RUI: Development of Unnatural Tryptophan Derivatives to Expand Tryptophan Function and to Study Biological Catalysis

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With this award, the Chemistry of Life Processes Program of the NSF Division of Chemistry is funding Dr. Adam Offenbacher of East Carolina University to addresses the challenge of developing a new set of chemical tools that allow tracking of electrons transported within and between proteins based on the amino acid tryptophan. Proteins are made up of amino acids, with the individual amino acid building blocks and their side chains conferring key structural and functional properties of proteins. In some cases, certain amino acids allow for communication between chemically distinct regions of proteins, sometimes by facilitating the shuttling of electrons between specific sites. The development of these chemical tools sets the stage for study of biological energy conversion and also holds potential to diversify biological function in select proteins that catalyze chemical reactions (enzymes). While a few chemically modified tryptophan derivatives have become accessible for such studies, these species lack desirable properties that enable easy characterization in complex biological systems. In this project, a highly collaborative approach, combining molecular biology, chemical biology, and theoretical chemistry, is employed to overcome these limitations and thoroughly characterize a series of tryptophan amino acid analogues having potentially valuable electron tracking capabilities. The project facilitates multi-faceted, hands-on research experiences primarily focused on the training of undergraduate students, most of whom are women or from underserved communities in the region, and who intend to continue their education in science, technology, engineering, and mathematics. 5-Hydroxyindole (5HOI) serves as the building block for the development of the unnatural tryptophan analogues targeted in these studies because it is easily accessible via a three-step synthesis from readily available precursors. The Offenbacher team then seeks to generate a collection of halogenated derivatives with tunable acidities, reduction potentials, and spectroscopic signatures for the study of biological proton-coupled electron transfer (PCET). Fluorinated 5HOI derivatives are converted to F(n)-L-5-hydroxytryptophan (F(n)-5HOW) using evolved tryptophan synthases in near quantitative yields. To enable incorporation into peptides using solid-state approaches, a one-pot, two-step chemoenzymatic synthesis of fluorinated FMOC-(9-fluorenylmethoxycarbonyl)-5HOW derivatives is being developed. As a proof-of-concept, these new F(n)-5HOW derivatives are to be incorporated site-specifically into model proteins using an orthogonal, tryptophanyl tRNA synthetase/tRNA pair. This study seeks to test the impact of the protein environment on the formal reduction potentials of these unnatural tryptophan sidechains using square wave voltammetry and to establish model spectroscopic signatures, characterized by electron paramagnetic resonance spectroscopy, of their oxidized sidechains. Density-functional theory is being utilized to quantify the electron density delocalization of F(n)-5HOW radicals formed through PCET processes that provide a physical basis for the unique spectroscopic properties observed.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.