UTILIZATION OF ISOPRENE ENZYMES FOR SYNTHESIS OF NON-NATURAL ISOPRENE ANALOGS

Abstract

Isoprenoids, also known as terpenoids, constitute the largest and most structurally diverse class of natural products, comprising over 50,000 compounds. These include essential primary metabolites such as sterols, carotenoids, and quinones, as well as a wide range of secondary metabolites with significant medicinal properties. All isoprenoids are biosynthetically derived from the universal five-carbon precursors dimethylallyl diphosphate (DMAPP) and its isomer isopentenyl diphosphate (IPP). While plants serve as a major source of isoprenoids, limitations such as slow growth rates, low yields, and labor-intensive extraction processes hinder their large-scale production.To overcome these challenges, considerable efforts have been devoted to metabolic engineering and chemoenzymatic approaches aimed at producing both natural and unnatural isoprenoids in heterologous hosts. This thesis focuses on the development of chemoenzymatic strategies for the synthesis and diversification of isoprenoid analogs, utilizing organic synthesis in conjunction with prenyltransferase (PT) enzymes—including both aromatic PTs and chain-elongating enzymes such as farnesyl diphosphate synthase (FPPS). A central aim of this research is the synthesis of novel isoprenoid precursors bearing chemoselective functional groups such as alkynes, alkenes, and azides, which facilitate late-stage functionalization—a key strategy in medicinal chemistry for expanding chemical diversity. While a broad range of alkyl pyrophosphate (alkyl-PP) donors with chain lengths of C5, C10, and C15 have been identified, synthetic access to these molecules—especially for late-stage modifications—remains limited. This work demonstrates that late-stage functionalization of complex isoprenoid intermediates, such as farnesyl diphosphate (FPP), significantly expands the utility of PTs and FPPS in drug discovery applications. FPP is a key intermediate in the biosynthesis of numerous biologically active compounds, including sterols, carotenoids, and antibiotics. However, the multistep synthesis of longer-chain prenyl donors from simple C5 precursors often suffers from low efficiency. This research addresses these limitations through the chemoenzymatic generation of structurally complex alkyl-PP compounds. Furthermore, this thesis explores the potential of PTs as biocatalysts for the late-stage diversification of complex natural products. PTs, particularly those derived from bacterial and fungal biosynthetic pathways, exhibit remarkable substrate promiscuity and can catalyze the transfer of diverse alkyl groups to aromatic acceptors. The versatility of this approach is demonstrated through the functionalization of two challenging natural products: daptomycin (Dap) and cannabigerolic acid (CBGA). Using the aromatic PT CdpNPT and a library of synthetic, non-native alkyl-PP donors, six novel Dap analogs were successfully synthesized. In addition, the mutant PT NphB M31S enabled selective C3 alkylation of olivetolic acid (OA), yielding 14 novel CBGA analogs. These results collectively highlight the value of chemoenzymatic late-stage functionalization as a powerful platform for the generation of structurally diverse isoprenoid analogs. This work expands the chemical space available for structure–activity relationship (SAR) studies and contributes to the development of novel bioactive compounds with potential pharmaceutical applications.