With a DBCO protection strategy in hand, we next investigated Click-Assisted NCL (CAN). We substituted the commonly used 4-mercaptophenylacetic acid (MPAA) thiol for methyl thioglycolate (MTG) during NCL, as MTG elutes in the void during RP-HPLC, avoiding co-elution with peptide. Additionally, MTG is compatible with one-pot desulfurizations. Although MTG has slower ligation kinetics compared to MPAA, we hypothesized that the effective concentration enhancement of the CAN strategy would render the choice of thiol additive less impactful. The non-templated control peptides STEVE (5a) and KENT (6a) were synthesized without SPAAC linkers to determine their in-solution NCL rate under representative conditions (Figure ). 5a was also designed with a C-terminal Val thioester, since they are known to be among the slowest in NCL due to steric hindrance. After 48 h, the ligation reaction was incomplete and several side products had formed, with only 29% of the total HPLC peak area corresponding to ligated product 7 (Figures and).