Chapter 4 Only a “Click” Away: Development of Arginine-Rich Peptide-Based Materials Using Click Chemistry Mariana Barbosa, Fabı´ola Costa, Ca´ tia Teixeira, M. Cristina L. Martins, and Paula Gomes Abstract The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) coupling is one of the most interesting chemoselective reactions that match the “click chemistry” concept. Both azide and alkyne moieties, easily incorporated into polymers and biomolecules, react selectively with each other yielding a stable triazole link. Thus, CuAAC “click” reaction has been widely explored as a powerful tool for the covalent linkage of biomolecules. In this context, this highly efficient and regioselective chemistry was selected for covalently immobilize arginine-rich peptides onto chitosan, a biopolymer with interesting bioactive properties. This chapter focuses on experimental procedures for immobilization of arginine-rich peptides by means of the aforementioned CuAAC reaction. Peptide grafting onto chitosan via CuAAC either on (1) ground chitosan powder (type 1 conjugation) or (2) preformed chitosan thin films (type 2 conjugation) is described, demonstrating the versatility of the selected coupling reaction. In addition, given its relevance in this particular context, solid-phase peptide synthesis of alkyne-modified peptides and conversion of chitosan amines into azides are also described. Overall, this work is intended to convey useful guidelines for the immobilization of arginine-rich peptides onto amine-functionalized polymers, by means of click chemistry via CuAAC. Key words Arginine, Azide-alkyne coupling, Chitosan, Click chemistry, Huisgen’s 1,3-dipolar cyclo- addition, Peptide, Polymer, Tethering, Bioconjugation, Peptide-based materials 1 Introduction Highly efficient chemistries toward development of tailor-made materials for specific applications have been widely explored in recent years, mostly aimed at creating new biomaterials with poten- tial application in biomedical engineering. The design and produc- tion of highly functional and specific biomaterials has been mainly driven by the need to enhance the therapeutic effectiveness of tissue regeneration approaches. Cells in vivo are enclosed in a three- dimensional (3D) extracellular matrix (ECM) composed of numer- ous biochemical and mechanical cues capable of modulating Olga Iranzo and Ana Cecı´lia Roque (eds.), Peptide and Protein Engineering: From Concepts to Biotechnological Applications, Springer Protocols Handbooks, https://doi.org/10.1007/978-1-0716-0720-6_4, © Springer Science+Business Media, LLC, part of Springer Nature 2020 37