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

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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.