Quantification of singlet oxygen from hematoporphyrin derivative by electron spin resonance

The mechanism of the generation and the quantitative analysis of singlet oxygen (O-1(2)) formed by the exposure of a hematoporphyrin derivative (HpD) to light was re-evaluated by electron spin resonance (ESR) combined with 2,2,6,6,-tetramethyl-4-piperidine (TMPD). The change from TMPD to 2,2.6,6,-tetramethyl-4-piperidine-N-oxide (TAN) has been reported to depend on singlet oxygen. However, we confirmed that this reagent also reacts with superoxide anion (O-2(-)) and hydroxyl radicals (OH). Therefore, the reactions between TMPD and O-1(2), O-2(-) and OH were re-examined using a kinetic approach. We found that the generatiation of TAN was proportional to the concentration of TMPD and HpD, as well as to the duration and strength of the illumination. The generation of TAN was enhanced in the presence of deuterium oxide. The generation of TAN was not inhibited by dimethyl-sulfoxide (DMSO) or superoxide dismutase (SOD). The reaction rate between TMPD and O-1(2) was determined to be 5.0 X 10(-7) M(-1)min(-1). The generation of O-1(2) from HpD was 2.7 X 10(-7) Mmin(-1) under our conditions. The competitive reaction observed between 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and TMPD for O-2(-) or OH shows that TMPD reacts with both forms of active oxygen, but gave no ESR signal. The second-order reaction rate constant of TMPD between O-2(-) and OH was calculated as 73M(-1)s(-1) and 1.5 X 10(9)M(-1)s(-1), respectively. The photochemical generation of O-1(2) from methylene blue, another sensitzer, was also demonstrated by this method. These results show that ESR signal of TAN can be used for the highly selective monitoring of O-1(2).