A new photolabile precursor of glycine with improved properties: A tool for chemical kinetic investigations of the glycine receptor

The synthesis and characterization of a new photolabile precursor of glycin e (caged glycine) is described. The alpha-carboxyl group of glycine is cova lently coupled to the alpha-carboxy-2-nitrobenzyl (alpha CNB) protecting gr oup. Photolysis of the caged glycine with UV light produces free glycine. A t 308 nm, the compound photolyzes with a quantum yield of 0.38. The absorpt ion spectrum and the pH dependence of a transient absorption produced after laser-flash illumination are typical for aci-nitro intermediates of alpha CNB-protected compounds. The time constant for the major component of the a ci-nitro intermediate decay (approximate to 84% of the total aci-nitro abso rbance) was determined to be 7 mu s at physiological pH. A minor component (approximate to 16%) decays with a rate constant of 170 mu s. The compound does not activate or inhibit the alpha(1)-homomeric glycine receptor transi ently expressed in HEK293 cells. After photolysis with a 10 ns pulse of 325 nm laser light, the glycine released from the caged compound activates gly cine-mediated whole-cell currents in the same cells. The rise of these curr ents can be measured in a time-resolved fashion and occurs on a millisecond to sub-millisecond time scale. It can be described with a single-exponenti al function over > 85% of the total current. The rate constant of the curre nt rise is about 2 orders of magnitude slower than the rate constant of cag ed glycine photolysis. Thermal hydrolysis of the alpha CNB-caged glycine ta kes place with a half-life of 15.6 h at physiological pH. The new caged gly cine is the first in a series of photoprotected glycine derivatives that ha s the required properties for use with chemical kinetic methods for investi gation of glycine-activated cell surface receptors. Photolysis is rapid and efficient with respect to the receptor reactions to be studied; hydrolysis in aqueous solution is sufficiently slow, and the compound is biologically inert. It will, therefore, be a useful tool for investigation of the proce sses leading to channel opening of glycine receptor channels and the effect s of mutations of the glycine receptor and of inhibitors on these processes .