Preparation and properties of S-nitroso-L-cysteine ethyl ester, an intracellular nitrosating agent

In this report, a protocol for the preparation of the hydrochloride of S-nitroso-L-cysteine ethyl ester (SNCEE·HCl; 2) is presented. The synthesis of 2 has been targeted because S-nitroso-L-cysteine (SNC; 2b), which is extensively used for trans-S-nitrosation of thiol-containing proteins, has a limited ability of crossing cellular membranes. The nitrosothiol 2 was prepared via direct S-nitrosation of the hydrochloride of L-cysteine ethyl ester (CEE·HCl; 1a) with ethyl nitrite. 2 is relatively stable in crystal form and when neutralized to SNCEE (2a) in aqueous solutions treated with chelators of metal ions. Traces of metal ions, however, triggered the decomposition of 2a to nitric oxide and a S-centered radical, which were detected by ESR spectrometry. In contrast to 2b, 2a is a lipophilic compound that was taken up by human neutrophils. The latter process was paralleled by inhibition of then NADPH oxidase-dependent generation of superoxide anion radicals, presumably via reaction(s) of intracellular trans-S-nitrosation. Intracellular accumulation of S-nitrosothiols was observed with 2a but not with 2b. It is expected that the use of 2a will be advantageous when intracellular reactions of trans-S-nitrosation are to be studied.