Synthesis of a new ß-amino acid with a 3-deoxy-L-ara furnaoside side chain: the influence of the side chain on the conformation of α/ß-peptides.

The important role of side chains in the stabilization of helical folds in peptidic foldamers containing C-linked carbo-ß-amino acids (ß-Caa), an interesting class of ß-amino acids, with carbohydrate side chains has been extensively elaborated. As a pragmatic approach to alleviate the interference of substituents in the side chains on the folding propensities of the peptides, they are often modified or removed. The present study reports the synthesis of a new ß-Caa with a 3-deoxy-L-ara furanoside side chain, [(R)-ß-Caa(da)], from D-glucose, and its use in the synthesis of α/ß-peptides in 1 : 1 alternation with D-Ala. The synthesis of peptides using (R)-ß-Caa(da), was facile unlike those from (R)-ß-Caa(a) having the L-ara furanoside side chain. The detailed NMR, molecular dynamics (MD) and CD studies on the new α/ß-peptides showed the presence of robust left-handed 11/9-mixed helices. The study demonstrates that the new (R)-ß-Caa(da), behaves differently compared to the other two related monomers, (R)-ß-Caa(x) with the D-xylo furanoside side chain and (R)-ß-Caa(a).

Three-residue turn in ß-peptides nucleated by a 12/10 helix.

A new three-residue turn in ß peptides nucleated by a 12/10-mixed helix is presented. In this design, ß peptides were derived from the 1:1 alternation of C-linked carbo-ß-amino acid ester [BocNH-(R)-ß-Caa(r)-OMe] (Boc=tert-butyloxycarbonyl), which consisted of a D-ribo furanoside side chain, and ß-hGly residues. The hexapeptide with (R)-ß-Caa(r) at the N terminus showed the 'turn' stabilized by a 14-membered NH(4)⋅⋅⋅CO(6) hydrogen bond at the C terminus nucleated by a robust 12/10-mixed helix, thus providing a 'helix-turn' (HT) motif. The turn and the helix were additionally stabilized by intraresidue electrostatic interaction between the furan oxygen in the carbohydrate side chain and NH in the backbone. However, the hexapeptide with a ß-hGly residue at the N terminus demonstrated the presence of a 10/12 helix through its entire length, which again showed the intraresidue interaction between NH and furan oxygen. The intraresidue NH⋅⋅⋅O-Me electrostatic interactions observed in the monomer, however, were absent in the peptides.

New helical folds in α-peptides with alternating chirality.

In α-peptides, the 8/10 helix is theoretically predicted to be energetically unstable and has not been experimentally observed so far. Based on our earlier studies on 'helical induction' and 'hybrid helices', we have adopted the 'end-capping' strategy to induce the 8/10 helix in α-peptides by using short α/ß-peptides. Thus, α-peptides containing a regular string of α-amino acids with alternating chirality were end capped by α/ß-peptides with 11/9-helical motifs at the termini. Extensive NMR spectroscopy studies of these peptides revealed the presence of a hitherto unknown 8/10-helical pattern; the H-bonds in the shorter pseudorings were rather weak. The approach of using short helical motifs to induce new mixed helices in α-peptides could provide avenues for more versatile design strategies.

Chirality and template-mediated induction of helical preferences in achiral ß-peptides.

This study describes chirality- or template-mediated helical induction in achiral ß-peptides for the first time. A strategy of end capping ß-peptides derived from ß-hGly (the smallest achiral ß-amino acid) with a chiral ß-amino acid that possesses a carbohydrate side chain (ß-Caa; C-linked carbo ß-amino acid) or a small, robust helical template derived from ß-Caas, was adopted to investigate folding propensity. A single chiral (R)-ß-Caa residue at the C- or N-terminus in these oligomers led to a preponderance of right-handed 12/10-helical folds, which was reiterated more strongly in peptides capped at both the C- and N-terminus. Likewise, the presence of a template (a 12/10-helical trimer) at both the C- and N-terminus resulted in a very robust helix. The propagation of the helical fold and its sustenance was found in a homo-oligomeric sequence with as many as seven ß-hGly residues. In both cases, the induction of helicity was stronger from the N terminus, whereas an anchor at the C terminus resulted in reduced helical propensity. Although these oligomers have been theoretically predicted to favor a 12/10-mixed helix in apolar solvents, this study provides the first experimental evidence for their existence. Diastereotopicity was found in both the methylene groups of the ß-hGly moieties due to chirality. Additionally, the ß-hGly units have shown split behavior in the conformational space to accommodate the 12/10-helix. Thus, end capping to assist chiralty- or template-mediated helical induction and stabilization in achiral ß-peptides is a very attractive strategy.

Synthesis of C-linked carbo-ß2-amino acids and ß2-peptides: design of new motifs for left-handed 12/10- and 10/12-mixed helices.

C-linked carbo-ß(2)-amino acids (ß(2)-Caa), a new class of ß-amino acid with a carbohydrate side chain having d-xylo configuration, were prepared from d-glucose. The main idea behind the design of the new ß-amino acids was to move the steric strain of the bulky carbohydrate side chain from the Cß- to the Cα-carbon atom and to explore its influence on the folding propensities in peptides with alternating (R)- and (S)-ß(2)-Caas. The tetra- and hexapeptides derived were studied employing NMR (in CDCl(3)), CD, and molecular dynamics simulations. The ß(2)-peptides of the present study form left-handed 12/10- and 10/12-mixed helices independent of the order of the alternating chiral amino acids in the sequence and result in a new motif. These results differ from earlier findings on ß(3)-peptides of the same design, containing a carbohydrate side chain with d-xylo configuration, which form exclusively right-handed 12/10-mixed helices. Quantum chemical calculations employing ab initio MO theory suggest the side chain chirality as an important factor for the observed definite left- or right-handedness of the helices in the ß(2)- and ß(3)-peptides.

Design of ß-amino acid with backbone-side chain interactions: stabilization of 14/15-helix in α/ß-peptides.

A new C-linked carbo-ß-amino acid, (R)-ß-Caa((r)), having a carbohydrate side chain with D-ribo configuration, was prepared from D-glucose by inverting the C-3 stereocenter to introduce constraints/interactions. From the NMR studies it was inferred that the new monomer may participate in additional electrostatic interactions, facilitating and enhancing novel folds in oligomeric peptides derived from it. The α/ß-peptides, synthesized from alternating L-Ala and (R)-ß-Caa((r)), have shown the presence of 14/15-helix by NMR (in CDCl(3), methanol-d(3) and CD(3)CN), CD and MD calculations. The hybrid peptides showed the presence of electrostatic interactions involving the intraresidue amide proton and the C3-OMe, which helped in the stabilization of the NH(i)···CO(i-4) H-bonds and adoption of 14/15-helix. The importance of such additional interactions has been well defined in recent times to stabilize the folding in a variety of peptidic foldamers. These observations suggest and emphasize that the side chain-backbone interactions are crucial in the stabilization of the desired folding propensity. The designed monomer thus enlarges the opportunities for the synthesis of peptides with novel conformations and expands the repertoire of the foldamers.

Synthesis and structural studies of homooligomers of geminally disubstituted ß(2,2)-amino acids with carbohydrate side chain.

A new class of geminally disubstituted C-linked carbo-ß(2,2)-amino acids (ß(2,2)-Caa) were prepared from d-glucose. The structures of homooligomeric di-, tetra-, and hexapeptides prepared from (S)-ß(2,2)-Caa were studied with NMR (in CDCl(3)), CD, and Molecular Dynamics calculations. These ß(2,2)-peptides have shown the presence of stable 6-membered (6-mr) NH(i)···CO(i) intra-residue H-bonded (C(6)) strands. It was found that the strand structures realized in these systems were additionally stabilized by the electrostatic interaction arising due to the proximity of amide proton (NH(i)) to the oxygen of the preceding methoxy group (O(Me)(i-1)) at the C3 carbon of the carbohydrate ring. The new ß(2,2)-Caa residues with additional support to H-bonding considerably expand the domain of foldamers.

Hybrid helices: motifs for secondary structure scaffolds in foldamers.

The concept of "hybrid helices" as a new motif for foldamers is presented. Hybrid helices can be realized by a combination of two or more different types of homologous and hybrid peptides, for example, beta-peptides and alpha/beta- and alpha/gamma-hybrid peptides, within the same oligomer. The different helix types of the various peptide foldamer classes are maintained and form a regular helix along the sequence of the oligomer. The transition from one helix type to another was found to be rather smooth with high compatibility of the different helix types. Such hybrid helices represent novel motifs of secondary structure scaffolds. They open up the possibility to change the direction of helix propagation in a subtle manner. Hybrid helices enrich the arsenal of defined foldamer structures for a structural and functional mimicry of native peptides and proteins.

Theoretical and experimental studies on alpha/epsilon-hybrid peptides: design of a 14/12-helix from peptides with alternating (S)-C-linked carbo-epsilon-amino acid [(S)-epsilon-Caa((x))] and L-ala.

An (S)-C-linked carbo-epsilon-amino acid [(S)-epsilon-Caa((x))] was prepared from the known (S)-delta-Caa. This monomer was utilized together with l-Ala to give novel alpha/epsilon-hybrid peptides in 1:1 alternation. Conformational analysis on penta- and hexapeptides by NMR (in CDCl(3)), CD, and MD studies led to the identification of robust 14/12-mixed helices. This is in agreement with the data from a theoretical conformational analysis on the basis of ab initio MO theory providing a complete overview on all formally possible hydrogen-bonded helix patterns of alpha/epsilon-hybrid peptides with 1:1 backbone alternation. The "new motif" of a mixed 14/12-helix was predicted as most stable in vacuum. Obviously, the formation of ordered secondary structures is also possible in peptide foldamers with amino acid constituents of considerable backbone lengths. Thus, alpha/epsilon-hybrid peptides expand the domain of foldamers and allow the introduction of desired functionalities via the alpha-amino acid constituents.

Synthesis and structure of alpha/delta-hybrid peptides--access to novel helix patterns in foldamers.

Stimulated by an overview on all periodic folding patterns of alpha/delta-hybrid peptides with 1:1 alternating backbone provided by ab initio molecular orbital theory, the first representatives of this foldamer class were synthesized connecting novel C-linked carbo-delta-amino acid constituents and L-Ala. In agreement with theoretical predictions, extensive NMR spectroscopic analyses confirm the formation of new motifs of 13/11-mixed helical patterns in these peptides supported by the rigidity of the D-xylose side chain in the selected delta-amino acid constituents. Relationships between possible helix types in alpha/delta-hybrid peptides and their counterparts in other 1:1 hybrid peptide classes and native alpha-peptides are discussed; these indicate the high potential of these foldamers to mimic native peptide secondary structures. The design of alpha/delta-hybrid peptides provides an opportunity to expand the domain of foldamers and allows the introduction of desired functionalities through the alpha-amino acid constituents.

Synthesis of beta-peptides with beta-helices from new C-linked carbo-beta-amino acids: study on the impact of carbohydrate side chains.

The design and synthesis of beta-peptides from new C-linked carbo-beta-amino acids (beta-Caa) presented here, provides an opportunity to understand the impact of carbohydrate side chains on the formation and stability of helical structures. The beta-amino acids, Boc-(S)-beta-Caa((g))-OMe 1 and Boc-(R)-beta-Caa((g))-OMe 2, having a D-galactopyranoside side chain were prepared from D-galactose. Similarly, the homo C-linked carbo-beta-amino acids (beta-hCaa); Boc-(S)-beta-hCaa((x))-OMe 3 and Boc-(R)-beta-hCaa((x))-OMe 4, were prepared from D-glucose. The peptides derived from the above monomers were investigated by NMR, CD, and MD studies. The beta-peptides, especially the shorter ones obtained from the epimeric (at the amine stereocenter C(beta)) 1 and 2 by the concept of alternating chirality, showed a much smaller propensity to form 10/12-helices. This substantial destabilization of the helix could be attributed to the bulkier D-galactopyranoside side chain. Our efforts to prepare peptides with alternating 3 and 4 were unsuccessful. However, the beta-peptides derived from alternating geometrically heterochiral (at C(beta)) 4 and Boc-(R)-beta-Caa((x))-OMe 5 (D-xylose side chain) display robust right-handed 10/12-helices, while the mixed peptides with alternating 4 and Boc-beta-hGly-OMe 6 (beta-homoglycine), resulted in left-handed beta-helices. These observations show a distinct influence of the side chains on helix formation as well as their stability.

Synthesis and conformational studies of peptides from new C-linked carbo-beta-amino acids (beta-Caas) with anomeric methylamino- and difluorophenyl moieties.

New C-linked carbo-beta-amino acids (beta-Caas), Cbz-(S)-beta-Caa-(NHBoc)-OMe (1) and Cbz-(R)-beta-Caa-(NHBoc)-OMe (2), with an additional amine group (methylamino group of NHBoc) at the C-1 position of the lyxofuranoside side chain and Boc-(S)-beta-Caa-(diFP)-OMe (3) and Boc-(R)-beta-Caa-(diFP)-OMe (4), with a C-difluorophenyl (diFP) moiety at the anomeric position of the lyxofuranoside side chain were prepared from D-mannose. Beta-peptides [tetra- and hexapeptides] were synthesized from these beta-Caas, 'epimeric' [at the amine stereocentre (C(beta))], using the concept of 'alternating chirality' to carry out their conformational studies [NMR (CDCl(3)), CD and MD]. In the monomer design, it was envisaged that the presence of an additional amine group in 1 or 2 would help in solubilizing the peptides in water, while, the C-difluorophenyl (diFP) moiety of 3 and 4 is expected to enhance the biological activity. The peptides having 1 and 2, though could not retain their 12-10-mixed helices in water, have shown moderate activity against gram positive and gram negative bacterial strains. The peptides prepared from 3 and 4, much against our expectations, did not display any biological activity.

