Discovery of a new class of potent pyrrolo[3,4-c]quinoline-1,3-diones based inhibitors of human dihydroorotate dehydrogenase: Synthesis, pharmacological and toxicological evaluation.

Twenty N-substituted pyrrolo[3,4-c]quinoline-1,3-diones 3a-t were synthesized by a cyclization reaction of Pfitzinger's quinoline ester precursor with the selected aromatic, heteroaromatic and aliphatic amines. The structures of all derivatives were confirmed by IR, 1H NMR, 13C NMR and HRMS spectra, while their purity was determined using HPLC techniques. Almost all compounds were identified as a new class ofpotent inhibitors against hDHODH among which 3a and 3t were the most active ones with the same IC50 values of 0.11 µM, about seven times better than reference drug leflunomide. These two derivatives also exhibited very low cytotoxic effects toward healthy HaCaT cells and the optimal lipophilic properties with logP value of 1.12 and 2.07 respectively, obtained experimentally at physiological pH. We further evaluated the comparative differences in toxicological impact of the three most active compounds 3a, 3n and 3t and reference drug leflunomide. The rats were divided into five groups and were treated intraperitoneally, control group (group I) with a single dose of leflunomide (20 mg/kg) group II and the other three groups, III, IV and V were treated with 3a, 3n and 3t (20 mg/kg bw) separately. The investigation was performed in liver, kidney and blood by examining serum biochemical parameters and parameters of oxidative stress.

UHPLC-Q-TOF analysis of pyrrolizidine alkaloids in North-Macedonian honey.

Honey contaminated with pyrrolizidine alkaloids (PAs) could pose a risk for human consumption, being a widely consumed food product. A fast and simple LC/MS method for the analysis of pyrrolizidine alkaloids in honey was optimised to collect occurrence data. The extraction efficiency was evaluated by a systematic study of multiple solvent mixtures and clean-up procedures. The best results for PA extraction were obtained using a formic acid/methanol mixture with subsequent clean-up by the QuEChERS method, resulting in a mean recovery range of 91.8-102%. The method validation showed satisfactory intra-day (RSD < 5.1%) and inter-day precision (RSD < 9.1%). The proposed method was applied to 14 samples. A total of six PAs and two N-oxides were detected, with levels between 89 and 8188 µg/kg. This assessment highlights the potential risk of intoxication and the need for further investigations regarding an effective quality system for manufacturers to control PAs in honey.

Bisphenol A monitoring during anaerobic degradation of papers with thermochromic prints in soil.

Pseudoestrogene bisphenol A (BPA) can be important ingredient of thermochromic inks, increasingly used materials in thermal printing paper, security printing, advertising, design and as temperature indicators in medicine and food industry. BPA mass fraction in thermochromic inks can be up to several percent. Hence, disposal of items with thermochromic prints pose a risk of environmental pollution. In this work BPA mass fraction was monitored during anaerobic degradation of papers with thermochromic prints in soil in both matrices: papers and soil. The degradation conditions simulated deeper layers of waste at a landfill site. Six types of papers with prints of thermochromic ink containing 2% of BPA were subjected to anaerobic degradation over up to 150 days. Initial mass fractions of BPA in papers decreased form (126-460) µg/g to (

Search for new antimicrobials: spectroscopic, spectrometric, and in vitro antimicrobial activity investigation of Ga(III) and Fe(III) complexes with aroylhydrazones.

The in vitro antimicrobial activity of Fe(III) and Ga(III) complexes with N'-(2,3-dihydroxy-phenylmethylidene)-3-pyridinecarbohydrazide (H2L1), N'-(2,4-dihydroxy-phenyl-methylidene)-3-pyridinecarbohydrazide (H2L2), N'-(2,5-dihydroxy-phenylmethylidene)-3-pyridinecarbohydrazide (H2L3), N'-(2-hydroxy-3-methoxyphenyl-methylidene)-3-pyridine-carbohydrazide (H2L4), N'-(2-hydroxy-4-methoxyphenylmethyl-idene)-3-pyridine-carbohydrazide (H2L5), and N'-(2-hydroxy-5-methoxyphenylmethylidene)-3-pyridinecarbo-hydrazide (H2L6) toward several Gram-positive strains of Staphylococcus aureus, a Gram-negative strain of Escherichia coli, and a yeast Candida albicans were investigated. Fe(III)-complexes do not possess antimicrobial activity against all tested strains at concentrations up to 10 mg mL-1. Ga(III) complexes with dihydroxy derivatives showed selective activity, while the broadest range of antibacterial and antifungal activities was observed for complex with 2-hydroxy-3-methoxy-derivative, ligand H2L5. In addition, the coordination properties of ligands H2L1-H2L3 in solution were investigated by UV-Vis spectroscopy. The stability constants (logK) for Ga(III)-H2L 1:1 complexes in MeOH/H2O 1/1 at pH 2.52 were determined, and amounted to 5.8, 5.68, and 4.7, respectively. Detailed characterization of complexes was performed by high-resolution mass spectrometry. The fragmentation pathways for dimer [Fe2(L1)2]2+, [Fe(HL)2]+, [Ga(HL2)2]+ and adduct ions are given. The comparison with analogue Ga(III) and Fe(III) complexes with compounds H2L4-H2L6 was made as well.

Investigation of Praziquantel/Cyclodextrin Inclusion Complexation by NMR and LC-HRMS/MS: Mechanism, Solubility, Chemical Stability, and Degradation Products.

Praziquantel (PZQ) is a biopharmaceutical classification system (BCS) class II anthelmintic drug characterized by poor solubility and a bitter taste, both of which can be addressed by inclusion complexation with cyclodextrins (CD). In this work, a comprehensive investigation of praziquantel/cyclodextrin (PZQ/CD) complexes was conducted by means of UV-vis spectroscopy, spectrofluorimetry, NMR spectroscopy, liquid chromatography-high-resolution mass spectrometry (LC-HRMS/MS), and molecular modeling. Phase solubility studies revealed that among four CDs tested, the randomly methylated ß-CD (RMßCD) and the sulfobutylether sodium salt ß-CD (SBEßCD) resulted in the highest increase in PZQ solubility (approximately 16-fold). The formation of 1:1 inclusion complexes was confirmed by HRMS, NMR, and molecular modeling. Both cyclohexane and the central pyrazino ring, as well as an aromatic part of PZQ are included in the CD central cavity through several different binding modes, which exist simultaneously. Furthermore, the influence of CDs on PZQ stability was investigated in solution (HCl, NaOH, H2O2) and in the solid state (accelerated degradation, photostability) by ultra-high-performance liquid chromatography-diode array detection-tandem mass spectrometry (UPLC-DAD/MS). CD complexation promoted new degradation pathways of the drug. In addition to three already known PZQ degradants, seven new degradation products were identified (m/z 148, 215, 217, 301, 327, 343, and 378) and their structures were proposed based on HRMS/MS data. Solid complexes were prepared by mechanochemical activation, a solvent-free and ecologically acceptable method.

A simple and sensitive LC-MS/MS method for determination and quantification of potential genotoxic impurities in the ceritinib active pharmaceutical ingredient.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used for quantification of four potential genotoxic impurities (PGIs) in the ceritinib active pharmaceutical ingredient. Chromatographic separation was achieved using a YMC-Triart C18 column, with 0.1% formic acid in water as mobile phase A and acetonitrile as mobile phase B in gradient elution mode at a 0.5 mL min-1 flow rate. Quantification of impurities was carried out using triple quadrupole mass detection with electrospray ionization in multiple reaction monitoring mode. The method was fully validated with good linearity over the concentration range of 0.5-5.0 ppm of the ceritinib test concentration for all four PGIs. The correlation coefficient obtained in each case was >0.998. The recoveries were found satisfactory over the range between 83.7 and 107.3% for all selected impurities. The developed method was able to quantitate all four PGIs at a concentration level of 1 ng mL-1 (0.5 ppm with respect to 2 mg mL-1 ceritinib).

Chiral separation of beta-blockers by high-performance liquid chromatography and determination of bisoprolol enantiomers in surface waters.

Beta-blockers are chiral compounds with enantiomers that have different bioactivity, which means that while one is active, the other can be inactive or even harmful. Due to their high consumption and incomplete degradation in waste water, they may reach surface waters and affect aquatic organisms. To address this issue we developed a chromatographic method suitable for determining beta-blocker enantiomers in surface waters. It was tested on five beta-blockers (acebutolol, atenolol, bisoprolol, labetalol and metoprolol) and validated on bisoprolol enantiomers. Good enantioseparation of all analysed beta-blockers was achieved on the Chirobiotic V column with the mobile phase composed of methanol/acetic acid/triethylamine (100/0.20/0.15 v/v/v) at a flow rate of 0.5 mL/min and column temperature of 45 °C. Method proved to be linear in the concentration range from 0.075 µg/mL to 5 µg/mL, and showed good recovery. The limits of bisoprolol enantiomer detection were 0.025 µg/mL and 0.026 µg/mL and of quantification 0.075 µg/mL and 0.075 µg/mL. Despite its limitations, it seems to be a promising method for bisoprolol enantiomer analysis in surface water samples. Further research could focus on waste water analysis, where enantiomer concentrations may be high. Furthermore, transferring the method to a more sensitive one such as liquid chromatography coupled with tandem mass spectrometry and using ammonium acetate as the mobile phase additive instead of acetic acid and triethylamine would perhaps yield much lower limits of detection and quantification.

Diverse coordination of aroylhydrazones toward iron(III) in solid state and in solution: spectrometric, spectroscopic and computational study.

The coordination properties of N'-(2-hydroxy-3-methoxyphenylmethylidene)-3-pyridinecarbohydrazide (H2L1), N'-(2-hydroxy-4-methoxyphenylmethylidene)-3-pyridinecarbohydrazide (H2L2) and N'-(2-hydroxy-5-methoxyphenylmethylidene)-3-pyridinecarbohydrazide (H2L3) toward Fe(III) ions were studied by computational, spectrometric (MS) and spectroscopic methods (UV-Vis, IR and Raman spectroscopy) in solid state and in solution. Free ligands were present in keto-amine form with intramolecular H-bond. In MeOH:H2O 1:1 system, the 1:1 complexes with Fe(III) were formed, characterized by lgK ≥ 6. The coordination to the metal ion was achieved via oxygen and azomethine nitrogen since the hydrolysis of hydrazone bond was suppressed. Unlike the 1:1 stoichiometry in methanolic solution, the composition of the complexes extracted to chloroform was Fe(L)(HL). The release of three protons upon complexation was determined by independent spectrophotometric measurements. The complexes isolated from MeOH/EtOH solution have also stoichiometry 1:2. However, depending on the position of the methoxy substituent, two types of complexes were formed. In Fe(H2L1)2Cl3 and Fe(H2L3)2Cl3, hydrazones acted as neutral ligands, while in Fe(HL2)2Cl the keto-enol tautomeric interconversion and release of one proton per ligand took place. All complexes were analyzed in gas phase as well, using triple quadrupole, ion trap and H/D exchange for determination of labile hydrogens. Based on the fragmentation pathways, the structural isomers were distinguished.

Antimicrobial assesment of aroylhydrazone derivatives in vitro.

