la chimie médicinale en permettant de faire des avancées vers le traitement des accouchements prématurés

Azabicycloalkanone synthesis by transannular cyclization

Une stratégie de synthèse efficace de différents composés de type azabicyclo[X.Y.0]alkanone fonctionnalisés a été développée. La stratégie synthétique implique la préparation de dipeptides par couplage avec des motifs vinyl-, allyl-, homoallyl- et homohomoallylglycine suivi d’une réaction de fermeture de cycle par métathèse permettant d’obtenir des lactames macrocycliques de 8, 9 et 10 membres, qui subissent une iodolactamisation transannulaire menant à l’obtention de mimes peptidiques bicycliques portant un groupement iode. Des couplages croisés catalysés par des métaux de transition ont été développés pour la synthèse d’acides aminés ω-insaturés énantiomériquement purs à partir de l’iodoanaline. L’étude du mécanisme suggère que l’iodure subit une attaque du coté le moins stériquement encombré de la lactame macrocyclique insaturée pour mener à l’obtention d’un intermédiaire iodonium. La cyclisation se produit ensuite par une route minimisant les interactions diaxiales et la tension allylique. L’iodolactamisation des différentes lactames macrocycliques insaturées a mené à l’obtention regio- et diastéréosélective d’acides aminés 5,5- et 6,6-iodobicycicliques. De plus, une imidate azabicyclo[4.3.1]alkane pontée de type anti-Bredt fut synthétisée à partir d’une lactame macrocyclique insaturé à neuf membres. Les analyses cristallographiques et spectroscopiques des macrocycles à 8, 9 et 10 membres, du composé iodobicyclique 5,5 ainsi que de l’imidate pontée, montrent bien le potentiel de ces dipeptides rigidifiés de servir en tant que mimes des résidus centraux de tours β de type I, II’, II et VI.An efficient strategy has been developed for the synthesis of different functionalized azabicyclo[X.Y.0]alkanone amino acids. The synthetic sequence features preparation of dipeptides by coupling respectively vinyl-, allyl-, homoallyl- and homohomoallylglycine building blocks, followed by ring closing metathesis to produced 8-, 9-, and 10-member unsaturated macrocyclic lactams, and transannular iodolactamization to make the bicyclic dipeptide surrogates bearing iodine on the lactam ring. Transition metal cross-coupling methods were developed to make enantiomerically pure ω-unsaturated amino acid building blocks from iodoalanine. Mechanistic considerations suggest that attack of iodine from the least hindered face of the unsaturated macrocyclic lactam provides an iodonium intermediate and cyclization occurs by a route that minimizes allylic strain and diaxial interactions. Iodolactamization of the unsaturated macrocyclic lactams gave regio- and diastereoselectively fused 5,5- and 6,6-iodo-bicylic amino acids. Moreover, an anti-Bredt bridgehead imidate azabicyclo[4.3.1]alkane was synthesized from a 9-membered unsaturated macrocyclic lactam precursor. X-ray crystallographic and spectroscopic analyses of the 8-, 9-, and 10-member macrocycles, as well as the 5,5-bicycle and bridgehead imidate demonstrate the potential of these constrained dipeptides to mimic the central residues of ideal type I, II’, II and VI β-turn geometry

Emerging roles for hyaluronidase in cancer metastasis and therapy

Hyaluronidases are a family of five human enzymes that have been differentially implicated in the progression of many solid tumor types, both clinically and in functional studies. Advances in the past five years have clarified many apparent contradictions, (1) by demonstrating that specific hyaluronidases have alternative substrates to hyaluronan (HA) or do not exhibit any enzymatic activity, (2) that high molecular weight HA polymers elicit signaling effects that are opposite those of the hyaluronidase-digested HA oligomers, and (3) that it is actually the combined overexpression of HA synthesizing enzymes with hyaluronidases that confers tumorigenic potential. This review examines the literature supporting these conclusions and discusses novel mechanisms by which hyaluronidases impact invasive tumor cell processes. In addition, a detailed structural and functional comparison of the hyaluronidases is presented with insights into substrate selectivity and potential for therapeutic targeting. Finally, technological advances in targeting hyaluronidase for tumor imaging and cancer therapy are summarized

Effect of colloidal interactions on formation of glasses, gels, stable clusters and structured films

Colloidal suspensions are ubiquitous because of their vast industrial and household usage. We demonstrate that interactions between colloidal particles play a crucial role in manipulating the phase behavior and thereby the macroscopic properties of a variety of colloidal materials, including structured films, gels, glasses and stable clusters. First, we examined films comprised of two different colloidal particles and investigated the impact of colloidal interactions in manipulating the extent of segregation in the dried films. A transport model was used to predict the volume fraction profiles of the particles as a function of film thickness, which showed that segregation could be altered by changing the particle interactions. Experimental studies were carried out using different charged latex particles and varying the pH to change the interactions, and the results from experiments and model show a very good agreement to capture the extent of segregation. Second, we studied the effect of adding low molecular weight adsorbing and non-adsorbing polymers to suspensions to modify the interparticle interactions. We studied the structural dynamics and bulk rheology of a disk-shaped clay colloid, laponite®, and polymer. Under basic conditions laponite® forms a repulsive colloidal glass. We show that low concentrations of an adsorbing polymer retards glass formation, whereas at higher concentrations an attractive glass is formed. Thus, we obtain a type of re-entrant glass transition, which is a first of its kind observed in anisotropic colloids with adsorbing polymer. On the other hand addition of a non-adsorbing polymer to laponite® suspensions triggers the formation of particle clusters, and increasing the concentration of polymer increases the strength of attraction between the particles and the size of the clusters. To further understand formation of stable clusters, we utilized population balance equations (PBE) models to study aggregation of charged colloids under quiescent conditions. We considered particles with a DLVO-type potential, where the interactions are a sum of van der Waals attraction and electrostatic repulsion. Under certain conditions, the net repulsion between large aggregates and a single particle acts as a barrier against further aggregation, and clusters reach a stable size. The PBE model was used to map out regimes of uncontrolled aggregation, controlled aggregation, and no aggregation as a function of ionic strength and colloid weight fraction. The model was tested using experimental data on charged latex particles with different colloid weight fractions and ionic strengths. The model was able to predict the regime of controlled aggregation and final size of aggregates very well. However, the rate of aggregation predicted by the model was much faster than observed experimentally. Finally, we explored aggregation of latex particles in a shear environment similar to that used in industrial toner production processes. We studied the effect of temperature, pH and coagulant concentration on aggregation and showed that there is a optimum variable space to have aggregates of controlled size and distribution

Polymer-Mediated Clustering of Charged Anisotropic Colloids

Formation of stable, dense nanoparticle clusters is interesting due to both the underlying physics and use of nanoclusters in applications such as digital printing, imaging and biosensing, and energy storage. Here, we explore formation of nanoparticle clusters in dispersions of the model disk-shaped colloid Laponite. Under basic conditions, the model disk-shaped colloid Laponite forms a repulsive glass in water due to strong electrostatic interactions. Addition of a nonadsorbing polymer, the sodium salt of poly(acrylic acid) (PAA), induces a depletion attraction between particles. Through dynamic light scattering (DLS) and rheology, we see that the polymer initially causes a transition from the glassy phase to an ergodic fluid. Samples at higher particle concentration age to a weak nonergodic state, while samples at lower Laponite remain as fluids. As the strength of attraction between particles is increased, we find an increase in the fast relaxation time measured via dynamic light scattering (e.g., slowing of the short-time diffusion of a single particle). While this may in part be attributed to an increase in the ionic strength, the aging behavior and glass-fluid transition we observe appear to be unique to the presence of polymer, suggesting that depletion plays an important role. DLS data on the fluid samples were consistent with two widely spaced diffusive relaxation modes, corresponding to motion of single particles and motion of large clusters, although other slow dynamic processes may be present. On the basis of the estimated volume fraction and depletion attraction, we believe the Laponite-PAA suspensions to be either fluids of stable dusters or glasses of clusters, although it is possible that the nonergodic state we observe is instead a gel of clusters. Additionally, the cluster size was found to be stable for at least 120 days and was directly related to the polymer concentration. This may serve as an important means of tuning cluster size in products and processes based on dense nanoparticle assemblies

A population balance equation model to predict regimes of controlled nanoparticle aggregation

Forming stable clusters or aggregates of nanoparticles is of interest in a number of emerging applications. While formation of unstable fractal aggregates and flocs has been well-studied with both experiments and theory, the conditions that lead to stable, finite-sized clusters is not as well understood. Here, we present an integrated experimental and modeling study to explore aggregation in concentrated attractive colloidal suspensions. A population balance equation (PBE) model is used to predict the aggregation dynamics of quiescent colloidal suspensions. A DLVO (Derjaguin-Landau-Verwey-Overbeek) type potential is used to describe the interparticle potential, with attractive interactions arising from van der Waals forces and long-range repulsive interactions caused by electrostatics. The PBE model includes a full calculation of stability ratio variations as a function of aggregate size, such that the energy barrier increases with increasing size. As the ionic strength is decreased, the model predicts three regimes of behavior: uncontrolled aggregation into large flocs, controlled aggregation into stable clusters, and no aggregation. The model is tested experimentally using latex particles at different salt concentrations and particle concentrations. When the Hamaker constant and surface potential are fit to aggregate size measurements collected at one salt concentration, the model accurately predicts the final mean aggregate size and regimes of aggregation at other salt concentrations and the same particle concentration. This result suggests that van der Waals and electrostatic forces are the dominant particle interactions in determining the final aggregate state. The mean aggregate size and aggregation regimes at different particle concentrations could be accurately predicted by adjusting the surface potential. This parameter adjustment is consistent with the expectation that increasing colloid weight fractions cause aggregates to have a more fractal nature and hence have a lower effective repulsion. However, the model predicts much faster aggregation rates than what are observed experimentally. This discrepancy may be due to hydrodynamic effects or another slow dynamical process which is not accounted for in the model. Nevertheless, this study presents the first PBE model that can successfully predict stable aggregate size and aggregation regimes of charged colloidal particles over a range of salt concentrations and particle concentrations. (C) 2013 Elsevier B.V. All rights reserved