Matrix Metalloproteinase-13 (MMP-13) belongs to a large family of proteolytic enzymes which are characterized by their ability to degrade the extracellular matrix components. MMP-13 appears to have a critical role in tumor invasion and metastasis. In this study, several fluorogenic probes specific for MMP-13 were designed and characterized. These synthesized probes could be modified with chelators to be applied for imaging MMP-13 in breast cancer and/or multiple myeloma models. The activity and selectivity of MMP-13 and other MMPs against these probes were studied through two approaches. It was found that these probes were cleaved by all MMPs, but MMP-13 showed the highest activity and selectivity towards these peptides. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

Visualizing proteins in living cells without perturbing biological function remains a key challenge in chemical biology. A chemical approach to this problem is the synthesis of small molecule fluorophores that react specifically with a protein of interest (POI). We have developed a site-specific labelling method based on a Fluorogenic Addition Reaction (FlARe). The FlARe probe’s fluorescence is quenched until it undergoes thiol addition with a small, genetically encoded dicysteine peptide tag fused to the POI. Recent blue coumarin probes were shown to be highly selective for target proteins over other cellular thiols; however, fluorogens that can label in the red and green channels of the fluorescence microscope are more desirable for cellular imaging, as red light is lower in energy and therefore less photo-toxic. In the work presented herein, we use DFT calculations to guide the design of red-shifted, PeT-quenched BODIPY based dimaleimide fluorogens. Driven by the preliminary results of a FlARe probe (YC29) that emitted in the red channel, we attempted to prepare the hit compound through a new synthetic approach to further evaluate kinetics and in cellulo labelling. Given the time available, this compound was unable to be synthesized through an SNAr or Pd-catalyzed approach. Alternatives probes lacking the red-shifting substituent were synthesized and evaluated in vitro and in cellulo. The fluorescent enhancement and reaction kinetics of these probes were evaluated in detail, in order to determine the suitability of their application to cellular labelling. A green-BODIPY fluorogen was synthesized that exhibits suitable kinetics for labelling and a dramatic fluorescent enhancement of ~800-fold upon tagging. This probe was successfully applied to the specific, fluorescent labelling of a nuclear histone protein in cellulo.

This dissertation is composed of two research projects. The first research project is aimed at using synthetic fluorogenic probes to study the possible or dominant linkages in biomass. These probes that mimic the linkages found in lignin-cellulosic biomass are designed to select the optimal fungi from direct evaluation process or could be tested against other microbials to screen candidates which can break ligno-hemicellulose bonds. For the first stage, these probes would be tested against white rot fungi extract. The white rot fungi are used for the first stage to see if releasing or degrading carbohydrates while keeping lignin largely intact is possible or not. These probes can help to answer fundamental questions, such as what could be the dominant linkages between lignin and hemicellulose, and what are the possible mechanisms for the cleavage of carbohydrates in biomasses. Understanding the linkages in these biomass will enable high efficient degradation or release of carbohydrates, primarily hemicelluloses, from biomass. The second project is focused on synthesizing new aminoglycoside analogs and exploring the potential to revive traditional antibacterial kanamycin as new types of antifungal agents. Aminoglycosides are widely used broad spectrum antibiotics. Although mainly used as antibacterial agents, there have been studies to show amphiphilic aminoglycoside derivatives could be possibly employed as antifungal agents. A concise and novel method for site-selective alkylation of tetra-azidokanamycin has been developed that leads to the divergent synthesis of three classes of kanamycin derivatives. These new amphiphilic kanamycin derivatives bearing alkyl chains length of 4, 6, 7, 8, 9, 10, 12, 14，16 have been synthesized and tested against bacteria and fungi. Surprisingly, the antibacterial effect of the synthesized kanamycin derivatives decline or disappear compared with the original kanamycin A, but some of the compounds show very strong activity as antifungal agents.

