Chemical tools for studying carbohydrates and carbohydrate processing enzymes

糖共轭化合物是一类普遍存在的生物分子，存在于所有生命王国中。然而，与其他领域相比，由于缺乏促进研究的化学工具和技术，糖共轭化合物的研究进展一直受到阻碍。广泛可用的强大的化学工具来研究蛋白质、核酸和被广泛认可的蛋白质修饰，如磷酸化，作为一个具有启发性的陪衬来强调这个问题。我们研究计划的一个主要目的是开发化学生物学工具来填补这一空白，从而促进对各种糖结合物及其构成它们的酶(糖基转移酶)和那些分解它们的酶(糖苷酶)的研究。在这里，我们概述了一个主要以化学为基础的研究计划，重点是创造使能的化学生物学工具。 首先，使用标准发色底物或荧光底物的体外酶化学动力学研究为糖苷水解酶如何作用于底物提供了至关重要的见解。然而，最终，这些酶在存在代谢物和蛋白质伙伴的复杂细胞环境中发挥作用。因此，我们将推进我们的开创性研究，为一系列糖苷酶合成明亮的荧光共振能量转移(FRET)猝灭底物。这些探针将以各种形式使用，以多种方式定量测量细胞内功能相关的人类酶的活性。 其次，活细胞酶学是一个前沿领域，其目标是直接在活细胞的自然环境中分析酶的活性。然而，这一新兴领域受到了一系列技术挑战的阻碍。在这里，我们的目标是开发两种糖苷酶的比率“笼式”底物，并利用这些底物在显微镜下建立第一个在活细胞中详细的酶动力学研究。这项工作将为了解内源性细胞因素如何影响细胞内的酶功能奠定基础。 第三，我们的目标是化学合成两种相关糖苷酶和糖基转移酶的“笼式”抑制剂。一旦这些最初不活跃的笼状抑制剂用光照射，光保护基团就会丢失，有效的抑制剂就会释放出来。这样的工具将允许对细胞和组织中的这些重要酶进行空间和时间控制。 总之，朝着这三个目标取得的进展应该会为观察和控制糖苷酶和糖基转移酶提供更好的工具。这些在细胞内使用的新工具应该会加速对这些酶和糖结合物的调节和功能重要性的生物学研究。这里应用的许多原理应该适用于其他碳水化合物加工酶，甚至更广泛地适用于其他超家族的酶。

Glycoconjugates are a ubiquitous class of biomolecules found in all kingdoms of life. Relative to other areas, however, advances in studying glycoconjugates have been hampered by a shortage of chemical tools and technologies to facilitate their investigation. The wide availability of robust chemical tools for studying proteins, nucleic acids, and widely recognized protein modifications such as phosphorylation, serves as an instructive foil to highlight the issue. A major thrust of our research program is directed toward developing chemical biology tools to fill this gap and thereby facilitate research into understanding various glycoconjugates and the enzymes that build them up (glycosyltransferases) and those that break them down (glycosidases). Here we outline a principally chemistry-based program of research focused on creation of enabling chemical biology tools. First, chemical kinetic studies of enzymes in vitro using standard chromogenic or fluorogenic substrates have provided critically important insights into how glycoside hydrolases act on their substrates. Ultimately, however, these enzymes function within the complex environment of the cell in the presence of metabolites and protein partners. Accordingly, we will advance our pioneering studies to synthesize bright fluorescence resonance energy transfer (FRET) quenched substrates for a suite of glycosidases. These probes will be usable in various formats to quantitatively measure the activities of functionally related human enzymes within cells in a multiplexed manner. Second, live cell enzymology is a frontier area having the goal of analyzing the activity of enzymes directly within their native milieu of living cells. This emerging field has, however, been hindered by a series of technical challenges. Here we aim to develop ratiometric “caged” substrates for two glycosidases and use these to establish the first detailed enzyme kinetic study in live cells by microscopy. This work will set the stage to understand how endogenous cellular factors influence enzyme function within cells. Third, we aim to chemically synthesize “caged” inhibitors of two related glycosidases and glycosyltransferases. Once these initially inactive caged inhibitors are irradiated with light the photoprotecting group is lost and the potent inhibitor is released. Such tools will permit spatial and temporal control over these important enzymes in cells and tissues. Together, progress toward these three aims should provide improved tools for observing and controlling glycosidases and glycosyltransferases. These new tools for use within cells should accelerate biological research into the regulation and functional importance of these enzymes and glycoconjugates. Many of the principles applied here should be applicable to other carbohydrate processing enzymes and, indeed, more broadly to other superfamilies of enzymes.