Importance of the ß5-ß6 Loop for the Structure, Catalytic Efficiency, and Stability of Carbapenem-Hydrolyzing Class D ß-Lactamase Subfamily OXA-143.

The class D ß-lactamase OXA-143 has been described as an efficient penicillinase, oxacillinase, and carbapenemase. The D224A variant, known as OXA-231, was described in 2012 as exhibiting less activity toward imipenem and increased oxacillinase activity. Additionally, the P227S mutation was reported as a case of convergent evolution for homologous enzymes. To investigate the impact of both mutations (D224A and P227S), we describe in this paper a deep investigation of the enzymatic activities of these three homologues. OXA-143(P227S) presented enhanced catalytic activity against ampicillin, oxacillins, aztreonam, and carbapenems. In addition, OXA-143(P227S) was the only member capable of hydrolyzing ceftazidime. These enhanced activities were due to a combination of a higher affinity (lower Km) and a higher turnover number (higher kcat). We also determined the crystal structure of apo OXA-231. As expected, the structure of this variant is very similar to the published OXA-143 structure, except for the two M223 conformations and the absence of electron density for three solvent-exposed loop segments. Molecular dynamics calculations showed that both mutants experience higher flexibility compared to that of the wild-type form. Therefore, our results illustrate that D224A and P227S act as deleterious and positive mutations, respectively, within the evolutionary path of the OXA-143 subfamily toward a more efficient carbapenemase.

Dealing with supramolecular structure for ionic liquids: a DOSY NMR approach.

Diffusion-ordered spectroscopy (DOSY) is arguably a powerful method for the NMR analysis of ionic liquids, since the self-diffusion coefficients for cations and anions can be measured straightforwardly. In this work, the dynamic-structural behaviour of imidazolium ionic liquids containing different anions has been investigated by experimental measurements of direct 1H diffusion coefficients in chloroform and water solutions. The influence of ion structure has been tested by using six IL salts formed by the association of different cations (1-n-butyl-3-methylimidazolium, 1,2,3-trimethylimidazolium and tetra-n-butylammonium) with different anion structures (prolinate, acetate and o-trifluoromehtylobenzoate). The influence of IL concentration (from 0.01 to 0.5 mol L-1) was also evaluated for BMI·Pro. The contact ion pairs (or aggregates) are maintained in both chloroform and water within the range of concentrations investigated. In the particular case of 1,2,3-trimethylimidazolium imidazolate (TMI·Im) containing confined water in DMSO the maintenance of the contact ion pairs depends on the water content which may even disrupt the IL supramolecular structure.

19 F NMR matrix-assisted DOSY: a versatile tool for differentiating fluorinated species in mixtures.

NMR is the most versatile tool for the analysis of organic compounds and, in combination with Diffusion-Ordered Spectroscopy ('DOSY'), can give information on compounds in complex mixtures without the need for physical separation. In mixtures where the components' diffusion coefficients are nearly identical, for example because of similar sizes, Matrix-Assisted DOSY ('MAD') can help separate the signals of different constituents, resolving their spectra. Unfortunately, DOSY (including MAD) typically fails where signals overlap, as is common in 1 H NMR. Using 19 F NMR avoids such problems, because the great sensitivity of the 19 F chemical shift to local environment leads to very well-dispersed spectra. Another advantage is the absence of any 19 F background signals from the matrices typically used, avoiding interference with the analyte signals. In this study, differentiation among fluorophenol and fluoroaniline isomers was evaluated using normal and reverse micelles-of sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB) and dioctyl sodium sulfosuccinate (AOT)-as matrices. These surfactants provide useful diffusion separation in these difficult mixtures, with all the solutes interacting with the matrices to different extents, in some cases leading to differences in diffusion coefficient of more than 30%. The best matrices for separating the signals of both acid and basic species were shown to be AOT and CTAB, which are useful over a wide range of surfactant concentration. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluation of the cytotoxicity on breast cancer cell of extracts and compounds isolated from Hyptis pectinata (L.) poit.

Hyptis pectinata is a herb popularly used in Brazil for the treatment of inflammations, pain, bacterial infections and cancer. In the present study, inflorescences (MPIn), leaves (MPL), branches (MPB), root (MPR) extracts and three compounds isolated from MPIn were assayed against breast tumor cell lines. The structures of the three compounds (pectinolide J, hyptolide and pectinolide E) were determined by means of spectroscopic analysis. Pectinolide J was isolated for the first time. The MPIn, MPL and MPR exhibited specific antiproliferative activity on tumor cell lines when compared to normal cell lines with IC50 of 52.01 ± 0.64, 45.91 ± 0.02 µg/mL and 82.84 ± 0.03 µg/mL, respectively. Although the isolated substances did not present good antiproliferative activity, when the three were associated, a greater biological effect was observed, suggesting a synergistic effect. Hyptolide (5.6 ± 0.4 µg/mL) showed IC50 sufficiently low to be considered as a drug prototype.