Photolabile 1-(2-Nitrophenyl)ethyl Phosphate Esters of Adenine Nucleotide Analogues. Synthesis and Mechanism of Photolysis

Article abstract of DOI:10.1021/ja00229a036

A general method is described for preparing photolabile 1-(2-nitrophenyl)ethyl esters of phosphate and thiophosphate compounds.The method is based on selective alkylation of weakly ionizing phosphate groups by a new alkylating agent, 1-(2-nitrophenyl)diazoethane.ATP and the widely used structural analogues of ATP, 5'-adenylyl imidodiphosphate (ATP(β,γNH)) and adenosine 5'-(3-thiophsophate) (ATP(γS)), were alkylated on the terminal (γ) phosphate group.ATP(γS) was alkylated on oxygen or on sulfur in approximately equal amounts.Photolysis of P³-1-(2-nitrophenyl)ethyladenosine 5'-triphosphate, commonly called "caged ATP", was analyzed spectroscopically at pH values close to neutral in aqueous solvents by use of laser pulse photolysis.The kinetics of formation of the three products, ATP, 2-nitrosoacetophenone, and H⁺, were each monitored, as well as the kinetics of formation and decay of an intermediate presumed to be an aci-nitro compound (apparent ε_406nm = 9.1 x 10³ M^-1 cm^-1).For caged ATP in the presence of 3 mM MgCl2, and aci-nitro intermediate and H⁺ formed first at > 10⁵ s^-1 followed by the decay of the intermediate at 86 s^-1 at pH 7.1, 21 deg C, and ionic strength 0.18 M.ATP, monitored by a biochemical assay, and 2-nitrosoacetophenone, monitored by a characteristic absorption band at 740 nm, were formed simultaneously with the decay of the intermediate under all conditions tested.The rate of decay of the aci-nitro intermediate was therefore used as a measure of the rate of release of the nucleotide analogues from their photolabile precursors.At pH 7.1, 0.18 M ionic strength, and 21 deg C the rate constants ranged from 35 to 250 s^-1 and displayed a similar dependence on pH as caged ATP.The steady-state quantum yields of the 1-(2-nitrophenyl)ethyl phosphate esters were in the range 0.49 - 0.63.The deleterious effect of 2-nitrosoacetophenone on biological materials can be avoided by having thiols present.The reaction kinetics of dithiothreitol and 2-nitrosoacetophenone was described by a two-step process, the first step having a rate constant of 3.5 x 10³ M^-1 s^-1 and the second 45 s^-1 at pH 7.0, 21 deg C, and ionic strength 0.18 M.