Trityl-nitroxide biradicals as unique molecular probes for the simultaneous measurement of redox status and oxygenation

Abstract

Novel trityl-nitroxide biradicals were synthesized and exhibited enhanced sensitivity and stability for rapid and simultaneous measurement of redox status and oxygenation by electron paramagnetic resonance spectroscopy.

Figure 1

(A) Molecular structure of trityl-nitroxide biradicals. (B) Experimental and simulated EPR spectra of TN1 and TN2 in PBS.

Figure 2

(A) EPR spectra obtained by reaction of TN1 (10 µM) with ascorbic acid (400 µM) in PBS (50 mM, pH 7.4). Gray lines shows the enlarged (5×) portion of the spectrum; (*) indicates the ¹³C hyperfine splittings. (B) Plot of EPR signal intensity as a function of time. For biradicals the singlet signals due to trityl radicals were monitored while the middle peak was monitored for TEMPO. Spectra were recorded with 5 mW microwave power and 0.2 G modulation amplitude.

Figure 3

(A) Plot of the linewidth of the trityl-hydroxylamine TN1-H and TN2-H as a function of O₂ concentration. (B) Plot of I_in/I_out for TN1-H as a function of O₂%. TN1-H and TN2-H were generated by mixing the biradical (50 uM) with ascorbate (2 mM) in PBS (50 mM, pH 7.4). See more details in the supporting information.

Figure 4

(A) Time-dependent EPR spectra obtained by incubating TN1 (50 µM) with fresh rat liver homogenate (1.8 mg protein/mL) in the presence of succinate (50 mM) in a sealed capillary. (B) Variations of EPR signal double integration (circle) and O₂ concentration (triangle) with time in the presence (unfilled) or absence (filled) of succinate. Spectra were recorded with 5 mW microwave power and 0.03 G modulation amplitude.