Photo-cleaving and photo-bleaching quantum yields of coumarin-caged luciferin

We applied quantitative bioluminescence experiments to evaluate the photo-cleaving quantum yields and cross sections of coumarin-caged luciferin quantitatively. We synthesized 112 mg of 6’-[(7-diethylaminocoumarin-4-yl)methoxy]-D-luciferin and determined its purity as 94%. It was found that the photo-cleaving quantum yield of coumarin-caged luciferin for 325-nm irradiation is 5.41×10−4 and for 405-nm irradiation is 6.27×10−5, and that the photo-bleaching quantum yield of coumarin-caged luciferin for 325-nm irradiation is 3.87×10−3 and for 405-nm irradiation is 5.83×10−4. Both photo-cleaving and photo-bleaching quantum yields of coumarin-caged luciferin with 325-nm irradiation are 10 times larger than those with 405-nm irradiation. These 10-fold differences were unexpectedly large, and indicate that the photo-cleavage and photo-bleaching occur not after the relaxation process from the second and third excited state to the first excited state but during the relaxation, which corresponds to the non-equilibrium situation.