Nucleotides and inorganic phosphates as potential antioxidants

Highly reactive OH radicals, formed in an iron-ion catalyzed Fenton reaction, are implicated in many pathological conditions. The quest for Fenton reaction inhibitors, either radical scavenger or metal-ion chelator antioxidants, spans the previous decades. Purine nucleotides were previously studied as natural modulators of the Fenton reaction; however, the modulatory role of purine nucleotides remained in dispute. Here, we have resolved this long-standing dispute and demonstrated a concentration-dependent biphasic modulation of the Fenton reaction by nucleotides. By electron spin resonance measurements with 0.1 mM Fe(II), we observed an increase of *OH production at low purine nucleotide concentrations (up to 0.15 mM), while at higher nucleotide concentrations, an exponential decay of *OH concentration was observed. We found that the phosphate moiety, not the nucleoside, determines the pro/antioxidant properties of a nucleotide, suggesting a chelation-based modulation. Furthermore, the biphasic modulation mode is probably due to diverse nucleotide-Fe(II) complexes formed in a concentration-dependent manner. At ATP concentrations much greater than Fe(II) concentrations, multiligand chelates are formed which inhibit the Fenton reaction owing to a full Fe(II) coordination sphere. In addition to natural nucleotides, we investigated a series of base- or phosphate-modified nucleotides, dinucleotides, and inorganic phosphates, as potential biocompatible antioxidants. Ap5A, inorganic thiophosphate and ATP-gamma-S proved highly potent antioxidants with IC50 values of 40, 30, and 10 microM, respectively. ATP-gamma-S proved 100 and 20 times more active than ATP and the potent antioxidant Trolox, respectively. In the presence of 30 microM ATP-gamma-S no *OH was detected after 5 min in the Fenton reaction mixture. The most potent antioxidants identified inhibit the Fenton reaction by forming full coordination sphere chelates.