Synthesis of (-)-Melodinine K: A Case Study of Efficiency in Natural Product Synthesis.

Efficiency is a key organizing principle in modern natural product synthesis. Practical criteria include time, cost, and effort expended to synthesize the target, which tracks with step-count and scale. The execution of a natural product synthesis, that is, the sum and identity of each reaction employed therein, falls along a continuum of chemical (abiotic) synthesis on one extreme, followed by the hybrid chemoenzymatic approach, and ultimately biological (biosynthesis) on the other, acknowledging the first synthesis belongs to Nature. Starting materials also span a continuum of structural complexity approaching the target with constituent elements on one extreme, followed by petroleum-derived and "chiral pool" building blocks, and complex natural products (i.e., semisynthesis) on the other. Herein, we detail our approach toward realizing the first synthesis of (-)-melodinine K, a complex bis-indole alkaloid. The total syntheses of monomers (-)-tabersonine and (-)-16-methoxytabersonine employing our domino Michael/Mannich annulation is described. Isolation of (-)-tabersonine from Voacanga africana and strategic biotransformation with tabersonine 16-hydroxylase for site-specific C-H oxidation enabled a scalable route. The Polonovski-Potier reaction was employed in biomimetic fragment coupling. Subsequent manipulations delivered the target. We conclude with a discussion of efficiency in natural products synthesis and how chemical and biological technologies define the synthetic frontier.

Two Tabersonine 6,7-Epoxidases Initiate Lochnericine-Derived Alkaloid Biosynthesis in Catharanthus roseus.

Lochnericine is a major monoterpene indole alkaloid (MIA) in the roots of Madagascar periwinkle (Catharanthus roseus). Lochnericine is derived from the stereoselective C6,C7-epoxidation of tabersonine and can be metabolized further to generate other complex MIAs. While the enzymes responsible for its downstream modifications have been characterized, those involved in lochnericine biosynthesis remain unknown. By combining gene correlation studies, functional assays, and transient gene inactivation, we identified two highly conserved P450s that efficiently catalyze the epoxidation of tabersonine: tabersonine 6,7-epoxidase isoforms 1 and 2 (TEX1 and TEX2). Both proteins are quite divergent from the previously characterized tabersonine 2,3-epoxidase and are more closely related to tabersonine 16-hydroxylase, involved in vindoline biosynthesis in leaves. Biochemical characterization of TEX1/2 revealed their strict substrate specificity for tabersonine and their inability to epoxidize 19-hydroxytabersonine, indicating that they catalyze the first step in the pathway leading to hörhammericine production. TEX1 and TEX2 displayed complementary expression profiles, with TEX1 expressed mainly in roots and TEX2 in aerial organs. Our results suggest that TEX1 and TEX2 originated from a gene duplication event and later acquired divergent, organ-specific regulatory elements for lochnericine biosynthesis throughout the plant, as supported by the presence of lochnericine in flowers. Finally, through the sequential expression of TEX1 and up to four other MIA biosynthetic genes in yeast, we reconstituted the 19-acetylhörhammericine biosynthetic pathway and produced tailor-made MIAs by mixing enzymatic modules that are naturally spatially separated in the plant. These results lay the groundwork for the metabolic engineering of tabersonine/lochnericine derivatives of pharmaceutical interest.