Model Tripeptide Research Articles

Site-Selective Functionalization of Molecularly Imprinted Nanoparticles to Recognize Lysine-Rich Peptides.

Sequence-selective binding of peptides has been a long-standing goal of chemists. As one of the most abundant amino acids in proteins, lysine plays an important role in protein functions as well as in antimicrobial and cell-penetrating peptides. Herein, we report molecularly imprinted nanoparticles (NPs) with high sequence selectivity for lysine-rich peptides. The NPs are prepared from molecular imprinting of cross-linkable surfactant micelles and postmodification of the imprinted pockets by photoaffinity labeling. The method allows carboxylic acids to be installed precisely near the lysine amino side chains, greatly enhancing the binding strengths of lysine-rich peptides. Small variations in the peptide sequence can be distinguished, and the binding affinity correlates positively with the number of lysine groups in model tripeptides. The method applies to complex lysine-rich biological peptides, achieving hundreds of nanomolar binding affinities and excellent binding specificities.

Development of QSARs for cysteine-containing di- and tripeptides with antioxidant activity:influence of the cysteine position.

Antioxidants agents play an essential role in the food industry for improving the oxidative stability of food products. In the last years, the search for new natural antioxidants has increased due to the potential high toxicity of chemical additives. Therefore, the synthesis and evaluation of the antioxidant activity in peptides is a field of current research. In this study, we performed a Quantitative Structure Activity Relationship analysis (QSAR) of cysteine-containing 19 dipeptides and 19 tripeptides. The main objective is to bring information on the relationship between the structure of peptides and their antioxidant activity. For this purpose, 1D and 2D molecular descriptors were calculated using the PaDEL software, which provides information about the structure, shape, size, charge, polarity, solubility and other aspects of the compounds. Different QSAR model for di- and tripeptides were developed. The statistic parameters for di-peptides model (R2train = 0.947 and R2test = 0.804) and for tripeptide models (R2train = 0.923 and R2test = 0.847) indicate that the generated models have high predictive capacity. Then, the influence of the cysteine position was analyzed predicting the antioxidant activity for new di- and tripeptides, and comparing them with glutathione. In dipeptides, excepting SC, TC and VC, the activity increases when cysteine is at the N-terminal position. For tripeptides, we observed a notable increase in activity when cysteine is placed in the N-terminal position.

Chirality and pH Influence the Self-Assembly of Antimicrobial Lipopeptides with Diverse Nanostructures.

Chirality plays a crucial role in the self-assembly of biomolecules in nature. Peptides show chirality-dependent conformation and self-assembly. Lipidation of peptides occurs in vivo and has recently been exploited in designed conjugates to drive self-assembly and enhance bioactivity. Here, a library of pH-responsive homochiral and heterochiral lipidated tripeptides has been designed. The designed lipopeptides comprise homochiral C16-YKK or C16-WKK (where all the amino acids are l-isomers), and two heterochiral conjugates C16-Ykk and C16-Wkk (where the two lysines are d-isomers). The self-assembly of all the synthesized lipopeptides in aqueous solution was examined using a combination of spectroscopic methods along with cryogenic-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS). Interestingly, it was observed that at acidic pH all the lipopeptides self-assemble into micelles, whereas at basic pH the homochiral lipopeptides self-assemble into nanofibers, whereas the heterochiral lipopeptides self-assemble into nanotapes and nanotubes. A pH switch was demonstrated using a thioflavin T fluorescence probe of β-sheet structure present in the extended structures at pH 8. We demonstrate that both chirality and pH in lipopeptides influence the self-assembly behavior of the model tripeptides, which also show promising bioactivity. Good cytocompatibility is observed in hemolytic assays and antimicrobial activity against both Gram-negative and Gram-positive bacteria is shown through the determination of minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) values and live/dead bacteria staining assay.

