Thèses sur le sujet « Bioconjugation reaction »

The research presented in this thesis focuses on the study of the reaction between boronic acids and diols and its evaluation as a possible "click" reaction, possibly applicable in bioconjugation and drug delivery. A key feature of this reaction is its reversibility at acidic pH, which could allow the release of a diol-containing drug from a bioconjugate in the acidic environment of late endosome/lysosome, possibly after undergoing receptor mediated endocytosis. Over the last two decades various studies have focused on the study of the conjugation of boronic acids to diols using Alizarin Red S as a fluorescence reporter. In this research we have presented an alternative method based on the batochromic shifts of Alizarin Red S absorbance; this method is particularly advantageous in complex systems with an elevated scattering, such as colloidal dispersions or for binding to complexed active compounds. We have therefore demonstrated that this method allows the determination of equilibrium constants between diols (e.g. catecholamines) and boronic acids. We have also demonstrated that the method allows to follow the kinetics of enzymatic reactions involving catechols; in particular, we have focused on cytochrome P450-mediated reactions such as the conversion of estradiol to 2-hydroxyestradiol using CYP1A2, or the demethylation of 3-methoxytyramine to dopamine using CYP2D6. Once we have established a reliable method for following this reaction on low molecular weight compounds, we have applied it to polymeric bioconjugates. Specifically, we have selected hyaluronic acid (HA) as a biocompatible and biodegradable polymeric backbone and produced derivatives containing boronic acids, catechols and dimethylated catechols (as negative controls). The resulting polymers where characterised via UV-Vis, 1H NMR and SLS, also qualitatively evaluating their cytotoxicity and enzymatic degradability. The conjugates with boronic acids showed the lowest cytotoxicity, and the highest degradability. The complexation of HA-boronic derivatives was then studied; using the same library of diols previously used with low molecular weight compounds, evaluating the effect of the presence of the polysaccharidic macromolecular chain.

Dynamic covalent chemistry (DCC) is currently exploited in several areas of biomedical applications such as in drug discovery, sensing, molecular separation, catalysis etc. Hydrazone and oxime chemistry have several advantages, such as mild reaction conditions, selectivity, efficiency, and biocompatibility and therefore, have the potential to be for bioconjugation applications. However, these reactions suffer from major drawbacks of slow reaction rate and poor bond stability under physiological conditions. In this regard, the work presented in this thesis focuses on designing novel bioconjugation reactions amenable under physiological conditions with tunable reaction kinetics and conjugation stability. The first part of the thesis presents different strategies of dynamic covalent reactions utilized for biomedical applications. In the next part, a detailed study related to the mechanism and catalysis of oxime chemistry was investigated in the presence of various catalysts. Aniline, carboxylate and saline were selective as target catalysts and their reaction kinetics were compared under physiological conditions (Paper I and II). Then we attempted to explore the potential of those chemistries in fabricating 3D hydrogel scaffolds for regenerative medicine application. A novel mild and regioselective method was devised to introduce an aldehyde moiety onto glycosaminoglycans structure. This involved the introduction of amino glycerol to glycosaminoglycans, followed by regioselective oxidation of tailed flexible diol without affecting the C2-C3 diol groups on the disaccharide repeating unit. The oxidation rate of the tailed flexible diol was 4-times faster than that of C2-C3 diol groups of native glycosaminoglycan. This strategy preserves the structural integrity of the glycosaminoglycans and provides a functional aldehyde moiety (Paper III). Further, different types of hydrazones were designed and their hydrolytic stability under acidic condition was carefully evaluated. The hydrazone linkage with the highest hydrolytic stability was utilized in the preparation of extracellular matrix hydrogel for delivery of bone morphogenetic proteins 2 in bone regeneration (Paper IV) and studied for controlled release of the growth factor (Paper III). In summary, this thesis presents a selection of strategies for designing bioconjugation chemistries that possess tunable stability and reaction kinetics under physiological conditions. These chemistries are powerful tools for conjugation of biomolecules for the biomedical applications.

Efficient chemical strategies that attach synthetic molecules to desired positions on protein surfaces are useful tools in the field of chemical biology and represent one major prerequisite for the development of new drugs and materials. Protein modification with polyethylene glycol (PEG) groups is indeed routinely performed on therapeutic proteins to improve serum half-life, or even cytotoxins or imaging agents are efficiently conjugated to cancer-targeting elements. In a typical approach, a synthetic functional group of interest is attached to a uniquely reactive amino acid group introduced by recombinant methods. Most bioconjugation reactions, however, do not reach full conversion. Therefore the development of a straightforward and reliable method to increase the extent of conversion into bioconjugates would be very helpful. In this perspective, we developed a generalizable combinatorial peptide library screening platform suitable for the identification of sequences displaying high levels of reactivity toward a desired bioconjugation reaction. This was achieved by using SAMDI MS technique (Self-Assembled Monolayer and Desorption/Ionization Mass Spectrometry) as a new, efficient and simple method for the evaluation of highly reactive amino acid motifs. The bioconjugation reaction we selected is the oxidative modification of electron-rich tyrosine residues performed using cerium(IV) ammonium nitrate (CAN) as oxidant reagent. The peptides were identified on a 361-member hexapeptide array, wherein the two N- and C-terminal residues to the target residue were varied. The arrays were prepared by immobilizing the peptides to a self-assembled monolayer of alkanethiolates on gold and could therefore be analyzed by mass spectrometry. We found that the most reactive peptides had either a serine N-terminal to the tyrosine residue or another tyrosine in proximity of the reactive site. Conversely, peptides displaying the lowest conversion level contained a positive charged residue: histidine, lysine or arginine, where the lowest relative activity was reached with arginine and leucine as C- and N- terminal residues, respectively. This study provides an important example of how synthetic peptide libraries can accelerate the discovery and optimization of protein bioconjugation strategies.

In the last decade considerable attention has been devoted to the rewarding use of Green Chemistry in various synthetic processes and applications. Green Chemistry is of special interest in the synthesis of expensive pharmaceutical products, where suitable adoption of “green” reagents and conditions is highly desirable. Our project especially focused in a search for new green radical processes which might also find useful applications in the industry. In particular, we have explored the possible adoption of green solvents in radical Thiol-Ene and Thiol-Yne coupling reactions, which to date have been normally performed in “ordinary” organic solvents such as benzene and toluene, with the primary aim of applying those coupling reactions to the construction of biological substrates. We have additionally tuned adequate reaction conditions which might enable achievement of highly functionalised materials and/or complex bioconjugation via homo/heterosequence. Furthermore, we have performed suitable theoretical studies to gain useful chemical information concerning mechanistic implications of the use of green solvents in the radical Thiol-Yne coupling reactions.

In the last decade considerable attention has been devoted to the rewarding use of Green Chemistry in various synthetic processes and applications. Green Chemistry is of special interest in the synthesis of expensive pharmaceutical products, where suitable adoption of “green” reagents and conditions is highly desirable. Our project especially focused in a search for new green radical processes which might also find useful applications in the industry. In particular, we have explored the possible adoption of green solvents in radical Thiol-Ene and Thiol-Yne coupling reactions, which to date have been normally performed in “ordinary” organic solvents such as benzene and toluene, with the primary aim of applying those coupling reactions to the construction of biological substrates. We have additionally tuned adequate reaction conditions which might enable achievement of highly functionalised materials and/or complex bioconjugation via homo/heterosequence. Furthermore, we have performed suitable theoretical studies to gain useful chemical information concerning mechanistic implications of the use of green solvents in the radical Thiol-Yne coupling reactions.

It is well established that protein glycosylation, a bioorthogonal process, is a posttranslational modification that profoundly affects protein folding, stability, immunogenicity, and biological properties and activities. In this regard, while native O- and N-glycosidic bonds of glycopeptides are prone to hydrolytic cleavage by O- and N-glycosidases, synthetic C- and S-analogs are expected to be stable toward such enzymatic degradation. Therefore, much effort has been devoted in the last decades to synthesizing C- and S-glycosyl amino acids and their assembly in glycopeptides. They with these non-native linkages can be used as probes for biochemical studies and leads in drug discovery, such as, for example, vaccines. In the first part of this thesis project we developed two routes to synthesis of S-glycopeptides. One route consists of S-glycosyl amino acid synthesis from photoinduced addition of sugar thiols to alkenyl glycine (TEC) followed by incorporation of this amino acid into a peptide. The second route, that is, specific for a cysteine containing peptide such as glutathione, involves peptide S-homoallylation followed by TEC with sugar thiol. We also demonstrated the selective propargylation of cysteine-containing peptides followed by photoinduced thiol-yne coupling with glycosyl thiols as an effective one-pot two-step platform for the dual glycosylation of peptides. By this strategy, we also performed the sequential glycosylation and biotinylation of peptides. In recent years, N-heterocyclic carbenes (NHCs) have attracted considerable interest due to their unique features, which allow them to be used as ligands for organometallic catalysis, reagents in the synthesis of heterocycles, and efficient organocatalysts in umpolung transformations. In the latter sub-area of research many efforts have been devoted to the realization of highly stereoselective versions of the classical benzoin and Stetter reactions through optimal pre-catalyst design, to the discovery of new transformations, and to the umpolung of electrophiles alternative to aldehydes and pyruvates, mainly acylsilanes and Michael acceptors. In this thesis, our group has recently demonstrated the capability of linear and cyclic dialkyl α-diketones to undergo polarity reversal under thiazolium carbene catalysis in benzoin-type and Stetter reactions, and thus act as a novel class of acyl anion precursors. Contrarily, it has been observed that diaryl α-diketones do not undergo polarity reversal in the presence of (benzo)thiazolium carbenes but are engaged in a novel multicomponent reaction with water to efficiently give medicinally relevant 1,4-thiazin-3-one heterocycles. The umpolung reactivity of diaryl 1,2-diones, however, can be effectively triggered by different NHCs.

Over the past decade, bioorthogonal chemistry has emerged as powerful tools used for tracking biomolecules within living systems. Despite the vast number of organic transformations in the literature, only select few reactions meet the stringent requirements of bioorthogonality. There is increasing demands to develop biocompatible reactions that display high specificity and exquisitely fast kinetics under physiological conditions. With the goal of increasing reaction rates as a means for reducing the concentrations of labelling reagents used for bioconjugation, we have developed metal-catalyzed and metal-free alkyne-nitrone cycloadditions as alternatives to azide-alkyne cycloadditions and demonstrate their applications for imaging cell surface proteins. The copper(I)-catalyzed alkyne-nitrone cycloaddition, also known as the Kinugasa reaction, is typically conducted with a Cu(I) catalyst in the absence of air. We have developed highly efficient micelle promoted multicomponent Kinugasa reactions in aqueous media to make the reaction faster and more efficient. Despite good product yields, the slow kinetics, limited substrate scope and competing side-reaction pathways precludes its practical applicability for biological labelling. We have designed and synthesized β-lactam alkyne probes obtained from these reactions for activity-based protein profiling of the activities of membrane proteins. Additionally, we report that alkyne tethered β-lactams serve as surface enhanced Raman spectroscopy (SERS) reporters bound to silver nanoparticles, and demonstrated that alkyne bound silver nanoparticles can be used for SERS imaging cell surface proteins. The strain-promoted alkyne-nitrone cycloaddition (SPANC) was also explored as a rapid alternative bioorthogonal reaction. We found that the reaction proceeded in high yield within aqueous media, and displayed rate enhancements that were 1-2 orders of magnitude faster than analogous reactions involving azides. The scope and kinetics of SPANC was evaluated in model reactions of various nitrones (acyclic and cyclic) with cyclooctynes, with the purpose of identifying stable nitrones that displayed intrinsically faster kinetics than azides in strain-promoted cycloadditions with cyclooctynes. Cyclic nitrones displayed good stability and exceptionally fast reactivity in these reactions. The SPANC reaction exhibited high selectivity in the presence of biological nucleophilic amino acid side chains and the presence of biological media did not adversely affect the reaction. We have utilized SPANC for highly specific labelling of proteins in vitro and for imaging ligand-receptor interactions on the surfaces of live cancer cells. The high selectivity, fast reaction rate, and aqueous compatibility of SPANC makes the reaction suitable for a variety of in vivo biological imaging applications.

Taking advantage of ATRP and using functionalized initiators, different functionalities were introduced in both α and ω chain-ends of synthetic polymers. These functionalized polymers could then go through modular synthetic pathways such as click cycloaddition (copper-catalyzed or copper-free) or amidation to couple synthetic polymers to other synthetic polymers, biomolecules or silica monoliths. Using this general strategy and designing these co/polymers so that they are thermoresponsive, yet bioinert and biocompatible with adjustable cloud point values (as it is the case in the present thesis), the whole generated system becomes "smart" and potentially applicable in different branches. The applications which were considered in the present thesis were in polymer post-functionalization (in situ functionalization of micellar aggregates with low and high molecular weight molecules), hydrophilic/hydrophobic tuning, chromatography and bioconjugation (enzyme thermoprecipitation and recovery, improvement of enzyme activity). Different α-functionalized co/polymers containing cholesterol moiety, aldehyde, t-Boc protected amine, TMS-protected alkyne and NHS-activated ester were designed and synthesized in this work.
In dieser Arbeit wurden mittels der ATRP Methode sowie durch Benutzung funktioneller Initiatoren verschiedene Funktionalitäten an der α- und ω-Position der synthetischen Polymere (Kettenenden) eingeführt. Diese funktionalisierten Polymere können durch modulare synthetische Methoden wie z.B. die “Klick-Zykloaddition” (kupferkatalysiert oder auch kupferfreie Methoden möglich), Amidierung mit anderen synthetischen Polymeren oder Biomolekülen, oder auch mit Silikatmonolithen gekuppelt werden. Den beschriebenen Strategien folgend und unter Benutzung von thermoresponsiven, bioinerten und biokompartiblen (Co-) Polymeren mit einstellbaren Trübungspunkten können mittels Temperaturänderungen leicht steuerbare, „smarte“ Polymersysteme für verschiedene Anwendungen hergestellt werden. Im Rahmen dieser Arbeit wurden speziell Anwendungen wie die Postfunktionalisierung (in situ Funktionalisierung mizellarer Aggregate mit Molekülen, die sowohl niedrige als auch höhere Molekulargewichte aufweisen), hydrophiles/hydrophobes Tuning von Polymeren, Chromatographie an Polymeren sowie Biokonjugation von Polymeren (Enzymthermoprezipitation und -Gewinnung, Enzymaktivitätsmodifizierung) genauer untersucht. Es wurden verschiedene α-funktionalisierte (Co-)Polymere, die Cholesterol, Aldehyde, t-Boc geschützte Amine, TMS-geschützte Alkine und NHS-aktivierte Ester entwickelt und hergestellt und mittels passender ATRP Initiatoren eingeführt.

La découverte et l’exploration des réactions bio-orthogonales pour le marquage spécifique d’entités biologiques est un défi majeur à portée de main depuis une dizaine d’années. Une variété de réactions bio-orthogonales a récemment été décrite, parmi lesquelles : les réactions de Diels-Alder entre des alcynes ou alcènes tendus et des tétrazines ou encore les cycloadditions entre alcynes tendus et azotures (SPAAC). Ces réactions biocompatibles sont aujourd’hui parmi les plus utilisées pour les applications de marquages in vivo ou in vitro. Récemment notre groupe et le groupe du Pr. Chin ont identifié une nouvelle réaction bio-orthogonale impliquant une sydnone et un alcyne tendu et conduisant à la formation d’un adduit pyrazole. Cette réaction a été nommée SPSAC par analogie à la réaction SPAAC.Le but de ces travaux de thèse a été, dans un premier temps, d’améliorer la cinétique de la réaction de SPSAC en incorporant différents substituants sur le noyau sydnone, de façon a montrer l’intérêt de cette réaction pour des applications en bioconjugaison en comparaison avec la réaction de SPAAC.Dans une deuxième partie et avec l’objectif de pousser plus loin le développement de cette réaction pour des applications en bioconjugaison, nous avons synthétisé des sondes sydnones pro-fluorescentes i.e. qui deviennent fluorescentes suite la réaction de SPSAC avec un alcyne tendu. La sonde la plus prometteuse a été utilisée pour effectuer le marquage sur gel d’une protéine modèle dans des milieux biologiques.Enfin les derniers travaux de cette thèse ont permis d’étendre les applications de la SPSAC à la chimie des matériaux. Une méthodologie de synthèse de sydnones tricycliques hautement conjuguées a été développée. Ces sydnones conduisent par réaction avec des diynes ou des arynes à des structures chirales complexes aux propriétés optiques intéressantes
The discovery and exploration of bio-orthogonal reactions for the specific labeling of biological entities is a major challenge. Up to now, a variety of bio-orthogonal reactions have been described, including the Diels-Alder reaction between strained alkynes or alkenes and tetrazines or the Strain Promoted Azide-Alkyne Cycloaddition (SPAAC). These “click reactions” are today the most popular tools for in vivo or in vitro chemical modifications of complex biomolecules.Recently, our group and Pr. Chin’s group have identified a new bio-orthogonal reaction involving sydnones and strained alkynes and leading to the formation of pyrazole adducts. This reaction, very similar to the SPAAC, was coined SPSAC for Strain Promoted Sydnone-Alkyne Cycloaddition.The aim of this PhD thesis was first to improve the kinetic properties of the SPSAC by incorporating various substituents on the sydnone ring in order to demonstrate the interest of using this reaction for bioconjugation applications.To extend the potential of this reaction for bio-labelling applications, we then investigated the synthesis of fluorogenic sydnone probes, i.e. sydnones that would emit fluorescence upon reaction with a strain alkyne. The most promising probe was involved in the fast fluorogenic labelling of a protein in a biological medium. This work is described in the second part of the manuscript.Finally, during the last part of my PhD, we extended the application of SPSAC to the field of material science. We developed a methodology enabling a straightforward access to highly conjugated tricycle sydnones. These sydnones, lead to complex chiral structures with interesting optical properties upon reaction with diynes or arynes

La ligation chimique implique la liaison des molécules de manière covalente pour former un nouveau complexe ayant les propriétés combinées de ses composants individuels. Ainsi, les composés naturels ou synthétiques avec leurs activités individuelles peuvent être conjuguer pour créer des substances possédant des caractéristiques uniques. Un domaine d' intérêt particulier à ces procédures est le marquage de protéines. Afin de simplifier et d'accélérer la découverte de nouvelles réactions de ligation bioselectives, nous avons conçu un système de screening rapide pour attribuer de la sélectivité et de la réactivité d'un groupement fonctionnel vers une série de dérivés d'acides aminés traçable. Une fonction chimique à propriétés prometteuse – 3-arylpropiolonitrile (APN) – a été identifiée. Les études comparatives ont démontré que cette technique offrait une meilleure sélectivité et stabilité par rapport à la technologie classique basée sur l’utilisation du groupement maléimide. L’utilisation de l’APN permet d’obtenir des bioconjugués propres et résistants à la décomposition, ce qui est d’une importance cruciale pour les applications médicales. Étude structure-réactivité nous a permis d'optimiser ses propriétés et de préparer une série de sondes fonctionnelles, dont un a été utilisé pour tester la sélectivité d'APN sur les mélanges modèles de peptides. De plus, les APN ont été trouvés à posséder une sélectivité élevée vers sélénocystéine: un acide aminé rare mais très important présent dans de nombreux enzymes actives. Une série des APN a été testée pour son activité inhibitrice envers une enzyme contenant sélénocystéine – thiorédoxine réductase – et s'est révélé posséder des activités élevées Enfin, une approche combinatoire de type split and mix a été développée visant à identifier des séquences peptidiques possédant la réactivité élevée avec les réactifs biosélectifs déjà connus
Chemical ligation involves the linking of molecules in covalent manner to form a novel complex having the combined properties of its individual components. Thus, natural or synthetic compounds with their individual activities can be chemically combined to create unique substances possessing carefully engineered characteristics. A field of especial interest in such ligation procedures is protein labeling.To accelerate the discovery of new bioselective ligation reactions, we designed a screening system for fast assigning of the selectivity and reactivity of a given functional group owards series of UVGtraceable amino acid derivatives. As a result of our screening a promising cysteineGselective scaffold–3Garylpropiolonitrile (APN)–was identified. Its remarkable selectivity, high reactivity and of both starting and addition products in aqueous and organic media represents an important advantage compared to methodologies classically used for cysteine tagging. StructureGreactivity study allowed us to optimise its properties and toprepare a series of funcional probes, one of which was used for!an accurate test of APN selectivity on model mixtures of peptides. Furthermore, APN were found to possess an elevated selectivity towards selenocysteine:ararebut very important amino!acid found in many active enzymes.A series of APN was tested for their inhibitory activity towards one of such selenocysteineGcontaining enzyme–thioredoxine reductase–and was found to possess promising activities, which however still must be!optimised.Lastly, a screening system devoted to the discovery of reagents reactivity towards a sequence of amino acid residue was elaborated and allowed us to determine presumable discrepancy in reactivity of APN depending on the amino acid residue neighbouring the cysteine moiety. Such difference in reactivity may represent an important advantage for bioconjugation, and is currently under further investigation

La mission centrale de la chimie verte est d'inventer de nouveaux procédés non polluants pour remplacer les technologies peu favorables à l'environnement. L'emploi de métaux de transition relativement non toxiques en quantités catalytiques associés à des ligands chiraux a permis de réaliser une avancée dans le domaine de la synthèse asymétrique. Dans ce travail, nous avons mis en évidence la possibilité de l'utilisation du peroxyde d'hydrogène, un oxydant vert et économique, en association avec des métalloporphyrines hydrosolubles (Fe, Mn) pour effectuer des réactions d'oxydation asymétrique (sulfoxydation, époxydation et hydroxylation). Ces systèmes représentatifs d'un modèle de l'effet " shunt " des enzymes monooxygénases dérivées de la famille cytochrome P450 sont très efficaces. Ils conduisent dans certains cas à des excès énantiomériques élevés (82%). D'autre part, nous avons développé les réactions de transfert de carbènes dans l'eau (insertion N-H et cyclopropanation asymétrique) catalysées par des porphyrines de fer. L'utilisation du fer comme métal a permis de surmonter plusieurs limites souvent rencontrées avec d'autres métaux (Ru, Rh) lors des réactions de transfert de carbènes dans l'eau. Comme application de la réaction d'insertion N-H, nous avons réalisé la bio-conjugaison régiosélective de l'insuline avec une conversion très élevée (90%).