Two H atoms in the solvated TCE molecule form intermolecular C—H⋯ hydrogen bonds with three N atoms in dbanth. π–π interactions link the dbanth molecules to form a one-dimensional columnar structure.The crystal structure of 1,1,2,2-tetrachloroethane (TCE)-solvated 1,13,14-triazadibenzanthracene  and one half of 1,1,2,2-tetrachloroethane (TCE), both of which are located on a crystallographic twofold rotation axis. The dihedral angle between the planes of the terminal benzene rings in dbanth is 3.59 (7)° owing to the steric repulsion between the H atoms in the two benzo groups and the H atom in the central pyridine ring of the anthridine skeleton. In the crystal, π–π interactions between pyridine rings [centroid–centroid distances = 3.568 (2) and 3.594 (2) Å] link the dbanth molecules to form a one-dimensional columnar structure along the c axis. The dbanth and TCE molecules are connected through weak bifurcated C—H⋯ hydrogen bonds.The asymmetric unit of the title compound, C However, there have been few reports on the crystal structures of 1,9,10-anthyridine derivatives. The crystal structure and intermolecular interactions of chlorobenzene-solvated 2,3,7,8-tetraphenyl-1,9,10-anthyridine have been reported  has been synthesized and its crystal structure has been reported phenol  and 3.594 (2) Å, respectively Fig. 2. The dbans Fig. 3. A twofoa,j]anthracene (dbanth) was synthesized via the reaction of 2,6-diamino-3,5-diiodopyridine with two equivalents of 2-formylbenzeneboronic acid using Pd(PPh3)4 as a catalyst according to a literature method  values set at 1.2Ueq of the parent atom.Crystal data, data collection, and refinement details are summarized in Table 210.1107/S2056989015009263/is5393sup1.cifCrystal structure: contains datablock(s) General, I. DOI: 10.1107/S2056989015009263/is5393Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015009263/is5393Isup3.cmlSupporting information file. DOI: 1401209, 1401209CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title compounds are the 3-ethyl, (I), and 3-isopropyl, (II), derivatives of amino phenyl carbonate. The main difference in the conformation of the two compounds is the angle of inclination of the phenoxycarbonyl ring with respect to the piperidine ring mean plane, with a dihedral angle of 2.05 (8)° in (I) and 45.24 (13)° in (II). 27H28N2O3, (I), and C28H30N2O3, (II), the conformation about the C=N bond is E. The piperidine rings adopt chair conformations with the attached phenyl rings almost normal to their mean planes, the dihedral angles being 85.82 (8) and 85.84 (7)° in (I), and 87.98 (12) and 86.42 (13)° in (II). The phenyl rings are inclined to one another by 52.87 (8)° in (I) and by 60.51 (14)° in (II). The main difference in the conformation of the two compounds is the angle of inclination of the phenoxycarbonyl ring to the piperidine ring mean plane. In (I), these two planes are almost coplanar, with a dihedral angle of 2.05 (8)°, while in (II), this angle is 45.24 (13)°. In the crystal of (I), molecules are linked by C—H⋯O hydrogen bonds, forming inversion dimers with R22(14) loops. The dimers are linked via C—H⋯π interactions forming a three-dimensional network. In the crystal of (II), there are no significant intermolecular interactions present.In the title compounds, C Such compounds have been used as antihistamines, anaesthetics, tranquilizers and hypotensive agents sp3 hybridization. The isopropyl group substituted at position 5 of the piperidine moiety is in an equatorial orientation.The molecular structure of compound (II)E)-3-methyl-1-methyl-2,6-diphenylpiperidin-4-one O-phenoxycarbonyl oxime via C-H⋯π interactions, forming a three-dimensional network rk Fig. 3.In the crystal of (II)ca. 44.9° in a very similar compound to those studied here, viz (E)-{[amino]oxy}(pyridin-3-yl)methanone piperidin-4-one oxime Uiso(H) = 1.5Ueq(C-methyl) and = 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S1600536814018893/su2763sup1.cifCrystal structure: contains datablock(s) I, II. DOI: 10.1107/S1600536814018893/su2763Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536814018893/su2763IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S1600536814018893/su2763Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S1600536814018893/su2763IIsup5.cmlSupporting information file. DOI: 1020223, 1020224CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Alteration of γδ T-cell distribution and function in peripheral blood is among the earliest defects during HIV-infection. We asked whether the polyfunctional response could also be affected, and how this impairment could be associated to CD4 T-cell count. To this aim, we performed a cross-sectional study on HIV-infected individuals. In order to evaluate the polyfunctional-Vγ9Vδ2 T-cell response after phosphoantigen-stimulation, we assessed the cytokine/chemokine production and cytotoxicity by flow-cytometry in HAART-treated-HIV+ persons and healthy-donors. During HIV-infection Vγ9Vδ2-polyfunctional response quality is affected, since several Vγ9Vδ2 T-cell subsets resulted significantly lower in HIV+ patients in respect to healthy donors. Interestingly, we found a weak positive correlation between Vγ9Vδ2 T-cell-response and CD4 T-cell counts. By dividing the HIV+ patients according to CD4 T-cell count, we found that Low-CD4 patients expressed a lower number of two Vγ9Vδ2 T-cell subsets expressing MIP-1β in different combinations with other molecules (CD107a/IFNγ) in respect to High-CD4 individuals. Our results show that the Vγ9Vδ2 T-cell-response quality in Low-CD4 patients is specifically affected, suggesting a direct link between innate Vγ9Vδ2 T-cells and CD4 T-cell count. These findings suggest that Vγ9Vδ2 T-cell quality may be indirectly influenced by HAART therapy and could be included in a new therapeutical strategy which would perform an important role in fighting HIV infection. Human γδ T-cells are critical components of the innate immune system and play important roles in the early response to invading pathogens. Vγ9Vδ2 T-lymphocytes, the major population among γδ T-cells, display beneficial roles, as they are able to control HIV infection  and exhiRecently, it was demonstrated that polyfunctional αβ T-cells are strongly associated with natural control of HIV disease, being higher in HIV-infected persons who spontaneously control the virus without any treatment regimen (eg. Long-Term Non-Progressor and Elite Controller) . HoweverIn the present paper, for the first time, Vγ9Vδ2 T-cell polyfunctionality has been evaluated by a cross-sectional study analyzing HIV-infected individuals under on HAART therapy, with different CD4 cell and viral load outcomes.Our study demonstrated for the first time that, similar to αβ T-cells, HIV infection heavily modulates Vγ9Vδ2 T-cell polyfunctional response, and this effect was associated to CD4 T-cell count. Moreover, we identified two Vγ9Vδ2 T-cell subsets that could be considered as negative prognostic factors to disease progression. A longitudinal study is in progress to confirm this hypothesis.3) and Low-CD4  patients. The mean ± s.d. of CD4 counts was 901.8 ± 335.0 cells/mm3 for H-CD4 patients and 82.2 ± 44.8 cells/mm3 for L-CD4 patients. Considering the total number of patients, the mean ± s.d. of plasma HIV RNA was 132,116 ± 504,600 copies/mL.Fifty two HIV+ patients under HAART therapy from the “Lazzaro Spallanzani” Institute for Infectious Diseases in Rome were enrolled in the study. Healthy HIV-uninfected age and gender-matched individuals were included as controls. Anonymised residual samples for routine CD4 analysis were used for the study. The study was approved by the “Lazzaro Spallanzani” National Institute for Infectious Diseases Ethical Committee , and a signed informed consent was obtained from the enrolled persons.3-1) response patterns for three functions, excluding the fully negative subset (Whole blood diluted with complete medium (1:2) was stimulated with Picostim, a specific Vγ9Vδ2 T-cell antigen . In all conditions CD107a-PE-Cy5 antibody  and Brefeldin A  were added at the beginning of culture and after 1 hour of stimulation, respectively. After 18 hours, cells were labeled with anti-CD3 Horizon V500 and anti-Vδ2 FITC (10 min. at 4°C); then a lysing/fixing solution was added to the cells, according to the manufacturer’s instructions (BD). After washing (PBS/BSA/NaN3 buffer), cells were incubated for 20 min. at room temperature (RT) with antibodies for intracellular staining (MIP-1β-PE and IFNγ PE-Cy7) prepared in a permeabilizing buffer, and then washed with 0.1% saponin/PBS/BSA/NaN3 solution. All antibodies were purchased from Becton-Dickinson . Samples were acquired on FACSCanto II flow cytometer (BD) and analyzed using FlowJo 6.4.7 software . FlowJo software, through the Boolean gate platform, allowed for creating the full array of possible combinations, equating to 7 . The statistical analysis of the polyfunctional responses was performed in SPICE version 5.22 using a permutation test based on χ2 test to compare pie charts. A p value less than 0.05 was considered statistically significant.First we analyzed the absolute number of Vγ9Vδ2 T-cells in the HD and HIV+ patients. As known, Vγ9Vδ2 T-cells were significantly lower in the HIV+ patients than HD  in the HD and HIV+ patients. The polyfunctional profile analysis showed a similar number of three-, bi- and mono-functional Vγ9Vδ2 T-cell subsets in the HD as well as in the HIV+ patients . InteresFinally, we analyzed the contribution of each unique functional subset to the global response according to the particular combination of expressed molecules. We obtained seven functional subsets derived from the combination of three molecules. As shown in We wondered whether the Vγ9Vδ2 T-cell number and functional capability was associated to CD4 T-cell count and/or plasma viral load. We found that CD4 T-cell count was positively correlated with both the Vγ9Vδ2 T-cell absolute number  and Low-CD4 (L-CD4) according to CD4 cell counts. As shown in Finally, we analyzed the contribution to the total response of Vγ9Vδ2 T-cells according to the cytokines produced, as well as the contribution of each unique functional subset in the High- and Low-CD4 patients. We found that the total MIP-1β expression was lower in the L-CD4 patients when compared to the H-CD4 subjects , and vertical lines show the minimum and maximum values. The Mann–Whitney U test was used to compare group medians and a p value less than 0.05 was considered statistically significant, ***p<0.001; **p<0,01; *p<0.05; >200 CD4 = patients with >200 CD4 cells/mmVγ9Vδ2 T-cells are a critical component of innate immunity. After phosphoantigen stimulation they are able to secrete cytokines and chemokines and to exhibit cytotoxicity activity against HIV-infected cells, thus exhibiting beneficial roles in controlling HIV infection .HIV infection causes an alteration of γδ T-cell distribution leading to a specific depletion of Vγ9Vδ2 T-cells in peripheral blood ,10. Althin vitro [It was largely demonstrated that αβ T-cell polyfunctionality is correlated to HIV disease control, being higher in HAART-naïve patients who spontaneously control disease progression  and in Hin vitro . As previn vitro  Vγ9Vδ2 Tin vitro  demonstrHere, for the first time, we demonstrated both the capacity of Vγ9Vδ2 T-cells to simultaneously perform different functions, including cytokine/chemokine secretion and cytotoxicity activity in healthy donors, and their impairment during HIV infection. Specifically, we showed that three-, bi- and mono-functional Vγ9Vδ2 T-cell subsets decrease in HIV infected patients in respect to healthy donors, indicating that polyfunctional capability is affected during HIV infection. Limited data are available in literature regarding γδ T-cell polyfunctional capability. Bridgett Ryan-Payseur  demonstrOur results are in line with Li et al. , by showInterestingly, we found that two specific unique functional subsets, both expressing MIP-1β (CD107a+MIP-1β+ and IFNγ+MIP-1β+), were modulated by CD4 cell counts which were inferior in patients with lower CD4 cell counts. It is known that MIP-1β production dominates HIV-specific CD8 T cell response , suggestIn conclusion, our data support the notion that a specific modulation of Vγ9Vδ2T-cell response capability is present in HIV-infected individuals, and is correlated to the course of HIV disease. These results highlight a supplemental beneficial role of antiretroviral treatment in allowing also the recovery of innate Vγ9Vδ2 T-cells function.As to the limitations of the present study, the limited number of HIV+-responding individuals suggests additional studies with larger cohorts, in order to confirm these results. Moreover, further studies including a longitudinal assessment of responses will help to elucidate whether Vγ9Vδ2 T-cell response capability modulations effectively contribute to protection in modifying HIV disease course, or whether they simply reflect a specific immune imbalance following HIV infection.


In the title compound, the planes of the two indole ring systems are approximately perpendicular to each other, with a dihedral angle of 89.3 (5)°. 27H21N3O6·C2H5OH, the indole ring systems are approximately perpendicular to each other, with a dihedral angle of 89.3 (5)°; the plane of the benzene ring is oriented with respect to the indole ring systems at 49.9 (5) and 73.4 (3)°. In the crystal, molecules are linked by N—H⋯O and O—H⋯O hydrogen bonds and weak C—H⋯π interactions into a three-dimensional supramolecular architecture. A void of 33.0 (7) Å3 is observed in the crystal structure. The solvent ethanol molecule acts as a donor, forming an O—H⋯O hydrogen bond, reinforcing the framework structure.In the title compound, C The yield was 90%. Crystals of the title compound suitable for X-ray analysis were obtained by slow evaporation of an ethanol solution.Methyl indole-2-carboxylate  was dissolved in 200 ml methanol; commercially available 3-nitrobenzaldehyde  was added and the mixture was heated to reflux temperature. Concentrated HCl (3.7 ml) was added and the reaction was left for 1 h. After cooling the white product was filtered off and washed thoroughly with methanol. The reaction can be followed by thin-layer chromatography (CHClUiso(H) = xUeq, where x = 1.5 for methyl and hydroxy, and x = 1.2 for all other H atoms.H atoms were positioned geometrically, with N—H = 0.86Å and O—H = 0.82Å, and C—H = 0.93, 0.96, 0.97 or 0.98 Å for aromatic, methyl, methene and methine H atom, respectively, and constrained to ride on their parent atoms, with 10.1107/S1600536814022296/xu5823sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S1600536814022296/xu5823Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814022296/xu5823Isup3.cmlSupporting information file. DOI: 1028397CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The third author's name is spelled incorrectly. The correct name is: Carlos Álvarez-Dardet. The correct citation is: Umaña-Peña R, Franco-Giraldo Á, Álvarez-Dardet C, Ruíz-Cantero MT, Gil-González D, et al. (2014) Assessment of the Association of Health with the Liberalisation of Trade in Services under the World Trade Organisation. PLoS ONE 9(7): e102385. doi:10.1371/journal.pone.0102385


Yarrowia lipolytica WSH-Z06. Six endogenous putative transporter genes, YALI0B19470g, YALI0C15488g, YALI0C21406g, YALI0D24607g, YALI0D20108g and YALI0E32901g, were identified. Transport characteristics and substrate specificities were further investigated using a carboxylate-transport-deficient Saccharomyces cerevisiae strain. These transporters were expressed in Y. lipolytica WSH-Z06 to assess their roles in regulating extracellular keto acids accumulation. In a Y. lipolytica T1 line over expressing YALI0B19470g, α-ketoglutarate accumulated to 46.7 g·L−1, whereas the concentration of pyruvate decreased to 12.3 g·L−1. Systematic identification of these keto acids transporters would provide clues to further improve the accumulation of specific organic acids with higher efficiency in eukaryotic microorganisms.Production of organic acids by microorganisms is of great importance for obtaining building-block chemicals from sustainable biomass. Extracellular accumulation of organic acids involved a series of transporters, which play important roles in the accumulation of specific organic acid while lack of systematic demonstration in eukaryotic microorganisms. To circumvent accumulation of by-product, efforts have being orchestrated to carboxylate transport mechanism for potential clue in  Light have been shed on economic feasibility of production of organic acids by microorganisms, expecially considering the finiteness of fossil raw materials34The metabolism and accumulation of α-ketoglutaric acid (α-KG) are subjected to a higher level of regulation than other organic acids involved in central carbon metabolismS. cerevisiae could be significantly improved with the combined expression of lactate dehydrogenase and the lactate transporter20Y. lipolytica orthologous gene of the carboxylate transporter have not yet been identified.Extracellular accumulation of non-target carboxylates is a common problem for the production of carboxylates16Y. lipolytica WSH-Z06, which exhibits flexibility in substrate specificity. The duplication of endogenous transporters might provide a powerful tool to ensure efficient carboxylate synthesis and maintain homeostasis of the intracellular environment. In this study, we observed that a competitive dual effect — a significant increase in α-KG production with a sharp decrease in pyruvate (PA) accumulation resulted from overexpression of YALI0B19470g. This result suggests a new and promising strategy for gene manipulation that can efficiently address α-KG accumulation. The identification of these transporters also have uncovered the mechanisms of extracellular accumulation of diverse organic acids in genome level, and provides new clues to orchestrate competition of extracellular accumulation between target and non-target carboxylates in yeast and other eukaryotic microorganisms.In this study, a number of carboxylate transporters were identified from Y. lipolytica CLIB122 genome were obtained from the UniProt database. Of these, 1104 proteins  protocols, in which ScJen1p and KlJen2p were used as query sequences2223In silico analysis of these protein sequences using the TMRPres2D tool revealed that the conserved sequence was located in the predicted transmembrane helices −1 for PA in α-KG- and PA-treated cells, respectively. After incubation in YPK or YPP medium for 1 h, the concentration of α-KG gradually decreased 1 h to 3 h, until the minimum of 0.85 μmol·(mg·DCW)−1 was reached. During this period, the content of PA also decreased to 0.23 μmol·(mg·DCW)−1  g·DCW)−1 .To assay the effects of exogenous carboxylates on the expression profiles of these potential transporters, the expression of these genes were examined by qRT-PCR, using RNA extracted at 0 h as the control. When intracellular α-KG levels increased, the expression of YALI0D20108g, YALI0C21406g, YALI0D24607g, and YALI0E32901g decreased by 3.72 ± 0.91-fold, 3.88 ± 0.34-fold, 2.47 ± 0.52-fold, and 1.60 ± 0.18-fold, respectively, whereas the expression of YALI0B19470g and YALI0C15488g increased by 9.39 ± 0.82-fold and 4.32 ± 0.83-fold, respectively. Exogenous PA had a similar effect on expression, albeit to different levels. When incubated with PA, expression of YALI0D20108g, YALI0C21406g, YALI0D24607g, and YALI0E32901g decreased by 5.83 ± 0.81-fold, 4.04 ± 1.04-fold, 6.44 ± 0.98-fold, and 15.88 ± 1.94-fold, respectively, whereas expression of YALI0B19470g and YALI0C15488g increased by 2.83 ± 0.21-fold and 9.72 ± 0.22-fold, respectively . Hence, S. cerevisiae CEN.PK2-1D. The ScJEN1 deletion mutant displayed reduced growth on lactate, acetate, PA, malate, and α-KG compared to the parental strain. To completely abolish the uptake by endogenous transporters, the Ady2p was also disrupted in W1 strain. In contrast, the W2 strain, in which both ScJEN1 and ScADY2 were disrupted, cannot grow on lactate, acetate, PA, malate, and α-KG containing media. Moreover, the parental strain as well as the W1 and W2 strains did not grow on citrate −1 PA, 3.14 ± 0.24 μmol·(mg·DCW)−1 malate, and 1.29 ± 0.12 μmol·(mg·DCW)−1 α-KG were present. In addition, acetate and citrate were not detected in cells incubated in the corresponding carboxylate-containing medium. As no carboxylates were detected in the double deletion cells, we concluded that ScJen1p and ScAdy2p deletions influenced the transport of PA, malate, and α-KG.To investigate the substrate specificity of these proteins, glucose-grown measured , reveali−1) occurring in the W4 strain. Among the set, only the W4 could accumulate PA intracellularly (0.17 μmol·(mg·DCW)−1). Based on these observations, we conclude that YALI0B19470g and YALI0D24607g encode proteins that transport dicarboxylates and tricarboxylates. In addition to PA, W4 cells (containing YALI0C15488p) also accumulated acetate, malate, α-KG, and citrate, indicating the corresponding protein also transported these carboxylates. In a similar manner, we conclude the proteins encoded by the corresponding genes transport the following carboxylates: YALI0C21406p for lactate, malate, α-KG, and citrate; YALI0D20108p for lactate, α-KG and citrate, and YALI0E32901p for acetate, malate, and citrate.The transport of α-KG was restored in all heterogeneous transporter-containing strains, with the highest accumulation (1.41 μmol·(mg·DCW)ACT1 was utilized as an endogenous controlACT1, no obvious relative changes of these transporters were observed  analysis was carried out. observed . This inobserved .Y. lipolytica WSH-Z06. The hp4d promoter was used to increase the transcription of these genes, and YALI0B19470g, YALI0C15488g, YALI0C21406g, YALI0D24607g, YALI0D20108g and YALI0E32901g expression was improved by 23.34 ± 2.67-fold, 8.53 ± 0.90-fold, 9.32 ± 0.82-fold, 11.79 ± 1.32-fold, 3.37 ± 0.49-fold and 10.50 ± 0.97-fold, respectively (As previously reported carboxylate transporter possess bi-functions for carboxylate influx and effluxectively . Due to ectively .in (μmol·(mg·DCW)−1), was also determined −1 for strains T1, T4, T5 and T6. The intracellular accumulation of PA decreased from 0.034 ± 0.006 μmol·(mg·DCW)−1 to 0.009 ± 0.002, 0.029 ± 0.003, 0.017 ± 0.002 and 0.025 ± 0.002 μmol·(mg·DCW)−1 in strains T1, T4, T5 and T6 respectively. Compared to the wild-type strain, the growth yield decreased from 11.17 ± 1.08 g·L−1 to 8.45 ± 0.72, 8.27 ± 0.89, 8.34 ± 0.76, 8.47 ± 0.61, 8.36 ± 0.88 and 7.40 ± 0.71 g·L−1 for strains T1, T2, T3, T4, T5 and T6, respectively, suggesting that enhanced carboxylate transportation caused the shift in carbon flux from cellular growth to carboxylate accumulation. The combination of increased carboxylate accumulation with decreased cell growth lead to a sharp increase in the yield of α-KG and PA, Yα-KG/DCW and YPA/DCW. Finally, the ratio of extracellular carboxylate to intracellular carboxylate increased, and maximum values of 2399.71 ± 241.63 for α-KG and 1685.20 ± 208.56 for PA were observed for the T1 strain. Based on these observations, overexpression of YALI0A9470g was considered the best strategy to enhance α-KG transport and reduce PA accumulation.The intracellular carboxylate content, Ctermined . The intThe object of the current work was to screen and identify carboxylate transporters, then determine whether the identified proteins regulate accumulation of non-target carboxylates. The duplication of transporters and the flexible substrate specificity demonstrated by the identified carboxylate transporters facilitated extracellular accumulation of carboxylates. Moreover, knowledge of the examined carboxylate transport mechanism is a prerequisite for improving carboxylate synthesis through metabolic engineering. The results provide new insights for regulating extracellular carboxylate accumulation in similar eukaryotic microorganisms.To circumvent the issue of PA accumulation, previous studies have focused mainly on the regulation of intrinsic forces that redistribute carbon flux from other intermediates to α-KG production. Expression of PA carboxylaseS. cerevisiae ScJEN1 and ScADY2 genes were identified as key carboxylate transporters28Kluyveromyceslactis, the presence of two carboxylate transporters, Jen1p and Jen2p, guaranteed efficient uptake of lactic acid as a substrate from a lactic-acid-producing habitatY. lipolytica harbored a powerful potential to use a wide range of substrates as a sources of carbon and energyThe carboxylate transport process of yeast is an intensively investigated fieldY. lipolytica, efficient carboxylate transport was achieved by the duplication of iso-functional transporters. Evolution analysis and motif identification confirmed that a precursor form of Jen1p, preJen1p, arose from the duplication of an ancestral Jen2pS. cerevisiae, the transport capacity and substrate affinity of Jen1p were determined by the conserved NXXS/THXS/TQDXXXT sequence29Y. lipolytica displayed different responses to exogenous carbon source, and single carboxylate induced multiple transporters.In The roles of these carboxylate transporters were assayed via double their copy numbers in the genomic DNA of α-KG producer. One more copy of each endogenous carboxylate transporter was observed . Our obs13Y. lipolytica WSH-Z06, an α-KG producing wild-type strain, was previously screened by our labXRP2 in the genome of Y. lipolyticahph was amplified from plasmid pUB4-CRE and was used to replace the URA3 sequence in plasmid p015Escherichia. coli JM109. S. cerevisiae CEN.PK2-1D  from EUROSARF  and the pY13-TEF1 plasmid was used for heterologous expression of potential transporters.All strains used in this study were summarized in E. coli was cultured in Luria broth (LB) medium  supplemented 100 mg·L−1 ampicillin when necessary. Yeast strains were cultured in YPD medium  or YNB medium 2SO4) supplemented with 50 μg·mL−1 uracil, leucine, tryptophan and histidine when necessary. The following media were used for carboxylate treatment: YPA 2SO4, 50 g·L−1 acetate, pH 7.0), YPL 2SO4, 50 g·L−1 lactate, pH 7.0), YPP 2SO4, and 50 g·L−1 PA, pH 7.0), YPM 2SO4, 50 g·L−1 malate, pH 7.0), YPK 2SO4, and 100 g·L−1 α-KG, pH 7.0), YPC 2SO4, 50 g·L−1 citrate, pH 7.0). The seed-culture media  and fermentation media 2SO4, 3 g·L−1; KH2PO4, 3 g·L−1; MgSO4·7H2O, 1.2 g·L−1; NaCl, 0.5 g·L−1; K2HPO4, 0.1 g·L−1; and thiamine-HCl 4 × 10−7 g·L−1, pH 4.5) were described previouslyY. lipolytica CLIB122 genomic DNA were obtained from UniProt (http://www.uniprot.org/). A genome-wide analysis using a transmembrane-helix sequence was performed following a method described previously38http://www.cbs.dtu.dk/services/TMHMM/). The predicted result was visualized using the TMRPres2D toolhttp://www.cbs.dtu.dk/services/SignalP/) to predict whether the sequence encoded a signal peptidehttp://blast.ncbi.nlm.nih.gov/) to identify orthologs that have been characterized as carboxylate transporters.The 6611 putative proteins encoded by Y. lipolytica WSH-Z06 were streaked onto YPD slants from glycerol stocks. Cells grown in glucose were harvested at the exponential phase. After a 2 h treatment in saline water, cells were transferred to 100 mL YPK or 100 mL YPP and incubated at 28°C. PA- or α-KG-treated cells were collected at regular time intervals for quantitative real-time PCR (qRT-PCR) analysis and determination of intracellular carboxylate content.The cells of −1 uracil, leucine, tryptophan, and histidine, were used to assay carboxylate transportation of S. cerevisiae CEN.PK2-1D cells and derivatives.YPA, YPL, YPP, YPM, YPK, and YPC media, supplemented as necessary with 50 μg·mLg for 10 min, and washed twice with cold distilled water. The dry cell weight was determined according to protocol described previously−1.Yeast cells were harvested by centrifugation at 10,000 × Y. lipolytica WSH-Z06 were harvested, centrifuged at 10,000 × g for 10 min, and immediately frozen in liquid nitrogen until RNA extraction. Total RNA was extracted using Trizol reagent , according to the manufacturer's instructions. cDNA was synthesized from 5 μg total RNA using the PrimeScript RT Reagent Kit Perfect Real Time . qRT-PCR was performed with the synthesized cDNA and primers listed in TaqTM Kit  and a LightCycler 480 II Real-time PCR instrument . All experiments were performed in triplicate and mean values were used for further calculations. Fold changes were determined by the 2-ΔΔCT method and normalized to the ACT1 geneThe cells of S. cerevisiae CEN.PK2-1D JEN1 and ADY2 genes, two disruption cassettes were constructed using a protocol described previouslyloxP-URA3-loxP and loxP-LEU2-loxP modules were amplified from plasmids pUG72 and pUG73, respectively, with oligonucleotides JEN-MF/JEN-MR, ADY-MF/ADY-MR, respectively , resulting in p0(hph)-470, p0(hph)-488, p0(hph)-406, p0(hph)-607, p0(hph)-108, and p0(hph)-901, respectively. These vectors were digested with AvrII, purified, and transformed into Y. lipolytica WSH-Z06 using a previously described protocolY. lipolytica T1, T2, T3, T4, T5, and T6, respectively, were screened on YPD plates containing 400 mg·L−1 hygromycin B and verified with the oligonucleotides listed in The YALI0B19470g, YALI0C15488g, YALI0C21406g, YALI0D24607g, YALI0D20108g, and YALI0E32901g open reading frames were PCR-amplified from ACT1 as the internal controlTaqTM Kit  and a LightCycler 480 II Real-time PCR instrument . All experiments were performed in triplicate and mean values were used for further calculations. Fold changes were determined by the 2−ΔΔCT method and normalized to the ACT1 geneIn order to determine the copy number of the integrative expression cassettes in the recombinant strains, a qPCR analysis was performed on the genomic DNA template using −1 for 144 h at 28°C.Shake flask culture was performed in 500 mL flasks containing 50 mL fermentation medium following the protocol stated previouslyg for 10 min. The supernatant was diluted 50 times and filtered through a membrane (pore size = 0.22 μm). α-KG, pyruvic acid, acetate, lactate, malate, and citrate present in the supernatant were simultaneously determined by HPLC  with an Aminex HPX-87H column . The mobile phase was 5 mmol L−1 sulfuric acid in distilled, de-ionized water filtered to 0.22 μm. The mobile phase flow rate was 0.6 mL min−1. The column temperature was maintained at 35°C, and the injection volume was 10 μL. α-KG, pyruvic acid, acetate, lactate, malate, and citrate were detected with a UV detector (wavelength at 210 nm)g for 10 min. The carboxylate content in supernatant was determined by HPLC followed the protocol above.Samples taken from shake flask culture were centrifuged at 10,000 × G.H.W. and Z.J.W. designed the experiments and wrote the paper. G.H.W. and L.P.R. performed the experiments. G.C.D. and J.C. conceived the project, analyzed the data and wrote the paper. C.M. analyzed the data.


Within each ribbon, the ligand molecule, acting in a μ2-mode, bridges two adjacent Li cations using both its N,O-bonding sites.In this one-dimensional coordination polymer, four symmetry-independent Li(C 5H3N2O2)(H2O)]·0.5H2O}n, comprises four symmetry-independent Li(C5H3N2O2)(H2O) units which form molecular ribbons running along the c-axis direction. Within each ribbon, the ligand molecule, acting in a μ2-mode, bridges two adjacent Li+ cations using both of its N,O-bonding sites. The coordination environment of each of the four Li+ cations can be described alternatively as either slightly distorted trigonal–bipyramidal or slightly distorted square–pyramidal. The ribbons are interconnected by a network of O—H⋯O hydrogen bonds.The title compound, {[Li(C II coordination polymers with this ligand  ligand constitutes the structural unit of the title polymeric compound, {[Li(C5H3N2O2)(H2O)]·0.5H2O}n. There are four such assemblies in the asymmetric unit. Linked into pairs, they form molecular ribbons in which the (C5H3N2O2) ligand bridges adjacent LiI cations using both its N,O bonding sites (μ2-bridging mode)  Fig. 1. The ribon Fig. 2.I cations show a penta-coordination mode which can be described by two alternative geometries: either trigonal–bipyramidal or square–pyramidal, both slightly deformed. For example, in the case of the Li1 cation, the equatorial plane of a trigonal bipyramid consists of atoms O13, N11 and N23 with Li1 0.0712 (5) Å out of this plane; atoms O11 and O22 are at the apices. On the other hand, the base of the square pyramid is formed by the O11, O22, N11 and N23 atoms [r.m.s. 0.0069 (1) Å], O13 is at the apex; the Li1 cation is 0.3989 (8) Å out of the base. A similar description can be made for the remaining three independent LiO3N2 groups. The Li—O and Li—N bond lengths  = 1.2Ueq(C). The H atoms of water molecules were found from the Fourier map and refined isotropically.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989014026735/bg2542sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989014026735/bg2542Isup2.hklStructure factors: contains datablock(s) I. DOI: 1037774CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The arsanilate anions and the water molecules of solvation are accommodated between the layers and are linked to them through a primary cation N—H⋯O(anion) hydrogen bond, as well as through water O—H⋯O hydrogen bonds to brucinium and arsanilate ions as well as bridging water O-atom acceptors, giving an overall three-dimensional network structure.In the structure of the brucinium salt of 4-aminophenylarsonic acid ( Strychnos alkaloid base brucine,  has been extensively employed as a resolving agent for chiral organic compounds  in 2-propanol/water was carried out, resulting in the formation of the crystalline hydrated title salt, C23H27N2O4+· C6H7AsNO3−·4H2O, and the structure is reported herein. The acid has biological significance as an anti-helminth in veterinary applications , all inter-associated through hydrogen bonds , C8(S), C12(S), C13(R), C14(R), C16(S) and the additional introduced (S) chiral centre at N19.The asymmetric unit of the title salt comprises a brucinium cation, a ds Fig. 1. Protonads Fig. 1 absoluteet al., 1985et al., 2002b-axis direction, with the previously described 21 propagation of the brucinium cations along the ca 12.3 Å axis  while two of the solvent water molecules (O1W and O3W) form hydrogen bonds with the carbonyl O25 atom of the the brucinium cation   -malate  = 1.2Ueq(N) or 1.5Ueq(O). Other H atoms were included in the refinement at calculated positions [C—H(aromatic) = 0.95 Å and C—H  = 0.97–1.00 Å] and treated as riding with Uiso(H) = 1.2Ueq(C). The absolute configuration determined for the parent strychnidinin-10-one molecule  global, I. DOI: 10.1107/S2056989016006691/lh5811Isup2.hklStructure factors: contains datablock(s) I. DOI: 1475231CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Both molecules possess inversion symmetry; however, the compounds crystallize in different crystal systems (triclinic versus monoclinic).2,5-Diazido-1,4-phenylene diacetate and dibutyrate are the first structurally characterized representatives with a  10H8N6O4, (I), and C14H16N6O4, (II), each contain half of the respective molecule which is completed by inversion symmetry. The two molecules differ in the ester moiety (acetate versus butyrate) and the crystal symmetry is different, i.e. triclinic for (I) and monoclinic for (II). The diazidophenylene moieties are essentially planar [maximum deviation of 0.0216 (7) Å for (I) and 0.0330 (14) Å for (II)], and the ester functionalities are almost perpendicular to these planes, making dihedral angles of 79.93 (3)° for (I) and 79.42 (6)° for (II). In the crystals of both (I) and (II), there are no significant intermolecular interactions present.The asymmetric units of the title compounds, C The diazidophenylene moieties do not differ significantly from planarity, with a maximum deviation of 0.0216 (7) Å in (I)trans positions to each other, deviate slightly from a linear arrangement, with an N—N—N angle of 173.01 (9)° for (I)The molecular structures of (I)There are no notable features in terms of π–π stacking interactions or hydrogen bonding in either structure. The crystal packing of (I)trans-diazidophenylene entity are listed, making the two examples presented herein the only ones reported so far.In the Cambridge Structural Database  and cooled to 288 K using an ice-water bath. NaN3  was dissolved in water  and added to the cooled and stirred solution of 1,4-benzoquinone in one portion. Stirring was stopped after 15 min and the flask was sealed and stored at 278 K overnight for crystallization. Vacuum filtration afforded a light-yellow solid, which was washed three times with water and dried in vacuo overnight to afford 2,5-diazidobenzene-1,4-diol . 1,4-Benzoquinone serves as starting material and as oxidation reagent in this reaction, resulting in a theoretical molar yield of only half of the applied starting material (50 mmol).Preparation of 2,5-diazidobenzene-1,4-diol: 1,4-benzoquinone  was dissolved in glacial acetic acid  in one portion and the reaction stirred until complete dissolution of the starting material. The reaction mixture was then allowed to cool to room temperature and stored overnight to allow 2,5-diazido-1,4-phenylene diacetate to crystallize. Vacuum filtration afforded light-orange crystals of compound (I)1H NMR : δ 6.89 , 2.33 ; 13C NMR : δ 168.3 (s), 140.0 (s), 129.3 (s), 115.3 (d), 20.4 (q).Preparation of 2,5-diazido-1,4-phenylene diacetate, (I)M) in one portion and the resulting suspension stirred for 45 min at this temperature. The reaction mixture was then allowed to cool to room temperature and stored for 5 days to allow 2,5-diazido-1,4-phenylene dibutyrate to crystallize. Vacuum filtration afforded yellow crystals of compound (II)1H NMR : δ 6.88 , 2.57 , 1.80 , 1.05 ; 13C NMR : δ 171.1 (s), 140.0 (s), 129.3 (s), 115.3 (d), 35.6 (t), 18.3 (t), 13.6 (q).Preparation of 2,5-diazido-1,4-phenylene dibutyrate, (II)Uiso(H) = 1.2Ueq(C).For both structures, (I)10.1107/S1600536814013762/su0008sup1.cifCrystal structure: contains datablock(s) general, I, II. DOI: 10.1107/S1600536814013762/su0008Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536814013762/su0008IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S1600536814013762/su0008Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S1600536814013762/su0008IIsup5.cmlSupporting information file. DOI: 1008063, 1008064CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The asymmetric unit of title compound contains two independent molecules which are built from three fused rings: a heptane ring, a cyclohexyl ring bearing a ketone and an alcohol group, and a three-membered cyclopropane ring bearing two Cl atoms. In the crystal, the molecules are linked by O—H⋯O and C—H⋯O hydrogen bonds, forming chains propagating along [100]. 16H24Cl2O2, contains two independent molecules (A and B) which are built from three fused rings, viz. a seven-membered heptane ring, a six-membered cyclohexyl ring bearing a ketone and an alcohol group, and a cyclopropane ring bearing two Cl atoms. In the crystal, the two molecules are linked via two O—H⋯O hydrogen bonds, forming an A–B dimer with an R22(10) ring motif. The A molecules of these dimers are linked via a C—H⋯O hydrogen bond, forming chains propagating along the a-axis direction. Both molecules have the same absolute configuration, i.e. 1S,3R,8R,10S, which is based on the synthetic pathway and further confirmed by resonant scattering [Flack parameter = 0.03 (5)].The asymmetric unit of the title compound, C The compound is built up from three fused rings: a seven-membered heptane ring, a six-membered cyclohexyl ring bearing a ketone and alcohol groups, and a three-membered propane ring bearing two Cl atoms  and q3 = 0.030 (5) Å. Owing to the disorder observed in molecule B within the seven-membered ring, the conformation of this ring is intermediate between boat and twist-boat  or chair and twist-chair , depending on the position of atom C6a or C6b.There are two molecules -2,2-dichloro-3,7,7,10-tetramethyltricyclododec-9-one prop-2-en-1-yl)-2-hydroxy-2-methyl-5-(prop-1-en-2-yl)cyclohexanone monohydrate -2,2-dichloro-3,7,7,10-tetramethyl-9,10-epoxytricyclododecane  was added at 273 K. The mixture was stirred at room temperature for 30 min and cooled to 273 K in an ice bath and 1.5 ml of an aqueous solution of CrO3  was added dropwise. The ice bath was removed and the mixture was stirred at room temperature for 1 h. The reaction mixture was extracted with dichloromethane (3 × 30 ml) and the organic layers were dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. The crude product was then purified on silica gel chromatography (230–400 mesh) using hexane/ethyl acetate (95:5) as eluent to give the title compound (yield 53%). Colourless plate-like crystals were obtained from a petroleum ether solution, by slow evaporation of the solvent at room temperature.To a solution of 0.4 g (1.319 mmol) of (1Uiso(H) = 1.5Ueq(O and C-methyl) and 1.2Ueq(C) for other H atoms. The disordered cycloheptane ring in molecule B was refined by splitting atoms C6a, C14a and C15a over two positions. The occupancy factors were initially refined and once the occupancy was correctly evaluated the values were held fixed with ratio 0.54:0.46. Atoms C5a and C7a were also split (C5a/C5b and C7a/C7b) and constrained to occupy the same site using EXYZ and EADP commands allowing then to locate the H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016006174/su5293sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989016006174/su5293Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016006174/su5293Isup3.cmlSupporting information file. DOI: 1473671CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Asac_1390 is composed of 490 amino acid residues and showed high sequence similarity to family 1 glycoside hydrolases from various thermophilic Crenarchaeota. The maximum activity was observed at pH 6.0 and 93°C. The half-life of the enzyme at 90°C was about 7 hours. Asac_1390 displayed high tolerance to glucose and exhibits hydrolytic activity towards cellobiose and various aryl glucosides. The hydrolytic activity with p-nitrophenyl (pNP) substrates followed the order pNP-β-D-galactopyranoside (328 U mg−1), pNP-β-D-glucopyranoside (246 U mg−1), pNP-β-D-xylopyranoside (72 U mg−1), and pNP-β-D-mannopyranoside (28 U mg−1). Thus the enzyme was actually a multifunctional β-glycosidase. Therefore, the utilization of Asac_1390 may contribute to facilitating the efficient degradation of lignocellulosic biomass and help enhance bioconversion processes.We expressed a putative  Lignocellulosic biomass, predominantly composed of cellulose, hemicellulose, and lignin, is the most abundant renewable resource on earth, and its degradation to soluble sugars is a key issue for the production of biobased chemicals and biofuels. Cellulose and hemicellulose consist of polysaccharides, and because of their recalcitrance, the enzymatic conversion of these substrates into simple sugars requires many steps.β-glucosidases (EC 3.2.1.21). Endoglucanases randomly cleave the internal bonds of cellulosic materials, releasing oligosaccharides of various lengths and thus generating new ends of the polysaccharide chains. The cellobiohydrolases act progressively on these chain ends generating short cellooligosaccharides or cellobiose. β-Glucosidases hydrolyze cellooligosaccharides or cellobiose to glucose units [β-xylanase (EC 3.2.1.8), β-xylosidase (EC 3.2.1.37), α-L-arabinofuranosidase (EC 3.2.1.55), endo-1,4-β-mannanase (EC 3.2.1.78), β-mannosidase (EC 3.2.1.25), and β-galactosidase (EC 3.2.1.23) [Microorganisms capable of degrading cellulose possess three types of enzymes which act synergistically , 2: (1) dases EC .2.1.21. se units . HemicelEC 3.2.1.5, endo-1β-glucosidases is very important in cellulose hydrolysis process because they perform a rate-limiting step by preventing the accumulation of cellobiose, which inhibits the activities of most endo- and exoglucanases [β-glucosidases are inhibited by glucose [β-glucosidases, with high specific activity and glucose tolerance, could improve the efficiency of cellulolytic enzyme complexes.The presence of ucanases . Moreove glucose . Therefoβ-glucosidases have been identified [β-glucosidases [Thermostable enzymes have several advantages in lignocellulose degradation processes because they are able to withstand rough reaction conditions at elevated temperature and allow elongated hydrolysis time due to higher stability and reduced risk of contamination. High temperature promotes high activity of these enzymes and increases the solubility of substrates in the aqueous phase. Although a number of thermostable entified –16, therosidases , 14.Acidilobus saccharovorans 345-15T (DSM 16705) is an anaerobic, organotrophic, thermoacidophilic crenarchaeon with a pH range from 2.5 to 5.8 (optimum at pH 3.5 to 4) and a temperature range from 60 to 90°C (optimum at 80 to 85°C), isolated from an acidic hot spring of Uzon Caldera, Kamchatka, Russia [ A. saccharovorans was sequenced [ A. saccharovorans on carbohydrates. Particularly, the gene Asac_1390 encoding putative β-galactosidase (EC 3.2.1.23) was identified [β-glycosidase., Russia . The comequenced , and theentified . In thisβ-galactosidase gene Asac_1390 was amplified from A. saccharovorans genomic DNA by PCR using primers F2_NcoI (TTCCATGGCAGTTACCTTCCCAAA) and R2_BglII (5′-TTAGATCTGGATCTACCAGGCGCT-3′); the PCR products were digested with NcoI and BglII and inserted into pQE60 (Qiagen) at NcoI and BglII sites, yielding the plasmid pQE60_Asac1390.The  Escherichia coli strain DLT1270 carrying plasmid pRARE2 (Novagen). Recombinant strain was grown at 37°C in Luria-Bertani medium (LB) supplemented with ampicillin and induced to express recombinant xylanases by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1.0 mM at OD600 approximately 0.6 and incubated further at 37°C for 20 hours. The grown cells were harvested by centrifugation at 5,000 g for 20 min at 4°C, washed with 0.1 M sodium phosphate buffer, pH 7.0, and resuspended in 20 mL of the same buffer with lysozyme (1 mg/mL). The cells were incubated 30 min at 4°C and then disrupted by sonication. The insoluble debris was removed by centrifugation at 5,000 g for 20 min at 4°C. The supernatant was then incubated in a water bath for 30 min at 75°C and then for 30 min at 85°C (double heat treatment) and cooled on ice. Denatured proteins of E. coli were removed by centrifugation at 12,000 g for 20 min at 4°C. The protein sample was dialysed against 25 mM phosphate buffer (pH 7.0) at 4°C for 3 h.Plasmid pQE60_Asac1390 was transformed intoThe purity of the purified protein was examined by SDS-PAGE (10%) and its concentration was determined by the Bradford method using bovine serum albumin (BSA) as a standard.β-galactosidase activity was analyzed using o-nitrophenyl-β-D-galactopyranoside  as substrate following modified Craven method [μg). The reaction was stopped with 0.375 mL of cold 1 M Na2CO3 solution. A blank, containing 0.05 mL of 0.1 M sodium phosphate buffer instead of enzyme solution, was used to correct for the thermal hydrolysis of oNPGal. The amount of released o-nitrophenol was measured at 420 nm. One unit (U) of enzyme activity was defined as the amount of enzyme required to liberate 1 μmol of o-nitrophenol per min under described conditions.The n method . 1.076 mβ-galactosidase activity, pH values were varied from 3.0 to 8.0 at 80°C using 0.1 M acetate buffer (pH 3.0 to 5.0) and 0.1 M sodium phosphate buffer (pH 6.0 to 8.0). Similarly, β-galactosidase assay was done at various temperatures (50–100°C) and pH 7.0 to determine the optimum temperature for Asac_1390 activity.To examine the effects of pH and temperature on β-galactosidase activity in 0.1 M sodium phosphate buffer (pH 7.0) at 50°C.The thermostability of Asac_1390 was assessed by preincubating the enzyme at 90°C in sodium phosphate buffer, pH 7.0. The samples were collected at the desired intervals and assayed for the residual  p-nitrophenyl-β-D-galactopyranoside , p-nitrophenyl-β-D-glucopyranoside (pNPGlu), p-nitrophenyl-β-D-xylopyranoside (pNPXyl), and pNP-β-D-mannopyranoside (pNPMan). The reactions were performed with 2.21 mM of each substrate in 0.1 M sodium phosphate buffer (pH 7.0) at 93°C and the activity was measured by release of pNP (at 405 nm) and oNP (at 420 nm).The substrate specificity of Asac_1390 was determined using oNPGal,μmol of glucose per minute under test conditions.When cellobiose was used as a substrate (0.5% w/v), the amount of glucose released was determined with a Sucrose/D-Glucose/D-Fructose Kit  according to the manufacturer's protocol. The cellobiose hydrolysis reactions were performed in 0.1 M sodium phosphate buffer (pH 6.0) at 50°C. In this assay, 1 U of activity is defined as the amount of enzyme which is required to release 1 Km (mM), Vmax (U/mg), kcat (c−1), and kcat/Km, were calculated from Hanes-Woolf plot of Michaelis-Menten equation.Various concentrations of oNPGal, pNPGal, pNPGlu, pNPXyl, and pNPMan  were used to determine kinetic parameters of Asac_1390. The reactions were performed in 0.1 M sodium phosphate buffer (pH 7.0) at 93°C. The enzyme kinetic parameters, β-galactosidase activity were investigated at the concentrations of glucose from 10 to 100 mM using 2.21 mM oNPGal at 80°C in 0.1 M phosphate buffer (pH 7.0). The relative activity was defined as the relative value to the maximum activity without glucose. The type of inhibition and inhibition constants for glucose were determined by fitting to Cornish-Bowden and Dixon plots [The effects of glucose on on plots  using vaβ-galactosidase Asac_1390 from A. saccharovorans exhibited 54–71% identities with the glycoside hydrolases from the thermophilic archaea of the genera Caldivirga, Sulfolobus, Vulcanisaeta, Thermoproteus, Ignisphaera, Thermoplasma, Thermosphaera, Picrophilus, Thermococcus, and Pyrococcus, annotated as β-galactosidases or β-glucosidases. A Blastp search of the amino acid sequence of Asac_1390 suggested that residues 1–46 contain a signature typical of glycosyl hydrolase (GH) family 1 (Pfam00232). Particularly, Asac_1390 is 63% identical to β-glycosidase from Sulfolobus acidocaldarius that was found to exhibit activities toward β-glucosides, β-galactosides, and β-fucosides [The amino acid sequence of a putative ucosides . The res E. coli. The recombinant enzyme was expressed with a yield of about 30% of the total soluble protein and was purified from crude E. coli extracts via two-step heat treatment to a purity of above 95%. The purified protein appeared in SDS-PAGE analysis as a single band with a molecular mass of approximately 55 kDa (Asac_1390 gene with the same sequence as that reported in GenBank (CP001742) was cloned and expressed iny 55 kDa , consistβ-galactosidase activity was examined over a pH range of 3.0 to 8.0 at 80°C. Maximum activity was observed at pH 6.0 in sodium phosphate buffer. At pH 5.0 and 7.0, the activity was approximately 70% of the maximum  at 50°C for 20 min . The Asaβ-galactosidases and β-glucosidases from hyperthermophilic archaea, including enzymes from Sulfolobus solfataricus  and T. kodakaraensis (18 h at 90°C).These values of the temperature and pH optima of Asac_1390 are typical for  pH 6.5, ), Pyroco pH 5.0, ), S. aci pH 5.5, ), and Th pH 6.5, ). In terβ-galactosidase activity during oNPGal hydrolysis were investigated at various concentrations of D-glucose. The addition of 10, 50, and 100 mM of sugar reduced the activity of Asac_1390 to 80%, 76%, and 65%, respectively. Thus, glucose had little effect on hydrolytic activity.The effects of glucose on Ki′ = 158 mM) was significantly lower than that for EI complex (Ki = 500 mM). Glucose was reported to be a competitive inhibitor of β-glycosidase from S. solfataricus [Ki of 96 mM, while it has little effect on the β-glucosidase from Pyrococcus furiosus with an apparent Ki of 300 mM [The Cornish-Bowden and Dixon plots demonstrated a mixed type of inhibition of Asac_1390 by glucose. The dissociation constant of the EIS complex (ataricus  with thef 300 mM .β-1-2 and β-1-4 linkages. The Michaelis-Menten constants (Km), turnover numbers (kcat), and catalytic efficiencies (kcat/Km) for oNPGal, pNPGal, pNPGlu, pNPXyl, and pNPMan are presented in kcat/Km for pNPGlu was much higher than that obtained with pNPGal, indicating that Asac_1390 was not a β-galactosidase as it was annotated, but rather a β-glucosidase.The hydrolytic activity of Asac_1390 was investigated with various aryl glycosides . For theβ-galactosidase and β-glucosidase activity (246–328 U mg−1 on different substrates), a high affinity with substrate (Km = 0.24 mM for pNPGlu), and high catalytic activity (kcat/Km = 1327 s−1 mM−1 for pNPGlu). These values are among the highest among archaeal enzymes of this class. Some more active β-galactosidases from thermophilic bacteria are known, but usually they are less thermostable  are less thermostable than Asac_1390 [Taking into account that some microbial GH1 family sac_1390 .β-galactosidase, this enzyme exhibits hydrolytic activity towards cellobiose and various aryl glucosides: high activity with pNPGal and pNPGlu, followed with pNPXyl and pNPMan. Such broad substrate specificity β-glycosidases are known among Archaea [ S. acidocaldarius and S. solfataricus [The broad substrate specificity of Asac_1390 is an interesting and important feature of this enzyme. Although initially annotated as  Archaea , includiataricus , 26. Anaataricus  could heβ-Xylosidases and β-glucosidases are responsible for the last steps of the hydrolysis of xylan and cellulose: cleavage of xylobiose to xylose [β-mannosidase is also useful since this enzyme participates in the production of fermentable sugar from another component of hemicellulosic materials, mannan [ Trichoderma reesei is a well-known cellulase-overproducing filamentous fungus which secretes several cellulolytic enzymes. However, β-glucosidase activity in T. reesei is partly mycelium-bound and obviously limits the enzyme performance in commercial T. reesei preparations [ T. reesei with highly active β-glucosidase, exhibiting also β-xylosidase, β-mannosidase, and β-galactosidase hydrolytic activities, could improve efficiency of processing of lignocellulose biomass. Asac_1390 is particularly suitable for these purposes due to its resistance to inhibition by the main reaction product, glucose.Multifunctionality of Asac_1390 makes it very promising for application in enzymatic hydrolysis of lignocellulose biomass. o xylose  and cell, mannan . For insarations . Supplem A. saccharovorans. This enzyme is optimally active at high temperature (93°C) and pH 6.0 and is highly thermostable. Asac_1390 is a multifunctional β-glycosidase exhibiting activities of β-glucosidase, β-galactosidase, β-xylosidase, and β-mannosidase. The broad substrate specificity and resistance to inhibition by glucose make the new enzyme promising for application in enzymatic degradation of lignocellulosic materials.In this study, we expressed and characterized a thermostable recombinant glycosyl hydrolase Asac_1390 from


The title fused heterocycles arose from an unexpected intramolecular cyclization reaction. Each molecule features an intramolecular O—H⋯O hydrogen bond. In the crystal, chains mediated by C—H⋯O interactions arise. H-thiazolopyrimidin-5-one, C13H8N2O3S, (1), and 6-(2-hydroxybenzyl)-3-methyl-5H-thiazolopyrimidin-5-one, C14H10N2O3S, (2), were synthesized when a chromone-3-carboxylic acid, activated with (benzotriazol-1-yloxy)tripyrrolidinylphosphonium hexafluoridophosphate (PyBOP), was reacted with a primary heteromamine. Instead of the expected amidation, the unusual title thiazolopyrimidine-5-one derivatives were obtained serendipitously and a mechanism of formation is proposed. Both compounds present an intramolecular O—H⋯O hydrogen bond, which generates an S(6) ring. The dihedral angles between the heterocyclic moiety and the 2-hydroxybenzoyl ring are 55.22 (5) and 46.83 (6)° for (1) and (2), respectively. In the crystals, the molecules are linked by weak C—H⋯O hydrogen bonds and π–π stacking interactions.The title compounds, 6-(2-hydroxybenzyl)-5 Even though some derivatives tested up to now have shown interesting anti-inflammatory -5H-thiazolopyrimidin-5-one (1) and 6-(2-hydroxybenzyl)-5H-thiazolopyrimidin-3-methyl-5-one (2), which will be screened for antimicrobial activity.A search made in the latest version  of the Cambridge Structural Database  and (2) are shown in Figs. 1viz. the hydroxybenzyl and the heterocyclic 5H-thiazolopyrimidin-5-one ring separated by a carbonyl spacer, as expected. In both compounds, the carbonyl O atoms are trans oriented with respect to each other, contributing to the establishment of an intramolecular O—H⋯O hydrogen bond between the o-hydroxyl group of the benzene ring and the carbonyl group of the spacer (Tables 1S(6) ring. Taken together, the benzene ring and hydrogen-bonded pseudo ring are roughly planar, the carbonyl oxygen atom deviates by 0.391 (3) and 0.055 (4) Å in (1) and (2), respectively from the least-square plane formed by the benzene ring atoms. The heterocyclic rings of both compounds are also almost planar, as expected; the maximum deviation from the best plane formed by the ten atoms of the thiazolopyrimidine moiety is 0.103 (1) Å for the carbonyl oxygen atom, O5, in (1) and 0.129 (1) Å for the same atom in (2). Thus, both molecules are twisted around the C6—C67 bond that links the ring systems, which are inclined to one another by 55.22 (5) and 46.83 (6)° for (1) and (2), respectively.The molecules of  the hydrogen-bond donor atom is C2 (of the heterocyclic group) and in (2) the hydrogen-bond donor atom is C64 (located in the exocyclic benzene ring).As noted above, the hydroxyl group is involved in intramolecular hydrogen bonding, which leaves it unavailable for participation in intermolecular hydrogen bonding. Thus, the molecules are linked 1) the molecules are linked by the C2—H2⋯O5 (x + y + z + C(6) chain, which runs parallel to [101] and results from the action of a c-glide at . Thus in the supramolecular structure of this compound, the molecules are linked by a C64—H64⋯O5 hydrogen bond, forming a C(9) chain, which runs parallel to the b-axis direction and results from the action of a 21 screw axis at  Table 2.1) there is a close contact between centrosymmetrically related rings containing atom C5 at  and  . In (2) the molecules stack above each other along the a-axis direction with unit translation of 3.931 (2) Å .Both molecules present aromatic π–π stacking contacts. In . Compounds (1) and (2) are herein characterized and the remaining are referred to by their CSD codes. GEFTES: 7-(methylsulfanyl)-5H-thiazolopyrimidin-5-one  of the Cambridge Structural Database revealed the existence of 11 deposited compounds containing the 51) and (2) were synthesized in moderate/high yields by a one-pot reaction using 4-oxo-4H-chromene-3-carboxylic acid as the starting material. Chromone-3-carboxylic acid was initially activated with benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluoridophosphate (PyBOP). Then the in situ formed intermediate reacts with the heteroamine (stoichiometry 1:1) giving rise to 5H-thiazolopyrimidin-5-one derivatives (1) (68%) and (2) (81%). From a mechanistic point of view, the 6-(2-hydroxybenzoyl)-5H-thiazolopyrimidin-5-one derivatives may have been obtained by a nucleophilic attack of primary heteroamine to the 2-position of the activated chromone with a subsequent opening of the pyran ring. Then, the heterocycle entities were obtained by a process involving an intramolecular reaction assisted by the nitrogen atom of the heterocycle moiety (see scheme below). Crystals were obtained by recrystallization from (1) in AcOEt (m.p. 454–456 K) in the form of colourless plates and from (2) in CH2Cl2 (m.p. 451–453 K) in the form of yellow blocks.Compounds (Uiso = 1.2Ueq(C) or 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015011044/hb7437sup1.cifCrystal structure: contains datablock(s) general, 1, 2. DOI: 10.1107/S2056989015011044/hb74371sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989015011044/hb74372sup3.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989015011044/hb74371sup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015011044/hb74372sup5.cmlSupporting information file. DOI: 1405409, 1405408CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Five closely related (2-chloroquinlin-3-yl)methyl ethers all exhibit different patterns of direction-specific intermolecular interactions, leading to the formation of different types of chain in four of them and sheets in the fifth. 18H13BrClNO3, (I), methyl 5-bromo-2-[(2-chloro-6-methylquinolin-3-yl)methoxy]benzoate, C19H15BrClNO3, (II), methyl 2-[(2-chloro-6-methylquinolin-3-yl)methoxy]benzoate, C19H16ClNO3, (III), which crystallizes with Z′ = 4 in space group P212121, and 2-chloro-3-[methyl]quinoline, C20H14ClNO, (IV), the non-H atoms are nearly coplanar, but in {5-[(2-chloroquinolin-3-yl)methoxy]-4-(hydroxymethyl)-6-methylpyridin-3-yl}methanol, C18H17ClN2O3, (V), the planes of the quinoline unit and of the unfused pyridine ring are almost parallel, although not coplanar. The molecules of (I) are linked by two independent π–π stacking interactions to form chains, but there are no hydrogen bonds present in the structure. In (II), the molecules are weakly linked into chains by a single type of π–π stacking interaction. In (III), three of the four independent molecules are linked by π–π stacking interactions but the other molecule does not participate in such interactions. Weak C—H⋯O hydrogen bonds link the molecules into three types of chains, two of which contain just one type of independent molecule while the third type of chain contains two types of molecule. The molecules of (IV) are linked into chains by a C—H⋯π(arene) hydrogen bond, but π–π stacking interactions are absent. In (V), there is an intramolecular O—H⋯O hydrogen bond, and molecules are linked into sheets by a combination of O—H⋯N hydrogen bonds and π–π stacking interactions.In the molecules of the title compounds, methyl 5-bromo-2-[(2-chloroquinolin-3-yl)methoxy]benzoate, C Quinoline itself has been found to possess antimalarial, anti-bacterial, antifungal, anthelminthic, cardiotonic, anticonvulsant, anti-inflammatory and analgesic activity Z′ = 1, and P212121 with Z′ = 4 for (III)x-coordinates of atoms Cl12 and Cl32 differ by ca 0.5 and their z-coordinates are almost identical, the y-coordinates of these two atoms differ by ca 0.13; similarly the x-coordinates of atoms Cl22 and Cl42 again differ by ca 0.5 but now the y-coordinates are almost identical, while the z-coordinates differ by ca 0.18. Hence it is not possible to identify even pseudosymmetry here. For compound (III)Z′ = 1, in space groups P21 and P21/c, respectively.As noted above, the molecular constitutions of compounds (I)–(III) are very similar: those of compounds (I)In compounds (I)–(III), the non-H atoms are almost co-planar, as shown by the relevant torsional and dihedral angles Table 1. It is ix parameter –(V) exhibits any internal symmetry and hence all are conformationally chiral. For compounds (I)C3 symmetry, such as a methyl group, is directly bonded to a group having approximate local C2 symmetry, such as an aryl ring, the rotational barrier between these two groups is extremely low, of the order of J mol−1 rather than the usual kJ mol−1 –(V) is determined by a variety of direction-specific intermolecular interactions, including both π–π stacking interactions and hydrogen bonds of C—H⋯N, C—H⋯O and C—H⋯π types, as well as O—H⋯N hydrogen bonds in compound (V)x, y, z) and the brominated ring of the molecule at  make a dihedral angle of 1.04°; the ring centroid separation is 3.6168 (10) Å, and the shortest perpendicular distance from the centroid of one ring to the plane of the other is 3.4132 (6) Å, with a ring-centroid offset of ca 1.20 Å. For the heterocyclic ring at  and the brominated aryl ring at , the corresponding values are 1.52 (9)°, 3.7454 (11) Å, 3.4357 (8) Å and ca 1.49 Å. The combination of these two stacking interactions links the molecules of (I)There are no hydrogen bonds of any kind in the crystal structure of compound (I)et al., 20091 screw axis. The pyridyl ring at  and the brominated aryl ring at (−x + y + z) make a dihedral angle of 3.87 (10)°: the shortest perpendicular distance from the centroid of one ring to the plane of the other is 3.3816 (9) Å, but the ring-centroid separation is 3.882 (12), resulting in a ring-centroid offset of ca 1.78 Å. Thus there is only a very modest overlap of these rings and a consequently weak stacking interaction: if this interaction is, in fact, regarded as significant, it links the molecules into a π-stacked chain running parallel to [010].The only short C—H⋯O contact in the structure of compound (II)cf. Figs. 3ca 1.03 and ca 1.06 Å, respectively. The only short C—H⋯N contact within the crystal structure of compound (III)Z′ = 4. One of the C—H⋯O interactions involves only molecules of type 1 which are related by the 21 screw axis along  ] Fig. 11, which rThere are no hydrogen bonds of the C—H⋯N or C—H⋯O types in the crystal structure of compound (IV)S(7)  chain running parallel to the acrylate, where Z′ = 2  chains to which the molecules of the second type are linked by O—H⋯N hydrogen bonds. Chains of C(6) type are formed also in N-{(2-chloro-3-quinlinyl)methyl]-4-fluoroaniline (hydroxy)methyl]acrylonitrile  chains by C—H⋯N hydrogen bonds. In the structure of 2-chloro-3-(dimethoxymethyl)-6-methoxyquinoline methyl acetate and (2-chloro-6-methylquinolin-3-yl)methyl acetate  together with potassium carbonate (2 mmol) and these mixtures were stirred at ambient temperature for 6–9 h, with monitoring by TLC. When each reaction was complete, ice-cold water (5 ml) was added and the resulting solid products were collected by filtration, washed with water and dried in air. Crystals suitable for single-crystal X-ray diffraction were obtained by slow evaporation, at ambient temperature and in the presence of air, of solutions in dichloromethane, with yields in the range 86–97%.For the synthesis of compounds (I)–(V), a mixture of 0.4 mmol of the appropriate quinoline derivative, 2-chloro-3-(chloromethyl)quinoline for compounds (I)Uiso(H) = 1.5Ueq(C) for the methyl groups, which were permitted to rotate but not to tilt, and 1.2Ueq(C) for other C-bound H atoms.Crystal data, data collection and structure refinement details are summarized in Table 3Uiso(H) = 1.5Ueq(O), giving O—H distances of 0.91 Å. For compound (III)x parameter  calculated −(I−)]/[(I+)+(I−)]. Use of the TWIN/BASF instructions in SHELXL2014  by the standard method  calculated using 102 quotients of type [(I+)−(I−)]/[(I+)+(I−)].The H atoms bonded to O atoms in compound (V)10.1107/S2056989015008233/su5121sup1.cifCrystal structure: contains datablock(s) global, I, II, III, IV, V. DOI: 10.1107/S2056989015008233/su5121Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015008233/su5121IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989015008233/su5121IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989015008233/su5121IVsup5.hklStructure factors: contains datablock(s) IV. DOI: 10.1107/S2056989015008233/su5121Vsup6.hklStructure factors: contains datablock(s) V. DOI: Click here for additional data file.10.1107/S2056989015008233/su5121Isup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015008233/su5121IIsup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015008233/su5121IIIsup9.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015008233/su5121IVsup10.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015008233/su5121Vsup11.cmlSupporting information file. DOI: 1061944, 992268, 992267, 1061945, 1061946CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title compounds, (I) and (II), are hydrazinecarbothioamide derivatives. In the crystal of (I), two independent molecules are linked by bifurcated N—H⋯O and C—H⋯O hydrogen bonds, forming two  13H13N3O2S·0.5H2O, (I), and C17H14ClN3OS, (II), are hydrazinecarbothioamide derivatives. Compound (I) crystallizes with two independent molecules (A and B) and a water molecule of crystallization in the asymmetric unit. The chromene moiety is essentially planar in molecules A and B, with maximum deviations of 0.028 (3) and 0.016 (3) Å, respectively, for the carbonyl C atoms. In (II), the pyran ring of the chromene moiety adopts a screw-boat conformation and the phenyl ring is inclined by 61.18 (9)° to its mean plane. In the crystal of (I), bifurcated N—H⋯O and C—H⋯O hydrogen bonds link the two independent molecules forming A–B dimers with two R21(6) ring motifs, and R22(10) and R22(14) ring motifs. In addition to these, the water molecule forms tetrafurcated hydrogen bonds which alternately generate R44(12) and R66(22) graph-set ring motifs. There are also π–π [inter-centroid distances = 3.5648 (14) and 3.6825 (15) Å] interactions present, leading to the formation of columns along the c-axis direction. In the crystal of (II), molecules are linked by pairs of N—H⋯S hydrogen bonds, forming inversion dimers with an R22(8) ring motif. The dimers are linked by C—H⋯π interactions, forming ribbons lying parallel to (210).The title compounds, C However, in compound (II)q = 0.314 (2) Å, θ = 116.4 (4)°, φ = 147.5 (5)° and ΔC2 = 0.7 (3)]. In compound (II)A and B, respectively, while the deviation of the Cl atom in compound (II)In compound (I)A and B of compound (I)The hydrazinecarbothioamide backbone is almost planar, with the maximum deviation being exhibited by atom N2 in both compounds; 0.025 (2) and 0.051 (2) Å, respectively, in molecules A and B, respectively, of compound (I)sp2 single-bond and S=Csp2 double-bond distances of ca 1.75 and 1.59 Å, respectively, indicating the presence of partial double-bond character , also indicating some degree of delocalization in both compounds. The S1=C11—N2—N1 torsion angles are 177.31 (16) and 174.29 (16)°, respectively, in molecules A and B of compound (I)trans to the azomethane nitrogen atom N1 in both compounds.Thiosemicarbazones exist in the thione form in the solid state and in solution they exist as an equilibrium mixture of thione and thiol forms  Å  and Cg5⋯Cg5ii = 3.6825 (15) Å .The water molecule of crystallization plays an important role in the hydrogen-bonding patterns of the three-dimensional network in compound (I)s Table 1. SimilarIn the crystal of compound (II)f Table 2. The dimf Table 2, formingH-chromen-3-yl]methylene}- N-phenylhydrazinecarbothioamide)copper methylene] hydrazinecarbothioamide (III) E)-2-[(4-chloro-2H-chromen-3-yl)methylene]-N-cyclohexylhydrazine carbothioamide (IV) A search of the Cambridge Structural Database : 1.19 g (0.01 mol) of N-ethylhydrazinecarbothioamide was dissolved in 20 ml of hot ethanol and to this was added 1.74 g (0.01 mol) of 4-oxo-4H-chromene-3-carbaldehyde in 10 cm3 of ethanol over a period of 10 min with continuous stirring. The reaction mixture was refluxed for 2 h and allowed to cool whereby a shiny white compound began to separate; this was filtered and washed thoroughly with ethanol and then dried in vacuo. The compound was recrystallized from hot ethanol (yield: 96%), giving colourless block-like crystals.Compound (II): 1.67 g (0.01 mol) of 4(N)-phenylthiosemicarbazide was dissolved in 20 ml of hot ethanol and to this was added 1.94 g (0.01 mol) of 4-chloro-2H-chromene-3-carbaldehyde in 10 ml of ethanol over a period of 10 min with continuous stirring. The reaction mixture was refluxed for 2 h and allowed to cool whereby a shiny yellow compound began to separate. It was filtered and washed thoroughly with ethanol and then dried in vacuo. The compound was recrystallized from hot ethanol (yield: 89%), giving colourless block-like crystals.Uiso(H) = 1.2Ueq(N). The C-bound H atoms in both molecules were included in calculated positions and treated as riding atoms: C—H = 0.93–0.97 Å with Uiso(H) = 1.5Ueq(C) for methyl H atoms and = 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details for compounds (I)10.1107/S2056989015003369/su5078sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989015003369/su5078Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015003369/su5078IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989015003369/su5078Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015003369/su5078IIsup5.cmlSupporting information file. DOI: 1016440, 1049914CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II atom in the title compound shows a square-planar NiN2O2 coordination with the imine N and phenolate O atoms of the two Schiff base ligands. C—H⋯O and C—H⋯π interactions result in the formation of sheets of molecules parallel to the ac plane.The Ni 15H14NO2)2], comprises an NiII cation, lying on an inversion centre, and a Schiff base anion that acts as a bidentate ligand. The NiII cation is in a square-planar coordination environment binding to the imine N and phenolate O atoms of the two Schiff base ligands. The N- and O-donor atoms of the two ligands are mutually trans, with Ni—N and Ni—O bond lengths of 1.9191 (11) and 1.8407 (9) Å, respectively. The plane of the methoxybenzene ring makes a dihedral angle of 84.92 (6)° with that of the phenolate ring. In the crystal, molecules are linked into screw chains by weak C—H⋯O hydrogen bonds. Additional C—H⋯O hydrogen bonds, together with C—H⋯π contacts, arrange the molecules into sheets parallel to the ac plane.The asymmetric unit of the title compound, [Ni(C The Ni1—N1 and Ni1—O1 bond lengths in the N2O2 coordination plane are 1.9191 (11) and 1.8407 (9) Å, respectively. These are similar to those observed in the other closely related NiII complexes with N2O2-coordinating Schiff base ligands nd Fig. 1. The N2OA⋯O2 interactions phenolate residues. No corresponding structures with a benzyl or substituted benzyl unit bound to the imino N atom were found. However, extending the search to allow additional substitution on the phenolate ring resulted in seven discrete structures including the closely related bis(2-[(E)-(4-fluorobenzyl)iminomethyl]-6-methoxyphenolato-κ2N,O1)nickel(II)  was dissolved in ethanol (15 ml). An ethanolic solution of nickel(II) acetate tetrahydrate  was added dropwise to the former solution and the mixture heated under reflux for 4 h, producing a green solid. The solid was filtered off, washed with ice-cold ethanol and air-dried at room temperature. The solid product was recrystallized from chloroform, yielding green crystals . Analytical data for [Ni(C28H30N2O4)]: C 66.82, H 5.23, N 5.19%; found: C 67.03, H 5.28, N 5.15%. IR : ν(C=N) 1605 (s), ν(C—N) 1391 (s), ν(C—O) 1325 (s), ν(Ni—O) 598 (w), ν(Ni—N) 437 (w).2 and 0.98 Å for CH3 hydrogens. The Uiso(H) values were constrained to be 1.5Ueq of the carrier atom for methyl H atoms and 1.2Ueq for the remaining H atoms. A rotating-group model was used for the methyl groups.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S160053681401650X/sj5419sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S160053681401650X/sj5419Isup2.hklStructure factors: contains datablock(s) I. DOI: 1014286CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The structures of these isolates were elucidated by extensive spectroscopic methods. The inhibitory activities of these isolates against five human cancer cell lines in vitro were also tested.Two dimeric abietane diterpenoids, salviwardins A and B (The online version of this article (doi:10.1007/s13659-015-0054-6) contains supplementary material, which is available to authorized users. Salvia is a rich source of diterpenoids with structural diversity +; positive HREIMS m/z 598.4029 .Orange powder; mentclass2pt{minimc 0.19, MeOH); UV (MeOH) λmax (log ε) 476 (3.25) nm; IR (KBr) νmax 3442, 2927, 2870, 1626, 1579, 1467, 1394, 1291, 1172, 1088, 1008 cm−1; 1H and 13C NMR data, see Tables m/z 619 [M + Na]+; positive HRESIMS m/z 597.3941 .Orange powder; mentclass2pt{minimc 0.26, CDCl3); UV (CDCl3) λmax (log ε) 336.5 (3.35) nm; IR (KBr) νmax 3430, 2927, 2871, 1722, 1633, 1423, 1330, 1271, 1172, 1023 cm−1; 1H and 13C NMR data see Table m/z 296 , 268 (26), 267 (94), 265 (100), 255 (18), 241 (35), 213 (54); positive HREIMS m/z 296.1775 .Colorless oil; 2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3--2,5-diphenyltetrazolium bromide (MTT) . Briefly, 100 μL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before test compound addition, while suspended cells were seeded just before test compound addition, both with an initial density of 1 × 105 cells/mL in 100 μL of medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cis-platin and paclitaxel (Sigma) as positive control. After the incubation, MTT (100 μg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 100 μL of 20 % SDS −50 % DMF after removal of 100 μL of medium. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by Reed and Muench’s method [The following human tumor cell lines were used: HL-60, SMMC-7721, A-549, MCF-7, and SW-480, which were obtained from ATCC . All cells were cultured in RPMI-1640 or DMEM medium , supplemented with 10 % fetal bovine serum  at 37 °C in a humidified atmosphere with 5 % COs method .Supplementary material 1 (PDF 1512 kb)Below is the link to the electronic supplementary material.


VI atom exhibits a pentagonal–bipyramidal N2O5 coordination environment. In the complex, the 1,2,4-triazole ligand is coordinated in a tridentate manner.The U 13H9N4O)(NO3)O2(H2O)]·CH3CN·H2O, the UVI atom is seven-coordinated in a distorted pentagonal–bipyramidal N2O5 manner by one tridentate triazole ligand, one monodentate nitrate anion and one water molecule in the equatorial plane and by two uranyl(VI) O atoms in the axial positions. In the crystal, the UVI complex molecule is linked to the water and acetonitrile solvent molecules through N—H⋯N, O—H⋯O and O—H⋯N hydrogen bonds, forming a sheet structure parallel to the bc plane. The sheets are further linked by an additional O—H⋯O hydrogen bond, forming a three-dimensional network.In the title compound, [U(C N-donor heterocyclic ligands is one of the fastest growing areas of coordination chemistry. 1,2,4-Triazoles and their derivatives can be assigned for such types of ligands. The presence of the 1,2,4-triazole ring in the organic ligand provides an additional site for coordination  and shorter than a Csp2—N single bond (1.347 Å) -1H-1,2,4-triazole-3,5-diyl]diphenol -1,2,4-triazole]bis(pyridine)dicopper  and [UO2(NO3)2]·2H2O (0.5 mmol) in acetonitrile (20 ml) was stirred for 20 min. The solution was left to evaporate slowly at room temperature. Red single crystals suitable for X-ray analysis were obtained after 2 d.A mixture of 3-(2-hydroxyphenyl)-5-(pyridin-2-yl)-1Uiso(H) = 1.5Ueq(O). All other H atoms were constrained to ride on their parent atoms, with Uiso(H) = 1.2Ueq.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901502438X/is5435sup1.cifCrystal structure: contains datablock(s) Global, I. DOI: 10.1107/S205698901502438X/is5435Isup2.hklStructure factors: contains datablock(s) I. DOI: 1443165CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A variety of 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxides have been developed as inhibitors of the enzyme protein tyrosine phosphatase 1B (PTP1B). For the title compound, there is the expected twisted relationship between the plane of the 1,2,5-thiadiazolidin-3-one 1,1-dioxide ring and the aryl ring to which it is attached, although the dihedral angle of 62.87 (8)° is substantially less than that seen in certain protein–ligand structures. 24H32N4O8S, (I), crystallizes as a zwitterion. The terminal amine N atom of the [(2-{2-[2-(2-ammonioethoxy)ethoxy]ethoxy}ethyl)carbamoyl] side chain is protonated, while the 1,2,5-thiadiazolidin-3-one 1,1-dioxide N atom is deprotonated. The side chain is turned over on itself with an intramolecular N—H⋯O hydrogen bond. The 1,2,5-thiadiazolidin-3-one 1,1-dioxide ring has an envelope conformation with the aryl-substituted N atom as the flap. Its mean plane is inclined by 62.87 (8)° to the aryl ring to which it is attached, while the aryl rings of the biphenyl unit are inclined to one another by 20.81 (8)°. In the crystal, molecules are linked by N—H⋯O and N—H⋯N hydrogen bonds, forming slabs lying parallel to (010). Within the slabs there are C—H⋯O and C—H⋯N hydrogen bonds and C—H⋯π interactions present.The title compound, C The ability of methyl and methoxy substituents to favor the twisted relationship between the 1,2,5-thiadiazolidin-3-one 1,1-dioxide and aryl rings in compounds like 2 and 3 has been studied computationally and the twisted relationship of these rings has been experimentally observed in the protein–ligand co-crystal structure of 4 with the enzyme PTP1B. However, to the best of our knowledge no crystal structures of free 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxides have been published. Herein, we describe the crystal structure of the title compound (I)4.A variety of 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxides have been developed as inhibitors of the enzyme protein tyrosine phosphatase 1B (PTP1B) .The title compound (I)In the crystal of (I)A search of the Cambridge Structural Database ethoxy]ethoxy}ethyl)carbamate and 3′--4′-methoxy--4-carboxylic acid via (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate. The precursors were synthesized according to published procedures  = 1.5Ueq(C) for methyl H atoms and = 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015003850/su5087sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989015003850/su5087Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015003850/su5087Isup3.cmlSupporting information file. DOI: 1051176CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Currently, more than 90% of commercial β-carotene is produced by chemical synthesis. Due to the growing public concern over food safety, the use of chemically synthesized β-carotene as food additives or functional cosmetic agents has been severely controlled in recent years. This has reignited the enthusiasm for seeking natural β-carotene in large-scale fermentative production by microorganisms.Bacillus subtilis, geranyl diphosphate synthase (GPPS2) from Abies grandis have been co-expressed in an engineered E. coli strain. To further enhance the production of β-carotene, the hybrid MVA  pathway has been introduced into an engineered E. coli strain, co-expressed with the optimized MEP pathway and GPPS2. The final genetically modified strain, YJM49, can accumulate 122.4±6.2 mg/L β-carotene in flask culture, approximately 113-fold and 1.7 times greater than strain YJM39, which carries the native MEP pathway, and YJM45, which harbors the MVA pathway and the native MEP pathway, respectively. Subsequently, the fermentation process was optimized to enhance β-carotene production with a maximum titer of 256.8±10.4 mg/L. Finally, the fed-batch fermentation of β-carotene was evaluated using the optimized culture conditions. After induction for 56 h, the final engineered strain YJM49 accumulated 3.2 g/L β-carotene with a volumetric productivity of 0.37 mg/(L · h · OD600) in aerobic fed-batch fermentation, and the conversion efficiency of glycerol to β-carotene (gram to gram) reached 2.76%.To increase β-carotene production by improving the isopentenyl pyrophosphate (IPP) and geranyl diphospate (GPP) concentration in the cell, the optimized MEP (methylerythritol 4-phosphate) pathway containing 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and isopentenyl pyrophosphate isomerase (FNI) from E. coli BL21(DE3) with the optimized MEP (methylerythritol 4-phosphate) pathway, the gene for GPPS2 from Abies grandis, the hybrid MVA  pathway and β-carotene synthesis genes from Erwinia herbicola.In this paper, by using metabolic engineering techniques, the more efficient biosynthetic pathway of β-carotene was successfully assembled in The online version of this article (doi:10.1186/s12934-014-0160-x) contains supplementary material, which is available to authorized users. Blakeslea trispora, a Rhodotorula glutinis mutant and the microalga Dunaliella salina , crtB (phytoene synthase), crtI (phytoene desaturase) and crtY (lycopene cyclase) from Erwinia herbicola [The plasmid pAC-BETA, kindly provided by Dr Francis X. Cunningham Jr, was used to produce β-carotene. The plasmid pAC-BETA contains all of the genes required for the synthesis of β-carotene, including erbicola , and retfni-1 gene was obtained by PCR using the primers fni-F1 (5′- CGCGGATCC AAGGAGATGACTCGAGCAGAACGAAAAAGA-3′) and fni-R1 (5′-CTAGGAG CTCTTATCGCACACTATAGCTTGA-3′) and Bacillus subtilis genomic DNA as a template. The isolated fni-1 gene fragment was excised using BamHI and SacI, followed by insertion into the corresponding sites of the vector pYJM45 to create pYJM47.The GPPS2 gene fragment was obtained by digestion of pGH/GPPS2 with NdeI and BglII and was then ligated into the corresponding sites of pYJM47 to create pYJM48.The dxs-1 gene was obtained by PCR using the primers dxs-F1 (5′-CTAGGACGTCAAGGAGATGGATCTTTTATCAATACAGGACC-3′) and dxs-R1 (5′-CCGCTCGAGTCATGATCCAATTCCTTTGTGT-3′) and Bacillus subtilis genomic DNA as a template. The isolated dxs-1 gene fragment was excised using AatII and XhoI, followed by insertion into the corresponding sites of the vector pYJM48 to create pYJM49  and incubated at 55°C for 15 min in the dark. Samples were then centrifuged at 14,000 rpm for 10 min, and the acetone supernatant containing β-carotene was transferred to a new tube. The β-carotene production in different engineered strains was analyzed using high performance liquid chromatography  with a UV/VIS detector at 454 nm using a Symmetry C18 column . Methanol, acetonitrile and dichloromethane (21:21:8) were used as the mobile phase at a flow rate of 1 mL/min at 30°C [To quantify β-carotene production, β-carotene was extracted from escribed . Cells w600 = 0.6 with 0.25 mM IPTG and further incubated at 30°C. The β-carotene extraction and analysis were performed as previously described [The strain was inoculated in either initial fermentation medium or optimized fermentation medium. When necessary, the cultures were supplemented with 100 mg/L ampicillin, 34 mg/L chloramphenicol and 50 μg/mL kanamycin. The cells were induced at ODescribed . Optimiz600 = 12 by adding 0.05 mM IPTG. IPTG was added every 8 h during the 80 h fermentation. Conversion efficiency (gram to gram) of glycerol to β-carotene was calculated with the following equation:Fed-batch cultures were carried out in a Biostat B plus MO5L fermentor  containing 3 L of the optimized fermentation medium  as described above. The temperature was initially maintained at 37°C, and 34°C after induction. The pH was controlled at 7.0, and the dissolved oxygen (DO) concentration was maintained at 20% saturation. During the course of fermentation, 40% glycerol was fed at a rate of 3 g/L/h. The cells were induced at ODs = weight of β-carotene (g); Gg = weight of glycerol (g).Where Y = conversion efficiency ; G


Journal of Experimental Botany, Vol. 66, No. 3 pp. 863–878, 2015, doi: 10.1093/jxb/eru448In figure 2 of the above paper, the Y-axis labels of the graphics D and E were incorrectly typed:–1 TW)’Graphic 2D: water use efficiency (WUE) was calculated on the basis of fresh weight and, therefore, the correct Y-axis label is ‘WUE (gFW·mls (mol H2O·m–2·s–1)’Graphic 2E: the units of stomatal conductance were incorrectly typed; the correct Y-axis label is ‘gThe corrected figure 2 is reprinted here:


The structure of the compound is remarkable, as the carboxylate group is coordinated in a chelating manner and no dimerization of the Ti(OiPr)3 groups through OR bridges was observed.The coordination polymer [Ti(O The structures of mono-substituted derivatives Ti(OR)3(CL) with β-diketonates 2, with bridging carboxylate ligands [3(OOCR′)(ROH)]2, where η1-carboxylate ligands are hydrogen-bonded to the coordinated alcohol at the neighboring titanium atom n (1) was obtained by reaction of Ti(OiPr)4 with an equimolar amount of 4-(imidazol-1-yl)butyric acid (L–H) in isopropanol (Scheme [Ti(O3N2H3)]n  was obta1 is a coordination polymer with mononuclear Ti(OiPr)3 units as connector and L as linker, coordinating through the carboxylate group to one Ti atom and the imidazolyl group to the next –N(2) distance is distinctly shorter than that observed in the Ti(OR)4(amine) adducts with primary amines, which are typically in the range 129–131 pm, or in Ti(OR)x derivatives with DHP ligands  n (1). Only few one-dimensional titanium-containing coordination polymers were hitherto structurally characterized 2(μ-DHP) with very bulky OR ligands [iPr)4 was reacted with di-, tri- and tetracarboxylic acids [Apart from the fact that a coordination polymer with Ti. Correlation spectra were recorded on a Bruker AVANCE DPX 300 . Both spectrometers were equipped with a 5-mm inverse-broadband probe head and a z-gradient unit. 2D experiments were measured with Bruker standard pulse sequences: HMBC (Heteronuclear Multiple Bond Correlation). 1H/15N HMBC spectra of 1 and L were recorded in CD2Cl2 and DMSO, respectively.All operations were carried out in a moisture- and oxygen-free argon atmosphere using Schlenk techniques. Isopropanol was dried by refluxing twice over sodium metal and distillation. The solvents for NMR spectroscopy (Eurisotop) were degassed prior to use and stored over molecular sieve. iPr)4 was obtained from ABCR and used as received. Ti(OiPr)4  was added to a solution of 590 mg of 4-(imidazol-1-yl)butyric acid (3.8 mmol) [3 of water-free isopropyl alcohol under argon. Crystals of 1 were obtained after 4 weeks . 1H NMR : δ = 1.21 , 1.83 , 2.05 , 3.78 , 4.70 , 6.74 , 7.17 , 7.59  ppm; 13C NMR : δ = 25.96 (CH3), 26.69 (CH2), 32.64 (CH2), 46.67 (CH2), 77.36 (CHMe2), 118.29 (CH), 129.75 (CH), 138.07 (CH), 186.50 (COO) ppm; IR (ATR): −1; IR (CHCl3): −1.Ti(O.8 mmol)  in 3 cm3α radiation (λ = 71.073 pm). Data were collected on a Bruker AXS SMART APEX II four-circle diffractometer with κ-geometry at 100 K with φ and ω-scans and 0.5° frame width (Table All measurements were performed using MoKion λ = 7.073 pm. F2 (SHELXL97) with anisotropic thermal parameters for all non-hydrogen atoms. Hydrogen atoms were inserted in calculated positions and refined riding with the corresponding atom. The carbon atoms of one OiPr ligand of 1 were disordered. Their two positions were refined with about 40 and 60 % occupancy.The structure was solved by the Patterson method (SHELXS97). Refinement was performed by the full-matrix least-squares method based on 1. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.CCDC-965819 contains the supplementary crystallographic data for


The anhydrous morpholinium salts of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) with 4-aminobenzoic acid, 3,5-dinitrobenzoic acid and 3,5-dinitrosalicylic acid, provide one example of a three-dimensional hydrogen-bonded network polymer and two of weakly inter-associated hydrogen-bonded cation–anion units. 9H17N2+·C7H6NO2− (I)], 3,5-dinitrobenzoic acid [1-aza-8-azoniabicyclo[5.4.0]undec-7-ene 3,5-dinitrobenzoate, C9H17N2+·C7H3N2O6−, (II)] and 3,5-dinitrosalicylic acid (DNSA) [1-aza-8-azoniabicyclo[5.4.0]undec-7-ene 2-hydroxy-3,5-dinitrobenzoate, C9H17N2+·C7H3N2O7−, (III)] have been determined and their hydrogen-bonded structures are described. In both (II) and (III), the DBU cations have a common disorder in three of the C atoms of the six-membered ring moieties , while in (III), there is additional rotational disorder in the DNSA anion, giving two sites  for the phenol group. In the crystals of (I) and (III), the cation–anion pairs are linked through a primary N—H⋯Ocarboxyl hydrogen bond . In (II), the ion pairs are linked through an asymmetric three-centre R12(4), N—H⋯O,O′ chelate association. In (I), structure extension is through amine N—H⋯Ocarboxyl hydrogen bonds between the PABA anions, giving a three-dimensional structure. The crystal structures of (II) and (III) are very similar, the cation–anion pairs being associated only through weak C—H⋯O hydrogen bonds, giving in both overall two-dimensional layered structures lying parallel to (001). No π–π ring associations are present in any of the structures.The anhydrous salts of the Lewis base 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) with 4-aminobenzoic acid [1-aza-8-azoniabicyclo[5.4.0]undec-7-ene 4-aminobenzoate, C Niphates digitalis  is an alkaloid isolated from the sponge Kaca 14), protonation of the N8 group of the six-membered hetero-ring of DBU is readily achieved and results in the formation of salts with carboxylic acids and phenols. The Cambridge Structural Database (2015 version) -4-nitrophenol   9H17N2+ C7H3N2O6− (II)9H17N2+ C7H3N2O7− (III)No reported crystal structures of salts with simple substituted benzoic acids are found, so in order to examine the hydrogen-bonding in crystals of the DBU salts with some common ring-substituted benzoic acids, a number of these were prepared. Suitable crystals were obtained with 4-aminobenzoic acid (PABA),  and , giving the anhydrous salts, CA) and a 4-aminobenzoate anion (B), (I)B), (II)B), (III)A—H⋯Ocarboxyl hydrogen bond  and were allowed to ride in the refinements, with Uiso(H) = 1.2Ueq(C). The amine and aminium H-atoms were located in difference-Fourier analyses and were allowed to refine with distance restraints [N—H = 0.90 (2) Å] and with Uiso(H) = 1.2Ueq(N). Disorder involving atoms C9A and C10A of the six-membered ring systems of both (II)A and C13A, with site occupancy factors of 0.735 (3)/0.265 (3) and 0.686 (4)/0.314 (4), respectively. Also in (III)B) having a SOF = 0.28, which was fixed in the final cycles of refinement. In the structure of (I)Kα X-rays.Crystal data, data collection and structure refinement details are given in Table 410.1107/S205698901600267X/pk2574sup1.cifCrystal structure: contains datablock(s) global, I, II, III. DOI: 10.1107/S205698901600267X/pk2574Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S205698901600267X/pk2574IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S205698901600267X/pk2574IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S205698901600267X/pk2574Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S205698901600267X/pk2574IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S205698901600267X/pk2574IIIsup7.cmlSupporting information file. DOI: 1453494, 1453493, 1453492CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: