Five copper(I) iodide coordination compounds were synthesized and characterized by single-crystal X-ray diffraction measurements; the resulting structures display a diverse array of structural features. N)(6-sulfanylidenepiperidin-2-one-κS)copper(I)], [Cu2I2(CH3CN)2(C5H7NOS)2] (I), bis(acetonitrile-κN)tetra-μ3-iodido-bis(6-sulfanylidenepiperidin-2-one-κS)-tetrahedro-tetracopper(I), [Cu4I4(CH3CN)4(C5H7NOS)4] (II), catena-poly[[(μ-6-sulfanylidenepiperidin-2-one-κ2O:S)copper(I)]-μ3-iodido], [CuI(C5H7NOS)]n (III), poly[[copper(I)]-μ3-iodido], [CuI(C5H7NS2)]n (IV), and poly[[copper(I)]-μ3-iodido], [CuI(C8H5NS2)]n (V). Compounds I and II crystallize as discrete dimeric and tetrameric complexes, whereas III, IV, and V crystallize as polymeric two-dimensional sheets. To the best of our knowledge, compound III is the first instance of an extended hexagonal [Cu3I3] structure that is not supported by bridging ligands. Structures I, II, and IV display weak to moderately strong Cu⋯Cu cuprophilic interactions [Cu⋯Cu internuclear distances range between 2.5803 (10) and 2.8485 (14) Å]. All structures except III display weak hydrogen-bonding interactions between the N—H of the ligand and the μ2 and μ3-I− atoms. Structure III contains classical N–H⋯O interactions between the SNO ligands that connect the molecules in a three-dimensional framework. Complex V features π–π stacking interactions between the aryl rings of the SNS6 ligands within the same polymeric sheet. In structure IV, there were three partially occupied solvent molecules of dichloromethane and one partially occupied molecule of acetonitrile present in the asymmetric unit. The SQUEEZE routine [Spek  iodide complexes are well known for displaying a diverse array of structural features even when only small changes in ligand design are made. This structural diversity is well displayed by five copper(I) iodide compounds reported here with closely related piperidine-2,6-dithione (SNS), isoindoline-1,3-dithione (SNS6), and 6-thioxopiperidin-2-one (SNO) ligands: di-μ-iodido-bis[(acetonitrile-κSpek 2015. Acta Cr These lO) Fig. 1, 2014 ▸.I crystallizes as a discrete dimer with a rhombic Cu2(μ2-I)2 core that resides on a crystallographic inversion center; thus, only one half of the dimer is symmetry-independent –118.719 (16)°; L = SNO or acetonitrile). The Cu⋯Cu internuclear distance of 2.7274 (6) Å is slightly shorter than the sum of the covalent radii (ca 2.87 Å) and is consistent with a weak cuprophilic interaction. The Cu—N and Cu—S distances (Table 1I are similar to the Cu—N and Cu—S distances in other discrete Cu2(μ2-I)2 dimers reported to the Cambridge Structural Database (CSD) and selected with moderate search criteria 4 core; the four Cu atoms form a distorted tetrahedron with μ3-I− atoms capping each of the tetrahedron faces  and 118.71 (2)°; L = SNO or acetonitrile]. The internuclear Cu⋯Cu distances vary between 2.5803 (10) and 2.8150 (11) Å (Table 1I) are indicative of weak to moderately strong cuprophilic interactions between Cu atoms in the tetrahedron. The Cu1—S and Cu2—N distances in II (Table 1I as a result of the increase from two μ2-I− to three μ3-I− atoms coordinating to each Cu center. The SNO ligand adopts an envelope conformation with a 47.5 (2)° dihedral angle between the planes defined by atoms C2–C3–C4 and C2–C1–N1–C5–C4.Complex es Fig. 3. The cenÅ Table 1, which  unit formula of [Cu(μ3-I)(SNO)]. The Cu atoms are coordinated by three μ3-I− atoms and one SNO ligand and have distorted tetrahedral geometries [I—Cu—I and I—Cu—S angles of 97.12 (4)–120.62 (4)°] –116.92 (2)°] with two short and one long Cu—I bonds  and 4.5148 (15) Å, which are outside the range of internuclear distances for cuprophilic interactions.Compound °] Fig. 4. The I− s Table 1. The polgs Fig. 6. The Cu⋯III, IV crystallizes with layered two-dimensional polymeric sheets with the symmetry-independent unit formula [Cu(μ2-I)(μ2-SNS)]2 2 rhombi where the center of each rhombus resides on a crystallographic inversion center. Thus, the symmetry-independent unit is best described as containing two structurally distinct [Cu2(μ2-I)2(μ2-SNS)2] half-dimers. The structures of the symmetry-independent Cu2(μ2-I)2 rhombi differ in two notable ways: first, while the Cu2(μ2-I)2 rhombus formed by Cu1, I1, and their symmetry-equivalents is slightly distorted, the rhombus formed by Cu2, I2, and their symmetry-equivalents is near ideal (Table 1ca 0.07 Å . These values are consistent with little to no Cu⋯Cu cuprophilic interaction in the Cu1 dimer while also indicating that there is a weak Cu⋯Cu cuprophilic interaction in the Cu2 dimer. For both half dimers, the Cu atom’s distorted tetrahedral [I—Cu—I angles between 115.06 (3) and 117.46 (3)° and S—Cu—I angles of 96.95 (4)–119.35 (6)°] coordination sphere is filled by two thione moieties from the μ2-SNS ligand; however, only one of these μ2-SNS ligands per Cu atom is symmetry-independent 2(μ2-SNS)] units. The propagation of these rings in the (001) crystallographic plane results in a mesh-like sheet structure, and the layering of these sheets perpendicularly to the (001) plane results in the presence of sizable solvent-accessible voids (ca 200 Å3) in the structure .In contrast to the monodentate SNO ligands in re Fig. 9. These vV also crystallizes as two-dimensional polymeric sheets with the symmetry-independent unit formula [Cu(μ2-I)(μ2-SNS6)]  and 120.16 (7)°]. Whereas the two S—Cu distances are almost identical, the two Cu—I distances are quite different  Å, see Fig. 12V than in III , is a stronger interaction; the second is between atoms N2iv—H2iv⋯I1  and is a weaker interaction (Table 2S(6) hydrogen-bonding motifs  −x + 1, −y + 1, −z + 2]. These hydrogen bonds are relatively strong  x, 1 − y, −z] within the same polymeric sheet. This interaction forms R5) and the phenyl rings with atoms C2i–C3i–C4i–C5i–C6i–C7i (R6) , is of moderate strength . These π–π stacking interactions, in tandem with the increased size of the SNS6 ligand relative to the SNS/SNO ligands, results in a tightly packed two-dimensional sheet (packing coefficient: 71.8%), which prevents the formation of the more mesh-like structure seen in IV (packing coefficient: 54.1%) 2 dimers with two neutral ligands binding with one nitrogen and one sulfur atom resulted in 17 matches. Only one had a homometallic [Cu(μ2-I)2(S)(N)]2 type structure where the S and N donors were part of monodentate ligands, which indicates that the coordination environment in I is a relatively unusual one. This structure, bis[(μ2-iodo)(acetonitrile)(triphenylthiophosphorane)copper(I)] 4 tetrahedrons with a mix of two Cu I3N coordination spheres and two I3S coordination spheres provided only one match, octakis(μ3-iodo)bis{μ2-bis[thio]methane-S,S′}tetrakis(acetonitrile)octacopper(I) acetonitrile tetrahydrofuran solvate 4 tetrahedrons. Two of the Cu centers in each tetrahedron have a (μ3-I)3(NCCH3) coordination sphere. The other two Cu centers have (μ3-I)3S coordination spheres with bridging bis[thio]methane ligands that tether the two tetrahedra together. The geometric parameters of this structure  are very similar to those in II.4(μ3-I) tetrahedra yielded 130 results for Cu4(μ3-I)4(L)4  tetrahedra with L as a neutral ligand. All of the resulting structures had identical first coordination spheres for each of the Cu centers . To the best of our knowledge, II is the first reported instance of a non-polymeric Cu4(μ3-I)4 tetrahedron with N and (non-bridging) S ligands.An additional, broader search for all non-polymeric CuIII: A search for structures containing Cu3(μ3-X)3 ring motifs that did not contain Cu4(μ3-X)4  tetrahedral motifs yielded 60 structures. Four of them contained Cu3(μ3-X)3 motifs and one of them 3 ring motif. This structure, catena-[bis[μ-5-(1-aminoethyl)tetrazolato]tetrakis(μ-iodo)copper(II)tetracopper(I)], contains four monovalent and one divalent symmetry-independent Cu centers that form a one-dimensional ribbon. This ribbon, in combination with the bridging (1S)-1-(5-tetrazolyl) ethylamine ligands, forms a three-dimensional network. There are a few other examples of copper halide extended structures based on Cu3(μ3-X)3 ring motifs that are both one-dimensional ]∞ structure reported by Blake et al. is the only two-dimensional sheet with hexagonal Cu3I3 rings and μ3-triazine linkers. To the best of our knowledge, III is the first instance of this extended hexagonal Cu3I3 structure that is not supported by bridging ligands.IV: A search for polymeric structures containing Cu2(μ2-X)2(μ2-S)  rhombi afforded 91 matches that included 35 polymeric homometallic structures. Among the 35 structures, 23 were two-dimensional polymers. Whereas there were no closely related matches for IV, a similar structure 2 rhombi and two μ2-1,3-dithiane ligands. The geometric parameters of the Cu2(μ2-I)2 rhombi are in good agreement with those in IV .V: A search for structures with Cu—X zigzag chains that did not contain the Cu2(μ2-X)2 rhombus afforded 112 matches. Among these, 56 were for polymeric homometallic structures and three of these [refcodes: AFUDUA . These structures have similar geometries to that of V except for the Cu–ligand distances.et al., 1984The ligands piperidine-2,6-dithione (SNS) and 6-thioxopiperidin-2-one (SNO) were purchased from Sigma–Aldrich and used as received. Isoindoline-1,3-dithione (SNS6) was prepared in a similar manner to that previously described in the literature  in dichloromethane with 10 mL of a colorless solution of CuI (0.502 mmol) in acetonitrile. Upon combination, the solution turned a bright-orange color. Vapor diffusion of the orange solution with diethyl ether afforded large, yellow, block-shaped crystals of I after three days.II) were small, yellow, plate-shaped crystals that co-crystallized with the larger yellow block-shaped crystals of I. The second by-product formed after exposing the initial orange solution from the reaction of 6-thioxopiperidin-2-one and CuI to air, and allowing that solution to slowly evaporate in air for approximately one week. After this time, small, red–orange crystals of III were obtained.Two by-products were also obtained from the reaction of 6-thioxopiperidin-2-one and CuI. The first  in dichloromethane over 10 mL of a colorless solution of CuI (1.00 mmol) in acetonitrile. Dark-red crystals of IV were obtained after one week.V were obtained in a similar manner to IV, with the exception that 1.00 mmol of isoindoline-1,3-dithione was used instead of piperidine-2,6-dithione.Black, needle-shaped crystals of I, the diffraction data were consistent with the space groups P1 and PE-statistics were consistent for the centrosymmetric space group PII–V, a combination of the systematic absences in the diffraction data and the E-statistics were used to assign the centrosymmetric space groups C2/c (II), Pbcn (III), P21/c (IV) and the non-centrosymmetric space group Cc (V).For structure via intrinsic phasing and refined by least-squares refinement on F2 followed by difference-Fourier synthesis. All non-hydrogen atoms were refined with anisotropic displacement parameters. Unless otherwise specified, all hydrogen atoms were included in the final structure-factor calculation at idealized positions and were allowed to ride on the neighboring atoms with relative isotropic displacement coefficients.The structures were solved I, II, and III were refined freely with a distance restraint for each N—H distance.The coordinates of the H atoms bound to N atoms in structures IV, there were three partially occupied solvent molecules of dichloromethane and one partially occupied molecule of acetonitrile present in the asymmetric unit. A significant amount of time was invested in identifying and refining the disordered molecules. Bond-length restraints were applied to model the molecules, but the resulting isotropic displacement coefficients suggested the molecules were mobile. In addition, the refinement was computationally unstable. The SQUEEZE option  and two larger (ca 196 Å3) solvent-accessible voids and is 27% of the unit-cell volume. The program calculated 155 electrons in the unit cell for the diffuse species. This corresponds to approximately one molecule of dichloromethane (42 electrons) that is 50% occupied and one molecule of acetonitrile (22 electrons) in the asymmetric unit. It is very likely that the solvent molecules are disordered over several positions. All derived results in Tables 1In structure Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020009676/zl2791sup1.cifCrystal structure: contains datablock(s) I, II, III, IV, V. DOI: 10.1107/S2056989020009676/zl2791Isup7.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020009676/zl2791IIsup8.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989020009676/zl2791IIIsup9.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2056989020009676/zl2791IVsup10.hklStructure factors: contains datablock(s) IV. DOI: 10.1107/S2056989020009676/zl2791Vsup11.hklStructure factors: contains datablock(s) V. DOI: 2016675, 2016674, 2016673, 2016672, 2016671CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports 10.1038/srep25739, published online 09 May 2016Correction to: -2 hr-1’ should read ‘µg m-2 hr-1’. The correct Figure 2 appears below as Figure This Article contains an error in Figure 2, where the units of the y-axis ‘mg m


Both compounds were characterized using IR, NMR, and single-crystal X-ray analysis.2-Ferrocenylcarbonylbenzoic acid (C 5H5)(C13H9O3)], 1, and 3-ferrocenylphthalide [systematic name: 3-ferrocenyl-2-benzofuran-1(3H)-one], [Fe(C5H5)(C13H9O2)], 2, have been synthesized and structurally characterized by single-crystal X-ray diffraction. The crystal structure of compound 1 was solved recently at room temperature . Here we report a redetermination of its crystal structure at 90 K with improved precision by a factor of about three. The molecular structures of both compounds exhibit a typical sandwich structure. In the crystal packing of compound 1, each molecule engages in intermolecular hydrogen bonding, forming a centrosymmetric dimer with graph-set notation R22 (8) and an O⋯O distance of 2.6073 (15) Å. There are weak C—H⋯O and C—H⋯π interactions in the crystal packing of compound 2. The phthalide moiety in 2 is oriented roughly perpendicular to the ferrocene backbone, with a dihedral angle of 77.4 (2)°.The title compounds, 2-(ferrocenylcarbonyl)benzoic acid, [Fe(C, Y. 2019. CSD Com In both structures, the Fe—C bond lengths towards the substituted carbon are shorter  than the remaining Fe—C bond lengths, similar to literature reports –1.431 (3) Å in 2. Similar to its carboxylate salts . The analogous angle in complex 2 is 3.31°. The Cp rings are essentially parallel in both complexes, making a dihedral angle of 2.45 (12)° in compound 1 and 1.14 (10)° in 2. The Fe⋯Cg distances in both compounds are in a similar range . The Cg—Fe—Cg angle in both structures is ca 178°. The carbonyl carbon, C11 in compound 1 bends toward the iron center with a distance of 0.163 (3) from the least-squares plane of the substituted Cp while the corresponding C11 atom in compound 2 bends slightly outward with a distance of 0.117 (4) Å from the plane of Cp. Similar bending can be seen in the N-imidazolyl derivative of compound 2 °. The phenyl ring in compound 1 is twisted away from the plane of the carbonyl (C=O) plane with a torsional angle O1—C11—C12—C13 of −112.41 (16)°. The aromatic ring of the phthalide moiety in compound 2 bends away from ferrocene and orients roughly perpendicular to the ferrocene backbone. The nine-atom phthalide plane of compound 2 inclines with the substituted Cp at a dihedral angle of 77.31 (7)°. This molecule contains a single asymmetric center at the C11 position in this racemic structure.A view of the molecular structures of the title compounds 1 are associated via classical hydrogen-bonding interactions between the carboxylic OH group of one molecule with the carbonyl oxygen of an adjacent molecule. The carboxylic acid groups are related via a crystallographic inversion center to form hydrogen bonds  with an et al., 19902  Å. There is a weak hydrogen-bonding interaction between the carbonyl oxygen O2 of the phthalide ring, and hydrogen H6 of the unsubstituted Cp with an H6⋯O2  at room temperature has been recently reported as a CSD Communication . To a stirred solution of phthalic anhydride  and AlCl3  in dichloroethane (60 mL), ferrocene  in dichloroethane (65 mL) was added dropwise. The reaction mixture was stirred for 2 h at room temperature, and the mixture poured into ice-cold water (400 mL). The product was extracted with dichloromethane (2 × 250 mL). The organic phase was collected and again extracted with 2 M NaOH (3 × 100 mL). The combined aqueous phase was acidified with conc. HCl until the pH dropped into the 2–3 range. The precipitate was filtered off, washed with water (200 mL), and dried under vacuum to give 1  as a red–brown crystalline solid. Crystals suitable for single-crystal X-ray diffraction were grown by slow evaporation, at ambient temperature, of a solution in a mixture of hexane and diethyl ether. M.p. 457–459 K [Lit. 459 K : 1652 (C=O), 1688 (C=O), 2600–3200 (OH). 1H NMR : δ 4.22  4.53 , 7.62–7.66 , 7.72–7.79 , 7.98 . 13C NMR : δ 70.7, 70.8, 72.9, 81.4 (Cp), 128.9, 130.3, 130.6, 130.7, 133.0, 143.8 (Ar), 167.7 (COOH), 200.1 (CO).3-Ferrocenylphthalide (2). In a 250 mL Schlenk flask, zinc powder  was activated by stirring it in a solution of CuSO4  in DI water (15 mL) for 10 minutes. The solution was decanted, and the residue was washed with water (50 mL). To the activated zinc, keto-acid 1  in NaOH solution (4.80 g in 30 mL of water) was added. The reaction mixture was allowed to reflux for 5 h, and then cooled to room temperature. The reaction mixture was filtered, and the filtrate acidified with conc. HCl. The resulting precipitate was collected, washed with water, and dried to give a viscous mass. The crude product was redissolved in dichloromethane (100 mL) and the acidic impurities extracted with 1 M NaOH (2 × 10 mL). The organic layer was collected, dried with anhydrous MgSO4, filtered, and the filtrate evaporated to dryness to give the title compound 2  as a pale-yellow solid. Crystals suitable for single-crystal X-ray diffraction were grown by slow evaporation, at ambient temperature, of a solution in a mixture of hexane and diethyl ether. M..p: 410–411 K. IR : 1760 (s); 1286 (s); 1068 (s). 1H NMR : δ 4.14 , 4.20 , 4.21 , 4.25 , 4.30 , 6.44 , 7.63 , 7.78–7.84 . 13C NMR : δ 66.7, 66.9, 68.2, 68.9, 79.6, 85.2, 123.4, 125.0, 126.1, 129.4, 134.1, 149.5, 169.5.Uiso(H) =1.2Ueq for the attached C atom. The coordinates of the OH hydrogen atom in 1 were refined with the O—H distance restrained to 0.88 (2) Å, and its Uiso value was assigned as 1.5Ueq of the O atom.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020008452/zl2783sup1.cifCrystal structure: contains datablock(s) 1, 2. DOI: 10.1107/S2056989020008452/zl27831sup2.hklStructure factors: contains datablock(s) 1. DOI: Click here for additional data file.10.1107/S2056989020008452/zl27831sup4.cdxSupporting information file. DOI: 10.1107/S2056989020008452/zl27832sup3.hklStructure factors: contains datablock(s) 2. DOI: 2011817, 2011818CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports 10.1038/s41598-019-42670-4, published online 25 April 2019Correction to: In this Article, the references for Figure 2c, 2d, and 2e are incorrect. As a result, the legend for Figure 2:13C records to regional records of palaeoclimate from key regions in and around Sundaland. (a) Cave stalagmite δ18O records from Borneo23,45. (b) δ13C values from fatty acids from Lake Towuti, Sulawesi39. (c) Guano δ13C record from Palawan23. (d) Aereal extent of Sunda shelf exposure38. (e) Guano δ13C record from Peninsular Malaysia23. (f) Guano (blue) and n-alkane records from Saleh Cave . Calibrated age ranges (2σ) from radiocarbon measurements are plotted. Highlighted area indicates the Last Glacial Maximum.”“Comparison of guano δshould read:13C records to regional records of palaeoclimate from key regions in and around Sundaland. (a) Cave stalagmite δ18O records from Borneo23,45. (b) δ13C values from fatty acids from Lake Towuti, Sulawesi39. (c) Guano δ13C record from Palawan24. (d) Aereal extent of Sunda shelf exposure39. (e) Guano δ13C record from Peninsular Malaysia24. (f) Guano (blue) and n-alkane records from Saleh Cave . Calibrated age ranges (2σ) from radiocarbon measurements are plotted. Highlighted area indicates the Last Glacial Maximum.”“Comparison of guano δ


N-methylated perimidines were prepared and studied by single-crystal X-ray analysis and 1H NMR spectroscopy.A series of unsubstituted, mono- and di- H-perimidine , 1-methyl-2-(pyridin-2-yl)-1H-perimidine , and 1,3-dimethyl-2-(pyridin-2-yl)-1H-perimidinium iodide  were synthesized under mild conditions and their structures were determined by 1H NMR spectroscopy and single-crystal X-ray analysis. The N-methylation of the nitrogen atom(s) at the perimidine moiety results in a significant increase of the interplane angle between the pyridin-2-yl ring and the perimidine system. The unsubstituted perimidine (1) forms a weak intramolecular N—H⋯N bond that consolidates the molecular conformation. In the crystal structures of 1–3, the molecular entities all are assembled through π–π and C—H⋯π interactions.The title compounds, 2-(pyridin-2-yl)-1 In all cases, the organic molecules occupy general positions and comprise an essentially flat perimidine system and the pyridyl ring. Depending on the number of N-substituents, the ring systems are twisted to a greater or lesser extent °, while the molecules of 2 and especially 3 show notably larger interplane angles  because of steric repulsion between the N-methyl group(s) and the pyridin-2-yl ring. The flat conformation of 1 may be stabilized by a weak intramolecular hydrogen bond between the perimidine N1—H1 donor group and the pyridyl N3 acceptor group  whereas in the molecular structure of 2 the pyridyl nitrogen atom participates in a weak intramolecular C12(sp3)—H12A⋯N3 contact . Compound 3 is a salt and its crystal consists of doubly N-methylated perimidinium cations and iodide counter-ions combined mainly through Coulombic interactions.The compositions and structures of the synthesized compounds were determined both by t Figs. 1–3 ▸ ▸. 1H NMR spectroscopic studies of 1–3 revealed correlations between the chemical shifts of some bands in the spectra and the mutual arrangement of the perimidine and pyridyl aromatics. In the 1H NMR spectrum of 1 in CDCl3, doublets at 6.36 and 6.91 ppm arise from the j and e protons, respectively, while the other protons of the perimidine core appear as complex multiplets in the range 7.06–7.25 ppm . A similar set of bands (corresponding to the same protons) with slightly different chemical shifts can be found in the 1H NMR spectrum of 2  whereas 1,3-dimethyl-2-(pyridin-2-yl)perimidinium iodide (3) demonstrates a reduced number of resonance signals  because the protons of the fused benzene rings become equivalent. The latter results from the above arrangement of the pyridyl ring almost orthogonal to the perimidine system.1, solvent-dependent resonance signals in the 1H NMR spectrum were detected. In DMSO-d6 as a solvent , the characteristic doublets arising from the protons j and e are now closer  while the integrated intensity of the signal of the N—H proton becomes lower (0.77 ppm) which may result from a weakening of the intramolecular N—H⋯N hydrogen bond by the polar solvent.For compound 1, molecules are assembled through parallel displaced π–π stacking interactions between the flat pyridyl and perimidine fragments distant by 3.295 (4) Å (C5⋯N1–C11centroid) and 3.302 (4) Å (N2⋯pycentroid), while the resulting offset stacks [centroid-to-centroid shift between the adjacent molecules in the stack 3.791 (4) Å] are grafted together in the resulting three-dimensional network by a C—H⋯π interaction [d(H⋯π) = 2.96 (2) Å] involving the pyridyl H15 atom and the centroid of the C2–C11 ring  Å] between the perimidine units  while the other is a pyridyl–pyridyl contact [distance between the C16 atom and the pyridyl ring 3.499 (3) Å] connecting the stacks together. Intermolecular contacts between the H12C atom and the C6–C11centroid [3.17 (2) Å] and between the H14 atom and C2–C11centroid [3.684 (19) Å] form a three-dimensional network in the crystal structure of 2  Å between the perimidine moieties], which form dense layers via C—H⋯π interactions .Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S205698902100013X/wm5594sup1.cifCrystal structure: contains datablock(s) 1, 2, 3. DOI: 10.1107/S205698902100013X/wm55941sup2.hklStructure factors: contains datablock(s) 1. DOI: Click here for additional data file.10.1107/S205698902100013X/wm55941sup5.molSupporting information file. DOI: 10.1107/S205698902100013X/wm55942sup3.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S205698902100013X/wm55942sup6.molSupporting information file. DOI: 10.1107/S205698902100013X/wm55943sup4.hklStructure factors: contains datablock(s) 3. DOI: Click here for additional data file.10.1107/S205698902100013X/wm55943sup7.molSupporting information file. DOI: Click here for additional data file.10.1107/S205698902100013X/wm55941sup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S205698902100013X/wm55942sup9.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S205698902100013X/wm55943sup10.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S205698902100013X/wm5594sup11.tifFig. S1. NMR spectrum for 1. DOI: Click here for additional data file.10.1107/S205698902100013X/wm5594sup12.tifFig. S2. NMR spectrum for 2. DOI: Click here for additional data file.10.1107/S205698902100013X/wm5594sup13.tifFig. S3. NMR spectrum for 3. DOI: Click here for additional data file.10.1107/S205698902100013X/wm5594sup14.tifFig. S4. NMR spectrum for 1 in DMSO solvent. DOI: 2051714, 2032890, 2032889CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Hydrilla verticillata–sulfur-based heterotrophic and autotrophic denitrification (HSHAD) process was developed in free water surface constructed wetland mesocosms for the treatment of nitrate-rich agricultural runoff with low chemical oxygen demand/total nitrogen (C/N) ratio, whose feasibility and mechanism were extensively studied and compared with those of H. verticillata heterotrophic denitrification (HHD) mesocosms through a 273-day operation. The results showed that the heterotrophic and autotrophic denitrification can be combined successfully in HSHAD mesocosms, and achieve satisfactory nitrate removal performance. The average NO3−-N removal efficiency and denitrification rate of HSHAD were 94.4% and 1.3 g NO3−-N m−3·d−1 in steady phase II (7–118 d). Most nitrate was reduced by heterotrophic denitrification with sufficient organic carbon in phase I (0–6 d) and II, i.e., the C/N ratio exceeded 4.0, and no significant difference of nitrate removal capacity was observed between HSHAD and HHD mesocosms. During phase III (119–273 d), sulfur autotrophic denitrification gradually dominated the HSHAD process with the C/N ratio less than 4.0, and HSHAD mesocosms obtained higher NO3−-N removal efficiency and denitrification rate (79.1% and 1.1 g NO3−-N m−3·d−1) than HHD mesocosms (65.3% and 1.0 g NO3−-N m−3·d−1). As a whole, HSHAD mesocosms removed 58.8 mg NO3−-N more than HHD mesocosms. pH fluctuated between 6.9–9.0 without any pH buffer. In general, HSHAD mesocosms were more stable and efficient than HHD mesocosms for NO3−-N removal from agricultural runoff during long-term operation. The denitrificans containing narG (1.67 × 108 ± 1.28 × 107 copies g−1 mixture-soil−1), nirS (8.25 × 107 ± 8.95 × 106 copies g−1 mixture-soil−1), and nosZ (1.56 × 106 ± 1.60 × 105 copies g−1 mixture-soil−1) of litter bags and bottoms in HSHAD were higher than those in HHD, which indicated that the combined heterotrophic and autotrophic denitrification can increase the abundance of denitrificans containing narG, nirS, and nosZ, thus leading to better denitrification performance. Agricultural runoff always has high nitrate and low chemical oxygen demand (COD)/total nitrogen (TN) (C/N) ratio. Increased nitrate availability in agricultural runoff may lead to serious eutrophication and deterioration of public health in many cases ,2,3. AmoBiological denitrification, occurring either heterotrophically or autotrophically, accounts for 60–90% of the total nitrate reduction ,8. Among+, Cl−, HCO3−, Ca2+, Cu2+, Fe2+, Fe3+, HCO3−, and SO42− on nitrate removal by combined sawdust-based heterotrophic and Fe-based autotrophic denitrification processes  (mg L−1), the NO3−-N concentration after the number n time of NaNO3 dosing 3−[NO-(n+1)bN] (mg L−1), the NO3−-N concentration before the number (n + 1) time of NaNO3 dosingt-test was used to evaluate the differences between the two mesocosms.The statistical analysis was carried out using SPSS software . The paired-sample H. verticillata (H. verticillata (0.17 and 0.23 g g−1 biomass−1) decreased to 0.09 and 0.04 g g−1 biomass–1 in HSHAD mesocosms and 0.15 and 0.03 g g−1 biomass−1 in HHD mesocosms, which indicated that sulfur does not have adverse effect on biomass decomposition. Lignin increased from 0.05 g g−1 biomass−1 to 0.22 g g−1 biomass−1 (HSHAD) and 0.15 g g−1 biomass−1 (HHD), respectively, at the same time  physical leaching and fragmentation, (2) extracellular enzyme hydrolysis, (3) aerobic and anaerobic catabolic activities of heterotrophic microorganisms . The higzed form . Crystalicillata . HemicelH. verticillata is C6H10O5, the stoichiometric equations of H. verticillate heterotrophic and sulfur autotrophic denitrification in the HSHAD process can be listed as Equations (4) and (5), respectively:Assuming the main component of the cellulose and hemicellulose in + will be consumed when 1.0 mol NO3−-N is denitrified by H. verticillata heterotrophic denitrification (H. verticillate pieces as carbon source), and 1.0 mol H+ will be provided when 0.78 mol NO3−-N is reduced to N2 by sulfur autotrophic denitrification. If the mole ratio of nitrate reduction by H. verticillata heterotrophic denitrification to that by sulfur autotrophic denitrification is 1:0.78, the H+ generated by sulfur autotrophic denitrification can be consumed simultaneously by H. verticillata heterotrophic denitrification. Thus, a pH adjustment is not needed. H. verticillata requirement for 1.0 mol NO3−-N reduction and sulfur requirement for 0.78 mol NO3−-N reduction are then determined. Cellulose and hemicellulose of H. verticillata are 0.17 and 0.23 g/g·raw pieces, which account for 40.1% of the pieces’ weight  are difficult to decompose and utilize [H. verticillata to sulfur was applied in this study.Two aspects should be noted for the HSHAD process. The first aspect is its ability to keep pH balanced without a pH buffer. According to Equations (4) and (5), 1.0 mol H’ weight , i.e., t utilize , the 5.3denitrificans and H. verticillata heterotrophic denitrificans grow well in the HSHAD system.The second aspect relates to the enhancement of denitrification performance. Theoretically, the synergistic improvement of the denitrification performance can be achieved if both the sulfur autotrophic −1 for HSHAD and HHD, respectively) in 1 day  was the long-term steady biological decay period, during which COD was released continuously at lower and steady rate from H. verticillata pieces to support the growth of the heterotrophic denitrificans [As depicted in in 1 day a. The rarificans ,50. In pp > 0.05), indicating that there was no significant difference of organic carbon dynamics between HSHAD and HHD mesocosms.The COD variation curve of HSHAD was similar to that of HHD (Four main fluorescence peaks of EEM fluorescence spectra were identified for water samples from different phases . EEM speH. verticillata/sulfur (or H. verticillata) mixture in the experimental mesocosms were gradually matured in Phase II, which corresponded to the steady COD concentration and nitrate removal , while the typical immature components contained tryptophan-like substances and soluble microbial byproduct-like substances (Peak C1 and C2) . The dec removal .H. verticillata attained the decline period.Almost no peak was found after 204 days of operation when the water medium of the mesocosms was displaced by the simulated agriculture runoff on the 204th day in phase III (data are not shown), which corresponded to the low COD concentration (around 10.0 mg/L) and denitrification rate , and it 3−-N removal performance of HSHAD and HHD is shown in 3−-N m−3·d−1 on the second day for HSHAD and 11.9 g NO3−-N m−3·d−1 on the first day for HHD, then it sharply dropped to 1.7 and 1.8 g NO3−-N m−3·d−1, respectively, on the sixth day  was 1.3 times higher than that of HHD mesocosms (0.3 g NO3−-N m−3·d−1).NOixth day b. During3−-N removal value (100.0%) was achieved on the second day. Then, the NO3−-N removal efficiency was maintained above 90.0% until the 118th day. No statistical differences were observed in NO3−-N removal efficiency between HSHAD and HHD mesocosms during phase I and II (p > 0.05)  was still 41.0% higher than that of HHD (28.6%), which might be caused by the combination of sulfur autotrophic denitrification.For both HSHAD and HHD mesocosms, the same maximum NO > 0.05) a, which 3−-N reduction by HHD was calculated using the stoichiometric equation of the heterotrophic denitrification process (Equation (4)). Theoretically, 912.8 mg NO3−-N can be reduced by HHD if cellulose and hemicellulose reduced  was 58.8 mg greater than that by HHD mesocosms (557.8 mg), which was attributed to sulfur autotrophic denitrification.Total NO reduced  in H. vewetlands . Total Ndenitrificans or activated sludge into heterotrophic and autotrophic denitrification systems [denitrificans was difficult to achieve without microbial inoculation probably because of the very different growth rates for both bacterial groups [In general, the HSHAD process was split into two major stages in this study. One stage occurred in the period 0–118 d (phase I and II), during which heterotrophic denitrification dominated at higher C/N ratio (above 4.0); the other stage spanned 119–273 d (phase III), during which sulfur autotrophic dentirification dominated at lower C/N ratio (less than 4.0), which was different from the simultaneous oxidation of sulfur (or thiosulfate) and organic matter by inoculating  systems ,58,59,60 systems , i.e., nl groups . To a gr42− concentrations for HSHAD, HHD, and control mesocosms were analyzed , SOanalyzed . No notaperation  with hig3−-N reduction in HSHAD mesocosms, 443.6 mg SO42− should be produced, based on Equation (5), which was lower than the measured value of 680.2 mg. This phenomenon suggests that SO42− generation did not completely obey the stoichiometric relationship due to sulfite and thiosulfate formation by some intermediate reaction processes [In fact, corresponding to the extra 58.8 mg NOrocesses ,62. 