Cyclin-dependent kinases (CDKs) play an important role in the cell-division cycle. Synthetic inhibitors of CDKs are based on 2,6,9-trisubstituted purines and are developed as potential anticancer drugs; however, they have low solubility in water. In this study, we proved that the pharmaco-chemical properties of purine-based inhibitors can be improved by appropriate substitution with the adamantane moiety. We prepared ten new purine derivatives with adamantane skeletons that were linked at position 6 using phenylene spacers of variable geometry and polarity. We demonstrated that the adamantane skeleton does not compromise the biological activity, and some of the new purines displayed even higher inhibition activity towards CDK2/cyclin E than the parental compounds. These findings were supported by a docking study, which showed an adamantane scaffold inside the binding pocket participating in the complex stabilisation with non-polar interactions. In addition, we demonstrated that β-cyclodextrin (CD) increases the drug's solubility in water, although this is at the cost of reducing the biochemical and cellular effect. Most likely, the drug concentration, which is necessary for target engagement, was decreased by competitive drug binding within the complex with β-CD.
- MeSH
- adamantan chemie MeSH
- antitumorózní látky chemie farmakologie MeSH
- beta-cyklodextriny chemie MeSH
- buňky K562 MeSH
- cyklin-dependentní kinasa 2 antagonisté a inhibitory MeSH
- inhibitory proteinkinas farmakologie MeSH
- lidé MeSH
- MFC-7 buňky MeSH
- puriny chemie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The advantages of using mixtures of organic solvents for the separation of labeled oligosaccharides on the amide stationary phase under hydrophilic interaction liquid chromatography conditions are presented. The effect of the type of buffer as well as solvent or their mixtures on retention of uracil, saccharide labeling reagents (2-aminobenzoic acid, 2-aminobenzamide, ethyl 4-aminobenzoate, procainamide), and corresponding labeled saccharides were evaluated. The successful isocratic separation of labeled isomeric trisaccharides (maltotriose, panose, and isomaltotriose) was achieved in the mobile phase consisting of a 90% (v/v) mixture of organic solvents (methanol/acetonitrile 60:40) and 10% (v/v) 30 mM ammonium formate, pH 3.3. Changing the volume ratio between methanol/acetonitrile from 60:40 to 50:50 (v/v) allowed to obtain the separation of di-, tri-, and tetrasaccharides labeled by ethyl 4-aminobenzoate in less than 10.5 min.
- MeSH
- acetonitrily chemie MeSH
- amidy chemie MeSH
- chemické techniky analytické přístrojové vybavení metody MeSH
- chromatografie kapalinová * MeSH
- formiáty chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- isomerie MeSH
- oligosacharidy izolace a purifikace MeSH
- ortoaminobenzoáty chemie MeSH
- rozpouštědla chemie MeSH
- sacharidy chemie MeSH
- Publikační typ
- časopisecké články MeSH
A novel and original application of salting-out assisted liquid-liquid extraction is presented. This technique was used to purify the final reaction products (quaternary ammonium salts) from unreacted components and by-products present in multiple excesses. The partition of two structurally related compounds as (2-aminoethyl)trimethylammonium salt (a labeling reagent) and a derivative of [2-(imidazoline-1-yl)ethyl]trimethylammonium salt (a final reaction product of N-acetylglucosamine labeling by (2-aminoethyl)trimethylammonium salt) between acetonitrile-rich and water-rich layers was monitored by hydrophilic interaction chromatography with electrospray ionization mass spectrometry. Despite the poor solubility of both highly polar substances in solutions containing a high concentration of acetonitrile, the main portion of the labeling reagent (72%) can be removed from the crude reaction mixture in the first extraction step using 95% acetonitrile/5% water as an extraction solvent. The purified final reaction product contained only 2% of the labeling reagent, and it was suitable for analysis by direct infusion mass spectrometry to confirm its identity. The capability of the suggested purification protocol to process small-volume highly salted reaction mixtures was also proven by analysis of saccharide mixture containing glucose, maltose, and maltotriose labeled by the positively charged tag.
- Publikační typ
- časopisecké články MeSH
Antibacterial antibiotic therapy has played an important role in the treatment of bacterial infections for almost a century. The increasing resistance of pathogenic bacteria to antibiotics leads to an attempt to use previously neglected antibacterial therapies. Here we provide information on the two recombinantly modified antistaphylococcal enzymes derived from lysostaphin (LYSSTAPH-S) and endolysin (LYSDERM-S) derived from kayvirus 812F1 whose target sites reside in the bacterial cell wall. LYSSTAPH-S showed a stable antimicrobial effect over 24-h testing, even in concentrations lower than 1 µg/mL across a wide variety of epidemiologically important sequence types (STs) of methicillin-resistant Staphylococcus aureus (MRSA), especially in the stationary phase of growth (status comparable to chronic infections). LYSDERM-S showed a less potent antimicrobial effect that lasted only a few hours at concentrations of 15 μg/mL and higher. Our data indicate that these antimicrobial enzymes could be of substantial help in the treatment of chronic MRSA wound infections.
- Publikační typ
- časopisecké články MeSH
In this work, we compare labeling by two negatively charged fluorescent labels, 8-aminopyrene-1,3,6-trisulfonic acid (APTS) and 8-(2-hydrazino-2-oxoethoxy)pyrene-1,3,6-trisulfonic acid (Cascade Blue hydrazide [CBH]). Effectiveness of the labeling chemistries were investigated by 4-hydroxybenzaldehyde and maltoheptaose followed by LC/UV-MS and CE/LIF analysis, respectively. The reaction yield of APTS labeling was determined to be only ∼10%. This is due to reduction of almost 90% of the analyte by sodium cyanoborohydride to alcohol, which cannot be further labeled via reductive amination. However, the CBH labeling provides ∼90% reaction yield based on the LC/UV-MS measurements. The significantly higher labeling yield was also confirmed by CE/LIF measurements. Finally, the more effective hydrazone formation technique of CBH was characterized and applied for N-linked glycan analysis by CE/LIF.
- MeSH
- aminace MeSH
- chromatografie kapalinová metody MeSH
- elektroforéza kapilární metody MeSH
- fluorescenční barviva chemie MeSH
- hmotnostní spektrometrie metody MeSH
- hydrazony chemie MeSH
- oligosacharidy analýza chemie MeSH
- pyreny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Capillary electrophoresis-mass spectrometry was applied for the analysis of oligosaccharides and N-linked glycans with an attached charge label facilitating electrophoretic migration and electrospray ionization efficiency. Several different labeling strategies have been tested with different tags and tagging reactions including reductive amination and hydrazone formation. However, a formation of multiple labeled N-linked glycans was observed by CE-MS in a positive ion mode when positively charged labels such as aliphatic amines containing a quaternary ammonium group were attached to N-linked glycans by reductive amination. A reaction mechanism explaining a side reaction occurring during the labeling and the multiple product formation was proposed and confirmed by using isotopically labeled N-acetylglucosamine. Finally, it was confirmed that derivatization of sugars via a hydrazone formation can be a simpler method with a high reaction yield suitable for high sensitive CE-ESI/MS analyses of N-linked glycans.
Residential areas in urban agglomerations and also in the countryside are often burdened with high concentrations of aerosol in winter, this originating from local combustion sources. Aerosol sources can be identified by a monitoring of organic markers of biomass burning. Abundant markers of biomass and softwood burning are levoglucosan and dehydroabietic acid, respectively. The aim of this research was to develop an analytical method for the determination of levoglucosan and dehydroabietic acid in aerosol over short time periods involving aerosol sampling into liquid samples, quantitative pre-concentration of analytes, and their determination by liquid chromatography - mass spectrometry. A Condensation Growth Unit - Aerosol Counterflow Two-Jets Unit (CGU-ACTJU) sampler was used for the quantitative collection of aerosol directly into water. Dehydroabietic acid was pre-concentrated from the aqueous phase by solid phase extraction (C-18). Afterwards, levoglucosan in water samples was concentrated on a vacuum evaporator. The detection limits of levoglucosan and dehydroabietic acid were 28 ng m-3 and 5.5 ng m-3, respectively. The results obtained by the developed method were compared with an independent determination of both markers in aerosol by means of the sampling of aerosols on a filter and subsequent analysis by GC-MS. The developed method demonstrated sufficient agreement with the independent determination for generated standard aerosol as well as for urban aerosol over an eight-day winter campaign. The presented method allows the monitoring of concentration changes in biomass burning markers in 2-h intervals.
The labeling by amino acids and peptides was investigated for sensitive and fast analyses of oligosaccharides and N-linked glycans by capillary electrophoresis-mass spectrometry (CE-MS). Peptide tags with a various number of histidine residues were tested for maltooligosaccharide labeling in order to investigate the effect of the size of labels and a number of charges on CE-MS analysis. Nevertheless, the reductive amination labeling of N-linked glycans by a hexahistidine tag resulted in a multiple products formation, therefore a peptide tag was modified by hydrazine functionality in order to perform labeling by hydrazone formation chemistry. This labeling approach significantly improved sensitivity with LOD of labeled maltopentaose determined to be 40 nmol/L and also significantly reduced separation time of neutral maltooligosaccharides and N-linked glycans released from bovine ribonuclease B. Furthermore, the labeling by this multi-cationic peptide hydrazine tag also allowed performing analysis of acidic glycans by CE-MS in a positive ion mode as demonstrated by separation of sialylated N-linked glycans released from bovine fetuin.
- MeSH
- elektroforéza kapilární metody MeSH
- fetuin A chemie MeSH
- histidin chemie MeSH
- hmotnostní spektrometrie metody MeSH
- oligopeptidy chemie MeSH
- oligosacharidy analýza chemie izolace a purifikace MeSH
- polysacharidy analýza chemie izolace a purifikace MeSH
- ribonukleasy chemie MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
