One chain type, an unanchored NEDD8 trimer, specifically bound to the second zinc finger domain of PARP‐1 and attenuated its activation. In cells in which Nedp1 is deleted, large amounts of tri‐NEDD8 constitutively form, resulting in inhibition of PARP‐1 and protection from PARP‐1‐dependent cell death. Surprisingly, these NEDD8 trimers are additionally acetylated, as shown by mass spectrometry analysis, and their binding to PARP‐1 is reduced by the overexpression of histone de‐acetylases, which rescues PARP‐1 activation. Our data suggest that trimeric, acetylated NEDD8 attenuates PARP‐1 activation after oxidative stress, likely to delay the initiation of PARP‐1‐dependent cell death.NEDD8 is a ubiquitin‐like protein that activates cullin‐RING E3 ubiquitin ligases (CRLs). Here, we identify a novel role for NEDD8 in regulating the activity of poly(ADP‐ribose) polymerase 1 (PARP‐1) in response to oxidative stress. We show that treatment of cells with H UBLs are small proteins of approximately 8 kDa molecular mass that become covalently linked to other proteins via an isopeptide bond, usually on lysine residues  polymerase 1 (PARP‐1) and attenuates its activation. PARP‐1 overactivation mediates cell death after oxidative stress, and our data suggest that tri‐NEDD8 prevents PARP‐1 hyperactivation to delay premature commitment to cell death.et al, et al, Nedp1 led to the accumulation of neddylated species that do not migrate at the ~ 100 kDa size of neddylated cullins in both cell lines . When exposed to cell extract, this resin efficiently enriched neddylated proteins from both NEDP1 KO cells and from WT cells, but it did not enrich for ubiquitylated proteins  and the NEDD8 E2 enzyme, UBE2M, but in the absence of any NEDD8 E3. The smallest protein in these in vitro reactions, aside from NEDD8 (8.5 kDa), was UBE2M, with a molecular mass of ~ 22 kDa. The smallest possible, non‐NEDD8‐NEDD8 conjugate in these reactions, a UBE2M‐NEDD8 linkage, would thus have a molecular mass of ~ 31 kDa. However, while this product was present in the reaction, conjugates were also formed at lower molecular masses that could only correspond to unanchored NEDD8 dimers, trimers and tetramers  inhibitor (MLN4924) alone, and not following treatment with the ubiquitin E1 enzyme (UBE) inhibitor (MLN7243) ation (PAR), including the poly(ADP‐ribose) polymerase PARP‐1 Fig . In factet al, et al, +) to covalently attach ADP‐ribose to various amino acids, including aspartic acid, glutamic acid, lysine, arginine and serine, that are present on itself and on its substrate proteins ation are known regulators of DNA repair and of the oxidative stress response. PARP‐1 senses DNA damage caused by oxygen radicals and aids in the early steps of DNA repair , together with its substrate receptor Kelch‐like ECH‐associated protein 1 (Keap1), is involved in the cellular response to oxidative stress. Oxidative stress inhibits the Cul3/Keap1‐dependent degradation of the transcription factor nuclear factor erythroid 2‐related factor 2 (Nrf2), which drives gene expression that protects against cell death , but not to its automodification and catalytic domain  Fig G. The DNet al, In addition to its roles in DNA repair, PARP‐1 also inhibits the transcription of some NF‐κB‐responsive genes, including CXCL10, independently of its catalytic activity, but via its ability to directly bind to DNA  Fig C to puriFrom these results, we hypothesized that the acetylation of NEDD8 may serve to mask the positively charged lysine residues of NEDD8 to facilitate its binding to the PARP‐1 Zn2 domain. To confirm whether changes in acetylation influence the interaction of NEDD8 with PARP‐1, we overexpressed the de‐acetylases HDAC1 or HDAC2 in NEDP1 KO cells, which indeed led to a decrease in the amount of NEDD8 trimer bound to exogenously expressed and precipitated GFP‐PARP‐1 Zn1 + 2 Fig C. Likewiin vitro NEDD8 chains by NEDP1 was strongly inhibited by the presence of even low amounts of H2O2  that occur following oxidation of DNA. Zn2 binds to DNA with the highest affinity , Sh α‐NEDP1 1:1,000 (DSTT S378D against AA1–212), Ms α‐DCUN1D1 1:1,000 (Sigma Clone 3D7), Sh α‐DCUN1D2 1:1,000 (DSTT S995C against AA1–60) incubated with recombinant DCNL1 (AA 1–45) peptide to block cross reaction, Sh α‐DCUN1D3 1:2,000 (DSTT S996C against AA1–60), Sh α‐DCUN1D4 1:2,000 (DSTT S997C against AA1–119), Sh α‐DCUN1D5 1:2,000 (DSTT S998C against AA1–124), Rb α‐NEDD8 1:1,000 (Abcam ab81264), Sh α‐UBE2M 1:1,000 [DSTT S432D against full length (mouse)], Sh α‐UBE2F 1:1,000 (DSTT S438D against AA1–70), Ms α‐Actin 1:1,000 , Rb α‐RBX1 1:200 (Thermo PA5‐16282), Rb α‐CAND1 1:1,000 (CST 7433S), Rb α‐CSN5 1:1,000 (Abcam ab12323), Rb α‐CSN8 1:1,000 (Abcam EPR5139), Rb α‐UBA3 1:1,000 (Epitomics 5157‐1), Ms α‐AppBp1/ULA1 1:1,000 (BD 611865), Rb α‐Cul1 1:1,000 (Life Tech 718700), Rb α‐Cul2 1:5,000 (Life Tech 700179), Sh α‐Cul3 1:1,000 , Sh α‐Cul3 1:1,000 (DSTT S464D AA544–768), Sh α‐Cul4A 1:3,500 (DSTT S087D AA1–124), Rb α‐Cul4A 1:1,000 (CST 2699S), Sh α‐Cul4B 1:1,000 (DSTT S070D AA1–162), Sh α‐Cul5 1:1,000 (DSTT S073D AA577–689), Ms α‐IκBα 1:1,000 (CST 4814), Ms α‐pIκBα 1:1,000 (CST 9246 clone 5A5), Ms α‐PAR Polymer 1:1,000 (Enzo Lifescience ALX‐804‐220‐R100 clone 10H), Rb α‐PARP‐1 1:1,000 (CST 9532), Rb α‐AIF 1:1,000 (Abcam ab 32516), Rb α‐NRF2 1:1,000 (Abcam ab62352), Rb α‐HIF‐1α 1:1,000 , Ms α‐FLAG M2 1:2,000 (Sigma F3165), Ms α‐GFP 1:5,000 (Abcam ab184519); Rb α‐PARG 1:1,000 (CST 66564S); Rb α‐Keap1 1:1,000 (CST 8047).2O2, Sigma (216763); Camptothecin, Sigma (C9911); Cycloheximide, Sigma (C1988); DPQ, Calbiochem (300270); CellTiter‐Glo, Promega (G7570); Olaparib, Cambridge Biosciences (CAY10621); PARG inhibitor PDD 17273, Tocris (5952); Sodium Butyrate, Sigma (303410); GFP‐Trap_A, Chromotek (gta), PARP‐1‐Trap_A, Chromotek (xta); phosSTOP, Sigma (4906845001); cOmplete EDTA‐free protease inhibitor cocktail (Roche‐11836170001); Pepstatin A, Sigma (P5318), Bestatin hydrochloride, Sigma (B8385); Deoxyribonuclease I, Sigma (D5025); LDS Sample Buffer, Life Technologies (NP0007); Colloidal Coomassie, Expedeon (ISB1L); IPTG, Formedium (IPTG025); Micrococcal Nuclease, NEB (M0247S).TNF‐α, PeproTech (300‐01A); HALO‐link resin, Promega (G1913); MLN7243, Active Biochemicals (A‐1384); MLN4924, Active Biochemicals (A‐1139); Hhis6‐HALO NEDP1(C163A) DSTT DU28042his6‐HALO NEDP1(C163A D29W A98K G99K) DSTT DU28008pCMV5‐NEDP1 DSTT DU28199GST‐NEDP1 DSTT DU23262Frt/TO N‐FLAG empty DSTT DU13236Frt/TO N‐GFP empty DSTT DU45825Frt/TO C‐GFP empty DSTT DU41574Frt/TO C‐FLAG empty DSTT DU43547GST empty DSTT DU49206et al, gRNAs for NEDP1 and CAND1 were generated by mutagenesis PCR of pEsgRNA (Munoz NEDP1 gRNA F 5′‐GGAAAGGACGAAACACCGCCCCGTAGTCTTGAGTTACAGTTTTAGAGCTAGAAAT‐3′NEDP1 gRNA R5′‐ATTTCTAGCTCTAAAACTGTAACTCAAGACTACGGGGCGGTGTTTCGTCCTTTCC‐3′CAND1 gRNA F 5′‐GGAAAGGACGAAACACCTCACCTAAAGTCCTTGTCGCGTTTTAGAGCTAGAAAT‐3′CAND1 gRNA R 5′‐ATTTCTAGCTCTAAAACGCGACAAGGACTTTAGGTGAGGTGTTTCGTCCTTTCC‐3′Full‐length PARP‐1 (NM_001618.3) was amplified from HUVEC cDNA and cloned into the N‐terminal GFP‐tagged vector. For domain mapping, individual zinc finger domains were cloned into the C‐terminal GFP vector and the catalytic domain was cloned into the N‐terminal GFP vector. GST‐PARP‐1 ZN1 + 2 was cloned in pGEX‐6P‐2 (GE Lifesciences). NSUN2 (NM_017755.5) was amplified from HUVEC cDNA and cloned into the N‐terminal FLAG empty vector. HDAC1 (NM_004964.2) and HDAC2 (NM_001527.3) were both amplified from HUVEC cDNA and cloned into the C‐terminal FLAG empty vector.PARP‐1 Full(NM_001618.3) F 5′‐ATGGCGGAGTCTTCGGATAAGCTCT‐3′PARP‐1 Full(NM_001618.3) R 5′‐TAGTACGCGGCCGCTTACCACAGGGAGGTCTTAAAATTGAATTTCAGTTTCAGC‐3′PARP‐1 Zn1 (AA1‐96) F 5′‐ATGGCGGAGTCTTCGGATAAGCTCT‐3′PARP‐1 Zn1 (AA1‐96) R 5′‐GTACTAGCGGCCGCGCCTGTCACTCCTCCAGCTTCCG‐3′PARP‐1 Zn2 (AA97‐215) F 5′‐TAGTACGGATCCAAAGGCCAGGATGGAATTGGTAGC‐3′PARP‐1 Zn2 (AA97‐215) R stop 5′‐GTACTAGCGGCCGCCTATCCATCCACCTCATCGCCTTTTCT‐3′PARP‐1 Zn2 (AA97‐215) R no stop 5′‐GTACTAGCGGCCGCTCCATCCACCTCATCGCCTTTTCT‐3′PARP‐1 Zn3 (AA216‐336) F 5′‐TAGTACGGATCCGTGGATGAAGTGGCGAAGAAGAAATCTA‐3′PARP‐1 Zn3 (AA216‐336) R 5′‐GTACTAGCGGCCGCTGGGGTTACCCACTCCTTCC‐3′PARP‐1 C‐terminal (AA336‐1014) F 5′‐TAGTACCCCGGGAAGGAATTCCGAGAAATCTCTTACCTCAA‐3′PARP‐1 C‐terminal (AA336‐1014) R5′‐TAGTACGCGGCCGCTTACCACAGGGAGGTCTTAAAATTGAATTTCAGTTTCAGC‐3′NSUN2(NM_017755.5) F 5′‐AATAATAGAATTCATGGGGCGGCGGTCGCGGG‐3′NSUN2(NM_017755.5) R 5′‐TATTATTGCGGCCGCTCACCGGGGTGGATGGACCCCC‐3′HDAC1(NM_004964.2) F 5′‐AAAGGATCCATGGCGCAGACGCAGGGCACCCGGAGGAAAGT‐3′HDAC1(NM_004964.2) R 5′‐TACGCGGCCGCGGCCAACTTGACCTCCTCCT‐3′HDAC2(NM_001527.3) F 5′‐TACGGATCCATGGCGTACAGTCAAGGAGGCGGCAAAAAA‐3′HDAC2(NM_001527.3) R 5′‐TACGCGGCCGCGGGGTTGCTGAGCTGTTCTGATTTGGTTC‐3′Ct−∆∆ method  for 4 h at 37°C and harvested with RNeasy  following the manufacturer's protocol. Following RNA purification, RNA concentration was measured with a NanoDrop (Thermo Fisher Scientific) and 1 μg of RNA was used to generate cDNA using the High‐Capacity cDNA Reverse Transcription Kit (Invitrogen #4368814). qPCRs were performed with Brilliant III Ultra‐Fast SYBR Green qPCR master mix (Agilent 600882) on a Bio‐Rad CFX96. The 218S F 5′‐GTAACCCGTTGAACCCCATT‐3′18S R 5′‐CCATCCAATCGGTAGTAGCG‐3′CXCL10 F 5′‐GCTGATGCAGGTACAGCGT‐3′CXCL10 R 5′‐CACCATGAATCAAACTGCGA‐3′IκBα F 5′‐GATCCGCCAGGTGAAGGG‐3′IκBα R 5′‐GCAATTTCTGGCTGGTTGG‐3′Nrf2 F 5′‐AGACGGTATGCAACAGGACA‐3′Nrf2 R 5′‐AGTTTGGCTTCTGGACTTGGA‐3′NQO1 F 5′‐TCACCGAGAGCCTAGTTCCG‐3′NQO1 R 5′‐TGGCATAGTTGAAGGACGTCC‐3′GAPDH F 5′‐GAAATCCCATCACCATCTTCCAGG‐3′GAPDH R 5′‐GTACCTCTTCCGACCCCGAG‐3′et al . GST‐NEDP1 and GST vectors were transformed into BL21 cells and purified with Glutathione Sepharose 4B . His6‐USP1/UAF1 was obtained from the Division of Signal Transduction Therapy (DSTT)  product, DU23056. The plasmids encoding GST‐PARP‐1 Zn1 + 2, GST‐PARP‐1 Zn1‐GFP and GST‐PARP‐1 Zn2‐GFP were transformed into BL21 Rosetta 2 (DE3)‐competent cells (Novagen 71400). Cells were grown in 2YT media supplemented with 100 μM ZnSO4, and protein expression was induced with 0.2 mM IPTG at 16°C for 20 h. Cells were pelleted by centrifugation, and pellets were resuspended in lysis buffer . Pellets were sonicated; then, 5 mM MgCl2 and DNase I were added to 200 Kunitz/ml; and the cell suspension was incubated with rotation at 4°C for 1 h. Cell lysate was spun at 20,000 × g for 30 min, and supernatant was collected. GST fusion proteins were then purified with Glutathione Sepharose 4B . The GST tag was cleaved from GST‐PARP‐1 Zn1‐GFP and GST‐PARP‐1 Zn2‐GFP with PreScission Protease . Bound DNA was removed from Zn1‐GFP and Zn2‐GFP via a Heparin column  as previously described  in a 37°C incubator with 5% CO2. Cell lines were routinely checked for mycoplasma contamination. siRNA‐mediated knockdown of DCNL1‐5 was carried out as previously described  supplemented with 10% FBS and 3 mM l, et al  and Munol . To immunoprecipitate the FLAG‐tagged proteins, cells were harvested as above in buffer A. Supernatants were incubated with anti‐FLAG‐M2 magnetic beads (Sigma) for 3 h at 4°C. Beads were then washed three times with 0.5 ml of buffer A without inhibitors and then resuspended in reaction buffer . Beads were split evenly between three tubes, and either GST‐NEDP1, his6‐USP1/UAF1 or no protein was added to each tube. Reactions were then incubated at 30°C for 30 min before being stopped with LDS sample loading buffer. To immunoprecipitate endogenous PARP‐1 and GFP fusion proteins, cells were harvested with buffer B: 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5% glycerol, 0.5% NP‐40, 5 mM MgCl2, 0.2 mM CaCl2, 1 μM pepstatin A, 1 μM bestatin supplemented with phosStop and cOmplete mini protease inhibitor. Lysates were sonicated for six pulses of 0.5 s. Subsequently, DNase I (200 Kunitz/ml) was added to lysates and incubated for 1 h at 4°C with rotation. Lysates were centrifuged at 4°C for 20 min at 17,000 × g, and supernatants were collected. GFP‐Trap beads or PARP‐1‐Trap beads were incubated with lysates for 3 h at 4°C with rotation. Next, agarose beads were washed three times with 0.5 ml of wash buffer . Bound proteins were eluted with LDS sample loading buffer and heated at 95°C for 5 min with 10 mM DTT. For GFP pulldown with HDAC overexpression, cells were lysed with buffer B with 2.7 mM KCL. For GFP pulldown after MNase treatment, cells were lysed with buffer B with 75 mM NaCl, 5 mM CaCl2, 15 mM IAA, 3 mM OPT and 4,000 units of MNase (NEB) per ml of lysate and incubated at 25°C for 30 min.Whole‐cell lysates were prepared with buffer A: 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5% glycerol, 0.5% NP‐40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 3 mM 1,10‐phenanthroline, and 15 mM iodoacetamide (IAA), supplemented with phosStop (Roche) and cOmplete mini protease inhibitor (Sigma). Lysates were sonicated and centrifuged at 4°C for 20 min at 17,000 × 2O2, cells were lysed directly in sample loading buffer  and DNA was fragmented by passage through a homogenizer column (Omega Bio‐tek). Proteins were subsequently reduced with 10 mM DTT at 95°C for 5 min before loading onto SDS–PAGE gels.For the analysis of Cullin neddylation, cells were lysed directly in urea loading buffer  and DNA was fragmented by passage through a homogenizer column (Omega Bio‐tek). Proteins were subsequently reduced with 10 mM DTT at 37°C for 10 min before loading onto SDS–PAGE gels. For the analysis of PAR polymer formation after H2O2 continuously for 24 h. Where indicated, cells were treated with DMSO, olaparib, or DPQ for 1 h before treatment with H2O2, and continuously thereafter. After 24 h, cell viability was determined using the CellTiter‐Glo assay (Promega). To assess the viability of U2OS cell lines after treatment with camptothecin, cells were plated at 5,000 cells per well of a 96‐well plate and 24 h later were treated with the indicated concentration of camptothecin at 37°C. After 48 h, cell viability was measured using the CellTiter‐Glo Assay. To analyse cell death after TNFα + cycloheximide treatment, U2OS cells were plated at 10,000 cells per well of a 96‐well plate. Twenty‐four hours after plating, cells were treated with cycloheximide (10 μg/ml) for 1 h and then treated with the indicated amount of TNF‐α. Twenty‐four hours after treatment, cell viability was measured using the CellTiter‐Glo Assay (Promega).For cell viability analysis of U2OS cell lines, cells were plated at 15,000 cells per well of a 96‐well plate and 24 h later were treated with the indicated concentration of HCell lysates were resolved on Bis‐Tris gels (homemade or NuPAGE 4–12%) with MOPS buffer (Formedium MOPS‐SDS5000) and transferred to nitrocellulose or 0.2‐μm PVDF membranes. Membranes were blocked for 1 h at RT with 5% milk, or with 5% BSA in TBST, and then incubated with the indicated antibodies overnight at 4°C. Membranes were then washed 3 × 10 min with TBST and incubated with HRP‐conjugated secondary antibodies for 1 h at RT and then washed 3 × 10 min with TBST. Membranes were incubated with chemiluminescent substrate (EMD Millipore WBKLS0500) and exposed to Konica Blue X‐ray film or, for quantification, were imaged using a ChemiDoc Imaging system (Bio‐Rad). Images were quantified with ImageJ (NIH).All statistical analysis was performed with GraphPad Prism using the statistical tests indicated in the figure legends.d‐lysine (P6407‐5 mg Sigma) and 24 h later were left untreated or treated with 600 μM H2O2 for 9 h at 37°C. Cells were then washed once with ice‐cold phosphate‐buffered saline (PBS) and then fixed with ice‐cold methanol for 1 min. Coverslips were washed three times with PBS and then blocked with 3% BSA in PBS for 1 h at room temperature. Next, coverslips were incubated with primary antibodies  diluted in PBS with 3% BSA for 1 h at room temperature. Coverslips were washed three times with PBS and then incubated with Alexa 488 conjugate anti‐rabbit secondary antibody  for 1 h at room temperature. DAPI  staining was performed for 5 min, followed by one wash with PBS. Coverslips were mounted onto slides with ProLong Gold Antifade reagent (Life Technologies) and imaged on a Zeiss LSM 710 confocal microscope. Quantification of nuclear AIF intensity was performed using ImageJ v1.50g .U2OS cells were plated on coverslips coated with 10 μg/ml Poly‐in vitro by incubating NAE (0.15 μM), UBE2M/2F (10 μM) and NEDD8 (20 μM) in reaction buffer  for 3 h at 30°C. Reactions were stopped by the addition of LDS sample loading buffer.NEDD8 chains were made 2 plates, scraped in ice‐cold PBS, collected and centrifuged at 500 × g for 5 min. Cell pellets were resuspended in buffer A without SDS, and lysates were spun at 17,000 × g for 20 min. The supernatant was collected and incubated with HALO‐Link resin (Promega) conjugated to HALO‐NEDP1 C163A or DAGC mutant for 3 h at 4°C. HALO resin was washed three times with buffer A and three times with 50 mM Tris–HCl without salt or detergent. Bound proteins were eluted with 100 μl of a 50:50 mixture of acetonitrile and 0.1% formic acid for a total of five elutions. Eluate was collected and dried in a speed vac (Thermo Scientific). Protein pellets were resuspended in LDS sample loading buffer and resolved by SDS–PAGE on a 4–12% Bis‐Tris gel . Gels were stained with InstantBlue colloidal Coomassie (Expedeon). Bands were then excised, washed with water, dehydrated in acetonitrile and rehydrated in 50 mM Tris–HCl pH 8.0. Gel slices were alkylated with 20 mM chloroacetamide, dehydrated in acetonitrile and then transferred to 50 mM triethylammonium bicarbonate. Gel bands were incubated with trypsin (5 μg/ml) overnight at 30°C. Peptides were extracted with acetonitrile, gel bands were incubated in 0.1% trifluoroacetic acid (TFA), and then, peptides were extracted two more times with acetonitrile. Extracted peptides were dried in a speed vac and then resuspended in 0.1% TFA/water. Samples were analysed on a LTQ Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific) coupled to an Ultimate 3000 UHPLC system with 15‐cm Acclaim PepMap 100 analytical column  with an addition Pepmap trapping column . Acquisition settings were lockmass of 445.120024, MS1 with 60,000 resolution, top 20 CID MS/MS using rapid scan, monoisotopic precursor selection, unassigned charge states and z = 1 rejected, dynamic exclusion of 60s with repeat count of 1. One‐hour linear gradients were performed from 5% solvent B to 35% solvent B . Raw files were processed in Proteome Discoverer 2.0 (Thermo Scientific), with Mascot 2.4.1 (Matrix Science),and subsequently processed in Scaffold 4.4.6 (Proteome Software) Searches were performed with a peptide tolerance of 10 ppm (monoisotopic) and a fragment tolerance of 0.60 Da (monoisotopic) or with MaxQuant v1.5.7.4 for label‐free quantitative and iBAQ analysis. Settings were fixed modifications of carbamidomethyl (C), variable modifications of oxidation (M), dioxidation (M), LRGG (K) and GlyGly (K). Protein identifications were filtered with a 1% FDR. Raw files were re‐searched separately with variable modification for acetylated peptides acetyl (K) or phosphorylated peptides phospho (STY); both searches included the fixed modifications of carbamidomethyl (C), variable modifications of oxidation (M) and dioxidation (M).For HALO pulldown for mass spectrometry analysis, NEDP1 KO HEK 293 cells were grown on 150‐cmin vitro neddylation reactions, the reactions were resolved on a 4–12% Bis‐Tris gel , stained with InstantBlue colloidal Coomassie (Expedeon), and processed for in‐gel trypsin digest, as described above.For analysis of g for 5 min. Cell pellets were washed two times with 10 ml of PBS and then lysed with 8 M urea and 0.5% CHAPS. Protein concentration was determined by BCA assay using the manufacturer's protocol . To remove salt and impurities, protein was precipitated with acetone. Four times the sample volume of −20°C acetone was added and the solution was incubated at −20 for 2 h. The sample was then centrifuged at 4°C for 20 min at 17,000 × g. The supernatant was decanted, and the pellet was washed with four volumes of 80% acetone, followed by centrifugation at 4°C for 5 min at 17,000 × g. The supernatant was decanted, the pellet was briefly centrifuged again, and residual acetone was removed. The pellet was dried at room temperature for 5 min. Pellets were resuspended with rehydration buffer . 65 mM DTT and trace bromophenol blue) to 30 μg/ml. Subsequently, 90 μg of protein was added to a 7 cm, pH 5–8 IPG strip (Bio‐Rad) and allowed to rehydrate on the strip for 14 h, followed by focusing for 12 h for a total of 22 kVhrs. For two‐dimensional electrophoresis, separation strips were equilibrated with equilibration buffer (Bio‐Rad) with 50 mM DTT for 15 min. The buffer was replaced and the strips equilibrated again for 15 additional minutes. IPG strips were loaded onto precast 4‐20% TGX gels (Bio‐Rad) and processed for Western blot analysis.To prepare lysate from wild‐type U2OS cells, one T‐150 flask was harvested with 5 ml trypsin‐EDTA (Life Technologies). Detached cells were collected with fresh media and centrifuged at 500 × For 2D gel electrophoresis analysis of GST pulldowns, USOS NEDP1 KO cells were harvested with lysis buffer B, as described above for the endogenous PARP‐1 immunoprecipitation. Next, 20 mg of lysate was used for each pulldown with 100 μg GST or 100 μg GST‐PARP‐1 Zn1 + 2 preconjugated to Sepharose 4B beads. The beads and lysates were incubated at 4°C for 2 h with rotation. The beads were then washed four times with wash buffer , followed by three washes with salt‐free buffer (50 mM Tris–HCl pH 7.5). Bound proteins were eluted with 8 M urea and 0.5% CHAPS and were processed for isoelectric focusing, as described above.MJK performed most experiments. RH designed NEDP1 constructs, performed HALO‐NEDP1 pulldowns and sample preparation for the HEK 293 mass spectrometry experiment and performed some cell viability experiments. OH performed microscopy of AIF translocation. RG processed mass spectrometry samples and performed database searches. RB performed 2D gel electrophoresis. MJK, RG, RH, MT and TK analysed mass spectrometry results. MJK, RH and TK designed experiments. MJK and TK wrote the manuscript with input from all authors. RH and TK conceived the project, and TK supervised the work.The authors declare that they have no conflict of interest.Expanded View Figures PDFClick here for additional data file.Table EV1Click here for additional data file.Review Process FileClick here for additional data file.


L′)FeCp*(CO)][PF6] [L′ = S—C(Me)=C(Me)—S—(CH2)3—S—C(Me)=C(Me)—S] is reported.The new structural NiFe hydrogenase model PF6 or [Ni(L′)FeCp*(CO)]PF6, is composed of the nickel complex fragment [Ni(L′)] coordinated as a metalloligand (using S1 and S4) to the [FeCp*(CO)]+ fragment, where (L′)2− is [S—C(Me)=C(Me)—S—(CH2)3—S—C(Me)=C(Me)—S]2− and where Cp*− is cyclo-C5(Me)5− (pentamethylcyclopentadienyl). The ratio of hexafluorophosphate anion per complex cation is 1:1. The structure at 150 K has orthorhombic (Pbcn) symmetry. The atoms of the complex cation are located on general positions (multiplicity = 8), whereas there are two independent hexafluorophosphate anions, each located on a twofold axis . The structure of the new dimetallic cation [Ni(L′)FeCp*(CO)]+ can be described as containing a three-legged piano-stool environment for iron [Cp*Fe(CO)‘S2’] and an approximately square-planar ‘S4’ environment for Ni. The NiS2Fe diamond-shaped substructure is notably folded at the S—S hinge: the angle between the NiS2 plane and the FeS2 plane normals is 64.85 (6)°. Largely because of this fold, the nickel–iron distance is relatively short, at 2.9195 (8) Å. The structural data for the complex cation, which contains a new unsaturated ‘S4’ ligand (two C=C double bonds), provide an interesting comparison with the known NiFe hydrogenase models containing a saturated ‘S4’-ligand analogue having the same number of carbon atoms in the ligand backbone, namely with the structures of [Ni(L)FeCp(CO)]+  and [Ni(L)FeCp*(CO)]+ , where (L)2− is [S—CH2—CH2—S—(CH2)3—S—CH2—CH2—S]2− and Cp− is cyclopentadienyl. The saturated analogues [Ni(L)FeCp(CO)]+ and [Ni(L)FeCp*(CO)]+ have similar Ni—Fe distances: 3.1727 (6), 3.1529 (7) Å (two independent molecules in the unit cell) and 3.111 (5) Å, respectively, for the two complexes, whereas [Ni(L′)FeCp*(CO)]+ described here stands out with a much shorter Ni—Fe distance [2.9196 (8) Å]. Also, [Ni(L)FeCp(CO)]+ and [Ni(L)FeCp*(CO)]+ show interplanar fold angles that are similar between the two: 39.56 (5), 41.99 (5) (independent molecules in the unit cell) and 47.22 (9) °, respectively, whereas [Ni(L′)FeCp*(CO)]+ possesses a much more pronounced fold [64.85 (6)°]. Given that larger fold angles and shorter Ni—Fe distances are considered to be structurally closer to the enzyme, unsaturation in an ‘S4’-ligand of the type (S—C2—S—C3—S—C2—S)2− seems to increase structural resemblance to the enzyme for structural models of the type [Ni(‘S4’)FeCpR(CO)]+ (CpR = Cp or Cp*).The complex cation in the title compound, (carbonyl-1κ The first ‘S4’ ligand used in this capacity featured a saturated two–three–two carbon linker, in L2− = [S—CH2—CH2—S—(CH2)3—S—CH2—CH2—S]2− FeCpR(CO)]+ model based on an analogous but unsaturated ‘S4’ ligand, namely L′2− = [S—C(Me)=C(Me)—S—(CH2)3—S—C(Me)=C(Me)—S]2− Fe(CpR)(CO)]+ complex in which the ‘S4’ ligand has a four-carbon linker in the remote portion of the backbone FeCp*(CO)]+ was obtained as solvent-free crystals containing the PF6− counter-ion. A drawing showing both cation and anion in this salt is shown below (see Supramolecular features), and the intramolecular structural features of the cation are discussed first. The structure of [Ni(L′)FeCp*(CO)]+ is shown in Fig. 34’ environment for Ni (sum of bond angles around Ni1 = 359.83°). Selected metal–ligand distances are Ni1—S1 = 2.1616 (11), Ni1—S2 = 2.1530 (12), Ni1—S3 = 2.1507 (11), Ni1—S4 = 2.1563 (12) Å, and Fe1—S1 = 2.3309 (12), Fe1—S4 = 2.3602 (12), Fe1—C11 = 1.768 (5), Fe1—C1 = 2.080 (4), Fe1—C2 = 2.107 (4), Fe1—C3 = 2.126 (4), Fe1—C4 = 2.138 (4), Fe1—C5 = 2.098 (4) Å. The intermetallic (Ni1—Fe1) distance is relatively short, i.e. 2.9195 (8) Å. The NiS2Fe diamond is markedly folded at the S—S hinge: the angle between the NiS2 plane and the FeS2 plane normals  is 64.85 (6)°, and this fold largely accounts for the short nickel–iron distance.[Ni(L′2− with those of literature complexes using the saturated ligand L2−. The structures of [Ni(L)FeCp(CO)]+, as the PF6− salt/ CH2Cl2 solvate FeCp*(CO)]+, as the PF6− salt FeCp(CO)]+ and [Ni(L)FeCp*(CO)]+ show Ni—Fe distances that are similar for the two, 3.1727 (6)/3.1529 (7) Å (two independent molecules in the unit cell) and 3.111 (5) Å, respectively, for the two complexes. The [Ni(L′)FeCp*(CO)]+ complex, on the other hand, has a much shorter Ni—Fe distance . Also, [Ni(L)FeCp(CO)]+ and [Ni(L)FeCp*(CO)]+ show interplanar fold angles that are similar for the two, 39.56 (5)/41.99 (5)° (two independent molecules in the unit cell) and 47.22 (9)°, respectively, while [Ni(L′)FeCp*(CO)]+ has a much larger fold angle of 64.85 (6)° (see above). The large fold angle and short Ni—Fe distance observed in the complex with the unsaturated ligand L′ match the structure of the enzymatic active site more closely than the angles/distances of the complexes containing the saturated ligand L. For eight structurally characterized enzymes, the dihedral angles range from 59 to 99° and the Ni—Fe distances range from 2.53 to 2.97 Å 2− can increase structural resemblance to the enzyme in models of the type [Ni(‘S4’)FeCpR(CO)]+. Structural similarity to the enzyme in models was, in alternative approaches, also favoured when additional donor atoms were incorporated into the ligand chain (such as ‘S3N2’) or where two bidentate chelate ligands were used instead of one large ‘S4’ ligand. FeCp*(CO)]+ model with four carbon atoms, instead of three, in the remote portion of the backbone  Å and 62.48 (4)°, respectively FeCp*(CO)]+ was shown to be active as a hydrogen-production catalyst Cp*(CO)]+ complex, with the unsaturated ‘S4’ ligand L′, might warrant deeper investigation. We conclude that the introduction of unsaturation in the ‘S4’ ligand led to a better structural model relative to the unsaturated ligand, highlighting a new variant of the classic [Ni(’S4’)FeCpR(CO)]+-type hydrogenase model.In the following discussion, we compare the structural features obtained with the unsaturated ligand L′)FeCp*(CO)]+ with hexafluorophosphate anions, without solvent molecules and without any solvent-accessible void. The ratio of hexafluorophosphate anion per complex cation is 1:1. The atoms of the complex cation are situated on general positions (multiplicity = 8), whereas there are two independent hexafluorophosphate anions, each situated on a twofold axis . A picture of the packing is shown in Fig. 4B⋯F4 = 2.55 Å; C15—H15B⋯F3i = 2.55 Å; C21—H21C⋯F4ii = 2.48 Å; C22—H22C⋯F1iii = 2.52 Å) and a C—H⋯O short contact (C14—H14A⋯O1 = 2.41 Å) .The structure results from packing of discrete cations +, they are not discussed further. LAZVUE FeCp(CO)]+ , MUDXOA FeCp*(CO)]+ , and SUWWAJ FeCp*(CO)]+ . These three complex cations are discussed in detail above.The Cambridge Crystallographic Database .The syntheses were performed in dried solvents under an inert atmosphere  using standard glove-box (MBraun) and Schlenk techniques. Deuterated NMR solvents were from Cambridge Isotopes.  using 1,3-dibromopropane. Na2[Ni(S2C2Me2)] was prepared from Ni(S2C2Me2)2 by reduction with excess sodium in THF , until the colour had changed from deep purple to brown–yellow. The subsequent alkylation of [Ni(S2C2Me2)]2− using 1,3-dibromopropane was performed analogously to the procedure described by Schrauzer and co-workers for the closely related Ni(S—C(Ph)=C(Ph)—S—(CH2)3—S—C(Ph)=C(Ph)—S). ][PF6]: This precursor for the iron part of the complex was prepared according to the general procedure for [Cp*Fe(CO)2(solvent)]+ given by Catheline & Astruc (1984[PF6]: Crude [Cp*Fe(CO)2(NCMe)][PF6]  was combined with 6 ml of acetonitrile and filtered through a glass filter frit. While purging with argon, the reaction was irradiated with UV–visible light  for 16 h. Under an inert atmosphere, a solution of 155 mg (0.46 mmol) of Ni(L′) in ca 7 ml of dichloromethane was added. The reaction mixture was heated under active argon flow to 325 K for 2 h. After cooling to room temperature, the volatiles were slowly removed under vacuum. The solid was dried under vacuum and stored in the glove-box. Yield of crude product: 253 mg (75%). 1H NMR  δ 1.60 ; δ 1.91 ; δ 1.96 ; δ 2.31 ; δ 2.0–3.7 . Note that the sample thus prepared showed a 1H NMR signal for metal-coordinated acetonitrile. The purpose of the prolonged photolysis was to remove all CO from iron, in order to selectively prepare [Ni(L′)FeCp*(NCMe)][PF6]. However, the sample obtained appeared to be a mixture of [Ni(L′)FeCp*(CO)][PF6] and [Ni(L′)FeCp*(NCMe)][PF6] and is thus referred to as [Ni(L′)FeCp*(CO/NCMe)][PF6]. Yet, crystallization from acetone yielded exclusively [Ni(L′)FeCp*(CO)][PF6], in crystalline form.[Ni(L′)FeCp*(CO)][PF6]: 11 mg of [Ni(L′)FeCp*(CO/NCMe)][PF6] were dissolved in 1.5 ml of acetone and filtered through 1 cm of Celite. Through solvent vapor diffusion, by placing the loosely capped vial into a larger vessel containing diethyl ether vapour (and some liquid), crystals of [Ni(L′)FeCp*(CO)][PF6] were grown within two days at 308 K.Crystallization of [Ni(Uiso(H) = 1.2Ueq(C) or 1.5Ueq(Cmethyl).Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2056989018010939/zl2735sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018010939/zl2735Isup2.hklStructure factors: contains datablock(s) I. DOI: 1859284CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, the molecules are linked via O—H⋯N and C—H⋯O hydrogen bonds, forming chains along [101].The title molecule is comprised of two non-coplanar benzene rings connected by an imino group in a  16H17NO4, the dihedral angle between benzene rings is 72.7 (2)°. The methoxy groups are rotated by 2.4 (2) and −4.9 (2) (benzilidene moiety) and by 5.6 (3)° (aniline moiety) relative to the adjacent benzene ring. In the crystal, the molecules are linked into chains along [101] through C—H⋯O and O—H⋯N hydrogen bonds.In the title compound, C The methoxy groups are almost co-planar with the planes of the adjacent aromatic rings .The molecular structure of the title molecule is shown on Fig. 1via C7—H7⋯O2ii and O2—H2⋯N1i hydrogen bonding methyl]phenol, (I)et al., 1992E)-5-methoxy-2-[(4-methoxyphenylimino)methyl]phenol, (II) methyl]phenol, (IV)   and 50 ml of distilled water. The reaction mixture was taken in a clean 250 ml round-bottom flask and stirred well with a magnetic stirrer. It was then refluxed for 7 h. The dark-yellow product that formed was separated by filtration, dried under vacuum and recrystallized from methanol solution upon slow evaporation for two days .4-Hydroxy-3,5-dimethoxybenzaldehyde  (0.05 mol) was added to a mixture of 50 ml of methanol and Uiso(H) = 1.2Ueq(C) or 1.5Ueq.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018013713/ld2146sup1.cifCrystal structure: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018013713/ld2146Isup3.cmlSupporting information file. DOI: 10.1107/S2056989018013713/ld2146Isup3.hklStructure factors: contains datablock(s) I. DOI: 1843910CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the original publication, values of the doses of insulin glargine, the most commonly used basal insulin analogue under the ‘Discussion’ section was incorrectly published.The sentence “…was similar (3100.314 ± 110.1298 U/kg body weight/day at baseline) to that in the Swedish study (0.33 ± 0.16 U/kg body weight/day)”.Should actually say “…was similar (0.31 ± 0.12 U/kg body weight/day at baseline) to that in the Swedish study (0.33 ± 0.16 U/kg body weight/day)”.


The asymmetric unit of the title compound consists of two independent molecules differing slightly in the conformations of the seven-membered rings and the butyl substituents. 16H20N2O2, consists of two independent molecules differing slightly in the conformations of the seven-membered rings and the butyl substituents, where the benzene rings are oriented at a dihedral angle of 34.56 (3)°. In the crystal, pairwise intermolecular C—H⋯O and complementary intramolecular C—H⋯O hydrogen bonds form twisted strips extending parallel to (012). These strips are connected into layers parallel to (111) by additional intermolecular C—H⋯O hydrogen bonds. The layers are further joined by C—H⋯π interactions. The Hirshfeld surface analysis of the crystal structure indicates that the most important contributions for the crystal packing are from H⋯H (65.5%), H⋯C/C⋯H (16.0%) and H⋯O/O⋯H (15.8%) interactions.The asymmetric unit of the title compound, C Rings B and D have boat conformations with slightly different Cremer–Pople puckering parameters . In the n-butyl substituents, the C13—C14—C15—C16 [177.96 (13)°] and C29—C30—C31—C32 [174.97 (12)°] chains also have slightly different torsion angles. The conformation of the 2-oxopropylidene units are partially determined by the intramolecular N—H⋯O hydrogen bonds (Table 1The asymmetric unit of the title compound consists of two independent molecules differing modestly in the conformations of the seven-membered s Table 1 The r.m.Bnz⋯OOxoprp (Bnz = Benzene and Oxoprp = 2-oxopropylidene) and complementary intramolecular C—HBnz⋯OBnzdzp  hydrogen bonds (Table 1Bnz⋯OOxoprp and C—HBnzdzp⋯OBnzdzp hydrogen bonds (Table 1Bnzdzp⋯π and C—HBty⋯π (Bty = n-butyl) interactions  analysis  contacts -4-(2-oxopropylidene)-4,5-dihydro-1H-benzo[b]diazepin-2(3H)-one (2.38 mmol) in 15 ml of dichloromethane were added 1.5 eq of 1-bromobutane, (3.57 mmol) of potassium hydroxide dissolved in water and 0.23 mmol of tetra-n-butyl ammonium bromide (BTBA). The mixture was kept under magnetic stirring at room temperature for 48 h. A little water was added and then the organic phase was extracted. The mixture obtained was chromatographed on a column of silica gel (eluent hexane/ethyl acetate 8/2) to give three products. The title compound was isolated as the major product in a yield of 77%.To a solution of (Uiso(H) = 1.5Ueq(C). The remaining H atoms were located in a difference-Fourier map and were freely refined. The crystal studied was twinned.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018014779/xu5946sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989018014779/xu5946Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018014779/xu5946Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018014779/xu5946Isup4.cmlSupporting information file. DOI: 1874203CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II and AuI cations to form a one-dimensional polymeric compound with free dicyanoaurate anions.Cyanide anions bridge Fe 2(C10H8N2)2(H2O)2][Au(CN)2]}n, the FeII ion, which is located on a twofold rotation axis, has a slightly distorted FeN4O2 octahedral geometry. It is coordinated by two phenylpyrazine molecules, two water molecules and two dicyanoaurate anions, the Au atom also being located on a second twofold rotation axis. In the crystal, the coordinated dicyanoaurate anions bridge the FeII ions to form polymeric chains propagating along the b-axis direction. In the crystal, the chains are linked by Owater—H⋯Ndicyanoaurate anions hydrogen bonds and aurophillic interactions [Au⋯Au = 3.5661 (3) Å], forming layers parallel to the bc plane. The layers are linked by offset π–π stacking interactions [intercentroid distance = 3.643 (3) Å], forming a supramolecular metal–organic framework.In the title polymeric complex, {[Fe(CN) Applying external stimuli, such as temperature, pressure, magnetic field, light irradiation or adding a guest can affect this kind of the compound and change their properties significantly , which consist of metal ions and organic ligand linkers, are studied intensively. Fe-based coordination polymers with N-donor bridging ligands are well known as compounds with switchable spin states  Å] in axial positions, and two N atoms of cyano bridges and two water O atoms of water molecules [Fe1—O1 = 2.122 (4) Å] in equatorial positions. The two CN− anions bridge the FeII and AuI cations [Fe1⋯Au1 = 5.244 (3) Å] to form a one-dimensional polymeric structure with bond lengths Fe1—N1 = 2.107 (5) Å and Fe1—–N2 = 2.117 (6) Å  is Σ|90 - Θ| = 8.53°, where Θ are the cis-N—Fe—O and cis-N—Fe—N angles in the coordination environment of the FeII atom.The structure of the title compound features a one-dimensional chain motif that runs parallel to the crystallographic s Figs. 1 and 2 ▸.water—H⋯N hydrogen bonds .The crystal packing features different types of weak interactions  in water (2.5 ml), the second was a mixture of water/acetonitrile  and the third layer was a solution of 2-phenylpyrazine  and [Fe(OTs)2]·6H2O  (OTs = p-toluenesulfonate) in acetonitrile (2.5 ml) with 0.3 ml of water. After two weeks, yellow crystals grew in the second layer; these were collected and maintained under the mother solution until measured.Crystals of the title compound were prepared by the slow diffusion method between three layers in a 10 ml tube. The first layer was a solution of K[Au(CN)Uiso(H) = 1.2Uiso(C). The idealized OH2 group was fixed using an AFIX 7 command that allowed the H atoms to ride on the O atom and rotate around the bond.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019009678/su5503sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989019009678/su5503Isup2.hklStructure factors: contains datablock(s) I. DOI: 1938914CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


C—H⋯O hydrogen bonds, weak C—H⋯π and weak offset π–π stacking interactions stabilize the packing.In the title pyridazinone derivative, the unsubstituted phenyl ring and the pyridazine ring are inclined to each other, making a dihedral angle of 17.41 (13)°, whereas the Cl-substituted phenyl ring is nearly orthogonal to the pyridazine ring [88.19 (13)°], C 21H19ClN2O3, is not planar. The unsubstituted phenyl ring and the pyridazine ring are inclined to each other, making a dihedral angle of 17.41 (13)° whereas the Cl-substituted phenyl ring is nearly orthogonal to the pyridazine ring [88.19 (13)°]. In the crystal, C—H⋯O hydrogen bonds generate dimers with R22(10) and R22(24) ring motifs which are linked by C—H⋯O interactions, forming chains extending parallel to the c-axis direction. The intermolecular interactions were investigated using Hirshfeld surface analysis and two-dimensional fingerprint plots, revealing that the most significant contributions to the crystal packing are from H⋯H (44.5%), C⋯H/H⋯C (18.5%), H⋯O/H⋯O (15.6%), Cl⋯H/H⋯Cl (10.6%) and C⋯C (2.8%) contacts.The title pyridazinone derivative, C A weak n Table 1. Weak aret al., 2016viz. 4-benzyl-6-p-tolylpyridazin-3(2H)-one  to 1.1730 (blue) a.u. The three-dimensional dnorm surface of the title molecule is illustrated in Fig. 3a. The pale-red spots symbolize short contacts and negative dnorm values on the surface and correspond to the C—H⋯O interactions . The convex blue regions symbolize hydrogen-donor groups and the concave red regions hydrogen-acceptor groups. π–π interactions are generally indicated by adjacent red and blue triangles in the shape-index map, as is the case for the title molecule.The shape-index map of the title molecule was generated in the range −1 to 1 Å Fig. 3b. The cc). The curvedness plot of the title complex shows large regions of green with a relatively flat (i.e. planar) surface area, indicating the presence of π–π stacking interactions, while the blue regions demonstrate areas of curvature.The curvedness map of the title complex was generated in the range −4.0 to 0.4 Å Fig. 3c. The ca, delineated into H⋯H, H⋯C/ C⋯H, H⋯O/O⋯H, H⋯Cl/Cl⋯H, C⋯C contacts associated with their relative contributions to the Hirshfeld surface in Fig. 4b–f, respectively. The most important intermolecular interaction is H⋯H, contributing 44.5% to the overall crystal packing, with the centre of the peak de = di = 1.18 Å . H⋯C/ C⋯H contacts, with a 18.5% contribution to the Hirshfeld surface, indicate the presence of the weak C—H⋯π interaction . H⋯O/O⋯H contacts arising from intermolecular C—H⋯O hydrogen bonding make a 15.6% contribution to the Hirshfeld surface and are represented by a pair of sharp spikes in the region de + di ∼2.35 Å The C⋯C contacts are a measure of π–\p stacking interactions and contribute 2.8% of the Hirshfeld surface. They appear as an arrow-shaped distribution at de + di ∼3.3 Å. Another contact to the Hirshfeld surface is from H⋯Cl/Cl⋯H interactions (10.6%).The overall two-dimensional fingerprint plot is illustrated in Fig. 4 Å Fig. 4b. H⋯C/ n Table 1. Two paint Fig. 4c. H⋯O/OH)-one in 30 ml of tetrahydrofuran (THF), potassium carbonate  was added. The mixture was refluxed for 1 h. After cooling, ethyl bromoacetate  was added and the mixture was refluxed for 8 h. The precipitated material was removed by filtration and the solvent evaporated under vacuum. The residue was purified through silica gel column chromatography using hexane/ethyl acetate (4:6 v/v). Slow evaporation at room temperature led to formation of single crystals with a yield of 70%.To a solution  of 4-(3-dichlorobenzyl)-6-phenylpyridazin-3(2Uiso(H) = 1.2Ueq(C)], C—H = 0.96 Å for methylene [Uiso(H) = 1.5Ueq(C)], C—H = 0.93 Å for aromatic [Uiso(H) = 1.2Ueq(C)] and C—H = 0.98 Å for methine [Uiso(H) = 1.2Ueq(C)] H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019007424/wm5505sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019007424/wm5505Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019007424/wm5505Isup3.cmlSupporting information file. DOI: 1917654CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal structure of the title compound, the molecules are linked by pairs of O—H⋯O hydrogen bonds, forming inversion dimers with an  15H15NO2, is a Schiff base that exists in the keto–enamine tautomeric form and adopts a Z configuration. The molecule is almost planar, with the two phenyl rings twisted relative to each other by 9.60 (18)°. There is an intramolecular N—H⋯O hydrogen bond present forming an S(6) ring motif. In the crystal, pairs of O—H⋯O hydrogen bonds link adjacent molecules into inversion dimers with an R22(18) ring motif. The dimers are linked by very weak π–π interactions, forming layers parallel to (The title compound, C RCH=N–R′) and are prepared by condensation reactions between amines and active carbonyl compounds -6-{[(2-hydroxy-4-methylphenyl)amino]methylidene}-4-methylcyclohexa-2,4-dien-1-one, was obtained in crystalline form from the reaction of 2-amino-5-methylphenol with 2-hydroxy-5-methylbenzaldehyde. We report here its synthesis conditions and the molecular and crystal structures, supplemented by Hirshfeld surface analysis.In the current study, a new Schiff base, . The molecule is almost planar, with an r.m.s. deviation of 0.1061 Å for the complete molecule except the H atoms [largest deviation 0.176 (3) Å for C8]. The two phenyl rings (C1–C6 and C9–C14) are inclined by 9.60 (18)°. The C1—O1 bond length [1.356 (3) Å] to the hydroxy group is in the normal range, while the C14=O2 bond length is comparatively elongated [1.302 (4) Å] due to the involvement of the carbonyl O atom in an intramolecular N—H⋯O hydrogen bond, forming an S(6) ring motif. The C6—N1 and C8=N1 bond lengths are 1.404 (4) and 1.310 (4) Å, respectively. Overall, the bond lengths in the title structure compare well with those of other keto–enamine tautomers known from the literature (see: Database Survey).The molecular structure of the title compound is illustrated in Fig. 1Cg1⋯Cg2 = 4.721 (2) Å; Cg1 and Cg2 are the centroids of the C1–C6 and C9–C14 rings, respectively], forming layers parallel to -2-[(2-hydroxyphenyliminio)methyl]phenolate fragment revealed 25 hits where this fragment adopts the keto–enamine tautomeric form. Nearly all bond lengths in the title structure are the same within standard uncertainties as the corresponding bond lengths in the structures of 2,4-dichloro-6-{[(2-methoxyphenyl)iminio]methyl}phenolate hydrate -[(2-hydroxyphenyl)iminio]methyl}-4-methylphenolate -4-hydroxy-2-[(2-hydroxyphenyl)iminiomethyl]phenolate -[(2-hydroxy-5-methylphenyl)iminio]methyl}-4-(trifluoromethoxy)phenolate  surface resolution. Fig. 4b shows the molecular electrostatic potential plotted over the three-dimensional Hirshfeld surface using the STO-3G basis set in the range −0.0975 to 0.2197 a.u. within the Hartree–Fock level of theory. The O—H⋯O hydrogen-bond donors and acceptors are shown as blue and red areas around the atoms related with positive (hydrogen-bond donors) and negative (hydrogen-bond acceptors) electrostatic potentials, respectively.A Hirshfeld surface analysis  points related to H⋯H contacts that represent a 55.2% contribution in the title structure. In Fig. 5c, two symmetrical wings on the left and right sides indicate C⋯H/H⋯C interactions with a contribution of 22.3%. Furthermore, there are O⋯H/H⋯O , C⋯C (4.9%) and C⋯N/N⋯C (2.6%) contacts.Fig. 5Fig. 6The title compound was prepared by refluxing a mixture of 2-hydroxy-5-methylbenzaldehyde  in ethanol (15 ml) and 2-amino-5-methylphenol  in ethanol (15 ml) for 5 h. Single crystals of the title compound for X-ray analysis were obtained by slow evaporation of an ethanol solution .Uiso(H) = 1.5Ueq. The C-bound H atoms were positioned geometrically and refined using a riding model with C—H = 0.93 and Uiso(H) = 1.2Ueq(C) for aromatic H atoms, and with C—H = 0.96 Å and Uiso(H) = 1.5Ueq(C) for methyl H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019006583/wm5504sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019006583/wm5504Isup2.hklStructure factors: contains datablock(s) I. DOI: 1902148CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


BM) grafts in F1 hybrid recipients is due to natural killer (NK) cell–mediated rejection triggered through “missing self” recognition. “Hybrid resistance” has usually been investigated in lethally irradiated F1 recipients in conjunction with pharmacological activation of NK cells. Here, we investigated BM‐directed NK‐cell alloreactivity in settings of reduced conditioning. Nonlethally irradiated (1‐3 Gy) or nonirradiated F1 (C57BL6 × BALB/c) recipient mice received titrated doses (5‐20 x 106) of unseparated parental BALB/c BM without pharmacological NK cell activation. BM successfully engrafted in all mice and multilineage donor chimerism persisted long‐term (24 weeks), even in the absence of irradiation. Chimerism was associated with the rearrangement of the NK‐cell receptor repertoire suggestive of reduced reactivity to BALB/c. Chimerism levels were lower after transplantation with parental BALB/c than with syngeneic F1 BM, indicating partial NK‐mediated rejection of parental BM. Activation of NK cells with polyinosinic–polycytidylic acid sodium salt poly(I:C), reduced parental chimerism in nonirradiated BM recipients but did not prevent hematopoietic stem cell engraftment. In contrast, equal numbers of parental lymph node cells were completely rejected. Hence, hybrid resistance leads to incomplete rejection of parental BM under reduced conditioning settings.Resistance to parental bone marrow ( Modifying conditioning in the hybrid resistance model reveals that lasting chimerism ensues after nonmyeloablative irradiation, even when no recipient irradiation is given, indicating that natural killer cell–mediated bone marrow rejection is incomplete in reduced conditioning settings. BMbone marrowGvHDgraft versus host diseaseLNlymph nodeNKnatural killerpoly(I:C)polyinosinic–polycytidylic acid sodium saltTBItotal body irradiationTLRtoll‐like receptor1Natural killer (NK) cells are large granular lymphocytes that serve as first‐line defense against pathogens and neoplastic cells.22.1CB6F1  and BALB/c (CD45.2) mice were purchased from Charles River and congenic B6.SJL‐Ptprca Pepcb/BoyJ (CD45.1) mice from Jackson Laboratory. F1 (CD45.1/CD45.2) mice were obtained by crossing male CD45.1 on C57BL/6 background with female CD45.2 BALB/c mice. All mice were housed under specific pathogen‐free conditions and female mice were used between 8 and 12 weeks of age. All animal experiments were approved by the internal review board of the Medical University of Vienna and by the Austrian Ministry of Science and Research (permission number GZ: BMWFW‐66.009/0028‐WF/V/3b/2015).2.26) of unseparated BALB/c (CD45.2) or CB6F1 (CD45.2) BM cells (d0). Bones  were flushed with a syringe and BM cells were collected in M199 medium (Sigma Aldrich) supplemented with 10 mM Hepes Buffer  and 50 μg/ml gentamycin .F1 (CD45.1/CD45.2) recipient mice received titrated doses (5‐20 × 102.36 BALB/c BM (d0) and poly(I:C) . Sixteen weeks after transplantation, BM cells were recovered from primary recipients and transplanted into secondary F1 mice conditioned with 11 Gy TBI (2 × 5.5 Gy). On the day of reconstitution, each secondary recipient was transplanted with 20 × 106 BM cells recovered from one chimera (i.v.).Primary recipients received 10 × 102.4Full‐thickness tail skin was grafted 4‐6 weeks after BM transplantation and visually inspected thereafter at short intervals. Grafts were considered to be rejected when less than 10% remained viable, as described earlier.2.5− CD45.2+ cells among CD45.1+ CD45.2+ plus CD45.1− CD45.2+ leukocytes (CD45.1− CD45.2+/(CD45.1− CD45.2+ + CD45.1+ CD45.2+) × 100). APC anti‐mouse CD45.1 (A20), PE anti‐mouse CD45.2 (104), FITC anti‐mouse Mac‐1 (M1/70), PE‐Cy7 anti‐mouse CD8 (53‐6.7), APC‐Cy7 anti‐mouse CD4 (RM4‐5), Pacific Blue anti‐mouse CD3 (17A2), FITC anti‐mouse CD49b (DX5), FITC anti‐mouse NK1.1 (PK136), PE anti‐mouse Ly49D (4E5), biotin anti‐mouse Ly49A (YE1/48.10.6) were purchased from Bio Legend. PE‐Cy7 anti‐mouse Ly49G2 (4D11) was purchased from eBioscience.The presence of donor cells was assessed at regular intervals by staining CD45.1 and CD45.2 on blood leukocytes. Donor chimerism was assessed as percentage of CD45.12.6t test with equal variances was used to compare chimerism levels. Total chimerism levels were compared between groups by using analysis of variance (ANOVA). The correlation between BM dose and chimerism level was assessed by a linear regression model. A P‐value below .05 was considered to denote statistical significance .Data were statistically analyzed with GraphPad Prism 5.0 . A 2‐sided Student's 36 BALB/c BM (d0). All recipients developed high levels of persistent multi‐lineage mixed chimerism   that bind the very same MHC molecule.+ Ly49A/G2− NK cells  male mice with BALB/c (CD45.2) female mice so that the resulting F1 generation coexpressed both CD45.1 and CD45.2, whereas donor BALB/c cells solely expressed CD45.2  7 days postinfusion by F1 recipient  is given.Irradiation promotes engraftment by creating space in the BM niche,So far the proliferation of recipient splenocytes shortly after BM transplantation served as surrogate marker for BM engraftment in hybrid resistance models using lethally irradiated mice.+ NK cells would obtain the expression of the inhibitory receptors Ly49A and/or Ly49G2. This adaptation extended over a period of 4 weeks, which approximately corresponds to the time of NK cell maturation in the BM.Even if allogeneic stem cells have sufficient space to engraft, one would expect NK cells to resist their engraftment unless very large BM doses are infused.Our data from the murine hybrid resistance setting suggest that NK‐mediated BM rejection is less potent in reduced conditioning settings than in lethal irradiation regimens, allowing stem cell engraftment with moderate BM doses even in nonirradiated recipients.American Journal of Transplantation.The authors of this manuscript have no conflicts of interest to disclose as described by the


A quantification of the intermolecular contacts in the crystal were estimated using Hirshfeld surface analysis and two-dimensional fingerprint plots.2-(4-Nitrophenyl)-2-oxoethyl picolinate was synthesized under mild conditions. The chemical and molecular structure was confirmed by single-crystal X-ray diffraction studies. The molecules are related by inversion into centrosymmetric dimers  14H10N2O5, was synthesized under mild conditions. The chemical and molecular structures were confirmed by single-crystal X-ray diffraction analysis. The molecules are linked by inversion into centrosymmetric dimers via weak intermolecular C—H⋯O interactions, forming R22(10) ring motifs, and further strengthened by weak π–π interactions. Hirshfeld surface analyses, the dnorm surfaces, electrostatic potential and two-dimensional fingerprint (FP) plots were used to verify the contributions of the different intermolecular interactions within the supramolecular structure. The shape-index surface shows that two sides of the molecules are involved with the same contacts in neighbouring molecules and curvedness plots show flat surface patches that are characteristic of planar stacking.2-(4-Nitrophenyl)-2-oxoethyl picolinate, C The torsion angle value of τ2 = −147.02 (11)° between the two carbonyl groups indicates a –anticlinal conformation. Likewise, owing to a substitution on the functional group, the title compound experiences steric repulsion between the substituent and adjacent carbonyl groups, which can influence the torsion angle [τ3 = 2.4 (2)%] and resulting in a +synclinal conformation. The bond lengths and angles are normal and the molecular conformation is characterized by a dihedral angle of 31.58 (8)° between the mean planes of the two aromatic rings. The nitro group lies nearly in the plane of the phenyl ring, as indicated by the torsion angle values of −4.7 (2) and −5.1 (2)° for C4—C3—N1—O4 and C2—C3—N1—O5, respectively.The molecular structure of the title compound is shown in Fig. 1B⋯O3 interactions stabilize the supramolecular architecture by forming Cg, N—O⋯Cg and Cg⋯Cg interactions. The hydrogen-bond geometry and lone pair-π interactions are listed in Table 2Cg⋯Cg interactions, i.e.Cg1⋯Cg1  and Cg2⋯Cg2 . These weak intermolecular interactions link the molecules to form a one-dimensional chain along the c axis and the molecules exhibit layered stacking  and shape-index (−1.0 to 1.0 a.u.), respectively. The calculated volume inside the Hirshfeld surface is 311.97 Å3 in the area of 305.78 Å3.Hirshfeld surfaces and fingerprint plots  = 1.2 or 1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019014105/jj2216sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989019014105/jj2216Isup2.hklStructure factors: contains datablock(s) I. DOI: 1449658, 1449658CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Quantum-mechanical (QM) calculations were performed at the MP2/aug-cc-pVDZ//B3LYP/6-311++G level in vacuum phase, along with Bader’s quantum theory of Atoms in Molecules (QTAIM). These processes involve transition states (TSs) with quasi-orthogonal structures (symmetry C1), which are highly polar, tight ion pairs ∙. Gibbs free energies of activation for the