7-羟基-3H-吩恶嗪-3-酮类荧光探针制备及过氧亚硝酸根离子检测和细胞荧光成像

Synthesis of 7-hydroxy-3H-phenoxazin-3-one–based fluorescent probes for peroxynitrite anion detection and cellular fluorescence imaging

摘要:

以7-羟基-3H-吩恶嗪-3-酮（试卤灵）和对甲氧基苯硫酚为原料，设计合成了两种荧光探针7-羟基-2-[(4-甲氧基苯基)硫基]-3H-吩恶嗪-3-酮（RSF-1）与7-羟基-1-[(4-甲氧基苯基)硫基]-3H-吩恶嗪-3-酮（RSF-2），将其用于过氧亚硝酸根阴离子（ONOO?）的检测，通过1HNMR和HRMS确证探针分子结构，采用细胞荧光成像实验，考察探针在活细胞和斑马鱼中ONOO?的荧光成像性能。结果表明，探针RSF-1荧光强度与ONOO?浓度（0~30 μmol/L）、探针RSF-2荧光强度与ONOO?浓度（0~3.6 μmol/L）均呈现良好的线性关系，两种探针对ONOO?的检测限分别为11和2.6 nmol/L。两种探针的硫原子在与ONOO?反应后，先被氧化为相应的亚砜，再一步转化为相应的砜，从而显著增强了荧光信号。RSF-2对ONOO?具有特异性荧光响应，不受活性氧/氮（H?O?, NO??, NO??等）、氨基酸、谷胱甘肽（GSH）、HSO??、H?S及SO?2?的干扰。RSF-2具有较低的细胞毒性，RAW264.7细胞与不同浓度（0~50 μmol/L）的RSF-2孵育12 h后的存活率均>84%。探针RSF-2可用于活细胞中外源性、内源性以及斑马鱼中ONOO?的成像分析。

Abstract:

Using 7-hydroxy-3H-phenoxazin-3-one (resorufin) and p-(methylthio)anisole as the starting material, two novel fluorescent probes 7-hydroxy-2-((4-methoxyphenyl)thio)-3H-phenoxazin-3-one (RSF-1) and 7-hydroxy-1-((4-methoxyphenyl)thio)-3H-phenoxazin-3-one （RSF-2）were designed and synthesized for the detection of peroxynitrite anion (ONOO?). The molecular structure of the probes was confirmed by 1HNMR and high-resolution mass spectrometry (HRMS). Cellular fluorescence imaging experiments assessed their performance for ONOO? imaging in living cells and zebrafish. The results demonstrate that the fluorescence intensity of both probes shows good linear relationships with ONOO? concentration (0~30 μmol/L for RSF-1 and 0~3.6 μmol/L for RSF-2). The detection limits for ONOO? were determined to be 11 nmol/L and 2.6 nmol/L for RSF-1 and RSF-2, respectively. Upon reaction with ONOO?, the sulfur atom in each probe is stepwise oxidized first to the corresponding sulfoxide and then to the sulfone, leading to a significant fluorescence enhancement. RSF-2 displays a specific fluorescence response to ONOO? unaffected by potential interferents such as reactive oxygen/nitrogen species (H?O?, NO??, NO??), amino acids, glutathione（GSH）, HSO??, H?S, or SO?2?. RSF-2 exhibits low cytotoxicity, with viability exceeding 84% for RAW264.7 cells incubated with probe concentrations ranging from 0 to 50 μmol/L for 12 hours. Consequently, probe RSF-2 proves suitable for imaging both exogenous and endogenous ONOO? in live cells and zebrafish.