Ágnes Révész

In‐Flight Epimerization of a Bis‐Tröger Base

In 1887, Julius Tr ger reported the synthesis of a nitrogen base, whose structure was determined only about 50 years later. In “Tr ger bases”, nitrogen atoms serve as chiral centers because the otherwise rapid nitrogen inversion is prevented by conformational strain. After Prelog and Wieland separated the enantiomers, the chirality and the rigid V-shape of Tr ger bases led to widespread applications in chemistry. A fundamental question in the chemistry of Tr ger bases concerns the mechanism of their pseudoepimerization, for which either a proton-catalyzed ring opening or a retro-Diels–Alder (RDA) sequence has been proposed (Scheme 1).

On the mechanism of the copper-mediated C-S bond formation in the intramolecular disproportionation of imine disulfides.

The mechanism of the copper-mediated disproportionation of aromatic imine disulfides to benzothiazoles in the gas phase is investigated by experimental and theoretical methods. Application of infrared multiphoton dissociation and hydrogen/deuterium exchange experiments combined with density functional theory (DFT) calculations of the relevant molecular structures and the associated infrared spectra allows the identification of the observed ionic intermediates. The theoretical investigation of the possible reaction pathways supported by collision-induced dissociation experiments provides a consistent mechanistic picture of the reaction catalyzed by a single copper(I) ion. Activation of the substrate proceeds via homolytic sulfur-sulfur bond cleavage, yielding metal complexes in the formal +3 oxidation state; carbon-sulfur coupling and hydrogen-atom transfer complete the transformation to the products. Exploratory studies demonstrate that in the gas phase, the disproportionation of the imine disulfide can also be mediated by other metal ions via different either homo- or heterolytic mechanisms without involving high-valent intermediates.

Composite Aromatic Boxes for Enzymatic Transformations of Quaternary Ammonium Substrates

Cation-π interactions to cognate ligands in enzymes have key roles in ligand binding and enzymatic catalysis. We have deciphered the key functional role of both charged and aromatic residues within the choline binding subsite of CTP:phosphocholine cytidylyltransferase and choline kinase from Plasmodium falciparum. Comparison of quaternary ammonium binding site structures revealed a general composite aromatic box pattern of enzyme recognition sites, well distinguished from the aromatic box recognition site of receptors.

Evolutionary and mechanistic insights into substrate and product accommodation of CTP:phosphocholine cytidylyltransferase from Plasmodium falciparum

The enzyme CTP:phosphocholine cytidylyltransferase (CCT) is essential in the lipid biosynthesis of Plasmodia (Haemosporida), presenting a promising antimalarial target. Here, we identified two independent gene duplication events of CCT within Apicomplexa and characterized a truncated construct of Plasmodium falciparum CCT that forms a dimer resembling the molecular architecture of CCT enzymes from other sources. Based on biophysical and enzyme kinetics methods, our data show that the CDP‐choline product of the CCT enzymatic reaction binds to the enzyme considerably stronger than either substrate (CTP or choline phosphate). Interestingly, in the presence of Mg2+, considered to be a cofactor of the enzyme, the binding of the CTP substrate is attenuated by a factor of 5. The weaker binding of CTP:Mg2+, similarly to the related enzyme family of aminoacyl tRNA synthetases, suggests that, with lack of Mg2+, positively charged side chain(s) of CCT may contribute to CTP accommodation. Thermodynamic investigations by isothermal titration calorimetry and fluorescent spectroscopy studies indicate that accommodation of the choline phosphate moiety in the CCT active site is different when it appears on its own as one of the substrates or when it is linked to the CDP‐choline product. A tryptophan residue within the active site is identified as a useful internal fluorescence sensor of enzyme–ligand binding. Results indicate that the catalytic mechanism of Plasmodium falciparum CCT may involve conformational changes affecting the choline subsite of the enzyme.

Binding Energies and Isomerization in Metallocene Ions from Threshold Photoelectron Photoion Coincidence Spectroscopy

Metallocene ions (Cp(2)M(+), M = Cr, Co, Ni) were studied by threshold photoelectron photoion coincidence spectroscopy (TPEPICO) to investigate the mechanism, energetics, and kinetics of the ionic dissociation processes. The examined energy-selected Cp(2)M(+) ions fragment by losing the neutral cyclopentadienyl ligand. In addition, CH and C(2)H(2) losses appear as minor channels, while the cobaltocene ion also loses an H atom. A possible isomerization pathway has also been observed for Cp(2)Ni(+), yielding a complex with pentafulvalene (C(10)H(8)) with a loss of H(2). In order to determine the 0 K appearance energies for the CpM(+) fragment ions, the asymmetric time-of-flight peak shapes and the breakdown diagrams of the energy-selected metallocene ions were modeled by both the rigid activated complex (RAC) Rice-Ramsperger-Kassel-Marcus (RRKM) theory and the simplified statistical adiabatic channel model (SSACM). The following appearance energies were obtained with SSACM, which is more reliable for loose transition states: 10.57 ± 0.14, 11.01 ± 0.13, and 10.18 ± 0.13 eV for M = Cr, Co, and Ni, respectively. These values combined with the corresponding adiabatic ionization energies yield M-Cp bond dissociation energies in Cp(2)M(+) ions of 5.04 ± 0.16, 5.77 ± 0.15, and 3.96 ± 0.15 eV. Density functional calculations at the B3LYP/6-311G(d,p) level of theory were used to determine the structures of these complexes and to provide parameters necessary for the analysis of the experimental data. The trends in the M-Cp bond energies can be related to the electronic structures of the metallocene ions based on a simple molecular orbital picture.

Identification and interconversion of diastereomeric oligo-Tröger bases probed by ion mobility mass spectrometry

Oligo-Tröger bases are auspicious scaffolds of molecular engineering, which motivates studies on the mechanism of their interconversion and on the facile determination of the relative configuration of their diastereoisomers. Protonated, sodiated, and argentated species of those compounds were therefore studied via ion-mobility mass spectrometry (IM-MS), allowing differentiation on the basis of the shapes of the ions. First, the isomerization was confirmed to be acid-catalyzed as it takes place readily in the case of protonated Tröger bases, whereas the metallated bases are configurationally stable. Second, the corrected arrival times of the various isomers of the cationized bases were found to show distinct differences in IM-MS, and their excellent correlation with the cross sections obtained from quantum chemical calculations paves the way toward the easy identification of diastereoisomers.

Stereocontrol in Diphenylprolinol Silyl Ether Catalyzed Michael Additions: Steric Shielding or Curtin–Hammett Scenario?

The enantioselectivity of amine-catalyzed reactions of aldehydes with electrophiles is often explained by simple steric arguments emphasizing the role of the bulky group of the catalyst that prevents the approach of the electrophile from the more hindered side. This standard steric shielding model has recently been challenged by the discovery of stable downstream intermediates, which appear to be involved in the rate-determining step of the catalytic cycle. The alternative model, referred to as the Curtin-Hammett scenario of stereocontrol, assumes that the enantioselectivity is related to the stability and reactivity of downstream intermediates. In our present computational study, we examine the two key processes of the catalytic Michael reaction between propanal and β-nitrostyrene that are relevant to the proposed stereoselectivity models, namely the C-C bond formation and the protonation steps. The free energy profiles obtained for the pathways leading to the enantiomeric products suggest that the rate- and stereodetermining steps are not identical as implied by the previous models. The stereoselectivity can be primarily controlled by C-C bond formation even though the reaction rate is dictated by the protonation step. This kinetic scheme is consistent with all observations of experimental mechanistic studies including those of mass spectrometric back reaction screening experiments, which reveal a mismatch between the stereoselectivity of the back and the forward reactions.

Potential steps in the evolution of a fused trimeric all-β dUTPase involve a catalytically competent fused dimeric intermediate.

Deoxyuridine 5′‐triphosphate nucleotidohydrolase (dUTPase) is essential for genome integrity. Interestingly, this enzyme from Drosophila virilis has an unusual form, as three monomer repeats are merged with short linker sequences, yielding a fused trimer‐like dUTPase fold. Unlike homotrimeric dUTPases that are encoded by a single repeat dut gene copy, the three repeats of the D. virilis dut gene are not identical due to several point mutations. We investigated the potential evolutionary pathway that led to the emergence of this extant fused trimeric dUTPase in D. virilis. The herein proposed scenario involves two sequential gene duplications followed by sequence divergence amongst the dut repeats. This pathway thus requires the existence of a transient two‐repeat‐containing fused dimeric dUTPase intermediate. We identified the corresponding ancestral dUTPase single repeat enzyme together with its tandem repeat evolutionary intermediate and characterized their enzymatic function and structural stability. We additionally engineered and characterized artificial single or tandem repeat constructs from the extant enzyme form to investigate the influence of the emergent residue alterations on the formation of a functional assembly. The observed severely impaired stability and catalytic activity of these latter constructs provide a plausible explanation for evolutionary persistence of the extant fused trimeric D. virilis dUTPase form. For the ancestral homotrimeric and the fused dimeric intermediate forms, we observed strong catalytic and structural competence, verifying viability of the proposed evolutionary pathway. We conclude that the progression along the herein described evolutionary trajectory is determined by the retained potential of the enzyme for its conserved three‐fold structural symmetry.

Selection of Collision Energies in Proteomics Mass Spectrometry Experiments for Best Peptide Identification: Study of Mascot Score Energy Dependence Reveals Double Optimum

Collision energy is a key parameter determining the information content of beam-type collision induced dissociation tandem mass spectrometry (MS/MS) spectra, and its optimal choice largely affects successful peptide and protein identification in MS-based proteomics. For an MS/MS spectrum, quality of peptide match based on sequence database search, often characterized in terms of a single score, is a complex function of spectrum characteristics, and its collision energy dependence has remained largely unexplored. We carried out electrospray ionization-quadrupole-time of flight (ESI-Q-TOF)-MS/MS measurements on 2807 peptides from tryptic digests of HeLa and E. coli at 21 different collision energies. Agglomerative clustering of the resulting Mascot score versus energy curves revealed that only few of them display a single, well-defined maximum; rather, they feature either a broad plateau or two clear peaks. Nonlinear least-squares fitting of one or two Gaussian functions allowed the characteristic energies to be determined. We found that the double peaks and the plateaus in Mascot score can be associated with the different energy dependence of b- and y-type fragment ion intensities. We determined that the energies for optimum Mascot scores follow separate linear trends for the unimodal and bimodal cases with rather large residual variance even after differences in proton mobility are taken into account. This leaves room for experiment optimization and points to the possible influence of further factors beyond m/ z.

Sensitive method for glycosaminoglycan analysis of tissue sections

A simple, isocratic HPLC method based on HILIC-WAX separation, has been developed for analyzing sulfated disaccharides of glycosaminoglycans (GAGs). To our best knowledge, this is the first successful attempt using this special phase in nano-HPLC-MS analysis. Mass spectrometry was based on negative ionization, improving both sensitivity and specificity. Detection limit for most sulfated disaccharides were approximately 1 fmol; quantitation limits 10 fmol. The method was applied for glycosaminoglycan profiling of tissue samples, using surface digestion protocols. This novel combination provides sufficient sensitivity for GAG disaccharide analysis, which was first performed using prostate cancer tissue microarrays. Preliminary results show that GAG analysis may be useful for identifying cancer related changes in small amounts of tissue samples (ca. 10 μg).