Carbohydrates take an essential place in the energy supply. Different pathologies, including cancers, are linked to the changes in sugar uptake, defining the corresponding sugar transporters as targets for diagnostics and therapy. Under tumorigenesis, the common trait is alterations in facilitative glucose transporters (GLUTs). Among GLUTs, GLUT5 stands out by being specialized in fructose transport and linked with the development and progression of cancers. Subsequently, molecular probes to analyze the GLUT activity are of interest for applications in disease diagnostics. To date, analysis of GLUT activity in cells has been approached using corresponding radiolabeled sugars as GLUT substrates. The challenges associated with radioactivity and experimental settings further drove the development of fluorescently-labeled tools. Glycoconjugation was found to facilitate the transport of fluorophores yielding the fluorescently-labeled molecular probes for GLUTs, including GLUT5-specific probes. However, background fluorescence limited their application as diagnostic tools. This work presents xanthene-based turn-on probes that enable an analysis of GLUT5 activity in live cells. Probes’ uptake efficiency, GLUT5-specificity, and the ability to discriminate between normal and cancer cells were established. The turn-on mechanism that relies on the activation of fluorescence in the acidic environment eliminates the fluorescence background and reflects the transport activity solely. The specificity of uptake through GLUT5 allows discrimination of GLUT5-positive cells. Moreover, the activation in the acidic environment also allowed discrimination<br/>of cancer vs. normal cells. Such dual discrimination promoted recognition of GLUT5-positive cancer cells, setting the basis for using these probes as cancer-specific diagnostic tools. The turn-on mechanism allows the analysis to be carried out in diverse settings, including microscopy, plate readers, and flow cytometers, providing a general tool applicable to different laboratory settings.

Enabling high-throughput analysis of GLUT5 activity in live cells with specific turn-on probes

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