HL1), crystallizes with three independent molecules in the asymmetric unit. Its reaction with Cu(Ac)2 produced a tetranuclear complex with a central tetrakis(μ-acetato)dicopper paddle-wheel moiety linked on either side via bridging acetate anions to a mononuclear copper(II)–(L1) complex.The title tridentate ligand pyrazine-2-carboxamide (HL1), crystallizes with three independent molecules  in the asymmetric unit. All three molecules are relatively planar , with the NH H atom forming three-centered (bifurcated) intramolecular N—H⋯N hydrogen bonds in each molecule. There is also an intramolecular C—H⋯O contact present in each molecule, involving the benzene ring of the quinoline unit and the amide carboxamide O atom. In the crystal, the three molecules stack in columns with the various molecules being linked by offset π-π interactions [intercentroid distances vary from 3.367 (5) to 3.589 (5) Å], forming layers parallel to the ab plane. The title complex, [Cu4(C42H44N8O16)]·2CH3OH, {hexa-μ-acetato-1:2κ2O:O′;2:3κ8O:O′;3:4κ2O:O′-dimethanol-1κO,2κO-bis[N-(quinolin-8-yl)pyrazine-2-carboxamide]-1κ3N,N′,N′′;4κ3N,N′,N′′-tetracopper(II) methanol disolvate} (I), was obtained by the reaction of HL1 with Cu(CH3CO2)2. It consists of a tetranuclear complex with a central tetrakis(μ-acetato)dicopper paddle-wheel moiety linked on either side via bridging acetato ions to a mononuclear copper(II)–(L1) complex; it crystallizes as a methanol disolvate. The complex possesses inversion symmetry, being located about a center of symmetry situated at the mid-point of the Cu⋯Cu bond of the paddle-wheel moiety. In the crystal, the complex molecules are linked by O—H⋯O hydrogen bonds, forming chains along the [01The title tridentate ligand, C HL1 have been synthesized in order to explore their super-exchange magnetic properties pyridine-2-carboxamide, has been reported [N-(quinolin-8-yl)pyrazine-2-carboxamidato]copper(II) monohydrate, a mononuclear complex with the ligand coordinating in a tridentate fashion  in the asymmetric unit, and their molecular structures are illustrated in Fig. 1b]pyrazine-5,7-diimine ° in B and 4.1 (4)° in C. For the three molecules, the r.m.s. deviations for the mean planes of the non-H atoms are 0.068, 0.055 and 0.06 Å, respectively. Inverted molecule A on molecule B has an r.m.s. deviation of 0.054 Å for the 19 non-H atoms, while inverted molecule B on molecule C has an r.m.s. deviation of 0.054 Å, and molecule A and molecule C have an r.m.s. deviation of 0.057 Å.The ligand HL1 with Cu(CH3CO2)2 produced a tetranuclear complex, I, with a central tetrakis(μ-acetato)-dicopper paddle-wheel moiety linked on either side via a bridging acetate anion to a mononuclear copper(II)–(L1) complex, illustrated in Fig. 2i bond  of the paddle-wheel moiety  was required. In the crystal, packets of the three molecules stack in the order (ABC), (ABC) etc . They are linked by offset π–π interactions, so forming layers lying parallel to the ab plane  Å, Cg is the centroid of the N4/C6–C14 ring, α = 0.04 (7)°, β = 25.7°, interplanar distance = 3.3684 (8) Å, offset 1.618 Å; symmetry code: (vi) −x + 1, −y, −z]. The chains are also linked by C—H⋯O hydrogen bonds, resulting in the formation of a supramolecular framework , are delineated into H⋯H at 43.0%, N⋯H/H⋯N at 14.5%, followed by C⋯H/H⋯C interactions at 11.8%. The contributions of the C⋯C and C⋯N interactions, which are 10.8 and 10.7%, respectively, are superior to the contribution of the O⋯H/H⋯O interactions at 8.1%. The relative percentage contributions of close contacts to the Hirshfeld surface for HL1 and for the individual molecules are similar, as indicated in Table 5The full two-dimensional fingerprint plots for L1 Fig. 8b, are det al., 2016HL1 yielded nine hits. The majority of these compounds are hetero bimetallic iron–manganese cyano complexes that exhibit super-exchange magnetic properties copper(II) has also been reported , and that in compound AYIFOF mentioned above.A search for complexes of the pyridine analogue of supporting information file S1). Eight of these compounds are polymeric structures, for example, the network structure catena-tetracopper(II)] tetracopper(II) bisimino] methyl}phenolato)tetracopper(II) [UJOWEX; Huang & Liu, 20162-acetato-O,O′)bistetrakis(triphenylphosphine-P)dicopper(I)dicopper(II) [CERTOI; Koman et al., 1984et al., 1986ca 2.604 to 2.669 Å; in I this distance, Cu2⋯Cu2i, is 2.6201 (6) Å. The Cu⋯Cu distance involving the two copper atoms bridged by a single acetato group varies from ca 3.772 to 5.441 Å. The longer distance is observed when only one O atom bridges the two copper atoms as in compound I, where distance Cu2⋯Cu1 is ca 5.147 Å, close to the distance of ca 5.392 Å observed in UJOWEX. A shorter distance is observed when one O atom bridges the two copper atoms and the second O atom coordinates to the second copper atom, in a  manner, as in CERTOI/CERTOI10 where this Cu⋯Cu distance is ca 3.772 Å.A search for the tetrakis(μ-acetato)dicopper paddle-wheel moiety gave 356 hits. Limiting the search for a tetrakis(μ-acetato)-dicopper paddle-wheel moiety bridged on either side by an acetato group to a second copper atom gave 15 hits for 14 structures (see Synthesis ofN-(quinolin-8-yl)pyrazine-2-carboxamide (HL1):2O (vapour temperature 355 K). The mixture was allowed to cool, and then 1,1′-carbonyldiimidazole  was added. After gas evolution had diminished, the solution was heated at reflux for 16 h. The reaction mixture was allowed to cool to RT and then added directly to a column  and eluted with CHCl3. On evaporation of the solvent the residue obtained was recrystallized from ethanol giving block-like colourless crystals of HL1 .A suspension of pyrazine-2-carboxylic acid  and 8-aminoquinoline  in 80 ml of 1,2-dichloroethane was distilled to azeotropically remove any solvated HSpectroscopic data for HL1 : 11.95 ; 9.41 ; 9.02 ; 8.93 ; 8.89 ; 8.48 ; 7.78 ; 7.69 . 13C NMR : 161.7 (C5), 150.3 (C13), 149.2 (C4), 144.9 (C1), 144.6 (C2), 144.5 (C3), 139.0 (C14), 137.7 (C11), 134.2 (C6), 128.8 (C10), 128.0 (C8), 123.7 (C12), 123.4 (C7), 117.0 C(9). IR : 1686 (vs), 1559 (s), 1533 (vs), 1485 (vs), 1471 (s), 1460 (s), 1425 (s), 1403 (s), 1384 (s), 1325 (s), 1129 (s), 1058 (s), 1020 (s), 830 (s), 796 (s), 763 (s), 741 (s), 710 (s), 599 (s). Analysis for C14H10N4O (Mr = 250.26 g mol−1); calculated (%) C 67.19, H 4.03, N 22.39; found (%) C 67.00, H 4.04, N 22.37.Synthesis of compound I:2·H2O  was added to a solution of HL1  dissolved in 15 ml of methanol. The green solution was stirred at room temperature for 30 min. It was then left to allow slow evaporation of the solvent giving finally green block-like crystals of I. The crystals were filtered off and washed with diethyl ether . IR : 3422 (s), 1624 (vs), 1581 (vs), 1430 (s), 1396 (vs).Cu(Ac)HL1 were measured at 223 K on a four-circle diffractometer assuming a C-centered unit cell and only one equivalent of data were measured; hence Rint = 0 and the h,k,l reflections for which h + k = 2n + 1 were not measured. For compound I, data were measured at 173 K on a Stoe IPDS1, a one-circle image-plate diffractometer. For compound I a small cusp of data is missing. This is common with data measured using the IPDS1 for monoclinic and triclinic crystal systems.Crystal data, data collection and structure refinement details are summarized in Table 6HL1 the NH H atoms could be located in a difference-Fourier map, but during refinement they were included in calculated positions and treated as riding: N—H = 0.87 Å with Uiso(H) = 1.2Ueq(N). The OH H atom of the coordinated methanol molecule in complex I was located in a difference-Fourier map and freely refined. The OH H atom of the solvent methanol molecule in I was included in a calculated position and treated as riding: O—H = 0.84 Å with Uiso(H) = 1.5Ueq(O). For HL1 and complex I the C-bound H atoms were included in calculated positions and treated as riding: C—H = 0.95–0.99 Å with Uiso(H) = 1.2Ueq(C).For ligand 10.1107/S2056989019005450/zl2755sup1.cifCrystal structure: contains datablock(s) HL1, I, Global. DOI: 10.1107/S2056989019005450/zl2755HL1sup2.hklStructure factors: contains datablock(s) HL1. DOI: 10.1107/S2056989019005450/zl2755Isup3.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989019005450/zl2755sup4.pdfCSD search S1. DOI: 1911375, 1911374CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Currently, the structural characteristics of the swine major histocompatibility complex (MHC) class I molecule, also named swine leukocyte antigen class I (SLA-I) molecule need to be further clarified.2-microglobulin and a cytotoxic T lymphocyte (CTL) epitope FMDV-AS64 (ALLRSATYY) derived from VP1 protein (residues 64–72) of Asia 1 serotype of foot-and-mouth disease virus (FMDV) was expressed, refolded, purified and crystallized. By preliminary X-ray diffraction analysis, it was shown that the diffraction resolution of the crystal was 2.4 Å and the space group belonged to P212121 with unit cell parameters a = 48.37, b = 97.75, c = 166.163 Å.A complex of SLA-I constituted by an SLA-2*HB01 molecule with swine βThis research will be in favor of illuminating the structural characteristics of an SLA-2 molecule associated with a CTL epitope derived from Asia1 serotype of FMDV. AB602431) coding for 275 amino acids in extracellular domain, a pair of primers was designed as shown in Table eviously . The two follows , 19: In 2m) complex was carried out as described previously [2m were renatured and refolded with the AS64 epitope according to a 1:1:3 molar ratio by using the gradual dilution method in a refolding buffer  at 4 °C. After refolding for 24 h, the soluble SLA-2*HB01-AS64-sβ2m complex was further concentrated and purified by chromatography separation on a Superdex 200 16/60 HiLoad size-exclusion column  followed by an anion-exchange chromatography Resource Q . SLA-2*HB01-AS64-sβ2m complex information is shown in Table The refolded SLA-I (SLA-2*HB01-AS64-sβeviously . Firstly2m complex proteins (~ 44 kDa) were further dialyzed against a crystallizing buffer  overnight at 4 °C and then concentrated to 15 mg/mL. Crystallizing assays were tried with Index and Crystal Screen kit (Hampton Research) at 4 °C and 18 °C by using the sitting-drop vapor diffusion method as previously described [2m were obtained at 14th day by using a protein concentration of 15 mg/mL in the Index solution No. 79  at 4 °C. Crystallizing condition is shown in Table The final purified SLA-2*HB01-AS64-sβescribed , 20. Two2m crystal was firstly soaked in reservoir solution supplemented with 17% (v/v) glycerol as a cryoprotectant for several seconds and then flash-cooled in a nitrogen stream at − 173 °C [The SLA-2*HB01-AS64-sβ− 173 °C . Data co− 173 °C .2m and the CTL epitope of AS64. The yield of the SLA-2*HB01-AS64-sβ2m complex is about 10%. It was shown four peaks in the chromatographic elution profile . The peak 7 detected in lane 7 with SDS-PAGE should be surplus sβ2m  and sβ2m light chain (12 kDa) (see inset in Fig. After purification to homogeneity by Superdex 200 16/60 HiLoad size-exclusion chromatography, it was shown that the heavy chain of SLA-2*HB01 was successfully refolded together with the light chain of sβ2m was concentrated to 15 mg/mL for crystal screening. Crystals occurred after 7 days in the Index solution No. 79 at 4 °C and then they were harvested at 14th d (Fig. 2m crystal used for data collection was 2.4 Å. The space group of the crystal belongs to P212121. The unit-cell parameters of the crystal were shown as a = 48.37, b = 97.75, c = 166.163 Å (Fig. The purified SLA-2*HB01-AS64-sβPreviously, we reported a multiple amino-acid sequence alignment between SLA-2*HB01 and other known MHC class I alleles in swine, human and mouse . Based o2m and a CTL epitope AS64 derived from the Asia1 serotype of FMDV was reported. It seems the space group type of SLA-2*HB01-AS64-sβ2m crystal is consistent with that of SLA-1 crystal but quite different from that of SLA-3 crystal, which indicates the structure of SLA-2 might be more similar to that of SLA-1 [In this work, the crystal of the SLA-2*HB01 molecule complexed with swine βof SLA-1 , 18. Recof SLA-1 . To learThe research data will be used to further elucidate the 3-dimentional structure of the SLA-2 molecules and design more refined viral epitopes.


