The Hydrogen Bond Mimic Approach:  Solid-Phase Synthesis of a Peptide Stabilized as an α-Helix with a Hydrazone Link

Abstract

Proteins are characterized by extensive hydrogen bonding that defines regular and irregular substructures. However, hydrogen bonds are weak and insufficient for stabilizing peptide conformation in water. Consequently, the biological activity of peptides is reduced. This led us to test whether a covalent mimic of the hydrogen bond could be used to stabilize peptide conformation in water. A solid-phase synthesis is described for replacing a main-chain hydrogen bond (NH f OdCRNH) with a hydrazone link (N-Nd CH-CH 2 CH 2 ) in peptides. The synthesis is easy to implement, rapid, and capable of high yields. The replacement of a putative (i + 4 f i) hydrogen bond with the hydrazone at the N terminus of acetyl-GLAGAEAAKA-NH 2 (1) to give [JLAZ]AEAAKA-NH 2 (2) converts it to a full-length R-helix in water at ambient temperature as indicated by NMR spectroscopy. The observation of weak d RN (i, i + 3), medium d NN (i, i + 1), and strong d R (i, i + 3) NOEs that span 2 establish the formation of a full-length R-helix in water. J RN coupling constants and amide proton chemical shifts and temperature coefficients are consistent with a model involving rapidly equilibrating extended and R-helical conformers. Substituting L-alanine with L-proline to give [JLPZ]AEAAKA-NH 2 (3) enhances R-helix nucleation and shifts the equilibrium further toward full-length R-helix. The hydrazone link displays many of the properties required of a hydrogen bond mimic and could find use as a general means for constraining peptides to a range of biologically relevant conformations.

Downsizing human, bacterial, and viral proteins to short water-stable alpha helices that maintain biological potency

“…The 116 residue HIV-Rev protein binds to RNA with Kd approximately 1 nM (28). Nociceptin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) is an agonist at only nM concentrations (25). We also found the helix-constrained compounds to be more stable in human serum than their linear analogues, the latter being typically degraded within 1 h whereas the constrained peptides were stable for >24 h, except for the exocyclic residues, which were cleaved by proteases over several hours.…”

“…1B and Table S5). This exceptional structural and chemical stability, achieved without incorporating any unnatural components, offers significant advantages over other methods being trialled for protein helix mimicry (5)(6)(7)(8)(9)(10).…”

“…This relatively low helicity for mimetic 26 versus mimetics 20, 22, and 24 is attributed to the unstructured "triggering" message domain at the N-terminus, as well as accumulation of positive charges on one helix face. Nevertheless, the induction of at least some helix structure in this peptide suggested that it should be more active than nociceptin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Function was investigated by nociceptin(1-17) induced intracellular ERK phosphorylation in mouse Neuro-2a neuroblastoma cells (Fig.…”

“…However, short synthetic peptides of this length are usually not thermodynamically stable helices in water and adopt only random structures (4). Techniques developed as generalized strategies for stabilising or mimicking short peptide α-helices have been limited by lack of helical structure in water, by sequence or context dependent helicity, by low biological potency or by poor target specificity (5)(6)(7)(8)(9)(10). Either these techniques have failed to induce α-helicity through an entire peptide, had complex synthetic routes that limited broad utility, or have not been applicable across a wide range of biological sequences and receptor types.…”

Effective α-Helix Stabilization via Hexenyl Propionate Cross-Link

“…Therefore, a variety of strategies to enforce short peptides to adopt an α-helical conformation via chemical modifications have been actively investigated. [3][4][5][6][7][8][9][10][11][12] These methods have been valuable chemical tools for better understanding protein folding, probing signal transduction pathways, and providing important information useful for the development of therapeutics.…”