Unlocking New Prenylation Modes: Azaindoles as a New Substrate Class for Indole Prenyltransferases.

Aza-substitution, the replacement of aromatic CH groups with nitrogen atoms, is an established medicinal chemistry strategy for increasing solubility, but current methods of accessing functionalized azaindoles are limited. In this work, indole-alkylating aromatic prenyltransferases (PTs) were explored as a strategy to directly functionalize azaindole-substituted analogs of natural products. For this, a series of aza-l-tryptophans (Aza-Trp) featuring N-substitution of every aromatic CH position of the indole ring and their corresponding cyclic Aza-l-Trp-l-proline dipeptides (Aza-CyWP), were synthesized as substrate mimetics for the indole-alkylating PTs FgaPT2, CdpNPT, and FtmPT1. We then demonstrated most of these substrate analogs were accepted by a PT, and the regioselectivity of each prenylation was heavily influenced by the position of the N-substitution. Remarkably, FgaPT2 was found to produce cationic N-prenylpyridinium products, representing not only a new substrate class for indole PTs but also a previously unobserved prenylation mode. The discovery that nitrogenous indole bioisosteres can be accepted by PTs thus provides access to previously unavailable chemical space in the search for bioactive indolediketopiperazine analogs.

Novel Homologs of Isopentenyl Phosphate Kinase Reveal Class-Wide Substrate Flexibility.

The widespread utility of isoprenoids has recently sparked interest in efficient synthesis of isoprene-diphosphate precursors. Current efforts have focused on evaluating two-step "isoprenol pathways," which phosphorylate prenyl alcohols using promiscuous kinases/phosphatases. The convergence on isopentenyl phosphate kinases (IPKs) in these schemes has prompted further speculation about the class's utility in synthesizing non-natural isoprenoids. However, the substrate promiscuity of IPKs in general has been largely unexplored. Towards this goal, we report the biochemical characterization of five novel IPKs from Archaea and the assessment of their substrate specificity using 58 alkyl-monophosphates. This study reveals the IPK-catalyzed synthesis of 38 alkyl-diphosphate analogs and discloses broad substrate specificity of IPKs. Further, to demonstrate the biocatalytic utility of IPK-generated alkyl-diphosphates, we also highlight the synthesis of alkyl-l-tryptophan derivatives using coupled IPK-prenyltransferase reactions. These results reveal IPK-catalyzed reactions are compatible with downstream isoprenoid enzymes and further support their development as biocatalytic tools for the synthesis of non-natural isoprenoids.

Acceptor substrate determines donor specificity of an aromatic prenyltransferase: expanding the biocatalytic potential of NphB.

Aromatic prenyltransferases are known for their extensive promiscuity toward aromatic acceptor substrates and their ability to form various carbon-carbon and carbon-heteroatom bonds. Of particular interest among the prenyltransferases is NphB, whose ability to geranylate cannabinoid precursors has been utilized in several in vivo and in vitro systems. It has therefore been established that prenyltransferases can be utilized as biocatalysts for the generation of useful compounds. However, recent observations of non-native alkyl-donor promiscuity among prenyltransferases indicate the role of NphB in biocatalysis could be expanded beyond geranylation reactions. Therefore, the goal of this study was to elucidate the donor promiscuity of NphB using different acceptor substrates. Herein, we report distinct donor profiles between NphB-catalyzed reactions involving the known substrate 1,6-dihydroxynaphthalene and an FDA-approved drug molecule sulfabenzamide. Furthermore, we report the first instance of regiospecific, NphB-catalyzed N-alkylation of sulfabenzamide using a library of non-native alkyl-donors, indicating the biocatalytic potential of NphB as a late-stage diversification tool. KEY POINTS: • NphB can utilize the antibacterial drug sulfabenzamide as an acceptor. • The donor profile of NphB changes dramatically with the choice of acceptor. • NphB performs a previously unknown regiospecific N-alkylation on sulfabenzamide. • Prenyltransferases like NphB can be utilized as drug-alkylating biocatalysts.

Indole C6 Functionalization of Tryprostatin B Using Prenyltransferase CdpNPT.

Tryprostatin A and B are prenylated, tryptophan-containing, diketopiperazine natural products, displaying cytotoxic activity through different mechanisms of action. The presence of the 6-methoxy substituent on the indole moiety of tryprostatin A was shown to be essential for the dual inhibition of topoisomerase II and tubulin polymerization. However, the inability to perform late-stage modification of the indole ring has limited the structure-activity relationship studies of this class of natural products. Herein, we describe an efficient chemoenzymatic approach for the late-stage modification of tryprostatin B using a cyclic dipeptide N-prenyltransferase (CdpNPT) from Aspergillus fumigatus, which generates novel analogs functionalized with allylic, benzylic, heterocyclic, and diene moieties. Notably, this biocatalytic functionalizational study revealed high selectivity for the indole C6 position. Seven of the 11 structurally characterized analogs were exclusively C6-alkylated, and the remaining four contained predominant C6-regioisomers. Of the 24 accepted substrates, 10 provided >50% conversion and eight provided 20-50% conversion, with the remaining six giving <20% conversion under standard conditions. This study demonstrates that prenyltransferase-based late-stage diversification enables direct access to previously inaccessible natural product analogs.