Highly selective determination of bioactive compounds based on intra molecular fluorescence resonance energy transfer detection.

建立了高选择性测定酪氨酸、色氨酸及其相关化合物(L多巴、儿茶胺、5-羟色胺等)的高效液相色谱方法。用市售的胺类荧光试剂按常规方法进行柱前衍生化。每个衍生物都提供了分子内荧光共振能量转移(FRET)，从酪氨酰或色氨酸部分(供体)到标记的荧光团(受体)；受体荧光在供体的激发下观察到280 nm。这些衍生物在反相柱上分离，然后通过监测它们的FRET有效地进行检测。通过对15种荧光试剂的筛选研究，筛选出ο-邻苯二甲醛(含2-巯基乙醇)、丹磺酰氯、4，5-二甲氧基-ο-邻苯二甲醛和DAMTC为高效荧光试剂，ο-邻苯二甲醛是一种高选择性、灵敏、实用的荧光试剂。与以往检测化合物的天然荧光或受体的典型荧光相比，FRET检测方法具有很高的选择性和灵敏度(检出限为20-70fmol)。该方法无需任何繁琐的前处理，即可成功地应用于人体尿样的测定。

I have developed highly selective HPLC method for the determination of tyrosine, tryptophan and their related compounds (L-DOPA, catechdlamines, 5-hydroxytryptamine, etc.). The compounds were precolumn-derivatized with a commercially available fluorogenic reagent for amines by usual manner. Each derivative afforded intramolecular fluorescence resonance energy transfer (FRET) from the tyrosyl or tryptophoryl moiety (donor) to the labeled fluorophore (acceptor) ; the acceptor fluorescence was observed with the excitation of the donor at 280 run. The derivatives were separated on a reversed-phase column and then effectively detected by monitoring their FRET. Through the screening study of 15 fluorogenic reagents, ο-phthalaldehyde (with 2-mercaptoethanol), Dansyl chloride, 4, 5-dimetholy-ο-phthalaldehyde and DAMTC afforded efficient FRET ; ο-phthalaldehyde was selected as a highy selective, sensitive and practical reagent. The FRET detection method was highly selective and sensitive (limits of detection ; 20-70 fmol on clumn) by comparison with the previous methods detecting native fluorescence of the compounds or typical fluorescence of the acceptor. This method could be successfully applied to human urine sample without any tedious pretre atment.