EPR STUDIES OF TRAPPED SINGLET OXYGEN (^lO₂) GENERATED DURING PHOTOIRRADIATION OF HYPOCRELLIN A

Abstract

Hypocrellin A, a peryloquinone derivative, has recently been isolated from the sacs of the fungus Hypocrella bambusae. This pigment, in combination with phototherapy, has been used in human medicine to cure various skin diseases. The generation of singlet oxygen during photoirradiation of Hypocrellin A (HA) was detected as an oxidation product of a sterically hindered amine (tetramethylpiperidine oxide; TEMPO) by electron paramagnetic resonance (EPR) spectroscopic techniques. Azide inhibited the EPR signal intensity in a dose-dependent manner with a quenching rate constant of 3.86 x 10(8) M-1s-1 in ethanol. Deuterated solvents, known to increase the lifetime of singlet oxygen, augmented the EPR signal intensity. The rate of production of singlet oxygen was dependent not only upon the concentration of HA and the time of irradiation but also on the oxygen content of the reaction mixture. The hyperfine splitting constant (aN = 16.3 G) and g-value (g = 2.0056) of the photoproduct of TEMP-singlet oxygen and TEMPO were found to be identical. This indicates that the nitroxide species detected by EPR spectroscopy generated by reacting TEMP with photogenerated 1O2 is TEMPO. The rate constant (kT) for the reaction of singlet oxygen with TEMP to form TEMPO radical was found to be 5.3 x 10(5) M-1s-1.

Reevaluation of analytical methods for photogenerated singlet oxygen

“…(9,10,21,22) Interestingly, when 4-hydroxy-TEMP was oxidized by singlet oxygen, ESR signals of TEMPOL and 4-oxo-TEMPO were observed. It was reported that TEMPOL was converted to 4-oxo-TEMPO when TEMPOL was oxidized by hydroxyl radical generated by photolysis of H 2 O 2 using 500 W ultraviolet (UV) light.…”

ESR study of singlet oxygen generation and its behavior during the photo-oxidation of P3HT in solution

“…The initial concentrations of P3HT (calculated using a monomer molecular weight of 166), TEMP, O 2 , and CB were 0.012 M, 5.7 × 10 −3 M, 1.6 × 10 −3 M, and 9.8 M, respectively. The values of k 1 = 1.2 × 10 −3 [11], k 3 = 5.3 × 10 5 [27], k 4 = 73 [24], k 5 = 2.1 × 10 3 [19], k 6 = 2.6 × 10 3 [28], and k 7 = 1.6 × 10 9 [19] were obtained from the literature, and the value of k 8 (=3.7 × 10 3 M −1 s −1 ) was estimated from the terminal X Y bond of the quencher molecule, as reported by Schweitzer and Schmidt [29]. The value of k 9 = 3.4 × 10 −5 s −1 was obtained from the decay of TEMPO measured in the dark reaction, as shown in Figure 4.…”

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia

“…1E-I). 2,2,6,6-Tetramethylpiperidine (TMP) was used to trap generated 1 O 2 , forming the detectable spin adduct TEMPOL, which produces a three line spectrum with hyperfine splitting of aN = 16.3 G [30]. Photodynamic irradiation under each O 2 condition led to significant increases in TEMPOL formation ( Fig.…”

“…Spectral acquisition settings were; microwave frequency 9.45 G, microwave power 4 mW, centre field 335.5 mT, sweep width 50 G, sweep time 100 s, time constant 1 s, modulation frequency 100 kHz and modulation width 1.25 G. The spectrum was acquired as a mean average of 3 spectral sweeps. The spin trap TMP (2,2,6,6-tetramethyl-4-piperidinol; Sigma-Aldrich) was used to trap 1 O 2 , forming TEMPOL (1-oxyl-2,2,6,6-tetramethyl-4-piperidinol) as the detectable spin adduct, which produces a three line spectrum with hyperfine splitting of aN = 16.3 G [30]. TMP was dissolved in 100% methanol to a concentration of 2 M. As a positive control for the trapping of 1 O 2 by TMP, commercially available PpIX (10 µM; Sigma-Aldrich) and TMP (50 mM in PBS, pH 7.4) were injected into a Jeol LC-11 flat cell and irradiated (hυ; 25 J/cm 2 ) in situ in the EPR cavity.…”