Native peptide folding dominates over stereoelectronic effects of prolyl hydroxylation in loop 5 of the macrocyclic peptide kalata B1

Kalata B1 (4) is a prototypical, 29-residue, Mobius cyclotide with a cis prolyl peptide bond in loop 5. Two analogs were synthesized in which Pro(24) was substituted by trans-4-hydroxy-L-proline (peptide 5) and cis-4-hydroxy-L-proline (peptide 6). Linear peptides were assembled by solid phase peptide synthesis using Fmoc/tBu chemistry. Head-to-tail cyclization was performed using HATU, side-chain protecting groups removed and the cyclic peptides 2 and 3 isolated by RP-HPLC. Oxidation led to the formation of peptides 5 and 6, each incorporating three disulfide bonds. Analysis of TOCSY and NOESY spectra of the purified peptides enabled assignment of the backbone amide and Ha resonances. These showed a striking correlation with those of native kalata B1, indicating that folding had produced the same disulfide bridge topology. While somewhat surprising that stereoelectronic effects introduced by the hydroxyl substituents in this key region of the peptide had little impact, this reflects the strong thermodynamic driving force toward formation of the cyclic cystine knot scaffold. (C) 2014 Elsevier Ltd. All rights reserved.