A two-photon ratiometric fluorescent probe for imaging and quantitative analysis of botanic glucosyltransferase: A key enzyme for the biosynthesis of bioactive glycosides

Botanic glucosyltransferases (GT, EC 2.4.1), are well known for the strong capability to transfer the active glucose to aglycone, regarded as the key functional enzymes for the biosynthesis of vital medicine substrates in plant. In the present work, according to the catalysis characteristics of GT, we designed a dual filtering strategy to obtain a sensitive two-photon ratiometric fluorescent probe (FNA, 4-hydroxy-N-(4-F-phenemyl)-1,8-naphthalimide) for the specific detection of GT. Using FNA for the real-time detection of endogenous GT, two-photon confocal microscopy images of various suspension cells and tissues of P. ginseng and P. notoginseng, were achieved successfully, and the real distribution of GT in medicinal plants was also revealed for the first time. Furthermore, using FNA, the rapid quality control method of medicinal materials was established, based on the real-time activity of endogenous GT, with an excellent linearity correlation between GT bioactivities and the total contents of glycosides. Therefore, these results revealed that GT was a key target enzyme for the biosynthesis of bioactive glycosides, and FNA could be successfully applied for monitoring the GT activity in plant cells and tissues, and for quality control of GT-rich herbal medicines.