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Joshua Baccile正在研究两种生物学上重要的化学实体：萜烯和戊烯化分子。它们是最大的生物分子类别之一，对基本生命过程至关重要，在临床上作为药物具有重要意义。萜烯和烯基化分子都部分或全部来源于两种结构相关的五碳结构单元，焦磷酸异戊烯基和焦磷酸二甲基烯基（分别为IPP和DMAPP）。因此，IPP和DMAPP是生物学中最重要的代谢前体。尽管如此，研究细胞中IPP和DMAPP的化学工具和策略仍然缺乏。Baccile博士旨在通过开发一套基于IPP和dapp的化学探针和方法来弥补这一科学差距，这些探针和方法用于萜烯和戊烯化分子的代谢标记以及以细胞特异性方式检测它们。所提议的工具和实验也将能够系统地评价IPP和DMAPP本身的作用，这目前是不可能的。除了化学创新，Baccile博士还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、教学和学习方面的创新目标。Baccile将努力通过一个专门的暑期项目，为来自社区大学的STEM（科学、技术、工程和数学）中代表性不足的学生扩大化学的参与。本研究项目旨在研究IPP和DMAPP探针的简易模块化合成，该探针利用类似“前药”的机制进行精确代谢标记。虽然聚糖和蛋白质的代谢标记已经采用了相关的策略，但这种方法在天然产物和翻译后修饰（PTM）研究中是一种创新。所提出的细胞类型特异性同位素标记和富集方法将使异质细胞群体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，五碳戊烯基PTMs被认为在生命中不存在，而不是已知的高阶蛋白质戊烯基化，特别是蛋白质法尼化（15个碳）和香叶基香叶基化（20个碳）。然而，用于明确排除这种五碳修饰的化学探针以前还没有经过测试。所提出的探针的成功应用将使鉴定五碳戊烯基修饰成为可能，这将是蛋白质戊烯化领域的一个范式转变。重要的调控机制和信号级联可能通过这种未知的PTM介导。我们注意到，提出的合成方法是模块化的，很容易适应其他焦磷酸盐代谢物。该项目是朝着创造资源来研究生物分子的生物合成、调控和功能的长期目标迈出的重要一步，重点是利用同位素标记和富集化学来理解界间信号。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识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.