Chemical Tools for Precision Metabolic Labeling and Detection of Terpenes and Prenylated Molecules

在化学系生命过程化学（CLP）项目的支持下，田纳西大学的约书亚·巴奇勒正在研究两类生物学上重要的化学实体，萜烯和异戊二烯化分子。它们是生物分子中最大的一类，对基本生命过程至关重要，并且作为药物在临床上很重要。萜烯和异戊二烯化分子都部分或全部衍生自两种结构相关的五碳结构单元，异戊烯基焦磷酸和二甲基烯丙基焦磷酸（分别为IPP和DMAPP）。因此，IPP和DMAPP是生物学中最重要的代谢前体之一。尽管如此突出，但缺乏研究细胞中IPP和DMAPP的化学工具和策略。Baccile博士的目标是通过开发一套基于IPP和DMAPP的化学探针和方法来弥合这一科学差距，用于萜烯和异戊二烯化分子的代谢标记及其以细胞特异性方式的检测。拟议的工具和实验还将能够系统地评估IPP和DMAPP本身的作用，这是目前不可能的。除了化学创新，博士Baccile将通过在实验室引入新的教学培训方法，整合学生研究指导，教学和学习的创新目标。 Baccile将奋进通过为社区大学STEM（科学、技术、工程和数学）领域代表性不足的学生提供专门的暑期项目来扩大对化学的参与。该研究项目旨在研究IPP和DMAPP探针的简单且模块化的合成，该探针利用“前药”样机制进行精确代谢标记。虽然相关策略已被用于聚糖和蛋白质的代谢标记，但这种方法在天然产物和翻译后修饰（PTM）研究中具有创新性。提出的细胞类型特异性同位素标记和富集方法将使异源细胞群中类异戊二烯途径的详细研究成为可能，这可能会改变我们对宿主-微生物相互作用和肿瘤发展的理解。目前，五碳异戊二烯基PTM被认为在生命中不存在，这与已知的公认的高级蛋白质异戊二烯化相反，特别是蛋白质法尼基化（15个碳）和香叶基香叶基化（20个碳）。 然而，设计用于明确排除这种五碳修饰的化学探针以前没有进行过测试。成功应用所提出的探针将使潜在的五碳异戊二烯基修饰，这将是一个范式转变，在蛋白质异戊二烯化领域的识别。重要的调控机制和信号级联反应可以通过这种未知的PTM介导。我们注意到，所提出的合成方法是模块化的，并且易于适应其他焦磷酸代谢物。该项目是朝着创造资源研究生物分子的生物合成、调节和功能的长期目标迈出的重要一步，重点是使用同位素标记和富集化学来理解王国间的信号传导。该奖项反映了NSF的法定使命，并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，被认为值得支持。

With the support of the Chemistry of Life Processes (CLP) Program in the Division of Chemistry, Joshua Baccile of the University of Tennessee is studying two biologically important classes of chemical entities, terpenes and prenylated molecules. They are among the largest classes of biomolecules and are both critical to basic life processes and clinically important as drugs. Terpenes and prenylated molecules both derive in part or in full, from the two structurally related five-carbon building blocks, isopentenyl pyrophosphate and dimethylallyl pyrophosphate (IPP and DMAPP, respectively). IPP and DMAPP are therefore among the most significant metabolic precursors in biology. Despite this prominence, chemical tools and strategies to study IPP and DMAPP in cells are lacking. Dr. Baccile aims to bridge this scientific gap through the development of a suite of IPP- and DMAPP-based chemical probes and methods for the metabolic labeling of terpenes and prenylated molecules and their detection in a cell-specific manner. The proposed tools and experiments will also enable a systematic evaluation of the roles of IPP and DMAPP themselves, which is not currently possible. In addition to the chemical innovation, Dr. Baccile will integrate objectives for innovation in student research mentorship, teaching, and learning through the introduction of new pedagogical training methods in the laboratory. Baccile will endeavor to broaden participation in chemistry through a dedicated summer program for underrepresented students in STEM (science, technology, engineering and mathematics) from community colleges.This research project sets out to investigate a facile and modular synthesis of IPP and DMAPP probes, which leverages a “prodrug”-like mechanism for precision metabolic labeling. While related strategies have been employed for metabolic labeling of glycans and proteins, this approach is innovative in natural product and post-translational modification (PTM) research. The proposed cell-type specific isotopic labeling and enrichment methods will enable detailed studies of the isoprenoid pathway in heterogenous cell populations, which could be transformative in our understanding of host-microbe interactions and tumor development. Currently, five-carbon prenyl PTMs are presumed to be absent in life in contrast to the well-established higher order protein prenylations known, specifically protein farnesylation (15 carbons) and geranylgeranylation (20 carbons). However, chemical probes engineered to definitively rule out such five-carbon modifications have not previously been tested. Successful application of the proposed probes will enable the potential identification of five-carbon prenyl modifications, which would be a paradigm shift in the field of protein prenylation. Significant regulatory mechanisms and signaling cascades could be mediated through this yet unknown PTM. We note that the proposed synthetic approach is modular and readily adapted to other pyrophosphate metabolites. This project is an important step toward the long-term goal of creating resources to study the biosynthesis, regulation, and function of biomolecules, with a focus on using isotopic labeling and enrichment chemistry to understand interkingdom signaling.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.