To demonstrate the general utility of CAN in CPS , we pursued a representative protein target -the E. coli 50S ribosomal subunit L32 (Figure ). L32 is an ideal CAN choice for several reasons. First, the 56-residue L32 contains a reasonably placed Thr-Ala ligation junction (Figure ). Similar to the Val thioesters used above, Thr thioesters are sterically hindered and exhibit slow NCL kinetics. Furthermore, L32 lacks any native Cys residues, so desulfurization must be performed after NCL to produce native L32. This protein allows us to test four reactions (SPAAC, CAN, desulfurization, and Ddap linker cleavage) in a one-pot fashion (Scheme 2). Finally, L32-C (45-57) conveniently bears a Lys residue near its N-terminus, and L32-N (2-44, removed initiator Met) contains Lys residues both proximal and distal to the C-terminal thioester, allowing us to investigate the impact of Ddap linker placement on CAN reaction rate (Scheme 2). Following the same procedure for the model peptide control reactions, we first performed a representative control NCL between peptide 12a (L32-N 2-44) and 13a (L32-C 45-57) that was nearly complete after 48 h (Figures , and S85-90), with 70% of the peak area corresponding to ligated 14a (Figure ). Notably, a significant amount of 12a was lost due to thioester hydrolysis.