Analytical Sciences

Reversible Fluorescence Derivatization of Amino Groups Using Dansylaminomethylmaleic Acid via Its Anhydride

Kayo SAKATA, Kenji HAMASE, Shigeki SASAKI, Minoru MAEDA and Kiyoshi ZAITSU

Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi, Higashi, Fukuoka 812-8582, Japan

A new derivatizing reagent, dansylaminomethylmaleic acid (DAM), was synthesized and utilized for the reversible fluorescence labeling of amino groups. The reagent DAM was dehydrated in the presence of (trimethylsilyl)ethoxyacetylene, a dehydrating agent, and the resulting anhydride reacted with amino groups, and could then be liberated under mild acidic conditions. With this reagent, benzylamine was derivatized as a model amino compound. The resultant fluorescent derivative, N-benzyl-2-dansylaminomethylmaleamic acid (BDAM), could be detected at 520 nm with fluorescence excitation at 340 nm. The fluorescence detection limit of BDAM (100 fmol, S/N=3) was smaller than that of intact benzylamine (200 pmol, S/N=10, absorbance at 254 nm). Under mild acidic conditions of pH 5.0, 82% of benzylamine was regenerated from BDAM within a day. With this reagent, insulin was successfully derivatized and 56% of insulin was regenerated. (Keywords: Reversible, fluorescence derivatization, acid anhydride, amino group)

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