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.