Three-residue turns in alpha/beta-peptides and their application in the design of tertiary structures.

A new three-residue turn was serendipitously discovered in alpha/beta hybrid peptides derived from alternating C-linked carbo-beta-amino acids (beta-Caa) and L-Ala residues. The three-residue beta-alpha-beta turn at the C termini, nucleated by a helix at the N termini, resulted in helix-turn (HT) supersecondary structures in these peptides. The turn in the HT motif is stabilized by two H bonds-CO(i-2)-NH(i), with a seven-membered pseudoring (gamma turn) in the backward direction, and NH(i-2)-CO(i), with a 13-membered pseudoring in the forward direction (i being the last residue)--at the C termini. The study was extended to generalize the new three-residue turn (beta-alpha-beta) by using different alpha- and beta-amino acids. Furthermore, the HT motifs were efficiently converted, by an extension with helical oligomers at the C termini, into peptides with novel helix-turn-helix (HTH) tertiary structures. However, this resulted in the destabilization of the beta-alpha-beta turn with the concomitant nucleation of another three-residue turn, alpha-beta-beta, which is stabilized by 11- and 15-membered bifurcated H bonds. Extensive NMR spectroscopic studies were carried out to delineate the secondary and tertiary structures in these peptides, which are further supported by molecular dynamics (MD) investigations.

Design of a "new motif" with beta-amino acids and alpha-aminoxy acids: synthesis of hybrid peptides with 12/10-helix.

Hybrid peptides are prepared from a C-linked carbo-beta-amino acid ester (R-beta-Caa) and an alpha-aminoxy acid (R-Ama) derived from S-lactic acid. Extensive NMR (in CDCl 3 solution), CD, and MD studies on the tetra- and hexapeptides led to identification of robust 12/10-mixed helices. The dipeptide repeat having an R-beta-Caa and an R-Ama thus provides a "new motif" to realize a 12/10-mixed helix, for the first time, in oligomers containing R-Ama. To understand the impact of side chains in the mixed helix formation, R-beta-Caa/Ama (with no substitution in Ama) and S-beta-hAla/R-Ama oligomers were investigated. NMR studies revealed the existence of 12/10-helices in these hybrid peptides, and the side chains of monomers were found to have a profound influence on their stabilities. These observations imply that the propensity of beta-amino acid to prefer a mixed 12/10-helix governs the structural behavior in these peptides. The structural consequences of the lone-pair repulsion between nitrogen and oxygen atoms result in a new and interesting structural motif which behaves like "pseudo" beta (3),beta(2)-peptides in generating 12/10-mixed helices.

12/10- and 11/13-mixed helices in alpha/gamma- and beta/gamma-hybrid peptides containing C-linked carbo-gamma-amino acids with alternating alpha- and beta-amino acids.

New classes of alpha/gamma- and beta/gamma-hybrid peptides have been synthesized with novel 12/10- and 11/13-mixed helical patterns, respectively. The alpha/gamma-peptides were derived from the dipeptide repeats with alternating arrays of l-Ala and gamma-Caa((l)) (C-linked carbo-gamma-amino acid from d-mannose), which generated a new 12/10-mixed helix, for the first time, without a beta-amino acid. The beta/gamma-peptides made from an alternating arrangement of beta-Caa((x)) (C-linked carbo-beta-amino acid) and gamma-Caa((x)) (C-linked carbo-gamma-amino acid from d-xylose), on the other hand, resulted in an unprecedented 11/13-helix. The secondary structures in these peptides have been ascertained from detailed NMR studies, and CD spectroscopy and molecular dynamics investigations provided additional support for the structures derived.

11/9-mixed helices in the alpha/beta-peptides derived from alternating alpha- and beta-amino acids with proteinogenic side chains.

alpha/beta-Hybrid peptides are prepared from amino acids with proteinogenic side chains on the basis of the concept of "alternating chirality", involving D-Phe and a beta3-hVal. Through the extensive NMR, CD, and MD studies, robust left-handed 11/9-mixed helices were identified in these peptides in CDCl3 solutions, wherein the 11/9-mixed helix was observed even in a small peptide with three residues.