Background. Human umbilical cord mesenchymal stem cells (UC-MSCs) can regulate the function of immune cells. However, whether and how UC-MSCs can modulate the function of Vγ9Vδ2 T cells has not been fully understood. Methods. The PBMCs or Vγ9Vδ2 T cells were activated and expanded with pamidronate (PAM) and interleukin-2 (IL-2) with or without the presence UC-MSCs. The effects of UC-MSCs on the proliferation, cytokine expression, and cytotoxicity of Vγ9Vδ2 T cells were determined by flow cytometry. The effects of UC-MSCs on Fas-L, TRAIL-expressing Vγ9Vδ2 T cells, and Vγ9Vδ2 T cell apoptosis were determined by flow cytometry. Results. UC-MSCs inhibited Vγ9Vδ2 T cell proliferation in a dose-dependent but cell-contact independent manner. Coculture with UC-MSCs reduced the frequency of IFNγ+ but increased granzyme B+ Vγ9Vδ2 T cells. UC-MSCs inhibited the cytotoxicity of Vγ9Vδ2 T cells against influenza virus H1N1 infected A549 cells and also reduced the frequency of Fas-L+, TRAIL+ Vγ9Vδ2 T cells but failed to modulate the apoptosis of Vγ9Vδ2 T cells. Conclusions. These results indicated that UC-MSCs efficiently suppressed the proliferation and cytotoxicity of Vγ9Vδ2 T cells and modulated their cytokine production. Fas-L and TRAIL were involved in the regulation. Cell contact and apoptosis of Vγ9Vδ2 T cells were not necessary for the inhibition. Furt T cells , 5, 6, N T cells , 7–9, ma T cells , and den T cells , 10. In  T cells . Because T cells , 13, myo T cells .γδ T cells have unique innate and adaptive immunity features and account for approximately 1%–5% of circulating T cells [γδ T cells can respond to exotic factors [γ9Vδ2 T cells, and the largest subset of γδ T cells can be activated by small nonpeptide phosphoantigens such as isopentenyl pyrophosphate (IPP) and pamidronate (PAM) [γ9Vδ2 T cells play an important role in host defense, especially in homeostasis and surveillance [γ9Vδ2 T cells regulate the process of innate immunity [γ9Vδ2 T cells and MSCs play regulating roles in GvHD and some autoimmune diseases [γ9Vδ2 T cells respond to allogeneic UC-MSCs has not been clarified. T cells , 16. γδ  factors , 18 and te (PAM) , 20 in ate (PAM) . Functioeillance . Previouimmunity , immune immunity , 23, canimmunity , 22, 24,immunity . Both Vγdiseases , 25, 26;γ9Vδ2 T cells in vitro. Our findings indicated that UC-MSCs inhibited the proliferation, cytokine production, and cytotoxicity of Vγ9Vδ2 T cells in vitro. Cell contact and apoptosis of Vγ9Vδ2 T cells were not necessary for the inhibition. Fas-L and TRAIL were involved in the regulation. We discussed the implications of our findings. In this study, we isolated and expanded UC-MSCs as well as peripheral blood mononuclear cells (PBMCs) from healthy donors and examined the regulatory effect of allogeneic UC-MSCs on the proliferation, cytokine production, and cytotoxicity of Vα-MEM medium  containing 15% heat inactivated fetal bovine serum , 100 units/mL of penicillin, and 100 μg/mL of streptomycin at 37°C in 5% CO2 incubator. When the cells reached 80% confluency, UC-MSCs were digested with 0.25% trypsin and harvested for subculture or irradiation procession. The expanded UC-MSCs were characterized for their abilities to self-renew and differentiate multiple lineages of cells (data not shown). UC-MSCs were irradiated by X-ray (30 Gy) on Precision X-ray, (X-RAD 320), and cocultured with PBMCs or Vγ9Vδ2 T cells.The experimental protocol was approved by the Institutional Review Board of Sichuan University. UC-MSCs were isolated from human umbilical cords, as described previously . UC-MSCsμg/mL of streptomycin, 2 μg/mL of pamidronate disodium (PAM), and 100 IU/mL of human recombinant IL-2 . The cells were exposed to freshly prepared PAM and IL-2 medium and then cultured with freshly prepared medium containing IL-2 every three days up to two weeks. The contained Vγ9Vδ2 T cells were purified by negative selection using a TCR Vγ9Vδ2 T-cell isolation kit, according to the manufacturer's instruction . Check the Vγ9Vδ2 T cells by flow cytometry. Vγ9Vδ2 T cells were gated on CD3+TCRγδ+ in lymphocytes.Human peripheral blood samples were obtained from healthy donors and PBMCs were isolated by Ficoll-Hypaque  density gradient centrifugation, as previously described . The iso6/well) were cocultured with 30 Gy-irradiated UC-MSCs at cell ratios of 5 : 1, 20 : 1, 80 : 1, or 320 : 1 for 48 hours to 14 days. In transwell system, PBMCs/Vγ9Vδ2 T cells were cultured at the top chamber of 24-well transwell plates  and 30 Gy-irradiated UC-MSCs were cultured at the bottom chamber of the plates.PBMCs were cocultured with UC-MSCs in 10% FBS RPMI 1640 medium containing PAM and IL-2 with or without transwell system. PBMCs (1.5∗106/mL) were labeled with 1 μM 5,6-carboxyfluorescein diacetate N-succinimidyl ester  for 15 minutes. After being washed, the labeled PBMCs were cocultured with UC-MSCs in triplicate at the cell ratios of 5 : 1, 20 : 1, 80 : 1, and 320 : 1 for 14 days with PAM and IL-2. The cells were exposed to freshly prepared medium every three days. The cells were stained PE-anti-TCRγδ and APC-cy7-anti-CD3  and the frequency of Vγ9Vδ2 T cell proliferation was determined by flow cytometry on a BECKMAN, FC 500, followed by analyzing the data with FlowJo software .The isolated PBMCs (1.0∗106/well) at the top chamber of 24-well transwell plates  were cocultured with 30 Gy-irradiated UC-MSCs at the bottom chamber of the plates at the cell ratios of 5 : 1, 20 : 1, or 80 : 1 for 14 days with PAM and IL-2. PBMCs alone at the top chamber served as controls. The PBMCs were stained with PE-anti-TCRγδ and APC-cy7-anti-CD3 to determine the proliferation of Vγ9Vδ2 T cells.Furthermore, the CFSE-labeled PBMCs  to examine the frequency of cytokine-expressing Vγ9Vδ2 T cells, respectively.Freshly isolated PBMCs were stimulated with PAM and IL-2 for 12 days and the cells were cocultured with, or without, allogeneic irradiated UC-MSCs at cell ratios of 5 : 1 or 80 : 1 for 48 hours. During the last 4-hour culture, the cells were treated with Brefeldin A. Subsequently, the cells were harvested, stained with APC-cy7-anti-CD3 and APC-anti-TCRHuman adenocarcinomic alveolar basal epithelial A549 cells provided by the laboratory were cultured in 10% FBS RPMI 1640 medium. Furthermore, A549 cells were cultured overnight and, after being washed with phosphate-buffered saline (PBS), the cells were infected with influenza virus H1N1 (A/PR/8/34)  at a mulγ9Vδ2 T cells were purified by negative selection, as described above. The purified Vγ9Vδ2 T cells (effector) were cocultured in triplicate with influenza virus- (H1N1-) infected A549 cells (target) at an effector-to-target (E/T) ratio of 10 : 1 for 5 hours. Subsequently, the cells were stained with APC-cy7-anti-CD3 and 7AAD (BD Biosciences) and the percentages of dead A549 cells in total CD3-A549 cells were determined by flow cytometry.Freshly isolated PBMCs were stimulated with PAM and IL-2 for 12 days and were cocultured with allogeneic irradiated UC-MSCs at cell ratios of 5 : 1 or 80 : 1 for 60 hours. The contained Vγ9Vδ2, and PE-anti-Fas-L or PE-anti-TRAIL. The frequency of Fas-L+ or TRIAL+ Vγ9Vδ2 T cells was determined by flow cytometry. In addition, some cells were stained with APC-cy7-anti-CD3, APC-anti-TCRγδ, FITC-Annexin-V, and 7-AAD to determine the frequency of apoptotic Vγ9Vδ2 T cells.Freshly isolated PBMCs were stimulated with PAM and IL-2 in the presence or absence of irradiated UC-MSCs at the cell ratio of 5 : 1 or 80 : 1 for 14 days. The cells were harvested and stained with APC-cy7-anti-CD3, APC-anti-TCRV t-test using GraphPad Prism software (version 5). A P value of <0.05 was considered statistically significant.Data are representative FACS charts or histograms or expressed as the mean ± SEM. The difference among groups was analyzed by ANOVA and post hocγ9Vδ2 T cells by flow cytometry analysis and found that peripheral blood Vγ9Vδ2 T cells accounted for 3%–8% of T cells (data not shown). Following stimulation with PAM and IL-2 for 14 days, the percentages of Vγ9Vδ2 T cells reached 40%–90% of cultured T cells. To determine the effect of UC-MSCs on the allogeneic Vγ9Vδ2 T cell proliferation, PBMCs were isolated from healthy donors and labeled with CFSE. The labeled PBMCs were stimulated with PAM and IL-2 in the presence or absence of different numbers of irradiated UC-MSCs for 14 days. Subsequently, the cells were stained with PE-anti-TCRγδ and APC-cy7-anti-CD3 and the percentages of Vγ9Vδ2 T cells were determined by flow cytometry  in the top chamber of 24-well transwell plates were cocultured with irradiated UC-MSCs in the bottom chamber of the plates in triplicate at the cell ratios of 5 : 1, 20 : 1, or 80 : 1 for 14 days. PBMCs alone in the top chamber served as controls. Subsequently, the PBMCs were stained with PE-anti-TCRγδ to determine the frequency of Vγ9Vδ2 T cells by flow cytometry. As shown in γ9Vδ2 T cells when both types of cells were cocultured together. Similarly, UC-MSCs also inhibited the proliferation of allogeneic Vγ9Vδ2 T cells when both types of cells were separately cultured in the top and bottom chambers and there was no significant difference in the inhibitory effects between these two different tests . Subsequently, the cells were harvested, stained with APC-cy7-anti-CD3 and APC-anti-TCRγδ, fixed, permeabilized, and intracellularly stained with FITC-anti-IFNγ, PE-anti-TNFα, PE-anti-IL-10, PE-anti-perforin, or FITC-anti-granzyme B. The percentages of TNFα+, IFNγ+, perforin+, granzyme B+, or IL-10+ Vγ9Vδ2 T cells were determined by flow cytometry. After being gated on CD3+TCRγδ+ cells, there was no significant difference in the frequency of TNFα+, perforin+, or IL-10+ Vγ9Vδ2 T cells in total Vγ9Vδ2 T cells regardless of the presence or absence of MSCs  were cocultured with influenza virus- (H1N1-) infected A549 cells (target) at an effector-to-target (E/T) ratio of 10 : 1 for 5 hours. Subsequently, the cells were stained with APC-cy7-anti-CD3 and 7AAD, and after being gated on CD3-A549 cells, the percentages of dead A549 cells in total A549 cells were determined by flow cytometry  [β, IL-10, and PGE2 are potent inhibitors of T cell immunity [γ9Vδ2 T cells.In this study, we found that UC-MSCs inhibit the proliferation of PAM/IL-2 stimulated V (TGF-β) , hepatic (TGF-β) , prostag (TGF-β) , IL-10 [ (TGF-β) , indolam (TGF-β) , 33, nit (TGF-β) , heme ox (TGF-β) , and hum (TGF-β) , and TGFimmunity , 36–38. γ9Vδ2 T cells may be similar to that with NK cells [γ Vγ9Vδ2 T cell responses and upregulate granzyme B expression, leading to the cytotoxicity against UC-MSCs by the granzyme-NKG2D pathway. Simultaneously, inhibitory factors, such as PGE2 [β [γ9Vδ2 T cells and the serine protease inhibitor 9 (SERPINB9) produced by UC-MSCs attenuates the activity of granzyme B-mediated cytotoxicity. Subsequently, inhibitory factors secreted by UC-MSCs control the function of Vγ9Vδ2 T cells by reducing IFNγ, Fas-L, and TRAIL expression in Vγ9Vδ2 T cells. Immunosuppressive functions of different sources of MSCs are varying and their functions are regulated by many other immunocompetent cells in vivo [γ9Vδ2 T cells.We speculate that the interaction of UC-MSCs with VNK cells . It is p as PGE2 , 33, TGF PGE2 [β , 33, NO  PGE2 [β , 40, sec in vivo . Therefoγ9Vδ2 T cells against virus infection are still incompletely understood, the protective role of Vγ9Vδ2 T cells has been proved in acute and chronic virus infections. Following the infection with different strains of influenza viruses, Vγ9Vδ2 T cells can secrete antiviral cytokines and directly kill virus-infected target cells [γ9Vδ2 T cells against HIN1 influenza virus in vitro. This result indicates that maybe in viral infection, especially in H1N1 influenza virus infection, UC-MSCs suppress the antiviral protection of Vγ9Vδ2 T cells. This needs to be proved in further studies.Even though the mechanisms underlying the cytotoxicity of Vet cells –44, whicet cells . UC-MSCsγ9Vδ2 T cell proliferation in a cell-to-cell contact-independent manner and modulated cytokine secretion by Vγ9Vδ2 T cells. Furthermore, UC-MSCs inhibited the cytotoxicity of activated Vγ9Vδ2 T cells against influenza virus-infected A549 cells. These regulations may be mediated by the inhibition of Fas-L and TRAIL expression byVγ9Vδ2 T cells but not by inducing the apoptosis of Vγ9Vδ2 T cells. Our novel data support that UC-MSCs can exhibit immune regulatory capacity by inhibiting Vγ9Vδ2 T cells and our findings may provide some clues for further research on the interaction between UC-MSCs and Vγ9Vδ2 T cells.In summary, our data indicated that human UC-MSCs inhibited the PAM/IL-2 stimulated V


However, WCO is certainly a renewable feedstock of bio-based materials. To get the rebirth of WCO, we have established a facile and high-yield method to convert WCO to bio-based zwitterionic surfactants with excellent surface and interfacial properties. The interfacial tension between crude oil and water could reach ultra-low value as 0.0016 mN m Most of the surfactants used in industrial fields are petroleum-based. Bio-based surfactants have attracted much attention from scientific and industrial fields due to their renewable feedstock and environmentally friendly application1234523451011121314151618203 as catalyst. Then the chemical modification of the carboxyl group of all fatty acids was accomplished by acyl chlorination with sulfoxide chloride, amidation with N, N-dimethyl-1,3- propylenediamine and quaternization with sodium chloroacetate to obtain bio-based zwitterionic surfactants, N-phenyl fatty amidopropyl-N,N-dimethylcarboxylbetaine (PFAPMB), yielded 84.9%. ESI HRMS (m/z): [M+Na]+ calcd for: C23H46N2O3 (C16:0), 421.3406, C25H50N2O3 (C18:0), 449.3719, C31H50N2O3 (C18:2:pheynl), 521.3719, C31H52N2O3 (C18:1:phenyl), 523.3875, C31H54N2O3 (C18:0:pheyl), 525.4134; found: 421.3416, 449.3722, 521.3674, 523.3884, 525.4045; C23H46N2O3 (C16:0) : C25H50N2O3 (C18:0) : C31H50N2O3 (C18:2:pheynl) : C31H52N2O3 (C18:1:phenyl) : C31H54N2O3 (C18:0:pheyl) = 0.24:0.17:0.11:0.80:1.0. 1H NMR : δ 7.159-7.116 (-C6H5), 6.921 (-CO-NH-), 4.144-4.090 (-CH=CH-), 3.852-3.847 (-NH-CH2-CH2-), 3.672-3.645 (-N(CH3)2-CH2-COO), 3.483 (-CH2-N(CH3)2-), 3.221 (-N(CH3)2-), 2.447-2.428 (-CH-C6H5), 2.142 (-CH2-CO-), 1.968-1.959 (-NH-CH2-CH2-CH2-N(CH3)2-), 1.578-1.563 , 1.255-1.238 (-(CH2)n-CH3), 0.890-0.856 (-CH2-CH3).The collected WCO was pretreated through filtration and centrifugation. The oil phase was acidified, filtered, washed with water and then dried. In the first step (as shown in oC was illustrated in −1 with the SFT at CMC (SFTCMC) of 28.4 mN m−1. The CMC of PFAPMB was three orders of magnitude lower than other bio-based surfactants and the max surface excess concentration was almost two times of others’Variation of the surface tensions (SFT) with the concentration of PFAPMB at 25.0 oC  were shown in Dynamic interfacial tensions (IFT) between Daqing crude oil , the efficiency of oil recovery will be remarkably enhanced22min) could reach 0.0016 mN m−1 at the concentration of 0.100 g L−1 PFAPMB. Considered in the application, achieving ultra-low interfacial tension is the primary goal, the lower usage of the surfactants is also preferred. So, the subsequent experiment selected the concentration of 0.020 g L−1 as the research basis, at which concentration the IFTmin was 0.0032 mN m−1. There is an interesting phenomenon, when the concentration of PFAPMB was much lower than single surfactant in our precious work1824When the interfacial tension between the surfactant solution and crude oil arrives at ultra-low level  and 30 mL NaOH methanol/water solution  were added into a round bottom flask. The reaction mixture was refluxed at 70 °C for 6 h. After the hydrolysis, the mixture was neutralized and further acidized using hydrochloric acid (6 M), until the pH value of the solution was about 2. Then 50 mL hot water was added to separate fatty acids and glycerol. The aqueous phase was removed. The oil phase was washed by hot water for 5 times, and the pH value of the aqueous phase was about 6. Then the oils phase was dried using cyclohexane as water-carrying agent. The acid values before and after the hydrolysis process were 1.37 mg KOH g−1 and 174.33 mg KOH g−1. So the hydrolysis efficiency was 98.0%. 5.00 g (18 mmol) of mix fatty acids and 2.40 g (18 mmol) of AlCl3 were added to 25 mL benzene in a round-bottom flask. The reaction mixture was refluxed at 65 °C, and anhydrous CaCl2 was placed in a drying tube above the reactor. After 6 h of reaction, the product was washed by 15 mL hydrochloric acids (1 M) for 3 times, then excess benzene was removed on a rotary evaporator giving Phenyl fatty acids (PFA), 5.90 g, yielded 98.0%. A solution of PFA  in trichloromethane (25 mL) was added dropwise to a drying flask with 1.45 mL SOCl2 (20 mmol) in it, and the mixture was stirred at 40 °C for 2 h. Then the mixture was distilled to remove the solvent and excess SOCl2. The residue was dissolved in 10 mL acetone, and 2.30 mL N,N-dimethyl-1,3-propanediamine (20 mmol) was slowly added in it at 0 °C. The reaction mixture was then warmed to 40 °C and stirred for 2 h. The excess diamine and solvent were removed on a rotary evaporator giving N-phenyl fatty amidopropyl–N,N-dimethylamine (PFAPMA), 6.76 g, yielded 91.5%. PFAPMA were quaternized using sodium chloroacetate with a molar ratio of 1:1.25 in a solvent mixture of methanol and water (vmethanol/vwater = 1:4). The final products were refluxed at 75 °C for about 12 h; the mixture was vaporized off under reduced pressure, then dissolved in ethanol and filtered. The filter liquor was distilled to remove ethanol and purified by recrystallization in ethyl acetate. 7.29 g precipitate, N-phenyl fatty amidopropyl-N,N-dimethylcarboxylbetaine (PFAPMB), was received and yielded 84.9%. ESI HRMS: m/z [M+Na]+ calcd for: 421.3406 (C16:0), 449.3719 (C18:0), 521.3719 (C18:2:pheynl), 523.3875 (C18:1:phenyl), 525.4134 (C18:0:pheyl); found: 421.3416, 449.3722, 521.3674, 523.3884, 525.4045. 1H NMR : δ 7.159-7.116 (-C6H5), 6.921 (-CO-NH-), 4.144-4.090 (-CH=CH-), 3.852-3.847 (-NH-CH2-CH2-), 3.672-3.645 (-N(CH3)2-CH2-COO), 3.483 (-CH2-N(CH3)2-), 3.221 (-N(CH3)2-), 2.447-2.428 (-CH-C6H5), 2.142 (-CH2-CO-), 1.968-1.959 (-NH-CH2-CH2-CH2-N(CH3)2-), 1.578-1.563 , 1.255-1.238 (-(CH2)n-CH3), 0.890-0.856 (-CH2-CH3).20.00 g WCO  in CDCl3 at room temperature.GC-MS spectra were recorded on an Agilent 6890N network GC system and 5975 inert Mass Selective Detector. ESI HRMS spectra were recorded on the Waters LCT Premier XE Mass Spectrometers. −1 at 25 °C.The surface tensions of the aqueous solution at different surfactant concentrations were measured by using a DCAT 21 tensiometer  with the plate method and the temperature was controlled at 25.0 ± 0.1 °C. Before each measurement, the quartz plate was briefly heated above an alcohol burner until glowing. In order to attain equilibrium conditions, the quartz plate should be cooled to room temperature and dipped in the solution for a while. The measurement should be repeated three times and an average value was obtained. The surface tension between air and double distilled water was 71.8 mN m−1. The volumetric ratio of water to oil in the spinning-drop tensiometer is about 2000. Daqing crude oil was used as oil phase. Surfactant solutions were prepared with Daqing oil field simulated formation water and the concentration of the surfactants was 0.020 g L−1. The formation water consists of 112.7 mg L−1 CaCl2, 42.8 mg L−1 MgCl2, 1597.1 mg L−1 NaCl, 17.0 mg L−1 Na2SO4, 381.6 mg L−1 Na2CO3 and 3176.0 mg L−1 NaHCO3. The total dissolved substance is 5327.2 mg L−1. The interfacial tension between Daqing crude oil and simulated formation water was 9.70 mN m−1 at 50 °C.Interfacial tensions between Daqing crude oil and water were measured by the spinning-drop method at 50 ± 0.1 °C using SVT 20 tensiometer . Equilibrium was affirmed to be obtained when successive values taken at 5 min intervals agreed to within 0.001 mN mo at 25 °C.The contact angles were measured by using the sessile drop technique. The apparatus consisted of a camera with a micro lens and a horizontal platform. The platform was connected to the thermostat to control temperature. Before measurement, the platform environment was preheated at 25 ± 1 °C by switching on the thermostat. When the temperature reached 25 °C, the solid substrate was placed in middle of the platform. A 2 μL solution drop was introduced onto the solid substrate through a microsyringe. The contact angles were measured by taking photographic images after adding the solution for 60 s. The contact angles were determined directly from the photographs by drawing tangent lines between the liquid drop and the solid surface. The measurements were repeated 5 times for every samples and the average angle value was figured out. The average contact angle on the three phase contact gas/double distilled water/solid was 92.04 −1 bio-based zwitterionic surfactants solution and 10 mL liquid paraffin in a graduated test tube. The time taken for separation of 3 mL and 5 mL aqueous phase solution was recorded (t/s). The recorded time was repeated for 3  imes.The emulsification properties of bio-based zwitterionic surfactants were obtained at room temperature. It was determined by giving vigorous downward stokes to a mixture of 10 mL 0.500 g LoCThe foaming property of bio-based zwitterionic surfactants was obtained according to the Ross-Miles test at 40 Ultimate biodegradation estimation of PFAPMB was determined by EPI Suite, BIOWIN3 biodegradation model, which is frequently used to estimate the degradation of organic chemicals3 , N, N-dimethyl-1, 3-propanediamine , Sodium chloroacetate  were used without further purification. All other chemicals used were of analytical reagent grade. WCO was collected at canteens and restaurants. The contents of fatty acids of WCO were analyzed by GC-MS.AlClThis study was designed by Q.-Q.Z., H.-Z.G., J.-F.L., S.-Z.Y. and B.-Z.M. Q.-Q.Z. performed the chemical synthesis of bio-based zwitterionic surfactants derived from WCO, measured the surface properties of the surfactants and analyzed the experimental results. B.-X.C. performed the interfacial tension measurements. W.-J.X. performed the hydrolysis of WCO. Q.-Q.Z. wrote the manuscript, assisted by all co-authors. All authors reviewed the final manuscript.How to cite this article: Zhang, Q.-Q. et al. The Rebirth of Waste Cooking Oil to Novel Bio-based Surfactants. Sci. Rep.5, 09971; doi: 10.1038/srep09971 (2015).


The coordination sphere of the FeIII atom is that of a slightly distorted octahedron. The crystal under investigation was a two-component pseudo-merohedral twin in the monoclinic system with a β angle close to 90°. Twin law [100/0-10/00-1] reduced the R1 residual [I > 2σ(I)] from 0.0769 to 0.0312, and the mass ratio of twin components refined to 0.8913 (5):0.1087 (5). In the crystal, molecules are arranged in sheets normal to [001] via non-classical C—H⋯O hydrogen bonding. No other significant intermolecular interactions are observed. The structure is a new polymorph of Fe(acac)3 and is isotypic with one polymorph of its gallium analog.In the structure of the title complex, [Fe(C For mul al. 1981; Shkol’n al. 1981; Iball & al. 1981; Kabak e al. 1996; Diaz-Ac al. 2001; Hu et a al. 2001; Stabnik al. 2007; Weng et al. 2011. For the al. 2005.5H7O2)3] = 0.031wR(F2) = 0.081S = 1.069058 reflections206 parametersH-atom parameters constrainedmax = 0.50 e Å−3Δρmin = −0.58 e Å−3ΔρAPEX2  used to solve structure: SHELXS2013  I, global. DOI: 10.1107/S2056989015021805/vn2103Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015021805/vn2103fig1.tif. DOI: The structure of the molecule showing the atom numbering, with displacement ellipsoids drawn at the 50% probability level.1437249CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The molecules are bound by O—H⋯O(OH) hydrogen bonds, notably in a zigzag C(2) chain along the short b (screw) axis, supplemented with an O—H⋯O(carbonyl) link along the a axis and other C(2) links.The structure determination of an oxyamine glycoside confirmed that it was obtained in the ring-closed β-pyranose configuration with a  12H23N5O6, solved using adequate data from a thin crystal plate, confirmed that this useful glycoconjugate was obtained in the ring-closed β-pyranose configuration with 4C1 conformation. The molecules are bound by O—H⋯O(OH) hydrogen bonds, notably in a zigzag C(2) chain along the short b (screw) axis, supplemented with an R22(12) O—H⋯O(carbonyl) link along the a axis and other C(2) links. The absolute configuration was not unambiguously determined but was known from the synthetic chemistry, which used natural 2-acetamido-2-deoxy-d-glucose as the starting material.The structure of the title compound, C The absolute configuration was not ambiguously determined but was known from the synthetic chemistry.The title compound crystallizes with one independent molecule in the asymmetric unit Fig. 1 in the CC(2) and C(8) types which combine to form a larger chain and rings e.g. R22(12), as shown on the right of Fig. 2The molecules are bound together with a comprehensive net of O—H⋯O hydrogen bonds, as well as one N—H⋯O(carbonyl) and one O—H⋯O(carbonyl) hydrogen bond Table 1. The basN-alkyl-N-(tetrahydro-2H-pyran-2-yl)oxyamines, and two structures were found, both of which are N-β-glycosyloxyamines, viz. an N-β-glucopyranosyloxyamine -N-(3-azidopropyl)-O-methylhydroxylamine was prepared as described in Munneke et al. . al. 2015 from 3-aUiso(H) = 1.5Ueq(C), but were allowed to rotate freely about the adjacent C—C bond. All other O,C-bound H atoms were placed in geometrically idealized positions and constrained to ride on their parent atoms with C—H distances of 0.99 (methylene) and 1.0 (tertiary) Å, O—H = 0.84 Å and with Uiso(H) = 1.2Ueq. The nitrogen H atom was located in a difference Fourier map and refined with Uiso(H) = 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016002164/lh5798sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989016002164/lh5798Isup2.hklStructure factors: contains datablock(s) I. DOI: 1451795CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Allium genus is currently classified. Apart from sulfur compounds, these are important biologically active molecules that are considered to be responsible for the observed activity of Allium species, including antifungal, cytotoxic, enzyme-inhibitory, and other. In this paper, literature data concerning chemistry and biological activity of steroidal saponins from the Allium genus has been reviewed.Steroidal saponins are widely distributed among monocots, including the Amaryllidaceae family to which the The online version of this article (doi:10.1007/s11101-014-9381-1) contains supplementary material, which is available to authorized users. Allium (Amaryllidaceae) is one of the largest monocot genera comprising more than 800 species , Costaceae (Costus), Dioscoreaceae (Dioscorea), Liliaceae (Lilium), Melanthiaceae (Paris), Smilacaceae (Smilax). Interestingly, these compounds have been reported as well in some dicotyledonous angiosperms: Zygophyllaceae , Solanaceae , Plantaginaceae  and Fabaceae (Trigonella).Apart from the Amaryllidaceae family, steroidal saponins are widely distributed in other monocot families: Asparagaceae , rhamnose (Rha), galactose , xylose (Xyl), and arabinose (Ara) units.Steroidal saponins from the genus Allium spirostanols is associated with the differences in the structure of aglycones, especially their oxygenation patterns and stereochemistry (Table trans (5α), or more rarely in a cis (5β) fusion (e.g. anzurogenin A [48] and C [58]). Δ5(6) unsaturation is considered to be a quite common feature . However, a double bond located at C25(27) was reported in the aglycones of saponins present in A. macrostemon and in one of the sapogenins identified in A. ursinum bulbs   , or C-27 (tuberoside L [104])  series. In the case of furostane-type sapogenins a double bond may also be located at 20(22)  or 22(23) (four furostanols from A. tuberosum)  unsaturation with an exception of schubertoside A [329]—Δ4(5), and one of the glycosides found in A. albopilosum with a saturated aglycone -cholest-5(6)-ene-1β,3β,16β,22-tetrol , or related sapogenins showing the same oxygenation pattern at C-1, C-3, C-16 and C-22 are most common   . Amongst tested spirostane saponins dioscin [135], isolated from A. ampleloprasum, seemed to be most potent, with an IC50 = 0.092 μg/mL against P388 cell line -spirost-5(6),25(27)-diene-3β-ol tetrasaccharide  (A. ursinum) -[α-l-Rha-(1 → 4)]-O-β-d-Glc) by an additional terminal rhamnose moiety. Both exhibited 100 % effect already at the concentration of 2 μg/mL on melanoma B16 and sarcoma XC. Similarly, deltonin [134] (diosgenin 3-O-β-d-Glc-(1 → 4)-[α-l-Rha-(1 → 2)]-O-β-d-Glc) isolated from A. schoenoprasum showed significant activity against HCT 116 and HT-29 cell lines with an IC50 = 0.40 and 0.75 μM, respectively -O-β-d-Glc sugar sequence attached to diosgenin is crucial for activity  and diosgenin 3-O-α-l-Rha-(1 → 2)-[β-d-Glc-(1 → 3)]-O-β-d-Glc [140] (A. senescens) were cytotoxic towards HeLa cells at the concentration of 50 μg/mL, whereas already at 5 μg/mL they exhibited 64.7 and 11.5 % inhibition, respectively  from the bulbs of A. karataviense exhibited considerable cytostatic activity with an IC50 value of 2.4 μg/mL as compared with etoposide (IC50 0.3 μg/mL)  on the HT-29 and HCT-116 cancer cell lines in the MTT assay -O-α-l-Rha 16-O-β-d-Gal [308], which exhibited in vitro cytotoxic properties (IC50 4.0–5.8 μg/mL) against two murine cell lines: WEHI 164 and J-774  exhibited much weaker activity when compared with 6-MP and were virtually inactive (>100 μg/mL)  -ene-3β,12β,22,26-tetrol 3-O-β-d-Glc-(1 → 2)-[β-d-Glc-(1 → 3)]-O-β-d-Glc-(1 → 4)-O-β-d-Gal [292] was found cytotoxic towards SF-268 cell line, while 26-O-β-d-Glc 5β-furost-20(22),25(27)-diene-3β,12β,26-triol 3-O-β-d-Glc-(1 → 2)-O-β-d-Gal [293] showed cytotoxicity towards SF-268 and NCI-H460 cell lines  showed concentration-dependent antifungal activity against a number of pathogens: Alternaria alternata, A. porri, B. cinerea, Fusarium oxysporum, F. solani, Pythium ultimum,R. solani, Trichoderma harzianum P1, T. harzianum T39 . Persicosides A [120] and B [121]—compounds isolated from A. ampeloprasum ssp. persicum, showed a statistically significant activity against P. italicum, A. niger and T. harzianum, higher than furostanol and cholestane compounds  showed antifungal activity, dependent on their concentration and the fungal species used: soil-borne pathogens , air-borne pathogens , antagonistic fungi (Trichodermaatroviride and T. harzianum), and a pathogen specific to the Allium genus—S. cepivorum -O-β-d-Glc-(1 → 4)-O-β-d-Gal [91] and β-chlorogenin 3-O-β-d-Glc-(1 → 2)-[β-d-Xyl-(1 → 3)]-O-β-d-Glc-(1 → 4)-O-β-d-Gal [80] exhibited antifungal activity on several Candida strains, including C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. kefyr, C. krusei, C. lusitaniae, and also on Cryptococcus neoformans, however β-chlorogenin glycoside was the most active compound with MFC from ≤6.25 to 25 μg/mL (as compared to amphotericin B 0.78–12.5 μg/mL). In another study the same compound isolated from A. porrum showed antifungal activity towards Fusarium culmorum (ED50 = 30 μg/mL) , β-chlorogenin glycoside as well, inhibited in vitro the growth of C. albicans (MIC 25 μg/mL) -O-β-d-Gal [87], and furostane-type ampeloside Bf1 [202] isolated from the same species did not inhibit the growth of Aspergillus niger; spirostanols showed weak activity against Candida albicans -ruscogenin glycosides), which are present mainly in the basal plates and roots of A. cepa Aggregatum group, to a different extent inhibited in vitro a wide range of plant pathogenic fungi: Alternaria ssp., Botrytis ssp., Colletotrichum spp., Curvularia lunata, Epicoccum nigrum, Fusarium ssp., Magnaporthe oryzae,S. cepivorum, and Thanatephorus cucumeris  the growth of Colletotrichum spp. isolates. It was also more effective against M. oryzae and S. cepivorum compared to alliospiroside B [170], however, its antifungal activity against B. cinerea, F. oxysporum and F. solani was relatively low.Alliospirosides A  (IC50 7.0 × 10−5 and 4.0 × 10−5 M respectively). Laxogenin glycosides exhibited significant activity only on cAMP phosphodiesterase, one of which, with an acetyl group in the saccharide moiety, was almost as potent as papaverine used as a positive control (IC50 3.3 × 10−5 and 3.0 × 10−5 M respectively).Saponin fraction isolated from the methanol extract of A. giganteum bulbs inhibited cAMP phosphodiesterase -5α-spirostane-2α,3β,5α,6β,24-pentaol 2-O-β-d-Glc [193] exhibited inhibitory activity almost equal to that of papaverine (IC50 4.1 × 10−5 and 3.0 × 10−5 M respectively). In the same study, furostanol saponins were revealed to be much more potent than the corresponding spirostanol glycosides. The results were in contrast to the previous studies of these authors which showed that furostanol glycosides were less active, exhibiting only weak inhibitory activity or none. The authors concluded that the anti-enzyme activity could be dependent on the number of hydroxyls in the A and B rings as in the present study the tested furostanol saponins contained several OH groups.Also, saponins isolated from A. karataviense and A. cepa as well as the products of chemical modifications of karatavioside A, were studied on a highly purified porcine kidney Na+/K+ATP-ase, in the concentration range from 1 × 10−4 to 1 × 10−7 M -induced human umbilical vein endothelial cell (HUVEC) injury  in a dose-dependent manner. Fistulosaponin A [250] was the most effective compound with a cell viability of 59.5 ± 3.0, 76.3 ± 3.3, 80.1 ± 3.6, 82.7 ± 4.1, 86.3 ± 4.6, and 78.2 ± 2.8 % for the six dose groups , respectively.Nine furostane saponins isolated by Lai et al. from 334], isolated from fruits of A. suvorovii and A. stipitatum, exhibited a statistically reliable hypotriglyceridemic activity in experimental hyperlipidemia caused by 1-day starvation, Triton WR-1339 and vitamin D2–cholesterol, when compared with lipanthyl .Furostanol saponins hirtifoliosides C1/C2  and B1/B2  were able to dose-dependently relieve acetylocholine- and histamine-induced contractions (50 % inhibition of contractions was seen at the concentration of 10−5 M)  -5α-furostane-3β,12β,22,26-tetrol 3-O-β-d-Glc-(1 → 2)-[β-d-Glc-(1 → 3)]-O-β-d-Glc-(1 → 4)-O-β-d-Gal [289] and 26-O-β-d-Glc (25R)-5α-furostane-3β,12α,22,26-tetrol 3-O-β-d-Glc-(1 → 2)-[β-d-Glc-(1 → 3)]-O-β-d-Glc-(1 → 4)-O-β-d-Gal [290] exhibited significant inhibitory activity on CD40L expression on the membrane of ADP stimulated platelets -[β-d-Glc-(1 → 3)]-O-β-d-Gal 6-O-β-d-Glc [78], isolated from the bulbs of A. ampeloprasum var. porrum, demonstrated in vivo antiinflammatory and gastroprotective effects in a carrageenan-induced oedema assay and by measuring acute gastric lesions induced by acidified ethanol -[β-d-Glc-(1 → 6)]-O-β-d-Glc-(1 → 4)-O-α-l-Rha-(1 → 4)-[α-l-Rha-(1 → 2)]-O-β-d-Glc [146], isolated from A. vineale, exhibited 100 % effect at 25 ppm in <24 h -5β-spirostane-1β,3β-diol [8] and 2,3-seco-porrigenin [64]. Despite a relatively low content of steroidal glycosides in Allium species, they are considered to contribute, in addition to sulfur compounds, to the overall biological activity of these plants. Undoubtedly, stability of saponins is their advantage as compared to fairly unstable sulfur compounds, thus, they in fact may be predominant active constituents of Allium products. Bearing this aspect in mind it seems highly feasible to develop antifungal Allium preparations against animal and plant pathogens. Also, reports on high in vitro cytotoxic activity of steroidal saponins from Allium species makes them potential candidates for further development as anti-cancer agents.Supplementary material 1 (DOC 410 kb)Supplementary material 2 (DOC 178 kb)Below is the link to the electronic supplementary material.


Reg (Regenerating gene) gene was originally isolated from rat regenerating islets and its encoding protein was revealed as an autocrine/paracrine growth factor for β cells. Rat Reg gene is activated in inflammatory conditions for β cell regeneration. In human, although five functional REG family genes  were isolated, their expressions in β cells under inflammatory conditions remained unclear. In this study, we found that combined addition of IL-6 and dexamethasone (Dx) induced REG Iα and REG Iβ expression in human 1.1B4 β cells. Promoter assay revealed that a signal transducer and activator of transcription- (STAT-) binding site in each promoter of REG Iα (TGCCGGGAA) and REG Iβ (TGCCAGGAA) was essential for the IL-6+Dx-induced promoter activation. A Janus kinase 2 (JAK2) inhibitor significantly inhibited the IL-6+Dx-induced REG Iα and REG Iβ transcription. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed that IL-6+Dx stimulation increased STAT3 binding to the REG Iα promoter. Furthermore, small interfering RNA-mediated targeting of STAT3 blocked the IL-6+Dx-induced expression of REG Iα and REG Iβ. These results indicate that the expression of REG Iα and REG Iβ should be upregulated in human β cells under inflammatory conditions through the JAK/STAT pathway. Regeneration of pancreatic β cells has been shown to occur at a basal rate in normal adult tissues and to increase under certain conditions such as pregnancy and obesity [β cell regeneration still remains to be elucidated.Decreased functional  obesity . HoweverReg (Regenerating gene) gene product, Reg protein, could be responsible for the regenerative process [ Reg gene was originally isolated from regenerating pancreatic islets from depancreatized rats, and it encodes a 16 kDa autocrine/paracrine growth factor for β cells [ Reg and Reg-related genes were isolated and revealed to constitute a multigene family, the Reg family, which consists of four subtypes  based on the primary structures of the encoded proteins of the genes [ Reg gene, Reg I, was the first isolated Reg gene from the regenerating islets [β cells in physiological conditions but is induced during islet regeneration [ Reg I gene induction, which occurs in response to inflammatory mediators such as interleukin- (IL-) 6 and glucocorticoid analogue dexamethasone (Dx) in rat RINm5F β cells [β cell regeneration. In human, five functional REG family genes  were isolated [ REG gene is expressed in β cells during regeneration is still obscure mainly because of their restrict supply of human islets. Induction of proliferation/regeneration in human β cells must be beneficial for both type 1 and type 2 diabetes treatment/prevention [ REG genes would play an important role in β cell proliferation/regeneration in human as the Reg I gene does in rodents. Since there are many differences in cell proliferation between rodent and human β cells [ REG genes in human β cells. Recently, β cell line, 1.1B4, was established and becomes available as human pancreatic β cells [ REG family genes in inflammatory conditions in 1.1B4 human β cells. process –6. The R β cells , 8. The he genes , 9, 10. g islets . In rat neration , 11, 12. β cells , is consisolated , 14–19. evention . The hum β cells , it is c β cells . In the β cell line  [μg/mL streptomycin  as described [2-95% air, water-saturated atmosphere at 37°C. Recombinant human IL-6, tumor necrosis factor- (TNF-) α, and interferon- (IFN-) γ were purchased from Roche Applied Science . Recombinant human IL-1β, IL-22, IFN-β, IL-8, and H2O2 were obtained from Wako Pure Chemical Industries. Recombinant human platelet-derived growth factor (PDGF) was obtained from PeproTech . Sodium palmitate  was prepared as albumin-bound form by stirring at 45°C with defatted bovine serum albumin . Dexamethasone (Dx) was purchased from MP Biomedicals . S-Nitroso-N-acetylpenicillamine (SNAP), a NO donor [Human 1.1B4 cells, an insulin-releasing human pancreatic ury, UK) , 23, andNO donor , and AG4NO donor , were ob REG family genes  upstream of a firefly luciferase reporter gene in pGL3-Basic vector . For construction of the promoter region mutants used in this study, the indicated sequences in the “−146” REG Iα/luciferase constructs and “−155” REG Iβ/luciferase constructs were substituted by primers using PCR. The cells were grown in 24-well plates and were transfected with reporter plasmids by lipofection. Briefly, 0.8 μg of each reporter plasmid and 0.08 μg of pCMV-SPORT-β-galactosidase , as an internal control, were mixed with Lipofectamine 2000 (Life Technologies) per well in a 24-well plate. After 24 h incubation, the cells were treated with indicated amount of stimulants, 100 nM Dx, 20 ng/mL IL-6, 300 U/mL IL-1β, 370 U/mL TNF-α, 100 U/mL IFN-γ, 60 U/mL IL-1β + 185 U/mL TNF-α + 14 U/mL IFN-γ, 10 ng/mL IL-22, 1500 U/mL IFN-β, 100 nM IL-8, and 1 U/mL PDGF, amino acids at a concentration five times greater than that in basal RPMI, 100 μM palmitate, 10 μM H2O2, 250 μM SNAP, 2 mM STZ, and 2 mM 3AB, and combinations thereof. After 24 h treatment, the cells were harvested and processed for luciferase assay as described [The reporter constructs were prepared by inserting the 5′-flanking regions of humanβ-actin (NM_001101) sense, 5′-GCGAGAAGATGACCCAGA-3′ and antisense, 5′- CAGAGGCGTACAGGGATA-3′; REG Iα (NM_002909) sense, 5′-AGGAGAGTGGCACTGATGACTT-3′ and antisense, 5′-TAGGAGACCAGGGACCCACTG-3′; REG Iβ (NM_006507) sense 5′-GCTGATCTCCTCCCTGATGTTC-3′ and antisense 5′-GGCAGCTGATTCGGGGATTA-3′; REG III (AB161037) sense 5′-GAATATTCTCCCCAAACTG-3′ and antisense 5′-GAGAAAAGCCTGAAATGAAG-3′; HIP/PAP (NM_138937) sense 5′-AGAGAATATTCGCTTAATTCC-3′ and antisense 5′-AATGAAGAGACTGAAATGACA-3′; REG IV (AY007243) sense 5′-ATCCTGGTCTGGCAAGTC-3′ and antisense 5′-CGTTGCTGCTCCAAGTTA-3′. All the PCR primers were synthesized by Nihon Gene Research Laboratories . Real-time PCR was performed using KAPA SYBR FAST qPCR Master Mix  and the Thermal Cycler Dice Real-Time System  as described [β-actin, REG III, and HIP/PAP and 40 cycles of 3 s at 95°C and 20 s at 64°C for REG Iα, REG Iβ, and REG IV. The level of target mRNA was normalized to the mRNA level of β-actin as an internal standard.Total RNA was isolated using the RNeasy Plus Mini Kit  from 1.1B4 cells, and cDNA was synthesized from total RNA as a template using High Capacity cDNA Reverse Transcription Kit  for the template of real-time PCR as described , 27, 28.escribed , 27, 28. REG Iα promoter; nt. 1048–1072 of J05412), probe M1, 5′-AGTGTGCAGTAGGAAAAGGCTCATA-3′.Nuclear extracts were prepared as described previously and stored at −80°C until use , 29. EMS2, 1 mM EDTA, 10% glycerol, 5 mM DTT, 1 mg/mL BSA, 0.7 mM PMSF, 50 ng/μL of poly(dI-dC) (Roche) for 10 min at room temperature, followed by an additional 30 min incubation with 32P-end-labeled probe at room temperature. For supershift assays, 1 μg of anti-signal transducer and activator of transcription (STAT)3 antibody  [DNA-protein binding reactions were performed by incubation of the nuclear extracts in a solution containing 10 mM HEPES (pH 7.8), 50 mM KCl, 5 mM MgClμg of anti-STAT3 antibody . The immune complexes were precipitated, eluted, reverse cross-linked, and treated with proteinase K. The resulting DNA samples were subjected to PCR amplification  of the REG Iα promoter using specific forward (5′-ACCTTGGACTTAGACAGCTTG-3′) and reverse (5′-ACCACGTCATTTAAGCAAAAGG-3′) primers designed to amplify the promoter region (−212 to −46 nucleotides in relation to the transcription start site). The PCR products were resolved by electrophoresis in a 2.5% agarose gel containing ethidium bromide for visualization.ChIP assay was performed using a ChIP-IT Express kit  following the manufacturer's instructions as described previously . Briefly Silencer Select predesigned small interfering RNAs (siRNAs) (Life Technologies) [ STAT3 gene. The sense sequence of siRNA (5′-GCACCUUCCUGCUAAGAUUtt-3′) was synthesized by Nihon Gene Research Laboratories. As a control, siRNA-scramble  [5 cells/mL) as described [RNAi was performed usingologies)  directedologies)  was alsoescribed .t-test using GraphPad Prism software .Results are expressed as mean ± SE. Statistical significance was determined by Student's  REG family gene transcription in human 1.1B4 β cells. As Reg gene expression was observed in the phase of transient β cell proliferation: for example, in pancreatic islets of diabetic BB/Wor//Tky rats during the remission phase of diabetes [β cells, during differentiation and proliferation in a mouse model of autoimmune diabetes [β, TNF-α, and IFN-γ [β cell toxic agents, such as palmitate [2O2 [ Reg gene expression in β cells, such as IL-6 [β [ REG Iα promoter activity in gastric endocrine carcinoma cells [ REG Iα promoter activity (columns 3 and 13). When IL-6 and the glucocorticoid analogue Dx were added together, the promoter activity was remarkably increased (columns 3 and 4). In contrast, the combination of IL-22 and Dx did not further increase the promoter activity (columns 13 and 14). The combination of IL-22 and IL-6 + Dx did not result in the synergistic response (columns 14 and 15). Treatments with other stimulants were ineffective or resulted in only small changes in promoter activity of REG Iα gene compared to IL-6 + Dx. These results showed that IL-6 + Dx was the most effective inducer for human REG Iα gene transcription in human β cells. IL-6 + Dx also enhanced the promoter activities of REG Iβ and HIP/PAP significantly; however, the fold increases were much smaller compared to that of REG Iα  were induced in human β cells under inflammatory conditions by increased levels of circulating IL-6 and glucocorticoids.We next analyzed the mRNA levels of intrinsic human Figures . On the  Figures . These r REG Iα induction by IL-6 + Dx, the reporter plasmids containing progressively deleted promoter fragments of REG Iα  upstream of luciferase gene were transfected into 1.1B4 cells. Deletion of the REG Iα promoter from position of −1190 to −146 retained a significant increase in response to IL-6 + Dx in luciferase activity  , significantly blocked the IL-6 + Dx-induced transcriptional activation of REG Iα in 1.1B4 cells  . Site-diCCGGGAA)  on the aB4 cells . Reg I gene for induction in response to IL-6 + Dx was the −81~−70 region, which corresponds to the −76~−65 region of human REG Iα gene [ Reg I gene and was involved in the active transcriptional DNA/protein complex formed by the stimulation of IL-6 + Dx. The DNA/protein complex formation was inhibited by the autopoly(ADP-ribosyl)ation of PARP in the complex. Thus, PARP inhibitors, such as 3AB, enhanced the DNA/protein complex formation for Reg I gene transcription [ REG Iα transcription, we treated 1.1B4 cells with IL-6 + Dx + 3AB and measured the promoter activities of the constructs “−146” and “−146 M.” As shown in  REG Iα transcription by IL-6 + Dx.In rat RINm5F cells, the cis-element of rat Iα gene . In RINmcription . To clarμM), for 4 h significantly inhibited the inducibility of REG Iα promoter by IL-6 + Dx  as shown in  REG Iβ transcription  ( REG Iα gene ( REG Iα promoter was induced by IL-6 + Dx in β cells.To further investigate whether STAT protein interacts with theCCGGGAA) , lane 2.CCGGGAA) , suggest Iα gene . These r REG Iα and REG Iβ was further demonstrated by siRNA. 1.1B4 cells were transfected with scrambled siRNA control or STAT3-specific siRNA and were stimulated with IL-6 + Dx. We confirmed that STAT3-siRNA suppressed both basal and IL-6 + Dx-induced STAT3 mRNA  is induced during pancreatic β cell regeneration [ REG genes  [ REG gene is involved in the β cell regeneration. In this study, we found that among five functional human REG genes, REG Iα and REG Iβ gene expressions were significantly enhanced by the combined addition of IL-6 and Dx in human β cells. The rat Reg I gene expression is activated by IL-6 + Dx [α protein promotes the proliferation of RINm5F cells and mouse islets [β cell proliferation.neration , 7. The  REG IV) , 50, 51.L-6 + Dx , and we e islets , 53. Thee islets , 31, the REG gene (REG Iα and REG Iβ) expression through the JAK/STAT3 pathway. It has been reported that in gastric cancer cells, IL-6 enhanced REG Iα transcription through the STAT3 activation [ REG Iα promoter activity by IL-6 was reported also in colon cancer cells [ REG Iα gene expression is not restricted in pancreatic β cells but rather common to diverse cell types. However, in β cells, Dx is required in addition of IL-6 for the induction, since IL-6 alone only slightly induced REG Iα expression. We recently reported that HGF gene transcription was also induced by IL-6 + Dx through STAT3 activation in pancreatic β cells [β cells. The role of glucocorticoids in the REG Iα induction is currently under investigation.We demonstrated that IL-6 + Dx induced the type Itivation , 55. In er cells  and in ser cells . Thus, i β cells . It is p REG Iα, were much larger than those of REG Iβ. The reason of the difference of inducibility between REG Iα and REG Iβ is not clear. The expression patterns of two genes are different in pancreas [ REG Iα gene expression but not REG Iβ gene expression was increased [ REG genes, these two genes should be regulated independently by other transcription factors under physiological and pathological conditions. Previous [ REG Iα gene rather than REG Iβ gene should be much more important for β cells in the circumstances in which β cells are damaged.The IL-6 + Dx-induced increases, in the promoter activity and the mRNA level ofpancreas . In addincreased . Thus, aPrevious  and curr REG Iα transcription via STAT3 activation in human colon cancer cells [ Reg I and Reg II in NOD mouse islets [β cells [ REG Iα and REG Iβ promoter activities in 1.1B4 cells. Therefore, STAT3 may not be fully activated by IL-22 nor IL-22 + Dx in human pancreatic β cells.IL-22 was reported to induceer cells  and to ie islets , 45, 60.[β cells . However Reg I gene for induction in response to IL-6 + Dx was the −81~−70 region [ Reg I promoter corresponds to the −76~−65 region of the human REG Iα gene, which is downstream of the STAT-binding site (−142~−134). In reporter assays, the promoter activity of the construct “−130” was not enhanced by the stimulation of IL-6 + Dx. Moreover, the combined addition of 3AB with IL-6 + Dx did not enhance the promoter activity of mutant construct “−146 M” as much as that of “−146.” These results suggest that although PARP was involved in the IL-6 + Dx-induced activation of REG Iα promoter, STAT binding to the −142~−134 region played a major role for the induction of REG Iα transcription in human β cells.In rat RINm5F cells, the cis-element of rat0 region . This re REG family genes, the expressions of REG Iα and REG Iβ were significantly enhanced by IL-6 + Dx. These results suggest that, under inflammatory conditions, human type I REG proteins could be increased and function as growth factors for β cells to facilitate proliferation. Furthermore, we showed that the JAK/STAT pathway was involved in the IL-6 + Dx-induced human type I REG gene transcription in β cells. Therefore, polymorphisms and/or mutations in REG Iα and REG Iβ promoters as well as other polymorphisms/mutations of factors involved in the JAK/STAT pathway may be involved in diabetes incidence/pathology.In this study, we found that among five human


N,N-dimethylpyridinium cations possess an extended chain conformation, while the bisnicklate(II) complex anion has a square-planar NiS4 geometry comprising four S-donor atoms from two bidentate chelate comprising maleonitriledithiolate ligands, with the Ni2+ cation lying on a crystallographic mirror plane. The crystal has a layered structure consisting of alternating cations and anions.In the title salt, the1-hexyl- 13H23N2)2[Ni(C4N2S2)2], consists of a 1-hexyl-N,N-dimethylpyridinium cation and one half of a [Ni(mnt)2]2− dianion  in which the Ni2+ cation lies on a crystallographic inversion centre. The square-planar coordination about Ni2+ comprises four S atoms from two bidentate chelate mnt2− ligands . The hydrocarbon chains of cations show trans-planar conformations and lie approximately parallel to the long molecular axis of the [Ni(mnt)2]2− anions, giving stacks along the c axis. The anions and cations form layers lying parallel to the bc plane. Only weak C—H⋯Ni and C—H⋯π associations are present in the crystal packing.The asymmetric unit of the title compound, (C M[dithiolene]2 complexes because of their application as building blocks in molecular-based materials showing magnetic, superconducting and optical properties 2]2− dianion, giving the title compound, (C13H23N2)2[Ni(C4N2S2)2], (I)Molecular solids based on transition metal dithiolene complexes have attracted much interest in recent years, not only regarding fundamental research of magnetic interactions and magneto-structural correlations but also in the development of new functional-molecule-based materials 2]2− dianion . The Ni2+ ion lies on a crystallographic inversion centre re Fig. 1. The comtrans-planar conformation, with a chain to pyridyl ring dihedral angle of 83.03 (19)°. The direction of the hydrocarbon chains is approximately parallel to the long molecular axis of the anions, with a dihedral angle between the molecular plane of the hydrocarbon chain and that of the anion  of 10.76 (18)° . Between the cation and anion there is a novel Ni1⋯H—C17ii interaction (H⋯Ni = 2.72 Å)  Fig. 2 2− dianions : to Cg1ii and Cg1iii (H⋯Cg = 2.77 Å) .In the crystal there are no formal hydrogen-bonding interactions. However, there are two weak C17—H⋯π associations to the chelate ring of the [Ni(mnt)2]2− complex dianions, chair-shaped organic compounds have been chosen as counter-cations and a series of compounds with segregated anion and cation stacks have been obtained N,N-dimethylpyridinium iodide (1.0 mmol) in methanol (10 mL) was added to the mixture and the red precipitate that was immediately formed was filtered off and washed with methanol. The crude product was recrystallized in acetone (20 mL) to give red block-shaped crystals which were used in the X-ray analysis.Disodium maleonitriledithiolate (2.0 mmol) and nickel(II) chloride hexahydrate (1.0 mmol) were mixed with stirring in water (20 mL) at room temperature. Subsequently, a solution of 1-hexyl-Uiso(H) = 1.2Ueq(aromatic or methylene) or 1.5Ueq(methyl).Crystal data, data collection and refinement details are summarized in Table 110.1107/S2056989016011191/zs2364sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989016011191/zs2364Isup2.hklStructure factors: contains datablock(s) I. DOI: 1491765CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title compound is a 1,4-diaroyl pyrazole derivative and has three aromatic rings. In the crystal, molecules are linked through stacking interactions between the pyrazole rings and between the naphthalene and phenyl rings, and through intermolecular C—H⋯O hydrogen bonds to form inversion dimers. 21H12Cl2N2O3, is a 1,4-diaroyl pyrazole derivative and has three aromatic rings. The dihedral angles between the naphthalene ring system and the pyrazole ring, the pyrazole and phenyl rings and the naphthalene ring system and the phenyl ring are 49.44 (13), 49.87 (16) and 0.58 (11)°, respectively. The phenolic proton forms an intramolecular O—H⋯O hydrogen bond with an adjacent carbonyl O atom. In the crystal, the molecules are linked through stacking interactions between the pyrazole rings [centroid–centroid distances = 3.546 (3)] and between the naphthalene ring system and the phenyl ring [centroid–centroid distances = 3.609 (4) Å] along the a-axis direction. The molecules are further connected through C—H⋯O hydrogen bonds, forming inversion dimers.The title compound, C The molecules are further connected through intermolecular C—H⋯O hydrogen bonds -N′-[methylene]-1-naphthohydrazide, is as follows: 1-naphthohydrazide (2.7 mmol) and 6,8-dichloro-3-formylchromone (2.7 mmol) were dissolved in 50 ml of benzene, and the mixture was refluxed with a Dean–Stark apparatus for 2 h with stirring. After cooling, the yellow precipitates were collected, washed with n-hexane and dried in vacuo (yield 18%). 1H NMR : δ = 7.60–7.64 , 7.78 , 8.03 , 8.11 , 8.23 , 8.26 , 8.48 , 8.98 , 12.17 . DART–MS calculated for [C21H12Cl2N2O3 + H+]: 411.030, found 410.905. Orange crystals of the title compound suitable for X-ray diffraction were obtained by slow evaporation of an ethyl acetate/acetone solution of the yellow precursor at room temperature.Preparation of the yellow precursor, (Uiso(H) = 1.2Ueq(C)]. The phenolic proton was located in a difference Fourier map, and refined using a riding model [O—H 0.84 Å with Uiso(H) = 1.5Ueq(O)].Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S1600536814024684/hb7275sup1.cifCrystal structure: contains datablock(s) General, I. DOI: 10.1107/S1600536814024684/hb7275Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814024684/hb7275Isup3.cmlSupporting information file. DOI: 1033534CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N--4-ethenyl-benzenamine and N--3-ethenyl-benzenamine (TBAMS) are novel (Co-)Monomers for intrinsic anti-microbial polymers. The secondary amines were prepared and polymerized to the corresponding water insoluble polymer. The antimicrobial activity was analyzed by the test method JIS Z 2801:2000. Investigations revealed a high antimicrobial activity against Staphylococcus aureus and Escherichia coli with a reduction level of >4.5 log10 units. Furthermore, scanning electron microscopy (SEM) of E. coli. in contact with the polymer indicates a bactericidal action which is caused by disruption of the bacteria cell membranes, leading to lysis of the cells.The use of biocidal compounds in polymers is steadily increasing because it is one solution to the need for safety and hygiene. It is possible to incorporate an antimicrobial moiety to a polymer. These polymers are referred to as intrinsic antimicrobial. The biocidal action results from contact of the polymer to the microorganisms, with no release of active molecules. This is particularly important in critical fields like food technology, medicine and ventilation technology, where migration or leaching is crucial and undesirable. The isomers  N-halamines  (poly(TBAEMA)) is a typical representative of a water-insoluble biocide, its structure is displayed in ®, Marl, Germany). According to the patent literature, poly(TBAEMA) exhibits inherent biocidal properties and has shown potential application in antifouling paints and coatings + was observed. The major fragments are at m/z 174 represents the (M-CH4)+, m/z 132 the (M-tert-butyl)+ (only observed after collision fragmentation of the isolated m/z 190) and m/z 117 for the loss of tert-butylamin. The structure was further confirmed by 1H and 13C NMR  that can be associated to the ammonium group in the hydrochloride. If the TBAMS hydrochloride is analyzed by GC-MS, the salt decomposes to HCl and TBAMS at the high temperature of the injector. Accordingly TBAMS was observed in the GC-MS chromatogram instead of TBAMS hydrochloride. As mentioned in For elemental analysis and further characterization of TBAMS the hydrochloride was prepared. The elemental