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Methylene
Kinds of Methylene Terms modified by Methylene Selected AbstractsNovel Spiroheterocycles by Aziridination of ,-Methylene-,- and -,-lactamsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 14 2007M. Antonietta Loreto Abstract New potentially bioactive ,-spiroaziridino-,- and -,-lactams have been prepared by treatment of ,-methylene-,- and -,-lactams with ethyl N -{[(4-nitrophenyl)sulfonyl]oxy}carbamate (NsONHCO2Et) in the presence of CaO. These compounds, through reductive aziridine ring opening, can be intermediates for the synthesis of ,- and ,-aminolactams, which are useful as conformational constraints in peptides. The above procedure has been successfully extended to one ,-methyleneoxindole to obtain a new spirooxindole derivative, a potential precursor of natural alkaloids. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] Synthesis of Methylene- and Alkylidenecyclopropane DerivativesADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 4 2010Gérard Audran Abstract Since the methylenecyclopropane moiety is found in many biologically active natural substances, the synthesis of methylene- and alkylidenecyclopropanes remains a considerable challenge. In addition, an attractive feature is their surprising stability, accompanied by a high level of strain, conferring on them an otherwise unattainable chemical reactivity. The growing interest in the chemistry of these compounds has in its turn stimulated the development of alternative approaches to their skeleton, aimed at selectively introducing structural and chemical diversification. The three principal methods to synthesize these important compounds are based on the formation of the cyclopropane ring, the use of preformed cyclopropanes, and the use of preformed methylene- and alkylidenecyclopropanes. , Abbreviations: Ac: acetyl; Ar: aryl; Bn: benzyl; Boc: tert -butoxycarbonyl; Box: bisoxazoline; BTMSA: bis(trimethylsilyl)amide; Bu: butyl; Bz: benzoyl; C: cyclo; Cod: cyclooctadiene; Cp: cyclopentadienyl; Cy: cyclohexyl; Dba: (E,E)-dibenzylideneacetone; DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene; DCE: 1,2-dichloroethane; de: diastereomeric excess; DEAD: diethyl azodicarboxylate; DMAc: N,N -dimethylacetamide; DME: 1,2-dimethoxyethane; DMF: dimethylformamide; DOSP: N - p -dodecylbenzenesulfonylprolinate; Dppb: 1,4-bis(diphenyl)phosphinoborane; Dppe: bis(diphenylphosphino) ethene; E: electrophile; ee: enantiomeric excess; Et: ethyl; Hex: hexyl; L: ligand; LDA: lithium diisopropylamide; LG: leaving group; MCPBA: 3-chloroperoxybenzoic acid; Me: methyl; MEM: methoxyethoxymethyl; MOM: methoxymethyl; Mp: morpholinyl; Ms: mesyl; Naph: naphthyl; NFSI: N -fluorobenzenesulfonimide; Ns: nosyl; Nu: nucleophile; Pent: pentyl; Ph: phenyl; PMB: p -methoxybenzoyl; Pr: propyl; Py: pyridyl; SEM: 2-(trimethylsilyl)ethoxymethyl; TASF: tris(dimethylamino)sulfonium difluorotrimethyl silicate; TBAF: tetra- n -butylammonium fluoride; TBS: tert -butyldimethylsilyl; TEA: triethylamine; Tedicyp: cis,cis,cis -1,2,3,4-tetrakis(diphenylphosphinomethyl)cyclopentane; Tf: trifluoromethanesulfonyl; TFP: tris(2-furyl)phosphine; THF: tetrahydrofuran; THP: tetrahydropyran; TMS: trimethylsilyl; Tol: tolyl; Ts: 4-toluenesulfonyl (tosyl). [source] Enantioselective Aza-Morita,Baylis,Hillman Reaction Using Aliphatic ,-Amidosulfones as Imine SurrogatesADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 4 2010Nacim Abermil Abstract The bifunctional catalyst 6,-deoxy-6,-acylamino-,-isocupreidine (1) served both as a base to trigger the in situ generation of N -sulfonylimine from readily available ,-amidosulfones and as a chiral nucleophile to initiate the enantioselective aza-Morita,Baylis,Hillman (aza-MBH) reaction. ,-Methylene-,-amino-,-alkyl carbonyl compounds, difficultly accessible previously, can now be synthesized in excellent yields and enantioselectivities. [source] Methylene blue-mediated photodynamic therapy induces mitochondria-dependent apoptosis in HeLa CellJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2008Yan Lu Abstract Methylene blue (MB), a widely studied reagent, is investigated in this work for its usage in photodynamic therapy (PDT). PDT has been proved to be highly effective in the treatment of different types of cancers. Previous studies showed MB has both high affinity for mitochondria and high photodynamic efficiency. To elucidate the effects of MB in PDT, we analyzed PDT-induced apoptosis in HeLa cells by introducing different doses of MB into the culture media. Our data showed that MB-mediated PDT triggered intense apoptotic cell death through a series of steps, beginning with photochemical generation of reactive oxygen species. The release of cytochrome c and activation of caspase-3 indicated that MB-PDT-mediated apoptosis in HeLa cells was executed by the mitochondria-dependent apoptotic pathway. Importantly, proteomic studies confirmed that expression levels of several mitochondrial proteins were altered in MB-PDT-induced apoptosis, including TRAP1, mitochondrial elongation factor Tu and peroxiredoxin 3 isoform b. Western blot data showed that phosphorylation of ERK1/2 and PKA were reduced in MB-PDT treated cells, indicating several signal molecules participating in this apoptotic cascade. Moreover, MB-PDT induced an increase in the strength of interaction between Bcl-xL and dephosphorylated Bad. This led to loss of the pro-survival function of Bcl-xL and resulted in mitochondria-mediated apoptosis. This study provides solid evidence of a strong induction by MB-PDT of a mitochondria-dependent apoptosis cascade in HeLa cells. J. Cell. Biochem. 105: 1451,1460, 2008. © 2008 Wiley-Liss, Inc. [source] Methylene as a possible universal footprinting reagent that will include hydrophobic surface areas: Overview and feasibility: Properties of diaizirine as a precursorPROTEIN SCIENCE, Issue 6 2004F.M. Richards No abstract is available for this article. [source] Methylene as a possible universal footprinting reagent that will include hydrophobic surface areas: Overview and feasibility: Properties of diazirine as a precursorPROTEIN SCIENCE, Issue 12 2000Frederic M. Richards Abstract Methylene is one of, if not the, most reactive organic chemical known. It has a very low specificity, which makes it essentially useless for synthesis, but suggests a possible role in protein footprinting with special importance in labeling solvent accessible nonpolar areas, identifying ligand binding sites, and outlining interaction areas on protomers that form homo or hetero oligomers in cellular assemblies. The singlet species is easily and conveniently formed by photolysis of diazirine. The reactions of interest are insertion into C-H bonds and addition to multiple bonds, both forming strong covalent bonds and stable compounds. Reaction with proteins and peptides is reported even in aqueous solutions where the vast majority of the reagent is used up in forming methanol. Species containing up to 5 to 10 extra : CH2 groups are easily detected by electrospray mass spectroscopy. In a mixture of a 14 Kd protein and a noninteracting 1.7 Kd peptide, the distribution of mass peaks in the electrospray spectra was close to that expected from random modification of the estimated solvent accessible area for the two molecules. For analysis at the single residue level, quantitation at labeling levels of one 13CH2 group per 10 to 20 kDa of protein appears to be possible with isotope ratio mass spectroscopy. In the absence of reactive solvents, photolysis of diazirine produces oily polymeric species that contain one or two nitrogen atoms, but not more, and are water soluble. [source] ChemInform Abstract: Chemoenzymatic Synthesis of Optically Active ,-Methylene-,-carboxy-,-lactams and ,-Lactones.CHEMINFORM, Issue 12 2010Annalisa Bertoli Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] ChemInform Abstract: Highly cis- and trans-Selective Alkyl Radical Addition to ,-Methylene-,-phenyl-,-butyrolactams.CHEMINFORM, Issue 26 2009Tomoko Yajima Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] ChemInform Abstract: Enantioselective Organocatalytic Approach to ,-Methylene-,-lactones and ,-Lactams.CHEMINFORM, Issue 12 2009Lukasz Albrecht Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] ChemInform Abstract: The Aza-Morita,Baylis,Hillman Reaction of N-Thiophosphoryl Imines Catalyzed by 1,3,5-Triaza-7-phosphaadamantane (PTA) , Convenient Synthesis of ,-Methylene-,-amino Ketone or Acid Derivatives.CHEMINFORM, Issue 1 2008Xinyuan Xu Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] Cross-Metathesis Between ,-Methylene-,-butyrolactone and Olefins: A Dramatic Additive Effect.CHEMINFORM, Issue 36 2007Joelle Moise Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source] Titanocene(III) Chloride Mediated Radical-Induced One-Pot Synthesis of ,-Methylene-,-butyrolactones.CHEMINFORM, Issue 33 2007Moumita Paira Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source] Synthesis of ,,,-Disubstituted ,-Methylene-,-butyrolactams Starting from the Baylis,Hillman Adducts.CHEMINFORM, Issue 21 2007Ka Young Lee Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source] Synthesis of Fused ,-Methylene-,-butyrolactone Derivatives Through Pyridine-Induced Addition of Phenols to Dimethyl Acetylenedicarboxylate.CHEMINFORM, Issue 40 2006Issa Yavari Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source] Synthesis and Biological Properties of New ,-Methylene-,-butyrolactones and ,,,-Unsaturated ,-Lactones.CHEMINFORM, Issue 31 2006Francesca Cateni Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source] Convenient Synthesis of Substituted ,-Methylene-,-valerolactones in Aqueous Medium Using Baylis,Hillman Chemistry.CHEMINFORM, Issue 20 2006Vijay Singh Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source] ,-Methylene-,-trichloroacetylamino Alkanoates from Trichloroacetimidates of the Baylis,Hillman Adducts.CHEMINFORM, Issue 11 2005Roberta Galeazzi Abstract For Abstract see ChemInform Abstract in Full Text. [source] Highly Diastereoselective 7-endo Radical Cyclization of Ethyl ,-Methylene-,-(bromomethyl)dimethylsiloxycarboxylates.CHEMINFORM, Issue 36 2004Hajime Nagano Abstract For Abstract see ChemInform Abstract in Full Text. [source] Studies on Bromination of Active Methylene by a Mixture of Hydrobromic Acid and Hydrogen Peroxide (or TBHP).CHEMINFORM, Issue 34 2003Vasudha H. Tillu Abstract For Abstract see ChemInform Abstract in Full Text. [source] Samarium(III) Iodide Promoted Three-Component Coupling Reactions of Aldehydes, ,-Haloketones, and Active Methylene or Methyl Compounds.CHEMINFORM, Issue 28 2003Yongmin Ma No abstract is available for this article. [source] Highly Enantioselective Syntheses of Functionalized ,-Methylene-,-butyrolactones via Rh(I)-Catalyzed Intramolecular Alder Ene Reaction: Application to Formal Synthesis of (+)-Pilocarpine.CHEMINFORM, Issue 44 2002Aiwen Lei Abstract For Abstract see ChemInform Abstract in Full Text. [source] ChemInform Abstract: Synthesis of ,-Methylene-,-butyrolactones: Ru-Catalyzed Cyclocarbonylation of Allenyl Aldehydes and Allenyl Ketones.CHEMINFORM, Issue 36 2002Suk-Ku Kang Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] Compositional effects on electrophoretic and chromatographic figures of merit in electrokinetic chromatography with cetyltrimethylammonium bromide/sodium octyl sulfate vesicles as the pseudostationary phase.ELECTROPHORESIS, Issue 5 2008Part 1: Effect of the phase ratio Abstract The effect of the phase ratio on the electrophoretic and chromatographic properties of unilamellar vesicles comprised of cetyltrimethylammonium bromide (CTAB) and sodium octyl sulfate (SOS) was investigated in EKC. The surfactant concentration of the vesicles was 0.9, 1.2, 1.5, and 1.8% w/v, with a mole ratio of 1:3.66 (CTAB/SOS). Results were compared to those obtained using SDS micelles at concentrations of 1.0% (w/v, 35,mM) and 1.5% (52,mM). The CTAB/SOS vesicles (0.9,1.8% w/v) provided a significantly larger elution range (5.7,,,tves/t0,,,8.7) and greater hydrophobic (methylene) selectivity (2.8,,,,CH2,,,3.1) than SDS micelles (3.1,,,tmc/t0,,,3.3; ,CH2,=,2.2). Whereas the larger elution range can be attributed to the 25% reduction in EOF due to the interaction of unaggregated CTAB cations and the negatively charged capillary wall, the higher methylene selectivity is likely due to the lower concentration of water expected in the CTAB/SOS vesicle bilayer compared to the Palisades layer of SDS micelles. For a given phase ratio, CTAB/SOS vesicles are somewhat less retentive than SDS micelles, although retention factors comparable to those observed in 1.0,1.5% SDS can be obtained with 1.5,1.8% CTAB/SOS. A linear relationship was observed between phase ratio and retention factor, confirming the validity of the phase ratio model for these vesicles. Unique polar group selectivities and positional isomer shape selectivities were obtained with CTAB/SOS vesicles, with both types of selectivities being nearly independent of the phase ratio. For four sets of positional isomers, the elution order was always para < ortho < meta. Finally, the thermodynamics of solute retention was qualitatively similar to that reported for other surfactant aggregates (micelles and microemulsions); the enthalpic contribution to retention was consistently favorable for all compounds, whereas the entropic contribution was favorable only to hydrophobic solutes. [source] Dynamic Stereochemical Behaviour of Congested Ruthenium(II) Complexes Containing Asymmetric Thioether Ligands Based on Pyridine and PyrimidineEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 30 2008Giuseppe Tresoldi Abstract The asymmetric thioethers L [L = 2-pyridylmethyl 2,-pyrimidyl sulfide (pps) and 2-(4-methylpyrimidyl) 2,-pyridylmethyl sulfide (mps)] reacted with cis -[RuCl2(N,N -L,)2] [L, = di-2-pyridyl sulfide (dps); 2,2,-bis(4-methylpyridyl) sulfide (4mdps); 2,2,-bis(5-methylpyridyl) sulfide (5mdps)] to give the five-membered-ring chelate complexes [Ru(N,N -L,)2(Npyridine,S -L)]++ as the major products (92,95,%). Because the sulfur and ruthenium atoms are stereogenic centres, with (R) and (S) and , and , configurations, respectively, four isomers, including the enantiomers were obtained. At low temperature and in the methylene region of the 1H NMR spectra, two AB systems due to the enantiomer couples ,S ,R (a) and ,R ,S (b) were observed with abundances of 77,89 and 6,18,%, respectively. Furthermore, NMR spectroscopic investigations showed that the hybrid polydentate ligands L change their coordination mode. Thus, although a and b largely predominate, a mixture of species containing L and the Ru(N,N -L,)2 unit in the ratio 1:1 are present. The four-membered-ring chelate complexes [Ru(N,N -L,)2(Npyrimidine,S -L)]++ (c), as minor species (abundance 1,8,%), are always observed, whereas the dinuclear species [{Ru(N,N -L,)2}2(,-L)2]+4 (d, e) are observed when L, = dps or 5mdps. In these cases, four AB systems are assigned to dinuclear species d and e containing two bridging L that act as Npyridine,S- or Npyridine,Npyrimidine -donor ligands. The 1H NMR spectra are temperature dependent in that at low temperature the complexes undergo inversion of the chiral centre of the coordinated sulfur atom (a [rlhar2] b) and the dimer (d, e) and monomer (c) are in equilibrium; at higher temperatures the complexes undergo a structural dynamic rearrangement, which involves exchange between the coordinated and uncoordinated N atoms (b [rlhar2] c). One-dimensional band-shape analysis of the exchanging methylene and methyl proton signals showed that the energy barriers for inversion of the sulfur centre are in the 50,53 kJ,mol,1 range, whereas those for the higher-temperatures process are in the 62,68 kJ,mol,1 range. The possible mechanisms of the processes are discussed. NMR spectroscopic findings suggest that inversion at the sulfur centre occurs without any bond rupture, whereas the exchange, at higher temperatures (b [rlhar2] c), is a dissociative process involving the breaking of a Ru,Npyridine bond.