The Study On The Synthesis Of Halotag Protein Targeted Fluorescent Probe For Nitric Oxide

Nitric oxide (NO) is an important biological signaling molecule which is related to diverse physiological and pathological processes in vivo. Fluorescence Imaging is a useful method for the investigation of NO. However, the ordinary diffusely distributed probes can detect the spatially averaged concentration of NO. To help study the biology and chemistry of NO intra-and intercellular, an ideal NO probes can be used to monitor the local, subcellular changes in NO levels. And the selective protein labeling approach such as HaloTag is a useful method. The paper introduces a novel HaloTag protein targeted fluorescent probe for detection and imaging of local NO.In this paper, we have prepared and improved the purification method of2-[2-(6-chloro-hexyloxy)-ethoxy]-ethylammonium, which is used as the HaloTag protein labeling reactive linker. Then6-carboxyfluorescein was synthesized and used to connect with the reactive linker to form diAcFAM ligand, which is used as the intracellular protein labeling probe. Bromoacetyl bromide was used to synthesize2-bromo-N-{2-[2-(6-chloro-hexyloxy)-ethoxy]-ethyl}-acetamide, which can be used as the linker of functional reporters contain amino group.Meanwhile,5,6-diaminofluorescein (DAF-2) and the diacetate (DAF-2DA) were synthesized as the fluorescent probes for NO. The substitution reaction between2-bromo-N-{2-[2-(6-chloro-hexyloxy)-ethoxy]-ethyl}-acetamide and DAF-2DA in DMF at room temperature with sodium iodide as catalyst and potassium carbonate as base afforded a mixture containing the desired product. HaloTag-DAF-2DA ligand was revealed by LC-MS analysis and obtained through preparative HPLC. The diacetate groups can be hydrolyzed by esterases after it permeate into cells. Then, the produced HaloTag-DAF-2can be immobilized by the preceding localized gene encode HaloTag protein and used for monitoring subcellular changes in NO levels. The emission of HaloTag-DAF-2was enhanced about85-fold after it was converted to HaloTag-DAF-2T. The compounds were characterized with1H NMR,13C NMR, HRMS, UV-vis absorption and fluorescence spectra.