Figure 6.

Detection of peroxynitrite. A, mechanism of 2′,7′-dichlorodihydrofluorescein (DCFH₂) oxidation. The reduced probe (DCFH₂) is oxidized by peroxynitrite-derived radicals and other one-electron oxidants yielding a radical intermediate (DCF^•−) that is oxidized by oxygen to yield fluorescent DCF. Thin arrows show alternative reactions and redox cycles. AH⁻ stands for ascorbate. B, boronate oxidation by peroxynitrite. The major pathway (>85%, above) consists of heterolytic cleavage of the peroxyl bond leading to phenol which, appropriately derivatized, is fluorescent. The minor radical pathway (below) involves homolytic cleavage of the peroxyl bond giving NO₂^• and a phenyl-type radical that yields the nitro-derivative (201). C, structures of boronate-derived probes. CBA and CBE indicate a boronic acid or a boronic pinacolate ester attached to a coumarin scaffold, respectively (193). Fl-B (194), FlAmBE (192), and FBBE (205) are boronic pinacolate ester derivatives linked to a fluorescein scaffold with structural modifications as shown. D, genetically-encoded boronate-based GFP for the detection of peroxynitrite. Nucleophilic attack by the peroxynitrite anion to the phenylalanine boron moiety results in the formation of tyrosine and fluorescence. Modified from Ref. 176.