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

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 gamma-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 delta-CH(2)-N(CH(3))- is exposed to the outside of the CLX molecule. The peptide contains two loops similar to beta-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.