Synthesis and characterization of an artificial glucosinolate bearing a chlorthalonil-based aglycon as a potent inhibitor of glucosinolate transporters

Abstract

Glucosinolates (GSLs) are specialized metabolites in plants of the order Brassicales. GSL transporters (GTRs) are essential for the redistribution of GSLs and also play a role in controlling the GSL content of seeds. However, specific inhibitors of these transporters have not been reported. In the current study, we described the design and synthesis of 2,3,4,6-tetrachloro-5-cyanophenyl GSL (TCPG), an artificial GSL bearing a chlorothalonil moiety as a potent inhibitor of GTRs, and evaluated its inhibitory effect on the substrate uptake mediated through GTR1 and GTR2. Molecular docking showed that the position of the β-D-glucose group of TCPG was significantly different from that of the natural substrate in GTRs and the chlorothalonil moiety forms halogen bonds with GTRs. Functional assays and kinetic analysis of the transport activity revealed that TCPG could significantly inhibit the transport activity of GTR1 and GTR2 (IC50 values (mean ± SD) being 79 ± 16 μM and 192 ± 14 μM, respectively). Similarly, TCPG could inhibit the uptake and phloem transport of exogenous sinigrin by Arabidopsis thaliana (L.) Heynh leaf tissues, while not affecting that of esculin (a fluorescent surrogate for sucrose). TCPG could also reduce the content of endogenous GSLs in phloem exudates. Together, TCPG was discovered as an undescribed inhibitor of the uptake and phloem transport of GSLs, which brings novel insights into the ligand recognition of GTRs and provides a new strategy to control the GSL level. Further tests on the ecotoxicological and environmental safety of TCPG are needed before using it as an agricultural or horticultural chemical in the future.