二氨基荧光素单取代功能化研究

Mono-substitution of Diaminofluorescein

一氧化氮(NO)是生物体内一种重要的信号分子。二氨基荧光素是一种能够有效检测NO的荧光探针化合物。通过结合"tagging"技术对二氨基荧光素进行单功能化可提高其NO探测的靶向性和空间分辨率。本文针对二氨基荧光素和HaloTag结合的问题,以邻苯二胺为模型化合物对反应条件进行优化,并用二氨基荧光素二乙酸酯验证了该系列反应条件的合理性,以高效液相色谱(HPLC)和液相色谱-质谱联用(LC-MS)分析了取代产物的组成和含量。实验结果表明,溴乙酰溴是二氨基荧光素和HaloTag之间最合适的连接子。采用优化的反应条件,目标单取代产物的产率可以达到34.8%。本研究为邻二氨基类一氧化氮荧光探针的单烷基取代功能化的应用奠定了基础。

Nitric oxide （NO） is an important biological signaling molecule. Diaminofluorescein （DAF- 2） is one of the most effective NO probes. Linking DAF-2 with other functionalities through tagging technique can greatly enhance its targeting effect and spatial sensitivity in detection of NO in vivo. In this work, ortho-aminobenzene is chosen as the model compound of DAF-2 to study the synthetic strategy of mono-substitution. Diacetyl ester of DAF-2 is used to verify the optimized reaction condition. High performance liquid chromatography （HPLC） and liquid chromatography-mass spectroscopy （LC-MS） were employed to determine the composition and content of substituted product. Results show that bromoacetyl bromide is the best linker between DAF-2 and HaloTag. Mono-HaloTag substituted DAF 2 is obtained with a yield of 34.8 %. This study paves a way for development and application of mono-substituted ortho- diamino type fluorescence probes for NO.