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  • 151.
    Cheruku, Pradeep
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Ali, Muhammad
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Neudoerfl, Joerg-M
    Andersson, Pher
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Development of new thiazole-based iridium catalysts and their applications in the asymmetric hydrogenation of trisubstituted olefins2008In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 6, no 2, p. 366-373Article in journal (Refereed)
    Abstract [en]

    New thiazole-based chiral N,P-ligands that are open-chain analogues of known cyclic thiazole ligands have been synthesized and evaluated in the iridium-catalyzed asymmetric hydrogenation of trisubstituted olefins. Chirality was introduced into the ligands through a highly diastereoselective alkylation using Oppolzer's camphorsultam as chiral auxiliary. In general, the new catalysts are as reactive and selective as their cyclic counterparts for the asymmetric hydrogenation of various trisubstituted olefins.

  • 152.
    Cheruku, Pradeep
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry.
    Church, Tamara
    Andersson, Pher
    Chemistry – An Asian Journal2008In: Phosphine-Free RuCp*-Diamine Catalyzed Hydrogenation of Imi-nes, Vol. 3, p. 1390-1394Article in journal (Refereed)
  • 153.
    Cheruku, Pradeep
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Church, Tamara
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Andersson, Pher
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Phosphine-Free Cp*Ru(Diamine) Catalysts in the Hydrogenation of Imines2008In: Chemistry - An Asian Journal, ISSN (Print)1861-4728.(Online)1861-471X, Vol. 3, no 8-9, p. 1390-1394Article in journal (Refereed)
    Abstract [en]

    We previously reported the phosphine-free Cp*Ru(diamine)-catalyzed hydrogenation of aryl methyl ketones. Herein we present the first report of ruthenium-diamine-catalyzed imine hydrogenation to form amines. The most effective catalyst, I/KOtBu, completely converted several imines to amines at room temperature. The effect of electron-donating and -with- drawing groups on the reaction was investigated using a suitable series of substrates. The asymmetric version of the reaction was studied for two substrates, and the chiral amine products could be obtained in moderate enantiomeric excess.

  • 154.
    Cheruku, Pradeep
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Church, Tamara L.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Trifonova, Anna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Wartmann, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Andersson, Pher G.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Access to chiral tertiary amines via the iridium-catalyzed asymmetric hydrogenation of enamines2008In: Tetrahedron Letters, ISSN 0040-4039, E-ISSN 1359-8562, Vol. 49, no 51, p. 7290-7293Article in journal (Refereed)
    Abstract [en]

    The asymmetric hydrogenation of N,N-dialkyl and N-alkyl-N-aryl enamines to chiral tertiary amines was studied. All the N,P-ligated iridium complexes investigated were active catalysts for the reaction, but only those with bicycle-supported oxazoline-phosphine ligands gave reasonable stereoinduction. The best catalyst produced a range of chiral tertiary amines in up to 87% ee.

  • 155.
    Cheruku, Pradeep
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Church, Tamara
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Trifonova, Anna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Wartmann, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Andersson, P G
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Access to Chiral Tertiary Amines via Iridium Catalyzed Asymmetric Hydrogenation Of Enamines2008In: Tetrahedron Letters, Vol. 49, no 51, p. 7290-7293Article in journal (Refereed)
    Abstract [en]

    The asymmetric hydrogenation of N,N-dialkyl and N-alkyl-N-aryl enaminesto chiral tertiary amines was studied. All the N,P-ligated iridium complexes investigated were active catalysts for the reaction, but only those with bicycle-supported oxazoline-phosphine ligands gave reasonable stereoinduction. The best catalyst produced a range of chiral tertiary amines in up to 87% ee.

  • 156.
    Cheruku, Pradeep
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Diesen, Jarle
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Andersson, Pher
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Asymmetric Hydrogenation of Di and Trisubstituted Enol Phosphinates with N,P-Ligated Iridium Complexes2008In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 130, no 16, p. 5595-5599Article in journal (Refereed)
    Abstract [en]

    The iridium-catalyzed asymmetric hydrogenation of various di- and trisubstituted enol phosphinates has been studied. Excellent enantioselectivities (up to >99% ee) and full conversion were observed for a range of substrates with both aromatic and aliphatic side chains. Enol phosphinates are structural analogues of enol acetates, and the hydrogenated alkyl phosphinate products can easily be transformed into the corresponding alcohols with conservation of stereochemistry. We have also hydrogenated, in excellent ee, several purely alkyl-substituted enol phosphinates, producing chiral alcohols that are difficult to obtain highly enantioselectively from ketone hydrogenations.

  • 157.
    Cheruku, Pradeep
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Gohil, Suresh
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Andersson, Pher
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Asymmetric Hydrogenation of Enol Phosphinates by Iridium Catalysts Having N, P Ligands2007In: Organic Letters, ISSN 1523-7060, E-ISSN 1523-7052, Vol. 9, no 9, p. 1659-1661Article in journal (Refereed)
    Abstract [en]

    Enol phosphinates, which are structural analogues of enol acetates, have for the first time been employed as substrates for Ir-catalyzed asymmetric hydrogenation. A number of enol phosphinates have been synthesized and reduced successfully with up to and above 99% ee.

  • 158.
    Cheruku, Pradeep
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Paptchikhine, Alexander
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Church, Tamara L
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Andersson, Pher G
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Iridium-N,P-ligand-catalyzed enantioselective hydrogenation of diphenylvinylphosphine oxides and vinylphosphonates2009In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 131, no 23, p. 8285-8289Article in journal (Refereed)
    Abstract [en]

    Diphenylvinylphosphine oxides and di- and trisubstituted vinylphosphonates have been employed as substrates in iridium-catalyzed asymmetric hydrogenations. Complete conversions and excellent enantioselectivities (up to and above 99% ee) were observed for a range of substrates with both aromatic and aliphatic groups at the prochiral carbon. We have also hydrogenated electron-deficient carboxyethylvinylphosphonates with excellent stereoselectivity (up to and above 99% ee). The hydrogenated products of both classes of substrates are synthetically useful intermediates.

  • 159.
    Christian, Kyle J.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Pharmaceutical Biochemistry.
    Lang, Matti A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Pharmaceutical Biochemistry.
    Raffalli-Mathieu, Francoise
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Interaction of heterogeneous nuclear ribonucleoprotein C1/C2 with a novel cis-regulatory element within p53 mRNA as a response to cytostatic drug treatment2008In: Molecular Pharmacology, ISSN 0026-895X, E-ISSN 1521-0111, Vol. 73, no 5, p. 1558-1567Article in journal (Refereed)
    Abstract [en]

    We describe a novel cis-element in the 5' coding region of p53 mRNA and its interaction with heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. This element is located in a putative hairpin loop structure, within the first 101 nucleotides downstream of the start codon. The binding of hnRNPC1/C2 is strongly enhanced in response to the DNA-damaging drug cisplatin [cis-diamminedichloroplatinum(II)] and the cytostatic transcriptional inhibitor actinomycin D (dactinomycin), both known inducers of apoptosis and p53. Strongly stimulated binding is observed in both nuclear and cytoplasmic compartments, and it is accompanied by a cytoplasmic increase of hnRNPC1/C2. Changes in hnRNPC1/C2 protein levels are not proportional to binding activity, suggesting qualitative changes in hnRNPC1/C2 upon activation. Phosphorylation studies reveal contrasting characteristics of the cytoplasmic and nuclear hnRNPC1/C2 interaction with p53 mRNA. Results from chimeric p53-luciferase reporter constructs suggest that hnRNPC1/C2 regulates p53 expression via this binding site. Our results are consistent with a mechanism in which the interaction of hnRNPC1/C2 with a cis-element within the coding region of the p53 transcript regulates the expression of p53 mRNA before and during apoptosis. In addition, we report that preapoptotic signals induced by transcriptional inhibition trigger the appearance of a truncated, exclusively cytoplasmic 43-kDa variant of p53 before apoptosis.

  • 160.
    Christian, Kyle
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Lang, Matti
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Raffalli-Mathieu, Françoise
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Interaction of Heterogeneous Nuclear Ribonucleoprotein C1/C2 with a Novel cis-Regulatory Element within p53 mRNA as a Response to Cytostatic Drug Treatment2008In: Molecular Pharmacology, ISSN 0026-895X, E-ISSN 1521-0111, Vol. 73, no 5, p. 1558-1567Article in journal (Refereed)
    Abstract [en]

    We describe a novel cis-element in the 5′ coding region of p53 mRNA and its interaction with heterogeneous nuclear ribonucleoprotein (hnRNP)C1/C2. This element is located in a putative hairpin loop structure, within the first 101 nucleotides downstream of the start codon. The binding of hnRNPC1/C2 is strongly enhanced in response to the DNA-damaging drug cisplatin [cis-diamminedichloroplatinum(II)] and the cytostatic transcriptional inhibitor actinomycin D (dactinomycin), both known inducers of apoptosis and p53. Strongly stimulated binding is observed in both nuclear and cytoplasmic compartments, and it is accompanied by a cytoplasmic increase of hnRNPC1/C2. Changes in hnRNPC1/C2 protein levels are not proportional to binding activity, suggesting qualitative changes in hnRNPC1/C2 upon activation. Phosphorylation studies reveal contrasting characteristics of the cytoplasmic and nuclear hnRNPC1/C2 interaction with p53 mRNA. Results from chimeric p53-luciferase reporter constructs suggest that hnRNPC1/C2 regulates p53 expression via this binding site. Our results are consistent with a mechanism in which the interaction of hnRNPC1/C2 with a cis-element within the coding region of the p53 transcript regulates the expression of p53 mRNA before and during apoptosis. In addition, we report that preapoptotic signals induced by transcriptional inhibition trigger the appearance of a truncated, exclusively cytoplasmic 43-kDa variant of p53 before apoptosis.

  • 161. Christopeit, Tony
    et al.
    Gossas, Thomas
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Characterization of Ca2+ and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor2009In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 391, no 1, p. 39-44Article in journal (Refereed)
    Abstract [en]

    The interactions between Ca2+ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca2+ or phosphocholine were injected. Binding of Ca2+ induced a 10-fold higher signal than expected from the molecular weight of Ca2+. It was interpreted to result from the conformational change that occurs on binding of Ca2+. Two sites with different characteristics were distinguished: a high-affinity site with K-D = 0.03 mM and a low-affinity site with K-D = 5.45 mM. The pH dependencies of the two Ca2+ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K-D = 5 mu M at 10 mM Ca2+. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and Suggest that Ca2+ is an efficient regulator under physiological conditions.

  • 162.
    Christopeit, Tony
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Stenberg, Gun
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Gossas, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Nyström, Susanne
    Baraznenok, Vera
    Lindström, Erik
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    A surface plasmon resonance-based biosensor with full-length BACE1 in a reconstituted membrane2011In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 414, no 1, p. 14-22Article in journal (Refereed)
    Abstract [en]

    A surface plasmon resonance (SPR) biosensor-based assay for membrane-embedded full-length BACE1 (β-site amyloid precursor protein cleaving enzyme 1), a drug target for Alzheimer's disease, has been developed. It allows the analysis of interactions with the protein in its natural lipid membrane environment. The enzyme was captured via an antibody recognizing a C-terminal His6 tag, after which a lipid membrane was reconstituted on the chip using a brain lipid extract. The interaction between the enzyme and several inhibitors confirmed that the surface was functional. It had slightly different interaction characteristics as compared with a reference surface with immobilized ectodomain BACE1 but had the same inhibitor characteristic pH effect. The possibility of studying interactions with BACE1 under more physiological conditions than assays using truncated enzyme or conditions dictated by high enzyme activity is expected to increase our understanding of the role of BACE1 in Alzheimer's disease and contribute to the discovery of clinically efficient BACE1 inhibitors. The strategy exploited in the current study can be adapted to other membrane-bound drug targets by selecting suitable capture antibodies and lipid mixtures for membrane reconstitution.

  • 163.
    Church, Tamara
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Byrne, Christopher M.
    Lobkovsky, Emil B.
    Coates, Geoffrey W.
    A New Multicomponent Reaction Catalyzed by a [Lewis Acid]+[Co(CO)4]- Catalyst:  Stereospecific Synthesis of 1,3-Oxazinane-2,4-diones from Epoxides, Isocyanates, and CO2007In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 129, no 26, p. 8156-8162Article in journal (Refereed)
    Abstract [en]

    The use of mechanistic information to develop a new, catalytic multicomponent reaction is described. The complex [(salph)Al(THF)2+[co(CO)4]-(1, salph = N,N-o-phenylenebis(3,5-di-tert-butylsalicylideneimine), THF = tetrahydrofuran), wich is known to carbonylate epoxides, aziridines, and B-lactones, was used to catalyze the synthesis of 1,3-oxazinane-2,4-diones from epoxides, isocyanates, and CO. Under optimized conditions, the reaction was both selective and high-yielding. 1,3-Oxazinane-2,4-diones were synthesized from a variety of epoxides and isocyanates, including some epoxides that do not undergo simple ring-expansion carbonylation. The best results were obtained using highly electrophilic isocyanates. The mechanism of the multicomponent reaction was investigated using labeling and stereochemsitry, and the data obtained were consistent with the 1-catalyzed formation of B-lactone and 1,3-oxazinane-2,4-dione from a common intermediate.

  • 164.
    Church, Tamara L.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Andersson, Pher G.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Iridium catalysts for the asymmetric hydrogenation of olefins with nontraditional functional substituents2008In: Coordination chemistry reviews, ISSN 0010-8545, E-ISSN 1873-3840, Vol. 252, no 5-7, p. 513-531Article, review/survey (Refereed)
    Abstract [en]

    Chiral iridium catalysts have now been used in the asymmetric hydrogenation of largely unfunctionallized olefins for a decade. Recently, they have also been applied to substrates with more exotic functional groups, including non-coordinating ones. These, unlike coordinating substituents, cannot direct asymmetric hydrogenation by rhodium- or ruthenium-based catalysts. This review discusses several classes of these less familiar substrates, outlines the progress that has been made toward their stereoselective hydrogenation, and highlights the role of iridium complexes in this emerging field. We hope this will inspire researchers to consider iridium-catalyzed asymmetric hydrogenation as a potential route to a broad range of chiral compounds.

  • 165.
    Church, Tamara L.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Rasmussen, Torben
    Andersson, Pher G.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry.
    Enantioselectivity in the Iridium-Catalyzed Hydrogenation of Unfunctionalized Olefins2010In: Organometallics, ISSN 0276-7333, E-ISSN 1520-6041, Vol. 29, no 24, p. 6769-6781Article in journal (Refereed)
    Abstract [en]

    The iridium catalyzed asymmetric hydrogenation of largely unfunctionalized olefins has been studied by DFT calculations using a full experimentally tested combination of ligand and substrate All possible diastereomeric pathways were considered within four different hydrogenation mechanisms The effect of a solvent continuum was also considered and both the gas phase and solvent continuum calculations favored the same mechanism This mechanism passed through Ir-III and Ir-V intermediates and was consistent with the sense of stereoselection observed experimentally Comparing the calculations to those performed on a model system permitted an evaluation of the model system s utility in representing the full one A simple general method for predicting the sense of stereoselection in iridium-catalyzed olefin hydrogenation was developed and tested against published data

  • 166.
    Co, Michelle
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Fagerlund, Amelie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Engman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Sunnerheim, Kerstin
    Department of Natural Sciences, Engineering and Mathematics, Mid Sweden University.
    Sjöberg, Per J. R
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Turner, Charlotta
    Dept of Organic Chemistry, Lund University.
    Extraction of Antioxidants from Spruce (Picea abies) Bark using Eco-Friendly Solvents2012In: Phytochemical Analysis, ISSN 0958-0344, E-ISSN 1099-1565, Vol. 23, no 1, p. 1-11Article in journal (Refereed)
    Abstract [en]

    Introduction-Antioxidants are known to avert oxidation processes and they are found in trees and other plant materials. Tree bark is a major waste product from paper pulp industries; hence it is worthwhile to develop an extraction technique to extract the antioxidants.

    Objective- To develop a fast and environmentally sustainable extraction technique for the extraction of antioxidants from bark of spruce (Picea abies) and also to identify the extracted antioxidants that are abundant in spruce bark.

    Methodology- A screening experiment that involved three different techniques, was conducted to determine the best technique to extract antioxidants.The antioxidant capacity of the extracts was determined with DPPH (2,2-diphenyl-2’-picrylhydrazyl) assay. Pressurised fluid extraction (PFE) turned out to be the best technique and a response surface design was therefore utilised to optimise PFE. Furthermore, NMR and HPLC-DAD-MS/MS were applied to identify the extracted antioxidants.

    Results- PFE using water and ethanol as solvent at 160 and 180°C, respectively, gave extracts of the highest antioxidant capacity. Stilbene glucosides such as isorhapontin, piceid and astringin were identified in the extracts.

    Conclusion-The study has shown that PFE is a fast and environmentally sustainable technique, using water and ethanol as solvent for the extraction of antioxidants from spruce bark.

  • 167. Coleman, V. A.
    et al.
    Knut, Ronny
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Molecular and condensed matter physics.
    Karis, Olof
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Molecular and condensed matter physics.
    Grennberg, H.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Jansson, U.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Materials Chemistry.
    Quinlan, R.
    Holloway, B. C.
    Sanyal, Biplab
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Materials Science, Materials Theory.
    Eriksson, Olle
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Materials Science, Materials Theory.
    Defect Formation In Graphene Nanosheets By Acid Treatment: An X-Ray Absorption Spectroscopy And Density Functional Theory Study2008In: Journal of Physics D: Applied Physics, ISSN 0022-3727, E-ISSN 1361-6463, Vol. 41, no 6, p. 062001-4Article in journal (Refereed)
    Abstract [en]

    In-plane defects have been introduced into graphene nanosheets by treatment with hydrochloric acid. Acid treatment induces bond cleavage in the C–C network via electrophilic attack. These resultant vacancy sites will then undergo further reactions with the surrounding ambient to produce C–O and C–H bonds. A σ* resonance at 287 eV in the carbon K-edge x-ray absorption spectra is observed with acid treatment and is assigned to C–O states. Theoretical modelling of a di-vacancy in a graphene bilayer reproduces all essential features of this resonance and in addition predicts a metallic conductivity of states around this vacancy. The possibility of engineering the properties of graphene via the routes explored here is an important step towards establishing strategies for building devices based on this material.

  • 168. Czyzewski, Michal
    et al.
    Bower, Justin
    Box, Matthew
    Ottosson, Henrik
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Steel, Patrick G.
    Silene equivalents through the rhodium-catalysed reactions of alpha-hypersilyl diazoesters: a computational and experimental study2011In: Chemical Science, ISSN 2041-6520, Vol. 2, no 12, p. 2367-2372Article in journal (Refereed)
    Abstract [en]

    The generation of silenes through the rhodium-catalysed decomposition of alpha-hypersilyl diazocarbonyl compounds has been explored both computationally and experimentally. This transformation proceeds via a pathway involving initial formation of the carbene, followed by rearrangement, initially to a silene and ultimately to a ketene. Density functional theory (DFT) calculations of model compounds suggested that silene formation was most preferential with electron donating substituents attached to the carbonyl group. The predictions were experimentally evaluated and hypersilyl diazoacetates provided an unusually long-lived species (t(1/2) > 40 h) that reacts as a formal silene equivalent. Further DFT calculations support the formation of an internally stabilised silene in the form of a 1,2-silaoxetene. Importantly the acylsilene-silaoxetene reaction is reversible and consequently this silene equivalent reacts with alpha,beta-unsaturated carbonyl compounds to form cyclic silyl enol ethers which have considerable potential for further synthetic transformations.

  • 169.
    Dahl, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Kinetic studies of NS3 and NS5B from Hepatitis C virus: Implications and applications for drug discovery2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The aim of these studies was to increase our understanding of the non-structural proteins 3 and 5B (NS3 and NS5B) from the hepatitis C virus (HCV), and thereby contribute to the development of new and better drugs against HCV.

    By studying NS3 with substitutions identified to be associated with resistance to NS3 inhibitors in clinical trials (R155Q, A156T and D168V) it was found that not all inhibitors were affected, indicating that cross-resistance can be avoided.

    Substitutions at position 526 and 528 in the helicase domain of this bifunctional enzyme were introduced and the effect on the protease was investigated. These substitutions affected protease inhibition, showing that the helicase can influence the protease.

    This interplay between the two domains is also involved in the discovered activation of the enzyme at low inhibitor concentrations. Being a case of "enzyme memory", the phenomenon stresses the importance of using full-length NS3 for enzymatic assays.

    Inhibitors with novel designs, with presumed increased stability in vivo, were developed and, even though they were found to be of low potency, provide alternative ideas of how to design an inhibitor.

    Detailed information about the interaction between NS3 and its protein cofactor NS4A or several protease inhibitors were determined using a direct binding assay. The rate constants of the inhibitor interactions were affected by NS4A and it was also possible to visualize time-dependent binding inhibitors. A good correlation between interaction data (Kd or koff) and inhibition data (Ki) or replicon data (EC50) was also seen.

    The same approach was used for studying the interactions between NS5B and several non-nucleoside inhibitors, providing information of the chemodynamics and giving insights into inhibitor design.

     

    Taken together, all these studies have resulted in new information about, and new tools with which to study, NS3 and NS5B. This is of great importance in the struggle to find new and potent drugs, leading to a cure for HCV infection.

    List of papers
    1. Resistance profiling of hepatitis C virus protease inhibitors using full-length NS3
    Open this publication in new window or tab >>Resistance profiling of hepatitis C virus protease inhibitors using full-length NS3
    2007 (English)In: Antiviral Therapy, ISSN 1359-6535, E-ISSN 2040-2058, Vol. 12, no 5, p. 733-740Article in journal (Refereed) Published
    Abstract [en]

    Background: The NS3 protease of hepatitis C virus (HCV) is a prime target for anti-HCV drugs but resistance towards inhibitors of the enzyme is likely to emerge because of mutations in the viral genome that modify the structure of the protein. Enzyme inhibition data supporting this is limited to studies with few compounds and analysis performed with truncated NS3.

    Experimental: The potential of HCV acquiring resistance towards NS3 protease inhibitors and the structural features associated with resistance has been explored with a series of inhibitors and by using full-length NS3 protease/helicase variants with amino acid substitutions (A156T, D168V and R155Q) in the protease domain.

    Results: The A156T and D168V substitutions did not influence the kinetic properties of the protease, whereas the R155Q substitution reduced the catalytic efficiency 20 times, as compared with the wild type. Inhibition studies revealed that these substitutions primarily affected the potency of compounds which effectively inhibit the wild-type enzyme, and had little effect on weak or moderate inhibitors. As a consequence, all compounds had similar inhibitory potencies to the substituted enzyme variants. An exception was VX-950, which inhibited the D168V enzyme more efficiently than the wild type. For this inhibitor, the present data correlated better with replicon data than data from assays with truncated enzyme.

    Conclusions: These results have provided a structural basis for designing inhibitors that may be less susceptible to resistance by three known mutations, and suggest that the present variants of full-length NS3 constitute effective models for resistance profiling of NS3 protease inhibitors.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-17089 (URN)000248546900004 ()17713156 (PubMedID)
    Available from: 2008-06-16 Created: 2008-06-16 Last updated: 2017-12-08Bibliographically approved
    2.
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    3. β-Amino acid substitutions and structure-based CoMFA modeling of hepatitis C virus NS3 protease inhibitors
    Open this publication in new window or tab >>β-Amino acid substitutions and structure-based CoMFA modeling of hepatitis C virus NS3 protease inhibitors
    Show others...
    2008 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 16, no 10, p. 5590-5605Article in journal (Refereed) Published
    Abstract [en]

    In an effort to develop a new type of HCV NS3 peptidomimetic inhibitor, a series of tripeptide inhibitors incorporating a mix of alpha- and beta-amino acids has been synthesized. To understand the structural implications of beta-amino acid substitution, the P(1), P(2), and P(3) positions of a potent tripeptide scaffold were scanned and combined with carboxylic acid and acyl sulfonamide C-terminal groups. Inhibition was evaluated and revealed that the structural changes resulted in a loss in potency compared with the alpha-peptide analogues. However, several compounds exhibited muM potency. Inhibition data were compared with modeled ligand-protein binding poses to understand how changes in ligand structure affected inhibition potency. The P(3) position seemed to be the least sensitive position for beta-amino acid substitution. Moreover, the importance of a proper oxyanion hole interaction for good potency was suggested by both inhibition data and molecular modeling. To gain further insight into the structural requirements for potent inhibitors, a three-dimensional quantitative structure-activity relationship (3D-QSAR) model has been constructed using comparative molecular field analysis (CoMFA). The most predictive CoMFA model has q(2)=0.48 and r(pred)(2)=0.68.

    Keywords
    hepatitis C, HCV, NS3, protease inhibitor, beta-amino acid, 3D-QSAR, CoMFA, docking
    National Category
    Pharmaceutical Sciences
    Identifiers
    urn:nbn:se:uu:diva-96053 (URN)10.1016/j.bmc.2008.04.005 (DOI)000256052400023 ()18434166 (PubMedID)
    Available from: 2007-09-06 Created: 2007-09-06 Last updated: 2018-01-13Bibliographically approved
    4. Hepatitis C Virus NS3 Protease Is Activated by Low Concentrations of  Protease Inhibitors
    Open this publication in new window or tab >>Hepatitis C Virus NS3 Protease Is Activated by Low Concentrations of  Protease Inhibitors
    2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 48, p. 11592-11602Article in journal (Refereed) Published
    Abstract [en]

    The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a   bifunctional enzyme with a protease and a helicase functionality   located in each of the two domains of the single peptide chain. There   is little experimental evidence for a functional role of this   unexpected arrangement since artificial single domain forms of both   enzymes are catalytically competent. We have observed that low   concentrations of certain protease inhibitors activate the protease of   full-length NS3 from HCV genotype 1a with up to 100%, depending on the   preincubation time and the inhibitor used. The activation was reduced,   but not eliminated, by increased ionic strength, lowered glycerol   concentration, or lowered pH. In all cases, it was at the expense of a   significant loss of activity. Activation was not seen with the   artificial protease domain of genotype 1b NS3 fused with a fragment of   the NS4A cofactor. This truncated and covalently modified enzyme form   was much less active and exhibited fundamentally different catalytic   properties to the full-length NS3 protease without (he fused cofactor.   The most plausible explanation for the activation was found to involve   a slow transition between two enzyme conformations, which differed in   their catalytic ability and affinity for inhibitors. Equations derived   based on this assumption resulted in better fits to the experimental   data than the equation for simple competitive inhibition. The mechanism   may involve an inhibitor-induced stabilization of the helicase domain   in a conformation that enhances the protease activity, or all improved   alignment of the catalytic triad in the protease. The proposed mnemonic   mechanism and derived equations are viable for both these explanations   and can serve as a basic framework for future studies of enzymes   activated by inhibitors or other ligands.

    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-98859 (URN)10.1021/bi9016928 (DOI)000272083900028 ()
    Available from: 2009-03-04 Created: 2009-03-04 Last updated: 2017-12-13Bibliographically approved
    5. Kinetic, mechanistic and chemodynamic characterisation of non-nucleoside hepatitis C virus NS5B polymerase inhibitors using SPR biosensor technology
    Open this publication in new window or tab >>Kinetic, mechanistic and chemodynamic characterisation of non-nucleoside hepatitis C virus NS5B polymerase inhibitors using SPR biosensor technology
    Show others...
    (English)Manuscript (Other academic)
    Abstract [en]

    Kinetic, mechanistic and chemodynamic aspects of the interaction between five non-nucleoside inhibitors and the HCV NS5B polymerase (genotype 2a) were assessed using SPR biosensor technology. The compounds were selected to represent different structural classes (benzothiadiazine, , α,γ-diketo acid, benzimidazole, thiophene carbocyclic acid and benzofuran), each known to interact with different binding sites. The viral polymerase interacted with the compounds with different kinetics and surprisingly also with different capacities. Cooperativity between the different allosteric inhibitor binding sites and the active site binding diketoacid was observed, but no cooperativity was seen between the allosteric sites. The interaction with diketoacid was stronger in phosphate buffer as compared to Tris buffer, indicating a phosphate ion-mediated interaction mechanism. The enzyme generally had reduced affinity for the inhibitors in the presence of RNA. Interaction parameters determined for human serum albumin revealed the propensity of the compounds to be distributed by HSA. This study provides important information for the design of optimized NS5B inhibitors and illustrates the complementarity of a biosensor-based analysis with inhibition studies, in particular for allosteric compounds with complex interaction mechanisms or when the target contains multiple ligand binding sites.

    Keywords
    Hepatitis C, NS5B, non-nucleoside inhibitors, biosensor
    Identifiers
    urn:nbn:se:uu:diva-98860 (URN)
    Available from: 2009-03-04 Created: 2009-03-04 Last updated: 2009-11-24
    6. Kinetic characterization of HCV NS3 inhibitors using an SPR biosensor
    Open this publication in new window or tab >>Kinetic characterization of HCV NS3 inhibitors using an SPR biosensor
    (English)Manuscript (Other academic)
    Abstract [en]

    An SPR biosensor-based assay for studies of the interactions with full-length NS3 protease from hepatitis C virus (HCV) has been developed. It was used to characterize the interaction kinetics for a series of NS3 protease inhibitors. Moreover, the interaction between NS3 and the NS4A cofactor could be studied.

    The KD of the NS3-NS4A interaction was 30 nM in the standard buffer. It was reduced 600-fold by increasing the ionic strength to 300 mM NaCl. By using surfaces with only NS3 or with NS3 and co-immobilised NS4A, the effect of this protein cofactor on the interaction with several protease inhibitors was investigated. NS4A increased the affinity for all compounds, between 2 to 40 times, indicating that the NS3-NS4A complex binds inhibitors better than only NS3. The obtained interaction data was also compared with inhibition data, revealing a very good correlation between koff or KD with Ki (r = 0.92 and r = 0.90 respectively) over a broad range of affinities and potencies, showing that this biosensor based assay is a good and powerful tool for detailed studies of NS3 protease inhibitors which can serve as a future cure for HCV infection.

    Keywords
    Hepatitis C virus, NS3, NS4A, protease, biosensor, SPR, inhibition
    Identifiers
    urn:nbn:se:uu:diva-98863 (URN)
    Available from: 2009-03-04 Created: 2009-03-04 Last updated: 2009-11-24
  • 170.
    Dahl, Göran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Gutiérrez Arenas, Omar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Hepatitis C Virus NS3 Protease Is Activated by Low Concentrations of  Protease Inhibitors2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 48, p. 11592-11602Article in journal (Refereed)
    Abstract [en]

    The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a   bifunctional enzyme with a protease and a helicase functionality   located in each of the two domains of the single peptide chain. There   is little experimental evidence for a functional role of this   unexpected arrangement since artificial single domain forms of both   enzymes are catalytically competent. We have observed that low   concentrations of certain protease inhibitors activate the protease of   full-length NS3 from HCV genotype 1a with up to 100%, depending on the   preincubation time and the inhibitor used. The activation was reduced,   but not eliminated, by increased ionic strength, lowered glycerol   concentration, or lowered pH. In all cases, it was at the expense of a   significant loss of activity. Activation was not seen with the   artificial protease domain of genotype 1b NS3 fused with a fragment of   the NS4A cofactor. This truncated and covalently modified enzyme form   was much less active and exhibited fundamentally different catalytic   properties to the full-length NS3 protease without (he fused cofactor.   The most plausible explanation for the activation was found to involve   a slow transition between two enzyme conformations, which differed in   their catalytic ability and affinity for inhibitors. Equations derived   based on this assumption resulted in better fits to the experimental   data than the equation for simple competitive inhibition. The mechanism   may involve an inhibitor-induced stabilization of the helicase domain   in a conformation that enhances the protease activity, or all improved   alignment of the catalytic triad in the protease. The proposed mnemonic   mechanism and derived equations are viable for both these explanations   and can serve as a basic framework for future studies of enzymes   activated by inhibitors or other ligands.

  • 171.
    Dahl, Göran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Sandström, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Åkerblom, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Effects on protease inhibition by modifying of helicase residues in hepatitis C virus nonstructural protein 32007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 22, p. 5979-5986Article in journal (Refereed)
    Abstract [en]

    This study of the full-length bifunctional nonstructural protein 3 from hepatitis C virus (HCV) has revealed that residues in the helicase domain affect the inhibition of the protease. Two residues (Q526 and H528), apparently located in the interface between the S2 and S4 binding pockets of the substrate binding site of the protease, were selected for modification, and three enzyme variants (Q526A, H528A and H528S) were expressed, purified and characterized. The substitutions resulted in indistinguishable Km values and slightly lower kcat values compared to the wild-type. The Ki values for a series of structurally diverse protease inhibitors were affected by the substitutions, with increases or decreases up to 10-fold. The inhibition profiles for H528A and H528S were different, confirming that not only did the removal of the imidazole side chain have an effect, but also that minor differences in the nature of the introduced side chain influenced the characteristics of the enzyme. These results indicate that residues in the helicase domain of nonstructural protein 3 can influence the protease, supporting our hypothesis that full-length hepatitis C virus nonstructural protein 3 should be used for protease inhibitor optimization and characterization. Furthermore, the data suggest that inhibitors can be designed to interact with residues in the helicase domain, potentially leading to more potent and selective compounds.

  • 172.
    Dahl, Göran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Sandström, Anja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Åkerblom, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Resistance profiling of hepatitis C virus protease inhibitors using full-length NS32007In: Antiviral Therapy, ISSN 1359-6535, E-ISSN 2040-2058, Vol. 12, no 5, p. 733-740Article in journal (Refereed)
    Abstract [en]

    Background: The NS3 protease of hepatitis C virus (HCV) is a prime target for anti-HCV drugs but resistance towards inhibitors of the enzyme is likely to emerge because of mutations in the viral genome that modify the structure of the protein. Enzyme inhibition data supporting this is limited to studies with few compounds and analysis performed with truncated NS3.

    Experimental: The potential of HCV acquiring resistance towards NS3 protease inhibitors and the structural features associated with resistance has been explored with a series of inhibitors and by using full-length NS3 protease/helicase variants with amino acid substitutions (A156T, D168V and R155Q) in the protease domain.

    Results: The A156T and D168V substitutions did not influence the kinetic properties of the protease, whereas the R155Q substitution reduced the catalytic efficiency 20 times, as compared with the wild type. Inhibition studies revealed that these substitutions primarily affected the potency of compounds which effectively inhibit the wild-type enzyme, and had little effect on weak or moderate inhibitors. As a consequence, all compounds had similar inhibitory potencies to the substituted enzyme variants. An exception was VX-950, which inhibited the D168V enzyme more efficiently than the wild type. For this inhibitor, the present data correlated better with replicon data than data from assays with truncated enzyme.

    Conclusions: These results have provided a structural basis for designing inhibitors that may be less susceptible to resistance by three known mutations, and suggest that the present variants of full-length NS3 constitute effective models for resistance profiling of NS3 protease inhibitors.

  • 173.
    Dahlstrand, Christian
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Yamazaki, Kaoru
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Kilså, Kristine
    Ottosson, Henrik
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Substituent Effects on the Electron Affinities and Ionization Energies of Tria-, Penta-, and Heptafulvenes: A Computational Investigation2010In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 75, no 23, p. 8060-8068Article in journal (Refereed)
    Abstract [en]

    The extent of substituent influence on the vertical electron affinities (EAs) and ionization energies (IEs) of 43 substituted tria-, penta-, and heptafulvenes was examined computationally at the OVGF/6-311G(d)//B3LYP/6-311G(d) level of theory and compared with those of tetracyanoquinodimethane (TCNQ) and tetrathiafulvalene (TTF) as representing strong electron-acceptor and -donor compounds, respectively. The substituents X at the exocyclic positions of the fulvenes were either NH2, H, or CN, while the substituents Y at the ring positions were H, CI, F, CN, or NH2. The variations of the EAs and lEs were rationalized by qualitative arguments based on frontier orbital symmetries for the different fulvene classes with either X or Y being constant. The minimum and maximum values found for the calculated EAs of the tria-, penta-, and heptafulvenes were 0.51-2.05, 0.24-3.63, and 0.53-3.14 eV, respectively, and for the IEs 5.27-9.96, 7.07-10.31, and 6.35-10.59 eV, respectively. Two of the investigated fulvenes outperform TCNQ (calcd EA = 2.63 eV) and one outperforms TTF (calcd IE = 6.25 eV) with regard to acceptor and donor abilities, respectively. We also evaluated the properties of bis(fulvene)s, i.e., compounds composed of a donor-type heptafulvene fused with an acceptor-type pentafulvene, and it was revealed that these bis(fulvene)s can be designed so that the IE and EA of the two separate fulvene segments are retained, potentially allowing for the design of compact donor-acceptor dyads.

  • 174.
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Fragment library screening and lead characterization using SPR biosensors2009In: Current Topics in Medicinal Chemistry, ISSN 1568-0266, E-ISSN 1873-4294, Vol. 9, no 18, p. 1725-1735Article in journal (Refereed)
    Abstract [en]

    The transition from high throughput screening of collections of drug-like compounds to screening of fragment libraries via lower throughput methods with high sensitivity has revolutionized early drug discovery. It is highlighting the need for sensitive biophysical techniques for interaction analysis rather than high throughput methods. Biosensors with SPR detection are well suited for this novel scenario. In less than 20 years the technique has been launched, established and become a highly informative method for a variety of applications in drug discovery. It is no longer limited to the detection of proteins or other high molecular weight analytes, but the detection of weakly interacting fragments is now feasible. This paper discusses the theoretical and experimental limitations for such applications and reviews a number of successful studies in the area of fragment-based lead discovery that have recently been published. It can be anticipated that the evolution of this young technique will be significantly influenced by the requirements for efficient fragment-based lead discovery.

  • 175.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Integrating surface plasmon resonance biosensor-based interaction kinetic analyses into the lead discovery and optimization process2009In: Future Medicinal Chemistry, ISSN 1756-8919, Vol. 1, no 8, p. 1399-1414Article in journal (Refereed)
    Abstract [en]

    Surface plasmon resonance biosensor technology has come of age and become an important tool for drug discovery. It is a label-free biophysical technique for the kinetic analysis of molecular interactions that provides exceptionally information-rich data. Recent improvements in sensitivity, experimental design, data analysis and sample throughput makes it suitable for use throughout the drug-discovery process. This article outlines the use of SPR biosensor technology for small-molecule drug discovery and exemplifies how it complements other techniques. The technology is especially valuable for fragment-based lead discovery since it has the required sensitivity and throughput for screening of fragment libraries. Hits can be identified with respect to multiple criteria, defined by the experimental design used for screening. Expansion of hits and subsequent characterization and optimization of leads can be performed with a variety of experiments exploiting the kinetic resolution of the technology. Leads identified by this strategy can therefore be extensively characterized with respect to their interactions, with their target as well as with nontarget proteins. Although it may take some time for the methods to become well established, and for the research community to reach proficiency and fully embrace the information-rich data that can be obtained, it can be predicted that this technology will be widely used for drug discovery within the near future. It is expected that the technology will be particularly important for fragment-based strategies and integrated with other experimental technologies as well as with computational methods.

  • 176.
    Danielson, U Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Esterbauer, Hermann
    Mannervik, Bengt
    Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases1987In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 247, no 3, p. 707-713Article in journal (Refereed)
    Abstract [en]

    The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism.

  • 177.
    Danielson, U. Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Jiang, Fanyi Y
    Hansson, Lars O.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Probing the kinetic mechanism and coenzyme specificity of glutathione reductase from the cyanobacterium Anabaena PCC 7120 by redesign of the pyridine-nucleotide-binding site1999In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 38, no 29, p. 9254-9263Article in journal (Refereed)
    Abstract [en]

    Glutathione reductase from the cyanobacterium Anabaena PCC 7120 contains a pyridine-nucleotide-binding motif differing from that of the enzyme from other sources and an insertion of 10 amino acid residues. Homology modeling was used to obtain a model of the enzyme structure. It revealed that in the Anabaena enzyme Lys(203) replaces Arg, found to interact with the 2'-phosphate of NADP(H) in the enzyme from other sources, and that it has an extra loop near the entrance of the pyridine-nucleotide-binding site. The steady-state and preequilibrium kinetic properties were characterized for the wild-type enzyme, a K203R, and a loop deletion mutant. All enzyme forms had higher catalytic efficiency with NADPH than with NADH, although the difference was less than for glutathione reductase from other sources. The specificity was most pronounced in the formation of the charge-transfer complex between the pyridine nucleotide and oxidized enzyme-bound FAD, as compared to later steps in the reaction. Unexpectedly, by replacing Lys(203) with Arg, the specificity for NADPH was diminished in the complete redox reaction. Ser(174) appears to interact with the 2'-phosphate of NADPH and introduction of arginine instead of lysine, therefore, has little effect on the interaction with this coenzyme. However, the efficiency in forming the charge-transfer complex between the pyridine nucleotide and oxidized enzyme-bound FAD was increased in the K203R mutant using NADPH but not with NADH. The lack of affinity toward 2',5'-ADP-Sepharose by the wild-type enzyme was not changed by replacing Lys(203) with Arg but deletion of the loop resulted in an enzyme that bound to the immobilized ligand. Removal of the loop increased the efficiency of the enzyme in the reductive half-reaction with both pyridine-nucleotides as well as in the overall catalytic mechanism.

  • 178.
    Danielson, U Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Kinetic independence of the subunits of cytosolic glutathione transferase from the rat1985In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 231, no 2, p. 263-267Article in journal (Refereed)
    Abstract [en]

    The steady-state kinetics of the dimeric glutathione transferases deviate from Michaelis-Menten kinetics, but have hyperbolic binding isotherms for substrates and products of the enzymic reaction. The possibility of subunit interactions during catalysis as an explanation for the rate behaviour was investigated by use of rat isoenzymes composed of subunits 1, 2, 3 and 4, which have distinct substrate specificities. The kinetic parameter kcat./Km was determined with 1-chloro-2,4-dinitrobenzene, 4-hydroxyalk-2-enals, ethacrynic acid and trans-4-phenylbut-3-en-2-one as electrophilic substrates for six isoenzymes: rat glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4. It was found that the kcat./Km values for the heterodimeric transferases 1-2 and 3-4 could be predicted from the kcat./Km values of the corresponding homodimers. Likewise, the initial velocities determined with transferases 3-3, 3-4 and 4-4 at different degrees of saturation with glutathione and 1-chloro-2,4-dinitrobenzene demonstrated that the kinetic properties of the subunits are additive. These results show that the subunits of glutathione transferase are kinetically independent.

  • 179.
    Danielson, U Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Paradoxical inhibition of rat glutathione transferase 4-4 by indomethacin explained by substrate-inhibitor-enzyme complexes in a random-order sequential mechanism1988In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 250, no 3, p. 705-711Article in journal (Refereed)
    Abstract [en]

    Under standard assay conditions, with 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrate, rat glutathione transferase 4-4 is strongly inhibited (I50 = 1 microM) by indomethacin. No other glutathione transferase investigated is significantly inhibited by micromolar concentrations of indomethacin. Paradoxically, the strong inhibition of glutathione transferase 4-4 was dependent on high (millimolar) concentrations of CDNB; at low concentrations of this substrate or with other substrates the effect of indomethacin on the enzyme was similar to the moderate inhibition noted for other glutathione transferases. In general, the inhibition of glutathione transferases can be explained by a random-order sequential mechanism, in which indomethacin acts as a competitive inhibitor with respect to the electrophilic substrate. In the specific case of glutathione transferase 4-4 with CDNB as substrate, indomethacin binds to enzyme-CDNB and enzyme-CDNB-GSH complexes with an even greater affinity than to the corresponding complexes lacking CDNB. Under presumed physiological conditions with low concentrations of electrophilic substrates, indomethacin is not specific for glutathione transferase 4-4 and may inhibit all forms of glutathione transferase.

  • 180. Darreh-Shori, Taher
    et al.
    Forsberg, Anton
    Modiri, Negar
    Andreasen, Niels
    Blennow, Kaj
    Kamil, Chelenk
    Ahmed, Hiba
    Almkvist, Ove
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Nordberg, Agneta
    Differential levels of apolipoprotein E and butyrylcholinesterase show strong association with pathological signs of Alzheimer's disease in the brain in vivo2011In: Neurobiology of Aging, ISSN 0197-4580, E-ISSN 1558-1497, Vol. 32, no 12, p. 2320.e15-2320.e32Article in journal (Refereed)
    Abstract [en]

    Recently, we reported that 3 of the known risk factors of Alzheimer's disease (AD), i.e., advanced age, apolipoprotein E (ApoE) epsilon 4, and female gender, are associated with differential levels of ApoE proteins and butyrylcholinesterase (BuChE) in the cerebrospinal fluid (CSF) of AD patients. The ApoE epsilon 4 allele and certain BuChE polymorphisms synergistically affect the conversion rate of mild cognitive impairment (MCI) to AD. Here, we investigated interrelationships between ApoE and BuChE levels, and pathological markers of AD in vivo. CSF from patients with probable AD, assessed for cerebral glucose metabolism (CMRglc; n = 50) and Pittsburgh compound B (PIB) retention (beta-amyloid [A beta] load, n = 29) by positron emission tomography (PET), was used for measurement of BuChE, ApoE, A beta, tau, phosphorylated tau (P-tau) and interleukin-1 beta (IL-1 beta) levels. Levels of ApoE and BuChE strongly correlated with CMRglc (fluorodeoxyglucose [FDG]-PET, r = 0.54, p < 0.0001, n = 50), cerebral A beta load (PIB retention, r = 0.73, p < 0.0001, n = 29), and CSF P-tau (r = 0.73, p < 0.0001, n = 33). High ApoE protein was tied to low CMRglc and high PIB retention and P-tau. BuChE levels had opposite relationships. Other CSF covariates were levels of interleukin-1 beta and A beta(42) peptide. The pattern of the patients' cognitive Z-scores strongly supported these observations. High ApoE protein was also linked to changes in 3 of the biodynamic properties of BuChE. In vitro analysis indicated that high ApoE protein levels were related to an increased pool of dormant BuChE molecules with an abnormally high intrinsic catalytic rate in CSF, which was "turned on" by excess A beta peptides. The findings suggest that abnormally high levels of ApoE may play a causative role in the pathological events of AD, particularly those involving the early cholinergic deficit in the AD brain, through modulation of cholinesterases activities, hence disturbing the acetylcholine-dependent activity of neurons and nonexcitable cells such as glial cells.

  • 181. de Kloe, Gerdien E
    et al.
    Retra, Kim
    Geitmann, Matthis
    Källblad, Per
    Nahar, Tariq
    van Elk, René
    Smit, August B
    van Muijlwijk-Koezen, Jacqueline E
    Leurs, Rob
    Irth, Hubertus
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    de Esch, Iwan J P
    Surface Plasmon Resonance Biosensor Based Fragment Screening Using Acetylcholine Binding Protein Identifies Ligand Efficiency Hot Spots (LE Hot Spots) by Deconstruction of Nicotinic Acetylcholine Receptor α7 Ligands2010In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 53, no 19, p. 7192-7201Article in journal (Refereed)
    Abstract [en]

    The soluble acetylcholine binding protein (AChBP) is a homologue of the ligand-binding domain of the nicotinic acetylcholine receptors (nAChR). To guide future fragment-screening using surface plasmon resonance (SPR) biosensor technology as a label-free, direct binding, biophysical screening assay, a focused fragment library was generated based on deconstruction of a set of α7 nAChR selective quinuclidine containing ligands with nanomolar affinities. The interaction characteristics of the fragments and the parent compounds with AChBP were evaluated using an SPR biosensor assay. The data obtained from this direct binding assay correlated well with data from the reference radioligand displacement assay. Ligand efficiencies for different (structural) groups of fragments in the library were correlated to binding with distinct regions of the binding pocket, thereby identifying ligand efficiency hot spots (LE hot spots). These hot spots can be used to identity the most promising hit fragments in a large scale fragment library screen.

  • 182.
    Dinér, Peter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry.
    Catalytic asymmetric chiral lithium amide-promoted epoxide rearrangement: a NMR spectroscopic and kinetic investigation2010In: Tetrahedron: asymmetry, ISSN 0957-4166, E-ISSN 1362-511X, Vol. 21, no 21-22, p. 2733-2739Article in journal (Refereed)
    Abstract [en]

    The lithium amide derived from the chiral diamine (1R,3S,4S)-3-(1-pyrrolidinyl)methyl-2-azabicyclo[2.2.1]heptane, has been reported to catalytically deprotonate cyclohexene oxide and other epoxides, yielding chiral allylic alcohols in excellent enantiomeric excess. In this work, 6Li, 1H and 13 C NMR spectroscopy have been used to study the aggregation of the chiral lithium amide in THF and the influence on the aggregation by the addition of additives, such as 1,8-diazabicyclo-[5.4.0]undec-7-ene (DBU). The activated complex under catalytic deprotonation of cyclohexene oxide, that is, with excess Li-DBU and free DBU, is built from one monomer of the chiral lithium amide, one molecule of epoxide and one additional molecule of DBU. The reaction order (0.97) obtained for the bulk base Li-DBU shows an inverse dependence on the concentration, suggesting a deaggregation of the initial mixed dimer to a monomer-based transition state containing a monomer of the lithium amide.

  • 183. Diéguez, Montserrat
    et al.
    Mazuela, Javier
    Pàmies, Oscar
    Verendel, J Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Andersson, Pher G
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Chiral pyranoside phosphite-oxazolines: a new class of ligand for asymmetric catalytic hydrogenation of alkenes2008In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 130, no 23, p. 7208-7209Article in journal (Refereed)
    Abstract [en]

    We have described the first successful application of a phosphite-oxazoline ligand library in the asymmetric Ir-catalyzed hydrogenation of several unfunctionalized olefins. The introduction of a bulky biaryl phosphite moiety in the ligand design is highly adventitious in the product outcome. By carefully selecting the ligand components, we obtained high activities (TOFs up to >1500 mol x (mol x h)(-1) at 1 bar of H2) and enantioselectivities (ee values up to >99%) and, at the same time, show a broad scope for different substrate types. So, this is an exceptional ligand class that competes favorably with a few other ligand series that also provide high ee values for tri- and disubstituted substrate types.

  • 184. Diéguez, Montserrat
    et al.
    Mazuela, Javier
    Pàmies, Oscar
    Verendel, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Andersson, Pher
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Biaryl phosphite-oxazolines from hydroxyl aminoacid derivatives: highly efficient modular ligands for Ir-catalyzed hydrogenation of alkenes2008In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, no 33, p. 3888-3890Article in journal (Refereed)
    Abstract [en]

    High enantioselectivities and activities are achieved in the Ir-catalyzed hydrogenation of several unfunctionalized olefins using modular biaryl phosphite-oxazoline ligands from hydroxyl aminoacid derivatives, the presence of a biaryl phosphite group is crucial to this success.

  • 185.
    Diéguez, Montserrat
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Mazuela, Javier
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Pàmies, Oscar
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Verendel, Johan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Andersson, Pher
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Chiral Pyranoside Phosphite - Oxazolines: A New Class of Ligand for Asymmetric Catalytic Hydrogenation of Alkenes2008In: J. Am. Chem. Soc., no 130, p. 7208-7209Article in journal (Refereed)
  • 186. Doi, Hisashi
    et al.
    Barletta, Julien
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Suzuki, Masaaki
    Noyori, Ryoji
    Watanabe, Yasuyoshi
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Synthesis of 11C-labelled N,N’-diphenylurea and ethyl phenylcarbamate by rhodium-promoted carbonylation reaction via [11C]-isocyanatobenzene using phenyl azide and [11C]carbon monoxide2004In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 2, no 21, p. 3063-3066Article in journal (Refereed)
    Abstract [en]

    The reaction with phenyl azide and [11C]carbon monoxide to give N,N'-diphenyl[11C]urea and ethyl phenyl[11C]carbamate has been studied with the aim of development of a new methodology for carbonylation using [11C]carbon monoxide with high specific radioactivity. The synthesis of 11C-labelled N,N'-diphenylurea from phenyl azide and [11C]carbon monoxide, with 1,2-bis(diphenylphosphino)ethane-bound Rh(I) complex at 120 degrees C at a pressure of 35 MPa in the presence of aniline was accomplished in 82% trapping efficiency and 82% conversion yield. This approach was also useful for the synthesis of ethyl phenyl[11C]carbamate with lithium ethoxide as a nucleophilic reagent giving 90% trapping efficiency and 76% conversion yield. These reactions can be considered to proceed via a [11C]isocyanate or a [11C]isocyanate-coordinated Rh complex to give the corresponding 11C-products. This protocol provides the chemical basis for the synthesis of [11C]urea and [11C]carbamate derived from [11C]isocyanates.

  • 187. Domínguez, José L
    et al.
    Christopeit, Tony
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Villaverde, M Carmen
    Gossas, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Otero, José M
    Nyström, Susanne
    Baraznenok, Vera
    Lindström, Erik
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Sussman, Fredy
    Effect of the Protonation State of the Titratable Residues on the Inhibitor Affinity to BACE-12010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 34, p. 7255-7263Article in journal (Refereed)
    Abstract [en]

    BACE-1 is one of the aspartic proteases involved in the cleavage of beta amyloid peptide, an initial step in the formation of amyloid plaques whose toxicity induces neuron death in Alzheimer's disease patients. One of the central issues in the search of novel BACE-1 inhibitors is the optimum pH for the binding of inhibitors to the enzyme. It is known that the enzyme has optimal catalytic activity at acidic pH, while cell active inhibitors may bind optimally at higher pH. In this work we determine the effect of the pH on the affinities of a set of inhibitors, with a variety of chemical motifs, for the ectodomain region of BACE-1 by a surface plasmon resonance (SPR) biosensor based assay. In order to understand the molecular interactions that underlie the diverse optimum pH for the binding of the various inhibitors as observed experimentally, we have calculated the titration curves for a set of BACE-1 ligand complexes. The results indicate that the pK(a) values of the titratable residues of the protein depend on the nature of the ligand involved, in disagreement with previous work. The enzyme-inhibitor structures with the resulting protonation states at pH values 4.5 and 7.4 served as the starting point for the prediction of the pH-dependent binding ranking. Our calculations reproduced the entire affinity ranking observed upon pH increase and most of the binding trends among inhibitors, especially at low pH. Finally, our cell-based assays indicate a possible correlation between high inhibitor affinity at both acidic and neutral pH values, with optimal cell response, a result that may open new venues for the search of potent BACE-1 inhibitors that are active at the cellular level.

  • 188. Dourado, Daniel F A R
    et al.
    Fernandes, Pedro Alexandrino
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Ramos, Maria João
    Glutathione Transferase A1-1: Catalytic Importance of Arginine 152010In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 114, no 4, p. 1690-1697Article in journal (Refereed)
    Abstract [en]

    Glutathione transferases (GSTs) are fundamental enzymes of the cell detoxification system. They catalyze the nucleophilic attack Of glutathione (GSH) on electrophilic substrates to produce less toxic compounds. The resulting Substrate can then be recognized by ATP-dependent transmembrane PUMPS and consequently expelled from the cell. Despite all the existing studies on GSTs, many aspects of the catalytic events are still poorly understood. Recently, using as a model the GSTAI-1 enzyme, we proposed it GSH activation mechanism. Resorting to the density functional theory (DFT), we demonstrated that a water molecule could assist a proton transfer between (lie GSH thiol and (x-carboxylic groups. after all initial conformational rearrangement of GSH, as evidenced by potential of mean force calculations. In this work to elucidate the catalytic role of Arg 15, a strictly conserved active site residue in class alpha GSTs. we analyzed the activation energy barrier and Structural details associated with the GSTAI-1 Mutants R15A, R15R epsilon, eta-c (an Arg residue with the epsilon-eta-nitrogens Substituted by carbons), and R 15Rneutral (a neutral Arg residue due to the a addition of a hydride in the zeta-carbon. A similar mechanism to the one used in Our GSH activation proposal was implemented.