Synthesis and photochemistry of photolabile N-glycine derivatives and effects of one on the glycine receptor

Abstract

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.

The free radical site in pea seedling copper amine oxidase probed by resonance Raman spectroscopy and generated by photolysis of caged substrate

“…Measurements over the range of 15-55°C gave linear Arrhenius plots and activation energies of 40 + 2 kJ mol -~ for both compounds. These uncaging rates are approximately an order of magnitude faster than those for N-(2-nitrobenzyl) cages of glycine [14], glutamine [15] and 7-aminobutyric acid [16], but slower than for the uncaging of N-(1-(2-nitrophenyl)ethyl)carbamoylcholine iodide [13]. The activation energy for decay of the aci-nitro intermediates of I and II is slightly smaller than the value of 55 kJ mol -] measured for caged ATP [11] under similar conditions.…”

“…2-Nitrobenzyl cages of benzylamine and phenethylamine were prepared by reaction of the amine with 2-nitrobenzaldehyde at room temperature for 12 h, followed by reduction with NaBH 4 at 0°C according to the procedures described by Billington et al [14]. The products were extracted and recrystallised from methanol as the HCI salts.…”

“…Copper containing amine oxidases (EC 1.4.3.6) have been shown to contain the quinone of TOPA (2,4,5-trihydroxyphenylalanine) as an active site cofactor [14]. This cofactor occurs in amine oxidases from yeasts, bacteria, plants and mammals.…”

“…'Caged' compounds have a functional group modified with a suitable photoremovable protecting or caging group, such as the 2-nitrophenyl substituent [13]. Rates of uncaging of 2-nitrobenzyl-substituted molecules, including amines, via aci-nitro intermediates vary widely [13][14][15][16] but in some cases occur on a sub-millisecond timescale, enabling kinetic measurements to be undertaken on timescales similar to or faster than those using the stopped flow technique. We describe steady-state resonance Raman spectroscopy of the free radical site in pea seedling copper amine oxidase (PSAO).…”

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

“…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.…”