Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by pathological deposition of misfolded self-protein amyloid beta (Aβ) which in kind facilitates tau aggregation and neurodegeneration. Neuroinflammation is accepted as a key disease driver caused by innate microglia activation. Recently, adaptive immune alterations have been uncovered that begin early and persist throughout the disease. How these occur and whether they can be harnessed to halt disease progress is unclear. We propose that self-antigens would induct autoreactive effector T cells (Teffs) that drive pro-inflammatory and neurodestructive immunity leading to cognitive impairments. Here, we investigated the role of effector immunity and how it could affect cellular-level disease pathobiology in an AD animal model.b (MHCII-IAb–KLVFFAEDVGSNKGA) tetramer binding. Aβ-Th1 and Aβ-Th17 clones were adoptively transferred into APP/PS1 double-transgenic mice expressing chimeric mouse/human amyloid precursor protein and mutant human presenilin 1, and the mice were assessed for memory impairments. Finally, blood, spleen, lymph nodes and brain were harvested for immunological, biochemical, and histological analyses.In this report, we developed and characterized cloned lines of amyloid beta (Aβ) reactive type 1 T helper (Th1) and type 17 Th (Th17) cells to study their role in AD pathogenesis. The cellular phenotype and antigen-specificity of Aβ-specific Th1 and Th17 clones were confirmed using flow cytometry, immunoblot staining and Aβ T cell epitope loaded haplotype-matched major histocompatibility complex II IAThe propagated Aβ-Th1 and Aβ-Th17 clones were confirmed stable and long-lived. Treatment of APP/PS1 mice with Aβ reactive Teffs accelerated memory impairment and systemic inflammation, increased amyloid burden, elevated microglia activation, and exacerbated neuroinflammation. Both Th1 and Th17 Aβ-reactive Teffs progressed AD pathology by downregulating anti-inflammatory and immunosuppressive regulatory T cells (Tregs) as recorded in the periphery and within the central nervous system.These results underscore an important pathological role for CD4+ Teffs in AD progression. We posit that aberrant disease-associated effector T cell immune responses can be controlled. One solution is by Aβ reactive Tregs.The online version contains supplementary material available at 10.1186/s12974-021-02308-7. Alzheimer’s disease (AD) is the most common form of age-associated dementia and has become the sixth leading cause of death in the United States , 2. AD ib-Aβ tetramer staining affirmed a distinctive cellular phenotype and antigen specificity. Aβ-reactive Th1 and Th17 Teffs, adoptively transferred into APP/PS1 mice demonstrated accelerated behavioral and pathological disease, thus supporting a role as disease perpetrators. This suggests an evolving role for CD4+ Teffs in AD progression with opportunities for therapeutic interventions by immune modulation.The peripheral adaptive immune system, comprised of cytokine secreting T cells, monocytes, and B lymphocytes, is activated by modified self-peptide fragments, such as Aβ and tau. These are engulfed and presented by antigen presenting cells (APCs) , 10. T c1–42 immunization of non-transgenic (non-Tg) mice. However, those cells were polarized for a short period prior to adoptive transfer with uncertain phenotypes  neurons in cortical brain lysates by western blot analysis. Co-expression of both pre- and post-synaptic proteins is essential for neuroplasticity, and therefore alteration of either protein level affects memory [p < 0.05, 45%) mice compared to non-Tg mice. Additionally, APP/PS1/Aβ-Th1 (p < 0.05), but not APP/PS1/Aβ-Th17 mice showed reduced expression of PSD95 compared to non-Tg mice. Thus, only presynaptic protein abnormalities were observed in APP/PS1/Aβ-Th17 mice, while pre-and post-synaptic protein densities were affected in APP/PS1/Aβ-Th1 mice. Overall, Aβ-Th1 and Aβ-Th17 cell-mediated exacerbation of neuronal synaptic disintegrity in amyloid-rich environments which contribute to Aβ-Teffs’ potential to accelerate memory impairment in APP/PS1 mice.Hippocampal neurogenesis correlates with memory function and neural plasticity; all affected during AD progression , 46. To s memory . ImmunobPrevious studies demonstrated a spectrum of effects for Aβ-reactive Teffs in animal models and AD patients , 48, 49.b-Aβ peptide tetramer reactive CD4+ T cells. Elevated Th1 and Th17 cytokines then weaken tight junctions of the BBB to allow peripheral inflammatory mediators, including TNFα, IL1β, and IL6, entrance into the brain serving to further activate resident microglia. We contend that reactive microglia contribute to aberrant adaptive immune responses by presenting cognate antigen and secreting pro-inflammatory cytokines [Systemic inflammation is operative in AD patients before the onset of memory impairment. This suggests an important role of peripheral immune activation in disease , 32, 33.ytokines . The dirPrevious studies reported decreased frequency and function of anti-inflammatory and immunosuppressive CD4+CD25+FOXP3+ Tregs in blood of AD patients , 58 and Microglia serve key roles in processing and presenting self-antigens including Aβ to T cells to maintain immune tolerance , 19. Non1–40 and Aβ1–42 as well as insoluble Aβ plaques without affecting full-length APP production. Aβ-Th17 cell treatment affected amyloid load and microglial responses in APP/PS1 mice brain to a lesser extent compared to Aβ-Th1 cells which might be due to their less extensive inflammatory responses as those induced by Aβ-Th1 cells in an amyloid-rich environment. The results were further supported by the previous study showing Th1 biased immune responses accountable for most of the CD4+ Teffs’ detrimental effects in APP/PS1 mice [In addition to affecting microglia responses, Aβ-Th1 cells significantly increased amyloid deposition in both cortex and hippocampus regions of brain as demonstrated by increased soluble AβPS1 mice . NeuroinPS1 mice  affectinPS1 mice , 9. FuncPS1 mice . AdoptivA previous study demonstrated that complete loss of peripheral adaptive immune system exacerbated amyloid deposition and neuroinflammation in 5XFAD-Rag mice and that reconstitution of this cell population restores AD pathology . LikewisDisease-reactive Aβ-Th1 and Aβ-Th17 cells transform a pro-inflammatory microenvironment to accelerate AD pathology in APP/PS1 mice. The detrimental effects of Aβ-Teffs are sped by limiting Treg activities in the periphery and CNS. Control of this neurodestructive environment represents a potential therapeutic strategy and can be sped by augmenting peripheral Treg numbers and function reactive against disease-specific protein.1–42 (50 µg)  emulsified in complete Freund’s adjuvant (Sigma Aldrich) containing Mycobacterium tuberculosis (1 mg/ml). The mice were boosted after 2 weeks with Aβ1–42 (50 µg) in incomplete Freund’s adjuvant (Sigma Aldrich). One week after the second immunization, the mice were killed, and spleen and lymph nodes  were harvested then mashed through 70-μm cell strainer to prepare single cell suspensions. The red blood cells were lysed with ammonium–chloride–potassium (ACK) lysis buffer . CD4+ T cells were enriched from single cell suspensions using an EasySep™ mouse CD4+ T cell isolation kit  and recovered CD4+ T cells  were cultured in vitro in the presence of monomeric Aβ1–42 (25 μg/ml) prepared as described before [7 feeder cells, i.e., splenocytes isolated from naïve female B6;129 mice and irradiated at 3200 Gy . Cells were propagated in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 25 mM HEPES, 1 mM sodium pyruvate, 1× nonessential amino acids, 55 nM 2-mercaptoethanol, 100 U/ml penicillin, and 100 µg streptomycin. Fresh culture media, Aβ, and feeder cells were provided weekly and the viable CD4+ T cells were counted before each passage. CD4+ T cells non-reactive to Aβ were eliminated over passages as evidenced by declined viable CD4+ cell count up to day 21 , and 3 µg/ml brefeldin A  for 12 h, then extracellular/intracellular staining was performed for flow cytometric analysis as described before [To induce cytokine production, 10d before . Cells w1–42 sequence, 1 to 15 amino acid region (DAEFRHDSGYEVHHQ) comprises B cell epitope while 15 to 30 amino acid region (KLVFFAEDVGSNKGA) comprises T cell epitope in the mice while in human it may be up to 15 to 42 amino acid region [1–42 presented by H-2b haplotypes (feeder cell matched), tetramers were constructed with I-Ab and the amino acid 15–30 (MHCII-IAb–KLVFFAEDVGSNKGA) conjugated to fluorophore BV421  Tetramer Core Facility, Emory University, Atlanta, GA). MHCII-IAb–PVSKMRMATPLLMQA tetramer with no Aβ specificity was used as control. For tetramer staining, 3 × 105 Aβ-Th1 or Aβ-Th17 cells were incubated with increased concentrations of MHCII-IAb–KLVFFAEDVGSNKGA Aβ tetramer  or MHCII-IAb–PVSKMRMATPLLMQA control tetramer (2.4 µg) for 3 h at 37 °C. After incubation, tetramer-stained T cells were reacted with anti-CD3e-PE and anti-CD4-APC-H7 antibodies for 30 min at room temperature, followed by live-dead staining with propidium iodide (0.5 µg/ml) for 5 min at room temperature. Stained T cells were analyzed with a LSR II flow cytometer and FACSDiva Software (BD Bioscience) at the University of Nebraska Medical Center Flow Cytometry Research Facility.From Aβd region . To eluc® mouse TCR a/b profiling kit . For the TCR sequence identification, PCR products generated were cloned into the pCR™ 4Blunt-TOPO® plasmid using Transform One Shot® Mach-T1™ competent cells following manufacturer’s instructions . Bacterial colonies were grown in LB agar media followed by DNA isolation and clean-up using monarch plasmid miniprep kit  and samples were submitted for Sanger sequencing with M13 primers at the University of Nebraska Medical Center Genomics Core Facility.Total RNA was isolated from the parent T cell clone (Aβ-Th1 cells) using RNAeasy mini kit . First strand complementary DNA (cDNA) was synthesized and TCRα and TCRβ chain variable region sequences were amplified using SMARTerbα with 256 residues (1–256) and MHCII-IAbβ with 264 residues (257–521). Aβ peptide had 42 residues (522–563), TCRα consisted of 274 residues (564–838) and TCRβ consisted of 305 residues (839–1144). The MHC–peptide residues were first modeled with the antibody modeling tools prediction and advanced homology modeling. The TCRα/β complex was modeled later and both models were analyzed separately for structural features. The single model was generated from the above-mentioned tools, the structural features were validated from the previously generated models. System preparation: All molecular dynamics (MD) simulations were performed on AMBER 18 software package [x = 192.98, y = 186.34 and z = 182.65. Total of 125,812 TIP3P water molecules were added to solvate the system [Unbiased explicit solvent MD simulation: Simulations were performed for 100 ns of time step on Nvidia V100-SXM2-16 GB Graphic Processing Unit using the PMEMD.CUDA module [The TCR-Aβ–MHCII (TCR–pMHC) complex was constructed with the fully automated structure preparation and homology modeling in the Schrodinger BioLuminate suite . The loo package . The pro package . The TCRe system . A suffie system . Unbiasedues 564–8 and TCRA module  was used6 Aβ-Th1 or Aβ-Th17 cells in 100 µl phosphate-buffered saline (PBS) were adoptively transferred to the APP/PS1 recipient mice, intravenously via tail vein using a 28-gauge needle affixed to a sterile tuberculin syringe, twice at 1-week intervals. Untreated and age-matched non-Tg mice served as control.All animal experiments were approved by the institutional Animal Care and Use Committee of University of Nebraska Medical Center. Transgenic mice overexpressing human APP695 with the Swedish mutation (Tg2576) were obtained from Drs. G. Carlson and K. Hsiao-Ashe through the Mayo Medical Venture . PS1 micTwo weeks after the second adoptive cell transfer, mice were submitted for radial arm water maze (RAWM) testing in a blinded fashion to assess memory impairment as previously described , 71. Bri1H magnetic resonance imaging (MRI)-compatible cradle using a bite bar. MRI was performed on a 7-Tesla scanner  with a Bruker-built quadrature mouse brain RF coil. Respiration and body temperature were monitored during scanning. A baseline glucose CEST (glucoCEST) MRI was acquired followed by 2DG (1 g/kg in PBS) injection via intraperitoneal catheter into the mice to monitor glucose signal in the brain over the time. GlucoCEST data were acquired using a rapid acquisition with relaxation enhancement (RARE) sequence (repetition time (TR)/echo time (TE) = 1600/16 ms, RARE factor = 8) with a continuous radio frequency (RF) for saturation with the power = 3 µT, duration = 1 s, saturation frequencies = − 1600 to 1600 Hz in steps of 80 Hz. A second CEST data with saturation RF power = 0.5 μT, and frequencies = − 300 to + 300 Hz were acquired for B0 inhomogeneity correction using WASSR [Mice were fasted for 24 h and fasting blood glucose concentrations were measured prior to the experiment using glucometer and test strips (ReliOn™ Prime) by collecting blood from tail vein puncture. Mice were anesthetized with isoflurane in the mixture of oxygen and the peritoneal cavity was cannulated for the injection of 2-deoxy glucose (2DG). Cannulated mice were fixed on a ng WASSR . The glu6 spleen or lymph node cells were stained for flow cytometric analysis as described above using antibodies: anti-CD3e-PE, anti-CD4-APC-H7, anti-CD8a-PE-Cyanine 5.5, anti-CD25-PE-Cy7 , anti-Tbet-eFluor 660, anti-RORγ-PerCp eFluor 710 and anti-FOXP3-Alexa Fluor 488 . For intracellular cytokine analysis, 1 × 106 spleen cells were stimulated with 20 ng/ml PMA and 1 µM ionomycin in presence of brefeldin A for 12 h and after incubation cells were stained with anti-CD3e-PE, anti-CD4-APC-H7, anti-IFNγ-FITC, anti-TNFα-eFluor 450, and anti-IL17a-Alexa Fluor 647 antibodies. To determine the frequency of Aβ reactive CD4+ T cells, 1 × 106 lymph node cells were stimulated with Aβ1–42 (25 μg/ml) in presence of feeder cells and IL2 (25 U/ml) for 5 days at 37 °C. On day 5, cells were collected by centrifugation and incubated with MHCII-IAb–KLVFFAEDVGSNKGA Aβ tetramer (6 µg) or MHCII-IAb–PVSKMRMATPLLMQA control tetramer (6 µg) for 3 h at 37 °C. After incubation, T cell-MHCII-Aβ tetramer complex was stained with anti-CD3e-PE, anti-CD4-APC-H7 antibodies and propidium iodide for flow cytometric analysis.On day 42, mice were terminally anesthetized with pentobarbital followed by spleen isolation in complete RPMI media and blood collection via cardiac puncture in the K3EDTA tubes . Lastly, mice were pericardially perfused with PBS and lymph nodes  and brain were harvested. Whole blood was stained while single cell suspension was prepared from spleen and lymph nodes. Either 50 μl of blood or 1 × 103 Tregs in 100 µl of media followed by addition of 50 × 103 CFSE-labeled Tresps into each well to obtain Tresp:Treg rations of 1:1, 1:0.5, 1:0.25 and 1:0.125, while wells with only Tresps served as controls. Mouse T cell activating CD3/CD28 Dynabeads  were added to each well at a bead:Tresp ration of 1:1 to induce Tresp proliferation. The immunosuppressive function of Tregs to inhibit proliferation of CFSE-stained Tresps was determined after 72 h incubation at 37 °C using flow cytometric analysis and is reported as Treg-mediated % inhibition.CD4+CD25+ Tregs and CD4+CD25− T responder cells (Tresps) were isolated from the mice spleen as described earlier  using Ea2-PCR array for Mouse Innate and Adaptive Immune Responses . Quantitative RT-PCR was performed using Mastercycler Realplex EP (Eppendorf) and data were analyzed using RT2 Profiler PCR array web-based data analysis software (Qiagen). Next, we conducted functional and pathway enrichment analysis of genes deregulated in APP/PS1 mice untreated or treated with Aβ-Teffs compared to non-Tg and APP/PS1 control mice. GSEA is a method to identify classes of genes or proteins that over-represented in a large set of genes or proteins [http://david.abcc.ncfcrf.gov/) [https://reactome.org/tools/reactome-fiviz) [http://string-db.org). KEGG is a database regarding genomes, biological pathways, diseases, drugs and chemical substances (http://www.genome/ad.jp/kegg) [http://www.reactome.org) is an open source, expert-authored, peer-reviewed, manually curated database of reactions, pathways and biological processes. STRING (http://string-db.org) database provides a critical assessment and integration of protein–protein interaction (PPI), including direct  and indirect  associations in a given organism [Hippocampal tissue was isolated from mouse brain and total RNA extracted with the RNAeasy mini kit . Recovered RNA was reverse transcribed into cDNA using a RevertAid First Strand cDNA Synthesis kit . One microgram cDNA was amplified using primer mix from the RTproteins . We condrf.gov/) , which ie-fiviz) , which ijp/kegg) . Reactomorganism .After transcardial perfusion, mouse brains were immediately harvested and divided into two hemispheres. The left was immediately frozen on dry ice for biochemical analysis and right was immersed in freshly depolymerized 4% paraformaldehyde in PBS for 48 h at 4 °C and cryoprotected by immersion in 15% then 30% sucrose for 24 h at 4 °C. Fixed brains were sectioned coronally with a Cryostat (ThermoFisher) with 30-μm-thick sections serially collected and stored at −80 °C. Immunohistochemistry was performed using antibodies against pan-Aβ , Iba1  and doublecortin (Dcx) . For immunodetection, biotin-conjugated anti-rabbit or anti-goat IgG secondary antibody was used followed by a tertiary incubation with Vectastain ABC Elite kit . 1% Thioflavin-S in 50% ethanol was used for counterstaining of compact amyloid plaque . For each of the immunohistochemical staining, six sections/slide were collected at eight intervals and were used for each of the experimental groups. Slides were masked and coded and Aβ occupied area was calculated using Cavalieri estimator probe (grid spacing 15 μm), while the number of Iba1-reactive microglia and Dcx-positive (Dcx+) neuroprogenitor cells were counted using the optical fractionator module of Stereo Investigator system (MBF Bioscience) as described earlier [g for 20 min at 4 °C, the supernatant collected and total protein quantified using micro-BCA kit . For Western blots, 80 μg or 20 μg of tissue protein was incubated with β-mercaptoethanol containing Laemmli buffer at 100 °C for 5 min, followed by electrophoresis on SDS-polyacrylamide gel and transferred to polyvinylidene fluoride membrane . The membranes were blocked in 5% skim milk/TBST and incubated with primary antibodies to 22C11 , 6E10 , arginase 1 , iNOS , synaptophysin , PSD95  and β-actin  at 4 °C overnight, followed by 60 min incubation in 5% skim milk/TBST with horseradish peroxidase-conjugated anti-rabbit, mouse, or goat secondary antibodies . Immunoreactive bands were detected using SuperSignal West Pico or Femto Chemiluminescent substrate, and images were captured using an iBlot750 Imager (Thermo Fisher Scientific). Immunoblots were quantified using ImageJ software (NIH) relative to β-actin expression.Brain cortical tissues were homogenized using lysis buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 50 mM EDTA, 1% Triton X-100, and a mixture of protease/phosphatase inhibitor . The lysate was centrifuged at 20,000×g for 20 min at 4 °C and the supernatant was analyzed for human Aβ1–40 and Aβ1–42 by ELISA  according to the manufacturer’s instructions.Mouse cortical tissues were homogenized in 50 mM Tris–HCl (pH 7.6) containing 150 mM NaCl and a protease/phosphatase inhibitor mixture. The homogenate was centrifuged at 20,000×p ≤ 0.05 was regarded as a significant difference.All data were normally distributed and presented as mean values ± standard errors of the mean (SEM). Comparisons of means between groups were analyzed by one-way ANOVA or two-way repeated measures ANOVA followed by Newman–Keuls post hoc test using GraphPad Prizm software version 8.0 . A value of Additional file 1: Figure S1. Development of monoclonal Aβ-Th1 and Aβ-Th17 cells. Figure S2. Modelling and explicit solvent molecular dynamics simulations. Figure S3. Adoptive transfer of Aβ-Teffs did not affect T cell frequency in APP/PS1 mice. Figure S4. Transcriptomic analysis of immune genes. Figure S5. Western blot images.


Some necessary and sufficient conditions for domains of attraction of multivariate extreme value distributions are shown by using dependence functions. The joint asymptotic distribution of multivariate extreme statistics is also shown. Multivariate extreme value distributions have been studied by many authors, and their contributions are summarized by Galambos  and ResnIn this paper, basic arithmetical operations arc always meant componentwise ,j=1,2,…,n, be a sample of size n, of a k-dimensional random vector with a distribution function F(x), The i-dimensional distribution function of the components jX1,jX1,…,jX1 will be denoted jF1j2−j1j−F(r)(jx(i)). We shall also use the notation k−1 and P∈, let F−1 (p)=inf {x:F (x)≥P}.Let , where Zin=max{iXl,…,inX}, i=1, 2,…k, and let us call nZ a multivariate extreme statistic.Let na>0, nb∈kR, n=1, 2,… such that (n−bnZ)/na converges in distribution to a random vector U with a nondegenerate distribution H , then F is said to be in the domain of attraction of H, F∈D(H) by symbol, and H is said to be a multivariate extreme value distribution. The convergence in distribution is equivalent to the conditionx, because multivariate extreme value distributions are continuous.If there exist We shall need the following lemma to prove a proposition in Sec. 2.Lemma 1.1Equation (1)is equivalent tofor all x such that 0<H(x)<I. k-dimensional distribution F,F. In this section, we derive necessary and sufficient conditions for domains of attraction in terms of the dependence function.For any Proposition 2.1i, iLet F be a k-dimensional distribution and let H be a multivariate extreme value distribution with univariate marginals H= l,… k Then the following statements are equivalent:F∈D(H).iF∈D (H1), i=1,…,k, andiF∈(iH) i=1,…,k, andiF∈D(iH), i=1,…,k, andProof, The proof is straightforward from Lemma 1.1, Theorem 5.2.3 and Lemma 5.4.1 of Galambos [Proposition 2.2i, iLet F be a k-dimensional distribution and let H be a multivariate extreme value distribution with univariate marginals H=1,…,k.F∈D (H) iif and only if F∈(iH), i= 1,…,k, and the functionsfor each fixed vector J(i)(i>1) and for all y∈1are finite, and the functionis a dependence function of H.The following inequalities hold.for a nonnegative integer r, wherenand D is a dependence function of a multivariate extreme value distribution.Proof. It is easily seen that for all s>0,Example 2.1 , wherex)=exp(−e−x).Proposition 2.3Let F and G be k-dimensional distri-butions and let H be a multivariate extreme value distribution.If, G∈D(H), thenIf F∈D (H), G1∈D(H1) i−1,…,k. andthen G∈D(H).H be a multivariate extreme value distribution with univariate marginals i, i=H1,…,k, Let *H;(x)=H1(x1)…kH(kx);and *H(x)=min{tH(tx), i=1 ,…, k}, then it holdsx∈kK. Both bounds, *H and H*, are multivariate extreme value distributions. Characterizations of these distributions are obtained by Takahashi [Let akahashi .F∈D(*H) and F∈D(*H). In this section we generalize his results.In the bivariate case Sibuya  obtains Proposition 3.1i be a univariate extreme value distribution, iLet F be a k-dimensional distribution and let H=1,…,k. Then the following statements are equivalent:F∈D(*H).iF∈D (iH), i=1,…,k and there exists y∈ksuch thatiF∈D(iH), i=1,…,k, andi∈D(iH). i=1,…,k, andFProof. The proof is straightforward from Theorems 2.2 and 4.1 and Corollary 2.4 of Takahashi [akahashi . □Remark. If k=2, we have the same result as Proposition 3.1 by Corollary 2.2 of Takahashi [akahashi .Example 3.1 , where *H=Λ(·)Λ(·).Proposition 3.2i be a univariate extreme value distribution, iLet F be a k-dimensional distribution and let H=1,…,k. Then the following statements are equivalent:F∈D(*H).iF∈D(iH), i=1,…,k, and there exists y∈ such thatiF∈D (iH), i=1,…,k. andProof. The proof is straightforward from Theorem 3.1 and Corollary 3.1 of Takahashi [akahashi . □In this section, we show the joint asymptotic distribution of several multivariate extreme statistics along the arguments in Sec. 2.3 of Leadbetter et al. . For simX1Y1),…, be a sequence of independent random vectors with common distribution F. The order statistics of the components will be denoted byi−0,1,…,r=1, definen i an (i+1)-th multivariate extreme statistic.Let >x2=.whereandProof. Definei,j,k,m=0,1,Thus we have the result. □Corollary 4.1Suppose thatfor some nondegenerate distribution H, Then, for fixed r≥1 and x1>…rxwhereis the (i+1)-th multivariate extreme statistic from the distribution H, i=0,…, r−1, nand H(αnx+βn)=H (x), n=1,2,…Example 4.1 Let F be the bivariate normal distribution with the correlation coefficient less than one. Theni+1)-th multivariate extreme statistic from the bivariate exponential distribution whose marginals are equal to the standard exponential distribution and they are independent. For the univariate case, it is a well known result (see Weissman [Weissman , Theorem


