Zoltán Kupihár

Enrichment of O-GlcNAc modified proteins by the periodate oxidation – hydrazide resin capture approach

A chemical derivatization approach has been developed for the enrichment of O-GlcNAc modified proteins. The procedure is based on the isolation technique used for N-glycoproteins with appropriate modifications because of the differences in the two types of glycosylation: a prolonged periodate oxidation is followed by hydrazide resin capture, on-resin proteolytic digestion, and release of the modified peptides by hydroxylamine. This enrichment strategy offers a fringe benefit in mass spectrometry analysis. Upon collisional activation, the presence of the open carbohydrate ring leads to characteristic fragmentation facilitating both glycopeptide identification and site assignment. The enrichment protocol was applied to the Drosophila proteasome complex previously described as O-GlcNAc modified. The O-GlcNAc modification was located on proteasome interacting proteins, deubiquitinating enzyme Faf (CG1945) and a ubiquitin-like domain containing protein (CG7546). Three other proteins were also found GlcNAc modified, a HSP70 homologue (CG2918), scribbled (CG5462) and the 205 kDa microtubule-associated protein (CG1483). Interestingly, in the HSP70 homologue the GlcNAc modification is attached to an asparagine residue of a N-glycosylation motif.

3-Substituted xanthines as promising candidates for quadruplex formation: computational, synthetic and analytical studies

Our computational studies suggest that 3-substituted xanthines are good candidates for tetrad and quadruplex structures. 3-Methylxanthine (3MX) has been synthesized from 7-benzylxanthine, and the existence of tetrameric and octameric aggregates of 3MX with NH4+, Na+ and K+ ions in the gas phase (MS) and in DMSO-d6 solution (NMR) has been observed. The “internal” H-bonds (N1H⋯O6) are stronger than the “external” ones (N7H⋯O2) in these clusters (NMR).

Neutral and positively charged new purine tetramer structures: a computational study of xanthine and uric acid derivatives

New tetramer structures, based on 9-methylxanthine (Xa), 9-methylxanthine protonated at N7 (XaH+) and 9-methyluric acid (Ua), were investigated by high-level density functional calculations. We have found that homo- and heterotetrads (XaH+)4, (XaH+–Xa)2, (XaH+–Ua)2 carrying positive charges can be formed by low barrier hydrogen bonds. Systems with zero charge [(Xa)4, (Xa–Ua)2, (Ua)4] were also constructed, investigated and compared to the guanine tetrad [(G)4]. It was shown that the new tetramers can bind cations and anions without the necessity of stacking interactions. Application of the calculated systems in higher-ordered structures (e.g. quadruplexes) is promising with or without intercalating ions.

Preparation of an asymmetrically protected phosphoramidite and its application in solid-phase synthesis of phosphopeptides

Abstract O-tert -Butyl- O ′--cyanoethyl- N , N -diisopropylphosphoramidite as a new global phosphorylation reagent and its application for solid-phase phosphopeptide synthesis via monoprotected phosphate-peptide ester during peptide synthesis are described.

Supramolecular H-bonded porous networks at surfaces: exploiting primary and secondary interactions in a bi-component melamine–xanthine system

The control over the formation of a bi-component porous network was attained by the self-assembly at a solid-liquid interface by exploiting both primary and secondary non-covalent interactions between melamine and N(3)-alkylated xanthine modules.

Synthesis and biological evaluation of triazolyl 13α-estrone-nucleoside bioconjugates

2′-Deoxynucleoside conjugates of 13α-estrone were synthesized by applying the copper-catalyzed alkyne–azide click reaction (CuAAC). For the introduction of the azido group the 5′-position of the nucleosides and a propargyl ether functional group on the 3-hydroxy group of 13α-estrone were chosen. The best yields were realized in our hands when the 3′-hydroxy groups of the nucleosides were protected by acetyl groups and the 5′-hydroxy groups were modified by the tosyl–azide exchange method. The commonly used conditions for click reaction between the protected-5′-azidonucleosides and the steroid alkyne was slightly modified by using 1.5 equivalent of Cu(I) catalyst. All the prepared conjugates were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7 and A2780) and the potential inhibitory activity of the new conjugates on human 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1) was investigated via in vitro radiosubstrate incubation. Some protected conjugates displayed moderate antiproliferative properties against a panel of human adherent cancer cell lines (the protected cytidine conjugate proved to be the most potent with IC50 value of 9 μM). The thymidine conjugate displayed considerable 17β-HSD1 inhibitory activity (IC50 = 19 μM).

Synthesis and analysis of peptide nucleic acid oligomers using Fmoc/acyl-protected monomers

The optimization of PNA oligomer synthesis has been accomplished employing Fmoc/acyl-protected monomers on TentaGel™ and Wang resins. Among the tested activating agents (CMP, BET, HATU) the latter was of choice in solid phase syntheses. “Leakage” of TentaGel™ resin greatly hampers the solution and MS analyses. Synthesis and acyl group deprotection steps have been separately examined using Wang resin. Optimal conditions also worked well on the CPG support. HPLC and MS analyses of the PNA oligomers were carried out under various conditions.

Supramolecular Ring Structures of 7-Methylguanine: A Computational Study of Its Self-assembly and Anion Binding

The density functional theory calculations of 7-methylguanine clusters revealed that stable ring assemblies can be formed with or without anions in the center position and hexameric clusters are the most stable and most planar ones. The coordination of anions (Cl−, Br−, NO3−) stabilizes and thus favors the formation of planar aggregates. We believe that the predicted planar structures stabilized by anions are good models for self-assembly structures formed at solid-liquid or solid-gas interfaces. Comparing the bonding and average H-bond energy to reference ribbon calculations we pointed out the presence of the previously introduced cooperativity effect in circular supramolecular structures of 7-methylguanine.

Electrospray ionization mass spectrometric analysis of highly reactive glycosyl halides.

Highly reactive glycosyl chlorides and bromides have been analysed by a routine mass spectrometric method using electrospray ionization and lithium salt adduct-forming agents in anhydrous acetonitrile solution, providing salient lithiated molecular ions [M+Li]+, [2M+Li]+etc. The role of other adduct-forming salts has also been evaluated. The lithium salt method is useful for accurate mass determination of these highly sensitive compounds.

A Novel and Convenient Method for the Synthesis of Free 5′-Thiol Modified Oligonucleotides

Abstract The synthesis of free 5′-thiol-modified oligonucleotides using a 4,4′,4″-trimethoxytrityl (TMTr)-protected linker and standard Poly-PakTM purification has been described.