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

有了这个奖项，NSF化学部的生命过程化学计划正在资助东卡罗莱纳大学的Adam Offenbacher博士，以解决开发一套新的化学工具的挑战，该工具允许跟踪基于氨基酸色氨酸的蛋白质内部和之间的电子传输。蛋白质由氨基酸组成，其中单个氨基酸构建块及其侧链赋予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化学上不同的区域之间的通信，有时通过促进电子在特定位点之间的穿梭。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蛋白质（酶）的生物功能多样化。虽然一些化学修饰的色氨酸衍生物已成为此类研究的可及对象，但这些物种缺乏使复杂生物系统易于表征的理想性质。在这个项目中，一个高度合作的方法，结合分子生物学，化学生物学和理论化学，是用来克服这些限制，并彻底表征一系列色氨酸氨基酸类似物具有潜在的有价值的电子跟踪能力。该项目促进了多方面的实践研究经验，主要侧重于本科生的培训，其中大多数是妇女或来自该地区服务不足的社区，并打算继续接受科学，技术，工程和数学教育。 5-羟基吲哚（5 HOI）作为这些研究中靶向的非天然色氨酸类似物的开发的构建单元，因为它可以通过三步合成从容易获得的前体容易地获得。 Offenbacher团队随后试图生成一系列具有可调酸度，还原电位和光谱特征的卤代衍生物，用于生物质子耦合电子转移（PCET）的研究。使用进化的色氨酸脱氢酶以接近定量的产率将氟化的5 HOI衍生物转化为F（n）-L-5-羟基色氨酸（F（n）-5HOW）。为了能够使用固态方法掺入肽中，正在开发氟化FMOC-（9-芴基甲氧羰基）-5HOW衍生物的一锅法、两步化学酶促合成。作为概念验证，这些新的F（n）-5HOW衍生物将使用正交的双酰tRNA合成酶/tRNA对位点特异性地掺入模型蛋白中。本研究旨在测试这些非天然色氨酸侧链使用方波伏安法的正式还原电位的蛋白质环境的影响，并建立模型的光谱签名，其特征在于通过电子顺磁共振光谱，其氧化侧链。密度泛函理论被用于量化通过PCET过程形成的F（n）-5HOW自由基的电子密度离域，为观察到的独特光谱特性提供了物理基础。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

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.