Rational tuning of the thiolate donor in model complexes of superoxide reductase: direct evidence for a trans influence in Fe(III)-OOR complexes

Iron peroxide species have been identified as important intermediates in a number of nonheme iron as well as heme-containing enzymes, yet there are only a few examples of such species either synthetic or biological that have been well characterized. We describe the synthesis and structural characterization of a new series of five-coordinate (N4S(thiolate))Fe(II) complexes that react with tert-butyl hydroperoxide ((t)BuOOH) or cumenyl hydroperoxide (CmOOH) to give metastable alkylperoxo-iron(III) species (N4S(thiolate)Fe(III)-OOR) at low temperature. These complexes were designed specifically to mimic the nonheme iron active site of superoxide reductase, which contains a five-coordinate iron(II) center bound by one Cys and four His residues in the active form of the protein. The structures of the Fe(II) complexes are analyzed by X-ray crystallography, and their electrochemical properties are assessed by cyclic voltammetry. For the Fe(III)-OOR species, low-temperature UV-vis spectra reveal intense peaks between 500-550 nm that are typical of peroxide to iron(III) ligand-to-metal charge-transfer (LMCT) transitions, and EPR spectroscopy shows that these alkylperoxo species are all low-spin iron(III) complexes. Identification of the vibrational modes of the Fe(III)-OOR unit comes from resonance Raman (RR) spectroscopy, which shows nu(Fe-O) modes between 600-635 cm(-1) and nu(O-O) bands near 800 cm(-1). These Fe-O stretching frequencies are significantly lower than those found in other low-spin Fe(III)-OOR complexes. Trends in the data conclusively show that this weakening of the Fe-O bond arises from a trans influence of the thiolate donor, and density functional theory (DFT) calculations support these findings. These results suggest a role for the cysteine ligand in SOR, and are discussed in light of the recent assessments of the function of the cysteine ligand in this enzyme.     

Separation of amino acid enantiomers VIA chiral derivatization and non-chiral gas chromatography

Two GC-MS methods for the enantioselective separation of the 20 proteinogenic amino acids are compared. Ethyl chloroformate and 2-chloropropanol were used to derivatize amino acid enantiomers. The diastereomers formed were separated on a non-chiral column by capillary gas chromatography. The separation performances were compared to those obtained when using non-chiral derivatization on a chiral column.     

Performance characteristics of an ion chromatographic method for the quantitation of citrate and phosphate in pharmaceutical solutions

The performance of an ion chromatographic method for measuring citrate and phosphate in pharmaceutical solutions is evaluated. Performance characteristics examined include accuracy, precision, specificity, response linearity, robustness, and the ability to meet system suitability criteria. In general, the method is found to be robust within reasonable deviations from its specified operating conditions. Analytical accuracy is typically 100 +/- 3%, and short-term precision is not more than 1.5% relative standard deviation. The instrument response is linear over a range of 50% to 150% of the standard preparation target concentrations (12 mg/L for phosphate and 20 mg/L for citrate), and the results obtained using a single-point standard versus a calibration curve are essentially equivalent. A small analytical bias is observed and ascribed to the relative purity of the differing salts, used as raw materials in tested finished products and as reference standards in the analytical method. The assay is specific in that no phosphate or citrate peaks are observed in a variety of method-related solutions and matrix blanks (with and without autoclaving). The assay with manual preparation of the eluents is sensitive to the composition of the eluent in the sense that the eluent must be effectively degassed and protected from CO(2) ingress during use. In order for the assay to perform effectively, extensive system equilibration and conditioning is required. However, a properly conditioned and equilibrated system can be used to test a number of samples via chromatographic runs that include many (> 50) injections.     

Diversification of catalytic function in a synthetic family of chimeric cytochrome p450s

We report initial characterization of a synthetic family of more than 3000 cytochrome P450s made by SCHEMA recombination of 3 bacterial CYP102s. A total of 16 heme domains and their holoenzyme fusions with each of the 3 parental reductase domains were tested for activity on 11 different substrates. The results show that the chimeric enzymes have acquired significant functional diversity, including the ability to accept substrates not accepted by the parent enzymes. K-means clustering analysis of the activity data allowed the enzymes to be classified into five distinct groups based on substrate specificity. The substrates can also be grouped such that one can be a "surrogate" for others in the group. Fusion of a functional chimeric heme domain with a parental reductase domain always reconstituted a functional holoenzyme, indicating that key interdomain interactions are conserved upon reductase swapping.     

Effect of Gel Structure on the In Vitro Gastrointestinal Digestion Behaviour of Whey Protein Emulsion Gels and the Bioaccessibility of Capsaicinoids

This study investigated the effect of gel structure on the digestion of heat-set whey protein emulsion gels containing capsaicinoids (CAP), including the bioaccessibility of CAP. Upon heat treatment at 90 °C, whey protein emulsion gels containing CAP (10 wt% whey protein isolate, 20 wt% soybean oil, 0.02 wt% CAP) with different structures and gel mechanical strengths were formed by varying ionic strength. The hard gel (i.e., oil droplet size d_4,3 ~ 0.5 μm, 200 mM NaCl), with compact particulate gel structure, led to slower disintegration of the gel particles and slower hydrolysis of the whey proteins during gastric digestion compared with the soft gel (i.e., d_4,3 ~ 0.5 μm, 10 mM NaCl). The oil droplets started to coalesce after 60 min of gastric digestion in the soft gel, whereas minor oil droplet coalescence was observed for the hard gel at the end of the gastric digestion. In general, during intestinal digestion, the gastric digesta from the hard gel was disintegrated more slowly than that from the soft gel. A power-law fit between the bioaccessibility of CAP (Y) and the extent of lipid digestion (X) was established: Y = 49.2 × (X − 305.3)^0.104, with R² = 0.84. A greater extent of lipid digestion would lead to greater release of CAP from the food matrix; also, more lipolytic products would be produced and would participate in micelle formation, which would help to solubilize the released CAP and therefore result in their higher bioaccessibility.     

Are the reduction and oxidation properties of nitrocompounds dissolved in water different from those produced when adsorbed on a silica surface? A DFT M05‐2X computational study

The reduction and oxidation properties of four nitrocompounds (trinitrotoluene [TNT], 2,4‐dinitrotoluene, 2,4‐dinitroanisole, and 5‐nitro‐2,4‐dihydro‐3H‐1,2,4‐triazol‐3‐one [NTO]) dissolved in water as compared with the same properties for compounds adsorbed on a silica surface were studied. To consider the influence of adsorption, cluster models were developed at the M05/tzvp level. A hydroxylated silica (001) surface was chosen to represent a key component of soil. The PCM(Pauling) and SMD solvation models were used to model water bulk influence. The following properties were analyzed: electron affinity, ionization potential, reduction Gibbs free energy, oxidation Gibbs free energy, and reduction and oxidation potentials. It was found that adsorption and solvation decrease gas phase electron affinity, ionization potential, and Gibbs free energy of reduction and oxidation, and thus, promote redox transformation of nitrocompounds. However, in case of solvation, the changes are more significant than for adsorption. This means that nitrocompounds dissolved in water are easier to transform by reduction or oxidation than adsorbed ones. Among the considered compounds, TNT was found to be the most reactive in an electron attachment process and the least reactive for an electron detachment transformation. During ionization, a deprotonation of adsorbed NTO was found to occur. © 2015 Wiley Periodicals, Inc.     

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands

We describe a general synthetic strategy for developing high‐affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full‐length protein to identify the best binder. We describe development of epitope‐targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies.     

Selective Enzymatic Oxidation of Silanes to Silanols

Compared to the biological world's rich chemistry for functionalizing carbon, enzymatic transformations of the heavier homologue silicon are rare. We report that a wild-type cytochrome P450 monooxygenase (P450_BM3 from Bacillus megaterium, CYP102A1) has promiscuous activity for oxidation of hydrosilanes to give silanols. Directed evolution was applied to enhance this non-native activity and create a highly efficient catalyst for selective silane oxidation under mild conditions with oxygen as the terminal oxidant. The evolved enzyme leaves C−H bonds present in the silane substrates untouched, and this biotransformation does not lead to disiloxane formation, a common problem in silanol syntheses. Computational studies reveal that catalysis proceeds through hydrogen atom abstraction followed by radical rebound, as observed in the native C−H hydroxylation mechanism of the P450 enzyme. This enzymatic silane oxidation extends nature's impressive catalytic repertoire.     

Student and Teacher Perspectives Across Mathematics and Science Classrooms: The Importance of Engaging Contexts

Substantial recent focus has been placed upon the competitiveness of American students in increasingly global economies and entrepreneurial enterprises. As concerns center on students’ educational preparedness and their efforts at continued learning, researchers acknowledge the importance of student engagement with school. In order to foster engaged learners, teachers must be able to determine and monitor their students’ levels of engagement. The current study examined the alignment of perceptions of engagement by students, teachers, and outside observers across middle and high school mathematics and science classrooms. Results indicated significant teacher‐student differences in perceptions of student cognitive engagement across mathematics and science classrooms with teachers consistently perceiving higher levels than students. Moreover, most effect sizes were moderate to large. A subsequent multi‐level analysis indicated that while teacher perceptions of student cognitive engagement were somewhat predictive of student reported cognitive engagement, academic engagement ratings by outside observers were not.