The first obstacle to developing CAN was generating a robust, SPPS-compatible method for installing azide and DBCO cycloaddition partners. While azide-containing peptides can routinely be synthesized via SPPS, DBCO peptides have only been synthesized using post-cleavage couplings, such as thiol-maleimide or amine-NHS ester. These post-cleavage workarounds are necessary because DBCO undergoes an inactivating rearrangement under the strongly acidic conditions, 95% trifluoroacetic acid (TFA), used for peptide deprotection and cleavage from the resin. This rearrangement has been demonstrated in DBCO and related biarylazacyclooctynones and is thought to result from an acid-catalyzed 5-endo-dig cycloisomerization. Longer peptides are unlikely to have a single primary amine or thiol for post-cleavage couplings, so DBCO needs to be incorporated during Fmoc-SPPS. While DBCO can withstand lower TFA concentrations (<30%), most commonly used protecting groups and resins require higher TFA for efficient removal. We set out to investigate the stability of DBCO in standard TFA cleavage conditions using the model peptides H-C(StBu)GK(DBCO)ENTWY-R (R = NH2 1a, OH 1b, or NHNH2 1c; see Table for all peptide sequences) and Ac-RRRYSTEVEK(DBCO)NV-NHNH2 2a (DBCO coupled to Lys side chains and Cys protected with StBu to prevent potential thiol-yne reactions). Our initial attempts to synthesize 1a via Fmoc-SPPS using a standard TFA peptide cleavage cocktail failed due to formation of an undetermined DBCO-related degradation product (Figure ). 2a was found to have the correct molecular weight via LC/MS analysis (Figure ). However, the DBCO moiety was unreactive when incubated with 6-azidohexanoic acid (Figure ), suggesting it had undergone acid-catalyzed 5-endodig cycloisomerization. We did observe formation of the triazole through SPAAC before resin cleavage (Figure ; see Table for symbols used to denote common modifications such as triazole), indicating that DBCO degraded during cleavage and not during on-resin coupling.