Takajo Tokuko, Kurihara Yoshinori, Iwase Kodai, Miyake Daiki, Tsuchida Kazunori, Anzai Kazunori
Faculty of Pharmaceutical Sciences, Nihon Pharmaceutical University.
Chem Pharm Bull (Tokyo). 2020;68(2):150-154. doi: 10.1248/cpb.c19-00770.
Singlet oxygen (O) is highly oxidative and exerts strong cytotoxic effects. We tried to establish the best combination of a singlet oxygen generation system and a detection method with ESR, for measurement of the quenching activities of various substances. The photosensitizing reaction of rose bengal or thermal decomposition of 4-methyl-1,4-etheno-2,3-benzodioxin-1(4H)-propanoic acid (endoperoxide, EP) was used for the generation of O, and a sterically hindered secondary amine, 2,2,6,6-tetramethyl-4-piperidone (TEMPD) or 2,2,6,6-tetramethyl-4-piperidinol (TEMP-OH), was used as the O detection probe. These secondary amines were oxidized by O to form stable nitroxide radicals, which were detectable by ESR. TEMPD was found to be readily oxidized by air, causing large background signals in comparison with TEMP-OH. The ESR signal obtained by the irradiation of rose bengal with visible light in the presence of TEMP-OH consisted of two kinds of nitroxide radical overlapping. In contrast, only a single nitroxide signal was observed when TEMP-OH was reacted with O generated from EP. Therefore, the best combination should be EP as the O generator and TEMP-OH as the detection probe. When using this combination, we found that the concentrations of some organic solvents such as dimethyl sulfoxide and acetonitrile should be kept constant for reliable quantification, because the concentrations of organic solvents affect the ESR signal intensity.
单线态氧（O）具有高度氧化性，能产生强烈的细胞毒性作用。我们试图建立单线态氧产生系统与电子自旋共振（ESR）检测方法的最佳组合，以测量各种物质的猝灭活性。孟加拉玫瑰红的光敏反应或4-甲基-1,4-亚乙基-2,3-苯并二恶英-1(4H)-丙酸（内过氧化物，EP）的热分解用于产生O，位阻仲胺2,2,6,6-四甲基-4-哌啶酮（TEMPD）或2,2,6,6-四甲基-4-哌啶醇（TEMP-OH）用作O检测探针。这些仲胺被O氧化形成稳定的氮氧自由基，可通过ESR检测到。发现TEMPD很容易被空气氧化，与TEMP-OH相比会产生较大的背景信号。在TEMP-OH存在下用可见光照射孟加拉玫瑰红获得的ESR信号由两种重叠的氮氧自由基组成。相比之下，当TEMP-OH与由EP产生的O反应时，只观察到一个单一的氮氧信号。因此，最佳组合应该是以EP作为O发生器，TEMP-OH作为检测探针。使用这种组合时，我们发现一些有机溶剂如二甲基亚砜和乙腈的浓度应保持恒定以进行可靠的定量，因为有机溶剂的浓度会影响ESR信号强度。
