Minimizing side reactions during amide formation using DIC and oxymapure in solid-phase peptide synthesis

• Safe Peptide Bond Formation using DIC and OxymaPure. • Minimization of Side-reactions. • Importance of the sequence of addition of reagents in a chemical reaction. • Five member ring formation as driven force for side-reactions. N,N’- diisopropylcarbodiimide (DIC) and OxymaPure are the reagents of choice for the formation of peptide bonds in Solid-Phase Peptide Synthesis (SPPS). The combined use of DIC and OxymaPure in model cases can be accompanied by a serious side reaction with the ultimate formation of oxadiazole and HCN. Here we demonstrate, in a practical case, that the first step of the reaction (formation of an adduct) takes place with approximately 1% respect to the amount of OxymaPure. Furthermore, we found that the second part of the side-reaction (formation of oxadiazole and HCN) can be minimized and even suppressed with an appropriate protocol for the sequence of reagent addition. Thus, the best conditions are preactivation of the Fmoc-amino acid with only DIC for 2–5 min followed by addition of the resulting mixture to the peptide-resin. OxymaPure can be added to the resin before adding the preactivation mixture or after first adding the preactivation mixture, the latter approach giving the best results. Ultimately, in standard research conditions, the combined use of DIC and OxymaPure as coupling method in SPPS is safe.