Fluorescein-5-thiosemicarbazide (FTSC) labeling for fluorescent imaging of pectin-derived oligogalacturonic acid transported in living cells by confocal microscopy

Abstract

Confocal microscopy is a powerful and effective tool for in vivo elucidation of the molecular basis of biological molecules. Chemical labeling of non-ultraviolet or non-fluorescent carbohydrate with fluorescent tag is an essential step that makes intra-cellular microscopic inspection possible. However, current labeling methods have proved unsuitable for functional pectin-derived oligogalacturonic acid due to the low fluorescence intensity and/or the formation of lactone byproducts. Here, fluorescein-5-thiosemicarbazide (FTSC) was employed for fluorescent labeling of pectin-derived oligogalacturonic acid under mild and neutral reaction conditions to avoid the generation of lactone byproducts. ESI–MS and LC–MS data displayed that pectin-derived pentagalacturonic acid, GalA5, could be quantitatively labeled with FTSC to afford thiosemicarbazones, without any lactone byproducts yielded. Moreover, GalA5-FTSC thiosemicarbazone showed good stability and desirable fluorescence characteristics and exhibited rather low levels of cytotoxicity against sensitive rat thymus cells. Additionally, we have achieved successful fluorescent imaging of GalA5-FTSC thiosemicarbazones transported in living SW-620 and HepG2 cells. The assay illustrates methodological significance in structure–activity elucidation study of functional carbohydrates especially acid oligo-/polysaccharides.