Among all the tested compounds, compound 5c with naphthalen-1-yl moiety exhibited the most potent antiproliferative activity against MCF-7 and A549 cell line, with IC50 values of 0.51 ± 0.03 µM and 0.33 ± 0.01 µM, respectively. The results of tubulin polymerisation assay shown that 5c exhibited a significant ability to inhibit tubulin polymerisation with IC50 value of 2.8 μM. Consistent with its antitubulin activity, 5c can significantly arrest the cell cycle at G2/M phase and induce apoptosis in MCF-7 cancer cells. Molecular docking study indicated that compound 5c inhibited tubulin polymerisation through interacting at the colchicine-binding site of tubulin. Furthermore, 5c exhibited low cytotoxic activity on human normal cell line.A new series of sulphonamide derivatives bearing naphthalene moiety were synthesised and evaluated for their antiproliferative and tubulin polymerisation inhibitory activities. These new compounds were evaluated for their  Then, condensation of compound 4 with appropriate commercially available aryl sulphonyl chloride in the presence of Et3N and DMAP in THF to generate the title compounds (5a-5e) in high yields. On the other hand, treatment of 3,4,5-trimethoxyaniline 1 with naphthalene-1-sulphonyl chloride 6 in the presence of Et3N in CH2Cl2 to afforded key intermediate 7, which reacted with commercially available benzyl halide in the presence of KI and K2CO3 to provide the title compounds (8a-8i). All target derivatives were fully characterised by 1H NMR, 13 C NMR, HRMS, and elemental analysis (see Supporting Information).The sulphonamide derivatives (5c was determined to be C27H27NO6S by high-resolution mass spectrum (HRMS) peak at m/z 516.1432 as [M + Na]+. The 1H NMR spectrum , 3.53 , and 3.63  ppm due to four methoxy groups on the phenyl rings. The methylene protons of –CH2– was appeared as singlet at δ 4.69 ppm . Two doublet peaks at δ 6.75 and δ 7.09 ppm with coupling constant of 8.4 Hz were attributed to the C-3′,5′-H and C-2′,6′-H of 4-methoxybenzyl moiety, respectively. According to the aromatic proton signal at at δ 6.11 ppm , there was a 3,4,5-trimethoxyphenyl in the structure. Seven aromatic hydrogen atoms of naphthalen-1-yl were appeared at δ 7.46–8.26 ppm . In 13 C NMR spectrum of 5c the signal at δ 53.37 ppm (C-11) was assigned to the carbon in methylene. The signals at δ 55.50 (OCH3-C-4′), 56.20 , and 60.61 (OCH3-C-4′′) ppm were attributed to the carbon in four methoxyl groups. The carbon of –CH2– was appeared at δ 53.37 ppm (C-11). In addition, there are 17 carbon signals at δ 107.41 , 114.18 , 125.24 (C-3), 125.36 (C-8), 127.40 (C-6), 128.15 (C-7), 128.59 (C-1′), 128.83 (C-9), 129.35 (C-5), 130.24 , 131.17 (C-2), 133.95 (C-1), 134.08 (C-1′′), 134.32 (C-10), 135.17 (C-4), 137.59 (C-4′′), 152.80 , 159.10 (C-4′).The molecular formula of compound δ 8.05 (H-5) to C-6, C-9 and C-4, from δ 7.65 (H-3) to C-1 and C-10, from δ 7.48 (H-7) to C-9 indicated the presence of a 1-naphthalene sulphonyl moiety. In addition, the correlations from the methoxy proton at δ 3.63 (H-OCH3-C-4′) to C-4′, from δ 3.33  to C-3′′ and 5′′, from δ 3.53 (H-OCH3-C-4′′) to C-4′′ and C-3′′ showed four methoxy substituents connected to C-4′, C-3′′, 5′′, and C-4′′, respectively. Furthermore, key HMBC correlations between δ 4.69 (H-11) and C-2′, C-6′ and C-1′′ showed that 4-methoxybenzyl and 3,4,5-trimethoxyphenyl were linked through N. Therefore, the spectral data were in full agreement with the expected structure of the compound 5c (Table S2).The HMBC correlations from the aromatic proton at 2.2.in vitro antiproliferative activities of these sulphonamide derivatives (5a-5e and 8a-8i) were evaluated against MCF-7 human breast cancer cells and A549 human non-small cell lung carcinoma cells by using CCK-8 method. Cisplatin, 5-fluorouracil (5-Fu), tamoxifen, and combretastatin A-4 (CA-4) were used as positive control. The results expressed as IC50 (µM) were summarised in 5a, 5c, 5e, and 8 b showed potent anticancer activity against both MCF-7 and A549 cell lines with IC50 between 0.33 ± 0.01 and 5.34 ± 0.31 µM. All these compounds were more potent than cisplatin (IC50 = 11.15 ± 0.75 µM), 5-Fu (IC50 = 11.61 ± 0.60 µM), tamoxifen (IC50 = 14.28 ± 0.40 µM), and CA-4 (IC50 = 5.55 ± 0.11 µM) on MCF-7 cancer cell line. In particular, compound 5c with a methoxyl group at the para position of phenyl ring and naphthalen-1-yl at the sulphonamide exhibited the most potent anticancer activity against MCF-7 and A549 cell lines .The 5a-5e, it can be seen that the aryl substituents on sulphonamides affected on antiproliferative activity of this class of compounds. Among these molecules, 5 b with a phenyl ring displayed low antiproliferative activity with IC50 value of > 30.0 µM. Introduction of electron-donating (5a and 5e) groups into the phenyl ring, results in significantly increased the antiproliferative activity. The replacement of phenyl ring with naphthalen-1-yl or naphthalen-2-yl led to compounds 5c and 5d, resulting in significantly increased the antiproliferative activity. In particular, compound 5c with naphthalen-1-yl moiety was found to be the most active compound in this series. These inhibitory results indicate that naphthalen-1-yl group seems to be the optimal substituent on the position.To study the structure-activity relationships (SAR) of this class of compounds, the aryl substituents on sulphonamides were discussed firstly. Based on the antiproliferative activity of compounds 8a-8i). Shifting methoxy group to the 3- position (8a) decreased the inhibitory activity. The replacement of the 4-methoxy (5c) with 4-methyl group (8 b) resulted in a slightly decrease of the inhibitory activity. Introduction of electron-withdrawing groups such as 3-fluoro (8c), 4-bromo (8d), 4-chloro (8e), 3-bromo (8f), 4-fluoro (8 g), 4-cyano (8 h), and 2-nitro (8i) into the phenyl ring, resulted in dramatically decrease the inhibitory activity. Compared the inhibitory activity of 5c (4-methoxy) and 8d (4-bromo) with 8a (3-methoxy) and 8f (3-bromo), the results were shown that the substituent group on the 4-position of the phenyl ring is more beneficial to the antiproliferative activity. The detailed SARs were summarised in To explore the effect of the substituents in phenyl ring on the inhibitory activity, various substituents including 3-methoxy, 4-methyl, 3-fluoro, 4-bromo, 4-chloro, 3-bromo, 4-fluoro, 4-cyano, and 2-nitro were introduced into the targeted compounds . The result was shown that compound 5c exhibited moderate cytotoxic activity against human normal liver cell (LO2) with IC50 value of 12.73 ± 3.26 µM. While, 5c displayed potent anticancer activity against MCF-7 and A549 cell lines with IC50 value of 0.51 ± 0.03 and 0.33 ± 0.01 µM, respectively. Hence, we could conclude that these compounds have good safety for potential application in the treatment of tumour cells.In order to verify the safety profile of this class of compounds, the most potent compound 2.3.in vitro tubulin polymerisation inhibitory activity of 5c was evaluated using tubulin polymerisation assay 5c or colchicine. The results shown that 5c exhibited a significant ability to inhibit tubulin polymerisation in a concentration-dependent manner with IC50 values of 2.8 µM, as compared to colchicine (IC50 = 9.3 µM). Besides, 5c and colchicine have similar effects on inhibit tubulin polymerisation, indicating that 5c was a microtubulin-destabilizing agent.To examine whether tubulin is the target of this class of compounds, the 2.4.,5c on the cell cycle of MCF-7 cancer cells was evaluated by using flow cytometry. As shown in 5c , the G2/M population in MCF-7 cancer cells increased from 21.19% (control) to 30.77% (0.125 µM), 57.50% (0.25 µM), and 82.22% (0.5 µM), respectively. The results indicate that compound 5c can arrest cell cycle at G2/M phase in a dose-dependent manner.Due to microtubules play an important role in eukaryotic cell division, tubulin polymerisation inhibitors can disrupt regulated cell cycle distribution and block the cell cycle in G2/M phase 2.5.,5c on cell apoptosis in MCF-7 cancer cells. As shown in 5c in the concentration of 0.125 µM, 0.25 µM, or 0.5 µM, the total numbers of early (the lower right quadrant) and late apoptotic cells (the upper right quadrant) were 18.67%, 33.1% and 50.3%, respectively, whereas that of control was only 8.4%. These results indicated that compound 5c effectively induced cell apoptosis in MCF-7 cells in a dose-dependent manner.Since many literatures have reported that tubulin polymerisation inhibitors are able to induce cellular apoptosis 2.6.5c with tubulin were performed. Colchicine was first docked into the colchicine binding site of tubulin. The result was shown that co-crystallized conformation of colchicine was reproduced approximately (RMSD: 1.10 Å), indicating that this protocol of molecular docking is credible. Then, the theoretical binding mode between 5c and tubulin was investigated, and the estimated binding energy was −9.6 kcal·mol−1. As shown in 5c adopted an “Y-shaped” conformation in the colchicine pocket of tubulin. Compound 5c located at the hydrophobic pocket, surrounded by the residues A/Ala-180, A/Val-181, B/Leu-248, B/Ala-250, B/Leu-255, B/Ala-316, B/Val-318, and B/Ala-354, forming a strong hydrophobic binding. Detailed analysis showed that the phenyl and naphthyl groups of 5c formed cation-π interactions with the residues Lys-254 and Lys-352, respectively. All these interactions helped 5c to anchor in the colchicine binding site of tubulin.To elucidate the binding mode of this class of compounds, molecular docking simulations of compound 2.7.5c and tubulin, molecular docking and molecular dynamics simulations were performed using the AutoDock vina 1.1.2 and Amber12 software package. The preferential binding mechanism of tubulin with 5c was determined by 30-ns molecular dynamics simulations based on the docking results. To explore the dynamic stability of the models and to ensure the rationality of the sampling strategy, the root-mean-square deviation (RMSD) value of the protein backbone based on the starting structure along the simulation time was calculated and plotted in To explore the potential binding mode between 5c and tubulin was shown in 5c adopted a compact conformation in the pocket of tubulin. The naphthyl group of 5c located at the hydrophobic pocket, surrounded by the residues A/Ala-180, A/Val-181, B/Val-238, B/Leu-248, B/Ala-250, B/Leu-255, B/Ala-316, B/Ala-317, B/Val-318 and B/Ala-354, forming a strong hydrophobic binding. Detailed analysis showed that the phenyl group of 5c formed a cation-π interaction with the residue Lys-254. It was shown that the residue Lys-254 (bond length: 2.7 Å) formed a hydrogen bond with 5c, which was the main interaction between 5c and tubulin. All in all, the above molecular dynamics simulation gave us rational explanation of the interaction between 5c and tubulin, which provided valuable information for further development of tubulin polymerisation inhibitors.The theoretical binding mode between 3.1H NMR, 13 C NMR, HRMS, and elemental analysis. All of the title compounds were screened for antiproliferative activity against human breast cancer cells (MCF-7) and human non-small cell lung carcinoma cells (A549) by using CCK-8 method. Among all synthesised compounds, compound 5c with naphthalen-1-yl moiety exhibited the most potent antiproliferative activity against MCF-7 and A549 cell line, with IC50 values of 0.51 ± 0.03 µM and 0.33 ± 0.01 µM, respectively. SAR studies suggested that the naphthalen-1-yl and 4-methoxybenzyl at the sulphonamide played an important role for the potent antiproliferative activity. Tubulin polymerisation assay revealed that compound was a microtubulin-destabilizing agent with IC50 value of 2.8 µM. Further mechanism evaluation demonstrated that 5c can significantly arrest the cell cycle at G2/M phase and induce apoptosis in MCF-7 cancer cells. Additionally, molecular modelling results showed that 5c binds well to the colchicine-binding site of tubulin. Hence, these results suggest that 5c could be used as a promising lead compound for further investigation in anticancer drug development.In summary, a new series of sulphonamide derivatives bearing naphthalene moiety have been synthesised and and characterised by 4.4.1.1 (10 mmol) and Et3N (20 mmol) in THF (50 ml) were added 4-methoxybenzoyl chloride 2 (10 mmol) and the reacting mixture was stirred at room temperature for 4 h. Then, the solvent was removed under reduced pressure, and water was added to the reaction mixture and extracted 3 times with ethyl acetate. The combined organic layers were dried, filtered, and concentrated. The residue was purified by chromatography on silica gel with EtOAc/petroleum ether = 1:1 to give compound 3 as white solid (yield 88%).To a solution of 3,4,5-trimethoxyaniline 4.2.4 (2 mmol) in anhydrous THF (10 ml) was stirred in an ice bath for 10 min. Then, a solution of compound 3 (1 mmol) in 10 ml of THF was added dropwise to the mixture. After completion of dropwise addition, the mixture was stirred at 0 °C for 30 min then refluxed at 70–80 °C for 2 h. After completion of the reaction, the solvent was evaporated and the organic product was extracted with ethyl acetate. The combined organic layers were dried over sodium sulphate and evaporated to afford black oil, which was used directly for the next step without further purification.A mixture of LiAlH4.3.4 (2 mmol), DMAP (0.2 mmol), and Et3N (2 mmol) in THF (50 ml) was added commercially available aryl sulphonyl chloride (2 mmol) and the reacting mixture was stirred at room temperature for 12 h. The solvent was evaporated and water was added to the reaction mixture. The organic material was extracted with ethyl acetate, and the organic layer was dried over Na2SO4 and concentrated under vacuum. The residue was purified by chromatography to give the title product 5a-5e.To a solution of compound 4.4.1 (10 mmol) and Et3N (10 mmol) in CH2Cl2 (100 ml) was added naphthalene-1-sulphonyl chloride 6 (10 mmol) and the mixture was stirred at room temperature for 6 h. Then, the solution was diluted with CH2Cl2 and washed with water. After the concentration of the solution, the residue was purified by silica gel column chromatography to give compound 7 as white solid (yield = 43%).To a solution of 3,4,5-trimethoxyaniline 4.5.7 (1.0 mmol), K2CO3 (2.0 mmol), KI (1.0 mmol), and commercially available benzyl halide (1.0 mmol) in acetone (10 ml) was stirred at reflux for 5 h. After completion of the reaction, the mixture was concentrated under reduced pressure and the residue was purified by chromatography to give the title compounds 8a-8i.A mixture of Click here for additional data file.


