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Compact Helices with Large (>10 atoms) H-bonded Rings

2.3.1.1 The Homologation Strategy: b- and g-Peptide Foldamers

Selecting (designing) the right monomeric units to build homo-oligomers that will ultimately show high (helical) folding propensity is obviously a critical and limiting step in the foldamer arena. The choice of b-amino acids and corresponding b-peptides by Seebach [3] and Gellman [1] was not pure serendipity. It was initially guided (for Seebach) by the resemblance of the b-peptide backbone to poly((R)-3-hydroxybutanoic acid) a biopolymer for which a right-handed helical conformation with about three units per turn had been proposed [39, 40], and (for Gellman) by the absence of H-bonding between nearest neighbor amide groups in model b-homoglycine (b-HGly) derivatives [41], a criterion used to evaluate the propensity of the b-peptide backbone to stabilize folds maintained by H-bonds between sequentially remote amide groups.

Fig. 2.1 The homologation approach to aliphatic oligoamide foldamers. (A) Peptide foldamers consisting of homologated a-amino acids generated by insertion of one (! b2 and b3-peptides) or two (! g4-peptides) CH2 groups, the amino acid side chain remaining unchanged. According to the nomenclature proposed by Balaram [49], the conformational space of b- and g-peptides can be described by the following sets of backbone torsion angles: (o, f, 0, c) and (o, f, 0i, 02, c), respectively. (B) The b-peptide 314 helical fold. Stereo view along the

Fig. 2.1 The homologation approach to aliphatic oligoamide foldamers. (A) Peptide foldamers consisting of homologated a-amino acids generated by insertion of one (! b2 and b3-peptides) or two (! g4-peptides) CH2 groups, the amino acid side chain remaining unchanged. According to the nomenclature proposed by Balaram [49], the conformational space of b- and g-peptides can be described by the following sets of backbone torsion angles: (o, f, 0, c) and (o, f, 0i, 02, c), respectively. (B) The b-peptide 314 helical fold. Stereo view along the helix axis and top view of the (M)-314-helix formed by b3-peptide 1 determined by NMR in CD3OH (adapted from [43, 44]). Average (f, 6, c) values are (-119°, +63°,-147°). Side chains have been omitted for clarity. (C) The g-peptide 2.614 helical fold. Stereo view along the helix axis and top view of a (P)-2.614-helical low energy conformer of 2 (from NMR data in pyridine-d5). Average (f, 61, 62, c), values calculated on residues 2-5 are (-127°, +66°, +64°, -140°). Side chains have been partially omitted for clarity (adapted from [45]).

Detailed NMR conformational analysis of b-peptides consisting of homologated a-amino acids generated by insertion of one CH2 group, the amino acid side chain remaining unchanged, revealed a stable (b3 ! b2-peptides) helical fold (314- or 14-helix) stabilized by 14-membered rings with H-bonds in a forward orientation (1 ! 3 H-bonds between NHi and C=Oi+2) (e.g. 1, [3, 42-44] Fig. 2.1).

Examination of the top view of the helix indicates that the side chains of residues i and i + 3 are located nearly on top of each other and suggests that hydro-phobic interactions between overlapping aliphatic side chains could play a significant role in stabilizing the overall structure. The distance between facing C(a) atoms at positions i and i + 3 is approximately 4.8 A. The helix is compact with a diameter of ca. 4.8 A slightly larger than that of the a-helix (4.2 A). Although less studied, their homologs with one additional methylene group inserted in the backbone of each residue, namely the g-peptides, have also been found to form stable helical secondary structures in solution [45-48]. g4-Peptide chains (e.g. 2) adopt a 2.614 helical structure stabilized by 1 ^ 4 H bonds between C=Oi and NHi+3 closing 14-membered pseudocycles [45]. While the a-helix of l-a-peptides and the 314 helix of the corresponding b3-peptides have opposite polarity and hel-icity, the insertion of two CH2 groups in the backbone of l-a-amino acids leaves these two helix parameters unchanged, both the a-helix and the 2.614 helix of the resulting g4-peptides being right-handed and polarized from N to C terminus. Both (M)-314 and (P)-2.614 helical backbones are characterized by a (+)-synclinal arrangement (gauche conformation) around ethane bonds (e.g. in 1 and 2, y, and y2 values are @+60° (±15°) (see Fig. 2.1 for definitions of torsion angles [49]).

