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The effects of inserting unsubstituted -amino acids into the strand segments of model -hairpin peptides was investigated by using four synthetic decapeptides, Boc-Leu-Val-Xxx-Val-D-Pro-Gly-Leu-Xxx-Val-Val-OMe: peptide 1 (Xxx=Gly), peptide 2 (Xxx=$\beta$Gly=$\beta$ahGly=homoglycine, -$\beta$glycine), peptide 3 (Xxx=$\gamma$Abu=-$\gamma$aminobutyric acid), peptide 4 (Xxx=$\delta$Ava=-$\delta$aminovaleric acid). $^1H\hspace{2mm}NMR$ studies (500 MHz, methanol) reveal several critical cross-strand NOEs, providing evidence for -$\beta$hairpin conformations in peptides 2-4. In peptide 3, the NMR results support the formation of the nucleating turn, however, evidence for cross-strand registry is not detected. Single-crystal X-ray diffraction studies of peptide 3 reveal a -$\beta$hairpin conformation for both molecules in the crystallographic asymmetric unit, stabilized by four cross-strand hydrogen bonds, with the $\gamma$Abu residues accommodated within the strands. The D-Pro-Gly segment in both molecules (A,B) adopts a type II -$\beta$turn conformation. The circular dichroism spectrum for peptide 3 is characterized by a negative CD band at 229 nm, whereas for peptides 2 and 4, the negative band is centered at 225 nm, suggesting a correlation between the orientation of the amide units in the strand segments and the observed CD pattern.

The incporation of beta-amino acid residues into the strand segments of designed beta-hairpin leads to the formation of polar sheets, since in the case of beta-peptide strands, all adjacent carbonyl groups point in one direction and the amide groups orient in the opposite direction. The conformational analysis of two designed peptide hairpins composed of alpha/beta-hybrid segments are described: Boc-Leu-betaPhe-Val-(D)-Pro-Gly-Leu-betaPhe-Val-OMe (1) and Boc-betaLeu-Phe-betaVal-D-Pro-Gly-betaLeu-Phe-betaVal-OMe (2). A 500-MHz 1H-NMR (nuclear magnetic resonance) analysis in methanol supports a significant population of hairpin conformations in both peptides. Diagnostic nuclear Overhauser effects (NOEs) are observed in both cases. X-ray diffraction studies on single crystals of peptide 1 reveal a beta-hairpin conformation in both the molecules, which constitute the crystallographic asymmetric unit. Three cross-strand hydrogen bonds and a nucleating type II' beta-turn at the D-Pro-Gly segment are observed in the two independent molecules. In peptide 1, the betaPhe residues at positions 2 and 7 occur at the nonhydrogen-bonding position, with the benzyl side chains pointing on opposite faces of the beta-sheet. The observed aromatic centroid-to-centroid distances are 8.92 A (molecule A) and 8.94 A (molecule B). In peptide 2, the aromatic rings must occupy facing positions in antiparallel strands, in the NMR-derived structure. Peptide 1 yields a normal "hairpin-like" CD spectrum in methanol with a minimum at 224 nm. The CD spectrum of peptide 2 reveals a negative band at 234 nm and a positive band at 221 nm, suggestive of an exciton split doublet. Modeling of the facing Phe side chains at the hydrogen-bonding position of a canonical beta-hairpin suggests that interring separation is approximately 4.78 A for the gauche+ gauche- (g+ g-) rotamer. A previously reported peptide beta-hairpin composed of only alpha-amino acids, Boc-Leu-Phe-Val-D-Pro-Gly-Leu-Phe-Val-OMe also exhibited an anomalous far-UV (ultraviolet) CD (circular dichroism) spectrum, which was interpreted in terms of interactions between facing aromatic chromophores, Phe 2 and Phe 7 (C. Zhao, P. L. Polavarapu, C. Das, and P. Balaram, Journal of the American Chemical Society, 2000, Vol 122, pp. 8228-8231).

Conformational studies on the synthetic 11-aa peptide t -butoxycarbonyl (Boc)-Val-Ala-Phe-α-aminoisobutyric acid (Aib)-( R )-β 3 -homovaline (βVal)-( S )-β 3 -homophenylalanine (βPhe)-Aib-Val-Ala-Phe-Aib-methyl ester (OMe) (peptide 1; βVal and βPhe are β amino acids generated by homologation of the corresponding l -residues) establish that insertion of two consecutive β residues into a polypeptide helix can be accomplished without significant structural distortion. Crystal-structure analysis reveals a continuous helical conformation encompassing the segment of residues 2–10 of peptide 1. At the site of insertion of the ββ segment, helical hydrogen-bonded rings are expanded. A C 15 hydrogen bond for the αββ segment and two C 14 hydrogen bonds for the ααβ or βαα segments have been characterized. The following conformational angles were determined from the crystal structure for the β residues: βVal-5 (φ = –126°, θ = 76°, and ψ = –124) and βPhe-6 (φ =–88°, θ = 80°, and ψ =–118). The N terminus of the peptide is partially unfolded in crystals. The 500-MHz 1 H-NMR studies establish a continuous helix over the entire length of the peptide in CDCl 3 solution, as evidenced by diagnostic nuclear Overhauser effects. The presence of seven intramolecular hydrogen bonds is also established by using solvent dependence of NH chemical shifts.

