Aib Residues Research Articles

Relevance of amphiphilicity and helicity on the antibacterial action of a histatin 5-derived peptide.

Peptide dhvar4, derived from the active domain of our salivary peptide histatin 5, bears a Phe residue in the middle of its hydrophilic face when folded into an α-helix. We then synthesized an analog with this Phe replaced by Lys and two analogs preserving Phe but bearing two and three α-aminoisobutyric acid (Aib) residues to stabilize the helical structure. The aim of this design was to verify which of the two features is more favorable to the biological activity. We performed a conformational study by means of circular dichroism and nuclear magnetic resonance, made antibacterial tests, and assessed the stability of the peptides in human serum. We observed that amphiphilicity is more important than helix stability, provided a peptide can adopt a helical conformation in a membrane-mimetic environment.

Sculpting Secondary Structure of a Cyclic Peptide: Conformational Analysis of a Cyclic Hexapeptide Containing a Combination of l-Leu, d-Leu, and Aib Residues.

We have previously reported that cyclo(l-Leu-d-Leu-Aib-l-Leu-d-Leu-Aib) (2), a cyclic hexapeptide consisting of heterochiral l-Leu and d-Leu (l-Leu-d-Leu) residues with achiral 2-aminoisobutyric acid (Aib) residues, forms a figure-8 conformation. In this study, we newly designed cyclo(l-Leu-d-Leu-Aib-d-Leu-l-Leu-Aib)+ (4), an epimer of 2, and examined the conformational differences between 2 and 4 by X-ray crystallographic analysis. Peptide 4 formed a planar cyclic conformation with an antiparallel β-sheet hydrogen-bonding pattern. This investigation demonstrates the potential to manipulate the molecular conformation of cyclic peptides by simply arranging the l- and d-amino acids and emphasizes that diverse conformations can be obtained by using cyclic peptides. Harnessing cyclic peptides as platforms for distinct molecular structures is a promising approach to expanding the chemical space for various applications.

Total synthesis of the natural, medium-length, peptaibol pentadecaibin and study of the chemical features responsible for its membrane activity.

Peptaibols are naturally occurring, antimicrobial peptides endowed with well-defined helical conformations and resistance to proteolysis. Both features stem from the presence in their sequence of several, Cα -tetrasubstituted, α-aminoisobutyric acid (Aib) residues. Peptaibols interact with biological membranes, usually causing their leakage. All of the peptaibol-membrane interaction mechanisms proposed so far begin with peptide aggregation or accumulation. The long-length alamethicin, the most studied peptaibol, acts by forming pores in the membranes. Conversely, the carpet mechanism has been claimed for short-length peptaibols, such as trichogin. The mechanism of medium-length peptaibols is far less studied, and this is partly due to the difficulties of their synthesis. They are believed to perturb membrane permeability in different ways, depending on the membrane properties. The present work focuses on pentadecaibin, a recently discovered, medium-length peptaibol. In contrast to the majority of its family members, its sequence does not comprise hydroxyprolines or prolines, and its helix is not kinked. A reliable and effective synthesis procedure is described that allowed us to produce also two shorter analogs. By a combination of techniques, we were able to establish a 3D-structure-activity relationship. In particular, the membrane activity of pentadecaibin heavily depends on the presence of three consecutive Aib residues that are responsible for the clear, albeit modest, amphiphilic character of its helix. The shortest analog, devoid of two of these three Aib residues, preserves a well-defined helical conformation, but not its amphipathicity, and loses almost completely the ability to cause membrane leakage. We conclude that pentadecaibin amphiphilicity is probably needed for the peptide ability to perturb model membranes.

Enantioselective conjugate addition to nitroolefins catalysed by helical peptides with a single remote stereogenic centre.

Two short pentapeptides rich in α-aminoisobutyric acid (Aib) residues have been shown to act as enantioselective organocatalysts for the conjugate addition of nucleophiles to nitroolefins. An L-alanine terminated peptide, (Aib)4(L-Ala)NHtBu, which has neither functionalised sidechains nor a highly designed reactive site, used an exposed N-terminal primary amine and the amide bonds of the backbone to mediate catalysis. Folding of this peptide into a 310 helical structure was observed by crystallography. Folding into a helix relays the conformational preference of the chiral alanine residue at the C-terminus to the primary amine at the N-terminus, 0.9 nm distant. The chiral environment and defined shape produced by the 310 helix brings the amine site into proximity to two exposed amide NHs. Reaction scope studies implied that the amine acts as a Brønsted base and the solvent-exposed NH groups of the helix, shown to weakly bind β-nitrostyrene, are needed to obtain an enantiomeric excess. Replacement of L-alanine with D-phenylalanine gave (Aib)4(D-Phe)NHtBu, a peptide that now catalysed the benchmark reaction with the opposite enantioselectivity. These studies show how achiral residues can play a key role in enantioselective catalysis by peptides through the promotion of folding.

Alpha-aminoisobutyric acid incorporated peptides for the construction of electrochemical biosensors with high stability and low fouling in serum

An effective strategy to construct low fouling electrochemical biosensors for assaying serum biomarkers was proposed based on specially designed α-aminoisobutyric acid (Aib) incorporated peptides. The Aib-peptides were designed to be of antifouling properties, and at the same to incorporate Aib residues in their interior to enhance the hydrolytic stability. In order to construct the electrochemical biosensor, two kinds of Aib-peptides labelled with biotin were modified on the electrode surface: One with cysteine terminal for easy attachment to the electrode modified with gold nanoparticles, the other with unique terminal peptide sequence for specific binding of immunoglobulin G (IgG), and they were connected through the streptavidin-biotin affinity system. Owing to the interposition of Aib residues, the peptides as well as the constructed biosensors showed excellent antifouling performances and enhanced stability against enzymatic degradation in serum. Furthermore, the IgG biosensor constructed with the Aib-peptides displayed a very low detection limit (29.5 pg mL−1) and a broad linear range (100 pg mL−1 - 10 μg mL−1), and it was able to assay IgG in clinical human sera with decent accuracy and reliability. This strategy provides a new path for the construction of stable antifouling biosensors based on functional peptides for practical biomarker assaying in real clinical samples.

Atomic insight into short helical peptide comprised of consecutive multiple aromatic residues.

The occurrence of sequential multiple aromatic residues in a helical sequence is rare compared to the β-sheet rich structure. Here, using helix promoting α-aminoisobutyric acid (Aib) residues, we unravel atomistic details of the helical secondary structure formation and the super helical assembly of two heptapeptides composed of sequential five and six phenylalanine (Phe) residues.

Cα-Methyl-l-valine: A Preferential Choice over α-Aminoisobutyric Acid for Designing Right-Handed α-Helical Scaffolds.

In synthetic peptides containing Gly and coded α-amino acids, one of the most common practices to enhance their helical extent is to incorporate a large number of l-Ala residues along with noncoded, strongly foldameric α-aminoisobutyric acid (Aib) units. Earlier studies have established that Aib-based peptides, with propensity for both the 310- and α-helices, have a tendency to form ordered three-dimensional structure that is much stronger than that exhibited by their l-Ala rich counterparts. However, the achiral nature of Aib induces an inherent, equal preference for the right- and left-handed helical conformations as found in Aib homopeptide stretches. This property poses challenges in the analysis of a model peptide helical conformation based on chirospectroscopic techniques like electronic circular dichroism (ECD), a very important tool for assigning secondary structures. To overcome such ambiguity, we have synthesized and investigated a thermally stable 14-mer peptide in which each of the Aib residues of our previously designed and reported analogue ABGY (where B stands for Aib) is replaced by Cα-methyl-l-valine (L-AMV). Analysis of the results described here from complementary ECD and 1H nuclear magnetic resonance spectroscopic techniques in a variety of environments firmly establishes that the L-AMV-containing peptide exhibits a significantly stronger preference compared to that of its Aib parent in terms of conferring α-helical character. Furthermore, being a chiral α-amino acid, L-AMV shows an intrinsic, extremely strong bias for a quite specific (right-handed) screw sense. These findings emphasize the relevance of L-AMV as a more appropriate unit for the design of right-handed α-helical peptide models that may be utilized as conformationally constrained scaffolds.

Conformationally Constrained Peptides with High Affinity to the Vascular Endothelial Growth Factor

The design of efficient vascular endothelial growth factor (VEGF) inhibitors is a high-priority research area aimed at the treatment of pathological angiogenesis. Among other compounds, v114* has been identified as a potent VEGF-binding peptide. In order to improve the affinity to VEGF, we built a conformational constrain in its structure. To this aim, Cα-tetrasubstituted amino acid Aib was introduced into the N-terminal tail, peptide loop, or C-terminal helix. NMR studies confirmed the stabilization of the helical conformation in proximity to the Aib residue. We found that the induction of the N-terminal helical structure or stabilization of the C-terminal helix can noticeably increase the peptide affinity to the VEGF. These peptides efficiently inhibited VEGF-stimulated cell proliferation as well. The insertion of the non-proteinogenic Aib residue significantly enhanced the stability of the peptides in the vitreous environment. Thus, these Aib-containing peptides are promising candidates for the design of VEGF inhibitors with improved properties.

Distance Dependence of Electron Transfer through Conformationally Constrained Peptides

Understanding and the ability of peptides to mediate long-range electron tunneling in proteins is an evergreen topic. In this context, the study of electron transfer (ET) through model peptides has been particularly useful, especially to assess the occurrence (or competition) of the superexchange and sequential hopping mechanisms as a function of the structure and length of the peptide bridge. In previous studies [1,2], we stressed the importance of dealing with peptides with a well-defined and rigid structure to properly correlate kinetic and structural data. This structural control was made possible by using model peptide bridges based on oligomers of the α-aminoisobutyric acid (Aib) residue. The Aib unit is characterized by a marked steric hindrance at the α-carbon, and this allows the preparation of very rigid peptides. Rigidity is ensured by a framework of intramolecular C=O···H-N hydrogen bonds. Consequently, these peptides possess a strong dipole moment along the main axis [3]. In previous studies, we used cyclic voltammetry to study the dissociative ETs in two series of Donor − Aib-peptide − Acceptor systems in which we changed the donor group, whereas the acceptor was kept constant (a dialkyl peroxide moiety, resulting in the cleavage of the O-O peroxide bond [4]). In this communication, we describe the outcome of dissociative ETs carried out using a new series of Donor − Aib-peptide − Acceptor system in which the acceptor was a perbenzoate moiety [5]. This change also affects the dissociative ET driving force. The same peptide-length dependent dissociative ETs were tested for a possible solvent effect.

Rational Design of Helix-Stabilized Antimicrobial Peptide Foldamers Containing α,α-Disubstituted Amino Acids or Side-Chain Stapling.

Antimicrobial peptides (AMPs) are expected to be good candidate molecules for novel antimicrobial therapies. Most AMPs exert their antimicrobial activity through disruption of microbial membranes due to their amphipathic properties. Recently, the helical peptide 'Stripe' was reported by our group, a rationally designed amphipathic AMP focused on distribution of natural cationic and hydrophobic amino acid residues. In this study, a set of Stripe-based AMP foldamers was designed, synthesized and investigated that contain α,α-disubstituted amino acids or side-chain stapling to stabilize their helical structures. Our results showed that a peptide containing 2-aminoisobutyric acid (Aib) residues exhibited potent antimicrobial activity against both Gram-positive S.aureus (MIC value: 3.125 μM) and Gram-negative bacteria (including a multidrug-resistant strain, MDRP, MIC value: 1.56 μM), without significant hemolytic activity (>100 μM). Electrophysiological measurements revealed that this peptide formed stable pores in a 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) bilayer but not in a dioleoylphosphocholine (DOPC) bilayer. The introduction of Aib residues into Stripe could be a promising way to increase the antimicrobial activity of AMP foldamers, and the peptide could represent a promising novel therapeutic candidate to treat multidrug-resistant bacterial infection.

