Dipeptides In Solution Research Articles

THERMODYNAMICS OF STEPWISE DISSOCIATION OF GLYCYL-D-PHENYLALANINE IN AQUEOUS SOLUTION

Recently, more and more drugs that are derivatives of oligopeptides have been introduced to the pharmaceutical market. The structural elements of these compounds are amino acids and dipeptides. When stabilizing functional groups are introduced into the peptide structure, as well as through the use of special delivery systems, oligopeptide derivatives can be used as innovative medicines. The calorimetric method was used to determine the heat of interaction of glycyl-D-phenylalanine with solutions of nitric acid and potassium hydroxide at 298.15K and the values of the ionic strength of the solution 0.5, 0.75 and 1.0 in the presence of KNO3. Measurements were carried out on a calorimeter with an isothermal shell and automatic recording of the temperature-time curve. The unit was equipped with a reaction vessel with a volume of 60 cm3. Calibration was performed by current at T = (293.15–308.15) ± 0.01 K and P = 100.5± 0.7 kPa. A thermistor KMT-14 was used as a temperature sensor. The temperature of the calorimetric cell was controlled in a thermostat equipped with a PID controller with an accuracy of 0.002 K. The temperature sensor of the thermostat was a platinum resistance thermometer. The calorimeter was calibrated by current. The volume of the calorimetric liquid is 42.83 ml. The enthalpy of stepwise dissociation of glycyl-D-phenylalanine at zero ionic strength was found by an equation with one individual parameter. Standard thermodynamic characteristics (ΔrH°, ΔrG°, ΔrS°) of acid-base interaction reactions in aqueous solutions of glycyl-D-phenylalanine are calculated. The effect of the background electrolyte concentration on the dissociation heats of glycyl-D-phenylalanine is considered. Knowledge of the thermochemical characteristics of dipeptides is necessary for understanding and subsequent modeling of the process of protein biosynthesis occurring in biological systems. Data on the thermodynamic characteristics of dipeptide solutions are needed in various fields where these compounds are used: pharmacology, medicine, food and cosmetic industries, for the development, justification and optimization of technological processes involving these compounds and their complexes with metals.

Method for non-invasive assessment of the effect of biologically active substances on the rate of the pH level restoration in the muscle after maximum load using 1H MRS

Purpose of the study: To determine the possibility of a non-invasive evaluation of the biologically active substances (BAS) effect on the rate of a pH level restoration in a muscle after a maximum load using 1H magnetic resonance spectroscopy (MRS).Materials and methods. Creatine monohydrate and beta-alanine were taken as tested biologically active substances, used according to the manufacturer's recommendations. At the first stage, calibration curves of a pH dependence on the magnitude of chemical shifts were plotted during assigning 1H spectra of model carnosine dipeptide solutions for non-invasive determination of intramuscular pH. Further experiments were carried out on laboratory animals (mice) using a 9 T NMR spectrometer Bruker Advance III WB 400MHz WB (Bruker, Germany). In experiments on volunteers the functional test pwc170 was used for assessing the ergogenic effects of biologically active substances on rectus quadriceps femoris. The test allows to achieve the level of myocytes cytoplasm acidification with lactate, and the effectiveness of functional biologically active substances on endurance, and also the function of aerobic systems by the muscle pH rate of recovery. Detection was performed using a high-field magnetic resonance imaging scanner (Philips Healthcare, Achieva 3.0T, North Braband, The Netherlands) and two SENSE Flex-L surface ring radiofrequency coils.Results. The effect of oral intake of creatine and beta-alanine on the restoration of rectus quadriceps femoris muscle pH after an acidification of the myocytes cytoplasm with lactate was evaluated using the 1H MRS method. Reproducible results with optimal signal-to-noise ratios and width of carnosine spectral peaks were achieved in volunteers using individual protocols and 1H MRS at 3T in the quadriceps femoris. Animal experiments have highlighted the need to develop and use more accurate techniques for voxel extraction and fat suppression during in vivo 1H spectroscopy to reliably capture the chemical shifts of carnosine peaks.Conclusion. The data obtained using 1H MRS on volunteers allow us to conclude that the developed method makes it possible to non-invasively assess the effect of biologically active substances on the rate of restoration of pH level in a muscle after a critical load in real time in vivo.

Molecular dynamics study of low molecular weight gel forming salt-triggered dipeptide

Molecular dynamics simulation method was used to study the aggregation of Na and Ca salts in different concentrations of Naphthalene-dipeptide (2NapFF) solutions. The results show that high-valence Ca2+ triggers the formation of a gel at a certain dipeptide concentration, and the low-valence Na+ system follows the aggregation law of general surfactants. The results also show that hydrophobic and electrostatic forces are the main driving forces for the formation of dipeptide aggregates, and that hydrogen bonds do not play a major role in the formation of dipeptide solution aggregates. Hydrophobic and electrostatic effects are the main driving forces for the formation of gels in dipeptide solutions triggered by Ca2+. Electrostatic attraction drives Ca2+ to form a weak coordination with four oxygen atoms on two carboxyl groups, which causes the dipeptide molecules to form a gel with a branched network structure.

Chiral analysis of linear protonated dipeptides by complexing with Cyclodextrins using Ion-Mobility Mass-Spectrometry and DFT structural calculations

The functions and applications of dipeptides have been reported to possess unique characteristics compared to that of proteins or amino acids. Naturally occurring amino acids, except for glycine, are chiral molecules; thus, for each type of dipeptide there are four possible isomers. Isomeric structural analysis of dipeptides is important for measuring the quality of dipeptide products, as well as understanding their chemical and biological functions in humans. In the present study, a rapid and simple method for isomeric recognition of protonated dipeptides H-D/l-Phe-D/L-X-OH, where X = hydrophobic amino acids, including alanine (Ala), isoleucine (Ile), leucine (Leu), proline (Pro), tryptophan (Trp), valine (Val), phenylalanine (Phe), and methionine (Met), was developed and investigated. Mixed solutions of dipeptides and cyclodextrins (CDs) were prepared for the production of gas phase, non-covalent complex ions by electrospray ionization (ESI). Trapped ion-mobility mass spectrometry (TIMS-MS) was subsequently used to investigate the corresponding ion mobilities (IM). The TIMS and density functional theory (DFT) results showed that the chiral structure of different dipeptides can be distinguished by β-CD, which indicates that different isomeric complexes have different collision cross sections (CCSs) and spatial conformations. The relative content of isomeric dipeptides mixtures was also obtained in the ratio range from 10:1 to 1:10 to measure the detection limits. Our work provides a direct analytical method for determining isomeric dipeptides, which could potentially have wide applications in the field of chiral recognition.

Structure Elucidation and Mitigation of Endogenous Interferences in LC-MS-Based Metabolic Profiling of Urine.

Liquid chromatography-mass spectrometry (LC-MS) is the main workhorse of metabolomics owing to its high degree of analytical sensitivity and specificity when measuring diverse chemistry in complex biological samples. LC-MS-based metabolic profiling of human urine, a biofluid of primary interest for clinical and biobank studies, is not widely considered to be compromised by the presence of endogenous interferences and is often accomplished using a simple "dilute-and-shoot" approach. Yet, it is our experience that broad obscuring signals are routinely observed in LC-MS metabolic profiles and represent interferences that lack consideration in the relevant metabolomics literature. In this work, we chromatographically isolated the interfering metabolites from human urine and unambiguously identified them via de novo structure elucidation as two separate proline-containing dipeptides: N,N,N-trimethyl-l-alanine-l-proline betaine (l,l-TMAP) and N,N-dimethyl-l-proline-l-proline betaine (l,l-DMPP), the latter reported here for the first time. Offline LC-MS/MS, magnetic resonance mass spectrometry (MRMS), and nuclear magnetic resonance (NMR) spectroscopy were essential components of this workflow for the full chemical and spectroscopic characterization of these metabolites and for establishing the coexistence of cis and trans isomers of both dipeptides in solution. Analysis of these definitive structures highlighted intramolecular ionic interactions as responsible for slow interconversion between these isomeric forms resulting in their unusually broad elution profiles. Proposed mitigation strategies, aimed at increasing the quality of LC-MS-based urine metabolomics data, include modification of column temperature and mobile-phase pH to reduce the chromatographic footprint of these dipeptides, thereby reducing their interfering effect on the underlying metabolic profiles. Alternatively, sample dilution and internal standardization methods may be employed to reduce or account for the observed effects of ionization suppression on the metabolic profile.

Modeling amide-I vibrations of alanine dipeptide in solution by using neural network protocol

Infrared spectroscopy is a powerful tool for the understanding of molecular structure and function of polypeptides. Theoretical interpretation of IR spectra relies on ab initio calculations may be very costly in computational resources. Herein, we developed a neural network (NN) modeling protocol to evaluate a model dipeptide’s backbone amide-I spectra. DFT calculations were performed for the amide-I vibrational motions and structural parameters of alanine dipeptide (ALAD) conformers in different micro-environments ranging from polar to non-polar ones. The obtained backbone dihedrals, C = O bond lengths and amide-I frequencies of ALAD were gather together for NN architecture. The applications of built NN protocols for the prediction of amide-I frequencies of ALAD in other solvation conditions are quite satisfactory with much less computational cost comparing with electronic structure calculations. The results show that this cost-effective way enables us to decipher the polypeptide’s dynamic secondary structures and biological functions with their backbone vibrational probes.

Identification and quantification of leucine and isoleucine residues in peptides using photoexcited tryptophan.

The molecular recognition ability of tryptophan (Trp) for isomeric amino acids, such as leucine (Leu) and isoleucine (Ile), and isomeric amino acid-containing dipeptides, such as Leu-Gly, Ile-Gly, Gly-Leu, and Gly-Ile (where Gly denotes glycine), was investigated using a tandem mass spectrometer equipped with an electrospray ionization source and cold ion trap. The ultraviolet photodissociation spectra of the cold gas-phase clusters of Leu and Ile with Na+Trp in the wavelength range of 265-290nm revealed that the relative intensities of Leu and Ile were only different in the wavelength range of 265-273nm; however, no differences in the relative intensities were observed when the wavelength exceeded 274nm. The molecular recognition ability of photoexcited Trp was used for the identification and quantification of Leu and Ile in dipeptides in solution. The mole fractions of Leu and Ile in dipeptides could be determined from the abundances observed in a single product ion spectrum of the cold gas-phase clusters of dipeptides with Na+Trp.

Conformational Free Energy Changes via an Alchemical Path without Reaction Coordinates.

We introduce a novel method called restrain-free energy perturbation-release (R-FEP-R) to estimate conformational free energy changes via an alchemical path, which for some conformational landscapes like those associated with cellular signaling proteins in the kinase family is more direct and readily converged than the corresponding free energy changes along the physical path. The R-FEP-R method was developed from the dual topology free energy perturbation method that is widely applied to estimate the binding free energy difference between two ligands. In R-FEP-R, the free energy change between two conformational basins is calculated by free energy perturbations that remove those atoms involved in the conformational change from their initial conformational basin while simultaneously growing them back according to the final conformational basin. Both the initial and final dual topology states are unphysical, but they are designed in a way such that the unphysical contributions to the initial and final partition functions cancel. Compared with other advanced sampling algorithms such as umbrella sampling and metadynamics, the R-FEP-R method does not require predetermined transition pathways or reaction coordinates that connect the two conformational states. As a first illustration, the R-FEP-R method was applied to calculate the free energy change between conformational basins for alanine dipeptide in solution and for a side chain in the binding pocket of T4 lysozyme. The results obtained by R-FEP-R agree with the benchmarks very well.

Automated Exploration of Free Energy Landscapes Based on Umbrella Integration.

We present a new approach for automated exploration of free energy landscapes on the basis of the umbrella integration (UI) method. The method to search points in the landscape relies on the normal distributions and gradients of the potential of mean force (PMF) obtained from UI calculations. We applied this approach to the alanine dipeptide in solution and demonstrated that the equilibrium and the transition states were efficiently found in the ascending order of the PMF values.

Metastable hydrogels from aromatic dipeptides.

We demonstrate that the well-known self-assembling dipeptide diphenylalanine (FF) and its amidated derivative (FF-NH2) can form metastable hydrogels upon sonication of the dipeptide solutions. The hydrogels show instantaneous syneresis upon mechanical contact resulting in rapid expulsion of water and collapse into a semi-solid gel.

Volumetric and conductometric studies on the interactions of dipeptides with potassium perfluoroalkanesulfonate in aqueous solution at different temperatures

Density and conductivity data for potassium perfluoroalkanesulfonate ((potassium perfluorobutanesulfonate (PFBS) and potassium perfluorooctanesulfonate (PFOS))+dipeptide+water) systems were determined at different temperatures. The standard partial molar volume (V2,ϕo), standard partial molar volume of transfer for dipeptide from water to aqueous potassium perfluoroalkanesulfonate solutions (ΔtVo), hydration number (NH), partial molar expansibility (Eϕo) and Hepler’s constant for the dipeptides have been calculated from density data. Electrical conductivity was used to estimate the limiting molar conductivity (Λo), Walden product (Λoη) and energy of activation of the transfer process (Ea) of potassium perfluoroalkanesulfonate in aqueous dipeptide solutions. Effects of temperature and the hydrocarbon chain length of the dipeptides on the volumetric and conductometric properties were examined. The results have been interpreted in terms of solute–solvent interactions and structural changes in the mixed solutions.

