Harnessing Cascade Biocatalysis for the Chemoenzymatic Synthesis of Unnatural Hapalindole-Type Metabolites and Further Exploration into their Biosynthesis

Abstract

This thesis presents results focused on the exploration of the enzymes responsible for the biosynthesis of the hapalindole-type metabolites. Hapalindole-type metabolites are a diverse group of indole alkaloids defined by their polycyclic ring system, various stereoisomers, unique functional groups and promising biological activities. Recently, the biosynthetic gene cluster responsible for their biosynthesis has been elucidated. This revealed a wide range of enzymes that included prenyltransferases, oxygenases, and cyclases. The cyclases, in particular, have drawn interest due to their ability to catalyze a unique three step cyclization cascade: 1) a Cope rearrangement, 2) 6-exo-trig cyclization, and 3) terminal electrophilic aromatic substitution (EAS) from a common indole C-3 geranylated (3-GC) intermediate. Utilizing cascade biocatalysis with a prenyltransferase and various cyclases, a chemoenzymatic route to produce unnatural hapalindole-type metabolites has been devised on both milligram and in vitro scales in reaction vessels and cell-free protein synthesis reactions. Further medicinal testing and semisynthetic modifications to this library could provide a drug candidate to combat increasing drug resistance. Another member of the hapalindole-type metabolites, the ambiguines, showcases similar biological activity but a greater amount of structural modifications. One of the most interesting is the addition of a fifth (E) ring. To date, it is unknown how this fifth ring is formed but it is proposed to arise from reactions catalyzed by Rieske-type oxygenases. To tackle this question, two routes were explored; one focusing on the Rieske-type oxygenases themselves and another exploring a chemoenzymatic synthesis of a pentacyclic ambiguine. While the Rieske-type oxygenases proved challenging to elucidate, the chemoenzymatic route has shown promise. To date, an optimized, efficient biocatalytic method for generating an unnatural ambiguine derivative, 12-epi-ambiguine H nitrile, has been developed. Efficient biosynthesis of the unnatural derivative is crucial to further synthetic chemistry or biocatalysis efforts to generate the E-ring. In addition, during early phases of this work, we uncovered an unexpected new metabolite of a one pot reaction previously unknown to the Stig Cyclases. This finding strengthens the Cope rearrangement hypothesis and provides a potential synthon towards total synthesis efforts of a diverse range of compounds. These works showcase the potential natural product enzymes have to produce complex metabolites and provide a new route to further develop the diverse library of already known natural products.