Brassica nigra plants showing symptoms caused by root-knot nematodes were detected in the municipality of La Joya, Arequipa Province, Peru. Based on morphology, esterase phenotypes, and species-specific characterized amplified region (SCAR) sequence, the causal agent was identified as Meloidogyne incognita. Pathogenicity was confirmed by a modified version of Koch’s postulates. To our knowledge, this is the first report of M. incognita infecting Brassica nigra in Peru. Brassica nigra (L.) W.D.J. Koch (black mustard) is important as a crop plant; it may have contributed to the evolution of several species in the genus Brassica . Among the plant-parasitic nematodes, the most important genus is In February 2020, many nematode galls  were obsn = 36 females) (n = 30), females (n = 10), and perineal patterns (n = 15); species molecular identification was confirmed by PCR species-specific characterized amplified region (SCAR) sequence for confirmation, using a primer set composed of inc-K14-F (5′-GGGATGTGTAAATGCTCCTG-3′) and inc-K14-R (5′-CCCGCTACACCCTCAACTTC-3′)  ; morpholCTTC-3′) .L) = 360.7 ± 21.3 (310-476) μm, a = 22.1 ± 4.3 (20.3-25.5), c = 8.4 ± 0.5 (4.9-9.5), stylet length = 11.3 ± 0.5 (9.1-12.2) μm, dorsal esophageal gland orifice to base of stylet (DGO) = 2.5 ± 0.4 (1.7-2.6) μm, tail length = 39.5 ± 1.0 (39.0-48.5) μm and hyaline tail terminus = 10.5 ± 0.5 (9.5-11.2) μm. Morphometrics of females: L = 655.5 ± 30.0 (550.4.5-700.5) μm, stylet length = 14.5 ± 0.5 (12.1-15.4) μm, and DGO = 3.5 ± 0.2 (2.8-4.0) μm. The perineal pattern of the female included a high and square dorsal arch with wavy striae bending toward area the lateral lines and the absence of distinct lateral line incisures. The overall morphology and morphometrics of this population appears similar to that of Meloidoyne incognita  of J2s: length  typical of ncognita . The PCRncognita .Brassica nigra plantlets were maintained in pots with 2 liters sterilized soil. In total, six replicates were inoculated with 3,000 eggs and J2s from the original population of M. incognita, in addition to a non-inoculated control. Eggs and J2 were extracted from infected roots using the 0.5% NaOCl (M. incognita.In greenhouse tests, 5% NaOCl  method aM. incognita parasitizing Brassica nigra in Peru. This finding may be important to Peruvian agriculture, since this nematode may damage black mustard plants becoming an additional problem for this crop. Additional surveys of black mustard may warrant the development of adequate control strategies for root-knot nematode if this nematode is widespread.To our knowledge, this is the first report of


The crystal structure of methanol imidazole featuring a tri-molecule hydrogen-bonded macrocycle is described. H-imidazole-1-methanol, C4H6N2O, are described. This compound comprises an imidazole ring with a methanol group attached at the 1-position affording an imine nitrogen atom able to receive a hydrogen bond and an alcohol group able to donate to a hydrogen bond. This imidazole methanol crystallizes with monoclinic (P21/n) symmetry with three symmetry-unique molecules. These three molecules are connected via O—H⋯N hydrogen bonding in a head-to-tail configuration to form independent three-membered macrocycles.The synthesis and the crystal structure of 1 N-heterocyclic carbenes (NHCs) , which are connected via head-to-tail hydrogen bonding. While each molecule is identical in formula and connectivity, it is the torsion angle of the methanol substituent with respect to the imidazole ring that distinguishes the unique symmetry of the three molecules. These deviations of torsion angle are isolated conformations essential to achieve the three-molecule hydrogen-bonded macrocycle as seen in Fig. 11, 2, and 3 to correspond with O1, O2, and O3 with the respective colors of light green, orange, and blue. The torsion angle is measured from the oxygen, methylene carbon, imidazole nitrogen, and atom C2 of the imidazole ring (the carbon between the two nitrogen atoms of the ring). The measured torsion angles are −102.0 (2), −80.2 (2), and 90.6 (2)° for molecules 1 (O1—C1—N1—C2), 2 (O2—C5—N3—C6), and 3 (O3—C9—N5—C10), respectively. Note that the blue molecule 3 has the methanol substituent rotated positively while the others are negative to achieve the geometry necessary to complete the hydrogen-bonded superstructure. While the isolated torsion angles observed are imparted by the hydrogen bonding, none of the bond angles, bond lengths, or torsion angles throughout each of the three asymmetric molecules is unusual when compared with structures deposited with the Cambridge Crystallographic Database accessed via Mogul  Å for O1—H1⋯N4 (light green to orange), 2.753 (2) Å for O2—H2A⋯N6 (orange to blue), and 2.715 (2) Å for O3—H3A⋯N2 (blue to light green). The resulting triangular supramolecule has a rough diameter of 9 to 9.5 Å and height of about 2.6 Å . A centroid was first calculated for the imidazole ring of molecule 1 using N1, C2, N2, C3, and C4; the distance from centroid-to-centroid across the inversion center is 3.7219 (3) Å and is within typical values for π-stacked systems  only AGAWIQ is also a substituted imidazole with a methylene linker to a possible hydrogen bond acceptor/donor. Because the methanol substituent for this molecule is at C4 of the imidazole ring, the same cyclic supermolecule does not form for this molecule even though hydrogen bonding is exhibited. One molecule forms the asymmetric unit for AGAWIQ with the hydroxyl oxygen being both a hydrogen-bond donor and acceptor to two different adjacent imidazole nitrogen atoms behaving as acceptor, non-protonated N, and donor, protonated N, respectively. The title molecule is not capable of this highly interconnected hydrogen-bonding network due to the methanol substitution being at N1 of the imidazole ring, removing the possibility of a nitrogen hydrogen-bond donor. The remaining seven hits included 3-methyl-3-pyrazolin-5-one  and stored at 277 K. The resulting product was analyzed at room temperature on a Bruker Avance II 400 MHz NMR spectrometer for 1H NMR, a Bruker Alpha II ATR for FT–IR, and Electrothermal Meltemp for melting point. 1H NMR : 5.40 , 6.93 , 7.08 , 7.34 . FT–IR (ATR): ν (cm−1) = 3135 (m), 3109 (m), 2811 (m), 2681 (m), 2492 (w), 1618 (w), 1509 (s), 1472 (m), 1459 (m), 1396 (m), 1342 (w), 1279 (m), 1229 (m), 1214 (s), 1107 (m), 1062 (s), 1036 (sh), 923 (m), 870 (w), 815 (m) 759 (m), 723 (s), 654 (m), 623 (m), and 439 (w), m.p. 328–330. A clear, colorless plate-like crystal of C4H6N2O was grown from a dioxane reaction mixture slurry and mounted on MiTeGen loop with Parabar oil.The synthesis of 1 al. 2010. Under aUiso(H) values were set to a multiple of Ueq(C) with 1.2 for CH and 1.5 Ueq(OH) for OH.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989022002614/pk2662sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989022002614/pk2662Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022002614/pk2662Isup3.cmlSupporting information file. DOI: 2157074CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


We have recently shown that a combined solution containing alpha-ketoglutarate (aKG) and 5-hydroxymethyl-furfural (5-HMF) might have anti-tumoral potential due to its antioxidative activities. The question arises if these substances have caspase-3- and apoptosis-activating effects on the cell proliferation in Jurkat and HF-SAR cells. Antioxidative capacity of several combined aKG + 5-HMF solution was estimated by cigarette smoke radical oxidized proteins of fetal calf serum (FCS) using the estimation of carbonylated proteins. The usage of 500 µg/mL aKG + 166.7 µg/mL 5-HMF showed the best antioxidative capacity to inhibit protein modification of more than 50% compared to control measurement. A Jurkat cell line and human fibroblasts (HF-SAR) were cultivated in the absence or presence of combined AKG + 5-HMF solutions between 0 µg/mL aKG + 0 µg/mL 5-HMF and different concentrations of 500 µg/mL aKG + 166.7 µg/mL 5-HMF. Aliquots of Jurkat cells were tested for cell proliferation, mitochondrial activity, caspase activity, apoptotic cells and of the carbonylated protein content as marker of oxidized proteins in cell lysates after 24, 48, and 72 h of incubation. The combined solutions of aKG + 5-HMF were shown to cause a reduction in Jurkat cell growth that was dependent on the dose and incubation time, with the greatest reductions using 500 µg/mL aKG + 166.7 µg/mL 5-HMF after 24 h of incubation compared to 24 h with the control , as well as after 48 h  and after 72 h . Cell growth was totally inhibited by the 500 µg/mL AKG + 166.7 µg/mL solution between 0 and 72 h of incubation compared to 0 h of incubation for the control. The mitochondrial activity measurements supported the data on cell growth in Jurkat cells: The highest concentration of 500 µg/mL aKG + 166.7 µg/mL 5-HMF was able to reduce the mitochondrial activity over 24 h (58.9%), 48 h (28.7%), and 72 h (9.9%) of incubation with Jurkat cells compared not only to the control incubation, but also to the concentrations of 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 375 µg/mL aKG 125 µg/mL 5-HMF, which were able to significantly reduce the mitochondrial activity after 48 h (28.7% or 35.1%) and 72 h (9.9% or 18.2%) compared to 24 h with the control (100%). A slight increase in cell proliferation was found in HF-SAR using the highest concentration (500 µg/mL aKG + 166.7 µg/mL 5-HMF) between 0 h and 72 h incubation of 140%, while no significant differences were found in the mitochondrial activity of HF-SAR in the absence or presence of several combined aKG + 5-HMF solutions. The solutions with 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 250 µg/mL aKG + 83.3 µg/mL 5-HMF showed a significantly higher caspase activity (51.6% or 13.5%) compared to the control (2.9%) in addition to a higher apoptosis rate (63.2% or 31.4% vs. control: 14.9%). Cell lysate carbonylated proteins were significantly higher in Jurkat cells compared to HF-SAR cells (11.10 vs. 2.2 nmol/mg). About 72 h incubation of Jurkat cells with 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 250 µg/mL aKG + 83.3 µg/mL 5-HMF reduced significantly the carbonylated protein content down to 5.55 or 7.44 nmol/mg whereas only the 500 µg/mL aKG + 166.7 µg/mL 5-HMF solution showed a significant reduction of carbonylated proteins of HF-SAR (1.73 nmol/mg). Clinical and pre-clinical studies of an anti-tumoral solution containing aKG, 5-HMF, N-acetyl-selenomethionine, and N-acetylmethionine for treating tumors ,2 showedThe reduction of oxidatively modified proteins generated by cigarette smoke  demonstrThe compound 5-HMF occurs in honey and apple juice and in even higher rates in dried fruits, caramel products, and coffees . BecauseGiven these reports, the present study was intended to check whether the addition of a combined solution of aKG + 5-HMF to leukemic cells would exert anti-tumoral, anti-proliferative, and apoptotic effects compared to HF-SAR.