The mutations also delayed CaSR internalization due to prolonged residency time of CaSR in clathrin structures that impaired or abolished endosomal signaling, which was predominantly mediated by Gαq/11. Thus, compartmental bias for CaSR-mediated Gαq/11 endomembrane signaling provides a mechanistic basis for multidimensional GPCR signaling.Spatial control of G-protein-coupled receptor (GPCR) signaling, which is used by cells to translate complex information into distinct downstream responses, is achieved by using plasma membrane (PM) and endocytic-derived signaling pathways. The roles of the endomembrane in regulating such pleiotropic signaling via multiple G-protein pathways remain unknown. Here, we investigated the effects of disease-causing mutations of the adaptor protein-2 σ subunit (AP2σ) on signaling by the class C GPCR calcium-sensing receptor (CaSR). These AP2σ mutations increase CaSR PM expression yet paradoxically reduce CaSR signaling. Hypercalcemia-associated AP2σ mutations reduced CaSR signaling via Gα  •q/11 and Gαi/o signaling by CaSR, a class C GPCRDisease-causing AP2σ mutants impair Gα•AP2σ mutants impair trafficking of the CaSR•The CaSR can signal by a sustained endosomal pathway•q/11 and Gαi/o for cell-surface and endosomal signalingCaSR differentially uses Gα q/11 and Gαi/o and from endosomes by using only Gαq/11. Adaptor protein-2 σ subunit (AP2σ) mutations impair CaSR internalization, leading to reduced sustained endosomal signaling and hypercalcemia in humans.Gorvin et al. show that the class C GPCR calcium-sensing receptor (CaSR) mediates signaling from plasma membranes using Gα For example, the CaSR, when stimulated by elevations in Ca2+e, signals predominantly via Gαq/11 to activate phospholipase C (PLC), with consequent hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2), to the second messengers inositol 1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG)  mobilization into the cytosol, and DAG activates protein kinase C (PKC) signaling cascades, including mitogen-activated protein kinase (MAPK) pathways (i/o to inhibit adenylate cyclase (AC) and reduce cyclic AMP (cAMP)  family is the largest family of signaling receptors, and GPCRs contribute significantly to fundamental cellular functions. The archetypal model of GPCR signaling has evolved from a single, cell-surface receptor activating a specific heterotrimeric G-protein pathway to a complex network in which receptors can activate multiple pathways, exhibit signal crosstalk, and display functional selectivity . This istabolism . Thus, ttabolism , forms dol (DAG) . IP3 actpathways . CaSR haP (cAMP) , Gα12/13modeling , and Gαsll lines .2+e  insertion and removal by endocytosis . The PM 2+e . Followi2+e . However2+e , have re+e , dopamine receptor D1 (DRD1), thyroid-stimulating hormone receptor (TSHR), vasopressin receptor 2 (V2R), and luteinizing hormone receptor (LHR)—and class B GPCRs  . These cAP2S1 constructs that also had silent mutations, which rendered them resistant to AP2σ-targeted small interfering RNA (siRNA), thereby allowing study of the mutant protein in the absence of endogenous protein. The presence of AP2σ mutant proteins or siRNA-resistant mutations did not affect expression of endogenous AP2α, AP2β, or AP2μ that with the σ subunit form the heterotetrameric AP2; general clathrin-mediated endocytic functions such as transferrin uptake; or internalization and signaling of another GPCR, the β2AR  values  or mutant  proteins were established, using appropriate pcDNA3.1-the β2AR . These sthe β2AR . All AP2) values . These r) values , demonst2+e-induced Ca2+i release of AP2σ mutant/CaSR-WT cells may be due to reduced calcium oscillations, and we assessed this by using single-cell microfluorimetry with the calcium-indicating dye Fura-2 in response to increasing concentrations (0–15 mM) of Ca2+e. CaSR-mediated Ca2+i oscillations were observed to occur from 1 to 5 mM Ca2+e, consistent with previous reports, but mutant cells were found to have reduced frequencies, with the AP2σ-C15 and AP2σ-L15 cells requiring higher Ca2+e concentrations to begin oscillating and AP2σ-H15 cells having oscillations with irregular amplitudes (2+i release activates transcription factors such as nuclear factor of activated T cells (NFAT) (1), an IP3 metabolite, revealed reduced IP1 in AP2σ mutant cells compared to AP2σ-WT cells (3-DAG pathway is impaired in AP2σ mutant cells.We hypothesized that Caplitudes A and S2.s (NFAT) . Investis (NFAT) B. SimilaWT cells , therebyq/11-mediated signaling also activates MAPK pathways -transformed lymphoblastoid cells from FHH3 patients with the AP2σ-R15C mutation  sensitive and therefore due to Gαi/o signaling, in HEK293 cells stably expressing CaSR (HEK-CaSR)  A. However Gαq/11 B. Moreov in cAMP B indicato Gαq/11 , but galo Gαq/11 C, therebl levels D and witl levels E and 2F.esponses G, consisq/11 and Gα12/13 signaling   A and S4. < 0.02)  using a  < 0.02) B. Furtheve cells C. Moreovruffling , thereby2+e) conditions or following exposure to 5 or 10 mM Ca2+e. Immediately before TIRF microscopy continuous recordings, cells were exposed to BTx with a fluorescent tag (BTx-594). AP2σ-WT and mutant cells expressed CaSR at the cell surface  was lost and that CaSR internalization measured by BTx-594 fluorescence was severely impaired , which contains the pleckstrin homology domain of PLC-delta (PH-PLC)  , or 2-aminoethoxydiphenyl borate (2-APB), which inhibits PLC, PKC, or the IP3 receptor (IP3R), respectively  . Before  = 8–15) . To furt pathway  on pERK1ectively . pERK1/2ectively , therebynd PTH1R , on the nd PTH1R . The pERd signal .Figure 62+e, SRE reporter responses were significantly higher in AP2σ-WT than in mutant cells, with peak expression occurring between 3 and 5 hr, in all cell lines  stage, whereas the AP2σ-H15 and AP2σ-L15 mutants inhibited CaSR internalization at the clathrin-coated vesicle (CCV) (Rab5-DN sensitive) stage. These milder effects of the AP2σ-C15 mutant on CaSR internalization still reduced Gαq/11 signaling, thereby indicating a possible threshold requirement for receptor occupancy within endosomes for activation of this G-protein pathway. In addition, the AP2σ-C15 mutant, but not AP2σ-L15 or AP2σ-H15, significantly affected Gαi/o signaling at high [Ca2+]e, i.e., 10 mM  pathways provides a paradigm for understanding how pleiotropic signaling pathways activated by a single GPCR can be resolved via spatially directed G-protein selectivity.Detailed methods and information on constructs, oligonucleotides, and antibodies can be found in the Informed consent was obtained from individuals using protocols approved by local and national ethics committees, London, UK (MREC/02/2/93).q, Gα11, Gα12, Gα13, βarrestin-1, and βarrestin-2 by CRISPR-Cas have been described   , cells w (Abcam) .1 assays were performed according to manufacturer’s instructions. For pERK1/2 AlphaScreen assays, cells were transfected with pEGFP-CaSR and treated with 0–10 mM CaCl2 for 5 min. For cAMP assays, cells were pre-treated with forskolin for 30 min. For inhibitor studies, cells were pre-treated with 300 ng/mL PTx or vehicle (ethanol) for 6 hr, 1 μM UBO-QIC or vehicle (DMSO) for 2 hr, or 15 μM gallein or vehicle (DMSO) for 15 min  0.1 mM CaCl2, (2) 5 mM CaCl2 for the whole experiment (constant), (3) 5 min pulse of 5 mM CaCl2 followed by 0.1 mM CaCl2 with vehicle (DMSO) for the duration of the experiment, or (4) 5 min pulse of 5 mM CaCl2 followed by 0.1 mM CaCl2 with 30 μM Dyngo-4a for the duration of the experiment. Cells were pre-incubated with 1 μM UBO-QIC or DMSO for 2 hr or 10 μM forskolin  and 300 ng/mL PTx (Sigma) or vehicle (ethanol diluent) for 6 hr (Transferrin assays were performed as described . IP1 assr 15 min . AlphaScr 15 min . Apoptosr 15 min . Cells wfor 6 hr . Lucifer2+e. Cells were imaged on a Nikon Eclipse E400 wide-field microscope using adapted protocols (2 concentrations. Fura-2 images were acquired using 340/380 nm excitation and 510 nm emission on μManager software (NIH). Methods for TIRF microscopy were adapted from previous studies (2 imaging solution. Images were captured at 10 frames/s in BSEP studies and 3 frames/s for clathrin studies. Images were acquired using CellˆR software (Olympus). Confocal imaging was performed in HEK293 cells using methods adapted from previous studies  following treatment with 0, 5, and 10 mM Carotocols . Single- studies . Images  studies . Images 2 test, Mann-Whitney U test, Pearson’s correlation coefficient, and F test were used to calculate statistical significance using GraphPad Prism 6 software. A p value < 0.05 was considered statistically significant. Statistical tests used are indicated in the methods in the Two-tailed unpaired t test, two-way ANOVA, χ


THS-55, and their structures were elucidated by extensive spectroscopic analysis. All of the compounds exhibited potent cytotoxicity in vitro against HPV-transformed HeLa cell line. Among them, compounds 6–7 were derived as natural products for the first time, and compound 5 (NADA) showed the highest potency. NADA inhibited the proliferation, arrested cell cycle distribution, and triggered apoptosis in HeLa cancer cells. Our molecular mechanic studies revealed NADA degraded the levels of E6/E7 oncoproteins through ROS-mediated ubiquitin-dependent proteasome system activation. This is the first report that demonstrates antimycin alkaloids analogue induces the degradation of high-risk HPV E6/E7 oncoproteins and finally induces apoptosis in cervical cancer cells. The present work suggested that these analogues could serve as lead compounds for the development of HPV-infected cervical cancer therapeutic agents, as well as research tools for the study of E6/E7 functions.Four new antimycin alkaloids ( E6/E7, have a close relationship with reactive oxygen species (ROS). ROS inducers can increase the enzymatic activities of the ubiquitin-conjugation system, and enhance the level of Ub-conjugates13Cervical cancer is the second most common malignancy among women, which is primarily resulted from humanpaplilloma virus (HPVs) infections, such as HPV-16 and HPV-1891011The sustained activation of cancer cell survive signaling through p53 and Rb, makes high-risk HPV E6/E7 oncoproteins potential drug targets for the treatment of cervical cancerStreptomyces sp. strain THS-55, was isolated from the marine sediment collected at the mangrove conservation in Hainan province, China. The crude extract of THS-55 showed a significant cytotoxicity against HeLa cells (with an inhibitory rate 79.4% at the concentration of 100 μg/mL) and an interesting HPLC profile. Further analysis of the crude extract led to the isolation and structural elucidation of four new antimycin alkaloids (1–4) and six known analogues including N-acetyl-deformylantimycin A , deformylated antimycin A2a (6), deformylated antimycin A1a (7)18 (8)6a (9)4a (10)6 and 7 were identified as natural products for the first time. The chemical structures of these isolated compounds are shown in We have studied the bioactive secondary metabolites from marine-derived actinomycetes1 was established as C24H32N2O9 based upon the observation of HRESIMS ion peak at m/z 493.2189 [M + H]+ . The 1D NMR data suggested the presence of five methyls, three methylenes, eight methines and eight quaternary carbons. The 1H and 13C NMR data  to C-8′ (δC 168.7)  . Therefo2) and G (3) have the same molecular formula C26H36N2O9, which were established on the basis of the HRESIMS analysis of ions at m/z 521.2487 [M + H]+ and 521.2482 [M + H]+, respectively. Compounds 2 and 3 were isolated as a mixture with the ratio (3:1). These two compounds were further separated by a chiral-phase column . Comparison of the NMR spectra of 2 with those of 1 revealed that the acetyl group attached to 8-O in the structure of 1 was replaced by an isobutyryl group in 2, which was further confirmed by analysis of the 1H-1H COSY and HMBC correlations of 2  and NADA (5) were also obtained as a mixture by a C18 semi-preparative HPLC column. Both of them were assigned the same molecular formula C29H42N2O9 on the basis of the HRESIMS analysis . The 1H and 13C NMR spectra of new compound 4 was very similar to those of antimycins A1aδH 2.24, δC 24.9), which was confirmed by the HMBC correlation (3-9′ (δH 2.24) to C-8′ (δC 168.7). Thus, the structure of 4 was determined as the acetylated version of antimycins A1a. Compounds 4 and 5 were separated from their mixture using a chiral-phase HPLC column .Antimycins H  and the NOESY correlations between H-3 and H-4, and between NH and H3–11  with those of the synthetic 2″′S-antimycin A3a (δH 1.190) and 2″′R-antimycin A3a (δH 1.197) which contained the same isovaleric acid side chainTo determine the absolute configurations of 6–7, had been previously obtained artificially by treating the antimycins with either hot hydrochloric acid or diisobutylaluminum hydrideThe deformylated antimycins, compounds 1–10 exhibit different cytotoxicity against HeLa cell line. Most of their IC50 values were in the nanomolar range with better activity than the commercial antimycin A (AMA)  analysis indicated that the acyl group (formyl or acetyl) attached to 3′-NH might be important for HeLa cytotoxicity, and the poor activity of compound 6 and 7 was because these are amine at physiological pH ammonium ions. Furthermore, a long group at R2 was clearly good for bioactivity and R1 seemed less important. Considering its potent activity against HeLa cell line, we selected NADA, the most potent compound against HeLa cells, to perform detailed cytotoxicity and mechanism studies. Besides NADA, other isolated compounds also showed different cytotoxicity towards other selected cell lines (Compound  A (AMA) . NADA anll lines .50 values of NADA against HeLa cells were 1.22, 0.11, and 0.02 μM after 24, 48, and 72 h treatment, respectively, presenting a concentration- and time-dependent manner . Interestingly, NADA also showed relative more potent cytotoxic effect against HPV-positive Siha and CaSki cells, while less cytotoxicity towards HPV-negative cell lines . The cytt manner . The cytt manner . In ordet manner .In order to analyze NADA stimulated apoptosis in HeLa cells, we monitored the morphological changes of nuclear chromatins after Hoechst 33342 staining. As shown in 2 consumption and mitochondrial respiration of HeLa cells in the presence of NADA. We found that, as the positive control AMA, NADA could inhibit the extracellular O2 consumption and subsequently disrupt the mitochondrial respiration of HeLa cells  probe. The percentage of the depolarized mitochondria was about 7% in the control group. When exposure to 0.05 and 0.1 μM NADA, the percentage of cells with depolarized MMP elevated to 19.11 and 26.84%, respectively, in a concentration-dependent manner , suggestLa cells .Considering HPV E6/E7 play a key role in the survival and the malignant phenotype of HPV-infected cancer cells, such as HeLa6338Under normal conditions, E6/E7 usually degrade via the UPS37It has been shown that ROS has a close relationship with the activity of UPSStreptomyces sp. and an inhibitor of mitochondrial electron transport chain that induces extra ROS in biological systems414346Streptomyces sp. THS-55. All these compounds showed cytotoxicity against HeLa cells, and the primary structure-activity relationship showed that the acyl group linked to 3′-NH seems to be essential to the cytotoxicity. Nonetheless, this hypothesis needs additional experimental evidences such as deformylation of these compounds and then reevaluation of their activities. The most active compound NADA possessed proliferation-inhibitory and proapoptotic effects in HeLa cells. Our results further revealed that HPV-positive cancer cells were more sensitive to NADA than the other HPV-negative cell lines  as an internal standard. ESIMS was measured on a Micromass Q-TOF Ultima Global GAA076 LC mass spectrometer. HRESIMS was measured on a Micromass EI-4000 (Autospec-Ultima-TOF). Semi-preparative HPLC was performed using an ODS column . Racemic mixtures were resolved on Chiralpak IA and Chiralpak IB columns . Column chromatography (CC) was performed on silica gel (200–300 mesh) and Sephadex LH-20.IR spectra were recorded on a Nicolet NEXUS 470 spectrophotometer in KBr discs. CD spectra were measured on JASCO J-715. UV spectra were recorded on Beckman DU640 spectrophotometer. Optical rotations were obtained on a JASCO P-1020 digital polarimeter. NMR spectra were recorded on an Agilent NMR spectrometer using CDClStreptomyces sp. based on 16S rDNA analysis (Genbank No. KM103736).The strain THS-55 was isolated from the sediment of mangrove, Sanya, China. It was classified as a 2HPO4, 0.05 g MgSO4·7H2O, 0.2 g KBr, 2 g CaCO3, dissolved in 1 liter of seawater, pH 7.0. Each flask was inoculated spores of the organism and incubated on a rotary shaker (180 r.p.m.) at 28 °C for 7 days. After cultivation, 80 L of whole broth was extracted three times with EtOAc. The organic extract was concentrated to dryness in vacuo to afford crude material (25.0 g).The strain THS-55 was cultivated in an Erlenmeyer flask (500 mL) containing 150 mL of a defined medium with the following components: 10 g glucose, 10 g soluble starch, 5 g peptone, 3 g yeast extract, 2 g beef extract, 2 g soy flour, 0.05 g K2Cl2-CH3OH. Fr.3, eluted with CH2Cl2 and 100:1 CH2Cl2-MeOH, was fractionated on a C-18 ODS column using a step gradient elution of MeOH/H2O and was separated into 6 subfractions (Fr.3.1-Fr.3.6). Fr.3.4 was further purified by Sephadex LH-20 (MeOH) and purified by semipreparative HPLC  to yield the compound compound 6 , Fr.3.4.1 and Fr.3.4.2. Fr.3.4.1 and Fr.3.4.2. were further purified by using a chiral-phase HPLC column respectively  to yield the compound 4 , compound 5 , and compound 7 . Fr.3.3 was also purified by as further purified by Sephadex LH-20 (MeOH) and purified by semipreparative HPLC  to yield the compound 1 , compound 8 , compound 9 , compound 10  and Fr.3.3.1. Fr.3.3.1 was further purified by using a chiral-phase HPLC column  to yield the compound 2 , compound 3 .The extract (25.0 g) was applied to a silica gel column chromatography and was separated into six fractions (Fr.1-Fr.6) using a step gradient elution of petroleum ether-acetone and CHAntimycin E (1): White powder; CD  λmax (Δε) 231.5 (+0.90) nm; UV (MeOH) λmax (log ε) 231 (2.74), 316 (0.60) nm; [α]D = +43.6 ; IR (KBr) νmax 3361, 2937, 1733, 1541, 1373, 1233, 1201, 754 cm−1; 1H and 13C NMR spectral data, see m/z 493.2189 [M + H]+ .Antimycin F(2): White powder; CD  λmax (Δε) 230.5 (+0.96) nm; UV (MeOH) λmax (log ε) 231 (2.95), 316 (0.71) nm; [α]D = +34.8 ; IR (KBr) νmax 3291, 2958, 1748, 1540, 1373, 1182, 1145, 749 cm−1; 1H and 13C NMR spectral data, see m/z 521.2487 [M + H]+ .Antimycin G (3): White powder; CD  λmax (Δε) 230.5 (+1.12) nm; UV (MeOH) λmax (log ε) 231 (2.95), 316 (0.71) nm; [α]D = +33.5 ; IR (KBr) νmax 3291, 2958, 1748, 1540, 1373, 1182, 1145, 749 cm−1; 1H and 13C NMR spectral data, see m/z 521.2482 [M + H]+ .Antimycin H (4): White powder; CD  λmax (Δε) 234.0 (+1.37) nm; UV (MeOH) λmax (log ε) 233 (2.81), 316 (0.64) nm; [α]D = +46.2 ; IR (KBr) νmax 3356, 2930, 1748, 1540, 1374, 1178, 1144, 753 cm−1; 1H and 13C NMR spectral data, see m/z 563.2947 [M + H]+ .N-acetyl-deformylantimycin A (5): White powder; CD  λmax (Δε) 234.0 (+1.27) nm; UV (MeOH) λmax (log ε) 233 (2.81), 316 (0.64) nm; [α]D = +42.2 ; IR (KBr) νmax 3356, 2930, 1748, 1540, 1374, 1178, 1144, 753 cm−1; 1H and 13C NMR spectral data, see m/z 563.2950 [M + H]+ . The raw NMR charts of these new compounds 1–5 are presented in Deformylated antimycin A2a(6): White powder; UV (MeOH) λmax (log ε) 231 (2.68), 316 (0.62) nm; [α]D = +17.7 ; IR (KBr) νmax 3385, 2931, 1748, 1540, 1457, 1184, 1144, 741 cm−1; 1H and 13C NMR spectral data, see + .Deformylated antimycin A1a(7): White powder; UV (MeOH) λmax (log ε) 231 (2.84), 316 (0.67) nm; [α]D = +19.6 ; IR (KBr) νmax 3566, 2925, 1748, 1541, 1457, 1186, 1143 cm−1; 1H NMR spectral data, see m/z 521.3 [M + H]+.Antimycin A18(8): White powder; UV (MeOH) λmax (log ε) 231 (2.72), 316 (0.63) nm; [α]D = +34.0 ; IR (KBr) νmax 3545, 2958, 1749, 1540, 1457, 1184, 1038 cm−1; ESIMS m/z 479.3 [M + H]+.Antimycin A6a(9): White powder; UV (MeOH) λmax (log ε) 231 (2.85), 316 (0.72) nm; [α]D = +28.0 ; IR (KBr) νmax 3366, 2933, 1748, 1540, 1457, 1362, 1185, 1144, 749 cm−1; ESIMS m/z 479.1 [M + H]+.Antimycin A4a(10): White powder; UV (MeOH) λmax (log ε) 231 (2.96), 316 (0.66) nm; [α]D = +32.0 ; IR (KBr) νmax 3366, 2933, 1748, 1540, 1557, 1362, 1181, 1144, 747 cm−1; ESIMS m/z 507.4 [M + H]+.Cell viability were assayed by 3--2, 5-diphenyltetrazolium bromide (MTT) methodsHeLa cells were plated onto 6-well plates (800 cells/well). After 24 h, NADA (0–2.56 μM) was added and incubated for 12 days. Cells were washed, fixed in methanol, and stained with Giemsa. Finally, colonies were scored and photographed.HeLa cells were seeded in six-well plates and incubated with NADA (0–0.1 μM) for 24 h, and then stained with Rho 123 (3 μg/mL), and the MMP were analyzed2 consumption assay kit , according to the manufacture’s protocols.HeLa cells were seeded 40,000/well. After 24 h, the inhibition on the mitochondrial respiration was assayed using the extracellular OHeLa cells were incubated with NADA (0–0.1 μM) for 24 h. After that, the cells were collected and washed in PBS and fixed in ice-cold 70% (v/v) ethanol overnight at −20 °C. The samples were prepared and analyzed as described previouslyHeLa cells were treated with NADA (0–0.1 μM) of for 24 h, the cells were washed twice with PBS and stained with Hoechst 33342 (5 μg/mL) for 30 min at 4 °C in the dark. Finally, the nuclei of apoptotic cells were detected by fluorescence microscopy.For immunoprecipitation, after treatment, HeLa cells were lysed. The supernatants were incubated with HPV-18 E6 antibody and protein A-Sepharose beads at 4 °C overnight. Subsequently, beads were washed and boiled in loading buffer for 12 min, and then, proteins were separated by 15% SDS-PAGE gels for ubiquitination analysis and detection of E6 protein with western blotting analysesHeLa cells were treated with 0.2 μM NADA for 2.5, 5, and 10 h, respectively. In another way, cells were pretreated, or not, with 2.5 mM, 5 mM Cat for 1 h followed by 0.2 μM NADA for 6 h. Then, cells were incubated with CM-H2DCFDA probes for 20 min and analyzed by flow cytometry.P < 0.05 or P < 0.01 were considered statistically significant.One-way ANOVA with Tukey’s post hoc test was implemented for all statistical analysis, and values were expressed as mean ± SD. Differences of How to cite this article: Zhang, W. et al. Marine Streptomyces sp. derived antimycin analogues suppress HeLa cells via depletion HPV E6/E7 mediated by ROS-dependent ubiquitin-proteasome system. Sci. Rep.7, 42180; doi: 10.1038/srep42180 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The packing motifs in the title compounds both feature C—H⋯O interactions but they show distinctly different Hirshfeld surface fingerprints. 9H8O3S, (I), and 2-oxo-1,3-benzoxathiol-6-yl acetate, C9H6O4S, (II), are described. Compound (I) is almost planar (r.m.s. deviation for the non-H atoms = 0.011 Å), whereas (II) shows a substantial twist between the fused-ring system and the acetate substituent [dihedral angle = 74.42 (3)°]. For both structures, the bond distances in the heterocyclic ring suggest that little if any conjugation occurs. In the crystal of (I), C—H⋯O hydrogen bonds link the molecules into [1-11] chains incorporating alternating R22(8) and R22(12) inversion dimers. The extended structure of (II) features C(7) [201] chains linked by C—H⋯O hydrogen bonds, with further C—H⋯O bonds and weak π–π stacking interactions connecting the chains into a three-dimensional network. Hirshfeld fingerprint analyses for (I) and (II) are presented and discussed.The crystal structures of 6-methoxy-1,3-benzoxathiol-2-one, C The rini  link generates inversion dimers featuring ii  bond is much weaker, but if it is considered significant, it generates a second inversion dimer , with adjacent molecules in the chain related by c-glide symmetry (plus translation)  1 − x, −y, 1 − z] bonds arising from the methyl group generate inversion dimers [In the crystal of (II)s Table 2 link then) Fig. 4. The C9—CrystalExplorer17 et al., 2007i.e. van der Waals interactions) are far more prominent in (I)When the fingerprint plots are decomposed into the separate types of contacts -4-benzenesulfonamide  as recently reported by Terra et al. Uiso(H) = 1.2Ueq(carrier) or 1.5Ueq(methyl carrier) was applied in all cases. The methyl groups were allowed to rotate, but not to tip, to best fit the electron density.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989017018072/nk2242sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989017018072/nk2242Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017018072/nk2242Isup4.cmlSupporting information file. DOI: 10.1107/S2056989017018072/nk2242IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989017018072/nk2242IIsup5.cmlSupporting information file. DOI: 1404758, 1404756CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The structures of the new isolates were determined through spectroscopic data analysis. Compounds 1–9, as well as compounds 10–18 previously isolated from the species, were measured for the activity against platelet aggregation induced by ADP or collagen. Three neoligans , one flavanone (14), and one alkaloid (16) showed inhibitory activity against ADP- or collagen-induced platelet aggregation as compared with tirofiban. The dihydrobenzofuran neolignans  are more potent than the benzofuran neolignan (13) and other types of neolignans (1–7). Glucosidation of the dihydrobenzofuran neolignans (11 and 12) is helpful for the activity.Two new neolignans selaginellol (1) and selaginellol 4′-O-β-d-glucopyranoside (2) were isolated from the whole plant of Selaginella moellendorffii. Several compounds from this plant showed the activity against platelet aggregation induced by ADP or collagen.Two new neolignans selaginellol (The online version of this article (doi:10.1007/s13659-016-0095-5) contains supplementary material, which is available to authorized users. Selaginella Beauv. Selaginella is a nearly worldwide genus of about 700 species, with 72 of them in China and more than 20 species used in traditional Chinese medicine + ion at m/z 415.1729 appropriate for a molecular formula of C21H28O7 requiring eight sites of unsaturation. The IR absorption signals revealed the presence of hydroxy (3428 cm−1) and aromatic  groups. The 1H NMR data of 1  and 3.70 ], and two 1,2,3,5-tetrasubstituted benzene rings . The 13C NMR data of 1 (Table δC 56.5 × 2 and 56.4), two phenyl rings, five methylenes including two oxygenated ones , and one methine (δC 45.4). According to above NMR signal characteristics  with that of a known analogue secodihydrodehydrodiconiferyl alcohol tetraacetate [The ons Fig.  exhibiteons Fig.  from H-2m/z 554.2365 [M]+ , the molecular formula of compound 2 was determined as C27H38O12 with nine degrees of unsaturation. The IR absorption signals showed the presence of hydroxy (3425 cm−1) and aromatic  groups. The NMR data ; δC 105.6, 75.9, 77.8, 71.1, 78.0, and 62.4] were observed. As demonstrated in the 1H–1H COSY, HMBC and ROESY correlations . The HMBC correlation from H-1″ to C-4′ indicated that the β-glucopyranosyl part was located at C-4′. According to our previously acidic hydrolysis of rel--3,3′,5-trimethoxy-4′,7-epoxy-8,5′-neoligna-4,9,9′-triol 4-O-β-d-glucopyranoside (11) [d-glucose. The absolute configuration of the aglycone was elucidated to be the same as that of selaginellol (1) by comparison of its ECD spectrum [Δε −0.37 (273)] with that of 1. Therefore, compound 2 is selaginellol 4′-O-β-d-glucopyranoside.According to the HREIMS ion at 3) +; positive ion HRESIMS m/z 415.1729 [M+Na]+ .Pale yellow oil (MeOH); [α]) 280 3.3, 228 ; UV (CH3OH) λmax (log ε) 274 (3.80) nm; ECD Δε  −0.37 (273), −6.17 (210), +3.20 (200); IR (KBr) vmax 3425, 1615, 1518, 1461, 1428, 1325, 1216, 1113, 1071 cm−1; 1H and 13C NMR data, see Table m/z 577 [M+Na]+; HREIMS m/z 554.2365 [M]+ .Pale yellow solid (MeOH); [α] Δε c 0.01, MeOH −8 platelets/mL. PRP in 270 μL was preincubated at 37 °C for 5 min in the cuvette with 20 μL of sample or vehicle , and then platelet aggregation was induced by 10 μL ADP (10 μM) or collagen (2.5 μg/mL). The maximum platelet aggregation rate was determined within 5 min with continuous stirring at 37 °C using four-channel aggregometer .In vitro platelet aggregation was conducted using the turbidimetric method with a minor modification , 17. Bri50 value was calculated as the concentration of inhibitor causing a 50 % inhibition of the aggregation using SPSS software.For each compound, five concentrations were chosen and a percentage inhibition-concentration curve was derived. From this curve the ICSupplementary material 1 (PDF 782 kb)Below is the link to the electronic supplementary material.


AbstractLongidoruschenisp. n., an amphimictic species recovered from the rhizosphere of Larixprincipis-rupprechtii and Pyracanthafortuneana in Shanxi and Beijing, China, is described and illustrated. The taxonomic position of L.chenisp. n. among other species within the genus was elucidated using morphometric and molecular data, and phylogenetic relationships were inferred using D2–D3 expansion domains of 28S and 18S rRNA genes by Bayesian Inference (BI) method. The new species is characterised by females with a medium body size (L = 4.9–6.6 mm), a lip region slightly expanded, broadly rounded frontally and laterally, the amphidial fovea broad and symmetrically bilobed at base, odontostyle long and slender (143–168 μm), odonthophore slightly swollen at the base, tail short bluntly conoid to rounded. Guide ring located far posterior from the oral aperture (70–93 μm). Males with two ad-cloacal pairs of supplements preceded by a row of 10–14 ventromedian supplements, with robust spicules measuring 111–126 μm along the median line. Three juvenile stages were present, tail shape of J1 elongate conoid while in J2 and J3 the tail gradually becomes bluntly rounded. Codes for identifying the new species are: A6-B3-C5-D2-E2-F3-G1-H1-I2-J2-K2. Longidoruschenisp. n. belongs to a group of species with a guide ring at the mid-odontostyle position that have a predominantly Asiatic origin. It differs from all of them by a combination of morphological characters and unique sequences of partial 18S and D2–D3 region of 28S rRNA genes. The percentage dissimilarities in partial 18S and D2–D3 28S rRNA genes of L.cheni to the closest species  were 1.5 %–1.8 % and 16.8–18.3 %, respectively.PageBreak Longidoruselongatus  was described almost one hundred and fifty years ago) continue to attract the attention of scientists due to their high species diversity, wide distribution, and economic importance. The valid Longidorus Micoletzky, 1922 species described to date reached 167  from Beijing, China. Five hundred grams (500 g) of soil were mixed and washed using a decanting and sieving technique (Brown and Boag 1988). The extract was left for two days on a Baermann funnel and the suspension was collected afterwards. Collected nematodes were examined under a stereomicroscope and Longidorus specimens were picked out and transferred to Syracuse dishes for storage. For morphometric studies the nematodes were killed, fixed with hot formalin, and processed PageBreakto glycerine . PCR tubes were centrifuged at 12000 rpm for 2 minutes and immediately frozen at -70 °C for at least 30 minutes. Subsequently, each tube was incubated for 65 °C for 3 hours and nematode was digested at 75 °C for 60 minutes and 95 °C for 10 minutes. Finally, the DNA suspensions were cooled down at 8 °C and stored at -20 °C until use. A total of 25 µl PCR mixture was prepared containing 2.5 µl LA buffer, 2 µl dNTP, 1.5 µl each primer  and 3 µl DNA template, 0.3 µl LATaq and 14.2 µl distilled water. All PCR reactions were conducted in the S1000 thermal cycler (BIO-RAD). Fragments of 18S and 28S region were amplified using two sets of primers: forward primer SSU_F_07 (5’ AAA GAT TAA GCC ATG CAT G 3’) and reverse primer SSU_R_81 (5’ TGA TCC ACC TGC AGG TTC AC 3’)   and forwC AC 3’) , respecthttp://guidance.tau.ac.il/ with default parameters (BI) algorithm implemented in MrBayes 3.2.5 was used for phylogenetic relationships reconstructions  57 sequences and 700 total characters for D2–D3 28S rRNA gene and b) 48 sequences and 993 total characters for 18S rRNA gene.The D2–D3 28S and 18S rDNA sequences were compared with those of other nematode species deposited in GenBank database using BLASTn similarity search tool. The homologous sequences nearest to those of the new species were aligned using the GUIDANCE2 Server available at rameters  and manurameters . Bayesiaructions . For furTaxon classificationAnimaliaNematodaLongidoridaehttp://zoobank.org/AD7993D5-AB3D-4436-863D-2D469EEE49CATwelve females, twelve males, fifty-two juveniles (J1-J3) from Shanxi province and ten females, four males, thirty juveniles (J1-J3) from Beijing.Measurements  distinctly separated by differences in the body length, odontostyle and replacement odontostyle length  was 844 bps and 856 bps, respectively. The sequences of both populations were identical. The phylogenetic relationships of L.cheni sp. n. with the closest species inferred from analyses of the partial 18S rDNA and D2–D3 expansion segments of 28S rDNA sequences using BI are presented in Figs Longidorus species of predominantly Asiatic origin in both phylogenetic reconstructions. In D2–D3 rDNA phylogenetic tree, L.cheni clustered in a PageBreakwell-supported clade comprising four species from China (L.juglans (MF318878), L.fangi (MF318883-84), Longidorus sp. (KF280150); one from Japan (L.jonesi (KF552069) and two species from North America, USA (L.diadecturus (AY601584) and Longidorus sp. (KF242342-43)). With exception of the species for which there are no descriptions, all mentioned species have a guide ring at mid-odontostyle area. Similarly, in the 18S rDNA phylogenetic reconstruction L.cheni sp. n. clustered with the same group of species  and L.litchii (AY687996) that has no D2–D3 rDNA sequence deposited in GenBank. The percentage dissimilarities of L.cheni to the closest species L.litchii, L.fangi, and L.juglans in 18S rRNA gene were 1.5 %, 1.8 %, and 1.8 %, respectively . Much higher were the pairwise percentage distances of L.cheni sp. n. to the closest species in D2–D3 28S rRNA gene ranging from 16.8–16.9 % (L.fangi and L.jonesi) to 18.3 % (L.juglans).The length of PCR products based on gel images of the amplification of partial 18S and D2–D3 region of 28S RNA genes of L.principis-rupprechtii) and Chinese firethorn (P.fortuneana) in mountainous region of Shanxi and botanic garden in Beijing, China, GPS coordinates 37°50'815"N, 111°27'253"E and 30°34'54.7"N, 114°15'40.9"E, respectively.Specimens were recovered from soil around the roots of a conifer  includes 12 females, 12 males and 52 juveniles deposited in the Nematode collection C602 Nematology laboratory of Zhejiang University, Hangzhou, China. One female, one male, three juveniles deposited at the nematode collection of the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria.The species is named after Prof. Pinsan Chen, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, one of the pioneer plant nematologists in China.Longidoruscheni sp. n. is an amphimictic species characterized by females with medium body size (L = 4.1–6.6 mm), assuming G-shape, lip region 16–23 μm wide, posteriorly situated guide ring (at 70–93 μm from PageBreakanterior end), long odontostyle (142–168 μm), odontophore base slightly swollen, tail short (24–33 μm) and bluntly conoid to rounded. Males abundant, spicules 111–123 μm long, ventromedian supplements 10–14. Three juvenile stages present. The tail of the first stage juvenile conoid, tail shape in the second and third stage juveniles gradually becoming rounded. Finally, the species have specific ribosomal sequences KY284157 and KF270638 for D2–D3 expansion domains of 28S rDNA, KF261570 and MG656980 for the 18S rDNA region. The identification codes of L.cheni sp. n. based on the polytomous key by Longidoruscheni sp. n. belongs to a group of species (L.jonesi-group) having guide ring at mid-odontostyle area  Xu & Hooper, 1990, L.ishigakiensis Hirata, 2002, L.jagerae Heyns & Swart, 1998, L.jonesi Siddiqi, 1962, L.juglans, L.laricis Hirata, 1995, L.litchii, L.macromucronatus Siddiqi, 1962, L.martini Merny, 1966, L.naganensis Hirata, 1995, L.orongorongensis Yeates, Van Etteger & Hooper, 1992, and L.waikouaitii Yeates, Boag & Brown, 1997 , males abundant vs males absent , different amphidial fovea shape (bilobed vs non-bilobed) (bilobed) ;L.laricis by females having a smaller a ratio (45.7–63.1 vs 83–108), males abundant vs males rare, longer spicules (101–124 vs 66.2 μm), different tail shape in J1  (1.8–2.4) ;L.litchii by females having a smaller a ratio (45.7–63.1 vs 72–84), wider lip region (17.5–23 vs 12.5–14 μm), smaller V ratio (40–49.4 vs 49–54), longer spicules (101–124 vs 68.5–71 μm), number of ventromedian supplements (10–14 vs 6–7), number of stages (3 vs 4), different tail shape in J1  (vs strongly flanged (72–3.42) , odontop flanged ;L.fangi by females having a smaller a ratio (45.7–63.1 vs 81–98), amphidial fovea shape (bilobed vs non-bilobed), longer odontostyle (142–168 vs 124–144 μm), lower c’ ratio in J1 (c’ = 1.02–1.46 vsc’ = 1.58–2.2) (.58–2.2) ;L.fursti by females having a smaller a ratio (45.7–63.1 vs 105–137), wider lip region (17.5–23 vs 14.5–16 μm), different amphidial pouch shape (bilobed vs non-bilobed), longer odontostyle (142–168 vs 99.5–108 μm), smaller V ratio (40–49.4 vs 51.5–53.6), lower c’ ratio in J1 (c’ = 1.02–1.46 vsc’ = 2.84–2.93) (84–2.93) ;L.himalayensis by females having a longer (L = 4.1–6.6 mm vs L = 3.42–3.9) and more plump body (a = 45.7–63.1 vsa = 97.8–112), a wider lip region (18–23 vs 14–15 μm), longer odontostyle (142–168 vs 115–125 μm), more posteriorly situated guide ring (70–91 vs 55–60 μm) (5–60 μm) ;L.ishigakiensis by females having a smaller a ratio (45.7–63.1 vs 106–130), wider lip region (18–23 vs 13–14 μm), different amphidial pouch shape (bilobed vs non-bilobed), smaller c’ ratio (c’ = 0.62–0.86 vsc’ = 1.0–1.2), different tail shape in J1 , males abundant vs males absent , more plump body (a = 45.7–63.1 vsa = 89–107), longer odontostyle (142–168 vs 95–109 μm), more anteriorly situated vulva (V = 40.0–49.4 vsV = 51.5–56.3), prerectal inclusions (absent vs present) ;L.jonesi by females having a longer body (L = 4.1–6.6 vs L = 3.17–3.8 mm) and odontostyle (142–168 vs 107–120 μm), more posteriorly situated guide ring (70–91 vs 57–66 μm), more anteriorly situated vulva (V = 40.0–49.4 vsV = 50–52.4) (50–52.4) ;L.martini by females having a longer body (L = 4.1–6.6 vs L = 3.18–4.29 mm) and odontostyle (142–168 vs 83–96 μm), more posteriorly situated guide ring (70–91 vs 51–66 μm), more anteriorly situated vulva (V = 40.0–49.4 vsV = 52–56) (= 52–56) ;L.diadecturus by females having a longer body (L = 4.1–6.6 vs L = 3.32–4.02 mm), odontostyle (143–168 vs 109–121 μm) and pharyngeal bulb (107–138 vs 62–83 μm), more posteriorly situated guide ring (70–91 vs 50–64 μm) (0–64 μm) ;L.orongorongensis by females having a shorter and more plump body , more posterior guide ring poPageBreaksition (70–91 vs 63–73 μm), smaller V ratio (40–49.4 vs 49–54), longer spicule (101–104 vs 84–87 μm) (4–87 μm) ;L.macromucronatus by females having a plumper body (a = 45.7–63.1 vsa = 94–105), a wider lip region (17.5–23 vs 14 μm), longer odontostyle (142–173 vs 117–128 μm), 3 vs 4 juvenile stages, differently shaped tail in J1 (broadly conoid vs sub-digitate c’ = 1.02–1.46 vsc’ = 0.63–0.8) (.63–0.8) ;L.waikouaitii by having differently shaped amphidial fovea , a longer odontostyle (142–173 vs 113–117 μm), more posterior position of the guide ring (70–91 vs 56.5–59.5 μm), males abundant vs males absent .L.cheni sp. n. are in agreement with the hypothesis about the common origin of Longidorus species having a guide ring at the mid-odontostyle area (L.litchii), all of them occurring in South East Asia , North India (three), New Zealand (two), South Africa , North America , and only one species (L.jonesi) reported from two regions . The highest number of species in South East Asia suggests this region as a probable centre of origin of Longidorusjonesi-group.Our findings on the morphology and genetics of yle area ; furtheria Table . This sh


Malbranchea cinnamomea strain FCH 10.5, isolated from compost at a waste treatment plant in Vietnam. The genome sequence contains 24.96 Mb with an overall GC content of 49.79% and comprises 9,437 protein-coding genes.We report here the annotated draft genome sequence of the thermophilic biomass-degrading fungus  Malbranchea cinnamomea is a thermophilic fungus belonging to the order Onygenales 2SO4, 0.8 g liter−1 CaCl2-2H2O, 0.6 g liter−1 MgSO4-7H2O, 6 g liter−1 Bacto peptone, 10 mg liter−1 FeSO4-7H2O, 3.2 mg liter−1 MnSO4-H2O, 2.8 mg liter−1 ZnSO4-7H2O, 4 mg liter−1 CoCl2−6H2O, 200 mL liter−1 Tween 80, and 20 g liter−1 glucose. DNA for sequencing was extracted with cetyltrimethylammonium bromide (CTAB) buffer  and purified from the supernatant by a combination of phenol-chloroform extraction and isopropanol precipitation  was used according to the manufacturer’s protocol for library preparation. Quality and yield after sample preparation were measured using Bioanalyzer (Agilent Technologies).Clustering and DNA sequencing using the Illumina cBot and HiSeq 2500 systems were performed according to the manufacturer’s protocols using a concentration of 8.0 pM of DNA, standard Illumina primers, and HiSeq control software HCS version 2.2.58. Image analysis, base calling, and quality checking were performed with the Illumina data analysis pipeline RTA version 1.18.64 and Bcl2fastq version 1.8.4.k-mer length of 64. Scaffolds shorter than 500 bp were removed.The 250-nt paired-end reads were trimmed for adapter sequences and filtered for sequence quality using our in-house tool FASTQFilter version 2.05. The short-read genome assembler based on De Bruijn graphs, Abyss version 1.3.7 . Furthermore, mapped mRNA-Seq reads were used by the CodingQuarry (For gene finding, two approaches were used and combined to obtain the presented data. The HMM-based algorithm Glimmer version 3.02  was traingQuarry  softwareM. cinnamomea FCH 10.5 whole-genome sequence has been deposited in DDBJ/EMBL/GenBank under the assembly accession no. FQSS00000000. The version described in this paper is the second version, FQSS02000000.The


Their structures were established on the basis of extensive spectroscopic analysis. Compounds 1 and 4 exhibited weak cytotoxic activity against five human cancer cell lines .Four hitherto unknown polysubstituted isoflavonoids, including three isoflavans: 7,4′-dihydroxy-8,2′,3′-trimethoxyisoflavan (The online version of this article (doi:10.1007/s13659-017-0121-2) contains supplementary material, which is available to authorized users. The 13C NMR spectrum (Table δC 31.4 (d), 31.5 (t), 70.5 (t), and three methoxy carbons at δC 60.6, 60.8, 61.0 (each q). The above NMR spectroscopic features were very similar to those of isoduartin  + . Comparison of the 1H and 13C NMR spectral data  to C-1′ (δC 119.9), C-2′ (δC 147.1), and C-3′ (δC 134.5), we could conclude that the emerging hydroxy was positioned at C-2′ . Five diagnostic ring C proton signals , an ABX-type aromatic proton system , two meta-coupled aromatic doublets [δH 5.88  and 5.99 ], and one methoxy signal [δH 3.74 ] were observed in the 1H NMR spectrum  confirmed the methoxy group at C-5  were established by comparison of the CD curves with those reported earlier for closely related analogues. All the CD spectra showed a negative Cotton effect in the transition region (215–240 nm), similar with those of 7-O-methylisomucronulatol, (3R)-isomucronulatol, and abruquinone L , a prenyl group , and a hydroxyethyl moiety  were also detected in the 1H NMR spectrum. The 13C NMR spectrum displayed a total of 22 carbon resonances, including two methyls, three sp3 methylenes, five sp2 methines, and 12 sp2 quaternary carbons. These spectroscopic features were very similar to those of cudraisoflavone L, which was recently isolated from the same genus and shared the same molecular formula with 4 D25 +11.0 ; IR (KBr) νmax: 3396, 2944, 1602, 1497, 1464, 1292, 1198, 1170, 1097, 1068, 1040, 1010, 963 cm−1; 1H NMR data: see Table 13C NMR data: see Table m/z 355 [M+Na]+; HRESIMS (pos.): m/z 355.1163 [M+Na]+ .White amorphous powder; UV (MeOH) λmax: 229 (sh), 277 nm; [α]D25 −13.2 ; IR (KBr) νmax: 3404, 2929, 1619, 1509, 1470, 1328, 1184, 1091, 1058, 1037, 1001, 804 cm−1; 1H NMR data: see Table 13C NMR data: see Table m/z 341 [M+Na]+; HRESIMS (pos.): m/z 341.1002 [M+Na]+ .White amorphous powder; UV (MeOH) λmax: 230 (sh), 279 nm; [α]D25 −24.0 ; IR (KBr) νmax: 3406, 2937, 1620, 1606, 1520, 1501, 1472, 1459, 1200, 1142, 1118, 1037, 974, 819 cm−1; 1H NMR data: see Table 13C NMR data: see Table m/z 289 [M+H]+; HRESIMS (pos.): m/z 289.1075 [M+H]+ .White amorphous powder; UV (MeOH) λmax: 212, 270, 346 (sh) nm; IR (KBr) νmax: 3425, 2926, 1644, 1582, 1522, 1435, 1380, 1306, 1209, 1116, 1083, 1030 cm−1; 1H and 13C NMR data: see Table m/z 399 [M+H]+; HRESIMS (pos.): m/z 399.1440 [M+H]+ .Yellowish amorphous powder; UV (MeOH) 2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of MTS . Briefly, 100 μL of adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 × 105 cells/mL in 100 μL medium. Each cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cisplatin and paclitaxel as positive controls. After the incubation, 20 μL MTS and 100 μL medium was added to each well after removal of 100 μL medium, and the incubation continued for 2–4 h at 37 °C. The optical density was measured at 492 nm using a Multiskan FC plate reader . The IC50 value of each compound was calculated according to the Reed and Muench method.Five human tumor cell lines  obtained from ATCC  were used in the cytotoxicity assay. All cells were cultured in RPMI-1640 or DMEM medium , supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere containing 5% COSupplementary material 1 (DOCX 525 kb)Below is the link to the electronic supplementary material.


