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Introducing Periodicity at the Level of a Dimer Unit in b-Peptides leads to a Remarkably Stable Mixed Helical Fold

Early studies by Seebach and co-workers established that, by analogy to b-helical a-peptides, b-peptides with an alternating substitution pattern adopt a mixed helical conformation [55, 144, 145]. The resulting structure which consists of 10- and 12-membered H-bonded pseudo rings alternating in forwards and backwards direction, respectively has only a small resulting macrodipole. This particularly robust mixed helical fold was subsequently observed for other b-peptides (Fig. 2.9) exhibiting periodicity at the level of a dimeric unit [146-148]. By Mixing b2- and b3-Amino Acids

The finding that the conformational preferences of "mixed" b-peptides composed of alternating b3- and b2-amino acid residues differed from that of the corresponding homopolymers consisting exclusively of b3- or b2-amino acid residues (i.e. the 314 helix) was largely unexpected [144]. In MeOH, b-peptides with (S)-

19 20 21

Fig. 2.9 Mixed 12/10- (70/72-) helix forming b-peptides [144-148].

19 20 21

Fig. 2.9 Mixed 12/10- (70/72-) helix forming b-peptides [144-148].

b2/b3 (or (S)-b3/b2) dipeptide repeats such as 14 and 15 did not display the CD-pattern characteristic of the 314 helix. Their CD spectra showed an intense maximum near 205 nm and no zero-crossing. The dispersion of the chemical shifts as well as the large 3J(H-C(a), H-C(b)) values (>10 Hz) in their NMR spectra recorded in pyridine and CD3OH indicated that at least one stable secondary structure was populated. ROESY experiments revealed a NOE pattern substantially different from that of the 314 helix with no i/i + 3 NOE crosspeaks and new i/i + 2 connectivities not compatible with the 314 helix. Restrained MD calculations based on NOEs and J values yielded a unique mixed helical structure with alternating intertwined 12- and 10-membered H-bonded rings. The helical screw sense was opposite to that of the related 314 helix and the overall macrodipole was strongly reduced because of alternating orientations of backbone carbonyl groups. The 12/10-structure of a low energy conformer of b2/b3-peptide 14 [55, 144] and detailed representations of the 12- and 10-membered pseudocycles are shown in Fig. 2.10. Comparison of the 10/12-helix turns with the corresponding 14-membered ring of the 314-helix reveals a common (+)-synclinal arrangement (y1 @ 60°) around the central C(a)-C(b) bond for each amino acid constituents. However, whereas both f and c angles are negative for b-amino acids in the regular 314-helix, b2-amino acid residues have a positive f value and b3-amino acid residues a positive c value in the 12/10-helix.

The tendency is that in the absence of any adjacent substituent on the two sides of an amide bond, the 12-membered turn is favored, the 10-membered being formed when the amide bond is flanked by substituted carbons (Fig. 2.10). In fully protected 15, the NH of residue 1 is engaged in the formation of an addi-

Fig. 2.10 The mixed 12/10 helical structure of b2/b3-dipeptide repeats. (A) View along the helix axis of a low energy conformer resulting from NOE-restrained modeling of 14 in pyridine [144, 145]. (B) Comparison of the 12- and 10-membered turns found in the 12/10 helix of 14 together with corresponding backbone dihedral angles.

Fig. 2.10 The mixed 12/10 helical structure of b2/b3-dipeptide repeats. (A) View along the helix axis of a low energy conformer resulting from NOE-restrained modeling of 14 in pyridine [144, 145]. (B) Comparison of the 12- and 10-membered turns found in the 12/10 helix of 14 together with corresponding backbone dihedral angles.

tional N-terminal 10-membered turn and the pattern of 10- and 12-membered turns is reversed. The strong stabilizing effect associated with N-terminus capping in mixed b-peptides was confirmed by CD studies in MeOH, the ellipticity value at 205 nm for 15 in MeOH being twice that of 14. In the case of longer b2/b3-peptides, the effect of removing the terminal protecting groups was even more pronounced with collapse of the band at 205 nm and restoration of a CD pattern, albeit weak, characteristic of the 314 helical conformation [145]. This observation may suggest the presence of equilibrating conformers and can be explained in term of unfavorable charge-pole interactions in the right-handed 12/ 10-arrangement of unprotected b2/b3-peptides, the positively charged amino terminus being rather a promoter of left-handed 314-helical structure. Information about the dynamic of (un)folding process in b2/b3-(b3/b2-) dipeptide repeats (protected or unprotected form) was gained by exploration of conformational ensembles produced by molecular dynamics (MD) simulations in explicit solvent using the GROMOS96 force-field. The results demonstrated reversible folding to the 12/10 helix and were consistent with experimental data (see also Chapter 6). [103, 104, 149-151]. Although alternate conformations such as the pure left-handed 314-helix and various partially folded conformations were also populated, the right-handed 12/10 helix was the predominant conformation in the simulation of b2/b3-(b3/b2-) dipeptide repeats in MeOH. Additional Substitution Patterns Stabilizing the Mixed 10/12- (12/10-) Helix

Theoretical studies at various levels of ab-initio MO theory (HF/6-31G* and B3LYP/ 6-31G*) provided further insight into the relative preference of b-peptides for the 314 and mixed 10/12-helices. Quantum mechanics calculations performed on un-substituted b-peptides (oligo-b-HGly peptides) revealed that the formation of the 10/12-helix is intrinsically favored over the 314 helix (by 21.4 and 4.8 kcal mol-1 in the gas phase and methanol solution for a protected hexapeptide) [152, 153]. Analysis of the influence of substitution patterns [151, 154] confirmed the intrinsic preference of (S)-b2/b3 dipeptide repeats for the right-handed 10/12-helical conformation and suggested other patterns of substitutions compatible with the formation of the 10/12-helix such as heterochiral dipeptide repeats (e.g. (R)-b3/ (S)-b3, (R)-b3/(S)-b2, (S)-b2/(R)-b2, (S)-b2/(R)-b3). This prediction was experimentally confirmed by the conformational analysis in solution of carbofuranosyl-b3-hexapeptides 16 and 17 made of regularly alternating (3R)- and (3S)-building blocks (Fig. 2.9) [147].

In MeOH, both peptides displayed a CD pattern characteristic of a right-handed 10/12- (12/10-) helical structure. More detailed structural analysis by NMR study in CDCl3 revealed that 16 and 17 adopt well-defined (P)-12/10 and 10/12 helical structures, respectively (Fig. 2.11).

It is noteworthy that the mixed b-peptide helical backbone can tolerate suppression of side chains every two residues. Thus, b-peptides made of alternating (3R)-carbofuranosyl-b3-amino acid and b-HGly (3-amino propionic acid) residues (18 and 19) and of (2S,3S)-b2' 3-(sugar) amino acid/b-HGly repeats (20), respectively

Fig. 2.11 Additional substitution patterns leading to mixed helices in b-peptides. Views along the helix axis of low energy conformers of (A) 16 and (B) 18 obtained by NOE-restrained modeling using NMR data obtained in CDCl3 [147, 148].

have been found to adopt well-defined right-handed (P)-12/10 (18 and 20) and (P)-10/12 (19) helical structures in nonpolar solvents such as CDCl3 and CD3CN [146, 148]. b-HGly thus behaves like b2-amino acid residues in b3/b2 repeats. This finding is not surprising if one keeps in mind that C(3) substitution is much more effective than C(2) in reducing the flexibility of the b-peptide backbone as already discussed in Section Inverting the configuration of b3-amino acid residues in b3/b-HGly repeats (18 ! 21) caused a switch in helix handedness facilitated by the conformational freedom ofb-HGly residues [148].

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