® Cell Culture; Corning Incorporated, 14831 New York, NY, USA) were used.Media: RPMI 1640, FBS, DMEM, antibiotic–antimycotic solution ; reagents: Cytofix–Cytoperm permeabilization Kit , FITC Active Caspase-3 Apoptosis Kit , WST-1 Cell Proliferation Reagent ; chemicals: alpha-ketoglutarate (Sigma), 5_hydroxymethyl-furfurale , guanidine-HCl, butyl-hydroxy-toluene , di-nitro-phenyl-hydrazine (DNPH) , and flasks  were dissolved in 10 mM DNPH containing 6 M guanidine pH 2.5 and incubated for 45 min at room temperature. Protein damage was measured spectro-photometically with the protein carbonyl assay according to Levine et al. [In vitro protein damage of FCS proteins (=control measurement) by cigarette smoke was assessed according to this setup (4): 50 mg/mL serum protein was dissolved in phosphate buffered saline. Total of 4 mL of protein solution was transferred to a suction bottle, which was connected to a water pump. Before smoking, two 100 µL aliquots of protein-solution were diluted with 1150 µL 10 mM PBS, pH 7.4, containing 40 µM BHT to obtain a 4 mg/mL BSA solution. About 1 mg of protein was precipitated by pipetting 250 µL of the 4 mg/mL protein solution with 250 µL 20% trichloric acid (TCA) solution. After smoking within 2 min further aliquots were diluted and precipitated with TCA. The glass was closed and incubated at 37 °C. After 15, 30, and 60 min aliquots were taken out, diluted, and precipitated. All precipitated samples (1 mg) were centrifuged at 5000× e et al. . Protein2 supply. The cell density was adjusted to about 1–2 × 106 cells/mL for all of the further experiments. The HF-SAR cell line was established from the normal human skin of a 2-year-old male [2 supply. The cell density was adjusted to about 1 × 106 cells/mL for the estimation of mitochondrial activity.The Jurkat-J6 cell line was procured from the National Center for Cell Sciences , and it had confluency > 80%. It is an immortalized line of human T-lymphocyte cells derived from acute T-cell leukemia. It is used to study acute T-cell leukemia. Jurkat-J6 cell line has the ability to produce IL-2 and that is why it is useful for different anti-cancer study . It was old male . The HF-® Cell Counter . Aliquots were used for further experiments.Cell proliferation experiments were carried out by incubating different aKG/5-HMF combinations  in an appropriate medium for 24, 48, and 72 h with the cells. The cell proliferation was estimated with the CASYA cytotoxicity assay (WST assay) was carried out to determine the viability of the Jurkat and HF-SAR cell lines. The WST-1 Cell Proliferation Reagent provided a simple and accurate method for measuring cell proliferation. The assay was carried out as described in the manufacturer’s instructions. In brief, cells  were seeded onto transparent 24-well plates and incubated for 0, 24, 48, and 72 h in the absence or presence of an aKG + 5-HMF combination . The cells were then washed twice with DPBS 1X and incubated in a fresh medium with 10% WST-1 reagent for 2 h. The absorbance was measured at 450 nm (690 nm was used as a reference wavelength and subtracted) in a spectrophotometric reader . 6 cells/mL was centrifuged (350× g) and re-suspended in PBS; the supernatant was removed, and the cells were washed again with cold PBS and then centrifuged (350× g). After removing the supernatant, the pellet was re-suspended in 500 µL of Cytofix–Cytoperm solution and incubated on ice for 20 min. After incubation for 72 h in the absence of any substances (negative control), in the presence of 4 or 0.4 µM CPT (positive control), or in the presence of either 250 µg/mL aKG + 83.3 µg/mL 5-HMF or 500 µg/mL aKG + 166.7 µg/mL 5-HMF, a population of 1.0 × 10g); the supernatant was removed, and the samples were re-suspended in the Perm/Wash buffer three times. The FITC–Caspase-3 activity was measured on an FACS Calibur®. The supernatant was collected, and 50 μL of reaction buffer and 5 μL of DEVD-AFC  were added and further incubated for 2 h at 37 °C. The resultant fluorescence was measured by using excitation and emission wavelengths of 495 and 519 nm, respectively.After the washing and centrifugation steps and the use of 500 µL of Perm/Wash buffer three times, the samples containing 100 µL of Perm/Wash buffer were incubated with 20 µL of antibody–FITC for 30 min in the dark. After incubation, the samples were washed with Perm/Wash buffer and centrifuged (350× 6 cells/mL was centrifuged (400 g), re-suspended in 0.5 mL of JC-1 solution , and incubated for 15 min and with 5% CO2 at 37 °C. After two washing steps with the assay buffer , the cells were re-suspended in 1 mL of assay buffer and analyzed with FACS. The aggregate dye was able to excite the monomer at 535 nm and the aggregate at 475 nm. After incubation for 72 h in the absence of any substances (negative control), in the presence of 4 or 0.4 µM CPT (positive control), or in the presence of either 250 µg/mL aKG + 83.3 µg/mL 5-HMF or 500 µg/mL aKG + 166.7 µg/mL 5-HMF, a population of 2.0 × 102 supply. After cooling to room temperature, the content of proteins were estimated at 562 nm (Spectra Max Photometer Pro 384). Samples were diluted to 1 mg/mL protein for the estimation of carbonylated proteins described above.Protein concentration of cell lysates were estimated according to the BSA Test Kit. About 25 µL of diluted albumin standards and cell lysates were transferred into 96 microtitration plates for triple estimations. After addition of the BCA reagent solution (50:1) the microtitration plate was covered with a top cover and incubated for 30 min on a plate shaker at 37 °C with a continuous 5% COt-tests where appropriate and indicated. Linear regression and exponential regression curves were calculated based on Pearson regression . All values are given as the mean values and standard deviations. Statistical significance was considered to be at p < 0.01, with high significance at p < 0.001.Group comparisons were made by using p < 0.01), also after 15 min , 30 min , and 60 min . At time points 30 and 60 min, this solution (500 µg/mL + 125 µg/mL 5-HMF) also showed a significantly lower carbonylated protein content compared to 500 µg/mL aKG + 62.5 µg/mL 5-HMF . The highest used combination 500 µg/mL + 250 µg/mL 5-HMF showed no significant difference compared to 500 µg/mL + 125 µg/mL 5-HMF, but the carbonyl proteins were higher with the highest combination solution. For the following experiments we have used the 500 µg/mL aKG + 166.7 µg/mL 5-HMF solution and its dilutions.p < 0.05). No significant changes were estimated between the control at 0 h and the Jurkat cells incubated in the presence of 500 µg/mL aKG + 166.7 µg/mL 5-HMF for 24 h. The cell growth at 48 h was significantly reduced compared to the control  by several different concentrations of the combination of aKG + 5-HMF: 125 µg/mL aKG + 41.7 µg/mL 5-HMF , 200 µg/mL aKG + 66.7 µg/mL 5-HMF , 250 µg/mL aKG + 83.3 µg 5-HMF , 375 µg/mL aKG + 125 µg/mL 5-HMF , and 500 µg/mL aKG + 166.7 µg/mL 5-HMF . After 72 h of incubation, the greatest combination, 500 µg/mL aKG + 166.7 µg/mL 5-HMF , showed a significant reduction compared to the control after 72 h , but did not show a reduction compared to the control cells after 0 or 24 h. A lesser reduction in the cell growth compared to the control after 72 h were obtained with 375 µg/mL aKG + 125 µg/mL 5-HMF , 250 µg/mL aKG + 83.3 µg/mL 5-HMF , 200 µg/mL aKG + 66.7 µg/mL 5-HMF , and 125 µg/mL aKG + 41.7 µg/mL 5-HMF .2 − 0.2335x + 81408) and the IC50% calculated for the 100 µg/mL AKG + 41.7 µg/mL 5-HMF solution, followed by 48 h of incubation  and the IC50% of the 200 µg/mL aKG + 66.7 µg/mL 5-HMF solution, and finally, the 24-h incubation  and the IC50% of around 375 µg/mL aKG + 125 µg/mL 5-HMF. These results showed also that the higher the incubation time with AKG + 5-HMF the lower concentrations are needed to reach the IC 50%. The decline of cell growth after 72 h incubation was higher compared to the 48 h and 24 h incubation.After correlating several concentrations of the combined AKG + 5-HMF solutions with cell growth B, all thp < 0.05) compared to 0, 24, and 48 h incubation.n = 5; p < 0.001). Using the 375 µg/mL aKG + 125 µg/mL 5-HMF solution  resulted in a 28% reduction compared to the control after 24 h.The mitochondrial activity of the Jurkat cells as it was expressed in the absorbance at 450 nm in the absence or presence of one of the dilutions of the aKG + 5-HMF solution is presented in n = 5; p < 0.001) showing the greatest reduction, followed by the 375 µg/mL aKG + 125 µg/mL 5-HMF solution , the 250 µg/mL aKG + 83.3 µg 5-HMF solution , and the 200 µg/mL aKG + 66.7 µg/mL 5-HMF solution . The lowest concentration, 125 µg/mL aKG + 41.7 µg/mL 5-HMF , showed no effects. The same trend could be seen after 72 h of incubation. While no effects compared to the control (0.495 ± 0.04) after 72 h were obtained when using 125 µg/mL aKG + 41.7 µg/mL 5-HMF , all the other used concentrations showed significant reductions: 200 µg/mL aKG + 66.7 µg/mL 5-HMF , 250 µg/mL aKG + 83.3 µg 5-HMF , 375 µg/mL aKG + 125 µg/mL 5-HMF , and 500 µg/mL aKG and 166.7 µg/mL 5-HMF . The mitochondrial activity in the Jurkat cells incubated for 72 h in the presence of 500 µg/mL aKG and 166.7 µg/mL 5-HMF was significantly lower than that with 48 and 24 h of incubation when using the same combined concentration (p < 0.001). A significant difference was also estimated between incubation for 72 h  and incubation for 24 h (0.136 ± 0.018) with the use of 375 µg/mL aKG + 125 µg/mL 5-HMF.The use of 48 h of incubation resulted in a higher reduction (of 36%) of the mitochondrial activity compared to that of the control after 48 h, with the 500 µg/mL aKG and 166.7 µg/mL 5-HMF solution , r = 0.98 for 48 h with a polynomial function (y = 5 × 10−7 x2 − 0.0008x + 0.3545), and r = 0.99 for 24 h with a linear function (y = −0.0002x + 0.1932). The IC50% was calculated for all functions, with nearly the same result of 250 µg/mL + 83.3 µg/mL 5-HMF. The decline of the mitochondrial activity was higher during 72 h incubation followed by 48 h incubation compared to 24 h incubation because of its different functions. The usage of 500 µg/mL aKG and 166.7 µg/mL 5-HMF and 375 µg/mL aKG + 125 µg/mL 5-HMF solutions showed a lower mitochondrial activity in favor of 72 h incubation followed by 48 h incubation compared to 24 h incubation.Mitochondrial activity at a level of nearly 30% was obtained after 48 h of incubation by using 375 µg/mL + 125.4 µg/mL 5-HMF and 500 µg/mL aKG + 166.7 µg/mL 5-HMF. Mitochondrial activity at levels of 10% and 20% remained after 72 h of incubation with 500 µg/mL aKG + 166.7 µg/mL 5-HMF and after 72 h of incubation with 375 µg/mL aKG + 125.4 µg/mL 5-HMF, respectively.The incubation of HF-SAR did not result in relevant differences between the different concentrations of aKG + 5-HMF and the incubation times compared to the control .n = 3; p < 0.001), but 500 µg/mL aKG + 166.7 µg/mL 5-HMF did so even more . While the activity with 500 µg/mL + 166.7 µg/mL 5-HMF was significantly higher than that of the positive control with 4 µM CPT , that of 250 µg/mL aKG + 83.3 µg/mL 5-HMF was lower .The loss of mitochondrial activity mostly induces caspase activity . Comparen=3; p < 0.001), but was significantly decreased compared to 4 µM CPT (50.7 ± 5.6%). The 500 µg/mL aKG + 166.7 µM 5-HMF combination showed a higher significance  compared to the control and to 4 µM CPT .The estimation of apoptotic cells  was signp < 0.01), but not of HF-SAR as presented in p < 0.01) in Jurkat cells and also in HF-SAR . Furthermore, the carbonylated protein content of Jurkat lysates showed a significantly higher content (11.1 ± 0.70 nmol/mg) compared to HF-SAR lysate  before incubation with 250 µg/mL aKG + 83.3 µg/mL 5-HMF or 500 µg/mL aKG + 166.7 µg/mL 5-HMF.After 72 h incubation with 250 µg/mL aKG + 83.3 µg/mL 5-HMF the carbonylated protein content of isolated membrane proteins of Jurkat cell line was significantly lower compared to 0 h  increased the caspase-3 activity by up to 51.6%, while 250 µg/mL aKG + 83.3 µg/mL 5-HMF increased it by up to 13.5%.The calculated molarities of aKG used here were 3.4 and 1.7 mM, and these were 5 to 25-fold lower than the aKG concentrations used in the study with osteosarcoma. aKG might regulate caspase-3 activity in Jurkat cells at lower dosages than in osteosarcoma cells, but we suggest that 5-HMF had a greater impact on caspase-3 activation than aKG in the aKG + 5-HMF solution.aKG is involved in autophagy, cell death, and cell apoptosis, which are regulated by p53 and oxoglutarate dehydrogenase , but 5-HCarbonylated proteins are a good and stabile marker for the detection of oxidative protein modification as a mechanism of redox signaling. Oxidative modification on proteins results from direct radical attack, like free radicals and RONS, or lipid peroxidation or glucose oxidized substances such as malondialdehyde, 4-hydroxy-nonenal. The oxidative modification takes place on specific amino acids, like cysteine or tyrosine, and can elicit changes in protein function and thereby mediating redox signaling, the so-called covalent redox signaling which differs from the classical receptor-ligand mediated signaling . aKG + 5Our data demonstrated that the combined solution of aKG + 5-HMF (1:3) showed an anti-proliferative impact on Jurkat cells in lowering mitochondrial-activity, increasing caspase-3-activity and apoptosis-inducing effects in Jurkat cells, which seems to be related to its antioxidative potential. However, no negative effect was obtained on HF-SAR in cell proliferation, whereas the mitochondrial activity was positively influenced. These results may thus provide a basis for further studies on cancer cells and on the individual compounds.


The title molecule is built up from a ferrocene unit disubstituted by an S-protected diphenylphosphine group and by a methylvinylketone chain. In the crystal, weak C—H⋯O and C—H⋯S interactions build a two-dimensional network. 5H5)(C21H18OPS)], are described. The molecule is built up from a ferrocene unit disubstituted by an S-protected diphenylphosphine group and by a methylvinylketone chain. The crystal structure features weak C—H⋯O and C—H⋯S interactions, which build a two-dimensional network. This structure is compared to that of the related disubstituted diphenylphosphino ferrocene.As a follow-up to our research on the chemistry of disubstituted ferrocene derivatives, the synthesis and the structure of the title compound, 2-(diphenylphosphinothioyl)-2-(3-oxobut-1-en-yl)ferrocene, [Fe(C This molecule has a planar chirality related to the occurrence of two different substituents on the Cp ring; however, as the space group is centrosymmetric, the two enantiomers R/S are present in equal numbers within the crystal. Two intramolecular C—H⋯S interactions occur cyclopentadienyl](η5-cyclopentadienyl)iron compound  to obtain the product as a red solid . Monocrystals suitable for X-ray diffraction analysis were obtained by slow diffusion of pentane into a dichloromethane solution of 4-(2-thiodiphenylphosphinoferrocenyl)-but-3-ene-one.To a solution of 2-(diphenylphosphinothioyl)ferrocenecarboxaldehyde  in acetone (40 mL) was added 2-picolylamine . The reaction mixture was refluxed for 24–36h with TLC monitoring of the consumption of aldehyde. After complete consumption, the reaction mixture was evaporated 1H NMR : δ 8.46 ; 7.90–7.80 ; 7.65–7.15 ; 6.28 ; 5.01 ; 4.65 ; 4.39 ; 4.07 ; 3.87 .13C NMR : δ 198. 16 ; 143.46 ; 134.93 ; 133.01 ; 132.03 ; 131.69 ; 131.54 ; 131.39 ; 128.40 ; 128.19 ; 126.89 ; 83.06 ; 77.44 ; 77.00 ; 71.87 ; 71.85 ; 69.90 ; 25.87 .31P NMR : δ 41.01.4)HRMS .M.p.: 441 K (dec).IR : νmax(solid)/cm−1: 1630 (s), 1607 (s), 1677 (w), 1364 (m), 1335 (m), 1264 (s), 1226 (m), 1165 (s), 1099 (s), 1055 (m), 987 (s), 863 (w), 832 (m), 822 (s), 760 (s), 7478 (m), 712 (s), 698 (s), 690 (s), 660 (s), 640 (s), 614 (sm), 583 (m), 534 (s).Uiso(H) = 1.2Ueq(CH aromatic) or Uiso(H) = 1.5Ueq(CH3). In the final difference-Fourier map, there is a large residual density, 1.43 e Å−3 in the vicinity (1.20 Å) of the H24A atom of the terminal methyl group; it is roughly located in the (100) plane; no chemically logical explanation could be found to explain this residual density.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698902100760X/zl5019sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698902100760X/zl5019Isup2.hklStructure factors: contains datablock(s) I. DOI: 2099273CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Our results suggest that the elimination of 2-5A might be the sole function of viral 2′,5′-PEs, thereby promoting viral escape from innate immunity by preventing or limiting the activation of RNase L.The 2′,5′-oligoadenylate (2-5A)-dependent endoribonuclease, RNase L, is a principal mediator of the interferon (IFN) antiviral response. Therefore, the regulation of cellular levels of 2-5A is a key point of control in antiviral innate immunity. Cellular 2-5A levels are determined by IFN-inducible 2′,5′-oligoadenylate synthetases (OASs) and by enzymes that degrade 2-5A. Importantly, many coronaviruses (CoVs) and rotaviruses encode 2-5A-degrading enzymes, thereby antagonizing RNase L and its antiviral effects. A-kinase-anchoring protein 7 (AKAP7), a mammalian counterpart, could possibly limit tissue damage from excessive or prolonged RNase L activation during viral infections or from self-double-stranded RNAs that activate OAS. We show that these enzymes, members of the two-histidine phosphoesterase (2H-PE) superfamily, constitute a subfamily referred here as 2′,5′-PEs. 2′,5′-PEs from the mouse CoV mouse hepatitis virus (MHV) (NS2), Middle East respiratory syndrome coronavirus (MERS-CoV) (NS4b), group A rotavirus (VP3), and mouse (AKAP7) were investigated for their evolutionary relationships and activities. While there was no activity against 3′,5′-oligoribonucleotides, they all cleaved 2′,5′-oligoadenylates efficiently but with variable activity against other 2′,5′-oligonucleotides. The 2′,5′-PEs are shown to be metal ion-independent enzymes that cleave trimer 2-5A (2′,5′-p Mamma–6–from ATP . The onlfrom ATP –15  superfamily, named for two His-ϕ-Thr/Ser-ϕ motifs (where ϕ is a hydrophobic residue) that degrade 2-5A and therefore function as potent RNase L antagonists , 20 Fig. Here, w3A3), producing mono- and diadenylates with 2′,3′-cyclic phosphoryl termini. Our findings suggest that the sole function of the viral 2′,5′-PEs may be to eliminate 2-5A, allowing some coronaviruses and rotaviruses to evade the antiviral activity of RNase L.The prototype of the 2′,5′-PEs is the mouse coronavirus (CoV) mouse hepatitis virus (MHV) accessory protein NS2 . However23–To probe the precise molecular mechanism by which 2′,5′-PEs allow some viruses to evade the antiviral effector RNase L, we further investigated MHV NS2, MERS-CoV NS4b (MERS-NS4b), the rotavirus group A (RVA) VP3 C-terminal domain (VP3-CTD), and mouse AKAP7 (muAKAP7) . A compaTo determine the phylogenetic relationships between the different 2′,5′-PEs, we constructed a tree for amino acid sequences containing the catalytic domains from coronavirus, rotavirus, and mammalian 2′,5′-PEs . 2′,5′-PBased on the phylogenetic relationship and functional relatedness, we further analyzed the sequence conservation by amino acid alignment. 2H-PE superfamily members are characterized by the presence of two H-ϕ-S/T-ϕ motifs, separated by an average of 80 amino acids (aa) (where ϕ represents a hydrophobic amino acid) . The aliAmong the sequences in the alignment, AKAP7 proteins of human, rat, and mouse origins shared the highest amino acid identity, ranging between 85 and 97% (88 to 97% similarity) see . NS2 pro10.1128/mBio.01781-21.7TABLE S1Table S1, PDF file, 0.2 MB.Catalytic domain sequence identity and similarity analysis of viral and cellular 2′,5′-PEs. The percent amino acid identity and similarity matrix is based on the alignment in Copyright © 2021 Asthana et al.2021Asthana et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the H182R, MHV NS2H126R, RVA VP3-CTDH718A, muAKAP7H93A;H185R, and human PDE12E351A served as the negative controls. The purity and identity of trimer 2-5A  were confirmed by high-performance liquid chromatography (HPLC)   and massy (HPLC)  to F. Iny (HPLC)  and MHV y (HPLC)  degradedy (HPLC)  and muAKy (HPLC)  gave fouy (HPLC)  results y (HPLC) . As expey (HPLC)  to J. Huy (HPLC)  5, p5′(C)5, or p5′(G)5 was not degraded (≤4%) 5 by >99%, while 2′-5′ p5′(rU)5, p5′(C)5, or p5′(G)5 was not degraded (≤7%) 5 at >95%, p5′(rU)5 at ∼40%, p5′(C)5 at ∼90%, and p5′(G)5 at ∼6%, while mutant RVA VP3-CTDH718A did not degrade any of the tested substrates 5 at >95%, p5′(C)5 at >95%, and p5′(G)5 at >90%, while mutant muAKAP7H93A;H185R did not degrade any of the tested substrates with the exception of 2′-5′ p5′(G)5 at ∼40% 5, p5′(C)5, or p5′(G)5 was not degraded (≤6%), suggesting that MERS-NS4b enzymatic activity is specific for the degradation of 2′,5′-oligoadenylates 5 was incubated with 0.2 μM MHV NS2 in the absence or presence of increasing concentrations of 2′-5′ p5′(rU)5 at 30°C for 10 min. The amounts of 2′-5′ p5′(rA)5 degraded by MHV NS2 in the presence of 0, 3.1, 10, 12.5, 25, 50, and 100 μM were determined by HPLC analysis (5 by MHV NS2 decreased as the amount of 2′-5′ p5′(rU)5 in the reaction mixture increased beyond 10 μM  (5 was able to bind MHV NS2 and competitively interfere with the ability of MHV NS2 to cleave 2′-5′ p5′(rA)5.To investigate the expanded substrate specificity of 2′,5′-PEs, we tested the possible cleavage of various 2′-5′- and 3′-5′-linked oligoribonucleotides by HPLC. Purified 2′,5′-PE proteins (1 μM) were incubated with either 2′-5′- or 3′-5′-linked pentaribonucleotide substrates (10 μM) at 30°C for 1 h. Wild-type MERS-NS4b specifically degraded 2′-5′ p5′(rA)ed (≤4%) . The cated (≤7%) . Mutant bstrates . Wild-ty at ∼40% . To ensuenylates . Becauseanalysis . Degradaof >1:1) . Our res10.1128/mBio.01781-21.1FIG S15 degradation by MHV NS2 decreases in the presence of 2′,5′-p(U)5. The substrate 2′,5′-p(A)5 was incubated with 0.2 μM MHV-NS2 wild-type protein in the absence or presence of the indicated concentrations of 2′,5′-p(U)5 at 30°C for 10 min. Samples were processed and analyzed by HPLC. 2′,5′-p(A)5 incubated under similar conditions in the absence of MHV-NS2 and 2′,5′-p(U)5 served as the nondegraded control. Experiments were performed three times (n = 3), and bars represent the standard errors of the means. Statistical significance was calculated using an unpaired t test (n = 3)  in GraphPad Prism (9.0.0) software. Download FIG S1, PDF file, 0.2 MB.2′,5′-p(A)Copyright © 2021 Asthana et al.2021Asthana et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01781-21.8TABLE S2Table S2, PDF file, 0.2 MB.MERS-NS4b-, MHV NS2-, RVA VP3-CTD-, and muAKAP7-mediated degradation of 5′-phosphorylated 2′-5′- or 3′-5′-linked pentaribonucleotide substrates. A total of 10 μM the indicated substrate was incubated with 1 μM wild-type or mutant 2′,5′-PE for 1 h at 30°C. Percent substrate degradation was calculated by measuring the areas under the peaks in the HPLC chromatograms. Results were reproduced in at least two independent experiments. Download Copyright © 2021 Asthana et al.2021Asthana et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01781-21.9TABLE S3Table S3, PDF file, 0.2 MB.MERS-NS4b-mediated degradation of 5′-phosphorylated 2′-5′- or 3′-5′-linked pentaribonucleotide substrates. A total of 10 μM the indicated substrate was incubated with 3 μM wild-type or mutant MERS-NS4b for 1 h at 30°C. Percent substrate degradation was calculated by measuring the areas under the peaks in the HPLC chromatograms. Results were reproduced in two independent experiments. Download Copyright © 2021 Asthana et al.2021Asthana et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 5, p5′(rU)5, and p5′(C)5. One micromolar enzyme was incubated with 10 μM the substrate at 30°C for 1 h. Wild-type MERS-NS4b and its mutant MERS-NS4bH182R, wild-type MHV NS2 and its mutant NS2H126R, RVA VP3-CTD and its mutant RVA VP3-CTDH718A, and wild-type muAKAP7 and its mutant muAKAP7H93A;H185R 5, and 3′-5′ p5′(C)5. . Our results suggest that all of the 2′,5′-PEs examined are highly specific for cleaving 2′,5′-linked oligoribonucleotides. Among the 2′,5′-linked substrates, MERS-NS4b and MHV NS2 are specific for cleaving 2′-5′-oligoadenylate, whereas RVA VP3-CTD cleaved, in order, 2′-5′ pA5 > pC5 > pU5 ≫ pG5, and muAKAP7 cleaved all of the 2′,5′-linked pentanucleotides with similar efficacy.We next tested the degradation activity of 2′,5′-PEs against 3′-5′-linked p5′(rA)3A;H185R  did not 3A3 (which served as a positive control) under similar conditions . cGAMP is a cyclic dinucleotide secondary messenger with mixed phosphodiester linkages between 2′-OH of GMP to the 5′-phosphate of AMP and 3′-OH of AMP to the 5′-phosphate of GMP, synthesized by cyclic GMP-AMP synthase (cGAS) in response to cytoplasmic double-stranded DNA (dsDNA) . cGAMP wnditions . Catalyt10.1128/mBio.01781-21.10TABLE S43A3. A total of 10 μM the indicated substrate was incubated with 1 μM wild-type or mutant 2′,5′-PEs for 1 h at 30°C. The substrates without enzyme incubated under similar conditions were used as undegraded controls. Percent substrate degradation was calculated by measuring the areas under the peaks in the HPLC chromatograms. Results were reproduced in two independent experiments. Download Table S4, PDF file, 0.2 MB.Incubations of 2′,5′-PEs with 2′,3′-cGAMP and 2′,5′-pCopyright © 2021 Asthana et al.2021Asthana et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2+ concentration of between 17 and 20 mM, of which only 5 to 22% may be free depending on the cellular compartment  , or biochemical analysis (by 5′-dephosphorylation) (Differences in the 2-5A cleavage products as determined by HPLC  suggesteylation) .m/z ratios of 597.25 , respectively. Intact 2-5A gave an m/z ratio of 584 for the doubly charged ion  The substrate 2′,5′-p3A3 (A) was degraded in the presence of NS4b into p3A>p and ApA (B). (B to D) No appreciable decrease in the amounts of ApA (area under the peak) after incubation for 1, 4, and 24 h, respectively. (E to H) HPLC chromatograms of the substrate pApA in the presence of NS4b at 0, 1, 4, and 24 h. (I) Table showing the amount of ApA or pApA degraded by NS4b as a function of time. Download FIG S2, PDF file, 0.2 MB.MERS-NS4b degrades 2′,5′-pCopyright © 2021 Asthana et al.2021Asthana et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 3A>p, A>p, ApA, adenosine, and an unknown intermediate (shown in gray)  degradation by RVA VP3-CTD proceeds via the formation of p3ApA>p and ApA intermediates. RVA VP3-CTD degrades p3ApA>p to form p3A>p (5′ product) and A>p (3′ product), whereas diadenylate (ApA) is further degraded to yield A>p (5′ product) and adenosine (3′ product). The complete degradation of p3A3 by RVA VP3-CTD results in the formation of p3A>p, A>p, and adenosine as end products (3A3 cleavage by RVA VP3-CTD was investigated in a time course experiment. RVA VP3-CTD (0.05 μM) was incubated with the p3A3 (200 μM) substrate at 30°C, and samples were collected at different time points. The substrate or product peaks at each time point were analyzed by calculating the percentages of the areas under the peaks of the HPLC chromatograms (3A3 is cleaved by RVA VP3-CTD to produce p3ApA>p (5′ product) and adenosine (3′ product). The intermediate species (p3ApA>p) is subsequently cleaved to produce p3A>p (5′ product) and A>p (3′ product). A minor fraction of p3A3 is cleaved by RVA VP3-CTD to produce p3A>p (5′ product) and ApA (3′ product). The diadenylate intermediate (ApA) is subsequently cleaved into A>p (5′ product) and adenosine (3′ product) (3ApA>p) is readily cleaved by RVA VP3-CTD, whereas ApA cleavage is slow, suggesting that 5′-triphosphorylated molecules are preferred over nonphosphorylated substrates (2 was incubated with 1 μM VP3-CTD at 30°C for 1 h. The results confirmed the formation of cleavage products corresponding to pA>p (5′ product) and adenosine (3′ product) (3A3 to produce p3A>p (5′ product) and ApA (3′ product)  . Based oectively . The depectively  and afteectively  dephosphly  , which iproduct) . Moreovebstrates  and S4. product) . Moreoveproduct) . The ApA10.1128/mBio.01781-21.3FIG S33A3 cleavage by RVA VP3-CTD. Purified 2′,5′-p3A3 (200 μM) was incubated with RVA VP3-CTD (0.05 μM) at 30°C. Samples were collected at 0 min (A), 2 min (B), 5 min (C), 10 min (D), and 30 min (E) and analyzed by HPLC. The percentages of the substrate or products at the indicated times were determined by calculating the areas under the peaks on the HPLC chromatograms. The right-hand side shows major and minor reactions proceeding at the indicated time points deduced from HPLC chromatogram analysis. Download FIG S3, PDF file, 0.2 MB.Time course of 2′,5′-pCopyright © 2021 Asthana et al.2021Asthana et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01781-21.4FIG S43A3 cleavage by RVA VP3-CTD. (A) The percentages of the substrate or the products at the indicated times were determined by calculating the areas under the peaks on the HPLC chromatograms obtained in the experiment from 3A3 by VP3-CTD. The minor reaction cleavage of ApA to A>p and Ado is inferred from incubations performed at a 20-fold-higher concentration of RVA VP3-CTD, 1 μM  at 30°C for 1 h. Samples were processed and analyzed by HPLC. The baseline buffer signal was subtracted from the samples. Download FIG S5, PDF file, 0.2 MB.RVA VP3-CTD degrades 2′,5′-linked diadenylate. The substrate 2′,5′-pApA (A) was incubated with 1 μM either wild-type RVA VP3-CTD (B) or its mutant RVA VP3 CTDCopyright © 2021 Asthana et al.2021Asthana et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01781-21.6FIG S63A3 cleavage by muAKAP7. Purified 2′,5′-p3A3 (200 μM) was incubated with muAKAP7 (1 μM) at 30°C. (A to E) Samples were collected at 0 min (A), 2 min (B), 10 min (C), 30 min (D), and 60 min (E) and analyzed by HPLC. The peaks were identified by comparison to the elution times of known standards. The percentages of the substrate or products at the indicated times were determined by calculating the areas under the peaks on the HPLC chromatograms. (F) Schematics showing the cleavage of 2′,5′-p3A3 by muAKAP7. The reaction intermediate is shown in gray. Download FIG S6, PDF file, 0.2 MB.Time course of 2′,5′-pCopyright © 2021 Asthana et al.2021Asthana et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2+ ions, as has been reported previously  and 2′,5′-PE family members are summarized in eviously . On the eviously . MERS-NSesterase  and 7.+, tRNAs, A5′p45′A, and mono- and poly-ADP-ribose are acceptors for the addition of 2′,5′-AMP residues from ATP by OAS. Also, OAS can add other 2′-terminal ribo- and deoxynucleotide monophosphates to 2-5A  with cyclic 2′,3′-phosphoryl groups and not 2′,3′-OH termini. These conclusions are based on analysis of 2-5A cleavage products by two types of HPLC columns (Dionex and C18) and, importantly, by mass spectrometry. In contrast, our previous studies based on a more limited analysis of the 2-5A cleavage products by one type of HPLC column (Dionex) misidentified these cleavage products of NS2, VP3-CTD, and AKAP7 as AMP and ATP , producing purine-2′,3′-cyclic phosphate-terminated oligonucleotides sensed by Toll-like receptor 8 (TLR8)  induced IFN-β expression through RIG-I, MDA5, and MAVS . Additio8 (TLR8) . The mam8 (TLR8) .The ability of viruses to evade or antagonize the IFN response contributes to viral tropism and disease pathogenesis. Accordingly, viruses have evolved or acquired diverse strategies to overcome inhibition by type I and type III IFNs, both of which induce the transcription of OAS genes , 2, 46. 24–The viral enzymes NS2, NS4b, and VP3-CTD are antagonists of innate immunity that support virus replication by eliminating 2-5A and preventing, or reducing, the activation of RNase L by 2-5A , 24–26. 24–Enzymes that degrade 2-5A have significance beyond antiviral innate immunity. Self-dsRNA activates the OAS-RNase L pathway, leading in some circumstances to apoptosis , 13. In NM_177966.5) was PCR amplified (using DNASU cDNA clone HsCD00296464 in vector pDONR221) with forward primer 5′-gaattcATGTGGAGGCTCCCAGGCGCTTCAA-3′ (with an EcoRI restriction site [in underlined lowercase type]) and reverse primer 5′-gtcgacTCATTTCCATTTTAAATCACATACAAGTGCTATGTGATCTTCAA-3′ . The PDE12E351A pGEX 6P-1 mutant  at the EcoRI and SalI restriction site, sequenced, and expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. To subclone the VP3 C-terminal domain (CTD) cDNA of rotavirus A strain RVA/Simian-tc/USA/RRV/1975/G3P (GenBank accession number EU636926.1) and its H718A mutant, we used codon-optimized constructs for expression in Sf9 insect cells (GenBank accession number KJ869109.1) (TACGCTGACGACCCCAACTACTTCATCG-3′ and reverse primer 5′-ccatggTTATTACTCGGACATGTCGAACACGGTGTCGTTCAA-3′ with an NcoI restriction site (in underlined lowercase type) were used for VP3-CTD. The wild-type and H718A RVA VP3-CTD proteins were expressed as fusions to maltose binding protein (MBP). Additional protein expression plasmid constructs were previously described, with sequences originating from MERS-CoV (MBP-NS4b and its mutant MBP-NS4bH182R) (H126R)   (cDNA te4bH182R) , MHV /pLysS . Wild-type and catalytically inactive mutants of AKAP7 and PDE12 were expressed as GST fusion proteins, and purification was performed by modification of a previous protocol  for 16 h at 22°C. Induced cell pellets were resuspended in buffer A . Pelleted cells were lysed by the addition of 200 μg/ml lysozyme, followed by sonication. Supernatants were collected after centrifugation at 12,000 × g for 40 min at 4°C in a Beckman JA-17 rotor. Supernatants were added to Pierce glutathione agarose  and incubated for 2 h at 4°C, followed by washes with buffer A. Digestions to release the GST tag were performed with PreScission protease  in a solution containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 1 mM DTT for 16 h at 4°C. Supernatants containing untagged protein were concentrated using Centriprep centrifugal filter devices  and loaded onto a Superdex 75 column on an Äkta pure 25L protein purification system  in a solution containing 20 mM HEPES (pH 7.5), 150 mM NaCl, and 1 mM DTT. Wild-type and mutant RVA VP3-CTD-expressing bacterial culture growth and IPTG induction conditions were the same as the ones described above, except that growth medium additionally included 2% glucose. Harvested bacterial cell pellets were suspended in buffer B  and lysed with lysozyme, followed by sonication. Supernatants were incubated with amylose resin  and washed three times with buffer, followed by elution with 100 mM maltose. Proteins were concentrated using Centriprep centrifugal filter devices  and further purified using size exclusion chromatography (SEC) on an Äkta pure 25L protein purification system  in buffer C . Wild-type and catalytic mutants of NS4b and MHV NS2 were purified as described previously (Proteins were expressed from pGEX-6P-1 or pMAL constructs in protocol . Single eviously . In addi35′A(2′p5′A)2  was synthesized from ATP by using histidine-tagged porcine OAS1 (3COO)2·4H2O, 50 mM KCl, 20% glycerol, and 7 mM β-mercaptoethanol]. Ten milliliters of beads was incubated with 10 mg of purified OAS protein for 2 h at 25°C, with intermittent vortexing. Beads were washed three times with buffer D by centrifugation at 3,000 × g at 4°C for 30 min. Beads were suspended in reaction mixtures containing 20 mM HEPES (pH 7.5), 20 mM Mg(CH3COO)2·4H2O, 20 mM KCl, 1 mM EDTA, and 10 mM ATP. The reaction mixtures were incubated in a shaking incubator set at 37°C at 120 rpm for 18 h. The supernatant was collected by centrifugation at 3,000 × g at 4°C for 30 min. The supernatant was heated at 95°C for 5 min and again centrifuged at 18,000 × g for 15 min at 4°C to remove the precipitate. To isolate individual 2-5A oligomers, the supernatants containing crude, unfractionated 2-5A oligomers were run on an HPLC instrument  equipped with a preparative Dionex column . Samples were injected and elution was performed at a flow rate of 3 ml/min in a stepwise gradient of 10 to 400 mM (0 to 120 min), 400 to 800 mM (121 to 125 min), and 10 mM (126 to 160 min) NH4HCO3 buffer (pH 7.8). Fractions were collected, lyophilized, and suspended in nuclease-free water.2-5A or pine OAS1 . The OASine OAS1 . Briefly3A3) with 2′-5′- or 3′-5′-phosphodiester linkages were commercially purchased. The oligonucleotide substrates 5′-pA2′p5′A2′p5′A2′p5′A2′p5′A-3′, 5′-pU2′p5′U2′p5′U2′p5′U2′p5′U-3′, 5′-pG2′p5′G2′p5′G2′p5′G2′p5′G-3′, 5′-pA3′p5′A3′p5′A3′p5′A3′p5′A-3′, and 5′-pU3′p5′U3′p5′U3′p5′U3′p5′U-3′ were purchased from Integrated DNA Technologies (IDT), while 5′-pC2′p5′C2′p5′C2′p5′C2′p5′C-3′, 5′-pC3′p5′C3′p5′C3′p5′C3′p5′C-3′, and 5′-pA2′p5′A-3′ were purchased from ChemGenes Corporation . Pentaribonucleotide substrates are shown as p5′(rN)5, where N represents an A, U, G, or C nucleotide. The A2′p5′A standard was prepared by incubating 5′pA2′p5′A with shrimp alkaline phosphatase  according to the manufacturer’s protocol. 2′,3′-Cyclic GMP-AMP (cGAMP), ATP, AMP, and adenosine were obtained from Sigma-Aldrich.RNA oligoribonucleotides . Where indicated, reactions were performed in the absence of MgCl2 with 2 mM EDTA added. Reactions were stopped by heating at 95°C for 5 min. Samples were centrifuged at 18,000 × g for 15 min at 4°C. Supernatants were collected and analyzed by HPLC. 2′,3′-cGAMP degradation assays were performed and analyzed under the same conditions as the ones described above. In all experiments, substrates incubated under similar conditions in the absence of enzyme served as controls.A total of 10 μM the substrates (with either a 2′-5′- or 3′-5′-phosphodiester linkage) was incubated with 1 μM enzyme. Final reactions were performed in a solution containing 20 mM HEPES buffer (pH 7.4), 1 mM DTT, and 10 mM MgCl18 analytical column . Eluent A was 50 mM ammonium phosphate buffer (pH 6.8), and eluent B was 50% methanol in water. Five microliters of processed samples was injected onto the C18 column, at a flow rate of 1 ml/min, and eluted with a linear gradient (0 to 40%) of eluent B over a period of 20 min and then 3 min of 40% eluent B, followed by equilibration to initial conditions (100% eluent A). The HPLC column was maintained at 40°C. Spectra were recorded at 256 nm. The products were identified either by comparison to the elution times of known standards or by mass spectrometry analysis. Alternatively, to test expanded substrate specificity, 10 μl of the processed samples was injected onto a Dionex DNAPac PA-100 analytical column at a flow rate of 1 ml/min and eluted with a linear gradient of 10 to 800 mM NH4HCO3 buffer (pH 7.8) over a period of 90 min, followed by 30 min of equilibration to initial conditions. Open Lab CDS software was used to analyze and calculate the areas under the peaks in HPLC spectra.The substrates and cleavage products were analyzed on a 1260 Infinity II Agilent Technologies HPLC instrument equipped with an Infinitylab Poroshell 120 CPurified substrates and cleavage product mixtures were dephosphorylated by incubation with SAP  at 37°C for 1 h according to the manufacturer’s protocol. Samples were prepared for subsequent analysis as described above.3A3) were collected by running samples on a Dionex DNAPac PA-100 analytical column as described above. The collected peaks were subjected to acetone precipitation, and supernatants containing cleavage products (from the HPLC peak) were collected and lyophilized. Lyophilized samples were suspended in 1 mM NH4HCO3 buffer (pH 7.8) and used for mass spectrometry analysis.The desired peak fractions  with a flow rate of 0.3 ml/min at 45°C. Mobile phases were mobile phase A (water containing 10 mM ammonium acetate and 20 mM ammonium hydroxide) and mobile phase B (methanol containing 10 mM ammonium acetate and 20 mM ammonium hydroxide). Mobile phase B at 0% was used at 0 to 2 min, and linear gradients were used starting from 0% mobile phase B to 100% B at 2 to 12 min and kept at 100% at 12 to 26 min and then from 100% B to 0% B at 26 to 27 min and kept at 0% mobile phase B for 8 min. The peaks shown in full scans were processed to locate and identify the cleavage products of the 2′,5′-p3A3 substrate using Xcalibur software v4.1. The standards adenosine, AMP, ATP, and adenosine-2′,3′-cyclic monophosphate sodium salt were run for reference.The prepared samples were subjected to mass spectrometry analysis. The LC-MS/MS analysis was carried out using a triple-quadrupole tandem mass spectrometer  equipped with an electrospray ionization (ESI) interface. The mass spectrometer was coupled to the outlet of the HPLC system that consisted of an ultrahigh-performance liquid chromatography (UHPLC) system  including an autosampler with a refrigerated sample compartment and an inline vacuum degasser. Xcalibur software was used for data processing. ESI mass spectrometric detection was performed in both the negative and positive ionization modes, with an ion spray voltage at 2.5 kV, sheath gas at 35 Arb (arbitrary relative unit for measurement of gas flow), and auxiliary (Aux) gas at 20 Arb. The ion transfer tube and vaporizer temperatures were set at 350°C and 250°C, respectively. Qualitative analysis was performed using a full scan at a range from https://mafft.cbrc.jp/alignment/server/). The MAFFT sequence alignment result was downloaded in Clustal format and visualized using Jalview 2.11.1.3 software. The sequence alignment was further processed on the MAFFT server to calculate the phylogenetic tree using the neighbor-joining method and the JTT (Jones-Taylor-Thornton) substitution model, and the tree was then visualized using Archaeopteryx.js software. The resultant fasta format output of the MAFFT multiple-sequence alignment was used to calculate the percentages of amino acid identity and similarity by the Sequence Identity and Similarity (SIAS) tool with default parameters (http://imed.med.ucm.es/Tools/sias.html). The names  of the aligned sequences are MHV NS2 , human coronavirus (HCoV) OC43 NS2 , human enteric coronavirus (HECoV) NS2 , equine coronavirus (ECoV) NS2 , Middle East respiratory syndrome coronavirus (MERS-CoV) NS4b , rat AKAP7δ/γ , mouse AKAP7 isoform 1 , human AKAP7γ , human rotavirus group A (RVA) WA-VP3 , simian RVA SA11-N5 , human rotavirus group B (RVB) Bang117 , bat coronavirus (BtCoV) SC2013 NS4b , and BtCoV HKU5 NS4b .The PDE domain sequences from different 2′,5′-PEs were used for creating a multiple-sequence alignment using MAFFT (Multiple Alignment using Fast Fourier Transform) version 7 , employihttps://mafft.cbrc.jp/alignment/server/. The alignment and phylogenetic tree construction tool is downloadable from https://www.jalview.org/. The Sequence Identity and Similarity (SIAS) tool with default parameters is available at http://imed.med.ucm.es/Tools/sias.html. The names (accession numbers) of the aligned sequences are MHV NS2 (UniProtKB/Swiss-Prot accession number P19738.1), HCoV OC43 NS2 (GenBank accession number AAT84352.1), HECoV NS2 (GenBank accession number ACJ35484.1), ECoV NS2 (GenBank accession number ABP87988.1), MERS-CoV NS4b (GenBank accession number AFS88939.1), rat AKAP7δ/γ (NCBI RefSeq accession number NP_001001801.1), mouse AKAP7 isoform 1 (NCBI RefSeq accession number NP_061217.3), human AKAP7γ (NCBI RefSeq accession number NP_057461.2), human RVA WA VP3 (GenBank accession number AFR77808.1), simian RVA SA11 N5 (GenBank accession number AFK09591.1), human RVB Bang117 (GenBank accession number ADF57896.1), BtCoV SC2013 NS4b (GenBank accession number AHY61340.1), and BtCoV HKU5 NS4b (NCBI RefSeq accession number YP_001039965.1).Multiple-sequence alignment software is available at


In the crystal structure of the title compound, intermolecular N—H⋯N and C—H⋯N hydrogen bonds between the molecules lead to sheets extending parallel to the (110) and  and (In the molecular structure of the title compound, C The dichlorophenyl ring (C11–C16) makes a dihedral angle of 80.82 (12)° with the mean plane of the 1,3,4,8-tetrahydro-2H-pyridopyrimidine ring system.In the molecular structure of the title compound, Fig. 1, the 1,4In the crystal, intermolecular N—H⋯N hydrogen bonds between the amine functions as donor groups and the nitrile N atoms as acceptor groups and intermolecular C—H⋯N hydrogen bonds lead to the formation of sheets extending parallel to (110) and  Table 1 and 4 ▸.CrystalExplorer17.5  and Fig. 7b) show the front and back sides of the three-dimensional Hirshfeld surface of the title molecule plotted over dnorm in the range −0.4776 to +1.4517 a.u., using a ‘high standard’ surface resolution colour-mapped over the normalized contact distance. The red, white and blue regions visible on the dnorm surfaces indicate contacts with distances shorter, longer and equal to the van der Waals separations. The red spots highlight the interatomic contacts, including the N—H⋯N and C—H⋯N hydrogen bonds.In order to visualize the intermolecular interactions in the crystal of the title compound, a Hirshfeld surface analysis -one -one -one . In the molecule of HECLUZ, the seven-membered pentamethylene ring adopts a twist-boat conformation. In the crystal, hydrogen bonds with 16-membered ring and three chain motifs are generated by N—H⋯N and N—H⋯O contacts. The amino group is located close to the nitrogen atoms, forming hydrogen bonds with In the crystal of VAMBET, molecules are linked To a dissolved mixture of 2-malononitrile  and malononitrile  in methanol (40 mL), 1,3-diaminopropane  was added and was stirred at room temperature for 10 min. Then 25 mL of methanol were removed from the reaction mixture that was left overnight. The precipitated crystals were separated by filtration and recrystallized from ethanol .1H NMR : 1.89 ; 3.13 ; 3.67 ; 5.31 ; 6.14 ; 6.78 ; 7.25 ; 7.42 . 13C NMR : 22.30 (CH2), 36.32 (Ar-CH), 38.62 (CH2), 42.92 (CH2), 51.70 (=Cquar), 55.06 (=Cquar), 121.61 (CN), 122.04 (CN), 129.56 (3CHarom), 138.25 (3Car), 152.11 (=Cquar), 154.17 (=Cquar).Uiso(H) = 1.2Ueq(C). All N-bound H atoms were located in a difference map  and they were refined with the constraint Uiso(H) = 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989021003583/wm5605sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021003583/wm5605Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021003583/wm5605Isup3.cmlSupporting information file. DOI: 2075706CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: