Synthesis, conformation, and immunosuppressive activity of CLX and its analogues

Abstract The CLX peptide isolated from flax seed has a sequence cyclo‐(PPFFILLX), where X is a nonproteinaceous amino acid residue, (2S,4R) 4‐amine‐N‐methylproline. Picur, B.; Lisowski, M.; Siemion, I.Z. Letters Pept Sci 1998, 5, 183–187. The structure of X strongly suggests that this natural amino acid plays a role of the dipeptide moiety with a nonplanar cis peptidomimetic bond. The X residue contains two asymmetrical carbons and thus can appear in four configurations: (2S,4R), (2S,4S), (2R,4S), and (2R,4R). All four diastereoisomers of X were synthesized and characterized as trifluoroacetates of 4‐phtalimido‐ N ‐methylproline benzylamides. Their full physicochemical characteristics are presented in this article. The synthesis of linear and cyclic analogues of CLX containing all four possible diastereoisomers of X was performed. Additionally, analogues with γ‐aminobutyric acid (GABA) and glycyl‐ N ‐methyl‐glycine dipeptide [G(Me)G] substituted for X were synthesized. The obtained peptides were purified using HPLC, examined by ESI/MS, and then studied by CD spectroscopy. They were also tested for immunosuppressive activity (PFC in vitro ). All of them revealed diverse immunosuppressive activity, however, lower than that of cyclolinopeptide A (CLA) Wieczorek, Z.; Bengtsson, B.; Trojnar, J.; Siemion, I.Z. Peptide Res 1991, 4, 275–283. and cyclosporine A (CsA). Ellis, G.P.; West, G.B. Progress Med Chem 1988, 25, 1–33. The structure of CLX with (2S,4R) 4‐amino‐ N ‐methylproline was determined by 2‐D NMR methods. All amide bonds are in the trans configuration. The cis peptidomimetic group δ‐CH 2 ‐N(CH 3 )‐ is exposed to the outside of the CLX molecule. The peptide contains two loops similar to β‐turns of type IV. Chou, P.Y.; Fasman, G.D. J Mol Biol 1977, 115, 136–715 and has the extended shape flanked by F3 and L7 residues with significant side chain flexibility. © 2003 Wiley Periodicals, Inc. Biopolymers 70: 497–511, 2003