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008) [source] Asymmetric Aza-Morita,Baylis,Hillman Reactions of Alkyl Vinyl Ketones with N -Protected Imines or In Situ Generated N -Protected IminesEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 21 2010Xiao-Yang Guan Abstract DABCO-catalyzed aza-MBH reactions of N -Boc imines with MVK and EVK have been thoroughly investigated in the paper. The asymmetric version of this aza-MBH reaction was also systematically investigated by using a chiral amine or a chiral phosphane catalyst. It was found that most of the N -protected imines are suitable substrates under the mild reaction conditions and are able to give the corresponding adducts in moderate yields with high ee values. The TQO- or LB1-catalyzed aza-MBH reactions of N -protected ,-amidoalkyl phenyl sulfones or ,-amidoalkyl p -tolyl sulfones with MVK could be well conducted, which provides a facile and direct route to obtain highly enantioselective aza-MBH adducts. The Boc protecting group of the aza-MBH product could be easily removed under acidic conditions to give the corresponding ,-methylene-,-amino ketone or ,-methylene-,-amino alcohol derivatives in good yields. [source] Highly Regio- and Stereoselective Diels,Alder Cycloaddition of Difluoro(methylene)cyclopropanesEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 6 2008Xiao-Chun Hang Abstract The Diels,Alder reactions of difluoro(methylene)cyclopropanes (F2MCPs) with cyclic dienes are described. These cycloaddition reactions exhibited complete regioselectivity and high endo -stereoselectivity. The obtained cycloadducts underwent a retro-Diels,Alder reaction to give the original dienophiles and dienes when heated, reflecting the reversible Diels,Alder reactivity of F2MCPs.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008) [source] Novel Spiroheterocycles by Aziridination of ,-Methylene-,- and -,-lactamsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 14 2007M. Antonietta Loreto Abstract New potentially bioactive ,-spiroaziridino-,- and -,-lactams have been prepared by treatment of ,-methylene-,- and -,-lactams with ethyl N -{[(4-nitrophenyl)sulfonyl]oxy}carbamate (NsONHCO2Et) in the presence of CaO. These compounds, through reductive aziridine ring opening, can be intermediates for the synthesis of ,- and ,-aminolactams, which are useful as conformational constraints in peptides. The above procedure has been successfully extended to one ,-methyleneoxindole to obtain a new spirooxindole derivative, a potential precursor of natural alkaloids. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] Enantioselective Butenolide Preparation for Straightforward Asymmetric Syntheses of ,-Lactones , Paraconic Acids, Avenaciolide, and Hydroxylated EleutherolEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 9 2006Stefan Braukmüller Abstract The naturally occurring ,-lactones (+)-methylenolactocin (13) and its enantiomer, (+)-protolichesterinic acid (14) and its enantiomer, (+)-rocellaric acid (15), and the methylene bis(,-lactone) (,)-avenaciolide (16) were synthesized with 95,98,% ees in very few steps. Enantiocontrol was imposed by the asymmetric dihydroxylation of trans -configured ,,,-unsaturated carboxylic esters (namely compounds 1i, 1j, and 1n) with AD mix-,® [for the levorotatory target structures, except for (,)-avenaciolide] or AD mix-,® [for the dextrorotatory target structures plus (,)-avenaciolide]. ,,,-Unsaturated carboxylic ester 1e required increased amounts of the oxidant and auxiliary to produce the hydroxy lactone R,R - 3e, a precursor of the naphtho-,-lactone (+)-9-hydroxyeleutherol (12; 96,% ee). (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source] XNA, (xylo Nucleic Acid): A Summary and New DerivativesEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 11 2005B. Ravindra Babu Abstract Fully modified homopyrimidine 2'-deoxy- xylo nucleic acid (dXNA) form triple helixes with complementary DNA/RNA with thermal stabilities comparable to those of the corresponding DNA:DNA and DNA:RNA duplexes. However, a single or few insertions of dXNA monomers in a DNA strand significantly lower duplex stabilities. The dXNA monomers are known to adopt predominantly an N -type furanose conformation in solution. With a desire to increase the binding affinity, seven sugar-modified XNA monomers (H, F, N, M, K, P and Q) have been synthesised and their effect on hybridization towards DNA and RNA complements studied. The introduction of 2'-fluoro and 2'-hydroxy substituents was expected to induce conformational restriction towards C3'- endo -type furanose conformation of monomer F derived from 1-(2'-deoxy-2'-fluoro-,- D -xylofuranosyl)thymine and monomer H derived from 1-(,- D -xylofuranosyl)thymine. The presence of functionalites facing the minor groove as in 1-(2'-amino-2'-deoxy-2'- N,4'- C -methylene-,- D -xylofuranosyl)thymine (monomer N), 1-[4- C -(N -methylpiperazinyl)methyl-,- D -xylofuranosyl]thymine (monomer P), 1-(4- C -piperazinylmethyl-,- D -xylofuranosyl)thymine (monomer Q), 1-(4- C -hydroxymethyl-,- D -xylofuranosyl)thymine (monomer M) and 9-(4- C -hydroxymethyl-,- D -xylofuranosyl)adenine (monomer K) was studied, with monomer N being locked in an N -type furanose conformation. Besides, an efficient synthesis of known xylo -LNA phosphoramidite 19, required for the incorporation of 1-(2'- O,4'- C -methylene-,- D -xylofuranosyl)thymine (monomer L) is described. For comparison, hydridization data of various XNAs reported in the literature are included in the discussion section. The thermal denaturation studies show that XNAs containing conformationally locked monomers (N and L) display improved binding affinity, and that partially modified DNA/XNA chimera, or fully modified XNA display preferential hybridization towards RNA complements. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005) [source] Kinetic mechanism for p38 MAP kinase ,FEBS JOURNAL, Issue 18 2005A partial rapid-equilibrium random-order ternary-complex mechanism for the phosphorylation of a protein substrate p38 Mitogen-activated protein kinase alpha (p38 MAPK,) is a member of the MAPK family. It is activated by cellular stresses and has a number of cellular substrates whose coordinated regulation mediates inflammatory responses. In addition, it is a useful anti-inflammatory drug target that has a high specificity for Ser-Pro or Thr-Pro motifs in proteins and contains a number of transcription factors as well as protein kinases in its catalog of known substrates. Fundamental to signal transduction research is the understanding of the kinetic mechanisms of protein kinases and other protein modifying enzymes. To achieve this end, because peptides often make only a subset of the full range of interactions made by proteins, protein substrates must be utilized to fully elucidate kinetic mechanisms. We show using an untagged highly active form of p38 MAPK,, expressed and purified from Escherichia coli[Szafranska AE, Luo X & Dalby KN (2005) Anal Biochem336, 1,10) that at pH 7.5, 10 mm Mg2+ and 27 °C p38 MAPK, phosphorylates ATF2,115 through a partial rapid-equilibrium random-order ternary-complex mechanism. This mechanism is supported by a combination of steady-state substrate and inhibition kinetics, as well as microcalorimetry and published structural studies. The steady-state kinetic experiments suggest that magnesium adenosine triphosphate (MgATP), adenylyl (,,,-methylene) diphosphonic acid (MgAMP-PCP) and magnesium adenosine diphosphate (MgADP) bind p38 MAPK, with dissociation constants of KA = 360 µm, KI = 240 µm, and KI > 2000 µm, respectively. Calorimetry experiments suggest that MgAMP-PCP and MgADP bind the p38 MAPK,,ATF2,115 binary complex slightly more tightly than they do the free enzyme, with a dissociation constant of Kd , 70 µm. Interestingly, MgAMP-PCP exhibits a mixed inhibition pattern with respect to ATF2,115, whereas MgADP exhibits an uncompetitive-like pattern. This discrepancy occurs because MgADP, unlike MgAMP-PCP, binds the free enzyme weakly. Intriguingly, no inhibition by 2 mm adenine or 2 mm MgAMP was detected, suggesting that the presence of a ,-phosphate is essential for significant binding of an ATP analog to the enzyme. Surprisingly, we found that inhibition by the well-known p38 MAPK, inhibitor SB 203580 does not follow classical linear inhibition kinetics at concentrations >,100 nm, as previously suggested, demonstrating that caution must be used when interpreting kinetic experiments using this inhibitor. [source] | |||||||||||||||||||||||||||||||||||||