SYNTHESIS AND CHARACTERIZATION OF A CAGED RECEPTOR-LIGAND SUITABLE FOR CHEMICAL KINETIC INVESTIGATIONS OF THE GLYCINE RECEPTOR IN THE 3-MU-S TIME-DOMAIN

Here we report the development and characterization of a new photolabi le protecting group for the carboxyl group of neurotransmitters, 2-met hoxy-5-nitrophenyl. The synthesis and characterization of a photolabil e derivative of beta-alanine, caged beta-alanine, are described. beta- Alanine can-activate the glycine receptor, a major inhibitory receptor in the mammalian central nervous system; the 2-methoxy-5-nitrophenyl derivative of beta-alanine combined with a laser-pulse photolysis meth od makes it possible to investigate the chemical kinetic mechanism of the receptor in the 3-mu s time domain. The derivative is photolyzed b y a laser pulse to release free beta-alanine within 3 mu s and with a product quantum yield of 0.2. In aqueous solution in the dark and at n eutral pH, the compound is more stable, by a factor of similar to 25, than the analogous derivative of glycine [Ramesh, D., Wieboldt, R., Ni u, L., Carpenter, B. K., & Hess, G. P. (1993) Proc. Natl. Acad, Sci, U .S.A. 90, 11074-11078]. 2-Methoxy-5-nitrophenyl-beta-alanine hydrolyze s in aqueous solution at neutral pH with a t(1/2) of approximately 1.5 h. Neither the 2-methoxy-5-nitrophenyl-beta-alanine nor the 2-methoxy -5-nitrophenol photolysis side product activates, inhibits, or potenti ates the response of glycine receptors in rat hippocampal neurons to g lycine. Photolysis of 2-methoxy-5-nitrophenyl-beta-alanine by irradiat ion with a 600-ns laser pulse at 333 nm releases beta-alanine, which t hen activates glycine receptor-channels on neurons equilibrated with t he caged compound, as detected by whole-cell current recording. Compar ed with the analogous derivative of glycine, in terms of quantum yield , photolysis rate, and stability, this new compound is nut only a bett er candidate for use in chemical kinetic investigations of the glycine receptor, but can also be used in determining the location of glycine receptors in neuronal cells.