Carbohydrate Bis-acetal-Based Substrates as Tunable Fluorescence Quenched Probes for Monitoring exo-Glycosidase Activity
Carbohydrate Bis-acetal-Based Substrates as Tunable Fluorescence Quenched Probes for Monitoring exo-Glycosidase Activity
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DOI：
10.1021/jacs.7b01948
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发表时间：
2017-06-28
影响因子：
15
通讯作者：
Vocadlo, David J.
中科院分区：
文献类型：
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作者：
Cecioni, Samy;Vocadlo, David J.
可调谐Forster共振能量转移(FRET)淬灭底物可用于监测相关生理环境中各种酶的活性。然而，FRET淬灭的胞外糖苷酶底物的开发受到其受限的口袋状活性部位的阻碍。在这里，我们报告了一种新型基板的设计，它克服了这一问题。这些基于双缩醛的底物(BABS)带有一个半缩醛苷元离开基团，该基团紧密地捆绑着荧光染料，也使它们远离活性部位的口袋。在糖苷键断裂后，被释放的半缩醛自发分解，导致荧光团和猝灭剂的分离。我们详细介绍了GlcNAc-BABS的合成和表征，显示出惊人的99.9%的猝灭效率。这些底物被人外糖苷酶O-GlcNAcase(OGA)有效地转化。我们发现半缩醛离开基团迅速分解，从而能够定量监测OGA的活性。我们预计这一策略将广泛用于底物探针的开发，以监测外糖苷酶，以及一系列其他具有受限制的口袋状活性部位的酶。
Tunable Forster resonance energy transfer (FRET)-quenched substrates are useful for monitoring the activity of various enzymes within their relevant physiological environments. Development of FRET-quenched substrates for exo-glycosidases, however, has been hindered by their constrained pocket-shaped active sites. Here we report the design of a new class of substrate that overcomes this problem. These Bis-Acetal-Based Substrates (BABS) bear a hemiacetal aglycon leaving group that tethers fluorochromes in close proximity, also positioning them distant from the active site pocket. Following cleavage of the glycosidic bond, the liberated hemiacetal spontaneously breaks down, leading to separation of the fluorophore and quencher. We detail the synthesis and characterization of GlcNAc-BABS, revealing a striking 99.9% quenching efficiency. These substrates are efficiently turned over by the human exo-glycosidase O-GlcNAcase (OGA). We find the hemiacetal leaving group rapidly breaks down, enabling quantitative monitoring of OGA activity. We expect this strategy to be broadly useful for the development of substrate probes for monitoring exo-glycosidases, as well as a range of other enzymes having constrained pocket-shaped active sites.