The reaction of S-nitroso-N-acetyl-d,l-penicillamine (SNAP) with the angiotensin converting enzyme inhibitor, captopril—mechanism of transnitrosation

Abstract

Kinetic studies involving the use of both stopped-flow and diode array spectrophotometers, show that the reaction between SNAP and captopril in the presence of the metal ion sequestering agent, EDTA, occurs in two well-defined stages. The first stage is a fast reaction while the second stage is slow. The first stage has been postulated to be transnitrosation, and the second stage involves the decay of the newly formed RSNO to effect nitric oxide (NO) release. Both stages are found to be dependent on captopril and H⁺ concentration. The rates of the transnitrosation increased drastically with increasing pH in the first stage, signifying that the deprotonated form of captopril is the more reactive species. In the case of the second stage the variation in pH showed an increase in rate up to pH 8 after which the rate remained unchanged. Both stages were clearly distinguishable and easily monitored separately. Transnitrosation is a reversible reaction with the tendency for the equilibrium to break down at high thiol concentration. Second-order rate constants were calculated based on the following derived expressions: −d[SNAP]/dt = k_f((K_SHCapSH[CapSH]_t)/(K_SHCapSH + [H⁺]))[SNAP]. k_f is the second-order rate constant for the forward reaction of the reversible transnitrosation process. At 37 °C, k_f = 785 ± 14 M⁻¹ s⁻¹, activation parameters ΔH_f^‡ = 49 ± 2 kJ mol⁻¹, ΔS_f^‡ = −32 ± 2 J K⁻¹ mol⁻¹. The activation parameters demonstrate the associative nature of the transnitrosation mechanism. The second stage has been found to be very complex, as a variety of nitrogen products form as predicted before. However, the following expression was derived from the initial kinetic data: rate = k₁K[SNOCap][CapS⁻]/(K[CapS⁻] + 1) to give k₁ = 13.3 ± 0.4 × 10⁻⁴ s⁻¹ and K = 5.59 ± 0.53 × 10⁴ M⁻¹, at 37 °C, where k₁ is the first-order rate constant for the decay of the intermediate formed during the reaction between SNOCap and the remaining excess CapSH present at the end of the first stage reaction. Activation parameters are ΔH₁^‡ = 37 ± 1 kJ mol⁻¹, ΔS₁^‡ = −181 ± 44 J K⁻¹ mol⁻¹.