4+-N, NO2−-N and TP during different phases were also analyzed. Average NO2--N concentrations were 1.7 (2.0), 2.9 (2.8) and 1.9 (2.0) mg L–1 for HSHAD (HHD) mesocosms in phase I, II, and III. By comparison with the data for HSHAD, HHD, and control, fast nutrient release from H. verticillata at the beginning (phase I) resulted in a sharp increase in NH4+-N and TP concentrations for both HSHAD and HHD mesocosms, which was similar with former research [4+-N and TP concentrations averaged 1.1 (1.2) and 1.0 (0.9) mg/L for HSHAD (HHD) mesocosms, which demonstrated that the initial NH4+-N and TP accumulation can be successfully removed after phase I. Presumably, the effluent NH4+-N, NO2−-N, and TP can be less likely accumulated if the experiment was operated in a continuous feeding mode.Concentrations of NHresearch ,63,64,653--N removal by sulfur autotrophic denitrification. pH had a closed relation with denitrificans growth, and therefore influenced denitrification performance [denitrificans is 7.0–8.0 [denitrificans is 7.7–8.6 [denitrificans growth  in the mesocosms were calculated using their corresponding genes copies to the weight of the DNA samples, and the average abundance of denitrifying genes with standard errors in bottom soil and in mixture from litter bags for HSHAD, HHD, and control mesocosms on the 273th day are illustrated and characterized in The total abundance of denitrifying genes   was higher than that in HHD mesocosms (1.6 × 106 ± 3.5 × 105 copies g−1 soil−1) (narG genes (3.2 × 108 ± 1.2 × 108 copies g−1 soil−1) in the bottom soil of HSHAD mesocosms was greater than that of HHD mesocosms (1.3 × 108 ± 2.8 × 107 copies g−1 soil−1)  (The xture−1) b. The no soil−1) c. Simila soil−1) a. The naxture−1) a,c.denitrificans genes content of the mesocosms was calculated using all the genes copies to the total weight of the litter bag mixture DNA sample and bottom DNA sample. The results showed that HSHAD could supply more favorable circumstances for the growth of denitrificans containing narG (1.7 × 108 ± 1.3 × 107 copies g−1 mixture-soil−1), nirS (8.2 × 107 ± 8.9 × 106 copies g−1 mixture-soil−1), and nosZ (1.6 × 106 ± 1.6 × 105 copies g−1 mixture-soil−1) than HHD, i.e., 1.0 × 108 ± 1.0 × 107, 4.9 × 107 ± 4.9 × 106 and 1.3 × 106 ± 1.8 × 105 copies g−1 mixture-soil−1, respectively  in control mesocosms were much less than those in HSHAD and HHD mesocosms, which demonstrated the positive role played by the H. verticillata carbon sources in the growth of denitrifying bacteria.The total ectively . It can nosZ genes was lower than that of narG and nirS genes, which was similar to previous studies [nosZ genes catalyzed the reduction of N2O to N2 and enhanced the growth of bacteria containing the nosZ gene, thus reduced the emission of N2O. However, it has been reported that approximately one-third of genome denitrifying bacteria have N2O reductase [The abundance of  studies ,70. The eductase .3−-N removal efficiency was attained because of inadequate influent organic carbon [3−-N removal efficiency (100%). In bioreactors, most HAD processes could achieve above 89.0% NO3−-N removal efficiency with the highest denitrification rate of 5050.0 g NO3−-N/m3·d  as the reported HAD bioreactors (89–100%). The highest denitrification rate of HSHAD (7.0 g NO3−-N m−3 d−1) was close to the double-layer permeable reactive barrier (9.5 g NO3−-N m−3 d−1) and the packed-bed bioreactor (5.0 g NO3−-N m−3·d−1) [3−-N m−3 d−1) [The nitrate removal performance of the HSHAD process was compared to that of other HAD processes, based on literature survey, and the packed fillers and the highest removal capacity of each process are listed in c carbon ,60; howe−-N/m3·d , and HSHm−3·d−1) ,21, but m−3 d−1) ,73,74. DTo effectively treat nitrate-rich agricultural runoff with low C/N ratio, the HSHAD process in free water surface constructed wetland mesocosms was put forward, and its performance was extensively evaluated and compared with that of HHD in mesocosms through a 273-day operation.3−-N reduction during the 0–118 days of operation with 4.0 or higher C/N ratio, while the latter process dominated during 119–273 days of the operation.Heterotrophic and autotrophic denitrification can be combined in the HSHAD process, i.e., the former process mainly dominated NO3−-N removal efficiency and denitrification rate of HSHAD mesocosms were 94.4% and 1.3 g NO3−-N m−3·d−1 in steady phase II (7–118 d). The HSHAD process was much more efficient and stable than the HHD process in the long-term operation. At the end of the experiment, the NO3−-N removal efficiency of HSHAD mesocosms (69.6%) was 41.0% higher than that of HHD mesocosms (28.6%). The rapid increase of NH4+-N, NO2−-N, and TP concentration in the beginning did not affect the HSHAD denitrification performance, and a pH buffer was not necessary for its moderate fluctuation throughout the operation.The average NOH. verticillate pieces heterotrophic and sulfur autotrophic denitrification led to the higher total abundance of denitrificans containing narG (1.67 × 108 ± 1.28 × 107 copies g−1 mixture-soil–1), nirS (8.25 × 107 ± 8.95 × 106 copies g−1 mixture-soil−1), and nosZ (1.56 × 106 ± 1.60 × 105 copies g−1 mixture-soil−1) in the litter bags and bottoms of HSHAD mesocosms than that of HHD, which thus resulted in better denitrification performance.The combination of


The cyclohex-2-one and the isoxazoline rings both exhibit an envelope conformation. The crystal packing features C—H⋯O, C—H⋯N and C—H⋯π interactions.The title compound is a pure diastereoisomer built up from a central five-membered dihydroisoxazole ring substituted in the 3 and 5 positions by a  17H18ClNO2, was prepared and isolated as a pure diastereoisomer, using column chromatography followed by a succession of fractional crystallizations. Its exact structure was fully identified via1H NMR and confirmed by X-ray diffraction. It is built up from a central five-membered dihydroisoxazole ring to which a p-chlorophenyl group and a cyclohex-2-enone ring are attached in the 3 and 5 positions. The cyclohex-2-one and isoxazoline rings each exhibit an envelope conformation. The crystal packing features C—H⋯O, C—H⋯N and C—H⋯π interactions, which generate a three-dimensional network.The title compound, C In the present work, we report the separation, identification by 1H NMR spectroscopy, and X-ray structural analysis of the slightly major diastereoisomer of the isoxazole obtained by the 1,3-dipolar cycloaddition of 4-chlorobenzonitrile oxide with (R)-carvone.In recent years, isoxazole and isoxazoline derivatives have been considered to be good drug candidates because of their broad spectrum of pharmaceutical activities, such as antitumoral  Å and φ2 = 138.1 (11)°. The puckering parameters for the cyclohexene ring, Q = 0.449 (3) Å, θ = 126.0 (4)° and φ = 189.2 (5)°, agree with an envelope conformation on C11. The mean plane of the isoxazole ring makes a dihedral angle of 13.4 (2)° with the C21–C26 benzene ring, whereas it makes a dihedral angle of 66.2 (1)° with the mean plane of the C11–C16 ring.The title compound is built up from a central five-membered dihydroisoxazol ring to which a ly Fig. 1. Atom C1i and C12—H12B⋯O13ii; symmetry codes as in Table 1a-axis direction, while the C—H⋯O interactions make a helical chain along the b-axis direction, forming a layer parallel to the ab plane -3--5-[carbonyl]-5-methyl-4,5-dihydroisoxazole compound -carvone, 1, was reacted with nitrile oxide, 2, generated in situ from the corresponding oxime according to our recently described methodology / diastereoisomeric mixture. The 1H NMR spectrum of 3 clearly shows a splitting of both the methyl and methylene groups in the α position of the newly formed stereogenic center of the isoxazole nucleus . The former gave rise to two singlets at 1.44 ppm and 1.48 ppm, respectively, while the latter is seen as two pairs of doublets, one at 2.90 and 3.20 ppm (J = 16.9 Hz) and the other at 2.75 and 3.30 ppm (J = 16.7 Hz). Integrating the corresponding 1H NMR signals allowed us to quantify the ratio of the diastereoisomereric mixture as 58:42. After several attempts at separation, either by column chromatography or a series of fractional crystallizations by slow evaporation from a chloroform solution of 3, we managed to separate the diastereoisomer 3a, the title compound, as pure single crystals suitable for crystallographic analysis. Its 1H NMR spectrum  is mainly characterized by the isoxazolic methyl group resonating as a singlet at 1.44 ppm, and the methylene group appeared as two doublets at 2.90 ppm (J = 16.9 Hz) and 3.20 ppm (J = 16.9 Hz).As shown in Fig. 3us Fig. 4a. The fum Fig. 4b is maiUiso(H) = 1.2Ueq(C) for methylene and methine or Uiso(H) = 1.5Ueq(C) for methyl H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989020001991/is5530sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989020001991/is5530Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020001991/is5530Isup3.cmlSupporting information file. DOI: 1983547CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


With the discovery of ScoE, a mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase from Streptomyces coeruleorubidus, a second route was identified. ScoE forms isonitrile from a glycine adduct, with both the nitrogen and carbon atoms coming from the same glycyl moiety. This reaction is part of the nonribosomal biosynthetic pathway of isonitrile lipopeptides. Here, we present structural, biochemical, and computational investigations of the mechanism of isonitrile formation by ScoE, an unprecedented reaction in the mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase superfamily. The stoichiometry of this enzymatic reaction is measured, and multiple high-resolution (1.45–1.96 Å resolution) crystal structures of Fe(II)-bound ScoE are presented, providing insight into the binding of substrate, (R)-3-((carboxylmethyl)amino)butanoic acid (CABA), cosubstrate α-ketoglutarate, and an Fe(IV)=O mimic oxovanadium. Comparison to a previously published crystal structure of ScoE suggests that ScoE has an “inducible” α-ketoglutarate binding site, in which two residues arginine-157 and histidine-299 move by approximately 10 Å from the surface of the protein into the active site to create a transient α-ketoglutarate binding pocket. Together, data from structural analyses, site-directed mutagenesis, and computation provide insight into the mode of α-ketoglutarate binding, the mechanism of isonitrile formation, and how the structure of ScoE has been adapted to perform this unusual chemical reaction.The isonitrile moiety is found in marine sponges and some microbes, where it plays a role in processes such as virulence and metal acquisition. Until recently only one route was known for isonitrile biosynthesis, a condensation reaction that brings together a nitrogen atom of  Furthermore, a study of the gene cluster from Streptomyces coeruleorubidus showed that isonitrile biosynthesis is catalyzed by the enzyme ScoE . The. The1). hosphate  2). Rec. Recl-Tryme ScoE . Notablyyme ScoE  Fig. 1)l-Trp/l-Tyme ScoE .Figure 1scoE gene encodes a nonheme iron(II) α-ketoglutarate (α-KG)-dependent dioxygenase (Fe(II)/αKG-dioxygenase) -3-carboxyaminobutanoic acid (CABA), to form an untethered product, (R)-3-isocyano butanoic acid (INBA)  . This prd (INBA)  4). Thi. ThiscoEd (INBA) . NotablyIndependent of the particular reaction, all Fe(II)/αKG-dioxygenases are thought to share a common mechanism of activation  5). Thi. Thi5). Streptomyces thioluteus based on a 13C-NMR spectrum obtained by using a 5-13C-CABA substrate (in vitro to determine whether it can be directly converted to INBA by ScoE. Regardless of the site(s) of hydroxylation, these mechanistic proposals would require two equivalents of α-KG and two equivalents of molecular oxygen. Chang and colleagues found that 4.5 equivalents of succinate were produced for every equivalent of isonitrile product generated by ScoE and suggested that this unexpectedly high succinate-to-isonitrile ratio could be due to uncoupling of α-KG cleavage from INBA production , showing that it is consistent with this family of enzymes . The strS. coeruleorubidus was purified heterologously from Escherichia coli, with the Fe(II) cofactor reconstituted in vitro and used to determine reaction stoichiometry. Product INBA formation was indirectly detected and quantified using 3,6-di-2-pyridyl-1,2,4,5-tetrazine (Py-tetrazine), which reacts with INBA to generate 3,5-di(pyridine-2-yl)-1H-pyrazol-4-amine (Py-aminopyrazole)  . No freeyrazole) . Enzymatyrazole) .The results from these stoichiometry quantification experiments show the expected 1:1:1 correlation between α-KG consumption, oxygen consumption, and succinate formation . CABA co2 begins . The release of [13C]-CO2 as a product of the ScoE catalyzed INBA formation was confirmed . Th. Th13C]-ng GC-MS . The + 163.0794, found: 163.0794 (0 ppm error). 1H NMR  δ 3.77 , 3.51 , 2.81 , 2.48 , 1.23 ; 13C NMR  δ 171.70, 168.51, 50.16, 49.94, 45.23, 16.30.In general, the synthesis of CABA was followed as previously reported . BromoacBA-6-13C  synthesiBA-6-13C . The pro 2Fe(SO4)2 was conducted in a sealed reaction chamber with an integrated oxygen electrode unit . The instrument was calibrated with 0.5 ml of 50 mM HEPES pH 8.0 at room temperature and a mixing rate of 50 RPM. The assay components were added to the reaction chamber except for the iron. The oxygen signal was allowed to equilibrate before addition of 90 μM (NH4)2Fe(SO4)2 with an airtight needle to initiate the reaction. Oxygen consumption was collected as a function of time, and the reaction was allowed to incubate until the profile reached a steady state. The difference between the starting and final oxygen concentrations was used to estimate O2 consumption. Two 100 μl aliquots of the reaction mixture were quenched with 200 μl of cold methanol and 200 μl of 667 μM 3,6-di(pyridine-2-yl)-1,2,4,5-tetrazine dissolved in cold methanol, respectively for coupled stoichiometry quantification experiments.To investigate Oin vitro assay containing the same reaction components as mentioned previously was quenched with 200 μl of 667 μM 3,6-di(pyridine-2-yl)-1,2,4,5-tetrazine dissolved in cold methanol, gently mixed, and incubated for 1 h at room temperature. The reaction mixture was vortexed briefly and centrifuged to remove aggregated protein. In total, 100 μl standards containing 50 mM HEPES pH 8, 500 μM CABA, 250 μM α-KG, and varying concentrations of 3,5-di(pyridine-2-yl)-1H-pyrazol-4amine (Py-aminopyrazole) were quenched using the same quench method to develop a standard curve using LC-HRMS. LC-HRMS analysis was performed on an Agilent Technologies 6545 Accurate-Mass Q-TOF LC-MS instrument and an Eclipse Plus C18 column (100 x 4.6 mm). Chromatography was performed using a linear gradient of 10 to 50% acetonitrile (vol/vol) with 0.1% formic acid over 12 min in water with 0.1% formic acid (vol/vol) at a flow rate of 0.5 ml/min. A Py-aminopyrazole standard was synthesized and characterized by NMR as previously described . A standard curve was used to quantify the production of INBA.A ScoE escribed  and was in vitro assay containing the same reaction components as mentioned previously was quenched with cold methanol, gently mixed, vortexed, and centrifuged to remove the aggregated protein. In total, 100 μl standards containing 50 mM HEPES pH 8 and varying concentrations of CABA, α-KG, and succinate were quenched with 200 μl of cold methanol to develop a standard curve using LC-HRMS. LC-HRMS analysis was performed on an Agilent Technologies 6545 Accurate-Mass Q-TOF LC-MS instrument and an Eclipse Plus C18 column (100 × 4.6 mm). Chromatography was performed using a linear gradient of 10 to 50% acetonitrile (vol/vol) with 0.1% formic acid over 12 min in water with 0.1% formic acid (vol/vol) at a flow rate of 0.5 ml/min. Extracted ion chromatograms (EICs) for CABA, α-KG, and succinate were generated using the following m/z: [M + H]+=162.0761 for CABA, [M − H]−=145.0142 for α-KG, and [M − H]−=117.0193 for succinate. The following m/z values were observed: [M + H]+=162.0763 for CABA (1.2 ppm error), [M − H]−=145.0142 for α-KG (0 ppm error), and [M − H]−=117.0193 for succinate (0 ppm error). Standard curves for CABA, α-KG, and succinate were utilized to quantify their respective consumption and productionA ScoE 2O2, the 0.5 ml ScoE enzymatic assay mentioned earlier was conducted with the same oxygen electrode unit. After the concentration of oxygen reached equilibrium, 120 U/ml of catalase was added to the reaction chamber. No appreciable increase of oxygen was observed when compared with adding solely water or buffer, indicating that no H2O2 was present in the reaction mixture. In addition, H2O2 was again undetected using the Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit by following the manufacturer’s instructions.To detect Hin vitro assay containing 50 mM HEPES pH 8, 500 μM CABA, 250 μM α-KG, 100 μM Apo-ScoE, and 90 μM (NH4)2Fe(SO4)2 was performed and incubated at room temperature for 10 min. This assay was adapted from previous work . Additionally, a Sigma-Aldrich Formate Assay Kit was utilized according to the manufacturer’s protocol, resulting in no detectable amounts of formate.A 200 μl in vitro assay containing 50 mM HEPES pH 8, 500 μM CABA, 250 μM α-KG, 100 μM Apo-ScoE, and 90 μM (NH4)2Fe(SO4)2 was performed and incubated at room temperature for 10 min in a sealed septum. This assay was adapted from previous work 2Fe(SO4)2, 2 mM (8 or unlabeled), 1 mM α-KG , and 100 μM ScoE. The reaction was performed in a 10-ml sealed headspace vial (Agilent) and initiated by adding the iron. One milliliter of the headspace gas was acquired using a gastight syringe (Hamilton) and injected into Agilent 5977A GCMS system equipped with a HP-5 ms column. Injector temperature was set at 120 °C, and helium was used as the carrier gas at a flow rate of 3 ml/min. The temperature gradient was as follows: 40 to 100 °C over 5 min. The mass spectrometer was operated in electron ionization mode with automatically tuned parameters, and the acquired mass range was m/z = 15 to 100. The CO2 and CO signals were confirmed using authentic standards, and the production of 13CO2 and 13CO was determined by extracting m/z = 45 and 29, respectively.For the detection of CO2; Coy Laboratory Products, Inc), following optimization of the previously published precipitant solution -ScoE with an off-site α-KG, were grown by the sitting drop vapor diffusion technique at 24 °C in an anaerobic environment -3-((carboxymethyl)amino)butanoic acid (CABA)-Fe(II)-ScoE, crystals that were grown as described above were transferred into 2 μl of a soaking solution containing 205 mM sodium acetate, 100 mM Tris pH 8.5, 24% (w/v) PEG 4,000, 1 mM CABA, and 250 μM Fe(II)Cl2 and lacking α-KG at 24 °C in an anaerobic environment .To obtain the structure of -ScoE with off-site α-KG.To obtain a structure of CABA-Fe(II)-ScoE with α-KG, crystals that were grown as described above were transferred into 2 μl of soaking solution containing 205 mM sodium acetate, 100 mM Tris pH 8.5, 24% (w/v) PEG 4,000, 1 mM CABA, 1 mM α-KG, and 250 μM Fe(II)Cl2.To obtain the structure of CABA-oxovanadium-ScoE, ScoE was cocrystallized with CABA and oxovanadium using the sitting drop vapor diffusion technique at 24 °C in aerobic conditions. A 1 μl aliquot of protein solution  sulfate) was added to 2 μl of precipitant solution  PEG 4000). Crystals formed within 1 week and reached a maximum size after approximately 2 weeks. Paraffin oil was used as a cryoprotectant for crystal harvesting. Crystals were manually looped and streaked through paraffin oil then flash-frozen in liquid Nmerge, CC1/2, I/σ, and data completeness in the highest resolution bin.Data for structures of CABA-Fe(II)-ScoE; and CABA-Fe(II)-ScoE with off-site α-KG; Fe(II)-ScoE with off-site α-KG were collected at the Advanced Photon Source  on beam line 24ID-C using a Pilatus 6M pixel array detector at a temperature of 100 K at a wavelength of 0.9791 Å in a single 180° wedge and were processed, indexed, integrated, and scaled in XDS . Data fofree calculations were chosen to be comprised of the same reflections as were used for refinement of the previously published crystal structure of ScoE for all data sets. Initial rounds of refinement included simulated annealing to reduce model bias from molecular replacement. Iterative rounds of model building were performed in Coot -oxo intermediate with succinate bound using Avogadro  and PyMOTo obtain further different configurations of Arg157, PyMOL’s mutagenesis tool was employed to generate different rotamers of Arg157 residue, starting with CS1. PyMOL found 22 backbone-dependent and 81 backbone-independent rotamers for Arg157 (and none for His299). Based on visual inspection and closest distance between the tail of succinate and side chain of Arg157, three backbone-dependent  and six backbone-independent  rotamers were selected for collecting dynamics .For nonstandard residues (succinate and CABA), we use the generalized AMBER force field (GAFF)  with res+ counterions for a total simulation of 42,923 atoms (4741 protein/substrate atoms).The protein structures were solvated in a periodic rectangular prism box with at least a 10 Å buffer of TIP3P  water an−1 and a random seed, and iii) 1-ns NpT equilibration using the Berendsen barostat with a pressure relaxation time of 2 ps. Production dynamics were collected for at least 250 ns for each structure  and His299 (the nitrogen atoms on sidechain ring). The minimum of the former four distances was labeled dR157 and the minimum of the latter four distances was labeled dH299  under the following accession codes: 6XN6 (CABA-Fe(II)-ScoE), 6XO3 (Fe(II)-ScoE with off-site α-KG), 6XOJ (CABA-Fe(II)-ScoE with off-site α-KG), and 6XPA (CABA-oxovanadium-ScoE).Atomic coordinates and structure factors have been deposited in the Protein Data Bank (The authors declare that they have no conflicts of interest with the contents of this article.


Psoriasis is a common chronic inflammatory skin disease. Keratinocytes hyperproliferation and excessive inflammatory response contribute to psoriasis pathogenesis. The agents able to attenuate keratinocytes hyperproliferation and excessive inflammatory response are considered to be potentially useful for psoriasis treatment. Daphnetin exhibits broad bioactivities including anti-proliferation and anti-inflammatory. This study aims to evaluate the anti-psoriatic potential of daphnetin in vitro and in vivo, and explore underlying mechanisms.HaCaT keratinocytes was stimulated with the mixture of IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α (M5) to establish psoriatic keratinocyte model in vitro. Cell viability was measured using Cell Counting Kit-8 (CCK-8). Quantitative Real-Time PCR (qRT-PCR) was performed to measure the mRNA levels of hyperproliferative marker gene keratin 6 (KRT6), differentiation marker gene keratin 1 (KRT1) and inflammatory factors IL-1β, IL-6, IL-8, TNF-α, IL-23A and MCP-1. Western blotting was used to detect the protein levels of p65 and p-p65. Indirect immunofluorescence assay (IFA) was carried out to detect p65 nuclear translocation. Imiquimod (IMQ) was used to construct psoriasis-like mouse model. Psoriasis severity  was scored based on Psoriasis Area Severity Index (PASI). Hematoxylin and eosin (H&E) staining was performed to examine histological change in skin lesion. The expression of inflammatory factors including IL-6, TNF-α, IL-23A and IL-17A in skin lesion was measured by qRT-PCR.Daphnetin attenuated M5-induced hyperproliferation in HaCaT keratinocytes. M5 stimulation significantly upregulated mRNA levels of IL-1β, IL-6, IL-8, TNF-α, IL-23A and MCP-1. However, daphnetin treatment partially attenuated the upregulation of those inflammatory cytokines. Daphnetin was found to be able to inhibit p65 phosphorylation and nuclear translocation in HaCaT keratinocytes. In addition, daphnetin significantly ameliorate the severity of skin lesion  in IMQ-induced psoriasis-like mouse model. Daphnetin treatment attenuated IMQ-induced upregulation of inflammatory cytokines including IL-6, IL-23A and IL-17A in skin lesion of mice.Daphnetin was able to attenuate proliferation and inflammatory response induced by M5 in HaCaT keratinocytes through suppression of NF-κB signaling pathway. Daphnetin could ameliorate the severity of skin lesion and improve inflammation status in IMQ-induced psoriasis-like mouse model. Daphnetin could be an attractive candidate for future development as an anti-psoriatic agent. Psoriasis is a common chronic inflammatory skin disease affecting 2–3% of the population worldwide . It is cNF-κB signaling, a crucial pathway to regulate a variety of cellular processes, including proliferation and inflammation , was fouDaphne odora var., exhibits broad bioactivities including anti-proliferation and anti-inflammation [Daphnetin , a natural coumarin compound isolated from ammation , 12. Dapammation . Daphnetammation . Daphnetammation . Daphnetammation . We haveammation . HoweverThe cocktail of IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α (M5) cytokines induces keratinocytes manifesting some features of psoriatic keratinocyte in vitro . TherefoThe cell viability of HaCaT keratinocytes treated with daphnetin was measured using CCK-8 assay. No significant toxicity was observed at concentrations below 20 μM after treatment with daphnetin for 96 h. Daphnetin at concentrations above 40 μM significantly decreased cell viability of HaCaT keratinocytes Fig. . TherefoThe mixture of IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α (M5) cytokines were used to simulate HaCaT keratinocytes to establish psoriatic keratinocyte model recapitulating some features of psoriasis in vitro –20. M5 . These rIn order to evaluate the effects of daphnetin treatment on M5-stimulated HaCaT keratinocytes, CCK8 was used to detect cell viability. The result showed that M5 stimulation for 72 h and 96 h significantly promoted proliferation of HaCaT keratinocytes. However, M5-induced hyperproliferation at 72 h and 96 h was attenuated in the presence of daphnetin Fig. a. M5 stiNF-κB signaling pathway plays crucial roles in the pathogenesis of psoriasis . In ordeTo evaluate the effects of daphnetin treatment on IMQ-induced psoriasis-like mouse model, mice was treated with topical daphnetin. The symptoms of erythema and scaling were induced after IMQ treatment. Vehicle cream treatment could not improve those symptoms. However, compared with vehicle group, topical application of daphnetin ameliorated the skin condition Fig. a. The seHistopathological results showed that daphnetin inhibited inflammatory cell infiltration to the dermis as compared with vehicle cream treatment Fig. d, g, sugPsoriasis is a complicated inflammatory skin disease mediated by various kinds of cells, including keratinocytes, T cells, endothelial cells, macrophages and dendritic cells . KeratinDaphne odora var., with broad pharmacological activities, including anti-proliferation and anti-inflammatory [Daphnetin is a natural coumarin compound isolated from ammatory , 17, 30.ammatory –20. In tIn order to investigate the effects of daphnetin on the inflammatory response of HaCaT keratinocytes, qRT-PCR was performed to measure the expression of cytokines IL-1β, IL-6, IL-8, TNF-α, IL-23A and MCP-1. IL-1β, IL-6 and TNF-α are important proinflammatory cytokins, especially TNF-α, involving in psoriasis pathogenesis , 32. TheNF-κB signaling pathway regulates a variety of cellular processes, including proliferation and inflammation . It has We further evaluated the effects of daphnetin on IMQ-induced psoriasis-like mouse model. IMQ, an agonist of Toll-like receptor 7/8 ligand, was found to be able to induce a dermatitis in mice closely resembling human psoriasis . Thus, IIn conclusion, daphnetin was able to attenuate proliferation and inflammatory response induced by M5 cytokines in HaCaT keratinocytes. These biological effects of daphnetin on HaCaT keratinocytes were associated with NF-κB signaling pathway inactivation. Moreover, daphnetin could ameliorate the severity of skin lesion and improve inflammation status in IMQ-induced psoriasis-like mouse model. These results indicated that daphnetin possesses anti-psoriatic potential, which make it an attractive candidate for future developing as an anti-psoriatic agent.DMSO (dimethyl sulfoxide) and Hoechst dye 33,258 for nucleus staining were purchased from Sigma-Aldrich . Daphnetin  was obtained from PharmaBlock Sciences  and dissolved in DMSO. Carbopol 940 and azone were purchased from Rhawn . IMQ cream was obtained from Sichuan Med-Shine Pharmaceutical Co., LTD . IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α cytokines were purchased from PeproTech . Cell Counting Kit-8 (CCK-8) was purchased from Dojindo . TRIzol™ reagent and primers were obtained from Invitrogen . RevertAid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher Scientific (USA). SYBR Green PCR Master Mix kit was purchased from TaKaRa (Japan). Rabbit anti-p65, anti-p-p65 (Ser536), anti-β-actin primary antibodies and Alexa Fluor 555-labeled anti-rabbit secondary antibody were purchased from Cell Signaling Technology .2 at 37 °C. Psoriasis-like keratinocytes model was established by adding M5 cocktail cytokins  into medium of HaCaT keratinocytes.Human HaCaT keratinocytes were obtained from Kunming Cell Bank of Type Culture Collection, Chinese Academy of Science . Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and cultured in incubator with 5% CO2 for 2 h. Absorbance value at 450 nm was measured using Multiskan Spectrum  and was directly proportional to the number of living cells.CCK-8 was used to measure cell viability. HaCaT keratinocytes were plated in 96-well plates and cultured for 24 h. Cells at 50–60% confluence were treated with daphnetin or M5 cytokines for 24–96 h. 10 μl of CCK-8 solution was added to each well and incubated at 37 °C with 5% COHaCaT keratinocytes were seeded in 6-well plates and cultured for 24 h. Cells at 60–80% confluence were treated with daphnetin (20 μM) for 24 h, and then stimulated with M5 (5 ng/ml) for 30 min. Cells was harvested and lysed with cell lysis buffer  containing 1 mM phenylmethyl-sulfonylfluoride (PMSF) for 20 min on the ice. Cell lysates were centrifuged for 15 min at 14,000 rpm. The concentration of supernatant protein was determined using BCA kit . About 15 μg of protein was separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinyl difluoride (PVDF) membranes, which subsequently were blocked using 5% nonfat milk at room temperature for 2 h. Next, the membranes were incubated with anti-p65 (1:1000), anti-p-p65 (1:1000) and anti-β-actin (1:1000) primary antibodies overnight at 4 °C. After being washed with TBS-T for three times, the membranes were incubated with HRP-conjugated secondary antibody at room temperature for 2 h. Finally, the membranes were incubated with chemiluminescence substrate  and transferred to ChemiDoc™ XRS + System  for visualizing of protein signals. The intensity of protein bands was measured using ImageJ software. β-actin served as an internal reference.HaCaT keratinocytes were plated onto coverslips and treated with daphnetin for 24 h, then stimulated with M5 cytokines for 30 min. Cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min. After being washed with PBS for three times, the cells were permeabilized with PBS containing 0.5% Triton X-100 for 15 min, and subsequently were blocked with PBS containing 1% bovine serum albumin (BSA) at room temperature for 30 min. Next, the coverslips were incubated with rabbit anti-p65 (1:500) primary antibody overnight at 4 °C. After being washed with PBS for three times, the coverslips were incubated with Alexa Fluor 555-labeled anti-rabbit secondary antibody (1:1000) for 2 h. Hoechst dye 33,258 was used to stain nucleus for 4 min. After that, the coverslips were washed with PBS for three times and mounted onto the microscope slides. Immunofluorescence was observed using Nikon Eclipse Ti-U fluorescent microscope.Female specific pathogen-free (SPF) BALB/c mice (6–8 weeks) were purchased from Hunan SJA Laboratory Animal Co., Ltd. . Mice were housed at constant levels of temperature and humidity with a 12-h light/dark cycle and allowed free access to food and water. All experiment protocols were approved by the Ethical Committees of Guilin Medical University .The composition of daphnetin gel (containing 1% daphnetin) is as follows: daphnetin 0.1 g, carbopol 940 0.2 g, azone 0.1 g, ethanol (96%) 3 g and distilled water q.s. to 10 g , 46. The2 daphnetin cream or vehicle cream (without daphnetin) was applied twice daily for 7 consecutive days [Mice were randomly divided into four groups (n = 6 per group): Vaseline group (Ctl), IMQ group (IMQ), IMQ + vehicle group (IMQ+Vehicle) and IMQ + daphnetin group (IMQ+Dap). A daily topical dose of 62.5 mg of IMQ cream or vaseline was used on the back skin of the mice for 7 consecutive days. A dose of 50 mg/cmive days . Erythem2 per sample.Skin tissues of mice were fixed in 4% paraformaldehyde solution and embedded in paraffin. The paraffin-embedded tissues were sectioned and stained with hematoxylin and eosin (H&E) for histological evaluation. Epidermal thickness was calculated by Image-pro Plus 6.0 software. The number of infiltrating cells in dermis was calculated with 5 randomly selected areas coverage of 0.04 mmHaCaT keratinocytes were seeded in 12-well plates and treated with daphnetin  or not for 2 h, and subsequently stimulated with M5 cytokines (2.5 ng/ml) or not for 24/72 h. Cells were harvested and RNA was extracted for qRT-PCR analysis of KRT6, IL-1β, IL-6, IL-8, TNF-α, IL-23A and MCP-1 mRNA levels. As shown in Fig. −ΔΔCt method [KRT6, 5′-GGGTTTCAGTGCCAACTCAG-3′ (forward) and 5′-CCAGGCCATACAGACTGCGG-3′ (reverse), 146 bp (product size);KRT1, 5′-CTTTTCTGCTGTTTCCCAATGAA-3′ (forward) and 5′-GGAAAGAACAAAGCAGGGTCATAG-3′ (reverse), 80 bp (product size);IL-1β, 5′-ATGATGGCTTATTACAGTGGCAA-3′ (forward) and 5′-GTCGGAGATTCGTAGCTGGA-3′ (reverse), 132 bp (product size);IL-6, 5′-ACTCACCTCTTCAGAACGAATTG-3′ (forward) and 5′-CCATCTTTGGAAGGTTCAGGTTG-3′ (reverse), 149 bp (product size);IL-8, 5′-ACTGAGAGTGATTGAGAGTGGAC-3′ (forward) and 5′-AACCCTCTGCACCCAGTTTTC-3′ (reverse), 112 bp (product size);TNF-α, 5′-CCTCTCTCTAATCAGCCCTCTG-3′ (forward) and 5′-GAGGACCTGGGAGTAGATGAG-3′ (reverse), 220 bp (product size);IL-23A, 5′-CTCAGGGACAACAGTCAGTTC-3′ (forward) and 5′-ACAGGGCTATCAGGGAGCA-3′ (reverse), 119 bp (product size);MCP-1, 5′-CAGCCAGATGCAATCAATGCC-3′ (forward) and 5′-TGGAATCCTGAACCCACTTCT-3′ (reverse), 190 bp (product size);GAPDH, 5′-CACATGGCCTCCAAGGAGTAA-3′ (forward) and 5′-TGAGGGTCTCTCTCTTCCTCTTGT-3′ (reverse), 75 bp (product size).Total RNA was extracted from HaCaT keratinocytes or mice skin tissue using TRIzol™ reagent according to the manufacturer’s instruction. First strand of cDNA was synthesized with 2 μg of total RNA using RevertAid First Strand cDNA Synthesis Kit. Quantitative Real-Time PCR (qRT-PCR) was performed with SYBR Green PCR Master Mix kit in CFX96 Touch™ Real-Time PCR Detection System . The qRT-PCR reaction procedure was 10 s for denaturation at 95 °C, 10 s for annealing at 60 °C and 10 s for extension at 72 °C, with 40 cycles in total. GAPDH served as internal reference. The relative expression was analyzed by the 2t method . PrimersKRT6, 5′-CTGTGAGTTTCTAATGGCCTGAGA-3′ (forward) and 5′-GAAACTTACATCACAGGACCAGTGA-3′ (reverse), 102 bp (product size);IL-6, 5′-CCTCTCTGCAAGAGACTTCCAT-3′ (forward) and 5′-AGTCTCCTCTCCGGACTTGT-3′ (reverse), 95 bp (product size);TNF-α, 5′-ATCCGCGACGTGGAACTG-3′ (forward) and 5′-ACCGCCTGGAGTTCTGGAA-3′ (reverse), 70 bp (product size);IL-23A, 5′-TCCTCCAGCCAGAGGATCACCC-3′ (forward) and 5′-AGAGTTGCTGCTCCGTGGGC-3′ (reverse), 160 bp (product size);IL-17A, 5′-CCTCACACGAGGCACAAGTG-3′ (forward) and 5′-CTCTCCCTGGACTCATGTTTGC-3′ (reverse), 68 bp (product size);GAPDH, 5′-AGCTTGTCATCAACGGGAAG-3′ (forward) and 5′-TTTGATGTTAGTGGGGTCTCG-3′ (reverse), 62 bp (product size).Primers sequences targeting genes in mice were as follows:t test. P value < 0.05 was considered statistically significant.Data were presented as mean ± standard error of mean (SEM) from at least three independent experiments. GraphPad Prism software was used for statistical analysis. Statistical significance analysis was performed using Student’s


In the crystal, molecules are linked by pairs of N—H⋯S hydrogen bonds, forming inversion dimers with an  7H5FN4S, the molecules are non-planar, with dihedral angle formed by least-squares planes of tetrazole and benzene rings of 59.94 (8) °. The crystal packing is formed by N—H⋯S hydrogen bonds, which link the molecules into centrosymmetric dimers with an R22(8) ring motif, and by the offset face-to-face π–π stacking interactions between the benzene rings, which join the dimers into layers parallel to (100). The Hirshfeld surface analysis shows that the most important contributions to the surface contacts are from N⋯H/H⋯N (21.9%), S⋯H/H⋯S (21.1%), H⋯H (14.6%), F⋯H/H⋯F (11.8%) and C⋯H/H⋯C (9.5%) interactions.In the crystal of the title compound, C The most common synthetic approach to construct tetrazoles, based on the reaction of nitriles with hydrazoic acid, was first discovered by Hantzsch & Vagt 1901. Up to ket al., 2019As a result of the considerable interest in this field, significant developments in the synthesis of tetrazoles have been attained, which were recently reviewed (Neochoritis H-tetrazole ring (N1–N4/C5) is essentially planar, with a largest deviation of 0.005 (1) Å for N3. The dihedral angle between the mean planes of the tetrazole and benzene rings is 59.94 (8)°. The bond dimensions are typical of similar compounds, with a distinct N2=N3 double bond.The molecule of the title compound Fig. 1 is non-pc glide plane. The hydrogen bonds and stacking interactions link the molecules into layers parallel to (100). Other short intermolecular contacts are collected in Table 2In the crystal, molecules are linked by pairs of N—H⋯S hydrogen bonds, forming centrosymmetric dimers with an et al., 2007CrystalExplorer17  are the major contributors to the Hirshfeld surface, while H⋯H (14.6%), F⋯H/H⋯F (11.8%) and C⋯H/H⋯C (9.5%) contacts make a less significant contribution. The contribution of the C⋯C (6.6%) (i.e. π–π stacking) contacts and other contacts such as N⋯N (2.8%), F⋯C/C⋯F (2.4%), N⋯C/C⋯N (2.4%), F⋯N/N⋯F (1.7%), S⋯N/N⋯S (1.7%), S⋯C/C⋯S (1.7%), F⋯F (1.5%) and S⋯S (0.4%) make a small contribution to the overall Hirshfeld surface.In order to investigate the intermolecular interactions in the crystal structure of the title compound in a visual manner, Hirshfeld surfaces -4,4,6-trimethyl-3,4-dihydropyrimidine-2(1H)-thione -thione -1H-1,2,4-triazol-5-yl)benzamide (2-fluorophenyl)methanone -5--2H-1,2,3-triazole -2-methyl-5--2H-1,2,3-triazole -3-(4-fluorophenyl)-1-prop-2-en-1-one -4H-1,2,4-triazole  interactions, forming supramolecular chains extending along the In the crystal structure of MITMOU, the supramolecular assembly is formed mainly by —H⋯ hydrogen-bond interactions. Initially, strong N—H⋯N hydrogen bonds link pairs of inversion-related molecules that act as slabs of infinite chains running along the [100] direction connected by a C—H⋯O hydrogen bond. Along the [010] direction, neighbouring chains are further connected by weak π–π interactions between two arene rings of adjacent molecules.The crystal structure of MITMIO is built by a combination of strong N—H⋯O and N—H⋯N hydrogen bonds, which form chains of molecules running along the [100] direction. Parallel inversion-related chains of molecules are further connected by weaker C—H⋯O interactions to build the molecular architecture along the [001] direction. Weak C—H⋯N interactions connect the molecules in order to complete the three-dimensional structure along the [010] direction.N-methylation of the PONWIA molecule, no hydrogen-bonding interactions were observed for structure PONWOG. The crystal structure of PONWOG shows a disorder due to a 180° flip of the benzothiophene ring system.In the crystal of PONWIA, the molecules are linked into chains by N—H⋯O hydrogen bonds with In the crystal of MESTAI, the asymmetric unit comprises two molecules with similar conformations. In the crystal, weak C—H⋯F interactions form chains of molecules and the chains are stacked to form layers parallel to (101).2 group and a triazole N atom, forming In JESTOR, molecules are linked principally by N—H⋯N hydrogen bonds involving the amino NH3 (29 mmol) in 50 mL of H2O 2-fluorophenylisothiocyanate (19.6 mmol) was added at 293 K. The reaction mixture was boiled for 2 h, cooled to 293 K; then the aqueous solution was filtered from undissolved impurities and a 10% aqueous solution of HCl was added to it with stirring to pH = 2. The precipitate of the title compound was filtered off, washed with water, and then the product was recrystallized from ethanol.To a solution of of NaNH-tetrazole-5(4H)-thione: yield 72% as white powder, m.p. 426 K. Analysis calculated for C7H5FN4S (%): C 42.85, H 2.57, N 28.56. Found (%): C 42.62, H 2.66, N 28.59. 1H NMR : δ = 7.73 , 7.68 , 7.55 , 7.45 . 13C NMR : δ = 162.76 (C=S), 156.39 (C—F), .1-(2-Fluorophenyl)-1Uiso(H) = 1.2Ueq(C). The N-bound H atom was located in a difference map and refined isotropically.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020007033/yk2131sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020007033/yk2131Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020007033/yk2131Isup3.cmlSupporting information file. DOI: 2005595CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Their structures including absolute configurations were elucidated by a combination of MS, NMR, ECD calculation, and single-crystal X-ray diffraction analysis. Compound 2 was an eburnea-type MIAs characterized by a rare chlorine atom while 5 possessed a novel pyranopyridine moiety. Their cytotoxicities against several human cancer cell lines were evaluated and compound 1 exhibited significant cytotoxicity with IC50 values of 0.8–7.4 μM.Four new monoterpenoid indole alkaloids, bousmekines A-D (The online version of this article (doi:10.1007/s13659-020-00278-6) contains supplementary material, which is available to authorized users. Bousigonia (Apocynaceae family) comprises only two species, B. angustifolia and B. mekongensis, both of them are distributed in southwestern China D20 − 22 . Its molecular formula, C25H30N2O4, was established by HRESIMS ion at m/z 423.2281 [M + H]+ , corresponding to nine degrees of unsaturation. IR absorptions implied the presence of carbonyl (1676 cm−1) function.Bousmekine A  and a nonprotonated quaternary carbon (δC 160.0). The key HMBC correlations of OMe (δH 3.81), H-9  and H-10  to C-11 (δC 160.0) indicated that the methoxy group was located at C-11  confirmed the other parts of 1 were the same as that of 3α-acetonyltabersonine -1. The ECD calculation results for -1 matched well with its experimental ECD spectrum finally established the absolute configuration of 1  was obtained as an optically active colorless crystal, [α]D20 =  − 98 . Its molecular formula was determined to be C19H21N2OCl by HRESIMS ion at m/z 329.1414 [M + H]+ , suggesting 10 unsaturation degrees. The 13C and DEPT spectra suggested that 2 possessed 19 carbons including one methyl, five methylenes, eight methines, and five nonprotonated carbons. The NMR data demonstrated that 2 was an eburnean-type alkaloid similar as meloyunine + , corresponding to nine degrees of unsaturation. Detailed analysis of its NMR spectra demonstrated that 3 was structurally related to eburnamine + . Compared its NMR data with 3 indicated that both compounds had the same skeleton. The coupling constant of H-16  demonstrated that H-16 took β orientation and thus compound 4 was assigned as the C-16 epimer of 3. Further 2D NMR spectra  and ECD data analysis confirmed the structure of 4, as shown in Fig. Bousmekine D (5) was obtained as an optically active colorless solid, [α]D20 − 66 . Its molecular formula, C10H13NO2, was established by HRESIMS analysis at m/z 180.1018 [M + H]+ , corresponding to five degrees of unsaturation. The 13C and DEPT spectra showed that 5 comprises 10 carbons, including two methyls, one methylene, five methines and two nonprotonated carbons.Bousmekine E , b (C-6 to C-7), and c (C-9 to C-10). HMBC correlations of H-6  to C-4 (δC 70.7), and of H-11  and H-7a  to C-3 (δC 135.9) established the linkage of a and b, which formed a pyran moiety. Meanwhile, HMBC correlations of H-2  to C-4, C-8 (δC 142.3), and C-10 (δC 148.4), and of H-9  to C-3 and C-7 (δC 34.7) finally established the connectivities of b, c, and the nitrogen atom. Thus, the planar structure of 5 was finally established as a novel pyranopyridine alkaloid -5  value of -5 was calculated by using density functional theory (DFT) method. The calculated [α] value for -5 was -67, which is close to its experimental value (–66), and thus the absolute configuration of 5 was assigned as .The ROESY correlation of H-4 and H-6 indicated that both protons took the same orientation and were arbitrarily designated as )-5 Fig. . To allo1 exhibited significant cytotoxicity with IC50 values of 0.8–7.4 μM  and paclitaxel (PXL) as positive controls . Among tμM Table .Table 3CNMR spectra were measured via Bruker AV-500 and Avance III 600 MHz, TMS was used as an internal standard. IR spectra were surveyed on a Bio-Rad FTS-135 with KBr pellets. A JASCO P-1020 digital polarimeter was used to get optical rotations, while the ECD spectral data were measured by an Applied Photophysics Chirascan Spectrometer. HRESIMS and ESI were surveyed on Aglient 1290 UPLC/6540 Q-TOF spectrometer. Silica gel , silica gel H , and Sephadex LH-20 , were used for column chromatography. Semi-preparative HPLC was carried out using a Shimadzu LC-20AT liquid chromatograph equipped with a YMC Triart C18 ExRS  reversed-phase column.B. angustifolia and B. mekongensis were collected in Xishuangbanna, Yunnan Province, People’s Republic of China, in July 2018. The samples were identified by Mr. Yu Chen, Kunming Botanical Garden. A specimen (no. ZY20180623 and no. ZY20180624) was deposited at State Key Laboratory of Phytochemistry and Plant Resource in West China, Kunming Institute of Botany, Chinese Academy of Science (CAS).The twigs and leaves of B. mekongensis (30.5 kg) were powdered and extracted three times with methanol. The extract was adjusted to 2–3 with hydrochloric acid (5%) and then extracted three times with petroleum ether. The water fraction was basified to pH 9–10 with sodium hydroxide (10%), then extracted with chloroform to get the crude alkaloids. The crude alkaloids (530 g) were separated on a silica gel column (100–200 mesh), and eluted with a gradient of CHCl3-MeOH (40:1 → 1:1) to yield five fractions (A-E). Fraction D (19.1 g) was purified by a RP-18 column  to give four subfractions (DI-DIV). Subfraction DII (5.2 g) was purified by a RP-18 column  and followed by semipreparative HPLC with MeOH/H2O  to give 1 . Subfraction DIV (4.9 g) was purified by a RP-18 column  and followed by semipreparative HPLC with MeOH/H2O  to afford 2 .The dried twigs and leaves of B. angustifolia (57.5 kg) were powdered and extracted three times with methanol. The crude alkaloids (730 g) were obtained according to the above method and were separated on a silica gel column (100–200 mesh) by CHCl3-MeOH (40:1 → 1:1) to yield 6 fractions (A-F). Fraction D (30.0 g) was purified by a RP-18 column  to give four subfractions (DI-DV). Subfraction DII (8.2 g) was purified by a RP-18 column  and followed by semipreparative HPLC with MeOH/H2O  to give 3  and 4 . Subfraction DIV (6.9 g) was purified by a RP-18 column  and followed by semipreparative HPLC with MeOH/H2O  to afford 5 .The dried twigs and leaves of 1): colorless solid; [α]D20 − 22 ; UV (MeOH) λmax (log ε): 327 (3.91) nm; ECD  λmax (∆ε) 210 (+ 5.33), 226 (+ 2.64) 241 (+ 4.63), 320 (− 3.43); IR (KBr) vmax 3441, 2963, 1676, 1617, 1500, 1438, 1263, 1113 cm−1; 1H and 13C NMR data  see Tables m/z 423.2281 [M + H]+ .Bousmekine A : colorle2): colorless crystal; [α]D20 − 98 ; UV (MeOH) λmax (log ε): 230 (4.27), 272 (3.65) nm; ECD  λmax (∆ε) 207 (+ 2.60), 231 (− 17.46); IR (KBr) vmax 3450, 2924, 1658, 1487, 1454, 1334, 1295, 1046 cm−1; 1H and 13C NMR data  see Tables m/z 329.1414 [M + H]+ .Bousmekine B : colorless solid; [α]D20 − 45 ; UV (MeOH) λmax (log ε): 223 (3.90) nm; ECD  λmax (∆ε) 200 (+ 1.15), 221 (− 1.47), 234 (+ 1.24); IR (KBr) vmax 3450, 2923, 1658, 1632, 1380, 1326, 1296 1011 cm−1; 1H and 13C NMR data  see Tables m/z 313.1912 [M + H]+ .Bousmekine C : colorless solid; [α]D20 + 18 ; UV (MeOH) λmax (log ε): 224 (4.33) nm; ECD  λmax (∆ε) 223 (+ 9.72); IR (KBr) vmax 3418, 2922, 2852, 1724, 1659, 1459, 1384, 1207, 1056 cm−1; 1H and 13C NMR data  see Tables m/z 313.1908 [M + H]+ .Bousmekine D (5): colorless solid; [α]D20 − 66 ; UV (MeOH) λmax (log ε): 259 (3.08) nm; IR (KBr) vmax 3442, 2918, 1736, 1647, 1542, 1467, 1384, 1261, 1048 cm−1; 1H and 13C NMR data  see Tables m/z 180.1018 [M + H]+ .Bousmekine E : 4(C19H21ClN2O)·C2H6O, M = 1361.37, a = 11.9505(3) Å, b = 12.3727(3) Å, c = 22.7041(6) Å, α = 90°, β = 95.5750(10)°, γ = 90°, V = 3341.15(15) Å3, T = 100.(2) K, space group P1211, Z = 2, μ(Cu Kα) = 2.091 mm−1, 73809 reflections measured, 13008 independent reflections (Rint = 0.1435). The final R1 values were 0.0675 (I > 2σ(I)). The final wR(F2) values were 0.1701 (I > 2σ(I)). The final R1 values were 0.0798 . The final wR(F2) values were 0.1860 . The goodness of fit on F2 was 1.030. Flack parameter = 0.104(12). Crystallographic data (excluding structure factor tables) for compound 2 have been deposited with the Cambridge Crystallographic Data Center as supplementary publication (deposit number CCDC 2026043). Copies of the data can be obtained free of charge by application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. + 44 (0) (1223) 336 033; e-mail: deposit@ccdc.cam.ac.uk.Bousmekine B  and one pyranopyridine alkaloid (5) were isolated from the twigs and leaves of two Bousigonia species: B. angustifolia and B. mekongensis. Among them, compound 2 was an eburnea-type MIAs and featured by a rare chlorine atom at C-14, while 5 was characterized by a novel pyranopyridine moiety. Moreover, compound 1 exhibited significant cytotoxicities against several human cancer cell lines with IC50 values of 0.8–7.4 μM. The findings enriched the diversity of secondary metabolites of the Bousigonia genus.In this investigation, four new MIAs Electronic supplementary material 2 (CIF 2240 kb)Below is the link to the electronic supplementary material.