D- and Unnatural Amino Acid Substituted Antimicrobial Peptides With Improved Proteolytic Resistance and Their Proteolytic Degradation Characteristics.

The transition of antimicrobial peptides (AMPs) from the laboratory to market has been severely hindered by their instability toward proteases in biological systems. In the present study, we synthesized derivatives of the cationic AMP Pep05 (KRLFKKLLKYLRKF) by substituting L-amino acid residues with D- and unnatural amino acids, such as D-lysine, D-arginine, L-2,4-diaminobutanoic acid (Dab), L-2,3-diaminopropionic acid (Dap), L-homoarginine, 4-aminobutanoic acid (Aib), and L-thienylalanine, and evaluated their antimicrobial activities, toxicities, and stabilities toward trypsin, plasma proteases, and secreted bacterial proteases. In addition to measuring changes in the concentration of the intact peptides, LC-MS was used to identify the degradation products of the modified AMPs in the presence of trypsin and plasma proteases to determine degradation pathways and examine whether the amino acid substitutions afforded improved proteolytic resistance. The results revealed that both D- and unnatural amino acids enhanced the stabilities of the peptides toward proteases. The derivative DP06, in which all of the L-lysine and L-arginine residues were replaced by D-amino acids, displayed remarkable stability and mild toxicity in vitro but only slight activity and severe toxicity in vivo, indicating a significant difference between the in vivo and in vitro results. Unexpectedly, we found that the incorporation of a single Aib residue at the N-terminus of compound UP09 afforded remarkably enhanced plasma stability and improved activity in vivo. Hence, this derivative may represent a candidate AMP for further optimization, providing a new strategy for the design of novel AMPs with improved bioavailability.

Exploring the Use of Helicogenic Amino Acids for Optimising Single Chain Relaxin-3 Peptide Agonists.

Relaxin-3 is a highly conserved two-chain neuropeptide that acts through its endogenous receptor the Relaxin Family Peptide-3 (RXFP3) receptor. The ligand/receptor system is known to modulate several physiological processes, with changes in food intake and anxiety-levels the most well studied in rodent models. Agonist and antagonist analogues based on the native two-chain peptide are costly to synthesise and not ideal drug leads. Since RXFP3 interacting residues are found in the relaxin B-chain only, this has been the focus of analogue development. The B-chain is unstructured without the A-chain support, but in single-chain variants structure can be induced by dicarba-based helical stapling strategies. Here we investigated whether alternative helical inducing strategies also can enhance structure and activity at RXFP3. Combinations of the helix inducing α-aminoisobutyric acid (Aib) were incorporated into the sequence of the relaxin-3 B-chain. Aib residues at positions 13, 17 and 18 partially reintroduce helicity and activity of the relaxin-3 B-chain, but other positions are generally not suited for modifications. We identify Thr21 as a putative new receptor contact residue important for RXFP3 binding. Cysteine residues were also incorporated into the sequence and cross-linked with dichloroacetone or α, α’-dibromo-m-xylene. However, in contrast to previously reported dicarba variants, neither were found to promote structure and RXFP3 activity.

Tumor Necrosis Factor-α Trimer Disassembly and Inactivation via Peptide-Small Molecule Synergy.

Aberrant signaling by tumor necrosis factor-α (TNFα) is associated with inflammatory diseases that can be treated with engineered proteins that inhibit binding of this cytokine to cell-surface receptors. Despite these clinical successes, there is considerable interest in the development of smaller antagonists of TNFα-receptor interactions. We describe a new 29-residue α/β-peptide, a molecule that contains three β-amino acid residues and three α-aminoisobutryic acid (Aib) residues, that displays potent inhibition of TNFα binding to TNFα receptor 1 (TNFR1) and rescues cells from TNFα-induced death. The complement of nonproteinogenic residues renders this α/β-peptide highly resistant to proteolysis, relative to all-α analogues. The mechanism of inhibitory action of the new 29-mer involves disruption of the trimeric TNFα quaternary structure, which prevents productive binding to TNFα receptors. Unexpectedly, we discovered that peptide-induced trimer disruption can be promoted by structurally diverse small molecules, including a detergent commonly used during selection procedures. The discovery of this synergistic effect provides a new context for understanding previous reports on peptidic antagonists of TNFα-receptor interactions and suggests new avenues for future efforts to block signaling via proteins with an active form that is oligomeric, including other members of the TNFα family.

Helical Peptides Design for Molecular Dipoles Functionalization of Wide Band Gap Oxides.

The use of helical hexapeptides to establish a surface dipole layer on a TiO2 substrate, with the goal of influencing the energy levels of a coadsorbed chromophore, is explored. Two helical hexapeptides, synthesized from 2-amino isobutyric acid (Aib) residues, were protected at the N-terminus with a carboxybenzyl group (Z) and at the C-terminus carried either a carboxylic acid or an isophthalic acid (Ipa) anchor group to form Z-(Aib)6-COOH or Z-(Aib)6-Ipa, respectively. Using a combination of vibrational and photoemission spectroscopies, bonding of the two peptides to TiO2 surfaces (either nanostructured or single-crystal TiO2(110)) was found to be highly dependent on the anchor group, with Ipa establishing a monolayer much more efficiently than COOH. Furthermore, a monolayer of Z-(Aib)6-Ipa on TiO2(110) was exposed for different binding times to a solution of a zinc tetraphenylporphyrin (ZnTPP) derivative terminated with an Ipa anchor group (ZnTPP-P-Ipa). Photoemission spectroscopy revealed that ZnTPP-P-Ipa partly displaced Z-(Aib)6-Ipa, forming a coadsorbed monolayer on the oxide surface. The presence of the peptide molecular dipole shifted the HOMO levels of the ZnTPP group to lower energy by ∼300 meV, in accordance with a simple parallel plate capacitor model. These results suggest that a mixed-layer approach, involving coadsorption of a strong molecular dipole compound with a chromophore, is a versatile method to shift the energy levels of such chromophores with respect to the band edges of the substrate.

Automated solid-phase concatenation of Aib residues to form long, water-soluble, helical peptides.

Iterative coupling of 2-aminoisobutyric acid (Aib) has been achieved rapidly and efficiently using automated solid-phase peptide synthesis, employing diisopropylcarbodiimide (DIC) in the presence of ethyl cyanohydroxyiminoacetate (Oxyma). This method has allowed the first total synthesis of the fungal antibiotic Cephaibol D, and enabled the synthesis of water-soluble oligomers of Aib containing up to an unprecedented sequence of 17 consecutive Aib residues. Conformational analysis of the Aib oligomers in aqueous solution shows a length dependence in their CD spectra, with oligomers of more than 14 Aib residues apparently adopting structured helical conformations.

Impact of Dehydroamino Acids on the Structure and Stability of Incipient 310-Helical Peptides.

A comparative study of the impact of small, medium-sized, and bulky α,β-dehydroamino acids (ΔAAs) on the structure and stability of Balaram's incipient 310-helical peptide (1) is reported. Replacement of the N-terminal Aib residue of 1 with a ΔAA afforded peptides 2a-c that maintained the 310-helical shape of 1. In contrast, installation of a ΔAA in place of Aib-3 yielded peptides 3a-c that preferred a β-sheet-like conformation. The impact of the ΔAA on peptide structure was independent of size, with small (ΔAla), medium-sized (Z-ΔAbu), and bulky (ΔVal) ΔAAs exerting similar effects. The proteolytic stabilities of 1 and its analogs were determined by incubation with Pronase. Z-ΔAbu and ΔVal increased the resistance of peptides to proteolysis when incorporated at the 3-position and had negligible impact on stability when placed at the 1-position, whereas ΔAla-containing peptides degraded rapidly regardless of position. Exposure of peptides 2a-c and 3a-c to the reactive thiol cysteamine revealed that ΔAla-containing peptides underwent conjugate addition at room temperature, while Z-ΔAbu- and ΔVal-containing peptides were inert even at elevated temperatures. These results suggest that both bulky and more accessible medium-sized ΔAAs should be valuable tools for bestowing rigidity and proteolytic stability on bioactive peptides.

Extent of Helical Induction Caused by Introducing α-Aminoisobutyric Acid into an Oligovaline Sequence.

The preferred conformations of a dodecapeptide composed of l-valine (l-Val) and α-aminoisobutyric acid (Aib) residues, Boc-(l-Val-l-Val-Aib)4-OMe (3), were analyzed in solution and in the crystalline state. Peptide 3 predominantly folded into a mixture of α- and 310-(P) helical structures in solution and a (P) α helix in the crystalline state.

Conformational properties, membrane interaction, and antibacterial activity of the peptaibiotic chalciporin A: Multitechnique spectroscopic and biophysical investigations on the natural compound and labeled analogs.

In this work, an extensive set of spectroscopic and biophysical techniques (including FT-IR absorption, CD, 2D-NMR, fluorescence, and CW/PELDOR EPR) was used to study the conformational preferences, membrane interaction, and bioactivity properties of the naturally occurring synthetic 14-mer peptaibiotic chalciporin A, characterized by a relatively low (≈20%), uncommon proportion of the strongly helicogenic Aib residue. In addition to the unlabeled peptide, we gained in-depth information from the study of two labeled analogs, characterized by one or two residues of the helicogenic, nitroxyl radical-containing TOAC. All three compounds were prepared using the SPPS methodology, which was carefully modified in the course of the syntheses of TOAC-labeled analogs in view of the poorly reactive α-amino function of this very bulky residue and the specific requirements of its free-radical side chain. Despite its potentially high flexibility, our results point to a predominant, partly amphiphilic, α-helical conformation for this peptaibiotic. Therefore, not surprisingly, we found an effective membrane affinity and a remarkable penetration propensity. However, chalciporin A exhibits a selectivity in its antibacterial activity not in agreement with that typical of the other members of this peptide class.

Constrained Cyclic β,γ‐Diamino Acids from Glutamic Acid: Synthesis of Both Diastereomers and Unexpected Kinetic Resolution

We describe here an efficient synthesis of both diastereomers of cyclic β,γ‐diamino acids starting from l‐glutamic acid, based on the Blaise reaction. We show that by changing the protecting group, we can access either the five‐membered‐ring lactam, which can be used as a β‐amino acid, or the six‐membered‐ring lactam, as a γ‐amino acid. We also discovered an interesting kinetic resolution during the synthesis that allowed easier separation of diastereomers. The products can be easily used in peptide synthesis, and a tetramer with alternating trans cyclic γ‐amino acid and AIB (2‐aminoisobutyric acid) residues was synthesized.

The Role of Terminal Functionality in the Membrane and Antibacterial Activity of Peptaibol-Mimetic Aib Foldamers.

Peptaibols are peptide antibiotics that typically feature an N-terminal acetyl cap, a C-terminal aminoalcohol, and a high proportion of α-aminoisobutyric acid (Aib) residues. To establish how each feature might affect the membrane-activity of peptaibols, biomimetic Aib foldamers with different lengths and terminal groups were synthesised. Vesicle assays showed that long foldamers (eleven Aib residues) with hydrophobic termini had the highest ionophoric activity. C-terminal acids or primary amides inhibited activity, while replacement of an N-terminal acetyl with an azide group made little difference. Crystallography showed that N3 Aib11 CH2 OTIPS folded into a 310 helix 2.91 nm long, which is close to the bilayer hydrophobic width. Planar bilayer conductance assays showed discrete ion channels only for N-acetylated foldamers. However long foldamers with hydrophobic termini had the highest antibacterial activity, indicating that ionophoric activity in vesicles was a better indicator of antibacterial activity than the observation of discrete ion channels.