The two N atoms and two O atoms around the CuII atom are trans to each other, with an O—Cu—O bond angle of 177.00 (9)° and an N—Cu—N bond angle of 165.63 (10)°. The average distances between the CuII atom and the coordinated O and N atoms are 1.905 (2) and 1.995 (2)Å, respectively.In the title complex, [Cu(C DOI: 10.1107/S1600536810037888/rk2236Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


II complex, [Ni(C9H10NO2)2(C10H14N2O)2(H2O)2], contains two dimethylaminobenzoate (DMAB), two diethylnicotinamide (DENA) ligands and two water molecules, all of them monodentate. The four O atoms in the equatorial plane around the NiII atom form a slightly distorted square-planar arrangement, while the slightly distorted octahedral coordination is completed by the two pyridine N atoms of the DENA ligands in axial positions. The NiII atom is displaced by 0.681 (1) Å out of the least-squares plane of the carboxylate group. The dihedral angle between the carboxylate group and the adjacent benzene ring is 5.61 (7)°, while the pyridine and benzene rings are oriented at a dihedral angle of 73.20 (4)°. An intramolecular O—H⋯O hydrogen bond results in the formation of a six-membered ring with a twisted conformation. In the crystal structure, intermolecular O—H⋯O and C—H⋯O hydrogen bonds link molecules into a three-dimensional network. Two weak C—H⋯π interactions are also present.The centrosymmetric title Ni N,N-diethylnicotinamide, an important respiratory stimulant, see: Bigoli et al. 2(C10H14N2O)2(H2O)2] = 0.029                           wR(F                           2) = 0.076                           S = 1.04                           4819 reflections253 parametersH atoms treated by a mixture of independent and constrained refinementmax = 0.47 e Å−3                        Δρmin = −0.41 e Å−3                        Δρ                                 APEX2  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008ORTEP-3 for Windows (Farrugia, 1997WinGX (Farrugia, 1999PLATON (Spek, 2009Data collection: 10.1107/S1600536809030098/xu2570sup1.cif            Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809030098/xu2570Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The Pt—P bond lengths of 2.2536 (8) and 2.2513 (8) Å and the Pt—Cl bond lengths of 2.3750 (8) and 2.3588 (8) Å are normal. This crystal form is a polymorph of a structure reported previously .The title complex, [PtClasai 1976. Bull. C DOI: 10.1107/S1600536808000603/pv2062Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Neighboring CoII atoms are linked together by two Cl bridges, forming a dinuclear CoII complex with inversion symmetry. There are intermolecular O—H⋯Cl hydrogen bonds and intermolecular π–π stacking interactions between adjacent bpy ligands [centroid–centroid distance = 3.617 (2) Å] in the structure.The title complex, [Co DOI: 10.1107/S1600536809027846/at2843Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The complexes are assembled into a three-dimensional network via C—H⋯O, C—H⋯π and π–π interactions. The mean interplanar distance between adjacent phenanthroline ligands is 3.399 (2) Å.In the crystal structure of the title compound, [Zn(C DOI: 10.1107/S1600536808006004/is2271Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The CoII cation is coordinated by two Cl− ions, one N atom from the 5-(4-pyridinio)tetrazolate zwitterion and three O atoms from three water molecules in a distorted octahedral geometry. In the crystal, molecules are linked into a three-dimensional network by N—H⋯Cl hydrogen bonds and O—H⋯O/N/Cl hydrogen bonds involving both coordinated and uncoordinated water molecules. Strong π–π stacking is present between parallel pyridinium and tetrazolate rings [centroid–centroid distances = 3.411 (2) and 3.436 (2) Å].The title compound, [CoCl DOI: 10.1107/S1600536809024337/ci2831Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Both the cationic and anionic portions of the starting proton-transfer compound are involved in the complexation. The NiII atom has a distorted octahedral geometry and is hexacoordinated by three O atoms and three N atoms from one phen fragment (as a bidentate ligand), one (pydc)2− unit (as a tridentate ligand) and one water molecule. In the crystal structure, extensive O—H⋯O, O—H⋯N and C—H⋯O hydrogen bonds with D⋯A distances ranging from 2.573 (2) to 3.385 (2) Å, π–π interactions between the phen ring systems  and intermolecular C—O⋯π interactions  connect the various components together.The title compound, [Ni(C DOI: 10.1107/S1600536808039378/om2263Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The other NiII ion is also six-coordinated, by four other pyridine N atoms from four other aminopyridine ligands and two carbonate O atoms to complete a distorted octahedral geometry. In the crystal structure, molecules are linked into an infinite three-dimensional network by O—H⋯O, N—H⋯Cl, N—H⋯O, O—H⋯N, C—H⋯O, C—H⋯N and C/N—H⋯π interactions involving the pyridine rings.In the title compound, [Ni DOI: 10.1107/S1600536808033321/at2653Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The apical Cu—O bond [2.3520 (16) Å] is much longer than the basal Cu—O and Cu—N bonds [1.9139 (14)–2.0136 (17) Å]. The carboxylate group bridges CuII atoms, forming a zigzag chain along the a axis.In the title compound, [Cu(C DOI: 10.1107/S1600536809037520/is2450Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The MoVI atom shows a distorted octahedral environment, with the phenanthroline N atoms and the two oxide groups forming the equatorial plane and the F atoms occupying the apical positions. Weak C—H⋯O and C—H⋯F hydrogen-bonding contacts and π–π interactions [centroid–centroid distance = 3.662 (1) Å] connect the complex molecules into a three-dimensional supramolecular framework.The title compound, [MoF DOI: 10.1107/S1600536809031626/si2192Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


A short intramolecular C—H⋯π interaction is observed involving a substituted η5-C5H4 ring and an ortho H atom of the benzene ring on the opposite side of the molecule. In the crystal structure, there are no classical hydrogen bonds: interactions comprise a short C6—H⋯π(C6) interaction involving substituted benzene rings and two C—H⋯O=C interactions per molecule.The title compound, [Fe DOI: 10.1107/S1600536809006278/bg2237Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials: interactive version of Fig. 2            Enhanced figure:


There is a mean deviation of 0.016 (4) Å from the least-squares plane defined by the nine constituent benzofuran atoms. The crystal structure is stabilized by aromatic π–π interactions between the benzene rings of neighbouring molecules [centroid–centroid distance = 3.689 (7) Å]and by a weak C—H⋯π interaction between an H atom of the methylene group bonded to the carboxylate O atom and the benzene ring of an adjacent molecule. In addition, the crystal structure exhibits weak non-classical intermolecular C—H⋯O hydrogen bonds. The chloroethyl group is disordered over two positions, with refined site-occupancy factors of 0.767 (6) and 0.233 (6).In the title compound, C DOI: 10.1107/S1600536809012847/sj2602Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Intermolecular C—H⋯Cl hydrogen bonds and π–π stacking interactions between the pyridine rings [centroid–centroid distances = 3.788 (1) and 3.957 (1) Å] are present in the crystal structure.In the title compound, [CoCl DOI: 10.1107/S1600536810036846/hy2353Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The terpyridyl motif in each fctpy ligand is coplanar, but the cyclopentadienyl ring is twisted by 9.5 (2)° out of coplanarity with each central pyridine. The two cyclopentadienyl rings of the ferrocenyl group are almost eclipsed with a deviation of 4.5 (1)°. In addition, intermolecular π–π interactions [centroid–centroid distance 3.753 (2) Å] are present between the cyclopentadienyl and outer pyridyl rings of the fctpy ligands. One of the perchlorate anions is equally disordered over two positions.In the title complex, [FeZn(C DOI: 10.1107/S1600536809023939/jh2081Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


It differs from the first orthorhombic polymorph [Pan, Lin & Zheng  ligand. In the present structure, the Cu atom is shifted from the mean plane of the dmp ligand by only 0.005 (1) Å, compared with 0.318 (6) Å in the orthorhombic form. Hydrogen-bonding and π–π stacking interactions (mean interplanar distance of 3.59 Å in the title compound) in the two different polymorphs are both essential to the supramolecular assembly.A new monoclinic polymorphic form of the title compound, [Cu(HCOheng 2005. Z. Kris DOI: 10.1107/S1600536808022812/fj2128Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Intermolecular O—H⋯O and N—H⋯O hydrogen bonding, as well as π–π stacking between parallel thiazole rings [centroid–centroid distance 3.531 (8) Å], helps to stabilize the crystal structure.In the crystal structure of the title compound, [Ni(C DOI: 10.1107/S1600536809017978/xu2518Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Together the ligands create a slightly distorted square-planar cordination environment for the Rh(I) atom. There are three molecules in the asymmetric unit and intramolecular P—Rh—P bite angles of 82.78 (5), 82.97 (6) and 83.09 (5)° are observed. The dihedral angles between the P—Rh—P and the X—Rh—X planes (X is the centroid of a double bond) are 14.7 (1), 14.8 (1) and 15.3 (1)°. The structure exhibits disorder of one cyclooctadiene ligand as well as one BF4 anion.The title compound, [Rh(C DOI: 10.1107/S1600536810019859/im2198Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


It was synthesized from 2,4,6,8-tetraphenyl-3,7-diazabicyclo[3.3.1]nonan-9-one and was crystallized from a methanol–ethanol solution over two years as a racemate. The C=N double bond [1.2868 (15) Å] is bent significantly out of the plane of the aromatic bicyclic ring system [N—C—C—C = −157.63 (12)°] and out of the plane of the phenyl ring bonded at the 1-position [N—C—C—C = 41.15 (16)°].The title compound, C DOI: 10.1107/S1600536810013619/bt5246Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Intramolecular C—H⋯O hydrogen bonding and intermolecular π–π stacking between parallel pyridine rings [centroid–centroid distance = 3.658 (3) Å] are present in the crystal structure.In the title compound, [CdI DOI: 10.1107/S1600536810012572/xu2734Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The anion is a six-coordinated complex with a distorted octahedral geometry around the FeIII atom. Extensive intermolecular O—H⋯O, N—H⋯O and C—H⋯O hydrogen bonds, involving the complex anion, (pydaH)+ counter-ion and two uncoordinated water molecules, and π–π [centroid-to-centroid distance 3.323 (11) Å] and C—O⋯π [O–centroid distance 3.150 (15) Å] interactions connect the various components into a supramolecular structure.The reaction of iron(II) sulfate heptahydrate with the proton-transfer compound (pydaH)(hypydcH) (pyda = pyridine-2,6-diamine; hypydcH DOI: 10.1107/S1600536808029280/hy2152Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The pyrrolidine ring is almost perpendicular to the cyclopentanone ring, making a dihedral angle of 81.91 (6)°. The molecular conformation is stabilized by an intramolecular O—H⋯N hydrogen bond and C—H⋯O interactions. The crystal structure is stabilized by intermolecular C—H⋯O interactions.In the title compound, [Fe(C DOI: 10.1107/S1600536809018583/bt2949Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The CuII center has a distorted square-pyramidal coordination with three N atoms of the 4′-ferrocenyl-2,2′:6′,2′′- terpyridine (fctpy) ligand and one 1,10-phenanthroline (phen) N atom in the basal plane and a second phen N atom in the apical position with an axial distance of 2.254 (4) Å. The disordered ClO4               − anion is weakly coordinated to the CuII ion with a Cu—O distance of 2.766 (11) Å. The two cyclopentadienyl rings of the ferrocenyl group are almost eclipsed with a deviation of 4.7 (1) °, and are involved in intermolecular π–π interactions with the outer pyridyl rings of the fctpy ligands [centroid–centroid distance = 3.759 (2) Å.].The title complex, [CuFe(C DOI: 10.1107/S1600536809020753/at2803Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The bis(diphenylarsino)methane ligand bridges an Ru—Ru bond and the monodentate phosphine ligand bonds to the third Ru atom. Both the phosphine and arsine ligands are equatorial with respect to the Ru3 triangle. In addition, each Ru atom carries one equatorial and two axial terminal carbonyl ligands. With regard to the three phosphine-substituted rings, the benzyl ring makes dihedral angles of 41.0 (3) and 43.9 (3)° with the other two benzene rings in molecule A; these angles are 49.8 (3) and 56.8 (3)° in molecule B. The dihedral angles between the two benzene rings are 76.1 (3) and 88.2 (3)° for the two diphenylarsino groups in molecule A and 71.3 (3) and 78.1 (3)° in molecule B. In the crystal packing, molecules are linked into chains via intermolecular C—H⋯O hydrogen bonds. Weak intermolecular C—H⋯π interactions further stabilize the crystal structure.The asymmetric unit of the title  DOI: 10.1107/S1600536809053045/sj2709Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The average Ni—N and Ni—O bond lengths are 2.131 (13) and 2.098 (11) Å, respectively. An intramolecular N—H⋯O interaction occurs. Relatively weak intermolecular C—H⋯O interactions between the ligands and the ClO4               − ions result in a chain extending along the b axis.In the title complex, [Ni(CH DOI: 10.1107/S1600536810034446/pv2321Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The Co—N bonds involving the thiocyanate ligands are significantly shorter than the other two Co—N bonds. The atoms of one of the ethoxy groups are essentially coplanar with the phenanthroline ring [N=C—O—C = 178.8 (4)°], while the other ethoxy group is slightly twisted from the phenanthroline ring plane [N=C—O—C = 167.2 (4)°]. In the crystal structure, there is a weak π–π stacking interaction between two symmetry-related phenanthroline rings with a centroid–centroid distance of 3.706 (4) Å.In the title complex, [Co(NCS) DOI: 10.1107/S160053680803496X/lh2718Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The coordination bonds of the 4,4′-substituted bipyridyl donors [Ru—N = 2.038 (3) and 2.051 (3) Å] are shorter than those of the 2,2′-bipyridyl donors [Ru—N1 = 2.065 (3)–2.077 (3) Å], due to the electron-withdrawing effects of the substituents at the 4,4′-positions. The angles between the pyridyl planes of the three bipyridyl ligands are 1.5 (2), 6.3 (3) and 8.7 (2)°, respectively. The cations are connected by anions via N—H⋯F interactions.In the title compound, [Ru(C DOI: 10.1107/S1600536809002360/kp2198Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The [ZnCl4]2− anions have a distorted tetrahedral geometry. The dihedral angles between the isoquinoline rings of the two cations are nearly equal [16.1 (2) and 16.3 (2)°]. In the crystal structure, the ordered linear formation is aggregated by weak intermolecular π–π stacking interactions between neighboring isoquinoline pyridine rings with a centroid–centroid distance of 3.779 (4) Å.The asymmetric unit of the title compound, (C DOI: 10.1107/S1600536809017693/bq2137Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The geometry of the resulting CuN2O4 coordination can be described as distorted octahedral. The the two pydc2− fragments are almost perpendicular to one another [77.51 (11)°]. To balance the charges, two centrosymmetric protonated butane-1,4-diammonium, (bdaH2)2+ cations are present. In the crystal structure, extensive O—H⋯O, N—H⋯O and C—H⋯O hydrogen bonds [D⋯A = 2.720 (2)–3.446 (3) Å], ion pairing, C—O⋯π [O⋯π = 3.099 (2) Å] and π–π stacking interactions between the pydc2− rings [centroid–centroid distance = 3.5334 (15) Å] contribute to the formation of a three-dimensional supramolecular structure.In the title compound, (C DOI: 10.1107/S1600536808011938/su2053Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


In the crystal structure, N—H⋯O hydrogen bonds link molecules in rows along a. Short intermolecular Cl⋯Cl interactions [3.4225 (5) Å] link these rows into sheets in the ac plane. Additional weak C—H⋯O and C—H⋯π interactions generate a three-dimensional network.In the title benzamide derivative, C DOI: 10.1107/S1600536808036313/sg2272Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The metal centre has a distorted octahedral coordination with the monoanionic Schiff base ligand occupying one equatorial and two axial coordination positions. The separation between V atoms is 3.214 (3) Å. In the crystal structure, there are N—H⋯O, C—H⋯O and C—H⋯π hydrogen bonds, and π–π interactions.In the title dinuclear compound, [V DOI: 10.1107/S160053680801787X/xu2429Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The crystal structure is stabilized by aromatic π–π interactions between the benzene rings of neighbouring molecules [centroid-to-centroid distance = 3.635 (3) Å], and by C—H⋯π interactions between a propyl methylene H atom and the furan ring of an adjacent molecule. In addition, the crystal structure exhibits weak intermolecular C—H⋯O hydrogen bonds.In the title compound, C DOI: 10.1107/S1600536809004735/gk2190Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Interactions of the π–π type are absent between cations in the stacks [centroid–centroid separation = 5.01 (5) Å]. Significant intermolecular Br–aryl interactions are present in the structure, especially an unusually short Br–ring centroid interaction of 3.78 (1) Å. The coordination geometry of the anion is approximately tetrahedral and a twofold rotation axis passes through the Co atom.In the crystal structure of the title compound, (C DOI: 10.1107/S160053680800439X/at2543Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The 2-fluorobenzoate ligands bridge Pb atoms, giving rise to a one-dimensional chain structure extending along the [100] direction. The polymeric chains are connected via C—H⋯O hydrogen bonds and π–π interactions, with an interplanar distance of 3.46 (1) Å. An intramolecular O—H⋯F interaction is also present.In the title compound, [Pb DOI: 10.1107/S1600536808022678/hy2143Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Face-to-face π-stacking interactions between inversion-related pyridine rings with centroid–centroid distances of 3.548 (3) and 3.662 (3) Å (perpendicular distances between the respective rings are 3.314 and 3.438 Å) are found. Intermolecular O—H⋯O hydrogen bonds between water molecules and L ligands form R               5               3(10), R               6               5(14) and R               5               5(12) rings and also a centrosymmetric cage-like unit of water molecules, which link eight adjacent NiII centers, forming a three-dimensional framework.In the title compound, [Ni(C DOI: 10.1107/S1600536809006515/si2152Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


II atom of the title complex, [Cu(C17H15N2S2)2], lies on a twofold rotation axis, and is in a distorted tetrahedral geometry with the two bidentate N2S2 Schiff bases. In the crystal structure, the molecules are interconnected into chains along the c axis by weak C—H⋯S intermolecular interactions. The crystal packing is further stabilized by C—H⋯π interactions.The Cu DOI: 10.1107/S1600536808002262/ci2558Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The dihedral between the naphthalene ring system and the bridging carbonyl C—C(=O)—C plane is 54.68 (6)°, far larger than that [12.54 (7)°] between the phenyl group and the bridging carbonyl group. The nitro group and the phenyl ring are almost coplanar [O—N—C—C torsion angle = 2.94 (19)°]. In the crystal, molecules are linked by C—H⋯π interactions and the phenyl rings are involved in a centrosymmetric π–π interaction with a perpendicular distance of 3.523 Å and a lateral offset of 1.497 Å. In addition, weak intermolecular C—H⋯O hydrogen bonds are formed between an H atom of one methoxy group and a nearby carbonyl O atom.In the title compound, C DOI: 10.1107/S1600536810005398/fl2290Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The observed Pt—I bond distances of 2.6094 (5) and 2.6130 (5) Å are in the expected range for PtI2 complexes. The C=C double bonds in the molecule differ significantly [1.373 (10) and 1.403 (10) Å]. As expected for a platinum(II) complex, the PtII atom is in a square-planar environment (ΣPtα= 359.71°).The monoclinic title complex, [PtI DOI: 10.1107/S1600536809029997/bg2283Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The AgI atom is coordinated in an almost linear fashion by two pyridyl N atoms from two nicotinate ligands. The linear coordination is augmented by weak interactions with three O atoms from one perchlorate anion, one uncoordinated water molecule and one carboxylate group. Two Pr atoms link two {Ag(nic)2}+ units into a ring, which is further extended into an infinite zigzag chain by sharing the Pr atoms. These chains are further connected into a three-dimensional network via weak Ag⋯O interactions, O—H⋯O hydrogen bonds, Ag⋯Ag interactions [3.357 (2) Å] and π–π interactions between the pyridyl rings [centroid–centroid distance = 3.685 (4) Å].In the title compound, {[Ag DOI: 10.1107/S1600536809001718/hy2177Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The bis(diphenylarsino)methane ligand bridges an Ru—Ru bond and the monodentate phosphine ligand bonds to the third Ru atom. Both the phosphine and arsine ligands are equatorial with respect to the Ru3 triangle. In addition, each Ru atom carries one equatorial and two axial terminal carbonyl ligands. The three phosphine-substituted benzene rings make dihedral angles of 73.5 (3), 57.2 (3) and 75.7 (3)° with each other in molecule A, while these angles are 60.7 (3), 86.8 (3) and 54.9 (3)° in molecule B. The dihedral angles between the two benzene rings are 87.3 (3) and 89.6 (3)° for the two diphenylarsino groups in molecule A and 85.6 (3) and 87.7 (3)° in molecule B. In the crystal packing, the molecules are linked into a three-dimensional framework via intermolecular C—H⋯O and C—H⋯Cl hydrogen bonds. Weak intermolecular C—H⋯π interactions furture stabilize the crystal structure. The crystal studied was an inversion twin, the refined ratio of twin components being 0.480 (7):0.520 (7).The asymmetric unit of the title  DOI: 10.1107/S1600536809053884/sj2702Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The benzofuran ring plane makes dihedral angles of 28.63 (6) and 31.55 (5)° with the 4-fluorophenyl and phenyl rings, respectively. Weak C—H⋯F and C—H⋯O hydrogen bonds and intermolecular C—H⋯π interactions are present in the crystal structure. The title crystal was refined as an inversion twin with a 0.39 (7):0.61 (7) domain ratio.In the title molecule, C DOI: 10.1107/S1600536810014613/fb2185Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The asymmetric unit contains two PbII atoms, four MBA ligands and two water molecules. Each PbII cation is heptacoordinated and chelated by four carboxylate O atoms from two MBA ligands. The Pb atoms are bridged through the carboxylate O atoms from another two MBA ligands, leading to a central Pb2O2 core. The Pb—O bond lengths are in the range 2.325 (3)–2.757 (4) Å. The intra- and interdimer Pb⋯Pb distances are 4.2942 (3) and 4.2283 (3) Å, respectively, indicating little direct metal–metal interaction. The coordinating water molecules and carboxylate O atoms are involved in extensive O—H⋯O hydrogen-bonding interactions. The complex has an extended ladder-like chain structure and the chains are assembled by hydrogen bonds and π–π interactions [centroid–centroid distance = 3.6246 (3) Å] into a three-dimensional supramolecular structure.The reaction of lead(II) acetate and 3-methylbenzoic acid (MBA) in aqueous solution yielded the title polymer, [Pb(C DOI: 10.1107/S1600536809019771/fj2218Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


H)-thiolate anions in the asymmetric unit of the title compound, Na+·C2H2N3S2               −·2H2O, which are almost perpendicular to each other [dihedral angle = 84.64 (6)°]. The two Na+ cations are in distorted fourfold coordinations by O atoms of the water molecules. The crystal structure is stabilized by N—H⋯S, O—H⋯N and O—H⋯S hydrogen bonds.There are two 5-amino-1,3,4-thiadiazole-2(3 DOI: 10.1107/S1600536809051897/pk2208Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