In the crystal, molecules are linked into inversion dimers with an via pairs of intermolecular C—H⋯O hydrogen bonds.The title chalcone derivative adopts an  23H15NO3, adopts an s-cis conformation with respect to the ethylene C=C and carbonyl C=O double bonds in the enone unit. The molecule is significantly twisted with a dihedral angle of 48.63 (14)° between the anthracene ring system and the benzene ring. In the crystal, molecules are linked into inversion dimers with an R22(10) graph-set motif via pairs of C—H⋯O hydrogen bonds. The intermolecular interactions were analysed and quantified by Hirshfeld surface analysis. The molecular structure was optimized and a small HOMO–LUMO energy gap of 2.55 eV was obtained using the DFT method at the B3LYP/6–311 G++ level of theory. This value is in close agreement with the experimental value of 2.52 eV obtained from the UV–vis analysis. The crystal used was a two-component merohedral twin with a refined ratio of 0.1996 (16):0.8004 (16).The title compound, C The molecular structure was optimized with the Gaussian09W software package  Å; 1.347 (DFT) Å] and C17=O1  double bonds in the enone unit (C15=C16—C17=O1) and the structure is twisted around the C14—C15 bond with a C1—C14—C15—C16 torsion angle of 51.1 (6)° and slightly deviated around the C17—C18 bond with a C16—C17—C18—C19 torsion angle of −15.6 (5)°. The corresponding values by DFT are 44.8 and 18.5°, respectively . These large twist angles are due to the bulkiness of the strong-electron-donor anthracene ring system . Furthermore, a large dihedral angle of 48.63 (14)° is observed between the anthracene ring system and the nitrobenzene ring ; this could diminish the electronic effect between the two ring systems : −x + 1, −y + 2, −z + 1; Table 1b-axis direction  with cut-off wavelength of 190 nm. Two major peaks at 253 and 427 nm were observed  and nitro substituent (NO2) functional groups  at 492.06 nm -1-(4-methoxyphenyl)prop-2-en-1-one -3-(anthracen-9-yl)-1-(4-bromophenyl)prop-2-en-1-one -3-(anthracen-9-yl)-1-(2-ethoxyphenyl)prop-2-en-1-one -3-(anthracen-9-yl)-1-(2-hydroxyphenyl)prop-2-en-1-one -3-(anthracen-9-yl)-1-(2-bromophenyl)prop-2-en-1-one  and WAFGOB are ortho-substituted ethoxy, hydroxy and bromo derivatives, respectively. Dihedral angles between the enone unit and the anthracene ring system and between the enone unit and the benzene ring are 81.6 (3) and 8.2 (4)°, respectively, for EMULIJ, 47.1 (3) and 22.9 (3)° for JAHPUG, 45.79 (10) and 20.88 (11)° for POPBAY, 82.49 (11) and 35.54 (13)° for KABHUS, 61.51 (9) and 14.56 (10)° [62.05 (9) and 11.04 (10)°] for UNUDUD, and 42.62 (16) and 63.00 (17)° for WAFGOB. The large dihedral angle of 82.49 (11)° between the enone unit and the anthracene ring system observed for KABHUS is due to the Z configuration of the molecule. Interestingly, EMULIJ with an E configuration also shows a large dihedral angle of 81.6 (3)° between the enone unit and the anthracene ring system, whereas the dihedral angle between the enone unit and the benzene ring is extremely small [8.2 (4)°].A search of the Cambridge Structural Database  and 9-anthracencarboxaldehyde (0.5 mmol) was dissolved in methanol (20 ml) and the solution stirred continuously. A catalytic amount of NaOH  was added to the solution dropwise until a precipitate formed and the reaction was stirred continuously for about 5 h at room temperature. After stirring, the solution was poured into 60 ml of ice-cold distilled water. The resultant crude product was filtered and washed several times with with distilled water until the filtrate turned colourless. The dried precipitate was further recrystallized to obtain the corresponding chalcone. Red plate-shaped single crystals suitable for X-ray diffraction were obtained by slow evaporation of an acetone solution.Uiso(H) = 1.2Ueq(C). Four outliers (002), (420), (300) and  I, mo_MFZ5_w_0m. DOI: 10.1107/S2056989019005243/is5513Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019005243/is5513sup3.docxComparison of bond lengths and angles between experimental and theoretical studies. DOI: Click here for additional data file.10.1107/S2056989019005243/is5513Isup4.cmlSupporting information file. DOI: 1905274CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The diffraction data confirmed the title compound as the main isomer produced in a coupling reaction. The structure and Hirshfeld surface analysis of the formed di-tetrazolyl chelate ligand are reported. 20H22N8O2, was synthesized by the coupling reaction of a sodium tetrazolate salt and dibromobutane in a molar ratio of 2:1. The reaction can produce several possible regioisomers and the title compound was separated as the major product. The X-ray crystallographic study confirmed that the title compound crystallizes in the monoclinic P21/c space group and possesses a bridging butylene group that connects two identical phenyl tetrazole moieties. The butylene group is attached not to the first but the second nitrogen atoms of both tetrazole rings. The dihedral angles between the phenyl groups and the adjacent tetrazolyl rings are 5.32 (6) and 15.37 (7)°. In the crystal, the molecules form centrosymmetric dimers through C—H⋯O hydrogen bonds between a C—H group of the butylene linker and the O atom of a methoxy group.The title compound, C The tilted fragment (N5–N8/C9–C15) interacts with the butyl bridge of a glide-related molecule through C19—H19CrystalExplorer17  to 1.4773 a.u. (blue). The 3D dnorm surface of the title complex is shown in Fig. 6a and 6b. The red spots indicate short contacts, i.e., negative dnorm values on the surface, which highlight the most important weak interactions: C17—H17A⋯O1i hydrogen bond (green dashed line), C4—H4⋯C1iv contact , C18—H18B⋯C5iii  and C16—H16A⋯C1v .To provide an overall view of the weak interactions between the molecules, a Hirshfeld surface analysis  and dibromobutane  were dissolved in acetonitrile and refluxed for 2 d. The resulting white solid was filtered and the solvent was removed under reduced pressure. The residue was purified by column chromatography on silica gel using hexane:acetone (1:1) as eluent. Three isomeric compounds were obtained, as shown in Fig. 1The synthesis scheme for the title compound is represented in Fig. 11H NMR : δ = 7.62 , 7.36 , 7.22 , 7.12 , 4.13 , 3.71 , 1.66 . 13C NMR : 156.56, 152.18, 133.10, 131.20, 120.80, 112.26, 111.91, 55.50, 46.63, 25.57 ppm.Spectroscopic data: Uiso(H) = 1.5Ueq(C) for methyl H atoms and Uiso(H) = 1.2Ueq(C) for all others. Two reflections (100 and 110) were omitted because of truncation by the beamstop.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019014877/fy2138sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019014877/fy2138Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019014877/fy2138Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019014877/fy2138Isup4.cmlSupporting information file. DOI: 1963337, 1963337CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Si,Si-diphenyl-, P-chlorofunctionalized diazaphosphasiletidine has been synthesized. The crystal structure of the highly moisture-sensitive four-membered heterocyclic molecular compound, which has a remarkably elongated P—Cl bond, is described and compared with related compounds and their arrangement of molecules in the solid state.A new  N,N′-di(tbutyl)-Si,Si-diphenylsilanediamide and PCl3 yielded an off-white solid. Sublimation of the crude product under reduced pressure at elevated temperature gave colourless prismatic crystals of the title compound, C20H28ClN2PSi, which crystallizes in the non-centrosymmetric monoclinic space group Cc. The asymmetric unit of the crystal structure contains one molecule and it is dominated by the central SiN2P four-membered ring, which is almost planar with a mean deviation of the atoms from the best plane of 0.014 Å. The angles between the plane defined by the silicon atom and the two nitrogen atoms and the best planes of the Si-phenyl groups are 85.1 (2) and 77.4 (2)°, with the tilt of the phenyl rings in the opposite direction. Both tert-butyl groups suffer from a two-position rotational disorder with site occupancies of 0.752 (6)/0.248 (6) and 0.878 (9)/0.122 (9). The P—Cl bond [2.2078 (17) Å] is remarkably elongated compared to the P—Cl distance in PCl3 .The chemical reaction of dilithium  The first synthesis was described in the year 1963 2PCl, namely 2-chloro-1,3-bis-4,4-dimethyl-1,3,2λ3,4-diazaphosphasiletidine 2PCl, namely 2-chloro-1,3-di-tert-pentyl-4,4-diphenyl-1,3,2λ3,4-diazaphosphasiletidine -4-chloro-4-methyl-1,3,2λ3,4-diazaphosphasiletidine. With respect to this impurity, an Si,Si-diphenyl-substituted diazaphosphasiletidine (C) has successfully been introduced to preparative chemistry to avoid problems related to the content of Si,P-bis(chloro)functionalized species present in samples of the Si,Si-dimethyl derivative. However, the crystal-structure determination of C suffered from severe disorder. All the aspects mentioned before persuaded us to focus on preparation of single crystals of the title compound suitable for structure determination. After extensive attempts, we were finally able to grow single crystals by slow sublimation in vacuo and confirmed its composition and its structure via X-ray diffraction.Diazaphosphasiletidines are heterocyclic compounds that contain an SiN2P four-membered ring, is almost planar. The nitrogen atoms exhibit a trigonal–planar coordination sphere [sums of bond angles 359.9° (N1) and 359.4° (N2)]. The phosphorus and silicon atoms bear the main ring strain [N1—Si1—N2 = 82.08 (19)° and N1—P1—N2 = 85.4 (2)°]. The Si–N bond lengths [Si1—N1 = 1.736 (4) Å and Si1—N2 =1.749 (4) Å] exceed the expected length of an Si—N single bond  compared to the P—Cl distance in PCl3  Å, due to dimerization]: Si—N = 1.743 (4) Å average (in the title compound) vs 1.7441 (17) Å in A, 1.7474 (14) Å in B and 1.7406 (15) Å in C ; P—N = 1.687 (4) Å vs 1.6856 (17) Å (A), 1.6815 (14) Å (B), 1.6910 (16) Å (C); P—Cl 2.2078 (17) Å vs 2.1813 (7) Å (A), 2.2498 (6) Å (B) (dimerization), 2.1965 (17) Å (C). The tert-butyl groups in the title compound are rotationally disordered (see Refinement).The asymmetric unit of the title compound contains one molecule Fig. 1. The cenc axis, but point in the opposite direction. The nearest intermolecular contact is between the Cl atom and the meta-H atom of one of the Si-bonded phenyl groups of a neighbouring molecule . In the figure, this contact is indicated by dashed lines. However, the geometric features of this contact  indicate that if at all, it is a borderline case of a directed bonding interaction.Fig. 2et al., 2016P-chlorofunctionalization. Of these, BADLUO phosphorane, VUHTOJ 5 complex DEXVAG. The structure of the only Si,Si-diphenyl-P-chloro derivative (C), 2-chloro-1,3-bis(2-methylbutan-2-yl)-4,4-diphenyl-1,3,2λ3,4-diazaphosphasiletidine 2ECl. Me2Si(NtBu)2AsCl contains dimers and in the antimony and the bismuth analogues the molecules are connected into chains via bridging Cl atoms 2AsCl 2SbCl -Si,Si-diphenylsilanediamine were dissolved in 60 ml n-pentane. 13.6 ml of a n-butyllithium solution  were added at 263 K. The reaction mixture was stirred for 24 h at room temperature. Cooling to 178 K and addition of 1.5 ml (16.8 mmol) PCl3 yielded an off-white suspension. This was stirred for 3 h. After filtration and removal of the solvent under reduced pressure, the crude product was obtained as an off-white solid. Sublimation at 333 K under reduced pressure yielded colourless crystals within a couple of hours (77% yield based on PCl3). 1H NMR : δ (p.p.m.) 1.17  = 0.9 Hz, 18H,C(CH3)3), 7.48 , 7.86 , 8.08 . 13C{1H} NMR : δ(p.p.m.) 32.9 , 52.6 , 128.3–136.3 . 31P{1H} NMR : δ (p.p.m.) 214.4 (s) EI–MS spectra were obtained using a Finnigan TSQ 7000 instrument. EI–MS: m/z (%) 390 (11) [M+], 375 (100) [M+—C(CH3)3]. IR spectra were measured using a Bio-Rad Excalibur FTS 3500 FT–IR spectrometer with ATR-unit, 4000–560 cm−1: 3070(w), 3050(w), 3026(sh), 3014(sh), 2956(vs), 2927(s), 2903(sh), 2868(m), 1964(vw), 1903(vw), 1827(vw), 1774(vw), 1588(w), 1429(s), 1305(vw), 1207(s), 1113(s), 1102(sh), 1055(s), 1042(sh), 889(vs), 820(w), 755(sh), 739(s), 696(s). Analysis calculated for C20H28ClN2PSi (326.56 g mol−1): C 61.44, H 7.22, N 7.17; found C 61.10, H 7.56, N 7.08, m.p.: 393.5 K.The title compound was prepared Fig. 3 accordinvia subsequent Fourier syntheses. In the refinement, a riding model was applied using idealized C—H bond lengths (0.95–0.98 Å) as well as H—C—H and C—C—H angles. In addition, the H atoms of the CH3 groups were allowed to rotate around the neighboring C—C bonds. The Uiso values were set to 1.5Ueq(Cmethyl) and 1.2Ueq(Car). To account for residual electron density in the regions of the two tert-butyl groups and for elongated anisotropic displacement ellipsoids of several carbon atoms that did not appear to be physically meaningful, a two-position disorder for each tert-butyl group was introduced with partial occupation sites for all carbon atoms but the tertiary ones C1 and C5 [occupancy ratio 0.752 (6):0.248 (6) ratio (group containing C1) and 0.878 (9):0.122 (9) ratio (C5); in Figs. 1Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2056989019002627/pk2614sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989019002627/pk2614Isup2.hklStructure factors: contains datablock(s) I. DOI: 1898427CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Isodon excisoides. Interestingly, these four diterpenoids were two pairs of tautomers that were first reported in plants. Their structures were further elucidated using various spectroscopic methods. The tautomerization phenomenon and mechanism for these two pairs of tautomers were emphatically described. The theoretical simulation results indicated that the diterpene tautomerization is greatly related to certain factors, including the existence of a transition state, the change of bond length and the level of conversion energy; the tautomerization for the two pairs of tautomers is mainly caused by proton transfer. For bioassays, the cytotoxicities of the tautomers against five human cancer cell lines were also investigated. The results indicated that each of the four diterpenoids showed significant cytotoxicity in at least three cell lines and could serve as potential anticancer agents for further investigation.Discovering anticancer drugs that do not have adverse side effects has been a developing research field worldwide in recent decades. In this work, four previously undescribed cytotoxic diterpenoids were isolated from the aerial parts of  Globally, cancer is the leading cause of morbidity and mortality. For certain types of cancer, chemotherapy drugs have been extensively used for treatment. However, resistance to chemotherapy and severe side effects are the drawbacks of these agents. Therefore, the development of new agents derived from plants has intensified.1. The plants of the Isodon genus are rich in diterpenoids. In addition, ent-kaurane diterpenoids, containing an enone system in ring D, are a class of characteristic components found in this genus, and they are mainly responsible for the reported cytotoxic activity2. To date, approximately 1000 diterpenoids from the Isodon genus have been shown to exhibit significant cytotoxicity8, and many diterpenoids with anticancer activities, such as oridonin, erycalyxin A and rabdophyllin G, have also been developed into new drugs10. Isodon excisoides, a common wild species, is a perennial herb mainly distributed in the western region of the Henan and Yunnan Provinces in China. Local inhabitants in the western mountainous region of Henan Province generally believe that I. excisoides is better for preventing esophageal cancer than that of Isodon rubescens from the same genus. Our previous studies showed that many diterpenoids from I. excisoides have anticancer activities2. As part of our ongoing search for anticancer diterpenoids, four new diterpenes, as two pairs of tautomers, were purified and identified from I. excisoides in this work. Moreover, the tautomerization phenomenon for both pairs of tautomers was also observed. It is worth mentioning that the tautomerism of diterpenoids was observed for the first time. Simultaneously, the tautomerization mechanism was emphatically discussed. Finally, their cytotoxicities against five human cancer cell lines were also investigated.Natural and synthetic tetracyclic diterpenoids exhibit interesting pharmacological activitiesent-kaurane skeleton tautomeric diterpenes [1a (45 mg) and 1b (30 mg) and 2a (42 mg) and 2b (31 mg)] , 1α,14β-dihydroxy-7α,20-diacetoxy-ent-kaur-15-one (1b), 1α,14β-diacetoxy-7α,20-dihydroxy-ent-kaur-16-en-15-one (2a), and 1α,7α-diacetoxy-14β,20-dihydroxy-ent-kaur-16-en-15-one (2b) . The UV spectrum of 1a showed an absorption maximum at 235 nm. The IR spectrum of 1a showed the presence of hydroxyl (3445 cm−1), carbonyl (1729 cm−1) and double bond (1649 cm−1) groups. The 1H, 13C-NMR and HSQC spectra of 1a, together with the results from an HMBC experiment showed the presence of one exocyclic double bond , two angular methyl groups , one ketone carbonyl (δC 207.0) and two acetoxyl groups . In addition, the other carbon signals were assigned to six methenes including three oxy-methines  and six methylene carbons including one oxy-methylene (δC 63.9), and three quaternary carbons . Considering the diterpenoids previously isolated from the plant, 1a was tentatively presumed to be a 7,20-non-epoxy-ent-kaurane skeleton, substituted with two hydroxyl groups and two acetoxyl groups.Compound 1H-NMR and 13C-NMR data of 1a were nearly identical to that of henryin, a known diterpene11, and the only difference found was in the moiety at C-14. The compounds 1a was a 14-acetylated compound of henryin. This hypothesis was further confirmed by the HMBC spectra of 1a. In the HMBC spectrum  with δC 146.3 (C-16), δC 207.0 (C-15) and δC 170.2 (-OOCCH3) revealed that the hydroxyl group at C-14 in henryin  had been replaced by an acetoxyl group in 1a . Thus, the basic skeleton of 1a was assumed to be 1,7-dihydroxy-14,20-diacetoxy-ent- kaur-16-en-15-one.The rum Fig. , the corThe relative configuration of the substituents was highlighted in a NOESY spectrum. The correlations of H-1 with H-5 and H-9, Me-18 with H-5, H-7 with H-5 and H-9, and H-13 with H-14 and H-16 indicated that H-1, H-5, H-7 and H-9 were positioned on the same side and that H-13, H-14 and H-16 were on the other side  spectrum of compound rve Fig. . Accordi1b was a white powder. The molecular formula of 1b was determined to be C24H34O7 by positive HRESIMS . The UV, IR, 1H, 13C-NMR, HSQC and HR-ESI-MS spectra of 1b, together with the results from an HMBC experiment, showed that 1b was an isomer of 1a. Comparing the NMR data of 1a and 1b  and δC 51.8 (C-5), δC 45.6 (C-10) and δC 168.2 (7-CH3COO-), and δH 4.79 (H-14) and δC 55.7 (C-9), δC 205.7 (C-15) and δC 146.1 (C-16). Thus, the planar structure of 1b was assumed to be 1,14-dihydroxy-7,20-diacetoxy-ent-kaur-16-en-15-one.Compound 1a and 1b was deduced from their similar NOESY correlations . The UV spectrum of 2a showed an absorption maximum at 235 nm. The IR spectrum of 2a showed the presence of hydroxyl (3445 cm−1), carbonyl (1730 cm−1) and double bond (1648 cm−1) groups. Together with the NMR data of 2a, the results showed 2a was also an analogue of 1a.Compound 1a and 2a  correlated with δC 25.4 (C-2), δC 47.3 (C-10), δC 55.9 (C-9) and δC 169.1 (1-CH3COO-); δH 4.44 (H-20β) correlated with δC 55.9 (C-9), and δH 4.23 (H-20α) correlated with δC 84.1(C-1). Thus, the planar structure of 2a was assumed to be 1,14-diacetoxy-7,20-dihydroxy-ent-kaur-16-en-15-one.Comparing the NMR data of compounds 2a was determined with the NOESY spectrum. The correlations of H-1 with H-5 and H-9, Me-18 with H-5, H-7 with H-5 and H-9, and H-13 with H-14 and H-16 indicated that they were positioned on the same side and that H-14, H-13, and H-16 were on the other side . The UV, IR and HR-ESI-MS spectra, together with the NMR data of 2b, showed that 2b was an isomer of 2a.Compound 2a and 2b  correlated with δC 25.4 (C-2), δC 47.3 (C-10), δC 55.9 (C-9) and δC 169.1 (1-CH3COO-); δH 4.44 (H-20β) correlated with δC 55.9 (C-9); δH 4.23 (H-20α) correlated with δC 84.1 (C-1). The NOESY and CD experiments showed that the configuration of the substituents of 2b is the same as that of 2a  of 1a initially accounted for 97.19% of the total chromatographic PA; the PA started to decrease after 24 h, and a corresponding increase in the PA of 1b was simultaneously detected. After 3 days, the PA of 1a significantly decreased, while the PA of 1b significantly increased. After 12 days, the reaction approached dynamic equilibrium, and the PA of 1a accounted for 68.20% of the total PA. After 16 days, the reaction reached dynamic equilibrium, and the PA ratio of 1a to 1b was 2:1. For a 1a solution incubated at 45 °C, the PA of 1a started to decrease after 7 h, and a corresponding increase in the PA of 1b was simultaneously detected. After 1 day, the PA of 1a significantly decreased, while that of 1b significantly increased. After 6 days, the reaction approached dynamic equilibrium, and the PA of 1b accounted for 63.94% of the total PA. After 9 days, the reaction reached dynamic equilibrium, and the PA ratio of 1a to 1b was also 2:1.As shown in Fig. 1b sample solution stored at 30 °C, the PA of 1b started to decrease after 7 h, and a corresponding increase in the PA of 1a was simultaneously detected. After 2 days, the PA of 1b significantly reduced and that of 1a significantly increased. After 9 days, the reaction approached dynamic equilibrium. After 12 days, the reaction reached dynamic equilibrium, and the PA ratio of 1a to 1b was 2:1. For a 1b sample solution incubated at 45 °C, the PA of 1b started to decrease after 1 h, and a corresponding increase in the PA of 1a was simultaneously detected. After 1 day, the PA of 1b was significantly reduced, while that of 1a significantly increased. After 5 days, the reaction approached dynamic equilibrium, and the PA of 1b accounted for 59.79% of the total PA. After 7 days, the reaction reached dynamic equilibrium, and the PA ratio of 1a to 1b was also 2:1.For a 1a was the preferential conformation in the pair of isomers that could interconvert, and the temperature affected both the stability and conversion speed of 1a to 1b and of 1b to 1a in a protic solvent, while their conversion rate was not affected. In addition, an interesting phenomenon was also found that under identical conditions, the conversion speed of 1b to 1a was faster than that of 1a to 1b in a protic solvent, and it increased with increasing temperature.All results showed that 2a and 2b were another pair of tautomers, of which 2a was the preferred conformation. Similarly, the conversion speed from 2a to 2b was faster than that from 2b to 2a, and the conversion speed was faster at higher temperatures. As Fig. 1b⇌1a was slower than that for 2b⇌2a, which could be related to the spatial structure of the compounds , which suggests that the optimal transitional state was obtained. The corresponding normal coordinates of the imaginary vibration modes for TS1 are shown in Fig. 1a and 1b, respectively.Furthermore, the tautomerization mechanism of the two pairs of tautomeric diterpenoids was investigated, and the transition state calculations for the reactions were conducted using density functional theory. There is only a virtual frequency of −971.70 cmThe minimum energy path (MEP) was also obtained through intrinsic reaction coordinate (IRC) calculations, which indicated that the transition state was correlated with both tautomers and that the transition state was located on the right reaction path.2a⇌2b reaction (TS2) was confirmed as well. There was only one virtual frequency of the transition state at −900.79 cm−1. A similar stretching vibration mode of H and C-C-O in TS2 could also be found  energy difference between 1a and 1b. The tautomeric reaction of 1a to 1b was a forward reaction while that of 1b to 1a was the reverse reaction. The activation energies (Ea) of the forward reaction and reverse reaction were 252.0 kJ·mol−1 and 241.5 kJ·mol−1, respectively. The results indicated that the tautomeric reaction had low activation energies and that their conversion proceeded easily.The curve of the energy change along the reaction pathway is shown in Fig. 2-OH on site C1 and -OAc on site C20 between 1a and 2a and between 1b and 2b was found in the experiment. To explain this observation, the theoretical transition states of the 1a⇌2a reaction (TS3) and 1b⇌2b reaction (TS4) were calculated as well. The structures of TS3 and TS4 are shown in Fig. 1a⇌2a and 1b⇌2b were obviously higher than those of 1a⇌1b and 2a⇌2b . The results are presented in Table The MTT test showed that the cytotoxicities of the four compounds against the five human cancer cell lines were very different. In the future, extensive studies should be conducted to reveal the structure-activity relationship of cytotoxic diterpenoids for the discovery of affective antitumour drugs.ent-kaurane skeleton diterpenes as 1a, 1b, 2a and 2b. The interconversion experiments between 1a and 1b and between 2a and 2b in methanol solutions confirmed that they exists as two pairs of tautomers. Further analysis using density functional theory showed that the tautomeric reaction was closely related to the existence of a transition state, the change in bond length and the level of conversion energy. It is thought that proton transfer was the major tautomerization reaction mechanism between each pair of diterpene tautomers. In addition, the four diterpenes exhibited potent cytotoxicities against three or more tested human cancer cells. The results could serve as a valuable reference for the tautomerization mechanism of other tautomers and the study of the structure-activity relationship on cytotoxicities as anticancer drug precursors.In this paper, we isolated and identified four new 7,20-non-epoxy-Optical rotation was measured using a SEPA-300 polarimeter . NMR spectra were recorded on a Bruker Advance III spectrometer . Orientation separation and HRESIMS data were acquired using a UPLC-LTQ orbitrap . Semi-preparative HPLC was performed on a Waters 600/Waters 2487  with a YMC (250 mm × 10 mm I.D. 5 μm) column. Column chromatography was performed either with silica gel , MCI gel CHP 20 P , or with ODS .I. excisoides were collected from Luanchuan County in Henan Province, China. The plant was authenticated by Xiao-Zheng Luo . A voucher specimen (No. 2011–0905) was deposited in the Laboratory of Research Center for Classic Chinese Medicines & Health Herbal Products.The aerial parts of I. excisoides (10 kg) were extracted 3 times with water (320 L × 1.5 h) at 100 °C and concentrated to 0.1 g/mL. The concentrate was subjected to a D-101 macroporous resin column and successively eluted with EtOH-H2O  to give four fractions (Fr. A-D). Fr. C  was purified with an MCI gel column (50 cm × 4 cm) and eluted with MeOH-H2O  to yield four subfractions (Fr. C1-C4). Fr. C2 (16 g) was subjected to a silica gel column and eluted with petroleum ether-Me2CO  to afford four fractions (Fr. C2a-C2d). Two tautomeric mixtures of Fr. 1 (1.0 g) and Fr. 2 (0.7 g) were crystallized from Fr. C2c using MeOH. 1a (45 mg) and 1b (30 mg) were isolated from the mixture of Fr. 1 (150 mg) by HPLC . The mixture of Fr. 2 (200 mg) was further purified by HPLC  to obtain 2a (42 mg) and 2b (31 mg) .The air-dried and powdered aerial parts of Rt : 27.46 min; infrared (IR) (cm−1): : 3472 , 2934 (C–H ʋ), 1730 (C=O ʋ), 1649 (C=C ʋ), 1368 (C–H ρ), 1272 (C–O ʋ), 1159, 1043 (C–C ʋ), 968 (=C–H ʋ), 868 (C–H ʋ). 1H (proton) and 13C (carbon) data are listed in Table 24H34O7 Na+, m/z 457.21967).White powder; UV (MeOH) λmax (log ɛ): 235 (3.8); TLC  Rf: 0.42; Rt : 29.31; infrared (IR) (cm−1): : 3445 , 2934 (C–H ʋ), 1731 (C=O ʋ), 1648 (C=C ʋ), 1368 (C–H ρ), 1242 (C–O ʋ), 1159, 1044 (C–C ʋ), 968 (=C–H ʋ), 866 (C–H ʋ). 1H (proton) and 13C (carbon) data are listed in Table 24H34O7 Na+, m/z 457.21967).White powder; UV (MeOH) λmax (log ɛ): 235 (3.8); Rt : 44.93; infrared (IR) (cm−1): : 3445 , 2935 (C–H ʋ), 1730 (C=O ʋ), 1648 (C=C ʋ), 1368 (C–H ρ), 1243 (C–O ʋ), 1158, 1044 (C–C ʋ), 968 (=C–H ʋ). 1H (proton) and 13C (carbon) data are listed in Table 24H34O7 Na+, m/z 457.21967).White powder; UV (MeOH) λmax (log ɛ): 235 (3.3); Rt : 48.38; Infrared (IR) (cm−1): : 3445 , 2934 (C–H ʋ), 1730 (C=O ʋ), 1648 (C=C ʋ), 1368 (C–H ρ), 1242 (C–O ʋ), 1158, 1044 (C–C ʋ), 968 (=C–H ʋ). 1H (proton) are 13C (carbon) data are listed in Table 24H34O7 Na+, m/z 457.21967).White powder; UV (MeOH) λmax (log ɛ): 235 (3.2); 1a, 1b, 2a and 2b were separately weighed, and each compound was divided into two equal portions, placed in amber sample vials, and dissolved in methanol to obtain sample solutions with concentrations of 1.0 mg/mL. The solutions were sealed and separately stored at 30 °C and 45 °C. Each sample solution was analysed by HPLC every hour until tautomerization occurred. Then, the analysis was performed every 24 h until equilibrium was achieved. Chromatographic analysis was performed on a Waters 2996/Waters 2487  equipped with a YMC C18 column  with acetonitrile-water (23–70) implemented as the mobile phase for isocratic elution. The flow rate was 1.0 mL·min−1, and the column temperature was 25 °C. The detection wavelength was 230 nm. The stability of 1a, 1b, 2a, 2b and the time taken to reach equilibrium under different temperatures were observed.Appropriate amounts of compounds 1a, 1b, 2a, and 2b and their transition states were calculated by means of the density functional theory (DFT) method at the 6–31 + g(d) level. The calculations were performed using the Gaussian 09 software package.Furthermore, the tautomerization mechanism of the two pairs of tautomeric diterpenoids was investigated, and transition state calculations for the reactions were conducted using density functional theory. Gaussview 5.0 was used to build the molecular structures of the tautomers. The transition state was found at the B3LYP/6–31 + g (d) level and was confirmed by vibration frequency analysis. Then, the minimum energy path (MEP) was determined by using intrinsic reaction coordinate (IRC) calculations, and the imaginary vibrational mode of the transition state was studied. The stability of compounds 2 at 37 °C. The cytotoxicity assay was performed according to the MTT method using 96-well microplates12.Five human cancer cell lines   were used for pharmacological experiments. All cells were cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum in a humidified atmosphere with 5% CO−5, 1 × 10−6, and 1 × 10−7 mol/L. The inhibitory rate of the cell growth was calculated according to the following formula: inhibition rate (%) = (OD control − OD treated)/OD control × 100. Finally, IC50 values were calculated using SPSS 16.0 statistical software.In the test, each tumour cell was exposed to the test compound at concentrations of 1 × 10Supplementary Information.


H17 cells, which are mediators of autoimmune conditions such as experimental autoimmune encephalomyelitis (EAE). RORγt also regulates thymocyte development and lymph node genesis. Here we show that the function of RORγt is regulated by its sumoylation. Loss of Sumo3, but not Sumo1, dampens TH17 differentiation and delays the progression of thymic CD8+ immature single-positive cells (ISPs). RORγt is SUMO3-modified by E3 ligase PIAS4 at lysine 31 (K31), and the mutation of K31 to arginine in mice prevents RORγt sumoylation, leading to impaired TH17 differentiation, resistance to TH17-mediated EAE, accumulation of thymic ISPs, and a lack of Peyer’s patches. Mechanistically, sumoylation of RORγt-K31 recruits histone acetyltransferase KAT2A, which stabilizes the binding of SRC1 to enhance RORγt transcription factor activity. This study thus demonstrates that sumoylation is a critical mechanism for regulating RORγt function, and reveals new drug targets for preventing TH17-mediated autoimmunity.RORγt controls the differentiation of T H17 cells, thymocyte development and lymphoid organogenesis. Here the authors show that the function of RORγt is regulated by PIAS4-mediated sumoylation that stabilize the interaction with SRC1 and KAT2A, to enhance the transcriptional activity of RORγt.The transcription factor RORγt is essential for the differentiation of T H17 cells, which secrete IL-17 and participate in both protective and pathological immunity1. The clearance of pathogens such as Citrobacter rodentium and fungus depends on robust protective TH17 immunity6. On the other hand, TH17 cells also mediate the pathological immune responses involved in autoimmune conditions, such as multiple sclerosis, colitis, and even autism, and the prevention of these conditions depends on inhibiting the formation and function of TH17 cells12. The critical function of RORγt has been demonstrated by severe immune deficiency in both mice13 and humans14 carrying mutated versions of the RORγt-encoding gene Rorc. In addition, RORγt enhances thymocyte survival and is thus essential for thymic T cell development. RORγt is also required for the biogenesis of secondary lymph tissues, including gut-associated Peyer’s patches18. Because RORγt is required for the generation of pathogenic TH17 cells responsible for autoimmunity, it is an attractive target for the development of drugs to control TH17-mediated immunological disorders20. It is thus important to understand the mechanisms regulating RORγt function.The transcription factor RORγt directs the differentiation of T22: an amino-terminal DNA-binding domain and a carboxyl-terminal ligand-binding domain. The very carboxyl terminal of the ligand-binding domain is an activation function 2 (AF2) motif responsible for recruiting steroid receptor coactivator 1 (SRC1) to nuclear receptors, which is required for RORγt-mediated transactivation of genes essential for TH17 differentiation25. Because RORγt is a transcription factor, previous studies have focused on the transcriptional aspects of RORγt function. However, the post-translational mechanisms that regulate RORγt function have long been neglected.As a member of the steroid nuclear receptor superfamily, RORγt has two conserved domains27 by regulating protein stability and by enabling new protein–protein interactions through the addition of new docking sites. Knockout of the E2 enzyme Ubc9 affects thymic T cell development and the expansion of regulatory T cells29, implicating sumoylation as an important regulator of these two processes. However, the roles of sumoylation in other aspects of T cell development and function, including TH17 differentiation, remain unknown.Sumoylation is a type of post-translational modification in which small ubiquitin-related modifier (SUMO) proteins are covalently attached to the lysines of target proteins. Mammals usually express three SUMO proteins: SUMO1, SUMO2, and SUMO3, which share approximately 50% amino acid sequence identity. Sumoylation is a multi-step reaction that is sequentially catalyzed by a SUMO-activating E1 enzyme, the single conjugating E2 enzyme Ubc9, and an E3 ligase. Sumoylation controls many aspects of cellular functionSumo3, but not Sumo1, impairs TH17 differentiation and delays progression of thymic immature single-positive (ISP) CD8+ cells, which are similar to phenotypes observed in Rorγt−/− mice. This work leads us to identify lysine 31 (K31) as a functional sumoylation site in RORγt. We find that mice expressing K31-mutant RORγtK31R are incapable of being sumoylated at K31 and exhibit multiple defective RORγt-dependent functions, including differentiation of TH17, induction of TH17-dependent experimental autoimmune encephalomyelitis (EAE), the progression of thymic ISP, and development of Peyer’s patches. Additional data attribute these effects to the defective recruitment of histone acetyltransferase KAT2A, which impairs the interactions between RORγt and co-activator SRC1. Finally, we identify the E3 ligase responsible for RORγt sumoylation to be PIAS (protein inhibitor of activated STAT) proteins form the largest family of sumoylation E3 ligases30, as the E3 ligase PIAS4 is able to bind and sumoylate RORγt at K31, and knockdown of PIAS4 impairs RORγt-dependent TH17 differentiation and progression of ISP, phenocopying the effects observed in RORγtK31R/K31R mice. Our study thus reveals sumoylation as a novel post-translational mechanism for regulating RORγt-dependent functions.Here, we demonstrate that the loss of Sumo1−/− and Sumo3-−/− CD4+ T lymphocytes . Deletion of Sumo1 compromised TH1 and Treg differentiation, but did not affect TH2 differentiation , CD4+CD8+ double-positive (DP), and CD4+ or CD8+ single-positive (SP). We did not detect obvious differences in the overall percentages of DN, DP, and SP populations in the thymi of WT versus Sumo1−/− mice , as well as a correspondingly lower percentage of mature TCRhiCD24loCD8+ cells, in the thymi of Sumo3−/−  did not affect SUMO3-RORγt levels, the K31R mutation (RORγtK11R) greatly reduced SUMO3-RORγt  failed to differentiate into TH17 cells. TH17 cell differentiation was rescued by WT RORγt and RORγtK11R, but not RORγtK31R , differentiated into CD4+CD8+ DP and CD4+ SP cells. However, retroviral expression of RORγtK31R failed to fully restore thymocyte development, indicated by more CD4−CD8− DN and CD8+ SP cells and fewer CD4+CD8+ DP and CD4+ SP cells   were cultured and stimulated with MOG35–55 in vitro under TH17- or TH1-polarizing conditions, and then adoptively transferred to Rag1−/− mice to induce EAE. Compared to their WT counterparts, RORγtK31R/K31R T cells that were expanded under TH17 conditions induced less severe EAE  in RORγtK31R/K31R thymi, similar to the frequency in Rorγt−/− thymi  K31 sumoylation-independent functions, including survival and cell cycle, and 2) K31 sumoylation-dependent functions, including the progression of ISPs and CD4 expression.To determine the function of K31 RORγt sumoylation in thymocyte development, we analyzed thymocytes from WT, ells Fig., but notells Fig., RORγt wice Fig. . These rymi Fig. . In addiymi Fig. , as wellymi Fig. . Not onlymi Fig. , suggesttro Fig. . As expeRORγtK31R/K31R thymocytes using ChIP-seq and RNA-seq assays. We observed similar expression patterns among biological replicates of WT or RORγtK31R/K31R thymocytes, as suggested by the color patterns in the clustered heat map shown in Fig. Rorγt−/− thymocytes were associated with significant changes in the expression of several important cell survival and cell cycle regulators33. The expression of several of these regulators, Pik3r3, Wee1, Bcl2, Myc, and Bcl2l1, was not significantly different between WT and RORγtK31R/K31R thymocytes , a histone acetyltransferase, is able to synergize with SRC1 to bind to nuclear receptorsbc9 Fig. . These rbc9 Fig. . On the lls Fig. , suggestion Fig.  and decr30. To identify the E3 responsible for RORγt sumoylation, we first monitored the interactions between RORγt and individual PIAS proteins  Sumo3−/− but not Sumo1−/− mice display defects in RORγt-regulated TH17 differentiation and thymocyte development ; (2) RORγt is SUMO3- but not SUMO1-modified at K31 in both TH17 cells and thymocytes; (3) mice expressing RORγtK31R exhibit multiple defective RORγt-dependent functions, including differentiation of TH17 cells, induction of TH17-mediated EAE, progression of ISPs in the thymus, and development of Peyer’s patches. We also identified PIAS4 as the E3 that catalyzes K31 sumoylation and regulates TH17 differentiation and progression of thymic ISPs. Therefore, we have demonstrated that post-translational sumoylation is a novel mechanism for modulating RORγt-dependent TH17 immunity that can be targeted by clinical therapies to enhance protective and inhibit pathogenic TH17 immunity.RORγt controls the function of T43. The ubiquitin E3 ligase, Itch, was found to bind and ubiquitinate RORγt for degradation and thus regulate TH17-dependent immune responses43, which explains why Itch−/− mice develop colitis. Another E3 ligase, UBR5, was also reported to regulate RORγt stability through the ubiquitin pathway42. However, the RORγt ubiquitination sites involved in the above two studies remain unknown. Meanwhile, we identified lysines 446 and 69 as ubiquitination sites through which RORγt-dependent TH17 differentiation can be controlled via degradation-independent mechanisms44. Therefore, we and others have demonstrated that TH17 immunity can be controlled through the ubiquitin pathway, which regulates RORγt stability and protein interactions. Although sumoylation can also regulate protein stability, our results do not support that K31 sumoylation affects RORγt stability. We showed that K31 sumoylation stimulates the recruitment of histone acetyltransferase KAT2A and co-activator SRC1 to RORγt. In addition, we showed that preventing K31 sumoylation reduces recruitment of SRC1 to the Il17f locus, suggesting that K31 sumoylation regulates the interaction between RORγt and its co-factors to activate Il17f expression.Previous studies have reported that RORγt function is regulated by ubiquitination, which is a post-translational modification similar to but distinct from sumoylation18. However, RORγt chromatin occupancy and target genes in thymocytes were not known, which limited understanding of the mechanisms responsible for RORγt-regulated thymocyte development. To address this need, we mapped genome-wide RORγt DNA-binding sites and identified RORγt target genes. Furthermore, we identified K31 as the sumoylation site of RORγt, which enabled us for the first time to dissect RORγt functions in thymus. One important function of RORγt is to regulate thymocyte survival by up-regulating anti-apoptotic Bcl-xL expression18. Our results showed that K31 sumoylation is actually not required to up-regulate Bcl-xL or to maintain thymocyte survival; however, it is specifically required for the progression of thymic ISPs. Our study thus separates RORγt functions and establishes a link between RORγt-regulated functions and RORγt target genes.RORγt has long been known to regulate thymocyte developmentH17 cells produce the effector cytokines IL-17A, IL-17F, IL-22, and GM-CSF to mediate pathological inflammation responsible for many types of autoimmune diseases; targeting TH17 cells is thus a potential treatment for these diseases45. Indeed, inhibiting the TH17 pathway is effective for treating psoriasis and multiple sclerosis47. Considering the essential function of RORγt in TH17 cells, pharmaceutical and academic scientists are developing RORγt inhibitors to treat TH17-dependent autoimmunity49. Unfortunately, such RORγt inhibitors can induce thymic lymphoma by inhibiting RORγt during thymocyte development50. Although K31 sumoylation is required for the progression of thymic ISP, it is not essential for regulating thymocyte survival or cell cycle progression, which are most likely responsible for the development of lymphoma observed in RORγt−/− mice51. Therefore, we expect that drugs targeting the K31 sumoylation pathway will inhibit TH17-mediated pathological immunity without interfering with thymocyte survival or cell cycle regulation, which could induce lymphoma in patients. Therefore, in addition to revealing a novel post-translational modification-based mechanism for regulating RORγt-dependent T cell function, our results also facilitate the development of a new category of RORγt-based drugs to treat TH17-mediated autoimmunity without serious side effects.TRORγtK31R/K31R mice were designed and generated by Biocytogen LLC. RORγtK31R/K31R mice are available at The Jackson Laboratory as Stock No. 032604. Rag1−/− (002216) mice were purchased from the Jackson Laboratory. The Rorγt−/− (RORC2−/−)18, Sumo1−/−52, and Sumo3−/− mice31 were bred and housed under specific pathogen-free (SPF) conditions in the Animal Resource Center at the Beckman Research Institute of City of Hope under protocols approved by the Institutional Animal Care and Use Committee. Mice were 10–12 weeks of age for EAE studies and 6–8 weeks of age for all other experiments, with littermates age-matched across experimental groups.Both the targeting vector and the knock-in Antibodies against RORγt , SRC1 , β-actin , GFP , KAT2A , HA , FLAG , SUMO1 , SUMO3 , and PIAS4  were used for immunoblot analysis. Phycoerythrin (PE)-indotricarbocyanine (Cy7)-conjugated anti-CD8 , PE-conjugated anti-RORγt , allophycocyanin (APC)-conjugated anti-IL-17A , PE-conjugated anti-Thy1.2 , PE-conjugated anti-CD24 , PE-conjugated anti-TCRβ , PE-indodicarbocyanine (Cy5)-conjugated anti-CD19 , PE-conjugated anti-CD11b , fluorescein isothiocyanate (FITC)-conjugated anti-CD4 , APC-conjugated anti-IL-4 , and APC-conjugated anti-Foxp3  antibodies were from eBioscience. Monoclonal antibodies against mouse CD3 (145-2C11), CD28 (37.51), IL-4 (11B11), IFN-γ (XMG1.2), and the p40 subunits of IL-12 and IL23 (C17.8), as well as PE-Cy7-conjugated anti-Ly6G , FITC-conjugated anti-IFN-γ , PE-conjugated anti-GM-CSF , FITC-Cy7-conjugated anti-CD45 , and PE-conjugated anti-CD25  antibodies, were purchased from BioLegend. Goat anti-hamster antibody was from MP Biomedicals. APC-conjugated anti-CD3  and FITC-conjugated anti-CD44  antibodies were from BD Pharmingen. Recombinant mouse IL-12, IL-4, IL-6, IL-23, and TGFβ were from Miltenyi Biotech. Recombinant mouse IL-2 was from Pepro Tech. The antibody against RORγt used for ChIP was a generous gift from Dan Littman at New York University.44. Point mutations of RORγt were generated using a site-directed mutagenesis kit from Agilent Technologies. pRK5-HA-ubiquitin , pCMV-sport2-mGCN5 , and constructs for expressing FLAG-PIAS  were obtained from Addgene. pCMV-FLAG-SUMO1, pCMV-FLAG-SUMO3, and pcDNA-UBC9 were generous gifts from Yuan Chen at the City of Hope. The LMP vector-based retroviral short hairpin RNA (shRNA)-expressing vectors were constructed using following oligonucleotide sequences: shKAT2A-1: TGCTGTTGACAGTGAGCGACCGCTATCTGGGCTACATCAATAGTGAAGCCACAGATGTATTGATGTAGCCCAGATAGCGGCTGCCTACTGCCTCGGA; shKAT2A-2: TGCTGTTGACAGTGAGCGCGCCAAGAATGCCCAAGGAATATAGTGAAGCCACAGATGTATATTCCTTGGGCATTCTTGGCATGCCTACTGCCTCGGA; shPIAS1-1: TGCTGTTGACAGTGAGCGAGGAACTAAAGCAAATGGTTATTAGTGAAGCCACAGATGTAATAACCATTTGCTTTAGTTCCGTGCCTACTGCCTCGGA; shPIAS1-2: TGCTGTTGACAGTGAGCGCCCGGATCATTCTAGAGCTTTATAGTGAAGCCACAGATGTATAAAGCTCTAGAATGATCCGGATGCCTACTGCCTCGGA; shPIAS4−1: TGCTGTTGACAGTGAGCGCGCTACAGAGGTTGAAGACGATTAGTGAAGCCACAGATGTAATCGTCTTCAACCTCTGTAGCATGCCTACTGCCTCGGA; shPIAS4-2: TGCTGTTGACAGTGAGCGCGAGCTGTATGAGACTCGCTATTAGTGAAGCCACAGATGTAATAGCGAGTCTCATACAGCTCTTGCCTACTGCCTCGGA.cDNA encoding RORγt or SRC1 was inserted into a XhoI/EcoRI-cut pMSCV vector+ T cells) or 5 ng/ml of recombinant IL-7 (for transducing CD4-CD8− thymocytes). Naïve CD4+ T cells were first activated with 0.25 µg/ml hamster anti-CD3  and 1 µg/ml hamster anti-CD28  in 24−well plates pre-coated with 0.2 mg/ml goat anti-hamster antibody for 24 h, then spin-infected with viral supernatants . The retroviral supernatant was also used to infect CD4−CD8− thymocytes that had been co-cultured with feeder OP9-DL4 cells  in the presence of recombinant IL-7 (5 ng/ml) for 24 h. After spin infection, the viral supernatant was replaced with culture media containing polarizing cytokines for in vitro differentiation (for transduced CD+ T cells) or 5 ng/ml of recombinant IL-7 for in vitro T cell development (for transduced CD4−CD8− thymocytes), as described below.Platinum-E packaging cells (Cell Biolabs) were plated in a 10-cm dish in 10 ml RPMI-1640 medium plus 10% FBS. 24 h later, cells were transfected with empty pMSCV or pLMP vectors or the appropriate retroviral expression plasmids with BioT transfection reagent (Bioland). After overnight incubation, the medium was replaced and cultures were maintained for another 24 h. Viral supernatants were collected 48 and 72 h later, passed through 0.4-μm filters (Millipore), and supplemented with 8 µg/ml of polybrene (Sigma-Aldrich) and 100 U/ml of recombinant IL-2 . Suspensions of 4 × 105 cells/ml Iscove’s modified DMEM (Cellgro) containing 2 mM L-glutamine, 50 mM 2-ME, 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% FBS were cultured in 24-well plates pre-coated with 0.2 mg/ml goat anti-hamster antibody for three days. The medium was supplemented with 0.25 µg/ml hamster anti-CD3, 1 µg/ml hamster anti-CD28, and polarizing cytokines: 2 ng/ml TGF-β, 20 ng/ml IL-6, 5 µg/ml anti-IL-4, and 5 µg/ml anti-IFNγ for TH17 differentiation; 20 µg/ml IL-12 and 5 µg/ml anti-IL-4 for Th1 differentiation; 10 ng/ml IL-4 and 10 µg/ml anti-IFNγ for TH2 differentiation; or 5 ng/ml TGF-β for Treg differentiation. For analysis, cells obtained from in vitro cultures were incubated for 4–5 h with 50 ng/ml PMA (Sigma-Aldrich), 750 ng/ml ionomycin (Sigma-Aldrich), and 10 µg/ml brefeldin A (BD Biosciences) in a tissue culture incubator at 37 °C, followed by intracellular cytokine staining.Naïve CD4−Thy1.2+CD4−CD8−populations were sorted using a FACSAria (BD Biosciences) and cultured at 5 × 105/ml overnight on an 80% confluent OP9-DL4 monolayer in flat-bottom 24-well culture plates with αMEM  supplemented with 20% FBS, 100 U/ml penicillin–streptomycin, 2 mM L-glutamine (Invitrogen Life Technologies), and 5 ng/ml recombinant murine IL-7. After 72 h, co-cultures were harvested for flow cytometry analysis.Thymocytes were stained with 7-AAD and antibodies against Thy1.2, CD4, and CD8. Specific 7-AAD+ T cells stimulated appropriately were stained for surface markers. Intracellular cytokines were stained with Fixation/Permeabilization solution . The expression of surface and intracellular markers were analyzed with FACSCanto (BD).Mouse thymi or spleens were homogenized by crushing with the head of a 1-ml syringe in a petri dish, followed by straining through a 40-μm nylon filter. Red Blood Cell Lysing buffer (Sigma-Aldrich) was used for red cell lysis. Cells isolated from thymi or spleens, co-cultures harvested from in vitro development, and CD4RORγtK31R/K31R mice, two separate samples were collected on different days, and thymocytes from four  were pooled each day. To determine the gene expression profile of TH17 cells, naive CD4+ T cells were enriched from WT or RORγtK31R/K31R mice and polarized under TH17 conditions for three days. Cells were processed for RNA isolation (Qiagen). Quality verification, library preparation, and sequencing were performed at the City of Hope Integrative Genomics Core Facility. Eluted RNAs were prepared for sequencing using Illumina protocols and sequenced on an Illumina HiSeq 2500 to generate 51-bp reads. Sequenced reads were aligned to the mouse mm10 reference genome using TopHat. Gene expression levels were quantified using HTSeq, and edgeR was utilized to identify differentially expressed genes (fold-change > 1.5 and FDR < 0.05). Gene expression abundance was quantified as fragments per kilobase of transcript per million fragments mapped (FPKM). Heat maps of differentially expressed genes were made with gplots using log2-transformed FPKM values.To measure gene expression in the thymi of WT or 7 cells were incubated with 1% formaldehyde to cross-link proteins with chromatin for 5 min at room temperature. 125 mM glycine was added to stop the cross-linking reaction. To shear genomic DNA into 200–500-bp fragments, cell lysates were sonicated using a water-bath sonicator (Covaris S200). Cell lysates were centrifuged  and incubated with specific antibodies (anti-RORγt from D. Littman or anti-SRC1 from Abcam) or IgG controls and protein A/G beads (Millipore). After extensive washing, DNA was eluted followed by reversion of the protein–DNA cross-linking. DNA was recovered for sequencing or qRT-PCR to quantify specific DNA fragments that were precipitated. Primers used for qRT-PCR are listed in Supplementary Table http://www.novocraft.com/). TDF files were generated for visualization on the Integrative Genomics Viewer53. The enrichment of RORγt binding sites across the genome was analyzed using MACS2 with ‘—nomodel—extsize 150’54. The irreproducible discovery rate (IDR) framework was utilized find reproducible peaks across replicates. Enriched known TF motifs in ChIP-seq peaks were identified by using HOMER (findMotifsGenome.pl)55.A total of 2 × 10β-actin, and all measurements were performed in triplicate.qRT-PCR was performed using SsoFast EvaGreen Supermix (Bio-Rad) in a CFX96 Real-Time PCR Detection System (Bio-Rad), using the primers listed in Supplementary Table RORγt−/−, or RORγtK31R/K31R mice and cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin–streptomycin, and 2 mM L-glutamine at 1 × 106 cells/ml. Thymocytes were incubated at 37 °C with 5% CO2. Dead cells were detected using Annexin V-PE and 7-AAD staining (BD Bioscience).Thymocytes were freshly isolated from WT, 35–55) peptide emulsion. On days 0 and 1 after immunization, mice were injected intraperitoneally with 200 ng Bordetella pertussis toxin. For TH17- or TH1-induced passive EAE, donor mice were immunized with MOG35–55 subcutaneously. 10 days later, cells were isolated from the spleen and lymph nodes and cultured with 20 µg/ml MOG35–55 for 3 days under either TH17-polarizing conditions (20 ng/ml rmIL23) or TH1-polarizing conditions . Rag1−/− recipient mice were then intraperitoneally transferred 3.0 × 107 MOG35–55-specific TH17 or TH1 cells. The severity of EAE was monitored and evaluated on a scale from 0 to 5 according to the Hooke Laboratories guidelines: 0 = no disease; 1 = paralyzed tail; 2 = hind limb weakness; 3 = hind limb paralysis; 4 = hind and forelimb paralysis; and 5 = moribund and death. When a mouse was euthanized because of severe paralysis, a score of 5 was entered for that mouse for the rest of the experiment.Active EAE was induced using an EAE induction kit, according to the manufacturer’s instructions . Briefly, mice were subcutaneously immunized with a 200-ml myelin oligodendrocyte glycoprotein 35–55  and heated at 90 °C for 10 min. Samples were then diluted (1:10) with lysis buffer and incubated with anti-RORγt at 4 °C overnight. Enrichment of ubiquitinated proteins was performed as previously described44. Briefly, cell lysates were incubated with equilibrated Agarose-coupled Tandem Ubiquitin Binding Entity 1 (Agarose-TUBE1) (LifeSensors) at 4 °C for 4 h. After incubation, beads were washed four times with lysis buffer, resolved using SDS-PAGE, and analyzed using Western blot.Cells were lysed in lysis buffer  supplemented with protease inhibitor cocktail (Sigma) and 1 mM PMSF. Cell extracts were incubated overnight with 1 µg of the relevant antibodies, and proteins were immunoprecipitated for an additional 1 h at 4 °C with protein A/G-Sepharose beads (milipore). To detect sumoylation, transfected HEK293T cells, primary thymocytes, or polarized Tt-tests and one-way analysis of variance (ANOVA) were used to compare experimental groups. A P-value of less than 0.05 was considered statistically significant.Prism software (GraphPad) was used for all statistical analyses. Two-tailed unpaired Student’s Supplementary InformationPeer Review file


Scientific Reports 10.1038/s41598-018-32862-9, published online 27 September 2018Correction to: In Figure 3C, the horizontal axis is incorrectly labelled as ‘Position (µm)’. This should read ‘Position (nm)’. The correct Figure 3C appears below as Figure


The synthesis and structures of a series of 6-aryl-1,3-diphenylfuvlenes with  varying methylation patterns on the 6-phenyl substituent are reported. A network of C—H⋯π ring interactions consolidates the packing in each structure. 25H20), 6-(4-methylphenyl)-1,3-diphenylfulvene (C25H20), 6-mesityl-3-diphenylfulvene (C27H24) and 6--1,3-diphenylfulvene (C29H28). The bond lengths are typical of those observed in related fulvenes. A network of C—H⋯π ring interactions consolidates the packing in each structure.The synthesis and crystal structures of a series of 6-arylfuvlenes  with varying methylation patterns on the 6-phenyl substituent are reported, namely 6-(3-methylphenyl)-1,3-diphenylfulvene (C In compound II, the phenyl substituents are rotated 31.83 (5), 20.92 (5) and 35.13 (5)° from the cyclopentadiene core for the 1-phenyl, 3-phenyl, and 6-phenyl substituents, respectively. For compound III, the phenyl substituents are rotated an average of 21.33 (13), 38.02 (13) and 57.22 (14)° from the cyclopentadiene core for the 1-phenyl, 3-phenyl, and 6-phenyl substituents, respectively. In fulvene IV, each phenyl ring is rotated further from the core of the fulvene molecule, likely because of the additional steric interactions provided by the pentamethylphenyl substituent. The phenyl substituents are rotated by 41.65 (7), 25.17 (7) and 64.15 (7)° from the cyclopentadiene core for the 1-phenyl, 3-phenyl, and 6-phenyl substituents, respectively.For fulvene I–IV is consolidated through a series of C—H⋯π ring interactions. In I, each molecule participates in C—H⋯π ring interactions with six other fulvene molecules. Each molecule acts as a C—H donor through the hydrogen atoms in the para position of each phenyl substituent, H10 and H16, as well as a meta hydrogen atom, H23, from the 6-(3-methylphenyl) substituent. Additionally, the π ring of the 3-phenyl and 6-(3-methylphenyl) substituents accept C—H interactions, with the latter accepting donations from both sides of the ring  substituents act as C—H acceptors, with the former accepting donations from both sides of the ring  substituent, H27C, serve as C—H donors for two separate fulvene molecules. The 6- π ring accepts C—H donation from two additional molecules fulvene (I) To a vigorously stirred solution of 1,3-diphenylcyclopentadiene  in absolute EtOH (25 ml), 3-methylbenzaldehyde  and pyrrolidine  were added. The reaction mixture was allowed to stir at room temperature for 22 h. The precipitate from the reaction mixture was vacuum filtered, washed with cold absolute EtOH (3 × 30 ml), and vacuum dried to give I as a dark-red solid . Red prisms suitable for single-crystal X-ray diffraction were obtained from diethyl ether solution by slow evaporation.1,3-diphenyl-6-(4-methylphenyl)fulvene (II) To a vigorously stirred solution of 1,3-diphenylcyclopentadiene  in absolute EtOH (8 ml), 4-methylbenzaldehyde  and pyrrolidine  were added. The reaction mixture was allowed to stir at room temperature for 24 h. The precipitate from the reaction mixture was vacuum filtered, washed with cold absolute EtOH (3 × 30 ml), and vacuum dried to give II as a dark-red solid . Red prisms suitable for single-crystal X-ray diffraction were obtained from diethyl ether solution by slow evaporation.3-diphenyl-6-mesitylfulvene (III) To a vigorously stirred solution of 1,3-diphenylcyclopentadiene  in absolute EtOH (50 ml), mesitylaldehyde  and pyrrolidine  were added. The reaction mixture was allowed to stir at reflux for 24 h. The reaction mixture was cooled to 278 K and the resulting precipitate was vacuum filtered, washed with cold absolute EtOH (3 × 30 ml), and vacuum dried to give III as a red–orange solid . Irregular red crystals suitable for single-crystal X-ray diffraction were obtained from pentane solution by slow evaporation.1,3-diphenyl-6-fulvene (IV) To a vigorously stirred solution of 1,3-diphenylcyclopentadiene  in absolute EtOH (50 ml), 2,3,4,5,6-pentamethylbenzaldehyde  and pyrrolidine  were added. The reaction mixture was allowed to stir at room temperature for 24 h. The precipitate from the reaction mixture was vacuum filtered, washed with cold absolute EtOH (3 × 30 ml), and vacuum dried to give IV as an orange solid . Orange needles suitable for single-crystal X-ray diffraction were obtained from ethyl acetate solution by slow evaporation.Uiso(H) = 1.2–1.5Ueq(C). The absolute structure of III was indeterminate in the present refinement. Compound III was refined as an inversion twin.Crystal data, data collection and structure refinement details are summarized in Table 610.1107/S205698901900700X/hb7824sup1.cifCrystal structure: contains datablock(s) I, II, III, IV. DOI: 10.1107/S205698901900700X/hb7824Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S205698901900700X/hb7824IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S205698901900700X/hb7824IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S205698901900700X/hb7824IVsup5.hklStructure factors: contains datablock(s) IV. DOI: Click here for additional data file.10.1107/S205698901900700X/hb7824Isup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S205698901900700X/hb7824IIsup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S205698901900700X/hb7824IIIsup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S205698901900700X/hb7824IVsup9.cmlSupporting information file. DOI: 1916092, 1916091, 1916090, 1916089CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Burkholderia cenocepacia TssA indicates a quaternary structure of 32 subunits in D16 symmetry.Analysis of the noncrystallographic symmetry of crystals of the C-terminal domain of  Burkholderia cenocepacia, a representative of clade 3, were expressed, purified and subjected to crystallization trials. Data were collected from crystals of constructs of the N-terminal and C-terminal domains. Analysis of the data from the crystals of these constructs and preliminary structure determination indicates that the C-terminal domain forms an assembly of 32 subunits in D16 symmetry, whereas the N-terminal domain is not involved in subunit assocation.TssA is a core component of the type VI secretion system, and phylogenetic analysis of TssA subunits from different species has suggested that these proteins fall into three distinct clades. Whilst representatives of two clades, TssA1 and TssA2, have been the subjects of investigation, no members of the third clade (TssA3) have been studied. Constructs of TssA from  Fractions containing the B.c His6-TssA Nt1 protein were concentrated using a 30 kDa molecular-weight cutoff concentrator and washed with 10 mM Tris pH 8.0 for crystallization.A B.c HisE. coli ER2523 cells (NEB Express) grown in Lennox broth containing 0.2% glucose , 2 × 500 ml of cells were grown as described above and harvested prior to induction. The cell pellets were then resuspended in selenomethionine minimal medium, harvested and the resuspension was added to 2 × 500 ml selenomethionine minimal medium  supplemented with 1.0 g l−1 MgSO4·7H2O, 4.0 mg l−1 thiamine, 100 mg l−1 each of l-lysine, l-phenylalanine and l-threonine, 50 mg l−1 each of l-isoleucine, l-leucine and l-valine, 40 mg l−1 seleno-l-methionine and 2.0 g l−1 glucose. Growth was continued for 1 h at 37°C prior to the induction of B.c MBP-TssA CTD expression by the addition of 0.3 mM IPTG and subsequent growth at 37°C overnight.The B.c MBP-TssA CTD fusion protein was overproduced in M Tris–HCl pH 8.0, 200 mM NaCl) and the cells were lysed by sonication. The cell lysate was cleared by centrifugation before being applied onto amylose resin (NEB) equilibrated with column-binding buffer. After washing the resin with column-binding buffer, the bound material was then eluted in the same buffer with the addition of 10 mM maltose. Fractions containing the fusion protein were concentrated and then digested with Factor Xa  in the presence of 2 mM CaCl2 at room temperature for ∼14 h to release the target protein from MBP. Following cleavage of the MBP, the products were separated by gel filtration on a Superdex 200 column  in 50 mM Tris–HCl pH 8.0, 500 mM NaCl. The B.c TssA CTD protein obtained from gel filtration was applied to a final amylose column to remove any uncleaved B.c MBP-TssA CTD fusion molecules. This step was added to avoid heterogeneity of the sample. The protein was then concentrated and buffer-exchanged into 5 mM Tris–HCl pH 8.0, 50 mM NaCl for crystallization. The purified B.c TssA CTD protein has an additional four non-native residues (ISHM) at the N-terminus following proteolytic release from the N-terminal MBP solubility tag . B.c His6-TssA Nt1 crystals grew in sitting-drop trial conditions consisting of 0.16 M calcium acetate, 0.08 M sodium cacodylate buffer pH 6.5, 14.4%(w/v) PEG 8000, 20%(v/v) glycerol. B.c TssA CTD produced crystals in sitting-drop trial conditions consisting of 0.1 M sodium chloride, 0.1 M Tris pH 8.0, 15%(v/v) ethanol, 5%(v/v) MPD. B.c SeMet-TssA CTD produced crystals in hanging-drop trial conditions consisting of 1 M ammonium sulfate, 2%(w/v) PEG 3350, 0.1 M bis-tris pH 6.0. Crystallization information is shown in Table 2B.c TssA protein constructs were set down into sitting-drop crystallization trials using a Matrix Hydra II Plus One robot, with 200 nl:200 nl drops and a 50 µl reservoir, or hanging-drop crystallization trials, and stored at 17°C. A selection of crystallization condition suites were used for screening, including JCSG+, ProPlex, PACT, Classics, MPD, AmSO2.3.6-TssA Nt1 crystals  on beamline I04-1 at Diamond Light Source. The data were processed to 1.87 Å resolution using the -3daii option in xia2 . Data-collection and processing statistics are shown in Table 3Data were collected from B.c HisM sodium chloride, 0.1 M Tris pH 8.0, 15%(v/v) ethanol, 25%(v/v) glycerol] on beamline I04 at Diamond Light Source. Data were processed to 3.79 Å resolution using the -3daii option in xia2  PEG 8000, 30%(v/v) glycerol and data collection on beamline I03 at Diamond Light Source. The data were processed to 2.01 Å resolution with fast_dp . Data-collection statistics are shown in Table 3B.c HisM ammonium sulfate, 3–4%(w/v) PEG 3350, 0.1 M bis-tris pH 6.0, 30%(v/v) glycerol] on beamline I03 at Diamond Light Source. The data were processed to 2.86 Å resolution using the -3d option in xia2 . Data-collection statistics are shown in Table 3Data were collected from B.c SeMet-TssA CTD crystals [cryoprotected in 1–1.2 3.3.1.The design of the constructs for the N- and C-terminal domains of B.c TssA was based on an analysis of the pattern of sequence conservation across the different clades of TssA and resulted in stable, soluble samples of the proteins encoded by these regions of the gene. Constructs of the predicted N-terminal domain (Nt1) and C-terminal domain (CTD) of B.c TssA produced crystals Fig. 1 which di3.2.6-TssA Nt1 crystals suggests that the crystals contain one molecule of ∼30.7 kDa in the asymmetric unit, with a VM of 2.31 Å3 Da−1 (Matthews, 1968et al., 2011VM suggests that the asymmetric unit contains between nine and 21 subunits (4.13–1.77 Å3 Da−1; Matthews, 1968et al., 2011et al., 2016et al., 2016bc plane every 11.25° (Fig. 2D16 symmetry. This suggests that the asymmetric unit contains eight subunits with an unusually high VM (4.64 Å3 Da−1; Matthews, 1968et al., 20116-TssA Nt1 crystals (Fig. 3Analysis of the Matthews coefficient of the B.c His5° Fig. 2. Conside5° Fig. 2, suggestls Fig. 3. Refinem