Browsing by Author "Department of Chemistry & Chemical Biology, School of Science"
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Item Analysis of Biofluids by Paper Spray Mass Spectrometry: Advances and Challenges(2016-03) Manicke, Nicholas E.; Bills, Brandon J.; Zhang, Chengsen; Department of Chemistry & Chemical Biology, School of ScienceAbstract Paper spray MS is part of a cohort of ambient ionization or direct analysis methods that seek to analyze complex samples without prior sample preparation. Extraction and electrospray ionization occur directly from the paper substrate upon which a dried matrix spot is stored. Paper spray MS is capable of detecting drugs directly from dried blood, plasma and urine spots at the low ng/ml to pg/ml levels without sample preparation. No front end separation is performed, so MS/MS or high-resolution MS is required. Here, we discuss paper spray methodology, give a comprehensive literature review of the use of paper spray MS for bioanalysis, discuss technological advancements and variations on this technique and discuss some of its limitations.Item Assigning the EPR Fine Structure Parameters of the Mn(II) Centers in Bacillus subtilis Oxalate Decarboxylase by Site-Directed Mutagenesis and DFT/MM Calculations(American Chemical Society, 2014-02-12) Campomanes, Pablo; Kellett, Whitney F.; Easthon, Lindsey M.; Ozarowski, Andrew; Allen, Karen N.; Angerhofer, Alexander; Rothlisberger, Ursula; Richards, Nigel G. J.; Department of Chemistry & Chemical Biology, School of ScienceOxalate decarboxylase (OxDC) catalyzes the Mn-dependent conversion of the oxalate monoanion into CO2 and formate. EPR-based strategies for investigating the catalytic mechanism of decarboxylation are complicated by the difficulty of assigning the signals associated with the two Mn(II) centers located in the N- and C-terminal cupin domains of the enzyme. We now report a mutational strategy that has established the assignment of EPR fine structure parameters to each of these Mn(II) centers at pH 8.5. These experimental findings are also used to assess the performance of a multistep strategy for calculating the zero-field splitting parameters of protein-bound Mn(II) ions. Despite the known sensitivity of calculated D and E values to the computational approach, we demonstrate that good estimates of these parameters can be obtained using cluster models taken from carefully optimized DFT/MM structures. Overall, our results provide new insights into the strengths and limitations of theoretical methods for understanding electronic properties of protein-bound Mn(II) ions, thereby setting the stage for future EPR studies on the electronic properties of the Mn(II) centers in OxDC and site-specific variants.Item Biomembrane-mimicking lipid bilayer system as a mechanically tunable cell substrate(Elsevier B.V., 2014-03) Lautscham, Lena A.; Lin, Corey Y.; Auernheimer, Vera; Naumann, Christoph A.; Goldmann, Wolfgang H.; Fabry, Ben; Department of Chemistry & Chemical Biology, School of ScienceCell behavior such as cell adhesion, spreading, and contraction critically depends on the elastic properties of the extracellular matrix. It is not known, however, how cells respond to viscoelastic or plastic material properties that more closely resemble the mechanical environment that cells encounter in the body. In this report, we employ viscoelastic and plastic biomembrane-mimicking cell substrates. The compliance of the substrates can be tuned by increasing the number of polymer-tethered bilayers. This leaves the density and conformation of adhesive ligands on the top bilayer unaltered. We then observe the response of fibroblasts to these property changes. For comparison, we also study the cells on soft polyacrylamide and hard glass surfaces. Cell morphology, motility, cell stiffness, contractile forces and adhesive contact size all decrease on more compliant matrices but are less sensitive to changes in matrix dissipative properties. These data suggest that cells are able to feel and respond predominantly to the effective matrix compliance, which arises as a combination of substrate and adhesive ligand mechanical properties.Item Cobalt-catalysed site-selective intra- and intermolecular dehydrogenative amination of unactivated sp3 carbons(Nature Publishing Group, 2015-03-10) Wu, Xuesong; Yang, Ke; Zhao, Yan; Sun, Hao; Li, Guigen; Ge, Haibo; Department of Chemistry & Chemical Biology, School of ScienceCobalt-catalysed sp2 C–H bond functionalization has attracted considerable attention in recent years because of the low cost of cobalt complexes and interesting modes of action in the process. In comparison, much less efforts have been devoted to the sp3 carbons. Here we report the cobalt-catalysed site-selective dehydrogenative cyclization of aliphatic amides via a C–H bond functionalization process on unactivated sp3 carbons with the assistance of a bidentate directing group. This method provides a straightforward synthesis of monocyclic and spiro β- or γ-lactams with good to excellent stereoselectivity and functional group tolerance. In addition, a new procedure has been developed to selectively remove the directing group, which enables the synthesis of free β- or γ-lactam compounds. Furthermore, the first cobalt-catalysed intermolecular dehydrogenative amination of unactivated sp3 carbons is also realized., Functionalizing unactivated carbon–hydrogen bonds is challenging, especially when using non-precious metals and dealing with sp3 hybridized carbons. Here, the authors report an intramolecular cobalt catalysed amination of C–H bonds of sp3 carbons, giving access to β- and γ-lactams.Item Comparison of Four Bleeding Risk Scores to Identify Rivaroxaban-treated Patients With Venous Thromboembolism at Low Risk for Major Bleeding(Wiley, 2016-02) Kline, Jeffrey A.; Jimenez, David; Courtney, Mark; Ianus, Juliana; Cao, Lynn; Lensing, Anthonie W. A.; Prins, Martin H.; Wells, Philip S.; Department of Chemistry & Chemical Biology, School of ScienceObjectives Outpatient treatment of acute venous thromboembolism (VTE) requires the selection of patients with a low risk of bleeding during the first few weeks of anticoagulation. The accuracy of four systems, originally derived for predicting bleeding in VTE treated with vitamin K antagonists (VKAs), was assessed in VTE patients treated with rivaroxaban. Methods All patients treated with rivaroxaban in the multinational EINSTEIN deep vein thrombosis (DVT) and pulmonary embolism (PE) trials were included. Major bleeding was defined as ≥2 g/dL drop in hemoglobin or ≥2-unit blood transfusion, bleeding in critical area, or bleeding contributing to death. The authors examined the incidence of major bleeding in patients with low-risk assignment by the systems of Ruiz-Gimenez et al. (score = 0 to 1), Beyth et al. (score = 0), Kuijer et al. (score = 0), and Landefeld and Goldman. (score = 0). For clinical relevance, the definition of low risk for all scores except Kuijer includes all patients < 65 years with no prior bleeding history and no comorbid conditions (current cancer, renal insufficiency, diabetes mellitus, anemia, prior stroke, or myocardial infarction). Results A total of 4,130 patients (1,731 with DVT only, 2,399 with PE with or without DVT) were treated with rivaroxaban for a mean (±SD) duration of 207.6 (±95.9) days. Major bleeding occurred in 1.0% (40 of 4,130; 95% confidence interval [CI] = 0.7% to 1.3%) overall. Rates of major bleeding for low-risk patients during the entire treatment period were similar: Ruiz-Gimenez et al., 12 of 2,622 (0.5%; 95% CI = 0.2% to 0.8%); Beyth et al., nine of 2,249 (0.4%; 95% CI = 0.2% to 0.8%); Kuijer et al., four of 1,186 (0.3%; 95% CI = 0.1% to 0.9%); and Landefeld and Goldman, 11 of 2,407 (0.5%; 95% CI = 0.2% to 0.8%). At 30 days, major bleed rates for low-risk patients were as follows: Ruiz-Gimenez et al., five of 2,622 (0.2%; 95% CI = 0.1% to 0.4%); Beyth et al., five of 2,249 (0.2%; 95% CI = 0.1% to 0.5%); Kuijer et al., three of 1,186 (0.3%; 95% CI = 0.1% to 0.7%); and Landefeld and Goldman, seven of 2,407 (0.3%; 95% CI = 0.1% to 0.6%). No low-risk patient had a fatal bleed. Conclusions Four scoring systems that use criteria obtained in routine clinical practice, derived to predict low bleeding risk with VKA treatment for VTE, identified patients with less than a 1% risk of major bleeding during full-course treatment with rivaroxaban.Item A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases(PNAS Online, 2016-07-26) Salmon, Melissa; Thimmappa, Ramesha B.; Minto, Robert E.; Melton, Rachel E.; Hughes, Richard K.; O’Maille, Paul E.; Hemmings, Andrew M.; Osbourn, Anne; Department of Chemistry & Chemical Biology, School of ScienceTriterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply.Item Copper-catalyzed diastereoselective aerobic intramolecular dehydrogenative coupling of hydrazones via sp3 C–H functionalization(Royal Society of Chemistry, 2015) Wu, Xuesong; Wang, Mian; Zhang, Guangwu; Zhao, Yan; Wang, Jianyi; Ge, Haibo; Department of Chemistry & Chemical Biology, School of ScienceTransition metal-catalyzed cross dehydrogenative coupling is an important tool for functionalization of the α Csp3–H bond of amines. Among this reaction category, copper-catalyzed selective C–C bond formation under atmospheric O2 is of considerable research interest and significant progress has been achieved in recent years. In comparison, development of the intramolecular version of this transformation is still in its infancy. Furthermore, diastereoselective cyclization with this transformation has not been achieved. Here, we describe the highly diastereoselective intramolecular dehydrogenative cyclization of N,N-disubstituted hydrazones by a copper-catalyzed sp3 C–H bond functionalization process. The reaction protocol utilizes O2 as the oxidant and shows great functional group compatibility. Computational studies suggest that a 5-center/6-electron disrotatory cyclization mechanism is probably involved in the process for controlling the diastereoselectivity. This work represents the first example of a copper-catalyzed, direct intramolecular diastereoselective coupling reaction via an iminium ion intermediate. Additionally, it provides an environmentally friendly and atom efficient approach to access substituted pyrazolines, an important structural unit in many biologically active compounds.Item Copper-promoted site-selective carbonylation of sp3 and sp2 C–H bonds with nitromethane(RSC, 2016-08) Wu, Xuesong; Miao, Jinmin; Li, Yanrong; Li, Guigen; Ge, Haibo; Department of Chemistry & Chemical Biology, School of ScienceCopper-promoted direct carbonylation of unactivated sp3 C–H and aromatic sp2 C–H bonds of amides was developed using nitromethane as a novel carbonyl source. The sp3 C–H functionalization showed high site-selectivity by favoring the C–H bonds of α-methyl groups. The sp2 C–H carbonylation featured high regioselectivity and good functional group compatibility. Kinetic isotope effect studies indicated that the sp3 C–H bond breaking step is reversible, whereas the sp2 C–H bond cleavage is an irreversible but not the rate-determining step. Control experiments showed that a nitromethyl intermediate should be involved in the present reaction.Item Detection of prohibited treatment products on racing tires using headspace solid phase microextraction (SPME) and gas chromatography/ mass spectrometry (GC/MS)(Royal Society of Chemistry, 2016-01) Kranz, William D.; Carroll, Clinton J.; Goodpaster, John V.; Department of Chemistry & Chemical Biology, School of ScienceA variety of commercial tire treatments are available that purport to help automobile tires better cling to the surface of a road or racetrack, raising concerns in the professional racing community that such products might be used to illicitly boost performance in competitive events. These tire treatments are reputed to cut lap times and improve handling and maneuverability. In some cases, the manufacturers even boast that their products are “undetectable” (i.e., impervious to the scrutiny of laboratory testing). In this study, a number of banned tire treatment products were evaluated principally by gas chromatography-mass spectrometry (GC-MS) using solid phase microextraction (SPME) as a pre-concentration technique. The chemicals off-gassed by each product were determined and grouped into two broad categories: ‘plasticizer-based’ tire treatment products and ‘hydrocarbon-based’ tire treatment products. This information was then applied to the analysis of genuine tire samples provided by the United States Auto Club (USAC), a professional racing association. Over the course of one year, 10 out of the 71 questioned samples tested positive for a prohibited treatment product. The manufacturers' claims regarding their products' invisibility to lab tests were largely proven to be unfounded: both the products themselves and the tires treated with them can be identified by a number of characteristic volatile compounds. These included known plasticizers such as pentanedioic acid diethyl ester, plasticizer-related compounds such as 2-ethyl-1-hexanol, and dearomatized distillates.Item Direct detection and quantification of microRNAs(Elsevier, 2009-04-01) Hunt, Eric A.; Goulding, Ann M.; Deo, Sapna K.; Department of Chemistry & Chemical Biology, School of Science