β-Ala Residues Research Articles

Exploring the space of histidine containing dipeptides in search of novel efficient RCS sequestering agents

The study reports a set of forty proteinogenic histidine-containing dipeptides as potential carbonyl quenchers. The peptides were chosen to cover as exhaustively as possible the accessible chemical space, and their quenching activities toward 4-hydroxy-2-nonenal (HNE) and pyridoxal were evaluated by HPLC analyses. The peptides were capped at the C-terminus as methyl esters or amides to favor their resistance to proteolysis and diastereoisomeric pairs were considered to reveal the influence of configuration on quenching. On average, the examined dipeptides are less active than the parent compound carnosine (βAla + His) thus emphasizing the unfavorable effect of the shortening of the βAla residue as confirmed by the control dipeptide Gly-His. Nevertheless, some peptides show promising activities toward HNE combined with a remarkable selectivity. The results emphasize the beneficial role of aromatic and positively charged residues, while negatively charged and H-bonding side chains show a detrimental effect on quenching. As a trend, ester derivatives are slightly more active than amides while heterochiral peptides are more active than their homochiral diastereoisomer. Overall, the results reveal that quenching activity strongly depends on conformational effects and vicinal residues (as evidenced by the reported QSAR analysis), offering insightful clues for the design of improved carbonyl quenchers and to rationalize the specific reactivity of histidine residues within proteins.

Enhanced cellular uptake and metabolic stability of lipo‐oligoarginine peptides

Developing efficient cellular delivery vectors is crucial for designing novel therapeutic agents to enhance their plasma membrane permeability and metabolic stability in cells. Previously, we engineered cell penetrating peptide vectors named as "lipo-oligoarginine peptides" (LOAPs) by conjugating a proper combination of fatty acid and oligoarginine that translocated into cell easily without adverse effect on cell viability. In the present study, we report a systemic evaluation of cellular uptake and metabolic stability of LOAPs in Jurkat cells by introducing different combination of D-Arg residues in the peptide backbone. The cellular uptake and intracellular fate, cell viability, and metabolic stability and proteolytic degradation patterns of various LOAPs consisted of different combination of L- and D-Arg sequences were confirmed by flow cytometry, cytotoxicity assay, and analytical RP-HPLC with MALDI-TOF mass. All investigated LOAPs penetrated the cell efficiently with low cellular toxicity. The LOAPs having D-Arg residues at their N-termini seemed to have better metabolic stability than the LOAPs having C-terminal D-Arg residues. The metabolic degradation patterns were similar among all investigated LOAPs. The major hydrolytic site was between lauroyl group and β-Ala residue. Without the lipid chain, the oligoarginine peptide was pumped out ofcells easily. The results presented in this study suggest that structurally modified LOAPs could be used as a novel CPP design toward improved therapeutic application.

Coordination abilities of difunctional, diaminophosphonic acid and its dipeptides towards Cu(II) ions

Phenylenedi(aminomethylphosphonic) acid ( L9) and its peptides with L-Ala and β-Ala residues were synthesized and their ability to bind Cu(II) ions in aqueous solution was studied. L9 forms complexes with metal to ligand ratios of 1:1 and 1:2 which are typical for aminophosphonic acids coordination mode 1x or 2 x { NH 2 , PO 3 2 - } . In the case of their dipeptide derivatives ( L10– L12), only one peptidyl chain is involved in the binding of metal ion with stoichiometry 1:1 and coordination pattern similar to that found in the simple phosphonic dipeptides. The influence of metal ion chelation on the properties of uncoordinated group in the same ligand molecule was found.

Novel Nociceptin Analogues: Synthesis and Biological Activity

Syntheses are described of the nociceptin (1–13) amide [NC(1–13)-NH2] and of several analogues in which either one or both the phenylalanine residues (positions 1 and 4), the arginine residues (positions 8 and 12) and the alanine residues (positions 7 and 11) have been replaced by N-benzyl-glycine, N-(3-guanidino-propyl)-glycine and β-alanine, respectively. The preparation is also described of NC(1–13)-NH2 analogues in which either galactose or N-acetyl-galactosamine are β-O-glycosidically linked to Thr5 and/or to Ser10. Preliminary pharmacological experiments on mouse vas deferens preparations showed that Phe4, Thr5, Ala7 and Arg8 are crucial residues for OP4 receptor activation. Manipulation of Phe1 yielded peptides endowed with antagonist activity but [Nphe1] NC(1–13)-NH2 acted as an antagonist still possessing weak agonist activity. Introduction of the βAla residue either in position 7 or 11 of the [Nphe1] NC(1–13)-NH2 sequence, abolished any residual agonist activity and [Nphe1, βAla7] NC(1–13)-NH2 and [Nphe1, βAla11] NC(1–13)-NH2 acted as competitive antagonists only. Modification of both Ala7 and Ala11 abolished the antagonist activity of [Nphe1]NC(1–13)-NH2 probably by hindering receptor binding. Changes at positions 10 and 11 gave analogues still possessing agonist activity. [Ser(βGal)10] NC(1–13)-NH2 displayed an activity comparable with that of NC(1–13)-NH2, [Ser(βGalNAc)10] NC(1–13)-NH2 and [βAla11] NC(1–13)-NH2 were five and 10 times less active, respectively.

Conformational properties of β-residue-containing oligopeptides in apolar solvent

Molecular dynamics simulations carried out on short linear peptides incorporating β-Ala residues, namely Boc–β–Ala–Phe–OMe (1) and Boc–Met–β–Ala–Phe–OMe (2), reveal a markedly different conformational-dynamical behaviour. The tripeptide (2), showing free energy minima deeper than the dipeptide is predicted to be characterized by long-lived conformers. The present theoretical results may help in rationalizing experimental (IR, NMR) results on (1) and (2) and definitely show the importance of a dynamical approach for a correct interpretation and prediction of the conformational behaviour in solution even for relatively small molecules.

β-Ala Containing Peptides: Potentials in Design and Construction of Bioactive Peptides and Protein Secondary Structure Mimics

In recent years, there has been increasing interest in de novo design and construction of novel synthetic peptides that mimic protein secondary structures, i.e., turns, helices and sheets. The unique structural influences exerted by unsubstituted, non-coded, non-chiral beta-amino acid, i.e., beta-alanine (beta-Ala; 3- or beta- aminopropionic acid) on peptide backbone, when inserted into peptide chain comprised alpha-amino acids, offer an excellent opportunity to design and construct diverse well-defined three-dimensional structures. Our current understanding of folding-unfolding behavior of the beta-Ala residues relies primarily from an examination of conformational preferences of a large number of short cyclic- as well as acyclic beta-Ala containing peptides investigated using single crystal X-ray diffraction analysis. In addition, theoretical conformational energy calculations and different spectroscopic techniques: 1H NMR, FT-IR and CD, have also been employed although, to a lesser extent. The obtainable results tend to reveal overwhelming preferences of the beta-Ala moiety for the folded gauche (mu approximately +/-65+/-10 degrees conformation in cyclic- and for an extended trans (mu approximately +/-165+/-10 degrees) as well as gauche (mu approximately +/-65+/-10 degrees) orientations in acyclic beta-Ala containing peptides. The results also indicate that in short linear beta-Ala containing peptides, the specific influence of selective neighboring side-chain substituents e.g. linear- or cyclic symmetrically C(alpha,alpha)-disubstituted glycines and other conformational constraints, may be significant in controlling the overall folded-unfolded topographical features across the two methylene units (-CbetaH2-CalphaH2-) of the beta-Ala residue. Taking into consideration the wide occurrence of beta-Ala moiety in animal and plant kingdoms and the remarkable structural versatility of the peptides incorporating beta-Ala residue(s), together with appreciable resistance towards enzymatic degradation, hold strong promise for biophysicists and biochemists not only to design molecules that fold to mimic protein secondary structures but also to develop potent peptide analogs and peptidomimetics displaying unique pharmaceutical properties.

Stability of the Cu(II) Complexes of Tripeptides, Cu(H−2L), in Dynamic Aspects; L = Tripeptides Composed of Various Combinations of α-, β-, and γ-Amino-Acid Residues. Stopped-Flow Kinetic Studies on the Reaction of Cu(H−2L) with Cysteine

Abstract The stability of the fused chelate-ring structures of Cu(H−2L) concerning dynamic aspects was evaluated based on the rate constant (k1+) for the formation of ternary complexes, Cu(H−1L)(Cys−), from Cu(H−2L) and cysteine (Cys), where L denotes tripeptides composed of various combinations of α-, β-, and γ-amino-acid residues. The ligand-exchange reaction was examined by the stopped-flow spectrophotometric method. The Cu(H−2L) species, having 5–6–5- and 6–5–5-membered fused-chelate rings, were kinetically fairly stable; k1+ = 2–3 × 104 M−1 s−1. Other Cu(H−2L) species, having 5–5–5-, 5–5–6-, 5–5–7-, 5–6–6-, and 6–5–6-membered rings, reacted rapidly with Cys to form the Cu(H−1L)(Cys−) species; k1+ ≥ 1 × 105 M−1 s−1. The Cu(H−1L)(Cys−) species, upon forming, successively reacted with Cys to afford a binary complex, Cu(Cys−)2. The Cu(H−1L)(Cys−) species, with the β-Ala residue at the N-terminus, were kinetically labile to be rapidly transformed to Cu(Cys−)2.

External chirality-triggered helicity control promoted by introducing a beta-Ala residue into the N-terminus of chiral peptides.

The noncovalent chiral domino effect (NCDE), defined as chiral interaction upon an N-terminus of a 3(10)-helical peptide, will provide a unique method for structural control of a peptide helix through the use of external chirality. On the other hand, the NCDE has not been considered to be effective for the helicity control of peptides strongly favoring a one-handed screw sense. We here aim to promote the NCDE on peptide helicity using two types of nonapeptides: H-beta-Ala-Delta(Z)Phe-Aib-Delta(Z)Phe-X-(Delta(Z)Phe-Aib)(2)-OCH(3) [Delta(Z)Phe = alpha,beta-didehydrophenylalanine, Aib = alpha-aminoisobutyric acid], where X as the single chirality is L-leucine (1) or L-phenylalanine (2). NMR, IR, and CD spectroscopy as well as energy calculation revealed that both peptides alone form a right-handed 3(10)-helix. The original CD amplitudes or signs in chloroform, irrespective of a strong screw-sense preference in the central chirality, responded sensitively to external chiral information. Namely added Boc-L-amino acid stabilized the original right-handed helix, while the corresponding d-isomer destabilized it or transformed it into a left-handed helix. These peptides were also shown to bind more favorably to an L-isomer from the racemate. Although similar helicity control was observed for analogous nonapeptides bearing an N-terminal Aib residue (Inai, Y.; et al. Biomacromolecules 2003, 4, 122), the present findings demonstrate that the N-terminal replacement by the beta-Ala residue significantly improves the previous NCDE to achieve more effective control of helicity. Semiempirical molecular orbital calculations on complexation of peptide 2 with Boc-(L or D)-Pro-OH reasonably explained the unique conformational change induced by external chirality.

An observation of non-superimposable stereogeometrical features in a non-chiral one-component β-Ala model peptide

This paper describes the chemical synthesis and crystal molecular conformation of a non-chiral β-Ala containing model peptide Boc-β-Ala-Acc 5-OCH 3. The analysis revealed the existence of two crystallographically independent molecules A and B, in the asymmetric unit. Unexpectedly, while the magnitudes of the backbone torsion angles in both molecules are remarkably similar, the signs of the corresponding torsion angles are reverse therefore, inclining us to suggest the existence of non-superimposable stereogeometrical features in a non-chiral one-component β-Ala model system. The critical μ torsion angle around C βH 2–C αH 2 bond of the β-Ala residue represents a typical gauche orientation i.e., μ=67.7° in A and μ=−61.2° in B, providing the molecule an overall crescent shaped topology. The observed conformation contrasts markedly to those determined for the correlated non-chiral model peptides: Boc-β-Ala-Acc 6-OCH 3 and Boc-β-Ala-Aib-OCH 3 signifying the role of stereocontrolling elements since the stereochemically constrained C α,α-disubstituted glycyl residues (e.g., Acc 5, Acc 6, and the prototype Aib) are known to strongly restrict the peptide backbone conformations in the 3 10/α-helical-regions ( φ≈±60 ±20°, ψ≈±30 ±20°) of the Ramachandran map. Unpredictably, the preferred, φ, ψ torsion angles of the Acc 5 residue fall outside the helical regions of the Ramachandran map and exhibit opposite-handed twists for A and B. The implications of the semi-extended conformation of the Acc 5 residue in the construction of backbone-modified novel scaffolds and peptides of biological relevance are highlighted. Taken together, the results indicate that in short linear β-Ala containing peptides specific structural changes can be induced by selective substitution of non-coded linear- or cyclic symmetrically C α,α-disubstituted glycines, reinstating the hypothesis that in addition to conformational restrictions, the chemical nature of the neighboring side-chain substituents and local environments collectively influences the stabilization of folding–unfolding behavior of the two methylene units of a β-Ala residue.

Influence of hydrophobic interactions on the conformational adaptability of the beta-Ala residue.

The chemical synthesis and X-ray crystal structure analysis of a model peptide incorporating a conformationally flexible beta-Ala residue: Boc-beta-Ala-Pda, 1 (C23H46N2O3: molecular weight = 398.62) have been described. The peptide crystallized in the crystal system triclinic with space group P21: a = 5.116(3) A, b = 5.6770(10) A, c = 21.744(5) A; alpha = 87.45 degrees, beta = 86.87 degrees, gamma = 90.0 degrees; Z = 1. An attractive feature of the crystal molecular structure of 1 is the induction of a reasonably extended backbone conformation of the beta-Ala moiety, i.e. the torsion angles phi approximately -115 degrees, mu approximately 173 degrees and psi approximately 122 degrees, correspond to skew-, trans and skew+ conformation, respectively, by an unbranched hydrophobic alkyl chain, Pda, which prefers an all-anti orientation (theta1 approximately -153 degrees, theta2 approximately ellipsis theta14 approximately +/-178 degrees ). The observation is remarkable because, systematic conformational investigations of short linear beta-Ala peptides of the type Boc-beta-Ala-Xaa-OCH3 (Xaa = Aib or Acc6) have shown that the chemical and stereochemical characters of the neighboring moieties may be critical in dictating the overall folded and/or unfolded conformational features of the beta-Ala residue. The overall conformation of 1 is typical of a 'bar'. It appears convincing that, in addition to a number of hydrophobic contacts between the parallel arranged molecules, an array of conventional N-HellipsisO=C intermolecular H-bonding interactions stabilize the crystal molecular structure. Moreover, the resulting 14-membered pseudo-ring motif, generated by the amide-amide interactions between the adjacent molecules, is completely devoid of nonconventional C-HellipsisO interaction. The potentials of the conformational adaptation of the beta-Ala residue, to influence and stabilize different structural characteristics have been highlighted.

Collateral Existence of Folded and Extended Conformations of the β-Ala Moiety in a Model Peptide

The single-crystal X-ray diffraction analysis of a nonchiral β-Ala-containing model peptide, Boc-β-Ala-Aib-OCH3 1 (β-Ala, 3-aminopropionic acid; Aib, α-aminoisobutyric acid), establishes the coexistence of distinctly different backbone conformations in two crystallographically independent molecules, A and B, in the asymmetric unit. Interestingly, the central μ torsion angle around the -Cβ–Cα- bond of the conformationally flexible β-Ala residue appears to be critical in dictating the overall distinct structural features, i.e., in molecule A it adopts a folded gauche conformation: μ = −71.0°, whereas it favors an extended trans conformation, μ = 161.2°, in molecule B. As expected, the stereochemically constrained Aib residue preferred an energetically favorable folded backbone conformation, the torsion angles being φ = 46.2° and ψ = 48.3° for molecule A and φ = −43.6° and ψ = −45.5° for molecule B, lying in the left-handed and right-handed helical regions of the Ramachandran map, respectively. Considering the signs as well as the magnitudes of the backbone torsional angles, molecule A typically folds into a pseudo type III′ β-turn-like structure while molecule B prefers an overall extended conformation. Entrapping the two dramatically distinct conformational characteristics in the crystalline state clearly suggests that the gauche and the trans effects of the β-Ala moieties are indeed energetically accessible to a short linear peptide and receive strong experimental support. The analyses permitted us to emphasize that in addition to conformational constraints of the neighboring residue, the chemical nature of the side-chain acyclic substituents and the “local environments” collectively seem to influence the stabilization of the folding–unfolding behavior of the two methylene units (-CONH-CH2-CH2-CONH-) in 1.

Entrapping unusual folding characteristics of the beta-Ala residues in a model peptide: Boc-beta-Ala-Aib-beta-Ala-NHCH3.

Crystal structure analysis of a model peptide: Boc-beta-Ala-Aib-beta-Ala-NHCH3 (beta-Ala: 3-amino propionic acid; Aib: alpha-aminoisobutyric acid) revealed distinct conformational preferences for folded [phi approximately 136 degrees, mu approximately -62 degrees, psi approximately 100 degrees] and semifolded [phi approximately 83 degrees, mu approximately -177 degrees, psi approximately -117 degrees] structures of the N-and C-terminus beta-Ala residues, respectively. The overall folded conformation is stabilized by unusual Ni...H-Ni+1 and nonconventional C-H...O intramolecular hydrogen bonding interactions.