In addition to the usual intermolecular C—H⋯O hydrogen bonding, short intramolecular C—H⋯S contacts and π–π stacking interactions [centroid–centroid distance = 3.762 (2) Å] contribute to the crystal packing.In the title compound, C DOI: 10.1107/S1600536808007356/rk2081Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


In the crystal structure, intermolecular N—H⋯Br hydrogen bonds link the molecules into centrosymmetric dimers. There are π–π contacts between the pyridine rings [centroid–centroid distances = 3.9662 (5) and 3.9321 (4) Å]. There also exists a C—H⋯π contact between the pyridine CH group and a pyridine ring.In the molecule of the title compound, [HgBr DOI: 10.1107/S1600536808038129/hk2575Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


An intermolecular π–π interaction between the pyridine rings [centroid–centroid distance = 3.900 (4) Å] is observed. The CdII atom has a distorted tetrahedral coordination.In the cation of the title compound, (C DOI: 10.1107/S1600536808030092/is2330Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The centroid–centroid distances of 3.809 (2) and 3.680 (2) Å between nearly parallel pyridine rings of the phen ligands and the benzene rings of dhba anions indicate that the dhba anions are involved in π–π stacking in the crystal structure. The face-to-face separation of 3.35 (3) Å between parallel phen ring systems also suggests π–π stacking between adjacent complex molecules. The crystal structure contains extensive O—H⋯O and C—H⋯O hydrogen bonding.In the crystal structure of the title compound, [Cd(C DOI: 10.1107/S1600536808018126/rk2092Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The crystal structure is stabilized by various π–π stacking interactions in which the benzene ring, a pyridine ring and the five-membered CuN2C2 ring are involved. The centroid–centroid distances range from 3.5631 (15) to 3.5666 (16) Å.In the title mononuclear complex, [CuCl DOI: 10.1107/S160053680801948X/lh2647Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The crystal structure is composed of centrosymmetric dimers lying about inversion centres. Both independent Sn atoms adopt distorted trigonal-bipyramidal SnC2O3 coordination geometries with the basal planes consisting of two C-atoms from the methyl groups and a bridging O atom. The Sn—C and Sn—O bond lengths lie in the ranges 2.090 (2)–2.104 (3) and 2.0241 (14)–2.2561 (15) Å, respectively. The central four-membered planar Sn2O2 ring [Sn⋯Sn distance = 3.2993 (2) Å] makes dihedral angles of 5.43 (11) and 59.50 (7)° with the methylphenyl groups, which are themselves oriented at a dihedral angle of 61.38 (8)°. Besides weak C—H⋯O and C—H⋯π interactions, the packing mainly features van der Waals forces between the molecules.The title compound, [Sn DOI: 10.1107/S1600536810036512/wm2399Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The asymmetric unit contains one 4-methylbenzoate anion, one nicotinamide (NA) ligand and one coordinated water molecule. The four O atoms in the equatorial plane around the MnII ion form a slightly distorted square-planar arrangement, while the slightly distorted octahedral coordination is completed by the two pyridine N atoms of the NA ligands in the axial positions. The dihedral angle between the carboxylate group and the attached benzene ring is 9.01 (7)°, while the pyridine and benzene rings are oriented at a dihedral angle of 42.44 (5)°. In the crystal structure, intermolecular O—H⋯O, N—H⋯O and C—H⋯O hydrogen bonds, and O—H⋯π and C—H⋯π interactions link the molecules into a two-dimensional network parallel to (001).In the mononuclear title complex, [Mn(C N,N-diethylnicotinamide, see: Bigoli et al. 2(C6H6N2O)2(H2O)2] = 0.035                           wR(F                           2) = 0.099                           S = 1.08                           3297 reflections204 parameters3 restraintsH atoms treated by a mixture of independent and constrained refinementmax = 0.73 e Å−3                        Δρmin = −0.38 e Å−3                        Δρ                                 APEX2  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008ORTEP-3 for Windows (Farrugia, 1997WinGX (Farrugia, 1999PLATON (Spek, 2009Data collection: 10.1107/S1600536810011815/ci5069sup1.cif            Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536810011815/ci5069Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


In the absence of any strong hydrogen bonds, the structure results from a large number of competing weaker interactions including Cl⋯Cl [3.4610 (5) Å] and C—H⋯Cl contacts and both (aryl) C—H⋯Br and N+—Csp               3—H⋯Br− cation–anion interactions.In the title compound, C DOI: 10.1107/S160053680902159X/jh2078Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The 2,6-dihydroxybenzoic acid molecule is disordered about an inversion center. The face-to-face separations of 3.540 (11) and 3.429 (8) Å between parallel phen ligands indicate the existence of π–π stacking between adjacent MnII complexes. Uncoordinated water molecules are linked with complex and dihydroxybenzoic acid molecules via O—H⋯Cl and O—H⋯O hydrogen bonds.In the crystal structure of the title compound, [MnCl DOI: 10.1107/S1600536808012427/ng2452Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The crystal structure is stabilized by N—H⋯O, N—H⋯N, O—H⋯O and O—H⋯N hydrogen bonds. In addition, weak π–π interactions are observed between symmetry-related thiadiazole ring systems .The title compound, C DOI: 10.1107/S1600536809021825/at2800Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The AgI atom is coordinated in an almost linear fashion by two pyridyl N atoms of two nic ligands. The linear coordination is augmented by weak interactions with one O atom from a half-occupied ClO4               − anion and a water molecule lying on a twofold axis. Two Ag(nic)2 units connect two La atoms, forming a cyclic unit. These units are further extended into an infinite zigzag chain. The chains are bridged by the disordered perchlorate ions via weak Ag—O [2.678 (2) Å] interactions. O—H⋯O hydrogen bonds, weak Ag⋯Ag [3.3340 (15) Å] interactions and π–π interactions between the pyridyl rings [centroid–centroid distance = 3.656 (2) Å] lead to a three-dimensional network.In the title complex, [Ag DOI: 10.1107/S1600536809026130/hy2200Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The molecule has a short intramolecular contact involving an aromatic H atom (Au⋯H = 2.64 Å); two adjacent molecules are linked by an Au⋯Hylid interaction (Au⋯H = 3.14 Å).The metal atom in the title ylid–gold(I) adduct, [Au(C DOI: 10.1107/S160053680904152X/ng2660Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The geometry of the resulting NiN2O4 coordination can be described as distorted octahedral. Considerable C=O⋯π stacking interactions are observed between the carboxylate C=O groups and the pyridine rings of the (pydc)2− fragments, with O⋯π distances of 3.1563 (12) and 3.2523 (12) Å and C=O⋯π angles of 95.14 (8) and 94.64 (8)°. In the crystal structure, a wide range of non-covalent interactions, consisting of hydrogen bonding , ion pairing, π–π [centroid-to-centroid distance = 3.4825 (8) Å] and C=O⋯π stacking, connect the various components to form a supramolecular structure.The reaction of nickel(II) nitrate hexahydrate, propane-1,2-diamine and pyridine-2,6-dicarboxylic acid in a 1:2:2 molar ratio in aqueous solution resulted in the formation of the title compound, (C DOI: 10.1107/S1600536808016309/su2054Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The CdII ion is hexacoordinated by four carboxylate O atoms [Cd⋯O = 2.280 (2)–2.404 (2) Å] from two chelating 4-methoxybenzoate anions, and two N atoms [Cd⋯N = 2.313 (2) and 2.332 (2) Å] from one chelating 2,2′-dimethyl-3,3′-(oxydiethylene)bis(1H-benzimidazole) ligand. In the crystal, molecules are linked by a weak intermolecular C—H⋯O hydrogen bond and an intermolecular C—H⋯π interaction.The title complex, [Cd DOI: 10.1107/S1600536810009505/lx2137Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The structure is consolidated by intermolecular O—H⋯O hydrogen bonding, as well as by π–π stacking interactions between adjacent naphthyl ring systems [centroid–centroid distance between parallel naphthoate rings is 3.768 (2) Å].In the title mononuclear complex, [Mg(C DOI: 10.1107/S1600536808006351/ng2419Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


II–HoIII complex ]diphenolato-1κ4               O               1,O               1′,O               6,O               6′:2κ4               O               1,N,N′,O               1′}trinitrato-1κ6               O,O′-holmium(III)nickel(II)), [HoNi(C20H22N2O4)(NO3)3], with the hexadentate Schiff base compartmental ligand N,N′-bisethylenediamine (H2               L), the Ho and Ni atoms are doubly bridged by two phenolate O atoms of the Schiff base ligand. The coordination of Ni is square-planar with the donor centers of two imine N atoms and two phenolate O atoms. The holmium(III) center has a tenfold coordination environment of O atoms, involving the phenolate O atoms, two ethoxy O atoms and two O atoms each from the three nitrates. Weak C—H⋯O and O⋯Ni [3.383 (4) Å] interactions generate a two-dimensional zigzag sheet.In the title heteronuclear Ni DOI: 10.1107/S1600536808013755/hg2401Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The asymmetric unit contains one 4-methylbenzoate (PMB) anion, one N,N-diethylnicotinamide (DENA) ligand and one coordinated water molecule. The four O atoms in the equatorial plane around the CoII ion form a slightly distorted square-planar arrangement, while the slightly distorted octahedral coordination is completed by the two pyridine N atoms of the DENA ligands in the axial positions. The dihedral angle between the carboxylate group and the attached benzene ring is 3.73 (14)°, while the pyridine and benzene rings are oriented at a dihedral angle of 77.28 (6)°. In the crystal structure, intermolecular O—H⋯O and C—H⋯O hydrogen bonds link the molecules into a two-dimensional network parallel to (001). The structure is further stabilized by π–π contacts between the pyridine rings [centroid–centroid distance = 3.544 (1) Å] and weak C—H⋯π interactions involving the benzene ring.In the centrosymmetric mononuclear title complex, [Co(C N,N-diethylnicotinamide, see: Bigoli et al. 2(C10H14N2O)2(H2O)2] = 0.038                           wR(F                           2) = 0.090                           S = 1.04                           4484 reflections234 parameters3 restraintsH atoms treated by a mixture of independent and constrained refinementmax = 0.86 e Å−3                        Δρmin = −0.55 e Å−3                        Δρ                                 APEX2  used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008et al., 2006WinGX (Farrugia, 1999PLATON (Spek, 2009Data collection: 10.1107/S1600536810013954/ci5076sup1.cif            Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536810013954/ci5076Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


There are three N-[(anthracen-9-yl)methyleneamino]thioureate ligands coordinated to the CoIII atom via three imine N and three thioamide S atoms. The Co—S and Co—N bond distances are in expected ranges . The endocyclic S—Co—N bond angles in the five-membered chelate rings range from 82.91 (7) to 85.33 (7)°. The structure contains four water molecules which are disordered over 12 sites and link the complex molecules into a three-dimensional network through N—H⋯O, O—H⋯O, O—H⋯N, and O—H⋯S hydrogen bonds.In the title complex, [Co(C DOI: 10.1107/S1600536808031425/pv2097Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The two inversion-related CdII ions are separated by 3.9920 (6) Å and are bridged by two O atoms from two nitrite ligands. There are two types of π–π stacking interactions involving symmetry-related pyrazole rings, with centroid–centroid distances of 3.445 (2) and 3.431 (2) Å.In the title centrosymmetric binuclear complex, [Cd DOI: 10.1107/S1600536809039841/lh2917Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


AID). Upon germinal centre exit, B cells differentiate into antibody‐secreting plasma cells. Germinal centre maintenance and terminal fate choice require transcriptional reprogramming that associates with a substantial reconfiguration of DNA methylation patterns. Here we examine the role of ten‐eleven‐translocation (TET) proteins, enzymes that facilitate DNA demethylation and promote a permissive chromatin state by oxidizing 5‐methylcytosine, in antibody‐mediated immunity. Using a conditional gene ablation strategy, we show that TET2 and TET3 guide the transition of germinal centre B cells to antibody‐secreting plasma cells. Optimal AID expression requires TET function, and TET2 and TET3 double‐deficient germinal centre B cells show defects in CSR. However, TET2/TET3 double‐deficiency does not prevent the generation and selection of high‐affinity germinal centre B cells. Rather, combined TET2 and TET3 loss‐of‐function in germinal centre B cells favours C‐to‐T and G‐to‐A transition mutagenesis, a finding that may be of significance for understanding the aetiology of B‐cell lymphomas evolving in conditions of reduced TET function.Upon activation by antigen, B cells form germinal centres where they clonally expand and introduce affinity‐enhancing mutations into their B‐cell receptor genes. Somatic mutagenesis and class switch recombination (CSR) in germinal centre B cells are initiated by the activation‐induced cytidine deaminase . Loss of TET function favors C‐to‐T and G‐to‐A mutagenesis during somatic hypermutation, a finding of potential significance for understanding the etiology of B‐cell lymphomas.TET (ten‐eleven‐translocation) enzymes promote gene expression by catalyzing the oxidation of 5‐methylcytosine in DNA. TET deficiency has been linked to defects in embryonic development and enhanced risk of myeloid malignancies, including B‐cell lymphomas. Here, Verena Labi and colleagues show using a conditional gene ablation strategy that TET function is vital for humoral immunity  As compared to mature naïve follicular (FO) B cells, TET2 and TET3 are substantially down‐regulated in antigen‐experienced GC B cells and plasma cells, a result in agreement with a recent report in human GC B cells During B‐cell development expression of both, TET2 Fig. A and TETDependent on the mouse strain and animal facility, young mice lacking only TET2 or TET3 display no or only moderate B‐cell phenotypes at steady state in vitro treatment of activated B cells with 5‐azacytidine augmented the appearance of plasmablasts in a division‐dependent manner B‐cell transit through the GC is accompanied by extensive DNA demethylation, focal methylation gains and an overall increased heterogeneity in DNA methylation patterns ;Tet2F/F;Tet3F/F mice in which physiologic germ‐line Cg1 transcription drives expression of the Cre‐recombinase Tet genes is expected in a majority of GC B cells upon IgG1‐priming. Of note, acute GC B cell‐specific Tet deletion circumvents indirect effects caused by extended TET‐deficiency during B‐cell development.Addressing the involvement of TET proteins in this process, we generated Cg1‐Crein vitro induced GC (iGC) B cells First, we used a co‐culture system that allows the generation and exponential growth of Tet deletion is complete as indicated by qRT‐PCR analysis  conjugated to chicken gamma globulin (NP‐CGG). NP‐specific GC B cells that form in response to alum‐adjuvanted immunogens predominately switch to IgG1 ;Tet2F/F;Tet3F/F GC B cells, suggesting that these cells proliferate normally  and high‐affinity (NP1.7‐BSA) α‐NP IgG1 were decreased similarly, resulting in no significant skewing of the affinity ratio in Cg1‐Cre;Tet2F/F;Tet3F/F mice  ;Tet2F/F;Tet3F/F (54%) GC B‐cell clones  in PBS in a volume of 200 μL per mouse. Animal procedures were approved by the Austrian Federal Ministry of Education, Science and Research (BMWF: 66‐011/0106‐WF/3b/2015 and 66‐011/0031‐V/3b/2019).The Cg1‐Cre 4)2  in PBS] was mixed with a similar volume of 1 mg·mL−1 NP15‐CGG , and the pH was adjusted to 6.5–7.0 using 10 m NaOH . The precipitate was washed three times with PBS at 2348 g for 15 s and finally resuspended in PBS to reach the initial volume of the Alum/NP‐CGG mixture.A freshly prepared 10% Alum solution  was generally used for cell preparation. Single‐cell suspensions were prepared by forcing murine spleen through 70 μB cells were enriched from splenic single‐cell suspensions using MagniSort Streptavidin Negative Selection Beads  as per manufacturer's instructions, and biotinylated antibodies against Ter119 , CD11b  and TCRβ .et al. m l‐glutamine  and 100 U·mL−1 penicillin/100 μg·mL−1 streptomycin . 3 × 106 feeder cells per 10 cm plate were treated for 2 h with 10 μg·mL−1 mitomycin C  in 6 mL feeder cell medium. Following five repeated washing steps with PBS, 1.5 × 106 B cells/10 cm dish were plated on 40LB feeder cells in 40 mL B‐cell medium: DMEM supplemented with 10% (v/v) FBS, 2 mm l‐glutamine, 10 mm Hepes , 1 mm sodium pyruvate , 1× nonessential amino acids , 100 U·mL−1 penicillin/100 μg·mL−1 streptomycin, 50 μm β‐mercapto‐ethanol  and 10 ng·mL−1 rIL‐4 . On day 3, 30 mL of medium containing IL‐4 were replaced. On day 4, iGC B cells were harvested and analysed. 1.5 × 106 iGC B cells were replated per 10 cm dish containing fresh mitomycin C‐treated 40LB feeder cells as detailed above, and cultivated in 40 mL of B‐cell medium containing either 10 ng·mL−1 rIL‐4 or 10 ng·mL−1 rIL‐21 . Thirty millilitre of medium containing the respective cytokines were replaced by fresh medium on day 7. The final analysis was conducted on day 8. Analyses (day 4 and 8) entailed collection of medium for ELISA and pellets for qRT‐PCR, cell counting (Trypan blue exclusion) and flow cytometric analysis. For analysis, iGC B cells were generally handled at room temperature and cultured at 37 °C in a humidified atmosphere containing 5% CO2.The iGC B cell culture was conducted as described by Nojima 2. Mitogenic stimuli were used as follows: 1 μg·mL−1 α‐CD40 , 25 ng·mL−1 IL‐4 , 10 ng·mL−1 IL‐21 (Peprotech 210‐21), 20 mg·mL−1 lipopolysaccharide  and 25 ng·mL−1 IL‐5 .Primary B cells were enriched from splenic single‐cell suspensions and cultivated in medium as described above for the iGC B‐cell culture at 37 °C in a humidified atmosphere containing 5% COin vitro, B cells were labelled with 10 μm Cell Proliferation Dye eFluor 450  as per manufacturer's instructions and placed in culture.To determine cell proliferation in the presence of mitogens 2‐FITC , αCD25‐PE , αcKit‐APC , αCD1d‐PE , αCD23‐PE/Cy7 , αCD38‐FITC , αCD38‐APC , αCD38‐eFluor450 , αCD95‐PE , αCD95‐PE/Cy7 , αCXCR4‐APC , αCD86‐PE/Cy7 , αIgG1‐FITC , αIgG1‐PE , CD138‐PE  and CD138‐BV510 . NP24‐PE  was used to detect NP‐binding B cells. Data were acquired on an LSRII cytometer (BD Biosciences) and analysed using flowjo software . Nonsinglet events were excluded from analyses using FSC‐H/FSC‐W and SSC‐H/SSC‐W characteristics.Flow cytometry analysis has been performed as described in Ref. m EDTA). Cells were harvested by addition of B‐cell medium and vigorous pipetting.To collect iGC B cells from 10 cm dishes, 9/10 of the medium was gently removed, and the cells were incubated for 5 min at 37 °C in 3 mL harvest buffer  and surface digested for 4 min at 37 °C. To stop trypsin digestion, 3 mL of staining buffer (described above for flow cytometry) were added, the cells were pelleted at 1800 r.p.m. for 4 min and the supernatant was removed. The cells were transferred to a U‐bottom 96 well plate and dead cells were labelled with the Fixable Viability Dye eFluor 780  as per manufacturer's instructions. Subsequently, cells were fixed and processed using the active Caspase 3 staining kit  as per manufacturer's instructions. Before staining with the active Caspase 3 antibody, the iGC B cells were incubated with 1 μg·mL−1 of αCD16/32 Fc‐Block  in 25 μL perm/wash buffer for 10 min at 4 °C. Subsequently another 25 μL of αIgE‐bio  diluted in perm/wash buffer was added, the cells were further incubated for 15 min at 4 °C and washed with perm/wash buffer at 568 g for 2 min. Next, the cells were incubated with 25 μL of antibody mix containing αIgM‐PeCy7 , αIgG1‐FITC , αCD19‐BV605  and Streptavidin‐APC  for 15 min at 4 °C. The cells were washed with perm/wash buffer at 2000 r.p.m. for 2 min and stained for active caspase 3 as per manufacturer's instructions.For CSR analysis, 5 × 105 iGC B cells were transferred to a U‐bottom 96 well plate and pelleted at 2000 r.p.m. for 2 min. Dead cells were labelled with the Fixable Viability Dye eFluor 780  as per manufacturer's instructions. Subsequently, cells were incubated with 1 μg·mL−1 of αCD16/32 Fc‐Block in 25 μL staining buffer (PBS/3%FBS) for 10 min. Next, 25 μL of staining buffer containing αCD19‐BV605  and αCD138‐BV421  was added. The cells were further incubated for 15 min, washed with 200 μL of staining buffer at 2000 r.p.m. for 2 min, and resuspended in 200 μL staining buffer for flow cytometry analysis.The processing of cells for plasmablast flow cytometry analysis was performed at room temperature in the dark. 5 × 10flowjo software (Tree Star). Nonsinglet events were excluded from analyses using FSC‐H/FSC‐W and SSC‐H/SSC‐W characteristics.Data were acquired on an LSRII cytometer (BD Biosciences) and analysed using −1 of αCD16/32 Fc‐Block in 500 μL staining buffer (PBS/3%FBS) for 10 min, washed and stained for 20 min with antibodies in a volume of 500 μL staining buffer. The sorted cell subsets were defined as follows: Bone Marrow: pro B cells (B220loCD19+AA4.1+IgM−CD25−ckit+), large pre B cells (B220loCD19+AA4.1+IgM−CD25+ckit−FSChi), small pre B cells (B220loCD19+AA4.1+IgM−CD25+ckit−FSClo) and immature IgM+ B cells (B220loCD19+AA4.1+IgM+). Spleen: T1 B cells (CD19+B220+AA4.1+CD23−IgMhi), T2 B cells (CD19+B220+AA4.1+CD23+IgMhi), FO B cells (CD19+B220+AA4.1−CD1d+IgM+), MZ B cells (CD19+B220+AA4.1−CD1dhiIgMhi), GC B cells (CD19+B220+CD138−CD95hiCD38lo/−), plasma cells/plasmablasts (B220−/loCD138hi), DZ GC B cells (CD19+B220+CD138−CD95hiCD38lo/−CXCR4hiCD86lo) and LZ GC B cells (CD19+B220+CD138−CD95hiCD38lo/−CXCR4loCD86hi). For sorting B‐cell division cycles cells were harvested, filtered through 70 μm mesh filters  and resuspended in staining buffer.Cell sorting has been performed as described in Ref. Cell sorting was carried out on a FACS Aria III (BD Biosciences). Nonsinglet events were excluded from analyses based on characteristics of FSC‐H/FSC‐W and SSC‐H/SSC‐W.in vitro‐cultivated B cells was isolated using Trizol reagent  as per manufacturer's instructions. DNA was removed using the RQ1 RNAse‐free DNase , and RNA was retrieved using GlycoBlue Coprecipitant  as per manufacturer's instructions. First‐strand cDNA was generated from 100 ng of total RNA using the iScript cDNA Synthesis Kit  and cDNA was amplified using the AceQ qPCR SYBR Green Master Mix  as per manufacturer's instructions. The qRT‐PCR was run on a StepOnePlus Real‐time PCR system  and melt curve analysis was performed for every run. The expression of individual mRNAs was normalized to HPRT with the following formula: fold induction = 2(−ΔCt), where ΔCt=Ct(target)−Ct(HPRT). The following primers were used: HPRT F: 5′‐GTCATGCCGACCCGCAGTC‐3′, HPRT R: 5′‐AGTCCATGAGGAATAAAC‐3′; TET2 F: 5′‐GCCAGAAGCAAGAAACCAAG‐3′, TET2 R: 5′‐TTGGAGCAATGACAGTAGCC‐3′; TET3 F: 5′‐AAGAGTCTGCTGGACACACC‐3′, TET3 R: 5′‐CTCCATGAGTTCCCGGATAG‐3′; AID F: 5′‐GGACTTCGGCCACCTTC‐3′, AID R: 5′‐CATCTCAGAAACTCAGCCACG‐3′. Ki67 F: 5′‐GAACAGACTTGCTCTGGCCTAC‐3′, Ki67 R: 5′‐ CTTCATAGGCATTCCCTCACTC‐3′.RNA from snap‐frozen cell pellets of FACS‐sorted B cells was isolated using the Quick‐RNA Micro Prep Kit  and DNase digestion as per manufacturer's instructions. RNA from snap‐frozen −1 capture antibody  was coated overnight onto 96‐well enzyme‐linked immunosorbent assay plates at 4 °C . Plates were washed three times with wash buffer (PBS containing 0.05% TWEEN 20), blocked with 100 μL per well 1% BSA in PBS for 4 h at room temperature and washed three more times with wash buffer. Subsequently, wells were incubated over night at 4 °C with 100 μL per well of mouse serum serially diluted 1 : 4 in blocking buffer (range 1 : 800 to 1 : 160 000). Plates were washed three times with wash buffer and incubated with 100 μL per well of HRP‐conjugated α‐mouse IgG1  or HRP‐conjugated α‐mouse IgM  for 4 h at room temperature. For detection, 100 μL of ABTS substrate solution per well  was incubated for 20 min. Absorbance was measured at 405 nm using a microplate reader .For NP‐specific serum IgG1 and IgM titres, ELISA was performed as described in zeiss zen blue 2.5 lite software (Zeiss).IHC was performed on 7 μm sections of murine spleens as described in Ref. ;Tet2F/F;Tet3F/F mice. Briefly, IgG1+ GC B cells were FACS‐sorted (CD19+B220+CD138−FashiCD38lo/−IgG1+), pelleted and total RNA was isolated using the Quick‐RNA Micro Prep Kit  including the DNase digestion step as per manufacturer's instructions. First‐strand cDNA was generated from 100 ng of total RNA using the Promega GoScript Reverse transcription system  and a gene‐specific primer for the IgG1 locus: Cg1 5′‐CATGGAGTTAGTTTGGGCAG‐3′. Subsequently, two rounds of semi‐nested PCRs were performed using the Herculase II Fusion DNA polymerase kit  as per manufacturer's instructions. Following each round of PCR, the gene product was run on an agarose gel and purified using the peqGold Gel extraction kit  as per manufacturer's instructions. For the first PCR, the following primers were used: V186.2 leader 5′‐AGCTGTATCATGCTCTTCTTGGCA‐3′, Cg1 5′‐CATGGAGTTAGTTTGGGCAG‐3′. For the second PCR, the following primers were used: V186.2 nested 5′‐CATGCTCTTCTTGGCAGCAACAG‐3′, Cg1 5′‐CATGGAGTTAGTTTGGGCAG‐3′. For cloning of VH186.2 segments, the pJet1.2 blunt vector was used and cleaved with EcoRV . The purified PCR products were ligated into the vector using T4 ligase  as per manufacturer's instructions. Using a standard transformation protocol, DH5α bacteria were transformed with the bulk of ligated plasmids, spread onto LB Agar/ampicillin plates and incubated over night at 37 °C. For each of the six mice, >10 colonies were transferred into a PCR Master Mix , and the very same colonies were selected on new LB agar plates. For the colony PCR, the following primers were used: pJet1.2 F 5′‐CGACTCACTATAGGGAGAGCGGC‐3′, pJet1.2 R 5′‐AAGAACATCGATTTTCCATGGCAG‐3′. Cycle conditions were: 1 cycle: 95 °C for 1 min; 30 cycles: 95 °C for 15 s, 56 °C for 15 s, 72 °C for 45 s; 1 cycle: 72 °C for 3 min. The PCR products were loaded onto an agarose gel, and colonies with correct insertions were grown in 2 mL LB medium over night. Plasmid DNA was extracted using the Monarch Plasmid Miniprep Kit  as per manufacturer's instructions. Subsequently, Sanger sequencing was performed using the pJet1.2 F primer detailed above. Sequences were aligned to the VH186.2 germ line sequence using VBASE2 (http://www.vbase2.org/). Mutation data were only used if the entire sequence was intact, and only unique sequences were included in the analysis. According to these criteria, two sequences from Cg1‐Cre;Tet2F/F;Tet3F/F GC B cells had to be excluded.Somatic hypermutation analysis was performed according to Ref. graphpad prism 7 software  using Student's t test when comparing two groups, One‐way ANOVA and Tukey post hoc test when comparing multiple groups and Mann–Whitney test for SHM analysis. The number of biological repetitions (n) is stated in each figure legend, and every experiment was performed at least twice. Differences between groups were considered statistically significant when P < 0.05. In figures, asterisks stand for: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.Results are always shown as mean and standard deviation (SD). Graphs were plotted and statistical analysis was performed with The authors declare no conflict of interest.Conceptualization: VL; Methodology: VL; Investigation: KS, SM, SH, ED, AA; Resources: VL, SH, ED, AV, KR; Writing – Original Draft: VL; Visualization: VL; Project Administration: VL; Funding Acquisition: VL.


One Cu atom is coordinated to the 2-ethoxypyrazine molecule and two bridging cyanide ligands, equally disordered over two sites. The second Cu atom is coordinated by two disordered over two sites bridging cyanide groups. Two copper–cyanide chains are connected through Cu⋯Cu contact.The title compound, {[Cu(EtOpz)(CN) 4H3OC2H5N2)][Cu(CN)]}n, there are two Cu atoms with different coordination environments. One CuI ion is coordinated in a triangular coordination geometry by the N atom of the 2-ethoxypyrazine molecule and by two bridging cyanide ligands, equally disordered over two sites exchanging C and N atoms, thus forming polymeric chains parallel to the c axis. The other Cu atom is connected to two bridging cyanide groups disordered over two sites with an occupancy of 0.5 for each C and N atom, and forming an almost linear polymeric chain parallel to the b axis. In the crystal, the two types of chain, which are orthogonal to each other, are connected by cuprophilic Cu⋯Cu interactions [2.7958 (13) Å], forming two-dimensional metal–organic coordination layers parallel to the bc plane. The coordination framework is further stabilized by weak long-range (electrostatic type) C—H⋯π interactions between cyano groups and 2-ethoxypyrazine rings.In the asymmetric unit of the title coordination compound, {[Cu(CN)(C The Cu1 atom is coordinated to the N atom of a 2-ethoxypyrazine molecule [Cu1—N5 = 2.090 (4) Å]. Two other coordination positions are occupied by bridging cyanide groups, which are equally disordered over two sites, exchanging C and N atoms [Cu1—C1/N1 = 1.905 (4) Å and Cu1—C2/N2 = 1.888 (4) Å], thus forming an irregular triangular coordination geometry where the copper ion is displaced from the centre . The Cu2 atom is coordinated by two cyanide ligands, which are also disordered over two sites with an occupancy of 0.5 for each C and N atom  to form an almost linear chain [C4/N4iii—Cu2—C3/N3 = 170.5 (2)°]. The two CuI centres are connected through a Cu⋯Cu interaction [Cu1—Cu2 = 2.7958 (13) Å] that could be interpreted as a cuprophilic contact  interconnected by Cu⋯Cu contacts and forming two-dimensional layers parallel to (100). The Cu⋯Cu contacts are almost perpendicular to the [Cu2(CN)] chains [C3/N3—Cu2—Cu1 = 89.8 (2)° and C4/N4iii—Cu2—Cu1 = 99.7 (2)°]. At the same time, the Cu2 atom occupies an axial position with respect to the triangular [N(CN)2] coordination environment of Cu1 [C1/N1—Cu1—Cu2 = 70.6 (2)° and C2/N2—Cu1—Cu2 = 87.6 (2)°]. The resulting metal–organic coordination framework is additionally stabilized by weak long-range (electrostatic-type) C—H⋯π interactions between cyanide groups and 2-ethoxypyrazine rings  Å are also observed .The crystal packing of the title compound Fig. 2 consistset al., 2016catena-[pentakis(μ2-cyano)tris(1-phenylpiperazine)pentacopper] (PhPip) and (CuCN)3(PhPip), associated by Cu⋯Cu pairwise cuprophilic interactions, with distances of 2.5586 (10) and 2.6441 (10) Å. A search of the CSD for two C—N—Cu—C—N fragments with a defined Cu⋯Cu distance less than 2.8 Å gave 80 hits, among which is an example close to the title structure, i.e.catena-[tetrakis(μ2-cyano)tetracopper(I)]  and 2.9644 (6) Å.A search of the Cambridge Structural Database  in 1 ml of H2O, the second layer was a H2O/EtOH mixture  and the third layer was a solution of 2-ethoxypyrazine  in 0.5 ml of EtOH. After two weeks, colourless block-shaped crystals had formed in the middle layer. The crystals were kept under the mother solution prior to measurement.Crystals of the title compound were obtained by slow diffusion within three layers in a 3 ml glass tube. The first layer was a solution of K[Cu(CN)Uiso(H) = 1.2Ueq(C) for aromatic hydrogens, C—H = 0.97 Å and Uiso(H) = 1.2Ueq(C) for the CH2 group, and C—H = 0.96 Å and Uiso(H) = 1.5Ueq(C) for the CH3 group. A rotating model was used for the methyl group. All cyano ligands are disordered over two sites with occupancies of 0.5. The coordinates of C and N atoms sharing the same sites and their displacement ellipsoids were constrained to be the same.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901901452X/rz5265sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698901901452X/rz5265Isup2.hklStructure factors: contains datablock(s) I. DOI: 1961274, 1961274CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ. DNA polymerase θ is a polymerase-helicase essential for microhomology-mediated end-joining (MMEJ) or alternative end-joining of DNA. Here the authors use biochemical and biophysical methods to reveal how full-length human DNA polymerase θ performs MMEJ at the molecular level. MMEJ functions during S and G2 cell-cycle phases and therefore acts on 3′ single-strand DNA (ssDNA) overhangs generated by 5′–3′ exonuclease resection of DSBs, similar to homologous recombination (HR) is a polymerase-helicase fusion protein Fig. , that is(HR)Fig. 7,8. The 11. Polθ-hel is related to HELQ/Hel308 helicases, which are involved in replication and repair13. Recent studies show that Polθ-hel unwinds short DNA in an ATP-dependent manner with 3′-5′ polarity, similar to HELQ/Hel30812. Polθ-hel also facilitates annealing of complementary ssDNA in an ATP-independent manner, and anneals RPA coated ssDNA by utilizing the energy of ATP14. Polθ-hel may also act as an anti-recombinase by counteracting RAD51 activity15. Polθ-pol is related to bacterial Pol I enzymes such as Klenow fragment, but contains an inactive 3′–5′ exonuclease domain16, and exhibits low fidelity DNA synthesis and translesion (TLS) synthesis activities21. The polymerase and helicase include unstructured motifs, and a disordered motif (loop 2) in Polθ-pol was shown to promote its ssDNA extension and MMEJ activities22.Polθ consists of a super-family 2 helicase (Polθ-hel), a disordered central domain (Polθ-cen), and an A-family polymerase (Polθ-pol) Fig. 1,10,11. 7, and that Polθ competes with HR15. Wyatt et al. also demonstrated that the efficiency of cellular MMEJ is positively correlated with 3′ ssDNA overhang length8. This report further demonstrated that Ku binds tightly to DNA ends with short 4 nt overhangs, but exhibits very low affinity for 70 nt overhangs8. This reveals a clear mechanistic difference between the respective substrate requirements for non-homologous end-joining (NHEJ) and MMEJ. How Polθ functions on long ssDNA overhangs to promote MMEJ, however, remains unknown.The ssDNA overhangs that Polθ functions on during MMEJ have become clearer in recent cellular studies. Wyatt et al. demonstrated that relatively long (≥ 45–70 nt) ssDNA overhangs promote MMEJ in cells Fig. . This is2. Recent biochemical studies similarly show that Polθ-pol can perform MMEJ on short ( < 12 nt) ssDNA23. Although these studies provide insight into the activity of Polθ-pol, they fail to recapitulate MMEJ on DNA with longer (45–70 nt) overhangs that support cellular MMEJ8. In vitro, Polθ-pol primarily performs snap-back replication on long ssDNA as a result of its intrastrand base-pairing activity22. For example, under physiological buffer conditions with Mg2+, Polθ-pol efficiently extends relatively long (≥ 26 nt) ssDNA by using the 5′ portion of the ssDNA as a template in cis  overhangs in cellular studies8, we hypothesized that the helicase domain within full-length Polθ (hereafter referred to as Polθ) promotes MMEJ by suppressing Polθ-pol intrastrand pairing. For instance, since Polθ-hel binds tightly to ssDNA and translocates along ssDNA with 3′–5′ directionality13, it may inhibit Polθ-pol intrastrand pairing by binding and/or translocating along ssDNA upstream from the polymerase in the context of the full-length protein  3′ overhangs are required for MMEJ activity by the isolated polymerase domain (Polθ-pol)cis Fig. . Consistcis Fig. 2,22. Becein Fig. .S. cerevisiae using a N-terminal 3xFLAG-tag  are equally deficient in MMEJ of long ssDNA  ssDNA overhangs to promote MMEJ in cells, we examined Polθ MMEJ of long ssDNA (70 nt and 100 nt) which models substrates with long 3′ overhangs Fig. . We find23. On longer 26 nt ssDNA with identical microhomology, Polθ-pol exclusively promotes ssDNAx byproducts  overhangs2, demonstrates it performs interstrand pairing without Polθ-hel. Polθ-pol, however, fails to perform efficient MMEJ of long ssDNA under various conditions  ssDNA which is almost entirely contained within its active site Fig. , leftSu and 3E16cts Fig. , right, cts Fig. -4F. Unexcts Fig. , yet is cts Fig. . This sucts Fig.  and 3E23 nt ssDNA8. Consistent with this, we find that decreasing ssDNA length to 45 nt while maintaining the same 6 bp microhomology significantly reduces Polθ MMEJ, yet, the full-length protein still shows significantly higher end-joining activity than Polθ-pol  would be extruded from the enzyme’s DNA exit channel  conjugated ssDNA  linker in place of the central domain (PolθΔcen) Fig. 27. PolθΔity Fig. , and is ity Fig. . Remarkaity Fig. . Togetheons Fig. . Controlons Fig. , right, ons Fig. , and is pol Fig. .2, and the addition of Polθ-hel in trans fails to stimulate Polθ-pol MMEJ of this substrate as expected  ssDNA compared to Polθ-pol Fig. , we test16. Our previous studies demonstrate that Polθ-hel exhibits similar activities to the related helicase Hel308, such as ATP-dependent 3′-5′ directional ssDNA translocation activity12. Based on this polarity, Polθ-hel was modeled facing towards the 5′ terminus. The model superimposes the ssDNA substrate from the Hel308 structure, in which the enzyme translocates along, onto Polθ-hel28. The primer strand from the Polθ-pol structure is also included with additional ssDNA linking the two substrates together, resulting in a long contiguous 31 nt ssDNA that is easily bound by the tethered enzymes. In this close configuration along ssDNA Polθ-pol and Polθ-hel fit well together and space is readily available for a long flexible linker (e.g. Polθ-cen). Although this model is speculative, it suggests bivalent ssDNA binding by the fusion protein which would be expected to be in the low nanomolar range.Because covalent attachment of Polθ-hel to Polθ-pol is the minimal requirement for stimulating Polθ-pol MMEJ on relatively long ssDNA substrates, this indicates close cooperation between these two domains on long ssDNA. To gain insight into how these linked enzymes cooperate, we modeled the structure of the minimal end-joining PolθΔcen protein on long ssDNA which combines the crystal structures of Polθ-pol and Polθ-hel attached by a flexible linker Fig. 13,16. Ou13, we further investigated the footprint of PolθΔcen on partial ssDNA (pssDNA) of similar size  following incubation in MMEJ buffer conditions lacking dNTPs and BSA. Without ssDNA, 81% of Polθ behaves as monomers and dimers, whereas 3% correspond to large ( > 1000 Å) multimers  connected by a flexible linker Fig. . The sphely Fig. . Despiteely Fig. –8.Fig. 731. The highly irregular shape of the assemblies suggests that, rather than droplets, the proteins form extended percolating networks; moreover, the fact that they are catalytically active indicates that the assemblies are permeable to the solvent and, possibly, large solutes. Based on the computational and EM evidence, we surmise that Polθ, and especially PolθΔcen, form large ( > 100 nm) gel-like complexes similar to others reported33, which accumulate DNA due to the high local concentration and accessibility of the polymerase and helicase domains.Overall, there is a close match between the morphology of the simulated complexes and the EM images Fig. . A peculUsing a structure function approach in vitro, we investigated how recombinant human full-length Polθ promotes MMEJ. On short ssDNA and short 3′ overhangs, Polθ-pol efficiently performs MMEJ due to its interstrand pairing activity Fig. . Thus, PPolθ-hel stimulates Polθ-pol end-joining independently from its ATPase function. This supports the notion that Polθ-hel upregulates Polθ-pol MMEJ on long overhangs by simply binding ssDNA 5′ proximal to the polymerase where it suppresses unproductive intrastrand pairing and snap-back replication by Polθ-pol Fig. . Polθ-he34.Polθ is unable to perform MMEJ or ssDNAx on short (≤ 26 nt) ssDNA Fig. . HoweverPolθ forms multimeric complexes upon binding DNA. Replacement of Polθ-cen with a short linker, however, results in multimerization of the enzyme into an extended gel-like phase, even without DNA. This suggests that Polθ-cen suppresses Polθ oligomerization by masking protein-protein interactions. Polθ DNA binding may alter the conformation of Polθ-cen which in turn enables Polθ oligomerization via protein-protein interactions. Our imaging data demonstrate that Polθ multimers facilitate DNA accumulation and MMEJ. In summary, this report reveals the importance of the unique polymerase-helicase architecture of Polθ which is essential for MMEJ.32P radiolabeled ssDNA in buffer ; the reaction was initiated by the addition of the indicated Polθ enzyme and was incubated for 45 min or as indicated at 37 °C. For analysis in nondenaturing gels, reactions were terminated by the addition of non-denaturing stop buffer  and incubated at 37 °C for at least 15–20 min. DNA was resolved in non-denaturing 11% or 12% polyacrylamide gels and analyzed by phosphorimager (Fujifilm FLA 7000). For time course experiments an aliquot of sample was removed from pooled reactions at the specified time point and transferred to tubes containing non-denaturing stop buffer. All quantified experiments were performed in triplicate and plotted with ± s.d. Quantification was performed using ImageJ Gel Analysis. For XmaI digestion assays, after initial incubation with the indicated Polθ enzyme the following buffer was added . 25 units of XmaI (New England Biolabs) was then added, as indicated, and incubated overnight at 37 °C. The reaction was then stopped using non-denaturing stop buffer and resolved as above.10 nM 5′-32P radiolabeled pssDNA in buffer ; the reaction was initiated by the addition of the indicated Polθ enzyme and incubated for 30 min at 37 °C. For analysis in denaturing gels, reactions were terminated by the addition of denaturing stop buffer (90% formamide and 50 mM EDTA). DNA was resolved in denaturing 15% polyacrylamide gels and analyzed by phosphorimager (Fujifilm FLA 7000).10 nM 5′-32P) ATP and 100 nM ssDNA (29 nt poly-dT) in buffer  at room temp for the indicated times. The reaction mixture was then spotted onto a TLC plate on PEI cellulose, which was developed in a buffer containing 1 M acetic acid and 0.25 M LiCl2 for 1.5 h. Plates were dried, then visualized by phosphorimager (Fujifilm FLA 7000).The indicated amounts of proteins were incubated with 10 μM ATP, 2 μCi of (γ-: 5 nM 5′-32P radiolabeled pssDNA (RP538/RP539) in buffer  was incubated with or without indicated Polθ enzyme for 15 min at 37 °C. After initial incubation, 4 units of EcoRI were added as indicated and incubated for 10 min at 37 °C. Reactions were terminated by the addition of non-denaturing stop buffer  and incubated at 37 °C for at least 15–20 min. DNA was resolved in non-denaturing 15% polyacrylamide gels and analyzed by phosphorimager (Fujifilm FLA 7000).T5 exonuclease assay: 8 nM 3′-Cy3 ssDNA (RP540Cy3) in buffer  was mixed with 10 nM PolθΔcen or Polθ-pol and was preincubated for 10 min at 37 °C. After initial incubation, T5 Exonuclease (New England Biolabs) to a final concentration of 0.1 U/ul or 0.5 U/ul was added to the reactions as indicated and incubated for 15 min at 37 °C. Reactions were terminated by the addition of denaturing stop buffer (90% formamide and 50 mM EDTA). DNA was resolved in denaturing 15% polyacrylamide gels and imaged at the Cy3 wavelength using FluorChem Q imager (Alpha Innotech). EcoRI endonuclease assay2, 2 mM ATP, 30 mM NaCl and 0.01% NP-40. Reactions comparing foci formation frequency were incubated for 10 min at room temp before imaging, excluding time course reactions which were imaged as indicated. Cy3 foci were identified using a Leica DMi8 scanning confocal microscope with a 63x objective at the Cy3 emission wavelength. Random fields were collected for each condition and quantified using ImageJ particle analysis. Time course reactions were imaged at a single fixed field and quantified over time using ImageJ particle analysis of each individual frame. End-joining reactions performed with confocal imaging were performed as written with the addition of 20 µM dNTPs. After confocal imaging, reactions were terminated by the addition of nondenaturing stop buffer  and incubated at 37 °C for 20 min. Reactions were resolved in non-denaturing 12% polyacrylamide gels and imaged at the Cy3 wavelength using FluorChem Q imager (Alpha Innotech).Glass bottom wells were coated with 30 mg/ml BSA for 30 min at room temp. For foci formation frequency comparisons 40 nM of Cy3 labeled DNA (RP334Cy3) was incubated with 3 nM of the indicated Polθ enzyme. For Rad52 confocal experiments 100 nM Cy3 labeled DNA (RP334Cy3) was incubated with the indicated amounts of Rad52. For time course confocal assays and end-joining confocal assays 15 nM of the indicated Polθ enzyme was incubated with 40 nM DNA (RP344Cy3). Reactions were performed with 25 mM Tris-HCl, pH 8.8, 1 mM DTT, 0.1 mg/ml BSA, 10% glycerol, 10 mM MgCl2, 10% glycerol, 0.1 mg/ml BSA, 1 mM ATP, 30 mM NaCl for at least 30 min at room temp. Reactions contained 10 nM FAM-conjugated ssDNA (RP316FAM5), and the indicated amounts of the indicated Polθ enzyme. A Clariostar (BMG Labtech) plate reader was used to measure fluorescence anisotropy. All experiments were performed in triplicate, normalized, and plotted with ± s.d.Binding reactions were performed at room temp in 25 mM Tris–HCl, pH 8.8, 1 mM DTT, 0.01% NP-40, 10 mM MgCl2, 10% glycerol, 0.1 mg/ml BSA, 1 mM ATP, 30 mM NaCl for at least 1 hr at room temp. Reactions contained 10 nM each of RP540Cy3 and RP541Cy5 and the indicated Polθ enzyme. A Clariostar (BMG Labtech) plate reader was used to measure FRET . All experiments were performed in triplicate, normalized, and plotted with ± s.d.Binding reactions were performed at room temp in 25 mM Tris-HCl, pH 8.8, 1 mM DTT, 0.01% NP-40, 10 mM MgCl2, 2 mM ATP, 0.01% NP-40 and 1 mM DTT. Incubations were carried out at 37 °C for 30 min and deposited onto freshly cleaved mica. After 15 s the mica surface was washed with milli Q water and dried with a stream of filtered air. Images were obtained on a NanoScope IV SFM  operating in tapping mode in air with a type J scanner. Silicon Nanotips were from AppNano . Images were collected at 3 μm × 3 μm and flattened to remove background slope using Nanoscope software. The size of proteins was measured from NanoScope images imported into IMAGE SXM 1.89 . Statistical analysis was done using QtiPlot (Version 0.9.8.9 svn 2288) and LibreOffice (Version: 5.1.6.2). The volume of proteins monomers, measured from SFM images of proteins deposited in 10 mM Tris–HCl, pH 8.8/100 mM KCl, were 100 nm3 for Polθ-hel and Polθ-pol, 210 nm3 for PolθΔcen and 320 nm3 for Polθ and PolθK121M. These values ( ± 50%) were used to normalize the volume of observed complexes. Quantification is presented as % of total proteins present in certain oligomeric state.Proteins (10 nM) with or without ssDNA (20 nM) were incubated in MMEJ buffer without dNTPs or BSA containing: 25 mM Tris–HCl, pH 8.8, 30 mM NaCl or 100 mM NaCl where indicated, 10 mM MgCl2, 2 mM ATP) at 37 °C for 30 min. Samples (4 μl) were applied to glow discharged lacey carbon grids  and incubated for 2 min at room temp. The grids were then washed with water three times followed by staining with 2% uranyl acetate for 10 s. Excess staining was removed by filter paper. The grids were air dried and imaged in a FEI Tecnai BioTwin Spirit transmission electron microscopy at Penn State University Cryo-Electron Microscopy facility.Polθ or PolθΔcen (200 nM) with or without ssDNA (2 μM) was incubated in 40 μl of MMEJ reaction buffer without dNTPs, BSA or detergent  or ATCC 208289/BJ5465 (a ura3-52 trp1 leu2-delta1 his3-delta200 pep4::HIS3 prb1-delta1.6 R can1 GAL + ), as previously described35. Colonies were picked and grown in SC-TRP with 2% raffinose at 30 °C. Starter cultures were expanded and grown to an OD600 of 0.6–1.0. Expanded cultures were grown to an OD600 of 1.6–2, diluted with YPR to OD600 of 0.8–1.0 and induced as follows. Expression was induced by the addition of 2% galactose for 5 hr at 30 °C. Cells were harvested by centrifugation at 6,000 rpm and washed with 50 mM HEPES pH 8.0 and 1 M Sorbitol then washed with lysis buffer . Cells were crushed in a freezer mill with liquid nitrogen. Frozen cell powder was stored at −80 °C until purification. Frozen cell powder was thawed and resuspended in lysis buffer. The resuspended cell powder was centrifuged at 92,000 g at 4 °C for 30 min. The clarified supernatant was re-centrifuged at 256,000 × g at 4 °C for 1 h. The supernatant was collected. Anti-FLAG M2 resin was washed with 0.1 M Glycine-HCl, pH 3.5; then with TBS buffer pH 7.4  then with lysis buffer by centrifugation at 1000 rpm at 4 °C for 5 min. In total 5 µg/ml 3xFLAG peptide (Sigma) and equilibrated anti-FLAG M2 resin (Sigma) were added to each tube and incubated at 4 °C. The resin was settled by centrifugation at 1,000 rpm at 4 °C for 5 min and flow-through was collected. The resin was washed with lysis buffer and then with wash buffer A . The resin was incubated in wash buffer A on ice for 15 min, then the resin was settled by centrifugation at 1,000 rpm at 4 °C for 5 min. The resin was then washed with wash buffer B . The resin was resuspended in elution buffer  with 500 µg/ml 3xFLAG peptide (Sigma) at 4 °C with rotation. A disposable 10 ml polypropylene column (Thermo Fisher) was washed with TBS buffer, pH 7.4  and then with elution buffer. The resin was loaded to the column and elution fractions were collected. The elution was dialyzed against dialysis buffer  overnight at 4 °C. Polθ, PolθK121M and PolθΔcen concentrations were determined by SDS gel analysis and by specific activity using Polθ-pol as a standard. Polθ, PolθK121M and PolθΔcen were stored in aliquots at −80 °C.Polθ-pol, Polθ-hel and RPA were purified as describedTemplates are as follows: Figs. 32P-5′-radiolabeled with T4 polynucleotide kinase (New England Biolabs) and (γ-32P) ATP (Perkin Elmer). RP469D: CTGTCCTGCATGATG;RP486: CACTGTGAGCTTAGTCACATTTCATCATGCAGGACAG;RP344: CACTGTGAGCTTAGGGTTAGCCCGGG;RP348: CACTGTGAGCTTAGGGTTAGAGCCGG;SJB108: GTTCTTCGGTCTCGAGGTGACTACAAGGATGACGACGACAAGGGCACTGTGAGCTTAGGGTTAGCCCGGG;SJB116: CACTGTGAGCTTAGGGTTAGGCGGCTTGCAGAGCACAGAGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATTATATGTGTGCCCGGG;RP514: GTTCTTCGGTCTCGAGGTGACTACAAGGATGACGACGACAAGGGCACTGTGAGCTTAGGGTTAGAAATTT;RP515: GTTCTTCGGTCTCGAGGTGACTACAAGGATGACGACGACAAGGGCACTGTGAGCTTAGGGTTAGCCGG;RP132: GACGTTGACTTAAAGTCTAACCTATAGGATACTTACAGCCATCGAGAGGGACACGGCGCATTCTCGAGCGTAC;RP132C: GCTCGAGAATGCGCCGTGTCCCTCTCGATGGCTGTAAGTATCCTATAGGTTAGACTTTAAGTCAACGTCGTAC;RP370: CACTGTGAGCTTAGGGTTAGGAATTC;RP316FAM5: /56-FAM/TTTTTTTTTTTTTTTTTTTTTTTTTTTTT;SJB123: GGTTAGCCCGGG;RP344Cy3: /5Cy3/TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT;RP343: CTAAGCTCACAGTG;SJB153: TATAATACCCAGCAAGTCAGCATC; SJB154: GGAATCTAGAGCACATTCTGCGGCC;SJB155: TCTGTGCTCTGCAAGCCGCCTAACC;SJB158: TATAATACCCAGCAAGTCAGCAT/3ddC/;SJB159: GGAATCTAGAGCACATTCTGCGGC/3ddC/;RP540Cy3: GCATATTCACTGTGAGCTTAGTGTTAGGACTCGG/3Cy3Sp/;RP541Cy5: GCATATTCACTGTGAGCTTAGTGTTACCGAGTCC/3Cy5Sp/;RP538: CGACAAGAGTCATGAATTCTTAGGGTTAGCCCGGG;RP539: CTAAGAATTCATGACTCTTGTCG;SJB089: CAATTCAGCAACTAATGTCATACCAGCTGAAGTTGGTGCAGAG;SJB090: CTCTGCACCAACTTCAGCTGGTATGACATTAGTTGCTGAATTG;SJB100: TTCTGCTAGCGGTGGTGGAGGAAGTGGAGGAGGCGGATCTGGTGGTGGCGGTAGCGGTTTTAAAGATAACTCTCCAATCTCAGATACTTC;SJB101: TCTTCTGCTAGCCATTTCAACCAAATCTTGTTGCAAG.Primer templates were 5′- phosphorylated on the shorter strand with T4 polynucleotide kinase (New England Biolabs) and ATP. Primer templates were annealed by mixture of a ratio of 1:2 of short to long strands then boiling and slow cooling to room temp. DNA was 3xFLAG-Polθ plasmid was derived from pRS424 (ATCC). Human Polθ nucleotide sequence was optimized for yeast and synthesized as 2 gene fragments by GenScript. Yeast optimized gene fragments were cloned into pRS424 sequentially. 3xFLAG-PolθK121M plasmid was derived from 3xFLAG-Polθ plasmid by site-directed mutagenesis using primers (SJB089 and SJB090). 3xFLAG-PolθΔcen was derived from 3xFLAG-Polθ plasmid by molecular cloning with PCR primers (SJB100 and SJB101).Polθ, Polθ-pol, and PolθΔcen were resolved by SDS/PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% milk in TBS with 0.1% Tween-20 and incubated with primary antibody against C-terminal portion of Polθ  in TBS/5% milk/0.1% Tween-20 for 1.5 hr at room temp, followed by incubation with HRP-conjugated secondary antibody  for 1 h at room temp. Bands were detected via chemiluminescence.13 and Polθ-pol 16. The Polθ-hel:ssDNA model was constructed by superposing Polθ-hel structure with the crystal structure of archaeal DNA helicase (Hel308):DNA complex28, and the strand in which Hel308 translocates on was included in the model. The template DNA in the Polθ-pol:DNA complex was removed and the 5′ end of the primer DNA was connected to the 3′-end of DNA in the Polθ-hel model. Polθ-hel was modeled facing upstream due to its 3′−5′ translocation activity.The PolθΔcen:ssDNA model was constructed by using the previously determined crystal structures of human Polθ-hel 3 linker (L), see Supplementary Fig. PolθΔcen is modelled by two large spheres, representing the polymerase (P) and helicase (H) domains, connected by a short flexible polymer chain made by 6 beads, representing the (GGGGS)T = 300 K and with a potential energy V, composed by the following interaction terms.Each bead is governed by a Langevin equation of motion at temp VP-P(r) = VH-H(r) = ε((σ/r)12-(σ/r)6), where σ = 5.6 nm is about twice the gyration radius of both P and H and ε = 10KBT is the interaction strength. The potentials VP-P and VH-H are truncated at r = 8 nm, which correspond to a cutoff distance of about 1.5 nm from their potential minimum, situated at 21/6σ. The distance 1.5 nm corresponds roughly to the Debye screening length for this system.The interactions between polymerase-polymerase and helicase-helicase are attractive, and are modelled with a Lennard-Jones potential r = 21/6σ.All other bead-bead interactions are instead only repulsive and modelled with a Lennard–Jones potential truncated at the potential minimum VP-H(r)), σ = 5.6 nm and ε = 10KBT. For linker-linker interactions (VL-L(r)), σ = 1 nm, corresponding to about 2.5 amino acids, and ε = 1KBT. For polymerase-linker and helicase-linker (VP-L(r) = VH-L(r)) σ = 3.3 nm and ε = 10KBT.σ and ε vary depending on the particle types. For polymerase-helicase interactions (VH = K(r-r0), where K = 750 KBT/nm2 and r0 = 1 nm for linker-linker bonds and r0 = 3.3 nm for linker-helicase and linker-polymerase bonds.The connectivity between the beads of each Polθ∆cen model , and the size range of the simulation boxes allow to reproduce the local concentration, size and shape of multimers observed in the EM data.To generate the assemblies of varying size, we initialized the system with proteins randomly distributed at densities of about 1–3 × 10BT and m = 900 Da. In simulation units, the integration timestep is dt = 0.01 and the friction coefficient is γ = 0.5. The simulation box is bounded by repulsive walls with a repulsive truncated Lennard–Jones potential, where the truncation is set at r = 21/6σ. A typical simulation is long 5–6 × 106 time units.The simulations are carried out with the software LAMMPS with reduced units σ = 1 nm, ε = 1KThe clusters generated by the self-assembly process have a peculiar elongated shape, which is also characteristic of the EM images. This structural feature is absent if the helicase-helicase or polymerase-polymerase attractive interactions are substituted with a truncated repulsive Lennard-Jones potential, see Supplementary Fig. Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Reporting SummarySource Data


Nature Communications 10.1038/s41467-018-03728-5; published online: 10 April 2018Correction to: The originally published version of this article contained an error in the name of the author Flóra Gölöncsér, which was incorrectly given as Flóra Göröncsér. This has now been corrected in both the PDF and HTML versions of the article.


In the title compound, intermolecular C—H⋯O, O—H⋯O and N—H⋯O hydrogen bonds link the molecules into a three-dimensional supramolecular network. 13H14N2O3, the dihydropyridazine ring (r.m.s. deviation = 0.166 Å) has a screw-boat conformation. The dihedral angle between its mean plane and the benzene ring is 0.77 (12)°. In the crystal, intermolecular O—H⋯O hydrogen bonds generate C(5) chains and N—H⋯O hydrogen bonds produce R22(8) motifs. These types of interactions lead to the formation of layers parallel to (12R24(8) motifs. Intermolecular interactions were additionally investigated using Hirshfeld surface analysis and two-dimensional fingerprint plots. The most significant contributions to the crystal packing are by H⋯H (43.3%), H⋯C/C⋯H (19.3%), H⋯O/H⋯O (22.6%), C⋯N/N⋯C (3.0%) and H⋯N/N⋯H (5.8%) contacts. C—H⋯π interactions and aromatic π–π stacking interactions are not observed.In the title compound, C This nitrogen heterocycle became a scaffold of choice for the development of potential drug candidates -6-(4-hydroxy-3-methoxystyryl)-4,5-dihydropyridazin-3(2H)-one, as well as an analysis of the Hirshfeld surfaces.In continuation of our studies related to molecular structures and Hirshfeld surface analysis of new heterocyclic derivatives  Å for the C3 atom; the C2 atom lies −0.177 (3) Å out of the plane in the opposite direction relative to the C3 atom. The dihedral angle between the dihydropyridazine ring mean plane and the benzene ring (C7–C12) is 0.77 (12)°, indicating an almost planar conformation of the molecule favouring delocalization over the C4—C5=C6—C7 bridge.In the title molecule Fig. 1, the coni hydrogen bonds between the phenolic OH group and the carbonyl O atom of a neighbouring molecule generate C(5) chains extending parallel to [101]. Likewise, N1—H1⋯O1ii hydrogen bonds between the N—H function of the dihydropyridazine ring and the carbonyl O atom generate centrosymmetric dimers with an A⋯O2iii and C13—H13C⋯O2iv hydrogen bonds with In the crystal, molecules are stacked in rows parallel to [100]. Notably, no significant C—H⋯π or π–π interactions are observed. O2—H2⋯O1fs Fig. 2.et al., 2016viz. 6-phenyl-4,5-dihydropyridazin-3(2H)-one -(−)-6-(4-aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one  to 2.2382 a.u. (blue). The surfaces mapped over relevant intermolecular contacts are illustrated in Fig. 3dnorm plotted onto the surface are shown for the H⋯H, H⋯C/C⋯H, H⋯O/O⋯H, C⋯N/N⋯C and H⋯N/N⋯H interactions in Figs. 4a)–(e), respectively. The overall two-dimensional fingerprint plot and those delineated into H⋯H, H⋯C/C⋯H, H⋯O/O⋯H, C⋯N/N⋯C and H⋯N/N⋯H contacts are illustrated in Figs. 5a)–(f), respectively. The largest interaction is that of H⋯H, contributing 43.3% to the overall crystal packing. H⋯C/C⋯H contacts add a 19.3% contribution to the Hirshfeld surface, with the tips at de + di ∼ 2.72 Å. H⋯O/O⋯H contacts make a 22.6% contribution to the Hirshfeld surface and are represented by a pair of sharp spikes in the region de + di ∼ 2.70 Å in the fingerprint plot. H⋯O/O⋯H interactions arise from intermolecular O—H⋯O hydrogen bonding and C—H⋯O contacts. The contributions of the other contacts to the Hirshfeld surface are negligible, i.e. C⋯N/N⋯C of 3.0% and H⋯N/N⋯H of 5.8%.Hirshfeld surface analysis was used to quantify the intermolecular interactions of the title compound, using To a solution of 6-(4-hydroxy-3-methoxyphenyl)-4-oxohex-5-enoic acid  in 20 ml of ethanol, an equimolar amount of hydrazine hydrate was added. The mixture was maintained under reflux until thin-layer chromatography (TLC) indicated the end of the reaction. After cooling, the precipitate which formed was filtered off, washed with ethanol and recrystallized from ethanol. Slow evaporation at room temperature led to the formation of single crystals of the title compound.Uiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) otherwise. The NH and OH hydrogens were located in a difference Fourier map and were constrained with N—H = 0.86 Å and Uiso(H) = 1.2Ueq(N), and O—H = 0.86 Å and Uiso(H) = 1.5Ueq(O), using a riding model.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019014130/wm5521sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989019014130/wm5521Isup3.hklStructure factors: contains datablock(s) I. DOI: 1959568, 1959568CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H-thiochromene (A) and 2-(2-bromo-5-fluorophenyl)-7-methoxy-3-nitro-2H-thiochromene (B) are described. In each crystal, the molecules are linked by hydrogen bonds and π–π stacking interactions.The synthesis and structure of 2-(2-bromo-5-fluorophenyl)-8-ethoxy-3-nitro-2 H-thiochromene  and 2-(2-bromo-5-fluorophenyl)-7-methoxy-3-nitro-2H-thiochromene , were prepared via the condensation reaction between 2-mercaptobenzaldehyde and nitrostyrene derivatives. In both compounds, the thiochromene plane is almost perpendicular to the phenyl ring. In the structure of A, molecules are assembled via π–π stacking and C—H⋯O and C—F⋯π interactions. In the crystal packing of B, molecules are linked by C—H⋯F, C—H⋯O, C—H⋯π and π–π interactions.Two thiochromene compounds containing Br and F atoms, namely 2-(2-bromo-5-fluorophenyl)-8-ethoxy-3-nitro-2 H-Chromenes (or 2H-benzopyrans) are heterocyclic compounds found in many natural plants. This class of molecules shows a wide variety of biological activities, such as anticancer, anti-inflammation and anti-HIV  and 2-(2-bromo-5-fluorophenyl)-7-methoxy-3-nitro-2H-thiochromene (B). Crystal structure determination can help to understand the role of halogenated substituents in the biological activity of these compounds.2A) crystallizes in the triclinic space group PB) crystallizes in the space group P21/c, both with one molecule in the asymmetric unit ° with phenyl ring C1–C6, while in B, this angle is 86.94 (8)°, which indicates that the 2-bromo-5-fluorophenyl ring is roughly perpendicular to the thiochromene plane. Both 2H-thiopyran rings have a screw-boat conformation, with atom C7 having the largest deviation from the best plane through atoms S1/C7–C11 . The C—S bond lengths are almost equal . The C11—S1—C7 bond angle is 102.5 (2)° in A and 100.47 (9)° in B. The N—O bond lengths in compound B [1.232 (2) and 1.221 (2) Å] are slightly longer than those in compound A [both 1.219 (5) Å]. The nitro group is situated in the thiochromene plane, as illustrated by the torsion angle O2—N8—C8—C9 of 1.3 (7)° in A and 9.5 (3)° in B.Compound  Å; symmetry code: (i) −x, −y + 2, −z; Cg3 is the centroid of the C10–C15 ring]. Neighbouring dimers interact through C—F⋯π and short Br1⋯H5ii interactions .In the crystal of s Table 1 and π–π B, two molecules form dimers through C—H⋯F hydrogen bonds (Table 2c direction through π–π interactions . Parallel chains interact via C—H⋯O and C—H⋯π interactions.In the crystal of compound s Table 2. These det al., 2016H-thiochromene derivatives, of which three contain halogen atoms [CSD refcodes IFOZIO  in toluene and the reaction mixture was stirred at room temperature for 2 h. After completion of the reaction, the solvent was evaporated under reduced pressure and the crude product was purified by flash chromatography on silica gel (yield 90%). Crystals suitable for single-crystal X-ray diffraction data collection were obtained by slow evaporation from an ethanol solution.To a round-bottomed flask was added 2-mercaptobenzaldehyde (1 equiv.), nitrostyrene (1 equiv.) and KUiso(H) = 1.2Ueq(C) for Csp2—H and Uiso(H) = 1.5Ueq(C) for Csp3—H. A rotating-group model was applied for methyl-group C17 in A and C16 in B.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019014178/vm2223sup1.cifCrystal structure: contains datablock(s) A, B, global. DOI: 10.1107/S2056989019014178/vm2223Asup8.hklStructure factors: contains datablock(s) A. DOI: 10.1107/S2056989019014178/vm2223Bsup9.hklStructure factors: contains datablock(s) B. DOI: Click here for additional data file.10.1107/S2056989019014178/vm2223Asup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019014178/vm2223Bsup5.cmlSupporting information file. DOI: 1950406, 1953121, 1950406, 1953121CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title compound has a nonplanar conformation. In the crystal, the anions are linked to the cations and the water molecules by N—H⋯O and O—H⋯O hydrogen bonds, forming a three-dimensional network. Face-to-face π–π stacking interactions are also observed. 2H10N22+·C21H13N3O8S2−·2H2O, the planes of the phenyl rings and the benzene ring of the 5-nitro-2-oxidobenzenesulfonate group are inclined to one another by 44.42 (11), 56.87 (11) and 77.70 (12)°. In the crystal, the anions are linked to the cations and the water molecules by N—H⋯O and O—H⋯O hydrogen bonds, forming a three-dimensional network. Furthermore, there are face-to-face π–π stacking interactions between the centroids of one phenyl ring and the benzene ring of the 5-nitro-2-oxidobenzenesulfonate group [centroid–centroid distance = 3.8382 (13) Å and slippage = 1.841 Å]. A Hirshfeld surface analysis was conducted to verify the contributions of the different intermolecular interactions.In the anion of the title hydrated salt, C DOI: 10.1107/S2056989018009118/qm2125Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018009118/qm2125Isup3.cmlSupporting information file. DOI: 1851087CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: