Department of Biobased Materials Science, Kyoto Institute of Technology, 1 Hashigami-Cho, Matsugasaki, Sakyo-Ku, Kyoto, 606-8585, Japan.
Anal Bioanal Chem. 2023 Aug;415(19):4661-4673. doi: 10.1007/s00216-023-04773-w. Epub 2023 May 31.
Itaconic acid (IA) and its derivatives produced by fungi have significant potential as industrial feedstocks. We recently developed a method for the detection of these compounds based on their terminal C-C double bonds. However, the presence of reducing agents, such as glucose and other fungal metabolites, leads to undesirable side reactions, and consequently, deteriorates the detection specificity. Therefore, we developed a fluorescence detection method for IA and its derivatives underpinned by a photoclick reaction. The photoclick reaction between conjugated IA and 5-(4-methoxyphenyl)-2-phenyl-2H-tetrazole under UV irradiation affords a fluorescent product. No fluorescence was detected when succinic acid was subjected to the reaction, indicating that a terminal C-C double bond is required to induce fluorescence. Optimal reaction conditions were determined to be a combination of 80% final dimethyl sulfoxide concentration, 30-s UV irradiation, and a pH of 2. Two weeks after the reaction at 4 °C, 89.0% of the initial intensity was retained, indicating that the reaction product was relatively stable. Glucose and kojic acid did not induce fluorescence after the reaction, indicating that these reducing agents did not affect fluorescence. IA was detected in a culture of Aspergillus terreus, and its quantification using the photoclick reaction was in agreement with the results obtained using high-performance liquid chromatography analysis. Interestingly, the IA derivative avenaciolide present in submillimolar quantities was also detectable in a culture of Aspergillus avenaceus using this method. The established method will enable the development of high-throughput screening methods to identify fungi that produce IA and its derivatives.
衣康酸（IA）及其真菌衍生的衍生物具有作为工业原料的巨大潜力。我们最近开发了一种基于其末端 C-C 双键的检测这些化合物的方法。然而，还原剂的存在，如葡萄糖和其他真菌代谢物，会导致不希望的副反应，从而降低检测的特异性。因此，我们开发了一种基于光点击反应的 IA 及其衍生物的荧光检测方法。在紫外光照射下，共轭 IA 与 5-(4-甲氧基苯基)-2-苯基-2H-四唑之间的光点击反应产生荧光产物。当琥珀酸进行反应时没有检测到荧光，表明末端 C-C 双键是产生荧光所必需的。确定的最佳反应条件是最终二甲基亚砜浓度为 80%、30 秒的紫外辐射和 pH 值为 2。在 4°C 下反应两周后，初始强度保留了 89.0%，表明反应产物相对稳定。葡萄糖和曲酸在反应后没有诱导荧光，表明这些还原剂不会影响荧光。IA 在土曲霉的培养物中被检测到，并且使用光点击反应进行定量与使用高效液相色谱分析得到的结果一致。有趣的是，使用这种方法还可以检测到在 Aspergillus avenaceus 培养物中存在的痕量衣康酸衍生物 avenaciolide。所建立的方法将能够开发高通量筛选方法来鉴定产生 IA 及其衍生物的真菌。
