Structurally two of the compounds differ only by the substituent on the benzene ring, i.e. 4-isopropylphenyl and o-tolyl, while two of the compounds differ only in that one has a pyrrole ring and one has an isoxazole ring.Three thiochromeno[4,3- 23H27NO2S, (I), and C21H23NO2S, (II), the pyrrole rings have envelope conformations with the C atom substituted by the benzene ring as the flap. In the third title compound, C20H21NO3S, (III), the isoxazole ring has a twisted conformation on the C—C bond substituted by the benzene ring and the carboxylate group. In all three compounds, the thiopyran ring has a half-chair conformation. The mean plane of the pyrrole ring is inclined to the mean plane of the thiopyran ring by 57.07 (9), 58.98 (9) and 60.34 (12)° in (I), (II) and (III), respectively. The benzene rings are inclined to one another by 73.26 (10)° in (I), 65.781)° in (II) and 63.37 (13)° in (III). In the crystals of all three compounds, there are no classical hydrogen bonds present. Only in the crystal of compound (I) are molecules linked by a pair of C—H⋯π interactions, forming inversion dimers. The isopropyl group in compound (I) is disordered over two sets of sites and has a refined occupancy ratio of 0.586 (13):0.414 (13).In the title compounds, C The thiopyran ring has a half-chair conformation, with the lowest asymmetry parameters ΔC2(S1—C7) = 8.34 (16) Å. The mean plane of the pyrrole ring makes dihedral angles of 57.07 (9) and 63.29 (10)° with the mean plane of the thiopyran ring and the benzene ring, respectively.The molecular structure of compound (I)q2 = 0.4673 (18) Å, φ2 = 223.5 (2)°. As in (I)The molecular structure of the compound (II)q2 = 0.466 (2) Å, φ2 = 275.7 (3)°. As in (I)H-chromenoisoxazole-3a-carbonitrile In the crystals of compounds (I)While a search of the Cambridge Structural Database -2-{[(2-formylphenyl)thio]methyl}-3-phenylacrylate (1 mmol) and sarcosine (1.2 mmol) in acetonitrile (10 ml), was added pyridine (0.2 mmol) and the mixture was refluxed until completion of the reaction (monitored by TLC). The crude product was subjected to column chromatography on silica gel (100–200 mesh) using petroleum ether–ethyl acetate (9:1) as eluent, which successfully provided the pure product as a colourless solid. The product was dissolved in chloroform and heated for 2 min. The resulting solution were allowed to evaporate slowly at room temperature and yielded colourless block-like crystals of compound (I)Compound (I)E)-2-{[(2-formylphenyl)thio]methyl}-3-(o-tolyl) acrylate (1 mmol) and sarcosine (1.2 mmol) in acetonitrile (10 ml) were reacted with pyridine following the same procedure as for compound (I)Compound (II)E)-2-{[(2-formylphenyl)thio]methyl}-3-(o-tolyl) acrylate(1 mmol) and N-methyl hydroxylamine hydrochloride (1.1 mmol) in acetonitrile (10 ml) were reacted with pyridine following the same procedure as for compounds (I)Compound (III)Uiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) for other H atoms. The isopropyl group in compound (I)Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015008063/su5106sup1.cifCrystal structure: contains datablock(s) global, I, II, III. DOI: 10.1107/S2056989015008063/su5106Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015008063/su5106IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989015008063/su5106IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989015008063/su5106Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015008063/su5106IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015008063/su5106IIIsup7.cmlSupporting information file. DOI: 1061279, 1061278, 1061277CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H-benzotriazol-1-ylmethyl)benzene shows an interesting three-dimensional assembly influenced by its non-planar molecular conformation.The crystal structure of 1,3-bis and 85.83 (10)° with the central aromatic ring and 57.08 (9)° with each other. The three-dimensional structure is controlled mainly by weak C—H⋯N and C—H⋯π interactions. The molecules are connected in inversion-related pairs, forming the slabs of infinite chains that run along the [-110] and [110] directions.The molecular structure of the title compound, C H-benzotriazol-1-ylmethyl)arene compounds are used as precursors for the synthesis of benzotriazolophanes, a class of positively charged cyclophanes that have the potential ability to trap anions and guest molecules with high electron density arene ligands has been of great interest. In these complexes, the metal center coordinates through the N3-nitrogen of the benzotriazole ring arene ligands have been determined: 2,6-bis(1H-benzotriazol-1-ylmethyl)pyridine benzene tetrahydrate phenyl ether benzene.Bis° and those between each benzotriazole moiety (N1–N3/C2–C7) and the central benzene ring are 88.74 (11) and 85.83 (10)° for the A and B groups, respectively. These values differ from the related structures 2,6-bispyridine, with a pyridine central ring, where the angle between the two benzotriazole units is 72.49 (6)° and those between the pyridine ring and the two benzotriazole units are 70.26 (6) and 57.70 (7)° benzene tetrahydrate, with para substitution, where the two benzotriazole units are parallel and the dihedral angle between each benzotriazole unit and the central benzene ring is 74.95 (9)°  −x + 1, −y + 1, −z + 1] interactions form slabs of infinite chains of molecules running along  interactions . Parallel chains interact through C7A—H7A⋯Cg1iii  . Since the chains run along the diagonal of the ab plane and a≃b, the 21 screw axis parallel to b transforms each chain into an orthogonal one, running along [110] . This orthogonal chain interacts with the initial one through C4B—H4B⋯Cg2iv  . In this way, each molecule displays four pairs of interactions with seven neighbouring molecules. This crystallographic three-dimensional organization differs from the related 1,4-bis(1H-benzotriazol-1-ylmethyl)benzene tetrahydrate where a two-dimensional network is observed benzene molecular structure with the possibility of any group replacing the 2,4,5,6-H atoms in the central benzene ring gave four hits, from which two have one additional arene substituent , one has the bis(1H-benzotriazole-1-ylmethanone) moiety instead of bis(1H-benzotriazol-1-ylmethyl) and the last one corresponds to a more complex molecular structure. When the search also considers heterocyclic compounds, two new hits (in addition to the first four structures) appear, one cyclic bipyridine and the related molecular structure 2,6-bis(1H-benzotriazol-1-ylmethyl)pyridine. A search for the 1,4-bis(1H-benzotriazol-1-ylmethyl)-substituted benzene ring gave four hits, one of which corresponds to a ligand with additional methyl groups at the 1,3,5,6-sites of the central benzene ring and the other to its corresponding palladium complex. The remaining two relate to the same compound, viz. 1,4-bis(1H-benzotriazol-1-ylmethyl)benzene tetrahydrate, a related molecular structure.A search of the Cambridge Structural Database  was added to a solution of 1H-benzotriazole  in toluene (60 mL), and the mixture was heated at reflux for 72 h. The resulting mixture was filtered, and the toluene solution was concentrated and cooled to give a white solid. Single crystals suitable for X-ray structure analysis were obtained by dissolving the compound in the minimum volume of dichloromethane, adding diethyl ether and cooling the solution to 277 K. The title compound formed colorless parallelepipeds. Yield: 668 mg (45%). M.p. 423–424 K. IR : ν 3058 (w), 3031 (w), 2979 (w), 2944 (w), 1613 (m), 1494 (m), 1452 (s), 1228 (s), 1159 (m), 1080 (s), 781 (s), 754 (s), 743 (s). 1H NMR : δ (p.p.m.) 8.04 , 7.72 , 7.47 , 7.38 , 7.36 , 7.32 , 7.25 , 5.95 . 13C NMR : δ (p.p.m.) 145.3 (C), 136.5 (C), 132.6 (C), 129.3 (CH), 127.5 (CH), 127.4 (CH), 127.2 (CH), 124.0 (CH), 119.2 (CH), 110.6 (CH), 50.7 (CH2). HRMS m/z (ESI) calculated for [C20H16N6+H]+: 341.1509; found 341.1532 [M+H]+.Ueq value of the parent atom.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016007805/bg2585sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016007805/bg2585Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016007805/bg2585Isup3.cmlSupporting information file. DOI: 1479416CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


MN2Cl2 bisphenoidal coordination geometry. In the crystals of both compounds, the polymer chains are linked via pairs of C—H⋯Cl hydrogen bonds, forming corrugated slabs parallel to the ac plane.The title complexes are isotypic one-dimensional coordination polymers. The metal ions are bridged by binding to the N atoms of the two pyridine rings, and have an  2(C18H14N4O4)]n, (I), and [HgCl2(C18H14N4O4)]n, (II), are, respectively, the cadmium(II) and mercury(II) complexes of the dimethyl ester of 5,6-bis(pyridin-2-yl)pyrazine-2,3-dicarboxylic acid. In both compounds, the metal ions are located on a twofold rotation axis and a second such axis bisects the Car—Car bonds of the pyrazine ring. The metal ions are bridged by binding to the N atoms of the two pyridine rings and have an MN2Cl2 bisphenoidal coordination geometry. The metal–Npyrazine distances are much longer than the metal–Npyridine distances; the difference is 0.389 (2) Å for the Cd—N bonds but only 0.286 (5) Å for the Hg—N bond lengths. In the crystals of both compounds, the polymer chains are linked via pairs of C—H⋯Cl hydrogen bonds, forming corrugated slabs parallel to the ac plane.The isotypic title one-dimensional coordination polymers, [CdCl The link to the pyrazine N atoms, N2 and N2i, is much weaker: Cd1⋯N1 = 2.7757 (17) Å and Hg1⋯N1 = 2.876 (5) Å. The difference in the metal–Npyrazine and metal–Npyridine bond lengths is 0.389 (2) Å for the Cd—N bonds but only 0.286 (5) Å for the Hg—N bonds (see Tables 1In compounds (I)4 parameter of 0.53, while for the Hg1 atom in (II)4 parameter = 0.30  have a bisphenoidal MN2Cl2 coordination environment.The fourfold coordination geometry of the metal atoms differ slightly, as illustrated in Fig. 3i/C1i/C2i) are not ideally planar [r.m.s. deviations are 0.096 and 0.092 Å for (I)Q) = 0.166 (2) Å, θ = 87.8 (7)°, φ = 270.0 (7)° for (I)Q) = 0.160 (6) Å, θ = 90 (2)°, φ = 270 (2)° for (II)x + y, −z + In both compounds, the pyrazine rings (N1/C1/C2/N1The pyridine rings (N2/C3–C7), are inclined to the pyrazine ring mean planes by 40.58 (10)° in (I)ca 100 and 160°, respectively, on coordination to the metal atom. While the pyrazine ring is ideally planar in the ligand (r.m.s. deviation = 0.032 Å), on coordination it is less planar with r.m.s. deviations of 0.096 and 0.092 Å for (I)It can be seen from Fig. 4via a pair of C—H⋯Cl hydrogen bonds, forming corrugated slabs parallel to the ac plane, as illustrated in Fig. 5In the crystals of both compounds, the polymer chains are linked et al., 2016MN2Cl2  four-coordinate metal ions yielded eight hits for M = cadmium and 52 hits for M = mercury. For the cadmium complexes, the Cd—Cl bonds are consistently longer than the Cd—Npyridine bonds, and the Cl—Cd—Cl bond angles are consistently larger than the N—Cd—N bond angle, as in compound (I)N}cadmium dichloridomercury(II)  has been reported on recently pyrazine-2,3-dicarboxylate (Synthesis of compound (I): CdCl2·2H2O  in 25 ml of dry MeOH was slowly added to a solution of 2LMe in 10 ml of dry MeOH. The colourless solution that formed was stirred at room temperature for 1 h, then filtered to remove any impurities. The filtrate was allowed to stand over several days until colourless square rod-like crystals were obtained . Elemental analysis for C18H14N4CdCl2O4 (Mw = 533.63); calculated C 40.51, H 2.64, N 10.50%; found C 40.49, H 2.53, N 10.59%. Selected IR bands : ν = 3066(w), 2997(w), 1751(vs), 1593(m), 1568(w), 1549(w), 1479(m), 1450(m), 1403(m), 1339(s), 1297(m), 1273(m), 1261(m), 1228(s), 1193(m), 1177(m), 1162(m), 1120(m), 1109(w), 1088(s), 1009(m), 974(m), 918(w), 827(m), 803(m), 789(m), 770(w), 758(m), 554(m).Synthesis of compound (II): 2LMe  was added in solid form to a solution of HgCl2·2H2O  in 25 ml of dry MeOH. The colourless solution immediately obtained was stirred at room temperature for 2 h, filtered to remove any impurity, and the filtrate allowed to evaporate slowly. After two days colourless needle-like crystals were obtained . Elemental analysis for C18H14N4Cl2HgO4 (Mw = 621.82); calculated C 34.77, H 2.27, N 9.01%; found C 34.79, H 2.44, N 9.03%. Selected IR bands : ν = 3065(w), 2997(w), 2951(w), 2882(w), 1747(vs), 1590(m), 1568(m), 1546(w), 1477(m), 1447(m), 1402(m), 1338(s), 1279(m), 1223(s), 1195(m), 1176(s), 1105(w), 1086(s), 1003(m), 973(w), 827(w), 802(m), 788(m), 769(m), 755(m), 553(m).Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H-atoms. For the mercury complex (II)Rint = 0.000 as only one equivalent was measured.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989016012093/zl2672sup1.cifCrystal structure: contains datablock(s) I, II, Global. DOI: 10.1107/S2056989016012093/zl2672Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989016012093/zl2672IIsup3.hklStructure factors: contains datablock(s) II. DOI: 1495931, 1495930CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Cellular oxidative stress serves as a common denominator in many neurodegenerative disorders, including Parkinson's disease. Here we use in-cell NMR spectroscopy to study the fate of the oxidation-damaged Parkinson's disease protein alpha-synuclein (α-Syn) in non-neuronal and neuronal mammalian cells. Specifically, we deliver methionine-oxidized, isotope-enriched α-Syn into cultured cells and follow intracellular protein repair by endogenous enzymes at atomic resolution. We show that N-terminal α-Syn methionines Met1 and Met5 are processed in a stepwise manner, with Met5 being exclusively repaired before Met1. By contrast, C-terminal methionines Met116 and Met127 remain oxidized and are not targeted by cellular enzymes. In turn, persisting oxidative damage in the C-terminus of α-Syn diminishes phosphorylation of Tyr125 by Fyn kinase, which ablates the necessary priming event for Ser129 modification by CK1. These results establish that oxidative stress can lead to the accumulation of chemically and functionally altered α-Syn in cells. α-synuclein is a protein linked to the occurrence of Parkinson's disease. Here, the authors use time-resolved in-cell NMR spectroscopy to study the repair of methionine-oxidized α-synuclein by endogenous cellular enzymes. To bettoisomers . We dete protein , which protein 34. This marotein 34.15N isotope-enriched α-Syn into mammalian A2780 and RCSN-3 cells for high-resolution in-cell NMR measurements. RCSN-3 cells are directly derived from rat substantia nigra neurons15N α-Syn displayed strong similarities with the disordered reference state of the N-terminally acetylated, monomeric protein (15N isotope-enriched α-Syn (Met-15N) into A2780 cells and acquired in-cell NMR spectra on the resulting cell samples. We clearly detected the cross-peak pattern of reduced Ac-Met1 and Met5, whereas NMR signals of Met116 and Met127 matched those of the oxidized states of both residues - protein , which e protein . To bettresidues . These fet5 ref.  - lysates . Time-re lysates , in supp lysates . Lysate ronments . They fuNext, we asked whether these properties were similarly displayed in other cell types. To this end, we extended our investigations to HeLa, SK-N-SH, B65 and SH-SY5Y cell lysates. We found that the stepwise repair of Met5 and Met1 occurred in an identical manner in these lysates and with comparable rates, whereas Met116 and Met127 remained oxidized .In vivo, methionine sulfoxide reduction depends on the interplay of a multicomponent system that includes enzymes of the methionine sulfoxide reductase family, MSRA and MSRB, and the NADPH-dependent thioredoxin (TRX)/TRX reductase (TRXR) complex  mouse modelGiven that C-terminal α-Syn sulfoxides are stably preserved in cells, they likely contribute to the accumulation of permanently altered protein species with possible roles in neurotoxicity171815N or 15N/13C isotopically enriched, N-terminally acetylated α-Syn was obtained by co-expressing the wild-type α-Syn plasmid (pT7-7) with the yeast NatB acetylase complexEscherichia coli BL21 (DE3) Express cells. Bacteria were grown in M9 minimal media supplemented with 0.5 g l−1of 15N NH4Cl (Sigma) and/or 2 g l−1 of 13C D-glucose (Sigma) until an OD600 of 0.6 was reached. After induction with 1 mM isopropyl-β-D-thiogalactoside (Roth), cells were grown for 12 h at 30 °C. For 15N/13C methionine-selective isotope-enriched samples, M9 medium contained natural abundance of NH4Cl, D-glucose and 0.3 mM of 15N/13C-enriched L-methionine −1)341H–15N NMR spectroscopy. Methionine-oxidized, N-terminally acetylated α-Syn was prepared by incubating 500 μM of recombinant protein with 4% H2O2 (Sigma)2O2. Complete peroxide removal was additionally verified with a colorimetric assay (Enzo Life Sciences). Quantitative methionine oxidation was confirmed using NMR spectroscopy (2D 1H–15N correlation spectra) in 20 mM phosphate buffer, pH 7.0, 150 mM NaCl and 10% D2O . Concentrations were assessed spectrophotometrically by absorption measurements at 274 nm .CD measurements were performed on a Jasco J-720 spectropolarimeter at 25 °C with 10 μM methionine-oxidized, N-terminally acetylated α-Syn in NMR buffer at pH 7.0. Far-ultraviolet CD spectra were collected using a 0.1-cm path-length cuvette. Eight scans were averaged and blank (buffer) spectra were subtracted from protein spectra to calculate ellipticity (mdeg).Overall, 200 μl of 500 μM methionine-oxidized, N-terminally acetylated α-Syn was separated on a Superdex 75 analytical column  connected to an AKTA purifier FPLC machine . Absorbance at 280 nm was plotted against elution volumes for SEC profiles.DLS was performed on a Malvern Zetasizer Nano operating at a laser wavelength of 633 nm at 25 °C in 3 × 3 mm cuvettes and with 50 μM of methionine-oxidized, N-terminally acetylated α-Syn in NMR buffer. Hydrodynamic radii were calculated based on volume-weighted analyses of scattered light, using the Malvern DTS software.Small unilamellar vesicles (SUVs) were reconstituted with pig brain polar lipids (Avanti) using freeze–thaw cycles and sonication. Briefly, the lipid powder was resuspended in NMR buffer and vortexed for 30 min at room temperature. Solutions were frozen on dry ice (5 min) and thawed at 37 °C for 5 min, five times before sonication at 4 °C for 20 min at 30% power settings (Bandelin Sonoplus), under a nitrogen stream15N/13C isotope-enriched (500 μM) protein in NMR buffer at pH 7.0. 2D (1H–15N) SOFAST-HMQC (ref. 1H) and 256 (15N) complex points for a sweep width (SW) of 16 p.p.m. (1H) and 26 p.p.m. (15N), with 16 scans. For 3D HNCACB and CBCACONH experiments, 1,024 (1H), 72 (15N) and 96 (13C) complex points for a SW of 16 p.p.m. (1H), 26 p.p.m. (15N) and 70 p.p.m. (13C), with 16 scans. Standard delays, pulse- and gradient shapes were used6515N isotope-enriched α-Syn at 30 °C for better signal-to-noise ratios.For NMR resonance assignment of reduced and fully methionine-oxidized α-Syn, we used MQC ref.  and thre1H) and 256 (15N) complex points, 128 scans and 60 ms recycling delays for α-Syn in A2780 cells, and 1,024 (1H) and 128 (15N) complex points, 2,048 (2 K) scans and 60 ms recycling delays for α-Syn in RCSN-3 cells. SW's were 16 p.p.m. (1H) and 26 p.p.m. (15N) for (U)-15N isotope-enriched oxidized α-Syn and 16 p.p.m. (1H) and 14 p.p.m. (15N) for 15N/13C methionine-isotope-enriched α-Syn. 13C decoupling was used for 15N/13C methionine-isotope-enriched α-Syn. One-dimensional (1D) 1H–15N SOFAST-HMQC spectra were acquired with identical settings and 4,096 (4 K) scans. NMR spectra were processed by zero-filling to four times the number of real points and by apodization with a sine-modulated window function and baseline correction in all dimensions.In-cell NMR spectra were acquired on 600 and 900 MHz Bruker Avance spectrometers, equipped with cryogenically cooled proton optimized triple resonance >‘inverse' NMR probe (TCI). All in-cell spectra were acquired at 10 °C. Parameters for 2D SOFAST-HMQC experiments were 1,024  and 128 (15N) complex points, 96 scans and recycling delays of 60 ms (acquisition time ∼20 min). NMR spectra were processed by zero-filling to four times the number of real points and by apodization with a sine-modulated window function and baseline correction in all dimensions. Processing and visualization of NMR spectra were performed with TOPSPIN 3.1 (Bruker) and SPARKY (UCSF). 1H–15N mean-weighted chemical shift differences were obtained using the equation [(ΔδH)2+(ΔδN/10)2]1/2  were determined for ∼110 unambiguously assigned, nonoverlapped cross-peaks of oxidized and reduced α-Syn by quantifying their intensities in the presence (I) and absence (I0) of SUVs. Error bars in the time-resolved reaction profiles represent the average experimental noise of NMR signals used to determine the repair kinetics.Time-resolved NMR experiments were performed on 600 and 750 MHz Bruker Avance spectrometers, equipped with cryogenically cooled proton optimized triple resonance >‘inverse' NMR probe (TCI). 1D and 2D 1/2 ref. , where ΔM1oxM5ox), oxidized Met1 and reduced Met5 (M1oxM5red) and reduced Met1 and Met5 (M1redM5red), as is evident from the three sets of NMR cross-peaks observed in our lysate time course experiments , which serves a good approximation along the published KM values of MSRA and MSRB enzymes, that is, ∼100 μM  and DMEM-Ham's F-12  growth media, respectively, supplemented with 10% fetal bovine serum  until 80% confluence. In all, 50–100 × 106 cells were collected for each in-cell NMR sample. Cells were detached with mild trypsin treatment  and resuspended in 2 ml of electroporation buffer . Fully oxidized, uniformly 15N isotope- or 15N methionine-enriched α-Syn (500 μM) was added to electroporation mixtures and aliquots (100 μl) were used for individual electroporations with an Amaxa nucleofector (Lonza) according to the manufacturer's instructions. Cells were re-seeded in 15-cm culture dishes and allowed to recover for 4 h at 37 °C (CO2 incubator). Cells were washed 3 × with pre-warmed PBS and collected with mild trypsin treatment, washed 2 × with pre-warmed growth media to remove excess trypsin and resuspended in pH-stable Leibovitz's L-15 medium  supplemented with 10% D2O. Finally, cells were transferred to NMR tubes for in-cell NMR experiments.In-cell NMR samples were prepared following a recently developed electroporation protocol6 cells were grown to ∼80% confluence at 37 °C, 5% CO2, in T175 flasks and were detached with mild trypsin treatment, washed twice with PBS and resuspended in NMR buffer with 1 × protease inhibitor mix . Cells were centrifuged for 5 min at 130g and the supernatant discarded. The cell pellet was lysed with five freeze–thaw cycles on dry ice/ethanol. Soluble lysate fractions were obtained by centrifugation at 16,000g for 10 min at 4 °C. Quantitative cell lysis was confirmed with Trypan Blue staining. Protein concentrations were determined using Bradford assays (Bio-Rad). Lysates were used immediately or aliquoted, snap-frozen in liquid nitrogen and stored at −80 °C until use. Repeated thawing and freezing of lysate aliquots was avoided to preserve enzyme activities−1 total protein concentrations and, unless otherwise specified, they were used at 10 mg ml−1 final concentrations. Samples for western blotting were boiled for 15 min in SDS-Laemmli buffer (Bio-Rad). Mammalian cell lines were as follows: A2780 , B65 , SH-SY5Y , RCSN-3 Typically, 20–50 × 10−1  with NMR buffer and supplemented with 20 mM dithiothreitol (DTT) (Applichem)15N or 15N/13C methionine-selective isotope-enriched, N-terminally acetylated α-Syn in duplicates at 25 °C. Consecutive 2D NMR experiments were started immediately after addition of α-Syn to cell lysates. Reaction volumes were 150 μl in 3-mm diameter Shigemi tubes.Lysate concentrations were adjusted to 10 mg mlSamples were separated with SDS–PAGE using 12 or 4–20% gradient gels (Bio-Rad) and transferred on polyvinylidene difluoride (PVDF) membranes. Blots were fixed with 4% paraformaldehyde for 15 min and washed thoroughly with PBS. Afterwards, blots were blocked in 5% non-fat dry milk in TBS-T for 1 h at room temperature and incubated with the following primary antibodies for 12 h at 4 °C: anti-α-Syn , anti-MSRA , anti-MSRB1 , anti-MSRB2 , anti-MSRB3 , anti-TRX , anti-TRXR  or anti-β-Actin . After washing in PBS and TBS (2 × 5 min each), membranes were incubated with horseradish peroxidase-coupled secondary antibodies for 1 h at room temperature and probed using SuperSignal West Pico or Femto chemiluminescent substrate (Thermo Scientific). Luminescence was detected and quantified on a Bio-Rad Molecular Imager and with the ImageLab software. Primary data and original western blot membranes are shown in 2 on poly-L-lysine-coated 25-mm cover slips. Slides were washed with PBS and fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton-X/PBS for 3 min. After 3 × 10 min PBS washes, samples were blocked with 0.13% gelatin from cold-water fish skin (Sigma-Aldrich) for 1 h. Slides were incubated for 2 h in 0.13% gelatin with anti-α-Syn antibody (1:500 Abcam ab51252), washed 3 × 10 min with PBS and incubated with anti-Rabbit IgG Atto647  for 1.5 h in 0.13% gelatin. Cells were washed 3 × 10 min with PBS and stained with 2 μg ml−1 4,6-diamidino-2-phenylindole (Invitrogen) in PBS for 15 min. Images were acquired at × 60 magnification.Immunofluorescence images of fixed cells electroporated with reduced or oxidized α-Syn were acquired on a Zeiss LSM ultraviolet confocal microscope. After electroporation, cells were recovered for 5 h at 37 °C, 5% COIn vitro phosphorylation reactions were carried out in duplicates at 25 °C with 250 U (1 U=1 pmol min−1) of the respective kinases and 25 μM of α-Syn in NMR buffer, at pH 7.0, supplemented with 1 mM ATP (Fermentas), MgCl2 and DTT according to the suppliers' instructions. Final reaction volumes were 150 μl in all cases.Fyn tyrosine kinase was purchased from SignalChem and Plk3 serine kinase from Millipore. How to cite this article: Binolfi, A. et al. Intracellular repair of oxidation-damaged α-synuclein fails to target C-terminal modification sites. Nat. Commun. 7:10251 doi: 10.1038/ncomms10251 (2016).Supplementary Figures 1-9 and Supplementary Table 1.


Mangifera pajang Kosterm is a plant species from the mango family (Anacardiaceae). The fruits are edible and have been reported to have high antioxidant content. However, the detailed phytochemical studies of the plant have not been reported previously. This study investigates the phytochemicals and biological activities of different parts of Mangifera pajang.The plant samples were extracted with solvents of different polarity to obtain the crude extracts. The isolated compounds were characterized using spectroscopic methods. The extracts and isolated compounds were subjected to cytotoxicity tests using human breast cancer (MCF-7), human cervical cancer (HeLa) and human colon cancer (HT-29) cells. The free radical scavenging activity test was conducted using the DPPH assay. Antimicrobial activity tests were carried out by using the disc diffusion method.Mangifera pajang led to the isolation of methyl gallate (1), mixture of benzaldehyde (2) and benzyl alcohol (3), mangiferonic acid (4), 3β-hydroxy-cycloart-24-ene-26-oic acid (5), 3β,23-dihydroxy-cycloart-24-ene-26-oic acid (6), lupeol(7) lupenone(8), β-sitosterol(9), stigmasterol(10), trans-sobrerol(11) and quercitrin (12). Crude ethyl acetate and methanol extracts from the kernel indicated strong cytotoxic activity towards MCF-7 and HeLa cells with IC50 values of less than 10 μg/mL, while petroleum ether, chloroform and ethyl acetate extracts of the stem bark showed strong to moderate activity against MCF-7, HeLa and HT-29 cancer cell lines with IC50 values ranging from 5 to 30 μg/mL. As for the antimicrobial assays, only the ethyl acetate and methanol extracts from the kernel displayed some inhibition against the microbes in the antibacterial assays. The kernel extracts showed highest free radical scavenging activity with IC50 values of less than 10 μg/mL, while the ethyl acetate and methanol extracts of leaves displayed only weak activity in the DPPH assays.Phytochemical investigation on the kernel, stem bark and leaves of Mangifera pajang have identified terpenoids and a flavonol derivative as major constituents. Bioassay studies have indicated that the crude extracts and isolated compounds have potential as naturally-derived anticancer and antimicrobial agents, besides possess high free radical scavenging activity.Phytochemical investigations on various parts of  Mangifera pajang Kosterm is also known as ‘bambangan’; a plant species from the mango group which can be found in Borneo Island  [Mangifera indica), the fruit of Mangifera pajang are rarely eaten as it is unpopular among the public in Peninsula Malaysia and it is relatively unknown as antioxidant source. The fruits are ovoid and light-brown coloured, while the peels are thick which constitute approximately 27% of the whole bambangan fruit. The bambangan fruit is also about three times larger than its more common counterpart, mango [Mangifera pajang.imantan) . Unlike t, mango . The pult, mango . In Sabat, mango . Previout, mango ,6. HowevThe plant was collected from Sabah, Malaysia and was identified by Dr Mohd Fadzelly Abu Bakar from Universiti Tun Hussein Onn Malaysia (UTHM). The voucher specimen was deposited at the herbarium, BORNEENSIS, Universiti Malaysia Sabah. The plant samples  were air-dried and ground into a fine powder prior to being used.Mangifera pajang were extracted successively, each three times with petroleum ether, chloroform, ethyl acetate and methanol respectively for three days, for each extract by cold maceration method. Small portions of each crude extract were put aside for bioassays and the rest were fractionated using the column chromatographic method. From the kernel of the plant, β-sitosterol (9) (7 mg) and a yellow oil containing mixture a of benzaldehyde (2) and benzyl alcohol (3) were obtained from petroleum ether (12.26 g) and chloroform extracts (1.30 g) while isolation work on the methanol extract (21.95 g) gave methyl gallate (1) (1.15 g). Various crude extracts; petroleum ether (18.08 g), chloroform (3.59 g), ethyl acetate (1.24 g) and methanol (5.04 g) extracts from stem bark of Mangifera pajang were fractionated using column chromatography which afforded mangiferonic acid (4) (3.10 g), 3β-hydroxy-cycloart-24-ene-26-oic acid (5) (2.27 g) and 3β,23-dihydroxy-cycloart-24-ene-26-oic acid (6) (10 mg), lupeol (7) (3.15 g), β-sitosterol (9) (9 mg), stigmasterol (10) (5 mg) and trans-sobrerol (11) (14 mg). In addition, fractionation of the leaves extract gave lupeol (7) (20 mg), lupenone (8) (16 mg), methyl gallate (1) (11 mg) and quercitrin (12) (1.29 g). The structures of the isolated compounds were elucidated using spectroscopic methods including infrared spectroscopy (IR), mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR).The ground kernel (180 g), leaves (500 g) and stem bark (330 g) of Methyl gallate (1): white solid, C8H8O5,m.p. 190–192°C (lit. m.p 188–189°C) [−1) νmax: 3347, 3100, 2850, 1686, 1441, 1466.EI-MS m/z (% intensity):184 . 1H and 13C NMR spectral data were in good agreement with the published data [8–189°C) . IR  and benzyl alcohol (3): yellow oil, IR  νmax: 3400, 1739, 1457. EI-MS m/z (% intensity) of benzaldehyde (C7H6O): 106 . EI-MS m/z (% intensity) of benzyl alcohol (C7H8O):108 .Mangiferonic acid (4): white solid, C30H46O3, m.p.185-188°C (lit. m.p. 188–192°C) [−1) νmax: 3306, 3067, 1681, 1642, 1454, 1376, 1106.EI-MS m/z (% intensity):454 .1H and 13C NMR spectral data were in good agreement with the published data [3β-Hydroxy-cycloart-24-ene-26-carboxylic acid (5): white solid, C30H48O3, m.p. 175–177°C (lit. m.p. 177–178°C) [−1) νmax: 3361, 2933, 2867, 1683, 1634, 1447, 1372, 1020. EI-MS m/z(% intensity):456 .1H and 13C NMR spectral data were in good agreement with the published data [3β,23-Dihydroxy-cycloart-24-ene-26-oic acid (6): white needle-shaped crystals, C30H48O4, m.p. 276-278°C (lit. m.p 279–281°C) [−1) νmax: 3318, 3101, 2933, 2870, 1670, 1439, 1374, 1043. EI-MS m/z(% intensity):472 . 1H and 13C NMR spectral data were in good agreement with the published data [Lupeol (7): white solid, C30H50O, m.p 199-210°C (lit. m.p 212-213°C) [−1) νmax: 3308, 2925, 2858, 1639, 1455, 1377, 1034. EI-MS m/z(% intensity):426 . 1H and 13C NMR spectral data were in good agreement with the published data [Lupenone (8): Colourless needle-shaped crystals, C30H48O, m.p. 166–168°C (lit. m.p 168-170°C) [−1) νmax: 2938, 2861, 1702, 1643, 1451, 1380, 869. EI-MS m/z(% intensity):424 . 1H and 13C NMR spectral data were in good agreement with the published data [8-170°C) . IR (UAThed data .β-Sitosterol (9): white needle-shaped crystals, C29H50O, m.p. 133–135°C (lit. m.p 136-138°C) [−1) νmax: 3413, 2935, 2860, 1673, 1455, 1372, 1047. EI-MS m/z(% intensity):414 . 1H and 13C NMR spectral data were in good agreement with the published data [6-138°C) . IR (UATStigmasterol (10): colourless needle-shaped crystals, C29H48O, m.p. 168 to 170°C (lit. m.p 167 - 169°C) [−1)νmax: 3429, 2928, 2857, 1711, 1460, 1375, 1058. EI-MS m/z (% intensity):412 . 1H and 13C NMR spectral data were in good agreement with the published data [- 169°C) . IR (UATmasterol : colourlTrans-sobrerol (11): white needle-shaped crystals, C10H18O2, m.p. 130 to 132°C (lit m.p 130–133°C) [−1) νmax: 3413, 2935, 2865, 1673, 1455, 1372, 1047.EI-MS m/z (% intensity):170 .1H and 13C NMR spectral data were in good agreement with the published data [0–133°C) . IR (UAThed data .Quercitrin (12): yellow solid, C21H20011, m.p. 188–190°C (lit. m.p 179–182°C) [−1) νmax: 3429, 1653, 1599, 1498, 1062, 814. EI-MS m/z (% intensity):302 (M+- Rham). 1H and 13C NMR spectral data were in good agreement with the published data [9–182°C) . IR (UATMangifera pajang were screened for cytotoxic activity against MCF-7 (human breast cancer), HeLa  and HT-29 (human colon cancer) cells according to the method described previously [50 values, which were defined as the concentration of the test samples that resulted in a 50% reduction of absorbance or a measure of 50% concentration of tested samples that was required to inhibit the growth of cancer cells. Extracts and isolated compounds that indicated IC50 values < 10 μg/mL, are considered to have significant cytotoxic activity against that particular cell line.Various crude extracts of eviously . The sto50 values were determined by plotting the percentage of inhibition against sample concentration, ranging from 500 to 7.81 μg/mL. The IC50 values are defined as the amount of antioxidants needed to decrease the initial concentration of DPPH by 50%.The scavenging activity of the crude extracts and constituents were determined by using 1,1- diphenyl-2-pycrylhydrazyl (DPPH) according to a procedure described previously . DPPH waMangifera pajang  were subjected to antibacterial and antifungal assays towards several targeted microbes including Methicillin resistant staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Salmonella choleraesuis and Bacillus subtilis for antibacterial screening while Candida albican, Aspergillus ochraceaus and Sacchoromyces cerevisiae were used for antifungal screening. The assays were carried out using the disc diffusion method [All crude extracts from n method . This inMangifera pajang have afforded several classes of constituents including aromatic esters, cycloartane and lupane triterpenes, a monoterpene, steroids and a flavonol glycoside.β-sitosterol (9) have been isolated from the kernel. Cycloartane triterpenes; mangiferonic acid (4), 3β-hydroxy-cycloart-24-ene-26-oic acid (5) and 3β,23-dihydroxy-cycloart-24-ene-26-oic acid (6), lupeol (7), β-sitosterol (9), stigmasterol (10) and a monoterpene identified as trans-sobrerol (11) were obtained from the stem bark. Meanwhile lupane triterpenes; lupeol (7) and lupenone (8) together with methyl gallate (1) and a flavonol glycoside; quercitrin (12) have been isolated from the leaves. The structure of compounds shown in Figure An aromatic ester; methyl gallate 1) and a mixture of benzaldehyde (2) and benzyl alcohol (3) together with  and a mi, lupeol , β-sitosm/z 106 and 108 corresponded to benzaldehyde (2), C7H6O and benzyl alcohol (3), C7H8O, respectively.Compound (1) was reported previously from ripe bambangan peel using HPLC-DAD and TSQ-ESI-MS analysis . The comMangifera indica [Mangifera pajang.Compounds (4), (5), and (6) which belong to cycloartane type triterpenes were obtained from the non-polar extracts of the stem bark. These compounds were previously isolated from a indica ,9 and thMangifera pajang. Lupeol (7) was obtained both from the stem bark and leaves while lupenone (8) was isolated only from the leaves. The EI-MS spectra of the compounds show molecular ion peaks at m/z 426 and 424 which corresponded to molecular formulae C30H50O (lupeol) and C30H48O (lupenone), respectively. Compound (9) and (10) which were identified as β-sitosterol and stigmasterol have also been isolated from the kernel and stem bark. The identities of the compounds were confirmed by comparing its physical and spectral data with the literature values [Two lupane triterpenes, lupeol (7) and lupenone (8) were obtained from the extracts of the stem bark and leaves of e values ,14.trans-sobrerol and quercitrin, respectively. Compound (11) is a monoterpene while compound (12) is a flavonol glycoside and both compounds have never been isolated from the genus Mangifera. Compound (11) was isolated from chloroform extract of the stem bark and appeared as white needle crystals with a melting point of 130 - 132°C and the spectral data were in good agreement with the published data [Myrsine seguinii [Compounds (11) and (12) were identified as hed data . Meanwhiseguinii .Phytochemical investigations on the kernel, stem bark and leaves of 50 values less than 10 μg/mL, and displayed strong to moderate activities towards the HT-29 cell line. In addition, petroleum ether and chloroform extracts of the stem bark also showed high cytotoxic activity towards MCF-7, HeLa and HT-29 cells with IC50 values of less than 15 μg/mL. In contrast, the ethyl acetate extract of the stem bark and the petroleum ether extract of the leaves displayed rather weak activity with IC50 values of more than 20 μg/mL. Similarly, most of the extracts from the leaves were not active with IC50 values being more than 30 μg/mL. Previous studies on the cytotoxic activity of Mangifera pajang have been reported on the ethanolic extract of its kernel towards MCF-7 (hormone-dependent breast cancer cells) and MDA-MB-231 (non-hormone dependent breast cancer cells) with IC50 values of 23 and 30.5 μg/mL, respectively [50 value of 10.5 ± 0.29 μg/mL. Meanwhile, 3β-hydroxy-cycloart-24-ene-26-oic acid (5) displayed moderate cytotoxic activity towards MCF-7 with an IC50 value of 13.03 ± 0.81 μg/mL. In contrast, weak cytotoxic activities were shown by lupeol (7), lupenone (8) and quercitrin (12) with IC50 values of 25.02 ± 0.71 , 27.01 ± 0.34 and 25.04 ± 0.72 μg/mL respectively, while mangiferonic acid (4) was inactive with IC50 > 30 μg/mL.Ethyl acetate and methanol extracts of the kernel showed strong cytotoxic activity towards MCF-7 and HeLa cell lines with ICβ-hydroxy-cycloart-24-ene-26-oic acid (5) with the IC50 value 6.27 ± 0.61 μg/mL. Apart from that, moderate activities were demonstrated by quercitrin (12), mangiferonic acid (4) and lupeol (7) with IC50 values of 11.93 ± 0.63, 16.51 ± 0.55 and 13.09 ± 0.80 μg/mL respectively. Other isolated constituents, methyl gallate (1) and lupenone (8) showed no activity with IC50 values > 30 μg/mL.The results from cytotoxic tests of the chemical constituents of the plant against human cervical cancer (HeLa) cells Table  indicate50 values of 3.82 ± 0.91 μg/mL. In addition, methyl gallate (1) and mangiferonic acid (4) have also demonstrated moderate cytotoxic activity with IC50 values of 18.07 ± 0.37 and 18.03 ± 0.75 μg/mL, respectively. However, no cytotoxic activity were shown by 3β-hydroxy-cycloart-24-ene-26-oic (5), lupeol(7) and lupenone (8) where the IC50 values were more than 30 μg/mL.Meanwhile, cytotoxic tests against human colon cancer (HT-29) cell Table  indicate50 value of more than 70 μg/mL) against human cervix adenocarcinoma cells (HeLa) and human fibroblast cells (L-132) [Some of the isolated compounds have been reported to show cytotoxic activities against several cancer cell lines. Methyl gallate (1) was shown to possess weak anticancer effects (IC (L-132) . However (L-132)  demonstr50 values of around 100 μg/mL towards three murine cancer cell lines  and also towards three human cancer cell lines .Mangiferonic acid (4) reported by Li et al.  showed i50 values more than 30 μg/mL against human colorectal cancer (HT-29) and mammary breast cancer (MDA-MB) cell lines. However, lupeol (7) was slightly toxic against the normal cell line (3 T3) with an IC50 value of 38.92 μg/mL while lupenone (8) did not demonstrate any cytotoxic effect against the 3 T3 cell line with IC50 values of more than 100 μg/mL [Meanwhile, anticancer activities of lupeol (7) as reviewed by Gallo and Sarachine,  found th00 μg/mL . Meanwhi00 μg/mL .All crude extracts from the kernel, stem bark and leaves were subjected to cytotoxic screening against MCF-7, HeLa and HT-29 cancer cells and the results are summarized in Table 50 values less than 6 μg/mL for the three cancer cell lines. This may due to the presence of the cytotoxic methyl gallate (1) constituent. Methyl gallate (1) which is an ester . Quercitrin (12) is a glycoside of quercetin with a rhamnose group attached to the hydroxyl group at C-3. However, the glycoside substituent on the ring did not enhance the cytotoxicity of quercitrin (12) as its parent flavonol structure; quercetin significantly inhibited human prostate cancer cell line PC-3 cell proliferation, whereas quercitrin (12) itself did not have any antiproliferation effect on the cancer cells [Quercitrin (12) obtained from extracts of leaves in polar solvents showed good anticancer activity with ICer cells .In addition, lupeol (7) and lupenone (8) together with most of leaf extracts exhibited weak cytotoxicity towards all cancer cell lines. Lupeol (7) is slightly more cytotoxic compared to lupenone (8) due to the presence of a hydroxyl group at C-3.The moderate to strong anticancer activities of the constituents of the plant have contributed to the overall cytotoxic properties of the plant extracts.50 values of less than 10 μg/mL, while polar (ethyl acetate and methanol) extracts of the leaves showed moderate to weak radical scavenging activity with IC50values 100 to 200 μg/mL. Previous studies have reported high scavenging activity of the fruit parts  of Mangifera pajang [Free radical scavenging activity was determined by DPPH assay , 3β-hydroxy-cycloart-24-ene-26-oic acid (5), lupeol (7) and lupenone (8) were not active with IC50 > 300 μg/mL. The strong scavenging activity of methyl gallate (1) was also reported previously [50 values of 2.8 μg/mL for methyl gallate in the DPPH free radical assay. However till date, there has been no previous report on free radical scavenging activity of mangiferonic acid (4) and 3β-hydroxy-cycloart-24-ene-26-oic acid (5).Further DPPH screening assay on the isolated compounds indicated that methyl gallate (1) showed the highest radical scavenging activity with ICeviously , where tMRSA with an inhibition zone of 21.5 mm, as compared with an inhibition zone of 23.0 mm given by the standard compound (streptomycin). In addition, methyl gallate (1) also exhibited moderate antimicrobial activities towards P. aeruginosa, S. choleraesuis and B. subtilis with inhibition zones 12.0, 15.5, and 12.5 mm, respectively. This was in agreement with previous reports on the potent antimicrobial properties of methyl gallate (1) [B. subtilis; Streptomyces viridochromogenes; S. aureus; Escherichia coli; Mucor miehei; Candida albicans. Meanwhile, there were no inhibition zones shown by quercitrin (12), mangiferonic acid (4), 3β-hydroxy-cycloart-24-ene-26-oic (5) and lupeol (7) towards the targeted microbes.In this study, only the isolated compound methyl gallate (1) demonstrated strong antibacterial activity towards late (1)  which shCandida albican, Aspergillus ochraceaus and Sacchoromyces cerevisiae. To our knowledge, this is the first antimicrobial activity study reported on Mangifera pajang.As for the antifungal tests, none of extracts and isolated compounds showed activity against The results of the agar diffusion assay indicated that most of the crude extracts did not show significant inhibition activity towards targeted microbes. Some crude extracts of different parts of the plant displayed either weak or moderate activities with inhibition zones between 6 and 13 mm.Mangifera pajang have demonstrated potential cytotoxic activity towards MCF-7, HeLa and HT-29 cancer cells, and the extract of kernel also displayed strong free radical scavenging activity. These are assumed to be due to the presence of bioactive constituents that were isolated including cycloartane and lupane triterpenes, together with the major constituent, methyl gallate. The plant species, Mangifera pajang could become a potential source for natural anticancer  and antioxidant agents. In particular, the major isolated constituent, methyl gallate could become a potential anticancer, antioxidant and antimicrobial agent.Extracts of the kernel, stem bark and leaves of


The α2δ ligand gabapentin interacts with α2δ-1, and inhibits calcium channel trafficking. However, α2-1 has also been proposed to play a synaptogenic role, independent of calcium channel function. In this regard, α2δ-1 was identified as a ligand of thrombospondins, with the interaction involving the thrombospondin synaptogenic domain and the α2δ-1 von-Willebrand-factor domain. Co-immunoprecipitation between α2δ-1 and the synaptogenic domain of thrombospondin-2 was prevented by gabapentin. We therefore examined whether interaction of thrombospondin with α2δ-1 might reciprocally influence 3H-gabapentin binding. We concentrated on thrombospondin-4, because, like α2δ-1, it is upregulated in neuropathic pain models. We found that in membranes from cells co-transfected with α2δ-1 and thrombospondin-4, there was a Mg2+ -dependent reduction in affinity of 3H-gabapentin binding to α2δ-1. This effect was lost for α2δ-1 with mutations in the von-Willebrand-factor-A domain. However, the effect on 3H-gabapentin binding was not reproduced by the synaptogenic EGF-domain of thrombospondin-4. Partial co-immunoprecipitation could be demonstrated between thrombospondin-4 and α2δ-1 when co-transfected, but there was no co-immunoprecipitation with thrombospondin-4-EGF domain. Furthermore, we could not detect any association between these two proteins on the cell-surface, indicating the demonstrated interaction occurs intracellularly.The α V1 and CaV2 voltage-gated calcium channels are associated with auxiliary β and α2δ subunits, which influence both the expression on the plasma membrane and the biophysical properties of the channels  are multi-domain secreted extracellular matrix proteins  with div2δ-1 demonstrated previously3H-gabapentin binding. We primarily concentrated on TSP4, as, like α2δ-183H-gabapentin binding to α2δ-1, which might influence the efficacy of this drug. We also performed co-immunoprecipitation and immunocytochemical experiments to examine whether α2δ-1 and TSP4 interacted with each other in this system.In the present study, our aim was to examine whether the gabapentin-sensitive interaction between TSPs and α3H-gabapentin, and only in the presence of Mg2+, whereas the isolated TSP4 EGF domains did not. Furthermore although we were able to demonstrate partial co-immunoprecipitation of α2δ-1 and full length TSP4, this did not occur for the EGF domains of TSP4. In immunocytochemistry experiments we could not demonstrate co-localisation of α2δ-1 and TSP4 at the cell surface of transfected cells, although both α2δ-1 and TSP4 could be detected intracellularly and TSP4 was secreted from transfected cells, in the proximity of cells expressing α2δ-1 on the cell surface.Our ligand binding experiments show that co-expression of full length TSP4 modestly reduced the binding affinity for 2δ-1 from brain2δ-1 could also be co-immunoprecipitated with a TSP2 synaptogenic region containing its EGF domains, when they were both co-expressed in HEK-293 cells. It was then shown that this co-immunoprecipitation was reduced in the presence of the α2δ ligand gabapentin3H-gabapentin binding to α2δ-1, we examined the effect of TSP4 because, like α2δ-1, it is implicated in neuropathic pain models2δ-1 and TSP4 together in tsA-201 cells and performed 3H-gabapentin binding assays on membrane preparations. The two main constructs used in these experiments were TSP4_HA (termed TSP4) or its truncated form containing the EGF domains  , or in the presence of 2 mM Mg2+  . In all  of TSP4 . Since T mM Mg2+ . We foun of EDTA . This is in Mg2+ . In contnly TSP4 .2δ-1 construct containing three point mutations in the metal ion dependent adhesion site (MIDAS) motif of the VWA domain2δ-1 MIDASAAA bound 3H-gabapentin with an affinity similar to WT α2δ-1 .The EGF-like domains of TSPs were identified previously as their synaptogenic region, and for TSP2 a domain including the EGF repeats was found to be the region interacting with α of Mg2+ . In a co2δ proteins are strongly concentrated in detergent-resistant membrane (DRM) fractions22232δ-1 in DRMs, as evidence of an interaction. When co-expressed with α2δ-1 in tsA-201 cells, only a very small proportion  and endogenous α2δ-1. However, in an experiment using adult rat brain tissue, we found that the 3H-gabapentin binding affinity was not altered by the presence of EDTA or MgCl2, either in crude brain membranes  binding to the VWA domain of α2δ-1 in a Mg2+ -dependent manner.We therefore examined whether there was a Mg2δ-1 and TSP4 in our system, and to understand where in the cell this putative interaction was occurring we also performed co-immunoprecipitation and immunocytochemistry. For co-immunoprecipitation studies, we expressed α2δ-1 and TSP4_HA together in tsA-201 cells. Full-length TSP4 was well-expressed , despite the clear presence of a neighbouring cell secreting TSP4  and Golgi apparatus. In permeabilised cells, when both TSP4 and α2δ-1 were transfected together, TSP4 was co-localised in part with both the ER marker PDI and the Golgi marker Golgin-97, but also showed extensive punctate localisation near the plasma membrane, which was not associated with ER or Golgi  with an HA tag on the C terminus, The tsA-201 cells were cultured as described previously102 inhalation and then decapitated. The whole brain minus cerebellum was homogenised on ice in a buffer containing the following (in mM): 20 HEPES, pH 7.4, 2 EDTA, EDTA-free protease inhibitor cocktail .Cell membrane and DRM fractions were prepared essentially as described previouslyg. The supernatant obtained was transferred to a 30 ml thick-walled ultracentrifuge tube (Beckman-Coulter). Membranes were pelleted in an ultracentrifuge (Beckman-Coulter) for 90 min at 60,000 × g, and resuspended as required. Aliquots were used to determine the protein concentration. For detection of secreted TSP4, medium was collected from transfected cells and concentrated 15-fold using 10 kDa cut-off filter .For membrane preparation, pellets obtained from cell harvesting, resuspended in ice-cold 10 mM Hepes plus protease inhibitors (pH 7.4), or brain homogenate, were mechanically lysed by 10–15 passages through a 23 gauge needle. Cell debris and non-lysed cells were pelleted for 15 min at 1,000 × g for 18 h at 4 °C (Beckman SW40 rotor). 1 ml fractions were subsequently harvested from the top to the bottom of the tube. When necessary, protein fractions from the gradient were washed free of sucrose by dilution into 25 volumes of PBS and centrifugation  to pellet the DRM material. The material was either used immediately or stored at −20 °C until required.For DRM preparation, the method used was essentially as described previously3H-gabapentin binding was performed essentially as described previously3H]-gabapentin , was aliquoted and stored at −80 °C. The specific activity was about 110 Ci/mmol and the concentration 1 mCi/ml. Non-radioactive gabapentin (U.S. Pharmacopea or Pfizer) was stored at 4 °C until use and all the solutions and the reagents were kept on ice during the preparation. Binding of [3H]-gabapentin was performed in a final volume of 250 μl at room temperature for 90 min. 50 μg/tube of membranes or 3 μg/tube of DRMs were incubated with various concentrations of [3H]-gabapentin in 10 mM HEPES/KOH, pH 7.4. Each experiment was carried out in triplicate, and the mean value used for subsequent analysis. Concentrations of [3H]-gabapentin higher than 20 nM were achieved by adding non-radioactive gabapentin and correcting the specific binding by the dilution factor  pre-soaked in 0.3% polyethyleneimine solution, in order to reduce non-specific binding to the filter paper. The samples were transferred to the filters by washing the tubes three times with Tris-HCl  using a Brandel Harvester (Brandel). The GF/B filter papers were then cut out and transferred to vials containing 5 ml of scintillant . Samples were equilibrated for 18 h and then the activity was counted in triplicate in a scintillation counter (Beckman Coulter) for 10 min per sample. Data were fit using the Hill- equation (y = Bmax*xn/(KDn + xn), where Bmax is the total number of binding sites; n, termed nH in the text, is the Hill coefficient and KD is the binding affinity. Hill coefficients were usually >1 and <1.5, which is very common for ligand binding studies, for reasons discussed previouslyNon-specific binding was determined in the presence of 100-fold or more excess of non-radioactive gabapentin (100 μM). A maximum level of non-specific binding of 25% of total was accepted. Total counts per minute were determined (in duplicate) to give an estimate of the activity of the [2δ-1 (unless stated) an anti-α2δ-1  was used. Where stated, a custom-made rabbit polyclonal antipeptide antibody directed against residues EPFPSAVTIKSWVDK . Other antibodies used were: anti-HA , anti-flotillin-1 , anti-GAPDH , anti-TSP4 antibody , rabbit polyclonal or mouse monoclonal anti-FLAG antibodies (Sigma), mouse Golgin-97 (Molecular Probes) and mouse Protein disulphide isomerase .The following primary antibodies were used: for α2δ-1 and TSP4_HA (or non-tagged TSP4 in g for 30 min at 4 °C. The supernatant WCL (1 mg protein) was pre-cleared for 2 h at 4 °C with 20 μl of Protein-G agarose beads (Invitrogen) to reduce the non-specific binding to the agarose beads (except in g for 30 s. and then incubated overnight with 5 μg/ml of rabbit anti-HA antibody or mouse anti-FLAG antibody at 4 °C. It was then incubated with Protein-G (or A/G-PLUS) beads for 2 h at 4 °C. A series of washes were carried out in order to reduce the non-specific binding to the beads  with rabbit anti-HA (1:500) for experiments where α2δ-1 and TSP4_HA were co-expressed; rabbit anti α2δ-1 (1:100) with mouse anti-Golgin-97 (1:500) or mouse anti-PDI (1:100) antibodies. Primary antibodies were incubated overnight at 4 °C, followed by the secondary antibodies, fluorescein isothiocyanate (FITC)-conjugated anti-mouse  or anti-rabbit Alexa Fluor 594 , for 1 h at room temperature. DAPI  was also used to visualise the nuclei. Cells were mounted in Vectashield  to reduce photobleaching. Cells were examined on a confocal laser scanning microscope .


The transcription factor CCAAT/enhancer binding protein α (C/EBPα) and microRNA (miRNA) let-7a-1 act as tumor suppressors in many types of cancers including lung cancer. In the present study, we aim to investigate whether let-7a-1 is a novel important target of C/EBPα in lung cancer cells.http://www.genomatix.de). Human lung cancer cell lines A549 and H1299, and human cervical cancer cell line Hela were used for transfection. Total RNA was extracted from cells using Trizol reagent and pri-let-7a-1 mRNA expression was measured using quantitative real-time polymerase chain reaction. Western blotting was performed to detect C/EBPα protein expression. To test whether C/EBP-α could up-regulate the expression level of let-7a at transcription level, dual-luciferase reporter gene assay was carried out. To determine whether C/EBPα could bind let-7a-1 promoter, electrophoretic mobility shift assay was employed. To further confirm the direct targeting let-7a-1 promoter by C/EBPα, chromatin immunoprecipitation was used.The DNA sequence of the 2.1 kb let-7a-1 promoter was analyzed with MatInspector 4.1 (Both C/EBPα and let-7a-1 were down-regulated in lung cancer A549 and H1299 cells, but up-regulated in Hela cells. Transfection and reporter gene assay showed that C/EBPα increased the expression of let-7a-1 at transcription level. Bioinformatics assay identified four putative C/EBP elements within let-7a-1 promoter. Dual-luciferase reporter gene, electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrated that these four elements mediated the up-regulation effect of C/EBPα on let-7a-1.The present study reveals that decreased C/EBPα contributes to the down-regulation of miRNA let-7a-1 in lung cancer cells. MicroRNA (miRNA) let-7 family plays pivotal roles in regulating carcinogenesis . The letHumans have 13 let-7 family members , includiThe CCAAT/enhancer binding protein α (C/EBPα) is a differentiation-inducing transcription factor that belongs to a family of basic region leucine zipper (bZIP) transcription factors, which includes six members  . C/EBPα http://www.genomatix.de). The MatInspector 4.1 software identifies putative transcription factor binding sites with weight matrices representing consensus recognition sequences for different transcription factors as defined in the MatInspector library . Protein concentration was determined using Bradford assay . Extracted protein (30 μg) was processed in a 10 % SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane. Protein levels of C/EBPα were measured by antibody raised against the C-terminus of human C/EBPα . Anti-β-Actin  was used as an internal control. Immunoblots were detected using an enhanced chemiluminescence kit  and visualized after exposure to X-ray film.Cells in 70–80 % confluence were co-transfected in 24-well plates. Each well contained 0.3 μg reporter gene plasmid, 0.02 μg of the internal control vector pGL4.74 [hRluc/TK] vector , 1 μl transfection agent, and 0.2 μg expression vector or si-RNA. At 48 h after the completion of the transfection procedure, all the cells underwent dual-luciferase reporter assay, following the protocol recommended by the manufacturer’s manual. The cells were lysed using 1× reporter lysis buffer and harvested by manual scraping. Luminescence was detected using a Mithras LB 940 . The firefly luciferase activity of the reporter gene plasmid was measure 1 (M1), and the renilla luciferase activity  of pGL4.74 [hRluc/TK] vector was measure 2 (M2). The relative luciferase activity was calculated as the ratio of M1/M2.At 24 h before transfection, H1299 cells were inoculated in 6-well plate. When in 70–80 % confluence, each well was transfected by 0.2 μg pcDNA3.1(+)-C/EBPα. At 48 h after transfection, the cells were collected and prepared for nuclear extracts as described previously . Double-6 H1299 cells and 2 μg of goat anti-C/EBPα  or control IgG  were used. The immunoprecipitated DNA was recovered and used as template for PCR using three pairs of primers complementary to the region flanking the C/EBPα motifs in let-7a-1 promoter -C/EBPα or pcDNA3.1(+). At 48 h after transfection, the cells were collected and ChIP assay was performed using ChIP Assay Kit  following the manufacturer’s protocol. For each immunoprecipitation reaction, 10All data were analyzed using SPSS 17.0 software package . All values were expressed as mean ± standard deviation from at least three independent experiments. Statistical significance was defined as P < 0.05.To determine the expression levels of C/EBPα and pri-let-7a-1, western blotting and qRT-PCR were used, respectively. The data showed that C/EBPα and pri-let-7a-1 levels in A549 cells were significantly higher than that in H1299 cells, but were significantly lower than that in Hela cells  , H1299 cells transfected with pcDNA3.1(+)-C/EBPα showed that all of four C/EBP sites within let-7a-1 promoter were indeed associated with C/EBPα immunoprecipitates (Fig. H. sapiens have been identified so far. As class II genes, expressions of many miRNAs are also regulated by enhancers or hormones [miRNAs are a group of gene regulators that play important roles in biologic processes such as cell proliferation, differentiation, and apoptosis. The dysregulation of miRNAs is associated with the development of cancers, including lung cancer , 28, 29.hormones –32. As aC/EBPα is a basic leucine zipper transcription factor that is expressed in many tissues, and plays an important role in regulating the balance between cell proliferation and cell differentiation , 33. LosOur previous study shows that C/EBPα enhances the promoter activity of let-7a-1 gene in lung cancer A549 cells . In the Bioinformatics assay has identified four putative C/EBP elements within let-7a-1 promoter. We infer that at least one of them may mediate the transactivation effect of C/EBPα. To confirm this, luciferase activity assay was performed in H1299 cells to analyze the function of the four putative CEBP elements. The results showedthat all of the four elements respond to ectopic expression of C/EBPα, and their luciferase activities are higher than that of C/EBP consensus sequence used as positive control. Among them, CEBP3 element has displayed the highest activity. Furthermore, in vitro binding assay shows that all of the four C/EBP elements and C/EBP consensus sequence have binding capacity with nuclear protein extracted from H1299 cells with ectopic C/EBPα expression. In addition, these binding complexes can be blocked by anti-C/EBPα antibody specifically. At last, ChIP assay demonstrates the direct interaction between C/EBPα and the four C/EBP elements. In summary, the present study identifies tumor suppressor miRNA let-7a-1 gene as a new direct target of transcription factor C/EBPα. Our results conclude that decrease or absence of tumor suppressor C/EBPα contributes to the transcriptional inhibition of let-7a-1.


III atom in the title molecule adopts a distorted octahedral coordination sphere, being C,N-chelated by two main 2-tert-butoxy-6-fluoro-3-(pyridine-2-yl)pyridine-4-yl ligands and O,O′-chelated by one ancillary pentane-2,4-dionato ligand.The Ir 14H14FN2O)2(C5H7O2)], is located on a twofold rotation axis, which passes through the IrIII atom and the central C atom of the pentane-2,4-dionate anion. The IrIII atom adopts a distorted octahedral coordination geometry, being C,N-chelated by two 2-tert-butoxy-6-fluoro-3-(pyridin-2-yl)pyridin-4-yl ligands and O,O′-chelated by the pentane-2,4-dionato ligand. The bipyridinate ligands, which are perpendicular to each other [dihedral angle between the two least-squares planes = 89.95 (5)°], are arranged in a cis-C,C′ and trans-N,N′ fashion relative to the central metal cation. Intramolecular C—H⋯O and C—H⋯N hydrogen bonds and intermolecular C—H⋯F hydrogen bonds as well as π—π interactions between neighbouring pyridine rings [centroid–centroid distance 3.680 (1) Å] contribute to the stabilization of the molecular and crystal structure, respectively.The title molecule, [Ir(C Therefore, the asymmetric unit consists of one Ir(III) atom on Wyckoff position 4e, one half of the acetylacetonate anion and one 2-tert-butoxy-6-fluoro-3-(pyridin-2-yl)pyridin-4-yl ligand. The IrIII atom is six-coordinated by the two main C,N-bidentate ligands and one ancillary O,O′-bidentate ligand, forming a distorted octahedral coordination sphere due to the narrow ligand bite angles, which range from 80.36 (7) to 88.65 (8)°. The C,N-bidentate ligands, which are perpendicular to each other [dihedral angle between the least-squares planes = 89.95 (5)°], are arranged in a cis-C,C′ and trans-N,N′ fashion. The Ir—C bond length of 1.9760 (19) Å is shorter than the Ir—N bond length of 2.0344 (16) Å due to the electronegative fluorine substituent 2(acac); dfpypy is a difuorinated bipyridine] (picolinate) °.The molecular structure of the title compound, Fig. 1t Table 1. The Ir—Cg1—Cg1iii = 3.680 (1) Å, Cg1 is the centroid of the N1, C6–C10 ring, symmetry code: (iii) −x, 1 − y, 2 − z] contribute to the stabilization of the crystal structure  = 0.95 Å, Uiso(H) = 1.2Ueq(C) for Csp2 H atoms, and 0.98 Å, Uiso(H) = 1.5Ueq(C) for methyl protons.Crystal data, data collection and crystal structure refinement details are summarized in Table 310.1107/S1600536814022934/wm5075sup1.cifCrystal structure: contains datablock(s) I, New_Global_Publ_Block. DOI: 10.1107/S1600536814022934/wm5075Isup2.hklStructure factors: contains datablock(s) I. DOI: 1029929CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The anhydrous morpholinium salts of phenoxyacetic acid, (4-fluorophenoxy)acetic acid and the isomeric acetic acid and acetic acid, provide three similar examples of one-dimensional hydrogen-bonded chain polymers and one of a cyclic hydrogen-bonded heterotetramer. H-1,4-oxazin-4-ium) phenoxyacetate, C4H10NO+·C8H7O3−, (I), morpholinium (4-fluorophenoxy)acetate, C4H10NO+·C8H6 FO3−, (II), and isomeric morpholinium acetate , (III), and morpholinium acetic acid , C4H10NO+·C8H5Cl2O3−, (IV), have been determined and their hydrogen-bonded structures are described. In the crystals of (I), (III) and (IV), one of the the aminium H atoms is involved in a three-centre asymmetric cation–anion N—H⋯O,O′ R12(4) hydrogen-bonding interaction with the two carboxyl O-atom acceptors of the anion. With the structure of (II), the primary N—H⋯O interaction is linear. In the structures of (I), (II) and (III), the second N—H⋯Ocarboxyl hydrogen bond generates one-dimensional chain structures extending in all cases along [100]. With (IV), the ion pairs are linked though inversion-related N—H⋯O hydrogen bonds [graph set R42(8)], giving a cyclic heterotetrameric structure.The anhydrous salts morpholinium (tetrahydro-2- H-1,4-oxazine) is an moderately strong base (paK = 8.33) and forms salts with a number of organic acids, some having medical applications, e.g. the salicylate , used as an analgesic, an antipyretic and an anti-inflammatory agent  and a phenoxyacetate anion (A) in (I)A) in (II)A) in (III)A) in (IV)A—O11A—C12A—C13A = 176.75 (14) and 176.53 (14)°, respectively. This antiperiplanar (180±30°) conformation is similar to those of the parent acids PAA °] and PFPA [−178.98 (17)°]  .The asymmetric units of (I)–(IV) comprise a morpholinium cation carboxyl hydrogen-bonding interaction is present, with the asymmetry in (I)In the crystals of both (I)a through an N1B—H⋯O14Ai hydrogen bond –(IV) are minor weak inter-unit C—H⋯O interactions: in (I)A—H⋯O4Bii –(III) compared to that in (IV) Tables 1–7 ▸ ▸. B—H⋯O14Ai hydrogen bond generates a centrosymmetric heterotetrameric ring structure  Fig. 8. For symOnly weak inter-unit C—H⋯O interactions to carboxyl or phenoxy O-atom acceptors are present in (IV)The title compounds (I)–(IV) were prepared by the dropwise addition of morpholine at room temperature to solutions of phenoxyacetic acid (150 mg), (4-fluorophenoxy)acetic (170 mg), acetic acid or acetic acid (220 mg), respectively, in 15 ml of ethanol. Room-temperature evaporation of the solutions gave either colourless plates of (III)Uiso(H) = 1.2Ueq(C). The aminium H atoms were located in difference Fourier analyses and were allowed to refine with distance restraints [N—H = 0.90 (2) Å] and Uiso(H) = 1.2Ueq(N).Crystal data, data collection and structure refinement details are given in Table 510.1107/S2056989015019842/su5227sup1.cifCrystal structure: contains datablock(s) global, I, II, III, IV. DOI: 10.1107/S2056989015019842/su5227Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015019842/su5227IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989015019842/su5227IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989015019842/su5227IVsup5.hklStructure factors: contains datablock(s) IV. DOI: Click here for additional data file.10.1107/S2056989015019842/su5227Isup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015019842/su5227IIsup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015019842/su5227IIIsup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015019842/su5227IVsup9.cmlSupporting information file. DOI: 1432389, 1432388, 1432387, 1432386CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Their structures were elucidated by extensive spectroscopic analyses and by comparison with literature data. The discovery of these new secondary metabolites enriched the family of cembranoids deduced from the title animal.Three new highly oxidative cembranoids, sarcophytrols D–F ( Sarcophyton D values with those reported in the literature.Samples of  (4) [5)  by compa1) was obtained as colorless oil. The molecular formula was established as C20H30O3 by HRESIMS (m/z 343.2248 [M + Na]+), sixteen mass units more than that of compound 4 +), was identical to that of 1. A detailed 2D NMR analysis of 2 and careful comparison with the NMR data of 1 , while the C-6 and C-9 signals upfield shifted . The observed differences can be rationalized when the two compounds are C-8 epimers, which was confirmed by the ROESY interaction of H-6 (δH 5.81)/Me-19 (δH 1.36)  established the molecular formula C20H32O4 (m/z 359.2188 [M + Na]+), 16 mass units more than that of sinugibberol (5)  column was also employed. Pre-coated silica gel GF254 plates  were used for analytical thin-layer chromatography (TLC). All solvents used were of analytical grade .Optical rotations were measured on a Perkin-Elmer 341 polarimeter. HRESIMS spectra were recorded on a Waters-Micromass Q-TOF Ultima Global electrospray mass spectrometer. NMR spectra were measured on a Bruker-DRX-500 spectrometer with the residual CHClS. trocheliophorum were collected by scuba at Yalong Bay, Hainan Province, China, in February 26, 2006, at a depth of −15 to −20 m, and identified by Professor R.-L. Zhou of South China Sea Institute of Oceanology, Chinese Academy of Sciences. The voucher sample is deposited at the Shanghai Institute of Materia Medica, CAS, under registration No. YAL-4.The soft corals S. trocheliophorum  were minced into pieces and exhaustively extracted with Me2CO at room temperature (3 × 1 L). The solvent-free Me2CO extract was partitioned between Et2O and H2O. The organic phase was evaporated under reduced pressure to give a dark brown residue (10 g), which was subjected to silica gel column chromatography (CC) and eluted with petroleum ether (PE) in Me2CO  to yield 14 fractions (A–M).The lyophilized bodies of 2O, 1:1) to give pure 4 (32.1 mg). Fraction F was subjected to Sephadex LH-20 CC , followed by repeated silica gel CC  to yield 6 sub-fractions (F1-F6). F2 gave compound 5 (2.3 mg) after silica gel CC . Fraction G was chromatographed over Sephadex LH-20 , followed by ODS  to yield 10 sub-fractions (G1-G10). After purification by RP-HPLC , G5 yielded pure 1 . Fraction H gave compound 3  after Sephadex LH-20 CC , CC on ODS  and RP-HPLC . Fraction I was subjected to Sephadex LH-20 CC  to give five sub-fractions (I1-I5). Fraction I4 was first split by CC on ODS  and then purified by silica gel  to afford pure 2 (3.1 mg).Fraction D was chromatographed over silica gel (PE/Et1): colorless oil; [α] +18.6 ; 1H and 13C NMR data, see Tables m/z 343.2248 [M + Na]+ .Sarcophytrol D : colorle2): colorless oil; [α] +48.6 ; 1H and 13C NMR data, see Tables m/z 343.2243 [M + Na]+ .Sarcophytrol E : colorless oil; [α] −12.7 ; 1H and 13C NMR data, see Tables m/z 359.2188 [M + Na]+ .Sarcophytrol F (


The structures of both complexes were deduced by a combination of instrumental analyses and molecular modelling. Variable-temperature magnetic susceptibility data suggested that 1 was made up of 56.8% high-spin (HS) and 43.2% low-spin (LS) Fe(II) atoms at 294 K and has a moderate antiferromagnetic interaction (J = −81.2 cm−1) between the two Fe(II) centres, while 2 was made up of 27.7% HS and 72.3% LS Fe(II) atoms at 300 K. The optical band gaps (Eo) for 1 were 1.9 eV (from absorption spectrum) and 2.2 eV (from fluorescence spectrum), electrochemical bandgap (Ee) was 0.83 eV, excited state lifetime (τ) was 0.67 ns, and formal redox potential (E′(FeIII/FeII)) was +0.63 V. The corresponding values for 2 were 3.5 eV (from absorption spectrum), 1.8 eV (from fluorescence spectrum), 0.69 eV, 2.8 ns, and +0.41 V.A dimeric iron(II) complex,  Polypyridyl complexes of Fe(II) are currently attracting the attention of researchers as potential photosensitizers in dye-sensitized solar cells (DSSC) –3. Theseπ-conjugated ligands, as they are expected to have better photosensitization  and redox properties.We noted that the Fe(II) complexes reported as potential DSSC materials were mostly mononuclear. Hence, we focused our research on multinuclear Fe(II) complexes, especially involving  trans-[Fe2(CH3COO)4(L1)2] (1) and a trinuclear iron(II) complex, [Fe3(CH3COO)4(H2O)4(L2)] (2) --triazine-2,4-diamine. The magnetic data of both complexes are also presented to establish the spin states of the Fe(II) centres. The main objective of this paper was to establish a correlation between band gaps and excited state lifetimes with the nuclearity and spin states of a complex, and types of conjugated ligands .This paper reports the syntheses, structural deduction, and determinations of band gaps and excited state lifetimes of a dimeric iron(II) complex,L2)] (2) . L1  LED-based Strobe Lifetime spectrofluorometer using the stroboscopic technique. The observed fluorescence decay was analyzed using PTI Felix GX data acquisition and analysis software. Data was recorded in 100 ps time intervals from 50 ns to 70 ns observation window. The instrument response function was measured from the scattered light and estimated to be about 1.5 ns . The measured transients were fitted to multiexponential functions convoluted with the system response function. The fitting procedure was based on the Marquardt algorithm, where the experimental data were compared to a model decay convoluted with the IRF. The fit was judged by the value of the reduced χ2. The cyclic voltammetric scans (CV) were recorded on a Gamry Instrument Reference 600 potentiostat/galvanostat/ZRA. The electrolyte was tetrabutylammonium tetrafluoroborate (TBATFB) (0.05 M), working electrode was glassy carbon, reference electrode was saturated calomel, and counter electrode was platinum wire. The initial and final voltages were 0 V, the potential range was +1.5 V to −1.5 V, and the scan rate was 100 mV s−1. The molarities of the samples were 0.005 M, and N2 was bubbled through the solutions prior to analyses.3,4-Bis(tetradecyloxy)benzaldehyde was a gift from Professor D. W. Bruce from the Department of Chemistry, University of York, United Kingdom. Other chemicals were commercially available and used as received. The elemental analyses  were carried out on a Thermo Finnigan Flash EA 1112. The 3) of 3,4-bis(tetradecyloxy)benzaldehyde , followed by a few crystals of p-toluenesulfonic acid. The reaction mixture was cooled to room temperature and left stirring overnight. The solvent was removed under reduced pressure, and the powder obtained was filtered and washed with ethanol. The yield was 5.6 g (98%).Aniline  was added to a magnetically stirred hot ethanolic solution (75 cm3) by heating at 50°C. 4,4′-Dimethyl-2,2′-bipyridine  was added to the hot solution, and the reaction mixture was flushed with nitrogen. Potassium t-butoxide  was added portionwise, and the reaction mixture was flushed again with nitrogen, heated to 80°C, and left stirring at this temperature for about 3 h. Hydrochloric acid (10%) was added to the cooled mixture until pH 7, followed by distilled water (200 cm3) and dichloromethane (300 cm3). The organic layer was washed with saturated sodium bicarbonate (200 cm3) and then with distilled water (200 cm3). It was dried over anhydrous sodium sulphate, and the solvent was removed under reduced pressure. The yield was 4.8 g (97%). 1H-NMR : 0.88 , 1.26–1.60 , 1.85 , 4.1 , 6.91 , 7.27 , 7.31 , 7.58 , 8.22 , 8.34 , 7.53 , and 8.53 . Selected IR bands (−1) were 2917 vs, 2850 vs, 1589 s, 1576 s, 1512 s, 1467 m, 1435 s, 1386 m, 1270 vs, 1237 s, 1209 m, 1165 m, 1135 vs, 822 m, and 808 m.aniline  was dissolved in DMF  was 6.75 , 7.43–7.52 , and 8.23–8.25 . Selected IR bands (−1) were 3292 (w), 3137 (w), 1620 (s), 1536 (s), 1389 (s), and 1257 (w) cm−1.An ethanolic solution of pyrrole-2-carboxaldehyde  was added to an ethanolic suspension of 2,4-diamino-6-phenyl-1,3,5-triazine . The mixture was refluxed in the presence of a few drops of glacial acetic acid for 2 h. The brownish solid formed was filtered from the hot reaction mixture, washed with cold ethanol, and dried in an oven at 80°C. The yield was 15.8 g (68%). 3COO)2]  and ascorbic acid  were dissolved in methanol (50 cm3) at room temperature, forming a black solution. A solution of L1  in chloroform (10 mL) was gradually added to the black solution. The reaction mixture was stirred at room temperature for 4 h, and then the solvents were evaporated off at room temperature. The dark purple powder formed was successively washed with distilled water, aqueous methanol (1 : 1), and methanol and dried in a warm oven for 1 h. The yield was 1.4 g (70%). Anal. Calc. for FeC86H138N2O8: C, 74.29; H, 10.48; N, 2.01%. Found: C, 74.96; H, 11.25; N, 1.84%. Selected IR bands (−1): 2917 (vs), 2849 (vs), 1686 (s), 1672 (m), 1596 (m), 1586 (m), 1511 (s), 1466 (m), 1390 (m), 1272 (vs), 1236 (m), 1165 (m), 1134 (vs), 1069 (m), 1053 (m), 1036 (m), 1018 (m), 1005 (m), 807 (s).[Fe(CH3COO)2]  was added to an ethanolic suspension of H2L2 , followed by about 0.1 g ascorbic acid as an antioxidant. The mixture was refluxed for 3 h, and the black solid formed was filtered off from the hot reaction mixture, washed with cold ethanol, and dried in an oven at 100°C. The yield was 2.8 g (97%). Anal. Calc. for Fe3C27H29N7O12: C, 39.8; H, 4.1, N, 12.0%. Found: C, 39.6; H, 3.8, N, 12.1%. Selected IR bands (−1) were 3296 (b), 1592 (w), 1532 (s), 1401 (s). (1), was obtained as dark purple powder in good yield (60%) from the reaction of L1 with [Fe(CH3COO)2] (mol ratio = 1 : 1). The powder was readily soluble in CH2Cl2, CHCl3, and toluene. Its structural formula was proposed based on the CHN elemental analytic data and IR spectrum, which showed peaks for C=O at 1686 cm−1, −1 and 1596 cm−1, −1, and peaks for L1. The Δ values for CH3COO ligand 4(H2O)4(L2)] (2), was obtained as a black solid from the reaction of H2L2 with [Fe(CH3COO)2] (mol ratio = 1 : 1). It was insoluble in most common organic solvents, except DMSO. As for 1, its structural formula was proposed based on the CHN elemental analytic data and IR spectrum, which showed peaks for coordinated H2O at 3296 cm−1, C=N at 1592 cm−1, −1, −1, and peaks for L2. The Δ value for CH3COO ligand was 131 cm−1, which suggests a chelating binding mode.The trinuclear complex, , where L3 = 4,4′-bis(tetramethoxy)styryl-2,2′-bipyridine, and for both monodentate and bidentate bridging CH3COO. The results ( trans-[Fe2(CH3COO)4(L3)2] with monodentate bridging CH3COO for 1 and good agreement for 2  peak at 544 nm (εmax = 2194 M−1 cm−1) and two weak d-d peaks at 1412 nm (εmax = 25.6 M−1 cm−1) and 1755 nm (εmax = 25.6 M−1 cm−1). The 1MLCT peak is assigned to t2g → π* electronic transition for LS Fe(II)  (τ = 50 ns)  τ = 0 ns [5],oseconds . 1 (− ) [The CV scan for 1  shows an 1  was +0.41 V (versus SCE), which was lower than 1, indicating a more facile oxidation of Fe(II). Finally, other peaks observed may be assigned to ligand-based redox processes.For CV scan  shows anEe) may be calculated using the relationship, Ee = |HOMO − LUMO|, where HOMO = (onset oxidation peak voltage + 4.4) eV and LUMO = (onset reduction peak voltage + 4.4). Hence Ee = 0.83 eV for 1 and Ee = 0.69 eV for 2. The higher Ee for 1 is consistent with the presence of insulating alkyl groups in this complex.The electrochemical bandgap (1) and trinuclear (2) complexes have LS and HS Fe(II) atoms. Complex 1 has a lower optical bandgap, higher electrochemical bandgap, shorter excited state lifetime, and higher formal redox potential than complex 2. Both complexes were potential DSSC materials, but 2 is better than 1 based on its longer excited lifetime and lower redox potential. It can be concluded from this work that higher nuclearity Fe(II) complexes are better potential as DSSC materials, while lower MLCT transition energies led to the formation of complexes with lower optical band gaps but also shorter excited state lifetimes, and the presence of insulating long alkyl chains resulted in higher electrochemical band gaps and higher formal redox potentials.Both dimeric (


Z)-5-[2-(benzo[b]thiophen-2-yl)-1-ethenyl]-1H-tetrazole methanol monosolvate, these chains involve both tetrazole and methanol, and are parallel to the b axis. In (Z)-5-[2-(benzo[b]thiophen-3-yl)-1-ethenyl]-1H-tetrazole, molecules are linked into chains parallel to the a axis by N—H⋯N hydrogen bonds between adjacent tetrazole rings.In both structures, molecules are linked into hydrogen-bonded chains. In ( Z)-5-[2-(Benzo[b]thiophen-2-yl)-1-ethenyl]-1H-tetrazole methanol monosolvate, C19H16N4O2S·CH3OH, (I), was prepared by the reaction of (Z)-3-(benzo[b]thiophen-2-yl)-2-acrylonitrile with tributyltin azide via a [3 + 2]cycloaddition azide condensation reaction. The structurally related compound (Z)-5-[2-(benzo[b]thiophen-3-yl)-1-ethenyl]-1H-tetrazole, C20H18N4O3S, (II), was prepared by the reaction of (Z)-3-(benzo[b]thiophen-3-yl)-2-acrylonitrile with tributyltin azide. Crystals of (I) have two molecules in the asymmetric unit (Z′ = 2), whereas crystals of (II) have Z′ = 1. The benzothiophene rings in (I) and (II) are almost planar, with r.m.s deviations from the mean plane of 0.0084 and 0.0037 Å in (I) and 0.0084 Å in (II). The tetrazole rings of (I) and (II) make dihedral angles with the mean planes of the benzothiophene rings of 88.81 (13) and 88.92 (13)° in (I), and 60.94 (6)° in (II). The dimethoxyphenyl and trimethoxyphenyl rings make dihedral angles with the benzothiophene rings of 23.91 (8) and 24.99 (8)° in (I) and 84.47 (3)° in (II). In both structures, molecules are linked into hydrogen-bonded chains. In (I), these chains involve both tetrazole and methanol, and are parallel to the b axis. In (II), molecules are linked into chains parallel to the a axis by N—H⋯N hydrogen bonds between adjacent tetrazole rings.( These chains are bidirectional, as the hydrogen atoms on the tetrazole rings and the methanol oxygen atom appear to be disordered over two positions. In the structure of (II)a axis by intermolecular N—H⋯N hydrogen bonds (Table 2Hydrogen bonding and the mode of packing of (I)s Table 2 between via its carbon atom to another carbon atom yielded 255 hits. Of these, only two were bonded to an sp2 carbon atom, namely 5-(2H-chromen-3-yl)-1H-tetrazole monohydrate -5-(dimethylamino)-2-(1H-tetrazol-5-yl)penta-2,4-dienenitrile methanol solvate R2Csp2H), 0.98 Å (RCH3), 0.84 Å (OH), 0.88 Å (Nsp2H). Uiso(H) values were set to either 1.2Ueq or 1.5Ueq  of the attached atom. Final models were checked using PLATON Pccn was also refined, but the overall quality of the refinement was not as good as the P21212 model given here. Indeed, the ADDSYM routine in PLATON . Other alternatives using space groups Pcc2, Pban, and Pna21 were much less satisfactory. Not surprisingly, the P21212 model was twinned by inversion, which was dealt with using standard SHELXL methods (TWIN and BASF commands).Refinement of (I)A and N1B. This assignment results in impossible clashes with symmetry equivalents about the twofold axis. Since there were suitable small difference map peaks for hydrogen atoms attached to atoms N4A and N4B as well as N1A and N1B, these hydrogen atoms were included as split over the two sites at half occupancy. Disorder of the tetrazole ring hydrogen atoms in this way also requires that the hydroxyl hydrogen atoms of the methanol molecules are disordered. Again, suitable  difference map peaks were apparent. Further evidence for the disorder is that the distances C11A—N1A, C11A—N4A and C11B—N1B, C11B—N4B are all very similar, indicating that the C=N double bond and C—N single bond in these rings are scrambled. Not surprisingly, convergence of the OH hydrogen-atom positions was rather problematic.The hydrogen on the tetrazole ring was initially placed solely on the atoms labelled N110.1107/S2056989016005600/hg5472sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989016005600/hg5472Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989016005600/hg5472IIsup3.hklStructure factors: contains datablock(s) II. DOI: 1472235, 1472234CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The crystal structures of three closely related benzothiazinone structures are reported. All three are conformationally similar having screw-boat puckering in the thiazine ring and C—H⋯π-type intermolecular interactions in the crystals. H-1,3-benzothiazin-4-one, namely 3-(4-methoxyphenyl)-2-phenyl-4H-1,3-benzothiazin-4-one, C21H17NO2S, (I), 2-phenyl-3-[4-(trifluoromethyl)phenyl]-2,3-dihydro-4H-1,3-benzothiazin-4-one toluene hemisolvate, C21H14F3NOS·0.5C7H8, (II), and 3-(3-bromophenyl)-2-phenyl-2,3-dihydro-4H-1,3-benzothiazin-4-one toluene hemisolvate, C20H14BrNOS·0.5C7H8, (III), were synthesized and their crystal structures determined. The hemisolvates differ in that in (II), the asymmetric unit comprises two molecules of the benzothiazinone compound and a toluene solvent molecule, whereas in (III), the unit comprises one benzothiazinone molecule and a half-occupancy toluene solvent molecule. All crystals are of racemic mixtures of the chiral 2-C atom of the thiazine moiety, which in all structures has a screw-boat puckering, with the puckering amplitude values within the range 0.575–0.603 Å. In all three structures, the benzene plane of the benzothiazine system makes a dihedral angle in the range 78.60 (5) to 98.40 (5)° with the unsubstituted benzene plane and in the range 70.50 (1) to 121.00 (5)° with the substituted benzene plane. The CF3 substituent group in one of the molecules of (II) shows positional disorder, with an occupancy ratio of 0.57 (3):0.43 (3). In the crystals of (I) and (II), weak intermolecular C—H⋯O interactions are present, giving in (I), molecules arranged in a plane parallel to (010), and in (II), chains along a. In addition, all three structures show weak C—H⋯π interactions involving various aromatic rings.Three ring-substituted 3-aryl analogs of 2-phenyl-2,3-dihydro-4 H-1,3-benzothiazin-4-one et al., 2013We have previously reported the crystal structures of 2,3-diphenyl-2,3-dihydro-43 substituent group in one of the molecules of (II)The three benzothiazinones Figs. 1–3 ▸ ▸ ea-axis direction  interaction of 3.8068 (10) Å is present with an interacting angle of 148°. In (II)Cg(toluene) distances of 3.5916 (7) and 3.6009 (7) Å, with interacting angles of 145 and 147°, respectively. In (III)Cg distances of 3.5802 (6) and 3.6823 (6) Å, with interacting angles of 156 and 153°, respectively  distance of 3.977 (1) Å and an interplanar angle of 8°.In (I)on Fig. 5. The cryon Fig. 5–3 ▸ ▸, et al., 2013H)-one has been reported in a patent application . Tetrahydrofuran or 2-methyltetrahydrofuran (2.3 ml) was added, the solid dissolved and the solution stirred. Pyridine  was added, followed by thiosalicylic acid . Finally, 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane 2,4,6-trioxide (T3P) in 2-methyltetrahydrofuran  was added. The mixture was stirred at room temperature and the reaction was followed using thin-layer chromatography. The mixture was then poured into a separatory funnel and dichloromethane and distilled water were added. The layers were separated and the aqueous layer was then extracted twice with dichloromethane. The organics were combined and washed with saturated sodium bicarbonate and then saturated sodium chloride. The organic extract was dried over sodium sulfate and concentrated under vacuum. The crude product was chromatographed on 30 g of flash silica gel using mixtures of ethyl acetate and hexanes, and then further purified as indicated below.A two-necked 25 ml round-bottomed flask was oven-dried, cooled under NRF = 0.52 (50% ethyl acetate/hexanes). Colorless block-shaped crystals suitable for the X-ray analysis were grown by slow evaporation from ethanol solution.Compound (I)RF = 0.56 (30% ethyl acetate/hexanes solution). Colorless needle-shaped crystals suitable for the X-ray analysis were grown by slow evaporation from toluene solution.Compound (II)RF = 0.39 (20% ethyl acetate/hexanes). A second crop of 0.30 g (12.7%) was obtained by slow evaporation of the mother liquor, giving colorless blocks suitable for the X-ray analysis.Compound (III)SHELXL2014 :0.43 (3). We tried to address the high R values (relative to Rint) by looking for twinning and using restraints but we have had no success in achieving respectable R values. In (III)Uiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) otherwise.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989016011002/zs2362sup1.cifCrystal structure: contains datablock(s) 1, II, III, I. DOI: Click here for additional data file.10.1107/S2056989016011002/zs2362Isup2.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016011002/zs2362IIsup3.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016011002/zs2362IIIsup4.cmlSupporting information file. DOI: 1491202, 1491201, 1491200CCDC references: crystallographic information; 3D view; checkCIF reportAdditional supporting information: