Newly synthesized photolabile derivatives of glutamate, caged glutamate, that release free glutamate on a microsecond time scale after a pulse of UV laser light are described. 2-Nitrobenzyl derivatives were attached to the amino or carboxyl groups of glutamate. Substitution with a -COj group at the benzylic carbon accelerates the photolysis reaction when compared to -H and -CH3 substituents. y-O-(aCarboxy-2.nitrobenzyl)glutamate is stable at neutral pH. In 100 mM phosphate buffer at pH 7.0, the compound is photolyzed at 308 nm with a quantum product yield of 0.14.

The gamma-aminobutyric acid (GABA) receptor is an abundant neuronal receptor in the mammalian and invertebrate nervous systems and is associated with an inhibitory chloride ion channel. GABA is the endogenous neurotransmitter for the receptor and can trigger both fast activation and a reversible desensitization of the receptor. A series of photolabile amine-linked o-nitrobenzyl derivatives of GABA were synthesized that photolyze rapidly to release free GABA. The photochemical properties of the GABA precursors were determined; the compounds undergo rapid photolysis, initiated with UV irradiation at 308 nm, and release free GABA on a millisecond time scale. The pH of the photolysis medium affects both the quantum yield and the rate of photolysis. For example, the quantum yield observed for N-(alpha-carboxy-2-nitrobenzyl)-gamma-aminobutyric acid increases from 0.06 at pH 5.0 to 0.1 at pH 10.5, and the half-life for the photolytic reaction decreases from 1.0 to 2.5 ms in the same pH range. Photolysis of the compounds induces rapid onset of transmembrane ion currents in mouse cortical neurons. The potential of the new compounds for use in rapid chemical kinetic investigations of the neuronal GABA receptor is demonstrated.

Three photolabile precursors of glycine containing a photosensitive 2-nitrobenzyl moiety attached to the amino group have been synthesized. When exposed to ultraviolet radiation between 308 and 350 nm, the compounds photolyze to release glycine, an important inhibitory neurotransmitter in the central nervous system. The identification of glycine as a photolysis product was determined by two different methods: separation of the photolyzed sample by thin-layer chromatography followed by a reaction with ninhydrin, and recognition of derivatized glycine using the Waters pico-tag method in conjunction with high-performance liquid chromatography. The photolysis of these compounds at 22 degrees C has been investigated, and the rate of decay of a transient intermediate in the reaction, which is assumed to reflect product release, has been measured. For N-(alpha-carboxy-2-nitrobenzyl)glycine this decay rate was found to be 940 s-1 at pH 6.8 and 600 s-1 at pH 7.5. Additionally, this compound was found to exhibit biological activity upon photolysis; cultured mouse spinal cord cells containing neuronal glycine receptors were used to detect the glycine liberation. The approach adopted here is useful in demonstrating the utility of photolabile precursors of neurotransmitters that have the protecting group linked to the neurotransmitter through the amino group. The rapid photolysis of such compounds to release free neurotransmitter is valuable in gaining access to chemical kinetic studies of neurotransmitter receptors. Previously, such studies have been limited because the available methods for neurotransmitter delivery did not give a sufficiently high time resolution.

A series of photolabile o-nitrobenzyl derivatives of serotonin (caged serotonin) were synthesized: the amine-linked serotonin derivatives N-(2-nitrobenzyl) serotonin (Bz-5HT) and N-(alpha-carboxy-2-nitrobenzyl) serotonin (N-CNB-5HT), and O-alpha-carboxy-2-nitrobenzyl) serotonin (O-CNB-5HT), which has the caging group attached to the phenolic OH group. All the derivatives released free serotonin when excited by 308-nm or 337-nm laser pulses. The time constant of serotonin release from N-CNB-5HT was 1. 2 ms, with a quantum yield of 0.08. This is too slow for rapid chemical kinetic measurements. O-CNB-5HT is suitable for transient kinetic investigations of the serotonin 5-HT(3) receptor. It released serotonin with a time constant of 16 micros and a quantum yield of 0.03. The biological properties of O-CNB-5HT were evaluated, and the applicability of the compound for kinetic studies of the 5-HT(3) receptor was demonstrated. O-CNB-5HT does not activate the 5-HT(3) receptor by itself, nor does it modulate the response of a cell when co-applied with serotonin. When irradiated with a 337-nm laser pulse, O-CNB-5HT released free serotonin that evoked 5-HT(3) receptor-mediated whole-cell currents in NIE-115 mouse neuroblastoma cells.

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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 ...

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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.