Mass spectrometry analysis for the determination of side reactions for cyclic peptides prepared from an Fmoc/t}Bu/Dmab protecting group strategy

Abstract

The Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Group (PSRG) proposed for their annual study that laboratory members prepare cyclo(Tyr-Glu-Ala-Ala-Arg-DPhe-Pro-Glu-Asp-Asn) according to the following synthetic pathway: (i) side-chain anchoring Fmoc-Asp(OH)-ODmab to a Rink amide resin; (ii) linear assembly; (iii) Dmab and Fmoc removal, respectively; (iv) on-resin cyclization with an uronium-based coupling reagent; (v) final cleavage/deprotection with TFA. Based upon this protocol, a variety of side-products were identified:(i) N-terminal guanidine formation; (ii) C-terminal piperidyl amide formation; and (iii) a novel C-terminal benzyl amide-guanidine derivative that formed due to a chemical reaction between the Dmab protecting group and the uronium-based coupling agent. The elemental composition and subsequent structure determination of this unexpected derivative was established by tandem mass spectrometry, i.e. low energy collision-induced dissociation experiments with fragment mass determination within 5 ppm.