A Multivalent entity, which could represent a protein, nanoparticle, polymer, virus or a lipid bilayer, has the ability to form multiple bonds to a substrate. Hence, a multivalent interaction can be strong, even if the individual bonds are weak. However, much more interestingly, multivalency enables the design of highly specific interactions using non-specific individual bonds. We attempt to rationalise multivalent effects using simple physical models complemented with numerical simulations. Based on physiochemical characteristics of multivalent binders, we aim to predict the overall strength of interaction and its sensitivity to variation in parameters. We start with a simple model of homo-multivalency, where all bonds are equivalent. Such systems can exhibit a super-selective response, which denotes the high sensitivity of the strength of multivalent binding to the number of accessible binding sites on the target surface. We present a theoretical analysis of systems of multivalent particles and show that a certain degree of disorder is necessary for super-selective behaviour. Moreover, we formulate a set of simple design rules for multivalent interactions that yield optimal selectivity. In the second stage, we expand the model to hetero-multivalency, accounting for multiple distinct types of binding partners. We consider targeting of cells based on a density profile of different membrane receptors types and demonstrate, that speci city towards a desired receptor density profile can be obtained. Hence, cells can be reliably targeted in the absence of specific markers. Crucially, we show that for optimal selectivity, individual bonds must be weak. Finally, we add information about specific geometry and positions of binding sites on the multivalent entity. We focus on molecular imprinting; the process whereby a polymer matrix is cross-linked in the presence of template molecules. The cross-linking process endows the polymer matrix with a chemical ‘memory’, such that the target molecules can subsequently be recognised by the matrix. We show how the binding multivalency and the polymer material properties affect the efficiency and selectivity of molecular imprinting.

This dissertation focuses on the synthesis and characterization of glyconanomaterials, as well as their applications in studying carbohydrate-protein interactions. A new and versatile method for coupling underivatized carbohydrates to nanomaterials including gold and silica nanoparticles was developed via the photochemically induced coupling reaction of perfluorophenylazide (PFPA). A wide range of carbohydrates including mono-, oligo- and poly-saccharides were conjugated to the nanoparticles with high yields and efficiency. New analytical methods were developed to determine the binding affinities of glyconanoparticles (GNPs) with lectins; these include fluorescence-based competition assay, dynamic light scattering (DLS) and isothermal titration calorimetry (ITC). Results showed that the multivalent presentation of carbohydrate ligands significantly enhanced the binding affinity of GNPs by several orders of magnitude compared to the free ligands. Systematic studies were carried out to investigate the impact of ligand presentation, i.e., the type and length of spacer linkage, the ligand density and the nanoparticle size on the binding affinity of the resulting glyconanoparticles. We used gold GNPs to study interactions with anti-HIV lectin cyanovirin-N (CV-N), and dye-doped silica nanoparticles for labeling glyans and developing high-throughput screening technique.

Les polymères synthétiques tels que les dendrons ou les dendrimères possèdent des propriétés structurales intéressantes. Leur monodispersité et leur synthèse étape par étape permet de contrôler et de caractériser totalement leur structure. De plus, la multivalence offre aux vecteurs une meilleure affinité d’interactions entre plusieurs copies d’un ligand lié au dendron/dendrimère et le récepteur désiré en comparaison au ligand seul. L’adamantane est une molécule rigide et stable dont plusieurs dérivés ont été commercialisés pour des applications thérapeutiques, principalement comme agents antiviraux. De plus, il est possible de le fonctionnaliser sur 4 positions symétriques via des synthèses organiques. Sa conformation 3D permet d’amoindrir les encombrements stériques entre les groupements fonctionnels. Nous avons alors choisi de combiner les propriétés de l’adamantane et des dendrons afin de construire de nouveaux vecteurs synthétiques. Leurs synthèses s’effectuent avec de hauts rendements et chaque nouveau composé a été totalement caractérisé par les différentes techniques d’analyses chimiques et structurales. Les dendrons à base d’adamantane polycationiques non cytotoxiques ont révélé une forte pénétration cellulaire permettant de mieux comprendre les mécanismes d’internalisation des dendrons. Ils ont également été évalués pour la formation de complexes avec un plasmide d’ADN. Des modifications sur leurs structures ont amélioré leur capacité à interagir avec l’acide nucléique grâce à la modification du point focal. Enfin, un peptide thérapeutique aux propriétés protectrices dans le lupus érythémateux disséminé, P140, a été couplé à un dendron à base d’adamantane à 3 branches et nous avons analysé les effets biologiques du trimère en comparaison avec le monomère. / Dendrons (wedge-shaped dendrimer sections) have been investigated as ideal nanoscale carrier molecules for the delivery of bioactive materials into the cells. Molecular engineering of these hyperbranched, monodisperse, well-defined structures can be easily performed using simple organic synthesis. Multivalency constituted by the multiple surface groups at the periphery of a dendron promotes higher binding affinity for ligand/receptor interactions. Adamantane molecule is a rigid structure consisting of four cyclohexane rings fused in chair conformation. The well-defined 3D conformation, the hydrophobicity and the lipophilicity provide to adamantane-based compounds favorable properties for their transport through biological membranes. In this context, the first part of this work was focused on the design and the synthesis of a novel type of polycationic dendrons based on adamantane, which are able to penetrate into cells without triggering cytotoxic effects. The next study of this Thesis concerned the investigation of our polycationic adamantane-based dendrons for gene delivery. We evaluated the capacity of the dendrons to complex a plasmid DNA. Hydrophobic compounds (biotin and cholesterol) were covalently bound to the focal point of the dendrons via “click” chemistry and the effects of the dendron generation, the peripheral cationic groups, and the hydrophobic modifications on the formation and stability of the complexes were studied. Finally, the dendrons constituted of an adamantane core, a focal point and three arms, were synthetized starting from a multifunctional adamantane derivative. We have coupled P140, a therapeutic peptide with protective properties in systemic lupus erythematosus, to an adamantane-based dendron and we have analyzed the biological effects of the resulting trimer compared to the monomer.

<p>Virtually all biologically relevant functions and processes are mediated by non-covalent, molecular recognition events, demonstrating astonishingly diverse affinities and specificities. Despite extensive research, the origin of affinity and specificity in aqueous solution - specifically the relationship between ligand binding thermodynamics and structure - remains remarkably obscure and is further complicated in the context of multivalent interactions. Multivalency describes the combinatorial interaction of multiple discrete epitopes across multiple binding surfaces where the association is considered as the sum of contributions from each epitope and the consequences of multivalent ligand assembly. Gaining the insight necessary to predictably influence biological processes with novel therapeutics begins with an understanding of the molecular basis of solution-phase interactions, and the thermodynamic parameters that follow from those interactions. Here we continue our efforts to understand the basis of aqueous affinity and the nature of multivalent additivity.</p><p>Multivalent additivity is the foundation of fragment-based drug discovery, where small, low affinity ligands are covalently assembled into a single high affinity inhibitor. Such systems are ideally suited for investigating the thermodynamic consequences of multivalent ligand assembly. In the first part of this work, we report the design and synthesis of a fragment-based ligand series for the Grb2-SH2 protein and thermodynamic evaluation of the low affinity ligand fragments compared to the intact, high affinity inhibitor by single and double displacement isothermal titration calorimetry (ITC). Interestingly, our investigations reveal positively cooperative multivalent additivity - a binding free energy of the full ligand greater than the sum of its constituent fragments - that is largely enthalpic in origin. These results contradict the most common theory of multivalent affinity enhancement arising from a "savings" in translational and rotational entropy. The Grb2-SH2 system reported here is the third distinct molecular system in which we have observed enthalpically driven multivalent enhancement of affinity.</p><p>Previous research by our group into similar multivalent affinity enhancements in protein-carbohydrate systems - the so-called "cluster glycoside effect" - revealed that evaluation of multivalent interactions in the solution-phase is not straightforward due to the accessibility of two disparate binding motifs: intramolecular, chelate-type binding and intermolecular, aggregative binding. Although a number of powerful techniques for evaluation of solution-phase multivalent interactions have been reported, these bulk techniques are often unable to differentiate between binding modes, obscuring thermodynamic interpretation. In the second part of this work, we report a competitive equilibrium approach to Molecular Recognition Force Microscopy (MRFM) for evaluation of ligand binding at the single-molecule level with potential to preclude aggregative associations. We have optimized surface functionalization strategies and MRFM experimental protocols to evaluate the binding constant of surface- and tip-immobilized single stranded DNA epitopes. Surprisingly, the monovalent affinity of an immobilized species is in remarkable agreement with the solution-phase affinity, suggesting the competitive equilibrium MRFM approach presents a unique opportunity to investigate the nature of multivalent additivity at the single molecule level.</p> / Dissertation

Diabetes Mellitus (DM) is a metabolic disorder in which the body fails to achieve glucose homeostasis, due to either insulin resistance or reduced insulin secretion or both. This inadequate glucose control leads to hyperglycemia which, if left unchecked, leads to secondary complications like nephropathy, neuropathy, retinal degeneration and other serious conditions. In non-disease state, normal glucose level in the blood is maintained by pancreatic β-cells, which secrete insulin. However, during diabetes development, there is loss of β-cell mass and function; resulting in decreased insulin secretion which is the ultimate cause of hyperglycemia. The ability to non-invasively monitor changes in the β-cell mass during the development or treatment of diabetes would be a significant advance in diabetes management. However, a primary limitation for analysis of β-cell mass and developing dysfunction is the lack of specificity of β-cell targeting agents. Our novel approach for achieving the required specificity for a usable β-cell targeted contrast agent is to target a set of receptors on the cell surface that, as a combination, are unique to that cell. Through genetic screening, Glucagon Like Peptide-1 Receptor (GLP-1R) and α2Adrenergic Receptor (α2AR) were chosen as a potential molecular barcode for β-cells since their combination expression is relatively unique to the β-cells. GLP-1R and α2AR are both G-protein couple receptors (GPCRs) that, apart from being a β-cell specific combination, play an important role in regulating fundamental downstream signaling pathways in β-cells. To target these receptors effectively, we synthesized a multivalent ligand composed of Yohimbine (Yhb), an α2 adrenergic receptor (α2AR) antagonist, linked to an active Glucagon-like Peptide 1 analog (GLP-1₇₋₃₆). In this manuscript, I describe the synthesis and characterization of binding selectivity and signaling ability of GLP-1/Yhb at the cellular level. Using high throughput binding assays, we observed high affinity binding of GLP-1/Yhb to βTC3 cells, a β-cell mimetic line expressing both receptors, at a Kd of ~3 nM. Using microscopy, we observed significant Cy5-tagged GLP-1/Yhb binding and rapid internalization in cells expressing the complementary receptor pair at low concentrations, as low as 1 nM and 5 nM. When one of the receptors was made inaccessible due to presence of saturating quantities of a single unlabeled monomer, GLP-1/Yhb-Cy5 failed to bind to the cells at low concentrations (<10 nM). Similarly, in cells where either GLP-1R or α2AR were knocked down (using shRNA), binding of GLP-1/Yhb was significantly reduced (≤half of cells with both receptors), indicating strong selectivity of the ligand to cells expressing the combination of receptors. We also observed that GLP-1/Yhb construct modulates downstream signaling inβ TC3 cells resulting in enhanced Glucose Stimulated Insulin Secretion (GSIS). In presence of stimulatory glucose, GLP-1/Yhb significantly potentiated GSIS with a half-maximal effective dose of 2.6 nM. Compared to GLP-1₇₋₃₆ alone or GLP-1₇₋₃₆ and Yhb monomers added together, only GLP-1/Yhb could significantly potentiate GSIS at 1 nM, demonstrating that GLP-1/Yhb could translate high affinity binding to increased efficacy for GSIS potentiation. Unlike for insulin secretion, high affinity divalent binding did not translate to increased cAMP production at low concentrations, with significant increases above baseline seen only at 10 nM and higher. Nevertheless, these data show that GLP-1/Yhb binds selectively to β-cells and affects signaling, demonstrating its potential for targeted β-cell imaging and therapy. Overall, our work indicates that synthetic heterobivalent ligands, such as GLP-1/Yhb can be developed to increase cellular specificity and sensitivity making them a strong candidate for both noninvasive imaging and targeted therapy.

