Design of Chiral ß-Double Helices from γ-Peptide Foldamers.

Chirality is ubiquitous in nature, and homochirality is manifested in many biomolecules. Although ß-double helices are rare in peptides and proteins, they consist of alternating L- and D-amino acids. No peptide double helices with homochiral amino acids have been observed. Here, we report chiral ß-double helices constructed from γ-peptides consisting of alternating achiral (E)-α,ß-unsaturated 4,4-dimethyl γ-amino acids and chiral (E)-α,ß-unsaturated γ-amino acids in both single crystals and in solution. The two independent strands of the same peptide intertwine to form a ß-double helix structure, and it is stabilized by inter-strand hydrogen bonds. The peptides with chiral (E)-α,ß-unsaturated γ-amino acids derived from α-L-amino acids adopt a (P)-ß-double helix, whereas peptides consisting of (E)-α,ß-unsaturated γ-amino acids derived from α-D-amino acids adopt an (M)-ß-double helix conformation. The circular dichroism (CD) signature of the (P) and (M)-ß-double helices and the stability of these peptides at higher temperatures were examined. Furthermore, ion transport studies suggested that these peptides transport ions across membranes. Even though the structural analogy suggests that these new ß-double helices are structurally different from those of the α-peptide ß-double helices, they retain ion transport activity. The results reported here may open new avenues in the design of functional foldamers.

Anion Tuned Structural Modulation and Nonlinear Optical Effects of Metal-Ion Directed 310 -Helix Networks.

Metals play an important role in the structure and functions of various proteins. The combination of metal ions and peptides have been emerging as an attractive field to create advanced structures and biomaterials. Here, we are reporting the anion-influenced, silver ion coordinated diverse networks of designed short tripeptide 310 -helices with terminal pyridyl groups. The short peptides adopted classical right-handed, left-handed and 310 EL -helical conformations in the presence of different silver salts. The peptides have displayed conformational flexibility to accommodate different sizes and interactions of anions to yield a variety of metal-coordinated networks. The complexes of metal ions and peptides have shown different porous networks, right- and left-handed helical polymers, transformation of helix into superhelix and 2 : 2 metal-peptide macrocycles. Further, the metal-peptide crystals with inherent dipoles of helical peptides gave striking second harmonic generation response. The optical energy upconversion from NIR to red and green light is demonstrated. Overall, we have shown the utilization of short 310 -helices for the construction of diverse metal-coordinated helical networks and notable non-linear optical effects.

Proteolytically Stable ααγ-Hybrid Peptides Inhibit the Aggregation and Cytotoxicity of Aß42.

The recent approval of antibody-based therapy for targeting the clearance of amyloid plaques fuels the research in designing small molecules and peptide inhibitors to target the aggregation of Aß-peptides. Here, we report that the 15-residue ααγ-hybrid peptide not only inhibits the aggregation of soluble Aß42 into fibrils but also disintegrates the aggregated Aß42 fibrils into smaller assemblies. Further, the hybrid peptide completely rescues neuronal cells from the toxicity of Aß42 at equimolar concentrations. The shorter 10- and 12-mer peptides showed weak aggregation inhibition activity, while the fully hydrophobic 15-mer ααγ-hybrid peptide analogue showed no aggregation inhibition activity. Further, the 15-mer ααγ-hybrid peptide showed resistance against trypsin digestion and also nontoxic to the neuronal cells. The CD revealed that the peptide upon interaction induces a helix-type conformation in the Aß42. This is in sharp contrast to the ß-sheet conformation of Aß42 upon incubation. The two-dimensional-NMR (2D-NMR) analysis revealed a large perturbation in the chemical shifts of residues at the N-terminus. The presence of 15-mer peptide at an equimolar concentration of Aß42 showed less tendency for aggregation and also exhibited nontoxicity to the neuronal cells. The results reported here may be useful in designing new therapeutics for Alzheimer's disease.

Foldamer Nanotubes Mediated Label-Free Detection of Protein-Small Molecule Interactions.

