Considering the objective of these experiments was to determine the amount of active DBCO following cleavage across a range of (MeCN)4CuBF4 equivalents, the analysis was simplified by synthesizing 3a with a Met-to-Nle substitution to give oxidationresistant 3c (Figure ). 3c was incubated with the indicated amounts of (MeCN)4CuBF4 in Figure through standard cleavage conditions for 3 h before ether precipitation and air drying. Crude 3c was then dissolved in HPLC buffer (0.1% TFA, 18% MeCN) and coupled with excess 6-azidohexanoic acid for 1 h at 37°C to determine the amount of reactive DBCO present (Figures and). DBCO protection was observed to increase until 5 equiv. (MeCN)4CuBF4. Surprisingly, 10 and 50 equiv. increased the amount of DBCO that failed to click with azide. This loss of reactivity may be attributed to excessive Cu(I) salts that are incompletely removed following ether precipitation, which could form a complex with the DBCO peptide, thus precluding efficient cycloaddition reactivity (Figures and). Based on these optimizations, the use of 5 equiv. of (MeCN)4CuBF4 was determined to be ideal for preventing the rearrangement of DBCO-containing peptides through standard cleavage conditions.