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for sequences) revealed that b- and g-amino acid residues can be substituted for their a-amino acid counterpart at discrete position in a-helical structures without major perturbation of the overall fold.

Experimentally defined backbone torsion angles of expanded 1 ^ 4 and 1 ^ 5 H-bonded units formed by hybrid segments in these peptides (marked in bold in Table 2.1) served as starting points to generate a series of energetically favorable models of a,o-hybrid helices. For example, the 11- and 13-membered H-bonded units encompassing the b3-HPhe-Aib (b, a) and the b-HGly-g-Gly (b, g) segments in X-ray structure of 26 [175] and 28 [174] provided appropriate geometrical parameters to model 11-b,a and 13-b,g-helices. It is worth noting that a b,g-dipeptide repeat is isostere to a a-tripeptide and that the 13-helical backbone proposed for b,g-peptides (f = -106°, 9 = 75°, c =-115° (b3-amino acid) and f =-117°, y1 = 66°, 92 = 62°, c = -120° (g-amino acid)), and also identified by quantum mechanics calculations [156] superimposes well to the a-helical backbone (RMSD value of 0.7 A). This interesting theoretical consideration will need to be verified experimentally.

Parallel to these semi-empirical studies, Reiser, Zerbe and colleagues [167] and Gellman [168-171] independently reported experimental evidence for periodic helix formation in short chain hybrid peptides consisting of alternating a- and b-amino acid residues (mixed helices reported by Sharma and Kunwar [157] have already been discussed in Section 2.4.3). In both cases, backbone pre-organization to enforce folding propensity was introduced at the b-amino acid positions with 2-aminocycloalkanecarboxylic acid residues. a,b-Peptides designed by Gellman and co-workers contained b-amino acid residues constrained with five-membered rings (S,S)-trans-ACPC and (S,S)-trans-APC (e.g. 29 and 30, Fig. 2.15).

Detailed NMR analysis of hybrid peptide 29 in CD3OH revealed a complex pattern of (i, i + 2) and (i, i + 3) inter-residue NOE connectivities that could be explained by assuming rapid interconversion between two helical conformations with H bonds in backward direction, namely a 11-helix (1 ^ 4 H bonds) and a 14/15-helix (1 ^ 5 H bonds) [168]. Although less plausible, a helical conformation with three-center backbone H bonds (both 11- and 14/15 H-bonded rings) was also proposed on the basis of the nonsequential NOEs observed. Factors governing helical folding propensity and helix type repartition in such a,b-peptide hybrids have been delineated. Increase in chain length seems to favor the 14/15 helical shape relative to the 11-helix [168]. The analogy to a-peptides which also show chain length-dependent conformational transition between 310 helix (1 ^ 4 H bonds) and a-helix (1 ^ 5 H-bonding pattern) is striking (Fig. 2.16).

a,a-Disubstitution of a-amino acids which is well known to promote helical folding among a-peptides [15] was shown to reinforce helix stability and to favor 11-helical folding of a,b-peptide hybrids [170]. Octamer 30 which consists of ACPC/Aib repeats adopts a perfect 11-helical fold in the solid state with all possible H-bonded rings present (Fig. 2.17A). In contrast, b substitution of a-aminoacid residues (e.g. Val, Ile, Thr) or substitution of acyclic b-amino acids for ACPC/APC residues is helix destabilizing [170]. Pre-organization with six-membered ring (S,S)-trans-ACHC residues does not support helix formation in

Fig. 2.15 Helix forming a,o-peptide hybrids [167-172].
Fig. 2.16 1 ^ 4 and 1 ^ 5 H-bond patterns of interconverting 11- and 14/15-helices in heterogeneous «.^-peptides such as 29 and analogy with H-bonding scheme of 31o and «-helical secondary structures.

Fig. 2.17 Helical secondary structures of peptide hybrids in the solid state (views along the helix axis and top views). (A) Right handed 11-helix formed by a,b-peptide 30 (adapted from [170]). The backbone torsion angles extracted for central residues 4 and 5 (f = -99.7°, y = 89.6°, c = -80.7° (b-amino acid) and f = -50.3°, c = -42.9° («-amino acid)) and those derived from the computer-generated model proposed by

Balaram (f = -105°, d = 80°, c = -73° (b-amino acid) and f = -62°, c = -44° («-amino acid) [172]) are in good agreement. (B) Right handed helical conformation (1 ^ 4 H-bonding pattern) of a,b,b-peptide 32 (adapted from [171]). (C) Right handed helical conformation (1 ^ 4 H-bonding pattern) of a,a,b-peptide 33 (adapted from [171]).

Fig. 2.17 Helical secondary structures of peptide hybrids in the solid state (views along the helix axis and top views). (A) Right handed 11-helix formed by a,b-peptide 30 (adapted from [170]). The backbone torsion angles extracted for central residues 4 and 5 (f = -99.7°, y = 89.6°, c = -80.7° (b-amino acid) and f = -50.3°, c = -42.9° («-amino acid)) and those derived from the computer-generated model proposed by

Balaram (f = -105°, d = 80°, c = -73° (b-amino acid) and f = -62°, c = -44° («-amino acid) [172]) are in good agreement. (B) Right handed helical conformation (1 ^ 4 H-bonding pattern) of a,b,b-peptide 32 (adapted from [171]). (C) Right handed helical conformation (1 ^ 4 H-bonding pattern) of a,a,b-peptide 33 (adapted from [171]).

a/b-peptide hybrids, probably because homogenous ACHC backbones favor H-bonding in the forward direction (see Section 2.3.1.2) [168].

In a related work [167], Reiser, Zerbe and colleagues used cis-b-aminocyclopro-panecarboxylic acids (cis-b-ACCs) substituted on the 3-position of the ring and investigated oligomers consisting of l-Ala/cis-b-ACC repeats (exemplified by hep-tamer 31). NMR studies in CD3OH and molecular modeling calculations led to the identification of a third helical fold (with 1 ^ 3 H-bonds) for a,b-peptide hybrids.

Introducing periodicity at the level of a trimer unit with b,b,«- and b,a,a-triad repeats (hexamer 32 and heptamer 33) successfully led to the identification of new helical secondary structures (Fig. 2.17B and C) [171]. While X-ray diffraction studies led to the characterization of helices with 1 ^ 4 H bonds, evidence for rapid interconversion between two helical conformations (1 ^ 4 and 1 ^ 5 H-bonding patterns) was gained from NMR studies in CD3OH.

Experimental evidence that short chain a,g-peptide hybrids also adopt helical secondary structures came from X-ray diffraction studies of tetramer 34 consisting of Aib-Gpn repeats [172]. The observed helical fold is stabilized by 12 H bonds in the backwards direction. By fixing both ethane bonds in a synclinal conformation (values for both 91 and d2 are close to 60°), b,b-disusbtitution (in Gpn) ensures conformational space restriction of g-amino acid residues.

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