A∙T(WC) / A∙T(rWC) ↔ A*∙Т(rwWC) / A*∙Т(wWC) tautomeric transitions  are lower than for the A∙T(H) / A∙T(rH) ↔ A*N7∙Т(rwH) / A*N7∙Т(wH) tautomerisations  . The (T)N3+H⋯N1-(A), (T)O4+H⋯N1-(A) / (T)N3+H⋯N1-(A) and (T)O2+H⋯N1-(A) H-bonds are found in the transition states TSA-·T+A·T(WC)↔A*·T(rwWC) / TSA-·T+A·T(rWC)↔A*·T(wWC). However, in the transition state TSA-·T+A·Т(H)↔A*N7·T(rwH) / TSA-·T+A·Т(rH)↔A*N7·T(wH), the (T)N3+H⋯N7-(A), (T)O4+H⋯N7-(A) / (T)N3+H⋯N7-(A) and (T)O2+H⋯N7-(A) H-bonds are supplemented by the attractive (T)O4+/O2+⋯N6-(A) van der Waals contacts. It was demonstrated that the products of the tautomerization of the classical A∙T DNA base pairs—A*∙Т(rwWC), A*N7∙Т(rwH) and A*N7∙Т(wH) (symmetry Cs)–further transform via double proton transfer into the energetically favorable wobble A∙T*(rwWC), A∙T*(rwH) and A∙T*O2(wH) base mispairs (symmetry Cs).In this paper we have theoretically predicted a novel pathway for the mutagenic tautomerization of the classical A∙T DNA base pairs in the free state, the Watson-Crick A·Т(WC), reverse Watson-Crick A·Т(rWC), Hoogsteen A·Т(H) and reverse Hoogsteen A·Т(rH) pairs,  Investigation of microstructural mechanisms for mutagenic tautomerization of the Watson-Crick DNA base pairs occupies an important place in molecular biophysics and molecular biology, enabling an understanding of the nature of genome instability –5. This N7 –to N7 nitrogen atom; T*–to O4 oxygen atom and T*O2 –to O2 oxygen atom. Löwdin proposed that the A∙T(WC)↔A*∙T*(L) and G∙C(WC)↔G*∙C*(L) transitions occur by double proton transfer (DPT) along neighboring intermolecular hydrogen (H) bonds via proton tunneling. These ideas have been prominent in the field of quantum biology and attracted much theoretical study of the mechanisms of spontaneous transitions and transversions arising during DNA replication [Löwdin , 4 firstlication .kT  for the G∙C(WC) DNA base pair [Recently, it has become clear that Löwdin’s mechanism does not provide the generation of sufficiently long-lived mutagenic tautomers of the DNA bases, which escape from the replicative DNA-polymerase transforming into their canonical tautomeric forms. The root cause of this observation is the absence of the reverse barrier of tautomerization ΔΔG in the A∙T(WC) DNA base pair and its small value in comparison with ase pair , 15–18.vice versa, which mechanism obviates the above difficulties. The chief difference of our mechanism from the Löwdin mechanism is that, in the process of mutagenic tautomerization through sequential proton transfer, the DNA bases shift laterally relative each other into the DNA minor or major grooves, leading to the wobble configuration which contains the mutagenic tautomers [In previous papers –27 we prautomers . Moreoveautomers , pyrimidautomers , 23 and automers –27 DNA bvice versa, which is the key to understanding the microstructural mechanisms for spontaneous transitions and transversions during DNA biosynthesis [This allows us to assume that it is the intrapair tautomeric transition of the wobble pairs from the main tautomeric form into the rare one with a WC configuration or close to it, and ynthesis –27. Theoynthesis –31.1) which are highly polar tight ion pairs ∙.This paper uses QM/QTAIM methods to explore new pathways for mutagenic tautomerization of the classical Watson-Crick A·Т(WC), reverse Watson-Crick A·Т(rWC), Hoogsteen A·Т(H) and reverse Hoogsteen A·Т(rH) base pairs with a remarkable biological meaning . TheThe geometries of all the investigated DNA base pairs and transition states (TSs) were optimized using the Gaussian’09 package . The B3LWe have performed investigations for the isolated H-bonded pairs of nucleotide bases, that adequately reflects the processes occurring in real duplex environment , 30, 31.el = electronic energy, while Ecorr = thermal correction to Gibbs free energy.The Gibbs free energy G for all structures was obtained in the following way:int were calculated at the MP2/6-311++G level of theory as the difference between the total energy of the base pair and energies of the monomers and corrected for the basis set superposition error (BSSE) [Electronic interaction energies ΔEr (BSSE) ,87 throur (BSSE) ,89 withor (BSSE) .Bader’s quantum theory of Atoms in Molecules (QTAIM) –96 was aThe energies of the attractive van der Waals contacts , 102 in Energies of conventional AH···B H-bonds were evaluated by the empirical Iogansen formula :EAH···Bsonances –114.The atom numbering scheme for the DNA bases is as per convention .These novel pathways for the mutagenic tautomerization of four biologically important A∙T DNA base pairs—Watson-Crick A·Т(WC), reverse Watson-Crick A·Т(rWC), Hoogsteen A·Т(H) and reverse Hoogsteen A·Т(rH) –49–are pvia proton transfer along intermolecular H-bonds as per currently known mechanisms for mutagenic tautomerization of WC pairs [Conformers of the A∙T base pairs remain plane symmetric structures along the entire IRC of tautomerization. This also holds for base pairs tautomerising WC pairs , 19, 49.WC) / A*∙Т(wWC) / A*N7∙Т(rwH) / A*N7∙Т(wH) tautomerisation reactions occur via the initial migration of proton localized at the N6 atom of the N6H2 amino group, leading to the formation of the A+∙Т- ion pair and significant change of the mutual orientation of the bases within the pair, i.e. mutual transformation of the cys / trans↔trans / cys-orientation of the N1H and N9H bonds relative to each other (1). Further proton transfers to the N1/N7 nitrogen atom causing the rotation of the base and formation of the terminal wobble base mispair. Each of these tautomeric conversions is followed by the asynchronous DPT along the intermolecular H-bonds in the wobble base mispairs (The A∙T(WC) / A∙T(rWC) / A∙T(H) / A∙T(rH) ↔ A*∙Т(rwch other . Our newmispairs .WC) / A*∙Т(wWC) / A*N7∙Т(rwH) / A*N7∙Т(wH) tautomerisation reactions, the TSs are highly polar (~ 6.8–12.7 D) tight ion pairs  ∙ ion pairs. In the TSA-·T+A·T(WC)↔A*·T(rwWC) / TSA-·T+A·T(rWC)↔A*·T(wWC) transition states of tautomerisation the (T)N3+H⋯N1-(A) (13.06 / 13.24) and (T)O4+ / O2+H⋯N1-(A)  are observed, while for the TSA-·T+A·Т(H)↔A*N7·T(rwH) / TSA-·T+A·Т(rH)↔A*N7·T(wH) transition states, the (T)N3+H⋯N7-(A) (8.98 / 8.46) and (T)O4+ / O2+H⋯N7-(A) (5.18 / 4.38) H-bonds are supplemented by attractive (T)O4+/O2+⋯N6-(A)  van der Waals contacts (+H⋯N1-/N7-(A) H-bonds  are significantly stronger than other specific contacts with increased ellipticity. The weakest among them are the attractive (T)O4+/O2+⋯N6-(A)  van der Waals contacts  / A∙T(rWC) / A∙T(H) / A∙T(rH) ↔ A*∙Т(rwl∙mol-1) . These Tcontacts , Table 2A*·T(rwWC)↔A·T*(rwWC), TSA*·T(wWC)↔A·T*O2(wWC), TSA*N7·Т(rwH)↔A·Т*(rwH) and TSA*N7·Т(wH)↔A·Т*O2(wH) of the DPT reactions are stabilized by the N6-H-N3 covalent bridge and one-single intermolecular H-bond—N1H⋯O4 (11.61), N1H⋯O2 (10.94), N7H⋯O4 (13.76) and N7H⋯O4 , accordingly , which are characterized by low energies ECH···O, estimated by the Espinose-Molins-Lecomte formula [C⋯O and dH⋯O) in comparison with the canonical H-bonds  BCPs of the intermolecular H-bonds range from 0.013 a.u. up to the 0.107 a.u.; the values of the Laplacian of the electron density Δρ at the  BCPs are positive for all intrapair H-bonds and lie within a wide range from 0.005 a.u. up to the 0.152 a.u., demonstrating that H-bonds are attractive closed-shell ineractions; the value of the ellipticity ε varies in the range 0.79–8.6·10−3 .A···B (2.574–3.103 Å), dH···B (1.442–2.293 Å) and ∠AH⋯B (139.2–179.3°)  .Interestingly, the energy of the intermolecular specific contacts  constitute only a minor part of the electronic energy of monomeric interactions for all these H-bonded structures ~14–0.87%) /A*∙Т(wWC) tautomerisations  is noticeably lower than for the A∙T(H)/A∙T(rH)↔A∙T*(rwH)/A∙T*O2(wH) tautomerisations  , achieving maximum values for each tautomeric transition at its TS  . The Gib2.65 D, aWC) reaction, occurs by participation of the dynamically unstable intermediate A*∙Т*(L) ↔A*∙Т. TheWC), A*N7∙Т(rwH) and A*N7∙Т(wH) mispairs (s) tautomerise further via the DPT mechanism along neighboring intermolecular H-bonds into the energetically-favorable plane-symmetric A∙T*(rwWC), A∙T*(rwH) and A∙T*O2(wH) DNA base mispairs, respectively N3H⋯N6(A) H-bonds, which, in fact, is a rate-limiting stage. It is noteworthy that the A*N7·Т(rwH)→A·Т*(rwH) and A*N7·Т(wH)→A·Т*O2(wH) tautomerisations are barrier-less  (WC)↔A∙T*(rwWC) (1.39) and A*·T(wWC)↔A·T*O2(wWC) (1.77) are significantly lower than for the novel tautomerisation reactions , but are comparable with the values for the other DPT reactions [syn↔A∙G*syn [-1 for A∙T↔A*∙T* [It should be noted that three out of four tautomerization processes of the A∙T base pairs do not complete with formation of the A*∙Т(rwmispairs . These pectively  and 2. Tl∙mol-1) , while teactions : from 2.↔A∙G*syn  to 10.29∙T↔A*∙T*  DPT tautО2 [N7·Т(rwH)↔A·Т*(rwH) and A*N7·Т(wH)↔A·Т*O2(wH) tautomeric equilibria are completely shifted to the right. For the two other cases, the following proportions are observed: A*·Т(rwWC) (6.9%) ↔ A·Т*(rwWC) (93.1%) and A*·Т(wWC) (83.6%) ↔ A·Т*O2(wWC) (16.4%).It is thus possible to say that the tautomerization processes described here terminate with the mutagenic tautomerization of both T and A DNA bases with further formation of the classical mutagenic tautomers Т*, Т*О2 , 106 andО2 , 114, re-1) and populations of the investigated base mispairs yield the order: A·Т(H) (0.00) < A·Т(rH) (0.22/0.63) < A·Т(WC) (1.05/0.21) < A·Т(rWC) (1.31/0.16) < A∙T*(rwWC) (8.83/3.77∙10−7) < A·Т*(rwH) (8.96/1.92∙10−7) < A*∙T(rwWC) (10.07/2.47∙10−8) < A*·T(wWC) (10.65/1.13∙10−8) < A·T*O2(wWC) (12.31/2.07∙10−9) < A·Т*O2(wH) (12.74/6.13∙10−10) < A*∙T*(L) (13.51/1.95∙10−10) < A*N7·Т(rwH) (24.69/1.08∙10−18) < A*N7·Т(wH) (25.70/2.24∙10−19). Notably, populations of the wobble A∙T*(rwWC), A·Т*(rwH), A*∙T(rwWC), A*·T(wWC), A·T*O2(wWC), A·Т*O2(wH) (12.74/6.13∙10−10) and A*∙T*(L) tautomerised states, fitting into the range of the frequencies of the spontaneous point mutations observed experimentally (10−11–10−9) [Gibbs free energies  –117, poiNotably, the methyl group of the T DNA base does not change its orientation during all these tautomerisation processes without exception. Moreover, the heterocycles of the DNA bases remain planar, despite their ability for out-of-plane bending –120.+ base occurs only in the TSA-·T+A·T(WC)↔A*·T(rwWC), TSA-·T+A·T(rWC)↔A*·T(wWC), TSA-·T+A·Т(H)↔A*N7·T(rwH) and TSA-·T+A·Т(rH)↔A*N7·T(wH) transition states. The maximum value of the non-planar dihedral angle reaches 2.5° (C2-N3-C4-C5), 3.1° (N1-C2-N3-C4), 3.7° (C2-N3-C4-C5) and 7.8° (N1-C2-N3-C4), respectively. Another structural feature of the protonated T+ base in these TSs is the deviation of the О4+H / О2+H hydroxyl group from the plane of the pyrimidine ring   .1) are highly polar tight ion pairs ∙. The tautomerization products—the A*∙Т(rwWC), A*N7∙Т(rwH) and A*N7∙Т(wH) pairs—further transform via concerted asynchronous double proton transfer into the energetically favorable wobble A∙T*(rwWC), A∙T*(rwH) and A∙T*O2(wH) mispairs (symmetry Cs), respectively. Moreover, it was established in our recent papers, that wobble A*∙T(rwWC) base mispair can also be formed from the reverse A∙T(rWC) base pair [N7·Т(wH) base mispair—from the Hoogsteen A∙T(H) base pair [Novel pathways for mutagenic tautomerization of four classical A∙T DNA base pairs, followed by the significant changes of base orientation within the pair, have been predicted by these QM results. The transition states with quasi-orthogonal structure Click here for additional data file.


CSNK2A1 inhibited TGFβ‐induced EMT. TGFβ signaling decreased CK2β but did not affect CK2α protein levels, resulting in a quantitative imbalance between the catalytic α and regulatory β subunits, thereby increasing CK2 activity. The decrease in CK2β expression was dependent on TGFBRI kinase activity and the ubiquitin–proteasome pathway. The E3 ubiquitin ligases responsible for TGFβ‐induced CK2β degradation were found to be CHIP and WWP1. Okadaic acid (OA) pretreatment protected CK2β from TGFβ‐induced degradation, suggesting that dephosphorylation of CK2β by an OA‐sensitive phosphatase might be required for CK2 activation in TGFβ‐induced EMT. Collectively, our results suggest CK2 as a therapeutic target for the prevention of EMT and metastasis of cancers.Transforming growth factor β (TGFβ) is overexpressed in advanced cancers and promotes tumorigenesis by inducing epithelial–mesenchymal transition (EMT), which enhances invasiveness and metastasis. Although we previously reported that EMT could be induced by increasing CK2 activity alone, it is not known whether CK2 also plays an essential role in TGFβ‐induced EMT. Therefore, in the present study, we investigated whether TGFβ signaling could activate CK2 and, if so, whether such activation is required for TGFβ‐induced EMT. We found that CK2 is activated by TGFβ treatment, and that activity peaks at 48 h after treatment. CK2 activation is dependent on TGFβ receptor (TGFBR) I kinase activity, but independent of SMAD4. Inhibition of CK2 activation through the use of either a CK2 inhibitor or shRNA against  CHIPcarboxyl terminus of Hsc70‐interacting proteinCK2protein kinase CK2CKDCSNK2A1 knockdownEMTepithelial–mesenchymal transitionHEKhuman embryonic kidneyNSCLCnon‐small cell lung cancerOAokadaic acidSKDSMAD4‐knockdownTGFBRIITGFβ type II receptorTGFBRITGFβ type I receptorTGFβtransforming growth factor βWWP1WW domain containing E3 ubiquitin protein ligase 11et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., et al., Transforming growth factor β (TGFβ) is a potent pleiotropic cytokine that regulates cell growth/differentiation, cell motility, extracellular matrix production, angiogenesis and cellular immune responses , and 100 U·mL−1 penicillin (Gibco). Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco's modified Eagle's medium (Gibco). All cells were cultured at 37 °C in 5% CO2. The CK2 inhibitor, emodin , and the reversible cell‐permeable proteasome inhibitor, MG132 (Sigma‐Aldrich), were prepared in 20 mm stock with dimethylsulfoxide (Sigma‐Aldrich). The TGFBRI‐specific inhibitor, SB431542 (Sigma‐Aldrich), was prepared in 10 mm stock with dimethylsulfoxide. The protein phosphatase inhibitor, okadaic acid , was prepared in 10 μm stock with dimethylsulfoxide. TGFβ  was prepared in 10 μg·mL−1 stock. pCMV5 TBRI‐His was a gift from Joan Massague (Addgene plasmid # 19161). pCMV5B‐TGFβ receptor I K232R was a gift from Jeff Wrana (Addgene plasmid # 11763). p3TP‐Lux  gene TGFβ response element and three collagenase I AP‐1 repeats in front of luciferase, was a gift from Joan Massague & Jeff Wrana (Addgene plasmid # 11767). pCMV5B‐Flag‐Smurf1 wt was a gift from Jeff Wrana (Addgene plasmid # 11752), pCMV5B‐Flag‐Smurf2 wt was a gift from Jeff Wrana (Addgene plasmid # 11746), p4489 Flag‐betaTrCP was a gift from Peter Howley (Addgene plasmid # 10865), pcDNA3‐HA2‐ROC1 was a gift from Yue Xiong (Addgene plasmid # 19897) and pCI HA NEDD4 was a gift from Joan Massague (Addgene plasmid # 27002). pCMV‐Tag3B‐WWP1‐myc was kindly provided by Dr Ceshi Chen (Kunming Institute of Zoology). Flag‐tagged pCMV‐Tag2C‐WWP1 was kindly provided by Dr Hyeon Soo Kim  cell line, A549 was cultured in Roswell Park Memorial Institute 1640 medium , supplemented with 10% fetal bovine serum (Gibco), 100 μg·mL2.2et al., Western blot analysis was performed as described previously . The beads were incubated with 100 μg of cell lysates in a final volume of 50 μL of kinase reaction buffer  of [γ‐32P]‐ATP]) for 20 min at 30 °C. The reactions were stopped by washing twice with 1 × kinase buffer. The samples were resuspended with 30 μL of 2 × SDS/PAGE sample‐loading buffer, subjected to 12% SDS/PAGE, stained with Coomassie Brilliant Blue, and dried on Whatman paper . 32P incorporation was detected by autoradiography.To evaluate intracellular CK2 activity, an 2.4CSNK2A1 . The shRNA sequences tested for SMAD4 knockdown were: sequence #1: TTCAGGTGGCTGGTCGGAAAGGATTTCCT; sequence #2: GCAGCCATAGTGAA GGACTGTTGCAGATA; sequence #3: CCAACATTCCTGTGGCTTCCACAAGTC AG; and sequence #4: GTCAGGTGCCTTAGTGACCACGCGGTCTT. We validated all constructs individually and found that constructs #1 and #2 were effective for SMAD4 knockdown. Subsequently, we used construct #1 for SMAD4 knockdown. Mission® esiRNA human STUB1 was purchased from Sigma‐Aldrich. Silencer® select pre‐designed siRNA targeting human WWP1  was purchased from Ambion (Thermo Fisher Scientific). The cells were transfected with siRNA using Lipofectamine® RNAiMAX  in accordance with the manufacturer's instructions.shRNA‐mediated knockdown of 2.5The cells were seeded in six‐well plates and cotransfected with p3TP‐Lux and pRL‐TK using ViaFect™ . Twenty‐four hours after transfection, the cells were treated with TGFβ for 24 h, washed with PBS and harvested. Cell lysates were prepared with 200 μL of Passive Lysis buffer (Promega). Aliquots (20 μL) of cleared lysate were analyzed for luciferase activity using a Dual‐luciferase® reporter assay system (Promega). The luciferase activity of p3TP‐Lux was normalized to that of pRL‐TK.2.6m Hepes (pH 7.9), 10 mm KCl, 0.1 mm EDTA, 1 mm dithiothreiltol, 1 mm phenylmethanesulfonyl fluoride, 1 × protease inhibitor cocktail and 1 mm sodium orthovanadate. The samples were adjusted to 0.6% Nonidet P‐40 (NP‐40), and vortexed vigorously for 10 s. Nuclei were pelleted by centrifugation at 10 000 × g for 30 s at 4 °C. The supernatants were collected and used as the cytoplasmic fraction. After washing the pellets with PBS, they were lysed in buffer C comprising 20 mm Hepes, pH 7.9, 0.4 m NaCl, 0.1 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethanesulfonyl fluoride, 1 × protease inhibitor cocktail and 1 mm sodium orthovanadate by sonication. The lysates were cleared by centrifugation at 10 000 × g for 20 min at 4 °C. The supernatants were collected and used as the nuclear fraction.The cells were allowed to swell in buffer A comprising 10 m2.7m Tris‐HCl, pH 7.4, 150 mm NaCl, 0.5% NP‐40) with cOmplete™ protease inhibitor cocktail (Roche Diagnostics). The cell lysates were pre‐cleared and then incubated with the appropriate antibodies for 1 h at 4 °C. The antibody–protein complexes were precipitated with Protein A/G‐Sepharose beads (Santa Cruz Biotechnology Inc.), washed, and resuspended in 40 μL of SDS/PAGE loading buffer.The cells were collected and lysed with 1 mL of immunoprecipitation lysis buffer  mutated to non‐phosphorylatable alanine residues or to phosphomimetic glutamic acids or to generate TGFBRI constitutively active (CA) mutant (threonine 204 is replaced with aspartic acid), mutagenesis was performed using a QuikChange site‐directed mutagenesis kit . All mutant constructs were confirmed by DNA sequencing. The mutagenic primer pairs used to generate mutants were: CK2β 3E (S2ES3ES4E): forward, 5′‐GACGTGAAGATGGAAGAAGAAGAGGAGGTGTCC‐3′; reverse, 5′‐GGACACCTCCTCTTCTTCTTCGATCTTCACGTC‐3′. CK2β 3A (S2A S3AS4A): forward, 5′‐GACGTGAAGATGGCAGCAGCAGAGGAGGTGTCC‐3′; reverse, 5′‐GGACACCTCCTCTGCTGCTGCCATCTTCACGTC‐3′. TGFBRI CA: forward, 5′‐GAACAATTGCGAGAGATATTGTGTTACAAG‐3′; reverse, 5′‐TCCGTA ACACAATATCTCTCGCAATTGTTC‐3′.2.9A cell migration assay was conducted using specific wound‐assay chambers purchased from ibidi GmbH . All experiments were performed in accordance with the manufacturer's instructions.2.10t‐test. P < 0.05 was considered statistically significant.Statistical comparisons of groups were performed using Student's 33.1et al., et al., in vitro kinase assay and western blot analysis were performed. CK2 activity peaked at 48 h after TGFβ treatment and the E‐to N‐cadherin switch was observed 24 h after TGFβ treatment  A549 cells. Previously, we reported that CKD could decrease cellular CK2 activity  A549 cells using shRNA. When SKD cells were treated with TGFβ, EMT was induced and CK2 was activated  A549 cells were generated using the CRISPR/Cas9 gene knockout system. Western blot analysis and in vitro kinase assay showed that with βKO, the E‐ to N‐cadherin switch was induced . In the present study, we treated cells with 2 nm OA and thus PP2A could be completely inhibited; however, this might not be the case for PP1. OA treatment protected CK2β from TGFβ‐induced degradation . Further experiments, including the generation of PPP2CA and PPP2CB double knockout A549 cells, are required to identify the phosphatase involved in TGFβ‐induced CK2β dephosphorylation.In non‐canonical TGFβ signaling, TGFBRI kinase‐dependent activation and interaction of phosphatase 2A with p70‐S6 kinase could result in the dephosphorylation and inactivation of the kinase, thereby inducing G1 arrest  on motility.Click here for additional data file.Fig. S2. Polyubiquitination of CK2β by TGFβ signaling.Click here for additional data file.Fig. S3. Screening of E3 ubiquitin ligases for CK2β degradation.Click here for additional data file.


A, B, C and D). The conformations of the molecules differ, as seen from the dihedral angles between the two pyridine rings in each molecule. They vary from 5.51 (9)° for molecule B to 25.25 (8)° for molecule A.The asymmetric unit of the title disubstituted 2,3′-bipyridine, contains four independent molecules  in the asymmetric unit. The dihedral angles between the two pyridine rings in each molecule are 25.25 (8)° in A, 5.51 (9)° in B, 11.11 (9)° in C and 16.24 (8)° in D. In the crystal, molecules A and B are linked by C—H⋯N hydrogen bonds to form layers extending parallel to the ab plane, while molecules C and D are linked by C—H⋯N hydrogen bonds forming –C–D–C–D– chains propagating along the b-axis direction. The layers and the chains are stacked alternately along the c axis through offset π–π and C≡N⋯π [N-to-pyridine-centroid distance = 3.882 (2) Å] interactions, resulting in the formation of a supramolecular framework.The title compound, C T1) compared with phenylpyridine-based CT1: 2.82 eV) is larger than that of alkoxy-functionalized analogue, 2′,6′-dimethoxy-2,3′-bipyridine (T1: 2.70 eV)  research area because developing blue phosphorescent materials remains a problem that has not been solved so far. Although there are a number of advantages in 2,3′-bipyridine ligands, incorporating the substituents into the ligand framework is difficult owing to the low selectivity and reactivity of the pyridine ring . The dihedral angles between the two pyridine rings in each molecule are 25.25 (8)° in A, 5.51 (9)° in B, 11.11 (9)° in C and 16.24 (8)° in D. In order to investigate the conformational similarity between the four molecules, the r.m.s. overlay fits of the 16 non-H atoms of each molecule were calculated using the AutoMolFit routine in PLATON , forming layers extending parallel to the ab plane, while the C and D molecules are connected through C—H⋯N hydrogen bonds  to from –C–D–C–D– chains propagating along the b-axis direction. The layers and chains stack alternately along the c axis, linked by intermolecular π–π stacking interactions, resulting in the formation of a supramolecular framework, as shown in Fig. 4Cg1⋯Cg2Di = 3.6741 (9) Å; Cg1⋯Cg2Div = 3.6546 (9) Å; Cg2⋯Cg1Div = 3.5888 (9) Å; Cg2B⋯Cg1Civ = 3.8196 (10) Å; Cg1 and Cg2 are the centroids of the N1/C1–C5 and N2/C6-C10 rings. Atoms and centroids labelled with suffixes B, C and D represent those of the molecules B, C and D, respectively]. In addition, intermolecular C≡N⋯π interactions between the cyano N atom of the D molecule and the N1B-containing pyridine ring of molecule B + .All experiments were performed under a dry NUiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018011532/xu5937sup1.cifCrystal structure: contains datablock(s) I, New_Global_Publ_Block. DOI: 10.1107/S2056989018011532/xu5937Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018011532/xu5937Isup3.cmlSupporting information file. DOI: 1862117CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A and B) in the asymmetric unit. They differ essentially in the conformation of the pyrrolidine and cyclopentene rings; respectively, twisted and flat in molecule A, but envelope and twisted in molecule B.The title compound crystallized with two independent molecules  in the asymmetric unit. In both molecules, the pyran and pyridine rings adopt envelope and chair conformations, respectively. The conformation of the pyrrolidine and cyclopentene rings differ in the two molecules; twisted and flat, respectively, in molecule A, but envelope and twisted, respectively, in molecule B. In both molecules, there is a C—H⋯N intramolecular hydrogen bond present. In both molecules, the oxygen atoms of the nitro groups are disordered as is the chlorine atom in molecule B. In the crystal, the B molecules are linked by C—H⋯O hydrogen bonds, forming –B–B–B– chains along [010], and by C—H⋯π interactions. The A and B molecules are also linked by a number of C—H⋯π interactions, resulting in the formation a supramolecular three-dimensional structure. In molecule A, the nitro group oxygen atoms are disordered over two positions with refined occupancy ratios of the nitro group oxygen atoms O3A and O4A in 0.59 (2):0.41 (2) while in molecule B one of the nitro O atoms is disordered over two positions with a refined occupancy ratio of 0.686 (13):0.314 (13) and the chlorine atoms is disordered over two positions with a refined occupancy ratio of 0.72 (3):0.28 (3).The title compound, C The mean plane of the five-membered pyrrolidine ring (N1/C12/C13/C21/C22) is inclined to the mean plane of the cyclopentene ring (C1/C2/C10–C12) by 87.30 (13) and 88.41 (11)° in molecules A and B, respectively. The benzene rings C27–C32 and C14–C19 are inclined to each other by 58.13 (13)° in molecule A and 57.13 (11)° in molecule B, while benzene rings C6–C11 and C3–C5/C33–C35 are inclined to each other by 10.20 (13)° in molecule A and 4.08 (13)° in molecule B. The mean plane of the pyrrolidine ring (N1/C12/C13/C21/C22) makes a dihedral angle with the mean plane of the pyran ring (O2/C13/C14/C19–C21) of 34.6 (2)° in molecule A and 29.65 (10)° in molecule B, and is inclined to the piperidine ring mean plane (N1/C22–C26) by 15.69 (12)° in molecule A and 12.36 (11)° in molecule B. The mean planes of the pyran and piperidine rings are inclined to each other by 37.06 (11) and 29.49 (10)° in molecules A and B, respectively. The mean plane of the pyrazine ring (N3/N4/C1/C2/C3/C4) makes a dihedral angle with the mean plane of the pyran ring (O2/C13/C14/C19–C21) of 63.42 (19)° in molecule A and 72.64 (10)° in molecule B. It is inclined to the pyrrolidine ring mean plane (N1/C12/C13/C21/C22) by 88.11 (1)° in molecule A and 86.69 (11)° in molecule B and is inclined to the piperidine ring mean plane (N1/C22–C26) by 77.24 (11)° in molecule A and 82.97 (11)° in molecule B.Chlorine atoms Cl1B molecules are linked by C—H⋯O hydrogen bonds, forming chains propagating along the b-axis direction -6a-nitro-6,6a,6 b,7,8,9,10,12a-octahydrospiroindolizine-12,3-indolin]-2-one  under a nitrogen atmosphere. The solution was refluxed for 20 h in a Dean–Stark apparatus to give the corresponding cycloadduct. After completion of the reaction, as indicated by TLC, the solvent was evaporated under reduced pressure. The crude product obtained was purified by column chromatography using hexane/EtOAc (6:4) as eluent (yield 86%). Colourless block-like crystals of the title compound were obtained by slow evaporation of a solution in ethanol.To a solution of indenoquinoxalinone (1.0 mmol) and pipacolinic acid (1.5 mmol) in dry toluene, was added 2-(4-chlorophenyl)-3-nitro-2Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 2A and O4A in A and O3B and O4B in B are disordered over two positions with refined occupancy ratios of O3A/O4A:O3A′/O4A′ = 0.59 (2):0.41 (2), and O3B/O4B:O3B′/O4B′ = 0.686 (13):0.314 (13). In molecule B, the chlorine atom Cl2 is disordered over two positions with a refined occupancy ratio of Cl2:Cl2′ = 0.72 (3):0.28 (3).In both molecules, the nitro group oxygen atoms O310.1107/S2056989019000975/su5475sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989019000975/su5475Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019000975/su5475Isup3.cmlSupporting information file. DOI: 1024832CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Out-of-hospital electrocardiogram (ECG) shows a sinus rhythm of 85 beats per minute (bpm), narrow QRS complex, and ST-segment elevations in leads V1–V4 (coved morphology in V1–V2), with a terminal negative T wave in V1–V2 (Fig. The patient met criteria for exertional heat stroke , and wasDiffuse ST-T deviations have been described in patients with heat stroke , but rigType 1 Brugada-like ECG pattern induced by exertional heat stroke.


The benzothiazine skeleton is not planar, with a maximum deviation of 0.3154 (11) Å from the least-squares plane. The molecule was expected to adopt mirror symmetry but slightly different conformational characteristics of the condensed benzothiazine ring lead to point group symmetry 1. 19H14N2O6S2·C3H7NO. The molecule was expected to adopt mirror symmetry but slightly different conformational characteristics of the condensed benzothiazine ring lead to point group symmetry 1. In the crystal, molecules form two types of stacking dimers with distances of 3.464 (2) Å and 3.528 (2) Å between π-systems. As a result, columns extending parallel to [100] are formed, which are connected to intermediate dimethylformamide solvent molecules by C—H⋯O interactions.The title molecule crystallizes as a dimethylformamide monosolvate, C R-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylates are highly reactive compounds -one 6,6,8,8-tetroxide (I)H-2λ6,1-benzothiazine-3-carboxylate  and S = 0.48, θ = 50.0°, Ψ = 22.8° for the C5–C6–C7–C8–N2–S2 ring (2). The S1 and C1 atoms deviate by 0.669 (2) and 0.207 (2) Å, respectively, from the mean-square plane of the remaining atoms in ring (1). The corresponding deviations in ring (2) are 0.668 (2) and 0.270 (2) Å, respectively.Both thiazine rings adopt a twist-boat conformation Fig. 1 with sliH-pyran-4-one ring (3) adopts a sofa conformation with puckering parameters S = 0.14, θ = 24.7°, Ψ = 22.6°. The deviation of C19 from the plane of the remaining atoms of (3) is 0.087 (2) Å. The C1=C2 and C5=C6 bonds [1.3571 (17) Å and 1.3529 (17) Å] are slightly elongated as compared to the mean value of 1.329 Å for a Csp2=Csp2 bond , H13⋯H18B = 2.28 Å (expected 2.34 Å), H13⋯H18C = 2.31 Å (expected 2.34 Å). These shortened contacts affect the very small pyramidalization of the nitrogen atoms; the sums of the bond angles centered at the N1 and N2 atoms are 354 and 356°, respectively.A further analysis of the molecular structure revealed the presence of other shortened intramolecular contacts: H9⋯H17In the crystal, molecules of (I)Ai and C18—H18B⋯O1Aii  and diphenyl oxide (10 ml) was maintained on a metal bath at 493 K for 3 h, then cooled and diluted with ethanol  = 1.5Ueq(C) for methyl groups and with C—H = 0.93 Å, Uiso(H) = 1.2Ueq(C) for all other hydrogen atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019008788/wm5508sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019008788/wm5508Isup2.hklStructure factors: contains datablock(s) I. DOI: 1935426CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Conventional O—H⋯O and N—H⋯O hydrogen bonding leads to a supramolecular layer in the crystal.A coordination geometry intermediate between square-planar and tetrahedral, defined by an N II complex, isolated as a dihydrate, [Cu(C21H14N3O3)2]·2H2O, reveals a highly distorted coordination geometry intermediate between square-planar and tetrahedral defined by an N2O2 donor set derived from two mono-anionic bidentate ligands. Furthermore, each six-membered chelate ring adopts an envelope conformation with the Cu atom being the flap. In the crystal, imidazolyl-amine-N—H⋯O(water), water-O—H⋯O, phenyl-C—H⋯O(nitro) and π(imidazolyl)–π(nitrobenzene) [inter-centroid distances = 3.7452 (14) and 3.6647 (13) Å] contacts link the components into a supramolecular layer lying parallel to (101). The connections between layers forming a three-dimensional architecture are of the types nitrobenzene-C—H⋯O(nitro) and phenyl-C—H⋯π(phenyl). The distorted coordination geometry for the CuII atom is highlighted in an analysis of the Hirshfeld surface calculated for the metal centre alone. The significance of the intermolecular contacts is also revealed in a study of the calculated Hirshfeld surfaces; the dominant contacts in the crystal are H⋯H (41.0%), O⋯H/H⋯O (27.1%) and C⋯H/H⋯C (19.6%).The crystal and molecular structures of the title Cu Complex (I)via an analysis of the calculated Hirshfeld surfaces.The title copper(II) complex, (I)N,O-chelated by two 2--4-nitrophenolate mono-anions. The resulting N2O2 donor set defines a highly distorted coordination geometry, as seen in the angles included in Table 14 is a geometric measure of the distortion of a four-coordinate geometry  Å out of the plane. The comparable parameters for the O2-chelate ring are 0.033 and 0.354 (3) Å, respectively. The dihedral angle formed between the two planar regions of the chelate rings is 49.38 (8)°. The dihedral angles between the best plane through the O1-chelate ring and each of the fused six- and five-membered rings are 9.18 (12) and 5.54 (14)°, respectively; the equivalent angles for the O2-chelate rings are 8.44 (8) and 2.71 (9)°, respectively. The N1-imidazol-2-yl ring forms dihedral angles of 41.20 (11) and 37.46 (10)° with the C10- and C16-phenyl substituents, respectively, and the dihedral angle between the phenyl rings is 59.92 (8)°, i.e. all indicating splayed relationships. A similar situation pertains to the N2-imidazol-2-yl ring, where the comparable dihedral angles formed with the C31- and C37-phenyl rings are 38.29 (10), 48.5 (9) and 50.84 (7)°, respectively. Finally, the nitro groups are not strictly coplanar with the benzene rings to which they are connected, as seen in the dihedral angles of 14.2 (4)° for C1–C6/N4/O3/O4 and 5.9 (3)° for C22–C27/N6/O5/O6.The crystallographic asymmetric unit of (I)W water molecule forms donor interactions to the coordinated O2 atom and to a symmetry-related O2W water molecule. The O2W water molecule connects to the coordinated O1 atom as well as to a nitro-O3 atom. Hence, the O2W water molecule is involved in four hydrogen-bonding interactions. The fourth contact involving the O1W water molecule, a C—H⋯O acceptor contact, is provided by the nitrobenzene ring. There is also a phenyl-C—H⋯O(nitro) contact of note, Table 2a). There are also π–π stacking and C—H⋯O interactions in the crystal, Fig. 3b). Within layers, there are π–π interactions occurring between the imidazolyl and nitrobenzene rings . The connections between layers along [010] are of the type nitrobenzene-C—H⋯O(nitro) and phenyl-C—H⋯π(phenyl), as detailed in Table 2As each component of the asymmetric unit has hydrogen-bonding functionality, conventional hydrogen bonds are found in the crystal of (I)CrystalExplorer17 , and O2W-water, Fig. 4b), molecules. In addition, the presence of faint-red spots near the O1W, O2W and H1W atoms in Figs. 4a) and 4(b) are indicative of the other contacts of these atoms with those of the CuII complex molecule (Table 2c)–(e).The Hirshfeld surface calculations for (I)e Table 2. The dondnorm-mapped Hirshfeld surface in Fig. 4c). The pair of faint-red spots appearing near the phenyl-C36 and H36 atoms, and also near the nitro-O5 atom on the surface indicating short interatomic contacts that characterize the weak C—H⋯O interaction, Table 3e). The donors and acceptors of this interaction are also evident as the blue bump and a bright-orange spot enclosed within the black circle on the Hirshfeld surface mapped with the shape-index property in Fig. 5a). The bright-orange region enclosed within a black circle in Fig. 5b) is also an indication of the O2W—H4W⋯Cg(C16–C21) contact. The Hirshfeld surfaces mapped over the calculated electrostatic potential for the water and complex molecules in Fig. 6dnorm illustrated in Figs. 4c)–(e), faint-red spots also appear near other atoms indicating their involvement in other short interatomic contacts, as summarized in Table 3The presence of a short interatomic C⋯C contact between atoms C22 and C28 Table 3 arises f2O4 donor set about the copper(II) centre in the complex molecule. This is performed by considering the Hirshfeld surface about the metal centre alone  and the small orange regions on the surface relatively far from the Cu—N bonds in Fig. 8b). The different curvature of the Hirshfeld surfaces coordinated by the N2O4 donor set in Figs. 8c) and 8(d) also support this observation. The Cu—O and Cu—N bonds are rationalized in the two-dimensional fingerprint plot taking into account only the Hirshfeld surface for the copper atom shown in Fig. 9de + di ∼ 2.0 Å for the Cu—N bonds (upper region) and the Cu—O bonds (lower region) are indicative of the distorted geometry , and those delineated into H⋯H, O⋯H/H⋯O, C⋯H/H⋯C, C⋯C and C⋯O/O⋯C contacts are illustrated in Figs. 10b)–(f), respectively. The percentage contribution from different interatomic contacts to the Hirshfeld surfaces of the complex molecule and for overall (I)de + di ∼ 1.9 Å in the fingerprint plot delineated into H⋯H contacts shown in Fig. 10b) is the result of the involvement of the H12 atom in a short interatomic H⋯H contact, Table 3W and H2W, Table 3de + di ∼ 1.8 Å in the plot delineated into O⋯H/H⋯O contacts, Fig. 10c), arise from the N—H⋯O hydrogen bond, while the merged points correspond to other interactions at greater interatomic distances. The significant contribution from interatomic C⋯H/H⋯C contacts (Table 4d). The presence of short interatomic C⋯C contacts are evident as the points near a rocket shape tip at de + di ∼ 3.2 Å in the respective delineated fingerprint plot, Fig. 10e), while the points corresponding π–π stacking between the imidazole and nitrobenzene rings are distributed about de = di = 1.7 Å in the plot. The small, i.e. 2.7%, contribution from C⋯N/N⋯C contacts to the surface is also due to these π–π stacking interactions (delineated plot not shown). The contribution of 3.2% from C⋯O/O⋯C contacts is due to the presence of short interatomic contacts involving nitro-O atoms, Table 2de + di ∼ 3.2 Å in the delineated plot of Fig. 10f). The contribution from other interatomic contacts to the surface summarized in Table 4The overall two-dimensional fingerprint plot for (I)s Table 4 to the Hs Table 4 and the H-imidazol-2-yl)phenolate ligands in the literature ; complex (IV) has crystallographic twofold symmetry. The final structure, a copper(II) complex –(VI) of 0.48, 0.53, 0.44, 0.37, 0.47 and 0.35, respectively.There are five crystal structures of copper complexes with related 2-In a typical procedure, benzil , ammonium acetate , 2-hydroxy-5-nitrobenzalaldehyde  and copper(II) borate  were ground in an agate mortar with a pestle. To this mixture, about 1.5 g of dried silica gel  was added and the reaction mixture was ground again for 30 min. The whole reaction mixture was then transferred to a 100 ml round-bottomed flask and heated at 130 °C with constant stirring for 4 h. The reaction mixture was then extracted with dry acetone and dried over MgSOUiso(H) values set at 1.2Ueq(C). The O- and N-bound H atoms were located in a difference Fourier map but were refined with distance restraints of O—H = 0.84 ± 0.01 Å and N—H = 0.88 ± 0.01 Å, respectively, and with Uiso(H) set at 1.5Ueq(O) or 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989019013720/hb7859sup1.cifCrystal structure: contains datablock(s) . DOI: 10.1107/S2056989019013720/hb7859Isup2.hklStructure factors: contains datablock(s) I. DOI: 1958158, 1958158CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: