Evaluation of biologically relevant short alpha-helices stabilized by a main-chain hydrogen-bond surrogate

Abstract

We previously reported the design and synthesis of a new class of artificial alpha-helices in which an N-terminal main-chain hydrogen bond is replaced by a carbon-carbon bond derived from a ring-closing metathesis reaction [Chapman, R. N.; Dimartino, G.; Arora, P. S. J. Am. Chem. Soc. 2004, 126, 12252-12253]. Our initial study utilized an alanine-rich sequence; in the present manuscript we evaluate the potential of this method for the synthesis of very short (10 residues) alpha-helices representing two different biologically relevant alpha-helical domains. We extensively characterized these two sets of artificial helices by NMR and circular dichroism spectroscopies and find that the hydrogen-bond surrogate approach can afford well-defined short alpha-helical structures from sequences that do not spontaneously form alpha-helical conformations.

Figure 1

Nucleation of short α-helices by replacement of an N-terminal i and i + 4 hydrogen bond (C=O–H–N) with a covalent link (C=X–Y–N). The hydrogen-bond surrogate-based (HBS) α-helices contain a carbon–carbon bond derived from an olefin metathesis reaction.

Figure 2

Unconstrained peptides and HBS α-helices used to evaluate the hydrogen-bond surrogate strategy.

Figure 3

(a and b) Circular dichroism spectra of unconstrained peptides 1 and 2 and their HBS analogues 3 and 4, respectively. (c) Effect of temperature on the stability of HBS α-helices 3 and 4. The CD spectra were obtained in 10% TFE/PBS.

Figure 4

NOESY correlation charts and cross-sections of NOESY spectra for 3 (a, b) and 4 (c, d). The alanine-3 residues in both artificial helices are N-alkylated. Filled rectangles indicate relative intensity of the NOE cross-peaks. Empty rectangles indicate NOE that could not be unambiguously assigned because of overlapping signals.

Figure 5

Temperature-dependent chemical shifts (a, c) and hydrogen–deuterium exchange plots (b, d) for backbone amide protons in 3 and 4.

Figure 6

NMR-derived structures of HBS α-helix -3. (a and b) Side and top views of 20 lowest energy structures, and (c) lowest energy structure showing hydrogen-bonding pattern within the artificial α-helix. -All carbon, nitrogen, and oxygen atoms are shown in green, blue, and red, respectively, with the exception of the trans alkene group which is shown in magenta.

Figure 7

Ramachandran plot representing the 20 lowest energy NMR structures. A vast majority of the values fall within the ideal α-helix region with the exception of the φ and ψ values for the Q1 residue. This map was generated within the MolMol molecular graphics program.

Scheme 1

Solid Phase Synthesis of Peptides 1 and 2 and HBS α-Helices 3 and 4