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

Abstract

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-thiotriphosphate) (ATP(7S)), 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 S'-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 ori-nitro compound (apparent ∊406nm= 9.1 X 103 M-1 cm-1). For caged ATP in the presence of 3 mM MgCl2, the 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 °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 °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 °C, and ionic strength 0.18 M. © 1988, American Chemical Society. All rights reserved.