Aroylhydrazones 1-13 were screened for antimicrobial and antibiofilm activities in vitro. N'-(2-hydroxy-phenylmethylidene)-3-pyridinecarbohydrazide (2), N'-(5-chloro-2-hydroxyphenyl-methylidene)-3-pyridinecarbohydrazide (10), N'-(3,5-chloro-2-hydroxyphenylmethylidene)-3-pyridinecarbohydrazide (11), and N'-(2-hydroxy-5-nitrophenylmethylidene)-3-pyridinecarbohydrazide (12) showed antibacterial activity against Escherichia coli, with MIC values (in µmol mL-1) of 0.18-0.23, 0.11-0.20, 0.16-0.17 and 0.35-0.37, resp. Compounds 11 and 12, as well as N'-(2-hydroxy-3-methoxyphenylmethylidene)-3-pyridinecarbohydrazide (6) and N'-(2-hydroxy-5- methoxyphenylmethylidene)-3-pyridinecarbohydrazide (8) showed antibacterial activity against Staphylococcus aureus, with the lowest MIC values of 0.005-0.2, 0.05-0.12, 0.06-0.48 and 0.17-0.99 µmol mL-1. N'-(2-hydroxy-5-methoxyphenylmethylidene)-3-pyridinecarbohydrazide (7) showed antifungal activity against both fluconazole resistant and susceptible C. albicans strains with IC90 range of 0.18-0.1 µmol mL-1. Only compound 11 showed activity against C. albicans ATCC 10231 comparable to the activity of nystatin (the lowest MIC 4.0 ×10-2 vs. 1.7 × 10-2 µmol mL-1). Good activity regarding multi-resistant clinical strains was observed for compound 12 against MRSA strain (MIC 0.02 µmol mL-1) and compounds 2, 6 and 12 against ESBL+ E. coli MFBF 12794, with the lowest MIC for compound 12 (IC50 0.16 µmol mL-1). Anti-biofilm activity was found for compounds 2 (MBFIC 0.015-0.02 µmol mL-1 against MRSA) and 12 (MBFIC 0.013 µmol mL-1 against EBSL+ E. coli). In the case of compound 2 against MRSA biofilm formation, MBFIC values were comparable to those of gentamicin sulphate, whereas in the case of compound 12 and EBSL+ E. coli even more favourable activity compared to gentamicin was observed.

Monitoring of selected pharmaceuticals in surface waters of Croatia.

Sulfonamides, macrolides, torasemide, fumagillin, and chloramphenicol were simultaneously analyzed in surface water samples by using solid-phase extraction (SPE) and reversed-phase (RP) liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS). In the pre-concentration and clean-up process, the pH value of samples and volume of the solvent for extraction of analytes from cartridge were optimized. Extraction recoveries were high with values in the range from 62 to 115%. Limits of quantification (LoQ) were in the range from 0.02 to 0.2 µg L-1. Repeatability of the method was evaluated at LoQ and expressed as relative standard deviation (RSD). Calculated RSDs were low with values in the range from 2.4 to 14.5%. The method was successfully applied for analysis of surface water real samples. Samples were collected along the rivers in Croatia on four sampling sites in 2012 in Danube catchment areas, 19 sampling sites in Danube and Adriatic catchment areas in 2013, and another 19 places in 2014. Altogether, 20 target compounds were analyzed in 148 water samples and detected in 31 samples in range (0.1-5.3) µg L-1 or in 20.1% of samples. The most frequent and highest concentrations were detected for macrolide antibiotics. This is the first attempt of such monitoring in surface waters in Croatia.

Biopharmaceutical characterization of praziquantel cocrystals and cyclodextrin complexes prepared by grinding.

Mechanochemical activation using several different co-grinding additives was applied as a green chemistry approach to improve physiochemical and biopharmaceutical properties of praziquantel (PZQ). Liquid assisted grinding with an equimolar amount of citric acid (CA), malic acid (MA), salicylic acid (SA) and tartaric acid (TA) gained in cocrystal formation, which all showed pH-dependent solubility and dissolution rate. However, the most soluble cocrystal of PZQ with MA was chemically unstable, as seen during the stability testing. Equimolar cyclodextrin complexes prepared by neat grinding with amorphous hydroxypropyl-ß-cyclodextrin (HPßCD) and randomly methylated ß-cyclodextrin (MEßCD) showed the highest improvement in drug solubility and the dissolution rate, but only PZQ/HPßCD product presented an acceptable chemical and photostability profile. A combined approach, by co-grinding the drug with both MA and HPßCD in equimolar ratio, also gave highly soluble amorphous product which again was chemical instable and therefore not suitable for the pharmaceutical use. Studies on Caco-2 monolayer confirmed the biocompatibility of PZQ/HPßCD complex and showed that complexation did not adversely affect the intrinsically high PZQ permeability (Papp(PZQ)=(3.72±0.33)×10-5cms-1 and Papp(PZQ/HPßCD)=(3.65±0.21)×10-5cms-1; p>0.05). All this confirmed that the co-grinding with the proper additive is as a promising strategy to improve biopharmaceutical properties of the drug.

Structural elucidation studies of 15-membered azalide macrocycles using H/D exchange and ESI-MS(n.).

A comprehensive study of fragmentation pathways of 15-membered azalide macrocycles using electrospray ionisation with multistage mass spectrometry (ESI-MS(n)) is presented in this work. Hydrogen/deuterium (H/D) exchange experiments and high-resolution mass spectrometry were used to investigate the proposed fragmentation pathways. In addition, the fragmentation patterns of sodium adduct ions [M+Na](+) of the macrocycles were also investigated as the presence of an alkali metal interacting with the aglycone ring influences the product-ion spectra.

Structural investigations of aroylhydrazones derived from nicotinic acid hydrazide in solid state and in solution.

Structural forms of aroylhydrazones derived from nicotinic acid hydrazide have been studied in the solid state by FT-IR spectroscopy and in solution by NMR, UV-Vis and ATR spectroscopy. The studied compounds were N'-benzylidene-3-pyridinecarbohydrazide (1), N'-(2,4-dihydroxyphenylmethylidene)-3-pyridinecarbohydrazide (2), N'-(5-chloro-2-hydroxyphenylmethylidene)-3-pyridinecarbohydrazide (3), and N'-(3,5-dichloro-2-hydroxymethoxyphenylmethylidene)-3-pyridinecarbohydrazide (4). The compound 1 adopted the most stable ketoamine form (form I, -CO-NH-N=C-) in the solid state as well as in various organic solvents. In mixtures of organic solvents with water the UV-Vis and ATR spectra implied intermolecular hydrogen bonding of 1 with water molecules. The presence of both tautomeric forms I and II (form II, -COH=N-N=C-) was proposed for the solid substance and highly concentrated solutions of 2, whereas form I was detected as the predominant one in diluted solutions. For compounds 3 and 4 a coexistence of forms I and III (form III, -CO-NH-NH-C=C-CO-) was noticed in the solid state and in polar protic organic solvents. The conversion to form III was induced by increasing the water content in the solvent mixtures. This process was the most pronounced for compound 4. When exposed to daylight, an appearance of a new band was observed during time in the UV-Vis spectrum of 4 in organic solvent/water 1/1 mixtures, which implied that tautomeric interconversion was most likely followed by E/Z isomerisation.

Structural investigation of aroylhydrazones in dimethylsulphoxide/water mixtures.

Molecular structures of aroylhydrazones derived from salicylaldehyde, o-vanilin and nicotinic acid hydrazide in DMSO and DMSO/H(2)O mixtures have been studied by NMR, UV-Vis, ATR and Raman spectroscopy. The addition of water to the system did not induce the tautomeric conversion of the existing form constituted of the ketoamino hydrazide part and the enolimino aldehyde part, but it was involved in the formation of hydrated molecules. Vibrational spectra (ATR and Raman) clearly indicated hydrogen bonding of the studied hydrazones through the carbonyl, amino and hydroxyl groups with water molecules. Increasing the water content conversion from E to Z isomer was not observed.

Urinary metabolites as biomarkers of human exposure to atrazine: atrazine mercapturate in agricultural workers.

Human exposure to atrazine and other triazine herbicides results in urinary excretion of traces of parent compounds and of their metabolites formed by N-dealkylation or conjugation with mercapturic acid. In contrast to N-dealkylated metabolites, which are not compound-specific, the measurement of atrazine mercapturate and unchanged atrazine provides an unambiguous confirmation of exposure to this herbicide. The aim of this study was to investigate the levels of these two compounds in a group of agricultural workers who may be considered representative for typical behaviour and procedures during the atrazine application in Croatia. The spot urine samples were collected at the beginning (samples A) and at the end (samples B) of a working day and 12h after exposure has ended (samples C). Atrazine and atrazine mercapturate were extracted from acidified urine samples (pH 2) with ethyl acetate and the extracts were analysed using high performance liquid chromatography-tandem mass spectrometry with a turbo ion spray (electrospray) ionization interface. The detection limits based on treatment of 2ml urine samples were 0.2ngml(-1) for both analytes. Atrazine was not detected in any of 27 analysed urine samples but traces of atrazine mercapturate were measured in about a third of pre-exposure and in all post-exposure urine samples in mass concentrations ranging from 0.3 to 10.4ngml(-1) (0.3 to 8.0µgg(-1) of creatinine). The metabolite concentrations in B and C group of post-exposure samples were not significantly different. The urinary atrazine mercapturate post-exposure concentrations were comparable to those reported for U.S. farmers engaged in a single field application of atrazine.

Vanadium-induced formation of thiadiazole and thiazoline compounds. Mononuclear and dinuclear oxovanadium(v) complexes with open-chain and cyclized thiosemicarbazone ligands.

Reactions of the salicylaldehyde 4-phenylthiosemicarbazone (H(2)L) with selected vanadium(iv) and vanadium(v) precursors ([VO(acac)(2)], [VO(OAc)(2)], VOSO(4), [V(2)O(4)(acac)(2)]) were investigated under aerobic conditions in different alcohols (methanol, ethanol, propanol). In all examined cases mononuclear alkoxo vanadium(v) complexes [VOL(OR)] (1) (OR = OMe, OEt, OPr) were isolated as major products. On prolonged standing, mother liquids afforded dinuclear vanadium(v) complexes [V(2)O(3)(L(cycl))(2)(OR)(2)] (3) (OR = OMe, OEt, OPr), where L(cycl)(-) represents 1,3,4-thiadiazole ligand, formed by vanadium-induced oxidative cyclization of H(2)L. When [VO(acac)(2)] or [V(2)O(4)(acac)(2)] were used as precursors, in addition to products 1 and 3, a thiazoline derivative HL(acac)(cycl) (2) was isolated. This compound, formed by a reaction between acetylacetone and H(2)L, represented the second type of cyclic product. The products were characterized by IR and NMR spectroscopies, TG analysis, and in some cases by single-crystal X-ray diffraction. To the best of our knowledge, compounds [V(2)O(3)(L(cycl))(2)(OR)(2)] represent the first structurally characterized dinuclear vanadium(v) complexes with a thiadiazole moiety acting as a bridging ligand. Complexes 1 and 3, when dissolved in an appropriate alcohol, underwent substitution of the alkoxo ligand as confirmed by XRPD. The kinetics of reactions in methanolic solutions was qualitatively studied by UV-Vis and ESMS spectrometries. Under the experimental conditions applied, a relatively slow formation of the mononuclear complex [VOL(OMe)] and an even slower formation of the cyclic species 2 were observed, whereas the presence of dinuclear compound [V(2)O(3)(L(cycl))(2)(OMe)(2)] in the reaction mixture could not be detected.

Spectrometric study of tautomeric and protonation equilibria of o-vanillin Schiff base derivatives and their complexes with Cu(II).

Electronic absorption and emission properties of a series of Schiff bases derived from 2-hydroxy-3-methoxybenzaldehyde and 2-aminopyridine, 2,3-diaminopyridine, 2,6-diaminopyridine, or 3-aminomethylpyridine were studied in solvents of different polarities. The interconversion of the enolimine to the ketoamine tautomeric form was observed for compound 1, 6-methoxy-2-(3-pyridylmethyliminomethyl)phenol, and the corresponding equilibrium constant was estimated in several solvents. Protonation constants of all the investigated compounds were determined spectrophotometrically in the methanol/water 1/4 system. The effect of copper(II) ions on absorption and on the emission spectra of these ligands was examined in the buffered dioxane/water 1/1 system (pH 5.8). Strong complexation of Cu(II) and formation of a 1:1 complex were observed for the bis-Schiff base derived from 2,3-diaminopyridine. The complex of copper(II) with compound 1 was isolated and characterized by elemental analysis, magnetic susceptibility measurement, UV-vis and IR spectrometry.

Structural characterization of PEGylated rHuG-CSF and location of PEG attachment sites.

Mass spectrometry structural characterization is an essential tool in validating the quality of PEG-rHu-proteins. However, in either case top-down or bottom-up fashion, the interference of high intensity PEG signals on MS detection or detrimental influence of PEG on protein structure, leads to incomplete structural characterization. We propose here a method that permits complete and reliable structural characterization of PEGylated recombinant human granulocyte-colony stimulating factor (rHuG-CSF). The approach includes on-column 2-methoxy-4,5-dihydro-1H-imidazole derivatization of digested PEG rHuG-CSF and subsequent LC/MS investigation. By comparing the LC/MS retention of derivatized and underivatized digested PEG rHuG-CSF, location of the PEG attachment within rHuG-CSF could be deduced. Besides, the protein sequence coverage and position of the disulfide bridges was fully deducible from the MS data interpretation. Additionally, ultra performance liquid chromatography-mass spectrometry-to-the-E (UPLC-MS(E)) was introduced for analysis of label-free digested PEG rHuG-CSF here to enable high resolution and mass accuracy of MS detection and facilitate deep structural insights of peptides.

Evaluation of recombinant human interferon alpha-2b structure and stability by in-gel tryptic digestion, H/D exchange and mass spectrometry.

Stability and structure of recombinant interferon alpha-2b (rHuINF alpha-2b) was studied by mass spectrometry (MALDI-TOF and Q-TOF MS), chromatography (LC-UV-FLD-DAD, LC-MS) and CD spectroscopy. Besides analysis of the substance according to Ph. Eur. methods, two additional mass spectrometric methods were developed. The aim of both methods was to estimate structure-stability relationship connected to methionine oxidation or protein degradation. Preservation or degradation of protein structure was confirmed by H/D exchange in four separate experiments. Kinetics of deuterium incorporation into macromolecule was monitored over 2670 min. Isoforms of rHuINF alpha-2b were separated by 2D gel electrophoresis. In-gel digestion with trypsin and mass spectrometric analysis, performed on four separated isoforms at the mass corresponding to the mass of rHuINF alpha-2b with oxidized methionines, confirmed oxidation of all methionines to a different extent. Another four isoforms observed in 2D gel are most likely dimers of the same macromolecules with scrambled disulphide bridges. Oxidation and dimerisation are consequences of protein interaction with oxidizing reagents in polyacrilamide gel.

Absorption and fluorescence spectra of ring-substituted indole-3-acetic acids.

The absorption and fluorescence spectra of indole-3-acetic acid (1), a plant growth regulator (auxin) and experimental cancer therapeutic, 29 ring-substituted derivatives and the 7-aza analogue (1H-pyrrolo[2,3b]pyridine-3-acetic acid) are compared. Two to four absorbance maxima in the 260-310-nm range are interpreted as overlapping vibronic lines of the 1La<--1A and 1Lb<--1A transitions. Two further maxima in the 200-230-nm region are assigned to the 1Ba<--1A and 1Bb<--1A transitions. 4- and 7-Fluoroindole-3-acetic acid exhibit blue shifts with respect to 1, most other derivatives show red shifts. All indole-3-acetic acids studied, with the exception of chloro-, bromo- and 4- or 7-fluoro-derivatives, fluoresce at 345-370 nm when excited at 275-280 nm. 7-Azaindole-3-acetic acid emits at 411 nm. The fluorescence quantum yield of 6-fluoroindole-3-acetic acid significantly exceeds that of 1 (0.3); the other derivatives have lower quantum yields. The plant-growth promoting activity of the ring-substituted indole-3-acetic acids studied correlates with the position of the 1Bb<--1A transition band.