analysis showed a good match of calculated C/N/H-ratios and experimentally determined values. The IR spectrum contains structural features  was raised to about 185 kDa in typical experiments. The polydispersity (MW/Mn) of 5.2 is rather high. A pronounced polydispersity (2.5–3) is typical for AIBN initiated radical reactions. The high polydispersity observed in the poly(TBAMS) polymer can be an indication, that grafting occurs during polymerization. Probably chain transfer occurs at the benzylic position of poly(TBAMS) and TBAMS. Poly(TBAMS) possesses a TG of about 68 °C, which is considerably higher than the TG of poly(TBAEMA). The poly(TBAMS) was soluble in ethanol and formed uniform, colorless and transparent films upon solvent evaporation. Glass transition temperatures can be further be adjusted by copolymerization to about 160 °C without the loss of the antimicrobial activity. The copolymer was synthetized using 42 mol % TBAMS, 42 mol % 4-vinylpyridine and 16 mol % 4-vinylbenzoic acid. The log reduction of this copolymer was 3.5 for S. aureus.Success of the polymerization was confirmed by  13C NMR . First e 13C NMR . The molTG of 218 °C is a poly-4-aminophenol conjugate [E. coli and S. aureus are good but cannot be directly compared to poly(TBAMS) as different methods were used to determine the reduction rates. Methylacrylamide based polymers and copolymers described by Dizman et al. also had comparingly high glass transition temperature of 169 °C for the homopolymer [TG, exhibited good antimicrobial properties.Chitosan is a well-studied intrinsic antimicrobial polymer, which is a possible candidate for technical application. The glass transition temperature varies over a broad range between 61 and 203 °C, depending on molecular weight, degree of deacetylation and residual water content ,34,35. Aonjugate . The antopolymer . UnfortuS. aureus determined on references samples without incubation was 1.6 × 105 CFU/mL and for E. coli was 4 × 105 CFU/mL.The antimicrobial activity of the polymer was analyzed by the test method JIS Z 2801:2000. The test method is based on the comparison of bacterial counts on reference and sample material after inoculation and incubation. The initial bacterial concentration of S. aureus on reference dishes is 5.5 log10 CFU/mL and for E. coli is 6.5 log10 CFU/mL after 2 h incubation time at 35 °C. Bacterial counts of S. aureus and E. coli on poly(TBAMS) were below the detection limit of 1 log10 CFU/mL and show a reduction level of 4.5 log10 units for S. aureus and 5.5 log10 units for E. coli respectively. Thus, the antimicrobial activity is proven against S. aureus and E. coli on poly(TBAMS). Poly(TBAMS) shows excellent antimicrobial properties towards the investigated microorganisms.The arithmetic mean of bacterial counts of E. coli after 12 h of contact pronounced morphological changes were observed, whereas cells in the blank petri dishes remained unchanged. Furthermore the cells show a exudate of fibrous and granular material after incubation with poly(TBAMS). The surface of treated bacteria appears to be wrinkled and rough if compared with untreated cells . In the case of the gram negative ed cells . AdditioN,N-dimethyl-aminomethylstyrene) bonded to a polystyrene fiber [The SEM images gave striking evidence that the mode of action of poly(TBAMS) is most likely due to disruption of cell walls. Very similar observations, concerning the morphological changes, have been made in the case of poly(TBAEMA) grafted on polyethylene , a poly.+-MS were recorded on a Bruker esquire 4000  instrument. Infrared spectra were recorded on a spectrum one  spectrometer using ATR technique. Differential scanning calorimetry analysis was carried out on a DSC821  system. 1H NMR and 13C NMR spectra were acquired on a Bruker Avance III NMR  spectrometer with an observing frequency of 400 MHz for 1H and 100 MHz for 13C, respectively. The samples were measured on the delta (δ) scale and are referenced to Trichloroethane  that was used as solvent.Melting points were recorded on a SPM30  using open capillaries. GC-MS were recorded on a QP2010plus  using a FS supreme 5 column . Detection was performed at 70 eV in EI mode. APCI2O (200 mL) and NaOH . After complete dissolution of the NaOH the solution was cooled to 25 °C the amine (1.05 mol) was added. The apparatus was put under inert atmosphere by purging with nitrogen. The heterogeneous mixture was heated to 70 °C using an external oil bath and chloromethylstyrene  in THF (150 mL) was added drop wise over the course of 75 min. After the addition was complete the reaction was stirred au 70 °C for additional 24 h. The organic phase was separated, dried over NaSO4 and filtered through a sintered glass funnel. Volatiles were removed under reduced pressure . Further workup was performed using fractional distillation.In a 1 L round bottom flask equipped with a reflux condenser and dripping funnel there were placed H1): Distillation: 135–140 °C, 20 mbar. Yield: 41.57g (65%). Purity: 96% . Analysis: EI-MS: M+ 175 m/z, 146 m/z, 117 m/z, 91 m/z, 58 m/z, 39 m/z. IR: 3309 cm−1·w, 3087 cm−1·w, 3006 cm−1·w, 2953 cm−1·m, 2930 cm−1·m, 2873 cm−1·m, 1629 cm−1·m, 1602 cm−1·w, 1582 cm−1·w, 1510 cm−1·m, 1456 cm−1·s, 1120 cm−1·s, 989 cm−1·s, 903 cm−1·s, 796 cm−1·s, 712 cm−1·s.Propyaminomethylstyrene (2): Distillation: 85–93 °C, 1 mbar. Yield: 52.82 g (81%). Purity: 95% . Analysis: EI-MS: M+ 175 m/z, 160 m/z, 117 m/z, 91 m/z, 65 m/z, 30 m/z.Isopropylaminomethylstyrene : Distillation 150–153 °C, 20 mbar. Yield: 46.06 g (66%). Purity: 96% . Analysis: EI-MS: M+ 189 m/z, 146 m/z, 117 m/z, 91 m/z, 65 m/z, 29 m/z.Butylaminomethylstyrene : Distillation: 140–145 °C, 21 mbar. Yield: 53.21g (76%). Purity: 95% . Analysis: EI-MS: M+ 189 m/z, 174 m/z, 160 m/z, 117 m/z, 91 m/z, 65 m/z, 39 m/z.Sec-Butylaminomethylstyrole : Distillation: 145–14 °C, 24 mbar. Yield: 51.55 g (74%). Purity: 95% . Analysis: EI-MS: M+ 189 m/z, 146 m/z, 117 m/z, 91 m/z, 65 m/z, 41 m/z.Isobutylaminomethylstyrole : Distillation: 117–123 °C, 1 mbar. Yield: 56.42 g (76%). Purity: 96% . Analysis: EI-MS: M+ 203 m/z, 146 m/z, 117 m/z, 43 m/z, 29 m/z.Pentylaminomethylstyrole : Distillation: 120–123 °C, 1 mbar. Yield: 59.65 g (76%). Purity: 95% . Analysis: EI-MS: M+ 203 m/z, 146 m/z, 117 m/z, 91 m/z, 41 m/z.3-Pentylaminomethylstyrole : Distillation: 140–145 °C, 9 mbar. Yield: 50.93 g (76%). Purity: 96% . Analysis: EI-MS: M+ 203 m/z, 146 m/z, 117 m/z, 41 m/z.Isopentylaminomethylstyrole : Distillation: 105–110 °C, 1 mbar. Yield: 55.12 g (74%). Purity: 96% . Analysis: EI-MS: M+ 203 m/z, 188m/z, 174 m/z, 117 m/z, 91 m/z, 42 m/z.−1·w, 3087 cm−1·w, 2963 cm−1·s, 2887 cm−1·s, 1630 cm−1·m, 1602 cm−1·w, 1582 cm−1·w, 1510 cm−1·m, 1460 cm−1·s, 1405 cm−1·m, 1380 cm−1·m, 1363 cm−1·m, 1289 cm−1·s, 1201 cm−1·s, 1093 cm−1·s, 988 cm−1·s, 903 cm−1·s, 827 cm−1·s, 796 cm−1·s, 712 cm−1·s.IR: 3320 cm10): Distillation: 132–140 °C, 1 mbar. Yield: 51.2 g (70%). Purity: 96% . Analysis: EI-MS: M+ 201 m/z, 172m/z, 158 m/z, 117 m/z, 91 m/z, 84 m/z, 65 m/z, 41 m/z.Cyclopentylaminomethylstyrole : Distillation: 135–137 °C, 1 mbar. Yield: 62.75 g (80%). Purity: 95% . Analysis: EI-MS: M+ 215 m/z, 172m/z, 158 m/z, 117 m/z, 98 m/z, 91 m/z, 55 m/z, 41 m/z.Cyclohexylaminomethylstyrole : Distillation: 165–170 °C, 2 mbar. Yield: 60.60 g (60%). Purity: 95% . Analysis: EI-MS: M+ 272 m/z, 257 m/z, 215 m/z, 173 m/z, 155 m/z, 117 m/z, 98 m/z, 58 m/z, 41 m/z.Tetramethylpiperidinaminomethylstyrole  in aqueous isoporopyl alcohol ). Finally, the polymer was dried in a vacuum drying cabinet  at 140 °C, 1 mbar until no more change in polymer mass was observed. Water soluble polymers were directly dried in vacuo after removal of volatiles  without prior precipitation in water.In a 250 mL round bottom flask the corresponding monomer (145.8 mmol) were dissolved in ethanol (60 mL), a solution of AIBN (0.37 g) in 2-butanone (10 mL) was added. The apparatus was put under inert atmosphere by purging with nitrogen. The contents of the flask were heated under constant stirring (300 rpm) to 60 °C with internal temperature control in a silicon oil bath for 48 h. The polymer was precipitated by slowly dripping the contents of the flask in to a stirred bath of 5 L H2O (300 mL) and NaOH  were placed. The solution was cooled down to room temperature and tert-butyl amine  was added. The apparatus was put under inert atmosphere by purging with nitrogen. The contents of the flask were heated to 60 °C with internal temperature control in a silicon oil bath. Vinylbenzyl chloride  in THF (200 mL) was added in a drop wise manner over the course of 4 h. Stirring and heating was continued over 20 h. After this the organic phase was separated using an extraction funnel. It was washed with H2O (2 × 50 mL) and dried over MgSO4. TBAMS was isolated by fractional distillation  as colorless oil with a high refractive index. The yield was 55.65 g (96%). Purity was 97% .In a 1 L three neck round bottom flask, equipped with reflux condenser, dripping funnel and magnetic stir bar H+ 189 m/z, 1.08% ;174 m/z, 26.23%; 117 m/z 100.00%; 91 m/z, 9.06%; 57 m/z, 3.76%; 41 m/z, 6.92%. IR: 3306 cm−1·w, 3088 cm−1·w, 2963 cm−1·s, 1630 cm−1·m, 1510 cm−1·m, 1479 cm−1·s, 1307 cm−1·m, 1228 cm−1·s; 1211 cm−1·s, 988 cm−1·s, 903 cm−1·s, 827 cm−1·s, 797 cm−1·s, 712 cm−1. 1H NMR: δ 0.99 ppm , δ 1.30 ppm 3), δ 3.80 ppm , δ 5.35 ppm and δ 5.87 ppm , δ 6.85 ppm , δ 7,42 ppm ; 13C NMR: δ 29.5 ppm (C–(CH3)), δ 47, 47.2 ppm (–CH2–NH), δ 50.7 ppm (NH–C(CH3)3), δ 114.1.7, 113 ppm (–CH=CH2), δ 124.9, 126.4 128.1, 128.8 ppm (aromatic CH), δ 126.4 128.6 ppm (aromatic CH of para isomer), δ 136.3, 136.9 137.2, 137.8 ppm (Quart-aromatic C), δ 141.8 142.3 ppm (–CH=CH2).Analysis: EI-MS: M2SO4) was introduced via a glass capillary in to the solution until the solution was slightly acidic. Stirring was continued for 2 h. After precipitation of the hydrochloride at −20 °C, the crystals were removed by filtration, washed with ice cold acetone (2 × 10 mL) and dried in vacuum. The crystals were recrystallized from isopropyl alcohol/toluene (10 mL/150 mL). After cooling to −20 °C and drying in vacuum TBAMS℘HCl  was obtained.In a 500 mL round bottom flask TBAMS  in Acetone (150 mL) was placed. The flask was nestled in an ice bath and stirred magnetically. A slow stream of dry HCl  was slowly added via a 250 mL dropping funnel under stirring. After all water was added the stirrer was turned off and the solution stored for 12 h to separate the polymer from the upper ethanol phase which was removed by decantation. The precipitated polymer was dissolved in ethanol (135 mL) and the aforementioned precipitation process was repeated. The liquid phase is then analyzed via TLC . The polymer remained at the start, whereas the Monomer had a retention factor value of approximately 0.5. The purification process was repeated until no more TBAMS can be observed via TLC. In a typical experiment, eight purification steps were necessary. The purified poly(TBAMS) solution was dried in a vacuum drying cabinet  at 70 °C, 1 mbar for 16 h and milled down using an ultra-centrifugal mill . A 200 μm sieve was applied, the mill is continuously cooled using liquid nitrogen. Yield: 20 g, 74%.TBAMS  was placed in a 500 mL two-neck round-bottom flask equipped with a reflux condenser . In a 30 mL glass crimp-top vial 2,2′-Azobisisobutyronitrile  was dissolved in 2-butanone (10 mL). This initiator solution was added to the monomer, followed by ethanol (150 mL). The flask was transferred into an oil bath at room temperature  which was also used to stir the mixture via a magnetic stirrer. The apparatus was flushed with nitrogen (purity 99.99999%) for at least 30 min to ensure an oxygen free atmosphere. The reaction solution was continuously stirred (approx. 300 rpm) during this time. Polymerization was carried out for 5 h at 55 °C and additionally for 22.5 h at 65 °C. After the solution was cooled to room temperature, HGPC measurements were performed on a Viscotec GPC max VE2001  machine equipped with two PSS SD LIN M 5 μm 8 × 300 mm columns  coupled in series using THF with 0.1% triethylamine as eluent and RI detection. Calibration was done with a Polystyrene standard (474-2520000 Da) .TG: 68° C ;GPC: Mw: 185588 Da, Mn: 3562 Da, MW/Mn: 5.2; IR: 2960 cm−1·s, 2921 cm−1·s, 1605 cm−1·m, 1443 cm−1·s, 1360 cm−1·s, 1228 cm−1·s, 1088 cm−1·m, 1019 cm−1·m, 810 cm−1·m, 792 cm−1 cm, 704 cm−1; 1HNMR: δ 0.4–2.5 ppm (–(CH3)3, NH, CH and CH2 from polymer backbone), δ 3.10–3.90 ppm (–CH2–NH), δ 6.00–7.2 ppm (aromatic H); 13C NMR: δ 29.4 ppm (–(CH3)3), δ 40.4 ppm (CH2 from polymer backbone), δ 41.5–45.5 ppm (CH from polymer backbone), δ 47.0–47.3 ppm (–CH2–NH), δ 50.8 ppm (–(CH3)3), δ 124.0–129.0 ppm (aromatic CH) δ138.8, 141.0, 143.9, 145.5 ppm (Quart-aromatic C).Analysis: d = 90 mm, h = 14.2 mm, VWR, Langenfeld, Germany) and dried in a vacuum drying cabinet  at 70 °C and 2 mbar for 1 h.For each sample 125 mg of purified polymer was dissolved in 3 mL ethanol under stirring. The solution was then cast in a petri dish without vents  and Escherichia coli (DSM No. 1576) were applied as test organisms. The inoculum was prepared by transferring a frozen culture to 10 mL nutrient broth , afterwards the broth was incubated at 37 °C for 24 h. In the beginning of each trial the inoculum was diluted in physiological saline solution with tryptone  to a final concentration of 105 cfu/mL. Samples and references were inoculated with 0.4 mL of this solution. To prevent evaporation and to standardize the contact area, the inoculums were covered loosely by sterile PE films (40 × 40 mm).The antimicrobial activity of purified poly(TBAMS) was tested according to the Antimicrobial products-Test for antimicrobial activity and efficacy (JIS Z 2801:2000). In each test three poly(TBAMS) samples and three blank petri dishes of the same size were used as reference. Three references were directly washed out with 10 mL soybean casein digest broth with lecithin polysorbat  to determine the starting concentration. The poly(TBAMS) samples and the remaining references were incubated at 35 °C and 90% humidity for 2 h. Afterwards all samples were washed in a similar manner. Viable counts were determined by counting the colonies on plate count agar  which were incubated for 48 h at 37 °C.Tx, reference) material 2 h after inoculation.The value of antimicrobial activity were calculated by subtracting the logarithmic value of viable counts on poly(TBAMS) from the logarithmic value of reference material after inoculation and incubation:10-reduction is ≥2.0 after 24 h at 35 °C.According to the JIS Z 2801:2000, a material can be characterized as antimicrobial, if the calculated logEscherichia coli in a complex medium , 0.84 g NaCl, and 0.1 g Trypton  and 100 mL using H2O, pH 7.4). Blank petri dishes were used as reference. The petri dishes were incubated at room temperature for 12 h. The bacteria suspension was transferred into a 15 mL centrifuge tube, the bacteria were fixed chemically using 2.5 mL of a aqueous 2% glutaraldehyde solution at 4 °C for 3 h. After this the suspension was centrifuged for 10 min at 400 rpm to harvest the bacteria. The supernatant was discarded and the bacteria were dehydrated for 15 min in a series of ethanol/water solutions of increasing ethanol content . The last drying step was repeated two times using absolute ethanol. Ethanol was removed in a vacuum exsiccator at 500 mbar. The pellets were transferred to a SEM sample holder. Gold was deposited on the specimens with an ion spatter. For scanning electron microscopy a Leo 982 “Gemini”  microscope was used.In a petri dish containing the poly(TBAMS) film there was added 4.5 mL of an overnight culture of E. coli and S. aureus was observed. First insights from the SEM experiments showed that the antimicrobial activity correlates with the disruption of the cell membranes. Further experiments elucidating the applicability of poly(TBAMS) as a antimicrobial additive in compounds are currently underway.The preparation and characterization of TBAMS and poly(TBAMS) was described in this study. The synthesis of TBAMS from commercially available starting materials was straightforward and high yielding. Polymerization to poly(TBAMS) generated a new intrinsic antimicrobial polymer with an acceptable glass transition temperature and good film building properties. Excellent antimicrobial action against


The protonated N atom and 2-amine group of the 5-fluorocytosinium (5FC) cation interact with the 3-hydroxypicolinate (3HAP) anion through a pair of nearly-parallel N—H⋯O hydrogen bonds, forming a robust  4H5FN3O+·C6H4NO3−, contains one 4-amino-5-fluoro-2-oxo-2,3-dihydropyrimidin-1-ium  cation and a 3-hydroxypicolinate (3HAP) anion. The 4-amino-5-fluoro-2-oxo-2,3-dihydropyrimidine molecule is protonated at one of the pyrimidine N atoms. The typical intramolecular N—H⋯F and O—H⋯O S(5) and S(6) hydrogen-bond ring motifs are observed in the cations and anions. The protonated N atom and 2-amine group of the 5FC cation interact with the 3HPA anion through a pair of nearly parallel N—H⋯O hydrogen bonds, forming a robust R22(8) ring motif. The ions are further linked by N—H⋯N, O—H⋯O, N—H⋯O and C—H⋯O hydrogen bonds, generating R22(7), R33(12) and R65(18) ring motifs, respectively, leading to supramolecular wave-like sheets parallel to (010). The crystal structure is further stabilized by C—H⋯π interactions, generating a three-dimensional architecture.The asymmetric unit of the title salt, C Candida and Cryptococcus  ring motif between the N4 amino group and the F atom of the 5-fluorocytosinum cation. These hydrogen-bonding parameters are similar to those observed in 5-fluorocytosinium salicylate  motif between the phenolic OH and carboxylate group, which is also observed in 3-hydroxypyridinium-2-carboxylate  interacts with the protonated N3 atom and the 4-amino group of the 5-fluorocytosinium moiety through a pair of N—H⋯O hydrogen bonds, forming a robust ds Fig. 3. The cryHot aqueous solutions of 5-fluorocytosine  and 3-hydroxypicolinic acid  were mixed in a 1:1 molar ratio. The resulting solution was warmed over a water bath for half an hour and then kept at room temperature for crystallization. After a week, colourless prismatic crystals were obtained.Uiso(H) = 1.2Ueq and Uiso(H) = 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S1600536814021898/tk5344sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S1600536814021898/tk5344Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814021898/tk5344Isup3.cmlSupporting information file. DOI: 1027535CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


R by resonant scattering.In the crystal structure of Brinzolamide, the various hydrogen bonds present lead to the formation of a bilayer structure. The absolute configuration of the asymmetric C atom was determined to be  12H21N3O5S3 thiazine-6-sulfonamide 1,1-dioxide], there exist three kinds of hydrogen-bonding interactions. The sulfonamide group is involved in hydrogen bonding with the secondary amine and the methoxy O atom, resulting in the formation of layers parallel to the bc plane. The layers are linked by an N—H⋯O hydrogen bond involving a sulfonamide O atom as acceptor and the secondary amine H atom as donor, which gives rise to the formation of a unique bilayer structure. The absolute structure of the molecule in the crystal was determined by resonant scattering [Flack parameter = 0.01 (4)].In crystal structure of the title compound, C The sulfonamide group is also involved in hydrogen bonding with the methoxy group thienothiazine 5,5-dioxide -6,6-dimethyl-4a,5,6,7-tetrahydro-4H-pyrrolothienothiazine 9,9-dioxide  Å for NH and 0.86 (1) Å for NH2 H atoms. The C-bound H atoms were included in calculated positions and treated as riding atoms: C—H = 0.95–1.00 Å with Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms. The absolute structure of the molecule in the crystal was determined by resonant scattering [Flack parameter = 0.01 (4)].Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016006022/su5292sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989016006022/su5292Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016006022/su5292Isup3.cmlSupporting information file. DOI: 1473394CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Here, we provide the information about the materials, methods, analysis and additional information related to our study published in Atherosclerosis  Cardiovascular diseases are the most common cause of death in the world, atherosclerosis being its main underlying disease. Information about the role of B cells during atherosclerotic process is scarce, but both proatherogenic and atheroprotective properties have been described in the immunopathology of this disease. Frequency and phenotype of B cell subpopulations were studied in wild type and apolipoprotein-E-deficient ( Specifications TableValue of the data•Similar alterations in frequency and phenotype of B cell subsets could be presented in murine models of other chronic inflammatory diseases.•The data can be used for further investigations concerning B cells biology in other lipid associated and metabolic diseases.•The data and procedures can be used by other scientists investigating the effects of dyslipidemia on murine B cells.•Characterization strategy for B cell subsets presented herein, can be used in other murine models.1apoE-/- and control mice  and Jackson Laboratory (ME), respectively. Mice were housed and kept under a specific pathogen-free environment, at the animal facility, Sede de Investigación Universitaria, Universidad de Antioquia . The institutional animal ethical committee at Universidad de Antioquia approved all the animal protocols and experiments. Water and a standard laboratory diet  or high fat diet   were given ad libitum to the animals.C57BL/6 WT and 2.22 exposition and heart, spleen, lymph nodes and aorta were harvested by dissection. Hearts were embedded in paraformaldehyde solution  at 4 °C during 48 h, and then in sucrose solution  for 24 h. Finally, heart tissues were stored at −20 °C in Shandon cryomatrix  until their use. Spleens, aortas and lymph nodes were collected in phosphate buffered saline  at 4 °C and immediately processed. Peripheral blood obtained by cardiac puncture was centrifuged at 10.000×g during 10 min at 4 °C to obtain serum. After centrifugation, serum was stored at −20 °C until used.Mice were euthanatized with COHeart tissues were serially sectioned in a CM-1850 Cryostat  for 6–7 µm thickness on slides with positive charge (Thermo scientific), and stained with Hematoxylin–Eosin (Sigma-Aldrich). Microphotografies were taken from aortic root and ascendant aorta at 4× with a digital camera Nikon DS-Fi1 coupled to a Nikon E2000 microscope . Images were analyzed with NIS Element Software (Nikon) to determinate the size of atherosclerotic lesions.2.3The following monoclonal antibodies were purchased from Becton Dickinson Bioscience : CD3e-FITC (145-2C11), CD4-FITC (RM4-5), CD5-PECy5 (53-7.3), CD8a-PE (53-6.7), CD11b-APC (M1/70), CD11c-APC (HL3), CD19-PECy7 (1D3), CD21-FITC (7G6), CD23-PE (B3B4), CD24-APC (M1/69), CD40-APC (3/23), CD45-APCCy7 (30-F11), CD49b/Pan-NK-FITC (DX5), CD80-APC (16-10A1), CD86-APC (GL1), CD95-PE (15A7), IgM-biotin (II/41), IgD-V450 (11-26c.2a), Ter-119-PE (TER-119), B220-Pacifc Blue (RA3-6B2), and the respective isotype controls. LIVE/DEAD Fixable Aqua Dead Cell Stain  was performed for viability study in each sample. Streptavidin PE  and V450 (BD Bioscience) were used for biotin labeled antibodies.2.4g, for 10 min at 4 °C. Erythrocytes were lysed with RBC Lysis Buffer  for 5 min with constant movement. Splenocytes were washed with PBS, resuspended in cold media and immediately used after obtention. Number of cells was determined in Neubauer camera. Viability  was evaluated by trypan blue exclusion and Live/Dead Fixable Aqua Dead Cell Stain Kit (Invitrogen).Spleens were perfused and macerated with syringe plunger  and cold media compound of RPMI 1640 Glutamax (Gibco) with 2% inactivated Fetal Bovine Serum  and 1% Penicillin/Streptomycin (Gibco) on ice. Cells were pelleted at 800×2.5g for 5 min at 4 °C and suspended in cold media. Cells were processed immediately after obtention and their number and viability was determined with trypan blue exclusion and Live/Dead Fixable Aqua Dead Cell Stain Kit . Blood contamination was determined with TER-119 .Aortas were processed as previously reported 2.66 splenocytes and 1–2×105 aortic cells) were washed in 1 ml of FACS buffer (0.01% sodium azide (Sigma-Aldrich) and 3% FBS in PBS, pH=7.35) and centrifuged at 600×g for 5 min, 4 °C. Cell pellets were incubated with blocking buffer  and 0.01% sodium azide in PBS, pH=7.35) for 15 min at 4 °C. Specific monoclonal antibodies were added in 100 µl FACS buffer and incubated for 20 min at 4 °C in dark. Cells marked with biotinylated antibodies were washed twice with FACS buffer and incubated with streptavidin for another 20 min at 4 °C in dark. Cells were washed two times with FACS buffer and immediately acquired in the flow cytometer FACS Canto-II (BD Biosciences). Data were analyzed using FlowJo software  and reported as percentages, absolute numbers and median fluorescence intensity (MFI).Cell suspensions  and stained with Hematoxylin-Eosine, anti- CD3, CD20, CD23, CD68 and CD79a. Images were acquired using the 4X, 10X, 20X objective on a Leica DM500 coupled with digital camera  using Leica LAS EZ software (Leica).2.8Total cholesterol serum levels were determined by conventional wet chemistry method using an A-15 automated biochemical analyzer  in the School of Veterinary, Universidad de Antioquia.2.9Serum IgM and IgG1 levels were measured by Enzyme-linked Immunosorbent Assay (ELISA) Ready-Set-Go! Kits (eBiosciences), following manufacturer׳s instructions and using an ELx800 Absorbance Microplate Reader  at 450 nm emission.2.10L-glutamine (Gibco), 0.05 mM 2-mercaptoethanol (Gibco), 1% Penicillin/Streptomycin in RPMI 1640 Glutamax. Two types of serum were used: 20% lipemic serum from apoE−/−HFD mice 20 weeks old, and 20% non-lipemic serum from WT-SLD mice 20 weeks old. Total splenocytes (2×106) were cultured in 1 ml of media for 5 and 12 hours at 37 °C and 5% CO2. Cells were washed with PBS and the immunofluoresce with specific antibodies was performed as previously described. Cells were fixed with a commercial solution  during 1 h and washed twice with PBS. Finally, cells were stained with Nile Red (Sigma-Aldrich) for 15 min at 4 °C in dark. Cells were washed twice with PBS and immediately acquired in the flow cytometer.Culture media containing 10 mM 2.11P-values <0.05 were considered statistically significant. The classical principal component analysis was performed with the Statgraphics Centurion XVI software .Differences between groups were examined using two ways ANOVA with Šidák´s multiple comparisons post-test. Differences in MFI of CD95 and Nile Red between groups were examined using non-parametric Mann–Whitney test. Spearman rank correlation coefficient test was performed to assess the association between two variables. Data was analyzed in Prism 6.0 software .


Three of five 6-mercaptopurine derivatives are isomorphous and accordingly their molecular and supramolecular structures are similar. In the remaining two derivatives, the purine and exocyclic phenyl rings are essentially planar, but that in the case of the three isomorphous compounds, these rings are twisted. viz. 2-[(9-acetyl-9H-purin-6-yl)sulfanyl]-1-(3-methoxyphenyl)ethan-1-one (1), C16H14N4O3S, 2-[(9-acetyl-9H-purin-6-yl)sulfanyl]-1-(4-methoxyphenyl)ethan-1-one (2), C16H14N4O3S, 2-[(9-acetyl-9H-purin-6-yl)sulfanyl]-1-(4-chlorophenyl)ethan-1-one (3), C15H11ClN4O2S, 2-[(9-acetyl-9H-purin-6-yl)sulfanyl]-1-(4-bromophenyl)ethan-1-one (4), C15H11BrN4O2S, and 1-(3-methoxyphenyl)-2-[(9H-purin-6-yl)sulfanyl]ethan-1-one (5), C14H12N4O2S. Compounds (2), (3) and (4) are isomorphous and accordingly their molecular and supramolecular structures are similar. An analysis of the dihedral angles between the purine and exocyclic phenyl rings show that the molecules of (1) and (5) are essentially planar but that in the case of the three isomorphous compounds (2), (3) and (4), these rings are twisted by a dihedral angle of approximately 38°. With the exception of (1) all molecules are linked by weak C—H⋯O hydrogen bonds in their crystals. There is π–π stacking in all compounds. A Cambridge Structural Database search revealed the existence of 11 deposited compounds containing the 1-phenyl-2-sulfanylethanone scaffold; of these, only eight have a cyclic ring as substituent, the majority of these being heterocycles.The crystal structures of five 6-mercaptopurine derivatives,  Here we describe the syntheses and characterization of five 6-mercaptopurine derivatives: 2- show that the degree of bending is higher in (1)–(4) than in (5). As regards the ethanone group, the C61—C62 bond lengths lie in the range 1.510 (4) Å (2) to 1.528 (4) Å, (4) and are normal for a Csp3—Csp3 bond while the C62—Ph bond lengths are shorter and lie in the range 1.474 (3) Å (2) to 1.496 (4) (4), suggesting that the electron density is delocalized from the phenyl ring.The Pu—S6 bond tends to be coplanar with the purine residue. In fact, the 6-mercaptopurine itself may appear in the thione form, 1, those between the mean plane of the purine ring and the plane defined by the S6—C61—C62—O6 atoms, θ2, and those between the mean planes of the phenyl ring and the plane defined by the S6—C61—C62—O6 atoms, θ3 are given in Table 61) and (5) are essentially planar. However, in the case of the three isomorphous compounds (2), (3) and (4), the purine and exocyclic phenyl rings are both twisted in the opposite direction from the plane of the bridging unit, resulting in a dihedral angle of approximately 38°. This is due to the rotations and bending around the bonds connecting the bridging unit to the purine and exocyclic phenyl rings as discussed above. The dihedral angles θ2 are higher than θ3; the former are mainly due to the rotations around the S6—C61 bond while the latter are mainly the result of the bending of the C62—Ph bond.The dihedral angles between the mean planes of the of the purine and phenyl ring, θ1). Hydrogen bonds for (2)–(5) are listed in Tables 72), (3) and (4) are isomorphous, their supramolecular structures follow similar patterns. Accordingly, hydrogen-bonding diagrams are given for (2) only. Atom C8 acts as a donor to O9 , via H8 forming an x + 1, y + z + via H61A, forming a C4 chain, which runs parallel to the b axis, Fig. 8y, 1/4). In (2), there is a short contact between C6 and the 4-methoxy atom O64 , the N9—H9⋯N9 , the pyrimidine ring (with centroid Cg2) and the benzyl ring (with centroid Cg3), it would be expected that π–π contacts were part of the supramolecular structure. Table 111)–(5). As may be seen in the Table, the pyrimidine ring establishes π–π contacts with the benzyl ring for all compounds. In (1), two molecules centrosymmetrically related across the inversion centre at  are involved in π–π stacking in which the purine ring stacks above the exocyclic phenyl ring. In (2), (3) and (4), the π–π stacking is between imidazole rings while in (1) and (5), the contact is between an imidazole ring and a benzyl ring. In particular, in (1) and (5) two molecules centrosymmetrically related across the centre of symmetry at  are involved in π–π stacking in which the purine rings stack above the exocyclic phenyl ring, Table 11Since those compounds have three rings, the imidazole ring -οne -one  and ETEWOP (1.82 Å). The supplementary figure also gives information about the distances of the –CH2– carbon atom to the best plane made up of the atoms of the heterocycles (CH2– distance). These values were computed in order to evaluate the degree of bending of the S—CH2 bond with respect to the main plane of the substituted rings. There are two main groups of compounds, one in which the distance is shorter than 0.3 Å and the other, which contains the CNH fragment in the heterocyclic ring, in which this distance is greater than 1.2 Å. As noted above, the sp3 character of the β-carbon atom of the ethanone fragment may also direct the relative positions of the acetophenone residue out of the main plane constituted by the substituted heteroaromatic ring. This is the case for SILGAW and IKAXOI. Thus, despite the small sample size, there is a wide range of adopted conformations.The 1)–(5) were obtained in moderate yields by a two-step synthetic strategy. Firstly, 6-mercaptopurine was alkylated using diverse monobromide acetophenone derivatives in DMF/potassium carbonate medium at room temperature –(4) under an argon atmosphere at room temperature sulfanyl]-1-(3-methoxyphenyl)ethan-1-one (1): overall yield: 48%; m.p. 432–435 K; 2-[(9-acetyl-9H-purin-6-yl)sulfanyl]-1-(4-methoxyphenyl)ethan-1-one (2): overall yield: 17%; m.p. 460–463 K; 2-[(9-acetyl-9H-purin-6-yl)sulfanyl]-1-(4-chlorophenyl)ethan-1-one (3): overall yield: 26%; m.p. 453–457 K; 2-[(9-acetyl-9H-purin-6-yl)sulfanyl]-1-(4-bromophenyl)ethan-1-one (4): overall yield: 10%; m.p. 449–451 K; 1-(3-methoxyphenyl)-2-[(9H-purin-6-yl)sulfanyl]ethan-1-one (5): overall yield: 55%; m.p. 461–464 K.The 6-mercaptopurine derivatives (Uiso = 1.2Ueq(C), C—H2(methylene), 0.99 Å, with Uiso = 1.2Ueq(C),C—H(methyl) 0.98 Å with Uiso = 1.5Ueq(C) and in (5) only, N—H, 0.88 Å, with Uiso = 1.2Ueq(C). The positions of the methyl groups were checked on a final difference map as was that of the N—H hydrogen atom in (5). In (4), the high difference map peaks were associated with the Br atom.Crystal data, data collection and structure refinement details are summarized in Table 1210.1107/S2056989016001833/hb7562sup1.cifCrystal structure: contains datablock(s) 1, 2, 3, 4, 5, global. DOI: 10.1107/S2056989016001833/hb75621sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989016001833/hb75622sup3.hklStructure factors: contains datablock(s) 2. DOI: 10.1107/S2056989016001833/hb75623sup4.hklStructure factors: contains datablock(s) 3. DOI: 10.1107/S2056989016001833/hb75624sup5.hklStructure factors: contains datablock(s) 4. DOI: 10.1107/S2056989016001833/hb75625sup6.hklStructure factors: contains datablock(s) 5. DOI: Click here for additional data file.10.1107/S2056989016001833/hb75621sup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016001833/hb75622sup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016001833/hb75623sup9.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016001833/hb75624sup10.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016001833/hb75625sup11.cmlSupporting information file. DOI: 1450945, 1450944, 1450943, 1450942, 1450941CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: