Solid-phase synthesis of backbone-cyclized β-helical peptides

Abstract

This paper describes the synthesis and purification of two 22-residue cyclic peptides, cyclo{[( l-Val- d-Val) 4-( l-Val- d-Pro-Gly)] 2-} 3 and cyclo{[( d-Leu- l-Leu) 4-( d-Leu- l-Pro-Gly)] 2-} 4, that were designed to fold into double-stranded antiparallel β-helical structures. Due to intramolecular hydrogen bonding and the conformational constraints imposed by the two reverse-turn segments ( d-Pro-Gly and l-Pro-Gly, respectively), the linear precursors to 3 and 4 (lin- 3 and lin- 4) were expected to adopt preorganized conformations that would bring the N and C termini close together and thereby favor ring closure. Precursors lin- 3 and lin- 4 were constructed by stepwise Boc solid-phase peptide synthesis using the commercially available alkanesulfonamide ‘safety-catch’ linker and cyclized head-to-tail via the method of cleavage-by-cyclization. The crude cyclic peptides were highly hydrophobic and contained minor impurities that could not be removed solely by reversed-phase HPLC (RP-HPLC); however, two-step purification—first by RP-HPLC with i-PrOH/water gradients, followed by gel-permeation chromatography (GPC) on Sephadex LH-20 with CHCl 3/MeOH—afforded both peptides in pure form (≥95% by 1H NMR) and in acceptable yield (23%). Subsequent 1H NMR experiments supported the expected structures of 3 and 4. The successful formation of the 66-membered rings of 3 and 4 is consistent with the notion of conformational preorganization in the linear precursors; furthermore, the protocols for synthesis and purification described should prove useful for preparing additional cyclic β-helical peptides, including longer peptides and peptides having polar residues.