Meints Lisa M, Poston Anne W, Piligian Brent F, Olson Claire D, Badger Katherine S, Woodruff Peter J, Swarts Benjamin M
Department of Chemistry and Biochemistry, Central Michigan University.
Department of Chemistry, University of Southern Maine.
J Vis Exp. 2017 Feb 17(120):54485. doi: 10.3791/54485.
Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other fields. For instance, trehalose analogues bearing detectable tags have been used to detect Mycobacterium tuberculosis and may have applications as tuberculosis diagnostic imaging agents. Hydrolytically stable versions of trehalose are also being pursued due to their potential for use as non-caloric sweeteners and bioprotective agents. Despite the appeal of this class of compounds for various applications, their potential remains unfulfilled due to the lack of a robust route for their production. Here, we report a detailed protocol for the rapid and efficient one-step biocatalytic synthesis of trehalose analogues that bypasses the problems associated with chemical synthesis. By utilizing the thermostable trehalose synthase (TreT) enzyme from Thermoproteus tenax, trehalose analogues can be generated in a single step from glucose analogues and uridine diphosphate glucose in high yield (up to quantitative conversion) in 15-60 min. A simple and rapid non-chromatographic purification protocol, which consists of spin dialysis and ion exchange, can deliver many trehalose analogues of known concentration in aqueous solution in as little as 45 min. In cases where unreacted glucose analogue still remains, chromatographic purification of the trehalose analogue product can be performed. Overall, this method provides a "green" biocatalytic platform for the expedited synthesis and purification of trehalose analogues that is efficient and accessible to non-chemists. To exemplify the applicability of this method, we describe a protocol for the synthesis, all-aqueous purification, and administration of a trehalose-based click chemistry probe to mycobacteria, all of which took less than 1 hour and enabled fluorescence detection of mycobacteria. In the future, we envision that, among other applications, this protocol may be applied to the rapid synthesis of trehalose-based probes for tuberculosis diagnostics. For instance, short-lived radionuclide-modified trehalose analogues (e.g., F-modified trehalose) could be used for advanced clinical imaging modalities such as positron emission tomography-computed tomography (PET-CT).
海藻糖的化学修饰版本，即海藻糖类似物，在生物学、生物技术和制药科学等领域有应用。例如，带有可检测标签的海藻糖类似物已被用于检测结核分枝杆菌，并且可能用作结核病诊断成像剂。由于海藻糖的水解稳定版本有潜力用作无热量甜味剂和生物保护剂，因此也在对其进行研究。尽管这类化合物在各种应用中颇具吸引力，但由于缺乏强大的生产途径，它们的潜力尚未得到充分发挥。在此，我们报告了一种详细的方案，用于快速高效地一步生物催化合成海藻糖类似物，该方案绕过了与化学合成相关的问题。通过利用来自嗜热栖热菌的耐热海藻糖合酶（TreT），海藻糖类似物可以在15至60分钟内从葡萄糖类似物和尿苷二磷酸葡萄糖一步高产率（高达定量转化）生成。一种简单快速的非色谱纯化方案，包括旋转透析和离子交换，可在短短45分钟内提供许多已知浓度的海藻糖类似物水溶液。在仍有未反应的葡萄糖类似物残留的情况下，可以对海藻糖类似物产物进行色谱纯化。总体而言，该方法为海藻糖类似物的快速合成和纯化提供了一个“绿色”生物催化平台，高效且非化学专业人员也可操作。为了举例说明该方法的适用性，我们描述了一种用于合成、全水相纯化以及将基于海藻糖的点击化学探针施用于分枝杆菌的方案，所有这些操作耗时不到1小时，并能够对分枝杆菌进行荧光检测。未来，我们设想，除其他应用外，该方案可应用于快速合成用于结核病诊断的基于海藻糖的探针。例如，短寿命放射性核素修饰的海藻糖类似物（如F修饰的海藻糖）可用于正电子发射断层扫描 - 计算机断层扫描（PET - CT）等先进临床成像模式。
