Advances in the Syntheses of Fusarium-Derived Natural Products: Progress Toward Immunomodulide and the Total Synthesis of Fusahexin

Abstract

Fungal natural products represent a chemically diverse and pharmacologically rich source of bioactive scaffolds, yet the majority remain incompletely characterized at the level of structure, biosynthesis, and biological function. Across the 12-membered cyclic tridepsipeptide family, to which Fusarium-derived immunomodulide belongs, biochemically characterized biosynthetic gene clusters remain the exception rather than the rule, leaving stereochemical assignment dependent on total synthesis in the absence of reliable biosynthetic prediction. These structural and biosynthetic uncertainties place practical limits on real-world applications for these compounds. The development of immunomodulide as a sustainable plant immune potentiator has been limited by the absence of a validated pharmacophore model and unresolved polyketide stereochemistry, and investigation into fusahexin's proposed role in Fusarium virulence and cell surface hydrophobicity has been limited by the absence of synthetic access to both the natural product and its biosynthetic precursor.

This thesis describes the use of total synthesis, comparative spectroscopic analysis, and biosynthetic investigation to resolve these structural and functional gaps for two macrocyclic fungal natural products. We have established the first pharmacophore model for the 12-membered cyclic tridepsipeptide class, identifying the native D-Trp/L-Pip macrolactam configuration as essential for immunomodulide's plant immunopotentiating activity and confirming that this activity translates into reduced bacterial colonization in planta. We have narrowed the stereochemical assignment of immunomodulide's polyketide-derived fragment from sixteen possible permutations to a single synthetic lead using comparative ¹³C NMR analysis, and have advanced its total synthesis to the macrolactamization precursor, providing a foundation for the future development of a lead scaffold for sustainable crop protection. Using a synthesis-supported workflow, CRISPR/Cas9 gene editing and comparative metabolomics, we have identified prefusahexin as the direct biosynthetic precursor to fusahexin and achieved the total syntheses of both natural products, with a reduction-triggered cyclization cascade delivering fusahexin's fused N,O-acetal in high yield. We have further shown that the fusahexin biosynthetic gene cluster is split across the genome and, through biomimetic oxidation of prefusahexin, implicated copper amine oxidases as the enzyme class responsible for the key oxidative transformation linking the two natural products. The synthetic and biosynthetic platforms presented in this thesis will be fundamental to pursuing further biological and translational investigations of these fungal natural products.