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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

Chemical labeling is an important tool for understanding protein structure and function. Biological research often requires the use of molecular labels that are covalently attached to facilitate detection or purification of the labeled protein and its binding partners. Although the number of probes have been developed for labeling of specific residues of proteins is substantial, there is still a need for new reagents with better reactivity, and selectivity. Moreover, these chemical probes should be able to label the protein of interest under mild biologically relevant conditions. Aryl diazonium salts have been utilized for selective modification of tyrosine residues. However, most diazonium compounds need to be generated in situ under strongly acidic conditions due to their instability1. Our group has previously shown that triazabutadienes can be used as precursors that can generate diazonium under mild acidification2 or photo-irradiation3. Current reported systems for bioconjugation of tyrosine require an additional step for fluorescent labeling4. To address this issue and reduce background fluorescence that is associated with fluorescent labeling, coumarin triazabutadiene-based fluorogenic probes were synthesized and tested for tyrosine specific labeling of proteins under mild acidic condition or photo-irradiation. Furthermore, a coumarin triazabutadiene-based cross-linker was synthesized with an azide functionality that can be used to attached the coumarin triazabutadiene warhead onto the surface of a protein. Upon the activation of the triazabutadiene group, by light or lowering the pH, this system can generate a coumarin diazonium salt on the surface of the protein. Such a system can find application in the study of protein-protein interactions and virus-protein interactions. A cyclooctyne triazabutadiene was synthesized to attach a cyclooctyne group on the tyrosine residues of proteins in biologically relevant pH, and 3-azido 7-hydroxy coumarin was made as a fluorogenic partner of the cyclooctyne triazabutadiene. It was demonstrated that this system can label tyrosine residue followed by a copper-free click reaction with the azido coumarin fluorophore. This system has been tested on model proteins and can be consider as one the first fluorogenic triazabutadiene systems that can be utilized for labeling of tyrosine under mild conditions. In conclusion, this dissertation demonstrates progress in developing fluorescent and fluorogenic triazabutadienes systems for labeling of tyrosine residues of proteins as well as fluorophore triazabutadiene cross-linker that can be used for studying protein-protein interaction, and virus-protein interactions. These systems offer a convenient tool to those wishing to study proteins, protein-protein interactions, and virus-protein interactions.

Fluorescent protein labelling is a powerful tool for the sensitive visualization of proteins in living cells, allowing the elucidation of their localization, trafficking and ultimately their cellular function. We have developed a novel labelling technique based on the genetic fusion of a protein of interest to a small helical peptide sequence containing two Cys residues (dC10). This tag can undergo an efficient reaction with small fluorogenic labelling agents composed of a fluorophore and a dimaleimide core (dM10) that confers high reaction specificity, and quenches the latent fluorescence through photo-induced electron transfer, until both of its maleimide groups have formed robust covalent bonds with the tag Cys thiol groups. Our initial efforts at intracellular protein labelling demonstrated the importance of the selectivity of the labelling reaction, which is dependent on the reactivity of the dC10 tag. To that end, we re-engineered the dC10 tag through rational protein design. Mutant libraries were prepared through combinatorial mutation at specific positions of the helical tag sequence, and screened for their fluorogenic reactivity. In this way, we identified a novel sequence for a next-generation dC10 tag that confers 10-fold greater selectivity that we then applied to in cellulo labelling. Subsequent mechanistic studies revealed the basis for this dramatic increase in reactivity. Current applications of this powerful labelling technique, including the site-specific chelation of lanthanide ions for NMR spectroscopy and site-specific covalent heavy-atom labelling for X-ray crystallography, will also be discussed.

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.

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