Huang Huazhang, Nishi Kosuke, Gee Shirley J, Hammock Bruce D
Department of Entomology and Cancer Research Center, University of California, Davis, California 95616, USA.
J Agric Food Chem. 2006 Feb 8;54(3):694-9. doi: 10.1021/jf0526083.
Esterases play a crucial role in industrial chemical synthesis, maintaining normal physiological metabolism and detoxifying exogenous ester-containing toxicants. To meet the rapidly increasing industrial need for all kinds of esterases, especially enantioselective esterases used to generate highly pure chiral compounds, general substrates are necessary for rapid screening, monitoring, purification, and characterization. In this study, general fluorescent substrates including phenolic derivatives and alpha-cyanoesters were evaluated for sensitivity in detecting esterases in buffer systems. Results with two different esterases and different incubation times suggested that the alpha-cyanoesters examined were significantly more sensitive at detecting esterases than the corresponding tested phenolic derivatives. More importantly, alpha-cyanoesters, containing a secondary alcohol, possess at least one chiral center; thus, they are tools to screen for enantioselective hydrolysis. Results indicated that the enantioselectivity of esterases toward general alpha-cyanoesters strongly depended on the esterase and the substrate, but the majority of esterases examined preferred S-isomers to their corresponding R-enantiomers. Most appealing was the very high enantioselectivity displayed in cytosolic esterases of the house fly. The potential utility of such esterases is discussed. In addition, the use of alpha-cyanoesters as chiral fluorescent substrates was demonstrated for monitoring in enantioselective esterases.
酯酶在工业化学合成、维持正常生理代谢以及对外源含酯毒物进行解毒过程中发挥着关键作用。为满足工业对各类酯酶迅速增长的需求，尤其是用于生成高纯度手性化合物的对映选择性酯酶，通用底物对于快速筛选、监测、纯化及表征而言是必不可少的。在本研究中，对包括酚类衍生物和α-氰基酯在内的通用荧光底物在缓冲体系中检测酯酶的灵敏度进行了评估。使用两种不同酯酶及不同孵育时间所得到的结果表明，所检测的α-氰基酯在检测酯酶方面比相应的受试酚类衍生物显著更灵敏。更重要的是，含有仲醇的α-氰基酯拥有至少一个手性中心；因此，它们是筛选对映选择性水解的工具。结果表明，酯酶对通用α-氰基酯的对映选择性强烈依赖于酯酶和底物，但所检测的大多数酯酶更倾向于S-异构体而非其相应的R-对映体。最引人注目的是家蝇胞质酯酶所表现出的非常高的对映选择性。讨论了此类酯酶的潜在用途。此外，还证明了使用α-氰基酯作为手性荧光底物来监测对映选择性酯酶。
