Kinetics Study of the Photocleavage of (Coumarin-4-yl)methyl Esters

Abstract

Photolabile coumarinylmethyl esters of biomolecules (caged compounds) are new tools for studying spatial and time-dependent aspects of signal transduction in living cells. Herein we describe a fluoresence spectroscopic method for the determination of the rate constants of the photolysis steps of such caged compounds using (6.7-dimethoxycoumarin-4-yl)methyl diethyl phosphate (DMCM-DEP) and sodium (6,7-dimethoxycoumarin-4-yl)methyl sulfate (DMCM-S). DMCM-DEP and DMCM-S are caged compounds which photorelease a proton, the corresponding acid anion, and the strongly fluorescent alcohol DMCM-OH upon excitation. The results of stationary and time-resolved measurements of the photochemistry and the luminescence of both caged compounds indicate that DMCM-OH is produced already during the excitation pulse. The quantitative analysis of the data demonstrates that the first step of the reactionheterolytic bond cleavage of the coumarinylmethyl ester leading to the ion pair of a DMCM cation and an acid anionis very fast with a rate constant of k1 ≈ 2 × 1010 s-1. Recombination of the ion pair occurs with a rate constant of krec ≈ 2.3 × 109 s-1 and is about 10 times faster than the competing hydrolysis reaction of the DMCM cation yielding DMCM-OH and a proton. Thus, both caged compounds belong to the fastest phototriggers known.