Martin Šafařík

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.

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.

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.

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.

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.

Glutamate carboxypeptidase II does not process amyloid-β peptide

The accumulation of amyloid‐β (Aβ) peptide is thought to be a major causative mechanism of Alzheimers disease. Aβ accumulation could be caused by dysregulated processing of amyloid precursor protein, yielding excessive amounts of Aβ, and/or by inefficient proteolytic degradation of the peptide itself. Several proteases have been described as Aβ degradation enzymes, most notably metalloendopeptidases, aspartic endopeptidases, and some exopeptidases. Recently a report suggested that another metallopeptidase, glutamate carboxypeptidase II (GCPII), can also cleave Aβ. 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 Aβ‐degrading activity of GCPII would compromise potential pharmaceutical use of GCPII inhibitors, because the enzyme inhibition might lead to increased Aβ levels and consequently to Alzheimers disease. Therefore, we analyzed the reported Aβ‐degrading activity of GCPII using highly purified recombinant enzyme and synthetic Aβ. We did not detect any Aβ 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.—Sedlák, F., Šácha, P., Blechová, M., Březinová, A., Šafařík, M., Šebestík, J., Konvalinka, J. Glutamate carboxypeptidase II does not process amyloid‐β peptide. FASEB J. 27, 2626‐2632 (2013). www.fasebj.org

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.

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 chromophores 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.

Electrophoresis of Derivatized Polyethylene Glycols: A Useful Method for Monitoring of Reactions on Soluble Polymeric Carrier

A useful method for monitoring of peptide synthesis on polyethylene glycol (PEG) was developed and is described in this paper. The course of reactions such as loading, acylation, detachment and others where a charge of reactant and product differ, can be easily determined. The loading of the first amino acid is quantified with errors fitting well to those from other known analytical methods.

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.