Hydrolysis of phosphotriesters: Determination of transition states in parallel reactions by heavy-atom isotope effects

The remote label method was used to measure primary and secondary O-18 isotope effects in the alkaline hydrolysis of O,O-diethylphosphorylcholine iodide (DEPC) and the primary O-18 effect in the alkaline hydrolysis of O,O-diethyl-m-nitrobenzyl phosphate (DEmNBP). Both the leaving group of interest (choline or m-nitrobenzyl alcohol) and ethanol can be ejected during hydrolysis due to the similarity of their pK values. The heavy-atom isotope effects were measured by isotope ratio mass spectrometry. Parallel reaction and incomplete labeling corrections were made for both systems. DEPC has a primary O-18 isotope effect of 1.041 +/- 0.003 and a secondary O-18 isotope effect of 1.033 +/- 0.002. The primary O-18 isotope effect for DEmNBP was 1.052 +/- 0.003. These large effects suggest a highly associative transition state in which the nucleophile approaches very close to the phosphorus atom to eject the leaving group. The large values are also indicative of a large compression, or general movement, on the reaction coordinate.