Dehydrogenation of coelenterazine to dehydrocoelenterazine using molecular sieves
Mitsuhiro Nakamura1, Kazuya Nakayama1, Kazuo Matsuda2
1Graduate School of Technology, Industrial and Social Sciences, Tokushima University, Tokushima, Japan.
Abstract:
Coelenterazine (CTZ) and dehydrocoelenterazine (dCTZ, a dehydrogenated form of CTZ) were first identified in the liver of the luminescent squid Watasenia scintillans. In this report, we demonstrate for the first time that CTZ can be successfully converted to dCTZ using molecular sieves (MS4A/Na+, MS13X/Na+, MS5A/Ca2+, and MS3A/K+) in isopropyl alcohol at 37 °C for 3 h. The conversion efficiency to dCTZ followed the order: MS4A/Na+ = MS13X/Na+ > MS5A/Ca2+ > MS3A/K+. Lower efficiencies observed with modified MS4A (TMS-MS4A, NH4+-MS4A, and H+-MS4A) suggest that cations in the molecular sieves play a crucial role in the dehydrogenation reaction of CTZ. Notably, MS4A recovered from the reaction mixture could be reused without thermal regeneration for dehydration. In purified dCTZ, no residual CTZ was detected using the luciferin-luciferase reaction, and dCTZ can be used as a substrate in luminescence assays to determine enzymatic conversion activity to CTZ.
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