C(6) hydrogen-bonded chains formed between the OH and C=O groups.The structures of two hydroxy-thiophenone derivatives related to the antibiotic thiolactomycin are presented. The main structural feature of both compounds is  H-thiophen-5-one, C6H8O2S, and 3-hydroxy-4-methyl-2H-thiophen-5-one, C5H6O2S. The main structural feature of both compounds is C(6) hydrogen-bonded chains formed between the OH and C=O groups. In achiral C5H6O2S, these chains propagate only by translation, corresponding to x + 1, y, z + 1. However, in contrast, for racemic C6H8O2S the hydrogen-bonded chains propagate through a −x + y + z operation, giving chains lying parallel to the crystallographic b-axis direction that are composed of alternate R and S enantiomers. The crystals of 3-hydroxy-4-methyl-2H-thiophen-5-one were found to be twinned by a 180° rotation about the reciprocal 001 direction. In the final refinement the twin ratio refined to 0.568 (2):0.432 (2).The structures of two hydroxy-thiophenone derivatives related to the antibiotic thiolactomycin are presented. These are the racemic 3-hydroxy-2,4-dimethyl-2 R)-4-hydroxy-3,5-dimethyl-5-thiophen-2-one, is a naturally occurring antibiotic isolated from Norcardia spp  1,  and 119.7 (3)°]. A search of the Cambridge Structural Database  hydrogen-bonded chain utilizing OH as the donor group and O1 as the acceptor group, see Tables 35 the chains propagate by translations corresponding to x + 1, y, z + 1. This propagation by translation alone gives the repeating pattern shown in Fig. 34 rings of the hydrogen-bonded unit are coplanar and all of the S atoms lie on the same side of the chain. When travelling along the b-axis direction, neighbouring chains bear their S atoms on different sides, giving the layered structure shown in Fig. 44 the chain propagates through a −x + y + z operation giving a chain lying parallel to the crystallographic b-axis direction. As shown in Fig. 5R and S enantiomers of the racemic chain having perpendicular relationships between the planes of their SC4 rings. This different chain geometry gives a very different packing arrangement from that of structure 5, see Fig. 65. This is a C—H⋯O contact between the CH2 group and the ketone O atom. Of the 4-hydroxy-thiophen-2-one structures described in the literature, both those of FIVKEA and BIHKIM (TLM) display the same C(6) hydrogen-bonded chain motif as 4 and 5. In both cases, the geometrical detail of the chain is similar to that found in 4, with the difference that both literature examples are enantiopure. The final structure, POXZOS, contains additional carboxylic acid and carbonyl groups and these strong hydrogen-bonding groups dominate the intermolecular contacts formed and so stop the formation of the otherwise common C(6) motif.The main supramolecular feature of both structures General synthesis of thiolactone analogues 4 and 5:M) was added until the solutions reached pH 5 before washing with ethyl acetate (3 × 50 ml) and drying over anhydrous magnesium sulfate. The mixtures were reduced under vacuum and precipitated using petrol (40/60) and diethyl ether to give the products as a solid. For 4, crystals suitable for crystallographic analysis were grown from a THF solution. For 5, crystals were grown from a toluene solution.Bromine (1.1 eq) was added to a stirring solution of the corresponding oxoester (1 eq.) dissolved in chloroform (50 ml) at 273 K. The mixtures were allowed to warm to ambient temperature and stirred for 20 h before removing the solvent under vacuum. The resulting crude mixtures were dissolved in THF (50 ml) before adding trimethylamine (1.1 eq.) and thioacetic acid (1.1 eq.) and stirring at ambient temperature for a further 18 h. The resulting mixtures were reduced under vacuum to give dark-orange oils that were vacuum filtered over silica using petrol (40/60) and diethyl ether (5:2) as eluent before removing the solvent. The mixtures were dissolved in ethanol (50 ml) before adding a solution of sodium hydroxide (2 eq.) dissolved in water (20 ml) and stirring for 24 h at 333 K. After cooling, HCl  Off-white solid : m.p. 408–409 K. 1H NMR (CDCl3) δ 4.12 , 1.57 . 13C NMR (CDCl3) δ 197.16, 177.63, 111.32, 77.26, 77.21, 77.00, 76.75, 42.99, 18.80, 7.62.3-Hydroxy-4-methyl-2H-thiophen-5-one (5)1H NMR (CDCl3) δ 3.94 , 1.68 . 13C NMR (CDCl3) δ 195.1, 175.2, 111.2, 32.1, 7.2.Off-white solid : m.p. 397–398 K. 2 and CH groups, respectively. Uiso(H) = 1.5Ueq(C) for methyl groups and 1.2Ueq(C) for the other CH groups. The H atoms of the hydroxy groups were refined isotropically. Data collection on 5 was carried out by the National Crystallography Service :0.432 (2).Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989020008269/lh5954sup1.cifCrystal structure: contains datablock(s) 4, 5, global. DOI: 10.1107/S2056989020008269/lh59544sup2.hklStructure factors: contains datablock(s) 4. DOI: 10.1107/S2056989020008269/lh59545sup3.hklStructure factors: contains datablock(s) 5. DOI: Click here for additional data file.10.1107/S2056989020008269/lh59544sup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020008269/lh59545sup5.cmlSupporting information file. DOI: 2011283, 2011284CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Tcra gene rearrangement has been extensively studied. The enhancer Eα plays an essential role in Tcra rearrangement by establishing a recombination centre in the Jα array and a chromatin hub for interactions between Vα and Jα genes. But the mechanism of the Eα and its downstream CTCF binding site (here named EACBE) in dynamic chromatin regulation is unknown. The Hi-C data showed that the EACBE is located at the sub-TAD boundary which separates the Tcra–Tcrd locus and the downstream region including the Dad1 gene. The EACBE is required for long-distance regulation of the Eα on the proximal Vα genes, and its deletion impaired the Tcra rearrangement. We also noticed that the EACBE and Eα regulate the genes in the downstream sub-TAD via asymmetric chromatin extrusion. This study provides a new insight into the role of CTCF binding sites at TAD boundaries in gene regulation.The regulation of T cell receptor  Tcrg, Tcrd and Tcrb genes undergo rearrangement in CD4− CD8− double negative (DN) thymocytes (Tcrb gene forms a pre-TCR heterodimer with pTα to drive cells into CD4+CD8+ double positive (DP) stage after several rounds of cell division. In DP cells, the Tcra gene undergoes several rounds of rearrangement on both alleles to generate TCRα, which replaces pTα to form αβTCRs that mediate positive and negative selection (+ or CD8+ single positive cells and migrate from thymus.T lymphocytes recognize fragments of antigen with highly diverse T cell receptors (TCR) which are generated by V(D)J recombination during T lymphocyte development in thymus ,2. V(D)Jymocytes . TCRβ prelection . Cells hTcra–Tcrd locus), and the Tcrd gene is located between the Vα and Jα gene arrays. The Tcrd gene also shares some V gene segments with the Tcra. The regulation of Tcra rearrangement has been extensively studied . EACBE−/− and LCR−/− mice were generated from strain C57BL/6 by Beijing Vitalstar Biotechnology Co., Ltd. EACBE−/− mice were bred with Rag1−/− or Rag2−/− mice to generate EACBE−/− × Rag1−/− or EACBE−/− × Rag2−/− mice. We crossed mice to get mice with C57BL/6 Tcra/d alleles. Mice were housed in a specific-pathogen-free facility managed by the Southern Medical University Division of Laboratory animal centre. Mice of both genders were included in all experiments and no differences on the basis of gender were noted. All mice were handled in accordance with protocols approved by the Southern Medical University Institutional Animal Care and Use Committee.Wild-type C57BL/6 mice were purchased from Guangdong Medical Animal Experimental Centre, Rag13, 0.1 mM EDTA, pH 7.4) for one minute. The reaction was quenched by adding MCAS buffer, pelleted by centrifugation, and resuspended in MACS buffer. To isolate DP thymocytes from Rag2−/− mice, mice were injected i.p. with 150 μg of anti-CD3ϵ antibody  at 3 weeks of age and thymi were harvested ten days later. For T cell activation, isolated PLN and spleen T cell was cultured in anti-CD3/CD28 coated plates and FACS analyze was performed at 0 and 24 h, respectively.Thymi, peripheral lymph nodes and spleens were generally collected from mice at 6 weeks of age and cells were filtered through a 40 μm nylon mesh and then pelleted. Red blood cells were removed by incubating in red cell lysis buffer , CD8α(53–6.7), CD44, CD25(PC61), Thy1.2(53–2.1). γδT cell were detected by using anti-γδT(GL3), CD45RG(C363.16A), CD3(145–2C11) and lineage (Lin) CD4(GK1.5), CD8a(53–6.7), NK-1.1(PK136), CD49b(DX5), Ly-6G/Ly-6C(RB6–8C5), CD11b(M1/70), TER-119(TER-119), TCRβ(H57–597), CD19, B220 (RA3–6B2). T cell activation analysis were done by staining with antibodies to CD4(RM4–5), CD8α(53–6.7), CD25(PC61), Thy1.2(53–2.1). Data were acquired on a BD FacsCanto II flow cytometer in 8-color configuration and cell sorting was conducted using a Beckman Coulter Astrios or MoFlo.7thymocytes were lysed in Trizol (ThermoFisher) per manufacturer's specifications and either stored at −80°C or used immediately for RNA extraction. Total RNA was subjected to template-switch 5′ RACE as described  primer in 8 μl nuclease-free water was heated to 65°C for 5 min and cooled down on ice for at least 5 min to snap-anneal the oligo(dT) primer. The reaction was then adjusted to 250 mM Tris–HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, 0.5 mM dNTPs and 5 mM dithiothreitol, before addition of 2 μl Superscript II (ThermoFisher), add 1 μl 25 uM 5′RACE adapter sequence (5′-GTCGCACGGTCCATCGCAGCAGTCACArGrGrG-3′) in a final volume of 20 μl. The reaction was incubated for 2 h at 50°C to synthesize cDNA and add RACE adapter by template switching. Reverse transcriptase was then inactivated by incubation at 85°C for 2 min.10escribed ,23, withPCR amplification of 5′RACE cDNA was performed as described , with moReads were obtained following quality filtering and adaptor trimming using trim_glore (cutadapt version 0.4.4_dev) with parameter ‘-paired -q 20′. Individual reads were analyzed with MIXCR v2.1.10 to derive unique clonotypes based on CDR3 homology. Reads that comprise the clonotypes of interest (with matching VJ genes to Mus Musculus library antibody) were extracted for further analysis. The corresponding germline sequences were retrieved from IMGT, the international ImMunoGeneTics database. The total number of clonotypes was standardized to one million, and then calculated V–J clonotype ratio as V–J pairing usage.7 cells were subjected to cross-linking by incubation for 10 min on ice in 10 ml of RPMI-1640 containing 10% FBS and 2% formaldehyde. The reaction was stopped by addition of glycine to 0.125 M and incubation for 5 min at room temperature. Cells were washed in PBS and lysed by incubation for 10 min on ice in 1 ml of cell lysis buffer ). Nuclei were pelleted, washed once with PBS, and lysed by incubation for 1 h at 37°C in 0.5 ml of 1× CutSmart digestion buffer (New England Biolabs) containing 0.3% SDS. Triton-X100 was then added to a final concentration of 2% and incubation was continued for an additional 1 h at 37°C. Chromatin was then digested by addition of 400 U MboI for overnight incubation at 37°C. The MboI enzyme was inactivated by adding 80 μl 10% SDS and incubating at 37 °C for 30 min. Samples were transferred into 15 ml falcons and incubated with 4860 μl sterile water, 700 μl 10× ligation buffer and 750 μl 10% Triton X-100 for 1 h at 37 °C. 200 U of T4 Ligases (New England Biolabs) were added and incubated overnight at 16°C. De-crosslinking was performed by adding 30 μl of 10 mg ml−1 proteinase K for 4 h at 65°C. The DNA was precipated with 0.6× isopropanol and washed with 70% ethnal twice. DNA was dissolved in 150 μl of diluted TE.3C sample preparation followed a previously described protocol with modification . In brie2, 1 mM DTT and 0.1 mM ATP, after which 200 U of T4 DNA ligase (New England Biolabs) were added for overnight incubation at 16°C. 4C libraries were then purified by phenol-chloroform extraction, precipitated with 0.6× isopropanol, and rehydrated in 200 μl of 10 mM Tris–HCl, pH 8.0, 0.1 mM EDTA. Each library was then used for two rounds of inverse PCR from each of five viewpoints. First-round PCR was conducted with the following primers at 0.2 μM: Eα up-F (5′-TGGCGATGAAGTTGACTTTGATC-3′) and Eα up-R (5′-CAGGCAGAGACTCTTCGACG-3′), and Eα down-F (5′-TGCCCATCATCCAGGTTCAGATC-3′) Eα down -R (5′-CTGGGTTTGCTGCACCTCAGT-3′), and TEAp-F (5′-ACACTCTCTTTTCACAGCTGATC-3′) TEAp-R (5′-GCGTTCTGATTTCCTTCACTTTG-3′), and Trav17-F (5′-GACAAGGAACACAAACCCCGATC-3′) Trav17-R (5′-CCTGCTGTAATAATGTAGGAGGGC-3′) and Trav1-F (5′-GCTTCTGACAGAGCTCCAGATC-3′) Trav1-R (5′-GGAGCGTGGAAATGCTGTAGAT-3′), andINT-F (5′-ATTCAGCAGTGCTTTGTGTAGATC-3′) INT-R (5′-GGTTTCTGTGGTTGGAGTAGACT-3′). PCR conditions were as follows: 3 min at 95°C, followed by 30 cycles of 15 s at 95°C, 15 s at 55°C and 30 s at 72°C, with a final extension for 5 min at 72°C. PCR products were precipitated with 2× ethanol, dissolve the pellet in 50 μl nuclease-free water. Products were run on a 2% agarose gel, cut bands of 300–700 bp, extracted DNA by gel extraction kit (Qiagen) following manufacturer's specifications. The library preparation and sequencing followed the protocol described above in repertoire sequencing.3C samples were digested overnight at 37°C with 10 U of NlaIII. The digested libraries were purified by phenol–chloroform extraction, precipitated with 0.6× isopropanol, and rehydrated in 7 ml 30 mM Tris–HCl, pH 8.0, 10 mM MgClk = 30.Reads were obtained following quality filtering and adaptor trimming using trim_glore (cutadapt version 0.4.4_dev) with parameter ‘-paired -q 20’. The first MboI enzyme fragment behind the viewpoint was extracted and mapped to mm10 by bowtie2 version 2.3.4.3 with parameter: –very-sensitive. Reads numbers were counted and normalized by the total mapped reads per sample after self-ligation remove, and then differential interactions were identified by 4C-ker with in situ Hi-C libraries of DP thymocytes from WT and EACBE−/− mice using the MboI restriction enzyme following the protocol described in  and sonicated to an average DNA fragment size of 300–500 bp (Q800R2 sonicator). 5 ug DNA dissolved in 130 μl 10 mM Tris–HCl (pH 8.0) was added into the pre-treated beads and incubated at RT for 15 min with rotation. The beads were then separated on a magnet and biotinylated DNA was bound to the streptavidin beads. After the ends of sheared DNA were repaired and the biotin from un-ligated ends was removed, adapters were added to the dA-tailed DNA fragments and PCR was performed with ten cycles using Illumina primers. Finally, DNA size selection was performed with 0.55–0.7× volume of VAHTS DNA Clean beads  to make the DNA length distribution between 300 and 500 bp. The library was quantified with Qubit and sequenced using an Illumina sequencing platform. Reads were obtained following quality filtering and adaptor trimming using trim_glore (cutadapt version 0.4.4_dev) with parameter ‘-paired -q 20’. Hi-C mapping, filtering, correction, and binning were performed with the HiC-Pro software v2.11.1. The reads were mapped to the mm10 mouse reference genome. TADs analysis was performed with the ‘Insulation Method’. A publicly available script (matrix2insulation.pl) was used to detect the TAD boundaries, with the following options: ‘–is 2000000 –ids 800000 –nt 0.3’. The script can be accessed through GitHub (https://github.com/dekkerlab/cworld-dekker).We generated ribed in  with sma7 thymocytes were subjected to cross-linking by incubation for 10min on ice in 10 ml of 1× PBS containing 10% FBS and 1% formaldehyde , ThermoFisher). The reaction was stopped by addition of glycine to 0.125 M and incubating for 5 min at room temperature. Cells were washed in PBS and lysed in by incubation for 5 min on ice in 1 ml of cell lysis buffer ). After centrifugation, the nuclei were suspended in 40 μl of nuclei lysis buffer  and were broken by vigorous pipetting. The volume was then adjusted to 400 μl with nuclease-free water and the suspension was sonicated using a Model 550 Sonic Dismembrator (Fisher Scientific). Chromosomal DNA was reduced to an average size of 300–500 bp as determined by agarose gel analysis. The composition of the chromatin solution was then adjusted by addition of Tris–HCl pH 8.0, Triton X-100 and NaCl to 25 mM, 1.1% (v/v) and 170 mM, respectively. Chromatin was pre-cleaned by incubation for 30 min at 4°C with 5μg of IgG  and 15μlprotein A/G magnetic beads  twice. Pre-cleaned chromatin corresponding to 1 × 106 thymocytes was incubated with 5 μg of specific antibody  or isotype-matched IgG for 16 h at 4°C, followed by addition of 15 μl protein A/G magnetic beads for an additional 4 h at 4°C. The supernatant was saved as the unbound fraction. Immunoprecipitates were washed by rocking for 5 min at 4°C twice each with 1 ml of the following buffers (containing protease inhibitors): (i) 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 20 mM Tris–HCl pH 8.0, 170 mM NaCl; (ii) 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8.0, 500 mM NaCl; (iii) 1% NP-40, 1% deoxycholic acid, 100 mM Tris–HCl pH 8.0, 500 mM LiCl; and with 4) 10 mM Tris–HCl pH 8.0, 1 mM EDTA. DNA/Protein/Ab/beads complexes were resuspended by50 μl of 50 mM Tris (PH 8.0), 10 mM EDTA,1% SDS, 0.2mg/ml Proteinase K, rocking on Thermo mixer(ThermoFisher) for 1 h at 55°C, then 4 h at 65°C. After reversal of cross-linking and deproteination, place tube on magnet and collect supernatant purify by PCR purification kit. The standard curve quantification with gradient-concentrate genomic DNAs was used for quantifying ChIP products. qPCR primer are list in Crosslink ChIP was performed essentially as previously described . 1 × 1076) were washed with cold PBS twice. Washed cells were then lysed in 200 μl of 80 mM NaCl, 10 mM Tris–HCl pH8.0, 10 mM sodium butyrate, 6 mM MgCl2, 1 mM CaCl2, 250 mM sucrose, 0.02% (vol/vol) NP40, 0.1 mM PMSF and 1× protease inhibitor cocktail for 5 min on ice. Centrifuge at 600 × g for 5 min at 4°C. Nuclei were pelleted and washed once with 10 mM NaCl, 10 mM Tris–HCl pH 8.0, 10 mM sodium butyrate, 3 mM MgCl2, 1 mM CaCl2 and 250 mM sucrose. Digestion was then performed to generate mainly mononucleosomes with a minor fraction of dinucleosomes, by incubation for 5 min at 37°C in 200 μl of the same buffer containing 6 U micrococcal nuclease (Worthington). The reaction was stopped by addition of 8μl of solution containing 0.2 M EDTA and 0.2 M EGTA. After centrifugation for 10 min at 18 000 × g, the supernatant was dilute into a final concentration of 16.7 mM Tris pH 8.0, 1.2 mM EDTA, 167 mM NaCl, 1.1% TritonX (vol/vol), 0.1 mM PMSF, and 1× protease inhibitor cocktail. Chromatin was then incubated overnight at 4°C with anti-acetylated H3 (Millipore 06-599), anti-trimethylated H3K4 (Millipore 04-745), anti-trimethylated H3K27  or control rabbit IgG . protein A/G magnetic beads  was added for an additional four hours incubation, after which immunoprecipitates were washed vigorously and DNAs were purified. The standard curve quantification with gradient-concentrate genomic DNAs was used for qPCR. Immunoprecipitated and input DNAs were quantified by real time PCR using a StepOne™ Real-Time PCR System  and a Hieff™ qPCR SYBR® Green Master Mix . For immunoprecipitation using anti-trimethylated H3K4 and anti-acetylation H3K27, analysis of B2m gene promoter was used as positive control and MageA2 gene promoter was used as negative control to normalize ratios of bound/input in different samples. Primers sequences are provided in Chromatin was prepared as previously described ,25. Thymhttp://hgdownload.cse.ucsc.edu/goldenPath/mm10/bigZips/chromFa.tar.gz). PCR duplicate reads were removed by sambamba markdup with parameter ‘ –r –overflow-list-size 600000 ’. We used bowtie2 version 2.3.4.3 with the default parameters to align the reads to the mouse reference genome. Peak calling was done using Homer. The ChIP-seq read coverage was calculated using deepTools bamCoverage with RPKM normalization. The processing of the external data  was same as the new data.ChIP-seq libraries were pooled and sequenced via paired-end 150-bp reads on a HiSeq X TEN platform (Novogene). Reads were obtained following quality filtering and adaptor trimming using trim_glore (cutadapt version 0.4.4_dev) with parameter ‘-paired -q 20’. Mouse genome sequence (mm10) was retrieved from the UCSC (RNA was extracted from unfractionated thymocytes using TRIzol (Invitrogen) according to the manufacturer's instructions. Contaminating genomic DNA was removed by incubation with 1U DNase I (New England Biolabs) for 10 min at 37°C. SuperScript reverse transcriptase (Invitrogen) and random hexamer primers were used to synthesize cDNA according to the manufacturer's instructions. Reverse transcription conditions were described previously . Real-tiTotal thymocytes were lysed by incubation in 10 mM Tris–HCl pH 8.0, 150 mM NaCl, 10 mM EDTA, 0.4% (wt/vol) SDS and 0.1 mg/ml proteinase K overnight at 37°C. Genomic DNA was prepared by phenol/chloroform extraction and ethanol precipitation. PCR conditions were as follows: 3 min at 95°C; 30 cycles of 30 s at 95°C, 30 s at 60°C, 1 min at 72°C; 5 min at 72°C. After agarose gel electrophoresis and transfer to nylon membranes, PCR products were detected by hybridization with Biotin-labeled oligonucleotide probes. Primer and probe sequences are provided in t-test, one-way ANOVA, or two-way ANOVA using Graphpad Prism software. P values of <0.05 were considered statistically significant. Sample sizes were estimated on the basis of initial experiments and measurements, rather than being predetermined on the basis of expected effect sizes. No data were excluded from analysis. There was no randomization of mice or ‘blinding’ of researchers to experimental groups.Data were analyzed by Tcra–Tcrd locus in DP thymocytes, we performed Hi-C assays with DP thymocytes obtained from anti-CD3 injected Rag1−/− mice. We observed that the enhancer Eα is located at the sub-TADs boundary which separates the Tcra–Tcrd locus and the downstream region including Dad1 gene , NPC , and neurons . This stTcra rearrangement, we generated an EACBE-deleted allele in which a 197bp DNA fragment containing two CTCF binding motifs was deleted by using CRISPR-Cas9 system, leaving the core Eα intact . We. WeTcra s Figure . To expls Figure . EACBE d−/− mice have normal thymocyte development with comparable numbers of thymocytes as WT mice  (Tcra rearrangement and repertoire formation.To learn the pre-selected 5′ RACE) . MiXCR i5′ RACE) . The Jα 5′ RACE) . When we5′ RACE) , consist5′ RACE) . We also5′ RACE) . In addi5′ RACE) . We also5′ RACE) . The resTcrb gene in WT and EACBE-deleted thymocytes. The results show that usage of Jβ and Vβ and combinations of Vβ-Jβ are exactly the same between WT and EACBE-deleted thymocytes J recombination by using qPCR to quantify Vα-to-Jα rearrangement in genomic DNA from WT and EACBE−/− thymocytes. In EACBE−/− thymocytes, the qPCR result revealed relatively normal rearrangement of Trav12, Trav14 and Trav13 to 5′ Jα gene segments, but impaired rearrangement of these Vα segments to the 3′ Jα segments  J recombination . The EACe Figure . We obsee Figure –C. We ale Figure . These odeleted) ,30, indiTcrd-unrearranged alleles.Previous reports showed that the Eα-EACBE region contacts proximal Vα genes in CTCF-dependent manner and regulates their histone modifications, germline transcription, and rearrangement ,31. H3K4Tcra–Tcrd locus, we performed a Hi-C experiment with DP thymocytes from EACBE−/− × Rag1−/− mice. The sub-TADs boundary disappeared, and the two sub-TADs merged in EACBE deficient DP thymocytes, while other regions kept intact  and EACBE right viewpoints showed increased interactions with the proximal region including the Dad1 gene in EACBE-deleted cells  in the downstream of the Cdh24 gene weren’t affected by the EACBE deletion  but may be facilitated by EACBE for distal genes (Ajuba and Cdh24).To determine the mechanism by which EACBE regulates gene expression in the downstream region, we also evaluated gene expression in EαE Figure –C. The rE Figure . This suWe also generated the LCR deleted allele, which deleted the region containing the two ATAC-seq peaks and the EACBE right Nipbl peak Figure , B. WhenTcra rearrangement requires establishment of the recombination center at the 5′ end of the Jα array and access of the proximal Vα genes to the recombination center J gene segments of antigen receptor genes , such asIgH , Sis/CerIgH  and H3 (at R8) to NF-κB and spliceosome proteins  HS2 to HS6 (NΔ26) including EACBE and four HS sites and observed lethality of homozygous mutant embryos due to the abnormal expression of the Dad1 gene (Dad1 are not affected in DP thymocytes. But we did see reduced expression of the Dad1 gene in the Eα-deleted mice.Hong NA ad1 gene . But ourCdh24 gene. The EACBE establishes a sub-TAD boundary which separates the Tcra–Tcrd locus and the downstream region. It helps the Eα core to regulate the select proximal Vα genes and normal Tcra rearrangement. It also makes the Eα able to regulate the far downstream genes via asymmetric chromatin extrusion. This study provides a new insight into the role of CTCF binding sites at TAD or sub-TAD boundaries in gene regulation.Here, we explored a role of the Eα CTCF binding site EACBE in the chromatin dynamic organization of the around 1Mb region from the proximal Vα region to the downstream 4C, RACE-seq, ChIP-seq and Hi-C data are available in GEO database as GSE145147. Rad21, Nipbl and CTCF ChIP-seq were downloaded from GEO database as GSE48763 and GSE41743.gkaa711_Supplemental_FileClick here for additional data file.


Many more H⋯O contacts between the stacks exist, which mostly involve the equatorial hydrogen and oxygen atoms. The highly polarized hydrogen atoms of the –SO2—CH2—SO2– moiety make no exceptionally short H⋯O contacts but clearly play a leading role in the formation of the stacks.In the title compound, the molecules stack parallel with the  4H8O4S2, has been determined to examine the intermolecular C—H⋯O hydrogen bonds in a small molecule with highly polarized hydrogen atoms. The crystals are monoclinic, space group Pn, with a = 4.9472 (5), b = 9.9021 (10), c = 7.1002 (7) Å and β = 91.464 (3)° with Z = 2. The molecules form two stacks parallel to the a axis with the molecules being one a translation distance from each other. This stacking involves axial hydrogen atoms on one molecule and the axial oxygen atoms on the adjacent molecule in the stack. None of these C—H⋯O contacts is particularly short . The many C—H⋯O contacts between the two stacks involve at least one equatorial hydrogen or oxygen atom. Again, no unusually short contacts are found. The whole crystal structure basically consists of a complex network of C—H⋯O contacts with no single, linear C—H⋯O contacts, only contacts that involve two (bifurcated), and mostly three or four neighbors.The crystal structure of 1,3-dithiane 1,1,3,3-tetraoxide, C The 1H NMR spectrum of the compound dissolved in DMSO shows a singlet, 2H, at δ = 5.238 ppm (highly polarized hydrogen atoms on C2 between the two SO2 groups); a triplet, 4H, at 3.370 ppm (moderately polarized hydrogen atoms on C4 and C6 with one adjacent SO2 group); and a pentet, 2H, at 2.260 ppm (relatively unpolarized hydrogen atoms on C5). See Li & Sammes  and decomposition temperatures  Å at 120 K. A general packing diagram is shown in Fig. 2n-glide, the only symmetry operator in space group Pn, curiously preserves the polarity of the stacks and creates a polar crystal with, for example, all of the axial oxygen atoms in the stacks pointing in the same a-axis direction  in glacial acetic acid (25 mL) and the mixture was heated to 323 K overnight. The white precipitate was separated on a Buchner funnel and washed with water (3 × 25 mL) and dried in air. Colorless, rod-like crystals of the compound were harvested from an evaporated KOH (0.5 M) solution of the the 1,3-disulfone. 1H NMR , δ: 5.238 , 3.370 , 2.260 ; 13C NMR , δ: 70.12 (C2), 50.09 (C4/C6), 17.60 (C4). HRMS  m/z found: 182.9803; calculated: 182.9786.To a stirred solution of 1,3-dithiane  in glacial acetic acid (25 mL) was added a solution of 30% HCrystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021000876/ey2004sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021000876/ey2004Isup2.hklStructure factors: contains datablock(s) I. DOI: 2058562CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reportshttps://www.doi.org/10.1038/s41598-020-76235-7, published online 05 November 2020Correction to: The Supplementary Information published with this Article contains errors. The reference numbers do not correspond to the correct references in the original version of the Article.In addition, the Supplementary Information contains a rounding error, where“Inter-item correlations measured with Cronbach’s α: 0.89 Session 1; 0.87 Session 2 (displacement items); 0.81 Session 1; 0.79 Session 2 .”should read:“Inter-item correlations measured with Cronbach’s α: 0.89 Session 1; 0.87 Session 2 (displacement items); 0.81 Session 1; 0.80 Session 2 .”The correct Supplementary Information file is provided below.Supplementary Information.


Here, we report a dication diradical that is based on two Se∴Se three-electron σ–bonds. In contrast, the dication of sulfur analogue does not display diradical character but exhibits a closed-shell singlet.The concept of odd-electron σ–bond was first proposed by Linus Pauling. Species containing such a bond have been recognized as important intermediates encountered in many fields. A number of radicals with a one-electron or three-electron σ-bond have been isolated, however, no example of a diradical based odd-electron σ-bonds has been reported. So far all stable diradicals are based on two  s/p-localized or π-delocalized unpaired electrons . Here, the authors report a dication diradical that is based on two Se∴Se three-electron σ-bonds.Stable diradicals are generally based on two  In general, there are two classes of stable radicals: s/p-localized and π-delocalized. In the former, the unpaired electron resides on an s/p-orbital of one atom, while in the latter, the unpaired electron is π-delocalized over two or more atoms. Apart from these two classes, there is the third class of radicals that have an unpaired electron delocalized in an σ orbital or antibonding σ* orbital between two atoms, leading to a one-electron σ-bond and a three-electron σ-bond ; however, no example of a diradical based odd-electron σ-bonds has been reported. In 2014, we isolated selenium and sulfur radical cations (NapSe2Ph2)+ and (NapS2Ph2)+ , which are of importance both in understanding of bonding nature and application as functional materials22+, Fig. 12+) does not display diradical character but exists as a closed-shell singlet instead.We now report a diradical  and +0.74, 1.04 V (2) 4] (ORF = OC(CF3)3)39 and NOSbF6 in CH2Cl2 to afford dications 12+ and 22+ in modest yields, respectively  bond is nearly perpendicular to the other at both sides of the naphthalene skeleton. Upon oxidation, in 12+ the two S–CPh bonds at the same side of the naphthalene skeleton are nearly linear (torsion angle ∠CPhSSCPh = 6°) and all four S–CPh bonds are coplanar to the naphthalene skeleton, while in 22+ two Se–CPh bonds at the same side are parallel and all Se–CPh bonds are nearly perpendicular to the naphthyl plane (∠CSeC = 100°). The average Se–C bond (Se–CPh and Se–CNap) lengths in 22+ are slightly shorter while ∠C–Se–C angles are slightly larger than those in neutral 2. The Se•••Se separation (2.905(1) Å) is shorter than that (3.054(2) Å) in 2, but longer than the Se–Se single bond length (ca. 2.34 Å)40. The Se–CPh bond alignment and structural parameters of 22+ are similar to those of (NapSe2Ph2)+30, indicating there is a three-electron σ-bond between two Se atoms, and the whole dication possesses two three-electron σ-bonds. In contrast, though the S•••S separation (2.774(2) Å) is also shorter than that (2.937(2) Å) in 1, it is much longer than a regular S–S single bond (ca. 2.05 Å) and comparable to those of molecules with weak intramolecular S•••S interactions41. The S–Cnap bond length (1.727(4) Å) of 12+ is also notably shorter than those of 1 (1.792(2) Å) and (NapS2Ph2)+ (1.768(3) Å)30. Moreover, the naphthalene skeleton of 12+ becomes quinoidal (C8–C9 1.351(6) Å).Crystals suitable for X-ray crystallographic studies were obtained by cooling solutions of neutral tetrachalcogenides and their oxidized species. Their crystal structures are shown in Fig. 12+•2[Al(ORF)4]− and 22+•2[Al(ORF)4]− solutions show characteristic absorptions at 710 and 610 nm, respectively, 4]− appears typical of a triplet state with the zero-field parameters D (136.0 G), E (53.0 G) and an anisotropic g factor  determined by spectral simulation. The giso value (2.0153) is slightly smaller than that of (NapSe2Ph2)•+ (2.0236)29. The average spin–spin distance was estimated to be 5.9 Å from the D parameter, which is comparable to the distance (6.6 Å) between the middle points of Se…Se bonds in the X-ray structure. The forbidden Δms = ±2 transition was not observed from the frozen solution due to the low spin concentration, but observed at the half region of the EPR spectrum on the powder sample of 22+•2[Al(ORF)4]− , confirming that 22+ has an open-shell singlet (OS) ground state. To exclude the intermolecular electronic interaction, frozen solution variable-temperature EPR spectroscopy was performed 43.The EPR spectrum Fig.  of the f4]− Fig. , indicat22+ Fig. . Careful.0 G), E 3.0 G andAT) versus 1/T gives the singlet–triplet gap ΔES–T of −0.14 kcal mol−1, which is close to that obtained from SQUID measurement, further confirming the intra-antiferromagnetic interaction. In contrast, both the frozen solution and powder samples of 12+ are EPR silent, which together with the diamagnetism observed by SQUID measurement , open-shell singlets (OS), and triplets at the (U)B3LYP/6-31+G level. 22+ has an OS ground state (22+-os) while 12+ has a closed-shell singlet ground state (12+-cs) (Supplementary Table 22+ (22+-cs) has a similar geometry to that of 12+-cs. However, a geometry optimization starting with 22+-cs does not reach 22+-os, probably due to a high energy barrier. The optimized geometries of these dications with the lowest energy reasonably agree with the X-ray crystal structures  calculations on neutral molecules and dications. We first used the crystal structure of ph bonds in 12+-cs are nearly coplanar with the naphthalene skeleton, leading to a quinoidal geometry reflected by the HOMO . In 22+-os, the spin density is mainly on Se atoms with an additional extension to the four phenyl rings and naphthalene skeleton  is in agreement with the value determined from SQUID measurement. Figure 2ρ(r), the bond paths and critical points of 12+ and 22+ in a plane that contains Ch  atoms and the naphthalene skeleton. It clearly shows the S–S and Se–Se bonding character, as indicted by the bond critical point between the Ch–Ch centers. Judging from the time-dependent DFT (TD-DFT) calculations  and HOMO-1 (α/β)→LUMO (α/β) (for 22+), respectively,  derived from the CASSCF density matrix and their occupation is given in Supplementary Fig. Since it is a complicated system to perform complete-active-space SCF (CASSCF) calculation that will take a large active space, we performed a CAS calculation with B3LYP optimized geometry to check whether we can call confidently that 1 and 2 with a naphthalene bridge underwent two-electron oxidations, which afforded room temperature stable dications 12+ and 22+. 12+ is shown to possess a closed-shell singlet ground state while 22+ is a diradical containing two Se∴Se three-electron σ-bonds with the electronic coupling of −0.29 kcal mol−1. The difference of electronic structures between two dications 12+ and 22+ is attributed to the easier pπ–pπ interaction between sulfur and carbon atoms than that between selenium and carbon atoms in terms of atomic size matching. The experimentally obtained geometry of 12+ may be rationalized by that two sulfur atoms from each side of the molecule loses one electron and form a quinoidal structure  is much longer than a typical S–S single bond (2.05 Å)41. Thus the geometry of 12+ is best described as the hybrid of resonance structures of I and II, which is supported by the calculated HOMO 4] (ORF = OC(CF3)3)39 were synthesized according to the literature procedures. NOSbF6, diphenyldisulfane (PhSSPh), diphenyldiselane (PhSeSePh), isochromenoisochromene-1,3,6,8-tetraone, and nBuLi  were purchased from Energy Chemical. CV was performed on an IM6ex electrochemical workstation, with platinum as the working and counter electrodes, Ag/Ag+ as the reference electrode and 0.2 M nBu4NPF6 as the supporting electrolyte. The NMR spectra were performed using a Bruker DRX-400 at room temperature in ppm downfield from internal Me4Si. EPR spectra were obtained using Bruker EMX-10/12 X-band variable-temperature apparatus. UV–Vis spectra were recorded on the Lambda 750 spectrometer. Element analyses of 12+•2[Al(ORF)4]− and 22+•2[Al(ORF)4]− were performed at Shanghai Institute of Organic Chemistry, the Chinese Academy of Sciences. Magnetic measurements were performed using a Quantum Design SQUID VSM magnetometer with a field of 0.1 T. X-ray crystal structures were obtained by using Bruker D8 CMOS detector at 193 K. Crystal data and structure refinement are listed in Supplementary Table All manipulations were carried out under an NPreparation of bis(phenylsulfane): A solution of nBuLi  in hexane was added dropwise to a solution of 1,4,5,8-tetrabromo naphthalene  in Et2O (150 ml) at −78 °C and stirring was maintained for 2 h. Then a solution of PhSSPh  in Et2O (20 ml) was added dropwise to the mixture. Then the resulting mixture was allowed to reach to room temperature and stirring was continued for 12 h. The crude product was treated with 0.10 M solution of sodium hydroxide (3 × 30 ml) and extracted with Et2O. The combined organic phase was dried over Na2SO4 and concentrated under vacuum. The crude product was purified by chromatography using petroleum ether: CH2Cl2 (10: 1) as the eluent to give 1.00 g of bis(phenylsulfane) (40%) as light yellow solid. 1H NMR δ 7.14  = 8.1 Hz, 2H, Ar-H), 7.26–7.32  7.60  = 8.1 Hz, 2H, Ar-H); 13C NMR δ 118.21, 128.24, 129.96, 132.89, 134.32, 134.35, 134.62, 137.09, and 137.36.Preparation of bis(phenylselane): By the procedure similar to the synthesis of the bis(phenylsulfane), a yellow solid is given. Yield: 1.01 g, 34.5%; 1H NMR  δ 7.10  = 8.16 Hz, 2H, Ar-H), 7.35–7.41  7.48  = 8.15 Hz, 2H, Ar-H), 7.56–7.58 ; 13C NMR δ 118.75, 129.32, 130.27, 132.34, 133.30, 133.52, 135.13, 135.72, and 136.59.Preparation of1: A solution of nBuLi  in hexane was added dropwise to a solution of bis(phenylsulfane)  in Et2O (120 ml) at −78 °C and maintained stirring for 2 h. Then a solution of PhSSPh  in Et2O (20 ml) was added dropwise to the mixture. Then the resulting mixture was raised to room temperature and kept stirring for 12 h. The crude product was treated with 0.10 M solution of sodium hydroxide (3 × 30 ml) and extracted with Et2O. The combined organic phase was dried over Na2SO4 and concentrated under vacuum. The crude product was purified by chromatography using petroleum ether: CH2Cl2 (5: 1) as the eluent to give 0.45 g (0.80 mmol) of 1 (40%) as a dark yellow solid. 1H NMR  δ 7.17–7.18 , 7.20  = 1.7 Hz, 4H, Ar-H), 7.21–7.23 , 7.24–7.25 , 7.26–7.28 , 7.29–7.30 ; 13C NMR  δ 127.49, 129.65, 131.31, 133.68, 135.07, 136.37, and 138.57.Preparation of2: By the procedure similar to the synthesis of 1, a yellow solid is given. Yield: 0.46 g, 30%; 1H NMR  δ 7.24–7.25 , 7.26–7.27 , 7.28–7.30 , 7.35–7.36 , 7.37–7.38 , 7.48  = 1.05 Hz, 4H, Ar-H); 13C NMR  δ 127.96, 129.77, 132.73, 133.31, 135.69, 135.95, and 138.64.Preparation of12+•2[Al(ORF)4]−: Under anaerobic and anhydrous conditions, CH2Cl2 (35 ml) was added dropwise to the mixture of 1 , NOSbF6  and Li[Al(ORF)4]  while stirring at room temperature. The resultant dark blue solution was stirred at room temperature for 12 h, and then filtered to remove the precipitate (LiSbF6). The filtrate was concentrated and stored at −40 °C for 24 h to afford yellow X-ray-quality crystals of 12+•2[Al(ORF)4]−. Isolated yield: 0.12 g, 24%; elemental analysis : C  H .Preparation of22+•2[Al(ORF)4]−: By the procedure similar to the synthesis of 12+•2[Al(ORF)4]−, black crystals are given. Isolated yield: 0.10 g, 19%; elemental analysis : C  H 2Cl2) effects, polarized continuum model was adopted in the calculation of the single point energies involved in the disproportionation and dimerization, and UV–Vis absorption spectrum. Wiberg bond order was calculated at the (U)B3LYP/6-31+G level with the Multiwfn program. These calculations were performed using Gaussian 16 A03 software. The electron density distribution was analyzed with Quantum Theory of Atom in Molecules method that was developed by Bader44. The multiconfigurational CASSCF46 calculations were performed on the (U)B3LYP/6-31+G optimized geometry of 22+-os with the def2-SVP basis set using ORCA 4.2.0 program47.Geometry optimization without symmetry constraint were performed using DFT at the (U)B3LYP/6-31+G level. Frequency results were examined to confirm stationary points as minima (no imaginary frequencies). The UV–Vis absorption spectrum was calculated on the optimized geometry using TD-DFT method at the (U)B3LYP/6-31+G level. To consider solvent (CHSupplementary Information


Knockdown of p38γ in established (U2OS) and primary human OS cells potently inhibited cell growth, proliferation, migration and invasion, while promoting cell apoptosis. Furthermore, CRISPR/Cas9-induced p38γ knockout inhibited human OS cell progression in vitro. Conversely, ectopic overexpression of p38γ in primary human OS cells augmented cell growth, proliferation and migration. Signaling studies show that retinoblastoma (Rb) phosphorylation and cyclin E1/cyclin A expression were decreased following p38γ shRNA knockdown and knockout, but increased after ectopic p38γ overexpression. Collectively, these results show that p38γ overexpression promotes human OS cell progression.Osteosarcoma (OS) is the most common primary bone malignancy in the adolescent population. Recent studies demonstrate that p38 gamma (p38γ) phosphorylates retinoblastoma (Rb) to promote cyclin expression, cell-cycle entry and tumorigenesis. Studying the potential function of p38γ in human OS, we show that  Osteosarcoma (OS) is the most common primary and malignant bone tumor detected in children, adolescents, and young adults . The curTo improve the outcome for OS patients there is an urgent need to understand the pathological mechanisms of OS progression –5. RecenExamining p38γ expression, a total of twelve (n=12) different OS tissues derived from primary human OS patients were tested. qPCR analysis of mRNA expression revealed that p38γ mRNA levels in OS tumor tissues (“T”) were increased over six fold compared to matched surrounding normal bone tissue (“N”) P<0.001, . Testingp38γ mRNA expression by greater than 95% (P<0.001 vs. control shRNA/shC), resulting in knockdown of p38γ protein in p38γ-shRNA-s1/s2-expressing OS1 cells (P<0.001 vs. shC). In contrast, p38γ-shRNA-s0 did not alter p38γ mRNA and protein expression in OS1 cells (P>0.05 vs. shC). The p38γ shRNAs failed to change expression of p38α mRNA (P>0.05 vs. shC) and protein (P>0.05 vs. shC) in OS1 cells.To investigate the impact of p38γ in OS cell function we generated three stable cell lines with shRNAs targeting p38γ. A set of three lentiviral p38γ shRNAs, with non-overlapping sequences, p38γ-shRNA-s0/s1/s2, were individually transduced into the primary human cells derived patient OS1. As shown, p38γ-shRNA-s1 and p38γ-shRNA-s2 reduced P<0.001). Examining cell viability, using a CCK-8 assay which reflects proliferative ability, p38γ silencing resulted in a 60-70% reduction in viability (P<0.001 vs. shC). In agreement, nuclear EdU incorporation (% vs. DAPI) was sharply decreased in p38γ-silenced OS1 cells (P<0.001 vs. shC). Corroborating these results, FACS analysis demonstrated that p38γ silencing disrupted cell cycle progression, causing G1-S arrest , proliferation  and migration . Collectively, these results show that in human OS cells p38γ silencing inhibited cell viability, growth, proliferation, migration and invasion.Further functional studies showed that silencing of p38γ potently inhibited OS1 cell P<0.001 vs. shC). Furthermore, increased levels of cleaved-caspase-3 and cleaved-PARP were also detected in p38γ-silenced OS1 cells (P<0.001 vs. shC). Confirming apoptosis, p38γ knockdown significantly increased the ratio of Annexin V-positive OS1 cells (P<0.001 vs. shC) and potently increased the ratio of TUNEL-positive nuclei . These results demonstrate that p38γ knockdown induces significant apoptosis activation in OS1 cells. The identical results were observed in primary OS2/ OS3 cells and in established U2OS cells, where shRNA-induced silencing of p38γ  and the ratio of TUNEL-positive nuclei (P<0.001 vs. shC).As inhibition of proliferation often results in apoptosis, we therefore examined apoptosis activation in p38γ-depleted OS cells. In p38γ-shRNA-s1/s2OS1 cells see  caspase-p38γ mRNA . The applied p38γ KO constructs failed to alter p38α expression , potently decreased in vitro cell migration and invasion (P<0.001 vs. Cas9 control cells), and caused significant apoptosis activation (P<0.001 vs. Cas9 control cells). Collectively, these results show that CRISPR/Cas9-induced p38γ KO inhibited OS1 cell proliferation, migration and invasion, while inducing apoptosis activation.To determine the effect of completely depleting p38γ, the CRISPR/Cas9 strategy was utilized to knockout (KO) p38γ. Two lenti-CRISPR/Cas9-GFPp38γ KO constructs(“sgRNA1”and “sgRNA2”) were individually transduced into the OS1 primary human OS cells. Stable p38γ KO OS1 cells were established, where 38γ mRNA  and prot38γ mRNA  expressiin vitro. A pLenti6-puro-GFP-p38γ expression vector (from Dr. Zheng at Nantong University  staining and Annexin V fluorescent-activated cell sorting (FACS), were described in our previous studies [Human OS cells with the applied genetic modifications were seeded into six-well plates . The difference between multiple groups was analyzed by ANOVA with multiple comparisons through Bonferroni post-hoc test, using a SPSS 21.0 software . A two-tailed unpaired T test (Excel 2017) was applied to test significance between two groups. Values of


T, was isolated from a 67-cm-long sediment core collected from the Daechung Reservoir at a water depth of 17 m, Daejeon, Republic of Korea. The cells of strain CHu50b-6-2T were aerobic non-motile and formed yellow colonies on R2A agar. The phylogenetic analysis based on 16S rRNA gene sequencing indicated that the strain formed a separate lineage within the family Microbacteriaceae, exhibiting 98.0%, 97.7% and 97.6% 16S rRNA gene sequence similarities to Glaciihabitans tibetensis KCTC 29148T, Frigoribacterium faeni KACC 20509T and Lysinibacter cavernae DSM 27960T, respectively. The phylogenetic trees revealed that strain CHu50b-6-2T did not show a clear affiliation to any genus within the family Microbacteriaceae. The chemotaxonomic results showed B1α type peptidoglacan containg 2, 4-diaminobutyric acid (DAB) as the diagnostic diamino acid, MK-10 as the predominant respiratory menaquinone, diphosphatidylglycerol, phosphatidylglycerol, and an unidentified glycolipid as the major polar lipids, anteiso-C15:0, iso-C16:0, and anteiso-C17:0 as the major fatty acids, and a DNA G + C content of 67.3 mol%. The combined genotypic and phenotypic data showed that strain CHu50b-6-2T could be distinguished from all genera within the family Microbacteriaceae and represents a novel genus, Lacisediminihabitans gen. nov., with the name Lacisediminihabitans profunda sp. nov., in the family Microbacteriaceae. The type strain is CHu50b-6-2T (= KCTC 49081T = JCM 32673T).A novel Gram-stain-positive bacterial strain, CHu50b-6-2The online version of this article (10.1007/s10482-019-01347-8) contains supplementary material, which is available to authorized users. Microbacteriaceae, 56 genera have been described validly in this family at the time of writing  collected from the Daechung Reservoir at a water depth of 17 m in Daejeon, South Korea. 1 g sediment sample was applied to serial dilution method. A 100 μl sub-sample (10−6 or 10−7) of the suspended material was spread onto modified 1/10 R2A agar  and incubated at room temperature (25 °C) for 4 weeks. One yellow colony, designated as CHu50b-6-2T, was isolated and subcultivated on R2A agar at 30 °C for further analysis. The colony characteristics were determined after growing for 5 days at 30 °C on R2A agar. Gram staining was performed using a Gram stain kit (Becton–Dickinson) and 3% KOH solution. The cell morphology and motility were examined under a phase-contrast microscope  and a transmission electron microscope  after negative staining with 2% (w/v) uranyl acetate using cells grown for 48 h on R2A agar.Strain CHu50b-6-22HPO4/NaH2PO4 buffer for pH 5–7 and Na2CO3/NaHCO3 buffer for pH 8–11 , and Biolog GN2 MicroPlate to determine carbon source utilization and to do enzyme activity assays as well as additional physiological tests following the manufacturer’s instructions. Duplicate antibiotic-susceptibility tests were conducted using filter-paper discs (6 mm) containing the following: amikacin (30 µg ml−1), ampicillin/sulbactam , chloramphenicol (30 µg ml−1), erythromycin (30 µg ml−1), gentamicin (30 µg ml−1), kanamycin (30 µg ml−1), lincomycin (15 µg ml−1), nalidixic acid (30 µg ml−1), rifampicin (30 µg ml−1), spectinomycin (25 µg ml−1), streptomycin (25 µg ml−1), teicoplanin (30 µg ml−1), tetracycline (30 µg ml−1), and vancomycin (30 µg ml−1). The discs were placed on R2A plates spread with a culture of strain CHu50b-6-2T and were then incubated at 30 °C for 2 days. Susceptibility was recorded as positive at zones with diameters greater than 10 mm.The cell growth was checked on R2A agar, trypticase soy agar , Luria–Bertani  medium, and nutrient agar. The growth temperature range was checked by incubating at 4, 8, 15, 20, 30, 37, and 45 °C on R2A agar. Salt tolerance was performed by adding different concentrations of NaCl to R2A agar. The pH growth was determined in R2A broth with a pH range of 5–11 at intervals of 1 pH unit. Different biological buffers were applied to adjust the pH values: NaT was cultured on R2A agar for 48 h to the late exponential phase. Harvesting of the cell mass was standardized in the instruction of MIDI . Separation and identification of the fatty acids were performed by GC (Hewlett Packard 6890), and the TSBA 6 database provided the Sherlock software 6.1. Extraction of isoprenoid quinone was carried out following the method described by Komagata and Suzuki (Glaciihabitans tibetensis KCTC 29148T was obtained from the KCTC (Korean Collection for Type Cultures), Frigoribacterium faeni KACC 20509T from the KACC , and Lysinibacter cavernae DSM 27960T from the DSMZ (German Collection of Microorganisms and Cell Cultures).For fatty acid profiling, strain CHu50b-6-2d Suzuki , and thed Suzuki . The isod Suzuki . The celd Suzuki . Genomicd Suzuki . Three rEscherichia coli position 8–27) and 1492R . The average nucleotide identity (ANI) was calculated using OrthoANI tool in the EZBioCloud  and recA-R (5′-GAR TCS TCS GGW AAG ACB AC-3′), were used for amplifying and sequencing . The cells were observed to assimilate N-acetyl-d-galactosamine, N-acetyl-glucosamine, l-arabinose, 2,3-butanediol, d-cellobiose, dextrin, d-fructose, d-galactose, d-gluconic acid, d-glucose, glycerol, inosine, inositol, α-keto butyric acid, dl-lactate, lactulose, maltose, mannitol, d-melibiose, pyruvic acid methyl ester, d-raffinose, rhamnose, d-ribose, d-sorbitol, sucrose, turanose, thymidine, uridine, and xylitol but not the rest . The cells were found to be positive for the following enzyme activities (API ZYM test strip): acid phosphatase, cystine arylamidase, esterase (C4), esterase lipase (C8), α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, leucine arylamidase, naphtol-AS-BI-phosphohydrolase, and valine arylamidase but the cells were found to be negative for the following enzyme activities: N-acetyl-β-glucosaminidase, alkaline phosphatase, α-chymotrypsin, α-fucosidase, β-glucuronidase, lipase (C14), α-mannosidase and trypsin (Table −1), chloramphenicol (30 µg ml−1), erythromycin (30 µg ml−1), kanamycin (30 µg ml−1), lincomycin (15 µg ml−1), rifampicin (30 µg ml−1), spectinomycin (25 µg ml−1), streptomycin (25 µg ml−1), teicoplanin (30 µg ml−1), and vancomycin (30 µg ml−1) but resistant to ampicillin/sulbactam , nalidixic acid (30 µg ml−1), gentamicin (30 µg ml−1), and tetracycline (30 µg ml−1). Strain CHu50b-6-2T could be differentiated from the closest species G. tibetensis by assimilating N-acetyl-glucosamine, l-arabinose, d-glucose, maltose, and d-mannitol and by activities of nitrate reduction, arginine dihydrolase, oxidase, α-chymotrypsin, lipase (C14), and trypsin; from F. faeni by assimilating N-acetyl-glucosamine and not assimilating citrate, by activities of arginine dihydrolase, alkaline phosphatase, and motility; from L. cavernae by not assimilating gluconate and d-mannose, by activities of nitrate reduction, arginine dihydrolase, oxidase, cystine arylamidase, α-galactosidase, β-galactosidase, and lipase (C14).Strain CHu50b-6-2T was deposited at DDBJ/EMBL/GenBank with the accession number PRJNA559971. The draft genome of strain CHu50b-6-2T was of 4,022,930 bp, containing 175 contigs, of which the largest was of 845,903 bp. The genome encoded 3975 genes, including 48 tRNAs and 7 rRNAs. The N50 value was 413,391 and the sequencing depth of coverage was 570X. The DNA G + C content calculated from the draft genome sequence was 67.3 mol% (Table S1). The ANI values of strain CHu50b-6-2T with G. tibetensis MP203T and F. faeni 801T were 73.1 and 73.4, respectively.The draft genome sequence of strain CHu50b-6-2T was compared with those of representative species within the family Microbacteriaceae. Strain CHu50b-6-2T showed over 97% 16S rRNA gene sequence similarities with G. tibetensis, F. faeni, L. cavernae, F. endophyticum, Parafrigoribacterium mesophilum, Cryobacterium arcticum, F. salinisoli, and Homoserinimonas aerilata, and less than 97% with the remaining members of the family Microbacteriaceae. According to maximum-likelihood, neighbour-joining, and maximum-parsimony tree analysis  data, for which our novel isolate showed DNA-DNA relatedness values of 31.2% , 29.7% , and 22.5%  with G. tibetensis KCTC 29148T, F. faeni KACC 20509T and L. cavernae DSM 27960T, respectively. For clearer delineation, the housekeeping gene, recA gene was applied as phylogenetic marker. The recA gene sequence of CHu50b-6-2T had 88.8%, 88.2%, 86.2%, 85.9%, and 85.4% similarities with Clavibacter michiganensis VKM Ac-1403T, Subtercola boreus DSM 13056T, Rathayibacter rathayi DSM 7485T, F. faeni KACC 20509T, and G. tibetensis KCTC 29148T respectively. Overall, phylogenetic analyses based on 16S rRNA, recA genes and upgma dendrogram based on the ANI values of genomic sequences revealed groups that are in good agreement with the currently recognized genera  of strain CHu50b-6-2anteiso-C15:0 (46.6%), iso-C16:0 (29.7%), and anteiso-C17:0 (14.5%) (Table T had a large amount of anteiso-C17:0 (14.5%) compared to the closet members G. tibetensis KCTC 29148T (3.6%), F. faeni KACC 20509T (5.1%) and L. cavernae DSM 27960T (1.8%), and a smaller amount of iso-C14:0 (1.6%) compared to L. cavernae DSM 27960T (22.2%). Although the overall polar lipid patterns were very similar, there were some differences in the unidentified phospholipids and the unidentified lipids between CHu50b-6-2T and the species G. tibetensis, F. faeni and L. cavernae. Strain CHu50b-6-2T had B1α type pepdidoglycan structure, which differed from phylogenetically related genera Glaciihabitans, Frigoribacterium, and Lysinibacter. Together with some other physiological results, it could be concluded that the strain CHu50b-6-2T differs from the close species G. tibetensis, F. faeni and L. cavernae .anteiso-C15:0, iso-C16:0, and anteiso-C17:0. The cell-wall peptidoglycan is B1α contains 2, 4-diaminobutyric acid as the diagnostic diamino acid. Phylogenetically, the genus belongs to the family Microbacteriaceae in the class Actinobacteria, being closely related to the genera Glaciihabitans, Frigoribacterium, and Lysinibacter. The type species is Lacisediminihabitans profunda.Cells are observed to be Gram-stain-positive, non-spore-forming, non-motile and rod-shaped. The predominant respiratory menaquinone is MK-10. The major polar lipids are diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), and an unidentified glycolipid (GL). The major fatty acids are Lacisediminihabitans profunda (pro.fun’da. L. fem. adj. profunda from the deep).β-galactosidase but negative for aesculin hydrolysis, indole production, glucose fermentation, urease, arginine dihydrolase or gelatin hydrolysis (API 20NE test strip). The G + C content of the genomic DNA is 67.3 mol%.In addition to the characteristics described above, the novel species has the following properties. Colonies on R2A are convex, circular with entire edges and yellow color. The cells are observed to be oxidase-negative but catalase-positive. Growth occurs on R2A at temperatures from 4 to 30 °C (optimum temperature 25–30 °C), but not at 37 °C. The pH range for growth is from pH 6–10 (optimum pH 7); however, there is no growth at pH 5 and 11. No growth was observed on TSA, LB, and NA media. The cells are positive for nitrate reduction and T (= KCTC 49081T = JCM 32673T) isolated from a 67-cm-long sediment core taken from the Daechung Reservoir, Republic of South Korea.The type strain is CHu50b-6-2Supplementary material 1 (PDF 1232 kb)Below is the link to the electronic supplementary material.


Scientific Reports 10.1038/s41598-020-76056-8, published online 05 November 2020Correction to: The Acknowledgements section in this Article was omitted. The Acknowledgements section should read:“J.S. was supported by a doctoral fellowship from the Direction Générale de l’Armement .”


H-isochromen-1-one.The title compound, 1--2,2,6,6-tetramethylpiperidine, was synthesized as a side-product during the synthesis of the intermediate, methyl 3,6-dimethoxy-2-(2-methoxy-2-oxoethyl)benzoate, necessary for the total synthesis of the isocoumarin 5,8-dimethoxy-3-methyl-1 17H27NO2, the piperidine ring has a chair conformation and is positioned normal to the benzene ring. In the crystal, molecules are linked by C—H⋯O hydrogen bonds, forming chains propagating along the c-axis direction.In the title compound, C Ceratocystis fimbriata species  was synthesized as a side product during the synthesis of the intermediate, methyl 3,6-dimethoxy-2-(2-methoxy-2-oxoethyl)benzoate (2) , is illustrated in Fig. 2The molecular structure of the title compound, 1--2,2,6,6-tetramethylpiperidine  through white to blue . The energy frameworks , O⋯H/H⋯O at 8.3%  and C⋯H/H⋯C at 7.6%  contacts. The intermolecular contacts are therefore dominated by dispersion forces . This is confirmed by the energy frameworks shown in Fig. 6−1 within 2 × 2 × 2 unit cells, and obtained using the wave function calculated at the HF/3-21G level of theory.A view of the Hirshfeld surface of 1% Fig. 5b, O⋯H/H3% Fig. 5c and C⋯6% Fig. 5d contacet al., 2016supporting information). A number of these structures involve heteroaryl and heterocyclic aluminium compounds, see for example CSD refcodes CEGLUY, CEGMF, CEGMEJ, CEGMIN, CEGMOT and CEGMUZ -2,2,6,6-tetramethylpiperidine °.A search of the Cambridge Structural Database  with tetramethylpiperidene (HTMP). It is a side product obtained during the synthesis of methyl 3,6-dimethoxy-2-(2-methoxy-2-oxoethyl)benzoate (2)  = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020005952/dj2006sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989020005952/dj2006Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020005952/dj2006sup3.pdfCSD search S1. DOI: Click here for additional data file.10.1107/S2056989020005952/dj2006Isup4.cmlSupporting information file. DOI: 2000223CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


II coordination polymer, [Co2(2-OHbenz)42.5(H2O)]n, constructed from 4,4′-bipyridine  and 2-hydroxybenzoate (2-OHbenz) are reported.The synthesis, crystal structure and physical properties of a novel one-dimensional zigzag chain-like Co II coordination polymer constructed from 4,4′-bipyridine  and 2-hydroxybenzoate (2-OHbenz) ligands, namely, catena-poly[[(μ-2-hydroxybenzoato-κ2O:O′)cobalt(II)]-μ-4,4′-bipyridine-κ2N:N′-[aquahemi(2-hydroxybenzoato-κO(2-hydroxybenzoato-κ2O:O′)cobalt(II)], [Co2(C7H5O3)4(C10H8N2)2.5(H2O)]n, has been synthesized by reacting cobalt(II) nitrate trihydrate, 4,4′-bpy and 2-hydroxybenzoic acid in a mixture of water and methanol at room temperature. There are two independent CoII centers, Co1 and Co2, in the asymmetric unit, revealing a distorted octahedral geometry with chromophore types of [CoN2O4] and [CoN2O3O′], respectively. The Co1 ions are doubly bridged by 2-OHbenz ligands with syn–anti coordination mode, generating a dinuclear unit. The bridging 4,4′-bpy ligands connect these dinuclear units and the mononuclear Co2 chromophores, providing a one-dimensional alternating zigzag chain-like structure. In the crystal, intermolecular hydrogen bonds, C—H⋯π and π–π stacking interactions are observed and these help to consolidate the packing. In addition, the physical properties of the title compound are reported.A novel one-dimensional zigzag chain-like Co As a result, a CoII coordination polymer containing 4,4′-bpy and 2-OHbenz, [Co2(2-OHbenz)42.5(H2O)]n, with a novel 1D alternating zigzag chain-like structure has been successfully synthesized and characterized and its crystal structure has been determined. Herein, we report the synthesis and crystal structure and physical properties of this compound.This work was undertaken as part of a search for new first-row transition-metal coordination polymers constructed from 4,4′-bpy and carboxylate ligands. The CoII atoms, two and a half of 4,4′-bpy ligands, four 2-OHbenz ligands and one water molecule –2.348 (15) and 2.1177 (16)–2.1568 (17) Å, respectively. Two Co1 centers are doubly bridged by two bridging 2-OHbenz ligands with a syn–anti coordination mode to form a discrete dinuclear unit. The dinuclear units are connected to Co2 atoms by the bridging 4,4′-bpy ligands, providing a one-dimensional zigzag chain-like structure along [101]  = −x + 1, −y, −z + 1] and Co1⋯Co2 distances are 4.099 (2) and 11.381 (2) Å, respectively.The asymmetric unit consists of two independent Cole Fig. 1. Both Coly Fig. 2. The Co11] Fig. 3. The Co1Cg1⋯Cg3i and Cg2⋯Cg4i being 3.965 (1) and 3.652 (1) Å, respectively, where Cg1, Cg2, Cg3 and Cg4 are the centroids of the N1/C1–C5, N2/C6–C10, N3/C11–C15 and N4/C16–C20 rings, respectively (NO3)2·4H2O, (phba = 4-hydroxybenzoate) 2(μ2-benz)2(benz)2]n, (benz = benzoate) 42]n 2·6H2O  in a 4:1 mixture of methanol and water (10 mL). The resulting solution was stirred for 20 min. Next, a solution of 2-OHbenzH  in methanol (5 mL) was slowly added dropwise and stirred over a period of 15 min. After that, the mixture was filtered. The filtered solution was left to stand without disturbance and allowed to slowly evaporate in the air. After five days, red crystals suitable for single crystal X-ray diffraction were obtained [56.18% yield based on cobalt(II) salt]. Elemental analysis; calculated for C53H42Co2N5O13: C 59.06, H 4.21, N 6.50%; found: C 59.14, H 3.99, N 6.41%. IR : 3087s, 1595s, 1485s, 1460s, 1459s, 1413m, 1389s, 1359s, 1308w, 1252m, 1218m, 1143w, 1068w, 1029w, 871w, 814s, 749s, 701w, 671w, 633w, 530w.A solution of 4,4′-bpy  in MeOH (5 mL) was slowly added into a solution of Co(NOsupporting information) shows a characteristic broad peak centered at 3087 cm−1, which is assigned to OH stretching vibrations of the water molecule and the hydroxyl groups of 2-OHbenz. Strong and sharp peaks at 1595 and 1485 cm−1 can be assigned as the asymmetric and symmetric COO− stretching vibrations of the chelating 2-OHbenz ligands, respectively. Peaks in the region of 600–1000 cm−1 are assigned to CH bending of the aromatic rings in the ligands  shows two broad bands in the visible region with the main peak centered about 515 nm (19.42 kK), which can be assigned to the ν3: 4Tg1 → 4Tg1(P) transition. There is a small peak as a shoulder at around 655 nm (15.27 kK), assigned to the ν2: 4Tg1 → 4Ag2 transition and a broad band centered about 1095 nm (13.24 kK), which can be assigned to the ν1: 4Tg1 → 4Tg2 transition. The characteristic bands of this electronic spectrum correspond to a distorted octahedral geometry for CoII compounds as confirmed by the X-ray structure  was used to check the phase purity of the bulk sample in the solid state. The measured PXRD pattern of the title compound closely matches the simulated pattern generated from the single-crystal X-ray diffraction data, confirming the title compound is pure.The PXRD pattern of the title compound  demonstrates the thermal stability of the title compound up to 160°C. The first weight-loss step of 27.37% is observed from 160 to 277°C and can be attributed to the loss of coordinated water and two 2-OHbenz molecules. The next step weight-loss step of 25.7% observed from 277 to 356°C corresponds to the loss of a coordinated 2-OHbenz molecule. Finally, the weight loss of about 36.33% from 356 to 520°C can be assigned to the removal of two and half of the 4,4′-bpy ligands. The residual product is assumed to be CoO.The TGA curve shown in  exhibit strong emission bands at 425 and 439 nm, respectively. However, no detectable emission can be observed for the title compound (λex = 340 nm). This complete PL quenching is the result of the low energy d–d transitions in the partially filled metal ion centers found for CoII compounds described above and reported elsewhere  = 1.2Ueq(C). O-bound H atoms were located in a difference electron-density map, and were refined with bond-length restraints of O—H = 0.84 (1) Å.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020009482/is5544sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020009482/is5544Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020009482/is5544sup3.pdfSupporting information file. DOI: 2015747CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


I complex possessing a Cu2S2 core and contains thiocyanate anions and allylthiourea derivatives as chelating and bridging ligands. The dimeric CuI complexes are linked by N—H⋯S hydrogen bonds, forming a network extending in two dimensions parallel to (100).The crystal structure of the title compound consists of a dimeric Cu 2(NCS)2(C9H16N2)2], was obtained from the reaction of copper(I) thiocyanate (CuSCN) with (N-prop-2-en-1-yl)piperidine-1-carbothioamide as a chelating and bridging thiourea ligand in chlorobenzene. The Cu2S2 core of the dimeric molecule is situated on a crystallographic inversion centre. The copper atom is coordinated by a thiocyanate nitrogen atom, each sulfur atom of the two thiourea ligands, and the C=C double bond of the ligand in a distorted tetrahedral geometry. The dimers are linked by N—H⋯S hydrogen bonds, forming a network extending in two dimensions parallel to (100).The title crystalline compound, [Cu N-substituted thiourea and N, N′-disubstituted thiourea, are well-known ligands to copper ions, such as for their structural relatedness of proteins in bioinorganic chemistry and controlling redox potentials of copper ions in electrochemistry. Recently, copper–thiourea complexes [Cu(tu)s] have been investigated as electronic materials, for precursors of copper sulfide to be applied as semiconductors piperidine-1-carbothioamide, as a hydrophobic bidentate ligand and report here the crystal structure of the title non-ionic CuI complex containing thiocyanates as coordinating anions.Thiourea and its derivatives, 2S2 central core is shown in Fig. 1i, and Cg1 . The four-coordinate geometry index, τ4 = [360° - (α + β)] / 141°, evaluated from the two largest angles (α < β), which has ideal values of 1 for a tetrahedral and 0 for a square-planar geometry  Å. The C14=C15 double bond is η2-π-coordinated to Cu, the bond being elongated to 1.351 (5) Å. The N atom of the piperidine ring (N4) shows no pyramidalization, with a displacement of 0.041 (3) Å from the plane of the bonded C atoms . The piperidine ring adopts a chair conformation with puckering parameters: Q = 0.573 (4), θ = 176.3 (4), and φ = 153 (6)  ring motif dicopper(I) dinitrate [Cu2(atu)2(CH3CN)2](NO3)2, a cationic analogue of the title compound with acetonitrile instead of thiocyanate and without the piperidine ring, shows a similar geometry around copper but has no crystallographic inversion centre because of the asymmetric packing of the nitrate anions (NO3)2 exhibits a complementary C—H⋯S interaction between discrete copper dimers forming a dimer of dimeric structures  Fig. 2. There iet al., 2016ConQuest piperidine-1-carbothioamide, itself and its metal complexes have not been reported. A survey for a Cu complex containing the 1-allylthiourea fragment as a κS-coordination ligand reveals 53 examples, which includes six examples of η2-π-coordination of an allyl group to Cu. All of these six examples are CuI complexes, which comprise four coordination polymers of 4-allyl-semicarbazide as ligands 2: C, 39.26; H, 5.27; N, 13.74; S, 20.96. Found: C, 38.72; H, 4.78; N, 13.59; S, 20.28.To a chlorobenzene solution (2.5 mL) containing copper(I) thiocyanate  and allyl isothiocyanate  in a 20 mL capped screw-tube bottle was slowly added piperidine  at 373 K under air and the mixture was stirred for 5 minutes. After that, it was left at room temperature. The pale-white precipitate formed in the bottle, and gradually changed to a pale-white solid containing single crystals. The mixture was filtered after 5 days to give a pale-white solid containing single crystals . Single crystals suitable for X-ray crystallographic analysis were selected in the product. Analysis calculated for  and refined as part of a riding model with Uiso(H) = 1.2Ueq(C). The N-bound H5 atom was located in the difference-Fourier map but was refined with a distance restraint of N—H = 0.86±0.01 Å, and with Uiso(H) set to 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020013146/yz2001sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020013146/yz2001Isup2.hklStructure factors: contains datablock(s) I. DOI: 2034488CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the title quinoxaline molecule, the dihedral angle angle between the benzene rings is 72.54 (15)°. In the crystal, molecules are connected into chains extending parallel to °. In the crystal, molecules are connected into chains extending parallel to (10The title quinoxaline molecule, C A Hirshfeld surface analysis was also performed.Given their importance in the pharmaceutical, chemical and industrial fields, the synthesis of quinoxaline and its derivatives has been a goal of chemists in recent years. Quinoxaline derivatives find use as anticancer  link the chains into a three-dimensional network structure  Table 1. Weak C2e Table 1.CrystalExplorer17.5  to 1.358 (blue) a.u., as illustrated in Fig. 4dnorm plot indicate intermolecular contacts involving the hydrogen bonds. The red spots identified in Fig. 4a) correspond to the intermolecular C—H⋯O bonds. Regions close to the sum of the van der Waals radii are shown in white. Fig. 4b) shows the shape-index surface, which can be used to detect the presence of π-stacking interactions. The absence of characteristic triangles indicates that no significant π–π interactions are present. Two-dimensional fingerprints were also generated in the range −1 to 1 Å -one as the main skeleton revealed the presence of three structures similar to the title compound, but with different substituents. These are: 1,4,6-tribenzoyl-3-(4-bromobenzyl)-1,4-dihydroquinoxaline-2-one -dione and 1,4-diallyl-6-nitroquinoxaline-2,3--dione on mild steel (MS) in 1.0 M HCl solution was investigated.A search of the Cambridge Structural Database . After stirring for 10 min, 0.5 mL (4.32 mmol) of benzyl chloride was added and the mixture was stirred at room temperature for 6 h. After filtration of the salts, the DMF was evaporated under reduced pressure and the residue obtained was dissolved in dichloromethane. The organic phase was then dried over Na2SO4 and concentrated. The mixture obtained was chromatographed on a silica gel column [eluent: hexane/ethylacetate (2/1)]. The crude product was recrystallized from ethanol as yellow crystals suitable for X-ray analysis (m.p. 493.5 K).To a solution of 6-methyl-1,4-dihydroquinoxaline-2,3-dione  in DMF (15 mL), were added potassium carbonate  and tetra-Uiso(H) = 1.5Ueq(C) for methyl, C—H = 0.96 Å and Uiso(H) = 1.2Ueq(C) for methylene, C—H = 0.93 Å and Uiso(H) = 1.2Ueq(C) for aromatic and C—H = 0.98 Å and Uiso(H) = 1.2Ueq(C) for methine H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989020009895/mw2165sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020009895/mw2165Isup3.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020009895/mw2165Isup3.cmlSupporting information file. DOI: 1936664CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Two new substituted propanedioate esters have been synthesized using a three-component solvent-free thermal reaction. The products, which are isomorphous, differ only in the presence of a bromophenyl group in one, where the molecules are fully ordered, as compared with a chlorophenyl group in the other, where the molecules exhibit two types of disorder. H-pyrazole-4-carbaldehyde and an aryl azide, forming two new C—C bonds in a single step. The products diethyl (RS)-2-[(4-bromophenyl)methyl]propanedioate, C24H25BrN2O5 (I), and diethyl (RS)-2-[(4-chlorophenyl)methyl]propanedioate, C24H25ClN2O5 (II), are isomorphous, with Z′ = 2 in space group P21/n. The two independent molecules in compound (I) are both fully ordered, while each of the independent molecules in compound (II) is disordered, but in different ways. In one molecule of (II), the N-phenyl ring is disordered over two sets of atomic sites having occupancies 0.635 (10) and 0.365 (10), and in the other molecule the ester function is disordered over two sets of atomic sites having occupancies 0.690 (5) and 0.310 (5). In both structures, the two independent molecules adopt different conformations and, in each structure, the molecules are linked into complex sheets by a combination of N—H⋯O, C—H⋯O and C—H⋯π(arene) hydrogen bonds. Comparisons are made with some related structures.Two new substituted propanedioate esters have been synthesized using a three-component solvent-free thermal reaction between diethyl propanedioate , 5-chloro-3-methyl-1-phenyl-1 Our expectation was that the methylene group of the ester component would undergo a condensation reaction with the carbaldehyde function to provide a new electron-deficient alkene system, which would then undergo a 1,3-dipolar cycloaddition with the aryl azide to provide pyrazole-substituted 1,2,3-triazoles. The reactions, carried out under thermal and solvent-free conditions, turned out to take an entirely different course, in which the azide group was lost and giving, instead of the anticipated products, the highly substituted esters diethyl (RS)-2-[(4-bromophenyl)methyl]propanedioate (I)RS)-2-[(4-chlorophenyl)methyl]propanedioate (II)3, and the H atoms in these by-products may well arise from thermal degradation of one or more of the reactants, particularly the ester component. However, despite the modest yields, compounds (I)Pyrazoles exhibit a very wide range of pharmacological and other biological activities, which have recently been extensively reviewed Compounds (I)R-configuration. The centrosymmetric space group confirms that both compounds have crystallized as racemic mixtures.All of the molecules contain a stereogenic centre, at atom C121 in the type 1 molecules Figs. 1 and 3 ▸ x2—Cx21 bond . Similar remarks apply to the conformations of the disordered ester units in compound (II)x2—Cx21—Cx31—Cx32 and Nx41—Nx42—Cx51—Cx52 4 Figs. 1 and 2 ▸.In compound (II)x, y, z) and the idealized riding site of the major occupancy atom H25A in molecule 2 at . However, the atom H25A forms part of a methyl group, and such methyl groups are likely to be undergoing extremely rapid rotations about the adjacent C—C bonds, particularly at ambient temperature , then molecule 2 at  will probably also be the minor-occupancy form. However, this does not imply any longer-range correlation between the disorder components, nor require any relationship between the disorder occupancy factors for the two independent molecules.In compound (II)cf. Acosta et al., 2009et al., 2012Compounds (I)n-glide plane at y = 0.75 are linked by a second, almost linear N—H⋯O hydrogen bond to form a et al., 1990et al., 1995n-glide plane at y = 0.75, resulting in a chain of rings running parallel to the [101] direction on Fig. 6. There iH-pyrazol-1-yl)methyl]malonate methyl]malonate (4-methoxyphenyl)methyl]propanedioate H-pyrazole-3,4-dicarboxylate derivatives   Fig. 5 was prep−1) 3150 , 1705 (ring C=O), 1690 (ester C=O); NMR (DMSO-d6) δ(1H) 2.21 , 2.30 , 2.36 , 2.54  3.98 , 7.1–8.6 .Compound (I)−1) 3230 , 1702 (ring C=O), 1605 (ester C=O); NMR (DMSO-d6) δ(1H) 1.78 , 2.30 , 2.45 , 2.83  4.02 , 6.8–8.6 .Compound (II)3), 0.99 Å (CH2) or 1.00 Å , and with Uiso(H) = 1.2Ueq(C). For the H atoms bonded to N atoms, the atomic coordinates were refined with Uiso(H) = 1.2Ueq(N) giving the N—H distances shown in Table 2Crystal data, data collection and refinement details are summarized in Table 310.1107/S2056989020011676/zl2798sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989020011676/zl2798Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020011676/zl2798IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989020011676/zl2798Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989020011676/zl2798IIsup5.cmlSupporting information file. DOI: 2025336, 2025335CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: