As culture conditions, maturation methods and antigen sources differ between the conducted studies, we analyzed the functional differences between IL-4 MoDC and IFNα MoDC, loaded with blebs, in a head-to-head comparison using commonly used protocols. Our data show that both MoDC types are potent (cross-) primers of CD8+ T cells. Whereas IFNα MoDC were more potent in their capacity to cross-present a 25-mer MART-1 synthetic long peptide (SLP) to a MART-1aa26-35 recognizing CD8+ T cell line, IL-4 MoDC proved more potent cross-primers of antigen-specific CD8+ T cells when loaded with blebs. The latter is likely due to the observed greater capacity of IL-4 MoDC to ingest apoptotic blebs. In conclusion, our data indicate the use of IFNα MoDC over IL-4 MoDC in the context of DC vaccination with SLP, whereas IL-4 MoDC are preferred for vaccination with bleb-derived antigens.Dendritic cells (DC) vaccination is a potent therapeutic approach for inducing tumor-directed immunity, but challenges remain. One of the particular interest is the induction of an immune response targeting multiple (unknown) tumor-associated antigens (TAA), which requires a polyvalent source of TAA. Previously, we described the preferred use of apoptotic cell-derived blebs over the larger apoptotic cell remnants, as a source of TAA for both in situ loading of skin-resident DC and in vitro loading of monocyte-derived DC (MoDC). Recent reports suggest that MoDC cultured in the presence of GM-CSF supplemented with IFNα (IFNα MoDC), as compared to IL-4 (IL-4 MoDC), have an increased capacity to cross-present antigen to CD8 Since the use of DC was first proposed for active anti-tumor vaccination by Banchereau and Steinman , strateg avidity .Monocytes are commonly differentiated into MoDC in the presence of GM-CSF and IL-4. Currently, there is growing interest in the use of MoDC differentiated in the presence of GM-CSF and interferon-α (IFNα), since IFNα MoDC have been described to be more potent in cross-presenting antigen –14, whicTo determine the preferred DC type to serve as a vehicle for AML-derived apoptotic bleb vaccines, we conducted a head-to-head comparison of IL-4 MoDC and IFNα MoDC. These MoDC types were cultured using the commonly used culture protocols , matured with a maturation cocktail consisting of IL-1β, IL-6, TNFα, PGE-2, and subsequently analyzed for their immunophenotype, endocytic capacity for apoptotic blebs and soluble antigen, and their ability to prime naive T cells, to cross-present, and to cross-prime antigen-specific T cells.+ T cell activation and induction, HL60 AML cells were retrovirally transduced with melanoma-associated antigen recognized by T cells (MART-1), as described previously [g, after which the resulting supernatant was spun down at 4000×g to isolate the apoptotic blebs. Next, the blebs were washed with PBS, and the protein concentration was determined using a ND-1000 Nanodrop spectrophotometer . The isolated blebs were stored in liquid nitrogen until use.In order to facilitate assessment of antigen-specific CD8eviously . ApoptotMonocytes were isolated from peripheral blood mononuclear cells (PBMC) of healthy donors, after informed consent, by magnetically activated cell sorting using CD14 Microbeads . Isolated monocytes were cultured in the presence of 800 U/ml GM-CSF , supplemented with either 500 U/ml IL-4  for the generation of IL-4 MoDC, or 1000 U/ml IFNα A/D (R&D Systems) for the induction of IFNα MoDC. IL-4 MoDC were cultured for 5 days and IFNα MoDC for 3 days, as most frequently described in literature –14, 16.5 MoDC were loaded with 40 µg of blebs in the presence of the differentiation cytokine cocktails , and 1 h after initiating loading, maturation was induced by IL-1β (10 ng/ml), TNFα , IL-6 , and PGE2 .After differentiation, MoDC were isolated and the immunophenotype was determined by flow cytometry, using FITC-labeled, PE-labeled, APC, Horizon V450, or PeCy7-labeled antibodies against HLA-ABC, HLA-DR, CD1a, CD14, CD36, CD40, CD80, CD83, CD86, CLEC9a, Lox-1, CD18/CD11b (complement receptor 3), and CD18/CD11c (complement receptor 4) , and the expression levels were subsequently analysed using flow cytometry ; the data were analyzed using FACS Diva software (BD Biosciences). MoDC cytokine production was analyzed, after overnight co-culture with irradiated CD40 ligand-expressing J558 cells and 1000 U/ml IFNγ , using an inflammatory cytokine bead array . For MoDC loading, 2 × 105 PKH26 red-labeled  MoDC. The percentage of double-positive cells was analyzed using flow cytometry (LSRFortessa™), as a measure of uptake.In order to determine uptake, 40 μg blebs were labeled with 0.5 μM carboxyfluorescein succinimidyl ester (CFSE) , and cultured overnight with 2 × 0Endocytosis of soluble proteins was analyzed by adding either dextran-FITC  or Lucifer Yellow  to immature IL-4 or IFNα MoDC for 1 h, after which the uptake was analyzed using flow cytometry (LSRFortessa™).+ cells using CD14 Microbeads (Miltenyi Biotec). PBL were stored in liquid nitrogen until further use. PBL were labeled with 1 µM CFSE (Invitrogen) and plated in a 96-well plate at 1 × 105 per well. Mature bleb-loaded MoDC were added to the wells at DC/PBL ratios of 1:5, 1:10, or 1:20, and CD3+CD4+ and CD3+CD8+ T cell proliferations were analyzed using flow cytometry after 6 days. The day 6 supernatant was analyzed for T cell cytokines, using a TH1/TH2/TH17 cytokine bead array .Peripheral blood lymphocytes (PBL) were isolated after informed consent from PBMC of healthy donors, by depleting CD14+ MoDC were loaded with different concentrations of a 25-mer MART-1aa16-40L SLP for 2 h, after which MoDC maturation was induced by adding IL-1β, IL-6, TNFα, and PGE-2. Blebs were loaded as described above, at 40 µg per 2 × 105 MoDC. After loading SLP or blebs overnight, MoDC were harvested and co-cultured for 5 h with a MART-1aa25-36 recognizing CD8+ T cell line , in the presence of 1 μl/ml GolgiStop™ (BD Biosciences). Next, the cells were washed and stained with an APC-labeled MART-1aa26-35 HLA-A2 dextramer  for 15 min, followed by 15 min of staining with Horizon V450 or FITC-labeled CD3 and CD8 mAbs (both BD Biosciences). After washing, the cells were fixed and permeabilized using BD Cytofix/Cytoperm™ solution (BD Biosciences), following manufacturer’s protocol. Finally, intracellular IFNγ (BD Biosciences) was stained for 30 min at 4 °C, after which the cells were washed and IFNγ production was analyzed using flow cytometry , as a measure of activation.HLA-A2+ from HLA-A2+ PBMC were differentiated into either IL-4 MoDC (5 days) or IFNα MoDC (3 days), and CD8+ and CD14CD8− cells were stored in liquid nitrogen for further use. IFNα MoDC or IL-4 MoDC were loaded with blebs or MART-1aa26-35L peptide, and plated at 1 × 105 per well. Autologous CD8+ T cells and irradiated CD14−CD8−cells were added to the loaded MoDC at 1 × 106 per well, supplemented with 20 U/ml IL-7, 10 ng/ml IL-12 (both Sanquin), and 10 ng/ml IL-6 (R&D systems) in a final volume of 2 ml in Yssel’s medium [CD14s medium . After 2aa26-35L peptide in serum-free RPMI, at concentrations ranging from 10 μM to 1 pM, in the presence of 3 μg/ml β2-microglobulin. 5 × 105 CD3+CD8+T cells were cultured with 5 × 105 peptide loaded JY, in the presence of 1 μl/ml GolgiStop™ (BD Biosciences) for 5 h, after which intracellular IFNγ production was assessed as described above.JY cells were loaded for 3 h with MART-1t test, Fisher’s exact test, or a Wilcoxon matched-pairs signed-rank test as indicated. Differences with p values ≤0.05 were regarded significant.Statistical analysis was performed with GraphPad Prism version 5 for Windows (GraphPad Software Inc.), using a paired two-tailed Student’s p = 0.04), and CD14 (p = 0.0005), as compared to IL-4 MoDC. Moreover, IFNα MoDC showed a higher expression of CD80 (p = 0.01) and CD86 (p = 0.03). Next, we assessed the MoDC phenotype upon maturation induction in the presence of apoptotic blebs. In contrast to the immature state, IL-4 MoDC displayed a significantly higher expression of both HLA class I (p = 0.0006) and HLA class II (p = 0.01) and CD1a (p = 0.009) following loading with blebs and subsequent maturation , CD80 (p = 0.002), CD83 (p = 0.001), and CD86 (p = 0.002), whereas IFNα MoDC retained a higher CD14 expression after maturation (p = 0.009). After inducing maturation, IFNα MoDC down-regulated CD1a, compared to their immature expression levels (p = 0.02), at levels lower than those on mature IL-4 MoDC (p = 0.002).We first assessed MoDC phenotype after a 3- or 5-day differentiation period for IFNα or IL-4 MoDC, respectively Fig. a. IFNα Mp = 0.0007; Fig. ., 55 %, p = 0.025, data not shown). In order to determine whether this difference in the capacity to ingest blebs could be explained by different expression levels of receptors involved in antigen uptake and cross-presentation, we analyzed the expression levels of CD36, Clec9a, Lox-1, complement receptors (CR)3, and CR4 on IFNα versus IL-4 MoDC  and a near-significant increase in the uptake of dextran-FITC (p = 0.06) by IL-4 MoDC, as compared to IFNα MoDC.As the ingestion of exogenous antigen by MoDC is essential for the (cross-) priming of antigen-specific T cells, we analyzed the capacity of IFNα and IL-4 MoDC to phagocytose apoptotic blebs. After overnight co-culture, a significantly higher percentage of IL-4 MoDC had ingested blebs as compared to IFNα MoDC, in an immature  and IL-12p70 (p = 0.01)  or with the MART-1 SLP, and maturation was induced 1 h post-loading. Loaded and matured MoDC were subsequently co-cultured for 5 h with a CD8+ cytotoxic T cell line, recognizing the MART-1aa26-35 immunodominant epitope in an HLA-A2 restricted fashion (MART-1 CTL), after which the percentage of IFNγ producing MART-1 CTL was determined by intracellular staining using flow cytometry. Unfortunately, this methodology proved to lack the sensitivity to assess cross-presentation efficiency of the MART-1aa26-35 epitope from blebs. While both IL-4 and IFNα MoDC were capable of cross-presenting the MART-1aa26-35 epitope from the MART-1 SLP, cross-presentation by IFNα MoDC was more efficient . These data demonstrate a clear trend toward a more efficient cross-presenting capacity of IFNα MoDC for soluble protein, in line with previous reports [We next determined the capacity of MoDC to cross-present a 25-mer MART-1 SLP (aa16-40L) and MART-1 from blebs. To this end, immature HLA-A2 reports , 14.Fig.aa26-35-specific CD8+ T cells. MoDC were loaded exogenously for 2 h with the 9-mer MART-1aa26-35L peptide, after which CD8+ T cells were primed and weekly restimulated with peptide-loaded IFNα and IL-4 MoDC, to analyze antigen-specific priming capacity in an autologous setting. From six healthy donors , we observed MART-1-specific CD8+ T cell expansion by specific HLA-A2 dextramer staining in 26/36 wells (72 %) and 21/36 wells (58 %) for peptide-loaded IL-4 MoDC versus IFNα MoDC, respectively  was higher in the IFNα MoDC versus IL-4 MoDC co-cultures  and the mean percentage of MART-1-specific dextramer-positive CD8+ T cells  was higher when CD8+ T cells were primed with IL-4 MoDC as compared to IFNα MoDC. However, CD8+ T cells primed with either MoDC type recognized their cognate antigen with a similar avidity . The differences in T cell proliferation by IL-4 MoDC could be a result of the more mature phenotype, in particular the increased expression levels of HLA class II, CD80, and CD83. Since we did not observe striking differences in production of cytokines that we tested  by matured MoDC, this is not likely to result in altered T cell proliferation, although we did observe more robust production of TNFα by IFNα MoDC after maturation.Our data clearly show that IL-4 MoDC are superior in taking up exogenous antigen, as the uptake of apoptotic blebs, pinocytosis, and receptor-mediated endocytosis were significantly increased, as compared to IFNα MoDC. This difference could not be related to differential expression of receptors previously reported to be involved in the uptake of apoptotic fragments, e.g., LOX-1, which we were unable to detect. The observed lack of LOX-1 expression on IFNα (as well as IL-4) MoDC as compared to earlier studies might be explained by the IFN α-subunit and/or source used for differentiation. Whether this also explains the observation that IL-4 MoDC were more potent in ingestion apoptotic blebs remains unclear, since this difference might also be explained by the used antigen source: apoptotic blebs instead of apoptotic cells. Additionally, the modalities used to induce MoDC maturation differ between the conducted studies and ours, which can result in functional differences. It will therefore be of interest to study the functional consequences of different activating stimuli on both types of MoDC in more detail. Moreover, since two types of DC are compared, some of the observed effects do not necessarily reflect a difference in IFNα MoDC, but could also reflect differences in the IL-4 MoDC. These differences in uptake did, however, not account for the observed higher priming efficiency of allogeneic CD4aa26-35 epitope derived from a 25-mer MART-1 SLP, similar to what was described previously for OVA [+ T cells when priming with MART-1aa26-35L peptide-loaded IFNα MoDC, the priming efficiency between IL-4 and IFNα MoDC was similar. However, the avidity of CD8+ T cells that were primed with peptide-loaded IL-4 MoDC was higher compared to that of CD8+ T cells primed with IFNα MoDC . The underlying mechanism leading to the higher avidity remains unclear, but it could be the result of the more mature phenotype of IL-4 MoDC, and possibly traces of the beneficial cytokines  as obseIn this study, we used IFNα as a differentiation-inducing cytokine, but it will be of interest to analyze the effect of adding IFNα in the maturation cytokine cocktail of IL-4 MoDC, on their cross-priming ability, since it has been shown that this has beneficial effects on CCR7 expression and T cell responses .+ T cell responses. However, the choice of either MoDC type as vaccination vehicle depends on the antigen source used. SLP-based therapies could benefit from using IFNα MoDC, whereas IL-4 MoDC appear better suited for cross-priming of CD8+ T cells to epitopes derived from apoptotic blebs, most likely due to their superior bleb uptake.We show that both IFNα and IL-4 MoDC are efficient primers of CD8


We report the syntheses and characterization of three solution-processable phenoxy silicon phthalocyanines (SiPcs). The π–π interactions between the aromatic SiPc cores were studied. In all three cases, the solubility of the molecules was increased by the addition of phenoxy groups while maintaining π–π interactions between the aromatic SiPc groups. 2-SiPc], C46H30N8O2Si, bis(2-sec-butylphenoxy)silicon [(2secBP)2-SiPc], C44H24I2N8O2Si, and bis(3-iodophenoxy)silicon [(3IP)2-SiPc], C52H42N8O2Si. Crystals grown of these compounds were characterized by single-crystal X-ray diffraction and the π–π interactions between the aromatic SiPc cores were studied. It was determined that (3MP)2-SiPc has similar interactions to previously reported bissilicon phthalocyanines [(345 F)2-SiPc] with significant π–π interactions between the SiPc groups. (3IP)2-SiPc and (2secBP)2-SiPc both experienced a parallel stacking of two of the peripheral aromatic groups. In all three cases, the solubility of these molecules was increased by the addition of phenoxy groups while maintaining π–π interactions between the aromatic SiPc groups. The solubility of (2secBP)2-SiPc was significantly higher than other bis-phenoxy-SiPcs and this was exemplified by the higher observed disorder within the crystal structure.We report the syntheses and characterization of three solution-processable phenoxy silicon phthalocyanines (SiPcs), namely bis(3-methylphenoxy)silicon [(3MP) The majority of these prototypes depend on fullerenes as acceptor molecules that are problematic due to their high manufacturing cost, low photovoltage generation and poor photochemical stability  are composed of a nitrogen-linked tetrameric diiminoisoindoline conjugated macrocycle that chelate a metal or metalloid through two covalent bonds and two coordination bonds 2-SiPc] as ternary additives in bulk heterojunction (BHJ) OPV devices - SiPc [(PDP)2-SiPc] were found to have very few π–π interactions between the aromatic SiPc core due to the large alkyl substituents silicon phthalocyanine [(2secBP)2-SiPc] and bis(3-iodophenoxy)silicon phthalocyanine [(3IP)2-SiPc] 2-SiPc 2-SiPc and (2secBP)2-SiPc were grown by slow diffusion of heptane into THF and were characterized by single crystal X-ray diffraction. (3MP)2-SiPc was also grown by slow diffusion of pentane into benzene and evaporation form chloroform, resulting in identical crystals as identified by X-ray crystallography. Fig. 22-SiPc, roughly 1.5 mm in size, grown by slow evaporation.Single crystals of (3MP)secBP)2-SiPc in the solid state 2-SiPc and other known bis-phenoxy-SiPc structures 2-SiPc and in contrast to the low disorder observed for the also highly soluble (3HS)2-SiPc)  normd and (b) shape index in Fig. 32-SiPc, Fig. 42-SiPc and Fig. 5secBP)2-SiPc. In all three figures, the regions shaded in red correspond to the contacts at distances shorter than the sum of the van der Waals radii while the white to blue are for the distances longer than the sum of the van der Waals radii. In each crystal, the close contacts  are readily identified on these maps and in all three cases they are different. For example for (3MP)2-SiPc  of the 3-methyl group on the phenoxy group experiences a contact of a distance of 2.341 Å . These observations are not consistent with our previous observations for various halo-phenoxy-BsubPcs such as 3-iodo-phenoxy BsubPc  is based on the two local principal curvatures of the HS, with concave regions shaded in red and convex regions shaded in blue . Unfortunately, similarly to previously reported carbazole derivatives  analysis 2-SiPc crystals which is very similar to the packing of previously reported bis SiPc [(345F)2-SiPc; Lessard, Grant et al., 20152-SiPc and (345FP)2-SiPc were determined to be 3.655 and 3.580 Å, respectively. In addition, the (3MP)2-SiPc exhibits a slip angle of 22.33/22.53° with a slight offset of 0.21° between the aromatic planes while (345F)2-SiPc has a less significant slip angle of 18.90° and exactly parallel (0° between planes) interacting isoindoline groups .Being interested in the stacking between aromatic macrocycles, we previously established 2-SiPc, which represents significant increases in π–π interaction between SiPc groups compared to the starting Cl2-SiPc molecule. (3IP)2-SiPc and (2secBP)2-SiPc on the other hand exhibit a parallel stacking of two of the peripheral aromatic groups. Of the SiPcs similar to (35F)2-SiPc and (246F)2-SiPc 2-SiPc 2-SiPc 2-SiPc has a more significant slip angle of 30.08° and completely parallel (0° between planes) and interacting aromatic groups 2-SiPc has a unique two-dimensional stacking where two peripheral aromatic groups will stack with an adjacent SiPc molecule and one of the same peripheral aromatic groups along with a third one will stack in a similar fashion but at 90° from the first interaction 2-SiPcs  which space out the molecules and increase the size of the unit cell.These results indicate that (3MP)Pc Fig. 6. (3IP)2-ps Fig. 6.  2-sec-butylphenol (98%) and 3-iodophenol (98%) were obtained from Sigma–Aldrich and chlorobenzene (99.5%) and chloroform  were obtained from Caledon Laboratories Ltd. All chemicals were used as received unless otherwise specified. Dichloro silicon phthalocyanine (Cl2-SiPc) was synthesized according to the literature 2-SiPcs and (2secBP)2-SiPcs were performed following the general procedure used to synthesize F10-SiPc· to Cl2-SiPc  in chlorobenzene (100 ml). The mixture was stirred and heated to 388 K overnight and cooled to room temperature. The product was then obtained by precipitation into isopropanol and filtered. The product was then dried in a vacuum oven overnight. Yield: 1.3g (80.2 mol%). DART Mass spectroscopy: calculated mass: 755.234, obtained mass: 755.236. (3IP)2-SiPcs and (2secBP)2-SiPcs were synthesized under similar conditions and crystals were again obtained by slow diffusion of heptane into a THF solution.The synthesis of (3MP)Uiso(H) = 1.2Ueq(C) or 1.5Ueq(Cmethyl).Crystal data collection and structure refinement details are summarized in Table 32-Si there appears to be pseudosymmetry with an approximate centre of symmetry. The c-glide reflections are weak but present and the P21/c structure refines only to ca R1 = 10% compared to 4.4% for the P21 structure. The crystal is an inversion twin with a ratio of components of 0.51 (4):0.49 (4).In (3MP)secBP)2-SiPc, electron density peaks were located that were believed to be highly disordered solvent molecules (possibly pentane/dichloromethane). Attempts made to model the solvent molecule were not successful. The SQUEEZE option  to the electron density was removed from the observed data. The density, the F(000) value, the molecular weight and the formula are given without taking into account the results obtained with SQUEEZE. Similar treatments of disordered solvent molecules were carried out by Stähler et al. 2-SiPc was a non-merehedral twin with a twin law determined by CELL_NOW  3MP2-SiPc, 3IP2-SiPc, 2secBP2-SiPc. DOI: 10.1107/S205698901600935X/hb75813MP2-SiPcsup3.hklStructure factors: contains datablock(s) 3MP2-SiPc. DOI: 10.1107/S205698901600935X/hb75813IP2-SiPcsup2.hklStructure factors: contains datablock(s) 3IP2-SiPc. DOI: 10.1107/S205698901600935X/hb75812secBP2-SiPcsup4.hklStructure factors: contains datablock(s) 2secBP2-SiPc. DOI: 10.1107/S205698901600935X/hb7581sup5.pdfSupporting information file. DOI: Click here for additional data file.10.1107/S205698901600935X/hb7581sup6.tifSupporting information file. DOI: Click here for additional data file.10.1107/S205698901600935X/hb7581sup7.tifSupporting information file. DOI: 1484189, 1484188, 1484187CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


S(7) graph-set motif. In the crystal, an intermolecular O—H⋯O hydrogen bond connects molecules into a dimer. The dimers are further linked into a sheet structure.In the title compound, intramolecular hydrogen bonding between the hydroxy and amino groups forms an  10H14Cl3NO5, the five-membered dioxolane ring adopts an envelope conformation. The C atom at the flap, which is bonded to the hydroxymethyl substituent, deviates from the mean plane of other ring atoms by 0.357 (5) Å. There are two intramolecular hydrogen bonds (O—H⋯N and N—H⋯O) between the hydroxy and amino groups, so that O- and N-bound H atoms involved in these hydrogen bonds are each disordered with equal occupancies of 0.50. The methyl 2-methylprop-2-enoate substituent also shows a disordered structure over two sets of sites with refined occupancies of 0.482 (5) and 0.518 (5). In the crystal, molecules are connected into a dimer by an O—H⋯O hydrogen bond. The dimers are further linked by N—H⋯O, C—H⋯N and C—H⋯O interactions, extending a sheet structure parallel to (In the title compound, C In the case of a diol, a cyclic orthoamide  may be afforded by controlling the reaction conditions though bis-imidates are usually produced. We have explored the rearrangement of the cyclic orthoamide prepared from a contiguous diol or triol, and have developed a novel strategy for the total synthesis of certain natural products  = 0.223 (3) Å and φ(2) = 111.7 (8)°. Atom C9 deviates from the mean plane of the other four atoms by 0.357 (5) Å. The hydroxy H atom has two possible positions and the amino H atoms have three possible positions, generating two types of intramolecular hydrogen bonds with an S(7) graph-set motif between the hydroxy and amino groups, N6—H6A⋯O12 and O12—H12B⋯N6  is disordered over two orientations with refined occupancies of 0.482 (5) and 0.518 (5).The molecular structure of the title compound is shown in Fig. 16 Table 1. The occA⋯O12i; Table 1C⋯O17Bii and N6—H6B⋯O17Aiv, C19A—H19C⋯N6iii and C19B—H19E⋯O12v; Table 1The crystal packing is stabilized by O—H⋯O hydrogen bonding (O12—H12a), are registered , 2-oxo, (c) and 2-alkoxy-2-alkyl (orthoester) derivative, (d), but its 2-amino-2-trichloromethyl derivative, (e), which is related to the title compound, (h), has not been reported.In the Cambridge Structural Database , gave 12 entries. These include two structures . N-bound hydrogen atoms in the structure of LIBHIO were refined as having an sp3 configuration and tilted towards chlorine atoms, whereas those in other 11 structures were refined assuming an sp2 configuration of the N atom.On the other hand, a search in CSD for a 2,2,2-trichloroethan-1-amine skeleton, .The title compound was derived from A and B with their geometries restrained, and the refined occupancies being 0.482 (5) and 0.518 (5), respectively.Crystal data, data collection and structure refinement details are summarized in Table 2Uiso(H) = 1.2Ueq(C) or 1.5Ueq(methyl C). The O-bound hydrogen atom has two possible positions. They were refined isotropically with Uiso(H) = 1.5Ueq(O) and the O—H distances restrained. The N-bound hydrogen atoms have three possible positions. They were refined isotropically with Uiso(H) = 1.2Ueq(N) and the N—H and H⋯H distances restrained. The site-occupation factors of the disordered H atoms of the hydroxy group (H12A and H12B) and one of the amino group (H6A) were uniquely assigned to 0.5 each based on the two possible patterns of the hydrogen-bonding linkages related by the twofold axis. The occupation factors of the other N-bound H6B and H6C atoms were assumed to be 0.75 each from a difference map.C-bound H atoms were positioned geometrically with C—H = 0.95–1.00 Å, and constrained to ride on their parent atoms with 10.1107/S2056989016002474/is5442sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989016002474/is5442Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016002474/is5442Isup3.cmlSupporting information file. DOI: 1452471CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


We found that miR-181a/b-1-deficiency had no effect on numbers of thymic γδ T cell or on their differentiation towards an IL-17- or IFN-γ-producing effector phenotype. Also, the composition of peripheral lymph node γδ T cells was not affected by miR-181a/b-1-deficiency. Dendritic epidermal γδ T cells were normally present in knock-out animals. However, we observed elevated frequencies and numbers of γδ NKT cells in the liver, possibly because γδ NKT cells can expand and replace missing αβ NKT cells in peripheral niches. In summary, we investigated the role of miR-181a/b-1 for selection, intrathymic development and homeostasis of γδ T cells. We conclude that miR-181a/b-1-dependent modulation of T cell selection is not critically required for innate development of γδ NKT cells or of any other γδ T cell subtypes.Thymic development of αβ T lymphocytes into invariant natural killer (NK) T cells depends on their selection via agonistic lipid antigen presented by CD1d. If successful, newly selected NKT cells gain effector functions already in the thymus. Some γδ T cell subsets also acquire effector functions in the thymus. However, it is not clear whether agonistic TCR stimulation is involved in thymic γδ T cell selection and development. Here we combine two genetic models to address this question. MiR-181a/b-1 Strong evolutionary conservation of γδ T cells in all jawed vertebrates suggests that these cells are essential for immune homeostasis and host competence against infections . In cont (DETCs) , 17 and  T cells  develop  T cells , 20. Int T cells . To datepidermis , 23. Fur T cells . There, pression –26. In celopment , 28, 29.elopment , 31 and elopment , 33.+CD8+ DP stage [Tcrd-H2BeGFP reporter mice [–/–deficient strain [We and others recently reported that the miR-181a/b-1 cluster is highly expressed during thymocyte development and positively regulates TCR signal strength , 34–36. DP stage . AccordiDP stage , 38, 39.t strain . Althougtm1Mal/J;[–/–(C57BL/6-Mirc14tm1.1Ankr) were already described. They were crossed to obtain miR-181a/b-1–/–x TcrdH2BeGFP mice. F1 C57BL/6 wild type mice  were already described [TcrdH2BeGFP  and miRescribed . Mice weAll experiments were conducted according to local and institutional guidelines approved by Lower Saxony State Office for Consumer Protection and Food Safety, file references 11/0533 and 12/0869.+ γδ T cells, was kindly provided by Bob Tigelaar, Yale. In the presence of unlabeled TCRγδ (GL3) antibody, 17D1 antibodies also recognize Vγ6+ γδ T cells in addition to Vγ5+ γδ T cells [hiCD117hiCD25–; DN2, CD44hiCD117hiCD25+; DN3, CD44–CD117–CD25+; DP, CD4+CD8+; γδ T cells, CD25–GFP+. Data were analyzed using FlowJo software 9.3.1 (Treestar).Antibodies directed against Vγ1 , IFN-γ , CD27 , anti-CD117 , anti-CD25 , anti-CD45.1  and Tcrβ  were purchased from Biolegend. Anti-CD45.2  was purchased from Miltenyi Biotec. Anti-CD24 , CCR6  and Vδ6.3/2  were from BD Biosciences. Antibodies directed against IgM , CD44 , NK1.1 , Tcrβ  and IL17A  were purchased from eBioscience. Anti-Vγ4 , subsequently labeled with Cy5, was kindly provided by Pablo Pereira, Paris. 17D1 antibody (rat IgM), here used for staining of Vγ6 T cells . Binding–CD8– thymocytes were separated by density gradient using Lympholyte M (Cedarlane laboratories). For the isolation of liver lymphocytes the organs were perfused to remove residual blood. The whole organs were cut into pieces and digested with 0.5mg/ml Collagenase D and 0.025mg/ml DNAseI. Digest was stopped by adding EDTA to a final concentration of 20mM. Digested organs were meshed through a 40μm Cellstrainer (Fisher Scientific). For isolation of lymphocytes from skin, mice were killed and backs of mice were shaved and subsequently treated with depilatory cream (Veet) to remove residual fur. Skin was cut into small pieces and incubated in RPMI supplemented with 1mg/ml Liberase (Roche) and 0,125mg/ml DNAseI for 60min at 37°C, shaking. During the last 15min, EDTA was added to a final concentration of 60mM. Digested skin was meshed through a 100μM Cellstrainer (Fisher Scientific). Liver and skin lymphocytes were separated by density gradient centrifugation using Percoll-gradients.Thymocytes were prepared by complement lyses  with anti-CD8 IgM (RL1.72), anti-CD4 IgM (M31) and 300μg DNAseI (Roche). CD4+CD3+.Mouse ears were shaved and dorsal and ventral halves were separated. Both were incubated in 0.5 M NH4SCN at 37°C for 20 min. Epidermal layers were peeled off and were fixed in 4% PFA at RT for 15 min, then rehydrated in TBS-T or PBS for 20 min at RT. Blocking was done with 8% rat serum for 30min at RT. The epidermal sheets were stained with CD3-Cy3 (clone 17A2) at RT for 2 hours and analyzed with an Olympus BX641 microscope. DETCs were identified as GFPRNA preparation and quantitative PCR was performed as described  using th–/– and C57BL/6 wild type  mice. Recipient C57BL/6 wild type (CD45.1) were lethally irradiated (9Gy) and reconstituted intravenously with 10x106 bone marrow cells in a 1:1 mixture of KO (CD45.2) and WT (CD45.1/CD45.2) cells within 4 hours after irradiation. Recipient mice were treated with Cotrimoxazol during the initial two weeks. Analysis was performed after 7 weeks.Bone marrow was isolated from miR-181a/b-1Statistical analyses were performed with GraphPad Prism (Version 4.03) using MannWhitney test. P-values <0.05 (*) were considered as significantly different.So far, the role of miR-181 family members in development and selection of γδ T cells was not investigated in detail. The miR-181a/b-1 cluster is differentially expressed across all DN immature stages of thymocyte development. It peaks with the highest copy number per cell and the highest relative expression of all miRNAs in DP thymocytes . However–/–mice the development of αβ NKT cells and most prominently that of CD1d-restricted semi-invariant so-called iNKT cells, is disturbed as indicated by massively reduced numbers of αβ KT cells [+ and Vγ4+ γδ T cells was lower in miR-181a/b-1–/–mice, whereas the frequency of Vγ6+ γδ T cells was increased compared to miR-181a/b-1-sufficient controls . Analysis of mixed bone marrow chimeras indicated that miR-181a/b-1–/– γδ NKT cells had no cell intrinsic advantage compared to miR-181a/b-1 sufficient γδ NKT cells  TCR antigens, it will be interesting to identify the factors that define the homeostasis of αβ NKT and γδ NKT cells, but not of NK cells or CD8+ αβ T cells, in the liver. Also, given the myriad functions of miR-181a/b-1 in T cell development, our study does not formally exclude a cell intrinsic function of miR-181a/b-1 in γδ NKT cell development and selection.On the other hand, we found no impairment of two potentially agonist-selected γδ T cell populations, namely DETCs and γδ NKT. For the former, our conclusions are based on microscopic analysis of adult epidermis. However, a thorough analysis of their early thymic ontogeny would be required to make a statement that DETC development and selection are not at all affected by the presence or absence of miR-181a/b-1. For the latter, our data underline that αβ NKT cells and γδ NKT cells share many but not all features. It was previously reported that αβ NKT cells and γδ NKT cells share some developmental requirements and home to similar anatomical sites . While bIn conclusion, this study investigated the impact of miR-181a/b-1 on γδ T cell development. We found that miR-181a/b-1-deficiency alters the γδ TCR repertoire to some degree, whereas tuning of TCR sensitivity by the miR-181a/b-1-cluster is not critically required for the development and expansion of γδ NKT cells.


Pna21 and is a polymorph of the same stoichiometric species, reported in Cc.A 5:1 mixture of 4-hydroxypyridine with benzene 1,3,5-tricarboxylic acid in methanol yields the title hydrogen-bonded framework compound. This compound crystallizes in the orthorhombic space group  9H6O6·3C5H5NO. This structure represents an orthorhombic polymorph of the previously reported C-centered, monoclinic structure .Slow co-crystallization of a solution of benzene-1,3,5-tricarboxylic acid with a large excess of 4-hydroxypyridine produces an interpenetrating, three-dimensional, hydrogen-bonded framework consisting of three 4-pyridone and one benzene-1,3,5-tricarboxylic acid molecules, C al. 2014. Acta Cr These are comparable with those for the previously reported polymorph of this compound . N3 forms hydrogen bonds to O3iii and O6iv related by translation along the crystallographic c-axis and the .Each of the pyridone molecules forms a hydrogen-bonded chain of symmetry-related molecules. N1 and N2 form hydrogen bonds to O1en Fig. 1. These Oks Fig. 2. The BTCet al., 2014Cc.A search in the Cambridge Structural Database  in methanol (3 mL) and benzene 1,3,5-tricarboxylic acid  in methanol (3 mL). The two solutions were combined and allowed to evaporate over 5 d yielding crystals suitable for diffraction studies. The crystallization process yields crystals of both the previously reported 1:1 co-crystal  was set to 1.2Ueq(C/N) and 1.5Ueq(O). The reliability for the correct enantiomorph of the space group is low, due to the use of Mo Kα radiation with a light atom structure. Analysis of the Flack x [0.1 (10); Flack, 1983y [0.2 (10); Hooft et al., 2008z [−0.2 (12); Parsons et al., 2013Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015017867/zl2644sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989015017867/zl2644Isup2.hklStructure factors: contains datablock(s) I. DOI: 1427116CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Leptaulus citroides Baill. (Cardiopteridaceae) led to the isolation of ethyl esters of three new triterpenoid saponins (1–3) and the known sesquiterpenoid cinnamosmolide (4). The structures of 1–3 were elucidated by extensive 1D and 2D NMR experiments and mass spectrometry. Compounds 1, 2, and 4 showed moderate cytotoxicity against the A2780 human ovarian cancer cell line with IC50 values of 2.8, 10.2 and 2.0 µM, respectively.Bioassay-guided fractionation of EtOH extracts obtained from the roots and wood of the Madagascan plant The online version of this article (doi:10.1007/s13659-015-0083-1) contains supplementary material, which is available to authorized users. Leptaulus citroides (Cardiopteridaceae) for investigation of antiproliferative natural products, since no previous chemical work has been reported on this genus. Bioassay-guided fractionation of the roots and wood extracts yielded ethyl esters of three new triterpenoid saponins (1–3) and the known sesquiterpenoid cinnamosmolide (4) . Her1) was obtained as an amorphous white powder. Its HRESIMS revealed a sodiated quasi-molecular ion peak at m/z 1223.5806 [M + Na]+, corresponding to the molecular formula C59H92O25. Compound 1 was assigned as an olean-12-ene triterpene derivative based on its 1D and 2D NMR spectra. The 1H NMR spectrum of the aglycone part of compound 1 displayed characteristic signals of six singlet methyl groups , an olefin group  and an aldehyde group . The position of the olefin group was confirmed by HMBC correlations of H3-27  to C-13 (δC 144.1), and H-12 to C-11 (δC 24.6)  was located at C-4 based on the HMBC correlation between the aldehyde proton and C-24 (δC 10.8), in addition to comparison of 1D NMR data of 1 with previously reported data of similar compounds , two methyl groups [δH 1.98  and 1.90 ], a carboxyl carbon  and two sp2 carbons , in agreement with the NMR data of related compounds  correlated with carbons at δC 102.7, 102.0, 107.5 and 104.8 were observed in the HSQC spectrum of 1, indicating the presence of four sugar units. The four sugar units were identified as β-glucuronopyranosyl (GlcA-1′-6′), β-galactopyranosyl , β-xylopyranosyl (Xyl-1′′′-5′′′), and β-xylopyranosyl (Xyl-1′′′′-5′′′′), by comparison of their 13C NMR data with those of apodytine B [β-d-xylopyranosyl-(1 → 2)-β-d-xylopyranosyl-(1 → 3)]-β-d-glucopyranosiduronate}--16,28-dihydroxy-22-{[(2Z)-2-methyl-1-oxo-2-buten-1-yl]oxy}-23-oxoolean-12-en-3-yl.The structure of the sugar moiety of ine B + corresponded to a molecular formula of C59H92O25. Further comparison of 1D NMR data of 2 suggested it is an isomer of 1 with the same sugar moiety and a similar aglycone to that of 1. The only difference between 1 and 2 was the position of the angeloyl and hydroxyl groups. The angeloyl group was assigned to C-16 in 2, as indicated by the deshielded signal of H-16 , and the hydroxyl group was assigned to C-22 due to the more shielded signal of H-22  compared to that of compound 1 [-β-d-xylopyranosyl-(1 → 2)-β-d-xylopyranosyl-(1 → 3)]-β-d-glucopyranosiduronate}--22,28-dihydroxy-16-{[(2Z)-2-methyl-1-oxo-2-buten-1-yl]oxy}-23-oxoolean-12-en-3-yl  was obtained as an amorphous white powder. The quasi-molecular ion peak at m/z 1301.5982 [M + Na]+ corresponded to a molecular formula of C64H94O26. Comparison of 1H and 13C NMR data with those of 2 indicated that they were closely related except that signals of the angeloyl group were replaced by those of a p-methoxycinnamoyl group in the spectra of 3. Thus the 1H NMR spectrum of 3 showed signals of a para-substituted benzene ring at δH 7.54  and 6.98 , two olefinic protons with E- configuration δH 7.75  and δH 6.35  and a methoxy group δH 3.84 . These signals suggested the occurrence of a 4-methoxycinnamoyl group, and this was confirmed by HMBC correlations between the methoxy group  with C7, and H-acyl-3  with C-acyl-4 (δC 128.2). Comparisons of chemical shifts and coupling constants of H-16 and H-22 in 2 and 3 suggested the 4-methoxycinnamoyl group was attached to C-16 [3) was determined as 3-O-{ethyl [β-d-galactopyranosyl-(1 → 2)][-β-d-xylopyranosyl-(1 → 2)-β-d-xylopyranosyl-(1 → 3)]-β-d-glucopyranosiduronate}--22,28-dihydroxy-16-[(E-4-methoxycinnamoyl)oxy]-23-oxoolean-12-en-3-yl  has also been reported to display antifungal and α-glucosidase inhibitory activities. +; C59H92O25Na+, .White powder; [α].0 c = 0.9, MeOH; α]D23 -7.7 ; UV (MeOH) λmax (log ε) 209 (6.11) nm; IR (film) νmax 3378, 1773, 1613, 1521, 1078 cm−1. 1H- and 13C-NMR: see Table m/z 1223.5781 [M + Na]+, C59H92O25Na+; .White powder; +, C64H94O26Na+; .White Powder; [α].6 c = 0.2, MeOH; o-tolylthiocarbamoyl)-thiazolidine-4(R)-carboxylates were prepared from d- and l-galactose, d- and l-glucose, d- and l-xylose, d- and l-galactose, d- and l-galactose, and d-glucuronic acid by reaction with l-cysteine methyl ester and o-tolylisothiocyanate as described [l-glucuronic acid was not available, the enantiomeric (o-tolylthiocarbamoyl)-thiazolidine-4(S)-carboxylate of d-glucuronic acid was prepared by reaction with d-cysteine methyl ester and o-tolylisothiocyanate. Compound 1 (2.0 mg) was treated with 3 M HCl for 4 h at 100 °C, and the solution was then neutralized with sodium bicarbonate and extracted thrice with EtOAc. The aqueous fraction was evaporated to dryness under reduced pressure. The resulting mixture of carbohydrates (0.7 mg) was then dissolved in 0.5 mL pyridine, 0.9 mg of l-cysteine methyl ester was added, and the mixture was heated at 60 °C for 1 h. o-Tolylisothiocyanate (0.9 mg) was then added to the mixture, which was again heated at 60 °C for 1 h. The reaction mixture was directly analyzed by reverse-phase HPLC on a Phenomenex Luna column , eluted with isocratic 0.1 % formic acid in CH3CN/H2O (15:85) at a flow rate of 2.5 mL/min for 5 min, followed by 0.1 % formic acid in CH3CN/H2O (25:75) for 30 min, and a wash with 100 % CH3CN for 10 min. The resulting chromatogram contained three major peaks with retention times of 25.29, 29.12, and 31.25 min, identical to those of the derivatives of d-xylose, d-galactose and d-glucuronic acid. Co-injection of each of the derivatives obtained by hydrolysis of 1 with its corresponding synthetic counterpart confirmed the identity of the compounds. The peak corresponding to the d-xylose derivative was approximately twice as large as that for the d-galactose derivative, consistent with the presence of two d-xylose units in 1.Authentic methyl 2--3-


Coumarins are a major group of plant secondary metabolites that serves as defense compounds against pathogens. Although coumarins can be obtained from diverse plant sources, the use of microorganisms to synthesize them could be an alternative way to supply building blocks for the synthesis of diverse coumarin derivatives.F6′H (encoding feruloyl CoA 6′ hydroxylase) and 4CL (encoding 4-coumarate CoA:ligase), were manipulated to increase the productivity of coumarins. Escherichia coli expressing the two genes was cultured in medium supplemented with hydroxycinnamic acids (HCs) including p-coumaric acid, caffeic acid, and ferulic acid, resulting in the synthesis of the corresponding coumarins, umbelliferone, esculetin, and scopoletin. Cell concentration and initial substrate feeding concentration were optimized. In addition, umbelliferone, and esculetin were synthesized from glucose by using a ybgC deletion mutant and co-expressing tyrosine ammonia lyase and other genes involved in the tyrosine biosynthesis pathway.Constructs harboring two genes, E. coli, several constructs containing F6′H and 4CL were made, and their ability to synthesize coumarin derivatives was tested. The solubility of F6′H was critical for the final yield. After optimization, 82.9 mg/L of umbelliferone, 79.5 mg/L of scopoletin, and 52.3 mg/L of esculetin were biosynthesized from the corresponding HCs, respectively in E. coli. Umbelliferone and esculetin were also synthesized from glucose using engineered E. coli strains. The final yields of umbelliferone and esculetin were 66.1 and 61.4 mg/L, respectively.To produce coumarin derivatives  in  The second group is hydroxycinnamic acids , which include p-coumaric acid, caffeic acid, and coumarin. The third group, the stilbenes, has a C6-C2-C6 skeleton, and includes resveratrol, piceatannol, and pallidol. The last group includes the flavonoids, which have a C6-C3-C6 skeleton, and includes quercetin, genistein, and apigenin. These plant phenolics are all synthesized from cinnamic acid derived from phenylalanine through the action of phenylalanine ammonia lyase (PAL). Malonyl-CoA supplies carbon to cinnamonyl-CoA to make the stilbenes and the flavonoids ) is a key step for the biosynthesis of coumarin  and IbF6′H2 [GenBank ID: AB636154]) were cloned using reverse transcription-polymerase chain reaction (RT-PCR). Total RNA from sweet potato tuber tissue was isolated using Plant Total RNA Isolation Kit , and cDNA was synthesized with Omniscript reverse transcript (Qiagen). The forward primers of IbF6′H1 and IbF6′H2 were 5′-ATGGCTCCAACACTCTTGAC-3′ and 5′-ATGAATCAAACACTCGCTGC-3′, respectively. The reverse primers were 5′-TCAGATCTTGGCGTAATCGA-3′ and 5′-TCAAATGTTGGCAAAATCGA-3′. The resulting PCR product of each gene was subcloned into pGEM-T easy vector  and sequenced. Each gene was then reamplified with the forward primer containing an EcoRI site, and the reverse primers containing a NotI site. The PCR product was subcloned into the EcoRI/NotI sites of pET-Duet (Novagen). Os4CL, which was cloned previously [CATATGGGGTCGGTGGCGGCGGAGGAGG-3′ and 5′-ATCTCGAGTTAGCTGCTTTTGGGCGCATC-3′ , and subcloned into the NdeI/XhoI sites of pET-Duet1 containing IbF6′H1 or IbF6′H2. Each gene in these constructs was controlled by an independent T7 promoter. The plasmids were called pE-pIbF6′H1-pOs4CL or pE-pIbF6′H2-pOs4CL  and a NotI site , and the reverse primer containing a XhoI site . The resulting PCR product was subcloned into the NotI/XhoI sites of pET-Duet1 containing IbF6′H1 or IbF6′H2. The resulting constructs, pE-pIbF6′H1-Os4CL and pE-pIbF6′H2-Os4CL contained a single promoter but an RBS site in front of each gene. pE-pIbF6′H1-Os4CL, and pE-pIbF6′H2-Os4CL were digested with EcoRI and XhoI, and the fragment containing IbF6′H1 and Os4CL or IbF6′H2 and Os4CL was subcloned into the EcoRI/XhoI sites of pGEX 5X-3 . The resulting constructs were named pG-pIbF6′H1-Os4CL, and pG-pIbF6′H2-Os4CL, respectively. To make the construct with two promoters and F6′H fused with GST, PCR was carried out using pE-pIbF6′H1-Os4CL, or pE-pIbF6′H2-Os4CL as a template using Pfu DNA polymerase with the primers, 5′-ATGAATTCGATGCCTTCAACAACACTCTCC-3′ for IbF6′H1  or 5′-ATGAATTCGATGATGCCTTCAACAACACTC-3′ for IbF6′H2  and 5′-TTAGCTGCTTTTGGGCGCATC-3′ for Os4CL. The resulting PCR product was digested with EcoRI and subcloned into the EcoRI/SmaI sites of pGEX 5X-3. The resulting constructs were pG-pIbF6′H1-pOs4CL and pG-pIbF6′H2-pOs4CL, respectively.eviously . p-Coumaeviously , was ampTAL gene from Saccharothrix espanaensis (SeTAL), aroG, tyrA, and the feedback-free versions of aroG (aroGfbr) and tyrA (tyrAfbr) were cloned previously [fbr-SeTAL-tyrAfbr construct, aroGfbr and tyrAfbr were introduced into the EcoRI/SalI and the NdeI/KpnI sites of pACYCDuet, respectively, and was named pA-aroGfbr-tyrAfbr. The SeTAL gene was cloned into the EcoRI/NotI sites of pACYDUet and named pA-SeTAL. SeTAL containing the T7 promoter and RBS was amplified with two primers flanking XhoI and NotI using pA-SeTAL as a template. The PCR product was digested with XhoI/NotI and ligated into the corresponding sites of pA-aroGfbr-tyrAfbr. The resulting construct was named pA-aroGfbr-SeTAL-tyrAfbr. pA-aroG-SeTAL-tyrA was constructed using the same method described above.The eviously . For theE. coli transformants containing pG-IbF6′H1-Os4CL or pG-IbF6′H2-Os4CL were grown in LB containing 50 μg/mL ampicillin for 16 h at 37°C. This culture was inoculated into fresh LB containing 50 μg/mL ampicillin and grown to an OD600 = 0.8. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM and the culture was grown for another 6 h at 25°C. Cells were harvested, and the cell concentration was adjusted to OD600 = 3 with 10 mL of fresh LB containing 50 μg/mL ampicillin in 100 mL flask. The substrate  was added at a concentration of 400 μM. The resulting culture was incubated at 30°C for 12 h with shaking. The effect of medium on the production of scopoletin was examined using LB, M9 (containing 2% glucose), or YM9 (M9 containing 2% glucose and 0.2% yeast extract). We used 10 mL of each medium in 100 mL flask.600 = 3. The mixture was incubated at 30°C for 12 h with shaking at 180 rpm. For the optimal cell density, cell density was adjusted to OD600 = 1, 2, 3, 5, or 10. Substrate was added at 0.7 mM, and the mixture was incubated at 30°C for 12 h with shaking at 180 rpm. The reaction scale was used as described above.To determine the substrate concentration to produce the highest yield of coumarin derivatives, substrate was added at 0.3, 0.4, 0.5, 0.7, 0.9, 1.2, or 1.5 mM. The cell density was ODThe culture (200 μL) was extracted twice with the equal volume of ethylacetate. The organic phase was recovered and evaporated to dryness. The remaining residue was dissolved in 60 μL of dimethylsulfoxide (DMSO) and analyzed using a Thermo Ultimate 3000 HPLC equipped with a photo diode array (PDA) detector and a Varian C18 reversed-phase column  by injecting 10 μL. The mobile phases consisted of 0.1% formic acid in water or acetonitrile. The program was: 15% acetonitrile at 0 min, 35% acetonitrile at 10 min, 90% acetonitrile at 12 min, 90% acetonitrile at 15 min, 15% acetonitrile at 15.1 min, and 15% acetonitrile at 20 min. The flow rate was 1 mL/min, and the separation was monitored at 290, 310, and 340 nm.Umbelliferone, scopoletin, and esculetin were purchased from Sigma  and used as standards to calculate yields of umbelliferone, scopoletin, or esculetin. The means and standard errors were calculated from triplicate experiments. Analysis of variance (ANOVA) was carried out using Tukey’s method with a significance level of P = 0.01 using 2010 Microsoft Office Excel.1H NMR  δ 7.87 , 7.52 , 6.85 , 6.75 , 6.17 .Mass spectrometry (MS) was performed as described previously . Structu1H NMR  δ 7.52 , 6.90 , 6.84 , 6.52 , 3.88 .Scopoletin: 1H NMR  δ 7.79 , 7.05 , 6.79 , 6.15 .Esculetin: 600 exceeded 1.0. The cells were collected by centrifugation. The cell density was adjusted to an OD600 of 1.0 with 10 mL of M9 medium supplemented with 1% yeast extract, 2% glucose, 50 μg/mL of ampicillin and chloramphenicol and 1 mM IPTG in 100 mL flask and then incubated at 30°C for 48 h with shaking. The reaction product was analyzed by HPLC.An overnight culture of each strain was inoculated into fresh LB medium containing 50 μg/mL of ampicillin, and chloramphenicol and the cells were grown at 37°C with shaking at 180 rpm until the OD600 = 0.5, 1. 1.5, 2.0, 2.5, or 3.0 with M9 medium containing 2% glucose, 1% yeast extract, 50 μg/mL chloramphenicol and ampicillin, and 1 mM IPTG. Each strain was grown at 30°C for 48 h and production of umbelliferone determined by HPLC. To determine the optimum incubation temperature, the cell concentration of B-CM11 was adjusted to OD600 = 2.5, and cells were then incubated at 20, 25, 30, and 37°C for 48 h with shaking at 180 rpm. The yield of umbelliferone from each culture was determined by HPLC. Esculetin production from glucose using B-CM12 was carried out using similar methods to that of umbelliferone production.To determine the optimal cell concentration for the production of umbelliferone, B-CM11 cells were grown and protein expression was induced as described above. Cells were harvested by centrifugation and cell concentrations were adjusted to OD


GSK-3 siRNA downregulation, or inhibition by small molecules, blocked PD-1 expression, resulting in increased CD8+ cytotoxic T lymphocyte (CTL) function. Mechanistically, GSK-3 inactivation increased Tbx21 transcription, promoting enhanced T-bet expression and subsequent suppression of Pdcd1 (encodes PD-1) transcription in CD8+ CTLs. Injection of GSK-3 inhibitors in mice increased in vivo CD8+ OT-I CTL function and the clearance of murine gamma-herpesvirus 68 and lymphocytic choriomeningitis clone 13 and reversed T cell exhaustion. Our findings identify GSK-3 as a regulator of PD-1 expression and demonstrate the applicability of GSK-3 inhibitors in the modulation of PD-1 in immunotherapy.Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway that regulates PD-1 expression has not been defined. Glycogen synthase kinase 3  is a serine-threonine kinase implicated in cellular processes. Here, we identified GSK-3 as a key upstream kinase that regulated PD-1 expression in CD8  •GSK-3 is a key upstream kinase that contributes to inhibition of PD-1 transcription•GSK-3 siRNAs or inhibitors block PD-1 transcription to thereby enhance CTL function•Tbx21 transcription, which represses PD-1 transcriptionGSK-3 inhibition enhances •Use of GSK-3 inhibitors in vivo downregulates PD-1 and enhances viral clearance The upstream pathway regulating PD-1 expression is not clear. Rudd and colleagues show that inhibition of the serine/threonine kinase GSK-3 upregulates T-bet expression, which decreases PD-1 expression and enhances CTL function. They demonstrate that the use of GSK-3 inhibitors in vivo inhibits PD-1 and enhances T cell clearance of viral infections. PD-1 isMV-Cl13)  and hepaMV-Cl13)  and in mMV-Cl13)  and corrMV-Cl13) . Blockinlearance . Checkpolearance  and in clearance .Two ligands, PD-L1 and PD-L2, have been identified for PD-1 , and PD-lck and ZAP-70 activate T cells  from the nucleus  that control PD-1 expression has been unclear. Here, we have identified GSK-3α and GSK-3β (hereafter referred to as GSK-3 collectively) as a key kinase that upregulated Tbx21 transcription for the downregulation of PD-1 and enhanced CD8+ cytolytic T cell function. We also demonstrated the use of small molecule inhibitors of GSK-3 to downregulate PD-1 for enhanced in vivo immunity involving the clearance of acute and chronic viral infections.Although certain transcription factors have been implicated in 257-264) as presented by H-2kb. T cells express the α and β isoforms of GSK-3 (2 = 0.9603)  A, iv. By2 = 0.9643). Incubation of OT-I T cells with SB415286 increased maximal killing by as much as 5-fold and shifted the killing curve by 10-fold . SB415286 also increased proliferation in response to OVA peptide to the same extent as when cells were cultured with anti-PD-1, as monitored by carboxyfluorescein succinimidyl ester (CFSE) labeling  in the absence and presence of GSK-3 inactivation . PD-1 bl+ T cells also increased cytolytic function (pdcd1 transcription by >85% reduction (siRNA KD of PD-1 expression in CD8function D. PD-1 seduction C and enhPdcd1 transcription (encodes PD-1) in different contexts .We next confirmed that GSK-3 inactivation acted on contexts . Anti-CDcontexts . Two-stecontexts . The preer 72 hr A. GSK-3 VA cells B. In add EL4-OVA C. A titrTbx21 (encodes T-box transcription factor T-bet) concurrent with Pdcd1 inhibition. This was observed in response to anti-CD3 ligation without affecting Tcf7-mediated transcription  into OT-I transgenic mice with and without SB415286 followed by the harvest of spleens and lymph nodes (LNs) at day 7 A or a re+ Murid herpesvirus 68 (MHV-68), an isolate of Murid herpesvirus 4 (MuHV-4)  was also significantly reduced from 6 × 104 to 1 × 104 at day 7 and from 2 × 104 to 4 × 103 at days 7 and 14  , in the (MuHV-4) . The pre(MuHV-4) . We foun(MuHV-4) A and S5B7 and 14 B. Quantipression C. Moreovcapacity D.7 to 8 × 105 ps-1) when assessed on days 3, 5, and 7. SB415286 reduced the maximum radiance to the same degree, and importantly, when used in combination, anti-PD-1 blockade had no further effect than SB415286, and vice versa. Measurement of viral titers at days 7 and 14 showed that anti-PD-1 blockade and SB415286 reduced viral titers to the same extent , GZMB , and IFN-γ  expression G. By con T cells . Cells e T cells I. Overal+ T cells during chronic infection are characterized by prolonged expression of PD-1 in response to the lymphocytic choriomeningitis virus variant LCMV-Cl13  (2 from control mice to 5 × 10−2), as reported in response to anti-PD-1 (33-41+CD8+ T cells (+ T cells (CD11a+CD49d+CD4+) from mice infected with LCMV-Cl13. LCMV-specific CD4+ T cells express CD11ahiCD49d+ (Exhausted virus-specific CD8CMV-Cl13 . This coCMV-Cl13 C. Mice w5 and 30 A. PD-1 e2 = 0.7) B. Consis2 = 0.7) C. When e2 = 0.7) D, mice snti-PD-1 . This wa T cells E. By con T cells F or PD-1 T cells G of CD4+hiCD49d+ .33-41+CD8+ T cells showed a significant increase in the percentage of cells expressing IFN-γ and tumor necrosis factor-alpha (TNF-α) at days 30 and 37 induced by SB415286  mice were housed at the Central Biological Services (Cambridge University). Mice were infected when 6–8 weeks old under the Home Office Project License 80/2189. Antibodies and GSK-3 inhibitors are described in the + cytolytic T cells were generated by incubating OT-I splenocytes with SIINFEKL peptide of OVA (OVA257-264) at 10 ng/mL for 5–7 days, usually in experiments using SB415286 such as in 257-264 peptide (Bachem) for 1 hr at 37°C and treated with mytomycin C (Sigma-Aldrich)  prior to mixing with primary T cells by co-culturing at a ratio of 1:5 of EL4 and T cells in order to generate cytotoxic T cells.Primary mouse T cells  were isolated from spleens and cultured in vitro in supplemented RPMI 1640 . T cell ocris Bioscience Boston Biochem) . 25 mM stock solutions were prepared in DMSO and diluted to a concentration of 1–10 μM in vitro. NSC 87877 was used at 50 μM (TBiochem)  and cycl6 T cells and suspended in 100 μL of nucleofector solution for T cells (Amaxa Biosystems). Cells were electroporated with a Nucleofector (Amaxa Biosystems), as previously described . 3.0–5.0 μg of siRNAs were added to 1 × 10escribed . In certescribed .91–99, GFNKLRSTL) (SIGMA-Genosys)  . CytotoxLuciferase reporter assays were performed with Dual-luciferase Reporter Assay System (Promega).Single-strand cDNA was synthesized with an RT-PCR kit (QIAGEN) and phases of PCR amplification were monitored to ensure a measurement of real-time transcription . ChIP  was injected i.v. into OT-I Tg mice with and without SB415286 (10 μg) on days 0 and 7. T cells were purified from spleens harvested on days 7 and 14.A.T. and C.E.R. conceived and designed experiments. A.T. conducted the majority of in vitro experiments and C.E.R. wrote the majority of the manuscript. A.T., P.G.S., and C.E.R. designed and A.T. conducted experiments on MHV-68, and J.A.H., A.T., and E.I.Z. designed and conducted LCMV-Cl13 experiments and contributed to writing the relevant section. K.C. conducted experiments on NFATc1 with A.T. and C.E.R.


This should read “β-Defensin 3”. The correct title is: Human β-Defensin 3 Exacerbates MDA5 but Suppresses TLR3 Responses to the Viral Molecular Pattern Mimic Polyinosinic:Polycytidylic Acid. The correct citation is: Semple F, MacPherson H, Webb S, Kilanowski F, Lettice L, McGlasson SL, et al. (2015) Human β-Defensin 3 Exacerbates MDA5 but Suppresses TLR3 Responses to the Viral Molecular Pattern Mimic Polyinosinic:Polycytidylic Acid. PLoS Genet 11(12): e1005673. doi:


H-chromen-2-one with bromine in acetic acid, the hydroxy group in involved in an intramolecular O—H⋯O hydrogen bond. In the crystal, π–π interactions between the rings of the bicycle pack molecules into stacks along the b axis.In a new coumarin derivative obtained from the reaction of 3-acetyl-4-hydroxy-2 11H6Br2O4, is a new coumarin derivative obtained from the reaction of 3-acetyl-4-hydroxy-2H-chromen-2-one with bromine in acetic acid. The hydroxyl group in involved in an intramolecular O—H⋯O hydrogen bond. In the crystal, π–π interactions between the rings of the bicycle [intercentroid distances = 3.498 (2) and 3.539 (2) Å] pack molecules into stacks along the b axis, and weak intermolecular C—H⋯O hydrogen bonds further link these stacks into layers parallel to the ab plane.The title compound, C H-chromen-2-one is one of the well-known 3-substituted-4-hydroxycoumarins, which form a class of fused-ring heterocycles and occur widely among natural products. Several natural products with the coumarinic moiety exhibit interesting biological properties such as anti-oxidant and antibacterial 3-Acetyl-4-hydroxy-2H-chromen-2-one b-axis direction on Fig. 2. The obson Fig. 2. Weak inon Fig. 2 further ne Fig. 3.H-chromen-2-one in acetic acid  set to 1.2Ueq of the parent atom.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989014027947/cv5480sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989014027947/cv5480Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989014027947/cv5480Isup3.cmlSupporting information file. DOI: 1040655CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


L1 and HL2, differ in their conformation with the pyridine ring being inclined to the pyrazine ring by 61.34 (6) and 84.33 (12)°, respectively. The crystal packing is also slightly different, with molecules of HL1 linked by pairs of N—H⋯N hydrogen bonds, forming inversion dimers, while for HL2 molecules are linked by N—H⋯N and C—H⋯N hydrogen bonds, forming chains along [010].The title molecules, H 11H10N4O (HL1) and C11H10N4O (HL2), are pyridine 2-ylmethyl and 4-ylmethyl derivatives, respectively, of pyrazine-2-carboxamide. HL1 was measured at 153 K and crystallized in the monoclinic space group P21/c with Z = 4. There has been a report of the same structure measured at room temperature but assumed to crystallize in the triclinic space group P-1 with Z = 4 . The chains are linked via C—H⋯O hydrogen bonds, forming sheets lying parallel to (100). Within the sheets there are π–π interactions involving neighbouring pyrazine rings [inter-centroid distance = 3.711 (15) Å]. Adjacent sheets are linked via parallel slipped π–π interactions involving inversion-related pyridine rings [inter-centroid distance = 3.6395 (17) Å], forming a three-dimensional structure.The title compounds, C al. 2008. Monatsh The same structure measured at room temperature was reported to crystallize in the triclinic space group PZ = 4 ] factor was rather high at 0.0699, hence it is possible that the choice of crystal system and space group are incorrect. However, this could not be confirmed when analysing the coordinates using the AddSymm routine in PLATON  . Within the sheets there are π–π interactions involving neighbouring pyrazine rings , forming a three-dimensional structure  , then extracted with CH2Cl2. The organic extract was dried over Na2SO4. The resulting yellow product was purified by recrystallization from hexane, or by column chromatography on silica gel using CH2Cl2 as eluant, giving finally colourless crystals (yield 80%).The precursor pyrazine-2-carboxylic acid methyl ester (2-pze) was prepared following the procedure described by Alvarez-Ibarra  al. 1994. 6.21 g L1 was prepared by refluxing 2-pze  and an excess of 2-(aminomethyl)pyridine  in 12 ml of methanol, for 6 h in a two-necked flask (50 ml). A yellowish oil remained when the methanol was evaporated off. The excess of 2-(aminomethyl)pyridine was eliminated by column chromatography on silica gel using CH2Cl2 as eluant . A yellow band of 2-(aminomethyl)pyridine remained on the column. After evaporation, the ligand could be recrystallized from diethyl ether, acetonitrile or ethyl acetate. HL1 is very soluble in MeOH and in CH2Cl2. Recrystallization from diethyl ether gave colourless blocks of HL1 . Analysis for C11H10N4O (rM = 214.46 g/mol) Calculated (%) C: 61.67 H: 4.71 N: 26.15; Found (%) C: 61.80 H: 4.76 N: 26.45. Spectroscopic data are available in the supporting information.The ligand HL2 was prepared using the same procedure as for HL1. 2-pze  with, this time, an excess of 4-(aminomethyl)pyridine  were refluxed in 20 ml of methanol, for 20 h in a two-necked flask (50 ml). 4-(aminomethyl)pyridine  was then added to the red solution. After 4 h the solution was evaporated to about 8 ml. HL2 crystallized out at room temperature. About 20 ml of diethyl ether was added to filtrate the product. It was then recrystallized from a mixture of 3 ml of methanol and 40 ml of diethyl ether to give colourless blocks . Anal. for C11H10N4O (rM = 214.46 g/mol) Calculated (%) C: 61.67 H: 4.71 N: 26.15 Found (%) C: 61.57 H: 4.75 N: 26.20. Spectroscopic data are available in the supporting information.HL1 and = 0.93 Å for HL2, with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S1600536814009519/hb0007sup1.cifCrystal structure: contains datablock(s) HL1, HL2, global. DOI: 10.1107/S1600536814009519/hb0007HL1sup2.hklStructure factors: contains datablock(s) HL1. DOI: Click here for additional data file.10.1107/S1600536814009519/hb0007HL1sup4.cmlSupporting information file. DOI: 10.1107/S1600536814009519/hb0007HL2sup3.hklStructure factors: contains datablock(s) HL2. DOI: Click here for additional data file.10.1107/S1600536814009519/hb0007HL2sup5.cmlSupporting information file. DOI: 1004272, 1004273CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Z′ = 2) which are linked into dimers by ab plane by weak intermolecular C—H⋯N interactions.The title compound crystallizes with two molecules in the asymmetric unit . Each molecule consists of a 2-hydroxy Schiff base moiety linked through a spacer to a 2-aminobenzothiazole moiety. Each molecule contains an intramolecular hydrogen bond between the –OH group and imine N atom, forming a six-membered ring. The two independent molecules are linked by a pair of C—H⋯O hydrogen bonds, forming dimers with an R22(20) ring motif. These dimers are further lined into sheets in the ab plane by weak intermolecular C—H⋯N interactions. The structure was refined as an inversion twinThe title compound, C Each molecule consists of a 2-hydroxy Schiff base moiety linked through a spacer to a 2-aminobenzothiazole moiety. This spacer is both planar  and very close to coplanar with both the Schiff base and 2-aminobenzothiazole end moieties . The molecules themselves are very close to planar, as is shown by the dihedral angles of 4.0 (3) and 6.3 (2) between the two end groups for molecules A and B, respectively. Each molecule contains an intramolecular hydrogen bond between the OH group and imine N atom, forming a six-membered ring.The title compound, Cab plane by weak intermolecular C—H⋯N interactions involving C15 and N2B, as shown in Fig. 2In addition to the intramolecular hydrogen bond mentioned above, the molecules are linked by a pair of C—H⋯O hydrogen bonds Table 1, formingE)-2-[(6-ethoxybenzothiazol-2-yl)iminomethyl]-6-methoxyphenol  salicylaldehyde and 0.746 g (4.10 mmol) 2-amino-6-methoxybenzothiozole was dissolved in 2 ml of acetonitrile in a vial. The mixture was reacted in a Biotage initiator eight mono mode microwave at 423 K for 2 min and then allowed to cool for 15 min. The resulting product was recrystallized from acetonitrile, filtered and then vacuum dried to afford 0.971 g (86% yield) of a yellow crystalline solid (m.p. 399–403 K). A sample was dissolved in ethanol and allowed to crystallize by slow evaporation to give yellow needles used for X-ray structural determination.1H NMR : δ 12.07 , 9.36 , 8.81 , 8.39 , 8.05 , 7.55 , 7.09 , 7.04 , 3.83 13C NMR : δ 55.07, 105.07, 115.46, 118.4, 121.2, 122.88, 125.26, 130.4, 132.44, 135.07, 145.59, 157.8 162.69, 165.36, 169.49isoU(H) = 1.5Ueq(C) for methyl H atoms and = 1.2eqU(C) for other H atoms. Phenol H atoms were located in a difference Fourier map and then refined as riding on their attached O atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015005228/hg5435sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989015005228/hg5435Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015005228/hg5435Isup3.cmlSupporting information file. DOI: 1053989CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N′2S is described. The S (Mg2Al4O8) and N′ (BeMgAl4O8) modules have the stacking sequence N′SSN′′.The crystal structure of a new polytype of magnesiotaaffeite-2 N′2S, ideally Mg3BeAl8O16 , is described in space-group symmetry P-3m1. It has been identified in a fragment of a mineral sample from Burma (Myanmar). The new polytype is composed of two Mg2Al4O8 (S)- and two BeMgAl4O8 (N′)-modules in a stacking sequence N′SSN′′ which differs from the N′SN′S-stacking sequence of the known magnesiotaaffeite-2N′2S polytype. The crystal structure can be derived from a close-packed arrangement of O atoms and is discussed with regard to its polytypism and its Cr3+ chromophore content.The crystal structure of a new polytype of magnesiotaaffeite-2 S) and nolanite (N′) modules , crystal structure  Å and three Mg1—O4 distances of 1.9296 (7) Å is more distorted than the Mg2 coordination environment, where the longer Mg2—O1 distance [1.9361 (13) Å] hardly differs from the three 1.9300 (7) Å Mg2—O5 distances. The average bond lengths at the tetrahedral sites, nominally occupied by Mg , and at the octahedral sites, nominally occupied by Al , are similar to the T-O (1.936 Å) and M-O (1.923 Å) distances reported for a natural Cr and V-bearing spinel from Burma with a small inversion parameter  Å] and three longer bonds [1.6615 (13) Å] with the O3-anions, while the tetrahedral angles are either 97.89 (9)° (O3—Be1—O3) or 119.45 (7)° (O7—Be1—O3). The Mg atom in the Mg3O6-octahedron exhibits a strong out-of-centre distortion, away from the Al3-cation, to which it has a distance of only 3.0580 (7) Å.The composition obtained by structure refinement is in good agreement with the composition obtained by electron microprobe analysis (EMPA). The calculated bond-valence sums agree reasonably well with the formal charges Table 1, and on e Table 1, which mO-layer  originates from Chaung-gyi, Mogok, Pyin-Oo-Lwin district, Burma (Myanmar). It has a red colour and a layered appearance , the empirical chemical formula was determined as Al7.86Be1.0Cr0.19Mg2.93O16. The corresponding oxide composition (in wt%) is MgO 20.82, Cr2O3 2.50, Al2O3 70.72, BeO 4.41, yielding a total of 98.45%.The studied natural sample of magnesiotaaffeite , 0.017 (5), 0.028 (5) and 0.048 (5), respectively. Two twin domains (twinning by merohedry) with volume fractions of 0.64 and 0.36 contribute to the total scattering intensity, related by reflection parallel to [1Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016010215/wm5296sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016010215/wm5296Isup2.hklStructure factors: contains datablock(s) I. DOI: 1487140CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The brown alga Sargassum ilicifolium was the source for the first isolation of the terpenoid C27-alcohol 1,1′,2-trinorsqualenol (1) from marine sources. From S. incisifolium we isolated the highly unsaturated glycolipid 1-O-palmitoyl-2-O-stearidonoyl-3-O-β-D-galactopyranosylglycerol (4) and we report the first full assignment of its 1H and 13C NMR data. Apo-9′-fucoxanthinone (8) along with 24-ketocholesterol (5), (22E)-3β-hydroxycholesta-5,22-dien-24-one (6), and saringosterol (7) were obtained from Turbinaria ornata. The crude extracts of all eight species of brown algae exhibited a pronounced antimicrobial activity against the Gram-positive bacteria Bacillus cereus, Staphylococcus aureus, and Streptococcus pneumoniae.Eight species of brown algae (Phaeophyceae) from the coast of Madagascar have been investigated for their chemical constituents. Fucosterol (The online version of this article (doi:10.1007/s13659-015-0068-0) contains supplementary material, which is available to authorized users. Spatoglossum sp., Sargassum sp. 1, Sargassum sp. 4, Zonaria sp., Chnoospora sp., and Spatoglossum sp. Andriamanantoanina and Rinaudo identified alginate polymers which are composed of (1 → 4)-β-d-mannuronic acid (M) and (1 → 4)-α-l-guluronic acid (G) units +). This information combined with 1H and 13C NMR data supported the molecular formula C27H46O. The 1H NMR spectrum displayed signals for 46 protons including five vinylic protons at δ = 5.09–5.15 ppm .Samples of brown seaweeds of 13C NMR and DEPT data of 1 revealed the presence of six methyl groups , five quaternary olefinic C-atoms , five olefinic CH moieties , one HO–CH2 group (C-1), and ten CH2 groups . A proton to carbon assignment could be achieved by an HSQC experiment. The location of the methyl groups and the olefinic moieties could be assigned on the basis of COSY, HMBC, and NOESY correlations. The E configuration of the double bonds in positions 4, 8, 12, and 16 was deduced from NOESY experiments by correlation of the methyl groups  with the respective allylic protons and absence of a correlation with the vinylic protons. The data obtained from 1D and 2D NMR experiments unambiguously led to the elucidation of compound 1 as -4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaen-1-ol, which is also known as 1,1′,2-trinorsqualenol, with full assignment of all 1H and 13C NMR signals. Our 1H and 13C NMR data are in good agreement with those of the synthetic product described by Gref and coworkers . Stigm alcohol . Djerass isomers . IsolatiS-i1–142 ntana H.  and othep of Guo . The preS. ilicifolium was found to be very active against the Gram-negative bacteria Shigella boydii and Klebsiella oxytoca, the Gram-positive bacteria Streptococcus pneumoniae and Staphylococcus aureus, and the yeasts Candida membranaefaciens, Trichosporon mucoides, and Cryptococcus neoformans . The methanol fraction was subjected to normal-phase column chromatography to give two fractions. Fraction 2 afforded a mixture of inseparable monogalactosyldiacylglycerols bearing fatty acid side chains with only few olefinic groups according to the intensity of vinylic signals in the 1H NMR spectrum. Fraction 1 was further purified by reversed-phase HPLC using THF–H2O (50:50–80:20) as mobile phase to afford 1-O-palmitoyl-2-O-stearidonoyl-3-O-β-D-galactopyranosylglycerol (4)  and carboxylic ester (1738 cm−1) groups. The 1D and 2D NMR spectra of 4 displayed signals for a carbon chain bearing four methylene-interrupted double bonds, a saturated carbon chain, a sugar moiety, a glycerol unit, and two carbonyl groups. The sugar residue could be identified as β-D-galactose by comparison of the 13C NMR shifts with those of various sugars listed in Ref. . Comparison of the 1H and 13C NMR data with those reported in the literature  confirm7) was obtained as a colorless, amorphous solid. Beside the signals for the steroidal backbone including the methyl groups, the 1H NMR spectrum revealed the presence of an additional double bond by three additional signals for olefinic protons. Signal doubling of the olefinic protons H-29  and the carbon atoms C-28  and C-29  indicated the presence of a 1:1 mixture of C-24 epimers. The 1H NMR data of the side chain are in agreement with those reported by Djerassi et al. D20 = –18.0 . The molecular formula was determined as C15H22O4 from the [M + NH4]+ ion at m/z = 284 in the ESI–MS in combination with the number and intensities of the 1H and 13C NMR signals. Compound 8 shows a characteristic IR absorption band at ν = 1937 cm−1 indicating the presence of an allene group. The 1H NMR spectrum displayed signals for 21 protons which could be assigned to five methyl, two methylene and two methine groups according to the DEPT data. The 13C NMR spectrum displayed resonances for 15 carbon atoms including two carbonyl groups at δ = 196.63 and 170.34 ppm in CDCl3 and at δ = 200.86 and 172.41 ppm in CD3OD and two oxygenated carbon atoms at δ = 72.05 and 67.37 ppm in CDCl3 and 72.38 and 69.13 ppm in CD3OD (Table 13C NMR (CDCl3) signals for the carbon atoms of the allene moiety appear at δ = 118.45 (C-6), 209.47 (C-7), and 100.91 ppm (C-8). Comparison of the NMR data with those reported for apo-9′-fucoxanthinone (8) by Kobayashi et al. confirmed the identity of the compound (Table 8) was first reported as an oxidative degradation product of fucoxanthin +.4E,8E,2E,16E-4,2) (1:1 mixture of C-24 epimers): Amorphous solid; mp = 119–120 °C -epimer: mp = 132–133 °C +, 790.6 [M + NH4 + Na − H]+; ESI–MS (+75 V): m/z = 773.7 [M + Na]+, 795.7 [M + 2Na − H]+; ESI–MS (−10 V): m/z = 749.4 [M–H]−, 771.4 [M + Na − 2H]−. 2D-NMR spectra  of compound 4: see Supplementary Material.1-5) (3:1 mixture with 6): Colorless amorphous solid; 1H NMR : δ (ppm) = 0.93 , 1.10 , 1.24–1.28* , 1.37–1.43* , 1.70–1.78* , 2.36–2.40 , 2.44–2.50 , 2.64 , *signals determined from HSQC spectrum; 13C NMR : δ (ppm) = 18.31, 18.38 , 18.51 (C-21), 29.84 (C-22), 35.39 (C-20), 37.24 (C-23), 40.83 (C-25), 215.48 (C-24); GC–MS : m/z (%) = 400 (50), 382 (41), 314 (33), 271 (33), 255 (35), 213 (58), 145 (47), 105 (52), 43 (100).24-Oxocholest-5-en-3β-ol (24-ketocholesterol) (E)-3β-Hydroxycholesta-5,22-dien-24-one (6) (1:3 mixture with 5): 1H NMR : δ (ppm) = 2.84 , 6.08 , 6.73 ; 13C NMR : δ (ppm) = 126.2* (C-23), 152.5* (C-22), 204.5 (C-24), *signals determined from HMBC spectrum; GC–MS : m/z (%) = 398 (5), 380 (7), 309 (6), 271 (9), 255 (9), 187 (24), 159 (22), 126 (100).(227) (1:1 mixture of C-24 epimers): Colorless amorphous solid; IR (ATR): ν (cm−1) = 3325 (br), 2959, 2931, 2865, 2850, 1667, 1459, 1431, 1375, 1055, 1021, 994, 957, 918, 798, 739; 1H NMR : δ (ppm) = 5.12  and 5.13 *, 5.17  and 5.18 *, 5.33–5.35 , 5.79  and 5.80 *; 13C NMR and DEPT : δ (ppm) = 71.80 (C-3), 112.84 and 112.93 (C-29)*, 121.70 (C-6), 140.73 (C-5), 142.46 and 142.55 (C-28)*. *Two sets of signals due to the presence of two epimers.Saringosterol (8): Amorphous solid; mp = 84–85 °C  D22 = −23.0   +, 289 [M + Na]+, 555 [2 M + Na]+. Anal. calcd for C15H22O4: C 67.64, H 8.33; found: C 67.94, H 8.52.Apo-9′-fucoxanthinone (78.5 °C) ; [α]D20 4, MeOH) , [α]D21 7 (EtOH) , [α]D = c = 0.6) , [α]D19 1, MeOH) , [α]D26 , CHCl3) ]; CD Δε 3 (257)] ; UV (MeOB. cereus (ATCC 13061), S. aureus (ATCC 11632), and S. pneumoniae (ATCC 6301); six Gram-negative bacteria: Enterobacter cloacae (ATCC 700323), Klebsiella oxytoca (ATCC 8724), Shigella boydii (ATCC 9204), E. coli, S. enteridis, P. aeruginosa (ATCC 9027); and against four yeast strains: C. albicans, C. membranaefaciens (ATCC 201377), Cryptococcus neoformans (ATCC 76484), and Trichosporon mucoides (ATCC 204094). The pathogens were supplied by the Laboratoire de Microbiologie de l’Environnement (LME), Centre National de Recherche sur l’Environnement (CNRE), Antananarivo, Madagascar. The crude extracts were dissolved in methanol with a concentration of 1 mg/mL. A sample (10 μL) of each solution was added via a pipette onto a sterile antibiotic filter disc of 6 mm diameter and oven dried at 40–50 °C. The discs were placed on Müller–Hinton agar plates which had been inoculated with the microorganisms mentioned above. The plates were incubated for 24 h at 37 °C for the bacteria and for 48 h at 25 °C for the yeast. The diameters of the inhibition zones generated around the discs were measured (Ø in mm). The tests were performed in triplicate and the mean values were determined. Methanol, used to dissolve the extracts, was checked for the absence of antimicrobial activity. The diameters of the halos of inhibition can be rationalized on a qualitative basis as follows: Ø < 7 mm: inactive, 7 mm ≤ Ø < 8 mm: slightly active, 8 mm ≤ Ø < 9 mm: significantly active, Ø ≥ 9 mm: very active.Antimicrobial assay: The antimicrobial activities were determined using the agar diffusion technique in Petri dishes. The crude brown algal extracts were tested for their antimicrobial activity against three Gram-positive bacteria: Plasmodium falciparum was determined by using the microfluorimetric assay previously reported [50 value in µg/mL.Antimalaria test: The antiplasmodial activity against the FCM29 strain of reported . The res3) is the major sterol of brown algae (Phaeophyceae) [2), 24-ketocholesterol (5), (22E)-3β-hydroxycholesta-5,22-dien-24-one (6), and saringosterol (7) have been isolated from S. ilicifolium and T.ornata, respectively. The allenic norterpenoid apo-9′-fucoxanthinone (8) was obtained from T.ornata. For the first time, 1,1′,2-trinorsqualenol (1) has been obtained as a pure compound from natural sources and moreover, its isolation from a marine organism (S. ilicifolium) is unprecedented. The glycolipid 1-O-palmitoyl-2-O-stearidonoyl-3-O-β-D-galactopyranosylglycerol (4) was obtained from the brown alga Sargassum incisifolium and for the first time, we report the complete assignment of its 1H NMR and 13C NMR data. Antimicrobial tests of the crude extracts of the various brown algae revealed a strong activity against Gram-positive bacteria, in particular against S. aureus and Streptococcus pneumonia, which may ascribed to the content of phytosterols.We present one of the first extensive studies on the non-polar chemical constituents of brown algae from the coast of Madagascar. Our results confirm the early reports that fucosterol (phyceae) . MoreoveSupplementary material 1 (PDF 1873 kb)


The absolute configuration of fuasiumin A has been established as  using a combination of RDC -based NMR and DFT-supported chiroptical spectroscopy. It is worth to note that in this study without the aid of the RDC analysis, an unambiguous determination of configuration and conformation was not feasible due to the excessive conformational possibilities of this open-chain compound.A new alkylpyrrole derivative, fusariumin A (The online version of this article (doi:10.1007/s13659-015-0084-0) contains supplementary material, which is available to authorized users. Fusarium is a large genus of filamentous fungi which widely distributed in soil and associated with plants. Most species are harmless saprobes, and are relatively abundant members of the soil microbial community. Some species infest rice, maize, oats, barley, and wheat as pathogens and produce mycotoxins in cereal crops that can affect human and animal health if they enter the food chain + was selected for MS/MS analysis, the characteristic fragmentation ion at m/z 290.2483 with the chemical composition C19H31NO was detected, which could be explained by the elimination of a C8H16O2 part, namely an octanoic acid . Therefore, the acyl at C-3′ was determined as an octanoyl group, and the left side chain extending from C-3′ was deduced with fourteen carbons  needed to be considered. Besides, two conformations for the H2′-C2′-N-C5 dihedral  were found from DFT calculations. Taken together, 12 (= 2 × 3 × 2) conformations were generated and DFT optimized for the two possible relative configurations  and , respectively . For all these 12 conformations of each configuration, the alkyl chain from C4′ to C14′ and from C2′′ to C8′′ was assumed to adopt an anti conformation, which is the energetically most preferable conformer -CLIP-HSQC -HECADE -HSQMBC -HECADE , -HSQMBC  and NOES]-HSQMBC  spectra,Fusarium sp. was collected in Botanic Garden of Kunming Institute of Botany, Chinese Academy of Sciences and identified by Prof. Zhu-Liang Yang . A voucher specimen has been deposited in the School of Pharmaceutical Sciences, South-Central University for Nationalities. The culture medium consists of potato , glucose (20 g), aneurine hydrochloride (10 mg), KH2PO4 (3 g), and MgSO4 (1.5 g) in deionized water (1 L). The pH was adjusted to 6.5 before autoclaving, and the fermentation was carried out in a shaker (150 rpm) at 25 °C for 25 days.The fungus 3-MeOH (1:1) to afford two fractions A1 and A2. The fraction A1 was purified by Preparative HPLC  to give fusariumin A (3.0 