Protection of DBCO peptide 1a from acid-mediated degradation using (MeCN)4CuBF4 in standard peptide cleavage conditions. This protection strategy generated a relatively clean crude peptide (dashed line in the LC/MS chromatogram) corresponding to the expected mass of 1a (observed mass: 1,401.9 Da, calculated average mass: 1,402.6 Da, Figure ). This crude peptide successfully underwent SPAAC when incubated with excess 6-azidohexanoic acid, as the expected triazole formed (solid line in the LC/MS chromatogram, 1a!; observed mass: 1,559.9 Da, calculated average mass: 1,559.8 Da). LC/MS Method C was used. After performing SPAAC on crude 3c, A280 peak areas of reacted (3c!) and unreacted peptide were integrated to calculate % reactive DBCO. ***Indicates higher equiv. of (MeCN)4CuBF4 that were found to complex with the DBCO peptide, preventing reaction with azide after cleavage. Although the % reactive DBCO is lower, this decrease is not due to 5-endo-dig-cycloisomerization. LC/MS chromatograms of crude 3c (before and after SPAAC) are shown in Figures . (1, 20, 50, 110, 230, 350, 470, 1430, and 2870 min). CAN ligations were fit to first-order rate constants. Note that the calculated rate constant for the 0.05 mM 8 CAN is slightly lower than expected due to the StBu protecting group being removed more slowly at the 10-fold lower TCEP concentration used. Importantly, the 8 CAN is complete after 4 h at both peptide concentrations. For the non-templated NCL, peak areas of both product (7) and STEVE reactant (5a^) were calculated using estimated extinction coefficients, as described in the supplemental methods. Each data point represents the average of two independent experiments, with the error bars corresponding to standard deviation. Relative peak area of the ligated product is shown at various time points (relative to TCEP addition). CAN ligations were fit to first-order rate constants. For the non-templated NCL, peak areas of both product (14a) and L32-N reactant (12a^) were calculated using estimated extinction coefficients, as described in the supplemental methods. Each data point represents the average of two independent experiments, with the error bars showing standard deviation.