During the total chemical synthesis of the water-soluble globular Haemophilus Influenzae DNA ligase Hin-Lig, we observed the surprising phenomenon of a soluble peptide segment that failed to undergo native chemical ligation. Based on dynamic light scattering and transmission electron microscopy experiments, we determined that the peptide formed soluble colloidal particles in a homogeneous solution containing 6 M Gn·HCl. Conventional peptide performance-improving strategies such as installation of a terminal/side-chain Arg tag or O-acyl isopeptide failed to enable the reaction, presumably due to their inability to disrupt the formation of soluble colloidal particles. However, a removable backbone modification strategy recently developed for the synthesis of membrane proteins did disrupt the formation of the colloids, and the desired ligation of this soluble but unreactive system was eventually accomplished. This work demonstrates that an appropriate solution dispersion state in addition to good peptide solubility is a prerequisite for successful peptide ligation.