One of the more attractive uses of CAN, and templated ligations in general, is the ability to perform ligations at lower peptide concentrations (i.e., the triazole-linkage renders the NCL an intramolecular reaction). To validate this property, we repeated CAN with 8 and 10 (±PEG8) at a 10-fold lower concentration (0.05 mM). We observed that a significant amount of the N-terminal Cys peptide was being capped (initially +74 Da, Figures ). The mass of this side product suggested that modification was the result of acylation from an MTG acyl donor. This acylation occurs negligibly in the 0.5 mM CAN reactions, indicating that acylation occurs on a small amount of peptide that only becomes problematic at lower peptide concentrations. Therefore, we surmised that reducing the MTG concentration would reduce this side reaction. A ten-fold reduction in MTG concentration without proportional reduction of TCEP concentration, however, led to significant Cys desulfurization (Figures ). Fortunately, a proportionate reduction of TCEP concentration eliminated this desulfurization. Using these conditions, CAN kinetics were similar for both the 0.05 and 0.5 mM reactions (Figures and). Compared to the non-templated NCL (0.4 mM 5a and 0.5 mM 6a), the 0.05 mM 8 CAN reached completion ~12x faster (4 h for CAN vs. 48 h for nontemplated NCL). Based on the 2 nd -order rate law, a 10-fold reduction in concentration of both peptides in the non-templated NCL would slow the reaction 100-fold, which is too slow to measure and would likely be dominated by side reactions. Therefore, the 0.05 mM 8 CAN reached completion ~1,200-fold faster than expected for the non-templated NCL at the same concentration.