Understanding the function and regulation of enzymes within their physiologically relevant milieu requires quality tools that report on their cellular activities. Here we describe a strategy for glycoside hydrolases that overcomes several limitations in the field, enabling quantitative monitoring of their activities within live cells. We detail the design and synthesis of bright and modularly assembled bis-acetal-based (BAB) fluorescence-quenched substrates, illustrating this strategy for sensitive quantitation of disease-relevant human α-galactosidase and α-N-acetylgalactosaminidase activities. We show that these substrates can be used within live patient cells to precisely measure the engagement of target enzymes by inhibitors and the efficiency of pharmacological chaperones, and highlight the importance of quantifying activity within cells using chemical perturbogens of cellular trafficking and lysosomal homeostasis. These BAB substrates should prove widely useful for interrogating the regulation of glycosidases within cells as well as in facilitating the development of therapeutics and diagnostics for this important class of enzymes.

了解酶在其生理相关环境中的功能和调节需要报告其细胞活动的质量工具。在这里，我们描述了一种糖苷水解酶的策略，该策略克服了该领域的一些限制，从而能够定量监测它们在活细胞内的活动。我们详细介绍了明亮和模块化组装的双缩醛基 (BAB) 荧光猝灭底物的设计和合成，说明了这种对疾病相关的人类 α-半乳糖苷酶和 α- N进行灵敏定量的策略-乙酰半乳糖胺酶活性。我们表明，这些底物可用于患者活细胞，以精确测量抑制剂对靶酶的参与和药理学伴侣的效率，并强调使用细胞运输和溶酶体稳态的化学干扰素量化细胞内活性的重要性。这些 BAB 底物应证明可广泛用于研究细胞内糖苷酶的调节以及促进这一重要酶类的治疗和诊断的开发。