Nagaoka H, Nohta H, Saito M, Ohkura Y
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Chem Pharm Bull (Tokyo). 1992 Sep;40(9):2559-61. doi: 10.1248/cpb.40.2559.
The fluorescence derivatization of nucleotides with 2-(5-chlorocarbonyl-2-oxazolyl)-5,6-methylenedioxybenzo++ furan in the presence of sodium azide and the separation of the derivatives by high-performance liquid chromatography are described. The reagent reacts with 5'-terminal hydroxyl groups of nucleotides to produce the corresponding fluorescent carbamates. The derivatives of mono- and oligonucleotides are separated by chromatography on a reversed phase column (TSKgel ODS-80TM) and the derivatives of octa- and deca-nucleotides on a size exclusion column (TSKgel G3000SWXL). The detection limits (signal-to-noise ratio = 3) are 0.8-6.0 pmol on column. 5'Phosphorylated nucleotide also gives a fluorescent derivative after alkaline phosphatase-mediated dephosphorylation.
描述了在叠氮化钠存在下，核苷酸与2-(5-氯甲酰基-2-恶唑基)-5,6-亚甲基二氧基苯并呋喃的荧光衍生化反应，以及通过高效液相色谱法分离衍生物的过程。该试剂与核苷酸的5'-末端羟基反应生成相应的荧光氨基甲酸酯。单核苷酸和寡核苷酸的衍生物在反相柱（TSKgel ODS-80TM）上进行色谱分离，八核苷酸和十核苷酸的衍生物在尺寸排阻柱（TSKgel G3000SWXL）上进行分离。柱上检测限（信噪比=3）为0.8-6.0皮摩尔。5'-磷酸化核苷酸在碱性磷酸酶介导的去磷酸化后也产生荧光衍生物。
