Nucleoside - 2′,3′/3′,5′-bis(thio)phosphate analogues are promising antioxidants acting mainly via Cu⁺/Fe ²⁺ ion chelation

We synthesized a series of adenine/guanine 2′,3′- or 3′,5′-bisphosphate and -bisphosphorothioate analogues, 1-6, as potential Cu⁺/Fe²⁺ chelators, with a view to apply them as biocompatible and water-soluble antioxidants. We found that electron paramagnetic resonance (EPR)-monitored inhibition of OH radicals production from H₂O₂, in an Fe²⁺-H₂O₂ system, by bisphosphate derivatives 1, 3, and 5 (IC₅₀ = 36, 24, and 40 μM, respectively), was more effective than it was by ethylenediaminetetraacetic acid (EDTA), by a factor of 1.5, 2, and 1.4, respectively. Moreover, 2′-deoxyadenosine-3′,5′-bisphosphate, 1, was 1.8- and 4.7-times more potent than adenosine 5′-monophosphate (AMP) and adenosine 5′-diphosphate (ADP), respectively. The bisphosphorothioate derivatives 2, 4, and 6 (IC₅₀ = 92, 50, and 80 μM, respectively), exhibited a dual antioxidant activity, acting as both metal-ion chelators and radical scavengers [2,2′-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay data indicates IC ₅₀ = 50, 70, and 108 μM vs 27 μM for Trolox]. Only 2′-deoxyadenosine-3′,5′-bisphosphorothioate, 2, exhibited good inhibition of Cu⁺-induced H₂O₂ decomposition (IC₅₀ = 78 vs 224 μM for EDTA). Nucleoside-bisphosphorothioate analogues (2, 4, and 6) were weaker inhibitors than the corresponding bisphosphate analogues (1, 3, and 5), due to intramolecular oxidation under Fenton reaction conditions. ¹H- and ³¹P NMR monitored Cu⁺ titration of 2, showed that Cu⁺ was coordinated by both 3′,5′-bisphosphorothioate groups, as well as N7-nitrogen atom, while adenosine-2′,3′-bisphosphorothioate, 6, coordinated Cu ⁺ only by 2′,3′-bisphosphorothioate groups. In conclusion, an additional terminal phosphate group on AMP/guanosine 5′-monophosphate (GMP) resulted in Fe²⁺-selective chelators highly potent as Fenton reaction inhibitors.