3,5-Difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), a small molecule of green fluorescent protein (GFP)-like fluorophore, has been widely used for sensing a variety of biological molecules in living systems. This study aimed to investigate the metabolic pathways and metabolic kinetics of this fluorophore in liver preparations from human and six commonly used experimental animals. Preliminary study has demonstrated that DFHBI is very stable in phase I metabolic system but this agent can be readily converted to its O-glucuronide by mammalian UDP-glucuronosyltransferases (UGTs) in the presence of UDPGA. It was also found that DFHBI-O-glucuronidation strongly reduced the fluorescence response of DFHBI, owing to that DFHBI-O-glucuronide could not form a fluorescent complex with Broccoli aptamer. Reaction phenotyping assays and chemical inhibition assays revealed that UGT1A1 played a key role in DFHBI-O-glucuronidation in the human liver, while UGT1A3, UGT1A7, UGT1A8, UGT1A9 and UGT1A10 contributed to a lesser extent. Further investigation demonstrated that DFHBI could also be O-glucuronidated by liver microsomes from all tested animal species, including human, mouse, rat, rabbit, dog, minipig and cynomolgus monkey, but the metabolic kinetics and DFHBI-O-glucuronidation rates in the liver microsomes from various species were much varied. Collectively, our findings suggested that O-glucuronidation was the major metabolic pathway of DFHBI in mammals, while UGT1A1 played a crucial role in DFHBI-O-glucuronidation in the human body. All these findings would be very helpful for deep understanding the metabolic fates of DFHBI in living systems, and also suggested that the metabolic stability of this fluorophore should be improved in future for imaging applications in living systems.

3,5-二氟-4-羟基亚苄基咪唑啉酮（DFHBI）是绿色荧光蛋白（GFP）类荧光团的小分子，已被广泛用于感测生命系统中的各种生物分子。这项研究旨在研究人类和六种常用实验动物肝脏制剂中该荧光团的代谢途径和代谢动力学。初步研究表明，DFHBI在I期代谢系统中非常稳定，但是在存在UDPGA的情况下，该试剂可以很容易地被哺乳动物UDP-葡萄糖醛酸转移酶（UGT）转化为其O-葡萄糖醛酸。人们还发现，DFHBI- ö -glucuronidation强烈降低DFHBI的荧光响应，由于该DFHBI- ö-葡糖醛酸苷不能与西兰花适体形成荧光复合物。反应表型分析和化学抑制分析表明，UGT1A1在人肝中的DFHBI- O-葡萄糖醛酸苷化中起关键作用，而UGT1A3，UGT1A7，UGT1A8，UGT1A9和UGT1A10的贡献程度较小。证明DFHBI还可以进一步调查Ø -glucuronidated通过肝微粒从所有测试的动物物种，包括人类，小鼠，大鼠，兔，犬，小型猪和食蟹猴，但代谢动力学和DFHBI- Ø -glucuronidation率在肝脏来自各种物种的微粒体差异很大。总的来说，我们的发现表明O-葡萄糖醛酸化是DFHBI在哺乳动物中的主要代谢途径，而UGT1A1在人体内DFHBI- O-葡萄糖醛酸化中起关键作用。所有这些发现对于深入了解DFHBI在生物系统中的代谢命运将非常有帮助，并且还建议在未来的荧光成像在生物系统中的应用中应改善该荧光团的代谢稳定性。