Chiral deaza-coelenterazine analogs for probing a substrate-binding site in the Ca2+-binding photoprotein aequorin

The Ca 2+ -binding photoprotein aequorin is a complex of apoAequorin (apoprotein) and ( S )-2-peroxycoelenterazine. Aequorin can be regenerated by the incubation of apoAequorin with coelenterazine and molecular oxygen (O 2 ). In this study, to investigate the molecular recognition of apoAequorin for coelenterazine using chemical probes, the chiral deaza-analogs of ( S )- and ( R )-deaza-CTZ (daCTZ) for coelenterazine and of ( S )-2- and ( R )-2-hydroxymethyl-deaza-CTZ (HM-daCTZ) for 2-peroxycoelenterazine were efficiently prepared by the improvement method. The chiral deaza-analogs of ( S )-daCTZ and ( S )-HM-daCTZ selectively inhibited the regeneration step to aequorin by binding the catalytic site of coelenterazine in the apoAequorin molecule. The crystal structures of the apoAequorin complexes with ( S )-daCTZ and ( S )-HM-daCTZ were determined, suggesting that the hydroxy moiety at the C6-hydroxyphenyl group and the carbonyl moiety of the imidazopyrazinone ring in coelenterazine are essential to bind the apoAequorin molecule through hydrogen bonding. Therefore, the chiral deaza-analogs of coelenterazine can be used as a probe to study the interaction between coelenterazine and the related proteins including photoprotein, luciferase, and coelenterazine-binding protein.