Figure 1.

Synthesis of circular peptide probes. (A) (1.) Cyclotides and probes were synthesized as linear thioester precursor peptides using a Boc-solid-phase peptide synthesis protocol. (2.) Peptide precursor was released from the resin and globally de-protected by hydrogen fluoride (HF) treatment. The cyclic native folded peptide was obtained after native chemical ligation potentially supported via the thia-zip mechanism described for cysteine-rich peptides in oxidative folding conditions. (3., 4.) Transthioesterification forms an intramolecular thiolactone intermediate. (5.) The proposed thia-zip mechanism brings the N-terminal cysteine residue in close proximity to the C-terminal thiolactone intermediate. (6., 7.) An irreversible S,N-acylshift forms a native peptide bond and recovers all thiols. (8.) The native cystine-knot fold is achieved in oxidative folding buffers. (B) Native folded cyclotides exhibit a shift in chromatographic retention compared to its reduced progenitor and all identified non-native folded species (marked with asterisks) as indicated by the A₂₈₀ UV traces of the peptide before and after oxidative folding. Synthesized and oxidative folded cyclotide probes [T20K]kB1 (C) and [T20K,W23Bpa]kB1 (D) were purified via preparative HPLC and evaluated for their molecular weight using MALDI-TOF mass spectrometry and for purity by analytical HPLC analysis. Mass spectra and UV chromatograms (A₂₈₀) are presented. Retention time and molecular weight (monoisotopic [M+H]⁺) are indicated (small inserts).