Besides NMR and X-ray diffraction (see Section 2.3.1.2), circular dichroism (CD) spectroscopy has been instrumental in studying parameters that influence the formation and stability of the b-peptide 14-helix. Typically, 14-helical b-peptides composed of acyclic amino acid residues display a common CD signature with one extremum at 215 nm (negative for (M)-helices and positive for (P)-helices) and the other extremum of opposite sign at 195 nm. In sharp contrast to a- and b-peptides, CD spectra of g-peptides were not very informative. g4-Peptides such as 2 which populate the 2.614 helical fold in solution do not display any characteristic CD signature in MeOH.

2.3.1.2 Imposing Backbone Conformational Restriction/Pre-organization for Optimal Helical Folding

Substantial stabilization of both the b- and g-peptide 14-helical fold has been achieved by increasing the level of pre-organization of b- and g-amino acid constituents. However, the rules formulated for a-peptides (see Section 2.2) do not necessarily apply (e.g. C(a)- (or C(b)-) tetrasubstitution of b-amino acid residues is not compatible with b-peptide 14-helix formation [43, 50-54]) and must be transposed. In particular, it has been shown that acyclic b2'3-amino acids of like configuration are more effective than their b3-counterparts in promoting requisite synclinal arrangement around C(a)-C(b) bonds in b-peptides (Fig. 2.2A) [43, 55]. With a y value fixed at approx. ±60°, trans-2-aminocyclohexyl carboxylic acid

Fig. 2.2 Optimal b-peptide backbone pre-organization for helix formation. (A) Acyclic b2'3 amino acid residues and (S,S)-ACHC residues which promote gauche conformation arouch the C(a)-C(b) bond are 14-helix stabilizers. The related five-membered ring ACPC promotes larger 0 values which are not compatible with the 14-helical conformation. (B) Comparison ofb-peptide 14- and 12-helices (f, 0, c) conformational space.

(M)-14-helix (values for 1, red closed circles, values for 2, green closed circles), (M)-12-helix (values for 3, blue closed circles). The enantiomeric positions in the (f, 0, c) space are shown as open circles. (C) Side view and top view of the (M)-12-helix formed by (R,R)-ACPC hexamer 3 as determined by X-ray diffraction (adapted from [59]). Average (f, 0, c) values for central residues are (88°,-85°, 98°).

Fig. 2.2 Optimal b-peptide backbone pre-organization for helix formation. (A) Acyclic b2'3 amino acid residues and (S,S)-ACHC residues which promote gauche conformation arouch the C(a)-C(b) bond are 14-helix stabilizers. The related five-membered ring ACPC promotes larger 0 values which are not compatible with the 14-helical conformation. (B) Comparison ofb-peptide 14- and 12-helices (f, 0, c) conformational space.

(M)-14-helix (values for 1, red closed circles, values for 2, green closed circles), (M)-12-helix (values for 3, blue closed circles). The enantiomeric positions in the (f, 0, c) space are shown as open circles. (C) Side view and top view of the (M)-12-helix formed by (R,R)-ACPC hexamer 3 as determined by X-ray diffraction (adapted from [59]). Average (f, 0, c) values for central residues are (88°,-85°, 98°).

(trans-ACHC), a cyclic b2 3-amino acid, in which the C(a)-C(b) bond is part of a 6-membered ring, is ideally pre-organized for 314 helix formation (Fig. 2.2A) [1, 56, 57]. In contrast, homo-oligomers (e.g. 3) consisting of the smaller ring size trans-2-aminocyclopentyl carboxylic acid (trans-ACPC) for which larger values of 0 only are accessible, adopt a stable 12- (2.512)-helix with a (1 ^ 4) H-bonding pattern that differs markedly from the 14-helix (Fig. 2.2) [58-60]. The 14- and 12-helices populated by (S,S)-ACHC and related (S,S)-ACPC oligomers, respectively have opposite polarity and helical screw sense. The (P)-12-helix display a CD pattern distinct from that of the corresponding 14-helix with a maximum at 204 nm, zero crossing at 214 nm and minimum at 221 nm.

b-Amino acids constrained with smaller rings such as cis-aminooxetane carboxylic acids have been shown to promote the formation of a 10-helical structure (e.g. 4) with H-bonds between neighboring amide units ((1 ! 2) H-bonding scheme) [61]. The 10-helix was later identified in short ACHC oligomers (tetramer), suggesting that it could represent a conformational intermediate in the folding pro-

cess toward the thermodynamically stable 14-helix [62]. The incorporation of cyclic o-amino acid units to fix the peptide backbone in a geometry favorable to helix formation has since been widely utilized (see also following sections) [63-64]. This approach was recently extended to higher oligoamides such as d-peptides [66-69]. Conformational search using the methods of ab initio MO theory identified several H-bonded helical backbones accessible to d-peptides [70]. Experimental validation came from studies with oligomers composed of carbohydrate-derived tetrahydrofuran amino acids with restricted rotation around C(a)-C(8) and C(b)-C(g) bonds. In chloroform solution, octamer 5 was found to adopt a well-defined 16-helical fold with 1 ^ 4 H-bonding pattern, reminiscent of the a-peptide p-helix [69].

Scheme 2.1

The intrinsic conformational preferences of substituted g-amino acid constituents of g-peptides derive in part from avoidance of destabilizing syn-pentane interaction [71, 72]. It is recognized that this effect plays a key role in fixing the bioactive conformation of a number of g-amino-acid-containing natural products such as Bleomycin A2 [73]. In g4-peptides five out of nine conformations generated by rotation around C(a)-C(b) and C(b)-C(g) bonds are free of syn-pentane interaction.

Adding substitutents at the 2-position (like configuration, see Fig. 2.3A) or at both 2 and 3-positions (like,like relative configuration) reduces to two the number of conformations devoid of syn-pentane interaction (conformation II in Fig. 2.3A is almost identical to that found in the g4-peptide 14-helical backbone) and thus reinforces optimal pre-organization for 14-helix formation (g2;4 and g2' 3;4-peptides) [45-48]. Other strategies to restrict the conformational space of the g-amino acid backbone, such as a,b-unsaturation (cis-vinologous g-peptides [74]), tetrasubtitution [oligomers of 1-(aminomethyl)cyclohexaneacetic acid (gabapentin, Gpn) [75]] cyclic g-amino acids (cis-g-amino-l-proline oligomers [76]) have been reported.

The g-peptide 14-helical backbone is characterized by large c values ranging from 120 to 150° (or —120 to —150°). This observation led to the finding that substituting urea for the CH2-CO-NH unit in g-peptide (substitution of nitrogen for C(a) in g-amino acids) can be used to rigidify the 14-helical fold by fixing the "c" angle to a value close to 170-180° (Fig. 2.3B) [77-80]. In methanol or pyridine solution, the resulting enantiopure N,N'-linked oligoureas (e.g. 6) adopt a well-

Fig. 2.3 (A) The two conformations free of destabilizing syn-pentane interaction [71, 72] in 2,4-disubstituted g-amino acid derivatives with like configuration. Conformation II is close to that found in the g4-peptide 14-helical backbone (see Fig. 2.1) (B) Structural analogy between the g4-peptide and N,N'-linked oligourea backbones. (C) (f, 91, c) and (f, y2, c) maps indicating g-peptide 14-helix and N,N'-linked oligourea 12,14-helix regions. 14-Helix [values for a g4- and a g2,34-peptide [45, 47], red closed circles (f, y1, c) and closed triangles (f, 02, c)], 12,14-helix [values for 6, green closed circles (f, y1, c) and closed triangles (f, 02, c)]. The

Fig. 2.3 (A) The two conformations free of destabilizing syn-pentane interaction [71, 72] in 2,4-disubstituted g-amino acid derivatives with like configuration. Conformation II is close to that found in the g4-peptide 14-helical backbone (see Fig. 2.1) (B) Structural analogy between the g4-peptide and N,N'-linked oligourea backbones. (C) (f, 91, c) and (f, y2, c) maps indicating g-peptide 14-helix and N,N'-linked oligourea 12,14-helix regions. 14-Helix [values for a g4- and a g2,34-peptide [45, 47], red closed circles (f, y1, c) and closed triangles (f, 02, c)], 12,14-helix [values for 6, green closed circles (f, y1, c) and closed triangles (f, 02, c)]. The enantiomeric positions in the (f, yn/02, c) space are shown as open circles/triangles. (D) The (P)-12,14-helical structure of N,N'-linked oligoureas. View along the helix axis and top view of a low energy conformer of nonamer 6 as determined by NMR spectroscopy and restrained molecular dynamics calculations in pyridine solution. Average (f, y1,02, c) values for central residues are (-105°, 55°, 88°,-168°). With an internal diameter of @3 A, the helix is particularly compact and is devoid of empty volume in the interior. Side chains have been partially omitted for clarity (adapted from defined 2.5 helical fold, reminiscent of the g4-peptide 14-helix (Fig. 2.3C and D). The structure is held by H-bonds closing both 12- and 14-membered rings (12,14-helix) and is characterized by a stable (+)-synclinal arrangement around the ethane bond. CD spectra recorded in MeOH display a characteristic signature with an intense maximum near 204 nm [79]. This is in contrast to related helical g4-peptides that do not exhibit any characteristic CD signature.

Alternatively, in a manner analogous to a-peptides (see Section 2.2), a helical backbone may be stabilized by creating a covalent linkage (e.g. disulfide bond) between two spatially proximal but sequentially remote side chains (e.g. i/i + 3 side chains in the 314 helix) [81].

2.3.1.3 Folding in an Aqueous Environment

The nature of the solvent can influence to a large extent the propensity of unnatural oligomers to adopt a given H-bonded fold. Conformational studies aimed at identifying new foldamers are often performed in apolar or moderately polar organic solvents (e.g. chloroform, MeCN, pyridine, trifluoroethanol (TFE), MeOH). However, determination of a folding pattern in an aqueous environment is highly relevant to applications of foldamers in biology.

Considerable efforts have been undertaken to address this issue in the case of 14- and 12-helical b-peptides. To increase water solubility of helical b-peptides composed of ACHC (14-helix promoter) or ACPC (12-helix promoter) oligomers while maintaining the level of backbone pre-organization, Gellman and coworkers developed amino-functionalized versions of trans-2-aminocycloalkane carboxylic acids. b-Peptides composed of alternating ACHC/DCHC [82] and ACHC/APiC [83] residues adopt a robust 14-helical conformation in aqueous solution (Fig 2.4A). Similarly, ACPC/APC [84], ACPC/AP [85], ACPC/3-aminomethyl-ACPC [86] repeats promote stable 12-helix formation (Fig 2.4A).

Alternatively, the introduction of a limited number of acyclic b3- or b2-Lys residues (1/3) in ACHC- and ACPC-peptides does not preclude the formation of stable 14- and 12-helical structures in aqueous solution [87-89]. Although helicity in water is intrinsically weaker in the absence of strong backbone pre-organization (e.g. b3-peptides), principles guiding the design of b3-peptides with high levels of 14-helicity in aqueous solution (e.g. 7, Fig. 2.4B) have recently been delineated by several groups [90-96]. They parallel those formulated for a-helical a-peptides (see Section 2.2) and include: (i) salt bridge or lactam formation between complementary charged i/i + 3 side chains (e.g. b3-HOrn/b3-HGlu; b3-HOrn/b3-HAsp; b3-HDab/b3-HAsp; Dab = 2,4-diaminobutyric acid; Orn = ornithine); (ii) maximization of electrostatic interactions with the helix macrodipole (e.g. by free charged termini, appropriate location of charged side chains, appropriate orientation of salt bridges) and (iii) introduction of b3-amino acids with high intrinsic 14-helix propensitiy. For noncharged b3-amino acids, 14-helix propensities have been found to differ significantly from a-helix propensities of corresponding a-amino acids. Ala is the most a-helix-stabilizing a-amino acid but the methyl side chain is one of the least 14-helical stabilizing. In contrast, branched side chains of Ile, Thr, and Val which display only moderate to low a-helix propensity are all

Fig. 2.4 b-Peptides that promote helix formation in water. (A) Using amino-functionalized versions of trans-2-aminocycloalcane carboxylic acids [82-86]. (B) De novo design of b3-peptide with high level of 14-helicity in aqueous solution by combining: salt bridges between i/i + 3 side chains, favorable electrostatic interactions with the helix macrodipole and side-chain branching (b3-HVal residues). Mean residue ellipticity of 7 measured by CD at 214 nm: ©214 = -13 320 deg cm"2 dmol-1 in 1 mM sodium phosphate/borate/citrate, pH 7.0 at 25 °C [95].

Fig. 2.4 b-Peptides that promote helix formation in water. (A) Using amino-functionalized versions of trans-2-aminocycloalcane carboxylic acids [82-86]. (B) De novo design of b3-peptide with high level of 14-helicity in aqueous solution by combining: salt bridges between i/i + 3 side chains, favorable electrostatic interactions with the helix macrodipole and side-chain branching (b3-HVal residues). Mean residue ellipticity of 7 measured by CD at 214 nm: ©214 = -13 320 deg cm"2 dmol-1 in 1 mM sodium phosphate/borate/citrate, pH 7.0 at 25 °C [95].

strongly 14-helix stabilizing. Intramolecular interhelical hydrophobic interactions (in a b3-peptide two-helix bundle) have also been shown to increase 14-helicity in aqueous solution [97].

If b-peptides are indisputably the best characterized helical aliphatic peptide fol-damer system in aqueous solution so far, some of the aromatic foldamers described in Chapter 1 fold into water even better than they do in organic solvents without any specific adjustment of their structure. It remains to be seen whether other foldamers based on remote intrastrand H-bonds can be designed to adopt robust helical structures in water.

2.3.1.4 Dynamics of b- and g-Peptide Helices: Evidence for Noncooperative Folding/Unfolding Processes

The unfolding and folding mechanisms of 14-helical b-, g-peptides and analogs have been investigated in polar solvents by various approaches. Temperature-dependent-CD and NMR measurements suggest that 14-helical b-peptides [44] and also 14-helical g2; 3;4-peptides [48] and helical N,N'-linked oligoureas [7779], undergo noncooperative unfolding upon heating in MeOH. For instance, the intensity of the extremum at 215 nm for b-peptide 1 decreases linearly (by ca 12% per 20 K) and noncooperative break-up of the structure is observed between 295 and 333 K. The scan, which is reversible, suggests that the unfolding and folding route of the helix must be reversible [44]. Insight into the dynamics of b-peptides and evidence for reversible folding were also provided by molecular dynamics (MD) simulations (GROMOS96 force-field [98], reviewed in Chapter 6) in explicit solvents and at different temperatures [99-104]. By simulating on a time scale that is long compared with the lifetime of any specific conformation (typically > 50 ns), it has been possible to determine the population and average lifetimes of the different conformations observed and to explore paths and rates of interconversion between the experimentally observed 14-helix conformation and (partially) unfolded conformations [102, 104]. In the course of a 50-ns simulation and irrespective of its initial conformation, b-peptide 1 folds rapidly (in the order of a few nanoseconds) into the experimentally observed 14-helix conformation (maximum lifetime @ 10 ns at 340 K) which is populated 50% of the time at 340 K [99, 101].

Recent progress towards the synthesis of the spin-labeled b2; 3-amino acid trans-b-TOAC (4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-3-carboxylic acid) and its incorporation into ACHC-peptides at i/i + 3 positions suggest that it might soon be possible to use electron spin resonance (ESR) to investigate further the structures and folding transitions in 14-helical b-peptides [105].

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