Validated spectrofluorimetric method for the determination of carbamazepine in pharmaceutical dosage forms after reaction with 4‐chloro‐7‐‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl)

A sensitive and simple spectrofluorimetric method has been developed and validated for the determination of the anti‐epileptic drug carbamazepine (CBZ) in its dosage forms. The method was based on a nucleophilic substitution reaction of CBZ with 4‐chloro‐7‐nitrobenzo‐2‐ oxa‐1,3‐diazole (NBD‐Cl) in borate buffer (pH 9) to form a highly fluorescent derivative that was measured at 530 nm after excitation at 460 nm. Factors affecting the formation of the reaction product were studied and optimized, and the reaction mechanism was postulated. The fluorescence–concentration plot is rectilinear over the range of 0.6–8 µg/mL with limit of detection of 0.06 µg/mL and limit of quantitation of 0.19 µg/mL. The method was applied to the analysis of commercial tablets and the results were in good agreement with those obtained using the reference method. Validation of the analytical procedures was evaluated according to ICH guidelines. Copyright © 2015 John Wiley & Sons, Ltd.     

Magnesium methoxide‐induced chemiluminescent decomposition of bicyclic dioxetanes bearing a 2′‐alkoxy‐2‐hydroxy‐1,1′‐binaphthyl‐7‐yl moiety

Bicyclic dioxetanes 2a–c bearing a 2′‐alkoxy‐2‐hydroxy‐1,1′‐binaphthyl‐7‐yl moiety were effectively synthesized and their base‐induced chemiluminescent decomposition was investigated by the use of alkaline metal (Na⁺ and K⁺) or Mg²⁺ alkoxide in MeOH. When 2a–c were treated with tetrabutylammonium fluoride (TBAF) in dimethyl sulfoxide (DMSO) as a reference system, they showed chemiluminescence as a flash of orange light (maximum wavelength λ_max^CL = 573–577 nm) with efficiency Φ^CL = 6–8 × 10^–2. On the other hand, for an alkaline metal (Na⁺ or K⁺) alkoxide/MeOH system, 2a–c decomposed slowly to emit a glow of chemiluminescence, the spectra of which were shifted slightly toward red from the TBAF/DMSO system, and Φ^CL (= 1.4–2.3 × 10^–3) was considerably decreased. In addition, Mg(OMe)₂ was found to play a characteristic role as a base for the chemiluminescent decomposition of 2a–c through coordination to the intermediary oxidoaryl‐substituted dioxetanes 13. Thus, Mg²⁺ increased Φ^CL to more than twice those with Na⁺ or K⁺, while it shifted λ_max^CL considerably toward blue (λ_max^CL = 550–566 nm). Copyright © 2012 John Wiley & Sons, Ltd.     

Application of NMR spectroscopy for assignment of the absolute configuration of 8‐tert‐butyl‐2‐hydroxy‐7‐methoxy‐8‐methyl‐9‐oxa‐6‐azaspiro[4.5]dec‐6‐en‐10‐one

In order to assign the absolute configurations of 8‐tert‐butyl‐2‐hydroxy‐7‐methoxy‐8‐methyl‐9‐oxa‐6‐azaspiro[4.5]dec‐6‐en‐10‐one ( 2a , 2b ), their esters ( 5a , 5b , 5c , 5d ) with (R)‐ or (S)‐2‐methoxyphenylacetic acid ( 4a , 4b ) have been synthesized. The absolute configurations of these compounds have been determined on the basis of NOESY correlations between the protons of the tert‐butyl group and the cyclopentane fragment of the molecules. The crucial part of this analysis was assignment of the absolute configuration at C‐5. Additionally, by calculation of the chemical shift anisotropy, δ^RS, for the relevant protons, it was also possible to confirm the absolute configurations at the C‐2 centres of compounds 2a , 2b and 5a , 5b , 5c , 5d . Chirality, 25:422–426, 2013.© 2013 Wiley Periodicals, Inc.     

Stereoselective Synthesis of (3R)‐3‐Alkyl‐4,1‐Benzoxazepine‐2,5‐Diones

Novel 3‐alkyl‐4,1‐benzoxazepine‐2,5‐diones were synthesized in good ee exploiting the chiral pool methodology, an economical way of asymmetric synthesis. Various anthranilic acids are coupled with different α‐haloacids to afford N‐acylated anthranilic acid intermediates which undergo cyclization to (3R)‐3‐alkyl‐4,1‐benzoxazepines‐2,5‐diones. Chirality 25:865–870, 2013. © 2013 Wiley Periodicals, Inc.     

Chromatographic profiles of tryptophan and kynurenine enantiomers derivatized with (S)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole using LC–MS/MS on a triazole‐bonded column

d ‐ and l ‐Tryptophan (Trp) and d ‐ and l ‐kynurenine (KYN) were derivatized with a chiral reagent, (S )‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS), and were separated enantiomerically by high‐performance liquid chromatography (HPLC) equipped with a triazole‐bonded column (Cosmosil HILIC) using tandem mass spectrometric (MS/MS) detection. Effects of column temperature, salt (HCO₂NH₄) concentration, and pH of the mobile phase in the enantiomeric separation, followed by MS detection of (S )‐DBD‐PyNCS‐d ,l ‐Trp and ‐d ,l ‐KYN, were investigated. The mobile phase consisting of CH₃CN/10 mM ammonium formate in H₂O (pH 5.0) (90/10) with a column temperature of 50–60 °C gave satisfactory resolution (R s) and mass‐spectrometric detection. The enantiomeric separation of d ,l ‐Trp and d ,l ‐KYN produced R s values of 2.22 and 2.13, and separation factors (α) of 1.08 and 1.08, for the Trp and KYN enantiomers, respectively. The proposed LC–MS/MS method provided excellent detection sensitivity of both enantiomers of Trp and KYN (5.1–19 nM).     

Posttranslational oxidative modification of (R)‐2‐(2,4‐dichlorophenoxy)propionate/α‐ketoglutarate‐dependent dioxygenases (RdpA) leads to improved degradation of 2,4‐dichlorophenoxyacetate (2,4‐D)

Microbial activities and the versatility gained through adaptation to xenobiotic compounds are the main biological forces to counteract environmental pollution. The current results present a new adaptive mechanism that is mediated through posttranslational modifications. Strains of Delftia acidovorans incapable of growing autochthonously on 2,4‐dichlorophenoxyacetate (2,4‐D) were cultivated in a chemostat on 2,4‐D in the presence of (R)‐2‐(2,4‐dichlorophenoxy)propionate. Long‐term cultivation led to enhanced 2,4‐D degradation, as demonstrated by improved values of the Michaelis–Menten constant K_m for 2,4‐D and the catalytic efficiency k_cat/K_m of the initial degradative key enzyme (R)‐2‐(2,4‐dichlorophenoxy)propionate/α‐ketoglutarate‐dependent dioxygenases (RdpA). Analyses of the rdpA gene did not reveal any mutations, indicating a nongenetic mechanism of adaptation. 2‐DE of enzyme preparations, however, showed a series of RdpA forms varying in their pI. During adaptation increased numbers of RdpA variants were observed. Subsequent immunoassays of the RdpA variants showed a specific reaction with 2,4‐dinitrophenylhydrazine (DNPH), characteristic of carbonylation modifications. Together these results indicate that posttranslational carbonylation modified the substrate specificity of RdpA. A model was implemented explaining the segregation of clones with improved degradative activity within the chemostat. The process described is capable of quickly responding to environmental conditions by reversibly adapting the degradative potential to various phenoxyalkanoate herbicides.     

Prokaryotic expression and antimicrobial mechanism of XPF‐St7‐derived α‐helical peptides

XPF‐St7 (GLLSNVAGLLKQFAKGGVNAVLNPK) is an antimicrobial peptide isolated from Silurana tropicalis. We developed an α‐helical segment of XPF‐St7 termed as XPF2. Using the XPF2 as a framework, we increased the positive net charge of XPF2 by amino acid substitutions, and thus obtained two novel antimicrobial peptides XPF4 and XPF6. These were each fused with an ubiquitin tag and successfully expressed in Escherichia coli. This ubiquitin fusion system may present a viable alternative for industrial production of antimicrobial peptides. XPF4 and XPF6 showed much better overall antimicrobial activity against both Gram‐negative and Gram‐positive bacteria than XPF2. The therapeutic index of XPF4 and XPF6 was 5.6‐fold and 6.7‐fold of XPF2, respectively. Bacterial cell membrane permeabilization and genomic DNA interaction assays were utilized to explore the mechanism of action of XPF serial peptides. The results revealed that the target of these antimicrobial peptides was the bacterial cytoplasmic membrane. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Selective recognition of thymidine homopolymer (poly T) oligonucleotide with cobalt(II)–4‐[(5‐chloro‐2‐pyridyl)azo]‐1,3‐diaminobenzene complex

The interactions of cobalt(II)–4‐[(5‐chloro‐2‐pyridyl)azo]‐1,3‐diaminobenzene (5‐Cl‐PADAB) complex with different kinds of homopolymer oligonucleotides in basic medium were investigated based on the measurements of resonance light scattering, UV–vis, circular dichroism spectra and dark field light‐scattering imaging. Experiments showed that only thymidine homopolymer (poly T) oligonucleotides with the length in the range of poly T₆ to poly T₁₈ could interact with the Co(II)–5‐Cl‐PADAB complex in alkaline conditions and cause evident color and spectral change. Thus, the binary complex of Co(II)–5‐Cl‐PADAB could be employed as a visual probe for selectively recognizing the poly T oligonucleotides. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of a novel fluorescent probe based on 7‐nitrobenzo‐2‐oxa‐1,3‐diazole skeleton for the rapid determination of vitamin B12 in pharmaceuticals

A new fluorescent probe, 4‐N,N‐di(2‐hydroxyethyl)imino‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (HINBD) was synthesized in a single step with reasonably good yield. The water‐soluble HINBD emits strongly in the visible region (λ_ex = 479 nm, λ_em = 545 nm) and is stable over a wide range of pH values. It was found that vitamin B₁₂ (VB₁₂) had the ability to quench the fluorescence of HINBD, and the quenched fluorescence intensity was proportional to the concentration of VB₁₂. A method for VB₁₂ determination based on the quenching fluorescence of HINBD was thus established. Interference effects of various substances, including sugars, vitamins, amino acids, inorganic cations and some organic substances have been studied. Under optimal conditions, the linear range is 0.0–2.4 × 10^–5 mol/L. The determination limit is 8.3 × 10^–8 mol/L. The method was applied to measure VB₁₂ in pharmaceutical preparations with satisfactory results. Copyright © 2013 John Wiley & Sons, Ltd.     

Synthesis and Biological Evaluation of Novel 1‐(4‐(Hydroxy(1‐oxo‐1,3‐dihydro‐2H‐inden‐2‐ylidene)methyl)phenyl)‐3‐phenylurea Derivatives

A series of novel phenylurea containing 2‐benzoylindan‐1‐one derivatives 3a  –  3j were synthesized from the reaction of phenylurea‐substituted acetophenones 1a  –  1j with phthalaldehyde 2 under mild reaction conditions in good yields. All synthesized compounds were characterized by spectroscopic methods. The obtained compounds ( 3a  –  3j ) were evaluated for anticancer activity against HeLa and C6 cell lines. Antiproliferative activity was determined by the BrdU proliferation ELISA assay, 3f and 3g were found to be most active compounds. The compounds were also screened for antimicrobial activity and all compounds showed remarkable activity against used microorganisms.     

Fluorescence enhancement of europium (III) perchlorate by 1,10‐phenanthroline on the 1‐(naphthalen‐2‐yl)‐2‐(phenylsulthio)ethanone complex and luminescence mechanism

A novel ligand, 1‐(naphthalen‐2‐yl)‐2‐(phenylsulthio)ethanone was synthesized using a new method and its two europium (Eu) (III) complexes were synthesized. The compounds were characterized by elemental analysis, coordination titration analysis, molar conductivity, infrared, thermo gravimetric analyzer‐differential scanning calorimetry (TGA‐DSC), ¹H NMR and UV spectra. The composition was suggested as EuL₅ · (ClO₄)₃ · 2H₂O and EuL₄ · phen(ClO₄)₃ · 2H₂O (L = C₁₀H₇COCH₂SOC₆H₅). The fluorescence spectra showed that the Eu(III) displayed strong characteristic metal‐centered fluorescence in the solid state. The ternary rare earth complex showed stronger fluorescence intensity than the binary rare earth complex in such material. The strongest characteristic fluorescence emission intensity of the ternary system was 1.49 times as strong as that of the binary system. The phosphorescence spectra were also discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Plasmid‐free T7‐based Escherichia coli expression systems

In order to release host cells from plasmid‐mediated increases in metabolic load and high gene dosages, we developed a plasmid‐free, T7‐based E. coli expression system in which the target gene is site‐specifically integrated into the genome of the host. With this system, plasmid‐loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid‐free system was proven in chemostat cultivation for 40 generations in a non‐induced and for 10 generations in a fully induced state. For this reason plasmid‐free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid‐free systems in upstream‐processing. Biotechnol. Bioeng. 2010. 105: 786–794. © 2009 Wiley Periodicals, Inc.     

The special optical properties and application of the heterocyclic compound diethyl 6‐anilino‐5H‐2,3‐dithia‐5,7‐diazacyclopenta(cd)indene‐1,4‐dicarboxylate

The heterocyclic compound diethyl 6‐anilino‐5H‐2,3‐dithia‐5,7‐diazacyclopenta(cd)indene‐1,4‐dicarboxylate (D1) was found to form highly emissive aggregates in polar solvents, and the aggregate emission can be tuned by the simple addition of water to a dimethylsulfoxide solution. A theoretical study based on Density functional theory (DFT) calculations, shows that intermolecular interactions of D1 with solvent may be potential factors in the fluorescence change. In addition, the phenyl ring in D1 plays an important role because of its response to solvent. In the non‐aggregated state, deprotonation of the N–H of D1 can proceed easily on the addition of base, and the deprotonated compound might interact with Ag⁺, resulting in a significant change in color and fluorescence quenching, which make it a potential chemosensor for the selective detection of trace amounts of Ag⁺. Copyright © 2013 John Wiley & Sons, Ltd.