Model Tripeptide Research Articles

CE-MS Identification of Amino Acid Sequence Inversion as a New Degradation Pathway of an Aspartyl Model Tripeptide

CE-MS was employed to identify two unknown degradation products of the model tripeptide Phe-α-Asp-Gly heated at 80 °C in aqueous solution at pH 7.4. Both compounds displayed essentially identical mass spectra indicating the presence of peptide diastereomers. The [M + H]+-ion at m/z 338 suggested a tripeptide composed of the amino acids Phe, Gly and Asp. The fragmentation pattern indicated that Phe was not located at the N-terminus. Subsequently, the linear peptide α-Asp-Phe-Gly and the branched peptide Asp(Gly)-Phe were synthesized and analyzed by CE-MS. The mass spectrum of synthetic α-Asp-Phe-Gly was identical to that of the unknown compounds confirming the structure of the degradation products. Asp(Gly)-Phe displayed a complex fragmentation pattern. In conclusion, amino acid sequence inversion represents another degradation pathway of Phe-α-Asp-Gly at pH 7.4 besides known reactions including isomerization, enantiomerization, cyclization to diketopiperazine derivatives and backbone hydrolysis. The mechanism of the rearrangement of the amino acid sequence is proposed to proceed via an aza-bridged intermediate.

Isolated β‐Turn Model Systems Investigated by Combined IR/UV Spectroscopy

The functionality of bioactive molecules sensitively depends on their structure. For the investigation of intrinsic structural properties, molecular beam experiments combined with laser spectroscopy have proven to be a suitable tool. Herein we present an analysis of the two isolated tripeptide model systems Ac-Phe-Tyr(Me)-NHMe and Boc-Phe-Tyr(Me)-NHMe. For this purpose, mass-selective combined IR/UV spectroscopy is applied to both substances in a molecular beam experiment. The comparison of the experimental data with DFT calculations, including different functionals as well as dispersion corrections, allows an assignment of both tripeptide models to β-turns formed independently from the protection groups and supported by the interaction of the two aromatic chromophores.

(S)-ABOC: A Rigid Bicyclic β-Amino Acid as Turn Inducer

In order to investigate the ability of the (S)-aminobicyclo[2.2.2]octane-2-carboxylic acid 1 (H-(S)-ABOC-OH) to induce reverse turns into peptides, two model tripeptides, in which this bicyclic unit was incorporated into the second position, were synthesized and analyzed by FT-IR, CD, NMR, and X-ray studies.

Tyrosinenitration affects thymidylate synthase properties

Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.

Nanomolar Binding of Peptides Containing Noncanonical Amino Acids by a Synthetic Receptor

This paper describes the molecular recognition of phenylalanine derivatives and their peptides by the synthetic receptor cucurbit[7]uril (Q7). The 4-tert-butyl and 4-aminomethyl derivatives of phenylalanine (tBuPhe and AMPhe) were identified from a screen to have 20-30-fold higher affinity than phenylalanine for Q7. Placement of these residues at the N-terminus of model tripeptides (X-Gly-Gly), resulted in no change in affinity for tBuPhe-Gly-Gly, but a remarkable 500-fold increase in affinity for AMPhe-Gly-Gly, which bound to Q7 with an equilibrium dissociation constant (K(d)) value of 0.95 nM in neutral phosphate buffer. Structure-activity studies revealed that three functional groups work in a positively cooperative manner to achieve this extraordinary stability (1) the N-terminal ammonium group, (2) the side chain ammonium group, and (3) the peptide backbone. Addition of the aminomethyl group to Phe substantially improved the selectivity for peptide versus amino acid and for an N-terminal vs nonterminal position. Importantly, Q7 binds to N-terminal AMPhe several orders of magnitude more tightly than any of the canonical amino acid residues. The high affinity, single-site selectivity, and small modification in this system make it attractive for the development of minimal affinity tags.

