Conformations and hydration of halopropionic acids studied by molecular dynamics and Raman optical activity.

Chiral 2-halopropionic acids and their derivatives were synthesized and their properties studied computationally using Raman and Raman optical activity (ROA) spectroscopy. For neat acids present as liquids small amount of water led to significant changes in the spectra, resulting even to flipping of some ROA band signs. We find this interesting for the role water plays in interpretation of vibrational optical activity spectra of biomolecules. Analysis of the results shows that when the water is present, it can change ROA band signs due to the changes in acidobasic equilibrium. Corresponding esters without acidic hydrogens do not exhibit such effects.

Photochemical synthesis of pink silver and its use for monitoring peptide nitration via surface enhanced Raman spectroscopy (SERS).

Oxidative stress may cause extended tyrosine posttranslational modifications of peptides and proteins. The 3-nitro-L-tyrosine (Nit), which is typically formed, affects protein behavior during neurodegenerative processes, such as Alzheimer's and Parkinson's diseases. Such metabolic products may be conveniently detected at very low concentrations by surface enhanced Raman spectroscopy (SERS). Previously, we have explored the SERS detection of the Nit NO2 bending vibrational bands in a presence of hydrogen chloride (Niederhafner et al., Amino Acids 53:517-532, 2021, ibid). In this article, we describe performance of a new SERS substrate, "pink silver", synthesized photochemically. It provides SERS even without the HCl induction, and the acid further decreases the detection limit about 9 times. Strong SERS bands were observed in the asymmetric (1550-1475 cm-1) and symmetric (1360-1290 cm-1) NO stretching in the NO2 group. The bending vibration was relatively weak, but appeared stronger when HCl was added. The band assignments were supported by density functional theory modeling.

Monitoring peptide tyrosine nitration by spectroscopic methods.

Oxidative stress can lead to various derivatives of the tyrosine residue in peptides and proteins. A typical product is 3-nitro-L-tyrosine residue (Nit), which can affect protein behavior during neurodegenerative processes, such as those associated with Alzheimer's and Parkinson's diseases. Surface enhanced Raman spectroscopy (SERS) is a technique with potential for detecting peptides and their metabolic products at very low concentrations. To explore the applicability to Nit, we use SERS to monitor tyrosine nitration in Met-Enkephalin, rev-Prion protein, and α-synuclein models. Useful nitration indicators were the intensity ratio of two tyrosine marker bands at 825 and 870 cm-1 and a bending vibration of the nitro group. During the SERS measurement, a conversion of nitrotyrosine to azobenzene containing peptides was observed. The interpretation of the spectra has been based on density functional theory (DFT) simulations. The CAM-B3LYP and ωB97XD functionals were found to be most suitable for modeling the measured data. The secondary structure of the α-synuclein models was monitored by electronic and vibrational circular dichroism (ECD and VCD) spectroscopies and modeled by molecular dynamics (MD) simulations. The results suggest that the nitration in these peptides has a limited effect on the secondary structure, but may trigger their aggregation.

Correction to: Rapid acidolysis of benzyl group as a suitable approach for syntheses of peptides naturally produced by oxidative stress and containing 3-nitrotyrosine.

This errata is for paper "Rapid acidolysis of benzyl group as a suitable approach for syntheses.

Quantitative Determination of Ala-Ala Conformer Ratios in Solution by Decomposition of Raman Optical Activity Spectra.

Raman optical activity (ROA) spectroscopy combined with quantum-chemical simulations is a sensitive method to determine the absolute configuration and conformation of chiral molecules in solutions. However, the precision of this approach varies for different systems. In the present study, the reliability and numerical stability of decomposing experimental spectra into calculated subspectra is tested on the Ala-Ala dipeptide. Molecular dynamics (MD) snapshots of Ala-Ala/water clusters are averaged to account for solvent effects and molecular flexibility. Multiple experiments with protonated, zwitterionic, and deprotonated dipeptide forms and natural and d2- and d8-isotopically labeled dipeptides are used to verify the results and estimate the overall accuracy. Although the precision is still limited by experimental noise and computational error, a very close match between the observed and theoretical spectral shapes has been achieved. This enabled quantitative determination of conformer populations with a typical dispersion of 10%. The spectroscopy also demonstrated how the conformation depends on pH. The ROA results were more consistent than the Raman ones. Typically, the ROA analysis was more resistant to artifacts in the experiment, such as incomplete baseline subtraction. Conformer ratios predicted by MD agree fairly but not fully with the experimental ones. This indicates minor deficiencies in the Amber force field, particularly for the protonated dipeptide. Overall, the combination of ROA experiment and computational chemistry appears to be a robust tool providing deep insight into molecular structure.

Reactivity of 9-aminoacridine drug quinacrine with glutathione limits its antiprion activity.

Quinacrine-the drug based on 9-aminoacridine-failed in clinical trials for prion diseases, whereas it was active in in vitro studies. We hypothesize that aromatic nucleophilic substitution at C9 could be contributing factor responsible for this failure because of the transfer of acridine moiety from quinacrine to abundant glutathione. Here, we described the semi-large-scale synthesis of the acridinylated glutathione and the consequences of its formation on biological and biophysical activities. The acridinylated glutathione is one order of magnitude weaker prion protein binder than the parent quinacrine. Moreover, according to log DpH 7.4 , the glutathione conjugate is two orders of magnitude more hydrophilic than quinacrine. Its higher hydrophilicity and higher dsDNA binding potency will significantly decrease its bioavailability in membrane-like environment. The glutathione deactivates quinacrine not only directly but also decreases its bioavailability. Furthermore, the conjugate can spontaneously decompose to practically insoluble acridone, which is precipitated out from the living systems.

Resolving Electronic Transitions in Synthetic Fluorescent Protein Chromophores by Magnetic Circular Dichroism.

The detailed electronic structures of fluorescent chromophores are important for their use in imaging of living cells. A series of green fluorescent protein chromophore derivatives is examined by magnetic circular dichroism (MCD) spectroscopy, which allows the resolution of more bands than plain absorption and fluorescence. Observed spectral patterns are rationalized with the aid of time-dependent density functional theory (TDDFT) computations and the sum-over-state (SOS) formalism, which also reveals a significant dependence of MCD intensities on chromophore conformation. The combination of organic and theoretical chemistry with spectroscopic techniques also appears useful in the rational design of fluorescence labels and understanding of the chromophore's properties. For example, the absorption threshold can be heavily affected by substitution on the phenyl ring but not much on the five-member ring, and methoxy groups can be used to further tune the electronic levels.

Rapid acidolysis of benzyl group as a suitable approach for syntheses of peptides naturally produced by oxidative stress and containing 3-nitrotyrosine.

3-Nitrotyrosine (Nit) belongs to products of oxidative stress and could probably influence conformation of neurodegenerative proteins. Syntheses of peptides require availability of suitable synthon for introduction of Nit residue. Common phenolic protection groups are more acid labile, when they are attached to Nit residue. We have found that Fmoc-Nit(Bn)-OH is a good building block for syntheses of Nit containing peptides by Fmoc/tBu strategy. Interestingly, the peptides containing multiple Nit residues can be available solely by use of Fmoc-Nit(Bn)-OH synthon. Bn is removed rapidly with ca 80 % trifluoroacetic acid in dark. The cleavage of Bn from Fmoc-Nit(Bn)-OH proceeds via pseudo-first order mechanism with activation barrier 32 kcal mol(-1) and rate k = 15.3 s(-1) at 20 °C. This rate is more than 2,000,000 times faster than that for cleavage of benzyl from Tyr(Bn).

Nonplanar tertiary amides in rigid chiral tricyclic dilactams. Peptide group distortions and vibrational optical activity.

We investigate amide nonplanarity in vibrational optical activity (VOA) spectra of tricyclic spirodilactams 5,8-diazatricyclo[6,3,0,0(1,5)]undecan-4,9-dione (I) and its 6,6',7,7'-tetradeuterio derivative (II). These rigid molecules constrain amide groups to nonplanar geometries with twisted pyramidal arrangements of bonds to amide nitrogen atoms. We have collected a full range vibrational circular dichroism (VCD) and Raman optical activity (ROA) spectra including signals of C-H and C-D stretching vibrations. We report normal-mode analysis and a comparison of calculated to experimental VCD and ROA. The data provide band-to-band assignment and offer a possibility to evaluate roles of constrained nonplanar tertiary amide groups and rigid chiral skeletons. Nonplanarity shows as single-signed VCD and ROA amide I signals, prevailing the couplets expected to arise from the amide-amide interaction. Amide-amide coupling dominates amide II (mainly C'-N stretching, modified in tertiary amides by the absence of a N-H bond) transitions (strong couplet in VCD, no significant ROA) probably due to the close proximity of amide nitrogen atoms. At lower wavenumbers, ROA spectra exhibit another likely manifestation of amide nonplanarity, showing signals of amide V (δ(oop)(N-C) at ~570 cm(-1)) and amide VI (δ(oop)(C'═O) at ~700 cm(-1) and ~650 cm(-1)) vibrations.

Glutamate carboxypeptidase II does not process amyloid-ß peptide.

The accumulation of amyloid-ß (Aß) peptide is thought to be a major causative mechanism of Alzheimer's disease. Aß accumulation could be caused by dysregulated processing of amyloid precursor protein, yielding excessive amounts of Aß, and/or by inefficient proteolytic degradation of the peptide itself. Several proteases have been described as Aß degradation enzymes, most notably metalloendopeptidases, aspartic endopeptidases, and some exopeptidases. Recently a report suggested that another metallopeptidase, glutamate carboxypeptidase II (GCPII), can also cleave Aß. GCPII is a zinc exopeptidase that cleaves glutamate from N-acetyl-L-aspartyl-L-glutamate in the central nervous system and from pteroylpoly-γ-glutamate in the jejunum. GCPII has been proposed as a promising therapeutic target for disorders caused by glutamate neurotoxicity. However, an Aß-degrading activity of GCPII would compromise potential pharmaceutical use of GCPII inhibitors, because the enzyme inhibition might lead to increased Aß levels and consequently to Alzheimer's disease. Therefore, we analyzed the reported Aß-degrading activity of GCPII using highly purified recombinant enzyme and synthetic Aß. We did not detect any Aß degradation activity of GCPII or its homologue even under prolonged incubation at a high enzyme to substrate ratio. These results are in good agreement with the current detailed structural understanding of the substrate specificity and enzyme-ligand interactions of GCPII.

Quinacrine reactivity with prion proteins and prion-derived peptides.

Quinacrine is a drug that is known to heal neuronal cell culture infected with prions, which are the causative agents of neurodegenerative diseases called transmissible spongiform encephalopathies. However, the drug fails when it is applied in vivo. In this work, we analyzed the reason for this failure. The drug was suggested to "covalently" modify the prion protein via an acridinyl exchange reaction. To investigate this hypothesis more closely, the acridine moiety of quinacrine was covalently attached to the thiol groups of cysteines belonging to prion-derived peptides and to the full-length prion protein. The labeled compounds were conveniently monitored by fluorescence and absorption spectroscopy in the ultraviolet and visible spectral regions. The acridine moiety demonstrated characteristic UV-vis spectrum, depending on the substituent at the C-9 position of the acridine ring. These results confirm that quinacrine almost exclusively reacts with the thiol groups present in proteins and peptides. The chemical reaction alters the prion properties and increases the concentration of the acridine moiety in the prion protein.

Three types of induced tryptophan optical activity compared in model dipeptides: theory and experiment.

The tryptophan (Trp) aromatic residue in chiral matrices often exhibits a large optical activity and thus provides valuable structural information. However, it can also obscure spectral contributions from other peptide parts. To better understand the induced chirality, electronic circular dichroism (ECD), vibrational circular dichroism (VCD), and Raman optical activity (ROA) spectra of Trp-containing cyclic dipeptides c-(Trp-X) (where X = Gly, Ala, Trp, Leu, nLeu, and Pro) are analyzed on the basis of experimental spectra and density functional theory (DFT) computations. The results provide valuable insight into the molecular conformational and spectroscopic behavior of Trp. Whereas the ECD is dominated by Trp π-π* transitions, VCD is dominated by the amide modes, well separated from minor Trp contributions. The ROA signal is the most complex. However, an ROA marker band at 1554 cm(-1) indicates the local χ(2) angle value in this residue, in accordance with previous theoretical predictions. The spectra and computations also indicate that the peptide ring is nonplanar, with a shallow potential so that the nonplanarity is primarily induced by the side chains. Dispersion-corrected DFT calculations provide better results than plain DFT, but comparison with experiment suggests that they overestimate the stability of the folded conformers. Molecular dynamics simulations and NMR results also confirm a limited accuracy of the dispersion-DFT model in nonaqueous solvents. Combination of chiral spectroscopies with theoretical analysis thus significantly enhances the information that can be obtained from the induced chirality of the Trp aromatic residue.

Ferric complexes of 3-hydroxy-4-pyridinones characterized by density functional theory and Raman and UV-vis spectroscopies.

Deferiprone and other 3-hydroxy-4-pyridinones are used in metal chelation therapy of iron overload. To investigate the structure and stability of these compounds in the natural aqueous environment, ferric complexes of deferiprone and amino acid maltol conjugates were synthesized and studied by computational and optical spectroscopic methods. The complexation caused characteristic intensity changes, a 300× overall enhancement of the Raman spectrum, and minor changes in UV-vis absorption. The spectra were interpreted on the basis of density functional theory (DFT) calculations. The CAM-B3LYP and ωB97XD functionals with CPCM solvent model were found to be the most suitable for simulations of the UV-vis spectra, whereas B3LYP, B3LYPD, B3PW91, M05-2X, M06, LC-BLYP, ωB97XD, and CAM-B3LYP functionals were all useful for simulation of the Raman scattering. Characteristic Raman band frequencies for 3-hydroxy-4-pyridinones were assigned to molecular vibrations. The computed conformer energies consistently suggest the presence of another isomer of the deferiprone-ferric complex in solution, in addition to that found previously by X-ray crystallography. However, the UV-vis and Raman spectra of the two species are similar and could not be resolved. In comparison to UV-vis, the Raman spectra and their combination with calculations appear more promising for future studies of iron sequestrating drugs and artificial metalloproteins as they are more sensitive to structural details.

Comparative syntheses of peptides and peptide thioesters derived from mouse and human prion proteins.

Prions are suspected as causative agents of several neuropathogenic diseases, even though the mode of their action is still not clear. A combination of chemical and recombinant syntheses can provide suitable probes for explanation of prions role in pathogenesis of neurodegenerative diseases. However, the prions contain several difficult sequences for synthesis by Fmoc/tBu approach. For that reason, the peptide thioesters as the key building blocks for chemical syntheses of proteins by native chemical ligation were employed. A scan of the mouse prion domain 93-231 was carried out in order to discover availability of derived thioesters as the suitable building blocks for a total chemical synthesis of the prion protein based probes. The synthesis on 2-chlorotritylchloride resin was utilized and after a deprotection of the samples for analysis, the peptide segments were purified and characterized. If the problems were detected during the synthesis, the segment was re-synthesized either using the special pseudoproline dipeptides or by splitting its molecule to two or three smaller segments, which were prepared easier. The protected segments, prepared correctly without any deletion and in sufficient amounts, were coupled either with EtSH after DIC/DMAP activation or with p-Ac-NH-Ph-SH using PyBOP activation to yield corresponding thioesters. In some special cases, the other techniques of thioester formation, like sulfonamide-safety catch and/or trimethylaluminium approach were utilized.

Tracking of the polyproline folding by density functional computations and Raman optical activity spectra.

Polyprolines offer many opportunities to study factors influencing peptide and protein folding and structure. Longer chains can adopt two well-defined forms (PPI and PPII), but shorter peptides are quite flexible. To understand in detail the dependence of the secondary structure on the length and the interplay between the side chain and main chain conformation, zwitterionic (Pro)(N) models (with N = 2, 3, 4, 6, 9, 12 and longer inhomogeneous chains) were studied by a combination of the Raman and Raman optical activity (ROA) spectroscopy with the density functional theory (DFT). Potential surfaces were systematically explored for the shorter oligoprolines, and Boltzmann conformational ratios were obtained both for the main chain and the proline ring puckering. The predictions were verified by comparison of the experimental and simulated ROA spectra. The conformer ratios extracted from a decomposition of the experimental ROA into scaled computed spectra well reproduced Boltzmann populations calculated from relative energies. For example, an "A" puckering of the proline ring was found prevalent, relatively independent of the length, whereas the cis-amide backbone form adopted by shorter peptides rapidly disappeared for N > 4. The results are consistent with previous NMR and vibrational circular dichroism (VCD) data. Delocalized exciton vibrations along the peptide chain often enhance the ROA signal, and can thus be used to indicate a longer regular peptide structure. The ROA technique appeared to be very sensitive to the ring puckering; less distinct spectral features were produced by changes in the main chain geometry.

Melectin MAPs: the influence of dendrimerization on antimicrobial and hemolytic activity.

The recently described antimicrobial peptide melectin (MEP, GFLSILKKVLPKVMAHMK-NH2) exhibits high antimicrobial activity against Gram-positive and Gram-negative bacteria. Here we describe the synthesis and biological activities of 23 new analogues of MEP. We studied the influence of dimerization and tetramerization (MAP-constructs of MEP) on the antimicrobial and hemolytic activities, as well as the role of Met in positions 14 and 17 of the peptide chain. Oxidation of the Met to Met(O) and Met(O2) decreases antimicrobial activity of all tested bacteria if the peptide is in the monomeric form, however, only to Staphylococcus aureus if in the form of dimer or tetramer. Dimerization and tetramerization increase the undesirable hemolytic activity of the peptides. Interestingly, substitution of Leu for Val in position 6 leads to the decrease of hemolytic activity. Introduction of the isosteric amino acid Nle into positions 14 or 17 or both leads to slight increase of hemolytic activity under preservation of high antimicrobial activities. Unfortunately, dimerization again leads to an increase of hemolytic activity.

Conformational properties of the Pro-Gly motif in the D-Ala-l-Pro-Gly-D-Ala model peptide explored by a statistical analysis of the NMR, Raman, and Raman optical activity spectra.

The Pro-Gly sequence in designed peptides and proteins is often used to mimic natural beta-hairpin turns. Shorter peptides containing this moiety, however, adopt multiple conformations, and their propensity to form the turn is not obvious. In this study, conformational flexibility of Pro-Gly was investigated with the aid of NMR, Raman scattering, and Raman optical activity (ROA). The spectra of a model tetrapeptide NH3+-D-Ala-L-Pro-Gly-D-Ala-CO2- were analyzed on the basis of statistical methods and density functional computations. Other peptide derivatives were adopted as supporting theoretical and experimental models. The results suggest that the loop conformation of the Pro-Gly core is not inherently stable in vacuum. On the other hand, in aqueous environment the propensity to form the beta-hairpin loops is an intrinsic property of the Pro-Gly sequence. It was observed also in a shorter Ac-D-Pro-Gly-NH-Me dipeptide. The attached alanine residues in the tetrapeptide stabilize the structure only partially. Thus an inclusion of the solvent in the calculations is important for correct description of peptide folding in the aqueous environment. The agreement of the optical spectra with the experiment was determined from overlaps between simulated and measured spectral curves. The comparison of the computed NMR, Raman, and ROA was hampered by experimental noise and limited accuracy of the computations. However, the statistical analysis of the spectroscopic data provided conformer distribution consistent with the computation of the relative energies. The combination of the NMR and Raman techniques with the quantum computations appeared very beneficial for the investigation of Pro-Gly conformational behavior, and can be recommended for future peptide folding studies.

Acridin-9-yl exchange: a proposal for the action of some 9-aminoacridine drugs.

The finding that several derivatives of 9-aminoacridine were deacridinylated in the presence of primary aliphatic amines during the solid phase synthesis of acridine-peptide conjugates prompted us to investigate the acridin-9-yl moiety transfer from a relatively low-molecular acridine source to a high-molecular carrier. The hydrophobic polymer was used as a model of hydrophobic core of biologically active proteins. While the alpha-amino group in the peptide was found to play the role of weak acridine acceptor, the epsilon-amino group of lysine appeared to serve as a moderate acceptor of acridine moiety. The covalent modification of the lysine residues side chain in the hydrophobic core of prion protein aggregates could thus explain the discrepancy between the ability of the acridine drug quinacrine to reduce efficiently the incidence of prion protein in cell culture and its weak prion binding affinity.