Helix Formation in Preorganized β/γ-Peptide Foldamers: Hydrogen-Bond Analogy to the α-Helix without α-Amino Acid Residues

Abstract

We report the first high-resolution structural data for the β/γ-peptide 13-helix (i,i+3 C=O···H-N H-bonds), a secondary structure that is formed by oligomers with a 1:1 alternation of β- and γ-amino acid residues. Our characterization includes both crystallophaphic and 2D NMR data. Previous studies suggested that β/γ-peptides constructed from conformationally flexible residues adopt a different helical secondary structure in solution. Our design features preorganized β- and γ-residues, which strongly promote 13-helical folding by the 1:1β:γ backbone.

Identification of new types of foldamers with strong and discrete secondary structural propensities is a subject of ongoing research.¹ These studies enhance our understanding of the relationship between local conformational preferences and molecular shape. In addition, new folding patterns can be valuable for specific applications.^2,3 Foldamers that contain more than one type of subunit, i.e., oligomers that have heterogeneous backbones, have been a subject of extensive recent interest.^1e Most examples involve combination of α-amino acid residues with other types of subunits, including those derived from β-⁴ or γ-amino acids⁵ or other building blocks.⁶ Heterogeneous backbones that do not include α-amino acid residues have received relatively limited attention,^5e,7 perhaps because α-amino acids are far more available than are other building blocks. Backbones with alternating β- and γ-amino acid residues (β/γ-peptides) are of particular interest because a β/γ-dipeptide has the same number of atoms between the N- and C-termini as an α-tripeptide.^5b An extended β/γ-peptide can in principle form a helix containing 13-membered ring backbone H-bonds (C=O(i)--H-N(i+3)) that are analogous to the 13-membered ring backbone H-bonds characteristic of the α-helix (C=O(i)--H-N(i+4)). However, Sharma, Kunwar et al.^5e have recently reported that flexible β/γ-peptides adopt a different type of helical conformation in solution. Here we show that β/γ-peptides containing appropriately preorganized subunits do indeed adopt the 13-helix in solution and the solid state.

The β/γ-peptide 13-helix is predicted by Hofmann et al.^5d to have g⁺,g⁺ or g⁻,g⁻ local conformations about the C_α−C_β (ζ) and C_β−C_γ (θ) bonds in the γ-residues and a C_α−C_β torsion angle of ~90° in the β-residues. Based on these predictions and available data for the conformational propensities of constrained β-and γ-residues in other contexts, we concluded that combining (R,R,R) γ-residue 1 (Figure 1), which has recently become available,^5i,8 with (R,R)-trans-2-aminocyclopentanecarboxylic acid (ACPC, 2) should favor formation of the left-handed β/γ-peptide 13-helix (the right-handed helix should be favored by residues with S configurations). This hypothesis was tested by preparation and analysis of tetramer 3, pentamer 4 and hexamer 5 (Figure 1).

Figure 1.

Structures of β/γ peptides 3, 4, 5 (arrows indicate H-bonds in the crystal structures of 3 and 4)

The crystal structure of β/γ-peptide 3 contains two molecules in the asymmetric unit; the two conformations are very similar (Figure 2). Each independent molecule forms one 13-atom H-bonded ring, involving the NH group of the second ACPC residue and the carbonyl of the N-terminal Boc group. The other possible 13-atom ring H-bond does not form in either case [N--O distance~ 4.9Å]; instead, each molecule contains an 8-atom ring H-bond involving the carbonyl of the first γ-residue and the NH group of second γ-residue. Despite this deviation from the 13-helical H-bonding pattern, the backbone torsion angles for the β- and γ-residues in 3 generally fall in ranges predicted by Hofmann et al.^5d for the β/γ-peptide 13-helix.⁹

Figure 2.

Crystal structures of 3 (left) and 4 (right): (top) views perpendicular to helical axis; (bottom) views along the helical axis.

Pentamer 4, containing β- and γ-residues with S configurations, adopts the right-handed 13-helix in the crystalline state. All three of the possible C=O(i)--H-N(i+3) H-bonds are formed (Figure 2). Table 1 compares backbone torsion angles for the β- and γ-residues in pentamer 4 with analogous values from the computational work of Hofmann et al.^5d and from the NMR analysis of flexible β/γ-peptides in organic solvent by Sharma, Kunwar et al.^5e The preorganized γ-residues in 4 display g⁺,g⁺ local conformations about the C_α−C_β (ζ) and C_β−C_γ (θ) bonds, and ψ and φ near −120°, with a somewhat wider distribution for the latter torsion angle. These values are consistent with the predictions for the 13-helical conformation from Hofmann et al.^5d In contrast, the helical conformations deduced via NMR for flexible β/γ-peptides feature opposite signs for the ζ and θ torsion angles (g⁻,g⁺), and opposite signs for the ψ and φ torsion angles. The helical conformation deduced for these flexible β/γ-peptides has a distinctive H-bonding pattern with two types of interaction: C=O_γ (i)--H-N_γ (i−1) and C=O_γ (i)--H-N_γ (i+3).

Table 1.

Backbone Torsion Angles (deg) ^† from β/γ Peptides

Peptides	residues	φ	θ	ζ	ψ
β/γ pentamer 4	β	−107.7	93.3		−128.3
	γ	−134.7	60.1	59.8	−121.0
	β	−133.6	113.5		−85.7
	γ	−147.3	57.9	46.5	−129.8
	β	−167.9	141.4		−155.0
computational Study ‡,5d	β	89.1	−94.1		121.9
	γ	124.9	−60.4	−62.2	132.0
flexible β/γ (NMR)5e	β	120	60		0
	γ	120	−60	60	−120

^†Nomenclature for the backbone torsion angles in β,γ-residues is as described in Figure 1.

^‡Average backbone torsion angles

Hexamer 5 did not produce high-quality crystals, but 2D ¹H NMR analysis in pyridine-d₅ solution indicated that the 13-helix is significantly populated under these conditions. Among the unambiguous NOEs involving backbone protons, six strong NOEs were observed between protons from residues that are not adjacent in the sequence: C_βH(1)--NH(3), C_βH(1)--C_αH(3), C_γH(2)--NH(4), C_βH(3)--NH(5), C_βH(3)--C_αH(5), and C_γH(4)--NH(6) (Figure 3). These NOEs are consistent with intramolecular proton-proton distances in the crystal structure of pentamer 4: C_βH(1)--NH(3) = 3.5 Å, C_βH(1)--C_αH(3) = 2.7 Å, C_γH(2)--NH(4) = 2.8 Å, C_βH(3)--NH(5) = 2.3 Å and C_βH(3)--C_αH(5) = 2.2 Å. Thus, the three NOE patterns observed for 5, C_βH(i)--NH(i+2) and C_βH(i)--C_αH(i+2) for β-residues and C_γH(i)--NH(i+2) for γ-residues, appear to be general indicators of β/γ-peptide 13-helical secondary structure.

Figure 3.

Characteristic NOEs patterns observed for the 1:1 β/γ-peptide hexamer 5 in pyridine-d₅.

The β/γ-peptide helix we have documented is interesting because of its relationship to the α-helix formed by pure α-residue backbones. Both helices contain 13-atom ring H-bonds. Detailed comparison of the two helices reveals further similarities: both have a rise-per-turn of 5.4 Å, and the radii are similar (2.5 vs. 2.3 Å).⁹ These parameters suggest that the β/γ-peptide 13-helix may be a promising scaffold for functional mimicry of natural α-helices.^2b,3

Our results show that appropriately preorganized residues promote the formation of the 13-helical conformation in short β/γ-peptides. This secondary structure was anticipated (along with alternative helices) in computational studies,^5c,d and hints of 13-helical propensity can be found in the local conformations observed in crystal structures for isolated β-γ segments,5b,g but the only previous analysis of β/γ-peptide oligomer folding indicated the formation of a different helical conformation, containing both 11- and 13-membered ring H-bonds.^5e Conformationally constrained β-amino acid residues have been shown to induce novel secondary structures,1a,e^,10 and the present studies highlight the prospect that constrained γ-amino acid residues will be similarly useful in controlling molecular shape.