The title cyano acid forms a honeycomb-like sheet structure, six molecules circumscribing anthracene, with carboxy hydrogen-bonded dimers linked by half of the possible  8H3Br2NO2·0.5C14H10, was self-assembled from a 2:1 mixture of the components in slowly evaporating dichloromethane. The molecules adopt a sheet structure parallel to (1-12) in which carboxy hydrogen-bonded dimers and anthracene molecules stagger in both dimensions. Within the sheets, six individual cyano acid molecules surround each anthracene molecule. Cyano acid molecules form one of the two possible R22(10) rings between neighboring cyano and bromo groups. Compared to the dichloro analog [Britton °.The title co-crystal, Ctton 2012. J. Chem In 2012, he found that the cyano acid molecules alone do not pack in this way, but slow evaporation of mixtures containing naphthalene or anthracene afforded 2:1 acid:hydrocarbon co-crystals roughly matching his proposed structure . Anthracene was the better fit, although it was too large to allow the ideal molecular arrangement. There is no obvious substitute for anthracene or naphthalene. Thus, we have prepared anthracene co-crystals with the dibromo analog in hopes that the larger Br bond and contact radii might close the CN⋯X gaps observed with Cl.Doyle Britton (1930–2015) published roughly 30 crystallographic articles on solid-phase cyano–halo interactions from variously substituted halobenzonitriles and isocyanides. Britton postulated that 3,5-dichloro-4-cyanobenzoic acid might assemble into a honeycomb-like sheet structure Fig. 1a via a The benzene (C2–C5/C7/C8) and anthracene . Overall, substitution of Cl atoms with Br atoms has facilitated the formation of half of the envisioned CN⋯X short contacts and also improved the coplanarity of the acid and hydrocarbon molecules, but anthracene is slightly too large to allow the ideal arrangement of cyano acid molecules. It is possible that upon substitution of Br atoms with I atoms, the improvements would continue and the envisioned sheet structure might occur. This possibility is currently being studied in our laboratory.The dihedral angle between the benzene and anthracene planes is 0.9 (2)°, which is much lower than 7.1° of the dichloro analog. As expected, d Table 1; these as Table 2; howevers Table 2. Insteads Table 2. In the s Table 2, whereass Table 2b. Overaet al., 2016i.e., Dubey & Desiraju, 2014et al., 2012et al., 1988et al., 2014A search of the Cambridge Structural Database : Bromine (3.9 mL) and then pyridine (5.7 mL) were added dropwise to ice-cold methanol (35 mL). This mixture was added dropwise to a solution of methyl 4-aminobenzoate + calculated for C8H7Br2NO2 331.8715, found 331.8709.Methyl 3,5-dibromo-4-cyanobenzoate (VI), adapted from the work of Toya et al. (1992Cyanide suspension: NaCN (680 mg) and CuCN (480 mg) and water (40 mL) were combined in a 400 mL beaker. After the solids dissolved, NaHCO3 (6.5 g) was added. The resulting suspension was cooled in an ice bath. Diazotization: Dibromo ester  was ground in a mortar and then combined with acetic acid (2.6 mL) in a round-bottomed flask. H2SO4 (0.6 mL) was added dropwise over 1 min, followed by a solution of NaNO2 (313 mg) in water (1.5 mL) over 30 min.. During the course of the additions, the reaction mixture was gradually warmed in an oil bath to 315 K. Cyanation: When no more starting material remained, as indicated by TLC, the diazotization mixture was removed from the heat and then added dropwise to the cyanide suspension. The ice bath was removed. The cyanation mixture was stirred overnight and then extracted with dichloromethane (3 × 20 mL). The combined organic portions were washed with water (20 mL), brine (20 mL), dried with Na2SO4, filtered, and then concentrated on a rotary evaporator. The resulting brown residue was separated by column chromatography. The desired fraction (Rf = 0.34 in 3:1 hexane:ethyl acetate on SiO2) was concentrated on a rotary evaporator, giving a tan powder . M.p. 410–411 K; 1H NMR  δ 8.264 , 3.974 ; 13C NMR  δ 163.4 (1C), 135.5 (1C), 132.6 (2C), 127.1 (2C), 122.5 (1C), 115.5 (1C), 53.5 (1C); IR  3076, 2955, 2229, 1732, 1429, 1263, 1124, 971, 748; MS  [M+Na]+ calculated for C9H5Br2NO2 341.8559, found 341.8547. al. 1992: Cyanide3,5-Dibromo-4-cyanobenzoic acid (VII), adapted from the work of Lepage et al.  were added. After being stirred for 10 min, the resulting mixture was separated by suction filtration, giving a light-brown powder . M.p. 423–425 K; 1H NMR 2SO) δ 14.120 , 8.232 ; 13C NMR 2SO) δ 163.9 (C1), 137.0 (C2), 132.1 , 126.7 , 120.6 (C5), 115.9 (C6); IR  3421, 3077, 2128, 1811, 1662, 1537, 1371, 1296, 1025, 825, 770, 748; MS  [M–H]− calculated for C8H3Br2NO2 303.8437, found 303.8443.Crystallization: 3,5-Dibromo-4-cyanobenzoic acid (100 mg) and anthracene (29 mg) were dissolved in dichloromethane (25 mL) in a loosely covered beaker. Most of the solvent was allowed to evaporate gradually over 3 d. The resulting colorless or pale-orange plate-shaped crystals were collected after decantation and then washed with several drops of ice-cold 1:3 dichloromethane:pentane.Uiso(H) set to 1.2Ueq(C) and 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017014815/lh5854sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017014815/lh5854Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017014815/lh5854Isup3.cmlSupporting information file. DOI: 1525811CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title molecule is a tetrasubstituted truxinic-type cyclobutane derivative with a central ring that is almost planar despite of being placed in a general position. The molecular structure of the dimer shows that the four benzene rings of the substituents are oriented in such a way that potential steric hindrance is minimized, whilst allowing some degree of intermolecular π–π interactions for crystal stabilization. E)-3-(2-nitrophenyl)-1-phenylprop-2-en-1-one, and, unexpectedly, the title compound, C30H22N2O6, which results from the syn head-to-head [2 + 2] cycloaddition of the chalcone. The molecular structure of the dimer shows that the four benzene rings of the substituents are oriented in such a way that potential steric hindrance is minimized, whilst allowing some degree of intermolecular π–π interactions for crystal stabilization. In the molecule, one nitro group is disordered over two positions, with occupancies for each part of 0.876 (7) and 0.127 (7).The condensation reaction of acetophenone (1-phenylethan-1-one) with 2-nitrobenzaldehyde in the molten state yielded the expected chalcone, ( An emblematic application of this large class of reactions is the synthesis of cage compounds such as cubane -chalcones may give four possible stereoisomers, namely syn/anti, head-to-head and head-to-tail  in solution . The title compound corresponds to the syn head-to-head stereoisomer , which could arise from a supra–supra bonding interaction between the singly occupied molecular orbital (SOMO) of one chalcone and the lowest unoccupied molecular orbital (LUMO) of the other one -3-(2-nitrophenyl)-1-phenylprop-2-en-1-one resulted in the title tetrasubstituted cyclobutane derivative  relative stereochemistry of the substituents is identical to that of β-truxinic acid, obtained by photodimerization of cinnamic acid . However, the departure from planarity is very small, the dihedral angle between the C1/C2/C3 and C1/C4/C3 mean planes being 3.6 (2)°. Some other rctt tetrasubstituted cyclobutane derivatives have a more marked butterfly conformation, which apparently results from steric restrictions imposed by bulky substituents °. In contrast, an intermolecular π–π contact is formed by parallel C11–C16 mnitobenzene rings related by an inversion centre. In that case, the separation between the rings is 3.883 (1) Å. These features seem to indicate that the molecular conformation is optimized in order to avoid steric hindrance, whilst at the same time allowing an efficient packing for the crystal stabilization.The molecule potentially belongs to the MOGUL . On the other hand, the average of absolute values for torsion angle defined by the four C atoms of the cyclobutane ring is 〈|δ|〉 = 2.52 (2)°. All these features support the conclusion reached by another research group who determined the structure of a closely related compound, namely a cyclobutane substituted by two benzoyl and two methoxyphenyl groups .A survey of the current organic sub-set of the CSD database , many of them (108) with the cyclobutane lying on a special position. The remaining 23% result formally from a head-to-head dimerization (known as truxinic type), and only one of them displays crystallographic symmetry  and solid NaOH pellets  were ground in an agate mortar with a pestle, at room temperature, for 23 min. The reaction proceeds exothermically (as noted by a rise in temperature of about 5–12 K). The progress of the reaction was monitored by TLC. After completion, the mixture was diluted with CH2Cl2 and washed with brine. The organic layer was separated, dried over MgSO4 and evaporated under reduced pressure. The crude product was purified by column chromatography using silica-gel and hexanes–ethyl acetate 4:1 as eluent, to give the expected chalcone and the title compound , as brown and colourless solids, respectively. Cyclobutane derivative: m.p. 517 K. FT–IR νmax/cm−1 1659 (C=O), 1557, 1348 (NO2). 1H NMR  δ/ppm: 7.81–7.33 , 5.22  and 4.87 . 13C NMR  δ/ppm: 44.3, 46.8, 125.0, 128.3, 129.4, 129.6, 130.4, 133.1, 134.7, 136.7, 137.5, 148.6, 196.9. HRMS (EI) calculated for C30H22N2O6 (M+) 506.1478; found 506.1477.A mixture of acetophenone , 22 bond, and was refined with two parts for the O atoms: O1A/O2A with occupancy 0.876 (7) and O1B/O2B with occupancy 0.124 (7). These four sites were restrained to have similar displacement parameters, with standard deviation of 0.04 Å2. The same restriction was applied to the O atoms of the other nitro group, O3/O4, given that this nitro group is probably also affected by disorder, although we were unable to refine a suitable model on the basis of the room temperature data for this group. The C-bound H atoms were treated as riding atoms in geometrically idealized positions: C—H 0.93–0.98 Å with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2056989017015936/ex2002sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017015936/ex2002Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017015936/ex2002sup3.txtexcel file for statistic analysis. DOI: Click here for additional data file.10.1107/S2056989017015936/ex2002Isup4.cmlSupporting information file. DOI: 1583527CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


O-isopropylidene-d-ribono-1,4-lactone and benzoyl chloride, the known absolute configuration for the lactone moiety of the ester substituent has been confirmed. The five-membered rings of the bicyclic lactone–dioxolane moiety both show envelope conformations and form a dihedral angle of 19.82 (7)° between the lactone ring and the benzene ring.In the title compound, obtained from the acylation reaction between 2,3- 15H16O6, obtained from the acylation reaction between 2,3-O-isopropylidene-d-ribono-1,4-lactone and benzoyl chloride, the known absolute configuration for the lactone moiety of the ester substituent has been confirmed. The five-membered rings of the bicyclic lactone–dioxolane moiety both show envelope conformations and form a dihedral angle of 19.82 (7)° between the lactone ring and the benzene ring. In the crystal, molecules of the acylated sugar are linked by very weak intermolecular C—H⋯O interactions, forming a three-dimensional network.In the title compound, C They have been widely employed as versatile chiral pools for the synthesis of biologically important molecules due to their abundance from sustainable resources as well as their low cost (Corma R),C3(S),C4(R)  with those for C3 , but this ring is slightly twisted about the C1—C2 bond. This is indicated by the comparative torsion angles C13—O2—C2—C3 for the dioxolane ring and C4—O4—C1—C2 of the lactone ring of 1.55 (18) and 6.87 (16)°, respectively. The dihedral angle between the mean plane of the benzene ring and that of the ester group (O6/C6/O5/C5) is 16.59 (9)°. All bond lengths and angles observed for (I)et al., 2004Compound (I)a axis. Several weak C—H⋯O interactions (Table 1The molecules of (I)s Table 1 are obseet al., 2016A search in the current version of the Cambridge Structural Database O-isopropylidene-d-ribono-1,4-lactone (II) with benzoyl chloride in pyridine followed by aqueous work-up and purification according to the reported method  and (III), and on the homogeneity of the reaction product.5-Ar), 1.00 Å (CH), 0.99 Å (CH2) or 0.98 Å (CH3) with Uiso(H) = 1.2Ueq(C) or 1.5Ueq(Cmethyl).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017002043/zs2372sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989017002043/zs2372Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017002043/zs2372Isup3.molSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017002043/zs2372Isup4.cmlSupporting information file. DOI: 1531628CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Thus, we define the relative importance of the spin–orbit and crystal field interactions, and explain the experimentally observed different ground states. We find optical absorption linewidths give a potential tool to identify spin–orbit coupled states, and show measurement of UV···UV super-exchange coupling in dimers by EPR. We show that observed slow magnetic relaxation occurs via two-phonon processes, with no obvious correlation to the crystal field.Determining the electronic structure of actinide complexes is intrinsically challenging because inter-electronic repulsion, crystal field, and spin–orbit coupling effects can be of similar magnitude. Moreover, such efforts have been hampered by the lack of structurally analogous families of complexes to study. Here we report an improved method to U≡N triple bonds, and assemble a family of uranium(V) nitrides. Along with an isoelectronic oxo, we quantify the electronic structure of this 5 Actinide electronic structure determination is fundamentally challenging. Here, the authors assemble a family of uranium(V)-nitrides and quantify the electronic structure of the molecules, defining the relative importance of spin orbit coupling and crystal field interactions. However, this is precisely the area where accurate models are needed because our understanding of core chemical concepts such as oxidation state, valence and the spin (S), orbital (L), and total angular momentum (J) quantum numbers become increasingly nebulous at the foot of the periodic table.In this nuclear age, there is a need for improved extraction agents for separations and recycling technologiesf1 complexes are appealing1112TIPS)(O)]  exploits the bulky TrenTIPS ligand to block disproportionation and stabilizes a wide range of novel uranium–ligand linkages14151617181920In recent years, many novel uranium molecules have become available, some accompanied by detailed spectroscopic and magnetic data456789567811III(TrenTIPS)] (2) by sodium azide to give the dinuclear contact ion pair (DCIP) complex [{UV(TrenTIPS)(μ-N)(μ-Na)}2] (3Na). Treatment of 3Na with 12-crown-4 ether (12C4) afforded the terminal nitride separated ion pair (SIP) complex [UV(TrenTIPS)(N)][Na(12C4)2] (4Na)V(TrenTIPS)(μ-N){Na(15C5)}]  was formed4Na could also be oxidized to [UVI(TrenTIPS)(N)] (6)222425262728303132333435363738394021914434446In 2012, we reported a molecular terminal uranium(V) nitride202122TIPS)(X)]n− complexes, in addition to their synthetic utility13C3) symmetry and the availability of a series in which only X and/or counter-ions vary. For example, we can study the influence of varying CF at uranium(V) by replacing O2− with N3− in isostructural and isoelectronic molecules: there are no other available uranium compound families where this is possible. However, extending the nitride family requires more reliable synthetic routes because, although the synthesis of 3Na is conceptually simple, it is not straightforward and sometimes fails for no obvious reason.These  (7) (ref. 8), or rubidium or caesium metals in benzene or toluene produces powders, from which 3Li, 3Na, 3K, 3Rb and 3Cs DCIP complexes [{U(TrenTIPS)(μ-N)(μ-M)}2] are consistently isolated in 35–57% crystalline yields (3M using sized-matched crown ethers to give the SIP complexes [U(TrenTIPS)(N)][M(crown)2] (4M): thus, 12C4 gives 4Na, 15C5 gives 4K and benzo-15-crown-5 (B15C5) gives 4Rb and 4Cs. Abstractions proceed quantitatively to give 4M as oils, with varying crystalline yields (21–95%). However, we were not able to prepare 4Li from 3Li with any abstracting ligand attempted (TIPS)(μ-N){M(crown)}] (5M) can be made from 3M, in crystalline yields of 34–66%, by mis-matching the crown to alkali metal, using variously 12C4 (5Li), 15C5 (5Na), dibenzo-18-crown-6  and 18-crown-6 . We note the synthetic utility of 4M, all of which can be oxidized with iodine to give 6 in crystalline yields of ∼60% (Treatment of − anion and M, and thus the anions have statistically indistinguishable U≡N distances (1.801(7)–1.825(15) Å, consistent with U≡N triple bonds5M have U–N–M cores, where M is capped by a crown ether. The U≡N distances vary from 1.803(5) to 1.840(3) Å, reflecting varying size and polarizing power of M. The U–N–M angles vary from linear for 5Na and 5Rb, residing on crystallographic threefold axes, to bent for 5Li, 5K and 5Cs (172.1(5), 162.76(16) and 148.8(3)°, respectively).Single crystal X-ray diffraction studies on geometry  and 3, w lengths .49 The Dab initio models, and finally parameterize the results to a CF and SOC Hamiltonian.To set up the quantitative analysis below, we first build a conceptual framework for the uranium nitride group, then develop this into f1 ground state with a SOC constant of λ ∼2,174 cm−1  model of a [UN]2+ species consists of the following: a σ-bond between a filled 2sp-hybridized nitride orbital and the metal-based lz=0 (5fσ) orbital; two π-bonds between the filled nitride 2p-orbitals and the uranium lz=±1 (5fπ) orbitals; a pair of non-bonding lz=±3 (5fϕ) uranium orbitals containing one electron; two non-bonding empty lz=±2 (5fδ) orbitals; two empty π*; and one empty σ* anti-bonding orbitals  and U(5f)/N(2sp) mixing but, despite this, the non-bonding  and anti-bonding orbitals have predominantly metal character (>80%). Following the approach of Eisenstein and Pryce532F  basis, where lz=±1 and 0 correspond to the π* and σ* orbitals, respectively. The equatorial electron density for each orbital is directly related to the lz quantum number, therefore they order as E±3<E±2<E±1<E0.The uranium(V) free-ion has a 5m−1 ref. , puttingorbitals . The π<σab initio calculations on the hypothetical [UN]2+ ion with the average experimental bond length of 1.84 Å. With a minimal active space of one electron in the 5f-orbitals the seven configuration state functions directly represent the relative orbital energies. Setting E±3=0 gives E±2 ∼1,300, E±1 ∼4,800 and E0 ∼6,300 cm−1  is calculated to be 4.20 .To estimate these energies, we performed Complete Active Space Self-Consistent Field 300 cm−1 . Includisymmetry . The calab initio results to a CF+SOC model with the Hamiltonian C∞v point group of [UN]2+, where the ab initio SOC-doublet energies gives λ=2,000 cm−1. The ground doublet has gz=4.21, which lies in between the SOC>>CF limit of gz=4.29) and the CF>>SOC limit of gz=4.00).We can parameterize the quations  yields trameters  from the3M–5M is the trigonal TrenTIPS ligand. As a first model, we performed ab initio calculations on the C3-symmetrized [U(NH3)(NH2)3(N)]− that mimics the amine and amide donors  an increase in the total spread of energies; (ii) the lz=±3 pair is split substantially ; (iii) the lz=±2 pair (which remains degenerate) falls below the barycentre of the |lz|=3 pair. The first effect is due to the additional axial ligand trans to the nitride, the second effect is due to the threefold equatorial field mixing the lz=±3 orbitals, and the last effect is due to the equatorial field destabilizing the in-plane lz=±3 orbitals. Thus, when SOC is introduced (jz≈±3/2) is at much lower energy than for [UN]2+  and (ii) gz of the ground doublet (still jz≈±5/2) is reduced to 3.80 and gx,y is no-longer zero. Due to the loss of C∞v symmetry, jz is not a good quantum number; however, owing to the strong axial potential, we use them as approximate labels.After the nitride, the next strongest perturbation in e donors . There atroduced : (i) theC3 point group. In addition to the q=0 terms, |q|=3 and 6 terms are allowed: these mix states with Δlz=3 and 6, respectively, hence mixing lz=±3 with 0, lz=±2 with ±1 and lz=+3 with −3  under |q|=6 will be most significant. Any mixing induced by |q|=3 would not induce any further loss of degeneracy and hence their effects can be parameterized with the q=0 terms. Therefore, to minimize the number of parameters, we have used a truncated Hamiltonian (neglecting |q|=3 terms), namely, the D3 Hamiltonian ab initio orbital energies (λ=0) gives the CF parameters CF+SOC parameterization requires new terms appropriate to the  with −3 . After S =0 ref. . Fittingrameters , and we κ=0.92 reproduces the ab initio ground state gz (NH2)3(N)]−, [U(TrenH)(N)]− and, finally, [U(TrenTIPS)(N)]−, show that lower symmetry perturbations introduce only small splittings within the lz=±2 and ±1 pairs compared with the dominant axial and C3 fields  for all compounds except 3Li and 3Na. This indicates an axial gz feature with small, but non-zero, gx,y ; this is most clear at Q-band, where the gz feature is split into a doublet . Spectra are dominated by ligand-to-metal charge transfer bands that tail in from the ultraviolet region to ∼12,000 cm−1. Five f–f transitions are observed at ∼4,700, ∼6,000, ∼6,900 and ∼8,750 cm−1 (ɛ ∼20–25 M–1 cm−1) with a shoulder on the ligand-to-metal charge transfer at ∼18,000 cm−1 (∼250 M–1 cm−1), in excellent agreement with the ab initio energies  lie in the narrow range 0.38–0.49 cm3 K mol−1: these are towards the lower end of the range associated with uranium(V) (0.25–1.3 cm3 K mol−1)χMT decreases slowly on cooling in the high-temperature range . The only outlier in this trend is 5K, which seems to behave more like the 4M series.For all compounds, the room temperature re range . For 4M,χMT is from the in-phase component of the ac susceptibility  is measured directly by the small oscillating field (∼1.5 G). For 3K, 3Rb, 3Cs and 5M (excluding 5K), measured in static field H=0 or 0.1 T, the frequency-independent part of χM′T(T) plateaus at low temperatures  fails to plateau for 4M and 5K (χM′T(T) to 0 K for 4M and 5K gives significantly lower values (0.1–0.2 cm3 K mol−1), implying that the lowest doublet is less magnetic. Low temperature magnetization (M) data mirror these trends (3M and 5M (excluding 5K) reach 0.6–0.8 μB mol−1 at 1.8 K and 7 T, while 4M and 5K reach lower values of 0.3–0.6 μB mol−1.A more accurate measurement of ty χM′T; , becauseM and 5K , implyine trends : 3M and 3Li, χM′T(T) decreases with increasing a.c. frequency below 10 K, reaching zero (0.1 T static field). This is accompanied by the appearance of peaks in the out-of-phase susceptibility (χM″) as a function of frequency and temperature (M(H) experiments.For all complexes, except perature . This sh3Li and 3Na behave differently from the other 3M complexes: χM′T(T) fails to plateau at low temperature (M(H) exhibits waist-restricted hysteresis that collapses below ∼0.5 T (The exchange-coupled dimers perature  and M(H)w ∼0.5 T .TIPS) framework . The transition to the first excited doublet is outside our spectral range, but all other states are observed. Interestingly, the f–f transition linewidths follow a consistent pattern (from low to high energy) of broad, sharp, sharp, broad and very broad. These transitions are to excited states derived primarily from |lz|=1, |lz|=2 or 3, |lz|=2 or 3, |lz|=1 and |lz|=0, respectively  than the latter. Hence, the assignments are consistent with the orbital parentage of the SO-coupled states. If this is characteristic for uranium(V) more widely, it could serve as a tool to directly evidence the nature of SO-coupled states, although it may only apply in cases with dominant axial CFs.Given the simplicity of our ectively . The brojz≈±5/2 and ±3/2), within a few hundred cm−1 of each other, well separated from all others by thousands of cm−1. In fact, the calculated separation of the two lowest doublets is of the same order as the errors in the calculations. Hence, it is feasible that their actual ordering could be reversed and dependent on small structural differences.  with an upper limit for gx,y<0.5, in agreement with ab initio values for the jz≈±5/2 doublet of 3.84 (8). The jz≈±3/2 doublet is calculated to have gz≈2.4 (3). Hence, the EPR spectra must arise from the jz≈±5/2 doublet in each case, and we initially assumed that this was the ground state. However, this is not compatible with the magnetic data in all cases.Importantly, the gross electronic structure is clear—there are two low-lying doublets  are 0.55 μB mol−1 and 0.18 cm3 K mol−1. The latter are consistent with experimental data for 4M and 5K , particularly allowing for uncertainty in gz. Hence, it appears for 4M and 5K that jz≈±3/2 is the ground state with ±5/2 as a low-lying excited state, consistent with the significant temperature dependence in χMT. The jz≈±5/2 state must be low enough that it is still sufficiently populated at<10 K to give the observed EPR spectrum. The experimental data for 3K, 3Rb, 3Cs and 5M (excluding 5K) are significantly higher . This implies that jz≈±5/2 is significantly lower in energy. Indeed, for 3K and 5Na, the data approach the calculated values for jz≈±5/2 and, along with the lack of temperature dependence in χMT, suggests that this is the ground state.The calculated magnetization for an isolated χMT(T) while simultaneously fitting all other experimental data  to the CF+SOC Hamiltonian  cm−1 above the ±2 pair, followed by lz=±1 and 0 at 3,500(200) and 13,800(400) cm−1, respectively. The splitting of the lz=±3 pair is 1,290(70) cm−1 , giving a gap between the lowest lying orbitals of only 200–300 cm−1, hence the subtlety of the electronic structure of these compounds. With SOC, the fit parameters give the ground state as jz≈±5/2 for 3K and 5Na with the ±3/2 state lying at 300–400 cm−1; however, this gap is poorly defined as we have no direct experimental handle. For all other complexes, the fits give the ground state as jz≈±3/2 with the ±5/2 state at 7–46 cm−1, where this gap is now better defined by the temperature dependence of χMT. Higher excited states are as in 11 and alkyl (R), alkoxide (OR), amide (NR2) and ketimide (NCR2) [UX6]− complexesf-orbitals span 11,361 (X=F), 6,307 (Cl), 5,310 (Br), 6,800 (R), 8,500 (OR), 7,400 (NR2) and 6,900 cm−1 (NCR2), the total breadth of the 5f-orbitals here spans a much larger 13,800 (400) cm−1 consistent with the strong axial CF of the nitride. The effective SOC constants are practically identical, where we find κλ=1,900(20) cm−1 compared with 1,910(70) cm−1 for [UX6]− .By modelling all the experimental data with a single model, we can discuss features of the experimental electronic structure. For the 5gz<2.2 with gx,y=0 for the jz≈±3/2 state, and 3.7<gz<4.0 with 0.14<gx,y<0.18 for the jz≈±5/2 state. With these values, the jz≈±5/2 state is EPR active , while the ±3/2 state is EPR silent . This is consistent with the observation that we do not observe EPR transitions corresponding to the jz≈±3/2 doublet regardless of the ground state. This is because in C3 symmetry the jz≈±5/2 state mixes with ±7/2 under the |q|=6 terms (jz=±1 component (the EPR selection rule). In contrast, jz≈±3/2 does not mix with any other state under |q|=6; even |q|=3 terms could only mix it with the other jz≈±3/2 state. No additional mixing is induced by SOC and there is no Δjz=±1 component. Although ab initio calculations give a small gx,y≠0 for this state, which must be due to low symmetry effects, our experiments are consistent with C3 symmetry.Our model gives 1.9<=6 terms ; Fig. 4,1  1 has a formal U=O double bond so gives a weaker axial CF than a formal U≡N triple bond; (ii) the equatorial CF due to the TrenTIPS ligand is unchanged; and (iii) the weaker axial CF of the oxo stabilizes the lz=±2 relative to ±3 orbitals, therefore stabilizing the jz≈±3/2 doublet relative to ±5/2 after SOC. Ab initio calculations on 1  complex 1 ref. . Compounons on 1  give theomplexes .3Li and 3Na show a pair of transitions centred on gz≈3.7  ions, is the Lines modelTo provide a unified description of the electronic structure, we have incorporated exchange into our CF+SOC framework for jzz to the EPR doublet gives ±0.77 cm−1 ; fitting .77 cm−1 ; Table 2at 0.5 T , the fieat 0.5 T .3M–5M, with the exception of 3Li, behave as SMMs. Magnetization relaxation times (τ) were determined by fitting χM″ data to a generalized Debye model (Ueff) assuming Orbach relaxation, that is, via an excited state by exchange of phonons with the lattice. For this mechanism, Ueff can be found from the slope of an Arrhenius plot of ln(τ) versus T−1 in the high-temperature regime. For 3M–5M, this gives Ueff=20–40 K: the top end of this range (5Na) would be among the highest reported for a uranium complex. These values are in the range of the lowest lying excited states determined above, hence Orbach relaxation via this state seems feasible.The first uranium SMMs were reported as recently as 2009 ref. , involvi14ye model . The usuT) and applied magnetic field (H) dependences. Simplified expressions are D and C are numerical coefficients). In the small d.c. fields and temperature range of our experiments, direct relaxation is negligible. Different presentations of relaxation data can help distinguish different mechanisms (τ−1(T), these profiles are switched. Examining such plots for 3M–5M  in or 3M–5M  shows thM  mono(oxo) and 14 uranium(V) nitride complexes by EPR and UV/Vis/NIR spectroscopies, super-conducting quantum interference device (SQUID) magnetometry, and modelling with CF and ab initio calculations.All manipulations were performed under a dry, oxygen-free nitrogen atmosphere using Schlenk-line and glove-box techniques, using dried and degassed solvents and reagents. The compounds were characterized by elemental analysis, single crystal X-ray diffraction, nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR), UV/Vis/NIR and EPR spectroscopies, and magnetic studies. See 7  in toluene (10 ml) was added to a cold (−78 °C) slurry of Li metal  in toluene (20 ml). The mixture was allowed to slowly warm to room temperature and then stirred for 5 days. Each day the mixture was sonicated for 1 h. After this time, a red precipitate had formed that was extracted with 60 ml hot (100 °C) toluene and filtered through a frit. The residue was washed with hot (100 °C) toluene (2 × 10 ml) and the combined extracts concentrated to ∼30 ml and stored at −30 °C to yield 3Li as a red crystalline solid. The product was isolated by filtration, washed with pentane (2 × 10 ml) and dried in vacuo. Yield 1.22 g, 35%. Anal. calcd for C66H150N10Li2Si6U2: C, 45.49; H, 8.68; N, 8.04. Found: C, 45.46; H, 8.62; N, 7.99. 1H NMR : δ 33.65 , 7.84 , −1.33 2), −2.74 2). FTIR ν cm−1 (Nujol): 1,591 (w), 1,302 (w), 1,137 (w), 1,052 (s), 965 (w), 932 (s), 882 (s), 737 (s), 672 (m), 633 (m), 567 (w), 545 (w), 516 (w). 29Si NMR and solution magnetic moment (Evans method) could not be conducted due to the insolubility of 3Li in aromatic solvent once isolated.A solution of 7  in toluene (10 ml) was added to a cold (−78 °C) toluene suspension of Na metal . The mixture was allowed to slowly warm to room temperature and then stirred for 5 days. Each day the mixture was sonicated for 1 h. After this time, a red precipitate had formed which was extracted and filtered through a frit using boiling toluene (30 ml). The residue was washed with boiling toluene (2 × 10 ml). The filtrate was concentrated to ∼10 ml and stored at −30 °C to yield 3Na as a red crystalline solid. The product was isolated by filtration, washed with pentane (2 × 10 ml) and dried in vacuo. Yield 1.32 g, 50%. The identity of 3Na was confirmed by comparison with previously reported methods.A solution of 7  and KC8 . The resulting mixture was allowed to warm to ambient temperature and stirred for a further 6 days to afford a dark brown suspension. After this time, the mixture was allowed to thoroughly settle (1 h) and carefully filtered to afford a dark brown solid that was washed with toluene (5 × 5 ml) until the washings obtained were colourless. The solid was then extracted into hot (70 °C) benzene and quickly filtered through a frit to remove the graphite precipitate. The filtrate was stored at 5 °C for 16 hs to yield dark red crystals of 3K that were isolated by filtration and dried in vacuo. Yield: 1.70 g, 57% . Anal. calcd for C66H150N10K2Si6U2: C, 43.88; H, 8.37; N, 7.75. Found: C, 44.18; H, 8.38; N, 7.62. FTIR ν cm−1 (Nujol): 1,344 (w), 1,134 (w), 1,069 (s), 1,056 (s), 992 (w), 932 (s), 880 (m), 851 (w), 780 (m), 746 (s), 671 (m), 626 (w), 571 (w), 545 (w), 515 (w). 1H, 29Si NMR and solution magnetic moment (Evans method) could not be obtained due to the insolubility of 3K in aromatic solvent once isolated.Toluene (15 ml) was added to a cold (−78 °C) stirring mixture of 7  in toluene (10 ml) was added to a cold (−78 °C) slurry of Rb metal  in toluene (10 ml). The mixture was allowed to slowly warm to room temperature and then stirred for 72 h. The resulting mixture was filtered and the remaining solid extracted with toluene (2 × 5 ml). The toluene extracts were combined and volatiles were removed in vacuo to afford a red-brown solid. Recrystallization from 10 ml hot (70 °C) benzene afforded 3Rb as a red crystalline solid on cooling to 5 °C. Yield: 1.90 g, 50% . Anal. calcd for C66H150N10Rb2Si6U2: C, 41.73; H, 7.96; N, 7.37. Found: C, 39.35; H, 7.29; N, 7.61. FTIR ν cm−1 (Nujol): 1,337 (w), 1,270 (w), 1,135 (m), 1,064 (s), 989 (m), 931 (s), 881 (m), 846 (m), 777 (s), 734 (s), 670 (m), 628 (w), 564 (w), 543 (w), 509 (w). 1H and 29Si NMR and solution magnetic moment (Evans method) could not be obtained due to the insolubility of 3Rb in aromatic solvent once isolated.A solution of 7  in benzene (10 ml) was added to a cold (−78 °C) slurry of Cs metal  in benzene (10 ml). The mixture was allowed to slowly warm to room temperature and then stirred for 72 h. The resulting mixture was filtered and the remaining solid extracted with hot benzene . The benzene extracts were combined and volatiles were removed in vacuo to afford a red-brown solid. Recrystallization from hot (70 °C) benzene (10 ml) afforded 3Cs as a red crystalline solid on cooling to 5 °C. Yield: 1.39 g, 36%. Anal. calcd for C66H150Cs2N10Si6U2: C, 39.75; H, 7.58; N, 7.02. Found: C, 40.12; H, 7.37; N, 7.02. 1H NMR : δ 37.54 , 8.19 , −5.14 2), −5.64 2). FTIR ν cm−1 (Nujol): 1,340 (w), 1,251 (w), 1,135 (m), 1,059 (s), 1,056 (s), 990 (m), 932 (s), 880 (m), 843 (m), 772 (s), 739 (s), 670 (m), 626 (w), 597 (m), 566 (w), 542 (w), 511 (w). μeff : 1.89 μB. 3Cs is only partially soluble in aromatic solvents meaning a reliable 1H NMR could not be obtained. Furthermore, a 29Si NMR spectrum could not be obtained.A solution of 3K  was suspended in 15 ml toluene and to it was added a solution of 15-crown-5  in toluene (10 ml) at room temperature. The red/brown mixture was stirred for a further 16 h and filtered. The solvent was removed in vacuo to yield a brown oil that crystallized within 1 h at room temperature. On occasions, storage at 5 °C was necessary to obtain crystalline material. Crystals of 4K were isolated by thorough washing with pentane (5 × 5 ml) and were dried in vacuo to afford a brown crystalline solid. Yield: 1.61 g, 95% . Anal. calcd for C53H115N5KO10Si3U: C 47.36; H 8.63; N 5.21. Found: C 47.47; H 8.44; N 4.93. 1H NMR : δ 39.10 , 11.92 , 8.45 , 4.82 , −5.58 2), −6.36 2). 29Si{1H} NMR : δ −15.13. FTIR ν cm−1 (Nujol): 1,302 (m), 1,260 (m), 1,124 (s), 1,042 (m), 935 (m), 882 (m), 856 (w), 741 (m), 723 (m), 670 (w), 625 (w), 565 (w), 506 (w). μeff : 1.58 μB.Complex 3Rb  and benzo-15-crown-5 . The resulting mixture was allowed to warm to room temperature with stirring over 16 h. Volatiles were removed in vacuo to afford a brown powder. Recrystallization of the powder from 10 ml warm (60 °C) toluene afforded 4Rb as a brown crystalline solid on cooling to ambient temperature. Yield: 0.25 g, 21% . Anal. calcd for C61H115N5O10RbSi3U: C, 49.29; H, 7.80; N, 4.71. Found: C, 48.03; H, 7.48; N, 4.64. 1H NMR : δ 38.62 , 11.00–5.00 , −5.45 2), −6.18 2). 29Si{1H} NMR : δ −15.80. FTIR ν cm−1 (Nujol): 1,595 (m), 1,296 (m), 1,217 (m), 1,127 (s), 1,077 (s), 1,042 (m), 934 (m), 882 (m), 851 (w), 739 (s), 670 (m), 627 (m), 542 (w), 507 (w). μeff : 2.46 μB.Toluene (20 ml) was added to a cold (−78 °C) mixture of 3Cs  and benzo-15-crown-5 . The resulting mixture was allowed to warm to room temperature with stirring over 16 h. Volatiles were removed in vacuo, and the product extracted into hot hexanes . The mixture was filtered and concentrated to ∼2 ml precipitating a sticky solid. The solid was isolated by filtration, suspended in pentane (5 ml) and stored at −80 °C for 16 h. The pentane was filtered away to afford 4Cs as a brown-black solid. A concentrated solution of 4Cs in hexanes afforded brown-black crystals suitable for single crystal X-ray diffraction studies over 48 h. Yield: 0.20 g, 50%. Anal. calcd for C61H115CsN5O10Si3U: C, 47.77; H, 7.56; N, 4.57. Found: C, 47.54; H, 7.55; N, 4.56. 1H NMR : δ 38.30 , 8.66 , 7.80 , 7.43 , 6.50–5.10 , −5.38 2, −6.25 2). 29Si{1H} NMR : δ −15.48 p.p.m. FTIR ν cm−1 (Nujol): 1,504 (m), 1,409 (w), 1,363 (m), 1,234 (m), 1,221 (m), 1,075 (s), 1,044 (s), 934 (s), 869 (w), 850 (m), 739 (s) 670 (m), 627 (w), 563 (w), 539 (w), 508 (w). μeff : 2.09 μB.Toluene (30 ml) was added cold (−78 °C) mixture of 3Li  and 12-crown-4 . The resulting mixture was allowed to warm to room temperature with stirring over 16 h. Volatiles were removed in vacuo to afford a sticky brown solid. This solid was then washed with hexanes (5 ml) and filtered to afford 5Li as a brown powder. Dark red crystals were grown from a concentrated solution of 5Li in toluene at room temperature. Yield 0.43 g, 41% . Anal. calcd for C41H91N5LiO4Si3U: C 47.01; H 8.76; N 6.69. Found: C 45.70; H 8.67; N 6.54. 1H NMR : δ 38.61 , 14.04 , 11.28 , 7.62 , −5.55 2), −6.74 2). 29Si{1H} NMR : δ −17.67. FTIR ν cm−1 (Nujol): 1,363 (w), 1,260 (s), 1,135 (s), 1,076 (s), 1,026 (s), 934 (m), 883 (m), 861 (w), 736 (s), 670 (w), 629 (w), 589 (w), 565 (w), 544 (w), 508 (w). μeff : 2.04 μB.Toluene (20 ml) was added to a cold (−78 °C) mixture of 3K  and dibenzo-18-crown-6 . The resulting red/brown mixture was allowed to warm to ambient temperature and stirred at room temperature for a further 16 h. The solvent was removed in vacuo to yield a pale brown solid that was extracted with hexanes (4 × 5 ml). The combined extracts were concentrated and stored at 5 °C for 16 h, yielding 5K as pale brown tablets. Yield: 0.56 g, 66% . Anal. calcd for C53H99KN5O6Si3U: C, 50.37; H, 7.90; N, 5.54. Found: C, 48.58; H, 7.72; N, 4.85. 1H NMR : δ 40.20 , 9.30 , 8.18 , 7.91 , 7.07 , −5.88 2), −7.24 2). 29Si{1H} NMR : δ −13.30. FTIR ν cm−1 (Nujol): 1,304 (s), 1,259 (s), 1,233 (m), 1,215 (m), 1,130 (s), 1,092 (m), 1,071 (s), 1,061 (s), 1,024 (m), 959 (w), 949 (w), 934 (m), 882 (w), 857 (w), 740 (s), 723 (s), 670 (w), 627 (w), 588 (w), 566 (w), 514 (w). μeff : 2.72 μB.Toluene (20 ml) was added to a cold (−78 °C) mixture of 3Rb  and 18-crown-6 . The resulting mixture was allowed to warm to room temperature with stirring over 16 h. Volatiles were removed in vacuo to afford a sticky brown solid. The solid was washed with hexanes (2 × 5 ml) and filtered to afford 5Rb as a brown powder. Crystalline material was obtained from a concentrated solution of 5Rb in hexanes. Yield: 0.37 g, 44% . Anal. calcd for C45H99N5O6RbSi3U: C, 44.52; H, 8.22; N, 5.77. Found: C, 38.36; H, 6.93; N, 7.86. 1H NMR : δ 41.91 , 9.86 , 8.27 , −6.37 2), −7.36 2). 29Si{1H} NMR : δ −11.88. FTIR ν cm−1 (Nujol): 1,352 (s), 1,284 (w), 1,116 (s), 1,079 (m), 1,013 (m), 987 (w), 962 (w), 935 (m), 882 (m), 866 (w), 840 (m), 740 (s), 671 (m), 627 (m), 564 (w), 542 (w), 509 (w). μeff : 1.89 μB.Toluene (20 ml) was added to a cold (−78 °C) mixture of 3Cs  and 18-crown-6 . The resulting mixture was allowed to warm to room temperature with stirring over 16 h. Volatiles were removed in vacuo and the resulting brown solid washed with hexanes (5 ml). The solid was extracted into toluene (5 ml), concentrated to 2 ml) and stored at −30 °C for 7 days to afford orange crystals of 5Cs. Yield: 0.14 g, 34%. Anal. calcd for C45H99CsN5O6Si3U: C, 42.85; H, 7.91; N, 5.55. Found: C, 42.80; H, 7.84; N, 5.32. 1H NMR : δ 40.10 , 10.89 , 7.91 , 3.61 , −5.88 2), −6.71 2). 29Si{1H} NMR : δ −13.32 p.p.m. FTIR ν cm−1 (Nujol): 1,351(m), 1,282 (w), 1,112 (s), 1,082 (m), 1,013 (m), 959 (w), 933 (m), 881 (w), 858 (w), 837 (s), 792 (s), 739 (m), 669 (m), 624 (w), 588 (w), 543 (w), 526 (w), 508 (w). μeff : 2.20 μB.Toluene (20 ml) was added to a cold (−78 °C) mixture of f electron are: The CF Hamiltonian employed here is a modified version of the Stevens operator equivalent formalism, where the operator equivalent factors . The relative energies of the excited states as determined by NIR were then fitted by allowing the SOC and CFPs to vary. During this procedure, the lowest two doublets were held in the chosen order by simultaneously fitting their characteristic gz values . In the event that they did not remain in the correct order, the CFPs were once again manually tweaked to correct the order and the process was repeated until good agreement with the optical data was obtained along with correct ordering of the lowest two states. Then, to achieve the correct gz value for the jz≈±5/2 state as measured by EPR, the orbital reduction parameter was ‘walked down' from 1 in steps of 0.02, where at each step the SOC and CFPs were allowed to vary to reproduce the optical data. Once a good estimate for the orbital reduction parameter was obtained, all free model parameters  were allowed to vary while simultaneously fitting the NIR, EPR and magnetic data. , under deposition numbers CCDC 1486679–1486689. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via How to cite this article: King, D. M. et al. Molecular and electronic structure of terminal and alkali metal-capped uranium(V) nitride complexes. Nat. Commun.7, 13773 doi: 10.1038/ncomms13773 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Notes, Supplementary Methods and Supplementary References.crystallographic information files


S,8aR)-1-isopropyl-4 b,8,8-trimethyl-7-oxo-4 b,7,8,8a,9,10-hexahydrophenanthren-2-yl acetate, was prepared by a direct acetylation reaction of naturally occurring totarolenone. In the crystal, molecules are linked to each other by C—H⋯O hydrogen bonds and C—H⋯π interactions, forming sheets parallel to the bc plane.The title compound,  and 0.793 (14), respectively.The title compound, C They exhibit a wide variety of interesting biological activities such as antitumour -1-isopropyl-4 b,8,8-trimethyl-7-oxo-4 b,7,8,8a,9,10-hexahydrophenanthren-2-yl acetate, 2, from naturally occurring totarolenone, 1, extracted from the heartwood of Tetraclinis articulata  assumes a half-chair conformation, as indicated by the total puckering amplitude QT = 0.55 (2) Å and spherical polar angle θ = 51.0 (2)° with φ = 136.0 (2)°. The major component of the cyclohexene ring exhibits a screw-boat conformation  while the minor component has a chair conformation .The molecular structure is built up from three fused six-membered rings Fig. 2. In the bc plane  as the main skeleton, revealed the presence of 75 structures. These include several compounds similar to the title compound. One with a similar conformation has a hydroxyl substituent in place of the acetate in the title compound , while six entries .A search of the Cambridge Structural Database using the 1,2,3,4,4a,9,10,10a-hexahydrophenanthren ring system Fig. 4a as the1  in acetic anhydride (10 mL) and sodium acetate (290 mg) was heated under reflux for 24 h. After cooling, the solution was extracted with ether (3 × 20 mL). The organic layer was washed with water, dried on anhydrous Na2SO4 and evaporated under reduced pressure. The obtained residue was chromatographed on silica gel column using hexane and ethyl acetate (95/5) as eluent, to give compound 2.A solution of totarolenone Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl). The carbonyl O atom is disordered over two sites having occupancies of 0.63 (7) and 0.37 (7). Atom C6 atom of the cyclohexene ring is disordered over two sites with an occupancy ratio of 0.793 (14):0.207 (14). The absolute structure was reliably determined based on the value of the Flack parameter [0.02 (5)].Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018005510/xu5922sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989018005510/xu5922Isup2.hklStructure factors: contains datablock(s) I. DOI: 1835842CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: