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

研究了用全氘化mevalonolactone （MVL-d_9）制备高氘化和多氘化类异戊二烯的新方法及其在分析类异戊二烯生物合成机理方面的潜力。使用MVL-d_9进行生物合成研究的方法在机械酶学方面是有价值的，特别是在类异戊二烯生物合成中观察到的涉及质子攻击和/或质子猝灭的关键转化。首先采用Sharpless不对称环氧化法制备了纯(R)-MVL-d_9对映体。这种研究类异戊二烯生物合成的方法具有通路切换和超氘化的特点，在阐明一种新型链霉菌二萜的反应机制方面显示出显著的优势。通过在双工程链霉菌中异源表达源自灰链霉菌的二萜环化酶基因，并通过MVL-d_9在体内标记，深入了解了香叶基香叶基二磷酸转化为氯代烷萜戊三烯的环化机制。灰链霉菌是一种独特的二萜类细胞毒性抗生素terpentecin的产生者。环化过程包括亲电质子化、阳离子环闭合、Wagner-Meerwein型重排和去质子化。一个关键特征是，标记的代谢物主要是氘化马赛克分子的混合物，提供了足够的信息，对每个异戊二烯单元的标记模式进行了密切的分析，主要是通过^1H NMR光谱。另外的例子是类胡萝卜素生物合成的研究。类胡萝卜素是由各种微生物和植物产生的重要天然色素。这些化合物的特异氘标记在生化和物理化学研究中是非常宝贵的。利用MVL-d_9基因工程大肠杆菌制备高度氘化的玉米黄质、番茄红素和β-胡萝卜素。

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.