Figure 1.

Enzymatic conversion of 1 into a fluorescent product selectively under hypoxic conditions. A. Fluorescence emission at 530 nm (λ_ex 340 nm). Each set of assays depicted in the bar graph consists of (from left to right): a control sample of compound 1 alone (0.8 mM) , a control reaction composed of xanthine oxidase (2.4 U/mL) and xanthine (2.4 mM, 3.2 mM, 4.0 mM and 6.4 mM) under aerobic conditions , a control reaction composed of xanthine oxidase (2.4 U/mL) and xanthine (2.4 mM, 3.2 mM, 4.0 mM and 6.4 mM) under anaerobic conditions , a control reaction composed of xanthine oxidase (2.4 U/mL), xanthine (2.4 mM, 3.2 mM, 4.0 mM and 6.4 mM) and the non-fluorescent electron acceptor, 1,2,4-benzotriazine 1,4-dioxide,⁵² (6.4 mM) under aerobic conditions , a control reaction composed of xanthine oxidase (2.4 U/mL), xanthine (2.4 mM, 3.2 mM, 4.0 mM and 6.4 mM) and the non-fluorescent electron acceptor, 1,2,4-benzotriazine 1,4-dioxide, (6.4 mM) under anaerobic conditions , a reaction composed of xanthine oxidase (2.4 U/mL), xanthine (2.4 mM, 3.2 mM, 4.0 mM and 6.4 mM) and 1 under aerobic conditions , a reaction composed of xanthine oxidase (2.4 U/mL), xanthine (2.4 mM, 3.2 mM, 4.0 mM and 6.4 mM) and 1 under anaerobic conditions . Reactions were incubated for 18 h in sodium phosphate buffer at (12 mM, pH 7.4) at 24 °C, diluted with aerobic sodium phosphate buffer (12 mM, pH 7.4), and the fluorescence measured (λ_ex 340 nm, λ_em 530 nm). B. Fluorescence spectra of reaction mixtures generated in the aerobic and anaerobic metabolism of 1 by xanthine oxidase (2.4 U/mL) and xanthine (6.4 mM) carried out as described in the Experimental Section and fluorescence spectrum of 6-aminoquinoline (2, 0.1 mM, λ_ex 340 nm, in sodium phosphate buffer, 10 mM, pH 7.4).