Metabolic carbohydrate engineering by exogenously added monosaccharide supplement is a technique of importance for studying physiological role of various glycans. Additionally, it also has the potential of developing new drug molecules for specific targeting. Lack of a spectroscopic reporting moiety in carbohydrates makes understanding their biochemical and physiological role very difficult. Towards this goal, we have modified glucose, with a propargyl group, wherein an azido coumarinyl profluorophore has been linked by "click chemistry." Here, we demonstrate the uptake and incorporation of this modified monosaccharide into bacteria, yeast and mammalian cells. We show that modification at C-2 (carbon numbered 2, according to IUPAC) position is tolerated best, and uptake is only slightly lower compared to glucose. In the presence of C-2-modified glucose, growth kinetics and cellular viability were also minimally affected in all the cell types used. Fluorescence spectroscopy of the labeled biomolecule and fluorescence imaging of the cells demonstrate that C-2-modified glucose is metabolically incorporated not only in the cell membrane but also accumulates in the nucleus. Such a fluorophore, incorporated into biomolecules, can be used as a tool to understand their structure-function relationship. Here, we show that the incorporation of such a fluorophore in a carbohydrate moiety may enable studying the physiological and biochemical processes associated with membranes. © 2012 Springer Science+Business Media, LLC.