Synthesis of Noncoded Amino Acids Bearing a Salicylaldehyde Tag for the Design of Reversible‐Covalent Peptides

AbstractInsertion of electrophilic species on the structure of small molecule ligands or peptides is a well‐known strategy to increase their binding affinity for the target protein of interest. Among these reactive units, the salicylaldehyde (SA) tag can form remarkably stable imine bonds with the ϵ‐amino group of lysine, a highly frequent residue in proteins. In this work, we describe the optimized synthesis of two new noncoded α‐amino acids, starting from l‐homoserine and featuring the SA tag on the side chain. One of these final compounds was successfully inserted into a model tripeptide through in‐solution synthesis. These building blocks will allow the versatile insertion of the SA tag at suitable position of peptide sequences, opening to a tailored design of Lys‐engaging peptide ligands.

Racemic Peptides from Amyloid β and Amylin Form Rippled β-Sheets Rather Than Pleated β-Sheets.

The rippled β-sheet was theorized by Pauling and Corey in 1953 as a structural motif in which mirror image peptide strands assemble into hydrogen-bonded periodic arrays with strictly alternating chirality. Structural characterization of the rippled β-sheet was limited to biophysical methods until 2022 when atomic resolution structures of the motif were first obtained. The crystal structural foundation is restricted to four model tripeptides composed exclusively of aromatic residues. Here, we report five new rippled sheet crystal structures derived from amyloid β and amylin, the aggregating toxic peptides of Alzheimer's disease and type II diabetes, respectively. Despite the variation in peptide sequence composition, all five structures form antiparallel rippled β-sheets that extend, like a fibril, along the entire length of the crystalline needle. The long-range packing of the crystals, however, varies. In three of the crystals, the sheets pack face-to-face and exclude water, giving rise to cross-β architectures grossly resembling the steric zipper motif of amyloid fibrils but differing in fundamental details. In the other two crystals, the solvent is encapsulated between the sheets, yielding fibril architectures capable of host-guest chemistry. Our study demonstrates that the formation of rippled β-sheets from aggregating racemic peptide mixtures in three-dimensional (3D) assemblies is a general phenomenon and provides a structural basis for targeting intrinsically disordered proteins.

Trifluoromethylthiolation of Tryptophan and Tyrosine Derivatives: A Tool for Enhancing the Local Hydrophobicity of Peptides.

The incorporation of fluorinated groups into peptides significantly affects their biophysical properties. We report herein the synthesis of Fmoc-protected trifluoromethylthiolated tyrosine (CF3S-Tyr) and tryptophan (CF3S-Trp) analogues on a gram scale (77-93% yield) and demonstrate their use as highly hydrophobic fluorinated building blocks for peptide chemistry. The developed methodology was successfully applied to the late-stage regioselective trifluoromethylthiolation of Trp residues in short peptides (66-80% yield) and the synthesis of various CF3S-analogues of biologically active monoamines. To prove the concept, Fmoc-(CF3S)Tyr and -Trp were incorporated into the endomorphin-1 chain (EM-1) and into model tripeptides by solid-phase peptide synthesis. A remarkable enhancement of the local hydrophobicity of the trifluoromethylthiolated peptides was quantified by the chromatographic hydrophobicity index determination method, demonstrating the high potential of CF3S-containing amino acids for the rational design of bioactive peptides.

Computational Investigation of Conformational Properties of Short Azapeptides: Insights from DFT Study and NBO Analysis.

Azapeptides have gained much attention due to their ability to enhance the stability and bioavailability of peptide drugs. Their structural preferences, essential to understanding their function and potential application in the peptide drug design, remain largely unknown. In this work, we systematically investigated the conformational preferences of three azaamino acid residues in tripeptide models, Ac-azaXaa-Pro-NHMe [Xaa = Asn (4), Asp (5), Ala (6)], using the popular DFT functionals, B3LYP and B3LYP-D3. A solvation model density (SMD) was used to mimic the solvation effect on the conformational behaviors of azapeptides in water. During the calculation, we considered the impact of the amide bond in the azapeptide models on the conformational preferences of models 4-6. We analyzed the effect of the HB between the side-chain main chain and main-chain main-chain on the conformational behaviors of azapeptides 4-6. We found that the predicted lowest energy conformation for the three models differs depending on the calculation methods. In the gas phase, B3LYP functional indicates that the conformers tttANP-1 and tttADP-1 of azapeptides 4 and 5 correspond to the type I of β-turn, the lowest energy conformation with all-trans amide bonds. Considering the dispersion correction, B3LYP-D3 functional predicts the conformers tctANP-2 and tctADP-3 of azapeptide 4 and 5, which contain the cis amide bond preceding the Pro residue, as the lowest energy conformation in the gas phase. The results imply that azaAsx and Pro residues may involve cis-trans isomerization in the gas phase. In water, the predicted lowest energy conformer of azapeptides 4 and 5 differs from the gas phase results and depends on the calculational method. For azapeptide 6, regardless of calculation methods and phases, tttAAP-1 (β-I turn) is predicted as the lowest energy conformer. The results imply that the effect of the side chain that can form HBs on the conformational preferences of azapeptides 4 and 5 may not be negligible. We compared the theoretical results of azaXaa-Pro models with those of Pro-azaXaa models, showing that incorporating azaamino acid residue in peptides at different positions can significantly impact the folding patterns and stability of azapeptides.

Inviting C5-Trifluoromethylated Pseudoprolines into Collagen Mimetic Peptides.

Numerous collagen mimetic peptides (CMPs) have been engineered using proline derivatives substituted at their C(3) and/or C(4) position in order to stabilize or functionalize collagen triple-helix mimics. However, no example has been reported so far with C(5) substitutions. Here, we introduce a fluorinated CMP incorporating trifluoromethyl groups at the C(5) position of pseudoproline residues. In tripeptide models, our CD, NMR, and molecular dynamics (MD) studies have shown that, when properly arranged, these residues meet the structural requirements for a triple-helix assembly. Two host-guest CMPs were synthesized and analyzed by CD spectroscopy. The NMR analysis in solution of the most stable confirmed the presence of structured homotrimers that we interpret as triple helices. MD calculations showed that the triple-helix model remained stable throughout the simulation with all six trifluoromethyl groups pointing outward from the triple helix. Pseudoprolines substituted at the C(5) positions appeared as valuable tools for the design of new fluorinated collagen mimetic peptides.

Electro-polarization of protein-like substances accelerates trans-cell-wall electron transfer in microbial extracellular respiration

Electrical stimulation has been used to strengthen microbial extracellular electron transfer (EET), however, the deep-seated reasons remain unclear. Here we reported that Bacillus subtilis, a typical gram-positive bacterium capable of extracellular respiration, obtained a higher EET capacity after the electrical domestication. After the electrical domestication, the current generated by the EET of B.subtilis was 23.4-fold that of the control group without pre-domestication. Multiple lines of evidence in bacterial cells of B.subtilis, their cell walls, and a model tripeptide indicated that the polarization of amide groups after the electrical stimulation forwarded the H-bonds recombination and radical generation of protein-like substances to develop extracellular electron transfer via the proton-coupled pattern. The improved electrochemical properties of protein-like substances benefited the trans-cell-wall electron transfer and strengthen extracellular respiration. This study was the first exploration to promote microbial extracellular respiration by improving the electrochemical properties of protein-like substances in cell envelopes.

Understanding the Role of Intramolecular Ion-Pair Interactions in Conformational Stability Using an Ab Initio Thermodynamic Cycle

Intramolecular ion-pair interactions yield shape and functionality to many molecules. With proper orientation, these interactions overcome steric factors and are responsible for the compact structures of several peptides. In this study, we present a thermodynamic cycle based on isoelectronic and alchemical mutation to estimate the intramolecular ion-pair interaction energy. We determine these energies for 26 benchmark molecules with common ion-pair combinations and compare them with results obtained using intramolecular symmetry-adapted perturbation theory. For systems with long linkers, the ion-pair energies evaluated using both approaches deviate by less than 2.5% in the vacuum phase. The thermodynamic cycle based on density functional theory facilitates calculations of salt-bridge interactions in model tripeptides with continuum/microsolvation modeling and four large peptides: 1EJG (crambin), 1BDK (bradykinin), 1L2Y (a mini-protein with a tryptophan cage), and 1SCO (a toxin from the scorpion venom).

