Low levels of type I interferon (IFN-I) are thought to be a driving force for immune activation and T-cell exhaustion in HIV-1 infected individuals on combination antiretroviral therapy (cART), though the causative mechanisms for persistent IFN-I signaling have remained unclear. Here, we show Rev–CRM1-dependent nuclear export and peripheral membrane association of intron-containing HIV-1 RNA, independent of primary viral sequence or viral protein expression, is subject to sensing and signaling via MAVS, resulting in IFN-I-dependent pro-inflammatory responses in macrophages. Additionally, HIV-1 intron-containing-RNA-induced innate immune activation of macrophages leads to upregulation of inhibitory receptor expression and functional immune exhaustion of co-cultured T cells. Our findings suggest that persistent expression of HIV-1 intron-containing RNA in macrophages contributes to chronic immune activation and T-cell dysfunction and that use of HIV RNA expression inhibitors as adjunct therapy might abrogate aberrant inflammation and restore immune function in HIV-infected individuals on cART. Type I Interferon is thought to be a driving force for immune activation and T cell exhaustion during HIV infection. Here the authors show that intron-containing HIV RNA induces innate immune activation resulting in associated T cell dysfunction. Despite long-term viral suppression by cART and restoration of CD4+ T-cell levels, immune activation, and inflammation persist in the majority of treated HIV-infected individuals, and is associated with excess risk of mortality and morbidity. Many factors have been attributed to cause this aberrant immune activation in vivo, such as bacterial endotoxin or co-infections4; however, a viral (HIV) etiology for the chronic inflammatory state has remained unclear. Persistent infection of myeloid cells, most likely tissue-resident macrophages, is postulated to contribute to chronic immune activation and HANAs7, though molecular mechanisms of how HIV-1 replication activates macrophages remain poorly understood.A hallmark of HIV-1 infection in vivo is systemic chronic immune activation+ and CD8+ T cells, and the phenotype is suppressed upon antagonism of IFN-I. These findings suggest that novel therapeutic strategies that suppress viral icRNA expression and IFN-I signaling cascades in tissue macrophages might have immunologic and therapeutic benefit in HIV-1 infected individuals on cART.In this study, we report that expression and Rev–CRM1-dependent nuclear export of intron-containing HIV-1 RNA (icRNA) activates host sensing mechanisms and type I interferon (IFN-I)-dependent pro-inflammatory responses via MAVS in productively infected macrophages. Additionally, the ability of cells to distinguish intron-containing HIV-1 RNA from self mRNA is dependent on the localization of non-self HIV icRNA at peripheral membrane sites. Interestingly, HIV-1 infection-induced activation of macrophages, in turn, leads to upregulation of inhibitory receptor (IR) expression and reduced effector function of co-cultured autologous CD48 whose expression is dramatically upregulated (fivefold) even upon low levels (<0.3 U ml–1) of IFN-α exposure  was inhibited in HIV-1-infected MDMs  results in induction of a myeloid cell specific ISG, CD169/Siglec1 Fig. 8 whose e11, MDMs infected with Nef, Env, Vpu, Vpr, or Vif-deficient mutants -binding-deficient CA mutant G89V  of the M10 mutant (M10-CTE) or the ∆RRE mutant (∆RRE-CTE) in the sense orientation, but not in the anti-sense orientation (M10-CTE-AS), rescued nuclear export of HIV-1 icRNA and Gag expression  expression  transcribed from the HIV-1 LTRion Fig. . Insertiion Fig. . Howeverion Fig. . Moreoveion Fig.  induced ion Fig. , Rev-indion Fig.  from an ion Fig. . A potenion Fig. , potentlion Fig.  and IP-1ion Fig. , further20. Establishment of productive HIV-1 infection in monocyte-derived dendritic cells (MDDCs) also upregulated CD169  specific to the Rev–RRE pathway dictates induction of pro-inflammatory signaling cascades. Since a subset of cytoplasmic HIV-1 icRNAs are trafficked to membrane-associated virus assembly sites for association with Gag and packaging into new virions15, we infected MDMs with a series of Gag mutants to potentially modulate localization of HIV-1 icRNA-associated RNPs  or deltaretrovirus (HTLVII)  production in mMA and mMA-fusion virus-transduced MDMs was similar to that observed upon infection with wild type HIV-1  located within the nef orf -7-Oxozeaenol)14, NF-κB (BAY11-7082)14, Ca2+-dependent signaling pathway 27, NLRP3 inflammasome (glybenclamide)28, and herbimycin A, an inhibitor of non-receptor tyrosine kinases including c-Abl, which is involved in MAVS activation29. None of the inhibitors were cytotoxic   in mice, chronic immune activation can result in T-cell exhaustiontry Fig. . Intereslls Fig. . While Llls Fig. , CD160 eure Fig. . CD160 ells Fig.  and CD16lls Fig.  in co-culls Fig. . Furtherlls Fig. , suggest32, such differences might also subject viral icRNA to detection by host innate immune sensing mechanisms.Whether HIV-1 infection of MDMs is subject to sensing has remained a matter of controversy. In this study, we show that a late step of HIV-1 replication, specifically, expression and Rev–CRM1-mediated nuclear export of viral icRNA to membrane-associated virus particle assembly sites in MDMs is subject to MAVS-dependent host sensing mechanisms and results in induction of pro-inflammatory responses and innate immune activation. Importantly, Rev–CRM1-dependent nuclear export specifically marks HIV-1 icRNA for detection by cytoplasmic sensing mechanisms, since access to alternative CTE-dependent nuclear export pathway failed to induce pro-inflammatory responses. There seems to be no sequence specificity for the sensing mechanism since inclusion of non-viral sequences (TagRFP) in HIV icRNA failed to abrogate production of pro-inflammatory responses. While the distinct trafficking pattern and composition of viral icRNA-containing RNPs has been hypothesized to confer translational advantage to HIV-1 mRNAs compared to bulk host mRNA33. We speculate that HIV-1 icRNA-containing RNPs are by default trafficked to the vicinity of peripheral membrane, and that tethering of HIV-1 icRNA to distinct peripheral membrane sites appeared to be a critical determinant for induction of innate immune sensing. Expression of mutant Gag that alters intrinsic peripheral membrane localization of icRNA (MLV-MA) or by preventing stable membrane association (HIV-1 MA-G2A mutant) abrogated sensing and/or induction of IFN-I responses. Although MAVS requirement for HIV-1-induced MDM activation has been postulated before34, viral determinant that was subject to sensing has remained unclear. Here, we identify that Rev–CRM1-dependent icRNA export pathway triggers MAVS-dependent innate immune sensing by a yet-to-be identified sensor that resides at or in the vicinity of HIV-1 Gag assembly site. Further studies are warranted to elucidate HIV-1 icRNA trafficking mechanisms in MDMs, and to identify the cytosolic sensor of HIV-1 icRNA in MDMs.It has been shown in neuronal cells that RNA-transporting granules  are trafficked to the vicinity of peripheral membrane sites distant from nucleus, and interestingly many of their components have also been found in HIV-1 RNP trafficking granules11, though results described in this report do not support the existence of a cryptic Gag protein sensor in myeloid cells. Furthermore, previous studies have reported that secreted viral proteins such as gp120 and Tat can cause microglia activation35. Differences in experimental system such as target cells and methods to express viral proteins (infections in the presence of SIVmac Vpx to degrade SAMHD1 or transient overexpression) might account for the discrepant findings.Expression of several viral proteins, including Gag, Tat and gp120 has been reported to activate myeloid cells including DCs, microglia, and macrophages. For instance, newly synthesized Gag is thought to activate DCs in a cell-intrinsic manner+ T cells. Importantly, the observed exhaustion phenotype of T cells was driven by IFN-I since the soluble IFN-I antagonist, B18R, abrogated the upregulation of IR expression on T cells, in agreement with recent in vivo findings on HIV-137 and LCMV39. Tissue-resident macrophages are important cellular targets of HIV and can remain persistently infected with HIV-1 even in individuals on cART7, and HIV-1 icRNAs have been detected in CD4+ T cells and alveolar macrophages from patients on long-term cART41. A recent study quantified a number of SIV RNA expressing cells in non-human primates on cART and revealed that 98% of those cells were found in gut tissues42. While most of the SIV RNA+ cells are presumably CD4+ T cells, previous estimates suggest myeloid cells including macrophages constitute up to ~35% of total CD45+ cells in gut tissues43. Although expression of viral RNAs might not lead to functional viral protein production, persistent expression of viral icRNA might trigger chronic innate immune activation in MDMs, that we demonstrate is a potent stimulus of IFN-I-dependent pro-inflammatory responses, contributing to T-cell exhaustion and dysfunction. Although a vast majority of proviruses in HIV+ individuals on cART are defective containing large internal deletions and/or APOBEC3G-mediated hypermutations44, these defective HIV proviruses remain transcriptionally competent encoding non-functional proteins40. Future studies will need to investigate whether icRNA expression in myeloid cells is correlated with IR expression in T cells in tissues of HIV/SIV-infected individuals on cART. Unfortunately, current cART regimen does not block virus RNA transcription in previously infected cells. Thus, the development and clinical application of inhibitors that decrease viral RNA expression such as Tat and Rev inhibitors46, especially in tissue macrophages, could alleviate systemic immune activation in HIV-infected individuals on cART.MDM sensing of HIV-1 icRNA expression resulted in low levels of IFN-I production that had negligible effects on virus replication, though the establishment of a IFN-I-dependent chronic inflammatory state might have detrimental consequences to the host. Here, we show that HIV-1-infection-induced MDM activation led to the upregulation of IRs on co-cultured T cells and functional impairment of IRnef orf) have been described previously48. HIV-1 mutants, Lai∆env∆vpr and Lai/YU-2env∆vpu, have been previously reported49. To create Lai∆envGFP∆vif, a vif-containing fragment from Lai∆env-luc∆vif (frame-shift insertions at the NdeI site in the vif orf)50 was replaced into the corresponding region of Lai∆envGFP. Lai∆envGFP∆Gag-pol was created by inserting frame-shift mutations at the ClaI site (nt 377) in the Env-expression construct that has a large deletion between N-terminus of CA and IN47, and the fragment containing the env and nef orfs was replaced with the corresponding portion of Lai∆envGFP. The CA G89V R9-GFP mutant and its parental virus R9-GFP were generous gifts from Dr. Christopher R. Aiken (Vanderbilt University School of Medicine). The PTAP- mutant and its parental virus R9 were generously provided by Dr. Wesley I. Sundquist . The following Gag mutants were generous gifts from Dr. Jaisri Lingappa (University of Washington): VK181/182AA, K158A, P224A, RS100/102AA, EE75/76AA, ∆p6, ∆NCp6, G2A. mMA12∆envGFP was created by replacing the luciferase gene of MHIV mMA12  with the gfp gene. Rev-deficient mutant (M10) was created by PCR-directed mutagenesis using the primer set (Supplementary Table StuI-AleI fragment in the env orf was deleted from Lai∆envGFP. The CTE was PCR-amplified using the primers (Supplementary Table BclI (nt 2011) and NheI (nt 3467) sites in a sense or anti-sense direction. HIV-1 MA-deficient mutants, ∆MA (lacking amino-acid residues 6 to 125) and ATG*, and the following Gag mutants were created by PCR mutagenesis  or overlapping PCR using the primers listed in Supplementary Table NcoI site was PCR-generated into at the Gag start codon of Lai∆envGFP  or at nt 378 (41 nt downstream of Gag starting codon) of Lai∆envGFP  or ATG* mutant . The latter two vectors have a portion of HIV-1 genome in order to enhance chimeric viral RNA incorporation into virions22 with (HIV-vec-fusion) or without (HIV-vec-plus) protein expression from the 41nt HIV-1 sequence. Then, an AatII site was PCR-generated just before the HIV-1 PR cleavage site into all three vectors. MA orfs PCR-amplified from HIV-1 Lai, HIV-2 Rod9 , SIVmac239 V1EGFP , SIVagmSab92018ivTF , SIVCPZTAN2.69 , pSIVgorCP2139  and FIV vector  were inserted between the above-mentioned NcoI and AatII sites of HIV-1-vec. MA orfs PCR-amplified from HTLVII plasmid , MHIV mMA12, and EIAV vector 51 were inserted between the above-mentioned NcoI and AatII sites of HIV-1-vec-plus. MA orfs PCR-amplified from MHIV mMA12 was also cloned in between the above-mentioned NcoI and AatII sites of HIV-1-vec-fusion to create mMA-fusion. TagRFP (Evrogen) was PCR-amplified and inserted into HIV-1-vec-plus. To visualize viral mRNA, the 24xMS2 stem loop cassettes from pCR4-24XMS2SL-stable 52 were inserted between the BclI (nt 2011) and NheI (nt 3467) sites in the pol orf of HIV-1-vec, mMA  and mMA-fusion. A DNA fragment encoding MS2-GFP-NLS, a fusion protein of the coat protein of bacterial phage MS2 and eGFP-NLS, was PCR-amplified from pMS2-GFP 24 and replaced with the gfp gene (in place of nef) of HIV-1-vec, mMA, or mMA-fusion with the 24xMS stem loops or Lai∆envGFP having no 24xMS stem loops. All of the mutants were verified by sequencing. A lentivector expressing ZsGreen, pHAGE-ZsGreen, is a generous gift from Dr. Darrell Kotton (Boston University), and HIV-1 packaging plasmid psPAX2, a SIV packaging plasmid containing SIVmac239 Vpx SIV3+ and VSV-G expression constructs have been previously described53. Lentiviral vectors (pLKO.1) expressing shRNAs were purchased from Sigma or generated by ligating a synthesized double strand DNA into a pLKO.1 cloning vector  digested with AgeI and EcoRI.HIV-1 replication competent molecular clones, Lai and Lai/YU-2env, single-round reporter constructs, Lai∆env and Lai∆envGFP  containing 10% heat-inactivated human AB serum  and recombinant human M-CSF  or containing 10% heat-inactivated fetal bovine serum supplemented with human GM-CSF  and IL-4 , respectively, for 5–6 days. HEK293T (ATCC), 293 ISRE-luc (Drs. Junzhi Wang and Xuguang Li), HeLa (NIH AIDS Reagent Program) and TZM-bl (NIH AIDS Reagent Program) were maintained in DMEM (Invitrogen) containing 10% heat-inactivated FBS (Invitrogen)54. All cell lines have been tested for mycoplasma contamination and confirmed negative. Cell viability was measured by a conventional MTT assay.Human monocyte-derived macrophages (MDMs) and monocyte-derived dendritic cells (MDDCs) were derived from beads-isolated CD1453. Single-round-replication competent viruses pseudotyped with VSV-G were generated from HEK293T cells via co-transfection of HIV-1∆env constructs and VSV-G expression plasmid. To express HIV-1 mutants which by themselves cannot infect MDMs, a mutant plasmid was co-transfected with the packaging construct (psPAX2) and a VSV-G expression vector into HEK293T53. Lentivector expressing ZsGreen or shRNA was also generated by co-transfecting HEK293T cells with psPAX2 and a VSV-G expression vector using calcium phosphate or TransIT-293 (Mirus). Virus-containing cell supernatants were harvested 2 days post-transfection, cleared of cell debris by centrifugation , passed through 0.45 µm filters, and purified and concentrated by ultracentrifugation on a 20% sucrose cushion ). The virus pellets were resuspended in PBS, aliquoted, and stored at −80 °C until use. The capsid content of HIV-1 was determined by a p24gag ELISA53 and virus titer was measured on TZM-bl55. VSV expressing dsRed upon infection was kindly provided by Dr. John Connor (Boston University).Replication competent viruses were derived from HEK293T cells via calcium phosphate transient transfectionGag or GFP expression by flow cytometry. In some experiments, MDMs or MDDCs were pretreated prior (at least 30 min) to infection with maraviroc , AZT , efavirenz , raltegravir , or treated 2–3 h post infection with flavopiridol , indinavir , B18R , calpeptin , glybenclamide , (5Z)-7-Oxozeaenol , BAY11-7082 , herbimycin A , and KPT-330 .MDMs seeded 1 day before infection or MDDCs were spinoculated with viruses (1 h at room temperature (RT) and 2300 rpm) in the presence of polybrene (Milipore) at various multiplicity of infection , cultured for 2–3 h at 37 °C, washed to remove unbound virus particles, and cultured for 3–6 days. Infection in MDMs or MDDCs was quantified by analyzing intracellular p24Gag content per 1 × 106 cells) in the presence of SIVmac Vpx-containing VLPs pseudotyped with VSV-G (SIV3+/G) and polybrene (Millipore) by spinoculation as described above. After culturing for 2 days, MDMs were cultured for another 5–7 days in the presence of puromycin  for selection before being used for infection or other experiments. Knockdown efficiency was quantified by qRT-PCR or western blotting. All the shRNA target sequences are listed in Supplementary Table To knockdown expression of host proteins, MDMs were transduced with lentivectors expressing shRNA  or to validate knockdown of host proteins , cell lysates containing 20−30 µg total protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and the membranes were probed with the following antibodies: a mouse anti-p24 antibody  or a rabbit anti-p24 antibody , a rabbit anti-p17 polyclonal antibody , a rabbit anti-MAVS polyclonal antibody , a rabbit anti-STING polyclonal antibody , a mouse anti-RIG-I antibody  or a rabbit anti-MDA5 antibody . Then, the membranes were stained with secondary antibodies, a goat anti-mouse-IgG-DyLight 680  or a goat anti-rabbit-IgG-DyLight 800 . As loading controls, actin was probed using a rabbit anti-actin antibody  or a mouse anti-actin antibody . Membranes were scanned with an Odessy scanner (Li-Cor). All uncropped images are shown in Supplementary Fig. 6 cells using a kit  and reverse-transcribed using oligo(dT)20 primer . Target mRNA was quantified using Maxima SYBR Green (Thermo Scientific) and normalized to GAPDH mRNA by the 2C-∆∆T method as described56. As a positive control for IFN-β mRNA expression, MDMs were treated with 1 µg ml–1 LPS (InvivoGen) for 2 h. For virion-associated viral RNA quantification, viral RNA was isolated from 140 µl cell-free culture supernatants using Qiamp Viral RNA mini kit (QIAGEN) and reverse-transcribed using random hexamers . Viral cDNA was then quantified using Maxima SYBR Green (Thermo Scientific) using pLai∆envGFP as standard. All the primers used for quantitative PCR are listed in the Supplementary Table Total mRNA was isolated from 1 × 1049.IP-10 in MDM culture supernatants was measured with a BD Human IP-10 ELISA Set (BD). Multiplex cytokine assay using a human cytokine 29-plex kit (Millipore) was performed using culture supernatants and was analyzed with a DropArray (Curiox Biosystems) and the MAGPIX System (Luminex). Bioactive IFN-I was measured by a bioassay using 293 ISRE-luc cell line 49 using FITC- or RD1-conjugated mouse anti-p24 monoclonal antibody . For T-cell exhaustion assay, MDM–PBMC co-culture was harvested, stained with Zombie-NIR  and a panel of makers designed for inhibitory receptor expression profiling57, and analyzed with FACS ARIA (BD). For intracellular IFN-γ staining, MDM–PBMC co-culture was unstimulated or stimulated with SEB  for 1 h, followed by culture with brefeldin A (BioLegend) for another 5 h, stained for inhibitory receptors as described above, fixed with FluoroFix Buffer (BioLegend), and stored in a staining buffer (2% NCS in PBS). Fixed cells were permeabilized with intracellular staining permeabilization wash buffer (BioLegend), stained with anti-human IFN-γ PE , and analyzed with FACS ARIA (BD). Data was analyzed with FlowJo software (FlowJo).CD169 expression on MDMs was quantified by staining with Alexa647-conjugated mouse anti-human CD169 antibody  and analyzed with BD LSRII (BD). Geo mean fluorescence intensity (MFI) was calculated and normalized to that of uninfected (mock) MDMs. In some experiments, intracellular p24 was stained as describedHeLa cells seeded on coverslips were transfected with a plasmid containing 24xMS2 binding loops and MS2-GFP-NLS by lipofectamine 2000 (Invitrogen). Cells were washed and fixed 24 h post-transfection with 4% parafolmaldehyde, permeabilized with 0.1% TritonX100, and stained with mouse anti-p24 antibody  followed by staining with Alexa594-conjugated anti-mouse-IgG antibody  and DAPI (Sigma). Cells were mounted on a slide glass with Fluoromount-G (Southern Biotech) and imaged with a confocal microscopy (Leica SP5). For co-localization analysis, more than 50 cells were imaged, and correlation between p24 signals and GFP signals in cytosol (DAPI negative) were analyzed with ImageJ (NIH).p-values were calculated using one-way ANOVA followed by the Tukey-Kramer post-test  or the Dunnett’s post-test (comparing to control (mock or wild type), symbols for p-values shown on each column), a Kruskal–Wallis test followed by the Dunn’s post-test , or a paired t-test . Symbols represent, *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant (p ≥ 0.05).All the statistics analysis was performed using GraphPad Prism 5. Two-tailed n) and statistical information of each figure are as follows. Figure n = 6, Fig. n = 5; Fig. n = 5, Day 2 n = 5, Day 3 n = 7, LPS n = 4; Fig. n = 5; Fig. n = 5, t = 1.815 degrees of freedom (df) = 4. Figure n: + / -  Nef 5, + / -  Env 5, + / -  Vpu 4, + / -  Vpr 3, + / -  Vif 5, F = 0.8536; Fig. n: mock 17: + / -  Nef 5, + / -  Env 5, + / -  Vpu 4, + / -  Vpr 3, + / -  Vif 5; Fig. n = 7, F = 2.618, df = 20; Fig. n = 7; Fig. n = 6; Fig. n = 5, F = 1.411, df = 27, n: WT 14, ∆MA 7, ATG* 7; Fig. n: mock 14, WT 13, ∆MA 7, ATG* 7; Fig. n: mock 12, WT 12, ∆MA 7, ATG* 5; Fig. n = 7, t = 0.9510, df = 6; Fig. n = 7, Fig. n: mock 8, WT 7, ∆Gag-pol 8. Figure n = 4; Fig. n: WT/M10/M10 CTE/M10 CTE-AS: 6, ΔRRE-CTE: 5; Fig. n: mock/WT/M10/M10 CTE/M10 CTE-AS = 6, ΔRRE-CTE = 5; Fig. n = 6, t = 0.07817, df = 5; Fig. n = 6; Fig. n = 4, t = 0.3356, df = 3; Fig. n = 4; Fig. n = 3; Fig. n = 4, t = 0.6931, df = 3; Fig. n = 4; Fig. n = 3; Fig. n = 9; Fig. n: KPT = 7, mock/EFZ/Ral/DMSO = 9; Fig. n = 5, t = 4.933, df = 4; Fig. n = 5. Figure n = 6, t = 1.339, df = 5; Fig. n = 6; Fig. n = 5; Fig. n = 7, F = 0.2156, df = 20; Fig. n = 7; Fig. n: HIV-1 = 6, mock/mMA/mMA-fusion = 7; Fig. n = 6, F = 1.837, df = 17; Fig. n = 7; Fig. n = 3; Fig. n: HIV-1/mMA = 50, mMA-fusion = 53. Figure n: MAVS = 4, STING = 3; Fig. n = 7, t = 0.1929, df = 6; Fig. n = 7, t = 0.6252, df = 6; Fig. n = 7; Fig. n: RIG-I = 4, MDA5 = 3; Fig. n = 6, t = 2.516, df = 5; Fig. n = 8, t = 0.05451, df = 7; Fig. n = 6; Fig. n = 8; Fig. n: DMSO1 = 13, (5Z)-7-Oxozeaenol/BAY11-7082/ DMSO2 = 9, Herbimycin A = 7, Calpeptin = 6, Glybenclamide = 5, F = 1.274, df = 57; Fig. n: DMSO1 = 13, (5Z)-7-Oxozeaenol/BAY11-7082/ DMSO2 = 9, Herbimycin A = 7, Calpeptin = 6, Glybenclamide = 4; Fig. n: DMSO1 = 12, (5Z)-7-Oxozeaenol/BAY11-7082/ DMSO2 = 9, Herbimycin A = 7, Calpeptin = 5, Glybenclamide = 4. Figure n = 6; Fig. n = 5. Supplementary Fig. n = 6; 1b, n = 3; 1c, n = 6; 1d–g, n = 5; 1h, i, n = 6; 1j, n = 3; 1k, n = 6; 1 l, m, n = 4; 1n, o, n = 6. Supplementary Figure n: mock 17: + / -  Nef 5, + / -  Env 5, + / -  Vpu 4, + / -  Vpr 3, + / -  Vif 5; Fig. n: mock 13; 2b, n = 3; 2c, n = 5, t = 0.3200, df = 4; 2d, n = 5; 2e, n = 4; 2f, g, n = 6; 2 h, n = 5; 2i, n = 5, t = 0.3056, df = 4; 2j, n = 5; 2k, n = 4. Supplementary Fig. n = 4; 3c, d, n = 6; 3e, n = 4; 3f, g, n = 4; 3 h, n = 9; 3i, n = 5. Supplementary Fig. n: WT = 7, 29/31KE/85YG/16EK/12LE/30LE/98EV = 5, 7A2T/21LS/34VE/69TR = 4, F = 0.5085, df = 50; 4b, n: WT = 7, 29/31KE/85YG/16EK/12LE/30LE/98EV = 5, 7A2T/21LS/34VE/69TR = 4, mock = 8; 4c, n: WT = 9, 29/31KE/85YG/16EK/12LE/30LE/98EV = 5, 7A2T/21LS/34VE/69TR = 4, mock = 9; 4f, n = 6, t = 0.3934, df = 5; 4g, h, n = 6; 4i, n: HIV-1 = 8, HIV-2 = 7, SIVmac/SIVagm/SIVcpz/SIVGor = 5, F = 0.2291, df = 34; 4j, k, n: HIV-1/mock = 8, HIV-2 = 7, SIVmac/SIVagm/SIVcpz/SIVGor = 5; 4 l, n: HIV-1 = 17, FIV = 6, HTLVII = 8, EIAV = 9; 4 m, n: HIV-1 = 15, FIV = 6, HTLVII = 8, EIAV = 9, mock = 20; 4n, n: HIV-1 = 9, FIV = 6, HTLVII = 8, EIAV = 9, mock = 13. Supplementary Fig. n: Scrambled = 18, MAVS/STING = 7, RIG-I = 5, MDA5 = 8; 5b, c, n = 7; 5d, n = 6; 5e, n = 8; 5f, n = 3; 5g, n: mock/DMSO = 13, (5Z)-7-Oxozeaenol/BAY11-7082 = 9, Herbimycin A = 7, Calpeptin = 6, Glybenclamide = 4; 5 h, n = 5; 5i, n: DMSO2/(5Z)-7-Oxozeaenol/BAY11-7082 = 6, Herbimycin A/DMSO1 = 5, F = 0.3910, df = 27. Supplementary Fig. n = 3.The numbers of experimental replicates (Supplementary Information


Scientific Reports 10.1038/s41598-018-22504-5, published online 09 March 2018Correction to: 18O (blue) curves. The correct Figure 4 appears below as Figure In Figure 4 the Ostracod curve does not align with δD (orange) and δ


The structures of new compounds were confirmed by the analysis of NMR and HRESIMS spectroscopic data. The biosynthetic origin of compounds 1–5 was postulated. Compounds 1–5 exhibited no antibacterial activity against Staphylococcus aureus and Escherichia coli at the dose of 100 μM.Three new heptelidic acid derivatives (The online version of this article (10.1007/s13659-018-0168-8) contains supplementary material, which is available to authorized users. Lentinellus are white rot, wood decay, and characterized with rough-walled and amyloid spores. Eighteen species and varieties of Lentinellus have been described all over the world. There have been eleven species reported in China +). The 1H, 13C NMR, and HSQC data (Table δC/δH 20.9/2.00), two doublet methyls , four methylenes containing two oxygenated methylenes , four methines, a trisubstituted double bond , 130.8), an oxygenated quaternary carbon (δC 76.3), and three carbonyl carbons . These data indicated that 1 shares the same sesquiterpenoid skeleton with hydroheptelidic acid (4) + 601.2615). The 1H, 13C NMR, and HSQC data of 2 revealed the presence of four doublet methyls , eight methylenes containing four oxygenated methylenes , eight methines, two pairs of trisubstituted olefinic carbons , two oxygenated quaternary carbon , and four carbonyl carbons   + 583.2517. The 1H and 13C NMR data of 3 revealed a similar dimeric structural feature as that of 2, except for the presence of two extra olefinic carbons  in 3, and the absence of a methine and an oxygenated quaternary carbon in 2. Two olefinic carbons were assigned at C-5′ and C-6′ by key HMBC corrections of H-12′ with C-5′, H2-8′ with C-5′ and C-6′, H2-7′ with C-5′ and C-6′. Further analysis of 2D NMR data , two dimeric esters (2–3), and two known heptelidic acid analogues (4–5), were isolated from the solid culture of L. ursinus. The current study enriches the secondary metabolites from this mushroom.In conclusion, three previously undescribed cadinane-type sesquiterpenes including one acetylated heptelidic acid derivative  as references. Mass spectra were obtained on an Agilent Accurate-Mass-Q-TOF LC/MS 6520 spectrometer. Optical rotations were recorded on a polarimeter with sodium light (589 nm) by using a Perkin-Elmer 241 polarimeter. UV and IR spectra were recorded on a Thermo Genesys-10S UV–Vis spectrophotometer and a Nicolet IS5FT-IR spectrophotometer, respectively.HPLC separation was conducted on Agilent 1200 HPLC system equipped with Agilent G1315D DAD detector, using a YMC-Pack ODS-A column . NMR spectra were measured on a Bruker Avance-500 spectrometer using solvent signals  by Junjie Han, and identified on the basis of the morphological characteristics and ITS sequences. The strain was cultured on PDA plates at 25 °C for 14 days. Agar plugs were inoculated into a 250 mL Erlenmeyer flask containing 100 mL PDB medium cultured at 25 °C on a rotary shaker at 180 rpm for 14 days. Large-scale cultivation was carried out in 20 × 500 mL Fernbach culture flasks each containing 80 g of rice and 100 mL of distilled water. Each flask was inoculated with 5 mL of culture medium and incubated at 25 °C for 40 days in dark.The strain of 2Cl2–MeOH  to give fifteen fractions (Fr.1–Fr.15).The cultivated rice substrate was extracted repeatedly with EtOAc (3 × 10 L) at room temperature and 20.2 g crude extract was obtained by evaporating solvent under vacuum. The crude extract was subjected to silica gel column chromatography (CC) using CH2Cl2–MeOH was separated by ODS CC eluting with MeOH–H2O gradient elution to give nineteen subfractions (Fr.7.1–Fr.7.19). The fraction Fr.7.4 (1.0 g) was purified by Sephadex LH20 CC eluted with 80% MeOH–H2O, followed by PR-HPLC (20% MeCN in H2O) to give 4 . The fraction Fr.7.6 (200.0 mg) was purified by PR-HPLC (28% MeCN in H2O) to afford 1  and 5 . Compound 2  and 3  were purified by PR-HPLC from Fr.7.11 (45% MeCN in H2O) and Fr.7.13 (50% MeCN in H2O), respectively.The fraction Fr.7 (4.0 g) eluted with 40:1 CHα]D25 62.9 (c 0.1 MeOH); UV (MeOH) λmax (log ε) 222 (3.3) nm; IR(neat) vmax 3432, 2959, 1778, 1719, 1465, 1310, 1233, 1170, 1019 cm−1; positive HRESIMS m/z [M+Na]+ 363.1412 .Colorless oil; + 601.2615 .Light yellow powder; c 0.2 MeOH); UV (MeOH) λmax (log ε) 222 (3.4) nm; IR(neat) vmax 3432, 2959, 1778, 1719, 1465, 1370, 1233, 1170, 1019 cm−1; positive HRESIMS m/z [M+Na]+ 583.2517 .Light yellow powder; S. aureus (ATCC 6538) and E. coli (ATCC 25922) were grown in Lysogeny Broth (LB) medium. The inhibition rate was calculated and plotted versus test concentrations to afford the MIC. MIC values were defined as the minimum concentration of compound that inhibited visible microbial growth.The antimicrobial assay was conducted as our previous described method . The bacElectronic supplementary material 1 (DOCX 1752 kb)Below is the link to the electronic supplementary material.


The structures of two new chalcone derivatives have been determined and are investigated using Hirshfeld surface analysis and molecular electrostatic potential techniques. E)-1-(anthracen-9-yl)-3-[4-(piperidin-1-yl)phenyl]prop-2-en-1-one, C28H25NO, (I), and (E)-1-(anthracen-9-yl)-3-[4-(diphenylamino)phenyl]prop-2-en-1-one, C35H25NO, (II), with the fused-ring system at the same position are described. In the crystals of (I) and (II), the molecules are linked via C—H⋯O hydrogen bonds into inversion dimers, forming R22(22) and R22(14) ring motifs, respectively. Weak intermolecular C—H⋯π interactions further help to stabilize the crystal structure, forming a two-dimensional architecture. The molecular structures are optimized using density functional theory (DFT) at B3LYP/6–311 G++ level and compared with the experimental results. The smallest HOMO–LUMO energy gaps of (I) (exp . 2.76 eV and DFT 3.40 eV) and (II) (exp . 2.70 eV and DFT 3.28 eV) indicates the suitability of these crystals in optoelectronic applications. All intermolecular contacts and weaker contributions involved in the supramolecular stabilization are investigated using Hirshfeld surface analysis. The molecular electrostatic potential (MEP) further identifies the positive, negative and neutral electrostatic potential regions of the molecules.The molecular and crystal structure of two new chalcone derivatives, ( The piperidine ring (N1/C24–C28) in (I)Q = 0.521 (4), Θ = 3.1 (3)° and φ = 221 (6)°. The enone moiety (O1/C15–C17) in compounds (I)s-trans configuration with respect to the C15=O1 and C16=C17 bonds. Both compounds (I)Compounds (I)et al., 2008et al., 2018abThe enone moiety for (I)via intermolecular C28—H28B⋯O1i interactions s Table 1, formingon Fig. 2. The crys Table 1. TogetheA⋯ O1i i Table 1 hydrogens Table 2 are alsoThe strongest absorption and smaller energy gap, particularly in the visible region, is important feature in the suitability for optoelectronic application. The electronic absorption and excitation properties of (I)et al., 2018The experimental energy band gaps for (I)normd, shape-index and ed b).Hirshfeld surface analysis assigns intermolecular interactions inside the unit-cell packing. The b as one distinct spike with a minimum value ed + id that is approximately less than the sum of van der Waals radii (2.4 Å). Furthermore, the intermolecular C—H⋯π interactions for compounds (I)ed + id ∼2.8 Å (I)ed + id ∼2.3 Å .Two-dimensional fingerprint plots as shown in Fig. 7 Å Fig. 7c.The molecular electrostatic potential (MEP) has become firmly established as an effective guide to molecular interactions. The importance of MEPs lies in the fact that it simultaneously displays molecular size and shape, as well as positive, negative and neutral electrostatic potential regions, in terms of colour grading and is useful in suties of the molecular structure and its physicochemical property relationship et al., 2016et al., 19752E)-1-(anthracen-9-yl)-3-[4-(propan-2-yl)phenyl]prop-2-en-1-one -1-(anthracen-9-yl)-3-(2-chloro-6-fluorophenyl)prop-2-en- 1-one -1,3-bis(anthracen-9-yl)prop-2-en-1-one. Other related compounds include 1-(anthracen-9-yl)-2-methylprop-2-en-1-one  and 4-(piperidin-1-yl)benzaldehyde (0.5 mmol) and 4-(diphenylamino)benzaldehyde (0.5 mmol) for compound (I)Uiso(H) = 1.2Ueq(C).Crystal data collection and structure refinement details are summarized in Table 310.1107/S2056989018006527/lh5873sup1.cifCrystal structure: contains datablock(s) I, II. DOI: 10.1107/S2056989018006527/lh5873Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989018006527/lh5873IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989018006527/lh5873sup4.pdfComparison between selected calculated (DFT) and experimental geometrical data. DOI: 1824550, 1817218CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


An N—H⋯O hydrogen bond connects the organic and water molecules. In the crystal, O—H⋯N hydrogen bonds link molecules into a two-dimensional network parallel to (100). Hirshfeld surface analyses and two-dimensional fingerprint plots were used to quantify the intermolecular interactions present in the crystal, indicating that the most important contributions for the crystal packing are from H⋯H (28.7%), C⋯H/H⋯C (27.1%), N⋯H/H⋯N (26.4%), C⋯N/N⋯C (6.1%), O⋯H/H⋯O (3.7%) and C⋯C (6.0%) interactions.In the title compound, C The O⋯H/H⋯O contacts, with a 3.7% contribution, appear with the points of low densities. Lastly, the C⋯N/N⋯C, C⋯C/C⋯C and O⋯C/C⋯O interactions in the structure with 6.1, 5.5 and 1.4% contributions, respectively, have symmetrical distributions of points.The overall two-dimensional fingerprint plot and those showing different contacts are characterized in Fig. 5et al., 2016H-benzo[d]imidazole-2yl)phenoxy]phthalonitrile phenol phenol phenoxy]phthalonitrile, 4-nitrophthalonitrile  and 2-(4-hydroxyphenyl)benzimidazole  were dissolved in DMF (15 mL) under argon. After stirring for 15 min, anhydrous K2CO3  was added portionwise over 2 h with efficient stirring. The suspension was maintained at 333 K for 24 h. The progress of the reaction was monitored by TLC using a CHCl3/EtOAc (10/1) solvent system. After the reaction was observed to be complete, the resulting mixture was poured into an ice–water mixture. The immediate precipitate was collected by filtration, washed with hot water, ethanol and diethyl ether and dried in vacuo. The desired pure compound was obtained in sufficient purity, yield: 96% (1.84 g). m.p. 421 K.The synthesis of the title compound Fig. 7 was desc al. 2018. 4-[4-(1Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018008745/qm2124sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018008745/qm2124Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018008745/qm2124Isup3.cmlSupporting information file. DOI: 1849358CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Activin A and follistatin exhibit immunomodulatory functions, thus affecting autoinflammatory processes as found in rheumatoid arthritis (RA). The impact of both proteins on the behavior of synovial fibroblasts (SF) in RA as well as in osteoarthritis (OA) is unknown.Immunohistochemical analyses of synovial tissue for expression of activin A and follistatin were performed. The influence of RASF overexpressing activin A on cartilage invasion in a SCID mouse model was examined. RASF and OASF were stimulated with either IL-1β or TNFα in combination with or solely with activin A, activin AB, or follistatin. Protein secretion was measured by ELISA and mRNA expression by RT-PCR. Smad signaling was confirmed by western blot.In human RA synovial tissue, the number of activin A-positive cells as well as its extracellular presence was higher than in the OA synovium. Single cells within the tissue expressed follistatin in RA and OA synovial tissue. In the SCID mouse model, activin A overexpression reduced RASF invasion. In human RASF, activin A was induced by IL-1β and TNFα. Activin A slightly increased IL-6 release by unstimulated RASF, but decreased protein and mRNA levels of follistatin.The observed decrease of cartilage invasion by RASF overexpressing activin A in the SCID mouse model appears to be mediated by an interaction between activin/follistatin and other local cells indirectly affecting RASF because activin A displayed certain pro-inflammatory effects on RASF. Activin A even inhibits production and release of follistatin in RASF and therefore prevents itself from being blocked by its inhibitory binding protein follistatin in the local inflammatory joint environment.The online version of this article (10.1186/s13075-019-1926-7) contains supplementary material, which is available to authorized users. A-subunits (βAβA) of inhibin A, which itself is formed by an α- and βA-subunit. The heterodimer consisting of an α- and βB-subunit is called inhibin B. Accordingly, two other forms of activin can be distinguished: activin B (βBβB) and activin AB (βAβB) [Activin A is a disulphide-linked homodimer composed of two βB (βAβB) , 2. BothB (βAβB) , but actB (βAβB) . In marrB (βAβB) , 6. In vB (βAβB) , 8. The B (βAβB) , 9. The B (βAβB) , but quiB (βAβB) , 12.The role of activin A and follistatin in chronic autoinflammatory disorders is not fully understood. There is evidence that activin A promotes allergic diseases  and inflDue to the clues pointing towards a role of activin A in RA, the aim of the study was to investigate the role of activin A and follistatin in the inflammatory and matrix degrading response of RASF and the known feedback loop between activin A and follistatin described for other cell types.RA/OA synovium and OA cartilage were obtained during knee replacement surgeries . RA patients fulfilled classification criteria of the American College of Rheumatology , 19. Hum2 [Synovium samples were snap-frozen, used for paraffin-embedding, or digested   for fibr2 .2O2 in 100% methanol. After blocking with 10% dry milk, the slides were incubated overnight with primary antibodies in 2.5% BSA at 4 °C: goat anti-human/mouse/rat polyclonal activin A , mouse anti-human monoclonal follistatin , goat anti-human polyclonal ACVR2A (A8081) and ACVR1B (A2455) , mouse anti-human monoclonal β2-microglobulin , and mouse anti-human monoclonal vimentin . Slides were incubated 30 min with secondary antibodies , and color development was performed with AEC substrate . For snap-frozen tissues, 5 μm acetone-fixed sections were used with the same procedure.Formalin-fixed 5 μm paraffin sections were deparaffinized, and antigen retrieval was performed with 4 M hydrochloric acid  or proteinase K (vimentin). The tissue was permeabilized with Triton X-100 and endogenous peroxidases blocked with 0.3% HRecombination vector pAdLox (digested with EcoR1) was used to generate adenoviral vectors with either the full-length activin A  or the GFP sequence inserted as a control. The adenoviral vectors were provided as a courtesy of the University of Pittsburgh. Based on previous experiments, a multiplicity of infection (MOI) of 100 was used for RASF or OASF transduction . The abs5 SF together with healthy areas of human OA cartilage in a carrier matrix  with up to four cartilage implants per animal [Female, 6-week-old Crl-scidBR mice  were kept under pathogen-free conditions with water and food ad libitum. Animal experiments were performed in accordance with the German Animal Welfare Act and approved by the local government authorities, RP Oberfranken, Germany, 621-2531.1-13/03. Animals underwent surgery with implantation of 1.5 × 10r animal . SCID mir animal , 22, 23.RASF or OASF were cultured for 48 h. The medium was replaced and cells stimulated with IL-1β or TNFα  with or without activin A/AB  or follistatin  for 15 h. Supernatants were centrifuged and stored at − 20 °C. As control, stimulation was performed under serum-free conditions.Cytokines, matrix-degrading proteinases (MMP), and growth factors were measured by enzyme-linked immunosorbent assay  or Luminex analysis for IL-6, IL-1β, TNFα, IL-10, VEGF, IL-12p40, GM-CSF, IFNγ, IL-8, IL-4, IL-2, and IL-5 using the Bead-based multiplex kit (R&D).RASF were harvested and total RNA extracted . Remaining DNA was removed using the RNase-free DNase Set (Qiagen). RNA concentrations were quantified  and RNA stored at − 80 °C.2, 0.5 mM spermidine, 1 mM dithiothreitol, 1 mM each dNTP , A260 unit random primer (Roche), 1.6 U/μl RNase inhibitor (Roche), and 1.3 U/μl AMV reverse transcriptase . Conditions were 25 °C 10 min, 42 °C 60 min, and 99 °C 5 min. cDNA was stored at − 20 °C.cDNA was synthesized using 150 ng RNA, 5 mM Tris-HCl , 50 mM KCl, 1 mM MgClE = 10–1/slope, E = 2.00 ± 0.05 was considered acceptable). PCR mixture includes 2 μl cDNA or water, 0.5 μM each primer, 10 μl 2xQuantiTect® SYBR® Green PCR Master Mix (Qiagen), and MgCl2 according to primer efficiency. PCR products were subjected to a melting curve analysis. Data were analyzed using the LightCycler analysis software (Roche). Primers include follistatin for: 5′-GTCGGGATGTTTTCTGTCCAG-3′ and rev: 5′-TGGCATAAGTGGCATTGTCAC-3′ .Real-time PCR was performed  using SYBR Green detection including melting curve analysis. 18S rRNA served as an endogenous control. Primer efficiencies were tested by the standard curve method (ACVR1) and activin A receptor type 2A (ACVR2A), standard PCR was performed using the Titan One-Tube RT-PCR system  followed by agarose gel electrophoresis (1%). Primers include ACVR1 for: 5′-AGCATCAACGATGGCTTCCA-3′, rev 5′-AGTGCTGTCTCCAACATTGG-3′; ACVR2A for: 5′-GGTGTACAGGCATCACAAGA-3′, rev: 5′-CCAAGAGACCACATTAGCCT-3′; 18S for: 5′-TCAAGAACGAAAGTCGGAG-3′, rev: 5′-GGACATCTAAGGGCATCACA-3′).For evaluation of activin A receptor type 1 (n = 3) were pre-incubated with serum-free medium for 2 h before stimulation with activin A for 10 min and lysed after stimulation ). Western blotting was performed with antibodies against total Smad2  and phosphorylated Smad2 . For detection, secondary goat anti-rabbit HRP-conjugated antibodies (Dako) and the ECL system  were used. Antibodies against activin A  and ACVR2A  were detected using secondary anti-mouse HRP-conjugated antibodies . Cyclophilin B served as the loading control.RASF . For comparisons with a single control group, one-way ANOVA followed by Dunnett’s post hoc test was performed. Multiple comparisons among several groups were performed by one-way ANOVA followed by Bonferroni’s post hoc test. For comparison of two groups with different treatments and increasing stimulation duration, two-way ANOVA followed by Bonferroni’s post hoc test was performed. The assessment of significance level for pair-wise comparisons was calculated by a Student two-tailed n = 4)  was comparable on cultured RA- and OASF by PCR  Fig. a, b. Cel 4) Fig. c. Activip < 0.05)  Fig. a. Due to05) Fig. c.Fig. 2En = 5) stimulated with activin A at 15 ng/ml showed suppression of follistatin protein secretion over time . Follistatin production in RASF was not altered by stimulation with 10 ng/ml TNFα, whereas 10 ng/ml IL-1β decreased the follistatin release from 2075 ± 474 to 1121 ± 380 pg/ml  after 15 h stimulation. (PPT 134 kb)Additional file 2:n = 3, experimental replicates). (PPT 220 kb)Synovial fibroblast stimulation with activin A; increasing suppression of follistatin release through RASF by activin A concentrations of 0.5 ng/ml and higher (


A and B) in the asymmetric unit. In the crystal, the A and B molecules stack head-to-tail in columns along the b-axis direction.This compound crystallizes with two independent molecules  in the asymmetric unit. The dihedral angles between mean planes of the methylphenyl ring and the naphthalene ring system are 78.32 (6) and 84.70 (6)° in molecules A and B, respectively. In the crystal, the antiferroelectric packing of molecules A and B is of an ABBAABB type along the b-axis direction. The crystal structure features N—H⋯O, O—H⋯O and weak C—H⋯O hydrogen bonds, which link the molecules into infinite chains propagating along the b-axis direction.The title compound, C The bond lengths in the methylphenyl rings and naphthalene ring systems of the two molecules are practically equal, while there are slight differences in bond angles, with for example N1—C7—C1 and N21—C27—C21 differing by 1.2° and the exocyclic angles C7—C11—C12 and C27—C211—C212 differing by 1.8°. The naphthalene ring systems are essentially planar with maximum deviations from the mean plane of 0.059 (1) Å (for C11) and −0.020 (1) and 0.020 (2) Å (for C211 and C213) in molecules A and B, respectively. The mean plane of the naphthalene ring system subtends a dihedral of angle of 78.32 (6)° with the methylphenyl ring in molecule A and 84.70 (6)° in B while the dihedral angles between the naphthalene ring system and the acetamide group is 55.98 (9)° in molecule A and 65.30 (9)° in B. This differences also exist between the mean plane of acetamide and phenyl rings which are about 80.63 (10)° for molecule A and 84.51 (10)° for molecule B. The methyl groups at C8 and C28 have a C–H bond eclipsed in the mean plane of the phenyl ring and they are oriented towards the acetamide group, as been observed in N-[(4-methylphenyl)methyl]acetamide  associated with hydrogen atoms is characterized by an extremity pointed to the origin along the a diagonal, which corresponds to di + de = 2.2 Å and represents 59.7% of all the intermolecular contacts. Fig. 5c illustrates C⋯H/H⋯C contacts between carbon and hydrogen atoms from inside and outside the Hirshfeld surface and vice versa, resulting from H⋯C short contacts. It accounts for 26.0% of the surface and is characterized by two symmetrical points with di + de = 2.6 Å. The plot of O⋯H/H⋯O contacts between hydrogen atoms located inside the Hirshfeld surface and oxygen from outside and vice versa is shown in Fig. 5d. These contacts account for 13.0% and are characterized by two symmetrical peaks with di + de = 1.8 Å; this reveals the presence of strong O⋯H contacts that are characteristic of C—H⋯O and O—H⋯O hydrogen bonds.The Hirshfeld surface analysis (m-tolyl)methyl]acetamide yielded four hits: methyl N-[(phenyl)methyl]carbamate (phenyl)methyl]acetamide (4-methylphenyl)methyl]acetamide (3-nitrophenyl)methyl]acetamide  , β-naphthol (2 mmol), acetamide (2.4 mmol) in the presence of a catalytic amount of phenylboronic acid (1.5 mmol) was heated at 393 K without solvent for 7 h (the reaction was monitored by TLC). After completion of the reaction, the solid mixture was allowed to warm to room temperature, then 5 ml of 96% ethanol was added while maintaining stirring for 10 min. The solid was filtered, washed with cold 96% EtOH, dried and recrystallized from ethanol.A mixture of −1) 3405, 2921, 2358, 1627, 1508, 1265, 1065, 748, 686, 623. 1H NMR : δ (ppm) 9.98 , 8.28 , 7.97 , 7.74 , 7.68 , 7.40–6.92 , 2.22 , 2.02 . 13C NMR : δ (ppm) 169.4, 153.1, 142.2, 137.0, 132.4, 129.0, 128.4, 127.7, 126.8, 126.6, 126.4, 123.2, 122.7, 122.4, 118.7, 118.6, 48.3, 22.9, 21.2.IR (KBr): ν (cmUiso(H) = 1.5Ueq(O). All other H atoms were positioned geometrically and refined as riding: N—H = 0.86, C—H = 0.93–0.96 Å with Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018008423/xu5928sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989018008423/xu5928Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018008423/xu5928Isup3.cmlSupporting information file. DOI: 1848011CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, the encapsulated 1,2-dichloroethane solvent is stabilized by C—H⋯π interactions and mediates the formation of an interlocked supramolecular polymer  99H158O10·C2H4Cl2, were grown from a 1,2-dicholoroethane/n-hexane solvent system. In the crystal, the encapsulated 1,2-dichloroethane solvent is stabilized by C—H⋯π interactions and mediates the formation of an interlocked supramolecular polymer via C—H⋯Cl interactions.Crystals of 1--2,3,4,5--pillar[5]arene, C The asymmetric unit contains half of the molecule and the whole structure is generated by twofold rotation symmetry  has been reported earlier . Within three days, crystals of the title compound of a suitable size for diffraction analysis had formed.The synthesis of 1--2,3,4,5 -pillar[5]arene (Uiso(H) = 1.2 or 1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018013415/dx2009sup1.cifCrystal structure: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018013415/dx2009Isup4.molSupporting information file. DOI: 10.1107/S2056989018013415/dx2009Isup5.hklStructure factors: contains datablock(s) I. DOI: 1868738CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H)-one, adjacent molecules are linked by a pair of N—H⋯O hydrogen bonds, forming inversion dimers with an In each asymmetric unit of the title compounds, one independent molecule is present. In the crystal structure of 4-benzyl-6-phenyl-4,5-dihydropyridazin-3(2 H)-one, C17H14N2O, (I), the phenyl and pyridazine rings are twisted with respect to each other, making a dihedral angle of 46.69 (9)°; the phenyl ring of the benzyl group is nearly perpendicular to the plane of the pyridazine ring, the dihedral angle being 78.31 (10)°. In methyl 2-acetate, C20H16Cl2N2O3, (II), the phenyl and pyridazine rings are twisted with respect to each other, making a dihedral angle of 21.76 (18)°, whereas the phenyl ring of the dichlorobenzyl group is inclined to the pyridazine ring by 79.61 (19)°. In the crystal structure of (I), pairs of N—H⋯O hydrogen bonds link the molecules into inversion dimers with an R22(8) ring motif. In the crystal structure of (II), C—H⋯O hydrogen bonds generate dimers with R12(7), R22(16) and R22(18) ring motifs. The Hirshfeld surface analyses of compound (I) suggests that the most significant contributions to the crystal packing are by H⋯H (48.2%), C⋯H/H⋯C (29.9%) and O⋯H/H⋯O (8.9%) contacts. For compound (II), H⋯H (34.4%), C⋯H/H⋯C (21.3%) and O⋯H/H⋯O (16.5%) interactions are the most important contributions.The asymmetric units of the title compounds both contain one nonplanar molecule. In 4-benzyl-6-phenyl-4,5-dihydropyridazin-3(2 The differences between (I) and (II) pertain to the addition of two chloro groups at the C2 and C6 ring positions of the benzyl group and N-alkylation of pyridazinone at the 2-position with an ethyl acetate group for (II). In (I), the phenyl ring (atoms C12–C17) and the pyridazine ring (N1/N2/C11/C10/C2/C1) are twisted with respect to each other, making a dihedral angle of 46.69 (9)°; the phenyl ring of the benzyl group (C4–C9) is nearly perpendicular to the pyridazine ring, with a dihedral angle of 78.31 (10)° , the phenyl ring (C11–C16) and the pyridazine ring (N1/N2/C17/C8/C9/C10) are twisted with respect to each other, making a dihedral angle of 21.76 (18)°; the phenyl ring (C1–C6) of the benzyl group is inclined to the pyridazine ring by 79.61 (19)°. The methoxy group in (II) is disordered over two sets of sites with an occupancy ratio of 0.626 (11):0.374 (11) , the carbonyl group has a C1=O1 bond length of 1.243 (2) Å, and the C1—N1 and C11—N2 bond lengths in the pyridazine ring are 1.363 (2) and 1.304 (2) Å, respectively. The corresponding values in (II) are 1.229 (5) Å for C17=O1, 1.388 (5) Å for C17—N2 and 1.299 (4) Å for C10—N1. The N1—N2 bond lengths in the structures are virtually the same, with values of 1.348 (2) Å in (I) and 1.353 (4) Å in (II).The molecular structures of compounds ° Fig. 1. In (II)1) Fig. 2. In (I),I), molecules are linked by C—H⋯O hydrogen bonds (Table 1a), and by a pair of N—H⋯O hydrogen bonds forming inversion dimers with an b). The dimers are linked by weak π–π interactions, with a centroid-to-centroid distance of 3.957 (2) Å for Cg1 and Cg2, where Cg1 is the centroid of the N1/N2/C11/C10/C2/C1 ring and Cg2 that of the C12–C17 phenyl ring. In this way, a three-dimensional network is formed.In the crystal of , molecules are connected via C—H⋯O hydrogen bonds between aryl groups and the carbonyl O1 atom -one skeleton yielded three hits, namely 4-(2-chloro-6-fluorophenyl)-2,5-dioxo-8-phenyl-1,2,3,4,5,6-hexahydropyridopyridazine -one  [1.363 (2) Å], with a value of 1.363 (2) Å for BARQOA, 1.364 (7) Å for BARQUG and 1.350 (2) Å for YOTVIN. The pyridazinone ring in each molecule is essentially planar, as in the structures of (I) and (II). The conformations of all three compounds resemble those of compounds (I) and (II), with the dihedral angles between the planes of the pyridazine and phenyl rings varying in the range 27.35–82.0°, compared to 46.69 (9) and 21.76 (18)° in (I) and (II), respectively.A search of the Cambridge Structural Database  to 1.3165 a.u. (blue) for (I) and of −0.2803 (red) to 1.5329 a.u. (blue) for (II). The red spots on the surface indicate the contacts involved in hydrogen bonding. Fig. 5a) illustrates the intermolecular N—H⋯O hydrogen bonding in (I), with dnorm mapped on the Hirshfeld surface. Likewise, C—H⋯O hydrogen bonding is visualized in Fig. 5b) for compound (II).Hirshfield surface analyses , represented in normal mode. H⋯H contacts clearly make the most significant contribution to the Hirshfeld surface (48.2%). A significant contribution of H⋯H interactions to the total HS (72.2%) was also reported by Ilmi et al. . The H⋯H interactions appear in the middle of the scattered points in the two-dimensional fingerprint plots, with a contribution to the overall Hirshfeld surface of 34.4% . The contributions (16.5%) from the O⋯H/H⋯O contacts, corresponding to the C—H⋯O interactions, are represented by a pair of sharp spikes characteristic of such hydrogen bonding .Similarly, Fig. 74% Fig. 7b. The cng Fig. 7d.I), sodium hydroxide  was added to a solution  of 6-phenyl-4,5-dihydropyridazin-3(2H)-one and benzaldehyde  in 30 ml of ethanol. The solvent was evaporated under vacuum and the residue was purified by silica-gel column chromatography using hexane/ethyl acetate (7:3 v/v). Colourless single crystals were obtained by slow evaporation at room temperature.For the preparation of compound (II), potassium carbonate  was added to a solution  of 4--6-phenylpyridazin-3(2H)-one in 30 ml of tetrahydrofuran (THF). The mixture was refluxed for 1 h. After cooling, ethyl bromoacetate  was added and the mixture was refluxed for 8 h. The solid material which formed was removed by filtration and the solvent evaporated in vacuo. The residue was purified by silica-gel column chromatography using hexane/ethyl acetate (4:6 v/v). Slow evaporation at room temperature led to colourless single crystals.For the preparation of compound (I), the N-bound H atom was located in a difference Fourier map and refined with N—H = 0.86 Å. For the refinement of structure (II), reflections with a θ angle greater than 28° were omitted from the refinement due to their very weak intensities. The methoxy group (O3—C20) in this compound was found to be disordered over two sets of sites and was refined with an occupancy ratio of 0.626 (11):0.374 (11)  = 1.5Ueq(C) for methyl H atoms or 1.2Ueq(C) otherwise.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019013707/wm5515sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989019013707/wm5515Isup4.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989019013707/wm5515IIsup5.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989019013707/wm5515Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019013707/wm5515IIsup5.cmlSupporting information file. DOI: 1958029, 1958030, 1958029, 1958030CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


It exhibits a pseudo-κ arrangement, never observed up to now in the BEDT-TTF family with thiocyanatocuprate(II) anions.The new compound (BEDT-TTF) 4]2– as the counter-anion, namely bis[bis(ethylenedithio)tetrathiafulvalenium] tetrathiocyanatocuprate(II) bis[bis(ethylenedithio)tetrathiafulvalene], (C10H8S8)2[Cu(NCS)4]·2C10H8S8 or (BEDT-TTF)4[Cu(NCS)4] was obtained during a galvanostatic electrocrystallization process. As previously observed with BEDT-TTF-based compounds with oxalatometallate anions, the BEDT-TTF molecules in (BEDT-TTF)4[Cu(NCS)4] exhibit the so-called pseudo-κ arrangement, with two BEDT-TTF molecules being positively charged and two electronically neutral. The bond lengths and angles in the two unique BEDT-TTF molecules differ slightly. The crystal structure consists of layers of BEDT-TTF molecules extending parallel to (001). The width of this layer corresponds to the length of the a axis [16.9036 (17) Å]. The BEDT-TTF layers are separated by layers of centrosymmetric square-planar [Cu(NCS)4]2– dianions.A new phase combining BEDT-TTF and [Cu(NCS) A structure determination of these crystals revealed a new salt-like compound, based on the BEDT-TTF donor and the [Cu(NCS)4]2– dianion, namely pseudo-κ-(BEDT-TTF)4[Cu(NCS)4].For several years, we have been interested in synthesizing molecular (super)conductors as nanoparticles 4], and thus is different from the well-known κ-phase (BEDT-TTF)2[Cu(NCS)2] 3] metallate anions, such as (BEDT-TTF)4[AM(C2O4)3]·solv.  , the other BEDT-TTF molecule is neutral (Q = 0.18). Not only do the bond lengths of the BEDT-TTF molecules in the title salt show some differences, but the overall shape of the molecules also differs. The BEDT-TTF molecule in the dimer deviates less from planarity  than the other BEDT-TTF molecule . Moreover, the outer ethylene groups tend to be more eclipsed in the molecule of the dimer whereas they tend to be more staggered in the other molecule 3]·solv. family.The asymmetric unit of the title salt contains two well-ordered BEDT-TTF molecules and one Cu(NCS)re Fig. 2. This re al. 1997, this alle Fig. 3. All thex] anions that are present as polymeric [Cu(NCS)x] entities, in the title compound discrete [Cu(NCS)4]2– units are observed. The CuII cation (which lies on a centre of inversion) of the [Cu(NCS)4]2– dianion adopts an almost regular square-planar CuN4 environment, with N—Cu—N angles close to 90 and 180°. Intramolecular bond lengths in the anion are in agreement with other tetrathiocyanatocuprates(II) ° for Cu—N1—C1; Fig. 2Contrary to what is observed in other BEDT-TTF compounds associated with [Cu(NCS)a axis and are separated from each other by layers of [Cu(NCS)4]2– dianions  Å, considering the least-squares planes of the molecule except for the terminal ethylene groups. In addition, there are two pairs of short S⋯S contacts within the dimer [S5⋯S8 = 3.461 (1) and S6⋯S7 = 3.515 (1) Å]. Each dimer is surrounded by six adjacent BEDT-TTF molecules Fig. 4. Four ofns Fig. 5.4[Cu(NCS)4] was prepared by galvanostatic electrocrystallization in an H-shaped cell, equipped with Pt electrodes and with a glass frit between the anodic and cathodic compartments. To the cathodic compartment were added EMIM(SCN)  and freshly distilled 1,1,2-TCE . Cu(NCS)  and EMIM(SCN)  were added to the anodic compartment, which was immediately filled with BEDT-TTF , previously dissolved in 10 ml of 1,1,2-TCE at 343 K. The current was set at 100 µA (current density of 318 µA cm−2). After 12 h, a black powder corresponding to the desired compound (BEDT-TTF)2[Cu(NCS)2] was harvested by filtration, together with some crystals of (BEDT-TTF)4[Cu(NCS)4], which were washed with 1,1,2-TCE and dried under vacuum.(BEDT-TTF)θmax = 47.42°). The hydrogen atoms of the ethylene bridges were placed in idealized positions and were refined with C—H = 0.99 Å and with Uiso(H) = 1.2Ueq(C). Reflections (100) and (110) were obstructed by the beam stop and thus were excluded from the refinement.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018015293/wm5468sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018015293/wm5468Isup2.hklStructure factors: contains datablock(s) I. DOI: 1876009CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Cynanchum paniculatum (Bge.) Kitag. Their structures were identified by spectroscopic methods including extensive 1D and 2D NMR techniques. All compounds were subjected to detect the anti-tobacco mosaic virus (TMV) activities and their cytotoxities against three human tumor cell lines . The results showed that compounds 1 and 5 exhibited potent protective activities against TMV, while 2, 4 and 6 had moderate effects on the SMMC-7721 cancer cells viability.Two new C21 steroidal glycosides, paniculatumosides H and I, together with four known ones were isolated from the roots of  Cynanchum paniculatum (Bunge) Kitag is a vivacious herb broadly distributed in China, Japan and Korea, whose dried roots have been used as a Chinese herbal medicine for the treatment of rheumatic arthralgia, epigastric pain, toothache, lumbago, traumatic injuries, and eczema + in the positive HRESI-MS . The IR spectrum displayed absorption bands for hydroxyl (3443 cm−1), carbonyl (1736 cm−1), olefinic (1635 cm−1) and carbon–oxygen bond (1081 cm−1). The 1H NMR spectrum of compound 1 showed the presence of two tertiary methyl groups at δH 0.92  and δH 1.53 , one olefinic proton at δH 5.38 , one olefinic deshielded proton at δH 6.24 , two oxygen-substituted methine protons at δH 3.44  and δH 5.29  and two methylene protons at δH 3.84  and δH 4.15 . By comparison of its 1H and 13C NMR spectra (Table 3) indicated that the aglycone of 1 is glaucogenin C, which also confirmed by correlations of H-19/H-2b, H-19/H-8, H-1b/H-3, H-21/H-16 and H-16/H-17 in the ROESY experiment , C-3 (+ 4.2) and C-4 (− 2.2). The sugar carbon signals  illustrated that the sugar belonged to 2,6-deoxy sugar because it contained four methines, one methylene, a terminal methyl group and a methoxyl group. In the sugar spin systems, the large coupling constants JH-3′/H-4′ (9.1 Hz) and JH-4′/H-5′ (9.3 Hz) disclosed their trans-diaxial relationship, which suggested that the sugar was oleandropyranose D20−21.5 ; IR (KBr) νmax 3443 (OH), 2919, 1736, 1635, 1384, 1081 cm−1; 1H and 13C NMR data, see Table m/z 527 [M + Na]+; HRESIMS m/z 527.2629 [M + Na]+ .White amorphous powder; [α]D20−52.0 ; IR (KBr) νmax 3433 (OH), 2922, 1736, 1632, 1383, 1070 cm−1; 1H and 13C NMR data, see Table m/z 657 [M + Na]+; HRESIMS m/z 657.3258 [M + Na]+ .White amorphous powder; [The half-leaf method was used to evaluate the anti-TMV activities as literatures reported . NingnanThe cytotoxicity of each compound on three cultured human cancer cell lines was tested by MTT assay. The cell lines used were SMMC-7721 (human hepatoma cells), MDA-MB-231 (triple-negative breast cancer cells), A549 (human lung cancer cell). Cell growth inhibition assay was performed as reported literatures . Cisplat