The incorporation of the -amino acid residues into specific positions in the strands and -turn segments of peptide hairpins is being systematically explored. The presence of an additional torsion variable about the C()C() bond () enhances the conformational repertoire in -residues. The conformational analysis of three designed peptide hairpins composed of /-hybrid segments is described: Boc-Leu-Val-Val-DPro-Phe- Leu-Val-Val-OMe (1), Boc-Leu-Val-Val-DPro-Gly-Leu-Val-Val-OMe (2), and Boc-Leu-Val-Phe-Val-DPro- Gly-Leu-Phe-Val-Val-OMe (3). 500-MHz 1H-NMR Analysis supports a preponderance of -hairpin conformation in solution for all three peptides, with critical cross-strand NOEs providing evidence for the proposed structures. The crystal structure of peptide 2 reveals a -hairpin conformation with two -residues occupying facing, non-H-bonded positions in antiparallel -strands. Notably, Val(3 ) adopts a gauche conformation about the C()C() bond (65) without disturbing cross-strand H-bonding. The crystal structure of 2, together with previously published crystal structures of peptides 3 and Boc-Phe-Phe-DPro-Gly- Phe-Phe-OMe, provide an opportunity to visualize the packing of peptide sheets with local −polar segments× formed as a consequence of reversal peptide-bond orientation. The available structural evidence for hairpins suggests that -residues can be accommodated into nucleating turn segments and into both the H-bonding and non-H-bonding positions on the strands.

Half a century has passed since the hydrogen-bonded secondary structures of polypeptides and proteins were first recognized. An extraordinary wealth of conformational information is now available on peptides and proteins, which are formed of alpha-amino acid residues. More recently, the discovery of well-folded structures in oligopeptides containing beta-amino acids has focused a great deal of current interest on the conformational properties of peptides constructed from higher homologues (omega) of alpha-amino acids. This review examines the nature of intramolecularly hydrogen-bonded conformations of hybrid peptides formed by amino acid residues, with a varying number of backbone atoms. The beta-turn, a ubiquitous structural feature formed by two residue (alphaalpha) segments in proteins and peptides, is stabilized by a 10-atom (C10) intramolecular 4--1 hydrogen bond. Hybrid turns may be classified by comparison with their alphaalpha counterparts. The available crystallographic information on hydrogen-bonded hybrid turns is surveyed in this review. Several recent examples demonstrate that individual omega-amino acid residues and hybrid dipeptide segments may be incorporated into the regular structures of alpha-peptides. Examples of both peptide helices and hairpins are presented. The present review explores the relationships between folded conformations in hybrid sequences and their counterparts in all alpha-residue sequences. The use of stereochemically constrained omega-residues promises to expand the range of peptide design strategies to include omega-amino acids. This approach is exemplified by well-folded structures like the C12 (alphagamma) and C14 (gammagamma) helices formed in short peptides containing multiply substituted gamma-residues. The achiral gamma-residue gabapentin is a readily accessible building block in the design of peptides containing gamma-amino acids. The construction of globular polypeptide structures using diverse hybrid sequences appears to be a realistic possibility.

The crystal structures of four protected beta-amino acid residues, Boc-(S)-beta3-HAla-NHMe (1); Boc-(R)-beta3-HVal-NHMe (2); Boc-(S)-beta3-HPhe-NHMe (3); Boc-(S)-beta3-HPro-OH (6) and two beta-dipeptides, Boc-(R)-beta3-HVal-(R)-beta3-HVal-OMe (4); Boc-(R)-beta3-HVal-(S)-beta3-HVal-OMe (5) have been determined. Gauche conformations about the C(beta)-C(alpha) bonds (theta approximately +/-60 degrees) are observed for the beta3-HPhe residues in and all four beta3-HVal residues in the dipeptides and . Trans conformations (theta is approximately 180 degrees) are observed for beta3-HAla residues in both independent molecules in and for the beta3-HVal and beta3-HPro residues in and , respectively. In the cases of compounds , molecules associate in the crystals via intermolecular backbone hydrogen bonds leading to the formation of sheets. The polar strands formed by beta3-residues aggregate in both parallel (1,3,5) and antiparallel (2,4 fashion. Sheet formation accommodates both the trans and gauche conformations about the C(beta)-C(alpha) bonds.