As a proof-of-concept for CAN, we then synthesized model peptides 5b and 6b, each functionalized with the traceless Lys linker Ddap, a single Lys (to enhance solubility), and azide or DBCO as described in Scheme 1B. Equal amounts of purified 5b and 6b were dissolved together in 6 M GnHCl at pH 3 to undergo SPAAC (Figures and S53-58). Importantly, to the best of our knowledge, this is the first demonstration of SPAAC in denaturing conditions using guanidine. HPLC analysis revealed the presence of two triazole cycloadduct peptides (both referred to as 8). In this instance, the two distinguishable isobaric peaks are attributed to the two anticipated triazole regioisomers (Figures and). Using comparable conditions to the non-templated reaction, the C-terminal hydrazide was converted to an MTG thioester in situ prior to performing CAN (0.5 mM 8, Figure ). Excitingly, the ligation to form 9 was complete at 4 h (Figures and S59-60), and the relative peak area of 9 from this CAN reaction was much higher (85%) compared to the non-templated control NCL (29%), attributable in part to significantly diminished side products (Figure ). We then cleaved the Ddap linkers of 9 in one pot via the addition of hydroxylamine to ~1 M at pH 6.75 for 2 h (Figures and). The native, ligated product without Ddap linkers was easily purified by HPLC to afford 7 (Figures and). CAN with 8 was then repeated for a more precise kinetic analysis (Figures and).