Development of chemoenzymatic platform for facile synthesis of alkyl-pyrophosphate analogs

化学酶天然产物多样化是制备药学相关分子的重要结构多样化策略之一；特别是当要多样化的分子结构复杂时，要用经典的化学合成方法来处理。我们的目标是开发一个基于戊烯基转移酶（PT）的化学酶平台，用于复杂天然产物支架的后期结构修饰，并产生具有潜在治疗价值的化合物。PTs转移烷基基团，其来源于活化的供体焦磷酸烷基（烷基- pps）。目前，烷基pp类似物的合成依赖于多步骤的合成和繁琐的纯化方法，将起始物质和其他副产物从所需产物中分离出来，从而限制了它们作为合成试剂的实际发展。因此，本提案寻求(i)通过酶工程方法开发一个通用的化学酶平台，用于两种不同类型激酶的烷基- pp类似物的合成和原位利用，以及（ii）通过生成fda批准的大环药物达托霉素的烷基多样化文库来验证该平台。拟议的研究将整合结构测定，高通量分析开发和结构指导设计，以揭示所选酶的结构-活性关系（SAR）。这项研究将产生高效，稳健的双酶偶联平台，以有效地产生新的烷基pp供体。将这些烷基pp类似物与不同的PTs偶联将提供前所未有的独特生物活性天然产物库，这是通过传统的有机合成不容易获得的。重要的是，这项研究有助于发现新的基于大环肽的药物先导物，用于下游药物评估和临床应用。

Chemoenzymatic natural product diversification is one of the important structural diversification strategies in use to generate pharmaceutically relevant molecules; especially when the molecule to be diversified is structurally complex to be handled by classical chemical synthesis. Our goal is to develop a prenyltransferase (PT)-based chemoenzymatic platform for the late stage structural modification of complex natural product scaffolds and generate compounds with potential therapeutic interest. PTs transfer alkyl groups, which are derived from their activated donors, alkyl pyrophosphates (alkyl-PPs). The current state-of-the-art of synthesis of alkyl-PP analogs relies upon multi-step synthesis and tedious purification methods to separate starting material and other byproducts from the desired product, thereby limiting their practical development as synthetic reagents. Therefore, this proposal seeks to (i) develop a general chemoenzymatic platform for the synthesis and in-situ utilization of alkyl-PP analogs by enzyme engineering approach of two different classes of kinases, and (ii) validate the platform via generating alkyl-diversified library of a FDA-approved macrocyclic drug, daptomycin. The proposed studies will integrate structure determination, high throughput assay development, and structure-guided design to expose structure-activity relationships (SAR) of the selected enzymes. This study will result in highly efficient, robust two-enzymes coupled platform engineered to be efficient in generating novel alkyl-PP donors. Coupling these alkyl-PP analogs with diverse PTs will offer unprecedented access to uniquely bioactive natural product libraries, which are not readily accessible via conventional organic synthesis. Importantly, this study contributes to the discovery of new macrocyclic peptide-based drug leads for downstream pharmaceutical assessment, and clinical use.