Induced Conformational Preferences in a Non-chiral β-Ala Residue: X-ray Diffraction, 1H NMR, FT-IR and CD Studies of Boc-β-Ala-D-Ala-NHCH 3 and Boc-β-Ala-L-Ala-NHCH 3

In order to demonstrate the conformational features that can be induced by a chiral residue in a β-Ala moiety, X-ray diffraction structure of Boc-β-Ala-D-Ala-NHCH 3 1 have been determined and compared with Boc-β-Ala-L-Ala-NHCH 3 2. An analysis of the crystal molecular conformations reveals the establishment of non-superimposable stereogeometrical features across β-Ala residues. The supramolecular assembly of the highly ordered L-shaped molecules, generated by the parallel arrangements of the peptide molecules, is stabilised by a network of intermolecular H-bonds between the CO and NH groups. The chiroptical investigations in solvents of varying polarities provide experimental evidence which strongly supports that the stereochemical preferences of the β-Ala residue can restrict significant conformational averaging.

An observation of a folded β-Ala conformation in a model peptide: Boc-L-Pip-β-Ala-NHCH 3, X-ray diffraction study

An X-ray crystallographic characterization of a ‘locally folded’ conformation of a β-Ala residue, in a short linear peptide: Boc-L-Pip-β-Ala-NHCH 3, has been described. The critical μ torsion angle between the methylene groups of the β-Ala adopts a typical gauche (g −) conformation. The urethane moiety is found in the uncommon type a. The influence of the geometrical variation of the Pip residue on β-Ala conformation has been emphasized.

Identification of novel stereogeometrical features in β-ala residue

Conformational investigations of β - alanine containing peptides, 1 and 2, in solution as well as in crystalline state, have revealed the preferences for chirality induced stereogeometrical features which restricted conformational averaging in solution. Evidences for the influences of these conformational features on circular dichroism (CD) spectral behavior of a chiral Ala residue have been provided.

β-Alanine and β-bends. X-Ray diffraction structures of three linear oligopeptides

A crystal-state structural analysis of t-Boc-L-Ala-β-Ala-NHMe, t-Boc-Aib-β-Ala-NHMe, and t-Boc-Aib-Aib-β-Ala-NHMe has been performed by X-ray diffraction. While the conformation adopted by t-Boc-L-Ala-β-Ala-NHMe and t-Boc-Aib-β-Ala-NHMe is essentially extended, t-Boc-Aib-Aib-β-Ala-NHMe is folded into two consecutive intramolecularly hydrogen-bonded structures of the i+ 3 →i type (β-bends), with Aib(1)–Aib(2) and Aib(2)–β-Ala(3), respectively, as corner residues. Owing to the presence of the β-aminoacid, the latter β-bend is characterized by an unusual C11 hydrogen-bonded ring. These results indicate that: (i) a β-aminoacid may be incorporated into a β-bend without a major perturbation of the overall geometry of this folded conformation, and (ii) the propensity of the β-Ala residue for β-bend formation is rather low, unless other conformational constraints (e.g. a preceding β-bend) are present in the linear peptide molecule.

Conformations in the Solid State and Solubility Properties of Protected Homooligopeptides of Glycine and β-Alanine

Abstract IR spectroscopic conformational analyses of Boc–Glyn–OBzl (n=3–7) and Boc–(β-Ala)n–OBzl (n=3–8) were performed in the solid state, suggesting the occurrence of the β-sheet structure in the higher oligomers (n=5–8). Solubility data indicate that insolubilities of Boc–Glyn–OBzl and Boc–(β-Ala)n–OBzl in high-polar solvents begin at hexa- and heptapeptide levels, respectively. Insolubility of protected homooligopeptides of Gly and β-Ala was estimated to be caused by β-sheet aggregation. The high potential for the β-sheet formation of Boc–Glyn–OBzl and Boc–(β-Ala)n–OBzl (n≥5) could clearly be attributed to the great freedom of the peptide backbone dihedral angles of each of the Gly and β-Ala residues in the β-sheet structure. The implications of a replacement of a few Gly residues with β-Ala residues in surface regions of proteins are also discussed.