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

通过这一奖项，美国国家科学基金会化学部门的生命过程化学项目资助了东卡罗来纳大学的Adam Offenbacher博士，以应对开发一套新的化学工具的挑战，该工具可以跟踪基于氨基酸色氨酸的蛋白质内部和之间的电子传输。蛋白质是由氨基酸组成的，单个氨基酸及其侧链决定了蛋白质的关键结构和功能特性。在某些情况下，某些氨基酸允许蛋白质的不同化学区域之间的通信，有时是通过促进电子在特定位置之间的穿梭。这些化学工具的发展为生物能量转换的研究奠定了基础，也有可能使催化化学反应的特定蛋白质(酶)的生物功能多样化。虽然一些经过化学修饰的色氨酸衍生物已经可以用于此类研究，但这些物种缺乏能够在复杂生物系统中轻松表征的理想特性。在这个项目中，结合了分子生物学、化学生物学和理论化学的高度协作的方法被用来克服这些限制并彻底表征一系列具有潜在有价值的电子跟踪能力的色氨酸氨基酸类似物。该项目促进了多方面的实践研究体验，主要侧重于培训打算继续接受科学、技术、工程和数学教育的本科生，这些本科生大多是妇女或来自该地区服务不足的社区。5-羟基吲哚(5HOI)是开发这些研究中目标的非天然色氨酸类似物的基础，因为它很容易通过三步合成从现成的前体中获得。然后，Offenbacher团队试图产生一系列具有可调酸度、还原电位和光谱特征的卤化衍生物，用于研究生物质子耦合电子转移(PCET)。利用进化的色氨酸合成酶以接近定量的产率将含氟的5HOI衍生物转化为F(N)-L-5-羟色氨酸(F(N)-5HOW)。为了能够通过固相方法将其结合到多肽中，正在开发一种一锅两步化学酶法合成含氟的Fmoc-(9-荧甲氧基羰基)-5HOW衍生物。作为概念验证，这些新的F(N)-5HOW衍生物将使用正交的色氨酰tRNA合成酶/tRNA对被特异性地结合到模型蛋白质中。本研究旨在利用方波伏安法测试蛋白质环境对这些非天然色氨酸侧链的形式还原电位的影响，并建立其氧化侧链的电子顺磁共振光谱特征模型。密度泛函理论正被用来量化通过PCET过程形成的F(N)-5HOW自由基的电子密度离域，这为观察到的独特光谱性质提供了物理基础。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.