To examine whether probes 1-4 fragment under reductive conditions to release the corresponding fluorescent dyes (5-7) both chemical and enzyme-mediated reductions were investigated. Treatment of compounds 1-4 with sodium dithionite, for between 10 and 60 mins, followed by HPLC analysis (Supporting Information) showed that all probes released the corresponding dyes, and the hydroquinone derivatives, in reductive conditions (Figures ). In contrast, negligible fragmentation of compounds 1-4 was observed in the absence of sodium dithionite, demonstrating that reduction of the indolequinone is required for the release of the fluorescent dye, and that the probes are stable to hydrolysis under these conditions. Next, we determined the ability of the fluorogenic probes to undergo bioreduction when treated with NADPH-cytochrome P450 reductase (PH51) (see Supporting Information). This class of enzymes is known to metabolize indolequinone derivatives and therefore provides a useful predictive model system to for cellular bioreduction of the probes. Compounds 2-4 were treated with NADPH-cytochrome P450 reductase and β-NADPH tetrasodium salts as co-factor, and the fluorescence spectra of the solution were recorded at regular intervals (Supporting Information and Figure ). In all cases a substantial increase in fluorescence was observed, but the time taken for fluorescence recovery (compared to the parent fluorophore) differed between probes. In hypoxia (0.1% O2) the resorufin-based probe 2 showed a 10-fold increase We were surprised to find that compound 1 only showed a weak increase in fluorescence after 90 mins, when treated at <0.1% O2 with the NADPH-cytochrome P450 reductase and β-NADPH (Figure ). Given the cyclic voltammetry data for this compound (vide supra) we proposed that the cytochrome P450 reductase was not only reducing the indolequinone, but also the DDAO resulting in a non-fluorescent product. HPLC analysis of the enzymatic reduction (Figure ) confirmed this hypothesis, with the reaction shown to produce both DDAO and its non-fluorescent reduced derivative (13). To investigate whether DDAO was susceptible to NADPH-cytochrome P450-mediated reduction, we treated this compound with the enzyme and β-NADPH in hypoxia (<0.1% O2) for 65 mins and observed the loss of fluorescence consistent with bioreduction occurring.