mg).The culture broth (18 L) was extracted with EtOAc for three times, and the organic layer was concentrated under reduced pressure to give a crude extract (8.0 g), which was subjected to silica gel column chromatography (CC) using a petroleum ether–acetone gradient (1:0 → 0:1) to afford fractions A–F. The fraction A was chromatographed first on a silica gel column eluted with petroleum ether–acetone , and then on a Sephadex LH-20 column eluted with CHClFusariumin A (1). colorless oil; [α]D21 = −108.6 ; IR (KBr) νmax 2926, 2855, 1741, 1668, 1467, 1164, 776 cm−1; 1H NMR  δ: 9.53 , 7.08 , 6.92 , 6.22 , 5.55 , 5.11 , 2.23 , 1.56 , 1.46 , 1.23 , 0.88 ; 13C NMR  δ: 179.9 , 172.9 , 131.5 , 127.7 , 125.6 , 110.0 , 75.4 , 54.2 , 34.2 , 31.9 ~ 22.6 (C-2 ~ C-11 and C-15 ~ C-19), 14.7 , 14.1 ; ESIMS (positive): m/z 456 [M + Na]+, 890 [2 M + Na + H]+, 434 [M + H]+, 290; HREIMS: m/z 433.3564 ; 290.2473 .N,N-dimethylacrylamide (1 M), N,N-methylenebisacryl- amide (0.03 M), and ammonium persulfate (8 mM) were dissolved in purified water followed by devolatilization, in vacuo, for 15 min. The stock solution was inserted into a gel cylinder  with an inner diameter of 2 mm and polymerized for 8–9 min at 70 °C. The gels were washed twice with aqueous HCl (0.02 M), three times with purified water (each time for 1–2 h) and finally dried under ambient conditions (air + room temperature) for at least 1 day.2-(Acrylamido)-2-methyl- propanesulfonic acid (1 M), The initial structure of each possible conformation was built with Discovery Studio 2.5 (Accelrys) and all trial structures were first minimized based on molecular mechanics calculations (CFF force field)  and foll1DCH couplings and the coordinate files obtained from DFT optimizations were given as the input data. Five independent members of the alignment tensor were determined by using the singular value decomposition (SVD) method. The goodness of fit between experimental and back-calculated RDCs was expressed in terms of the Cornilescu quality factor Q [The fitting procedure of the experimental RDC data was performed by using the MSpin program (Mestrelab Research) .10 experfactor Q .Time-dependent DFT (TD-DFT) with the basis set B3LYP/6-31G(d) was used to calculate the spin-allowed excitation energies, rotatory and oscillator strengths of the lowest 50 excited states. The calculations were performed with Gaussian09  by using36 with DMSO as the solvent.The optical rotation dispersion calculations were performed at the four wavelengths 365, 436, 546, 578 and 589 nm by using the optimized structures as input coordinates. The calculations were carried out with the basis set B3LYP/6-31G(d) by using the IEFPCM solvent continuum model as implemented in Gaussian09Supplementary material 1 (DOCX 2068 kb)


A 2:1 organic salt formed from pyrazole and picric acid was obtained from methanol solution. In the crystal, N—H⋯O hydrogen bonds give rise to a hydrogen-bonded chain along [100]. Adjacent [100] chains are linked by a weak C—H⋯O interaction. H-pyrazol-2-ium 2,4,6-trinitrophenolate–1H-pyrazole (1/1)], H(C3H4N2)2+·C6H2N3O7−, consists of one picrate anion and one hydrogen-bonded dimer of a pyrazolium monocation. The H atom involved in the dimer N—H⋯N hydrogen bond is disordered over both symmetry-unique pyrazole molecules with occupancies of 0.52 (5) and 0.48 (5). In the crystal, the component ions are linked into chains along [100] by two different bifurcated N—H⋯ hydrogen bonds. In addition, weak C—H⋯O hydrogen bonds link inversion-related chains, forming columns along [100].The asymmetric unit of the title organic salt [systematic name: 1 A short O3(nitro)⋯O3(nitro)  distance of 2.913 (2) Å is also observed et al., 2016viz. SASKII, SASLAB, SASKUU, SASLUB .Pyrazole  and picric acid  were dissolved in a 2:1 molar ratio in 95% methanol (50.0 ml). The mixture was stirred for an hour at 323 K and then cooled to room temperature and filtered. The resulting yellow solution was kept in air for two weeks. Needle-like yellow crystals of (I)Uiso(H) = 1.2Ueq(C). H atoms bonded to N atoms were refined with a constraint of dN—H = 0.86 (1) Å and Uiso(H) = 1.2Ueq(N). Atoms H4A and H6A were found in difference Fourier maps and refined as disordered using the PART command  global, I. DOI: 10.1107/S2056989016008215/lh5810Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016008215/lh5810Isup3.cmlSupporting information file. DOI: 1480969CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In (I), in which the metal atom lies on a twofold rotation axis, the dithiocarbamate ligand coordinates with approximately equal Sn—S bond lengths and the ipso-C atoms of the Sn-bound phenyl groups occupy cis-positions in the resulting octahedral C2S4 donor set. A quite distinct coordination geometry is noted in (II), arising as a result of quite disparate Sn—S bond lengths. Here, the four S-donors define a trapezoidal plane with the ipso-C atoms lying over the weaker of the Sn—S bonds so that the C2S4 donor set defines a skewed trapezoidal bipyramid. The packing of (I) features supramolecular layers in the ab plane sustained by methylene-C—H⋯π(Sn–aryl) interactions; these stack along the c-axis direction with no specific interactions between them. In (II), supramolecular chains along the b-axis direction are formed by methylene-C—O(ether) interactions; these pack with no directional interactions between them. A Hirshfeld surface analysis was conducted on both (I) and (II) and revealed the dominance of H⋯H interactions contributing to the respective surfaces, i.e. >60% in each case, and other features consistent with the description of the molecular packing above.The crystal and molecular structures of two diphenyltin bis(dithiocarbamate)s, [Sn(C This symmetry is reflected in the near equivalence of the associated C1—S bond lengths with the difference between them being 0.024 Å, Table 1trans to the ipso-carbon atom. The overall coordination geometry is based on an octahedron with the ipso-carbon atoms occupying mutually cis positions. The methoxyethyl group is approximately perpendicular to the S2CN core as seen in the value of the C1—N1—C3—C4 torsion angle of 93.8 (2)°. The residue itself is almost planar and adopts an extended conformation as seen in the C5—O1—C4—C3 torsion angle of 175.27 (19)°.The asymmetric unit of (I)2S4 donor set. However, the dithiocarbamate ligand is coordinating with significantly greater values of ΔS, i.e. 0.48 and 0.46 Å for the S1- and S3-ligands, respectively, with the Sn—Sshort bonds in (II)long bonds in (II)bond lengths in (II)4 donor atoms define a trapezoidal plane. The tin-bound ipso-carbon atoms are disposed over the weaker Sn—S bonds so that the coordination geometry is skewed trapezoidal bipyramidal. Reflecting the significant disparity in the Sn—S bonds, there are large differences in the associated C—S bonds with the shorter of these being associated with the weakly coordinating sulfur atoms, Table 12CN atoms with the greatest deviation being for the O1-containing residue, i.e. the C1—N1—C5—C6 torsion angle is −81.5 (3)°. For each dithiocarbamate ligand, the residues lie to either side of the S2CN plane, and each is as for (I)The molecule in (II)PLATON b axis, Fig. 4a. These pack into the three-dimensional architecture of the crystal with no directional intermolecular interactions between them, Fig. 4b.In the molecular packing of (II)A more detailed analysis of the molecular packing in (I)dnorm, de, shape-index, curvedness and electrostatic potential with the aid of Crystal Explorer 3.1 dnorm (not shown). By contrast, in (II)As evident from Fig. 5de, di) ranges, i.e. 1.2 to 2.7 Å for (I)The overall two-dimensional fingerprint plots for (I)b, H⋯H contacts clearly make the most significant contributions to the Hirshfeld surfaces of both (I)b), all the points are situated at  distances equal to or greater than their van der Waals separations i.e. 1.2 Å, reflecting zero propensity to form such intermolecular contacts. By contrast, for (II)de, di) distances less than 1.2 Å, with the peak at de = di ∼1.2 Å, resulting from short interatomic H⋯H contacts, Table 5de + di ∼2.5 Å in Fig. 7c. The small contribution, i.e. 4.7%, due to analogous interactions in (I)As evident from the data in Table 4de + di ∼2.9 Å in the fingerprint plot delineated into C⋯H/H⋯C contacts for (I)d. The presence of these interactions are also indicated through the pale-orange spots present on the Hirshfeld surface mapped over de, shown within the blue circle in Fig. 8a, and bright-red spots over the front side of shape-indexed surfaces identified with arrows in Fig. 8b. The reciprocal of these C—H⋯π contacts, i.e. π⋯H—C contacts, are seen as blue spots near the ring in Fig. 8b. The fingerprint plot for (II)de, di) distances greater than their van der Waals separations, confirming the absence of these interactions, Fig. 7d. The conformations of dithiocarbamate ligands in both (I)i.e. 3.0 Å. This observation is despite the nearly symmetrical distributions of points in the respective plots for both (I)e, and the significant percentage contributions to their Hirshfeld surfaces a, and distorted octahedral, as in (I)c. Less common are five-coordinate, trigonal–bipyramidal species, arising as one dithiocarbamate ligand is monodentate, Fig. 9b, are found, for example, in the structure of (t-Bu)2Sn(S2CNMe2)2 CH2CH2]2Sn(S2CNMe)2 2, and eight adopt the motif shown in Fig. 9a, including both independent molecules of Ph2Sn[S2CN(Me)Hex]2  structures included in the CSD and eight of these adopt the motif shown in Fig. 9v/v) ratio. This mixture was allowed to slowly evaporate at room temperature yielding colourless crystals of (I)−1): 1,497 ν(C—N), 988 ν(C—S), 523 ν(Sn—C), 389 ν(Sn—S). 1H NMR (CDCl3): δ 7.28–8.00 , 3.97 , 3.69 , 3.44 , 3.36 . 13C NMR (CDCl3): δ 199.88 (S2C), 128.24–151.24 (Sn–Ar), 69.96 (OCH2), 59.06 (NCH2), 57.84 (OCH3), 45.45 (NCH3).Synthesis of (I)−1): 1,482 ν(C—N), 985 ν(C—S), 571 ν(Sn—C), 381 ν(Sn—S). 1H NMR (CDCl3): 7.38–7.89 , 4.07 , 3.77 , 3.35 (OCH3). 13C NMR (CDCl3): δ 200.16 (S2C), 128.26–150.89 (Sn–Ar), 69.90 (OCH2), 59.02 (NCH2), 56.72 (OCH3).Compound (II)Uiso(H) set to 1.2–1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 610.1107/S2056989016011385/hb7599sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989016011385/hb7599Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989016011385/hb7599IIsup3.hklStructure factors: contains datablock(s) II. DOI: 1492701, 1492700CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The self-assembled cations form a homo-synthon R21(6) motif which is further linked with the sulfonate anion via N—H⋯O hydrogen bonds to generate an R32(10) ring motif. The three motifs are fused together and extended as supramolecular ribbons along the b-axis direction. Adjacent ribbons are further linked via N—H⋯O hydrogen bonds to form an annulus, with an R44(20) ring motif, resulting in a tunnel-like arrangement propagating along [010]. There are slipped parallel π–π stacking interactions [inter-centroid distance = 3.6539 (7) Å], between the tunnel-like polymer chains, forming slabs parallel to (100). In the title salt, C The cation undergoes self-association via a pair of bifurcated N—H⋯ hydrogen bonds, forming a homo-synthon, i of the cation  Å [Cg is the centroid of ring N1/C2/N3/C4–C6; the dihedral angle between the ring planes = 1.86 (6)°; perpendicular separation = 3.2501 (5) Å; symmetry code: (iii) −x + 1, y, −z + Adjacent tunnels interact by off-set aromatic π–π stacking interactions which are observed between symmetry-related pyrimidine rings of the cations with a centroid–centroid distance p-toluene sulfonate but only three hits for the 2,6-diamino-4-oxo-1,3-dihydropyrimidin-1-ium cation. These include the sulfate monohydrate  was warmed at 323 K for 30 min over a water bath. The mixture was cooled slowly and kept at room temperature and after three weeks light-yellow needle-shaped crystals were obtained.A hot methanolic solution (20 ml) of 2,6-diamino-4-hydroxy pyrimidine  and Uiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015006787/su5107sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989015006787/su5107Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015006787/su5107Isup3.cmlSupporting information file. DOI: 1057933CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: