Ladderlike polyethers have intrigued scientists since brevotoxin B's characterization by the Nakanishi and Clardy research group in 1981. Studies of these compounds have been challenging however due to these compounds only being accessible through complex multistep reactions or challenging extractions from microorganisms like dinoflagellates. Hence, the challenge the proposed research will address is the development of a new strategy toward the rapid synthesis of libraries of ladder polyether scaffolds. This project plans to address the challenging syntheses of ladderlike polyethers by making related structures via the Ring-Opening Metathesis Polymerization (ROMP) of oxanorbornene epoxide. If the proposed monomer can undergo ROMP, the epoxide groups on the resulting polymer will be utilized in a cascade cyclization initiated by an oxygen-based nucleophile. This cascade reaction should form fused 5-7 membered rings that alternate and resemble ladderlike polyether molecules. If our compound of interest can be made, its physiological properties will be further studied in lab as a conjugate of ladderlike polyethers such as brevetoxin B.

Enzymes have recently been incorporated into multiple high-value industrial syntheses, demonstrating the utility of enzymes as highly selective catalysts for practical industrial processes. However, the current scope of non-biological enzymatic reactions is narrow and new reactions and reaction pathways need to be engineered. The goal of our work in the Zalatan lab is engineering enzymes as catalysts in carbon-hydrogen bond functionalization reactions, a transformation critical for practical industrial synthesis where selective catalysis is still a major challenge. Importantly, we are interested in exploring more efficient and informed engineering approaches by establishing structure-function relationships with the enzymes that we work with. Our model system for this work is the non-heme iron(II) 2-oxoglutarate dependent oxygenase superfamily (Fe(II)-2OGs). I use a high-throughput microfluidics based kinetic assay to determine key sites that we can target for mutagenesis and directed evolution in a candidate Fe(II)-2OG found to catalyze a new reaction. Overall, we expect that this work will enable new directions and principles for engineering Fe(II)-2OGs, and that lessons learned here can then be extended to additional industrially-relevant enzyme families.

Protein design originally began as a field intended to demonstrate our understanding of how natural proteins, which are one of the four macromolecules, are created. Over the last three decades, advances in the protein design literature have enabled us to routinely engineer novel, de novo proteins. We focus on a class of these small proteins known as Miniproteins, which are attractive candidates to study not only due to their size, but also other advantageous properties that can be modified and applied as therapeutics, virus-inhibitors, and other biological applications. In our lab, we work with Miniproteins termed mFAPs that bind to fluorescent molecules and enhances fluorescence up to 100-fold compared to non-bounded ligands. However, the existing designs of mFAPs are not as efficient compared to existing biosensors and lack explanation as to why, on the molecular level, this is the case. Using molecular dynamic (MD) simulations, we have found and validated that atomic-level, structural features, such as particular side chains of amino acids, explain why some mFAPs are brighter than others. Currently, we are developing a high-throughput, low-cost algorithm that can scan for amino acid mutations that bias the certain amino acid side chains into these configurations.

My study will draw on changes in the effectiveness of a chromophore's electron distribution (in the ground and excited states) to the Quantum Yield of a particular protein (mini-Fluorescence Activating Proteins) of interest. The DFHBI ligand has a -1 net charge, with a unique partial charge for each of its atoms in the molecule. When an incoming photon interacts with the chromophore, it consumes that photon and kicks an electron into a higher energy state. This process allows the other electrons in the chromophore to shift around and generate new partial charges. And when the electron comes back down to the ground state, in which the electron discharges a photon, fluorescence happens. Using the previous parameters (not accounting for the change in partial charges in the excited state) of existing quantum mechanics simulations, I compared and contrasted them with newly generated simulations to discover how the distribution of partial charges affects Molecular Dynamic simulations. By simulating electrons in the ground and excited states, we can see if these computational simulations are a better predictor in modeling the possibility of electrostatics in mFAP playing a role in our ability to predict and better its quantum yield.