Three‐Residue Turns in α/β‐Peptides and Their Application in the Design of Tertiary Structures

A new three‐residue turn was serendipitously discovered in α/β hybrid peptides derived from alternating C‐linked carbo‐β‐amino acids (β‐Caa) and L ‐Ala residues. The three‐residue β‐α‐β 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 (γ 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 (β‐α‐β) by using different α‐ and β‐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 β‐α‐β turn with the concomitant nucleation of another three‐residue turn, α‐β‐β, 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.     

Three‐Residue Turn in β‐Peptides Nucleated by a 12/10 Helix

A new three‐residue turn in β peptides nucleated by a 12/10‐mixed helix is presented. In this design, β peptides were derived from the 1:1 alternation of C‐linked carbo‐β‐amino acid ester [BocNH‐(R)‐β‐Caa_(r)‐OMe] (Boc=tert‐butyloxycarbonyl), which consisted of a D ‐ribo furanoside side chain, and β‐hGly residues. The hexapeptide with (R)‐β‐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 β‐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.     

One‐Handed Helical Screw Direction of Homopeptide Foldamer Exclusively Induced by Cyclic α‐Amino Acid Side‐Chain Chiral Centers

Chiral cyclic α,α‐disubstituted amino acids, (3S,4S)‐ and (3R,4R)‐1‐amino‐3,4‐(dialkoxy)cyclopentanecarboxylic acids ((S,S)‐ and (R,R)‐Ac₅c^dOR; R: methyl, methoxymethyl), were synthesized from dimethyl L ‐(+)‐ or D ‐(?)‐tartrate, and their homochiral homoligomers were prepared by solution‐phase methods. The preferred secondary structure of the (S,S)‐Ac₅c^dOMe hexapeptide was a left‐handed (M) 3₁₀ helix, whereas those of the (S,S)‐Ac₅c^dOMe octa‐ and decapeptides were left‐handed (M) α helices, both in solution and in the crystal state. The octa‐ and decapeptides can be well dissolved in pure water and are more α helical in water than in 2,2,2‐trifluoroethanol solution. The left‐handed (M) helices of the (S,S)‐Ac₅c^dOMe homochiral homopeptides were exclusively controlled by the side‐chain chiral centers, because the cyclic amino acid (S,S)‐Ac₅c^dOMe does not have an α‐carbon chiral center but has side‐chain γ‐carbon chiral centers.     

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/β‐peptides. Thus, α‐peptides containing a regular string of α‐amino acids with alternating chirality were end capped by α/β‐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.     

Synthesis of β‐Peptides with β‐Helices from New C‐Linked Carbo‐β‐Amino Acids: Study on the Impact of Carbohydrate Side Chains

The design and synthesis of β‐peptides from new C‐linked carbo‐β‐amino acids (β‐Caa) presented here, provides an opportunity to understand the impact of carbohydrate side chains on the formation and stability of helical structures. The β‐amino acids, Boc‐(S)‐β‐Caa_(g)‐OMe 1 and Boc‐(R)‐β‐Caa_(g)‐OMe 2 , having a D ‐galactopyranoside side chain were prepared from D ‐galactose. Similarly, the homo C‐linked carbo‐β‐amino acids (β‐hCaa); Boc‐(S)‐β‐hCaa_(x)‐OMe 3 and Boc‐(R)‐β‐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 β‐peptides, especially the shorter ones obtained from the epimeric (at the amine stereocenter C_β) 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 β‐peptides derived from alternating geometrically heterochiral (at C_β) 4 and Boc‐(R)‐β‐Caa_(x)‐OMe 5 (D ‐xylose side chain) display robust right‐handed 10/12‐helices, while the mixed peptides with alternating 4 and Boc‐β‐hGly‐OMe 6 (β‐homoglycine), resulted in left‐handed β‐helices. These observations show a distinct influence of the side chains on helix formation as well as their stability.     

Helix Nucleation by the Smallest Known α‐Helix in Water

Cyclic pentapeptides (e.g. Ac‐(cyclo‐1,5)‐[KAXAD]‐NH₂; X=Ala, 1 ; Arg, 2 ) in water adopt one α‐helical turn defined by three hydrogen bonds. NMR structure analysis reveals a slight distortion from α‐helicity at the C‐terminal aspartate caused by torsional restraints imposed by the K(i)–D(i+4) lactam bridge. To investigate this effect on helix nucleation, the more water‐soluble 2 was appended to N‐, C‐, or both termini of a palindromic peptide ARAARAARA (≤5 % helicity), resulting in 67, 92, or 100 % relative α‐helicity, as calculated from CD spectra. From the C‐terminus of peptides, 2 can nucleate at least six α‐helical turns. From the N‐terminus, imperfect alignment of the Asp5 backbone amide in 2 reduces helix nucleation, but is corrected by a second unit of 2 separated by 0–9 residues from the first. These cyclic peptides are extremely versatile helix nucleators that can be placed anywhere in 5–25 residue peptides, which correspond to most helix lengths in protein–protein interactions.     

Fine Tuning of β‐Peptide Foldamers: a Single Atom Replacement Holds Back the Switch from an 8‐Helix to a 12‐Helix

Cyclic homologated amino acids are important building blocks for the construction of helical foldamers. N‐aminoazetidine‐2‐carboxylic acid (AAzC), an aza analogue of trans‐2‐aminocyclobutanecarboxylic acid (tACBC), displays a strong hydrazino turn conformational feature, which is proposed to act as an 8‐helix primer. tACBC oligomers bearing a single N‐terminal AAzC residue were studied to evaluate the ability of AAzC to induce and support an 8‐helix along the oligopeptide length. While tACBC homooligomers assume a dominant 12‐helix conformation, the aza‐primed oligomers preferentially adopt a stabilized 8‐helix conformation for an oligomer length up to 6 residues. The (formal) single‐atom exchange at the N terminus of a tACBC oligomer thus contributes to the sustainability of the 8‐helix, which resists the switch to a 12‐helix. This effect illustrates atomic‐level programmable design for fine tuning of peptide foldamer architectures.     

Efficient Access to Enantiopure γ4‐Amino Acids with Proteinogenic Side‐Chains and Structural Investigation of γ4‐Asn and γ4‐Ser in Hybrid Peptide Helices

Hybrid peptides composed of α‐ and β‐amino acids have recently emerged as new class of peptide foldamers. Comparatively, γ‐ and hybrid γ‐peptides composed of γ⁴‐amino acids are less studied than their β‐counterparts. However, recent investigations reveal that γ⁴‐amino acids have a higher propensity to fold into ordered helical structures. As amino acid side‐chain functional groups play a crucial role in the biological context, the objective of this study was to investigate efficient synthesis of γ⁴‐residues with functional proteinogenic side‐chains and their structural analysis in hybrid‐peptide sequences. Here, the efficient and enantiopure synthesis of various N‐ and C‐terminal free‐γ⁴‐residues, starting from the benzyl esters (COOBzl) of N‐Cbz‐protected (E)‐α,β‐unsaturated γ‐amino acids through multiple hydrogenolysis and double‐bond reduction in a single‐pot catalytic hydrogenation is reported. The crystal conformations of eight unprotected γ⁴‐amino acids (γ⁴‐Val, γ⁴‐Leu, γ⁴‐Ile, γ⁴‐Thr(OtBu), γ⁴‐Tyr, γ⁴‐Asp(OtBu), γ⁴‐Glu(OtBu), and γ‐Aib) reveals that these amino acids adopted a helix favoring gauche conformations along the central C^γ? C^β bond. To study the behavior of γ⁴‐residues with functional side chains in peptide sequences, two short hybrid γ‐peptides P1 (Ac‐Aib‐γ⁴‐Asn‐Aib‐γ⁴‐Leu‐Aib‐γ⁴‐Leu‐CONH₂) and P2 (Ac‐Aib‐γ⁴‐Ser‐Aib‐γ⁴‐Val‐Aib‐γ⁴‐Val‐CONH₂) were designed, synthesized on solid phase, and their 12‐helical conformation in single crystals were studied. Remarkably, the γ⁴‐Asn residue in P1 facilitates the tetrameric helical aggregations through interhelical H bonding between the side‐chain amide groups. Furthermore, the hydroxyl side‐chain of γ⁴‐Ser in P2 is involved in the interhelical H bonding with the backbone amide group. In addition, the analysis of 87 γ⁴‐residues in peptide single‐crystals reveal that the γ⁴‐residues in 12‐helices are more ordered as compared with the 10/12‐ and 12/14‐helices.     

Synthesis and Conformational Analysis of Cyclic Homooligomers from Pyranoid ε‐Sugar Amino Acids

New pyranoid ε‐sugar amino acids were designed as building blocks, in which the carboxylic acid and the amine groups were placed in positions C2 and C3 with respect to the tetrahydropyran oxygen atom. By using standard solution‐phase coupling procedures, cyclic homooligomers containing pyranoid ε‐sugar amino acids were synthesized. Conformation analysis was performed by using NMR spectroscopic experiments, FTIR spectroscopic studies, X‐ray analysis, and a theoretical conformation search. These studies reveal that the presence of a methoxy group in the position C4 of the pyran ring produces an important structural change in the cyclodipeptides. When the methoxy groups are present, the structure collapses through interresidue hydrogen bonds between the oxygen atoms of the pyran ring and the amide protons. However, when the cyclodipeptide lacks the methoxy groups, a U‐shape structure is adopted, in which there is a hydrophilic concave face with four oxygen atoms and two amide protons directed toward the center of the cavity. Additionally, we found important evidence of the key role played by weak electrostatic interactions, such as the five‐membered hydrogen‐bonded pseudocycles (C₅) between the amide protons and the ether oxygen atoms, in the conformation equilibrium of the macrocycles and in the cyclization step of the cyclic tetrapeptides.     

α‐Aminoxy Oligopeptides: Synthesis,Secondary Structure,and Cytotoxicity of a New Class of Anticancer Foldamers

α‐Aminoxy peptides are peptidomimetic foldamers with high proteolytic and conformational stability. To gain an improved synthetic access to α‐aminoxy oligopeptides we used a straightforward combination of solution‐ and solid‐phase‐supported methods and obtained oligomers that showed a remarkable anticancer activity against a panel of cancer cell lines. We solved the first X‐ray crystal structure of an α‐aminoxy peptide with multiple turns around the helical axis. The crystal structure revealed a right‐handed 2₈‐helical conformation with precisely two residues per turn and a helical pitch of 5.8 Å. By 2D ROESY experiments, molecular dynamics simulations, and CD spectroscopy we were able to identify the 2₈‐helix as the predominant conformation in organic solvents. In aqueous solution, the α‐aminoxy peptides exist in the 2₈‐helical conformation at acidic pH, but exhibit remarkable changes in the secondary structure with increasing pH. The most cytotoxic α‐aminoxy peptides have an increased propensity to take up a 2₈‐helical conformation in the presence of a model membrane. This indicates a correlation between the 2₈‐helical conformation and the membranolytic activity observed in mode of action studies, thereby providing novel insights in the folding properties and the biological activity of α‐aminoxy peptides.     

1H‐Azepine‐4‐amino‐4‐carboxylic Acid: A New α,α‐Disubstituted Ornithine Analogue Capable of Inducing Helix Conformations in Short Ala‐Aib Pentapeptides

A very efficient synthesis of orthogonally protected 1H‐azepine‐4‐amino‐4‐carboxylic acid, abbreviated as Azn, a conformationally restricted analogue of ornithine, was realized. It was obtained on a gram scale in good overall yield in five steps, three of which did not require isolation of the intermediates, starting from the readily available 1‐amino‐4‐oxo‐cyclohexane‐4‐carboxylic acid. Both enantiomers were used for the preparation of pentapeptide models containing Ala, Aib, and Azn. Conformational studies using both spectroscopic techniques (NMR, CD) and molecular dynamics on model 5‐mer peptides showed that the (R)‐Azn isomer possesses a marked helicogenic effect.