Influence of stereochemistry on proton transfer in protonated tripeptide models

Vectorial proton transfer among carbonyl oxygen atoms was studied in two models of tripeptide via quantum chemical calculations using the hybrid B3LYP functional and the 6-31++G basis set. Two principal proton transfer pathways were found: a first path involving isomerization of the proton around the double bond of the carbonyl group, and a second based on the large conformational flexibility of the tripeptide model where all carbonyl oxygen atoms cooperate. The latter pathway has a rate-determining step energy barrier that is only around half of that for the first pathway. As conformational flexibility plays a crucial role in second pathway, the effect of attaching methyl groups to the alpha carbon atoms was studied. The results obtained are presented for all four possible stereochemical configurations.

Morphological Transformation between Nanofibers and Vesicles in a Controllable Bipyridine–Tripeptide Self‐Assembly

Tuning structures: Stimulus-responsive peptide self-assembly requires a balance of conformational change and structural continuity of stable β sheets. In an amphiphilic bipyridine–tripeptide model, temperature, and ultrasound switch a reversible morphological transformation between vesicles and nanofibers (see picture) through the synergistic effects of terminal β-sheet-forming peptides, flexible linkers, and rotatable bipyridine groups.

Synthesis and hydrolytic stability of novel 3-[ 18F]fluoroethoxybis(1-methylethyl)silyl]propanamine-based prosthetic groups

Two new silicon-based prosthetic groups, derived from 3-[ethoxybis(1-methylethyl)silyl]propanamine, have been prepared in good yields. These silicon groups bearing an acid or an azide group were coupled to a model tripeptide (Leu-Gly-Gly) either through a classical amide bond formation or through “click chemistry” via the Huisgen cycloaddition. The radiolabelling with fluorine-18 by substitution of the ethoxy group at silicon has been carried out with success in 51–54% decay corrected radiochemical yields. Radiolabelled peptides were easily prepared by direct 18F-fluorination of the silicon-bearing tripeptide or by coupling the peptide with a radiolabelled silicon-based prosthetic group. Their stabilities in physiological medium were studied and proved poor.

Arginine‐Facilitated α‐ and π‐Radical Migrations in Glycylarginyltryptophan Radical Cations

We have used model tripeptides GXW (with X being one of the amino acid residues glycine (G), alanine (A), leucine (L), phenylalanine (F), glutamic acid (E), histidine (H), lysine (K), or arginine (R)) to study the effects of the basicity of the amino acid residue on the radical migrations and dissociations of odd-electron molecular peptide radical cations M(·+) in the gas phase. Low-energy collision-induced dissociation (CID) experiments revealed that the interconvertibility of the isomers [G(·)XW](+) (radical centered on the N-terminal α-carbon atom) and [GXW](·+) (radical centered on the π system of the indolyl ring) generally increased upon increasing the proton affinity of residue X. When X was arginine, the most basic amino acid, the two isomers were fully interconvertible and produced almost identical CID spectra despite the different locations of their initial radical sites. The presence of the very basic arginine residue allowed radical migrations to proceed readily among the [G(·)RW](+) and [GRW](·+) isomers prior to their dissociations. Density functional theory calculations revealed that the energy barriers for isomerizations among the α-carbon-centered radical [G(·)RW](+), the π-centered radical [GRW](·+), and the β-carbon-centered radical [GRW(β)(·)](+) (ca. 32-36 kcal mol(-1)) were comparable with those for their dissociations (ca. 32-34 kcal mol(-1)). The arginine residue in these GRW radical cations tightly sequesters the proton, thereby resulting in minimal changes in the chemical environment during the radical migrations, in contrast to the situation for the analogous GGW system, in which the proton is inefficiently stabilized during the course of radical migration.

Peptide synthesis catalysed by a haloalkaliphilic serine protease from the archaeon Natrialba magadii (Nep)

Haloarchaeal proteases function optimally in high salt (low water activity); thus, they offer an advantage over the nonhalophilic counterparts as biocatalysts for protease-catalysed peptide synthesis. The haloalkaliphilic archaeon Natrialba magadii secretes a solvent-tolerant protease, Nep (Natrialba magadii extracellular protease). In this work, the ability of Nep to catalyse peptide synthesis was examined. The tripeptide Ac-Phe-Gly-Phe-NH(2) was synthesized using Ac-Phe-OEt and Gly-Phe-NH(2) substrates as building blocks in the presence of Nep, 30% (v/v) dimethyl sulfoxide (DMSO) and 1.5 or 0.5 mol l(-1) NaCl. Purification and identification of the peptide product was achieved by RP-HPLC and ESI-MS, respectively. The native as well as the recombinant enzyme produced in Haloferax volcanii (HvNep) was similarly effective as catalysts for the synthesis of this model tripeptide with yields of up to 60% and without secondary hydrolysis of the product. HvNep catalysed the synthesis of various tripeptides with preference for those having aromatic amino acids in the P1 site. Nep is able to catalyse peptide synthesis under different salt concentrations in the presence of DMSO. The catalytic property of Nep in peptide synthesis combined with overproduction of this protease in Hfx. volcanii anticipates the potential applicability of this haloarchaeal protease in biotechnology.

Preparation and Use of Cysteine Orthoesters for Solid-Supported Synthesis of Peptides

Synthesis of a chiral cysteine derivative 2 with the carboxyl protected by an acid-labile 4-methyl-2,6,7-trioxabicyclo[2.2.2]octyl (OBO) orthoester is reported. A disulfide anchoring strategy is used to link the sulfur of this OBO cysteine derivative onto modified trityl polystyrene resin for synthesis of peptides having C-terminal cysteine (Cys) residues. Fmoc-based solid phase peptide synthesis affords model tripeptides without significant epimerization. The approach is used to make the orally active analgesic crotalphine and its Cys1 diastereomer.

Conformations and vibrational spectra of a model tripeptide: change of secondary structure upon micro-solvation

Mid-infrared (IR) hole burning spectra of the model tripeptide Z-Aib-Pro-NHMe (Z = benzyloxycarbonyl) in gas phase and its micro-clusters with one and two methanol molecules are presented. To establish a relation between experimental spectra and the underlying conformations, calculations at the DFT [B3LYP/6-311++G(d,p)] level of theory are performed. In particular, the intra-peptide and the peptide-methanol hydrogen bonds can be identified from spectral shifts in the amide I, II, and III regions. While the unsolvated tripeptide as well as its one-methanol cluster prefer a gamma-turn structure, a beta-turn structure is found for the two-methanol cluster, in agreement with previous condensed phase studies. Comparison of measured and simulated spectra reveals that the favorable methanol binding sites are at the head and tail parts of the tripeptide. The interconversions between gamma-turn and beta-turn structures are governed by potential barriers below 10 kJ mol(-1) inside one of the low energy basins of the potential energy surface.

Novel Tripeptide Model of Nickel Superoxide Dismutase

Nickel superoxide dismutase (Ni-SOD) catalyzes the disproportionation of superoxide to molecular oxygen and hydrogen peroxide, but the overall reaction mechanism has yet to be determined. Peptide-based models of the 2N:2S nickel coordination sphere of Ni-SOD have provided some insight into the mechanism of this enzyme. Here we show that the coordination sphere of Ni-SOD can be mimicked using the tripeptide asparagine-cysteine-cysteine (NCC). NCC binds nickel with extremely high affinity at physiological pH with 2N:2S geometry, as demonstrated by electronic absorption and circular dichroism (CD) data. Like Ni-SOD, Ni-NCC has mixed amine/amide ligation that favors metal-based oxidation over ligand-based oxidation. Electronic absorption, CD, and magnetic CD (MCD) data collected for Ni-NCC are consistent with a diamagnetic Ni(II) center bound in square-planar geometry. Ni-NCC is quasi-reversibly oxidized with a midpoint potential of 0.72(2) V (vs Ag/AgCl) and breaks down superoxide in an enzyme-based assay, supporting its potential use as a model for Ni-SOD chemistry.

