Legumain, an asparaginyl endopeptidase and cysteine protease, cleaves proteins at asparagine residues under acidic conditions (pH<5.5) and is an important part of many biological processes. Legumain is synthesized as an inactive proenzyme and undergoes activation through pH-based autocatalytic cleavage. Legumain’s substrates in the proteome are not well documented so my project focuses on better understanding: (1) legumain’s substrates and (2) legumain’s cleavage mechanism. To achieve this goal, I developed a plasmid for the most efficient legumain purification by inserting a wild-type legumain gene into a C-terminally-positioned His-tagged plasmid. Protein tags, such as His-tags, are commonly used for protein isolation and analysis, helping with studying enzymes like legumain. I will then use the purified legumain to understand cleavage motifs and substrates it interacts with through proteomics and LC-MS based peptide cleavage assays. I have successfully made the C-term legumain His-tag construct and expressed it in human embryonic kidney cells, however, western blot analysis showed that the C-term His-tag was possibly cleaved due to legumain activating in acidic conditions. This finding diverges from the previous hypothesis that the C-term His-tag would remain intact after transfection and basic lysis buffer treatment. My next steps are to generate an N-term His-tag that may better preserve the tag for future protein purification experiments and downstream application to elucidating legumain’s impact on the proteome.

Hypoxic microenvironments–regions of low oxygen–are common in tumors. These regions facilitate tumor development and prevent effective treatment by many chemotherapies, contributing to poor patient prognosis. Hypoxia-activated prodrugs (HAPs), consisting of a hypoxia-activated molecule and an anticancer drug, aim to combat these issues. By design, HAPs activate and release anticancer drugs in hypoxic environments via bioreductive pathways, allowing for improved targeting of aggressive cancer cells and reduction of the toxicity of anticancer drugs in normal cells; the toxic prodrugs in HAPs are inactive at typical cellular oxygen levels. Derivatives of indolequinone and nitroimidazole are both under established classes of hypoxia-activated molecules. Attachment of these groups to proteolysis-targeting chimeras (PROTACs) and panobinostat, a lysine deacetylase (KDAC) inhibitor, will improve the specificity and effect of the prodrugs. Through the synthesis of a hypoxia-activated indolequinone derivative and its attachment to a PROTAC, as well as the synthesis of the anticancer drug panobinostat and its attachment to a hypoxia-activated nitroimidazole, multiple pathways for improved anticancer drug targeting will be explored. The finished and purified HAPs will be tested in cell cultures to confirm function and minimized toxicity. Future research should aim to develop syntheses for HAPs to improve synthetic yields, increase the specificity of release in hypoxia, and enable stable delivery of prodrugs to cancerous regions.

Optimizing polymer synthesis requires addressing challenges such as low monomer conversion, side reactions affecting yield, and difficulties regulating molecular weight to enhance polymer properties. Redox-switchable catalysis seeks to overcome these issues by utilizing complex metal catalysts that switch between oxidation states via the use of chemical triggers to polymerize a monomer selectively. A ferrocene-based aluminum complex, (salfan-H2)Al(OiPr) with (salfan-H2 = 1,1’-di(2,4-bis-tert-butyl-salicylamino)ferrocene), demonstrated efficient catalytic activity with defined redox and protonation control. Our study explores the reactivity of (salfan-H2)Al(OiPr) with novel epoxide monomers: cyclohexadiene oxide, ethoxy vinyl glycidyl ether, glycidyl propargyl ether, and phenyl glycidyl ether. Using these monomers, we can evaluate if the catalyst can be used for ring-opening polymerization and to form controlled sequence copolymers. We have synthesized the four epoxide monomers, yielding two grams of product each. Initial polymerization studies demonstrate that one orthogonal state of the catalyst forms a homopolymer. The next step is to leverage the orthogonality of the redox state of polymerization selectivity to synthesize copolymers. Ultimately, with successful copolymerization with carbon dioxide, the novel multiblock copolymers will have a valuable microstructure.

Nitrogen-containing heterocycles are particularly prominent in bioactive natural products and pharmaceutical agents, but their synthesis can pose many difficulties. This project aims to explore an approach to synthesizing seven-membered azacyclic allenes, which are a particularly understudied strained intermediate and would provide easy access to a variety of more functionalized azacyclic products. These seven-membered allenes are especially interesting because of their increased conformational flexibility in comparison to allenes in smaller rings. The proposed reaction generates allenes in situ and traps them in cycloaddition reactions. The project aims to evaluate the reactivity of these allenes in different types of cycloadditions, and the products will be confirmed using NMR, IR spectroscopy, and mass spectroscopy.

JBIR-141 is a natural product derived from the soil bacteria Streptomyces. This compound has demonstrated to be an inhibitor of transcription factor FoxO3a activity which plays a critical role in the persistence of chronic myeloid leukemia (CML) stem cells following treatment with tyrosine kinase inhibitors. In luciferase gene reporter assays, JBIR-141 has emerged as a novel peptidomimetic, exhibiting a potent cellular IC₅₀ value of 23 nM. Additionally, it demonstrates strong cytotoxicity against ovarian, mesothelial, and lymphoma cancer cell lines, making JBIR-141 a promising lead compound for potential CML therapies. Our work aims to establish a full, efficient laboratory synthesis of JBIR starting from affordable, commercially available chemicals. Currently, our synthetic route consists of a convergent strategy linking three complex fragments decorated with oxazoline, nitrosohydroxylamine, and tetramic acid motifs whose sensitivities we must be cognizant of in our syntheses and purifications. Our isolated compounds have been purified via flash column chromatography and characterized by proton and carbon nuclear magnetic resonance (NMR) spectroscopy. After establishing the total synthesis of JBIR-141, we plan to further explore a metal binding form of our product which can potentially enhance biological activity. We also wish to explore structural derivatives of JBIR that could exhibit improved or comparable cytotoxicity.