Development of a selective novel fluorescent substrate for sodium-dependent transporters

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.

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.

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.

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.