Development of miniaturized lab-on-chip devices for the detection of rapid and specific small molecule-protein binding interactions at very low concentrations holds significant importance in drug discovery and biomedical applications. Here, the label-free detection of small molecule-protein interactions is reported on the surface functionalizable nanotubes of α,γ-hybrid peptide helical foldamers using nanoscale capacitance and impedance spectroscopy. The 12-helix conformation of the α,γ-hybrid peptide observed in the single crystals, self-assembled into nanotubes in an aqueous environment with exposed cysteine thiols for small molecule conjugation. The binding of streptavidin to the covalently linked biotin on the surface of nanotubes was detected at the picomolar concentrations. No change in the capacitance and impedance were observed in the absence of either immobilized biotin or protein streptavidin. The functionalizable hybrid peptide nanotubes reported here pave the way for the label-free detection of various small molecule protein interactions at very low concentrations.

Cyclization of N-Boc-(E)-α,ß-unsaturated γ-amino acid active esters into N-Boc-(Z)-α,ß-unsaturated γ-lactams through E → Z isomerization.

Here, we are reporting the spontaneous transformation of the active esters of N-Boc protected E-α,ß-unsaturated γ-amino acids into the corresponding Z-α,ß-unsaturated γ-lactams with concomitant E → Z isomerization in the presence of a weak base. No cyclization was observed in the absence of the base. Analysis revealed that amide γ-NH is crucial for both lactamization and E → Z isomerization. This mild transformation provides easy access to the synthetically challenging α,ß-unsaturated γ-lactams and also gives new insights into the E → Z isomerization of double bonds.

Stereoselective synthesis of backbone extended π-conjugated amino esters.

Utilization of the Wittig reaction to synthesize conjugative multiple double bonds is rare. We examined the utility of the Wittig reaction to construct conjugative two and three carbon-carbon double bonds on the N-protected amino acid backbone. The ethyl esters of N-Boc amino acids with multiple carbon-carbon double bonds in the backbone were isolated in excellent yields with exceptional E-selectivity of the double bonds. The allylic alcohols of α,ß-unsaturated γ-amino esters were selectively synthesized from the DIBAL-H and BF3·OEt2. The allylic alcohols were transformed into aldehydes using IBX oxidation. Using this protocol, we synthesized ethyl esters of N-Boc-(E,E)-α,ß,γ,δ-unsaturated ε-amino acids with various side-chain functionalities and ethyl esters of N-Boc-(E,E,E)-α,ß,γ,δ,ε,ζ-unsaturated η-amino acids with excellent yields. We speculated the exceptional E-selectivity is probably due to the stabilization of the planar transition state of the Wittig reaction with the double bond p-orbitals. No racemization was observed in the synthesis of amino acids. The reported process may serve as an excellent route to synthesize multiple conjugative carbon-carbon double bonds.

A cationic amphiphilic peptide chaperone rescues Aß42 aggregation and cytotoxicity.

Directing Aß42 to adopt a conformation that is free from aggregation and cell toxicity is an attractive and viable strategy to design therapeutics for Alzheimer's disease. Over the years, extensive efforts have been made to disrupt the aggregation of Aß42 using various types of inhibitors but with limited success. Herein, we report the inhibition of aggregation of Aß42 and disintegration of matured fibrils of Aß42 into smaller assemblies by a 15-mer cationic amphiphilic peptide. The biophysical analysis comprising thioflavin T (ThT) mediated amyloid aggregation kinetic analysis, dynamic light scattering, ELISA, AFM, and TEM suggested that the peptide effectively disrupts Aß42 aggregation. The circular dichroism (CD) and 2D-NMR HSQC analysis reveal that upon interaction, the peptide induces a conformational change in Aß42 that is free from aggregation. Further, the cell assay experiments revealed that this peptide is non-toxic to cells and also rescues the cells from the toxicity of Aß42. Peptides with a shorter length displayed either weak or no inhibitory effect on Aß42 aggregation and cytotoxicity. These results suggest that the 15-residue cationic amphiphilic peptide reported here may serve as a potential therapeutic candidate for Alzheimer's disease.

Metal-Coordinated Supramolecular Polymers from the Minimalistic Hybrid Peptide Foldamers.

Availing the peptide folded architectures to design metal-coordinated frameworks and cages is restricted due to the scarcity of readily accessible short and stable secondary structures. The secondary structures, α-helix and ß-sheets, play significant roles in stabilizing tertiary folds of proteins. Designing such helical structures from the short sequences of peptides without having any steric restrictions is exceptionally challenging. Here we reveal the short α,γ-hybrid tripeptide sequences that manifest stable helical structures without having any sterically constrained amino acids. These short hybrid tripeptides fold into helices even in the presence of two typically ß-sheet favoring Val residues. The hybrid helix consisting of terminal pyridine units coordinates with the metal ions and drives the helical polymerization. Depending on the sequence and the position of N in pyridine moieties, these peptides form selective metallogels with Ag+ and Cu2+ ions. The X-ray diffracted analysis of the peptide single crystals obtained from the gel matrix reveals that the helical structure is maintained during the self-assembly process. Further, by varying the counter anion, a 3D helical crystalline coordination polymer with permanent porosity is generated. The findings reported here can be used to design new functional metal-foldamer coordinated polymers.

Structural insight into hybrid peptide ε-helices.

Unique ε-helical organizations (11-helices) from ß,γ-hybrid peptides composed of chiral ß3-amino acids along with achiral 3,3- or 4,4-dimethyl substituted γ-amino acids are disclosed.

Structural Investigation of Hybrid Peptide Foldamers Composed of α-Dipeptide Equivalent ß-Oxy-δ5 -amino Acids.

Due to their equivalent lengths, δ-amino acids can serve as surrogates of α-dipeptides. However, δ-amino acids with proteinogenic side chains have not been well studied because of synthetic difficulties and because of their insolubility in organic solvents. Recently we reported the spontaneous supramolecular gelation of δ-peptides composed of ß(O)-δ5 -amino acids. Here, we report the incorporation of ß(O)-δ5 -amino acids as guests into the host α-helix, α,γ-hybrid peptide 12-helix and their single-crystal conformations. In addition, we studied the solution conformations of hybrid peptides composed of 1:1 alternating α and ß(O)-δ5 -amino acids. In contrast to the control α-helix structures, the crystal structure of peptides with ß(O)-δ5 -amino acids exhibit α-helical conformations consisting of both 13- and 10-membered H-bonds. The α,δ-hybrid peptide adopted mixed 13/11-helix conformation in solution with alternating H-bond directionality. Crystal-structure analysis revealed that the α,γ4 -hybrid peptide accommodated the guest ß(O)-δ5 -amino acid without significant deviation to the overall helix folding. The results reported here emphasize that ß(O)-δ5 -amino acids with proteinogenic side chains can be accommodated into regular α-helix or 12-helix as guests without much deviation of the overall helix folding of the peptides.

Ambidextrous α,γ-Hybrid Peptide Foldamers.

Molecular chirality is ubiquitous in nature. The natural biopolymers, proteins and DNA, preferred a right-handed helical bias due to the inherent stereochemistry of the monomer building blocks. Here, we are reporting a rare co-existence of left- and right-handed helical conformations and helix-terminating property at the C-terminus within a single molecule of α,γ-hybrid peptide foldamers composed of achiral Aib (α-aminoisobutyric acid) and 3,3-dimethyl-substituted γ-amino acid (Adb; 4-amino-3,3-dimethylbutanoic acid). At the molecular level, the left- and right-handed helical screw sense of α,γ-hybrid peptides are representing a macroscopic tendril perversion. The pronounced helix-terminating behaviour of C-terminal Adb residues was further explored to design helix-Schellman loop mimetics and to study their conformations in solution and single crystals. The stereochemical constraints of dialkyl substitutions on γ-amino acids showed a marked impact on the folding behaviour of α,γ-hybrid peptides.

Direct Transformation of N-Protected α,ß-Unsaturated γ-Amino Amides into γ-Lactams through a Base-Mediated Molecular Rearrangement.

Here, we are reporting a single-step transformation of N-protected α,ß-unsaturated γ-amino amides into 5,5-disubstituted γ-lactams through a base-mediated new molecular rearrangement. In contrast to the known N- to C(O) cyclization of saturated γ-amino acids into corresponding γ-lactams, the new rearrangement involves the cyclization between N-terminal Cγ- to C-terminal amide N. The cyclization process was initiated by the migration of double bond from α,ß â†’ ß,γ position. The enamine-imine tautomerization of the new ß,γ-double bond and subsequent 5-exo-trig cyclization of terminal amide leads to the formation of N-protected 5,5-disubstituted γ-lactam. The structures of various γ-lactams obtained from the rearrangement were studied in single crystals. Overall, the results reported here demonstrate the facile and single-step transformation of N-protected α,ß-unsaturated γ-amino amides into γ-lactams and provided an excellent opportunity to construct small-molecule peptidomimetics.

Impact of substituent effects on the design of ß-sheet mimetics and ß-double helices from (E)-vinylogous γ-amino acid oligomers.

Here we report the design, single crystal conformations, impact of the substituents and structural differences of two structurally important motifs, ß-sheets and ß-double helices. Though ß-sheets are common structural motifs in protein structures, ß-double helices are not common in proteins and peptides. We found that both ß-sheet mimetics and ß-double helices can be constructed from the homooligomers of α,ß-unsaturated γ-amino acids. Results suggested that introducing gem-dialkyl substitutions at the γ-carbon of the homooligomer of α,ß-unsaturated γ-amino acids resulted in the ß-double helix conformation, while the same oligomer with monosubstitution at the γ-carbon displayed a ß-sheet structure.

Design of Helical Peptide Foldamers through α,ß â†’ ß,γ Double-Bond Migration.

The direct transformation of nonhelical α,γ-hybrid peptides composed of alternating α- and E-vinylogous amino acids into 12-helical structures through a base-mediated α,ß â†’ ß,γ double-bond migration is reported. The conformations of double-bond-migrated new 12-helices were studied in single crystals and in solution. Instructively, the 12-helices reported here were found to be acid labile, and they completely break down into the corresponding amino acid derivatives upon treatment with acids.

Divergent Supramolecular Gelation of Backbone Modified Short Hybrid δ-Peptides.

The ordered supramolecular assemblies of short peptides have been recently gaining momentum due to their widespread applications in biology and materials sciences. In contrast to the α-peptides, limited success has been achieved from the backbone modified peptides. The proteolytic stability and conformational flexibility of the backbone modified peptides composed of ß-, γ-, and δ-amino acids can be explored to design ordered supramolecular gels and self-assembled materials. In this article, we are reporting the divergent supramolecular gels from a new class of short hybrid dipeptides composed of conformationally flexible new ß(O)-δ5-amino acids. The hybrid dipeptide composed of ß3- and ß(O)-δ5-Phe showed the formation of transparent gels from the aromatic solvents, while the dipeptide composed of ß(O)-δ5-Phe showed the thixotropic gel in phosphate buffered saline (PBS). In contrast, no organic or hydrogels were observed from the dipeptides composed of alternating α- and ß(O)-δ5-Phe as well as γ4 and ß(O)-δ5-Phe. The organogelation property displayed by the ß3,ß(O)-δ5-Phe dipeptide was further explored to recover the oil spills from the oil-water mixture. The thixotropic hydrogels displayed by the ß(O)-δ5, ß(O)-δ5-Phe dipeptide was further utilized as matrix along with cell culture medium to grow the cells in 2D-cell culture. Replacing the backbone -CH2- with "O" in the δ-Phe leads to the drastic change in the supramolecular behavior of δ-peptides. Overall, the short dipeptides from different backbone modified amino acids showed the divergent gelation properties and these properties can be further explored to design new functional biomaterials.

Metal-Helix Frameworks from Short Hybrid Peptide Foldamers.

The potential of structured peptides has not been explored much in the design of metal-organic frameworks (MOFs). This is partly due to the difficulties in obtaining stable secondary structures from the short α-peptide sequences. Here we report the design, crystal conformations, coordination site dependent different silver coordinated frameworks of short α,γ-hybrid peptide 12-helices consisting of terminal pyridyl moieties and the utility of metal-helix frameworks in the adsorption of CO2 . Upon silver ion coordination the 12-helix terminated by the 3-pyridyl derivatives adopted a 2:2 macrocyclic structure, while the 12-helix terminated by the 4-pyridyl derivatives displayed remarkable porous metal-helix frameworks. Both head-to-tail intermolecular H-bonds of the 12-helix and metal ion coordination have played an important role in stabilizing the ordered metal-helix frameworks. The studies described here open the door to design a new class of metal-organic-frameworks from peptide foldamers.

Inhibition of ß-Amyloid Aggregation through a Designed ß-Hairpin Peptide.

Designing peptide-based drugs to target the ß-sheet-rich toxic intermediates during the aggregation of amyloid-ß 1-42 (Aß1-42) has been a major challenge. In general, ß-sheet breaker peptides (BSBPs) are designed to complement the enthalpic interactions with the aggregating protein, and entropic effects are usually ignored. Here, we have developed a conformationally constrained cyclic BSBP by the use of an unnatural amino acid and a disulfide bond. We show that our peptide strongly inhibits the aggregation of Aß1-42 in a concentration-dependent manner. It stabilizes the random coil conformation of Aß1-42 monomers and inhibits the secondary structural transition to a ß-sheet-rich conformation which allows Aß1-42 to oligomerize in an ordered assembly during its aggregation. Our cyclic peptide also rescues the toxicity of soluble aggregates of Aß1-42 toward neuronal cells. However, it significantly loses its potency in the conformationally relaxed acyclic form. It appears that limiting the loss of conformational entropy of the BSBP ligand can play a very important role in the attainment of conformations for precise and tight binding, making them a potent inhibitor for Aß1-42 amyloidosis.

Artificial ß-Double Helices from Achiral γ-Peptides.

Double helices are not common in polypeptides and proteins except in the peptide antibiotic gramicidin A and analogous l,d-peptides. In contrast to natural polypeptides, remarkable ß-double-helical structures from achiral γ-peptides built from α,ß-unsaturated γ-amino acids have been observed. The crystal structures suggest that they adopted parallel ß-double helical structures and these structures are stabilized by the interstrand backbone amide H-bonds. Furthermore, both NMR spectroscopy and fluorescence studies support the existence of double-helical conformations in solution. Although a variety of folded architectures featuring distinct H-bonds have been discovered from the ß- and γ-peptide foldamers, this is the first report to show that achiral γ-peptides can spontaneously intertwine into ß-double helical structures.

Modulating the Structural Properties of α,γ-Hybrid Peptides by α-Amino Acid Residues: Uniform 12-Helix Versus "Mixed" 12/10-Helix.

The most important natural α- and 310 -helices are stabilized by unidirectional intramolecular hydrogen bonds along the helical cylinder. In contrast, we report here on 12/10-helical conformations with alternately changing hydrogen-bond directionality in sequences of α,γ-hybrid peptides P1-P5 [P1: Boc-Ala-Aic-Ala-Aic-COOH; P2: Boc-Leu-Aic-Leu-Aic-OEt; P3: Boc-Leu-Aic-Leu-Aic-Leu-Aic-Aib-OMe; P4: Boc-Ala-Aic-Ala-Aic-Ala-Aic-Ala-OMe; P5: Boc-Leu-Aic-Leu-Aic-Leu-Aic-Leu-Aic-Aib-OMe; Aic=4-aminoisocaproic acid, Aib=2-aminoisobutyric acid] composed of natural α-amino acids and the achiral γ4,4 -dimethyl substituted γ-amino acid Aic in solution and in single crystals. The helical conformations are stabilized by alternating i→i+3 and i→i-1 intramolecular hydrogen bonds. The experimental data are supported by ab initio MO calculations. Surprisingly, replacing the natural α-amino acids of the sequence by the achiral dialkyl amino acid Ac6 c [P6: Boc-Ac6 c-Aic-Ac6 c-Aic-Ac6 c-Aic-Ac6 c-Aic-Ac6 c-CONHMe; Ac6 c = 1-aminocyclohexane-1-carboxylic acid] led to a 12-helix with unidirectional hydrogen bonds showing an entirely different backbone conformation. The results presented here emphasize the influence of the structure of the α-amino acid residues in dictating the helix types in α,γ-hybrid peptide foldamers and demonstrate the consequences for folding of small structural variations in the monomers.

Potent Antimicrobial Activity of Lipidated Short α,γ-Hybrid Peptides.

Herein we report the potent antimicrobial activity of α,γ-hybrid lipopeptides composed of 1:1 alternating α- and γ-amino acids. Along with their potent antimicrobial activity against various Gram-positive and Gram-negative bacteria, these hybrid lipopeptides were found to be less hemolytic. Studies into the mechanism of action revealed that these short cationic lipopeptides bind and disrupt the bacterial cell membrane. Time-kill kinetics analyses revealed that the potent α,γ-hybrid lipopeptides completely inhibit bacterial growth in less than 20 minutes. Overall, the promising antimicrobial activity along with lower hemolytic activity displayed by these α,γ-hybrid lipopeptides make them well suited for further exploration into the design of potent lipopeptide antibiotics.