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

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 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 method makes it possible to investigate the chemical kinetic mechanism of the receptor in the 3-microseconds time domain. The derivative is photolyzed by a laser pulse to release free beta-alanine within 3 microseconds 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 approximately 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-beta-alanine hydrolyzes in aqueous solution at neutral pH with a t1/2 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 potentiates the response of glycine receptors in rat hippocampal neurons to glycine. Photolysis of 2-methoxy-5-nitrophenyl-beta-alanine by irradiation with a 600-ns laser pulse at 333 nm releases beta-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.

Ncm, a Photolabile Group for Preparation of Caged Molecules: Synthesis and Biological Application

“…A plausible explanation is neighboring group participation: The α-amino/ammonio group can perform intramolecular general acid-base catalysis and can stabilize the tetrahedral intermediate resulting from nucleophilic attack on the carbonyl [ 36 , 43 ]. This view rationalizes the observation that β-amino esters hydrolyze more slowly than their α counterparts [ 44 ], and that O -Ncm-Gly hydrolyzes more quickly than γ- O -Ncm-Glu.…”

Identification of chemical synapses in the pharynx of Caenorhabditis elegans

“…This information is expected to be basic to an understanding of how a circuit of cells communicating by chemical signals controls the rhyth- mical contractions of a muscle like the pharynx. The caged neurotransmitters required for these studies can be synthesized using published procedures (14,16,21,24,25) or can be obtained from Molecular Probes.…”

“…Additionally, using caged neurotransmitters (14,16,20,21), one can obtain spatial resolution regarding the location of receptors in neurons and muscle cells (reviewed in refs. 22 and 23) because the laser light used for photolysis can be focused to a spot with a size on the order of micrometers, and the photolysis of the caged neurotransmitters occurs in the microsecond time region (14,16,21,24,25). Laser-pulse photolysis of caged neurotransmitters, therefore, provides both high temporal (reviewed in ref.…”

Achieving synaptically relevant pulses of neurotransmitter using PDMS microfluidics

“…In contrast to photoactivation of “caged” neurotransmitter (Niu et al, 1996), this method does not require use of expensive reagents or radiation sources, is not constrained by the existing library of photoactivatable compounds, and most importantly, provides better control over the rate of neurotransmitter application and washout. Solution switching is typically accomplished by reversibly translating parallel control and neurotransmitter-containing solution streams generated from an array of glass capillary tubing across stationary cells or excised membrane patches.…”