Abstract：
OBJECTIVE: To synthesize, characterize, and validate 6FGA, a fluorescent glucose modified with a Cyanine5.5 at carbon-6 position, for probing the function of sodium-dependent glucose transporters, SGLT1 and SGLT2.
METHODS: The synthesis of fluorescent glucose analogue was achieved through \"click chemistry\" of Cyanine5.5-alkyne and 6-azido-6-deoxy-d-glucose. Cell system studies were conducted to characterize the in vivo transport properties.
RESULTS: Optical analyses revealed that 6FGA displayed similar spectral profiles to Cyanine5.5 in DMSO, allowing for concentration determination, thus supporting its utility in quantitative kinetic studies within biological assays. Uptake studies in cell system SGLT models, LLC-PK1 and HEK293 cells, exhibited concentration and time-dependent behavior, indicating saturation at specific concentrations and durations which are hallmarks of transported-mediated uptake. The results of cytotoxicity assays suggested cell viability at micromolar concentrations, enabling usage in assays for at least 1 h without significant toxicity. The dependence of 6FGA uptake on sodium, the co-transported cation, was demonstrated in LLC-PK1 and HEK293 cells. Fluorescence microscopy confirmed intracellular localization of 6FGA, particularly near the nucleus. Competition studies revealed that glucose tends to weakly reduce 6FGA uptake, although the effect did not achieve statistical significance. Assessments using standard SGLT and GLUT inhibitors highlighted 6FGA\'s sensitivity for probing SGLT-mediated transport.
CONCLUSIONS: 6FGA is a new fluorescent glucose analog offering advantages over existing probes due to its improved photophysical properties, greater sensitivity, enabling subcellular resolution and efficient tissue penetration in near-infrared imaging. 6FGA presents practicality and cost-effectiveness, making it a promising tool for nonradioactive, microplate-based assays at investigating SGLT-mediated glucose transport mechanisms.
METHODS: The synthesis of fluorescent glucose analogue was achieved through \"click chemistry\" of Cyanine5.5-alkyne and 6-azido-6-deoxy-d-glucose. Cell system studies were conducted to characterize the in vivo transport properties.
RESULTS: Optical analyses revealed that 6FGA displayed similar spectral profiles to Cyanine5.5 in DMSO, allowing for concentration determination, thus supporting its utility in quantitative kinetic studies within biological assays. Uptake studies in cell system SGLT models, LLC-PK1 and HEK293 cells, exhibited concentration and time-dependent behavior, indicating saturation at specific concentrations and durations which are hallmarks of transported-mediated uptake. The results of cytotoxicity assays suggested cell viability at micromolar concentrations, enabling usage in assays for at least 1 h without significant toxicity. The dependence of 6FGA uptake on sodium, the co-transported cation, was demonstrated in LLC-PK1 and HEK293 cells. Fluorescence microscopy confirmed intracellular localization of 6FGA, particularly near the nucleus. Competition studies revealed that glucose tends to weakly reduce 6FGA uptake, although the effect did not achieve statistical significance. Assessments using standard SGLT and GLUT inhibitors highlighted 6FGA\'s sensitivity for probing SGLT-mediated transport.
CONCLUSIONS: 6FGA is a new fluorescent glucose analog offering advantages over existing probes due to its improved photophysical properties, greater sensitivity, enabling subcellular resolution and efficient tissue penetration in near-infrared imaging. 6FGA presents practicality and cost-effectiveness, making it a promising tool for nonradioactive, microplate-based assays at investigating SGLT-mediated glucose transport mechanisms.
摘要：
目的：合成，表征，并验证6FGA，在碳-6位用氰化物5.5修饰的荧光葡萄糖，用于探测钠依赖性葡萄糖转运蛋白的功能，SGLT1和SGLT2。
方法：荧光葡萄糖类似物的合成是通过“点击化学”氰化5.5-炔和6-叠氮基-6-脱氧-d-葡萄糖实现的。进行细胞系统研究以表征体内转运特性。
结果：光学分析表明，6FGA在DMSO中显示出与Cyanine5.5相似的光谱图，允许浓度测定，从而支持其在生物测定中的定量动力学研究中的应用。细胞系统SGLT模型中的摄取研究，LLC-PK1和HEK293细胞，表现出浓度和时间依赖性行为，表明特定浓度和持续时间的饱和度，这是运输介导的摄取的标志。细胞毒性测定的结果表明，在微摩尔浓度下的细胞活力，使得在测定中使用至少1小时而没有明显的毒性。6FGA摄取对钠的依赖性，共转运的阳离子，在LLC-PK1和HEK293细胞中得到证实。荧光显微镜证实6FGA的细胞内定位，特别是在细胞核附近。竞争研究表明，葡萄糖倾向于弱降低6FGA的摄取，虽然效果没有达到统计学意义。使用标准SGLT和GLUT抑制剂的评估强调了6FGA对探测SGLT介导的转运的敏感性。
结论：6FGA是一种新的荧光葡萄糖类似物，由于其改进的光物理性质，与现有探针相比具有优势，更高的灵敏度，在近红外成像中实现亚细胞分辨率和有效的组织穿透。6FGA具有实用性和成本效益，使其成为非放射性的有希望的工具，基于微孔板的试验研究SGLT介导的葡萄糖转运机制。
方法：荧光葡萄糖类似物的合成是通过“点击化学”氰化5.5-炔和6-叠氮基-6-脱氧-d-葡萄糖实现的。进行细胞系统研究以表征体内转运特性。
结果：光学分析表明，6FGA在DMSO中显示出与Cyanine5.5相似的光谱图，允许浓度测定，从而支持其在生物测定中的定量动力学研究中的应用。细胞系统SGLT模型中的摄取研究，LLC-PK1和HEK293细胞，表现出浓度和时间依赖性行为，表明特定浓度和持续时间的饱和度，这是运输介导的摄取的标志。细胞毒性测定的结果表明，在微摩尔浓度下的细胞活力，使得在测定中使用至少1小时而没有明显的毒性。6FGA摄取对钠的依赖性，共转运的阳离子，在LLC-PK1和HEK293细胞中得到证实。荧光显微镜证实6FGA的细胞内定位，特别是在细胞核附近。竞争研究表明，葡萄糖倾向于弱降低6FGA的摄取，虽然效果没有达到统计学意义。使用标准SGLT和GLUT抑制剂的评估强调了6FGA对探测SGLT介导的转运的敏感性。
结论：6FGA是一种新的荧光葡萄糖类似物，由于其改进的光物理性质，与现有探针相比具有优势，更高的灵敏度，在近红外成像中实现亚细胞分辨率和有效的组织穿透。6FGA具有实用性和成本效益，使其成为非放射性的有希望的工具，基于微孔板的试验研究SGLT介导的葡萄糖转运机制。
