Chemoenzymatic Transformations of Indole and Azaindole Containing Natural Product Analogs Using Promiscuous Prenyltransferases

Abstract

The overall aim of my work was to expand the practical utility of prenyltransferase-based biocatalysis as a tool in organic synthesis, natural product diversification, and drug discovery efforts. Aromatic prenyltransferases (PTs) serve a key role in the biosynthesis of countless bioactive natural products (NPs), by catalyzing the transfer of a 5 carbon prenyl (dimethylallyl) moiety from an alkyl pyrophosphate “donor” onto an aromatic “acceptor” substrate via electrophilic aromatic substitution. Many PTs have been observed to have remarkably broad substrate scopes, capable of accepting allylic, benzylic, heterobenzylic, and diene containing alkyl donors and transferring them to a range of phenolic and indole-containing substrates. Having evolved to carry out highly regioselective and chemoselective alkylation in complex chemical environments, PTs represent a powerful untapped source of late-stage functionalization catalysts for both natural and synthetic drug diversification efforts. Chapter 1 introduces and highlights the significance of indole-containing NPs and the diverse spectrum of biological activity that arises from substituted indole scaffolds. A brief overview of known synthetic methods for indole functionalization is covered, focusing on the challenges associated with functionalizing the benzenoid portion of indole. Subsequently, indole-modifying enzymes are introduced, and the structure, function, and mechanism of indole PTs is described as a foundation to this dissertation work. Chapter 2 demonstrates the utility of PT-based natural product diversification, in which the cytotoxic prenylated tryptophan-containing cyclic dipeptide tryprostatin B (TPS-B) was synthesized and chemoenzymatically alkylated using the promiscuous PT CdpNPT. Using a library of 66 synthetic alkyl-pyrophosphate donors, 24 unique donors were accepted by CdpNPT in the presence of TPS-B to generate novel NP analogs. 11 of these chemoenzymatically produced TPS-B analogs were isolated for structure elucidation via nuclear magnetic resonance (NMR) to reveal high selectivity for indole C6 alkylation. Cytotoxicity assays revealed that the TPS-B analogs produced in this work have a potency similar to their parent NPs. This work demonstrates that PT-based biocatalysts can be used for the late-stage diversification of NPs which provides direct access to NP analogs not accessible through current synthetic methods. Chapter 3 outlines the synthesis and utilization of a series of azaindole-substituted tryptophan analogs (Aza-Trp) to probe the compatibility of tryptophan-PTs with medicinally relevant indole isosteres. Synthetic tryptophan-mimetic substrates containing additional aromatic N atoms at the 2, 4, 5, 6, and 7 positions were prepared and screened as substrates for the indole C4-PT FgaPT2. These results identified 4-azatryprophan and 5-azatryptophan as previously unreported substrate classes for aromatic PTs. After structural elucidation of the prenylated products, we discovered FgaPT2 catalyzed N-prenylation of the 6-membered ring of these azaindole substrates to form a cationic N-prenylpyridinium products. Not only is this the first report of chemoenzymatic prenylation of azaindole substrates, but this work uncovered a previously undocumented PT-catalyzed reaction. Chapter 4 reports the synthesis and evaluation of azaindole-substituted tryptophan-proline cyclic dipeptides (Aza-CyWP) as substrates for aromatic PTs, with the goal of chemoenzymatically producing azaindole-containing analogs of this privileged NP core scaffold with enhanced aqueous solubility and altered bioactivity. We discovered that the indole-C2 PT which has the native function of TPS-B biosynthesis, FtmPT1, was capable of prenylating all 5 azaindole-containing substrates. However, these synthetic substrates were found to alter the regioselectivity of prenylation in an azaindole iso-dependent manner, resulting in a total of 7 fully characterized N1, C2, and C3 prenylated products. Additionally, the highly promiscuous PT utilized in Chapter 2, CdpNPT, was found to accept the 7-azaindole containing Aza-CyWP, to produce cyclized C3-reverse prenylated and N1 prenylated products. Using the common medicinal chemistry strategy of isosteric replacement, azaindole containing NP analogs were successfully generated using indole PTs. This work represents the first step toward the application of PT-based biocatalytic functionalization of synthetic heterocyclic scaffolds commonly utilized in structure-activity relationship studies.