To investigate CAN for L32, L32-N (2-44) was functionalized with Ddap followed by a single Lys and 6-azidohexanoic acid at Lys 37 (closer distance, 12b, L32-close) or Lys (farther distance, 12c, L32-far) (Scheme 2). Nle was substituted for the sole Met , as cleavage cocktails containing NH4I additive were found to cause azide degradation (Figure ), and we desired to avoid Met oxidation during peptide cleavage. L32-C (45-57) was functionalized at Lys This new method to directly synthesize DBCO peptides via Fmoc-SPPS will be a useful tool for many peptide chemists. For example, peptide and protein nanoparticle conjugates have exciting biomedical applications (e.g., drug delivery and in vivo imaging), but there is a need to develop additional methods enabling site-specific incorporation of peptides onto nanoparticles. DBCO peptides can be easily conjugated to azide-functionalized nanoparticles, and this method will enable larger and more sequence-diverse DBCO peptides to be incorporated. Additionally, several methods exist to incorporate azides into proteins via engineered tRNA synthetases or Met replacement with azide-containing analogs, enabling DBCO peptides to tag recombinant azido-proteins for various applications (e.g., proteome labeling and imaging). Although cyclooctynes can be genetically encoded into proteins, these methods are more challenging to implement compared to azide. The finding that (MeCN)4CuBF4 prevents acid-catalyzed 5-endo-dig cycloisomerization in DBCO, and perhaps other alkyne-derived isomerization reactions, 105 is an important observation.