To investigate the impact of linker length on the CAN reaction rate, peptides 5c and 6c were each functionalized with Ddap, a single Lys, and a PEG8 linker before addition of their respective SPAAC partners. The PEG8 spacers double the total extended distance between the triazole-linked peptides. Using the same conditions as with 5b and 6b, the PEG8 peptides first underwent SPAAC to give triazole-linked 10 (2.5 mM both peptides at 37°C for 2 h, Figures S66-71) before performing CAN at 0.5 mM to give ligated product 11 (Figures ). This CAN with PEG8 spacers was ~3-fold slower than the non-PEGylated CAN (Figure ).