title: A new photolabile precursor of glycine with improved properties: A tool for chemical kinetic investigations of the glycine receptor.
abstract: The synthesis and characterization of a new photolabile precursor of glycine (caged glycine) is described. The alpha-carboxyl group of glycine is covalently coupled to the alpha-carboxy-2-nitrobenzyl (alphaCNB) protecting group. Photolysis of the caged glycine with UV light produces free glycine. At 308 nm, the compound photolyzes with a quantum yield of 0.38. The absorption spectrum and the pH dependence of a transient absorption produced after laser-flash illumination are typical for aci-nitro intermediates of alphaCNB-protected compounds. The time constant for the major component of the aci-nitro intermediate decay ( approximately 84% of the total aci-nitro absorbance) was determined to be 7 micros at physiological pH. A minor component ( approximately 16%) decays with a rate constant of 170 micros. The compound does not activate or inhibit the alpha(1)-homomeric glycine receptor transiently expressed in HEK293 cells. After photolysis with a 10 ns pulse of 325 nm laser light, the glycine released from the caged compound activates glycine-mediated whole-cell currents in the same cells. The rise of these currents 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-exponential function over >85% of the total current. The rate constant of the current rise is about 2 orders of magnitude slower than the rate constant of caged glycine photolysis. Thermal hydrolysis of the alphaCNB-caged glycine takes place with a half-life of 15.6 h at physiological pH. The new caged glycine is the first in a series of photoprotected glycine derivatives that has the required properties for use with chemical kinetic methods for investigation 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 processes leading to channel opening of glycine receptor channels and the effects of mutations of the glycine receptor and of inhibitors on these processes.
authors.affiliation: Department of Molecular Biology and Genetics, 217 Biotechnology Building, Cornell University, Ithaca, New York 14853-2703, USA.
authors.forename: C
authors.initials: C
authors.lastname: Grewer
authors.forename: J
authors.initials: J
authors.lastname: Jäger
authors.forename: B K
authors.initials: BK
authors.lastname: Carpenter
authors.forename: G P
authors.initials: GP
authors.lastname: Hess
mesh_terms: D002460:Cell Line; D004355:Drug Stability; D005998:Glycine / Q000031:analogs & derivatives / Q000138:chemical synthesis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010648:Phenylacetates / Q000138:chemical synthesis; D010777:Photochemistry; D010782:Photolysis; D018009:Receptors, Glycine / Q000502:physiology*; D013997:Time Factors
chemical_list: D010648:Phenylacetates; D018009:Receptors, Glycine; C405350:alpha-carboxy-2-nitrobenzylglycine; D005998:Glycine