Conjugation of CRAMP18–35 Peptide to Chitosan and Hydroxypropyl Chitosan via Copper-Catalyzed Azide–Alkyne Cycloaddition and Investigation of Antibacterial Activity

We developed a synthesis strategy involving a diazo transfer reaction and subsequent click reaction to conjugate a murine cathelicidin-related antimicrobial peptide (CRAMP_18-35) to chitosan and hydroxypropyl chitosan (HPC), confirmed the structure, and investigated the antimicrobial activity. Chitosan azide and HPC-azide were prepared with a low degree of azidation by reacting the parent chitosan and HPC with imidazole sulfonyl azide hydrochloride. CRAMP_18-35 carrying an N-terminal pentynoyl group was successfully grafted onto chitosan and HPC via copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. The chitosan-peptide conjugates were characterized by IR spectroscopy and proton NMR to confirm the conversion of the azide to 1,2,3-triazole and to determine the degree of substitution (DS). The DS of the chitosan and HPC CRAMP_18-35 conjugates was 0.20 and 0.13, respectively. The antibacterial activity of chitosan-peptide conjugates was evaluated for activity against two species of Gram-positive bacteria, <i>Staphylococcus aureus</i> (<i>S. aureus</i>) and <i>Enterococcus faecalis</i> (<i>E. faecalis</i>), and two species of Gram-negative bacteria<i>, Escherichia coli</i> (<i>E. coli</i>) and <i>Pseudomonas aeruginosa</i> (<i>P. aeruginosa</i>). The antimicrobial peptide conjugates were selectively active against the Gram-negative bacteria and lacking activity against Gram-positive bacteria.