Photolysis of a protecting group for the carboxyl function of neurotransmitters within 3 microseconds and with product quantum yield of 0.2.

Abstract

The synthesis of a photosensitive blocking group for the carboxyl function of neurotransmitters, in this case glycine, is reported. The compound, 2-methoxy-5-nitrophenyl glycine ester (caged glycine), is photolyzed by a laser pulse at 308 or 337 nm within 3 ps and with a product quantum yield of 0.2. The compound is hydrolyzed in water with a time constant i of 6.1 min at pH 7.1 and 3 hr at pH 4.0. Mouse cerebral cortical neurons containing glycine receptors were used in biological assays. A cell-flow device, in which solutions of caged glycine at pH 4.0 were mixed with buffer to give a final pH of 7.1, was used to equilibrate the compound with receptors on the cell surface. Neither the caged compound nor the 2-methoxy-5-nitrophenol photolysis product affected the glycine receptors or modified their response to glycine.When cells equilibrated with caged glycine are irradiated by a laser pulse at 337 nm, glycine receptor channels are opened, as detected in whole-ceOl current recordings. The approach described may be used in the synthesis and characterization of photolabile precursors of neurotransmitters and other compounds that contain carboxyl groups and for kinetic investigations ofneurotransmitter receptors in central nervous system cells in the microsecond time domain.o-Nitrobenzyl derivatives have been used as photoremovable blocking reagents for a variety of functional groups in organic chemistry (1)(2)(3). Use of the o-nitrobenzyl blocking group for derivatives of ATP (4, 5), inositol polyphosphate (6), calcium chelators (7, 8), amino acids (9, 10), and proton chelators (11) Thus, it became possible to equilibrate receptors on a cell surface with caged carbamoylcholine, liberate carbamoylcholine by a laser pulse within about 120 ,us, and investigate the kinetics of the formation of acetylcholine receptorchannels on a microsecond time scale. However, photolysis of analogous N-caged derivatives of y-aminobutyric acid (GABA) (19) and glycine (20), which activate specific inhibitory receptors in the central nervous system, occurs in the millisecond time region and with low product quantum yield (<0.05). Extension of our approach for making chemical kinetic measurements in the microsecond-to-millisecond time region to the receptors for these central nervous system neurotransmitters required more suitable caged compounds. The report of photochemical hydrolysis of nitrophenyl acetates by Kuzmic et al. (21) suggested a new type of photosensitive blocking group for the carboxyl function of amino acids, including glycine and GABA and the excitatory neurotransmitters glutamic acid and N-methyl-D-aspartic acid (NMDA). Here we report the synthesis of 2-methoxy-5-nitrophenyl glycine ester and describe its photolysis, induced by a pulse oflight at either 308 or 337 nm, within 3 ,s and with a product quantum yield of 0.2. We also show that both the caged compound and the photolysis product, 2-methoxy-5-nitrophenol, are biologically inert when tested with mouse cortical neurons containing glycine receptors, and ...

Mechanisms of photoswitch conjugation and light activation of an ionotropic glutamate receptor

“…A major advance has been the development of photolysable cages for soluble ligands (2). The caged ligand is allowed to slowly diffuse into tissue in its inert form, and a powerful light flash cleaves a photolabile protecting group, releasing the active ligand rapidly (within microseconds) (3,4). This provides fast on-rates that are complemented with reasonably fast off-rates, which depend on native binding affinity, diffusion, sequestration, and breakdown.…”

Synthesis, Photophysical, Photochemical and Biological Properties of Caged GABA, 4‐[[(2H‐1‐Benzopyran‐2‐one‐7‐amino‐4‐methoxy) carbonyl] amino] Butanoic Acid¶

“…Storage of stock solution did not seem to affect the ability to produce responses on uncaging, as we observed no obvious differences in response amplitudes between using fresh and stored stock solutions (fresh, 125‐160 pA; 1 day old, 100‐450 pA; 11 day old, 40‐700 pA). Finally, our recordings also indicate that BC204 is also stable at physiological pH, a basic requirement for using caged compounds in longer experiments (34). In our recordings lasting up to 1 h, we found no indication for spontaneous degradation of BC204 in ACSF at pH 7.4, allowing us to reuse BC204‐containing ACSF in another slice or to keep a slice in BC204‐containing ACSF for 2 h before starting another recording.…”

{7‐[Bis(carboxymethyl)amino]coumarin‐4‐yl}methoxycarbonyl Derivatives for Photorelease of Carboxylic Acids, Alcohols/Phenols, Thioalcohols/Thiophenols, and Amines

“…Hydrolysis of 1 b is dependent upon pH. As expected [20] the compound is more stable at lower pH. We found that 17 % of 1 b was hydrolysed in citrate/phosphate buffer at pH 4.0 within 24 h. Because hydrolysis in the dark is often a serious problem it seems that 1 b is less suitable as caged compound and requires some care to minimize contamination by free amino acid.…”