Correction to Balanced Protein-Water Interactions Improve Properties of Disordered Proteins and Non-Specific Protein Association.

In our paper1 we did not properly acknowledge the contribution by Head-Gordon and co-workers2 (ref 41 of our manuscript). These authors used a similar modification of the mixing rules for protein–water interactions, although the correction was applied on a per-atom basis to both the characteristic distance (sigma) and energy (epsilon) parameters rather than using a single scaling factor for epsilon as we did. Head-Gordon and co-workers used extensive solvation free energy data on model organic compounds to fit their parameters, whereas we used FRET efficiencies for a single unfolded protein (cold shock protein) to estimate our scaling parameter. Overall, we reached qualitatively similar conclusions regarding the solvation free energies of force fields with Amber 943 Lennard-Jones parameters being too unfavorable, as reported previously by these authors.2 The more systematic approach of Nerenberg et al. showed a mean signed error of solvation free energies close to zero for their optimized force field.2 We would also like to make a specific correction to the sentence on page 5115, which is inaccurate: “Recently, a full parametrization of protein Lennard-Jones parameters to reproduce solvation free energies showed some promising results, but unfortunately resulted in very unstable folded proteins”. Instead, it should read as follows: “Recently, a rescaling of protein–water Lennard-Jones parameters to reproduce solvation free energies showed some promising results for correcting protein–protein interactions using concentrated solutions of glycine and leucine dipeptides. In conjunction with a 12-10 van der Waals (vdW) term to replace the standard 12-6 vdW potential to model the nonelectrostatic part of the hydrogen bonding between the backbone carbonyl oxygens and amide protons, this modification resulted in stable folded proteins.”

Production and Quantitative Determination of Thymofer Drug Substance

Immunomodulating thymofer drug substance was prepared as an aqueous solution (0.0148%) of the Fe(II) coordination complex with isoleucine-tryptophan. The drug substance was produced by mechanical mixing of equimolar amounts of powdered dipeptide and iron(II) sulfate. The physicochemical parameters of the preparations obtained from the resulting drug substance and that prepared by mixing aqueous solutions of the dipeptide and iron salt did not differ. Quality control methods for thymofer drug substance were developed for the main parameters. Use of the developed preparation could avoid the steps of preparing the dipeptide and iron-salt solutions and mixing them to produce the finished dosage form.

An improved procedure for determination of the mean aggregation number of micelles

In this paper, a theory of fluorescence quenching in micelles which enables a dynamic approach to the evaluation of the aggregation numbers of micelles is presented. This method is based on a detailed kinetic model of quenching of fluorescent probe developed by Tachiya (1975, 1982) and takes into account that a part of quenchers are associated with micelles but the remaining quenchers are in the aqueous phase. The approach presented is an improvement on a previous fluorescence quenching method (Turro and Yekta, 1978) and is applied to determine the aggregation number of sodium dodecyl sulfonate (SAS) in aqueous dipeptide solution using cetylpyridinium chloride as quencher. The values of aggregation number and association constant for quencher–micelle association are presented.

Thermodynamics of mixed-ligand complex formation of zinc nitrilotriacetate with amino acids and dipeptides in solution

The isothermal calorimetry, pH-potentiometric titration and 1H and 13C NMR methods has been used to study the mixed-ligand complex formation in the systems Zn2+–Nta3––L− (L=His, Orn, Lys, GlyGly, AlaAla) in aqueous solution at 298.15K and the ionic strength of I=0.5 (KNO3). The thermodynamic parameters of formation of the mixed complexes have been determined. The relationship between the probable coordination modes of the complexone and amino acid or dipeptide molecules in the mixed-ligand complex and the thermodynamic parameters has been discussed.

How Accurately Do Current Force Fields Predict Experimental Peptide Conformations? An Adiabatic Free Energy Dynamics Study

The quality of classical biomolecular simulations is inevitably limited by two problems: the accuracy of the force field used and the comprehensiveness of configuration space sampling. In this work we tackle the sampling problem by carrying out driven adiabatic free energy dynamics to obtain converged free energy surfaces of dipeptides in the gas phase and in solution using selected dihedral angles as collective variables. To calculate populations of conformational macrostates observed in experiment, we introduce a fuzzy clustering algorithm in collective-variable space, which delineates macrostates without prior definition of arbitrary boundaries. With this approach, we calculate the conformational preferences of small peptides with six biomolecular force fields chosen from among the most recent and widely used. We assess the accuracy of each force field against recently published Raman or IR-UV spectroscopy measurements of conformer populations for the dipeptides in solution or in the gas phase.

Exploring the Conformational Dynamics of Alanine Dipeptide in Solution Subjected to an External Electric Field: A Nonequilibrium Molecular Dynamics Simulation

In this paper, we investigate the conformational dynamics of alanine dipeptide under an external electric field by nonequilibrium molecular dynamics simulation. We consider the case of a constant and of an oscillatory field. In this context, we propose a procedure to implement the temperature control, which removes the irrelevant thermal effects of the field. For the constant field different time-scales are identified in the conformational, dipole moment, and orientational dynamics. Moreover, we prove that the solvent structure only marginally changes when the external field is switched on. In the case of oscillatory field, the conformational changes are shown to be as strong as in the previous case, and nontrivial nonequilibrium circular paths in the conformation space are revealed by calculating the integrated net probability fluxes.

Dipeptide Aggregation in Aqueous Solution from Fixed Point-Charge Force Fields.

The description of aggregation processes with molecular dynamics simulations is a playground for testing biomolecular force fields, including a new generation of force fields that explicitly describe electronic polarization. In this work, we study a system consisting of 50 glycyl-l-alanine (Gly-Ala) dipeptides in solution with 1001 water molecules. Neutron diffraction experiments have shown that at this concentration, Gly-Ala aggregates into large clusters. However, general-purpose force fields in combination with established water models can fail to correctly describe this aggregation process, highlighting important deficiencies in how solute–solute and solute–solvent interactions are parametrized in these force fields. We found that even for the fully polarizable AMOEBA force field, the degree of association is considerably underestimated. Instead, a fixed point-charge approach based on the newly developed IPolQ scheme [Cerutti et al. J. Phys. Chem.2013, 117, 2328] allows for the correct modeling of the dipeptide aggregation in aqueous solution. This result should stimulate interest in novel fitting schemes that aim to improve the description of the solvent polarization effect within both explicitly polarizable and fixed point-charge frameworks.

Densities, electrical conductances, and spectroscopic properties of glycyl dipeptide + ionic liquid ([C12mim]Br) + water mixtures at different temperatures

Densities, conductivity, fluorescence and UV–vis absorption spectroscopy of glycyl dipeptide+ionic liquid (1-dodecyl-3-methylimidazolium bromide ([C12mim]Br))+water mixtures have been measured at different temperatures. From density data, the standard partial molar volume (V2,ϕ0), standard partial molar volume of transfer for three glycyl dipeptides from water to aqueous [C12mim]Br solutions (ΔtVo), partial molar expansibility (Eϕ°) and Hepler's constant have been calculated. Through the electrical conductivity measurements, the critical micellar concentration (ccmc) and a series of thermodynamic parameters of micellization of [C12mim]Br in aqueous glycyl dipeptide solutions are evaluated. Effects of temperature and hydrocarbon chain length of the glycyl dipeptides on the volumetric properties of dipeptides and critical micelle concentration (ccmc) of [C12mim]Br were examined. The pyrene fluorescence spectra were used to study the change of micropolarity produced by the interactions of [C12mim]Br with glycyl dipeptides, and the aggregation behavior of [C12mim]Br. From the results of UV–vis absorption spectra, the binding constant between dipeptide and [C12mim]Br above the ccmc was determined.

Interactions of dipeptides with Triton X-100 in aqueous solution: A volumetric and spectroscopic study

The interactions of dipeptides with Triton X-100 in aqueous solution have been investigated by means of density, fluorescence spectroscopy and UV–vis spectroscopy. The standard partial molar volume (V2,ϕo), standard partial molar volume of transfer for dipeptide from water to aqueous Triton X-100 solution (ΔtVo) and partial molar expansibility (Eϕo) have been calculated from density data. Fluorescence spectroscopy was used to estimate the critical micellar concentration (ccmc) and micelle aggregation number of Triton X-100 in aqueous dipeptide solutions. Effects of temperature and hydrocarbon chain length of dipeptides on the volumetric properties of dipeptide and critical micelle concentration (ccmc) of Triton X-100 were examined. The pyrene fluorescence spectra were also used to study the change of micropolarity produced by the interactions of Triton X-100 with dipeptides. From the results of UV–vis absorption spectra, the binding constant between dipeptide and Triton X-100 above the ccmc was determined. The results have been interpreted in terms of solute–solvent interactions and structural changes in the mixed solutions.