In the crystal, no interactions stronger than van der Waals forces are found between the molecules that neither suffer from chair conformation disorder nor from rotational disorder of the tert-butyl groups. Characteristic structural features are the partial flattening of the ‘cyclohexane-chair’ conformation at the heteroatom side of the six-membered ring and the length of the weakened P—Cl bond [2.2869 (6) Å].Sublimation  tert-butyl-2-chloro-1,3,2-diazaphosphorinane, C11H24ClN2P (1), were obtained by sublimation in vacuo slightly above room temperature. The asymmetric unit of the monoclinic crystal structure of the six-membered N-heterocyclic compound is defined by one molecule in a general position. The six-membered ring of the molecule adopts a cyclohexane-like chair conformation; the chair at one side is to some extent flattened as a result of the approximately trigonal–planar coordination of both nitrogen atoms. In detail, this modified chair conformation is characterized by an angle of 53.07 (15)° between the plane defined by the three carbon atoms and the best plane of the two nitrogen atoms and the two carbon atoms bound to them, and an angle of 27.96 (7)° between the latter plane and the plane defined by the nitrogen and phosphorus atoms. The tert-butyl groups are oriented equatorially and the chloro substituent is oriented axially. The P—Cl bond length of 2.2869 (6) Å is substantially longer than the P—Cl single-bond length in PCl3 [2.034 Å; Galy & Enjalbert  Å, excluding a significant influence on the P—Cl bonding.Colourless blocks of 1,3-di-bert 1982. J. Soli P-chlorofunctionalized N-heterocyclic phosphanes (NHPCls) received considerable attention, mainly as precursors of N-heterocyclic phosphenium ions (NHPs) that are valence isoelectronic compounds of the well-known N-heterocyclic carbenes (NHCs) 2>E and the related NHPs. The fourth ring member >E, joining the class-defining Cl—P<(NR)2 fragment, is an >SiR2 group in most cases 2>P–Cl, for instance, have been known since the early 1970s  the partially flattened chair conformation of the central six-membered heterocycle  and 355.8 (N2)], in combination with the axial orientation of the chloro substituent 2>P–Cl shows almost identical bonding at the phosphorus atom  with an enforced chair conformation, P—Cl = 2.0754 (11) Å and in the 2-chloro-1,3,2-dioxaphophorinane derivative [(–O)2 >P—Cl] described by Pavan Kumar & Kumara Swamy  to 2.244 (3) Å. Further geometric details of 1 are given in the supporting information. C—C and C—N bond lengths, as well as endocyclic and exocyclic bond angles, are as expected taking into account the main structural characteristics given above. Finally it should be noted that the crystal structure determination described here confirms the suggestions of Hutchins et al.  Å, symmetry related by the c glide plane . Fig. 3Inspection of the intermolecular distances gives no evidence for interactions stronger than van der Waals forces in the crystal of et al., 2016i.e. exclusively single bonds in the six-membered ring) yielded only one structure -1,4,8,11-tetraazacyclotetradecane calix(4)arenes FEMLOZ and FEMLUF dichlorotitanium trimethylsilylamino-2,4-di-tert-butyl-1-[2-]imino-3-thio-1,2,4,3-thiadiazaphosphetidine 2 fragment and allowing for any kind of bonding in the ring gave 16 hits including eight with three carbon atoms. In addition to DEHZOH mentioned before, these include 2-chloro-1-(2′-chloroethyl)-3-methyl-5,6-benzo-1,3,2-diazaphosphorin-4-one . 16 ml of an n-butyllithium solution  were slowly added at 263 K. Half an hour later, the reaction mixture was allowed to reach room temperature and the resulting pale-yellow suspension was stirred for 16 h. 2.92 g of PCl3 (21.3 mmol) were added dropwise over a period of 15 minutes at 195 K. To complete the reaction, the yellow reaction mixture was stirred for another hour with cooling and finally for two h at room temperature. Subsequently, the LiCl precipitate was filtered off and, after removal of the solvent under reduced pressure, the crude product was obtained as a yellow solid. Colourless block-shaped crystals suitable for X-ray structure determination were obtained by sublimation in a vacuum (3·10−2 mbar) at 313 K , by NMR analysis proved to be pure substance. 1H-NMR  δ 3.16–3.07 , 1.90–1.80 , 1.34 .The title compound was prepared under an argon atmosphere in oven-dried glassware using standard Schlenk techniques, modifying a published procedure  as well as H—C—H and C—C—H angles. In addition, the H atoms of the CH3 groups were allowed to rotate around the neighbouring C—C bonds. The Uiso values were set to 1.5Ueq(Cmethyl) and 1.2Ueq(Cmethylene).Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S2056989019004195/pk2615sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019004195/pk2615Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019004195/pk2615Isup3.cmlSupporting information file. DOI: 1906304CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Acetoin (AC) and 2,3-butanediol  as highly promising bio-based platform chemicals have received more attentions due to their wide range of applications. However, the non-efficient substrate conversion and mutually transition between AC and 2,3-BD in their natural producing strains not only led to a low selectivity but also increase the difficulty of downstream purification. Therefore, synthetic engineering of more suitable strains should be a reliable strategy to selectively produce AC and 2,3-BD, respectively.alsS and alsD) and 2,3-BD biosynthesis pathway genes  derived from Bacillus subtilis 168 were successfully expressed in non-natural AC and 2,3-BD producing Corynebacterium crenatum, and generated recombinant strains, C. crenatum SD and C. crenatum SDA, were proved to produce 9.86 g L−1 of AC and 17.08 g L−1 of 2,3-BD, respectively. To further increase AC and 2,3-BD selectivity, the AC reducing gene (butA) and lactic acid dehydrogenase gene (ldh) in C. crenatum were then deleted. Finally, C. crenatumΔbutAΔldh SD produced 76.93 g L−1 AC in one-step biocatalysis with the yield of 0.67 mol mol−1. Meanwhile, after eliminating the lactic acid production and enhancing 2,3-butanediol dehydrogenase activity, C. crenatumΔldh SDA synthesized 88.83 g L−1 of 2,3-BD with the yield of 0.80 mol mol−1.In this study, the respective AC  contains supplementary material, which is available to authorized users. As important platform compounds, acetoin (AC) and 2,3-butanediol  are widely used in food, medicine and chemical industries –4. In adα-acetolactate synthase (ALS), α-acetolactate decarboxylase (ALDC) and 2,3-butanediol dehydrogenase (BDH) to increase the titer of AC (0.87 g L−1) and 2,3-BD (1.12 g L−1) in Escherichia coli [Bacillus subtilis and Saccharomyces cerevisiae, in which the productivity of AC from glucose was increased by approximately 62.9% and 58%, respectively [S. cerevisiae, 2,3-BD was further increased to 96.2 g L−1 [−1 by reducing the biosynthesis of lactic acid and acetic acid in Klebsiella oxytoca [bdhA, pta and acoA in B. subtilis and increased the yield of AC to 0.49 g g−1 [E. coli [Serratia marcescens, Klebsiella pneumoniae, Enterobacter and S. cerevisiae [Cyanobacteria, and successfully produced AC and 2,3-BD using CO2 [Corynebacterium glutamicum was also engineered for 2,3-BD production by expressing gene cluster of 2,3-BD biosynthetic pathway from Lactococcus lactis [budABC cluster from K. pneumoniae [−1 of 2,3-BD, respectively. After multistage modification, C. glutamicum could also be used to produce optically pure d-(−)-AC [In recent years, consolidate efforts have been made towards increasing AC and 2,3-BD production by inactivation of competitive pathways or overexpression of key genes of AC and 2,3-BD biosynthesis pathways . Nielsenof AC 0.8 g L−1 an.2 g L−1 . On the  oxytoca . Wang et2,3-BD 1. g L−1 in[E. coli . Meanwhirevisiae . Took thsing CO2 . In addis lactis  or the beumoniae , which sd-(−)-AC . However+/NADH levels in E. coli and B. subtilis [Corynebacterium crenatum SYPA5-5 as the host since the following reasons: C. crenatum, genetically close to C. glutamicum, has the advantages of growing fast, non-sporulation, and easy gene editing [C. crenatum was demonstrated that catalyze both AC and diacetyl (DA) for 2,3-BD formation [C. crenatum SYPA5-5 should be a reliable strategy to selectively produce AC and 2,3-BD, respectively.Compared to AC and 2,3-BD fermentation, AC and 2,3-BD biocatalysis generally present a higher yield and selectivity , 20. Effsubtilis , 22. How editing . In addiormation . TherefoalsS and alsD) and 2,3-BD biosynthesis pathway genes  from B. subtilis 168 were overexpressed in C. crenatumΔldh and C. crenatumΔbutAΔldh, respectively, in which glucose was specifically transformed into AC or 2,3-BD in one-step biocatalysis , C. crenatum/pXMJ19-alsD (C. crenatum D), C. crenatum/pXMJ19-bdhA (C. crenatum A), C. crenatum/pXMJ19-alsSD (C. crenatum SD), and C. crenatum/pXMJ19-alsSD-bdhA (C. crenatum SDA), respectively.Here, an artificial operon including C. crenatum recombinants  comparable to C. crenatum A (0.38 and 0.19 U mg−1). In addition, although AR/BDH was a bidirectional reversible enzyme [B. subtilis present a higher AR activity than BDH activity in C. crenatum A and C. crenatum SDA. With ALS, ALDC, and (or) AR/BDH, the recombinant C. crenatum can catalyze the pyruvate to either AC or DA, thus allowing the respective conversion of glucose to AC and 2,3-BD.The SDS-PAGE was used to confirm the expression of ALS (AHAS), ALDC, and AR/BDH in crenatum , 26. Meae enzyme , 27, the−1 glucose as the sole carbon source were first performed in shack flask to investigate the metabolic impact of ALS, ALDC, and BDH expression. Since C. crenatum is a facultative anaerobe, cell growth under different dissolved oxygen condition often leads to generate different products [C. crenatum shows a fast growth rate of biomass when using glucose as the substrate, and is diffusely used in the yielding of sundry amino acids such as l-glutamic acid and l-arginine under sufficient oxygen supply conditions [l-arginine was also determined at the end of fermentation in this study. The results are shown in Table Batch fermentations with 120 g Lproducts , 29. Prenditions . TherefoC. crenatum WT yield 23.6 g L−1l-arginine, and no AC and 2,3-BD were detected in the fermentation broth. Theoretically, overexpression of alsS, alsD, and bdhA gene, respectively, cannot produce AC and 2,3-BD in C. crenatum. However, except for little AC and 2,3-BD producing, C. crenatum S also produced 2.17 g L−1 of unexpected DA, which could not be accumulated in B. subtilis [α-acetolactate under natural oxidative aerobic conditions [C. crenatum could catalyze DA to AC and then convert to 2,3-BD [C. crenatum S without expression of ALDC. Moreover, it has been reported that DA is a bacteriostatic food additive that can inhibit bacteria growth [C. crenatum S consumed only about 36 g L−1 glucose and then suspended the fermentation while accumulated comparable amount of DA, leading to a low efficiency of the multiple-step reaction from pyruvate to AC and 2,3-BD.As expected, subtilis . This minditions . In addio 2,3-BD . This exalsSD operon with one promoter into C. crenatum, co-expression of ALS and ALDC effectively converted pyruvate to 13.59 g L−1 AC and 10.61 g L−1 2,3-BD, respectively. To enhance 2,3-BD production, bdhA was further expressed in C. crenatum SD and the generated C. crenatum SDA dramatically increased 2,3-BD titer by 43%. These results indicated that ALS and ALDC are both important for AC and 2,3-BD biosynthesis in C. crenatum. In addition, enhanced heterogeneous BDH activity was confirmed the positive effect on 2,3-BD biosynthesis. However, C. crenatum SD and C. crenatum SDA still produced comparable amount of l-arginine as by-product, resulting in a low yield of AC and 2,3-BD. Therefore, l-arginine biosynthesis must be decreased.On the contrary, after introducing the l-Arginine as growth factor is necessary for C. crenatum growth, which cannot be directly depressed by gene knock-out. l-arginine production by C. crenatum needs high level of dissolved oxygen, while there is no strict demand on dissolved oxygen for the acid-butanediol fermentation. Therefore, we attempted to produce AC and 2,3-BD using resting cell bioconversion with glucose as substrate by C. crenatum SD and C. crenatum SDA, respectively.l-arginine was detected during the resting cell bioconversion process (data not shown). C. crenatum SD converted 100 g L−1 glucose to 9.86 g L−1 AC and 12.76 g L−1 2,3-BD, respectively. Similarly, C. crenatum SDA further accumulated 2,3-BD to 17.08 g L−1 with a high 2,3-BD/AC ratio. However, the content of organic acids, especially lactic acid, were also detected in both C. crenatum SD and C. crenatum SDA, which was consistent with previous study that C. crenatum can produce various organic acids such as succinic acid, acetic acid and lactic acid in the case of limited dissolved oxygen [As demonstrated in Fig. d oxygen . As the d oxygen , 33, thel-arginine formation, some pyruvate was still converted to lactic acid. In addition, unexpected high yield of 2,3-BD was still produced by C. crenatum SD. To further decrease by-products and enhance the AC and 2,3-BD selectivity, blocking the competitive synthesis pathways in C. crenatum was considered to be necessity. The homologous lactate dehydrogenase (ldh) and butanol dehydrogenase (butA) genes in C. crenatum were knocked out to increase respective AC and 2,3-BD selectivity. The suicide vectors pK18-Δldh . C. crenatumΔbutAΔldh SD consumed 94 g L−1 glucose after 60 h with an uptake rate of 1.57 g L−1 h−1, which was significantly higher  than that of C. crenatum WT and C. crenatumΔbutAΔldh, indicating that the expression of heterologous AC biosynthesis pathway strongly promoted glucose for AC production. Consequently, AC production dramatically increased from 9.86 to 23.56 g L−1, about 2.39-fold than before. Meanwhile, only a small amount of 2,3-BD (1.28 g L−1) and lactic acid (1.83 g L−1) were detected, resulting in an AC molar yield of up to 0.51 mol mol−1. Consistence with that of C. crenatumΔbutAΔldh, acetic acid and succinic acid has hardly changed  which co-expresses both homologous and heterologous AR/BDH. Generally, compared with C. crenatum SDA as the positive control, C. crenatumΔldh SDA resting cell converted more glucose to 2,3-BD. After 60 h, C. crenatumΔldh SDA consumed 95 g L−1 glucose with an uptake rate of 1.58 g L−1 h−1. With overexpression of AR/BDH and depressing LDH, 2,3-BD production increased from 17.08 to 25.93 g L−1, about 52% higher than the control. Meanwhile, lactic acid was decreased about 95%, leading to a high 2,3-BD molar yield of 0.55 mol mol−1  and succinic acid (1.1 g L−1) were slightly higher than C. crenatum SDA.To further extend the products chain for 2,3-BD production, pXMJ19-C. crenatum, repeated batch resting cell bioconversion were performed in 5 L bioreactors, and the results are shown in Fig. −1 of glucose as the substrate. In the first two batch conversions, AC production was stably increased by C. crenatumΔbutAΔldh SD. However, AC productivity of the third batch has gradually declined. Finally, after total 60 h of bioconversion, 76.93 g L−1 of AC with the yield of 0.67 mol mol−1 was produced by C. crenatumΔbutAΔldh SD . Therefore, further strategies should be developed to maintain the cell viability and stability during the whole bioconversion process.To investigate the long-term stability and performance of recombinant E. coli and B. subtilis can be used as host for biocatalytic synthesis of AC and 2,3-BD, in which the highest yield of AC and 2,3-BD can be reached to 0.98 mol mol−1 and 0.96 mol mol−1. However, most of these biocatalysts cases were carried out using AC or 2,3-BD as a substrate, which are not suitable for industrial production at all. Although 2,3-BD and AC bioconversion from glucose were achieved by K. pneumoniae and B. subtilis, two step batch strategy still cannot selectively separate mixed -2,3-BD and (3S)-AC [C. crenatum as the only host for AC and 2,3-BD production directly from glucose. After depressing competition pathways and overexpressing AC and 2,3-BD biosynthesis genes from B. subtilis, one-step biosynthesis of AC and 2,3-BD, respectively, were achieved in C. crenatumΔbutAΔldh SD and C. crenatumΔldh SDA without additional complex nutrients.Currently, microbial fermentation is still the main method for producing AC and 2,3-BD from various substrates. However, its relatively long fermentation period and low productivity and substrate conversion rate are currently obstructed the industrial application , 34, 35. (3S)-AC . In this−1 AC and 2,3-BD produced by microbial fermentation, further improvements of C. crenatum are necessary for industrial application. Although depressing ldh and butA and enhancing AC or 2,3-BD biosynthesis pathway activity, C. crenatumΔbutAΔldh SD and C. crenatumΔldh SDA showed a higher AC and 2,3-BD selectivity, respectively, some pyruvate still converted to acetic and succinic acid. Therefore, increased AC and 2,3-BD production can be realized by disruption of the acetate and succinate biosynthesis pathway, as demonstrated in K. oxytoca and B. subtilis [l-arginine by site-directed mutation of the key enzyme (NAGK) of C. creantum, and overexpressed the l-arginine operon, which effectively increased the yield of l-arginine by 41.7% [butA) gene might be further improved the AC and 2,3-BD yield and selectivity in C. creantum. In addition, although the repeated batch biocatalysis showed a high average productivity for AC and 2,3-BD production, the catalytic efficiency significantly deceased after only two batches, which can be further improved by cell immobilization to increase the cell viability and stability [C. creantum.Compared to usual 100–120 g Lsubtilis , 12. Morby 41.7%  and 29% by 41.7% . Therefotability , 45. On C. crenatum SYPA5-5 to overexpress alsS, alsD and/or bdhA from B. subtilis 168 for selective AC and 2,3-BD biosynthesis from glucose. After depressing competition pathways, recombinant C. crenatumΔbutAΔldh SD and C. crenatumΔldh SDA further increased AC and 2,3-BD production to 76.93 g L−1 and 88.83 g L−1, respectively. Overall, respective non-natural AC and 2,3-BD biosynthesis pathway constructed in this study efficiently reduced the by-products accumulation and improved AC and 2,3-BD selectivity in C. crenatum. The optimal selection of AC and 2,3-BD biosynthesis strategies with further metabolic engineering should lead to the development of a promising microbial cell factory for AC and 2,3-BD production.In this study, we successfully engineered B. subtilis 168, E. coli BL21 and JM109, C. crenatum SYPA5-5 were stored in our laboratory. E. coli BL21 and JM109 were host strains of recombinant plasmid pXMJ19 and pk18, respectively. pXMJ19 is the shuttle expression vector of E. coli and Corynebacterium. pK18mobsacB carrying sacB gene is used for gene integration and knockout in C. crenatum [All strains, plasmids and primers involved are shown in Additional file crenatum .Table 5pFastPure Gel DNA Extraction Mini Kit, FastPure Plasmid Mini Kit and FastPure Bacteria DNA Isolation Mini Kit were all purchased from Vazyme . Antibiotics, restriction endonucleases and other tool enzymes such as high-fidelity 2× ExTaq DNA polymerase all purchased from Shenggong Biological . All other reagents of analysis grade or higher quality were obtained from Wuxi Reagent Company.E. coli. LBG (LB + 0.5% glucose) medium was used for preincubation of C. crenatum. C. crenatum competent medium for electroporation: LB medium was supplemented with 3% glycine and 0.1% tween 80. Shake flask fermentation medium (g L−1): (NH4)2SO4 40, yeast extract 8, KH2PO4 1.5, KCl 1, MnSO4·H2O 0.02, FeSO4·7H2O 0.02, MgSO4·7H2O 0.5, CaCO3 30. Seed medium of 5 L bioreactor (g L−1): yeast extract 20, MgSO4·7H2O 0.5, (NH4)2SO4 20, KH2PO4 1.5. Fermentation medium of 5 L bioreactor (g L−1): (NH4)2SO4 20, yeast extract 20, KH2PO4 1.5, MnSO4·H2O 0.02, FeSO4·7H2O 0.02, MgSO4·7H2O 0.5. Resting cell bioconversion medium (g L−1): K2HPO4·3H2O 0.5, KH2PO4 0.5, MnSO4·H2O 4.2, MgSO4·7H2O 0.5, FeSO4·7H2O 6. E. coli was cultured at 37 °C, 180 r min−1. C. crenatum was cultured at 30 °C, 180 r min−1 or 250 r min−1.Luria–Bertani (LB) medium was used to culture butA gene fragment was obtained by PCR using the C. crenatum SYPA5-5 genome as template and PbutA1F and PbutA2R as primers. The PCR product was connected to pMD18-T cloning vector and sent to Shenggong Biological (Shanghai) for sequencing. Firstly, PbutA1F, PbutA3R and PbutA2R, PbutA4F primers were used for the first round of PCR using the C. crenatum SYPA5-5 genome as template to obtain two PCR products. Secondly, the deleted gene fragment ΔbutA was obtained by fusing the two PCR products in the overlap-extension PCR and PbutA1F and PbutA2R were used . The washed cells were suspended in 5 mL of Tris–HCl buffer and lysozyme was added to treat the cell wall for 3–4 h. Cell disruption was accomplished by sonication for 30 min under ice bath conditions. Cell debris was removed by centrifugation at 4 °C, 12,000 r min−1 for 30 min and the supernatant was harvested for measurement of intracellular enzyme activity. Protein concentration in supernatant was determined by Bradford method [Recombinant d method . ALS/AHAd method , 50, AR/d method  and LDH d method  enzyme a−1 glucose for resting cell bioconversion. 30 g L−1 CaCO3 was added during transformation to neutralize the organic acids produced. When resting cell bioconversion was performed in 5 L bioreactor, 4 L recombinant C. crenatum cultures were collected and resuspended in 2 L resting cell bioconversion medium. Resting cell bioconversion was performed in three batches at 30 °C, 250 r min−1. The converted solution was kept at pH 7.0 by feeding 50% NH3·H2O automatically. After the end of each batch of transformation, cells were harvested by centrifugation and then washed 3 times using Tris–HCl buffer. Resting cell bioconversion was continued by resuspending the cells in 2 L resting cell bioconversion medium containing 100 g L−1 glucose.The bacterial solution was inoculated into 10 mL LBG medium at a ratio of 1% and incubated for about 12 h. 3 mL bacterial liquid was transferred to 30 mL fermentation medium and cultured for 24 h. The cells in the fermentation medium were harvested and suspended in resting cell bioconversion medium containing 100 g L562 = 0.375 g L−1 DCW). The concentration of glucose in conversion solution and fermentation broth was determined using SBA-40E biosensor analyzer. The concentration of AC and 2,3-BD in conversion solution and fermentation broth was measured using Agilent gas chromatography [For biomass analysis, the absorbance of cell cultures were measured at 562 nm with distilled water as the blank control. The dry cell weight (DCW) can be converted by using the equation (1 ODtography . Amino atography  and orgatography  were anaAdditional file 1: Figure S1. SDS-PAGE analysis of ALS, ALDC, and BDH in recombinant C. crenatum. The following samples and markers are shown: M: Protein marker; Lane 1: whole cell protein of C. crenatum WT; Lane 2: whole cell protein of C. crenatum S; Lane 3: whole cell protein of C. crenatum D; Lane 4: whole cell protein of C. crenatum A; Lane 5: whole cell protein of C. crenatum SD; Lane 6: C. crenatum SDA.Additional file 2: Figure S2. Identification of pK18-Δldh by enzyme digestion. The following samples and markers are shown: M1: λDNA/HindIII marker; M2: DL2000 marker; Lane 1: pK18-Δldh digested with EcoRI; Lane 2: pK18-Δldh digested with EcoRI and HindIII.Additional file 3: Figure S3. Identification of pK18-ΔbutA by enzyme digestion. The following samples and markers are shown: M1: λDNA/HindIII marker; M2: DL2000 marker; Lane 1: pK18-ΔbutA digested with EcoRI; Lane 2: pK18-ΔbutA digested with EcoRI and HindIII.Additional file 4: Figure S4. A: PCR identification of ldh gene knockout strains and recovery strains. M: DL2000 marker. Lane 1: ldh gene recovery strain. Lane 2 and 3: ldh gene knockout strains. B: PCR identification of butA gene knockout strains and recovery strains. M: DL2000 marker. Lane 1 and 4: butA gene recovery strains. Lane 2 and 3: butA gene knockout strains. C: PCR identification of ldh gene and butA knockout strain. M: DL2000 marker. Lane 1: PCR amplification of ΔbutA. Lane 2: PCR amplification of Δldh.Additional file 5: Table S1. Dry cell weight of C. crenatumΔldh, C. crenatumΔbutA and C. crenatumΔbutAΔldh. Results are shown as the mean ± standard of three replicates.Additional file 6: Table S2. Strains and plasmids used in this study. Kanamycin resistance is labeled as KmR, and chloramphenicol resistance is labeled as CmR.Additional file 7: Figure S5. PCR amplification of the knockout fragment ΔbutA.Additional file 8: Figure S6. PCR amplification of the knockout fragment Δldh.


P21/c) symmetry and a distorted tetrahedral geometry around the nickel center, with the cyclooctadiene ligand coordinated in a κ2,η2 fashion. The bidentate NHC ligand is not planar, with a C(carbene)—Ni—C(carbene) angle of 91.51 (12)°, resulting in the mesityl groups being on the same side of the cyclooctadiene (COD) ligand.The complex at 100 K has monoclinic ( 25H28N4)(C8H12)]·C4H8O or (MesNHC2Me)Ni(COD), which contains a bidentate N-heterocyclic carbene (NHC) ligand with mesityl aryl groups is reported. The complex at 100 K has monoclinic (P21/c) symmetry and a distorted tetrahedral geometry around the nickel center, with the cyclooctadiene ligand coordinated in a κ2,η2 fashion. The bidentate NHC ligand is not planar, with a C(carbene)—Ni—C(carbene) angle of 91.51 (12)°, resulting in the mesityl groups being on the same side of the cyclooctadiene (COD) ligand. One molecule of tetrahydrofuran (THF) is co-crystallized with the nickel complex and has positional disorder.The crystal structure of the title compound, [Ni(C The distances between the nickel center and the (MesNHC2Me) ligand are 1.909 (3) Å for Ni1—C1 and 1.916 (3) Å for Ni1—C4. These are slightly shorter than the analagous distances of 1.938 (3) and 1.953 (3) Å, respectively, reported for (DippNHC2Me)Ni(COD) Ni(COD) of 1.374 (4), 1.387 (4), 1.379 (4), and 1.386 (4) Å, respectively Ni(COD) of 1.383 (5), and 1.355 (5) Å, respectively Ni(COD) of 1.502–1.529 Å Ni(COD) and THF are present in the unit cell, as depicted in Fig. 3A⋯H32A and H27B⋯H31B, respectively). Standard deviations for distances including hydrogen atoms are not listed because hydrogen atoms were positionally fixed. The methyl group at the para position of the mesityl fragment is oriented towards the aryl ring of the mesityl of the neighboring molecule, with a distance of 2.72 Å between the aryl ring centroid (C8–C13) and the nearest methyl hydrogen atom (H15C). The THF molecule is closest to the backbone of the (MesNHC2Me) ligand, such that the molecules are 3.527 (17) Å apart from one oxygen atom (O1) to the next nearest carbon atom (C36) Ni(COD) (tBu = tert-butyl) and (DippNHC2Me)Ni(COD) phenyl) Ni(COD) is coordinated in a κ1,η2 fashion.A survey of the Cambridge Structural Database -3,3′-methylene-diimidazolium dibromide were synthesized according to literature procedures -3,3′-methylenediimidazolin-2,2′-diylidene}nickel(0)cyclooctadiene was synthesized by the following method. A 20 mL scintillation vial was charged with 0.203 g  of 1,1′-di(mesityl)-3,3′-methylene-diimidazolium dibromide, approximately 10 mL of tetrahydrofuran and a stirbar. 1.80 mL  of 0.5 M potassium bis(trimethylsilyl)amide in toluene were added dropwise to the solution while stirring, resulting in a color change to blue–green. The mixture was stirred for approximately five h, resulting in a clear orange–brown solution, which was filtered through a glass frit with celite. The filtrate was transferred to a new 20 mL glass scintillation vial and stirred while adding 0.090 g  of bisnickel(0). The mixture was stirred for 4–12 h, resulting in a clear dark red–orange solution. The solvent was removed in vacuo, and the orange solid was washed with pentane (3–5 washes of approximately 10 mL), resulting in 0.151 g (78%) of an orange solid identified as {1,1′-di(mesityl)-3,3′-methylenediimidazolin-2,2′-diylidene}nickel(0)cyclooctadiene. Single crystals suitable for X-ray analysis were grown from a dilute solution of pentane with a drop of tetrahydrofuran. 1H NMR : δ = 1.96–2.12 , 2.13 , 2.17 , 4.07 , 4.68 , 6.12 , 6.42 , 6.84 . 13C NMR : δ = 18.43 (CH3o-mesityl), 21.14 (CH3p-mesityl), 32.51 (CH2-COD), 61.31 (CH2), 74.17 (CH-Ni-COD), 118.18 (CH-Im), 119.81 (CH-Im), 128.94 (m-CH-Ar), 136.22 (o-C-Ar), 137.89 (p-C-Ar), 138.91 (i-C-Ar), 205.37 (N2C-Im).1--1SHELXL and refined as riding with distances of 0.95 Å for C—H, 0.99 Å for CH2 and 0.98 Å for CH3. Methyl H atoms were allowed to rotate but not to tip to best fit the experimental electron density. Uiso values of riding H atoms were set to 1.2 times Ueq(C) for CH and CH2, and 1.5 times Ueq(C) for CH3. The positions of the hydrogen atoms on the portions of the COD ligand directly bound to nickel and attached to C26, C29, C30, and C33 were determined from the difference map. Positions and isotropic displacement parameters were refined, but the associated C—H atom distances were restrained to be similar to each other by using a SADI command of SHELXL .Crystal data, data collection and structure refinement details are summarized in Table 2SHELXL was applied for O1′ through C37′ and O1 through C34 to make bond distances and angles equivalent with standard deviations of 0.02 and 0.04 Å for 1,2- and 1,3 distances, respectively). Uij components of ADPs of the disordered atoms were restrained to be similar to each other with an esd of 0.01 Å2 for atoms closer to each other than 2.0 Å (SIMU command of SHELXL), resulting in a final close-to-equal site occupancy ratio of 0.502 (13) to 0.498 (13).The two moieties of the disordered THF molecule were restrained to have similar geometries  I. DOI: 10.1107/S2056989018012252/zl2737Isup2.hklStructure factors: contains datablock(s) I. DOI: 1864396CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In this compound, C⋯F and CH⋯F/O contacts, present between the cations and anions, extend the structure into a three-dimensional supramolecular network.The title compound, [Fe(C 27H41N10O3)](PF6)2, the tripodal tris{4-[1-(2-methoxyethyl)imidazol-2-yl]-3-azabut-3-enyl}amine ligand is coordinated to an FeII ion through the nitrogen atoms of three imidazole and three imino groups. The Fe atom exhibits a distorted octahedral geometry. In the crystal, l and d antipodes are arranged in layers in the bc plane. Weak C⋯F and C—H⋯F/O contacts exist between the ligands of the complex cation and the PF6− anions, generating a three-dimensional network. At 120 K, the FeII ion is in a low-spin state, with an average Fe—N bond distance of 1.970 (2) Å. On heating, the FeII ion converts to the high-spin state, as demonstrated by magnetic susceptibility measurements.In the complex cation of the title compound, [Fe(C II spin-crossover (SCO) complexes, which can change between high-spin  and low-spin  electronic states on application of physicochemical stimuli. The LS-to-HS conversion involves an electron transfer between the eg and t2g orbitals and is strongly coupled to structural changes in the coordination sphere of the FeII ions, affecting the Fe–ligand bond lengths and angles imidazol-2-yl]-3-azabut-3-enyl}amine, which can be crystallized in the presence of hexafluoridophosphate anions  to balance the charge. In the complex cation, the FeII ion is wrapped by three 1-(2-methoxyethyl)-imidazol-2-ylimino moieties, defining a pseudo-octahedral [FeN6] coordination environment /24  . At 300 K, the χMT value is close to 1.8 cm3 K mol−1, displaying at this temperature an incomplete transition of the FeII ion to the paramagnetic high-spin state (S = 2). On cooling, a gradual decrease of χMT value down to 0.07 cm3 K mol−1 is observed corresponding to an almost compete transformation to the diamagnetic low-spin state (S = 0). This corroborates well with the observed short average Fe—N bond length at 120 K and identifies the low-spin state of the central iron(II) ion.Variable-temperature magnetic susceptibility measurements were performed on single crystals (20 mg) of the title compound using a Quantum Design MPMS2 superconducting quantum interference device (SQUID) susceptometer operating at 1 T in the temperature range 2–300 K. Experimental susceptibilities were corrected for the diamagnetism of the holder (gelatine capsule) and of the constituent atoms by the application of Pascal’s constants. The magnetic behaviour of the compound recorded at 1 K minet al., 2016II ion wrapped by a tripodal ligand with a tris{imidazol-2-yl-3-azabut-3-enyl}amine fragment yielded 29 hits, for which the Fe—N bond lengths lie in the ranges 1.926–2.016 and 2.151–2.286 Å for the low-spin and high-spin spin states of the FeII ion, respectively.A search of the Cambridge Structural Database  in absolute ethanol (5 mL) was added dropwise to a boiling solution of 1-(2-methoxyethyl)imidazole-2-carbaldehyde , tris(2-ethanolamine)amine  and [NBu4]PF6  in 5 ml of absolute ethanol. The resulting dark red–purple solution was stirred for 5 min. After standing for several days under ambient conditions, well-shaped red needles of the title compound were formed. Elemental analysis for C27H41F12FeN10O3P2 (found): C, 36.58, H, 4.98, N, 15.55%; : C, 36.77, H, 4.85, N, 15.32.A filtered solution of FeClUiso(H) = 1.2–1.5Ueq(parent atom). The highest peak is located 1.21 Å from atom C24 and the deepest hole is located 0.65 Å from atom P2.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019001531/cq2029sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019001531/cq2029Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019001531/cq2029Isup3.cdxSupporting information file. DOI: 1893862CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: