Synthesis and Characterization of a Caged Receptor Ligand Suitable for Chemical Kinetic Investigations of the Glycine Receptor in the 3-μs Time Domain

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Abstract
Here we report the development and characterization of a new photolabile protecting group for the carboxyl group of neurotransmitters, 2-methoxy-5-nitrophenyl. The synthesis and characterization of a photolabile derivative of β-alanine, caged β-alanine, are described. β-Alanine can activate the glycine receptor, a major inhibitory receptor in the mammalian central nervous system; the 2-methoxy-5-nitrophenyl derivative of β-alanine combined with a laser-pulse photolysis method makes it possible to investigate the chemical kinetic mechanism of the receptor in the 3-μs time domain. The derivative is photolyzed by a laser pulse to release free β-alanine within 3 μs and with a product quantum yield of 0.2. In aqueous solution in the dark and at neutral pH, the compound is more stable, by a factor of ∼25, than the analogous derivative of glycine [Ramesh, D., Wieboldt, R., Niu, L., Carpenter, B. K., & Hess, G. P. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11074−11078]. 2-Methoxy-5-nitrophenyl-β-alanine hydrolyzes in aqueous solution at neutral pH with a t1/2 of approximately 1.5 h. Neither the 2-methoxy-5-nitrophenyl-β-alanine nor the 2-methoxy-5-nitrophenol photolysis side product activates, inhibits, or potentiates the response of glycine receptors in rat hippocampal neurons to glycine. Photolysis of 2-methoxy-5-nitrophenyl-β-alanine by irradiation with a 600-ns laser pulse at 333 nm releases β-alanine, which then activates glycine receptor-channels on neurons equilibrated with the caged compound, as detected by whole-cell current recording. Compared with the analogous derivative of glycine, in terms of quantum yield, photolysis rate, and stability, this new compound is not only a better 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.