Synthesis and Characterization of HNO Donors

HPLC and mass spectrometry–grade acetonitrile (ACN), and a bicinchoninic acid assay kit were purchased from Thermo Fisher Scientific. DMSO-d₆, D₂O, and ¹⁵N-labeled hydroxylamine hydrochloride were purchased from Cambridge Isotope Laboratories. DTT, β-mercaptoethanol, and 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) were of the highest purity available and obtained from Sigma-Aldrich. The syntheses of the HNO donor, Angeli’s salt (Na₂N₂O₃, AS) and ¹⁵N-labeled Angeli’s salt (¹⁵N-AS), were performed as previously described (Bonner and Ravid, 1975 (link); Hughes and Cammack, 1999 (link)). The HNO donor, 2-methylsulfonyl benzene N-hydroxy sulfonamide (2-methylsulfonyl Piloty’s acid, 2-MSPA; Toscano et al., 2011 ; Sabbah et al., 2013 (link); Zhu et al., 2015 (link)), the ¹⁵N-labeled HNO donor, 2-methylsulfonyl benzene ¹⁵N-hydroxy sulfonamide (¹⁵N-2-MSPA; Toscano et al., 2011 ; Sabbah et al., 2013 (link); Zhu et al., 2015 (link)), and the donor byproduct, 2-(methylsulfonyl)benzenesulfinic acid (2-MSSA), were gifts from Cardioxyl Pharmaceuticals. Dodecylphosphocholine (DPC) was purchased from Avanti Polar Lipids. Milli Q water was used for all purifications and experiments.