Modulating Refolding and Collagen Binding of Collagen Hybridizing Peptides through Peptoid Incorporation

Collagen hybridizing peptides (CHPs) selectively bind denatured collagens (dColl), offering a powerful platform for diagnostics and targeted drug delivery. However, the rapid self-trimerization of GPO-derived CHPs (O = hydroxyproline) limits their applications. Here, we present peptoid-based strategies to reduce the triple helix stability and refolding speed of (GPO)₉ by introducing derivatives of Nasp (N-carboxymethyl glycine). Systematic substitution studies revealed that N^α-C^β branched Nsce (N-(S)-carboxyethyl glycine) destabilized the triple helix primarily through steric hindrance rather than electrostatic repulsion. Triple Nsce substitutions not only drastically reduced the refolding propensity but also compromised dColl binding. In contrast, the nonbranched Nasp residue, under the same conditions, slowed refolding while enhancing gelatin binding compared to (GPO)₉. The CHP incorporating Nasp stained dColl associated with skin melanoma in histological sections as effective as (GPO)₉. Our findings demonstrate that CHPs incorporating peptoid residues may enable fine-tuning of CHP folding and binding kinetics for the development of next-generation CHPs.