Synthesis and characterization of water-soluble macrocyclic peptides stabilizing protein α-turn

Abstract

Short peptides composed of naturally occurring amino acids are usually unstructured in aqueous media. The installation of covalent constraints within their side chains or backbones, resulting in the formation of macrocyclic peptides, is an appealing approach to stabilize them in defined secondary structures. Therefore, with the objective to stabilize α-turn conformation, we designed, synthesized and characterized constrained 13-membered macrocyclic peptides. Their design was inspired by previous work using the replacement of a hydrogen bond by a covalent bond, for the stabilization of α-helical secondary structures. Their synthesis employed our recently published solid-phase method based on Fukuyama-Mitsunobu alkylation reactions. We report herein an optimized synthesis leading to three water-soluble 13-membered macrocyclic peptides 10a-c, including respectively two, one and zero glycine residues. They were characterized by CD and NMR, which indicated the presence of equilibrating conformers. The detailed conformational analysis was based on extensive NMR and molecular dynamics studies. We found that the peptide without glycine residues 10c was mostly present as slowly interconverting conformers whereas the peptide with two glycine residues 10a was mostly present as rapidly interconverting conformers. We did not find a good match between the conformers of 10a and α-turns occurring in proteins, due to the high flexibility of the glycine backbone. Interestingly, we found that the major conformer of 10c accurately matched the "non-classical" or "tight" α-turn of type II-α, with a RMSD value of 0.42 Å for heavy atoms constituting the macrocycle. This is, to the best of our knowledge, the first molecule reported to mimic this type of α-turn found in proteins.

H-Bond Surrogate-Stabilized Shortest Single-Turn α-Helices: sp2 Constraints and Residue Preferences for the Highest α-Helicities

“…Various structural modifications have been designed to overcome this entropic disfavor and to create a proclivity for the α-helical conformation in short peptides. These include the use of helix-nucleating templates, ,− unnatural amino acids, ,− metal clamps, − noncovalent − and covalent ,,,,− side-chain linkers, and covalent hydrogen-bond surrogates (HBSs). ,− Among these, the HBS strategy presents the advantage of not perturbing the native side-chain molecular recognition surface, which is essential for eventual functional mimicry. But an efficient HBS model is yet to be designed for constraining tetrapeptides in single α-helical turns with predictable high helicities.…”

Bicyclic Pyrrolidine-Isoxazoline γ Amino Acid: A Constrained Scaffold for Stabilizing α-Turn Conformation in Isolated Peptides

“…In the amide bond region, two negative minima at 225 and 208 nm, with the same intensity, and a slightly positive band at 200 nm were also observed. The presence of the exciton splitting of the π → π * transition band suggested that a α-turn conformation was present (Wang et al, 2018), although in short peptides it is difficult to correctly evaluate the intensity of Cotton effects with respect to secondary structure motifs (Chin et al, 2002; Wang et al, 2018).…”

“…Our results indicated that (+)-(3a R 6a S )- 1 enantiomer, in combination with glycine, is effective in stabilizing the α-turn conformation in peptides (Figure 1). This structural motif occurs quite often in many key sites of proteins, such as enzyme active site, and metal binding domains (Wintjens et al, 1996), although few molecules are known to mimic or stabilize it on isolated peptides (Kelso et al, 2004; Hoang et al, 2011; Krishna et al, 2014; Wang et al, 2018.). Our results make thus the unnatural γ-amino acid 1 of particular interest for future development of bioactive peptidomimetics.…”

Large Scale Synthesis of Native Turn and Helix Mimics Stabilized by a Generic Hydrogen Bond Surrogate

“…In these models, the i+1 st residue of the turn is also replaced, apart from the hydrogen bond. Broussy and co-workers 46 , Alewood and co-workers 47 and Prabhakaran and co-workers 48 developed the propane, substituted-propane and an additional exocyclic sp² hybridization of i+1 st residue nitrogen respectively, as HBS models. Here, exclusively the hydrogen bond is replaced by the HBS model and all residues in the HBS-constrained turn are retained.…”

“…Add 10.1 g of potassium carbonate (26.4 mmol, 2 eq.) 46. Then add 2.03 ml of thiophenol (19.8 mmol, 1.5 eq.)…”