Two variable semi‐empirical and artificial neural‐network‐based modeling of peptide mobilities in CZE: The effect of temperature and organic modifier concentration

This work was focused on investigating the effects of two separation influencing parameters in CZE, namely temperature and organic additive concentration upon the electrophoretic migration properties of model tripeptides. Two variable semi-empirical (TVSE) models and back-propagation artificial neural networks (ANN) were applied to predict the electrophoretic mobilities of the tripeptides with non-polar, polar, positively charged, negatively charged and aromatic R group characteristics. Previously published work on the subject did not account for the effect of temperature and buffer organic modifier concentration on peptide mobility, in spite of the fact that both were considered to be influential factors in peptide analysis. In this work, a substantial data set was generated consisting of actual electrophoretic mobilities of the model tripeptides in 30 mM phosphate buffer at pH 7.5, at 20, 25, 30, 35 and 40 degrees C and at four different organic additive containing running buffers (0, 5, 10 and 15% MeOH) applying two electric field strengths (12 and 16 kV) to assess our mobility predicting models. Based on the Arrhenius plots of natural logarithm of mobility versus reciprocal absolute temperature of the various experimental setups, the corresponding activation energy values were derived and evaluated. Calculated mobilities by TVSE and back-propagation ANN models were compared with each other and to the experimental data, respectively. Neural network approaches were able to model the complex impact of both temperature and organic additive concentrations and resulted in considerably higher predictive power over the TVSE models, justifying that the effect of these two factors should not be neglected.

TDDFT diagnostic testing and functional assessment for triazene chromophores

A simple diagnostic test based on orbital overlap [M. J. G. Peach et al., J. Chem. Phys., 2008, 128, 044118] may be used to help judge the reliability of excitation energies in time-dependent density functional theory (TDDFT) when using generalized gradient approximation (GGA) and hybrid functionals. Orbital plots are used to illustrate the test for a model tripeptide and for 4-(N,N-dimethylamino)benzonitrile, which are representative of systems containing low- and high-overlap charge-transfer excitations. The scheme is then applied to a series of triazene chromophores in solvent, highlighting the relationship between overlap and oscillator strength and its implications for theoretical absorption spectra. No low-overlap excitations are observed with a hybrid functional; a single one is identified using a GGA. To assess the diagnostic test and to judge functional performance, gas phase triazene TDDFT excitations are compared with correlated ab initio values. The diagnostic test correctly identifies two low-overlap problematic GGA excitations. However, it does not identify another problematic excitation where the electron is excited to a spatially extended orbital, which necessarily has reasonable overlap with the occupied orbital; an improved diagnostic quantity is required for such cases. The best agreement between TDDFT and correlated ab initio excitations is obtained using a Coulomb-attenuated functional; the errors are significantly smaller than from the GGA and hybrid functionals. The study provides further support for the high quality excitations from Coulomb-attenuated functionals, negating the need for diagnostic tests.

Effect of conformational degrees of freedom on the charge transfer in model tripeptide

An extensive conformational dependence of the intramolecular charge transfer (both hole and electron) between intermediate residues of the model tripeptide in gas phase has been studied. The charge transfer integral, spatial overlap integral and site-energy for both hole and electron transfer between the intermediate residues in the tripeptides were calculated using the fragment orbital method. The site-energies and the charge transfer integrals have been calculated for different conformation of the glycine tripeptide by varying the dihedral angles ( ϕ and ψ) along the α-carbon atom of amino acid subgroups. Electronic structure calculations show that the charge transfer integral between intermediate residues is strongly depending on the nature of the conformation of the peptide. The calculations indicate that the charge transfer is maximum at the particular conformation of the intermediate amino acid residues.

Theoretical Analysis of Secondary Structures of β-Peptides

Unlike alpha-amino acids, peptides formed from beta-amino acids (beta-peptides) display stability toward enzymatic degradation and may form turns and helices with as few as four residues. Because both the C alpha and C beta of the beta-amino acid may bear substituents, a large number of beta-amino acids can be synthesized. Beta-peptides form various well-defined secondary structures, including 14-helix, 12-helix, 10/12-helix, 10-helix, 8-helix, turn structures, sheets, and hairpins. For all of these reasons, beta-amino acids have been increasingly used as building blocks for molecular design and pharmaceutical applications. To explain the conformational features of beta-peptides, several quantum mechanics and molecular dynamics studies that rationalize the observed conformational features have been reported. However, a systematic account that unifies various factors critical to the conformational features is still lacking. In this Account, we present a detailed analysis of the conformational features of various beta-peptides. We start by studying the basic local conformational features of beta-peptides using di- and tripeptide models. Then, various secondary structures of unsubstituted beta-peptides with differing numbers of residues are investigated using a repeating unit approach to derive the intrinsic backbone conformational features. We find that the 10/12-helix is intrinsically most stable for the beta-peptide backbone. The 14-helix, 12-helix, and 10-helix structures have similar stabilities for beta-peptide backbones of four to six residues. The substituent effects on the stabilities of beta-peptide secondary structures are then analyzed. Combined with the substituent effect and the intrinsic backbone preferences, all experimental observations of secondary structure formation can be understood. For example, the 10/12-helix is favored for like-beta(2)/beta(3)-peptides, unlike-beta(3)/beta(3)-peptides, and beta(3)/beta-hGly-peptides because these substitution patterns do not cause steric problems for the 10/12-helix. Beta(3)-peptides, beta(2)-peptides, and beta (2,3)-peptides favor the 14-helix because the substituents in these peptides benefit the 14-helix the most but significantly destabilize the 10/12-helix. Because the 10/12-helix is intrinsically favored and has two favorable positions in each residue for substituents, many more hybrid beta-peptides are predicted to exist in this secondary structure, which suggests the need for further experiments. These results are valuable for determining the best use of these building blocks in the design of well-structured molecules with desirable chemical functions.

Artificial Neural Network Modeling of pH Dependent Structural Descriptor-Mobility Relationship for Capillary Zone Electrophoresis of Tripeptides

The aim of this work was to study the structural descriptor-mobility relationship of representative tripeptides in capillary zone electrophoresis (CZE) with the change of such separation parameters as pH, applied voltage and separation length in respect to their influence on electrophoretic migration properties. At the present stage of the work, the ionic charge was considered as structural descriptor. A multivariable linear regression (MLR) model and a back-propagation artificial neural network (BP-ANN) were applied to predict the electrophoretic mobilities of the model tripeptides with non-polar, polar, positively charged, negatively charged and aromatic R group characteristics. Here we present a comprehensive analysis on electrophoretic mobilities measured at pHs 2.5, 4.5, 7.5 and 9.5 at two different capillary lengths of 10 cm and 30 cm, as well as four applied electric field strengths ranging from 100 to 400 V/cm to teach and evaluate our mobility predicting models. The anticipated mobilities predicted by MLR and BP-ANN were compared to each other and to the experimental data, respectively. The BP-ANN model resulted in considerable higher precision in predictability that of the MLR method.

Structural dependencies of protein backbone 2JNC′ couplings

Protein folding can introduce strain in peptide covalent geometry, including deviations from planarity that are difficult to detect, especially for a protein in solution. We have found dependencies in protein backbone (2)J(NC') couplings on the planarity and the relative orientation of the sequential peptide planes. These dependences were observed in experimental (2)J(NC') couplings from seven proteins, and also were supported by DFT calculations for a model tripeptide. Findings indicate that elevated (2)J(NC') couplings may serve as reporters of structural strain in the protein backbone imposed by protein folds. Such information, supplemented with the H-bond strengths derived from (h3)J(NC') couplings, provides useful insight into the overall energy profile of the protein backbone in solution.

Investigation of Secondary Structure Elements by IR/UV Double Resonance Spectroscopy: Analysis of an Isolated β-Sheet Model System

An isolated beta-sheet model system is investigated in a molecular beam experiment by means of mass- and isomer-selective IR/R2PI double resonance spectroscopy as well as ab initio and DFT calculations. As the exclusive intermolecular assembly, a beta-sheet motif is formed by spontaneous dimerization of two isolated peptide molecules. This secondary structure is produced from the tripeptide model Ac-Val-Tyr(Me)-NHMe without any further environment to form the binding motif which is analyzed by both the characteristic amide A and I vibrations. The experimental and theoretical investigations yield the assignment to an antiparallel beta-sheet model. The result of this detailed spectroscopic analysis on an isolated beta-sheet model indicates that there are intrinsic properties of a beta-sheet structure which can be formed without a solvent or a peptidic environment.