Analysis of genomic methylation level using micellar electrokinetic chromatography with UV detection

Analytical methods for quantification of 5′‐methylcytosine in genomes are important tools to investigate epigenetic changes in gene expression during development, differentiation, aging, or cancer. Here, we report a novel genomic methylation content assay based on enzymatic hydrolysis of DNA and MEKC separation of 5′‐deoxyribonucleoside monophosphates (dNMP) using the cationic surfactant CTAB as pseudostationary phase. Calf Thymus DNA was used during method development to determine electrophoretic parameters and electrolyte composition for a complete separation between 2′‐deoxycytosine‐5′‐monophosphate and 2′‐deoxy‐5′‐methylcytosine 5′‐monophosphate (d5mCMP). Methylated and not methylated oligonucleotides were used to confirm the identity of each peak and evaluate analytical parameters of the method. The LOD of the method was found to be 12.5 pmol/μL for d5mCMP.     

Analysis of veterinary drug residues in cheese by ultra‐high‐performance LC coupled to triple quadrupole MS/MS

A simple, reliable, and fast multiresidue method has been developed for the determination of 17 veterinary drugs belonging to several families (macrolides, sulfonamides, and anthelmintics) in cheese at trace levels. Ultra‐high‐performance LC coupled to MS/MS has been used for the analysis of these compounds in less than 9 min. Veterinary drug residues have been extracted from cheese samples using a QuEChERS (quick, easy, cheap, effective, rugged, and safe)‐based extraction procedure without applying any further clean‐up step. Matrix‐matched calibration was used for quantification and recoveries were calculated at three concentration levels (10, 50, and 100 μg/kg). The obtained values ranged from 70 to 110% for the selected compounds except for tylosin and josamycin at 100 μg/kg (111.7 and 112.7%, respectively). Intra‐ and interday precision were also evaluated and RSDs were lower than 25% in all the cases. LOQs ranged from 0.3 μg/kg (for thiabendazole, oxfendazole, mebendazole, josamycin, and fenbendazole) to 10.5 μg/kg (abamectin), whereas decision limit and detection capability ranged from 2.3 (thiabendazole) to 11.3 (abamectin) and 4.2 (thiabendazole) to 14.3 μg/kg (abamectin), respectively. Finally, 13 samples were analyzed and traces of thiabendazole were detected in two different cheeses.     

Optimization of microwave‐assisted extraction of analgesic and anti‐inflammatory drugs from human plasma and urine using response surface experimental designs†

The performance of microwave‐assisted extraction and HPLC with photodiode array detection method for determination of six analgesic and anti‐inflammatory drugs from plasma and urine, is described, optimized, and validated. Several parameters affecting the extraction technique were optimized using experimental designs. A four‐factor (temperature, phosphate buffer pH 4.0 volume, extraction solvent volume, and time) hybrid experimental design was used for extraction optimization in plasma, and three‐factor (temperature, extraction solvent volume, and time) Doehlert design was chosen to extraction optimization in urine. The use of desirability functions revealed the optimal extraction conditions as follows: 67°C, 4 mL phosphate buffer pH 4.0, 12 mL of ethyl acetate and 9 min, for plasma and the same volume of buffer and ethyl acetate, 115°C and 4 min for urine. Limits of detection ranged from 4 to 45 ng/mL in plasma and from 8 to 85 ng/mL in urine. The reproducibility evaluated at two concentration levels was less than 6.5% for both specimens. The recoveries were from 89 to 99% for plasma and from 83 to 99% for urine. The proposed method was successfully applied in plasma and urine samples obtained from analgesic users.     

Cycloalkane and alicyclic heterocycle complexation by new switchable resorcin[4]arene-based container molecules: NMR and ITC binding studies

The synthesis and structural characterization of novel, "molecular basket"-type bridged cavitands is reported. The resorcin[4]arene-based container molecules feature well-defined cavities that bind a wide variety of cycloalkanes and alicyclic heterocycles. Association constants (K(a)) of the 1:1 inclusion complexes were determined by both (1)H NMR and isothermal titration calorimetry (ITC). The obtained K(a) values in mesitylene ranged from 1.7×10(2) M(-1) for cycloheptane up to 1.7×10(7) M(-1) for morpholine. Host-guest complexation by the molecular baskets is generally driven by dispersion interactions, C-H···π interactions of the guests with the aromatic walls of the cavity, and optimal cavity filling. Correlations between NMR-based structural data and binding affinities support that the complexed heterocyclic guests undergo additional polar C-O···C=O, N-H···π, and S···π interactions. The first crystal structure of a cavitand-based molecular basket is reported, providing precise information on the geometry and volume of the inner cavity in the solid state. Molecular dynamic (MD) simulations provided information on the size and conformational preorganization of the cavity in the presence of encapsulated guests. The strongest binding of heterocyclic guests, engaging in polar interactions with the host, was observed at a cavity filling volume of 63 ± 9%.     

Unprecedented 1,3-diaza[3]ferrocenophane scaffold as molecular probe for anions

The guanidine unit in the guise of 2-aminoimidazole in the new structural motif 2-arylamino-1,3-diaza[3]ferrocenophane 4 acts as a binding site for anions. The electrochemical behavior of this compound has been studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) and was found to exhibit a quasi reversible oxidation peak, associated to the Fe(II)/Fe(III) redox couple (Ep = 440 mV), and a non-reversible oxidation wave (Ep = 817 mV), probably associated to the oxidation of the C═N unit present in the guanidine bridge. Recognition of AcO(-), PhCO(2)(-), F(-), Cl(-), and Br(-) anions by the free receptor and the less basic anions Br(-), Cl(-), and NO(3)(-) by its monoprotonated form takes place by unusual redox-ratiometric measurements and spectroscopic ((1)H NMR and UV-vis) changes.     

The phase coexistence method to obtain surface free energies and nucleation barriers: a brief review

ABSTRACT

A recently developed method where one analyses the finite size effects associated with liquid–solid phase equilibria including vapour–crystal coexistence is briefly reviewed. It is shown that the estimation of the chemical potential of the vapour surrounding the crystal as function of the crystal volume yields information on the bulk coexistence conditions, when an extrapolation to the thermodynamic limit is performed. Estimating the pressure of the fluid surrounding the crystal nucleus in the finite simulation box and the volume of this nucleus that coexists with the fluid in thermal equilibrium, an estimate for the total surface excess free energy can be obtained, which to a very good approximation is independent of the size of the simulation box. The free energy barrier against homogeneous nucleation of crystals thus can be estimated as a function of the nucleus volume. Monte Carlo simulations for the soft effective Asakura–Oosawa model of colloid-polymer mixtures which form face-centered cubic colloidal crystals are used to exemplify this method, computing the surface excess free energy of these crystals over a wide range of crystal volumes, without the need to characterise the non-spherical crystal shape. A possible extension of these concepts to heterogeneous nucleation is also briefly discussed.     

Synthesis,Chiral Resolution and Enantiomers Absolute Configuration of 4-Nitropropranolol and 7-Nitropropranolol

We recently identified 6-nitrodopamine and other nitro-catecholamines (6-nitrodopa, 6-nitroadrenaline), indicating that the endothelium has the ability to nitrate the classical catecholamines (dopamine, noradrenaline, and adrenaline). In order to investigate whether drugs could be subject to the same nitration process, we synthesized 4-nitro- and 7-nitropropranolol as probes to evaluate the possible nitration of the propranolol by the endothelium. The separation of the enantiomers in very high yields and excellent enantiopurity was achieved by chiral HPLC. Finally, we used Riguera’s method to determine the absolute configuration of the enantiomers, through double derivatization with MPA and NMR studies.     

Development of Red-Shifted and Fluorogenic Nucleoside and Oligonucleotide Diarylethene Photoswitches

The reversible modulation of fluorescence signals by light is of high interest for applications in super-resolution microscopy, especially on the DNA level. In this article we describe the systematic variation of the core structure in nucleoside-based diarylethenes (DAEs), in order to generate intrinsically fluorescent photochromes. The introduction of aromatic bridging units resulted in a bathochromic shift of the visible absorption maximum of the closed-ring form, but caused reduced thermal stability and switching efficiency. The replacement of the thiophene aryl unit by thiazol improved the thermal stability, whereas the introduction of a benzothiophene unit led to inherent and modulatable turn-off fluorescence. This feature was further optimized by introducing a fluorescent indole nucleobase into the DAE core, resulting in an effective photoswitch with a fluorescence quantum yield of 0.0166 and a fluorescence turn-off factor of 3.2. The site-specific incorporation into an oligonucleotide resulted in fluorescence-switchable DNA with high cyclization quantum yields and switching efficiency, which may facilitate future applications.     

Probing α-Amino Aldehydes as Weakly Acidic Pronucleophiles: Direct Access to Quaternary α-Amino Aldehydes by an Enantioselective Michael Addition Catalyzed by Brønsted Bases

The high tendency of α-amino aldehydes to undergo 1,2-additions and their relatively low stability under basic conditions have largely prevented their use as pronucleophiles in the realm of asymmetric catalysis, particularly for the production of quaternary α-amino aldehydes. Herein, it is demonstrated that the chemistry of α-amino aldehydes may be expanded beyond these limits by documenting the first direct α-alkylation of α-branched α-amino aldehydes with nitroolefins. The reaction produces densely functionalized products bearing up to two, quaternary and tertiary, vicinal stereocenters with high diastereo- and enantioselectivity. DFT modeling leads to the proposal that intramolecular hydrogen bonding between the NH group and the carbonyl oxygen atom in the starting α-amino aldehyde is key for reaction stereocontrol.     

Persicamidines—Unprecedented Sesquarterpenoids with Potent Antiviral Bioactivity against Coronaviruses

A new family of highly unusual sesquarterpenoids (persicamidines A–E) exhibiting significant antiviral activity was isolated from a newly discovered actinobacterial strain, Kibdelosporangium persicum sp. nov., collected from a hot desert in Iran. Extensive NMR analysis unraveled a hexacyclic terpenoid molecule with a modified sugar moiety on one side and a highly unusual isourea moiety fused to the terpenoid structure. The structures of the five analogues differed only in the aminoalkyl side chain attached to the isourea moiety. Persicamidines A–E showed potent activity against hCoV-229E and SARS-CoV-2 viruses in the nanomolar range together with very good selectivity indices, making persicamidines promising as starting points for drug development.