Macromolecular tool box to elucidate CLAVATA3/Embryo Surrounding Region-Related-RLK binding, signaling and downstream effects.

Plant peptides communicate by binding to a large family of receptor-like kinases (RLKs) and they share a conserved binding mechanism, which may account for their promiscuous interaction with several RLKs. In order to understand the in vivo binding specificity of CLAVATA3/Embryo Surrounding Region-Related peptide family, we have developed a novel set of CLAVATA 3 (CLV3) based peptide tools. After carefully evaluating the CLE peptide binding characteristics, using solid phase synthesis process, we have modified the CLV3 peptide and attached a fluorophore and a photoactivable side group. We observed that the labeled CLV3 shows binding specificity within CLAVATA1 clade of RLKs while avoiding the distantly-related PEP RECEPTOR clade, thus resolving the contradictory results obtained previously by many in vitro methods. Furthermore, we observed that the RLK-bound CLV3 undergoes clathrin-mediated endocytosis and gets trafficked to vacuole via ARA7-labeled endosomes. Additionally, modifying CLV3 for light-controlled activation enabled spatial and temporal control over CLE signalling. Hence, our CLV3 macromolecular toolbox can be used to study rapid cell specific down-stream effects. Given the conserved binding properties, in the future our toolbox can also be used as a template to modify other CLE peptides.

Late-stage C-H Functionalization of Tryptophan-Containing Peptides with Thianthrenium Salts: Conjugation and Ligation.

Bioorthogonal late-stage diversification of structurally complex peptides bears enormous potential for drug discovery and molecular imaging, among other applications. Herein, we report on a palladium-catalyzed C-H arylation of tryptophan-containing peptides with readily accessible and modular arylthianthrenium salts. Under exceedingly mild reaction conditions, the late-stage diversification of structurally complex peptides was accomplished. The tunability and ease of preparation of arylthianthrenium salts allowed the expedient stitching of tryptophan-containing peptides with drug, natural product, and peptidic scaffolds by forging sterically congested biaryl linkages. The robustness of the palladium catalysis regime was reflected by the full tolerance of a plethora of sensitive and coordinating functional groups. Hence, our manifold enabled efficient access to highly decorated, labelled, conjugated, and ligated linear and cyclic peptides.