Original Article

Discovery of a novel calcium-sensitive fluorescent probe for α-ketoglutarate

Authors: GAN Lin-lin1,2, CHEN Lin-hai1,2, NAN Fa-jun1,2
1 Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
2 University of Chinese Academy of Sciences, Beijing 100049, China
DOI: 10.1038/aps.2017.75
Received: 27 February 2017
Accepted: 28 March 2017
Advance online: 17 July 2017


α-Ketoglutarate (α-KG), a pivotal metabolite in energy metabolism, has been implicated in nonalcoholic fatty liver disease (NAFLD) and several cancers. It is recently proposed that plasma α-KG is a surrogate biomarker of NAFLD. Here, we report the development of a novel “turn-on” chemosensor for α-KG that contains a coumarin moiety as a fluorophore. Using benzothiazole-coumarin (BTC) as inspiration, we designed a probe for calcium ion recognition that possesses a unique fluorophore compared with previously reported probes for α-KG measurement. This chemosensor is based on the specific Schiff base reaction and the calcium ion recognition property of the widely used calcium indicator BTC. The probe was synthesized, and a series of parallel experiments were conducted to optimize the chemical recognition process. Compared to the initial weak fluorescence, a remarkable 7.6-fold enhancement in fluorescence intensity (I/I0 at 495 nm) was observed for the conditions in which the probe (1 μmol/L), α-KG (50 μmol/L), and Ca2+ (100 μmol/L) were incubated at 30 °C in EtOH. The probe displayed good selectivity for α-KG even in an environment with an abundance of amino acids and other interfering species such as glutaric acid. We determined that the quantitative detection range of α-KG in EtOH was between 5 and 50 μmol/L. Finally, probe in serum loaded with α-KG (10 mmol/L) showed a 7.4-fold fluorescence enhancement. In summary, a novel probe for detecting the biomarker α-KG through a typical Schiff base reaction has been discovered. With further optimization, this probe may be a good alternative for detecting the physiological metabolite α-KG.
Keywords: nonalcoholic fatty liver disease; biomarker; α-ketoglutarate; fluorescent probe; calcium-sensitive; benzothiazole-coumarin