Oryza sativa (rice) is one of the most important staple crops in the world. In the present study, we isolated and elucidated the structures of specialized metabolites from rice by using MS/MS and NMR. Thirty-six compounds, including five new flavonoids and eight rare flavonolignan isomers, were isolated from the rice leaves. The MS/MS spectral data of the isolated compounds, with a detailed interpretation of MS fragmentation data, will facilitate metabolite annotation of the related phytochemicals by enriching the public mass spectral data depositories, including the plant-specific MS/MS-based database, ReSpect.Metabolomics plays an important role in phytochemical genomics and crop breeding; however, metabolite annotation is a significant bottleneck in metabolomic studies. In particular, in liquid chromatography–mass spectrometry (MS)-based metabolomics, which has become a routine technology for the profiling of plant-specialized metabolites, a substantial number of metabolites detected as MS peaks are still not assigned properly to a single metabolite. The online version of this article (doi:10.1007/s11306-013-0619-5) contains supplementary material, which is available to authorized users. In recent years, the use of metabolomics has rapidly developed in the fields of phytochemical genomics and crop breeding. It has been used in the investigation of plant biological mechanisms related to genetic and/or environmental factors   due to the genetic polymorphism. Unequivocal structures of such metabolites are useful for understanding gene-to-metabolite correlations , which will help to analyze metabolomic studies of rice and its related species, and facilitate the annotation of plant metabolites  were grown in plastic pots containing granular soil ; after approximately 10 weeks of incubation, shoots were collected, lyophilized, and stored at −80 °C until use  to afford nine fractions (Fr.1–9). These fractions were purified using semipreparative HPLC performed under the following conditions: column, Cadenza CD-C18 or Unison UK-C18 columns, Imtakt 150 × 10 mm i.d.; particle size, 3 μm; solvents, water and methanol or acetonitrile, containing 0.1 % v/v formic acid; and flow rate, 3.0 mL/min. The following compounds were obtained: 1 (4.52 mg), 2 (12.69 mg), 3 (2.07 mg), 4 (2.57 mg), 5 (1.15 mg), 6 (0.94 mg), 7 (1.51 mg), 8 (1.23 mg), 9 (0.71 mg), 10 (2.53 mg), 11 (1.63 mg), 12 (3.93 mg), 13 (2.74 mg), 14 (0.58 mg), 15 (0.96 mg), 16 (1.89 mg), 17 (0.22 mg), 18 (0.09 mg), 19 (0.65 mg), 20 (0.20 mg), 21 (0.28 mg), 22 (0.64 mg), 23 (1.04 mg), 24 (0.76 mg), 25 (2.31 mg), 26 (2.20 mg), 27 (2.04 mg), 28 (1.25 mg), 29 (0.64 mg), 30 (0.29 mg), 31 (0.63 mg), 32 (2.29 mg), 33 (4.84 mg), 34 (4.99 mg), 35 (1.13 mg), and 36 (2.70 mg). For details regarding the isolation procedures from rice, see Supplementary data file S1.The leaf powder of rice (90 g) was extracted with 90 % methanol as described in a previous study  C18 column , and the column temperature was set at 40 °C. The mobile phase consisted of A (0.1 % v/v formic acid in water) and B (0.1 % v/v formic acid in acetonitrile). The gradient conditions of the mobile phase were as follows: 0 min, 99.5 % A; 10.0 min, 20 % A; 10.01 min, 0.5 % A; 12.0 min, 0.5 % A; 12.1 min, 99.5 % A; and 14.5 min, 99.5 % A. The flow rate was 0.30 mL/min. UV–visible absorption spectra of samples were determined using a photodiode array (PDA) detector in the range of 200–600 nm. The sample injection volume was 1 μL.m/z. Nitrogen was used as the nebulizer and auxiliary gas; argon was utilized as the collision gas. The ESI source was operated in positive and negative ionization modes with a capillary voltage of 3 kV, sampling cone voltage of 25 V, cone gas flow of 50 L/h, desolvation gas flow of 800 L/h, desolvation temperature of 450 °C, source temperature of 120 °C, and CID energy ramped from 10 to 50 eV. Tandem MS analysis was performed using fast data directed analysis (FastDDA), which is rapid automated, intelligent MS/MS data acquisition for targeted qualitative analyses. Data acquisition and processing were performed with the MassLynx 4.1 software.MS detection was performed on a Waters Xevo G2 QTOF mass spectrometer with an electrospray ionization (ESI) interface (Waters). Full scan mass spectra were recorded through a range of 50–1,500 1H-NMR was measured of 64 or 128 scans and at a receiver gain of 11.3 using standard pulse sequences. 1D 13C-NMR, and two-dimensional (2D) NMR experiments, 1H–1H correlation spectroscopy (COSY), 1H–13C heteronuclear single quantum coherence spectroscopy (HSQC), and 1H–13C heteronuclear multiple bond connectivity spectroscopy (HMBC) were obtained using standard pulse sequences. Dimethylsulfoxide-d6 or methanol-d4 was used as solvent, and tetramethylsilane (TMS) was used as an internal standard. The samples were added to 5 mm Shigemi micro NMR tubes . NMR data were acquired and processed with the TopSpin software .The NMR spectra were recorded on a Bruker 600 MHz spectrometer with a DCH CryoProbe . One-dimensional (1D) http://prime.psc.riken.jp/) and are freely available.All data acquired by LC–QTOF-MS/MS were uploaded to DROP Met in PRIMe (6–9 and 24), were isolated and assigned from the leaves of rice using MS/MS and NMR methods  have been isolated from rice leaves. Those 36 compounds have been assigned in LC-PDA chromatogram of rice leaves extract . Herein, we report the structural elucidation of new flavonoids and analysis of the MS/MS fragmentation patterns of isolated compounds by using high-resolution QTOF mass spectrometry with an ESI source. In the MS/MS analysis, the ramped collision energies mode was used to obtain a combined spectrum from fragments detected at various collision energies -162]+, corresponding to the loss of a hexose group, and a major product ion at m/z 331 [(M + H)-162-176]+, representing the loss of hexose and glucuronosyl groups. The 1H-NMR spectrum of compound 6 indicated an A2-type aromatic proton signal at δ 7.37 ; meta-coupled proton signals at δ 6.52  and 7.01 ; an aromatic proton signal at δ 7.07 ; two methoxy proton signals at δ 3.89 ; and two anomeric proton signals at δ 5.33  and 4.48   spectra, these data indicated that compound 6 was tricin glucopyranosyl-glucuronopyranoside. The relatively large coupling constant values of anomeric protons suggested that the configuration of glucose and glucuronic acid were β forms. In addition, in the HMBC spectrum, the anomeric proton signals δ 5.33 (H-1′′) and 4.48 (H-1′′′) showed long-range correlation with the carbon signals at δ 162.5 (C-7) and 82.5 (C-2′′), respectively, suggesting that the glucuronosyl was located at the C-7 of aglycone and that glucose was located at the C-2 of glucuronosyl -β-d-glucuronopyranoside.Compound 7 was obtained as a yellow amorphous powder. HR-ESI-QTOF-MS gave the molecular formula C26H26O15. The MS/MS spectra of the precursor ion at m/z 579 [M + H]+ showed a product ion at m/z 493 [(M + H)-86]+, corresponding to the loss of a malonyl group, and a major product ion at m/z 331 [(M + H)-86-162]+, corresponding to the loss of malonyl and hexose groups. The 1H-NMR spectrum of compound 7 indicated an A2-type aromatic proton signal at δ 7.37 ; three aromatic proton signals at δ 6.45 , 6.73 , and 7.06 ; two methoxy proton signals at δ 3.89 ; and a sugar of the anomeric proton signal at δ 5.10   spectra, indicated that compound 7 was tricin malonyl-glucopyranoside. Furthermore, in the HMBC spectrum, the anomeric proton signal δ 5.10 (H-1′′) showed long-range correlation with the carbon signal at δ 162.7 (C-7), suggesting that the glucose was located at C-7. The sugar proton signal at δ 4.15 (H-6′′) showed correlation with the carbon signal at δ 167.4 (C-1′′′), suggesting that the malonyl moiety was located at the C-6 of glucose -β-d-glucopyranoside.Compound 8 was obtained as a yellow amorphous powder. The molecular formula of compound 8 was determined as C34H34O16 with HR-ESI-QTOF-MS. The MS/MS spectra of the precursor ion at m/z 699 [M + H]+ gave a major product ion at m/z 331 [(M + H)-206-162]+, representing the loss of sinapoyl and hexose groups. The fragment ion of the sinapoyl moiety at m/z 207 was also observed ; meta-coupled proton signals at δ 6.52  and 6.89 ; an aromatic proton signal at δ 6.96 ; two methoxy proton signals at δ 3.88 ; and an anomeric proton signal at δ 5.15 , which were similar to those of compounds 6 and 7 ; two methoxy proton signals at δ 3.71 ; and two olefinic proton signals at δ 6.44  and 7.47 , suggesting the presence of a trans-sinapoyl moiety. Furthermore, in the HMBC spectrum, the anomeric proton signal δ 5.15 (H-1′′) showed a long-range correlation with the carbon signal at δ 162.6 (C-7), suggesting that the glucose was located at C-7. The sugar proton signal at δ 4.10 (H-6′′) showed correlation with the carbon signal at δ 166.2 (C-9′′′), suggesting that the sinapoyl moiety was located at the C-6 of glucose -sinapoyl)-β-d-glucopyranoside.Compound 9 was obtained as a yellow amorphous powder. The molecular formula of compound 9 was established as C34H38O17 by HR-ESI-QTOF-MS. The MS/MS spectra of the precursor ion at m/z 719 [M + H]+ gave a product ion at m/z 557 [(M + H)-162]+, corresponding to the loss of a hexose group, and a product ion at m/z 331 [(M + H)-226-162]+, corresponding to the loss of syringylglyceryl and hexose groups. The product ion at m/z 539 was formed by the loss of glucose and a water molecule from the precursor ion at m/z 719. In addition, a major product ion was observed at m/z 209, which was formed by the loss of a water molecule from the syringylglyceryl moiety. The 1H-NMR spectrum of compound 9 indicated an A2-type aromatic proton signal at δ 7.26 ; meta-coupled proton signals at δ 6.10  and 6.34 ; an aromatic proton signal at δ 6.64 ; two methoxy proton signals at δ 3.96 ; and an anomeric proton signal at δ 4.57 . Moreover, the 1H-NMR spectrum of compound 9 was similar to that of compound 15, except for an A2-type aromatic proton signal at δ 6.81  and six proton signals at δ 3.84 , corresponding to two methoxyl groups of the syringylglyceryl moiety  spectra, these data indicated that compound 9 was a flavonolignan glycoside with tricin as the aglycone. In addition, the coupling constant of JH-7′′, H-8′′ was 5.5 Hz, suggesting that compound 9 was of the threo type because the coupling constant between the adjacent protons of the threo form is known to be larger than that of the erythro form  spectra. However, these compounds did not exhibit Cotton effects, presumably due to conformational mobility  showed long-range correlation with the carbon signal at δ 82.0 (C-7′′), suggesting that the glucose was located at C-7′′. The syringylglyceryl proton signal at δ 4.55 (H-8′′) showed correlation with the carbon signal at δ 140.7 (C-4′), suggesting that the location of the syringylglyceryl moiety was at C-4′  ether 7′′-O-β-d-glucopyranoside.Compound 24 was obtained as a yellow amorphous powder. The molecular formula was found to be C26H28O15 by HR-ESI-QTOF-MS. The MS/MS spectra of the precursor ion at m/z 581 [M + H]+ showed a product ion at m/z 419 [(M + H)-162]+, corresponding to the loss of a hexose group. In addition, characteristic fragment ions of C-glycoside were also observed. The fragment ions at m/z 401, 383, and 365 were formed by the loss of water molecules from the C-glycoside fragment at m/z 419. Product ions at m/z 329 [(M + H)-162-90]+ (0.2X+) and 353 [(M + H)-162-66]+ (0.4X+-2H2O) were formed by cross-ring cleavages of a sugar residue from m/z 419. The 1H-NMR spectrum of compound 24 indicated ortho-coupled proton signals at δ 6.87  and 7.382 ; three aromatic proton signals at δ 6.37 , 6.56 , and 7.375 ; and two anomeric proton signals at δ 4.58  and 4.19   spectra, these data indicated that compound 24 was luteolin glucopyranosyl-arabinoside. The relatively large coupling constant values of anomeric protons suggested that the configuration of the glucose was the β form and of the arabinose was the α form  and 4.19 (H-1′′′) showed a long-range correlation with the carbon signals at δ 108.3 (C-6) and 78.7 (C-2′′), respectively, suggesting that the arabinosyl moiety was located at the C-6 of aglycone and glucosyl at the C-2 of arabinose -α-l-arabinoside.Compound 1 in the positive and negative ionization modes showed a protonated molecular ion at m/z 331 and a deprotonated molecular ion at m/z 329, respectively. MS/MS spectra of compounds 2–5 and 10–17 in the positive mode showed diagnostic fragment ions of tricin at m/z 331, suggesting that these compounds were tricin derivatives. For the detailed MS/MS analysis of these compounds, see Sect. 3.3. Furthermore, on comparison of the 1H- and 13C-NMR spectral data (Supplementary data file S2) with those in the literature, these compounds were assigned as tricin (1)      ether (10)  ether (11)  ether 7-O-β-d-glucopyranoside (12)  ether 7-O-β-d-glucopyranoside (13)  ether 7′′-O-β-d-glucopyranoside (14)  ether 7′′-O-β-d-glucopyranoside (15)  ether 9′′-O-β-d-glucopyranoside (16)  ether (17) -162]+, corresponding to the loss of a hexose group. MS/MS spectra of compound 19 showed major fragment ions at m/z 509 [(M + H)-146]+ and 347 [(M + H)-146-162]+, indicating the loss of deoxyhexose and hexose groups. Furthermore, comparing the 1H-NMR spectral data with those in the literature, they were assigned as syringetin 3-O-β-d-glucopyranoside (18)   were assigned as apigenin 6-C-α-l-arabinosyl-8-C-β-l-arabinoside (20)     were assigned as isoscoparin 2′′-O-(6′′′-(E)-feruloyl)-glucopyranoside (25) -p-coumaroyl)-glucopyranoside (26) -feruloyl)-glucopyranoside (27) -p-coumaroyl)-glucopyranoside (28)      -162]−, suggesting the presence of a hexose group. Compound 34 was assigned as salicylic acid 2-O-β-d-glucopyranoside   and deoxyhexose (m/z 146) from the precursor ion are common fragmentations, which are formed by rearrangement reactions at the interglycosidic bonds  showed a higher relative abundance of a tricin aglycone fragment ion at m/z 331 [(M + H)-162]+ (Y0+) than tricin 7-O-β-d-glucopyranoside (2) in their MS spectra and lower relative abundance of ions at m/z 493 than compound 2 in the MS and MS/MS spectra . These results suggested that the glucose at the 5-position was lost more readily than at the 7-position. Our results are in agreement with earlier studies on luteolin 5-O-glucoside and luteolin 7-O-glucoside  in the positive ionization mode showed a major fragment ion at m/z 493 [(M + H)-146]+ (Y1+), which was formed by the loss of rhamnose, whereas tricin 7-O-neohesperidoside (5) produced only a very low abundance of the Y1+ ion. Compounds 4 and 5 both showed the base peak of aglycone fragment ions at m/z 331 (Y0+), which were formed by the loss of rutinose and neohesperidose moieties, respectively. These results indicated that the Y0+/Y1+ ratio was higher for 1 → 2 linked neohesperidose [rhamnosyl (1 → 2)-glucose] than for 1 → 6 linked rutinose [rhamnosyl (1 → 6)-glucose] in the positive ionization mode . The fragment ions (Y1−) formed by the loss of rhamnose were not observed. Compound 4 produced a relatively higher level of the aglycone fragment ion than compound 5, suggesting that the rutinose was more readily lost than the neohesperidose in the negative ionization mode .The MS/MS spectra of tricin 7-O-(erythro-β-guaiacylglyceryl) ether (10) and tricin 4′-O-(threo-β-guaiacylglyceryl) ether (11) in the positive ionization mode showed the same protonated molecular ions at m/z 527. Both MS/MS spectra showed major fragment ions of aglycone at m/z 331 [(M + H)-196]+, corresponding to the loss of a guaiacylglyceryl group. Similarly, MS/MS data of tricin 4′-O-(erythro-β-guaiacylglyceryl) ether 7-O-β-d-glucopyranoside (12) and tricin 4′-O-(threo-β-guaiacylglyceryl) ether 7-O-β-d-glucopyranoside (13) showed major product ions at m/z 527 [(M + H)-162]+, corresponding to the loss of glucose and aglycone fragment ions, and at m/z 331 [(M + H)-162-196]+, indicating the loss of glucose and guaiacylglyceryl groups  ether 7′′-O-β-d-glucopyranoside (14), tricin 4′-O-(threo-β-guaiacylglyceryl) ether 7′′-O-β-d-glucopyranoside (15), and tricin 4′-O-(erythro-β-guaiacylglyceryl) ether 9′′-O-β-d-glucopyranoside (16) showed major aglycone fragment ions at m/z 331 and a lower amount of product ions at m/z 527. In addition, compounds 14, 15, and 16 showed fragment ions at m/z 509 [(M + H)-162-18]+, which were formed by the loss of one water molecule from ions at m/z 527 . These results suggested that fragment ions at m/z 509 were characteristic fragments of flavonolignans 14, 15, and 16, which have a glucose located at the 7′′- or 9′′-position of the guaiacylglyceryl group. In the negative ionization mode, flavonolignans 10–16 also showed similar fragment patterns with neutral loss of guaiacylglyceryl and glucose groups .The MS/MS spectra of the flavonolignans tricin 4′-C-glycosides differ from those of O-glycosides; loss of water molecules and cross-ring cleavages of sugar residues are characteristic fragments of C-glycosides, whereas the neutral loss of a sugar moiety can be observed in O,C-glycosides  and chrysoeriol 6-C-α-l-arabinosyl-8-C-β-l-arabinoside (21) in the positive ionization mode showed the loss of one, two, and three water molecules from precursor ions at m/z 535 and 565, leading to product ions at m/z 517, 499, and 481, respectively, for compound 20 and product ions at m/z 547, 529, and 511, respectively, for compound 21. The cross-ring cleavage of the sugar residue of C-glycoside yielded many characteristic product ions, such as m/z 445 (0.2X+) and 469 (0.4X+-2H2O) of compound 20 as well as m/z 475 (0.2X+) and 499 (0.4X+-2H2O) of compound 21 (Vukics and Guttman m/z 473 (0.3X−), 443 (0.2X−), 383 (0.3X−-90 or 0.2X−-60), and 353 (0.2X−-90) of compound 20 as well as m/z 503 (0.3X−), 473 (0.2X−), 413 (0.3X−-90 or 0.2X−-60), and 383 (0.2X−-90) of compound 21 . These results suggested that the loss of m/z 90 (0.2X) and 60 (0.3X) are characteristics of flavonoid C-pentosides -feruloyl)-glucopyranoside (25), isoscoparin 2′′-O-(6′′′-(E)-p-coumaroyl)-glucopyranoside (26), isovitexin 2′′-O-(6′′′-(E)-feruloyl)-glucopyranoside (27), and isovitexin 2′′-O-(6′′′-(E)-p-coumaroyl)-glucopyranoside (28) in the positive ionization mode showed fragment ions of the C-glycoside at m/z 463 and 433, which were formed by the neutral loss of glucose and acyl substituents (feruloyl or coumaroyl moiety). Fragment ions of the feruloyl moiety at m/z 177 and coumaroyl moiety at m/z 147 were also observed , 367 (0.4X+-2H2O), and 343 (0.2X+), which were formed by the cross-ring cleavage of the sugar residue of the C-glycoside fragment at m/z 463 . However, the MS/MS spectra of compounds 25–28 in the negative ionization mode showed different fragment patterns compared with those for the positive ionization mode . Product ions at m/z 623 and 593 were formed by the loss of feruloyl or coumaroyl moieties. C-glycoside fragment ions at m/z 443 and 413 were formed by the neutral loss of glucose and a water molecule from ions at m/z 623 and 593, respectively. The MS/MS spectra also showed ferulic acid and coumaric acid ions at m/z 193 and 163, respectively. The major fragment ions at m/z 323 and 293 (0.2X−) were formed by cross-ring cleavages of sugar residues of C-glycoside fragments at m/z 443 and 413, respectively, which are similar to those observed in the positive ion mode. These results suggested that the loss of m/z 120 (0.2X) is characteristic of flavonoid C-hexosides known phytochemicals. Using this strategy, 36 compounds, including five new flavonoids and eight rare flavonolignan isomers, were isolated and identified from rice. The unique MS/MS fragment patterns of flavonoid Supplementary material 1 (DOCX 663 kb) Procedures for isolation from rice1H- and 13C-NMR spectral data of known compoundsSupplementary material 2 (DOCX 45 kb) Supplementary material 3 (DOCX 207 kb) LC-PDA chromatogram of rice leaf extracts at 340 nm Supplementary material 4 (DOCX 2339 kb) MS/MS spectra of isolated compounds Supplementary material 5 (XLSX 19 kb) MS/MS and on-line UV spectral data of isolated compoundsBelow is the link to the electronic supplementary material.


Heligmosomoides polygyrus are of particular interest as the excretory–secretory products (termed HES) of this parasite contain both heat-labile and heat-stable components with immunomodulatory effects. We used MALDI-TOF-MS and LC–MS/MS to analyse the repertoire of N- and O-linked glycans released from Heligmosomoides polygyrus excretory–secretory products by PNGase A and F, β-elimination and hydrazinolysis revealing a broad range of structures including novel methylhexose- and methylfucose-containing glycans. Monoclonal antibodies to two immunodominant glycans of H. polygyrus, previously designated Glycans A and B, were found to react by glycan array analysis to a methyl-hexose-rich fraction and to a sulphated LacDiNAc  structure, respectively. We also analysed the glycan repertoire of a major glycoprotein in Heligmosomoides polygyrus excretory–secretory products, VAL-2, which contains many glycan structures present in Heligmosomoides polygyrus excretory–secretory products including Glycan A. However, it was found that this set of glycans is not responsible for the heat-stable immunomodulatory properties of Heligmosomoides polygyrus excretory–secretory products, as revealed by the inability of VAL-2 to inhibit allergic lung inflammation. Taken together, these studies reveal that H. polygyrus secretes a diverse range of antigenic glycoconjugates, and provides a framework to explore the biological and immunomodulatory roles they may play within the mammalian host.Glycan molecules from helminth parasites have been associated with diverse biological functions ranging from interactions with neighbouring host cell populations to down-modulation of specific host immunity. Glycoproteins secreted by the intestinal nematode  Indeed,Indeed, as the range and complexity of helminth-associated glycans become increasingly well-characterised, it is already clear that many specific glycans and carbohydrate motifs fulfil critical and important biological roles in the host–parasite relationship . Most imHeligmosomoides polygyrus, which reproduces the chronic infection pattern of both human and veterinary parasites , that are highly immunomodulatory -1 and -2, which are members of a large multi-gene CAP-domain family (Pfam00188) expressed in many phyla including nematodes, cestodes and chordates  is unable to down-regulate allergic lung inflammation.22.1Hp monoclonal antibodies and generation of secondary infection immune sera were as reported previously (Nippostrongylus brasiliensis (NES) was prepared as previously reported  assay were generally very low and in the range of 0.01–0.1 U/μg of protein. Silver staining was carried out as previously described -treated HES was performed as described previously . For com2.318 reverse-phase (RP) column , with (glycan-containing) flow-through  and wash (2 ml of water) combined, partially lyophilised to remove ACN, then applied to carbon columns . Carbon columns were washed with 6 ml of water and the PNGase F-released glycan pool eluted with 3 ml of 25% ACN and 3 ml of 25% ACN/0.05% trifluoroacetic acid (TFA), then lyophilised. Remaining peptides and glycopeptides on the C18 RP column were eluted with 4 ml of 30% ACN/0.1% TFA and 4 ml of 60% ACN/0.1% TFA then lyophilised. These were then resuspended in 0.1 M sodium acetate buffer pH 5 and treated with PNGase A  for 24 h at 37 °C. Liberated PNGase A-sensitive glycans and (glyco)-peptides were separated as above with C18 RP and carbon columns. PNGase F and A-released glycan pools were resuspended in 50 μl of water, which was mixed with 50 μl of water with 2-aminobenzoic acid  labelling mix  followed by 2 h incubation at 65 °C. Labelled glycans were then cleaned up using Biogel P-10  in 75% ACN, washed with 80% ACN, then eluted with water.HES (150 μg), VAL-2 or VAL-3 (both 10 μg) were incubated with trypsin-coupled Sepharose for 16 h at 37 °C with shaking. Peptides within the supernatant were then treated with PNGase F  for 24 h at 37 °C as described previously . The rea2.44 and incubated for 24 h at 40 °C. Samples were neutralised on ice with 4 M acetic acid, lyophilised, then boric acid was removed by repeated evaporations (seven+) in 1% acetic acid in methanol. Released glycans were purified with C18 RP and carbon columns as for N-glycans in Section Following PNGase F and A treatment, remaining (glyco)-peptides were resuspended in 200 μl of 0.1 M NaOH/1 M NaBH2.5HES (100 μg) was dialysed into 50 mM ammonium acetate pH 7.5 and lyophilised, then twice resuspended in water and re-lyophilised, before addition of 100 μl of HF  for 48 h at 4 °C as previously described . Samples2.6O-Glycans were released from lyophilised HES (500 μg) using the Ludger Liberate Hydrazinolysis Glycan Release Kit  according to the manufacturer’s instructions . Sample acidification (0.1% TFA) during the release procedure prevents glycan peeling/degradation . Fractions were dissolved in 20 μl of 1× spotting buffer  with 10% DMSO in 384-well V-bottom plates  at half log dilutions 0.03–10 μM. Samples (five of each) were printed on epoxysilane-coated glass slides  and probed with mAb (10 μg/ml) or polyclonal mouse serum (1/100), and analysed as before . Arrays were probed as described elsewhere  or positive ion (permethylated β-elimination-released O-glycans) reflectron mode with 2,5-dihydroxy benzoic acid (DHB) (20 mg/ml in 20% ACN) as matrix. N-glycans were eluted directly onto target plates with matrix following Zip-Tip C18. MS spectra were annotated using Glyco-Peakfinder (http://www.glyco-peakfinder.org/). For LC–MS analysis, glycans were applied to a RP column  using an Ultimate 3000 nano-LC system (both Dionex/LC Packings) at room temperature. The column was equilibrated with eluent A (0.1% formic acid/water) at flow rate 200 nl/min. Following sample injection, conditions were changed to 10% solvent B (0.1% formic acid/95% ACN) with a gradient to 60% B over 45 min, then isocratic elution for 10 min. The LC column was coupled to an Esquire HCT Ultra ESI-IT-MS  equipped with an online nanospray source in positive-ion mode. Conditions were as in m/z 300–1800 and 140–2200 were registered in MS and MS/MS mode, respectively. MS/MS spectra were manually interpreted using Bruker Daltonics DataAnalysis software .Glycans were analysed as described  with an 2.9+CD11b+SiglecF+SShi eosinophil infiltration as a marker of allergic inflammation  which includes several VAL antigens. Glycan B is present on high molecular weight species which migrate diffusely on SDS–PAGE as well as a ∼65-kDa molecule distinct from the VAL proteins , suggesting that Glycan A is a novel structure specific to H. polygyrus.Panels of mAbs directed against Glycans A and B were used to screen an extensive glycan array , v5.1) containing 610 native and synthetic sugars, predominantly related to mammalian glycosylation. Similar screening has previously been performed with polyclonal antisera against the related parasites ontortus  and Tricspiralis , as wellspiralis . First, spiralis . These mIn contrast to anti-Glycan A mAbs, all three anti-Glycan B mAbs tested  showed 3.2O- rather than N-linked glycosylation, and that antibodies to these glycans dominate the humoral response of infected mice  substitution as previously suggested to be present in HES (m/z 691.5 [M+2H]2+ (F1H3N3) and m/z 698.7 [M+2H]2+ (meF1H3N3); F, deoxyhexose/fucose; H, hexose; N,N-acetylhexosamine; meF, methylated fucose; O-methylated fucose residue, as previously reported in some other nematode glycans  of the glycan species at m/z 738.4 and 912.5 [M+Na]+.We then analysed total HES de units A. PredomO-glycans liberated from HF-treated HES. HF cleaves off α1,3-linked fucose, as well as other relatively labile moieties such as PC (m/z 1086.6 (F2H2N1), as well as the minor deoxyhexose-containing glycan species observed at m/z 953.5 (F1H1N2), 841.5 (F1H3), and 708.4 (F1H1N1), with the emergence of m/z 779.5 (H1N2), possibly derived from m/z 953.5 (F1H1N2) (+). In contrast, HF treatment left the dominant peaks of m/z 942.5 (H3N1) and 738.4 (H2N1), the latter with increased relative intensity due to defucosylation of F1-2H2N1. Only a single, minor signal was left of a glycan species with a deoxyhexose residue (m/z 912.5 (F1H2N1) after HF treatment. Possibly, this signal remains due to incomplete removal of both fucoses from the major F2H2N1 glycan. These observations suggest that most deoxyhexoses in HES O-glycans are α1,3-linked fucose and not α1,4/6-linked fucose  B , N2-AA (fractions 17–19) and F1H1-AA (fractions 23–24).RP fractionation of the labelled hydrazinolysis-released ructures . Togetheructures A and LC–ructures B–D revea1H1N1-AA, H2N1-AA, F1H3-AA, F1H2N1-AA, F1H1N2-AA; 3N1-AA species . Instead, fractions 15–16 contained a glycan with the putative structure of meH1H2N1-AA, where meH represents a methylated hexose residue, based on LC–MS/MS fragment differences of 176 Da (hexose + 14) (N-glycans the O-glycans contain methylated species. We discount the possibility of this mass representing a hexuronic acid residue (U) as earlier analysis of permethylated β-elimination material detected H3N1-AA but no U1H2N1-AA structures  B, confirructures . Interescing end . This is samples . However1N1-AA (fractions 20–21), meH1H1N1-AA , meH1H1-AA (fraction 23) and F1meH1-AA (fraction 26). Moreover, a methyl-fucosylated species meF1H1-AA (fractions 25–26) was deduced from the LC–MS/MS spectrum of m/z 462.5 [M+H]+, indicating the presence of the 160-Da methylated fucose monosaccharide (m/z 344 fragment ion (trimethyl hexose + AA label) in LC–MS/MS spectra of fractions 19, 22 and 26 are suggestive of the presence of trimethylated hexose and fucose residues, respectively . Both IgG . This was affinity purified from HES using a specific mAb, alongside the related VAL-3 protein that lacks Glycan A , in neither case did we detect PNGase F- or A-released N-glycans by MALDI-TOF-MS (data not shown). Permethylated O-glycans liberated from VAL-2 by β -elimination were readily detected  particularly prominent and m/z 942.6 (H3N1) also strongly represented. In all, 10 distinct structures were determined by LC–MS/MS, including one with m/z 779.6 (H1N2) that was not observed in total HES. Other differences to total HES O-glycans included the complete absence from VAL-2 of the m/z 1086.6 (F2H2N1) species, confirming this structure is not Glycan A, a conclusion already supported by the HF-sensitivity of this glycan  and H1N1 (m/z 512.3 [M+H]+), no O-linked glycans were detected in VAL-3 (data not shown), which is consistent with VAL-3 having very few serine or threonine residues predicted to be O-glycosylated . PNGasedetected B, revealotal HES A, with ms glycan B. In conosylated .3.6HES is able to both inhibit in vitro DC activation in response to inflammatory stimuli  and in vO-glycans linked to VAL-2.We did, however, find that in accordance with our previous publication , heat-tr4H. polygyrus is a widely studied model of chronic helminth infection in which host immunity is profoundly down-modulated by a range of parasite secreted products, termed HES . However, this is not detected in the (non-permethylated) hydrazine-released glycan pool, and instead a HexNAc with two bound hexoses and a 176 Da molecular species is seen. Whilst this could represent either a methylated hexose or a hexuronic acid, we view the latter as unlikely since we failed to detect a permethylated hexuronic acid in the β-elimination material. Additionally, the presence of methylated fucoses in the enzymatically released N-glycans (as well as in the hydrazinolysis released O-glycans) provides further evidence for this modification Glycan methylation has previously been found in bacteria, fungi and plants  bridged by a glycosylated serine/threonine linker region , as did polyclonal antibodies from immune mice. This reactive fraction contains methylated di- and tri-saccharides. Interestingly, it was previously found that methylated hexoses are also immunodominant antibody targets following infection with the nematode Toxocara canis  and galectins expressed by innate cells (e.g. DCs and macrophages), direct the host immune response (H. polygyrus, similar interactions through one or more of the many host pathogen pattern recognition receptors may prove to be important in the ability of this parasite to establish in the mammalian host.Taken together, our results establish that response . Thus, Lresponse , and delresponse  as well response . LeX conresponse , and areresponse . Althoug


In the crystal, enantiomers aggregate with each other and are linked by O—H⋯N hydrogen bonds, forming chiral 21-helical columnar structures from C(5) chains along the b-axis direction. Left- and right-handed 21 helices are formed from -2-amino-1,2-diphenylethanol and -2-amino-1,2-diphenylethanol, respectively.In the title racemic compound, C R,2S)-ADE (cis-isomer) , and can be purchased without difficulty. It was considered that cis- and trans-ADE have different properties and play different roles in diastereomer salt separations. In fact, co-crystal structures with cis-ADE enantiomers have been found in previous reports. The racemic structure of trans-ADE has been reported  , Fig. 1trans-ADE  and 54.01 (10)°, respectively. However, in cis-ADE the hydroxyl group against the opposed phenyl ring adopts a gauche conformation [O1—C1—C2—C9 = −67.39 (11)°] compared to a trans conformation in trans-ADE. Thus a tweezer-like motif bent against the phenyl groups is seen in cis-ADE versus a projected motif in trans-ADE. The arrangements are similar to those found in the diastereomer salts with cis-enantiomers, except for -2-ammonio-1,2-diphenylethanol  chains along the b-axis direction -ADE and -ADE, respectively. The hydrophobic columnar structures surrounded by phenyl groups are consolidated by the C—H⋯π and N—H⋯π interactions, forming slabs parallel to the ab plane (Table 2R22(10) ring dimers from the O—H⋯N hydrogen bonds observed in the crystal structure of trans-ADE  Å] hydrogen bonds, forming chiral 2n Table 2: Left- ae Table 2. This iscis-Enantiomers of 2-amino-1,2-diphenylethanol (ADE) were purchased from Sigma–Aldrich Co. Ltd. Equivalent weights were mixed in a bottle. Plate-like colourless crystals of the title racemic compound were obtained by vapour-phase diffusion of an aqueous ethanol solution at 297 K.2 and OH H atoms were freely refined. The C-bound H atoms were included in calculated positions and treated as riding atoms: C—H = 0.93–0.98 Å with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015022318/su5243sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989015022318/su5243Isup2.hklStructure factors: contains datablock(s) I. DOI: 1438134CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The structures of these new compounds were determined by HRESI-MS, NMR, CD spectra and X-ray diffraction analysis. Compound 1 displayed moderately inhibitory ratio (48.5%) compared with the positive compound NSC-87877 (81.6%) at the concentration of 0.022 μg/mL. Compounds 7–10 possess 3, 4-seco-ent-kaurane skeleton containing a disaccharide moiety with an unusual linkage at C-2′ to C-1′′ instead of the common linkage at C-6′ to C-1′′, and this is the first report in 600 more ent-kauranes found in nature, which might be originated from ent-kaurane diterpenoids through post-modified reactions of Baeyer-Villiger oxygenation and glycosylation.Fimbriatols A–J (1–10), ten new  Ent-kaurane diterpenoids are a big member of terpenoids with diverse structural features. More than 600 ent-kaurane diterpenoids have been isolated from different plants, especially from the genus Isodon (=Rabdosia)12ent-kaurane diterpenoids such as neoadenoloside Aent-kaurane diterpenoids. According to the structure features, Professor Fujita ever divided this member of diterpenoids into four groups: ent-kauranes, 6,7-seco-ent-kauranes, 8,9-seco-ent-kauranes, and others in 1984ent-kauranes into nine groups including C-20 non-oxygenated ones, C-20 oxygenated ones, 6,7-seco-ent-kauranes, 8,9-seco-ent-kauranes, 8,15-seco-ent-kauranes, 15,16-seco-ent-kauranes, 7,20-seco-ent-kauranes, ent-kaurane dimers, miscellaneous ent-kauranesent-kauranes (ent-kaurane diterpenoids have never found in nature (kauranes 7. Althoun nature .Flickingeria fimbriata (B1.) Hawkes is a medicinal plant distributed in the south of China including Hainan, Guangxi, and Yunnan Provinces. Its whole herb has been used as tonic and antipyretic agents in the Minority Nation Li, in Hainan province. In addition, this medicinal plant  was often used as the equivalent of Pale Ephemerantha (“Shi Hu”) in the medicinal market. Previous chemical investigations of this plant obtained pimarane-type diterpenoids, several phenanthrenes, steroids, and bibenzyls78910111314ent-kaurane diterpenoids fimbriatols A–J (1–10) including four 3, 4-seco-ent-kauranes (7–10) were isolated  indicating the presence of five degrees of unsaturation. Detailed analysis of the 1HNMR, 13CNMR, and HMQC data for 1 -16,17-dihydroxy-phyllocladan-3-one. This result confirmed that the stereo-center of C-5 was S-configuration. Thus the absolute configuration of compound 1 was determined to 4R, 5S, 8S, 13R, 16R1 indeed possessed the skeleton of ent-kauranes.Compound ta for 1  and 2 reelations . The cor protons , which e spectra , which d1 was obtained for X-ray crystallography, and the Flack factor was -0.1, which determined the stereochemistry of 1 as -16, 17, 19- trihydroxy-ent-kaur-3-one , indicating the molecular formula of 2 as C20H32O5 (five degrees of unsaturation) with one more oxygen atom than that of 1. Analysis of NMR data revealed one additional oxygenated methine  present in NMR spectra in 2 compared with that of 1. The 1H–1H COSY and HMBC correlations confirmed that the CH2-6 in 1 was oxygenated to the corresponding oxymethine unit in 2. The relative configuration was established by analysis of coupling constant and NOESY correlations. The small coupling constant between H-5 and H-6 (J ≈ 0 Hz) together with the NOESY correlations from H-5, H-6 to CH3-18 implied the cis relationship of this two protons. The other NOESY correlations were same as those of 1  (The molecular formula of  = 10.8) .4 possessed the same as molecular formula as that of 2 and 3 on the basis of HRESIMS  implying that 4 was an isomer of 2 and 3. The NMR data mainly including the 2D NNR experiments suggested that the hydroxyl group was attached at C-9 not at C-6 found in 2 and 3. The NOESY correlation revealed the same relative configuration as those of 1–3 except for the 9-OH.Compound 5 suggested the same molecular formula as compounds 2–4. The NMR spectra revealed the similar structure feature as those present in 1 except the additional hydroxyl group was connected at C-11, which was confirmed by H–1H COSY and HMBC correlations. The small coupling constant (J ≈ 0 Hz) between H-9 (singlet) and H-11 implied that the dihedral angel was 90°. The NOESY correlations from H-11 with CH3-20, and H-1a revealed that the 11-OH possessed the β-configuration (The HRESIMS of guration .+ Δ + 0.8) of compound 6 afforded the molecular formula as C20H32O5 same as that of compounds 2–5. The 1H NMR spectra displayed three methyl groups present in 6, implying that the 19-methyl was not oxidized. The 1H–1H COSY and HMBC correlations confirmed that both C-1 and C-6 contained one hydroxyl group, respectively. The hydroxyl group for C-1 was determined to be α-configuration on the basis of the NOESY correlations from H-1 to H-5 and H-9, and hydroxyl group at C-6 was established as β-configuration on the basis of NOESY correlations of H-6 with CH3-19 and CH3-20. In addition, the large coupling constant between H-5 and H-6 (J ≈ 10.8 Hz) supported the trans relationship of these two protons.The HRESI MS . Analysis of the 1H and 13C NMR data of 7 revealed the significant difference compared with those of 1  and a double of exocyclic olefinic carbons  signals, whereas the keto carbonyl (C-3) and the oxymethylene (C-19) were disappeared. These differences implied that the carbon-bond of C-3/C-4 might be oxidized to shape the corresponding carboxylic group, and then dehydration reaction at C-19 formed the exocyclic double bond. The 2D NMR experiments especially the HMBC correlations from CH2-1 and CH2-2 to C-3 (δC 174.7) and from CH3-18 to C-4, C-5 and C-19 (exocyclic olefinic carbon) confirmed the above-mentioned postulation . Comparison of the NMR data of compound 8 and 7 revealed that the same structural fragments were found in 8 except for an additional oxyethyl and an acetonide group present in 8 +; Δ − 3.0 mmu] and NMR data  suggested a β-configured glucose unit, whereas the coupling constant for the apiose anomeric proton  and the chemical shift value for the anomeric carbon signal at δ 109.9 suggested the β-configured apiose unit. The linkage of the sugar moieties was determined by HMBC correlations. The HMBCcorrelations from CH2-17 to C-1′, in turn, from H-1′ to C-17 implied the connection of C-17 with C-1′ by an oxygen atom. The linkage between C-2′ and C-1′′ by an ether bond was confirmed by HMBC correlations from H-1′′ to C-2′, and from H-2′ to C-1′′. The chemical shifts of the glucose and apiose moiety were assigned through 1H–1H COSY, HMQC and HMBC spectra. Thus the planar structure of 9 was established. The relative configuration of 9 was same as that of 7 by analysis of the NOESY correlations.The molecular formula of NMR data . The 1H 10 was established as C32H52O13 by analysis of its HRESIMS  with 14 mass units more than that of compound 9. The 1H and 13C NMR spectra of 10 displayed resonances nearly identical to those of 9 except for an additional OMe group, suggesting that 10 was the oxymethyl derivative of 9. Analysis of HMBC data confirmed the above observations, and permitted the OMe unit to be connected with the carboxyl group (C-3). The relative configuration of 10 was same as those of 7 and 9 by analysis of its NOESY correlations. Due to the different groups found in compounds 7–10, the CD spectra of compunds 7–10 were different from those compounds 1–6  and cytotoxic activities without any effects.In addition, compounds ent-kaurane diterpenoids with diverse structurally features have been isolated from different plant121–6 are different from all known analogues by possessing highly oxidized sites, whereas 7–10 contain the 3, 4-seco-ent-kaurane skeleton, which is the first report in all ent-kaurane diterpenoids. Mono-glycosylated ent-kaurane diterpenoids are ever isolated from different resource, whereas 9 and 10 contained a disaccharide moiety at C-17 with the unusual linkage at C-2′ to C-1′′ not the common linkage at C-6′ to C-1′′, which was the first report in all known ent-kaurane diterpenoids. Recently, Hu et al. confirmed that a flavin-dependent monooxygenase catalyzed a keto group to the corresponding ester moiety by Baeyer-Villiger reaction in Aspergillus clavatus7–10 might be biosynthesized from ent-kaurane diterpenoids firstly through Baeyer-Villiger oxidation reaction to cleave the carbon bond between C-3 and C-4, and then through hydration, dehydration and glycosylation reactions to form 9 and 10 by post-modification  Hawkes. Our result further diversifies the structural feature of ent-kaurane diterpenoids.Up to date, more than 600 fication . Conside1H and 13C NMR data were acquired with a Bruker 600 spectrometer using solvent signals  as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HRESIMS data were acquired using a LTQ Orbitrap XL mass spectrometer.Optical rotations were measured on a Perkin-Elmer 241 polarimeter, and UV data were recorded on Beckman Coulter DU 800 spectrometer. IR data were recorded using a Shimadzu FTIR-8400S spectrophotometer. Flickingeria fimbriata (B1.) Hawkes was collected from Changjiang, Hainan Province, People’s Republic of China, in August 2008. The sample was identified by Mr. Rong-tao, Li , and a voucher specimen (FF2008-1) has been deposited in the herbarium of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Beijing.The plant of F. fimbriata were extracted with 95% EtOH to afford a crude extract after evaporation under vacuum. The extract was suspended in H2O and then partitioned sequentially with petroleum ether, CH2Cl2, EtOAc, and n-BuOH, successively. The CH2Cl2 and EtOAc extracts were subjected to column chromatography over silica gel to obtain 10 new ent-kaurane diterpenoids named fimbriatols A–J (1–10), which were characterized by HRESIMS, NMR and X-ray diffraction experiments.The air-dried and smashed stems of F. fimbriata (3.5 kg) were extracted with 95% EtOH (3 × 14 L) to afford a crude extract (330 g) after evaporation under vacuum. The extract was suspended in H2O (2.0 L) and then partitioned sequentially with petroleum ether (3 × 3.0 L), CH2Cl2 (3 × 3.0 L), EtOAc (3 × 3.0 L), and n-BuOH (3 × 3.0 L), successively. The CH2Cl2 extract (80.0 g) was subjected to column chromatography over silica gel (80–100 mesh), eluted with petroleum ether-acetone (10:1~1:1) as the mobile phase to yield eight fractions (C1~C8). E4 (3.2g) was subjected to column chromatography over silica gel (100–200 mesh) repeatedly to obtain 8 (16 mg). E6 (18.0 g) eluted with petroleum ether-acetone (3:2) was subjected to column chromatography over silica gel (100–200 mesh), eluted with ether-acetone (50:1~4:1) repeatedly, and then recrystallization to afford compounds 1 (3.0 g) and 7 (15 mg), respectively. E7 (18.0 g) eluted with petroleum ether-acetone (1:1) was subjected to column chromatography over silica gel (100–200 mesh), Sephadex LH-20, ODS and HW-40C (ethanol: H2O = 1:1) to afford compounds 2 (2.0 mg), 3 (2.0 mg), and 5 (10.0 mg), respectively. The EtOAc extract (10.0 g) was subjected to column chromatography over silica gel (100–200 mesh), eluted with CH2Cl2-CH3OH (50:1~3:1) to yield eight fractions (E1~E7). E6 (1.0 g) was subjected to column chromatography over ODS and HW-40C to 10 (14.6 mg). E7 (1.5 g) was subjected to column chromatography over silica gel (100–200 mesh), then Sephadex LH-20, ODS and HW-40C to 9 (73.0 mg). E7 (6.0 g) eluted with petroleum ether-acetone (1:1) was subjected to column chromatography over silica gel (100–200 mesh), eluted with ether-acetone (7:3~0:1) and Sephadex LH 20 column chromatography to obtain 4 (5.0 mg) and 6 (7.5 mg), respectively.The air-dried and smashed stems of R, 16R)-16, 17, 19- trihydroxy-ent-kaur-3-one (1): white crystal; mp 194–196 °C; [α]20D -20.0 ; UV (MeOH) λmax (log ε) 205 (0.5), 282.5 (0.027) nm; IR (KBr) νmax 3372 (OH), 2928 (CH), 1693 (C=O) cm−1; 1H and 13C NMR see m/z 337.2380 [M + H]+ .Fimbriatol A -6, 16, 17, 19- tetrahydroxy-ent-kaur-3-one (2): white crystal; mp 220–222 °C. c 0.05, MeOH); UV (MeOH) λmax (log ε) 210 (0.16), 292.5 (0.018) nm; IR (KBr) νmax3200 (OH), 2922 (CH), 1696 (C = O) cm−1; 1H and 13C NMR see 2; HRESI-MS m/z 375.2061 [M + Na]+ .Fimbriatol B -6, 16, 17, 19- tetrahydroxy-ent-kaur-3-one (3): white crystal; mp. 226–228 °C; c 0.075, MeOH); UV (CH3OH) λmax (log ε) 210 (0.30) nm; IR (KBr) νmax 3381 (OH), 2931(CH), 1696 (C=O) cm−1; 1H and 13C NMR see m/z 375.2164 [M + Na]+ .Fimbriatol C -9, 16, 17, 19- tetrahydroxy-ent-kaur-3-one (4): white crystal; mp. 116–118 °C; c 0.07, MeOH); UV (CH3OH) λmax(log ε) 212 (0.26) nm; IR (KBr) νmax 3367 (OH), 2945(CH), 1685 (C=O) cm−1; 1H and 13C NMR see m/z 375.2169 [M + Na]+ .Fimbriatol D -11, 16, 17, 19- tetrahydroxy-ent-kaur-3-one (5): white crystal; mp. 202–204 oC; c 0.06,MeOH); UV (CH3OH) λmax(log ε) 212 (0.26), 282.5 (0.015) nm; IR (KBr) νmax 3382 (OH), 2931 (CH), 1694 (C=O) cm−1; 1H and 13C NMR see m/z 375.2167 [M + Na]+ .Fimbriatol E -1, 6, 16, 17- tetrahydroxy-ent-kaur-3-one (6): amorphous powder; mp. 284–286 oC; c 0.055,MeOH); UV (CH3OH) λmax(log ε) 206 (0.27), 285 (0.022) nm; IR (KBr) νmax 3378 (OH), 2928 (CH), 1708 (C=O) cm−1; 1H and 13C NMR see m/z 375.2134 [M + Na]+ .Fimbriatol F (17): white crystal; mp 236–238 °C; [α]20D -68.3 ; UV (CH3OH) λmax(log ε) 204 (1.48), 237 (0.021) nm; IR (KBr) νmax 3437 (OH), 2925(CH), 1706 (C=O) cm−1; 1H and 13C NMR data see m/z 359.2198 [M + Na]+ .Fimbriatol G (8): white crystal; mp 200-202 °C; [α]20D -19.5 ; UV (CH3OH) λmax(log ε) 205 (5.0) nm; IR (KBr) νmax 2926(CH), 1734 (C = O) cm−1; 1H and 13C NMR see m/z 404.2903 [M]+ .Fimbriatol H (9): brown oil; [α]20D - 80 ; UV (CH3OH)λmax (log ε) 202 (1.2) nm; IR (KBr) νmax 3401 (OH), 2929 (CH), 1705 (C = O) cm−1; 1H and 13C NMR see Tabled 3; HRESI-MS m/z 653.3179 (M + Na)+ .Fimbriatol I (10): brown oil; [α]20D - 66 ; UV (CH3OH)λmax (log ε) 202 (1.263) nm; IR (KBr) νmax 3376 (OH), 2929 (CH), 1730 (C = O) cm−1; 1H and 13C NMR see m/z 667.3328 (M + Na)+ .Fimbriatol J (9 and 10 (1mg each) were put in 1 N HCl (0.25 mL) and stirred at 80 °C for 4 h. Then after cooling, the solution was dried by blowing air2Cl2 . The CH2Cl2 layer was analyzed by GC (Agilent technologies 7890B system) using an capillary HP-5 column (30 m*250 μm*0.25 μm). Temperature was maintained at 50 °C for 1min, then raised to 100 °C at the rate of 5 °C/min (5 min), and raised to 265 °C at the rate of 5 °C/min (11 min). Peaks of the hydrolysate of 9 and 10 were detected at 15.25, 15.32, 16.49 min, and 15.25, 15.32, 16.49 min, respectively. Peaks of D-apiose, were 15.27, 16.49 min, and D- and L-glucose were 15.24, and 15.30 min, respectively.Compounds 2O (10: 1) yielded colorless prisms of 1. A crystal (0.08 × 0.14 × 0.52mm) was separated from the sample and mounted on a glass fiber, and data were acquired with a a Rigaku MicroMax 002 + diffractometer with graphite-monochromated Mo Kα radiation and a graphite monochromator. Structure analysis was made using the SHELXL97 program. Crystallographic data (excluding structure factors) for 1 in this paper have been deposited with the Cambridge Crystallographic Data Centre (deposition number CCDC 1063873). Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: + 44 12 23336033 or e-mail: deposit@ccdc.cam.ac.uk). Crystal data: C20H32O4·H2O, M = 336.47, space group P212121; unit cell dimensions a = 6.083(3), b = 11.417(3), c = 26.629(6)Å, monoclinic, V = 1849.4(12)Å3, Dc = 1.273g/cm3, Z = 4. A total of 592 maps and 3145 independent reflections were collected in the range of 0° < Θ < 180°, of which 2995 observable reflections [|F|2>2σ|F|2)], completeness to Θ max was 96.7%; non-hydrogen atoms were refined anisotropically. Hydrogen atoms were located in Fourier difference maps and refined with idealized geometries and riding constraints. The final indices were R1 = 0.0377, wR2 = 0.0977, S = 1.049.Crystallization from petroleum MeOH: HEscherichia coli cells growing in 1 l LB media at OD600 of near 0.8 were inducted with 0.5 mM IPTG at 16 °C for 16 h. Centrifuged culture pellets were extracted by sonication in PBS buffer with 100 mM PMSF. Extracts were clarified by centrifugation 13,000 rpm for 30 min at 4 °C to yield the soluble extract. GST-tagged proteins present in the soluble extracts were purified by GST-affinity chromatography and dialyzed overnight against buffer containing 50 mM Tris–HCl (pH 7.5), and 0.1 M NaCl.The DNA fragment encoding DUSP26 was subcloned from human DUSP26 cDNA  into the expression vector pGEX-4T-2 as a fusion with a N-terminal GST-tag and a thrombin protease cleavage site. For proteins expression, In vitro phosphatase assay used for determining whether compounds are DUSP26 inhibitors was established and optimized based on methods described previously222O. The compound 1 were solubilized in HPLC grade DMSO with concentration at 0.022 μg/mL. All reactions were performed at a final concentration of 1% DMSO, while the enzyme activity was not affected by this concentration of DMSO.The purified DUSP26 (10 nM), and a candidate inhibitor were incubated in the reaction mixture containing 50 mM Tris–HCl (pH 7.5), 1% DMSO, for 15 min at 37 °C. Reactions were initiated by addition of pNPP and the incubation time was 30 min at 37 °C. This enzymatic reaction was stopped with the addition of 0.2 M NaOH. The phosphatase activities were then monitored by measuring the absorbance changes due to the hydrolysis of the substrate at 405 nM. The activities of DUSP26 with and without candidate inhibitors were represented as Ai and A0. The value of Ai/A0 × 100% was the residual activity (R.A.) of DUSP26. All experiments were performed in triplicate.How to cite this article: Ding, G. et al. Fimbriatols A-J, Highly Oxidized ent-Kaurane Diterpenoids from Traditional Chinese Plant Flickingeria fimbriata (B1.) Hawkes. Sci. Rep.6, 30560; doi: 10.1038/srep30560 (2016).


Integrins are a family of heterodimeric (α+β) adhesionreceptors that play key roles in many cellular processes. Integrins are unusual inthat their functions can be modulated from both outside and inside the cell.Inside-out signaling is mediated by binding adaptor proteins to the flexiblecytoplasmic tails of the α- and β-integrin subunits. Talin is one well-knownintracellular activator, but various other adaptors bind to integrin tails, including14-3-3-ζ, a member of the 14-3-3 family of dimeric proteins that have a preferencefor binding phosphorylated sequence motifs. Phosphorylation of a threonine in the β2integrin tail has been shown to modulate β2/14-3-3-ζ interactions, and recently, theα4 integrin tail was reported to bind to 14-3-3-ζ and associate with paxillin in aternary complex that is regulated by serine phosphorylation.Here, we use a range of biophysical techniques tocharacterize interactions between 14-3-3-ζ and the cytoplasmic tails of α4, β1, β2and β3 integrins. The X-ray structure of the 14-3-3-ζ/α4 complex indicates acanonical binding mode for the α4 phospho-peptide, but unexpected features are alsoobserved: residues outside the consensus 14-3-3-ζ binding motif are shown to beessential for an efficient interaction; in contrast, a short β2 phospho-peptide issufficient for high-affinity binding to 14-3-3-ζ. In addition, we report novel14-3-3-ζ/integrin tail interactions that are independent of phosphorylation. Of theintegrin tails studied, the strongest interaction with 14-3-3-ζ is observed for theβ1A variant. In summary, new insights about 14-3-3-ζ/integrin tail interactions thathave implications for the role of these molecular associations in cells aredescribed.  •Integrin tails are important for bidirectional signalingacross the membrane.•14-3-3-ζ binding to integrin tails has been studied usingbiophysical techniques.•Residues outside the 14-3-3 binding motif contribute toaffinity in α4 but not in β2.•Phosphorylation-independent 14-3-3-ζ interactions withintegrin tails are reported. Integrins are membrane-spanning heterodimeric receptors, associatedwith a wide-range of normal functions, including cell adhesion, migration anddifferentiation, as well as disease.The 14-3-3 family consists of highly conserved acidic proteins of~ 30 kDa molecular size that are expressed inall eukaryotic cells. In humans, seven distinct 14-3-3 isoforms  have been described, all of which form homodimers or heterodimers. They areinvolved in the regulation of many signaling pathways, including cell cycleprogression, programmed cell death and cytoskeletal dynamics. The family is alsoassociated with a number of human diseases, such as cancer and neurologicaldisorders.etal.,Recently, the structure of 14-3-3-ζ in complex with a phosphorylatedpeptide from the β2 integrin tail was determined.nuclear magneticresonance) and ITC , revealedthat the 14-3-3-ζ/α4 interaction is dependent not only on phosphorylation but also onresidues outside the central 14-3-3-ζ binding motif. We found no evidence for aternary 14-3-3-ζ/paxillin/α4 complex or a binary paxillin/α4 complex. We also carriedout a biophysical characterization of β-integrin tail interactions with 14-3-3-ζ. Allβ-integrin tails tested bound to 14-3-3-ζ in a phosphorylation-independent mannerthrough an epitope proximal to the transmembrane domain. In the case of β2 integrintail, this region is well separated from the previously characterizedphosphorylation-dependent 14-3-3-ζ binding site. Studies of 14-3-3-ζ variantsprovided further clues about the key determinants of the interactions.Here, we aimed to gain insight into the association of integrincytoplasmic tails with 14-3-3 proteins. Our initial focus was characterizing theinteraction between the α4 integrin tail and 14-3-3-ζ. We first solved the crystalstructure of 14-3-3-ζ in complex with a phosphorylated α4-derived peptide and definedits binding mode. The use of complementary techniques, particularly NMR orS1011D, with unlabeled 14-3-3-ζ produced changes of α4 resonances. As shown inWe used the α4 phospho-mimetic, S1011D, to explore thephosphorylation dependence of 14-3-3-ζ binding to the consensus RQYKSIL motif inthe α4 tail.Kd could not be determined by ITC.However, the α4 S1011D construct had a Kdvalue of approximately 60 μM, and the affinity of thephospho-serine containing α4-30pS was also strong(Kd ~ 24 μM). These results are consistent with aprominent role for phosphorylation in the regulation of 14-3-3 interactions andshow that the phospho-serine-to-aspartic substitution is a suitablephospho-mimetic for the 14-3-3-ζ/α4 association. Crucially, we observed that α4residues outside the consensus 14-3-3 binding motif are also necessary forefficient association, as illustrated by the 14-3-3-ζ/α4-11pS interaction thatgave a Kd value of over 300 μM (an ~ 15-fold reduction in affinity compared toα4-30pS).The affinities of 14-3-3-ζ/α4 interactions were measured by ITCfor the α4 wt and S1011D samples, as well as for two synthetic peptides containingphospho-serine residues α4-30pS and α4-11pS  in the α4 peptide  serverin vivoThe interactions of α4 and 14-3-3-ζ with paxillin were alsoinvestigated. The association of paxillin with α4 has been detected usingpull-down experiments with glutathione 15N heteronuclear single quantum coherence (HSQC) experiments ofunphosphorylated β2 integrin tail in the absence or presence of 14-3-3-ζ wererecorded. Addition of 14-3-3-ζ caused selective broadening of β2 resonances with 14-3-3-ζin an analogous manner to the experiments with α4. NMR sonancesa, revealsonancesb, withesonancesc. This iKd values of 8 μM and4 μM were obtained for β2-45pT and β2-11pT, respectively showed anintensity reduction for several resonances upon 14-3-3-ζ addition. Mapping theintensity changes reveals that binding occurs to β1A and β3, primarily in theN-terminal region, in a similar way to β2 . Thepattern of resonance intensity changes is more widespread in β7 . Our results are consistent with a study that detected aphosphorylation-independent interaction between 14-3-3-β and β1A by yeasttwo-hybrid experiments.NMR We also explored whether the phosphorylation-dependent 14-3-3-ζbinding site seen for β2 is present in other β-integrin tails. As illustrated inKd ~ 280 μM, than β2, β3 and α4  with 0.5 mM IPTG overnight at 37 °C. M9 minimal media containing15NH4Cl and 13C glucose were used to prepare labeled integrin tails. The proteinswere purified under denaturing conditions  by Co2 + affinity resin (Thermo Scientific) eluting with 200 mM imidazole. Samples were then dialyzed against 50 mM sodium phosphate, 150 mM NaCl, 1 mMethylenediaminetetraacetic acid and 1 mM DTT, cleaved with 3Cprotease at 4 °C and further purified by reverse phase HPLCusing a C18 column (Jupiter).Integrin cytoplasmic tails were cloned into a modified pET16bvector with an N-terminal His2 + affinitychromatography, cleaved to remove the His10 tag and furtherpurified by size-exclusion chromatography in NMR buffer [20 mMsodium phosphate and 50 mM NaCl (pH 6.5)],using a Superdex75 column .14-3-3-ζ was expressed from the same pET16b vector in ananalogous manner . Cells were harvested by centrifugation; resuspended in 50 mM sodium phosphate, 150 mM NaCl and 0.035%β-mercaptoethanol (pH 7.0); and disrupted by sonication. Thesoluble fraction was purified by CoIntegrin tail and 14-3-3-ζ mutants were generated according tothe QuikChange site-directed mutagenesis protocol (Stratagene) and produced andpurified in the same way as wt proteins.2O, and experiments were performed at 25 °C. Titration experiments were collected on a 500-MHz spectrometerusing a water flip-back-embedded gradient enhanced 15N–1H HSQC pulse sequence.15N–13C integrin tails of 0.6 mM concentration were used to measure CBCA(CO)NH and CBCANHexperiments on a Bruker 500-MHz instrument equipped with a cryogenic probe head.Data were processed using NMRPipeSamples were prepared in NMR buffer (pH 6.5)with 5% D200 instrument  were performed at 298 K as follows: the cell  contained the different integrin tail fragments, and the syringe contained 14-3-3-ζ. A firstinjection of 2 μl was followed by 15 injections of 2.5 μl, with a stirring speed of 1500 rpm and a delaybetween injections of 180 s. A blank titration was performed byinjecting 14-3-3-ζ into buffer to take the heat of dilution into account.Experiments were repeated twice for each sample. Raw data were processed andfitted with MicroCal Origin software using a one-site model where the errorfunction is calculated as the sum of the squared deviations between the data andthe model curve.Phosphorylated α4 synthetic peptides were purchased from GLBiochem Ltd. (Shanghai). All recombinant proteins and synthetic peptides wereprepared in 20 mM sodium phosphate and 50 mMNaCl (pH 6.5), and concentrations were determined by UVabsorbance at 280 nm. ITC experiments using aVP-ITCFor fluorescence polarization experiments, integrin tails werelabeled with fluorescein-5-maleimide (Invitrogen) linked to an engineeredC-terminal Cys residue. Measurements were performed using 96-well microplates(Corning) in a PHERAstar FS microplate reader (BMG Labtech). Fluorescein-labeledintegrin tails at 1 μM concentration were excited at 485 nm, and polarization was recorded at 520 nm.Samples for crystallization trials contained 10 mg/ml (0.35 mM) 14-3-3-ζ and 2.6 mg/ml(0.7 mM) α4-30pS peptide in 20 mM Tris–HCland 50 mM NaCl (pH 7.5). Crystals were grownby the sitting-drop vapor diffusion method at 4 °C using 1:1ratios of sample and 0.1 M Na Hepes (pH 7.5)and 25% polyethylene glycol 2000 monomethyl ether mother liquor. For datacollection, crystals were soaked in the same buffer plus 15% glycerol and flashfrozen in liquid nitrogen.Rfree. Initial structure determinationwas performed by molecular replacement using Phaser2O02Rwork/Rfree,and the quality of the structure was assessed using MolProbity.Data were collected at the Diamond Light Source beamline I04-1;the diffraction data extended to 2.2 Å resolution. Data wereindexed and integrated using MOSFLM and merged using SCALA from the CCP4 programsuite.4HKC, andchemical shift resonance assignments for the α4 and β2 integrin cytoplasmicdomains have been deposited in the Biological Magnetic Resonance Bank underaccession numbers 18718 and 187191871818719.Atomic coordinates for the 14-3-3-ζ/α4 phosphorylated peptidecomplex have been deposited in the Protein Data Bank under accession number1433_a4_complexThe following are the supplementary materialsrelated to this article.


Exercise-associated hyponatremia (EAH), rhabdomyolysis and renal failure appear to be a unique problem in ultra-endurance racers.i.e. multi-stage mountain biking, 24-h mountain biking, 24-h ultra-running and 100-km ultra-running).We investigated the combined occurrence of EAH and rhabdomyolysis in seven different ultra-endurance races and disciplines (n = 13) and four (4 %) ultra-runners (four men) from the normonatremic group (n = 100) showed rhabdomyolysis following elevated blood creatine kinase (CK) levels > 10,000 U/L without the development of renal failure and the necessity of a medical treatment. Post-race creatine kinase, plasma and urine creatinine significantly increased, while plasma [Na+] and creatine clearance decreased in hyponatremic and normonatremic athletes, respectively. The percentage increase of CK was higher in the hyponatremic compared to the normonatremic group (P < 0.05). Post-race CK levels were higher in ultra-runners compared to mountain bikers (P < 0.01), in faster normonatremic (P < 0.05) and older and more experienced hyponatremic ultra-athletes (P < 0.05). In all finishers, pre-race plasma [K+] was related to post-race CK (P < 0.05).Two (15.4 %) ultra-runners (man and woman) from hyponatremic ultra-athletes (+] and CK concentration in both hypo- and normonatremic ultra-athletes.Hyponatremic ultra-athletes tended to develop exercise-induced rhabdomyolysis more frequently than normonatremic ultra-athletes. Ultra-runners tended to develop rhabdomyolysis more frequently than mountain bikers. We found no association between post-race plasma [Na Exertional rhabdomyolysis, muscle cell breakdown with release of toxic chemicals into plasma and urine, is known as a relatively common response to prolonged strenuous exercise as running –21, but Factors proposed for the development of acute renal failure include dehydration secondary to inadequate fluid intake and/or diarrhoea or vomiting , 20, 27,+] and/or [Ca++] concentrations and reduce the stability of the cell membrane, or through direct mechanism decrease cell membrane stability through extracellular fluid osmotically drawn into the muscle cell [+] falls during endurance exercise, the intracellular [Ca++] is impaired and increases of [Ca++] lead to cellular injury, because arginine vasopressin is not able to restore the [Na+]/[Ca++] balance [+] is depleted and transferred to the extracellular compartment and myocytes are injured [+] induces rhabdomyolysis , plasma [K+], plasma and urine creatinine and plasma CK. Plasma [Na+], plasma [K+], plasma creatinine and plasma CK were determined using biochemical analyzer Modular SWA P300 . Samples of urine were collected in one Sarstedt Monovette for urine (10 ml) and sent to the laboratory. CK was determined in urine samples using biochemical analyzer Modular SWA P300 .At each race, blood and urine measurements were provided during registration the day before each race. Post-race measurements were upon completion of the race. Blood samples were drawn from the seated athlete into heparinized tubes via an antecubital vein. One Sarstedt S-Monovette  for chemical analysis was cooled, sent to the laboratory and analysed within 6 h. Blood samples were obtained to determine pre- and post-race plasma , plasma [K+], plasma creatinine, urine creatinine and creatinine clearance) are shown in Table Of the 145 ultra-runners and ultra-mountain bikers included in this study, 113 (81.8 %) finishers underwent body mass measurements and provided pre and post-race blood and urine samples. Thirty-two ultra-athletes did not finish their race or they did not undergo all measurements. Thirteen (11.5 %) finishers developed EAH, the remaining subjects  compared to mountain bikers (n = 82) (P <0.05). There was no difference in post-race plasma [Na+] between ultra-runners and mountain bikers (P > 0.05). The highest average post-race CK levels were in 24RUNners , follow by 100RUNners , 24MTBers  and SMTBers (918 ± 818 U/L). In 24RUNners post-race increase of CK was significantly higher than in 24MTBers .We combined the racers by each ultra-endurance disciplines to four categories  Tables  and 6. Te Tables  and 6. Pn = 13) and the normonatremic (n = 100) group of ultra-athletes. An average race performance (absolute order in each race) was 38.5 ± 47.7 in the hyponatremic group and 49.5 ± 43.3 in the normonatremic group without significant difference between both groups. Race performance correlated negatively to post-race  and percentage change  in CK only in the normonatremic group. An average age of hyponatremic athletes was 39.5 ± 6.9 years, normonatremic athletes 38.6 ± 8.5 years. Age was associated with post-race  and percentage change  in CK in the hyponatremic group. The number of years as an active runner (biker) was related to the percentage change  in CK only in the hyponatremic group. Neither the number of finished ultra-marathons nor the total number of hours spent by training weekly, nor gender was related to post-race CK concentration (P > 0.05) in both groups. In the hyponatremic group, CK increased by 3,456 ± 4,982 U/L and 1.9 ± 2.9 %, respectively, from baseline (P < 0.01). In the normonatremic group CK increased by 1,831 ± 4,073 U/L and 1.1 ± 2.8 %, respectively, from baseline (P < 0.01). The percentage increase of CK was higher in the hyponatremic group (P < 0.05)  .We compared the hyponatremic n = 3 and the+] decreased by 5.8 ± 2.1 mmol/L (4.1 ± 1.5 %) (P < 0.01). In the normonatremic group, post-race plasma [Na+] decreased by 1.2 ± 2.4 mmol/L, (0.8 ± 1.7 %) (P < 0.01). Percentage change in plasma [Na+] was significantly higher in hyponatremic ultra-athletes (P < 0.05). Plasma [K+] remained stable post-race (P > 0.05) in hyponatremic ultra-athletes. Post-race plasma [K+] decreased by 5.1 ± 0.9 mmol/L post-race, (1.3 ± 25.3 %) (P < 0.05) in normonatremic ultra-athletes. Pre-race plasma [K+] was not related to post-race plasma [K+] in all finishers, hyponatremic or normonatremic ultra-athletes (P > 0.05). Post-race plasma creatinine increased by 1.1 ± 0.5 mg/dL (39.5 ± 71.0 %) (P < 0.05) in hyponatremic finishers. Post-race plasma creatinine increased by 1.1 ± 0.3 mg/dL (25.2 ± 34.1 %) (P < 0.01) in normonatremic finishers. Post-race urine creatinine increased by 7.9 ± 7.0 mmol/L (216 ± 287 %) (P < 0.01) in the hyponatremic group. Post-race urine creatinine increased by 9.5 ± 5.7 mmol/L (229 ± 235 %) (p < 0.01) in the normonatremic group. Post-race plasma creatine clearance decreased by 2,454 ± 2,428 ml/min (21.7 ± 18.4 %) (P < 0.01) in the hyponatremic group. Plasma creatine clearance decreased by 1,913 ± 1,815 ml/min (18.0 ± 15.6 %) (P < 0.01) in the normonatremic group. In comparison of the normonatremic and the hyponatremic group percentage change in plasma [K+], plasma and urine creatinine and creatine clearance did not differ significantly between both groups.In the hyponatremic group, post-race plasma  decreased in cases 1 and 2 with rhabdomyolysis and EAH and cases 3 and 5 with rhabdomyolysis within the hyponatremic and the normonatremic group with a significantly higher increase in the hyponatremic group. In different kinds of the present races limited by various numbers of participant’s plasma [Na+] decreased in all ultra-disciplines. Hyponatremia as the most common electrolyte disorder associated with ultra-running and muscle-cell swelling with mechanical stress caused by running (footrace) may result in skeletal muscle damage, rhabdomyolysis, or acute renal injury [+] and CK concentrations were negatively correlated in 161-km ultra-runners . In a recent study of Hoffman and Stuempfle [+] and CK concentration was also evident, however, without the difference between the hyponatremic and the normonatremic group due to wide variability in creatine kinase concentrations. Similarly, in the study of Hoffman et al. [+] did not reach statistical significance.Plasma  ≥ 5.5 mmol/L) is important to remove [K+] from the body [+] into mild (5.5 - 6.5 mmol/L), moderate (6.5 - 7.5 mmol/L) and severe (>7.5 mmol/L) hyperkalemia [+] depletion due to cell swelling eventually induces rhabdomyolysis [+] 5.2 mmol/L and case 2 plasma [K+] of 4.6 mmol/L. A study of four athletes with EAH and rhabdomyolysis showed plasma [K+] in the range from 4.1 mmol/L to 4.9 mmol/L [+] were higher than in the study of Bruso et al.  ranged from 4.8 mmol/L to 5.0 mmol/L, only case 6 reached 6.7 mmol/L . Another finding was the post-race decrease of plasma [K+] in all cases (1–5), except case 6. The reason could be probably pre-race mild to severe hyperkalemia in cases 1–5 due to the range of pre-race plasma [K+] from 5.9 mmol/L to 8.1 mmol/L (case 4 with 8.1 mmol/L). On the contrary, case 6 presented with a pre-race level of 4.9 mmol/L and therefore probably exhibited post-race increase of plasma [K+]. The average post-race plasma [K+] in the present normonatremic finishers was in the range from 3.8 mmol/L to 8.2 mmol/L and twenty-seven (27 %) athletes developed levels higher than 5.5 mmol/L. Their average pre-race plasma [K+] was 5.4 ± 1.2 mmol/L (range 3.5 mmol/L to 9.2 mmol/L) with fifteen (15 %) ultra-athletes with pre-race level ≥ 5.5 mmol/L. Hyponatremic finishers had an average post-race plasma [K+] in the range from 4.4 mmol/L to 6.5 mmol/L with four (30.8 %) finishers with a level higher than 5.5 mmol/L. Even in five (38.5 %) hyponatremic finishers pre-race plasma [K+] reached levels of ≥ 5.5 mmol/L. Post-race plasma [K+] significantly decreased in both genders in 24MTBers and 24RUNners and non-significantly in male 100RUNners despite the limitation of different number of finishers in each ultra-endurance discipline.The interesting finding was that the present cases 1 and 2 showed pre-race values of plasma [K+] showed an average value of 6.7 mmol/L and 6.2 mmol/L in male and female 24RUNners, respectively, 5.5 mmol/L and 6.2 mmol/L in male 24MTBers and male 100RUNners, respectively. On the contrary, the lowest pre-race level was 4.4 mmol/L in male SMTBers, 4.7 mmol/L in female SMTBers, 5.2 mmol/L in female 100RUNners, and we simultaneously found a significant increase in post-race plasma [K+]. Overall, despite the genders and the various numbers of racers in each ultra-disciplines 57.9 % 24RUNners, 42.0 % of 24MTBers, 16.7 % 100RUNners and 6.3 % of SMTBers showed pre-race hyperkalemia and 47.4 % 100RUNners, 28.1 % SMTBers, 20 % 24MTBers and 16.7 % 24RUNners developed post-race hyperkalemia.Another finding was that pre-race plasma [K+] and post-race CK levels in the present finishers. The ultra-athletes with higher pre-race plasma [K+] developed post-race higher CK levels. It also appears that races with a higher occurrence of pre-race hyperkalemia tended to a lower occurrence of hyperkalemia post-race and conversely. However, there was no significant relationship between pre-race plasma [K+] and post-race plasma [K+] considering all present ultra-athletes. The presence of mild pre-race or post-race hyperkalemia in some present hyponatremic and normonatremic cases supports either excessive [K+] ingestion pre-race or during the race, ingestion of NSAID, reduced renal excretion or tissue breakdown as in rhabdomyolysis [+] and CK concentration. We have to take into account that some results could be also caused by pseudohyperkalemia from leakage of [K+] from the intracellular space during or after blood sampling in field conditions  134 mmol/L) developed also high pre- and post-race levels of plasma [K+] (5.5 and 5.7 mmol/L), minus 3.8 percentage change in body mass; the highest percentage decrease (minus 74.1 %) of creatine clearance within all finishers; however with post-race CK just 1,197 U/L. The 26-year old normonatremic SMTBer showed plasma [Na+] 140 mmol/L, pre- and post-race levels of plasma [K+] 3.6 and 4.9 mmol/L, plus 0.6 % percentage change in body mass, the second highest percentage decrease (minus 70. 4 %) of creatine clearance within all finishers; however similarly with post-race CK only 1,168 U/L. Both current cases presented post-race without the development of renal failure and the necessity of a medical treatment. Neumayr et al. investigated the effect of marathon cycling on renal function in recreational and professional road cyclists [+] and the lowest concentrations of creatine clearance; however, both with low post-race CK concentrations. Moreover, these hyponatremic multi-stage bikers showed a higher percentage increase in post-race CK levels than normonatremic biker. In the study of Hoffman et al.  and the restoring of cellular volume [We have to take into account that the number of present ultra-athletes in various disciplines and gender representation was different. Further we did not observe a urine dipstick for a urine colour, blood and myoglobin. However, direct measurement of myoglobin is less reliable and not clinically useful . Moreover volume . Peak CKr volume . In the r volume , 23. We The value of CK as a prognostic tool is limited, due to the wide inter-individual range of CK levels. In situations where the diagnosis of EAH is uncertain, fluid restriction during or after the race is contraindicated in case of dehydration and rhabdomyolysis with impending acute kidney injury , 29, 41.The present hyponatremic ultra-athletes tended to develop exercise-induced rhabdomyolysis more than normonatremic ultra-athletes. Ultra-runners tended to develop rhabdomyolysis more than mountain bikers. The present work does not clarify the mechanism that might be involved in the suggested link between rhabdomyolysis and EAH.Written informed consent was obtained from all testing subjects for the publication of this report and any accompanying images.


H-imidazole. The FeCl2 and FeBr2 complexes are isotypic, while the third compound, also an FeBr2 complex, crystallizes as a diethyl ether disolvate.The title compounds are iron(II) dihalide complexes of the bulky arylimidazole ligand 1--1 2(C15H20N2)4], (I), [FeBr2(C15H20N2)4], (II), and [FeBr2(C15H20N2)4]·2C4H10O, (IIb), respectively, all have triclinic symmetry, with (I) and (II) being isotypic. The FeII atoms in each of the structures are located on an inversion center. They have octahedral FeX2N4  coordination spheres with the FeII atom coordinated by two halide ions in a trans arrangement and by the tertiary N atom of four arylimidazole ligands [1--1H-imidazole] in the equatorial plane. In the two independent ligands, the benzene and imidazole rings are almost normal to one another, with dihedral angles of 88.19 (15) and 79.26 (14)° in (I), 87.0 (3) and 79.2 (3)° in (II), and 84.71 (11) and 80.58 (13)° in (IIb). The imidazole rings of the two independent ligand molecules are inclined to one another by 70.04 (15), 69.3 (3) and 61.55 (12)° in (I), (II) and (IIb), respectively, while the benzene rings are inclined to one another by 82.83 (13), 83.0 (2) and 88.16 (12)°, respectively. The various dihedral angles involving (IIb) differ slightly from those in (I) and (II), probably due to the close proximity of the diethyl ether solvent molecule. There are a number of C—H⋯halide hydrogen bonds in each molecule involving the CH groups of the imidazole units. In the structures of compounds (I) and (II), molecules are linked via pairs of C—H⋯halogen hydrogen bonds, forming chains along the a axis that enclose R22(12) ring motifs. The chains are linked by C—H⋯π interactions, forming sheets parallel to (001). In the structure of compound (IIb), molecules are linked via pairs of C—H⋯halogen hydrogen bonds, forming chains along the b axis, and the diethyl ether solvent molecules are attached to the chains via C—H⋯O hydrogen bonds. The chains are linked by C—H⋯π interactions, forming sheets parallel to (001). In (I) and (II), the methyl groups of an isopropyl group are disordered over two positions . In (IIb), one of the ethyl groups of the diethyl ether solvent molecule is disordered over two positions (occupancy ratio = 0.5:0.5).The title compounds, [FeCl However, despite this, only a very few of them contain iron(II), except the tridentate diimine pyridine complex b). Compound (I)L1 in Fig. 7b) were isolated when bisimidazoliummethylene tetrabromidoferrate(III)  differs due to the presence of solvent diethyl ether molecules. The whole molecule of each compound, (I)b), is generated by inversion symmetry  coordination sphere. It is coordinated by the tertiary N atoms of four imidazole ligands [1--1H-imidazole], in the equatorial plane, while the axial positions are occupied by the halogen ions. In (I)b), the Fe—Br1 bond lengths are 2.7040 (5) and 2.7422 (3) Å, respectively. The Fe—N1 and Fe1—N3 bond lengths are 2.190 (3) and 2.161 (3) Å in (II)b). In each molecule, all of the imidazole C-bound H atoms are involved in intramolecular C—H⋯halogen hydrogen bonds (see Tables 1The structures of (I)b), the corresponding angles are 87.0 (3) and 79.2 (3)°, and 84.71 (11) and 80.58 (13)°, respectively. The imidazole rings (N1/N2/C1–C3 and N3/N4/C16-C18) of the two independent ligand molecules are inclined to one another by 70.04 (15), 69.3 (3) and 61.55 (12)° in (I)b), respectively, while the benzene rings (C4–C9 and C19–C24) are inclined to one another by 82.83 (13), 83.0 (2) and 88.16 (12)°, respectively. The various dihedral angles involving (IIb) differ slightly from those in (I)In the two independent ligands of (I)b), molecules are linked via pairs of C—H⋯halogen hydrogen bonds, forming chains along the a axis diiron(II) iron(II) has also been reported   in 20 ml of THF was added 0.09 g (2.3 mmol) of NaH 60% and 0.01 g (0.1 mmol) of tBuOK, and the reaction mixture was heated at 340 K for 8 h. The solution was then filtered and the solvent evaporated under vacuum yielding an orange solid. Yellow crystals were obtained by slow diffusion of diethyl ether into a THF solution of the isolated orange solid. UV–vis : 364, 290. Compounds (II)b) were prepared in a similar manner. To a solution of (L2)  in 20 ml of THF was added 0.09 g (2.3 mmol) of NaH 60% and 0.01 g (0.1 mmol) of tBuOK at 273 K, and the reaction mixture was heated at reflux for 8 h. The solution was then filtered and the solvent evaporated under vacuum yielding a yellow–brown solid. Yellow crystals were obtained by slow diffusion of diethyl ether into a THF solution of the isolated yellow–brownish solid. UV–vis : 292. Two types of crystals were obtained: yellow plates for (II)b).The synthesis of the precursors bisimidazolium methylene tetrachlorido- and tetrabromidoferrate(III)  = 1.5Ueq(C-methyl) and = 1.2Ueq(C) for other H atoms. In (I)b), one of the ethyl groups of the diethyl ether solvent molecule is disordered over two positions (occupancy ratio fixed at 0.5:0.5).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S1600536814014056/wm0006sup1.cifCrystal structure: contains datablock(s) I, II, IIb, Global. DOI: 10.1107/S1600536814014056/wm0006Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536814014056/wm0006IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S1600536814014056/wm0006IIbsup4.hklStructure factors: contains datablock(s) IIb. DOI: 1008173, 1008174, 1008175CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


E)-3-aryl-1-prop-2-en-1-ones all have nearly planar molecular skeletons. C—H⋯O hydrogen bonds are present in only three of the structures but short Br⋯Br, Br⋯O and Cl⋯Cl contacts are also present in some of the structures.Six closely related (2 E)-1-(5-chlorothiophen-2-yl)-3-(4-ethylphenyl)prop-2-en-1-one, C15H13ClOS, (I), and (2E)-1-(5-bromothiophen-2-yl)-3-(4-ethylphenyl)prop-2-en-1-one, C15H13BrOS, (II), are isostructural in space group P-1, while (2E)-1-(5-chlorothiophen-2-yl)-3-(4-ethoxyphenyl)prop-2-en-1-one, C15H13ClO2S, (III), and (2E)-1-(5-bromothiophen-2-yl)-3-(4-ethoxyphenyl)prop-2-en-1-one C15H13BrO2S, (IV), are isostructural in space group P21/c. There are no hydrogen bonds of any kind in the structures of compounds (I) and (II), but in the structures of compounds (III) and (IV), the molecules are linked into C(7) chains by means of C—H⋯O hydrogen bonds. In the structure of (2E)-3-(4-bromophenyl)-1-(5-chlorothiophen-2-yl)prop-2-en-1-one, C13H8BrClOS, (V), there are again no hydrogen bonds nor π–π stacking interactions but in that of (2E)-1-(5-bromothiophen-2-yl)-3-(3-methoxyphenyl)prop-2-en-1-one, C14H11BrO2S, (VI), the molecules are linked into C(5) chains by C—H⋯O hydrogen bonds. In each of compounds (I)–(VI), the molecular skeletons are close to planarity, and there are short halogen⋯halogen contacts in the structures of compounds (II) and (V) and a short Br⋯O contact in the structure of compound (VI). Comparisons are made with the structures of some similar compounds.The structures of six chalcones containing 5-halogenothiophen-2-yl substituents are reported: (2 They display a wide range of pharmacological properties including antibacterial (Tang PP21/c. Although the unit-cell dimensions for compound (V)Compounds (I)PP21/c. In the molecules of each of (I)–(V), the 5-halogenothienyl unit adopts the same orientation, with S11—C12—C1—C2 torsion angles close to 180° (Table 1Although the molecules of compounds (I)–(VI) all lie in general positions, the non-H atoms of the molecular skeletons are quite close to being co-planar, apart from the terminal C atoms of the ethyl groups in compounds (I)–(IV), as shown by the dihedral angles in Table 1° Table 1. There iet al., 1996In each of compounds (III)et al., 1987The bond distances in compounds (I)–(VI) all lie within the usual ranges (Allen i = 3.4917 (5) Å and C15—Br15⋯Br15i = 151.37 (8)° . The Br ⋯Br distance is significantly shorter than the van der Waals contact distance of 3.70 Å c-glide symmetry into zigzag C(7) ii , the geometrical parameters are Cl⋯Clii = 3.4825 (11) Å and C—Cl⋯Clii = 167.83 (10)°. The Cl⋯Cl distances is thus just at the van der Waals contact distance 3.48 Å 1 screw axis along  chain running parallel to the [010] direction  Å and C15—Br15⋯O33iii = 167.21 (7)° .A single C—H⋯O hydrogen bond Table 2 links thon Fig. 8. Two chaPP21/c, and there appear to be some interesting connections between the space groups and the nature of the direction-specific intermolecular interactions manifested in the various structures. Thus although all six of the compounds described here contain carbonyl groups, only in compounds (III)P21/c. Of the three 5-bromothienyl derivatives reported here, a short Br⋯Br contact occurs only in compound (II)PAll of the compounds reported here crystallize either in space group E)-3-aryl-1-(5-chlorothiophen-2-yl)-prop-2-en-1-one derivatives closely related to compounds (I)–(VI) have been reported recently, usually in the form of brief reports on single structures in which no comparisons with related compounds were made, and sometimes with little or no mention of the supramolecular assembly. It is thus of interest briefly to compare the supramolecular assembly in these compounds with that in compounds (I)–(VI). Compound (VII) (see Scheme below) is isomeric with compound (V)P21/c  Å (not 3.698 (1) Å as stated in the original report), is larger than the sum, 3.55 Å  and  are strictly parallel with an interplanar spacing of 3.5113 (8) Å: the ring centroid separation is 3.6535 (11) Å, corresponding to a ring-centroid offset of 1.009 (2) Å, so leading to the formation of a centrosymmetric π-stacked dimer   chain running parallel to the [001] direction, and secondly that inversion-related pairs of molecules are linked by a π–π stacking interaction involving the phenyl rings of the molecules at  and , with interplanar spacing 3.4465 (10) Å, ring-centroid separation 3.749 (3) Å and ring-centroid offset 1.475 (3) Å. The combined effect of these two types of interaction is the formation of a sheet lying parallel to (100); see Fig. 10The original report on compound (XI)  chains are formed in the structure of compound (XIII) built from C—H⋯O hydrogen bonds  which were described (Prabhu w/v solution). The mixtures were all stirred at ambient temperature for 4 h, and then poured into ice-cold water (250 ml): the resulting solid products were collected by filtration and dried in air at 323 K. Crystals suitable for single-crystal X-ray diffraction were grown by slow evaporation, at ambient temperature and in the presence of air, of solutions in acetone: melting points: (I)For the synthesis of each compound, an equimolar mixture (0.01 mol of each component) of the appropriate 2-acetyl-5-halogenothiophen and the appropriately-substituted benzaldehyde was dissolved in a mixture of methanol (20 ml) and aqueous sodium hydroxide solution (5 ml of 30% 3) or 0.99 Å (CH2), and with Uiso(H) = kUeq(C), where k = 1.5 for the methyl groups, which were permitted to rotate but not to tilt, and 1.2 for other H atoms. The low-angle reflections  global, I, II, III, IV, V, VI. DOI: 10.1107/S2056989015015534/su5193Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015015534/su5193IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989015015534/su5193IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989015015534/su5193IVsup5.hklStructure factors: contains datablock(s) IV. DOI: 10.1107/S2056989015015534/su5193Vsup6.hklStructure factors: contains datablock(s) V. DOI: 10.1107/S2056989015015534/su5193VIsup7.hklStructure factors: contains datablock(s) VI. DOI: Click here for additional data file.10.1107/S2056989015015534/su5193Isup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015015534/su5193IIsup9.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015015534/su5193IIIsup10.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015015534/su5193IVsup11.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015015534/su5193Vsup12.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015015534/su5193VIsup13.cmlSupporting information file. DOI: 1419530, 1419529, 1419528, 1419527, 1419526, 1419525CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N-glycosylation at the Fc region, which is therefore regarded as a crucial factor in the design and production of therapeutic antibodies. NMR spectroscopy plays a unique role in the characterization of conformational dynamics and intermolecular interactions of IgG-Fc in solutions. Here, we report NMR assignments of the glycosylated Fc fragment (Mr 53 kDa), cleaved from a chimeric antibody with human IgG1 constant regions, which was produced in Chinese hamster ovary cells with uniform 13C- and 15N-labeling.The Fc portion of immunoglobulin G (IgG) recruits complements and its cognate receptors, thereby promoting defensive mechanisms in the humoral immune system. These effector functions critically depend on The online version of this article (doi:10.1007/s12104-014-9586-7) contains supplementary material, which is available to authorized users. H2 and CH3 domains. The CH2 domain possesses a conserved N-glycosylation site, Asn297, at which a biantennary complex-type oligosaccharide is expressed with microheterogenieties characterized by the presence and absence of the non-reducing terminal galactose, fucose, sialic acid, and bisecting N-acetylglucosamine residues.Immunoglobulin G (IgG) is a multifunctional glycoprotein composed of an Fc region and two Fab regions, which are connected through the hinge region  cleaved from a chimeric antibody with human IgG1 constant regions that was expressed by Chinese hamster ovary (CHO) cells with uniform 13C- and 15N-labeling.In an extension of these studies, we herein report NMR assignments of the glycosylated version of Fc fragment  in which glucose, sodium pyruvate, succinic acid, and amino acids were replaced by 1 g/L [15N/13C]algal amino acid mixture, 2 g/L D-[13C6]glucose, 110 mg/L [13C3]pyruvic acid sodium salt, 59 mg/L [13C4]succinic acid, 149 mg/L L-Arg·HCl, 42.5 mg/L L-Asn·H2O, 24 mg/L L-Cys, 450 mg/L L-Gln, 17 mg/L L-His·HCl·H2O, 27 mg/L L-Tyr, and 7 mg/L L-Trp. Amino acid-selective labeling of IgG1 was performed using the modified Nissui NYSF 404 medium  in which selected amino acid components were substituted with isotopically labeled analogs, as described previously Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc, the Fc fragment was treated with a recombinant Streptococcus pneumoniae β1,4-galactosidase (CALBIOCHEM), according to the literature  was kindly provided by Dr. Lawrence Chasin . An anti-CCR4 chimeric antibody (designated KM3060), with human IgG1/κ constant regions, was produced in a CHO cell line as described previously (Yamaguchi and Kato 1980 was 2O. NMR spectra were acquired at 42 or 52 °C using DMX500 (Bruker BioSpin), AVANCE800 (Bruker BioSpin), and ECA-920 (JEOL) spectrometers. Chemical shifts of 1H were referenced to DSS (0 ppm), and 13C and 15N chemical shifts were referenced indirectly using the gyromagnetic ratios of 13C, 15N, and 1H .For NMR measurements, the Fc fragment was dissolved in 0.5 ml of 5 mM sodium phosphate buffer (pH 6.0) containing 50 mM NaCl and 10 % (v/v) D1H–15N HSQC spectral data of uniformly or selectively 13C/15N-labeled IgG1-Fc, and 3D spectral data obtained with the following experiments: HNCA, HNCO, HN(CA)CO, CBCA(CO)NH, and HNCACB. All NMR data were processed using NMRPipe software , Cβ (84 %), CO (80 %), HN (99 %), and N (99 %) (except for N of prolines). The spectral assignments at lower temperatures could be extrapolated by observing progressive spectral changes, depending on temperature, as exemplified by the spectrum at 42 °C . The present spectral assignments indicate that a cluster of amino acid residues in the vicinity of the N-glycans, i.e. Gln295-Thr299 exhibit significant chemical shift differences in comparison with the previously reported assignments of human Fc produced in Escherichia coli  under the accession number 25224.The assignments for the Supplementary material 1 (PPT 615 kb)Below is the link to the electronic supplementary material.


The two chloride anions are not coordinating to Pd2+ but are connected to the complex cations through N—H⋯Cl hydrogen bonds. C—H⋯Cl hydrogen bonds lead to a three-dimensional linkage of cations and anions.In the title salt, the Pd 3H4N2)4]Cl2, was obtained unexpectedly by the reaction of palladium(II) dichloride with equimolar amounts of 1-chloro-1-nitro-2,2,2-tris(pyrazolyl)ethane in methanol solution. The Pd2+ cation is located on an inversion centre and has a square-planar coordination sphere defined by four N atoms of four neutral pyrazole ligands. The average Pd—N distance is 2.000 (2) Å. The two chloride anions are not coordinating to Pd2+. They are connected to the complex cations through N—H⋯Cl hydrogen bonds. In addition, C—H⋯Cl hydrogen bonds are observed, leading to a three-dimensional linkage of cations and anions.The title salt, [Pd(C The formation of free pyrazole could only be explained by a solvolytic degradation of the starting material. This degradation must have taken place to a large extent as the isolated yield was 64% 3 Fig. 1 in metha3H4N2)4]Cl2 is presented here. An elemental analysis of the title compound was also in very good agreement with the composition C12H16Cl2PdN8. In this case, too, the yield was fairly high (56%), indicating a far-reaching decomposition of the starting material. Apparently, the ligand degradation of azolyl-nitrochloroalkanes in the presence of transition metal salts is a more common phenomenon than originally anticipated.We have now carried out a closely related reaction of 1-chloro-1-nitro-2,2,2-tris(pyrazolyl)ethane with palladium(II) dichloride in methanol solution. Structure determination of the yellow reaction product using X-ray analysis surprisingly again revealed the presence of a homoleptic pyrazole complex. The structure of the resultant title compound,   analog [Mn(pyrazole)4Cl2] 4Cl2] show related features. In the structures with M = Mn and Cu and an octahedral coordination of the metal cation, the crystal structures likewise exhibit N—H⋯Cl and C—H⋯Cl hydrogen bonds which, in combination, yield three-dimensional networks.In the title compound, the crystal packing is stabilized by two N—H⋯Cl hydrogen bonds Table 1 between ns Fig. 3. Weaker et al., 1998et al., 1986M(pyrazole)4Cl2] have previously been reported for M = Mn, Fe, Co, Ni, and Cu . Analysis calculated for C12H16Cl2PdN8: C 32.06%; H 3.59%; N 24.92%; Cl 15.77%; found: C 31.55%; H 3.38%; N 25.13%; Cl 15.25%. IR (KBr): 3090vs, 2977vs, 2371m, 1798w, 1772w, 1632w, 1518m, 1487m, 1472s, 1401m, 1367s, 1312m, 1264m, 1251m, 1209w, 1181vs, 1169m, 1139s, 1123s, 1078vs, 1052vs, 983m, 956m, 913m, 908m, 899m, 886m, 878m, 779vs, 739s, 615s, 606s cm−1.Solid palladium(II) dichloride  was added to a solution of 1-chloro-1-nitro-2,2,2-tris(pyrazolyl)ethane  in methanol (100 ml). After stirring for 48 h at room temperature, a small amount of unreacted PdClUiso(H) = 1.2Ueq(C). The hydrogen atoms attached to nitrogen were refined with a restrained distance N—H = 0.88 (2) Å and with Uiso(H) = 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S160053681402460X/wm5084sup1.cifCrystal structure: contains datablock(s) I, New_Global_Publ_Block. DOI: 10.1107/S160053681402460X/wm5084Isup2.hklStructure factors: contains datablock(s) I. DOI: 1033485CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: