Anomer-specific Raman biosensing reveals β-selective induction of metabolic signatures at 1130 and 1603 cm-1 in endothelial cells.

Glucose metabolism is central to cellular homeostasis and vascular integrity in endothelial cells (ECs). Although glucose exists as α- and β-anomers, their distinct metabolic effects are rarely investigated. In this study, stereoselective cellular responses were examined using glucose analogs bearing alkyne (3-OPG, 6-OPG) and acetonitrile (3-OACN) reporter groups. Anomeric purity was verified using Raman and NMR spectroscopy, while intracellular accumulation and biotransformation were monitored through Raman imaging combined with chemometric analysis. Only the β-enriched anomers of 3-OPG and 3-OACN induced a distinct Raman response characterized by bands at 1130 and 1603 cm-1, whereas the α-enriched anomer of 3-OPG and the C6-modified 6-OPG remained spectrally silent regarding these biomarkers. The appearance of these bands was accompanied by a reduction or disappearance of the expected reporter-group signal, indicating intracellular transformation of the probes. Furthermore, inhibition studies demonstrated that the characteristic Raman signature persisted following the inhibition of glucose-6-phosphate dehydrogenase, suggesting that it is more closely associated with cellular uptake, intracellular retention, and the accumulation of probe-derived species than with selective metabolism through the pentose phosphate pathway. Collectively, these findings reveal a pronounced anomer-dependent cellular response and support a role for the anomeric ratio in shaping the intracellular processing and accumulation of glucose analogs in ECs. These findings uncover a previously unrecognized anomer-dependent metabolic-chemical gating mechanism underlying the formation of Raman-active intracellular species in ECs. Ultimately, this work provides a robust foundation for high-precision, non-invasive biosensing of cellular probe uptake, retention, and biotransformation in both physiological and pathological contexts.