Department of Chemistry, Simon Fraser University , Burnaby, British Columbia, Canada.
Department of Molecular Biology and Biochemistry, Simon Fraser University , Burnaby, British Columbia, Canada.
J Am Chem Soc. 2017 Jun 28;139(25):8392-8395. doi: 10.1021/jacs.7b01948. Epub 2017 Jun 20.
Tunable Förster 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.
可调节的Förster 共振能量转移（FRET）猝灭底物可用于监测各种酶在其相关生理环境中的活性。然而，外糖苷酶的 FRET 猝灭底物的发展受到其受限的口袋状活性位点的阻碍。在这里，我们报告了一类克服此问题的新型底物的设计。这些双缩醛基底物（BABS）带有半缩醛离去基团，可将荧光团紧密连接在一起，同时将其定位远离活性位点口袋。糖苷键断裂后，释放的半缩醛会自动分解，导致荧光团和猝灭剂分离。我们详细描述了 GlcNAc-BABS 的合成和表征，揭示了惊人的 99.9%猝灭效率。这些底物可被人类外糖苷酶 O-GlcNAcase（OGA）有效转化。我们发现半缩醛离去基团迅速分解，能够定量监测 OGA 活性。我们预计这种策略将广泛用于开发用于监测外糖苷酶以及具有受限口袋状活性位点的一系列其他酶的底物探针。