The combat against viral diseases has been, and still is, a major challenge in the field of drug development. Viruses are intracellular parasites that use the host cell ma-chinery for their replication and release. Therefore it is difficult to target and destroy the viral particle without disturbing the essential functions of the host cell. This thesis describes studies towards antiviral agents targeting adenovirus type 37 (Ad37), which causes the severe ocular infection epidemic keratoconjunctivitis (EKC). Cell surface oligosaccharides serve as cellular receptors for many pathogens, including viruses and bacteria. For EKC-causing adenoviruses, cell surface oligo-saccharides with terminal sialic acid have recently been shown to be critical for their attachment to and infection of host cells. The work in this thesis support these re-sults and identifies the minimal binding epitope for viral recognition. As carbo-hydrate–protein interactions in general, the sialic acid–Ad37 interaction is very weak. Nature overcomes this problem and vastly improves the binding affinity by presenting the carbohydrates in a multivalent fashion. Adenoviruses interact with their cellular receptors via multiple fiber proteins, whereby it is likely that the ideal inhibitor of adenoviral infections should be multivalent. This thesis includes design and synthesis of multivalent sialic acid glycoconjugates that mimic the structure of the cellular receptor in order to inhibit adenoviral attachment to and infection of human corneal epithelial (HCE) cells. Synthetic routes to three different classes of sialic acid conjugates, i.e. derivatives of sialic acid, 3’-sialyllactose and N-acyl modified sialic acids, and their multivalent counterparts on human serum albumine (HSA) have been developed. Evaluation of these conjugates in cell binding and cell infectivity assays revealed that they are effective as inhibitors. Moreover the results verify the hypothesis of the multivalency effect and clearly shows that the power of inhibition is significantly increased with higher orders of valency. Potential inhibi-tors could easily be transferred to the eye using a salve or eye drops, and thereby they would escape the metabolic processes of the body, a major drawback of using carbohydrates as drugs. The results herein could therefore be useful in efforts to develop an antiviral drug for treatment of EKC.

<p>Antivascular targeting is a promising strategy for tumor therapy. This strategy overcomes many of the transport barriers and has shown efficacy in many preclinical models, but targeting epitopes on tumor vasculature can also promote accumulation in healthy tissues. We used Elastin-like Polypeptide (ELP) to form block copolymers (BCs) consisting of two separate ELP blocks seamlessly fused at the genetic level. ELPBCs self-assemble into spherical micelles at a critical micelle temperature (CMT), allowing external control over monovalent unimer and multivalent micelle forms. We hypothesized that thermal self-assembly could trigger specific binding of ligand-ELPBC to target receptors via the multivalency effect as a method to spatially restrict high-avidity interactions. We termed this approach Dynamic Affinity Modulation (DAM). The objectives of this study were to design, identify, and evaluate protein-based drug carriers that specifically bind to target receptors through static or dynamic multivalent ligand presentation.</p> <p>ELPBCs were modified to include a low-affinity GRGDS or GNGRG ligand and a unique conjugation site for hydrophobic compounds. This addition did not disrupt micelle self-assembly and facilitated thermally-controlled multivalency. The ability of ligand-ELPBC to specifically interact with isolated AvB3 or CD13 was tested using an in vitro binding assay incorporating an engineered cell line. RGD-ELPBC promoted specific receptor binding in response to multivalent presentation but NGR-ELPBC did not. Enhanced binding with multivalent presentation was also observed only with constructs exhibiting CMT < body temperature. This study establishes proof-of-principle of DAM, but ELPBC requires thermal optimization for use with applied hyperthermia. Static affinity targeting of fluorescent ligand-ELPBC was then analyzed in vivo using intravital microscopy (IM), immunohistochemistry (IHC), and custom image processing algorithms. IM showed increased accumulation of NGR-ELPBC in tumor tissue relative to normal tissue while RGD-ELPBC and non-ligand ELPBC did not, and IHC verified these observations. This study shows (1) multivalent NGR presentation is suitable for static multivalent targeting of tumors and tumor vasculature, (2) multivalent RGD presentation may be suitable for DAM with thermal optimization, and (3) ELPBC micelles may selectively target proteins at the tumor margin.</p> / Dissertation

Effective detection and treatment of tumor malignancies depends upon identifying targets – molecular markers that differentiate cancer cells from healthy cells. Current cancer therapies involve targeting overexpressed specific gene products. An alternative approach is proposed here: to specifically target combinations of cell-surface receptors using heteromultivalent ligands (htMVLs). There are about 2500 genes encoding for cellsurface proteins in the human genome that can potentially be targeted. Taken as sets, there can be ~ 10⁶ two-receptor combinations and ~ 10⁹ three-receptor combinations available. Our group envisions that using cell-surface protein combinations that are expressed on a cancer cell but not on a normal cell, multivalent constructs displaying complementary ligands of weak affinities can be assembled. These multivalent ligands should bind with high avidity to cancer populations in vivo, and provide a degree of specificity not seen with current approaches. As a proof-of-concept, a series of multivalent ligands were designed and synthesized for a model system consisting of the human Melanocortin subtype 4 receptor (hMC4R) and the Cholecystokinin subtype 2 receptor (CCK-2R). Modeling studies on GPCR dimers predicted that a minimum linker span of 20 - 50 Å would be required to non-covalently crosslink these two receptors. The multivalent ligands were assembled using a modular parallel synthesis approach and using solidphase chemistries. A variety of linkers were explored ranging from highly rigid to highly flexible, and using natural and/or synthetic building blocks. Ligand binding affinities were evaluated using a lanthanide based competitive binding assay in cells that expressed both receptors (bivalent binding) vs those that expressed only one of the receptors (monovalent binding), and were demonstrated to have enhanced binding affinities of up to nearly two orders of magnitude. The promising ligands were further explored by synthesizing fluorescently labeled and/or lanthanide chelate labeled monovalent and heterobivalent ligands designed for in vitro and in vivo studies. More explorative work using these labeled constructs is in progress. To the best of our knowledge, the author believes this is the first such demonstration of a 'synthetic htMVL' directed recruitment and crosslinking of two heterologous cell-surface receptors. This receptor combination approach opens up new possibilities for single cell imaging, cancer detection and therapeutic intervention, and can provide a revolutionary new platform technology with which to direct therapeutics to defined cell populations.

abstract: ABSTRACT Manipulation of biological targets using synthetic or naturally occurring organic compounds has been the focal point of medicinal chemistry. The work described herein centers on the synthesis of organic small molecules that are targeted either to cell surface receptors, to the ribosomal catalytic center or to human immunodeficiency virus reverse transcriptase. Bleomycins (BLMs) are a family of naturally occurring glycopeptidic antitumor agents with an inherent selectivity towards cancer cells. DeglycoBLM, which lacks the sugar moiety of bleomycin, has much lower cytotoxicity in cellular assays. A recent study using microbbuble conjugates of BLM and deglycoBLM showed that BLM was able to selectively bind to breast cancer cells, whereas the deglyco analogue was unable to target either the cancer or normal cells. This prompted us to further investigate the role of the carbohydrate moiety in bleomycin. Fluorescent conjugates of BLM, deglycoBLM and the BLM carbohydrate were studied for their ability to target cancer cells. Work presented here describes the synthesis of the fluorescent carbohydrate conjugate. Cell culture assays showed that the sugar moiety was able to selectively target various cancer cells. A second conjugate was prepared to study the importance of the C-3 carbamoyl group present on the mannose residue of the carbohydrate. Three additional fluorescent probes were prepared to improve the uptake of this carbohydrate moiety into cancer cells. Encouraged by the results from the fluorescence experiments, the sugar moiety was conjugated to a cytotoxic molecule to selectively deliver this drug into cancer cells. The nonsense codon suppression technique has enabled researchers to site specifically incorporate noncanonical amino acids into proteins. The amino acids successfully incorporated this way are mostly &#945;-L-amino acids. The non-&#945;-L-amino acids are not utilized as substrates by ribosome catalytic center. Hoping that mutations near the ribosome peptidyltransferase site might alleviate its bias towards &#945;-L-amino acids, a library of modified ribosomes was generated. Analogues of the naturally occurring antibiotic puromycin were used to select promising candidates that would allow incorporation of non-&#945;-L-amino acids into proteins. Syntheses of three different puromycin analogues are described here. The reverse transcriptase enzyme from HIV-1 (HIV-1 RT) has been a popular target of HIV therapeutic agents due to its crucial role in viral replication. The 4-chlorophenyl hydrazone of mesoxalic acid (CPHM) was identified in a screen designed to find inhibitors of strand transfer reactions catalyzed by HIV-1 RT. Our collaborators designed several analogues of CPHM with different substituents on the aromatic ring using molecular docking simulations. Work presented here describes the synthesis of eight different analogues of CPHM. / Dissertation/Thesis / Ph.D. Chemistry 2013

Les bambusurils, BU[4] et BU[6], sont des oligomères cycliques apparentés aux cucurbiturils, CBs, constitués respectivement de 4 et 6 motifs glycolurils. Les bambusurils diffèrent des CBs par la présence de glycolurils difonctionnalisés.Les BU[6] ont la capacité d'encapsuler des anions dans leur cavité, propriété intéressante pour la décontamination d'effluents, par exemple.Une nouvelle famille de bambusurils, les allylbambusurils, qui possèdent des groupements allyles sur leur portail macrocyclique, a été développée. Leur post-fonctionnalisation par oxydation, métathèse croisée ou réaction thiol-ène a été étudiée. Par réaction thiol-ène, des BU[4] et BU[6], fonctionnalisés respectivement par 8 ou 12 thiols d'intérèt, ont été obtenus. Les BU[6] sont toujours isolés avec un halogènure à l’intérieur de leur cavité. Une méthode utilisant l’hexafluoroantimonate d’argent a été mise au point pour les décomplexer. L'affinité de ces nouveaux BU[6] exempts d'anion, pour différents halogénures, a été évaluée par RMN 1H.Des glycobambusurils ont été synthétisés par réaction thiol-ène en présence de sucres fonctionnalisés par des thiols. Ces glycoBUs donnent accès à des plateformes multivalentes de valence 8 pour les BU[4] et 12 pour les BU[6]. Le pouvoir inhibiteur de ces nouvelles plateformes a été testé sur l'enzyme WaaC, une heptosyltransferase présente dans la paroi bactérienne. Les tests enzymatiques montrent que ces glycobambusurils sont des plateformes multivalentes prometteuses. / Bambusurils, BU[4] and BU[6] are cyclic oligomers that belong to the cucurbiturils family, CBs, assembled respectively by 4 and 6 glycoluril units. Bambusurils are different from cucurbiturils because of their difunctionalized glycolurils. BU[6] are able to encapsulate anions inside their cavity and this property can be useful for the treatment of effluents.A new family of BUs, the allylbambusurils having allyls groups on their macrocyclic portal, has been developed. Their postfunctionalization by oxidation, cross metathesis and thiol-ene coupling has been studied. BU[4] and BU[6] functionalized by respectively 8 and 12 thiols of interest have been prepared.BU[6] are always obtained with an halide inside the cavity. A method using silver hexafluoroantimonate has been developed to remove this halide. Binding constants of these new empty bambusurils have been determined towards severals halide by 1H NMR.Glycobambusurils have been synthesized by thiol- ene coupling with thiosugars. These glycoBUs can lead to multivalent platforms of valency up to 8 for BU[4] and 12 for BU[6]. Inhibition activity of these new platforms has been tested on WaaC enzyme, an heptosyltransferase found in bacterial cell wall. Enzymatic tests show that these glycobambusurils are promising multivalent platforms.