Synthesis and hyperpolarisation of eNOS substrates for quantification of NO production by 1H NMR spectroscopy

Abstract Hyperpolarization enhances the intensity of the NMR signals of a molecule, whose in vivo metabolic fate can be monitored by MRI with higher sensitivity. SABRE is a hyperpolarization technique that could potentially be used to image nitric oxide (NO) production in vivo . This would be very important, because NO dysregulation is involved in several pathologies, including cardiovascular ones. The nitric oxide synthase (NOS) pathway leads to NO production via conversion of l -arginine into l -citrulline. NO is a free radical gas with a short half-life in vivo (≈5 s), therefore direct NO quantification is challenging. An indirect method – based on quantifying conversion of an l -Arg- to l -Cit-derivative by 1 H NMR spectroscopy – is herein proposed. A small library of pyridyl containing l -Arg derivatives was designed and synthesised. In vitro tests showed that compounds 4a – j and 11a – c were better or equivalent substrates for the eNOS enzyme (NO 2 − production = 19–46 μM) than native l -Arg (NO 2 − production = 25 μM). Enzymatic conversion of l -Arg to l -Cit derivatives could be monitored by 1 H NMR. The maximum hyperpolarization achieved by SABRE reached 870-fold NMR signal enhancement, which opens up exciting future perspectives of using these molecules as hyperpolarized MRI tracers in vivo .