‘CAGED’ Compounds to Probe the Dynamics of Cellular Processes: Synthesis and Properties of some Novel Photosensitive P-2-Nitrobenzyl Esters of Nucleotides
Photochemical release of substrates or regulatory molecules is a developing technique that overcomes problems such as diffusional delays in the kinetic study of biological processes involving macromolecular assemblies. A wide range of phosphate esters such as nucleotides and nucleotide analogues can be alkylated on their phosphate groups with 1-(2-nitrophenyl)diazoethane and l-(3,4- dimethoxy-6-nitrophenyl)diazoethane. The phosphate esters so formed are generally biologically inert and may be photolyzed to starting material with near-UV (300-360 nm) irradiation. Flash photolysis of these ‘caged’ compounds with a pulsed laser or arc lamp releases up to several millimolar of a biologically active compound. Subsequent to photon absorption, dark reactions occur within the caged compounds before the active compound is released and measurement of the rate of these processes is necessary to determine the time resolution of the technique. P-l-(2-Nitrophenyl)ethyl esters of nucleoside 3′,5′-cyclic phosphates photolyze at 5 s∓1 at 21°C in aqueous solution at pH 7 and 11 M ionic strength. Below pH 7 the photolysis is acid catalyzed. P-l-(3,4-dimethoxy-6-nitrophenyl)ethyl esters of Pi and ATP photolyze more slowly than the corresponding P-l-(2-nitrophenyl)ethyl esters. The P-1-(3,4-dimethoxy-6-nitrophenyl)ethyl esters of cyclic nucleotides are unstable in water at pH 7. However the P-l-(3,4- dimethoxy-6-nitrobenzyl)esters of cyclic nucleotides appear to photolyze more rapidly than their P-1-(2-nitrobenzyl)ester counterparts, though with low quantum yield. The implications of these results for the photolysis mechanisms and the application of 2- nitrobenzyl phosphate esters in biological research is discussed.
KeywordsEthyl Ester Cyclic Nucleotide Phosphate Ester Photolysis Rate Cyclic Phosphate
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