Mevalonolactone-d_9 ; A Versatile Tool for Biosynthetic Study of Isoprenoids. Synthesis and Its Application

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A new practical approach to the preparation of highly- and multiply deuterated isoprenoids, and its potential for analyzing the biosynthetic mechanism of isoprenoids are investigated by using fully deuterated mevalonolactone (MVL-d_9). The methodology for biosynthetic studies using MVL-d_9 is meritorious for mechanistic enzymology, particularly, the key transformation involving proton attack and/or proton quench as observed in isoprenoids biosynthesis. At first, enantiomeric pure (R)-MVL-d_9 was synthesized using Sharpless asymmetric epoxidation. This methodology for the investigation of isoprenoid biosynthesis featuring pathway switching and hyperdeuteration has shown significant advantages in elucidating the reaction mechanism of a novel Streptomyces diterpene. Insight into the cyclization mechanism involved in the conversion of geranylgeranyl diphosphate into a clerodane hydrocarbon terpentetriene was obtained by heterologous expression in doubly engineered Streptomyces lividans of a diterpene cyclase gene derived from Streptomyces griseolosporeus, a producer of a unique diterpenoid cytotoxic antibiotic terpentecin, and by in vivo labeling with MVL-d_9. The cyclization involved electrophilic protonation, cationic ring closure, Wagner-Meerwein type rearrangements, and deprotonation. A key feature was that the labeled metabolite as a mixture of predominantly deuterated mosaic molecules provided sufficient information that close analysis of the labeling pattern for each individual isoprene unit was achieved primarily by ^1H NMR spectroscopy.Additional examples were investigation for carotenoid biosynthesis. Carotenoids are important natural pigments produced by various microorganisms and plants. Specific deuterium-labeling of these compounds is invaluable in biochemical and physiochemical research. Preparation of highly deuterated zeaxanthin, lycopene, and β-carotene using engineered Escherichia coli with MVL-d_9 was performed.