G-quadruplexes in oncogene promoters provide putative targets for transcriptional regulation. The structure of a putative G-quadruplex sequence (S1: GGAGAAGGAGGAGGTGGAGGAGGAGGG) in potassium solution in the her2 promoter has been resolved mainly through nuclear magnetic resonance (NMR) spectroscopy. By application of various NMR spectra, we proved the formation of a four-layer G-quadruplex composing of two G-tetrads and two G/A-mixed planes with a four-residues loop (A3-G4-A5-A6). Further evidence from a luciferase reporter assay, Q-RT-PCR and Western blotting indicates that S1 G-quadruplex formation can repress her2 promoter activity, and a selected G-quadruplex ligand cβ can enhance the repression by down regulating her2 transcription and expression. These findings provide a G-quadruplex target and perspective implications in her2 transcriptional regulation. So far, the therapeutic strategies for treatment of breast cancer caused by her2 overexpression include using antibodies such as Herceptin® to block her2 protein ligand binding6 and chaperone Hsp90 antagonists to prevent stabilization of the active her2 conformation at the cell surface8. Alternate strategy is to suppress her2 expression by directly targeting her2 oncogene promoter. Studies have shown that triplex formation by a pyrimidine-rich oligonucleotide can inhibit her2 transcription in vitro9, and Bis-peptide nucleic acids targeting the polypurine tract of the her2 promoter can prevent her2 gene expression11.The proto-oncogene her2  encodes a transmembrane growth factor receptor. The her2 gene is amplified in 25 to 30 percent of human primary breast cancers15. Numerous studies have demonstrated that potential G-quadruplexes prevalently exist in human genomes19, and those involved in telomeres and oncogene promoters provide therapeutic targets for anti-cancer drug design29. There is a G-rich sequence  is located 7 nt down-stream31, giving potent biological relevance of this G-rich sequence.G-quadruplex is a higher-order DNA/RNA structure that is formed by guanine-rich sequences36. Previous studies by Katahira demonstrated that a GGA repeat sequence d(GGA)8 similar to her2 S1 can form a T:H:H:T G-quadruplex composed of two guanine tetrads (T) stacked onto two guanine-adenine heptads (H)38. Further studies showed the T:H:H:T G-quadruplex formed in the c-myb promoter can act as a target of transcriptional regulation40. In the present study, we probed the her2 G-quadruplex structure formed by an original G-rich sequence (S1) mainly through NMR spectroscopy. Our result revealed that S1 forms a similar T:H:H:T G-quadruplex composing of two G-tetrads and two G/A-mixed planes. But unlike d(GGA)8, there is a four-residues loop (A3-G4-A5-A6) linking the first G-tetrad and the second G/A-mixed plane in S1. Based on the structure, we found a synthetic small molecule cβ that can selectively bind to the her2 G-quadruplex with respect to its corresponding duplex DNA. Further results from luciferase assay, quantitative real-time reverse transcription polymerase chain reaction (Q-RT-PCR) and western blotting indicated that formation of the S1 G-quadruplex can repress the activity of the her2 promoter, and the G-quadruplex ligand cβ can further down-regulate her2 mRNA transcription and decrease her2 protein expression. Our work provides a G-quadruplex structure formed by the G-rich sequence in human her2 proto-oncogene promoter as well as a new strategy of her2 transcriptional regulation.Nuclear magnetic resonance (NMR) spectroscopy has been widely used as a powerful tool in the determination of quadruplex structures formed in human telomeres and oncogene promoters44. The CD spectrum of S1 gives a strong positive absorption at 260 nm and another high-intensity positive band at 210 nm in 100 mM potassium solution 8. The formation of S1 G-quadruplex is also supported by ESI mass spectrometry. The mass spectrum of S1 (Mw = 8673.7) in 100 mM ammonium solution reveals a base peak (100%) of a complex ion formed by one molecule of S1 and three ammonium ions . As ammonium ions have a similar atomic radius to potassium ions and are located between adjacent G-tetrad planes47, the existence of the complex ion [S1 + 3NH4+-8H+]5− indicates that S1 forms an intramolecular G-quadruplex with four planes. Furthermore, polymerase stop assays were performed to investigate the S1 G-quadruplex formation in a DNA template strand, and the results demonstrate that S1 G-quadruplex is formed and arrests Taq polymerase in a potassium-dependent manner  spectroscopy is used to evaluate the formation and directions of strands of S1 G-quadruplex. Numerous studies have shown that parallel G-quadruplexes are characterized by a dominant positive CD absorption around 260 nm, whereas anti-parallel ones have a negative CD absorption at 260 nm and a positive absorption at 290 nm, and both quadruplex types show an additional characteristic positive peak at 210 nmion Fig. , suggestTo investigate which guanines participated in the G-tetrad core structure, we performed DMS-induced strand cleavage for S1. As Fig. 15N, 13C-isotope labelling approach49. The chemical shifts of exchangeable imino and amino protons were gained through 15N-1H-HSQC spectra  The characteristic NOE correlations between imino and H8 protons reveal the G-tetrad composition Fig. . Figure 51. In Fig. Guanine and adenine can form a sheared G:A base-pair structure of which a strong GNH21/22-AH8 interaction can be observed in the NOE spectrumThe strong NOE interactions of aromatic H2 and sugar protons between adenines indicate the arrangement of the two G/A-mixed planes. The existence of strong NOE correlations  and H8 (7.17 ppm) resonance of G2. The single residue T15 links the upper and the lower half motif. The 5′-terminal G27 stacks on the outer G-tetrad.Figure The stacking between the G-tetrads and G/A-mixed planes are shown in Fig. 837 were shown in Fig. 8, all of the sequential G-G-A motifs participating in the G-tetrads and G/A-mixed planes are similar with a single A residue linking the tetrad and G/A-mixed plane; while in her2 S1 G-quadruplex, except for the single A residue, there is a four-residues (A3-G4-A5-A6) loop which links the first G-tetrad and the second G/A-mixed plane. This result provides further implications of formation of G-quadruplexes with G/A-mixed planes by non-sequential GGA repeats.The schematic structures of G-quadruplexes formed by her2 S1 sequence and d(GGA)5−, m/z = 1972.2), and the free Q1 ion cannot be detected. While the ESI mass spectra of the mixtures of the other small molecules and the G-quadruplex (Q1) reveal no complex ions but free Q1 , while no complex duplex DNA ion  and cβ were observed, indicating that cβ selectively recognized the her2 G-quadruplex over the duplex DNA. Further evidence from a polymerase stop assay supported this result. When cβ was added to the system, the ratio of the fully-elongated band to the sum of arrested and fully-elongated band of primer extension using the template containing S1 G-quadruplex decreased to 80%  and a mutant (MU) sequence of S1  in the her2 proto-oncogene promoter. The A3-G4-A5-A6 loop in the S1 G-quadruplex provides further evidence of four-layer G-quadruplex formation with G/A-mixed planes by non-sequential GGA repeats. We also demonstrated that the S1 G-quadruplex can inhibit her2 promoter activity, and a selected S1 G-quadruplex ligand cβ can suppress her2 transcription and expression. Since the elevated expression of her2 has been shown to be strongly associated with the development and progression of aggressive breast cancers, our study provides a putative G-quadruplex target for breast cancer treatment. Further efforts may be made on the study of the her2 S1 G-quadruplex ligands, either bind to the tetrad core or the loops, to stabilize the G-quadruplex and hence repress the her2 oncogene expression for related pathologies regulation.15N and 13C) oligonucleotides were synthesized by Takara Biotech.  using nucleotides labelled with 15N and 13C from Cambridge Isotope Laboratories, Inc. . Small molecules  were synthesized in our lab54. DNA samples for NMR spectroscopy were purified by successive dialysis against 0.5 M KCl solution and then against water58. For a stable G-quadruplex solution generation, purified S1 was diluted in K+ buffer containing 75 mM KCl, 25 mM K2HPO4-KH2PO4 (pH 7.0) at a final concentration of 1.0 mM strands for NMR spectra scanning.Unlabeled oligonucleotides were purchased from Sangon Biotech. . Site-specific labelled (64. Resonances were assigned unambiguously by 1D-15N-1H-HSQC and 1D-13C-1H-HSQC experiments using a site-specific low-enrichment (6–8%) fully 15N, 13C-labeling approach49. Assignments were confirmed through standard 2D NMR experiments including TOCSY (τm of 120 ms) and NOESY . Spectra in water solutions were performed with water suppression using watergate W5 pulse sequence with gradients. Relaxation delays were set to 2 s. The acquired sizes were set to 1024 × 320, and the final spectral sizes were 1024 × 1024. 2,2-dimethylsilapentane-5-sulfonic acid (DSS) was used as an internal standard for the 1H chemical shifts assignments.NMR spectra were recorded on Bruker Advance 800 MHz, and Varian VNMRS 600 MHz spectrometers equipped with cryogenic probes at 25 °Chttp://mestrelab.com/). Cross-peaks were assigned and integrated using MestReNova peak fitting function and volume integration. The inter-proton distance restraints were obtained from a 2D NOESY spectrum with a mixing time of 200 ms using the average volume integral of the H1′-H2′′ cross-peaks (2.20 Å) as a reference63. A certain value of bound was added to each of the calculated distance, that is 0.8 Å added for the calculated distance of 0.0–2.7 Å, 1.5 Å added for 2.7–3.2 Å, 2.0 Å added for 3.2–4.0 Å, and 2.5 Å added for 4.0–6.0 Å. Further 1.0 Å was added for the imino H1 and aromatic H8 pairs in the G-tetrad core and 1.8 Å was used as a lower bound distance for all proton pairs38. Atoms participating in experimentally identified G-tetrads were restrained with distances corresponding to ideal hydrogen bond geometry60. Dihedral angle restraints for the β and ε torsion angles were derived from 31P-1H-HSQC spectra with coupling constants of 5 to 25 Hz as described previously38. Thus, the β torsion angles of G2, A3, A6, G8, G11, G17, G20 and G23 were constrained to 180 ± 20°, and the ε torsion angles of all residues except G2, A6 and G27 were constrained to −120 ± 45°. The γ torsion angles of A3, G4, A5, A6, G7, G10, G13, G14, G15, G16, G18, G19, G22, G23, G25 and G27 were constrained to 60 ± 20° derived from the relative intensities of H3′–H5′/H5′′ and H4′–H5′/H5′′, and χ torsion angles of the anti-residues G2, A5, A6, A9, G10, G13, G17, A18, G20, A21, A24 and G25 were constrained to 220 ± 40° derived from the relative intensities of H8/H6-H1′ as described previously60.NMR spectra were processed and analyzed using MestReNova software  version 1.3 program4OAc solution (pH 6.9) and pre-incubated at 37 °C for 8 h to generate the G-quadruplex. Each of the synthetic small molecule ligands were dissolved in 1:9 (v/v) DMSO/CH3OH solution to a stock concentration of 500 μM and added to the DNA samples for binding affinity investigation. Duplex DNA were generated by mixing 10 μM S1 and the equivalent of its complementary cytosine-rich strand (designated C1). For each sample, 25% (v/v) methanol was added to improve electrospray efficiency. Negative ion mode was used with a spray voltage of 2.7 kv and a capillary temperature of 140 °C. The infusion rate was 2 μl/min, and scans lasting 5 min were summed for each spectrum.ESI-MS experiments were performed using the Finnigan LCQ Deca XP Plus ion-trap mass spectrometer . S1 (10 μM) was dissolved in 100 mM NHCD experiments were performed with a JASCO J-815 CD spectrometer . S1 (10 μM) was dissolved in 30 mM Tris-HCl (pH 7.4) buffer with 100 mM or without KCl. Samples were pre-annealed at 90 °C for 10 min and then slowly cooled down to 25 °C (more than 8 h) before being tested in a 0.1 cm path-length cuvette. The scanning wavelength range was from 200 to 400 nm, and three times scans were averaged for each spectrum.68. 3′-6-FAM labelled oligonucleotides  before being treated with 10% (v/v) DMS for 1.5 min at 25 °C. The reaction was quenched by adding 100 μl stop solution . The DMS-treated DNA samples were extracted with a Tris-phenol-chloroform solution (pH 8.0) and then precipitated using ethanol at −80 °C before being cleaved by 10% (v/v) piperidine at 90 °C for 30 min. The cleaved DNA samples were separated on a 20% sequencing gel at 1500 v for 4 h in a 4 °C cold room and imaged using a GE Healthcare Typhoon9400 gel scanner .DMS footprinting experiments were performed as followsleotides  were dil69. Briefly, the her2 promoter S1 sequence was inserted into a general template and annealed with a 5′-6-FAM labelled primer (sequences listed in supporting information Table Polymerase stop assays were performed as follows52. An approximately 500 bp sized fragment of the human her2 promoter (from −414 to +78) including the S1 sequence was generated via PCR from Hela cells and inserted into a pGL4.10-basic vector  for construction of the wild-type (WT) plasmid. The mutant plasmid (MU) containing S3 instead of S1 was also constructed as described, and both plasmids were verified via automated sequencing.The luciferase assay experiments were performed as follows5 cells/well into a 24-well plate and incubated at 37 °C overnight. To verify the influence of the mutation on promoter activity, 400 ng WT or MU plasmids were transfected into MCF-7 cells using Lipofectamine 2000  as a transfection reagent. p-RL-TK vector (40 ng) was co-transfected as an internal control. To verify the effect of the small molecule ligand on her2 promoter activity, 0–800 nM cβ was mixed with the WT plasmid and transfected into MCF-7 cells. Luciferase activities were measured using a dual luciferase reporter assay system  according to the manufacturer′s protocol. Relative luciferase activities were acquired by normalizing the ratio of firefly luciferase activity to renilla luciferase activity of the mutant construct with that of the wild-type construct.The cultured MCF-7 cells were plated at a density of 1 × 1052. Cultured MCF-7 cells were split into a 6-well plate at a density of 8 × 105 cells/well and incubated at 37 °C overnight. Powdered cβ was dissolved in DMSO to generate a 10 mM stock solution. A mixture of this stock solution and PEI  at a mass ratio of 1:0.8 was generated and diluted with complete growth medium to form a working solution (0–100 μM), which was used to treat MCF-7 cells at 37 °C overnight. Cells were harvested, and total RNA was extracted using Trireagent  according to the manufacturer′s instructions. TransScript II All-in-One-First-Strand cDNA SuperMix kits  were used to generate cDNA. Forward and reverse primers  and incubated at 37 °C overnight. Powdered cβ was dissolved in DMSO to generate a 10 mM stock solution and mixed with her2 S1 DNA solution (0.5 mM) at a volume ratio of 4:1 to generate a cβ-DNA mixture before annealing at 90 °C. A serial volume of this cβ-DNA mixture was diluted using complete growth medium to a working concentration of 0–50 μM for cβ. Lipofectamine 2000  was used as a transfection reagent, and DMSO was the control. The samples were harvested using a general procedure and then loaded on a 10% SDS gel and transferred to a nitrocellulose membrane at 45 v for 18 h in a 4 °C cold room. The samples were then blocked with 5% non-fat milk at room temperature for 1 h and probed with specific primary antibodies at 4 °C overnight. Target proteins were visualized via treatment with HRP-linked secondary antibodies.Western blotting experiments were performed as described previouslySupporting Information


Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide with limited treatment options. Mutation of β-catenin is one of the most frequent genetic events along hepatocarcinogenesis. β-catenin mutations can be in the form of point mutation or large N-terminal deletion. Studies suggested that different β-catenin mutations might have distinct oncogenic potential.We tested the oncogenic activity of β-cateninS45Y, one of the most frequent point mutations of β-catenin, and ∆N90-β-catenin, a form of β-catenin with a large N-terminal deletion, in promoting HCC development in mice. Thus, we co-expressed β-cateninS45Y or ∆N90-β-catenin together with c-Met into the mouse liver using hydrodynamic injection.We found that both β-catenin mutations were able to induce HCC formation in combination with c-Met at the same latency and efficiency. Tumors showed similar histological features and proliferation rates. However, immunohistochemistry showed predominantly nuclear staining of β-catenin in c-Met/∆N90-β-catenin HCC, but membrane immunoreactivity in c-Met/β-cateninS45Y HCC. qRT-PCR analysis demonstrated that both ∆N90-β-catenin and β-cateninS45Y induced the same effectors, although at somewhat different levels. In cultured cells, both ∆N90-β-catenin and β-cateninS45Y were capable of inducing TCF/LEF reporter expression, promoting proliferation, and inhibiting apoptosis.Our study suggests that β-cateninS45Y and ∆N90-β-catenin, in combination with the c-Met proto-oncogene, have similar oncogenic potential. Furthermore, nuclear staining of β-catenin does not always characterize β-catenin activity. Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, with up to 1 million cases diagnosed and almost 800,000 deaths globally each year , 3. ThusTERT) promoter region, TP53, ARID1A, ARID2, and CTNNB1 genes. Among them, mutations of CTNNB1, which encodes β-catenin, were found in 13–43% of HCCs [Recent genomic studies provide a comprehensive genetic landscape of human HCCs. The most frequent genetic alterations found in this tumor type include mutations of telomerase reverse transcriptase . Activation of c-Met is frequently found in human HCCs . RecentlIn the present study, we investigated the oncogenic potential of the weak β-catenin mutation (β-cateninS45Y) and the strong β-catenin mutation (∆N90-β-catenin) in inducing HCC formation in mice. Our investigation demonstrates that both alterations can equally promote HCC development in combination with the c-Met protooncogene.Two mouse HCC cell lines, HCC3–4 and HCC4–4, which were derived from c-Myc transgenic mice , were cuThe constructs used for mouse injection including pT3-EF1α, pT3-EF1α-c-Met, pT3-EF1α-β-cateninS45Y , pT3-EF1α-∆N90-β-catenin , and pCMV/sleeping beauty transposase (pCMV/SB) have been described previously , 17. AllWild-type (WT) FVB/N mice were obtained from The Jackson Laboratory . Hydrodynamic injection was performed as described previously . In brieMouse liver lesions were fixed in 4% paraformaldehyde overnight at 4 °C and embedded in paraffin. Hematoxylin & Eosin (H&E) staining on 4 μm liver sections was performed to determine the time of appearance and characteristics of neoplastic foci. For immunohistochemistry, antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) by placement in a microwave oven on high for 10 min, followed by a 20-min cool down at room temperature. After a blocking step with the 5% goat serum and Avidin-Biotin blocking kit , the slides were incubated with the following primary antibodies: mouse anti-β-catenin , mouse anti-Glutamine Synthetase , β-catenin (D10A8) XP® Rabbit mAb , anti-Myc tag , Ki-67  overnight at 4 °C. Slides were then subjected to 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity and subsequently the biotin conjugated secondary antibody was applied at a 1:500 dilution for 30 min at room temperature. Signal was detected using the Vectastain ABC Elite kit (Vector Laboratories Inc.) and developed using DAB . Sections were counterstained with hematoxylin solution  and passed through the dehydration process and covered-slipped.Tissue were homogenized with a Polytron and cells were washed in PBS and lysed in M-PER™ Mammalian Protein Extraction Reagent (ThermoFisher Scientific) containing the Halt™ Protease Inhibitor Cocktail (ThermoFisher Scientific). Protein concentration was quantified using the Pierce™ Microplate BCA Protein Assay Kit (ThermoFisher Scientific). Membranes were blocked in in 5% non-fat milk for 1 h and incubated with the following primary antibodies: β-catenin , p-β-catenin , Myc-tag , Glutamine Synthetase , c-Met , p-Met , cyclin D1 , β-actin  and GAPDH . Each primary antibody was followed by incubation with secondary antibody  for 1 h. After appropriate washing, bands were revealed with the Super Signal West Dura Kit (ThermoFisher Scientific).-ΔCt, wherein the ΔCt value of each sample was calculated by subtracting the maximum Ct value of the target gene from the average Ct value of the rRNA gene.Total RNAs were isolated from frozen mouse tissue samples using the Quick-RNA™ MiniPrep . Reverse transcription was conducted according to the manufacturer’s instructions . The mouse primers used for PCR analysis were synthesized by Integrated DNA Technologies . The sequences of the primers were as follows: ribosomal RNA (rRNA):5’-CGGCTACCACATCCAAGGAA-3′ (Forward) and 5’-GCTGGAATTACCGCGGCT-3′ (Reverse); Alpha Fetoprotein (AFP): 5’-TCTGCTGGCACGCAAGAAG-3′ (Forward) and 5’-TCGGCAGGTTCTGGAAACTG-3′ (Reverse); Glypican 3 (GPC3): 5’-CAGCCCGGACTCAAATGGG-3′ (Forward) and 5’-CAGCCGTGCTGTTAGTTGGTA-3′ (Reverse); axis inhibition protein 2 (AXIN2): 5’-GCTCCAGAAGATCACAAAGAGC-3′ (Forward) and 5’-AGCTTTGAGCCTTCAGCATC-3′ (Reverse); G-protein coupled receptor 5 (LGR5): 5’-ACCGAGCCTTACAGAGCCT-3′ (Forward) and 5’-GCCGTCGTCTTTATTCCATTGG-3′ (Reverse); MYC proto-oncogene (c-Myc): 5’-TGTACCTCGTCCGATTCC-3′ (Forward) and 5’-CATCTTCTTGCTCTTCTTCAG-3′ (Reverse); Cyclin D1 (CCND1): 5’-CGTGGCCTCTAAGATGAAGGA-3′ (Forward) and 5’-CCTCGGGCCGGATAGAGTAG-3′ (Reverse); Glutamine Synthetase (GS): 5’-CAGGCTGCCATACCAACTTCA-3′ (Forward) and 5’-TCCTCAATGCACTTCAGACCAT-3′ (Reverse); T-Box3 (TBX3): 5’-CAGGCAGCCTTCAACTGCTT-3′ (Forward) and 5’-GGACACAGATCTTTGAGGTTGGA-3′ (Reverse); Ornithine Aminotransferase (OAT): 5’-GGAGTCCACACCTCAGTCG-3′ (Forward) and 5’-CCACATCCCACATATAAATGCCT-3′ (Reverse); Leukocyte cell-derived chemotaxin-2 (LECT2): 5’-CCCACAACAATCCTCATTTCAGC-3′ (Forward) and 5’-ACACCTGGGTGATGCCTTTG-3′ (Reverse). Quantitative real-time polymerase chain reaction was performed with 100 ng of cDNA, using an ABI Prism 7000 Sequence Detection System and TaqMan Universal PCR Master Mix (ThermoFisher Scientific). Cycling conditions were: 10 min of denaturation at 95 °C and 40 cycles at 95 °C for 15 s and at 52 °C for 1 min. Quantitative values were calculated by using the PE Biosystems Analysis software and expressed as N target (NT). NT = 2HCC3–4 and HCC4–4 cells were plated in triplicate in 24-well plate at 80–90% confluency. Cells were transfected using the Lipofectamine 2000 reagents (Invitrogen). In brief, each well was transfected with 600 ng of pT3-EF1α (empty vector control) or pT3-EF1α-β-cateninS45Y or pT3-EF1α-∆N90-β-catenin, together with 200 ng of TOPFlash plasmid DNA or the negative control FOPFlash, as well as 8 ng of pRL-CMV. Cells were harvested 48 h post transfection. Luciferase activity was measured using the Dual-Luciferase® Reporter Assay System (Promega), according to the manufacturer’s protocol. Experiments were repeated at least three times in triplicate.HCC3–4 and HCC4–4 cells were plated in triplicate in 24-well plate at 80–90% confluency. Cells were transfected using the Lipofectamine 2000 reagents (Invitrogen). Proliferation and apoptosis were assessed using the BrdU Cell Proliferation Reagent  and the Cell Death Detection Elisa Plus Kit , respectively, following the manufacturers’ protocol. Experiments were repeated at least three times in triplicate.P < 0.05 was considered significant.Statistical analysis was performed using Student’s t-test and Tukey-Kramer test. The data were expressed as the mean ± SD of at least three independent experiments. First, we investigated the oncogenic potential of “weak” β-catenin mutant (S45Y) and “strong” β-catenin mutant (∆N90-β-catenin) proto-oncogenes. However, since overexpression of β-cateninS45Y or ∆N90-β-catenin alone does not lead to tumor formation , 17, 19,Grossly, numerous tumor nodules could be found throughout the liver parenchyma of c-Met/β-cateninS45Y and c-Met/∆N90-β-catenin mice Fig. . HistoloCollectively, our results suggest that β-cateninS45Y and ∆N90-β-catenin cooperate with c-Met to promote HCC formation in vivo at similar latency and efficiency.Next, we performed IHC of β-catenin in normal liver as well as HCCs from c-Met/β-cateninS45Y and c-Met/∆N90-β-catenin mice. Using a widely used mouse monoclonal antibody against β-catenin, we found that, in normal liver, β-catenin staining could only be detected at the hepatocyte membrane Fig.  and b. IAt the molecular level, using Western blotting, we found that both c-Met/β-cateninS45Y and c-Met/∆N90-β-catenin HCC lesions showed expression of ectopically injected human c-Met (Fig. Next, we investigated whether c-Met/β-cateninS45Y and c-Met/∆N90-β-catenin mouse HCCs showed similar expression patterns of Wnt/β-catenin canonical target genes. A total of 8 genes were analyzed via qRT-PCR, including the pan-β-catenin targets Axin2, LGR5, c-Myc, and Cyclin D1 Fig. , as wellIn summary, our study shows that β-catenin displays distinct staining patterns in c-Met/β-cateninS45Y and c-Met/∆N90-β-catenin mouse HCCs. However, the staining patterns were not associated with β-catenin activity, as both c-Met/β-cateninS45Y and c-Met/∆N90-β-catenin mouse HCCs demonstrated high and similar expression of β-catenin target genes.Subsequently, we evaluated the transcriptional activity of β-cateninS45Y versus ∆N90-β-catenin with the widely used TCF/LEF reporter constructs. Specifically, two mouse HCC cell lines isolated from c-Myc transgenic mice, HCC3–4 and HCC4–4 cells , were usFinally, we assessed the consequence of the transfection of β-cateninS45Y and ∆N90-β-catenin constructs in terms of proliferation and apoptosis in HCC3–4 and HCC4–4 cells. Noticeably, both mutant forms of β-catenin were able to induce higher proliferation and lower apoptosis than untreated and empty vector transfected cells Fig. . Once agTERT promoter mutation and TP53 mutation, in HCCs [Somatic mutations of β-catenin are one of the most frequent genetic alterations along hepatocarcinogenesis. Specifically, in the most recently released TCGA dataset based on whole-exome sequencing of 363 HCC cases, β-catenin mutation was identified in 27% cases . Survey  in HCCs . Trunk m in HCCs  and are  in HCCs . Thus, iIn this study, we compared the oncogenic potential of β-cateninS45Y and ∆N90-β-catenin mutations in combination with c-Met proto-oncogene in promoting HCC development using mouse modeling. In a previous study, β-cateninS45Y was characterized as a “weak” mutant, whereas ∆N90-β-catenin was characterized as a “strong” mutant . We founPerhaps the most intriguing results from our current study is that ∆N90-β-catenin/c-Met mouse HCC demonstrated strong nuclear β-catenin staining while β-cateninS45Y/c-Met showed predominantly membranous β-catenin immunolabeling Fig. . It is wPrevious studies showed that approximately 10% of human HCCs have concomitant β-catenin mutations along with c-Met overexpression . β-catenOur study suggests that β-cateninS45Y and ∆N90-β-catenin have similar oncogenic potential. Furthermore, nuclear staining of β-catenin does not always characterize β-catenin activity.


Three ammonium salts of 2-amino-4-nitrobenzoate are described. Based on N—H⋯O and O—H⋯O hydrogen-bonding involving the different constituents, supramolecular chains, tubes and double-layers are found in their crystals. 4H12NO+·C7H5N2O4−, (I), C6H16NO+·C7H5N2O4−, (II), and C4H12NO3+·C7H5N2O4−, (III), are described. The common feature of these salts is the presence of the 2-amino-4-nitrobenzoate anion, which exhibit non-chemically significant variations in the conformational relationships between the carboxylate and nitro groups, and between these and the benzene rings they are connected to. The number of ammonium-N—H H atoms in the cations increases from one to three in (I) to (III), respectively, and this variation significantly influences the supramolecular aggregation patterns in the respective crystals. Thus, a linear supramolecular chain along [100] sustained by charge-assisted tertiary-ammonium-N—H⋯O(carboxylate), hydroxy-O—H⋯O(carboxylate) and amino-N—H⋯O(carboxylate) hydrogen-bonds is apparent in the crystal of (I). Chains are connected into a three-dimensional architecture by methyl-C—H⋯O(hydroxy) and π–π interactions, the latter between benzene rings [inter-centroid separation = 3.5796 (10) Å]. In the crystal of (II), a supramolecular tube propagating along [901] arises as a result of charge-assisted secondary-ammonium-N—H⋯O(carboxylate) and hydroxy-O—H⋯O(carboxylate) hydrogen-bonding. These are connected by methylene- and methyl-C—H⋯O(nitro) and π–π stacking between benzene rings [inter-centroid separation = 3.5226 (10) Å]. Finally, double-layers parallel to (100) sustained by charge-assisted ammonium-N—H⋯O(carboxylate), ammonium-N—H⋯O(hydroxy) and hydroxy-O—H⋯O(carboxylate) hydrogen-bonds are apparent in the crystal of (III). These are connected in a three-dimensional architecture by amine-N—H⋯O(nitro) hydrogen-bonds.The crystal and molecular structures of the title molecular salts, C Na+, K+ H4 H3 2C=N(+)H2 H2, i.e. R = Me, n-Bu 2O CH2CH2N(+)H3  and amines substituted with hydroxy groups, i.e. each of Me2N(CH2CH2OH), (t-Bu)N(H)CH2CH2OH and (HOCH2)3CNH2 are described whereupon the anhydrous 1:1 salts, i.e. [Me2N(+)H(CH2CH2OH)]L (I)t-Bu)N(+)H2(CH2CH2OH)]L (II)2)3CN(+)H3]L (III)Despite being tetramorphic °. Similarly, the nitro group lies out of the plane of the benzene ring, forming a dihedral angle of 6.6 (3)°. A dis-rotatory relationship between the carboxylate and nitro substituents is indicated by the dihedral angle between them of 11.5 (4)°. An intramolecular amine-N1—H⋯O1(carboxylate) hydrogen-bond is noted which closes an S(6) loop, Table 22N(+)(H)CH2CH2OH cation, the N3—C8—C9—O5 torsion angle of −71.15 (19)° is indicative of a −syn-clinal conformation.The molecular structures of the constituent ions in (I)t-Bu)N(+)H2(CH2CH2OH) cation is relatively rare, being reported for the first time in its salt with sulfathiazolate only in 2012 2)3CN(+)H3 cation exhibits N3—C8—C9—O5, N3—C8—C10—O6 and N3—C8—C11—O7 torsion angles of −59.01 (18), −49.84 (19) and −58.12 (18)°, respectively, indicating −syn-clinal relationships.The anion in (III)As expected from the chemical compositions of (I)–(III), significant charge-assisted hydrogen-bonding is apparent in their respective crystals. Geometric data characterizing these and other identified interactions are collated in Tables 2via charge-assisted ammonium-N3—H⋯O(carboxylate) and hydroxy-O—H⋯O(carboxylate) hydrogen-bonds to form a nine-membered {⋯OCO⋯HNC2OH} heterosynthon. These are connected into a linear, supramolecular chain along the a-axis direction via amino-N—H⋯O(carboxylate) hydrogen-bonds, Fig. 4a). The chains are linked along the b axis via π–π interactions between benzene rings , and methyl-C—H⋯O(hydroxy) interactions link molecules along the c-axis to consolidate the three-dimensional packing, Fig. 4b).As indicated in Fig. 12OH} heterosynthon, observed in (I)a). However, in (II)b). As seen from Fig. 5b), the benzene rings are aligned to be proximate and, indeed, they interact via π–π stacking with the inter-centroid separation being 3.4944 (9) Å . The carboxylate-O2 atom forms two hydrogen-bonds. The connections between the tubes are of the type methylene- and methyl-C—H⋯O(nitro), involving both nitro-O atoms, as well as π–π stacking between benzene rings . A view of the unit-cell contents is shown in Fig. 5c), highlighting the intra- and inter-tube π–π stacking along the c-axis direction.In the crystal of (II)bc-plane are formed as a result of charge-assisted ammonium-N3—H⋯O(carboxylate), ammonium-N3—H⋯O(hydroxy) and hydroxy-O—H⋯O(carboxylate) hydrogen-bonds. The ammonium-N3—H3N atom is bifurcated, forming two weak ammonium-N3—H⋯O(hydroxy) hydrogen-bonds. A view normal to the plane of the double-layer and a side-on view are shown in Fig. 6a) and (b), respectively. From the latter, the intra-layer region comprises the ammonium groups, each of which forms four N—H⋯O hydrogen-bonds to carboxylate and hydroxy groups on either side. Each hydroxy group of the cation forms a hydroxy-O—H⋯O(carboxylate) hydrogen-bond with a carboxylate-O atom derived from a different anion, and each accepts an ammonium-N—H atom derived from a different cation. Each carboxylate-O atom forms two hydrogen-bonds, the O1 accepts hydrogen-bonds from different hydroxy groups, and the O2 atom accept hydrogen-bonds from hydroxy and ammonium groups. Projecting to either side of the double-layer are the nitrobenzene groups, Fig. 6c) and (d). These provide the links to construct the three-dimensional architecture, i.e. via amine-N—H⋯O(nitro) interactions, involving both nitro-O atoms.In the crystal of (III)i.e. chain in (I)The obvious trend from the present study is the increase in dimensionality of the supramolecular aggregation pattern, Chemical context, a number of ammonium salts of the anion derived from 2-amino-4-nitrobenzoic acid have now been described. The key conformational indicators for the anion are the dihedral angles formed between CO2/C6/NO2. The smallest dihedral angles between the CO2/C6, C6/NO2 and CO2/NO2 pairs of least-squares planes of 3.44 (14), 0.69 (11) and 3.2 (2)° are found for the anion in the salt with H3N(+)CH2CH2N(+)H3 , 12.6 (3) and 26.73 (14)°, respectively, are found in the N(+)H4 H2 H3 : 3500–2700 , 1632, 1537, 1433, 1381, 1346, 1329, 1279, 1263, 1209, 1140, 1099, 1072, 1022, 918, 858, 823, 785, 731, 692, 684, 577, 513, 486.Preparation of dimethyl(2-hydroxyethyl)ammonium 2-amino-4-nitrobenzoate (I)tert-butyl(2-hydroxyethyl)ammonium 2-amino-4-nitrobenzoate (II)tert-butyl(2-hydroxyethyl)amine (1 mmol) in methanol (10 ml). The reaction mixture was refluxed for 15 mins, and then maintained at room temperature. Crystals of (II)−1): 3550–2700 , 1640, 1446, 1370, 1351, 1329, 1269, 1221 1137, 1085, 1034, 858, 8245, 739, 687, 587.Preparation of −1): 3700–2400 , 1647, 1431, 1350 1250, 1146, 1115, 1063, 1010, 872, 827, 736, 689, 596, 578 511, 484, 1549, 1356.Preparation of tris(hydroxymethyl)methylammonium 2-amino-4-nitrobenzoate (III)Uiso(H) set to 1.2–1.5Ueq(C). The O- and N-bound H atoms were located from difference maps, but refined with O—H = 0.84±0.01 Å and Uiso(H) = 1.5Ueq(O), and with N—H = 0.86–0.88±0.01 Å and Uiso(H) = 1.2Ueq(N), respectively. In the refinement of (II)i.e. (0 2 0), was omitted from the final cycles of refinement.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989018015578/hb7785sup1.cifCrystal structure: contains datablock(s) I, II, III, global. DOI: 10.1107/S2056989018015578/hb7785Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989018015578/hb7785IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989018015578/hb7785IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989018015578/hb7785Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018015578/hb7785IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018015578/hb7785IIIsup7.cmlSupporting information file. DOI: 1876928, 1876927, 1876926CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Tinospora sinensis in virtue of their special structures and activities. Herein, an analytical method was developed for rapid screening and identification of diterpenoids in T. sinensis using high-performmance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (HPLC-LTQ-Orbitrap) in negative ion mode. Two diterpenoid reference standards were first analyzed to obtain their characteristic ESI-MS/MS fragmentation patterns. Then, based on the extracted ion chromatogram (EIC) data-mining method and characteristic fragmentation pathways analysis, diterpenoids in T. sinensis were rapidly screened and identified. After that, an important parameter, Clog P, was adopted to discriminate between the isomers of diterpenoids. As a result, 63 diterpenoids were characterized from the extract of T. sinensis, including 10 diterpenoids and 53 diterpenoid glycosides. Among them, 15 compounds were tentatively identified as new compounds. Finally, target isolation of one diterpenoid glycoside named tinosineside A was performed based on the obtained results, which further confirmed the deduced fragmentation patterns and identified diterpenoid profile in T. sinensis. The results demonstrated that the established method could be a rapid, effective analytical tool for screening and characterization of diterpenoids in the complex systems of natural medicines.Diterpenoids are considered the major active compounds in  Tinospora sinensis (Kuanjinteng in Chinese) is commonly known as ‘Gurch’ and belongs to the family Menispermaceae, which is mainly distributed in the tropical parts of the eastern hemisphere. It has been traditionally used as folk medicine for treating debility, dyspepsia, rheumatism, gonorrhea, fever, inflammation, syphilis, ulcer, bronchitis, jaundice, urinary disease, skin disease and liver disease −, [M − H − H2O]−, [M − H − CO2]− and [M − H − H2O − CO2]−. Columbin produced a fragment ion at m/z 329 through losing carbonyl, and the ion at m/z 329 loss of a CO2 produced its ESI-MS2 base peak ion at m/z 285. The ion at m/z 245 [M − H − 112]− resulted from a Retro-Diels-Alder (RDA) cleavage fragmentation at the 1,4-position of the A-ring. Moreover, the product ion at m/z 245 generated the minor ion at m/z 217 by loss of one molecule of CO.Columbin and isocolumbin yielded their [M − H]2 base peak ion at m/z 339 by neutral loss of H2O. After a RDA cleavage at A-ring and successive fracture of C9–11 , the prominent ion at m/z 151 was generated  of 29 and 51 generated their [M − H]− ions at m/z 357.13326 (C20H21O6). They were unambiguously assigned as isocolumbin and columbin, respectively, by comparison of their retention times, literature data and MS fragmentation data with their standards − ion at m/z 417.15439 (C22H25O8). In CID mode, it further generated [M − H − CH3]−, [M − H − H2O − CO]− and [M − H − H2O − CO − CH3]− ion at m/z 402, m/z 371 and m/z 356, respectively. The ESI-MS2 base peak ion at m/z 181 (C11H17O2) was generated by RDA cleavage fragmentation from 1,4-position of the A-ring and fracture of C9–11 (C6H4O3). The product ion at m/z 181 loss of a methyl generated the prominent ion at m/z 166. Therefore, compound 32 was tentatively deduced as tinocapilactone B [Compound actone B .40 and 41 produced their [M − H]− ions at m/z 373.12817 (C20H21O7). Both of their deprotonated molecular ions generated a series of fragment ions at m/z 358 [M − H − CH3]−, m/z 343 [M − H − CO − H2]−, m/z 325 [M − H − 48 amu]− and m/z 313 [M − H − 60 amu]−, respectively. In addition, compound 41 generated two minor ions at m/z 305 and m/z 261. By comparison with the MS fragmentation patterns obtained from the reference standards, compounds 40 and 41 were plausibly characterized as 8-hydroxycolumbin and 6-hydroxycolumbin, respectively.Compounds 57 showed the [M − H]− ion at m/z 329.13835 (C19H21O5). Its ESI-MS2 base peak ion at m/z 314 was generated by loss of a methyl. The major fragment ions at m/z 311 and m/z 285 were yielded by neutral loss of H2O and CO2, respectively. Moreover, the product ion at m/z 285 generated the predominant product ion at m/z 191 due to the overall fracture of C9–11 (C6H6O). According to the literature data, it was tentatively identified as tinosponone [Compound osponone .60 and 61 produced their respective [M − H]− ion at m/z 329.17473 (C20H25O4) and m/z 347.18530 (C20H27O5). Both of their deprotonated molecular ions generated [M − H − H2O]−, [M − H − CO2]− and [M − H − H2O − CO2]− ions at m/z 311, m/z 329, m/z 285, m/z 303 and m/z 267, m/z 285, respectively. By comparing with the literature data, compounds 60 and 61 were tentatively identified as tinotufolin D and -6-2-(3-furanyl)ethyl-2a,3,4,5,5a,6,7,8,8a,8b- decahydro-2a,3-dihydroxy-6,7,8b-trimethyl-2H-naphtho-1,8-bcfuran-2-one, respectively [Compounds ectively .62 gave a [M − H]− ion at m/z 331.19038 (C20H27O4). Its MS2 spectrum produced the fragment ion at m/z 313, which involves the loss of H2O. The molecular ion yielded a series of fragment ions at m/z 287 [M − H − CO2]−, m/z 283 [M − H − H2O − H2CO]− and m/z 271 [M − H − CH4 − CO2]−, suggesting the presence of -COOCH3. The base peak ion at m/z 285 was generated by losing water and carbon monoxide. The [M − H − C6H6O]− ion at m/z 237 was generated by the fracture of C9-11 (C6H6O). Moreover, by comparison with the fragmentation patterns obtained from the two reference standards, compound 62 was tentatively identified as a new compound.Compound 63 generated a [M − H]− ion at m/z 363.21660 (C21H31O5). It generated a serial of fragment ions at m/z 345 [M − H − H2O]−, m/z 334 [M − H − 29 amu]−, m/z 317 [M − H − H2O − CO]− and m/z 295 [M − H − 2H2O − OCH4]−. By comparison with the literature data, compound 63 was tentatively identified as tinotufolin C [Compound 1 and 24 produced their [M – H]− ions at m/z 765.29642 (C37H49O17). After the CID cleavage, both of their further fragmentations resulted in a [M − H − CO2]− ion at m/z 721 and a [M − H − H2O − CO]− ion at m/z 719, which were consistent with the characteristic fragmentation pathways of diterpenoids. According to the fragmentation patterns and the values of Clog P, compounds 1 and 24 were plausibly characterized as -2-(acetoxymethyl)-6--2-(furan-3-yl)-9,10-dimethoxy-7-(methoxycarbonyl)-6,10-dimethyl-4-oxododecahydro-1H-benzo[f]isochromen-6-yl)-oxy)tetrahydro- 2H-pyran-3,4,5-triyl triacetate and rumphioside D, respectively.Compounds 2, 6 and 39 produced their [M − H]− ions at m/z 537.19665 (C26H33O12). They all produced the [M − H − H2O]− and [M − H − H2O − CO]− ions at m/z 519 and m/z 491. In addition, compounds 2 and 39 also yielded the [M − H − 162]− ions at m/z 375 due to the overall fracture of dehydrated glucose, which was the ESI-MS2 base peak of compound 2. As well as we known, [M − H − 162]− was the characteristic ion of glycosides. Furthermore, compounds 2, 6 and 39 generated the ions at m/z 490 [M − 2H − H2O − CO]−, m/z 357 [M − H − Glc]−, m/z 327 [M − H − Glc − 2CH3]−, m/z 297 [M − 2H − CO − Glc − OCH3]−; m/z 492 [M − 2H − CO2]−, m/z 490 [M − 2H − H2O − CO]−, m/z 469 [M − 2H − H2O − CO − OCH3]−, m/z 297 [M − 2H − CO − Glc − OCH3]−; m/z 493 [M − H − CO2]−, m/z 357 [M − H − Glc]−, m/z 341 [M − H − O − Glc]−, m/z 329 [M − H − CO − Glc]−, m/z 297 [M − 2H − CO − Glc −OCH3]−, respectively. By comparison with the literature data, compounds 2, 6 and 39 were tentatively assigned as cordioside, cordifoliside D and borapetoside A, respectively − ions at m/z 597.21778 (C28H37O14). Firstly, they were divided into two categories based on whether they produced the [M − H − 112]− ion at m/z 486 by the RDA cleavage from A-ring. Compound 47 generated its ESI-MS2 base peak ion at m/z 433 by losing dehydrated glucose. In addition, it also yielded [M − H − 2H2O]− ion at m/z 561, [M − H − H2O − CO]− ion at m/z 551, [M − H − 112]− ion at m/z 486, [M − H − dehydrated Glc]− ion at m/z 435, and so on. As far as we knew, there were no related literatures reported. And thus, compound 47 was finally deduced to be a new compound. Compounds 3, 8, 14, 20, 25, 34, 43 and 49 yielded their [M − H − H2O − CO]− ions at m/z 551, which was the ESI-MS2 base peak of compounds 20 and 34, and it also a high intense characteristic fragment ion of compound 49. Compared with the prominent ions of them, there was no difficulty to deduce that compounds 34 and 49 have two lactone rings, which were different to the other compounds. Furthermore, the fragment ions further validated the above deduction. Finally, according to their Clog P values, they were tentatively identified and differentiated. By comparison with the bibliography and MS fragmentation data, compounds 3, 8, 14, 20, 25, 34, 43 and 49 were tentatively identified . Both of their deprotonated molecular ions yielded [M − H − H2O]− ion at m/z 665, [M − H − H2O − CO]− ion at m/z 637, [M − H − C4H4O]− ion at m/z 615, [M − H − dehydrated Glc]− ion at m/z 520, respectively. Moreover, compound 4 also dissociated into fragment ions [M − H − CO2]− at m/z 639, [M − H − Glc − dehydrated xyl]− at m/z 370 and [M − H − Glc − dehydrated xyl − CO2 − H2O]− at m/z 309. According to the literature, compounds 4 and 46 were plausibly characterized as -15,16-epoxy-2-hydroxy-6-O-{β-d-xylopyranosyl(1→6)–d-glucopyranosyl}-cleroda-3,13(16),14-trien-17,12-olid-18-oic acid methyl ester and -15,16-epoxy-2-hydroxy-6-O-{β-d-glucopyranosyl-(1→6)-α-d-xylopyran-osyl}-cleroda-3,13(16),14-trien-17,12-olid-18-oic acid methyl ester, respectively [Compounds ectively .5 and 18 produced their [M − H]− ions at m/z 513.19665 (C24H33O12). The dominant characteristic fragment ions were presented at m/z 333 [M – H − 180]− and m/z 351 [M − H − 162]− as the ESI-MS2 base peak corresponding to the cleavage from glucopyranosyl. After the CID cleavage, both of their further fragmentation resulted in [M − H − H2O]− at m/z 495 and [M − H − Glc − CO]− at m/z 305. The product ion at m/z 305 of compound 18 further generated the predominant ion at m/z 287 by losing one molecular of water. By comparison with the literature data and MS fragmentation data, compounds 5 and 18 were tentatively identified as 1-deacetyltinosposide A and -2-(furan-3-yl)-6,9,10–trihydroxy-10b-methyl-7--3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)decahydro-1H-benzo[f]isochromen-4(2H)-one, respectively − ions at m/z 567.20721 (C27H35O13). They all produced the [M − H − H2O − CO]− and [M − H − Glc]− ions at m/z 521 and m/z 405. Besides, compounds 7 and 10 generated m/z 552 by losing a methyl, and the ion at m/z 341 was generated by losing a series of fragment ions of glucose residue, 2H2O, and CO. Compounds 7 and 15 were also generated [M − H − Glc − CO]− ions at m/z 359, which was the ESI-MS2 base peak of compound 7. Meanwhile, compound 7 generated the minor ion at m/z 329. Compound 17 produced its ESI-MS2 base peak by losing two molecular of methyl, one molecular of water and carbonyl at m/z 491. Combined with bibliography data and fragmentation pathways, these four compounds were tentatively deduced as tinospinoside D, amritoside C, rumphioside A and rumphioside F, respectively − ions at m/z 551.21230 (C27H35O12). Compounds 9, 13 and 19 produced their ESI-MS2 base peak ions at m/z 389 [M − H − dehydrated Glc]−, and further generated a series of fragment ions by losing methyl, water, carbon dioxide units at the mean time. According to the literature and the values of Clog P, compounds 9, 13 and 19 were plausibly characterized as borapetoside B, tinosposinenside A and -15,16-epoxy-2-hydroxy-6-O-(β-d-glucopyranosyl)-cleroda-3,13(16), 14-trien-17,12-olid-18-oic acid methyl ester, respectively − ions at m/z 535.18100 (C26H31O12). Compound 12 produced its ESI-MS2 base peak ion at m/z 373 by loss of a dehydrated glucose and then generated two minor ions at m/z 345 [M − H − dehydrated Glc − CO]− and m/z 313 [M − H − O − dehydrated Glc − CO2]−. While compound 21 generated a series of fragment ions at m/z 520 [M − H − CH3]−, m/z 517 [M − H − H2O]−, m/z 373 [M − H − dehydrated Glc]− and m/z 329 [M − H − dehydrated Glc − CO2]−. According to the fragmentation patterns and the values of Clog P, compounds 12 and 21 were tentatively characterized as -2-(furan-3-yl)-6a,9b-dimethyl-6--3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)decahydro-1H-9,7-(epoxymethano) oxirenobenzoisochromene-4,11(2H)-dione and palmatoside F, respectively.Compounds 16 produced its [M − H]− ion at m/z 521.20173 (C26H33O11). A high intense characteristic fragment ion was present at m/z 359 [M – H − 162]− as its ESI-MS2 base peak corresponding to the cleavage of the dehydrated glucose. The product ion at m/z 359 successively generated the predominant product ions at m/z 344 and m/z 341 by losing a methyl and one molecular of water. According to the literature data, compound 16 was tentatively identified as furanoid diterpene glycoside [Compound lycoside .22 generated its [M − H]− ion at m/z 527.21230 (C25H35O12). Upon CID mode, its further fragmentation resulted in [M − H − H2O]− ion at m/z 509, [M − H − H2O − CH3]− ion at m/z 494, [M – H − H2O − CO]− ion at m/z 481, [M − H − Glc]− ion at m/z 347, and [M − H − Glc − CH2O − CO − CH4]− ion at m/z 273. As far as we knew, there were no related literatures reported. Thus, compound 22 was tentatively deduced to be a new compound.Compound 23, 31 and 37 yielded their [M − H]− ions at m/z 555.20721 (C26H35O13). Compound 31 produced its ESI-MS2 base peak ion at m/z 513 [M − Ac]−, and the m/z 513 ion generated its predominant ions at m/z 495 [M − H − CH2CO − H2O]− and m/z 333 [M − H − CH2CO − dehydrated Glc]− due to the overall fracture of a hydroxy and glycoside bond. The ion at m/z 333 generated two minor ions at m/z 315 and m/z 305 through losing H2O and CO, respectively. Besides, its [M − H]− ion also yielded characteristic fragment ion at m/z 393 [M − H − 162]− by losing a dehydrated glucose. By comparison with the literature data, compound 31 was tentatively identified as tinosineside A [23 and 37 produced their fragment ions at m/z 537 [M − H − H2O]−, m/z 513 [M − CH3 − CO]−, m/z 495 [M − H − CH3 − COOH]− and m/z 393 [M − H − dehydrated Glc]−. Furthermore, compound 23 generated the fragment ions at m/z 375 [M − H–H2O–Glc]−, m/z 305 [M − H–dehydrated Glc–CH4 − CO − CO2]− and m/z 297 [M − H − H2O − Glc − CO2 − CH4]− or [M − H − 2H2O − Glc − CH2CO]−. According to the fragment ions and the values of Clog P, compounds 23 and 37 were tentatively identified as amritoside A and -1-(2-(furan-3-yl)-2-hydroxyethyl)-2-hydroxy-5-(methoxycarbonyl)-1-methyl-7--3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-1,2,3,4,6,7,8,8-octahydronaphthalene-2-carboxylic acid, respectively.Compounds neside A . Compoun26 produced its [M − H]− ion at m/z 523.21738 (C26H35O11). Its ESI-MS2 base peak ion at m/z 361 was yielded by loss of a dehydrated glucose. In addition, it generated a series fragment ions, such as m/z 508, m/z 505, m/z 347, m/z 343 and m/z 329, involving the loss of a methyl group, a water molecule, and glucose. By comparing with the fragmentation patterns obtained from two reference standards, compound 26 was tentatively identified as sagittatayunnanoside D. Similarly, based on the literature data, compound 27 was tentatively identified as borapetoside H [Compound toside H .28 and 33 exhibited their [M − H]− ions at m/z 549.19665 (C27H33O12). Both of their deprotonated molecular ions generated a serial of fragment ions at m/z 531 [M − H − H2O]−, m/z 503 [M − H − H2O − CO]−, m/z 481 [M − H − 2H2O − CH3OH]− and m/z 387 [M − H − dehydrated Glc]−, respectively. Meanwhile, a product ion at m/z 513 [M − H − 2H2O]− was observed in the ESI-MS/MS spectra of compound 33, suggesting there was two hydroxy groups at the skeleton. According to the literature data and the values of Clog P, compounds 28 and 33 were tentatively identified as -15,16-epoxy-2-oxo-6-O-(β-d-glucopyranosyl)–cleroda-3,13(16),14-trien-17,12 -olid-18-oic acid methyl ester and tinoscorside C, respectively [Compounds ectively .30 generated its [M − H]− ion at m/z 519.18608 (C26H31O11). The dominant characteristic fragment ion was present at m/z 357 [M − H − 162]− as its ESI-MS2 base peak, corresponding to the cleavage from the dehydrated glucose. Meanwhile, the product ions [M − H − H2O]− (lose of water) at m/z 501 and [M − H − 46]− (lose of water and carbon monoxide) at m/z 473 were monitored, which were in accordance with the diterpenoids glycosides cracking patterns, suggesting this compound might be tinoside.Compound 36 and 44 yielded their [M − H]− ions at m/z 535.21738 (C27H35O11). Both of their deprotonated molecular ions generated a serial of fragment ions at m/z 520 [M − H − CH3]−, m/z 488 [M − 2H − CO − H2O]−, m/z 373 [M − H − dehydrated Glc]−, m/z 359 [M − H − dehydrated Glc − CH2]−, m/z 358 [M − H − dehydrated Glc − CH3]−, m/z 355 [M − H − Glc]− and m/z 341 [M − H − Glc − CH2]−. According to the literature data and the values of Clog P, compounds 36 and 44 were tentatively identified as borapetoside C and tinocrisposide [Compounds isposide .38 gave a [M − H]− ion at m/z 581.22286 (C28H37O13). Its major ions in the MS2 spectrum were m/z 563 [M − H − H2O]−, m/z 535 [M − H − H2O − CO]−, m/z 419 [M − H − dehydrated Glc]−, m/z 373 [M − H − Glc − CO]− and m/z 343 [M − Glc − CH2CO2]−, suggesting this compound might contain the glucose and acetyl fragments. Comparison with the parent nucleus and fragmentation patterns, compound 38 was tentatively identified as tinosposinenside B.Compound 42 exhibited its [M − H]− ion at m/z 537.23303 (C27H37O11). Its ESI-MS2 base peak ion at m/z 375 was produced by losing dehydrated glucose, and the product ion at m/z 375 further generated two minor ions at m/z 360 and m/z 357 by losing CH3 and H2O, respectively. According to the fragmentation pathways, compound 42 was tentatively identified as boropetoside G. Likewise, compound 48 was tentatively defined as tinosposinenside C.Compound 45 produced its [M − H]− ion at m/z 597.20002 (C28H37O12S), which exhibited the characteristic fragment ion [M − H − C8H14O6S]− at m/z 359 suggesting the overall fracture of side chain. S-diterpenoids glycosides were very rare in Tinospora, and there only two compounds were reported in this family so far [45 was plausibly described as cordifolide A.Compound y so far . By comp50 and 59 produced their [M − H]− ions at m/z 623.23343 (C30H39O14). According to the respective fragmentation pathways and the values of Clog P, compounds 50 and 59 were tentatively identified as 6′-O-lactoylborapetoside B and 2-O-lactoylborapetoside B, respectively.Compounds 52–54 produced their respective [M − H]− ion at m/z 517.17043 (C26H29O11), m/z 491.19117 (C25H31O10) and m/z 715.28077 (C33H47O17). None but compound 53 exhibited the [M − H + HCOOH]− adduct ion at m/z 537.19665 (C26H33O12). Take compound 52 for example, its major ions in ESI-MS2 spectrum were m/z 499 [M − H − H2O]−, m/z 473 [M − H − CO2]−, m/z 471 [M − H − H2O − CO]−, m/z 455 [M − H − CO2 − H2O]−, m/z 355 [M − H − dehydrated Glc]−, m/z 162 −C6H10O5 and m/z 151 –C5H11O5, suggesting this compound might contain glucose and undergo a RDA coverage from the A-ring at the 1,4-position. According to the above analysis, compounds 52–54 were tentatively defined as tinospinoside E, tinosporaside and sagittatayunnanoside B, respectively.Compounds 56 and 58 generated their respective [M − H]− ion at m/z 671.29094 (C32H47O15) and 509.23812 (C26H37O10). The defference between of these molecules was 162 Da, suggesting that compound 56 had one more glucose unit than compound 58. Both of them generated the same ESI-MS2 base peak ions at m/z 329 due to the overall fracture of glycoside bond. Furthermore, the product ion at m/z 329 generated a predominant ion at m/z 301 by splitting off CO. Comparing with the parent nucleus and respective fragmentation pathways, compounds 56 and 58 were plausibly identified as sagittatayunnanoside C and sagittatayunnanoside A, respectively.Compounds − at m/z 555.20721 . It firstly produced the ESI-MS2 base peak ion at m/z 513 [M − Ac]− and further generated the ions at m/z 495 and m/z 375 by losing one molecule of water and dehydrated glucose. The ion at m/z 351 generated minor ions at m/z 333, m/z 315 and m/z 307 through losing H2O, 2H2O and CO2, respectively. Besides, its [M − H]− ion also yielded characteristic fragment ion at m/z 393 [M − H − 162]− by losing a dehydrated glucose. The fragmentation pathway was consistent with deduced of compound 31, which further proved the validity of the results  and then authenticated by Professor Chun-sheng Liu in Beijing University of Chinese Medicine.The reference standards of columbin and isocolumbin (purity over 98%) were procured from Tauto Biotech .The structures of these standards were presented in The standard solutions of columbin and isocolumbin were prepared in methanol at appropriate concentrations.T. sinensis were weighed accurately (0.13 g) and refluxed with a tenfold excess of ethanol/water  three times, and placed in 20 mL of methanol/water . The mixture was then extracted in an ultrasonic bath at room temperature for 0.5 h, and the same solvent was added to compensate for the weight lost during the extraction. The extract was filtered and evaporated to near dryness, then placed on a C18 Solid Phase Extraction (SPE) column , which was washed with 4 mL distilled water and 4 mL methanol. The methanol eluent was filtered through a 0.22 µm membrane for analysis. All of the solutions were stored at 4 °C and brought to room temperature before analysis.Powdered dried alcoholic extracts of B) and 0.1% (v/v) formic acid in water (A) with the elution gradient set as follows: 0–5 min, 8–12% B; 5–25 min, 12–16% B, 25–45 min, 16–25% B; 45–75 min, 25–46% B; 75–80 min, 46–58% B; 80–95 min, 58–65% B; 95–105 min. The flow rate was set as 1.0 mL/min.HPLC analysis was carried out on a DIONEX Ultimate 3000 UHPLC system  with a binary pump and an autosampler. The samples were separated on a Sunfire C18 column  at room temperature. The mobile phase consisted of acetonitrile  flow, 30 arb.; auxiliary gas (nitrogen) flow, 10 arb.; electrospray voltage, 3.0 kV; capillary voltage, −35 V; tube lens voltage, −110 V. The resolution of Orbitrap analyzer was set at 30,000 with data-dependent ESI-MS2 analysis triggered by the three most abundant ions from one-stage mass spectrometry scanning. Collision-induced dissociation (CID) was performed in LTQ with an activation q of 0.25 and activation time of 30 ms. The isolation width was 2 amu, and the normalized collision energy was set to 35%.A LTQ-Orbitrap XL mass spectrometer  was connected to the HPLC system via an electrospray ionization (ESI) interface. The effluent was introduced into the ESI source in a post-column splitting ratio of 1:4. Full scan data acquisition was performed from P parameter obtained from Chemdraw to distinguish their polarity. And the exact mass error of all determined compounds was within 3 ppm.A Thermo Xcalibur 2.1 workstation was used for the data acquisition and processing, In order to obtain as many fragment ions of the diterpenoids as possible, the peaks detected with intensity over 30,000 were selected for identification. The chemical formulas for all parent ions of the selected peaks were calculated from the accurate mass using a formula predictor by setting the parameters as follows: C (0–50), H (0–100), O (0–30), S (0–2), N (0–2). Other elements were not considered because they are rarely present in diterpenoids. Furthermore, diterpenoids isomers were differentiated by an important Clog 2O, and then successively extracted with CHCl3, EtOAc, n-BuOH and MeOH. The n-BuOH extract was passed through an AB-8 macroporous resin column and then washed with H2O, 30% EtOH, 50% EtOH, 70% EtOH and 95% EtOH. The 30% EtOH fraction was further purified by silica gel column chromatography with elution by CHCl3–MeOH  to give the diterpenoid glycoside named tinosineside A as a white powder.The air-dried stems (10.0 kg) were extracted three times with tenfold excess of 70% EtOH under reflux for 2 h each at 80 °C. The combined extract was evaporated under reduced pressure to obtain a crude residue. This residue was further dispersed in HT. sinensis. According to the characteristic fragmentation pathways of two reference standards, all diterpenoids were divided into two categories, namely diterpenoid aglycones and diterpenoid glycosides. Furthermore, an important parameter Clog P was used to differentiate the isomers of diterpenoids. As a result, 63 diterpenoids were preliminarily identified, including 48 known compounds and 15 new compounds. These compounds included 10 diterpenoid aglycones, 53 diterpenoid glycosides. Finally, target isolation of one diterpenoid glycoside named tinosineside A was performed based on the obtained results, which further confirmed the deduced fragmentation patterns and identified profile of diterpenoids in T. sinensis. This represents the first systematic report of diterpenoids in T. sinensis. The results indicated that the established method could be employed as a rapid, effective technique to screen and identify diterpenoids in T. sinensis. The study also provided significant guidance for the analysis of other herbal medicines or preparations.In this study, a sensitive HPLC-LTQ-Orbitrap coupled with EIC data-mining method was established for the rapid characterization of diterpenoids in


H-pyridine-2-selenenyl dibromide and its cycloadduct with cyclopentene were studied by X-ray diffraction.Bromination of bis(pyridin-2-yl) diselenide in methylene chloride was carried out and the mechanism is proposed. The molecular and crystal structures of 1 H-Pyridine-2-selenenyl dibromide, C5H5NSeBr2, 1, is a product of the bromination of bis(pyridin-2-yl) diselenide in methylene chloride recrystallization from methanol. Compound 1 is essentially zwitterionic: the negative charge resides on the SeBr2 moiety and the positive charge is delocalized over the pyridinium fragment. The C—Se distance of 1.927 (3) Å is typical of a single bond. The virtually linear Br—Se—Br moiety of 178.428 (15)° has symmetrical geometry, with Se—Br bonds of 2.5761 (4) and 2.5920 (4) Å, and is twisted by 63.79 (8)° relative to the pyridinium plane. The Se atom forms an intermolecular Se⋯Br contact of 3.4326 (4) Å, adopting a distorted square-planar coordination. In the crystal, molecules of 1 are linked by intermolecular N—H⋯Br and C—H⋯Br hydrogen bonds, as well as by non-covalent Se⋯Br interactions, into a three-dimensional framework. -2,3,3a,9a-Tetrahydro-1H-cyclopentaselenazolopyridinium-9 bromide, C10H12NSe+·Br−, 2, is a product of the cycloaddition reaction of 1 with cyclopentene. Compound 2 is a salt containing a selenazolopyridinium cation and a bromide anion. Both five-membered rings of the cation adopt envelope conformations. The dihedral angle between the basal planes of these rings is 62.45 (11)°. The Se atom of the cation forms two additional non-covalent interactions with the bromide anions at distances of 3.2715 (4) and 3.5683 (3) Å, attaining a distorted square-planar coordination. In the crystal, the cations and anions of 2 form centrosymmetric dimers by non-covalent Se⋯Br interactions. The dimers are linked by weak C—H⋯Br hydrogen bonds into double layers parallel to (001).1 We isolated a compound with the same melting point as that previously obtained by the Japanese authors only after recrystallization from methanol. In our opinion, it is the lower melting point product that is the 2-pyridylselenenyl bromide  1 Fig. 1.a]pyridin-4-ium derivatives via heterocyclization of unsaturated compounds and 2-pyridinesulfenyl, selenenyl and tellurenyl chlorides with ring closure through the nitrogen atom of the pyridyl fragment azolo. Thus, the selenium atom adopts a distorted square-planar coordination.Compound nt Fig. 5. The C2—2, C10H12NSeBr, is a salt containing a selenazolopyridinium cation and a bromide anion  Å from the plane through the other ring atoms. The cyclopentane fragment has the usual envelope conformation with the C2 carbon atom deviating from the plane through the other ring atoms by 0.648 (4) Å. The dihedral angle between the basal planes of the two five-membered rings of the cation is 62.45 (11)°. The selenium atom of the cation forms two additional non-covalent interactions with the bromide anions at distances of 3.2715 (4) Å [Se4⋯Br1] and 3.5683 (3) Å [Se4⋯Br1], affording a distorted square-planar coordination.Compound on Fig. 6. The fiv2 has two asymmetric C3A and C9A carbon atoms. The crystal of the compound is racemic with the following relative configurations of the centers – rac-3aSR,9aRS.Cation 1, molecules are linked by intermolecular N—H⋯Br and C—H⋯Br hydrogen bonds –3.556 (1) Å.A search of the Cambridge Structural Database  Å observed in the crystal of 2-Pyridineselenenyl bromide (1*). A solution of bromine  in ethylene chloride (10 ml) was added to a solution of bis(pyridin-2-yl)diselenide  in methylene chloride (10 ml) at room temperature. After 30 min, the solvent was removed under vacuum. The residue was washed with diethyl ether. Yield 0.93 g (98%), bright-yellow powder, m.p. 373–375 K. Analysis calculated for C5H4BrNSe (%): C, 25.35; H, 1.70; N, 5.91. Found (%): C, 25.31; H, 1.68; N, 5.89.1H-Pyridine-2-selenenyl dibromide (1). Compound 1* was recrystallized from methanol. Yield 0.59 g (92%), orange crystals, m.p. 388–390 K. Analysis calculated for C5H5Br2NSe (%): C, 18.89; H, 1.59; N, 4.41. Found (%): C, 18.81; H, 1.55; N, 4.37.2,3,3a,9a-Tetrahydro-1H-cyclopentaσelenazolopyridinium-9 bromide (2). A solution of cyclopentene  in ethyl acetate (5 ml) was added to a solution of 1  in ethyl acetate (10 ml) at room temperature. The reaction mixture was kept at room temperature for 24 h, then the solvent was removed under vacuum. The crude white solid was recrystallized from methylene chloride. Single crystals suitable for X-ray diffraction analysis were obtained by recrystallization from methylene chloride. Yield 0.133 g (87%), white powder, m.p. 463–465 K. Analysis calculated for C10H12BrNSe (%): C, 39.29; H, 3.91; N, 4.52. Found (%): C, 39.38; H, 3.97; N, 4.59. 1H NMR : δ = 8.98 , 8.20 , 7.74 , 5.78 , 4.66 , 2.34, 2.09, 1.71 .1 was localized in the difference-Fourier map and refined isotropically with fixed displacement parameters [Uiso(H) = 1.2Ueq(N)]. The other hydrogen atoms in 1 and 2 were placed in calculated positions with C—H = 0.95–1.00 Å and refined using a riding model with fixed isotropic displacement parameters [Uiso(H) = 1.5Ueq(C) for the CH3-groups and 1.2Ueq(C) for the other groups].Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019004997/yk2121sup1.cifCrystal structure: contains datablock(s) global, 1, 2. DOI: 10.1107/S2056989019004997/yk21211sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989019004997/yk21212sup3.hklStructure factors: contains datablock(s) 2. DOI: 1909603, 1909602CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In addition, weak C—H⋯π (ring) interactions, which only involve the minor disorder component. Inversion-related Cl⋯Cl halogen bonds and C—Cl⋯π (ring) contacts also help to stabilize the packing.In the crystal, the cations and anions stacked along the  16H14Cl2N3S+·Br−, the central thiazolidine ring adopts an envelope conformation. The phenyl ring is disordered over two sites with a refined occupancy ratio of 0.541 (9):0.459 (9). In the crystal, C—H⋯Br and N—H⋯Br hydrogen bonds link the components into a three-dimensional network with the cations and anions stacked along the b-axis direction. Weak C—H⋯π interactions, which only involve the minor disorder component of the ring, also contribute to the molecular packing. In addition, there are also inversion-related Cl⋯Cl halogen bonds and C—Cl⋯π (ring) contacts. A Hirshfeld surface analysis was conducted to verify the contributions of the different intermolecular interactions.In the cation of the title salt, C The dihedral angle between the mean plane of the central thiazolidine ring and the 2,3-dichlorobenzene ring (C5–C10) is 16.0 (2)° while this plane subtends angles of 79.1 (3) and 86.7 (4)° with the major and minor components (C11–C16 and C11/C12′–C16′), respectively, of the disordered phenyl ring. The dihedral angle between the two disorder components of the ring is 7.6 (4)° and these components are oriented to the 2,3-dichlorobenzene ring by 64.8 (3) and 72.4 (4)°, respectively. The N2—N1—C4—C5 bridge that links the thiazolidine and 2,3-dichlorobenzene rings has a torsion angle of 175.1 (4)°.In the cation of the title salt Fig. 1, the cenCg3iv and C7—Cl2⋯Cg4iv contacts  and inside  the surface, respectively]. These represent both the overall two-dimensional fingerprint plots and those that represent H⋯H, Cl⋯H/H⋯Cl, C⋯H/H⋯C and Br⋯H/H⋯Br contacts, respectively . The most significant intermolecular interactions are the H⋯H interaction (25.4%), which appear in the central region of the fingerprint plot with de = di ≃ 1.2 Å . The reciprocal Cl⋯H/H⋯Cl interactions appear as two symmetrical broad wings with de + di ≃ 2.8 Å and contribute 19.1% to the Hirshfeld surface . The reciprocal C⋯H/H⋯C and Br⋯H/H⋯Br interactions with 18.2% and 16.2% contributions are present as sharp symmetrical spikes at diagonal axes de + di ≃ 2.7 and 2.4 Å, respectively . The percentage contributions of other intermolecular contacts are less than 6% in the Hirshfeld surface mapping : 0.179 (3). The system of hydrogen bonds connecting two cations and two anions into 12-membered rings is identical in the racemic and in the optically active crystals. YITCEJ : C, 44.57; H, 3.27; N, 9.75. Found: C, 44.51; H, 3.23; N, 9.72%. 1H NMR  : 4,62 ; 4.96 ; 5.59 ; 7.38–8.50 ; 8.35 ; 10.56 . 13C NMR: 46.62, 55.68, 127.28, 127.99, 128.48, 128.96, 129.11, 132.27, 132.41, 132.51, 133.04, 137.24, 145.89, 168.92. MS (ESI), m/z: 351.24 [C16H14Cl2N3S]+ and 79.88 Br−.Yield 89%, m.p. 521 K. Analysis calculated for CUiso(H) = 1.2Ueq. The phenyl ring in the cation is disordered over two positions with a site occupancy ratio of 0.541 (9):0.459 (9). Using DFIX, the bond distances in the two disorder components of the phenyl ring were set to 1.40 Å. Corresponding displacement parameters were also held to be the same using EADP.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989018010496/sj5561sup1.cifCrystal structure: contains datablock(s) global. DOI: Click here for additional data file.10.1107/S2056989018010496/sj5561globalsup2.cmlSupporting information file. DOI: 1857411CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H-benzimidazol-2-yl)aniline CdII complex is reported in which the CdII atom lies on a twofold rotation axis and is coordinated by four N atoms, provided by two bidentate 2-(1H-benzimidazol-2-yl)aniline ligands, and two nitrato O atoms, forming a distorted octahedral geometry.The synthesis and crystal structure of the title 2-(1 3)2(C13H11N3)2], the CdII atom lies on a twofold rotation axis and is coordinated by four N atoms and two O atoms, provided by two bidentate 2-(1H-benzimidazol-2-yl)aniline ligands, and two nitrato O atoms, forming a distorted octahedral geometry [range of bond angles around the Cd atom = 73.82 (2)–106.95 (8)°]. In the ligand, the dihedral angle between the aniline ring and the benzimidazole ring system is 30.43 (7)°. The discrete complex molecule is stabilized by an intramolecular N—H⋯O hydrogen bond. In the crystal, intermolecular N—H⋯O hydrogen bonds link the molecules, forming a three-dimensional network.In the title compound, [Cd(NO The Cd—N and Cd—O bond lengths  are comparable with those of other Cd complexes aniline or 2-(2-aminophenyl)-1H-benzimidazole with a transition metal. These include Ni ·4H2O  in ethanol (20 ml) was added a solution of 2-(1H-benzimidazol-2-yl)aniline  in ethanol (10 ml) at 333 K. After 24 h of stirring, the title complex was obtained as a white powder. The powder was filtered off and washed with ethanol. Colourless crystals of the title complex were obtained by slow evaporation of the methanol solvent at room temperature within two weeks.Chemicals were obtained commercially in reagent grade and used as received. Solvents were dried using standard procedures described in the literature. To a stirred solution of Cd(NO2 groups were located in a difference-Fourier map and refined freely [refined distances: N—H = 0.75 (3)–0.86 (3) Å]. Other H atoms were positioned geometrically and refined using a riding model, with C—H = 0.93 Å, and with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019012416/is5523sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019012416/is5523Isup2.hklStructure factors: contains datablock(s) I. DOI: 1951821CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Mutations in germline-encoded Vγ4 CDR2 and HV4 loops, but not in somatically recombined CDR3 loops, drastically diminished binding and T cell responsiveness to BTNL3-BTNL8-expressing cells. Conversely, CDR3γ and CDR3δ loops mediated LES TCR binding to endothelial protein C receptor, a clonally restricted autoantigen, with minimal CDR1, CDR2, or HV4 contributions. Thus, the γδ TCR can employ two discrete binding modalities: a non-clonotypic, superantigen-like interaction mediating subset-specific regulation by BTNL/BTN molecules and CDR3-dependent, antibody-like interactions mediating adaptive γδ T cell biology. How these findings might broadly apply to γδ T cell regulation is also examined.Butyrophilin (BTN) and butyrophilin-like (BTNL/Btnl) heteromers are major regulators of human and mouse γδ T cell subsets, but considerable contention surrounds whether they represent direct γδ T cell receptor (TCR) ligands. We demonstrate that the BTNL3 IgV domain binds directly and specifically to a human Vγ4  •+ TCRs via its IgV domainBTNL3 binds directly and specifically to Vγ4•The superantigen-like binding mode focuses on germline-encoded TCR regions•In contrast, γδ TCR binding to a clonally restricted antigen is CDR3-mediated•Mutagenesis indicates parallels with BTN3A1-mediated activation of Vγ9Vδ2 T cells + TCRs via a superantigen-like binding mode that is focused on germline-encoded TCR regions.Butyrophilin (BTN) and butyrophilin-like (BTNL) molecules powerfully influence selection and activation of specific γδ lymphocyte subsets, but whether they directly bind the γδ TCR has remained contentious. Willcox et al. show that BTNL3 directly binds to human Vγ4 The firinocytes . Subsequ T cells . More repulation . Btnl1 apulation , whereinpulation  that canithelium , are maj T cells  and are  disease . More ge disease . Thus, d disease .Soon after their discovery, when γδ T cells were first found to express a limited number of V regions, the existence of a range of host-encoded ligands that might mediate subset-specific γδ T cell selection and/or activation was hypothesized . ClearlyHere we provide unequivocal evidence for direct binding of a γδ TCR to a BTNL protein. We show that a human Vγ4 TCR binds the BTNL3 IgV domain via germline-encoded regions, somewhat analogous to superantigen binding to an αβ TCR. In contrast, binding of a clonally restricted ligand to the same Vγ4 TCR was critically influenced by the CDR3 regions of the γδ TCR, consistent with its adaptive biology. Thus, we highlight two distinct and complementary modalities for ligand interaction: one involving a BTNL molecule in Vγ region-specific regulation of tissue-restricted γδ T cell subsets and the other involving highly specific, clonally restricted ligand recognition, underpinning adaptive γδ T cell responses. Moreover, the two binding modes may extend to BTN3A1-mediated regulation of the human blood Vγ9Vδ2 subset by P-Ags, suggesting broad significance of the BTNL modality that we outline.+ γδ TCRs to 293T cells expressing BTNL3.8 heterodimers (293T.L3L8) led to CD69 upregulation and TCR downregulation, consistent with TCR triggering  of BTNL3 IgV for a Vγ4 TCR, LES, was ∼15–25 μM  at 25°C (−1 at 20°C) and marginally entropically unfavorable   D and 1E.Vγ3+ TCR B and S1CConsistent with our finding that BTNL3 IgV, but not BTNL8 IgV, directly bound Vγ4 TCRs, soluble Vγ4 TCR binding to 293T cells transduced with FLAG -BTNL3 and hemagglutinin (HA) -BTNL8 heterodimer constructs was abrogated by anti-FLAG antibody, presumably because of steric hindrance, but was only marginally affected by anti-HA antibody D. Anti-F+ T cells to Btnl1.6 was more potently abrogated by mutations in the V region of Btnl6 compared with Btnl1, suggestive of Btnl6 being a direct ligand for Vγ7  cells expressing a mouse Vγ7 TCR were stimulated with MODE-K cells transduced with FLAG -Btnl1 and HA -Btnl6 constructs, in which case the anti-HA antibody more potently abrogated J76 activation A. This w for Vγ7 . Indeed, for Vγ7 B in a do for Vγ7 C, consisAlthough our current and previous data suggested that multiple Vδ chains are compatible with Vγ4-mediated recognition of BTNL3.8, we could not exclude an effect of TCRδ. Compounding this, a recent study suggested that a certain Vγ4 TCR was not able to mediate BTNL3.8-driven responses in a cellular assay and suggested that particularly long Vδ1 CDR3 sequences might explain this observation . To addr+ T cells  , as was recently considered  Figure A. Upon i binding A–S4D. MoT BTNL3) E and S4FT BTNL3) G and S4H BTNL3.8 F. Altoge BTNL3.8   confirmed a strong potential for BTNL3 IgC-BTNL8 IgC interchain heterodimer interactions  seems unlikely given comparisons of equivalent amino acids in BTNL3 IgV and BTNL8 IgC and in BTNL3 IgC and BTNL8 IgV  corresponding to those  implicated in the BTNL3 interaction with Vγ4 HV4  in CRA123 cells. Again, the SSLEQ and YEMAL mutants displayed cell surface expression similar to WT BTN3A1+BTN3A2, while the DF mutants showed decreased expression, particularly when both BTN3A1 and BTN3A2 carried this mutation  region sequences , in the 1-ATPase . Support1-ATPase .In the most extreme generality, the existence of distinct, parallel ligand recognition modes might be a feature of many antigen receptors. Thus, there are αβ TCRs that can engage endogenous or exogenous superantigens via germline-encoded subdomains and peptide-MHC complexes largely via recombination-dependent CDR3 subregions . Moreoveb.willcox@bham.ac.uk.For additional information about reagents and resources, contact the lead contact, Benjamin E. Willcox, at E. coli BL21 (DE3) strain (NEB). Protein expression was induced by addition of 0.5mM isopropyl-b-D-1-thiogalactopyranoside and culture for 4 hours at 37°C. The bacterial cell pellet was harvested by centrifugation at 7500 g for 20 min, resuspended in PBS and lysed by mechanical disruption using an Emulsiflex C3. The overexpressed inclusion body protein was isolated by centrifugation at 44000 g for 30 min. The pellet was washed three times in 50mM Tris, pH8, 0.5% Triton X-100, 2 mM DTT, and 0.1% sodium azide, and once in 50 mM Tris, pH8, 2 mM DTT, and 0.1% sodium azide, then solubilised in 8 M urea, 50 mM MES, pH 6.5, 1 mM EDTA, 2 mM DTT. BTNL3 or BTNL8 inclusion body proteins were further reduced by addition of fresh 20 mM DTT for 30 min at 37°C immediately prior to refolding. BTNL3 IgV was refolded by dilution in 100 mM Tris, 400 mM L-Arginine-HCl, 2 mM EDTA, 6.8 mM cystamine, 2.7 mM cysteamine, 0.1 mM PMSF, pH 8, overnight at 4°C. BTNL8 IgV was refolded as described for Skint1 IgV , Vγ2+ TCR, Vγ3+ TCR and mouse Vγ7+ TCR) and soluble EPCR were generated in Drosophila S2 cells and purified by nickel chromatography as previously described (cDNA encoding wt BTNL3 or BTNL8 IgV domains (Q18 to V131), or BTNL3 IgV incorporating the described mutations, were generated as gblocks (Integrated DNA Technologies) including the sequence for a C-terminal 6x Histidine tag, cloned into the pET23a expression vector (Novagen), and were overexpressed in int1 IgV . The refescribed . Mutant escribed . TCRs weescribed .SPR was performed as described  on a BIACalorimetric measurements were carried out using an iTC200 instrument . All experiments were performed at 20°C, with proteins purified in 20mM Tris pH 8, 50 mM NaCl. Typically, TCR proteins were contained in the calorimeter cell at a concentration of 70 μM, into which BTNL3 proteins at a ten-fold higher molar concentration were titrated as 20 × 2 μl injections. All ITC binding experiments were corrected for heats of dilution. Binding data were analyzed by fitting the binding isotherm to a single independent binding site model using Origin software.293T and MODE-K cells were maintained in DMEM supplemented with 4.5 g/L D-glucose, L-glutamine, 10% heat-inactivated fetal calf serum (FCS) and 1% penicillin-streptomycin (Complete DMEM). JRT3 and Jurkat 76 (J76) were maintained in RPMI 1640 with L-glutamine, 10% heat-inactivated FCS and 1% penicillin-streptomycin (Complete RPMI). All cell culture reagents were from Thermo Fisher. Polyclonal Vγ9Vδ2 T cell lines were generated from PBMC from healthy donors after informed consent as described previously .Transduced 293T and MODE-K cells were grown in Complete DMEM supplemented with 1 μg/mL puromycin (Sigma-Aldrich) alone or in combination with 500 ng/mL hygromycin B (Thermo Fisher). Polyethylenimine was from Polysciences. Zoledronate (Zol) was from Sigma-Aldrich.R cassettes. OE-PCR was likewise used to mutate BTN3A1 and BTN3A2, which were subsequently cloned into pCSIGPW.Plasmids encoding hu17 (human Vγ4Vδ1), huPB (human Vγ9Vδ2) and mo5 (murine Vγ7Vδ2-2) TCRs, and BTNL and Btnl variants, and BTN3A1 and BTN3A2 have been described . Chimerigag-pol pCR/V1 , HIV-1 l pCR/V1  and VSV-v pHIT/G  using PE5 JRT3/J76 transductants or PBMC-derived polyclonal Vγ9Vδ2 T cells were mixed in 96-well plates with 1.5 × 105 293T or MODE-K cells, followed by co-culture for 5 h. 293T transiently expressing BTNL3 and BTNL8 (293T.L3L8) were used in blocking experiments. 48 h post-transfection, 293T cells were harvested and pre-incubated for 30 min at 4°C with α-FLAG, α-HA or IgG control antibodies . JRT3 were subsequently added and the cells were co-cultured for 3h at 37°C in the presence of the antibodies. For blocking experiments of murine Btnl molecules, MODE-K stably expressing Btnl1+Btnl6 (MODE-K.l1.l6) were pre-incubated for 60 min at 37°C with α-FLAG, α-HA or IgG control antibodies in 96-well plates. J76-mo5 were added to the wells and cells were co-cultured for 5h in the presence of the antibodies. 293T-CRA123 cells were transiently transfected with BTN3 constructs. Media was replaced 24 h post-transfection with Complete DMEM supplemented with Zol (10 μM). Cells were maintained for 16 h, washed twice, and co-cultured with JRT3 transductants or PBMC-derived polyclonal Vγ9Vδ2 T cell lines as described above.0.5 × 10Flow cytometry was performed using the following antibodies from BioLegend, unless otherwise stated. Antibodies to the following human molecules were used: CD69-AF647 (FN50), CD69-PE (FN50), CD3-BV421 (OKT3), γδTCR-PeCy7 , CD45-PacificBlue (HI30), TCRVδ2-FITC (B6). Antibodies to the following murine molecules were used: TCRδ-PerCPe710 (GL3), TCRδ-APC (GL3), Vγ7-AF647 . Other antibodies were as follows: DYKDDDDK-PE , HA-AF647 (16B12). 6xHis-APC . Data were acquired on BD Canto II or Fortessa cytometers. sTCR staining was performed as in Flow cytometry data were analyzed in FlowJo  and Prism . Structural figures were generated in PyMOL . For SPR and ITC, data were analyzed in BIAevaluation  and Origin 2015 (OriginLab).Molecular models of the BTNL-3 and BTNL-8 IgV-IgC domains were derived from the I-TASSER server . BTNL-3 Flow cytometry data were analysed in FlowJo  and Prism . Structural figures were generated in PyMOL . For SPR and ITC, data were analysed in BIAevaluation  and Origin 2015 (OriginLab).There is no data or availability to report.


The crystal structure of a binuclear mixed-valence ytterbium complex containing bridging and terminal 2-anthracene-substituted phenoxide ligands is described. One of the bridging ligands shows a π-interaction between an anthracene bond and the ytterbium(II) centre. 1) with divalent Yb[N(SiMe3)2]2·2THF in THF–toluene mixtures affords the mixed-valence YbII–YbIII dimer {[2-(anthracen-9-yl)phenolato-κO]bis(tetrahydrofuran)ytterbium(III)}-tris[μ-2-(anthracen-9-yl)phenolato]-κ4O:O;κO:1,2-η,κO-{[2-(anthracen-9-yl)phenolato-κO]ytterbium(II)} toluene trisolvate, [Yb2(C20H13O)5(C4H8O)2]·3C7H7 or [YbIII(THF)2(OPhAn)](μ-OPhAn)3[YbII(OPhAn)]·3C7H7 (2), as the major product. It crystallized as a toluene trisolvate. The Yb—O bond lengths in the crystal structure of this dimer clearly identify the YbII and YbIII centres. Interestingly, the formally four-coordinate YbII centre shows a close contact with one anthracene C—C bond of a bridging OPhAn ligand, bringing the formal coordination number to five.Reaction of 2-(anthracen-9-yl)phenol (HOPhAn,  The advantage of this method is that it avoids the formation of additional complexes  often favoured during metathesis reactions Fig. 1  is shown in Fig. 2d and C19d are depicted.The structure of complex 2 are connected . This is well within the wide range recognized for weak YbII–arene interactions  after correction to five-coordination.The Yb1—O(THF) distances [2.306 (3) and 2.309 (6) Å] are typical of six-coordinate YbII centre prefers to satisfy its electron deficiency by coordination of an anthracene π-bond (or the anthracene C—H bonds) rather than coordinating a further molecule of THF.It appears that the Yb2 are C—H⋯π interactions, both intra- and intermolecular (see Table S2 in the supporting information). Interstitial space is filled with three toluene molecules of solvation .The only noteworthy interactions observable in the crystal structure of complex 3]2(THF)2 was prepared by analogy to the procedure of Hitchcock et al. (20023)2 and YbI2(THF)x, and recrystallized from hot hexane. NMR spectra were recorded on a Bruker AV III 300 MHz Spectrometer in sealable Teflon-valved tubes and were referenced to residual solvent resonances. The line widths at half maximum (ν1/2 in Hz) were measured for all paramagnetic resonances and are reported below. Elemental analyses were performed by Canadian Microanalytical Ltd.All solvents were purchased from Sigma–Aldrich Chemicals and dried by distillation from sodium under nitrogen. The 2-(anthracen-9-yl)phenol was purified by recrystallization from hot toluene, while Yb[N(SiMe) al. 2002 using Na2 is illustrated in Fig. 53]2(THF)2  in toluene (10 ml). The deep-orange solution darkened to red on stirring overnight. The solution was filtered through Celite on a sintered glass frit and the filtrate was evaporated to dryness under reduced pressure. The red solid was recrystallized from a mixture of toluene and hexane at 143 K, yielding deep-orange crystals . 1H NMR : δ 88.4 , 49.4 , 47.9 , 12.86 , 11.70 , 10.93 , 10.00 , 9.30 , 1.26 , 0.96 , −2.77 , −3.89 , −5.38 , −11.2 , −11.4 , −16.0 , −24.4 , −77.2 . Analysis calculated for C129H105O7Yb2 (%): C 73.30, H 5.01; found: C 72.55, H 4.87.The reaction scheme for the synthesis of complex Uiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 2e–C20e:O7/C1f–C20f, was disordered and modelled in two positions, with an occupancy ratio of 0.533 (3):0.467 (3), using rigid groups and restraints. A metal-coordinated THF molecule, O6a/C21a–C24a:O6b/C21b–C24b, is also disordered and was modelled in two positions, with an occupancy ratio of 0.542 (9):0.458 (9), using restraints. Three toluene solvent molecules are also present and are all disordered over two positions; they were modelled with rigid groups and restraints; the C29 and C43 toluene molecules were modelled at 50% occupancy each and the C36 toluene molecule was modelled with an occupancy ratio of 0.682 (9):0.318 (9).One bridging ligand, O7/C1d; the r.m.s. deviation is 0.11 e Å−3.The largest peak in the final difference electron density synthesis is near atom Yb1 and the largest hole is near atom C1910.1107/S205698901901154X/su5510sup1.cifCrystal structure: contains datablock(s) Global, I. DOI: 10.1107/S205698901901154X/su5510Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S205698901901154X/su5510sup3.pdfCSD search S1: CSD refcodes and references; C-H...pi interactions: Table S2. DOI: 1947717CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title compound crystallized from an equimolar mixture of 2-amino-6-bromopyridine and 2,3,5,6-tetrafluorobenzoic acid in absolute ethanol. 5H6BrN2+·C7HF4O2−, contains one pyridinium cation and one benzoate anion. In the crystal, the aminopyridinium cationic unit forms two hydrogen bonds to the benzoate oxygen atoms in an R22(8) motif. Two pyridinium benzoate units are hydrogen bonded through self-complementary hydrogen bonds between the second amine hydrogen and a carboxylate O with a second R22(8) motif to form a discrete hydrogen-bonded complex containing two 2-amino-6-bromopyridinium moieties and two 2,3,5,6-tetrafluorobenzoate moieties. The 2-amino-6-bromopyridinium moieties π-stack in a head-to-tail mode with a centroid–centroid separation of 3.7227 (12) Å and adjacent tetrafluorobenzoates also π-stack in a head-to-tail mode with a centroid–centroid separation of 3.6537 (13) Å.The asymmetric unit of the co-crystal salt 2-amino-6-bromopyridinium 2,3,5,6-tetrafluorobenzoate, C It has been shown that these hydrogen bonds can be diverted by O—H⋯N hydrogen bonding to pyridines, often supported by a non-conventional pyridine C—H⋯O hydrogen bond . The interaction of the more basic pyridines, for example 4-pyridine, with carboxylic acids most often yields charge-assisted hydrogen-bonded salts . Similarly, the combination of 2-aminopyridines and benzoic acids has been demonstrated to be a reliable supramolecular synthon resulting in the formation of charge-assisted hydrogen-bonded complexes shown in Fig. 1d if Fig. 2. The bonif Fig. 1 are almoc axis.In the co-crystal salt (I)if Fig. 3. The twoCg1⋯Cg1i distance of 3.6537 (13) Å  and there is a close C—F⋯π interaction with a Cg1⋯F3ii distance of 3.1640 (17) Å .There are two interactions that involve the tetrafluorobenzoate Fig. 4. Adjacenb axis  Å and a shortest perpendicular interplanar distance of 3.2547 (8) Å .The 2-aminopyridinium groups form offset alternating head-to-tail π-stacks parallel to the is Fig. 5 with a Civ distance of 3.867 (2) Å .There is one short contact to the bromine with a C9⋯Br1et al., 2016Conquest pyridine and carboxylic acids revealed only four neutral co-crystals and 54 structures corresponding to the pyridinium carboxylate as shown in Fig. 1c. A similar search for co-crystals formed between 2-aminopyridines with benzoic acids yielded 41 hits, of which 40 feature charge-assisted aminopyridinium carboxylate hydrogen-bonded co-crystals as the result of proton transfer shown in Fig. 1d. The structure that is reported to form a neutral hydrogen-bonded complex corresponds to the co-crystal formed between 2-aminopyridine and 4-aminobenzoic acid [refcode WOPCOV; Chandrasekaran & Babu, 2014et al., 2016A search of the Cambridge Crystallographic Database  of each component were added to a screw-capped vial and 3 mL of ethanol added to effect a clear colorless solution that was allowed to slowly concentrate over two weeks. A homogeneous mass of crystals was obtained.Uiso(H) = 1.2Ueq(N). The aromatic H atoms were included in the refinement at calculated positions with C—H = 0.95 Å and Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019001294/pk2612sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989019001294/pk2612Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019001294/pk2612Isup3.cmlSupporting information file. DOI: 1893117CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: