| Nitroxyl (HNO), a nitrogen monoxide with distinct chemistry and biology, has gained interest as a potential treatment of congestive heart failure through the ability of the HNO donor, Angeli's salt (AS), to evoke positive inotropic effects in canine cardiac muscle. The release of nitrite during decomposition limits the use of AS requiring other HNO sources. Acyloxy nitroso compounds hydrolyze to HNO, while the rate of decomposition of these compounds can change through variation of the ester portion of the molecule. A number of cyclohexanone acyloxy nitroso compounds that contain different R groups on the ester that include a t-butyl group to increase stability, long side chains and double bonds to increase lipophilicity, and oxygen in the ring to increase solubility , were synthesized and characterized as HNO donors.;The greatest variation of HNO release came from 1-nitrosocylohexyl acetate, 1-nitrosocyclohexyl pivalate and 1-nitrosocyclohexyl trifluoroacetate, where under buffered conditions, the trifluoroacetate is a fast HNO donor, the acetate is a slow HNO donor, and the pivalate does not release HNO. Ultraviolet-visible spectroscopy and mass spectrometry show the hydrolysis rate depends on pH and ester group structure with the observed rate being trifluoroacetate > acetate > pivalate. Under all conditions, the trifluoroacetate rapidly hydrolyzes to HNO. A combination of spectroscopic, kinetic and product studies show that addition of thiols increases the decomposition rate of the acetate and pivalate leading to hydrolysis and HNO. Under conditions that favor thiolates, the thiolate directly reacts with the nitroso group yielding oximes without HNO formation. Biologically, the trifluoroacetate behaves like AS demonstrating thiol-sensitive nitric oxide-mediated soluble guanylate cyclase-dependent vasorelaxation, suggesting HNO-mediated vasorelaxation. The slow HNO donor, the acetate, demonstrates inotropic properties as well as weak thiol-insensitive vasorelaxation, indicating HNO release kinetics determine HNO bioavailability and activity. These results show that acyloxy nitroso compounds represent new HNO donors capable of vasorelaxation depending on HNO release kinetics.;To increase the bioavailability of acyloxy nitroso compounds the synthesis of the water-soluble 4-nitrosotetrahydro-2H-pyran-4-yl acetate and 4-nitrosotetrahydro-2H-pyran-4-yl pivalate allows for pig liver esterase (PLE)-catalysis increasing the rate of decomposition and HNO release. The pivalate derivative does not release HNO, but the addition of PLE catalyzes hydrolysis (t1/2 = 39 min) and HNO formation (65% after 30 minutes). In the presence of PLE, this compound converts metmyoglobin (Mb) to iron nitrosyl Mb and oxyMb to metMb indicating these compounds only react with heme proteins as HNO donors. The pivalate in the presence and the absence of PLE inhibits aldehyde dehydrogenase (ALDH) with IC50 values of 3.5 and 3.3 microM, respectively, in a time-dependent manner. Reversibility assays reveal reversible inhibition of ALDH in absence of PLE and partially irreversible inhibition with PLE. Liquid chromatography-mass spectrometry (LC-MS) reveals formation of a disulfide upon incubation of an ALDH peptide without PLE and a mixture of disulfide and sulfinamide in the presence of PLE. A dehydroalanine residue forms upon incubation of this peptide with excess AS. These results identify acyloxy nitroso compounds as unique HNO donors capable of thiol modification through direct electrophilic reaction or HNO release, and will assist in the level of knowledge that is crucial for understanding the reactions of HNO with thiol and heme containing proteins, which are specific for treatment of heart failure. |
