Browsing by Author "Department of Chemistry and Chemical Biology, School of Science"
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Item DNA Cleaving "Tandem-Array" Metallopeptides Activated With KHSO5: Towards the Development of Multi-Metallated Bioactive Conjugates and Compounds(Bentham Science, 2014) Lewis, Mark A.; Williams, Katie M.; Fang, Ya-Yin; Schultz, Franklin A.; Long, Eric C.; Department of Chemistry and Chemical Biology, School of ScienceAmino terminal peptides of the general form Gly-Gly-His have been used to introduce single sites of metal binding and redox activity into a wide range of biomolecules to create bioactive compounds and conjugates capable of substrate oxidation. We report here that Gly-Gly-His-like peptides linked in a tandem fashion can also be generated leading to multi-metal binding arrays. While metal binding by the native Gly-Gly-His motif (typically to Cu(2+), Ni(2+), or Co(2+)) requires a terminal peptide amine ligand, previous work has demonstrated that an ornithine (Orn) residue can be substituted for the terminal Gly residue to allow solid-phase peptide synthesis to continue via the side chain N-δ. This strategy thus frees the Orn residue N-α for metal binding and permits placement of a Gly-Gly-His-like metal binding domain at any location within a linear, synthetic peptide chain. As we show here, this strategy also permits the assembly of tandem arrays of metal binding units in linear peptides of the form: NH2-Gly-Gly-His-[(δ)-Orn-Gly-His]n-(δ)-Orn-Gly-His-CONH2 (where n = 0, 1, and 2). Metal binding titrations of these tandem arrays monitored by UV-vis and ESI-MS indicated that they bind Cu(2+), Ni(2+), or Co(2+) at each available metal binding site. Further, it was found that these systems retained their ability to modify DNA oxidatively and to an extent greater than their parent M(II)•Gly-Gly-His. These findings suggest that the tandem array metallopeptides described here may function with increased efficiency as "next generation" appendages in the design of bioactive compounds and conjugates.Item Improving the confidence of “questioned versus known” fiber comparisons using microspectrophotometry and chemometrics(Elsevier, 2016-11) Sauzier, Georgina; Reichard, Eric; van Bronswijk, Wilhelm; Lewis, Simon W.; Goodpaster, John V.; Department of Chemistry and Chemical Biology, School of ScienceMicrospectrophotometry followed by chemometric data analysis was conducted on pairs of visually similar blue acrylic fibers, simulating the “questioned versus known” scenarios often encountered in forensic casework. The relative similarity or dissimilarity of each pair was determined by employing principal component analysis, discriminant analysis and Fisher’s exact test. Comparison of fibers from within each set resulted in a correct inclusion result in 10 out of 11 scenarios, with the one false exclusion attributed to a lack of reproducibility in the spectra. Comparison of fibers from different sets resulted in a correct exclusion result in 108 of 110 scenarios, with two sets that shared identical dye combinations being indistinguishable. Although the presented methods are not infallible, they may nonetheless provide a path forward for forensic fiber examiners that has a more scientifically rigorous basis on which to support their findings in a court of law.Item Mapping explosive residues on galvanized pipe bomb fragments using total vaporization solid phase microextraction (TV-SPME)(Royal Society of Chemistry, 2015-12) Bors, Dana; Goodpaster, John V.; Department of Chemistry and Chemical Biology, School of ScienceSolid phase microextraction (SPME) is a popular sampling technique whereby analytes are sorbed to a coated fiber and subsequently desorbed into an analytical instrument. In headspace SPME, analytes partition between the sample, the headspace above the sample, and the SPME fiber coating. In total vaporization SPME (TV-SPME), sample extracts are heated until both the solvent and analytes completely vaporize, whereupon the analytes partition between the vapor phase and the SPME fiber. In this study, TV-SPME using a polyethylene glycol fiber was coupled with fast gas chromatography/mass spectrometry to identify components of double-base smokeless powder (DBSP). Nitroglycerin (NG), diphenylamine (DPA) and ethyl centralite (EC) were separated in under 5 min. For NG, the optimal sample volume (70 μL), extraction temperature (60 °C) and extraction time (20 min) resulted in a method that was over twelve fold more sensitive than traditional liquid injection and with a detection limit below 1 ppb. This method was then used to quantify DBSP residue on post-blast debris from five galvanized steel pipe bombs. The mean concentration of NG on the fragments was 0.25 ppm (w/w). An average of 1.01 mg of NG was recovered from the devices. Finally, the distribution of NG could be “mapped” by tracking the original locations of each fragment within the device. These maps showed that the distribution of NG was far from uniform. In fact, the concentration of the NG on fragments originating from the end caps was several fold higher than in other locations. This finding can help guide the selection of bomb fragments for chemical analyses in real-world scenarios.Item Mechanistic and Structural Insight to an Evolved Benzoylformate Decarboxylase with Enhanced Pyruvate Decarboxylase Activity(MDPI, 2016-12) Andrews, Forest H.; Wechsler, Cindy; Rogers, Megan P.; Meyer, Danilo; Tittmann, Kai; McLeish, Michael J.; Department of Chemistry and Chemical Biology, School of ScienceBenzoylformate decarboxylase (BFDC) and pyruvate decarboxylase (PDC) are thiamin diphosphate-dependent enzymes that share some structural and mechanistic similarities. Both enzymes catalyze the nonoxidative decarboxylation of 2-keto acids, yet differ considerably in their substrate specificity. In particular, the BFDC from P. putida exhibits very limited activity with pyruvate, whereas the PDCs from S. cerevisiae or from Z. mobilis show virtually no activity with benzoylformate (phenylglyoxylate). Previously, saturation mutagenesis was used to generate the BFDC T377L/A460Y variant, which exhibited a greater than 10,000-fold increase in pyruvate/benzoylformate substrate utilization ratio compared to that of wtBFDC. Much of this change could be attributed to an improvement in the Km value for pyruvate and, concomitantly, a decrease in the kcat value for benzoylformate. However, the steady-state data did not provide any details about changes in individual catalytic steps. To gain insight into the changes in conversion rates of pyruvate and benzoylformate to acetaldehyde and benzaldehyde, respectively, by the BFDC T377L/A460Y variant, reaction intermediates of both substrates were analyzed by NMR and microscopic rate constants for the elementary catalytic steps were calculated. Herein we also report the high resolution X-ray structure of the BFDC T377L/A460Y variant, which provides context for the observed changes in substrate specificity.Item Palladium-Catalyzed Decarboxylative Alkoxycarbonylation of Potassium Aryltrifluoroborates with Potassium Oxalate Monoesters(RSC, 2016-02) Miao, Jinmin; Fang, Ping; Jagdeep, Sahota; Ge, Haibo; Department of Chemistry and Chemical Biology, School of SciencePalladium-catalyzed decarboxylative alkoxycarbonylation of potassium aryltrifluoroborates with potassium oxalate monoesters in the presence of potassium persulfate was performed under mild conditions. A number of benzoyl esters with a wide variety of substituents at different positions were efficiently synthesized with this method. Mechanism of the palladium-catalyzed decarboxylative carbonylation of aryltrifluoroborates was studied, and a radical-mediated Pd(II)/Pd(IV) catalytic cycle was proposed.Item Phosphonodifluoropyruvate is a mechanism-based inhibitor of phosphonopyruvate decarboxylase from Bacteroides fragilis(Elsevier, 2017-08) Pallitsch, Katharina; Rogers, Megan P.; Andrews, Forest H.; Hammerschmidt, Friedrich; McLeish, Michael J.; Department of Chemistry and Chemical Biology, School of ScienceBacteroides fragilis, a human pathogen, helps in the formation of intra-abdominal abscesses and is involved in 90% of anaerobic peritoneal infections. Phosphonopyruvate decarboxylase (PnPDC), a thiamin diphosphate (ThDP)-dependent enzyme, plays a key role in the formation of 2-aminoethylphosphonate, a component of the cell wall of B. fragilis. As such PnPDC is a possible target for therapeutic intervention in this, and other phosphonate producing organisms. However, the enzyme is of more general interest as it appears to be an evolutionary forerunner to the decarboxylase family of ThDP-dependent enzymes. To date, PnPDC has proved difficult to crystallize and no X-ray structures are available. In the past we have shown that ThDP-dependent enzymes will often crystallize if the cofactor has been irreversibly inactivated. To explore this possibility, and the utility of inhibitors of phosphonate biosynthesis as potential antibiotics, we synthesized phosphonodifluoropyruvate (PnDFP) as a prospective mechanism-based inhibitor of PnPDC. Here we provide evidence that PnDFP indeed inactivates the enzyme, that the inactivation is irreversible, and is accompanied by release of fluoride ion, i.e., PnDFP bears all the hallmarks of a mechanism-based inhibitor. Unfortunately, the enzyme remains refractive to crystallization.