Phytohormone (+)-7-iso-Jasmonoyl- L-isoleucine (JA-Ile) causes various physiological activities in planta, such as immune response, growth inhibition and enhancing of biosynthesis of secondary metabolites. In the jasmonic acid (JA) responses, JA-Ile induces Protein-Protein interaction (PPI) between F-box protein (COI1) and JAZ repressor. As previously reported, COI1 and 12 subtypes of JAZ proteins are coded in Arabidopsis thaliana. However, biological roles of each JAZ subtype have been remained unclear due to the high genetic redundancy of JAZ genes, and subtype selective agonist for COI1/JAZ co-receptor would be a potential tool to reveal JA signaling pathway. The natural phytotoxin coronatine (1) exhibits various promising biological activities similar to JA-Ile. We have already reported that a stereoisomer of 1 shows moderate subtype selectivity. From this result, we anticipated that other stereoisomers of 1 might show selectivity to other subtypes. Therefore, we planned to construct a focused stereochemical library of 1. In this presentation, we will report practical synthesis of coronatine which was also confirmed to be flexible enough to provide all the stereochemical isomers. We have also previously developed novel in vitro co-immunoprecipitation assay system for screening of COI1/JAZ agonists with fluorescence-epitope-conjugated JAZ degron peptides. By using this method, we have discovered the candidate of JAZ subtype-selective agonist from derivatives of 1. In this work, for development of more convenient and accurate detection method, we optimized the fluorescence molecule in the JAZ1 peptide, and succeeded in detection of this PPI by fluorescence anisotropy change, We also confirmed that such anisotropy change could be observed with other subtypes of JAZ degron peptides, and these results implied that our new method can be applied to the high throughput screening system for agonists of COI1/JAZs. In this presentation, we will also report the evaluation of JAZ-selectivity of all stereoisomers of 1 by using the new fluorescence anisotropy-based screening system.
