Structural Diversification of Hapalindole and Fischerindole Natural Products via Cascade Biocatalysis

Hapalindoles and related compounds (ambiguines, fischerindoles, welwitindolinones) are a diverse class of indole alkaloid natural products. They are typically isolated from the Stigonematales order of cyanobacteria and possess a broad scope of biological activities. Recently the biosynthetic pathway for assembly of these metabolites has been elucidated. In order to generate the core ring system, l-tryptophan is converted into the cis-indole isonitrile subunit before being prenylated with geranyl pyrophosphate at the C-3 position. A class of cyclases (Stig) catalyzes a three-step process, including a Cope rearrangement, 6-exo-trig cyclization, and electrophilic aromatic substitution, to create a polycyclic core. The formation of the initial alkaloid is followed by diverse late-stage tailoring reactions mediated by additional biosynthetic enzymes to give rise to a wide array of structural variations observed in this compound class. Herein, we demonstrate the versatility and utility of the Fam prenyltransferase and Stig cyclases toward the core structural diversification of this family of indole alkaloids. Through the synthesis of cis-indole isonitrile subunit derivatives, and with the aid of protein engineering and computational analysis, we have employed cascade biocatalysis to generate a range of derivatives and gained insights into the basis for substrate flexibility in this system.