Conjugates of adenosine mimetics and arginine-rich peptides serve as inhibitors and fluorescent probes but not as long-lifetime photoluminescent probes for protein arginine methyltransferases.

The conjugates of an adenosine mimetic and oligo-l-arginine or oligo-d-arginine (ARCs) were initially designed in our research group as inhibitors and photoluminescent probes targeting basophilic protein kinases. Here, we explored a panel of ARCs and their fluorescent derivatives in biochemical assays with members of the protein arginine methyltransferase (PRMT) family, focusing specifically on PRMT1. In the binding/displacement assay with detection of fluorescence anisotropy, we found that ARCs and arginine-rich peptides could serve as high-affinity ligands for PRMT1, whereas the equilibrium dissociation constant values depended dramatically on the number of arginine residues within the compounds. The fluorescently labeled probe ARC-1081 was displaced from its complex with PRMT1 by both S-adenosyl-l-methionine (SAM) and S-adenosyl-l-homocysteine (SAH), indicating binding of the adenosine mimetic of ARCs to the SAM/SAH-binding site within PRMT1. The ARCs that had previously shown microsecond-lifetime photoluminescence in complex with protein kinases did not feature such property in complex with PRMT1, demonstrating the selectivity of the time-resolved readout format. When tested against a panel of PRMT family members in single-dose inhibition experiments, a micromolar concentration of ARC-902 was required for the inhibition of PRMT1 and PRMT7. Overall, our results suggest that the compounds containing multiple arginine residues (including the well-known cell-penetrating peptides) are likely to inhibit PRMT and thus interfere with the epigenetic modification status in complex biological systems, which should be taken into consideration during interpretation of the experimental data.

Macrocyclic versus open-chain carbazole receptors for carboxylate binding.

The anion recognition properties of six synthetic acyclic and macrocyclic carbazole-based receptors have been studied by 1H-NMR as well as with COSMO-RS calculations towards acetate, benzoate, lactate, sorbate and formate. The receptors differed by the number and geometry of hydrogen-bond donor (HBD) sites, the nature and length of the linker(s) between the HBD sites and the cyclic or non-cyclic nature. The binding ability of the receptors is strongly influenced by the structure and steric variables of the receptors and anions. It was found that when urea was replaced with the flexible diglycolyl as the connecting linker between carbazole subunits, the carboxylate binding affinity of the receptor decreased significantly. The effects of the receptors' structure on anion binding have been investigated and several intriguing cases have been identified and analysed. The current findings shed light on carboxylate anion binding and contribute to the systematic synthesis of receptors with beneficial functional selectivity for carboxylate anions.

LogP determination for highly lipophilic hydrogen-bonding anion receptor molecules.

Lipophilicity, usually expressed as octanol-water partition coefficient (logPo/w), is an important property in biomedical research, drug design and technology. However, high logPo/w values of complex hydrogen-bonding molecules are not easy to measure or calculate. Exemplary problematic molecules are prospective active components (ionophores) of polymeric sensor membranes - the working elements of ion-selective electrodes. High lipophilicities of the membrane components are crucial for the sensor lifetime. In this work, lipophilicities of a wide range of urea-, carbazole- and indolocarbazole-based anion receptor molecules (some newly synthesized) and two common plasticizers were determined using a chromatography-based approach and/or the COSMO-RS method. Very high logPo/w values, up to around 20, i.e. far beyond directly experimentally accessible range, were obtained. The agreement between the two approaches ranged from very good to satisfactory. Based on these results, simple fragment-based equations were developed for quick lipophilicity estimation without any specialized software. Membrane-water partition coefficients for the studied compounds were modeled. Limitations and biases of the used methods are discussed.

Design, synthesis and application of carbazole macrocycles in anion sensors.

Carboxylate sensing solid-contact ion-selective electrodes (ISEs) were created to provide a proof-of-concept ISE development process covering all aspects from in silico ionophore design to functional sensor characterization. The biscarbazolylurea moiety was used to synthesize methylene-bridged macrocycles of different ring size aiming to fine tune selectivity towards different carboxylates. Cyclization was achieved with two separate strategies, using either amide synthesis to access up to -[CH2]10- macrocycles or acyl halides to access up to -[CH2]14- macrocycles. Seventy-five receptor-anion complexes were modelled and studied with COSMO-RS, in addition to all free host molecules. In order to predict initial selectivity towards carboxylates, 1H NMR relative titrations were used to quantify binding in DMSO-d 6/H2O solvent systems of two proportions - 99.5%:0.5% m/m and 90.0%:10.0% m/m, suggesting initial selectivity towards acetate. Three ionophores were selected for successful sensor prototype development and characterization. The constructed ion-selective electrodes showed higher selectivity towards benzoate than acetate, i.e., the selectivity patterns of the final sensors deviated from that predicted by the classic titration experiments. While the binding constants obtained by NMR titration in DMSO-d 6/H2O solvent systems provided important guidance for sensor development, the results obtained in this work emphasize the importance of evaluating the binding behavior of receptors in real sensor membranes.

A Selective Biligand Inhibitor of CK2 Increases Caspase-3 Activity in Cancer Cells and Inhibits Platelet Aggregation.

Cancer cells express high levels of CK2, and its inhibition leads to apoptosis. CK2 has therefore emerged as a new drug target for cancer therapy. A biligand inhibitor ARC-772 was constructed by conjugating 4-(2-amino-1,3-thiazol-5-yl)benzoic acid and a carboxylate-rich peptoid. ARC-772 was found to bind CK2 with a Kd value of 0.3 nm and showed remarkable CK2 inhibitory selectivity in a panel of 140 protein kinases (Gini coefficient: 0.75 at c=100 nm). ARC-775, the acetoxymethyl ester prodrug of ARC-772, was efficiently taken up by cells. Once internalized, the inhibitor is activated by cellular esterase activity. In HeLa cancer cells ARC-775 was found to activate caspase-3 (an apoptosis marker) at sub-micromolar concentrations (EC50 =0.3 µm), a 20-fold lower extracellular concentration than CX-4945, the only CK2 inhibitor under clinical trials. At micromolar concentrations, ARC-775 was also found to inhibit ADP-induced aggregation of human platelets. The overall results of this study demonstrate that oligo-anionic biligand inhibitors have good potential for drug development.

Allosteric Effect of Adenosine Triphosphate on Peptide Recognition by 3'5'-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits.

The allosteric influence of adenosine triphosphate (ATP) on the binding effectiveness of a series of peptide inhibitors with the catalytic subunit of 3'5'-cyclic adenosine monophosphate dependent protein kinase was investigated, and the dependence of this effect on peptide structure was analyzed. The allosteric effect was calculated as ratio of peptide binding effectiveness with the enzyme-ATP complex and with the free enzyme, quantified by the competitive inhibition of the enzyme in the presence of ATP excess, and by the enzyme-peptide complex denaturation assay, respectively It was found that the principle "better binding-stronger allostery" holds for interactions of the studied peptides with the enzyme, indicating that allostery and peptide binding with the free enzyme are governed by the same specificity pattern. This means that the allosteric regulation does not include new ligand-protein interactions, but changes the intensity (strength) of the interatomic forces that govern the complex formation in the case of each individual ligand. We propose that the allosteric regulation can be explained by the alteration of the intrinsic dynamics of the protein by ligand binding, and that this phenomenon, in turn, modulates the ligand off-rate from its binding site as well as the binding affinity. The positive allostery could therefore be induced by a reduction in the enzyme's overall intrinsic dynamics.

Experimental Basicities of Superbasic Phosphonium Ylides and Phosphazenes.

Experimental basicities of some of the strongest superbases ever measured (phosphonium ylides) are reported, and by employing these compounds, the experimental self-consistent basicity scale of superbases in THF, reaching a pKα (estimate of pKa) of 35 and spanning more than 30 pKa units, has been compiled. Basicities of 47 compounds (around half of which are newly synthesized) are included. The solution basicity of the well-known t-Bu-N═P4(dma)9 phosphazene superbase is now rigorously linked to the scale. The compiled scale is a useful tool for further basicity studies in THF as well as in other solvents, in particular, in acetonitrile. A good correlation between basicities in THF and acetonitrile spanning 25 orders of magnitude gives access to experimentally supported very high (pKa > 40) basicities in acetonitrile, which cannot be directly measured. Analysis of structure-basicity trends is presented.

Covalent attachment of polymeric monolith to polyether ether ketone (PEEK) tubing.

A new method of reproducible preparation of vinylic polymeric monolithic columns with a key step of covalently anchoring the monolith to PEEK surface is described. In order to chemically attach the polymer monolith to the tube wall, methacrylate functional groups were introduced onto PEEK surface by a three-step procedure, including surface etching, surface reduction and surface methacryloylation. The chemical state of the modified tubing surface was characterized by attenuated total reflectance infrared (ATR-IR) spectroscopy. It was found that the etching step is the key to successfully modifying the PEEK tubing surface. Poly(styrene-co-divinylbenzene) monoliths were in situ synthesized by thermally initiated free radical copolymerization within the confines of surface-vinylized PEEK tubings of dimensions close to ones conventionally used in HPLC and UHPLC (1.6 mm internal diameter, 10.0-12.5 cm length). Adhesion test was done by measuring the operating pressure drop, which the prepared stationary phases can withstand. Good pressure resistance, up to 140 bar/10 cm (flow rate 0.5 mL min(-1), acetonitrile as a mobile phase), indicates strong bonding of monolith to the tubing wall. The monolithic material was proven to have a permeability of 1.7 × 10 (-14) m(2), applying acetonitrile-water 70:30 (v/v) as a mobile phase. The column performance was reproducible from column to column and was evaluated via the isocratic separation of a series of alkylbenzenes in the reversed-phase mode (acetonitrile-water 70:30, v/v). The numbers of plates per meter at optimal flow rate were found to be between 26 000 and 32 000 for the different analytes.

2,5-Dihydroxybenzoic acid solution in MALDI-MS: ageing and use for mass calibration.

2,5-Dihydroxybenzoic acid (DHB) is one of the most widely used and studied matrix compounds in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. However, the influence of ageing of the DHB solution on the MALDI mass spectra has not been yet systematically studied. In this work, the possible changes occurring in the acidified acetonitrile/water solution of the MALDI matrix compound DHB during 1-year usage period have been monitored with MALDI-Fourier transform ion cyclotron resonance mass spectrometer (MALDI-FT-ICR-MS) and attenuated total reflectance Fourier transform infrared (ATR-FT-IR) spectroscopy. No significant ageing products have been detected. The ability of the aged DHB solution to act as a MALDI matrix was tested with two materials widely used in art and conservation - bone glue (a proteinaceous material) and shellac resin (a resinous material) - and good results were obtained. A number of peaks in the mass spectra measured from the DHB solution were identified, which can be used for internal calibration of the mass axis.

Alternative eluent composition for LC-MS analysis of perfluoroalkyl acids in raw fish samples.

A wide range of anthropogenic pollutants that possess serious environmental and health risks are known. One type of these harmful substances that have become a focus of interest during the past decade are perfluoroalkyl acids (PFAAs), which are extensively used in industry for different purposes. Due to the harmful effects that these compounds might cause in living organisms, EFSA and EU CONTAM panel have issued a monitoring program for PFAAs in foodstuffs. This has given rise to intense research dedicated to the analysis of PFAAs over the past few years. This work focuses on chromatographic analysis of three PFAAs in fish. The analytes, perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorooctanesulfonic acid (PFOS), are commonly associated with the production of fluoropolymers. Fluorinated alcohols are used as eluent components, and their possible advantages as eluent modifiers in LC-MS analysis of PFAAs, alternative retention mechanism and enhanced ionization efficiency, are examined. The analyzed fish samples originating from Estonian fresh and marine waters had low contents of PFAAs.

NMR method for simultaneous host-guest binding constant measurement.

An NMR-based relative binding affinity measurement method has been developed in which differences in the binding affinities of different hosts toward a particular guest (ΔlogK(ass) values) are measured in the same solution. As an advancement, the method allows the simultaneous determination of several ΔlogK(ass) values in a single run. As a proof of principle, the method was used to measure binding affinity differences of a number of indolocarbazole- and urea-based synthetic receptors toward acetate ion in DMSO-d6/H2O (99.5%:0.5% m/m). As a result, a binding affinity scale containing 33 receptors and spanning 2.32 log units with excellent self-consistency (consistency standard deviation = 0.01 log unit) was created. Together with the very good agreement of the results with those obtained by UV-vis spectrophotometry, this demonstrates the high accuracy of the method and the fact that the NMR and UV-vis techniques can be used interchangeably (in spite of the very different concentrations used in these techniques). Additionally, it was found for symmetrical receptor molecules from the same compound family that there is a correlation between the acetate binding affinity of a receptor and the (15)N chemical shift of the nitrogen atoms of its binding centers.

Accurate method to quantify binding in supramolecular chemistry.

An approach for accurate and comparable measurement of host-guest binding affinities is introduced whereby differences in binding strength (ΔlogKass values) are measured between two host molecules toward a particular guest under identical solvent conditions. Measuring differences instead of absolute values enables obtaining highly accurate results, because many of the uncertainty sources (the solvation/association state of the guest in solution, deviations in solvent composition, etc.) cancel out. As a proof of concept, this method was applied to the measurement of the binding strength of 28 synthetic anion receptors toward acetate in acetonitrile containing 0.5% water. The receptors included differently substituted indolocarbazoles, ureas, thioureas, and some others. Possible deprotonation of more acidic receptors of each compound class by acetate was checked by measuring their acidities (ΔpKa values) relative to acetic acid in the same solvent. A self-consistent (consistency standard deviation 0.04 log units) binding affinity scale ranging for around 2.7 log units was constructed from the results. Absolute logKass values were found by anchoring the scale to the absolute logKass values of two receptor molecules, determined independently by direct measurements. This new approach is expected to find use in accurate quantification of a wide range of binding processes relevant to supramolecular chemistry.

Analysis of dammar resin with MALDI-FT-ICR-MS and APCI-FT-ICR-MS.

Comprehensive analysis of high-resolution mass spectra of aged natural dammar resin obtained with Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) using matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure chemical ionization (APCI) is presented. Dammar resin is one of the most important components of painting varnishes. Dammar resin is a terpenoid resin (dominated by triterpenoids) with intrinsically very complex composition. This complexity further increases with aging. Ten different solvents and two-component solvent mixtures were tested for sample preparation. The most suitable solvent mixtures for the MALDI-FT-ICR-MS analysis were dichloromethane-acetone and dichloromethane-ethanol. The obtained MALDI-FTMS mass spectrum contains nine clusters of peaks in the m/z range of 420-2200, and the obtained APCI-FTMS mass spectrum contains three clusters of peaks in the m/z range of 380-910. The peaks in the clusters correspond to the oxygenated derivatives of terpenoids differing by the number of C(15)H(24) units. The clusters, in turn, are composed of subclusters differing by the number of oxygen atoms in the molecules. Thorough analysis and identification of the components (or groups of components) by their accurate m/z ratios was carried out, and molecular formulas (elemental compositions) of all major peaks in the MALDI-FTMS and APCI-FTMS spectra were identified (and groups of possible isomeric compounds were proposed). In the MALDI-FTMS and APCI-FTMS mass spectrum, besides the oxidized C(30), triterpenoids also peaks corresponding to C(29) and C(31) derivatives of triterpenoids (demethylated and methylated, correspondingly) were detected. MALDI and APCI are complementary ionization sources for the analysis of natural dammar resin. In the MALDI source, preferably polar (extensively oxidized) components of the resin are ionized (mostly as Na(+) adducts), whereas in the APCI source, preferably nonpolar (hydrocarbon and slightly oxidized) compounds are ionized (by protonation). Either of the two ionization methods, when used alone, gives an incomplete picture of the dammar resin composition.