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One-pot fluorescent labeling of saccharides with fluorescein-5-thiosemicarbazide for imaging polysaccharides transported in living cells
Authors:Zhang Ying  Wang Zhongfu  Zhang Xiaorui  Zhou Wenxia  Huang Linjuan
Affiliation:aKey Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, 229 Northern Taibai Road, Xi’an 710069, PR China;bBeijing Institute of Pharmacology and Toxicology, 27 Tai-Ping Road, Haidian District, Beijing 100850, PR China
Abstract:A simple and efficient procedure for the fluorescent labeling of saccharides is a prerequisite step for imaging the transport of polysaccharides in living cells. We report a one-pot strategy for the fluorescent labeling of saccharides with fluorescein-5-thiosemicarbazide (FTSC), which introduces the thiosemicarbazide group of FTSC to the aldehyde group at the reducing end of saccharides to form stable amino derivatives via reductive amination. The Glc-FTSC derivative was characterized by HPLC–MS, HRESIMS and NMR spectroscopy. Saccharides were quantitatively labeled with FTSC at 75 °C for 1 h under optimum reaction conditions. Fluorescence studies illustrated that the conjugation of FTSC to saccharides did not change its florescence properties (λex = 495 nm, λem = 517 nm), presenting desirable compatibility with commonly used fluorescence equipment. Polysaccharide AAG-FTSC derivatives exhibited rather low levels of cytotoxicity against rat thymus cells, and the fluorescent labeling procedure had slight impact on their anti-tumor activity. Results indicate that the assay neither introduces discernible cytotoxicity against living cells nor obviously alters the functional activities of polysaccharides, and provides a convenient, highly efficient fluorescent labeling approach for imaging the transport of polysaccharides in living cells.
Keywords:Abbreviations: HPLC, high-performance liquid chromatography   ESIMS, electrospray-ionization mass spectrometry   MALDI-TOFMS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry   HPLC-ESIMS, high performance liquid chromatography coupled with electrospray-ionization mass spectrometry   Glc, glucose   Lac, lactose   FTSC, fluorescein-5-thiosemicarbazide   DMSO, dimethyl sulfoxide   MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide   3H-TdR, thymidine-[methyl-3H]   FITC, fluorescein isothiocyanate   NaBH3CN, sodium cyanoborohydride   PBS, phosphate buffered solution   HepG2, human hepatoma   ACC, acinar cells carcinoma   Glc-FTSC, FTSC labeled glucose   DP, degree of polymerization   G1, saccharide with degree of polymerization of 1   LPS, lipopolysaccharide   AAG, polysaccharides separated from Auricularia auricula-judae   AAG-FTSC, FTSC labeled AAG   FI, fluorescent intensity   HRESIMS, high-resolution electrospray-ionization mass spectroscopy   NMR, nuclear magnetic resonance
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