Structures of rhenium(I) complexes with 3-hydroxyflavone and benzhydroxamic acid as O,O′-bidentate ligands and confirmation of π-stacking by solid-state NMR spectroscopy

The synthesis and crystal structures of two new rhenium(I) complexes obtained utilizing benzhydroxamic acid (BHAH) and 3-hydroxyflavone (2-phenylchromen-4-one, FlavH) as bidentate ligands, namely tetraethylammonium fac-(benzhydroxamato-κ2 O,O′)bromidotricarbonylrhenate(I), (C8H20N)[ReBr(C7H6NO2)(CO)3], 1, and fac-aquatricarbonyl(4-oxo-2-phenylchromen-3-olato-κ2 O,O′)rhenium(I)–3-hydroxyflavone (1/1), [Re(C15H9O3)(CO)3(H2O)]·C15H10O3, 3, are reported. Furthermore, the crystal structure of free 3-hydroxyflavone, C15H10O3, 4, was redetermined at 100 K in order to compare the packing trends and solid-state NMR spectroscopy with that of the solvate flavone molecule in 3. The compounds were characterized in solution by 1H and 13C NMR spectroscopy, and in the solid state by 13C NMR spectroscopy using the cross-polarization magic angle spinning (CP/MAS) technique. Compounds 1 and 3 both crystallize in the triclinic space group P\overline{1} with one molecule in the asymmetric unit, while 4 crystallizes in the orthorhombic space group P212121. Molecules of 1 and 3 generate one-dimensional chains formed through intermolecular interactions. A comparison of the coordinated 3-hydroxyflavone ligand with the uncoordinated solvate molecule and free molecule 4 shows that the last two are virtually completely planar due to hydrogen-bonding interactions, as opposed to the former, which is able to rotate more freely. The differences between the solid- and solution-state 13C NMR spectra of 3 and 4 are ascribed to inter- and intramolecular interactions. The study also investigated the potential labelling of both bidentate ligands with the corresponding fac-99mTc-tricarbonyl synthon. All attempts were unsuccessful and reasons for this are provided.

Use of Bispecific Antibodies in Molecular Velcro Assays Whose Specificity Approaches the Theoretical Limit of Immunodetection for Bordetella pertussis

ABSTRACT A bispecific monoclonal antibody (bsMAb) that detects Bordetella pertussis, the causative agent of whooping cough, and horseradish peroxidase (HRPO) has been developed by use of the quadroma technology. A quadroma, P123, was produced by fusing two well-characterized hybridomas against the bacterium and the enzyme and was subcloned to obtain a stable bsMAb-secreting cell line. The quadroma was theoretically expected to produce up to 10 different molecular species of immunoglobulins, so secreted bispecific antibody was complexed with excess HRPO and the HRPO-bsMAb complex was purified in one step by benzhydroxamic acid-agarose affinity cochromatography. An ultrasensitive homosandwich molecular “velcro” enzyme-linked immunosorbent assay for the detection of B. pertussis whole bacteria with HRPO-bsMAb was established in both microplate and nasopharyngeal swab formats. This assay demonstrates a high sensitivity that approaches the theoretical limit of detection of one bacterium. This new nanoprobe can be used to develop a new generation of assays that are simple, inexpensive alternatives to quantitative PCR and that can be used by clinical laboratories. This strategy of homosandwich assays with solid-phase monospecific antibodies and solution-phase bsMAb with specificity for the same repeating surface determinants can be applied to generate ultrasensitive immunodiagnostic assays for viruses and bacteria.

Temperature-dependent alterations of respiration in leaves of two selected vascular plant species: the role of the alternative oxidase pathway

Effects of higher temperatures on respiration and activity of alternative oxidase (AOX) were studied in mature leaves of Ajuga reptans L. and Rhodiola rosea L. Total respiration in both A. reptans and R. rosea increased exponentially with the increasing temperature of 10 °C to 35 °C. Respiration in the presence of benzhydroxamic acid (BHAM) also increased exponentially in accordance with the increasing temperature in the leaves of both A. reptans and R. rosea. Relative activity of the alternative pathway decreased significantly in the leaves of A. reptans with increasing temperatures. However, an increase in the relative activity of the alternative pathway was detected in the leaves of R. rosea. Thermoresistance of the alternative respiratory pathway was considered to be higher in R. rosea compared to A. reptans. We suppose that A. reptans and R. rosea have different mechanisms regulating partitioning of electrons to the alternative respiratory pathway.

Haem-linked interactions in horseradish peroxidase revealed by spectroscopic analysis of the Phe-221→Met mutant

A gene encoding a Phe-221-to-Met substitution in the haem enzyme horseradish peroxidase has been constructed and expressed in Escherichia coli. In the wild-type enzyme the side chain of Phe-221 is tightly stacked against the imidazole ring of His-170, which provides the only axial ligand to the haem iron atom. The Phe-221 → Met enzyme is active, and forms characteristic complexes with typical peroxidase ligands (CO, cyanide, fluoride), and with benzhydroxamic acid. Significant differences between the mutant and wild-type enzymes can be detected spectroscopically. These include a change in the Fe(III) resting state of the enzyme to an unusual quantum mechanically mixed-spin haem species, a marked decrease in the pKa of the alkaline transition and a reduction in enzyme stability at alkaline pH for both Fe(III) and Fe(II) forms. The perturbation of the haem pocket in the mutant can be attributed to several factors, including the increased steric freedom and solvent accessibility of the His-170 ligand, as indicated by 1H-NMR data, and the loss of the πŐπ interaction between His-170 and Phe-221.