17 O NMR spectroscopy-assisted in vitro bioactivity studies of the intermediates formed via Na 2 S and RSNO cross-linking reactions.

The cross-linking reaction between sulfide and S -nitrosothiol moieties has been intensively investigated and thionitrite/thionitrous acid (SNO - /HSNO) as well as nitrosopersulfide (SSNO - ) were reported to be the intermediates that could serve as reservoirs for nitric oxide (NO). However, debate still exists regarding the stability and biological activity of SNO - /HSNO and SSNO - . In order to investigate the chemical properties and biological activity of SNO - and SSNO - , we set out to re-characterize the reaction intermediates using UV-Vis and 15 N NMR spectroscopy techniques, as well as a new 17 O NMR approach. The effects of SNO - and SSNO - on cellular NO and cGMP levels were assessed via cell culture experiments, and also the effects of SNO - and SSNO - on cell proliferation, migration, and capillary-like structure formation were evaluated with human umbilical vein endothelial cells (HUVEC). Through this work, the characteristic peaks and half-lives of SNO - and SSNO - were elucidated under various preparation conditions. The biological assays demonstrated that SSNO - increased the cellular NO and cGMP levels and also facilitated cell proliferation, migration and stimulated angiogenesis, while in contrast SNO - did not exhibit these effects.