One-pot fluorescent labeling of saccharides with fluorescein-5-thiosemicarbazide for imaging polysaccharides transported in living cells |
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Authors: | Zhang Ying Wang Zhongfu Zhang Xiaorui Zhou Wenxia Huang Linjuan |
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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 |
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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. |
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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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