Chemical synthesis of the EPF-family of plant cysteine-rich proteins and late-stage dye attachment by chemoselective amide-forming ligations.

Chemical protein synthesis can provide well-defined modified proteins. Herein, we report the chemical synthesis of plant-derived cysteine-rich secretory proteins and late-stage derivatization of the synthetic proteins. The syntheses were achieved with distinct chemoselective amide bond forming reactions - EPF2 by native chemical ligation (NCL), epidermal patterning factor (EPF) 1 by the α-ketoacid-hydroxylamine (KAHA) ligation, and fluorescent functionalization of their folded variants by potassium acyltrifluoroborate (KAT) ligation. The chemically synthesized EPFs exhibit bioactivity on stomatal development in Arabidopsis thaliana . Comprehensive synthesis of EPF derivatives allowed us to identify suitable fluorescent variants for bioimaging of the subcellar localization of EPFs.