Despite major advances in the diagnosis and treatment of cancer, there remains a paucity of biomarkers for early detection (poor selectivity and specificity). Legumain [asparaginyl endopeptidase (AEP); EC 3.4.22.34] is a potential cancer biomarker and molecular target for imaging and therapy. Legumain is a lysosomal protease, active at acidic pH (4.0 - 6.5) with a remarkably restricted substrate specificity, uniquely cleaving an asparagine (Asn) at the P1 position, and is overexpressed in various solid tumours. A novel legumain-targeted first generation fluorogenic rhodamine-B based peptide substrate Rho-Pro-Ala-Asn~PEG-AQ (SM9) has been developed for diagnostic application in the early detection of tumours, which exploits the enzyme's proteolytic specificity. The fluorogenic probe SM9 is an efficient FRET substrate, in which an aminoanthraquinone acts as a ‘black hole' quencher of rhodamine fluorescence that is restored on incubation with recombinant (rh)-legumain. Importantly, confocal microscopy studies have revealed localization of SM9 in the lysosomes of PC3 prostate cancer cells. The design principles have been extended to a second generation orthogonally functionalised, legumain-activated dual fluorogenic gadolinium-based magnetic resonance imaging (MRI) contrast agent Rho-Pro-Ala-Asn~Lys-[DOTA-(Gd3+)]-PEG-AQ (SM32). Furthermore, towards the development of selective and targeted theranostic (therapeutic and diagnostic) anticancer agents, a novel third generation fluorogenic legumain substrate prodrug Rho-Pro-Ala-Asn~Propyl-Pip-Propyl-AQ (ALS5) that incorporates a cytotoxic, lysosomotropic anthracenedione ALS1, has been designed. Activation of prodrug ALS5 by rh-legumain in vitro directly afforded the cleavage product and active drug ALS1. Confocal microscopy studies have shown that ALS5 and active ALS1 (at 1 μM) were also localized in the lysosomes of PC-3 cells. Furthermore, ALS1 induced morphological changes and apoptosis in PC-3 cells, as measured by fluorescence microscopy, and staining with Annexin V and DAPI using flow cytometry. All novel legumain-activated oligopeptide substrates and intermediate compounds have been fully characterised by high resolution mass spectrometry and NMR spectroscopy. Selected compounds have been further characterised by HPLC. The molecular probes and prodrugs have the potential to be used as diagnostic tools to define the legumain expression in tumour biopsies and provide prognostic information of value in determining patient-focussed treatment options, with extension to therapeutic strategies to improve tumour targeting.

L'étude de la dynamique des protéines est essentielle pour comprendre les processus biologiques. Notre laboratoire a développé une nouvelle classe de protéines fluorescentes semi-synthétiques, appelée Fluorescence-Activating and absorption-Shifting Tag (FAST). Cette thèse de doctorat présente le développement de nouveaux systèmes FAST avec diverses propriétés pour l'imagerie multiplexée. Nous avons développé une série de fluorogènes permettant de modifier la couleur de FAST de vert-jaune à orange et rouge. Au delà de l’application de l’imagerie multi-couleurs, ces fluorogènes permettant un échange dynamique des couleurs grâce à la liaison réversible de FAST, ouvrant de nouvelles perspectives pour le développement de méthodes d’imagerie sélective reposant sur la dynamique de systèmes réactifs. Pour étendre davantage les propriétés spectrales de FAST vers le rouge lointain, nous avons développé une nouvelle série de fluorogènes rouges, pour lesquels nous avons sélectionné par une stratégie d'évolution dirigée basée sur le yeast display et la cytométrie en flux de nouveaux tags protéiques capables d’interagir avec ces fluorogènes et d’activer leur fluorescence. Nous avons enfin développé de nouveaux fluorogènes capables de former des complexes fluorescents avec FAST, mais incapables de traverser la membrane plasmique, ce qui permet de détecter sélectivement les protéines membranaires. / Studying protein activities could help us to understand the complex mechanisms controlling cells and organisms. Our laboratory recently developed Fluorescence-Activating and absorption-Shifting Tag (FAST), a small fluorogen-based reporter enabling to fluorescently label fusion proteins in living cells. My PhD thesis presents the developments of new FAST systems with various properties for multiplexed imaging. We report a collection of fluorogens enabling to tune the fluorescence color of FAST from green-yellow to orange and red. Beyond allowing multicolor imaging of FAST-tagged proteins in live cells, these fluorogens enable dynamic color switching because of FAST’s reversible labeling, opening great prospects for the design of selective imaging methods relying on dynamic systems. In order to further expand the spectral properties of FAST to red, we also designed and developed a library of red fluorogenic dyes, for which we engineered specific protein binders by applying a directed evolution strategy based on the yeast display technology and high-throughput fluorescence activating cell sorting (FACS). We finally developed novel fluorogens able to form fluorescent complexes with FAST, but incapable of crossing the plasma membrane, which makes it possible to selectively detect FAST-tagged cell-surface proteins.

Cette thèse traite de la conception de sondes fluorogènes incorporant des bras espaceurs auto-effondrables répondant à l’activité enzymatique.Ces travaux commencent par la synthèse d’une sonde modèle à trois composantes pour détecter l’activité de la Leucine AminoPeptidase (LAP). Le cœur de cette sonde est un espaceur cyclisant efficace (t1/2 cyclisation≈7sec) qui unit un substrat enzymatique à un fluorophore précipitant avec une stabilité exemplaire (pas de dégradation sur 15h d’incubation). Appliquée sur des cellules vivantes, cette sonde produit des précipités fluorescents qui marquent durablement les cellules. L’élaboration de sondes pour d’autres enzymes a cependant soulevé l’importance d’abaisser le seuil de solubilité du fluorophore.Cette thèse se penche également sur deux nouvelles conceptions de sondes à la spécificité améliorée. Alors que la première tente de réutiliser les efforts déployés pour obtenir des inhibiteurs hautement sélectifs, la seconde est basée sur deux transformations successives par deux enzymes indépendantes. Si la première solution a échoué jusqu’alors, la seconde nous a effectivement permis de différencier des populations cellulaires.Enfin, ce manuscrit détaille le développement d’une nouvelle génération d’espaceur permettant l’affinement de certaines propriétés des sondes. Focalisés sur l’amélioration de l’hydrosolubilité, les premiers exemples sont très prometteurs. Particulièrement, une sonde pour Péncilline G Amidase possède une hydrosolubilité 3500 fois supérieure à celle de son analogue commercial. Véritables multiprises chimiques, ces espaceurs devraient permettent de relever certains des grands défis de la chimie médicinale moderne. / This thesis concerns the design and evaluation of fluorogenic molecular probes that respond to enzyme activity via the help of self-immolative spacers.This work starts with the synthesis of a model three-component probe that detects the activity of Leucine AminoPeptidase (LAP). The heart of this probe is an efficient cyclizing spacer (t1/2 cyclization≈7sec) that links a specific enzyme substrate to a precipitating fluorophore with an exemplary stability (no false positive signal over 15h incubation). When incubated with live cells, this construct is processed by active LAP to yield fluorescent precipitates which lead to long-term cell-tagging. However, probes susceptible to other enzyme activity have indicated the interest in further reduction of the solubility threshold of ELF®97.This manuscript also describes two new strategies to improve the specificity of the probes. While the first tries to take advantage of the efforts made to develop highly selective inhibitors, the second is based on two consecutive transformations by two independent enzymes. The first strategy has not yet been successfully applied in our hands, but the second has led to a first prototype that allowed discriminating between different cell lines.Lastly, this thesis relates the design and synthesis of a new generation of cyclizing spacer which opens up a great number of possibilities to optimize probes’ properties. For example, a probe targeting Pencilline G Amidase and containing such a spacer possesses a hydrosolubility 3500 times higher than its commercial analogue. As true “molecular hubs”, these spacers may turn out to address the big challenges of modern imaging agent and prodrug development.

Cette thèse de doctorat traite de la conception et de l’évaluation de sondes magnétogènes et fluorogènes pour la détection in vivo d’activités enzymatiques.Des molécules capables d’acquérir un moment magnétique à partir d’un état diamagnétique et en réponse à l’action d’une enzyme seraient d’un grand intérêt pour l’imagerie moléculaire par résonance magnétique. Deux exemples de telles sondes magnétogéniques avaient été mis au point précédemment, l’un pouvant opérer en conditions physiologiques, l’autre nécessitant une acidification du milieu pour devenir paramagnétique. En préparant de nouveaux analogues du premier exemple, j’ai pu trouver une molécule dont la fragmentation a lieu trois fois plus rapidement que la molécule originale. J’ai ensuite travaillé à la conception de sondes dérivées du deuxième exemple et répondant à des activités enzymatiques ; de telles molécules permettraient de réaliser la quantification in vitro d’une activité enzymatique à des fins diagnostiques. À ce titre, j’ai participé à l’élaboration de deux preuves de concept de dispositifs dédiés à la mesure du temps de relaxation longitudinale de micro-volumes. J’ai enfin entamé le développement de nouveaux complexes s’inspirant du second exemple mais capables de fonctionner dans le milieu biologique.Le deuxième volet de mes travaux porte sur la réalisation de sondes fluorogènes précipitantes pour des activités glycosidases. Une sonde pour la leucine aminopeptidase profitant des propriétés exceptionnelles de stabilité, de luminescence et de solubilité du fluorophore ELF®-97 avaient démontré une grande efficacité pour marquer rapidement des cellules HeLa. J’ai mis au point une nouvelle architecture de sondes qui permet le ciblage de glycosidases via l’utilisation d’un tandem d’espaceurs cyclisants. Deux sondes ont été préparées, l’une pour la beta-galactosidase, l’autre pour la cellulase. La première a prouvé son bon fonctionnement pour marquer les cellules exprimant l’enzyme en bénéficiant d’une grande sensibilité. La seconde a pu être utilisée pour quantifier l’activité cellulase sécrétée par des levures, avec l’objectif d’obtenir un moyen économiquement intéressant de produire du bioéthanol à partir des déchets végétaux. / This PhD thesis deals with the design and evaluation of magnetogenic and fluorogenic probes for the in vivo detection of enzyme activities.Molecules capable of switching from a diamagnetic to a paramagnetic state in response to an enzyme stimulus would be of great interest for molecular magnetic resonance imaging. Two examples of such magnetogenic probes had been designed in a previous work : one can operate in physiological conditions, whereas the other needs an acidification of the water medium to become paramagnetic. I prepared new analogues of the first probe ; one molecule displayed fragmentation three times faster than the original compound. Then I designed and synthesized probes derived from the second example and responsive to enzyme activities ; such molecules are suitable for the in vitro quantification of enzyme biomarkers for diagnosis purposes. I participated to the conception of two proofs of concept of devices dedicated to the measurement of longitudinal relaxation times in micro-volumes. Finally, I started the development of a new family of molecules inspired by the second example but able to work at the physiological pH.I also worked on precipitating fluorogenic probes for the detection of glycosidase activities. A former probe for leucine aminopeptidase, based on the exceptional characteristics of the fluorophore ELF-97 in terms of solubility, luminescence and stability, had demonstrated great efficiency to label live HeLa cells. I designed a new architecture of probes responding to glycosidases via an original tandem of selfimmolative spacers. Two probes have been prepared, one targets beta-galactosidase and the second detects cellulase. The first probe performed a fast and sensitive labelling of beta-galactosidase-expressing cells. The second molecule was employed successfully to quantify the cellulase activity secreted by yeasts, which will be useful for the high-throughput screening of yeasts capable of producing bioethanol from vegetal waste.

L'imagerie de biomolécules, et plus particulièrement des glycanes, au sein d'organismes vivants, représente un incroyable challenge. Cependant, des progrès significatifs ont été réalisés ces dix dernières années grâce au développement de la stratégie du rapporteur chimique bioorthogonal. Cette technique implique, dans un premier temps, l'incorporation d'une fonctionnalité chimique non native (rapporteur) au sein de biomolécules complexes. Ce rapporteur peut ensuite réagir sélectivement avec une sonde moléculaire spécifique permettant ainsi la détection de la biomolécule ciblée. Afin d'imager ces biomolécules en temps réel, les sondes fluorogènes, des réactifs non fluorescents qui deviennent fluorescents après réaction avec le rapporteur chimique, ont été récemment développées. Ces dernières années, la cycloaddition 1,3-dipolaire sans métaux, entre les cylooctynes et les azotures, a été élégamment employée comme réaction bioorthogonale pour l'imagerie de biomolécules au sein d'organismes vivants. Cependant, l'azoture présente certaines limitations, notamment il peut être réduit par les thiols cellulaires. En conséquence, nous avons étudié l'utilisation de 1,3-dipôles plus stables, en tant que nouveaux rapporteurs chimiques bioorthogonaux pour l'imagerie de biomolécules par fluorescence. De plus, nous avons aussi développé de nouvelles sondes fluorogènes bioorthogonales ayant de bonnes propriétés de fluorescence, telles que de hautes valeurs de rendement quantique et des longueurs d'ondes d'émission déplacées dans le rouge, pour une potentielle application chez l'animal. / Imaging biomolecules, such as glycans, in living systems remains a formidable chemical challenge. However, significant progress has been made over the past ten years, with the ground-breaking development of the bioorthogonal chemical reporter strategy. In this context, complex biomolecules are fitted with a non-native chemical functionality (reporter) that can react selectively with a complementary bioorthogonal probe for detection. In order to image these biomolecules in real time, fluorogenic probes, non fluorescent reagents that produce highly fluorescent products, have recently been developed. This last decade, the metal-free 1,3-dipolar cycloaddition between cyclooctynes and azides have been elegantly employed for biomolecules imaging in living systems. However, azides suffer from limitations such as their reduction by endogenous cellular thiols. Consequently, we have investigated the use of more stable 1,3-dipoles as new chemical reporters for fluorescent biomolecule imaging. In addition, we also developed novel bioorthogonal fluorogenic probes with improved fluorescence properties such as high quantum yields and red-shifted fluorescence emission for potential applications in living animals.

Due to the system does not recognize equations and formulas the resumo and abstract can be found in the PDF file. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Devido ao sistema não reconhecer equações e fórmulas o resumo e abstract encontra-se no arquivo em PDF.

L’imagerie moléculaire optique joue maintenant un rôle essentiel dans le diagnostic pré-clinique et le développement de médicaments. En effet, c’est un outil précieux dans la détection et le suivi de cellules vivantes que ce soit en utilisant de simples agents de marquage ou des sondes plus développées, dites « intelligentes » et activées uniquement par une interaction spécifique avec le bio-analyte ciblé. Ce travail de thèse a consisté à développer des outils synthétiques innovants afin d’optimiser les paramètres physico-chimiques et les propriétés optiques des sondes luminescentes. Ceci dans le but de répondre à la problématique complexe de l’imagerie dans le contexte in vivo. Nous avons notamment travaillé sur des aspects de pro-fluorescence et de chémiluminescence. De nouveaux pro-fluorophores à phénol basés sur une architecture originale de type bis-coumarinique ont été développés. De plus, nous avons mis en place une méthode d’hydrosolubilisation généralisable aux fluorophores à phénol de type coumarine et xanthène. Nos recherches en chémiluminescence ont permis la synthèse de nouveaux chémiluminophores couplés à des fluorophores organiques afin d‘augmenter l’efficacité d’émission de chémiluminescence dans le rouge. Enfin, nos travaux ont permis de mettre en place les premières « cassettes » chémiluminescentes basées sur une architecture de type 1,2-dioxétane. / Optical molecular imaging is now playing a pivotal role both in pre-clinical diagnosis and drug development. Indeed, this is a valuable tool for the real time detection and monitoring of living cells either through the use of structurally simple labels or more recently by means of sophisticated fluorescent probes, called “smart” probes and only activatable upon specific interaction with the targeted bio-analyte. The aim of this PhD work was the design of new synthetic tools aimed at optimizing physico-chemical and optical properties of fluorescent probes intended for challenging in vivo imaging applications. We have focused on the pro-fluorescence and chemiluminescence approaches. New phenol-based pro-fluorophores have been developed by using an original bis-coumarinic scaffold. In the context of the chemistry of fluorophores, we have also investigated a general method for the water-solubilisation of phenol-based fluorophore belonging to the coumarin and xanthene families. Our research in chemiluminescence has led the synthesis of new chemiluminophores covalently linked to fluorescent organic dyes aimed at increasing the emission efficiency in the red region of such chemiluminophores. Thus, the first chemiluminescent “energy transfer cassettes” based on a 1,2-dioxetane scaffold have been obtained.