Propargylamine Amino Acids as Constrained Nε-Substituted Lysine Mimetics.

Herein, alkylated propargylamines are reported as constrained lysine mimetics and constructed in a single step using a copper(I)-catalyzed A3-coupling reaction. Using multiple secondary amines, the reaction allowed the generation of a structurally diverse set of N-Fmoc protected amino acid derivatives. In addition, the A3-reaction was applied on solid phase via the assembly of short model tripeptides. Moreover, the internal alkyne moiety allowed further functionalization toward novel 1,4,5-trisubstituted 1,2,3-triazole-based amino acids.

Rapid and Highly Selective Fluorescent Labeling of Peptides via a Thia-Diels-Alder Cycloaddition: Application to Apelin.

Herein, we describe a catalyst-free thia-Diels-Alder cycloaddition for the chemoselective labeling of fully deprotected phosphonodithioester-peptides in solution with fluorophores functionalized with an exocyclic diene. The reaction was optimized on the model tripeptide 1 containing a lysine residue, which enabled its rapid and straightforward labeling with three different fluorophores (fluorescein, lissamine rhodamine B, and squaraine) in very mild conditions (H2O/iPrOH, 37 °C, 1 h). The reaction was then successfully applied to the chemoselective labeling of fully deprotected apelin-13 with squaraine dye. The resulting fluorescent ligand 18 exhibited a high affinity (0.17 ± 0.03 nM) for apelinR. It enabled the development of time-resolved FRET-based competition assays for high-throughput screening and drug discovery. Thanks to its fluorogenic properties, ligand 18 was also successfully involved in the live-cell optical imaging of apelinR in no-wash conditions.

Amino Acid-Viologen Hybrids: Synthesis, Cucurbituril Host-Guest Chemistry, and Implementation on the Production of Peptides.

We present herein the development of a series of viologen–amino acid hybrids, obtained in good yields either by successive alkylations of 4,4′-bipyridine, or by Zincke reactions followed by a second alkylation step. The potential of the obtained amino acids has been exemplified, either as typical guests of the curcubituril family of hosts (particularly CB[7]/[8]) or as suitable building blocks for the solution/solid-phase synthesis of two model tripeptides with the viologen core inserted within their sequences.

Hydrogen Bonding and Solvation of a Proline-Based Peptide Model in Salt Solutions.

The hydrogen bonding of water and water/salt mixtures around the proline-based tripeptide model glycyl-l-prolyl-glycinamide·HCl (GPG-NH2) is investigated here by multi-wavelength UV resonance Raman spectroscopy (UVRR) to clarify the role of ion–peptide interactions in affecting the conformational stability of this peptide. The unique sensitivity and selectivity of the UVRR technique allow us to efficiently probe the hydrogen bond interaction between water molecules and proline residues in different solvation conditions, along with its influence on trans to cis isomerism in the hydrated tripeptide. The spectroscopic data suggest a relevant role played by the cations in altering the solvation shell at the carbonyl site of proline., while the fluoride and chloride anions were found to promote the establishment of the strongest interactions on the site of proline. This latter effect is reflected in the greater stabilization of the trans conformers of the tripeptide in the presence of these specific ions. The molecular view provided by UVRR experiments was complemented by the results of circular dichroism (CD) measurements that show a strong structural stabilizing effect on the β-turn motif of GPG-NH2 observed in the presence of KF as a co-solute.

Sequence sensitivity and pH dependence of maleimide conjugated N-terminal cysteine peptides to thiazine rearrangement.

Thiazine formation during the conjugation of N‐terminal cysteine peptides to maleimides is an underreported side reaction in the peptide literature. When the conjugation was performed at neutral and basic pH, we observed the thiazine isomer as a significant by‐product. Nuclear magnetic resonance (NMR) spectroscopy confirmed the structure of the six‐membered thiazine and ultra‐high performance liquid chromatography (UHPLC) combined with tandem mass spectrometry (MS/MS) allowed for facile, unambiguous detection due to a unique thiazine mass fragment. Furthermore, substitution of various amino acids adjacent to the N‐terminal cysteine in a tripeptide model system resulted in different rates of thiazine formation, albeit within the same order of magnitude. We also determined that varying the N‐substitution of the maleimide affects the thiazine conversion rate. Altogether, our findings suggest that thiazine rearrangement for N‐terminal cysteine‐maleimide adducts is a general side reaction that is applicable to other peptide or protein systems. Performing the conjugation reaction under acidic conditions or avoiding the use of an N‐terminal cysteine with a free amino group prevents the formation of the thiazine impurity.

Formation of Pyrazines in Maillard Model Systems: Effects of Structures of Lysine-Containing Dipeptides/Tripeptides.

At present, most investigations involving the Maillard reaction models have focused on free amino acids (FAAs), whereas the effects of peptides on volatile products are poorly understood. In our study, the formation mechanism of pyrazines, which were detected as characteristic volatiles in sunflower seed oil, from the reaction system of glucose and lysine-containing dipeptides and tripeptides was studied. The effect of the amino acid sequences of the dipeptides and tripeptides on pyrazine formation was further highlighted. Four different dipeptides and six tripeptides were selected. The results showed that the production of pyrazines in the lysine-containing dipeptide models was higher than that in the tripeptide and control models. Compounds 2,5(6)-Dimethylpyrazine and 2,3,5-trimethylpyrazine were the main pyrazine compounds in the dipeptide models. Furthermore, the C- or N-terminal amino acids of lysine-containing dipeptides can exert an important effect on the formation of pyrazines. In dipeptide models with lysine at the C-terminus, the content of total pyrazines followed the order of Arg−Lys > His−Lys; the order of the total pyrazine content was Lys−His > Lys−Arg in dipeptide models with N-terminal lysine. Additionally, for the tripeptide models with different amino acid sequences, more pyrazines and a greater variety of pyrazines were detected in the tripeptide models with N-terminal lysine/arginine than in the tripeptide models with N-terminal histidine. However, the total pyrazine content and the percentage of pyrazines in the total volatiles were similar in the tripeptide models with the same amino acids at the N-terminus. This study clearly illustrates the ability of dipeptides and tripeptides containing lysine, arginine and histidine to form pyrazines, improving volatile formation during sunflower seed oil processing.

Molecularly Imprinted Polymeric Receptors with Interfacial Hydrogen Bonds for Peptide Recognition in Water

Protein receptors bind their peptide ligands by a combination of hydrophobic and hydrogen-bonding interactions to achieve high affinity and selectivity. Construction of synthetic receptors for peptides by the same principle, however, is challenging because of the complexity of the guest molecules and subtle structural differences among closely related sequences. Molecular imprinting of peptides in surface–core doubly-cross-linked micelles yielded hydrophobic pockets complementary to the hydrophobic side chains of peptides. Amide-functionalized bis­(acrylamide) cross-linkers such as N,N′-methylene­bis­(acrylamide) (MBAm) used in the core cross-linking installed a layer of hydrogen bonds at the surfactant/water interface and was found to enhance the molecular recognition of peptides in water, particularly hydrophilic ones rich in polar residues. An extremely strong imprinting effect was obtained, with the imprinted/nonimprinted ratio ranging from 3000 to 10000 for model tripeptides. These hydrogen bonds allowed distinction of closely related peptide sequences and enabled a general, simple, one-pot preparation of highly selective receptors for binding complex hydrophilic peptides with <200 nM affinity in aqueous buffer.

Quantum chemical mass spectrometry: Ab initio study of b2 -ion formation mechanisms for the singly protonated Gln-His-Ser tripeptide.

Both amide bond protonation triggering peptide fragmentations and the controversial b2 -ion structures have been subjects of intense research. The involvement of histidine (H), with its imidazole side chain that induces specific dissociation patterns involving inter-side-chain (ISC) interactions, in b2 -ion formation was investigated, focusing on the QHS model tripeptide. To identify the effect of histidine on fragmentations issued from ISC interactions, QHS was selected for a comprehensive analysis of the pathways leading to the three possible b2 -ion structures, using quantum chemical calculations performed at the DFT/B3LYP/6-311+G* level of theory. Electrospray ionization ion trap mass spectrometry allowed the recording of MS2 and MS3 tandem mass spectra, whereas the Quantum Chemical Mass Spectrometry for Materials Science (QCMS2 ) method was used to predict fragmentation patterns. Whereas it is very difficult to differentiate among protonated oxazolone, diketopiperazine, or lactam b2 -ions using MS2 and MS3 mass spectra, the calculations indicated that the QH b2 -ion (detected at m/z 266) is probably a mixture of the lactam and oxazolone structures formed after amide nitrogen protonation, making the formation of diketopiperazine less likely as it requires an additional step for its formation. In contrast to glycine-histidine-containing b2 -ions, known to be issued from the backbone-imidazole cyclization, we found that interactions between the side chains were not obvious to perceive, neither from a thermodynamics nor from a fragmentation perspective, emphasizing the importance of the whole sequence on the dissociation behavior usually demonstrated from simple glycine-containing tripeptides.

Accounting for the temperature dependence of 13C spin\u2013lattice relaxation of methyl groups in the glycyl\u2013alanyl-leucine model system under MAS with spin diffusion

The difficulties in quantitatively modeling the temperature dependence of spin–lattice relaxation in a model isotope-enriched peptide are explored as a prelude to obtaining dynamics parameters for motions in proteins from such measurements. The degree to which this can be handled by adding spin diffusion to a bath in standard rate matrix relaxation theory is studied using a small tri-peptide model system, glycyl–alanyl-leucine (GAL). We observe in this molecule that the relaxation of backbone carbons CO and Cα is not dominated by local fluctuations of the 13C–1H dipolar couplings, but rather by 13C–13C spin diffusion to nearby methyl relaxation sinks. A treatment of the methyl relaxation itself, which ignores 13C–13C spin diffusion effects back to the otherwise slowly relaxing bath, provides poor agreement between theory and experimental data obtained for the temperature dependence of the methyl relaxation rates. Closed form approximate spectral densities and relaxation rates for a methyl group during magic angle spinning are obtained to compute the needed transition rates. These average computed rates, in conjunction with an extended form of the Solomon equations, are found to adequately model the temperature dependence of the methyl relaxation rates when spin diffusion is included. The barrier to rotation for the alanine methyl in GAL is determined to be 3.5 kcal mol−1.

Modeling pH-Dependent NMR Chemical Shift Perturbations in Peptides

Modeling the pH dependence of protein and peptide chemical shifts outside the range of physiological values (6.5–7) is key to understanding structure-function relationships of these systems. These capabilities are largely not available in current chemical shift prediction software. In this study, we utilize a combination of molecular dynamics and quantum mechanics to investigate the through-space and through-bond contributions of protonation-dependent chemical shift perturbations (CSPs) in model tripeptides. By altering the protonation state of the titratable group in the tripeptides, we observe a notable difference in the conformational ensembles and attendantly compute significant CSPs for all nuclei near the site of protonation. We thus demonstrate the ability to recapitulate experimental pH-dependent CSPs with good agreement (R = 0.85, 0.99, and 0.98 for 13C, 15N, and 1H, respectively). Broadly, we provide the groundwork for incorporating pH effects into empirical and semiempirical chemical shift predictors.