M. tuberculosis GmhA enzyme catalyzes the isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-α-manno-heptose-7-phosphate in GDP-D-glycero-α-D-manno-heptose biosynthetic pathway. The D-glycero-α-D-manno-heptose is a major constituent of lipopolysaccharide and contributes to virulence and antibiotic resistance to mycobacteria. In current study, we have performed the structural and biochemical analysis of M. tuberculosis GmhA, the first enzyme involved in D-sedoheptulose 7-phosphate isomerization in GDP-D-α-D-heptose biosynthetic pathway. The MtbGmhA enzyme exits as tetramer and small angle X-ray scattering analysis also yielded tetrameric envelope in solution. The MtbGmhA enzyme binds to D-sedoheptulose 7-phosphate with Km ~ 0.31 ± 0.06 mM−1 and coverts it to D-glycero-D-α-manno-heptose-7-phosphate with catalytic efficiency (kcat/Km) ~ 1.45 mM−1 s−1. The residues involved in D-sedoheptulose 7-phosphate and Zn2+ binding were identified using modeled MtbGmhA + D-sedoheptulose 7-phosphate + Zn2+ structure. To understand the role in catalysis, six site directed mutants of MtbGmhA were generated, which showed significant decrease in catalytic activity. The circular dichroism analysis showed ~ 46% α-helix, ~ 19% β-sheet and ~ 35% random coil structures of MtbGmhA enzyme and melting temperature ~ 53.5 °C. Small angle X-ray scattering analysis showed the tetrameric envelope, which fitted well with modeled MtbGmhA tetramer in closed conformation. The MtbGmhA dynamics involved in D-sedoheptulose 7-phosphate and Zn2+ binding was identified using dynamics simulation and showed enhanced stability in presence of these ligands. Our biochemical data and structural knowledge have provided insight into mechanism of action of MtbGmhA enzyme, which can be targeted for novel antibiotics development against M. tuberculosis. The GDP-D-glycero-α-D-manno-heptose is a key building block of lipopolysaccharide in mycobacteria and blocking of its biosynthetic pathway leads to high antibiotic susceptibility and reduced virulence of mycobacteria. The enzymes involved in GDP-D-α-D-heptose biosynthetic pathway offer an attractive target for novel antibiotics development. The GDP-D-α-D-heptose in mycobacteria is synthesized in four steps, 1) isomerization of D-sedoheptulose 7-phosphate into D-glycero-D-α-manno-heptose-7-phosphate by GmhA enzyme (2) Phosphorylation of D-glycero-D-α-manno-heptose-7-phosphate at C1 position by HddA enzyme, which forms D-glycero-D-α-manno-heptose-1,7-bisphosphate (3) Removal of phosphate at C7 position in D-glycero-D-α-manno-heptose-1,7-bisphosphate by GmhB enzyme, which leads to D-glycero-D-α-manno-heptose-1-phosphate and (4) modification of the phosphate at C1 position to form a phosphodiester linkage with GDP by HddC enzyme and leads to GDP-D-glycero-α-D-manno-heptose  is the first enzyme involved in D-sedoheptulose 7-phosphate isomerization and converts it to D-glycero-D-α-manno-heptose-7-phosphate. The MtbGmhA gene was cloned in pET28a expression vector  from Superdex 200 (16/60) column, identified based on molecular mass standard  are shown in Fig. MtbGmhA enzyme and all mutant proteins were purified like wild type enzyme and eluted as tetramer ] as a function of momentum transfer vector Q, Guinier plot using globular approximation, Kratky plot and pair-distribution function [P(R)] were calculated  ~ log10Q). These data showed no inter particulate effect or aggregation of protein, as lack of upwards or downwards points were observed, when data approached to 0 nm−1. The Fig. MtbGmhA in solution. The Guinier plot analysis yielded the radius of gyration RG ~ 2.38 ± 0.11 nm, quite similar to theoretical RG of MtbGmhA tetramer. The Fig. max) ~ 6.35 nm and RG ~ 2.45 nm for MtbGmhA tetramer , the intensity at zero angle (I0) ~ 464 a. u. was observed for 1.0 kDa in 1 h exposure. Using it as control, the relative intensity of MtbGmhA protein (conc. ~ 1.2 mg/ml) has yielded the molecular mass ~ 93.2 kDa, which corresponds to MtbGmhA tetramer  ~ 1.30 ± 0.03, a measure of similarity between individual solutions. The NSD showed the high similarity between dummy residue shape of solved models. The modeled MtbGmhA tetramer in closed state fitted well into SAXS envelope (χ2 ~ 1.17)  , performed the coupling assay using all three enzymes and monitored the release of phosphate. As control, a phosphate standard curve was calculated using 0–50 μM phosphate, dissolved in coupling reaction buffer having no MtbGmhA enzyme. Following kinetic parameters, Km ~ 0.31 ± 0.06 mM−1, kcat ~ 0.45 ± 0.02 s−1 and catalytic efficiency (kcat/Km) ~ 1.45 mM−1 s1 were obtained for MtbGmhA enzyme  than MtbGmhA enzyme and other GmhA orthologues. The H. pylori GmhA was observed as dimer in solution, while tetramers were observed for GmhA enzymes from M. tuberculosis, E. coli and B. pseudomallei. It is not clear how oligomeric state of GmhA enzyme affects its catalytic efficiency.The MtbGmhA + S7P + Zn2+ complex and identified the six residues involved in D-sedoheptulose 7-phosphate binding. we have generated the six MtbGmhA mutants, performed coupling assay and release of free Pi was monitored , Ser121 → Gly mutation , Thr122 → Ala mutation , Ser123 → Ala mutation , Ser126 → Ala mutation  were observed. The Asn53 forms bifurcated hydrogen bonds with O4 and O9 atoms of D-sedoheptulose 7-phosphate and Asn53 → Gly mutation leads to ~ 3.5 fold decrease in catalytic efficiency. The Gln173 forms hydrogen bond with O2 atom of D-sedoheptulose 7-phosphate and its mutation to Ala leads to ~ 24 fold decrease in catalytic efficiency. The bar diagram showing the catalytic efficiency of wild type and six MtbGmhA mutants are shown in Fig. As seen in Table Escherichia coli and P. aeruginosa GmhA structures10, the Glu68 and His183 acted as acid and base in enzyme catalysis, which converted the D-sedoheptulose 7-phosphate to D-glycero-D-α-manno-heptose-7-phosphate. In B. pseudomallei GmhA structure12, His183 was buried behind Zn2+ in the active site and Glu68 and Gln175 bind to Zn2+ in such a way, that their side chains acted as acid and base in enzyme catalysis.In MtbGmhA + D-sedoheptulose 7-phosphate + Zn2+ tetramer, the Glu66 from one monomer and Gln173 from another monomer bind to D-sedoheptulose 7-phosphate and Zn2+ and may act as acid and base in enzyme catalysis  data was collected on MtbGmhA enzyme in 25–90 °C range with 10 °C step  was obtained using I-TASSER (Iterative Threading ASSEmbly Refinement) server, which used the PDB-2X3Y  as the best input template . The I-TASSER server also used the PDB-2I2W  as another template for MtbGmhA modeling. However, current template did not yield suitable MtbGmhA model, as having following parameters . The D-sedoheptulose 7-phosphate and Zn2+ ions were docked into MtbGmhA tetramer using GLIDE module of Schroedinger program  were used as reference to validate the results obtained in docking analysis of both ligands.The MtbGmhA monomer consists of central five stranded parallel β-sheets, flanked by eight α-helices and forms a helix-beta-helix sandwich  involved in D-sedoheptulose 7-phosphate and Zn2+ binding were found quite conserved. The active site residues e. g. Gln173 from one monomer and Glu66 from another monomer (shown as #), which bind to C1-O1 and C2-O2 of D-sedoheptulose 7-phosphate were also quite conserved. Other MtbGmhA residues involved i in substrate binding e. g. Asn53 (fully conserved), Gly55 , Ser121 (S → T in 3TRJ), Thr122 (fully conserved), Ser123 (S → R in 2YVA), Ser126 (fully conserved ), Gln173 (fully conserved) and Glu66  have shown degree of conservation with 11 other GmhA orthologues. In Francisella tularensis, Ser121 was replaced with Thr121, however not involved in catalytic cleft formation16. Similarly, Ser123 → Arg mutation in DiaA helps in timely initiation of chromosomal replication during cell cycle. In DiaA structure, important residues involved in enzyme catalysis were Ser52, Ala53, Arg71, Pro72, Asn83, Lys101, Leu190, and Phe19117.The Fig. MtbGmhA residues , L117, G124, A132, A136, G148, Gly151, Gly152, Asp159, Pro165, Ile172, Glu174, His176) were fully conserved in sequence alignment with 11 GmhA orthologues.In addition, other MtbGmhA model have yielded following r.m.s. deviation e. g. 1TK9 14 , 2X3Y 12 , 5LU7 18 , 1X92 10 , 3BJZ10 , 2YVA17  , 5BY211 (Sedoheptulose 7-phosphate isomerase from Colwellia psychrerythraea strain 34H) , 5IOL 19 , 3TRJ 16, 1X94 14 , 1I2W 20   to MtbGmhA, when compared to structures of 10 GmhA orthologues.Structural superposition of 11 GmhA orthologues on MtbGmhA tetramer in (i) Apo (ii) D-sedoheptulose 7-phosphate bound and (iii) D-sedoheptulose 7-phosphate + Zn2+ bound state and analyzed the dynamics involved in D-sedoheptulose 7-phosphate and Zn2+ binding  on starting structure (Grey) have yielded RMSD ~ 1.3 Å for 705 Cα atoms, indicating quite similar structure, except minor changes were observed in the loop regions of protein  were observed for amino acid stretches, 20–30, 50–60, 70–80, 105–115, 120–130, 150–155 and 160–170 of MtbGmhA tetramer  during 1 ns simulation  in MtbGmhA tetramer was quite stable during simulation period and found ~ 2.6 nm  on starting complex structure (grey) have yielded the RMSD ~ 1.1 Å for 740 Cα atoms   of complex was quite stable and found ~ 2.2 nm . These data showed that D-sedoheptulose 7-phosphate binding to MtbGmhA tetramer enhanced the overall stability of enzyme.Figure MtbGmhA + D-sedoheptulose 7-phosphate + Zn2+ tetramer was performed to examine the combined effect of Zn2+ and D-sedoheptulose 7-phosphate binding to MtbGmhA. Figure MtbGmhA + D-sedoheptulose 7-phosphate + Zn2+ (Blue) on starting complex structure (Grey) (RMSD ~ 0.5 Å for 752 Cα atoms). Overall structure of MtbGmhA tetramer was quite stable except minor difference in the loop region of the enzyme. The D-sedoheptulose 7-phosphate and Zn2+ ions were quite stable in four subunits of MtbGmhA tetramer, except minor movement of Zn2+ ions occurred in different subunits. It appears that Zn2+ binding further enhanced the stability of MtbGmhA + D-sedoheptulose 7-phosphate tetramer and essential for D-sedoheptulose 7-phosphate binding by MtbGmhA enzyme.Dynamics simulation on MtbGmhA + D-sedoheptulose 7-phosphate + Zn2+ tetramer during the entire simulation period   was quite stable and found ~ 2.2 nm  was amplified from H37Rv genomic DNA by polymerase chain reaction and cloned into pET28a expression vector (Novagen) using NdeI and HindIII restriction sites. The pET28a-MtbGmhA plasmid was confirmed by restriction-digestion and gene sequencing analysis. The pET28a-MtbGmhA plasmid was transformed in E. coli BL21(DE3) cells and cells were grown at 37 °C in luria bertani media (50 µg/ml kanamycin as antibiotic) till OD600 ~ 0.6–0.7. The cell culture was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside and grown for another 4 h. The MtbGmhA protein overexpressed in soluble fraction of the cell.The g for 10 min at 4 °C and suspended in lysis buffer containing  Glycerol, 3 mM β-mercaptoethanol, 1 mM Phenylmethylsulfonyl fluoride, 1 mM Benzamidine hydrochloride, 10 mM Imidazole and 0.2 mg/ml Lysozyme). The cells were homogenized, disrupted by sonication and lysate was centrifuged at 25,000 × g to collect the supernatant. The supernatant was loaded on Ni–NTA column , pre-equilibrated with buffer-A . The column was washed with 0–25 mM gradient of imidazole and eluted the protein in buffer-A + 250 mM imidazole. The eluted protein was concentrated and loaded on Superdex 200 column  pre-equilibrated with buffer . The peak fractions were pooled and concentrated using 10 kDa cutoff ultracentrifugal device . The purified MtbGmhA was analyzed on 12% SDS-PAGE and mass spectrometry. Protein concentration was determined using absorbance at 280 nm and stored at − 80 °C. The recombinant MtbGmhA contains 216 residues e.g. 6 residues from 6xHis tag and 14 residues from vector at N-terminal and 196 residues of MtbGmhA enzyme.The cells were harvested by centrifugation at 10,000 × Stratagene) was used to generate the six mutants of MtbGmhA. All mutants were purified with protocol used for wild type MtbGmhA purification21. All MtbGmhA mutants were confirmed by gene sequencing.QuickChange II XL site directed mutagenesis protocol  was used for small angle X-ray scattering data collection. The small angle X-ray scattering data on Lysozyme  was collected for beam intensity estimation and comparison with MtbGmhA data. After data collection, the MtbGmhA enzyme was checked on SDS-PAGE analysis to see if any degradation occurred during data collection. SAXStreat software was used for beam position correction. 120 µl of MtbGmhA was loaded in quartz capillary flow cell and exposed to X-ray beam (λ = 1.5418 Å).The small angle X-ray scattering data was collected by using SAXSpace instrument  at 10 °C using line collimation optics. The 22 and data containing scattering intensity (I) as a function of scattering vector Q,  were obtained. The Kratky plot [I(Q)Q2 vs Q] was obtained from intensity profile. The radius of gyration (Rg) and radius of cross section (Rc) were estimated from scattering intensity at low Q region using Guinier analysis. Additionally, using RG and RC values, the length of an ellipsoidal structure L were estimated using the L = [12(RG2 − RC2)] ½ relationship. The GNOM45 program was used to estimate the shape and size of scattering entities, which consider both low and high Q data. Indirect Fourier transformation of scattering data over measured Q range was computed as a pairwise distribution function of interatomic vectors23. This analysis also provided RG and Io from the second moment and start of P(R) as well as Dmax (maximum diameter).The small angle X-ray scattering data was collected in triplicate and averaged. SAXSquant 4.2.4 software was used to remove the buffer contribution and desmearing the line collimation. The small angle X-ray scattering data were processed using ATSAS 2.8.3 suite and ScÅtterMtbGmhA were generated using DAMMIF program v. 1.1.224, averaged the aligned model and filtered at a given cutoff volume using DAMAVER v. 5.0 program25. The MtbGmhA tetramer was superimposed on SAXS envelope using the SUPCOMB v. 2.3 program26. The SAXS curve was calculated from the atomic model using the CRYSOL v. 2.8.3 program27. The PyMOL v. 2.3 program28 was used for structural visualization and to generate all superimposed structures. The curve fitting and data plotting were performed using Graphpad-Prism 5 program29.Ten independent models of MtbGmhA were determined by coupling reaction using MtbHddA, MtbHddA and MtbGmhB enzymes and release of Pi was monitored . We prepared the 200 µl of reaction mixture containing  in 20 mM HEPES buffer pH 8.0, 10 mM MgCl2, 10 mM KCl. 10 mM ATP and 0–3 mM D-sedoheptulose 7-phosphate were added in the reaction mixture and incubated at room temperature for 30 min. The Pi concentration was measured using 50 μl of Pi colorlock Gold and 0.5 μl of Accelerator (Innova Biosciences Ltd) in the reaction mixture. After 2 min, 20 μl of stabilization reagent was added in each sample and absorbance was taken at 630 nm. All reactions were performed in triplicate and 11 different concentrations of D-sedoheptulose 7-phosphate were used for each reaction. The kinetic parameters were determined by fitting the data to Michaelis–Menten equation using GraphPad Prism version 6.0.2 .The activity of wild type and six mutants of MtbGmhA was performed using Autolab Esprit Surface Plasmon Resonance equipment31. The gold surface [self-assembled monolayer of 11-mercaptoundecanoic acid (11-MUA)] was first activated by EDC —NHS  coupling. 50 µM of MtbGmhA was immobilized on gold surface using 20 mM sodium acetate buffer, pH 4.2. After immobilization, the surface was blocked with 100 mM ethanolamine pH 8.5. The binding experiment was performed at 25 °C in running buffer . The D-sedoheptulose 7-phosphate substrate was injected at 30 µl/min for 5 min, followed by dissociation for 5 min. After each experiment, the sensor surface was regenerated using 50 mM NaOH.The D-sedoheptulose 7-phosphate binding to MtbGmhA, MtbHddA and MtbGmhB, 50 µM of MtbGmhB was immobilized on gold surface activated by EDC-NHS coupling using 20 mM sodium acetate buffer, pH 4.2. The surface was blocked with 100 mM Ethanolamine pH 8.5. 50 µM of MtbGmhA was injected at 30 µl/min for 5 min followed by dissociation for 5 min. Similar protocol was used for interaction analysis between MtbHddA and MtbGmhB and MtbGmhA and MtbHddA enzymes. All experiments were performed at 25 °C. The sensograms were collected, processed, analyzed using BIAevaluation 32 and fitted with Langmuir 1:1 model using formulaeq is binding response at steady state, Rmax is maximal binding response, CA is the concentration of analyte and KA is equilibrium association constant.For interaction analysis between MtbGmhA enzyme33. A far UV spectrum on MtbGmhA was collected using Chirascan spectropolarimeter (Applied Photophysics) with quartz cuvette of 1 mm path length. The MtbGmhA enzyme was transferred in Sodium phosphate buffer, pH 7.5. The circular dichroism data was collected in 200–260 nm range at 25 °C. The spectra from baseline was subtracted from protein spectra and obtained values were averaged for each dataset. The mean residue ellipticity (θ) was calculated from the observed spectra, as a function of wavelength. The K2D2 program was used to estimate the secondary structure of MtbGmhA34. Similar protocol was used for CD data collection on all six MtbGmhA mutants. The theoretical secondary structure prediction on MtbGmhA was performed using various programs e.g., SOPMA35, CFSSP36, GOR37, PHD38, SIMPA9639, DSC40, HNN41, RaptorX42, JPred43 and PsiPred44.Circular dichroism analysis was performed to estimate the secondary structure of MtbGmhA was recorded at 222 nm from 25 to 90 °C range in 10 °C step. The melting temperature was calculated from scattering profile using polynomial fitting, taking temperature corresponding to half denaturation45. A graph of (ΔA/ΔT) versus (Tavg) was plotted using Sigma Plot program. A bell-shaped melting curve was observed, and peak corresponds to melting temperature of the protein.For thermal stability analysis, the circular dichroism spectra of MtbGmhA sequence was retrieved from UniProt (P9WGG1) database and sequences of 11 GmhA orthologues were retrieved from RCSB database46 The MtbGmhA sequence was aligned with 11 GmhA orthologues using MultAlin47 and ESPript 3.048 programs.The MtbGmhA tetramer and was ranked by the C-score and TM-score49. The MtbGmhA sequence (1–196 residues) was given as input and server retrieved the template proteins of similar folds from Protein Data Bank by LOMETS 50. Additional steps were performed to remove the steric clashes and refined the global topology of the MtbGmhA models.I-TASSER server was used to model the B. pseudomallei12) as the best template for MtbGmhA modeling . The best MtbGmhA model was obtained having following parameters (C-score = 0.21 and TM-score = 0.88 ± 0.07), which indicated a reliable model with correct global topology. The I-TASSER server also yielded PDB-2I2W  as another template for MtbGmhA modeling, however following parameters  did not warrant a reliable MtbGmhA model in “open state”.I-TASSER server identified the PDB-2X3Y  scoring function was used in docking calculation.Docking of D-sedoheptulose 7-phosphate and Zn2+ bound models of MtbGmhA tetramer were performed using GROMACS package 4.5  using CHARMM all-atom force field parameters  Apo (ii) D-sedoheptulose 7-phosphate bound and (iii) D-sedoheptulose 7-phosphate + Zn54 method with 0.16 FF grid spacing and 4th order B-spline interpolation for the reciprocal sum space. Periodic boundary conditions were applied in all directions. Rigid body displacements and rotations were removed from all trajectories. The PRODRG server55 was used to generate the topology of the D-sedoheptulose 7-phosphate. PyMol and Plot256 programs were used for generating the figures and all simulation trajectories. The MtbGmhA tetramers obtained after dynamics simulation exhibited good stereochemistry. The structural validation on all models was performed using various servers e.g., ERRAT57, Verify3D58, and PROCHECK59.The 1 nm cutoff was used for the treatment of Van der Waals interactions and long-range electrostatic interactions and simulated using PMESupplementary information.


The 6-methyl-2,2,4-trioxo-2H,4H-1,2,3-oxathiazin-3-ide anion adopts an envelope conformation with the S atom as the flap. In the crystal, the anions and cations are held together by N—H⋯O, N—H⋯N, O—H⋯O and C—H⋯O hydrogen bonds, thus forming a three-dimensional structure. The Hirshfeld surface analysis and fingerprint plots reveal that the crystal packing is dominated by O⋯H/H⋯O (43.1%) and H⋯H (24.2%) contacts.The asymmetric unit of the title compound, C These aet al., 2016H,4H-1,2,3-oxathiazin-3-ide hemihydrate -one -iminium 6-methyl-4-oxo-4H-1,2,3-oxathiazin-3-ide 2,2-dioxide hemihydrate  are apparent around the oxygen atom O4, which participates in the N—H⋯O contacts, and around the nitrogen atom N1, which participates in the N—H⋯N contacts ts Fig. 2. The fints Fig. 2.Potassium acesulfamate (1 mmol) was dissolved in 15 mL ethanol and heated to 348 K. To this solution 1 mmol of 2-amino-3-hydroxypyridine in 15 mL of ethanol was added slowly under continuous stirring. After the addition, the solution was stirred for another 6 min at the same temperature. The compound thus formed was separated from the solution and then recrystallized from ethanol solution at room temperature. The red needle-shaped crystals obtained were filtered, washed with ethyl acetate and dried, yield 91%.Uiso(H) = 1.5Ueq(C) for methyl and with Uiso(H) = 1.2Ueq(C) for other C atoms, C—H = 0.96 Å for methyl and 0.93 Å for sp2-hybridized C atoms. All other H atoms were located from the difference map and refined freely.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020003813/yk2127sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020003813/yk2127Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020003813/yk2127Isup3.cmlSupporting information file. DOI: 1920140CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Integrin α4β7 binds MAdCAM-1, mediating rolling adhesion of lymphocytes on blood vessel walls when inactive and firm adhesion when activated, thereby controlling two critical steps of lymphocyte homing to the gut. By contrast, integrin αEβ7 mediates the adhesion of lymphocytes to gut epithelial cells by interacting with E-cadherin. Integrin β7 blocking antibodies have shown efficacy in clinical management of inflammatory bowel disease (IBD); however, fully blocking β7 function leads to the depletion of colonic regulatory T (Treg) cells and exacerbates dextran sulfate sodium (DSS)-induced colitis by evoking aberrant innate immunity, implying its potential adverse effect for IBD management. Thus, a better therapeutic strategy targeting integrin β7 is required to avoid this adverse effect.β4β7 activation in vivo by creating mice that carry in their integrin β7 gene a mutation (F185A) which from structural studies is known to lock α4β7 in its resting state. Lymphocytes from β7-F185A knock-in (KI) mice expressed α4β7 integrins that could not be activated by chemokines and showed significantly impaired homing to the gut. The β7-F185A mutation did not inhibit αEβ7 activation, but led to the depletion of αEβ7+ lymphocytes in the spleen and a significantly reduced population of αEβ7+ lymphocytes in the gut of KI mice. β7-F185A KI mice were resistant to T cell transfer-induced chronic colitis, but did not show an increased susceptibility to DSS-induced innate colitis, the adverse effect of fully blocking β7 function.Herein, we inhibited integrin α4β7 activation is a potentially better strategy than fully blocking α4β7 function for IBD treatment.Our findings demonstrate that specific inhibition of integrin α Mice were weighted weekly and observed for signs of illness as reported previously [CD4eviously . At weekAcute colitis was induced by the provision of 2% (wt/vol) DSS with molecular mass of 36–50 kDa  in drinking water for a total of 5 days (days 0–5), followed by regulatory drinking water (days 6–15). Mice were assessed daily for body weight, diarrhea, and bloody stool. The disease activity index (DAI) and histological damage were assessed by trained individuals blinded to the treatment groups, as reported previously , 59. DAIt test or two-way ANOVA as indicated. *P < 0.01, **P < 0.005, and ***P < 0.001 were considered statistically significant in all figures.Statistical analyses were performed with GraphPad PRISM software 5.0 . Significances were determined by two-tailed Student’s Additional file 1: Figure S1. The secondary lymphoid tissues other than GALT appear normal in β7-F185A KI and β7-KO mice. Representative histological sections of the peripheral lymph node (PLN), mesenteric lymph node (MLN) and spleen (SP) of WT, β7-F185A KI (KI) and β7-KO (KO) mice were analyzed by hematoxylin and eosin staining. Scale bars, 500μm. Figure S2. Expression of integrins β7 in splenic lymphocytes of WT and β7-F185A mice. (A) Quantitative PCR analysis of integrin β7 expression in splenic lymphocytes from WT and KI mice. Results are normalized to GAPDH. (B) Total (cell surface plus intracellular) protein expression of integrin β7 in splenic lymphocytes was determined by flow cytometry using permeabilized cells. Data are mean ± s.d. of at least 3 independent experiments (A-B). Figure S3. Impaired adhesion and transmigration of Jurkat T-β7 F185A cells. (A) Expession of β7 and CCR9 in Jurkat T-β7 WT and Jurkat T-β7 F185A cell lines were determined by flow cytometry. (B) Adhesion of Jurkat T-β7 WT, Jurkat T-β7 F185A and Jurkat T cells to MAdCAM-1 substrates at 1 dyn/cm2 or 2 dyn/cm2 before and after chemokine stimulation. (C) Transmigration of Jurkat T-β7 WT, Jurkat T-F185A and Jurkat T cells through MAdCAM-1-coated insert. Data are mean ± s.d. of at least 3 independent experiments (B C). *** P < 0.001; ns, not significant (Student’s t-test). Asterisk in B indicates the changes of total adherent cells. Figure S4. β7-F185A mutation does not affect αEβ7-mediated cell adhesion to E-cadherin substrates. (A) Expession of β7 and αE in Jurkat T-αEβ7 WT and Jurkat T-αEβ7 F185A cell lines were determined by flow cytometry. The numbers within the panels show the specific mean fluorescence intensities of FIB504 (anti-β7) and M290 (anti-αE) mAbs. (B) Adhesion of Jurkat T-αEβ7 WT, Jurkat T-αEβ7 F185A and Jurkat T cells to the immobilized E-cadherin substrates (40 μg/ml) at 1dyn/cm2 before and after chemokine stimulation. αEβ7-E-cadherin binding was inhibited by pre-treatment of cells with 10 μg/ml αE blocking antibody M290. Data are mean ± s.d. of at least 3 independent experiments (A-B).*** P < 0.001; ns, not significant (Student’s t-test). Figure S5. Integrin αE+ lymphocytes in spleen, SI and colon. (A) Expression of αE in WT, +/-, KI and KO splenic lymphocytes was determined by flow cytometry. (B-C) Expression of αE in intestinal IEL and LPL was detected by flow cytometry. The numbers within the panels (A-B) show the percentage of αE+ lymphocytes. The numbers within the table (C) show the specific mean fluorescence intensities of M290 (anti-αE) mAb in αE+ lymphocytes. Data are mean ± s.d. of at least 3 independent experiments (A-C). AAP < 0.05; AAAP < 0.001 (Student's t-test in C). SP, spleen; SI, small intestine; IEL, intraepithelial lymphocyte; LPL, lamina propria lymphocyte.Additional file 2: Table S1. The analysis of integrin β7-high splenic lymphocytes subsets in WT mice. Table S2. The list of primers used for genotyping. Table S3. The list of primers used for real-time quantitative PCR analyses


S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid) moiety. Variations include 9-O-desmethylAdda (DMAdda) and 9-O-acetylDMAdda (ADMAdda) which, unless targeted, may go undetected. Therefore, reference standards were prepared of [ADMAdda5]MCs and [DMAdda5]MCs, which were analyzed using multiple approaches. The cross-reactivities of the [DMAdda5]- and [ADMAdda5]MC standards were similar to that of MC-LR when analyzed with a protein phosphatase 2A (PP2A) inhibition assay, but were <0.25% when analyzed with an Adda enzyme-linked immunosorbent assay (ELISA). Oxidative cleavage experiments identified compounds that could be used in the analysis of total MCs/NODs in a similar fashion to the 2R-methyl-3S-methoxy-4-phenylbutanoic acid (MMPB) technique. Products from oxidative cleavage of both the 4,5- and 6,7-ene of Adda, DMAdda and ADMAdda were observed, and three oxidation products, one from each Adda variant, were chosen for analysis and applied to three field samples and a Nostoc culture. Results from the oxidative cleavage method for total Adda, DMAdda, and ADMAdda were similar to those from the Adda-ELISA, PP2A inhibition, and LC-MS/MS analyses, except for the Nostoc culture where the Adda-ELISA greatly underestimated microcystin levels. This oxidative cleavage method can be used for routine analysis of field samples and to assess the presence of the rarely reported, but toxic, DMAdda/ADMAdda-containing MCs and NODs.Microcystins (MCs) and nodularins (NODs) exhibit high structural variability, including modifications of the Adda (3  •Toxic ADMAdda/DMAdda microcystins (MCs) exhibit low cross-reactivity with Adda ELISA.•A method for oxidative cleavage was expanded to test for ADMAdda/DMAdda MCs.•Two sites of oxidative cleavage of the Adda/DMAdda/ADMAdda were observed.•Oxidation products were identified and analytically tested to quantitate ‘total MCs’. Howevervariants . MCs witvariants , making 5]MCs even when arginine is present MC-LR from the National Research Council Canada (NRC) ; a reference material (RM) of MC-RY [d-Asp3]MC-RR, [d-Asp3]MC-LR, MC-HtyR, MC-LF, MC-LW, MC-HilR  and; RMs of MC-YR, MC-LA, MC-LY, and [d-Leu1]MC-LR produced by GreenWater Laboratories . Internal standards included d5-MC-LF and d7-MC-LR from Eurofins Abraxis .Standards for calibration included: certified reference materials (CRMs) of NOD-R, MC-LR, MC-RR, and MC-LR (3) and [ADMAdda5]MC-LR (4) with >95% purity. The peak containing 5 and 6 from the initial semi-preparative HPLC step was further purified  and MC-LHar (6) with ≥95% purity. Final fractions containing purified 3–6 were evaporated and the residue dissolved in water (1 mL) for characterization.The residue was dissolved  and [ADMAdda5]MC-LHar (4)  for 3 d at ambient temperature. The products were purified as in section 5]MC-LR (1) and [DMAdda5]MC-LHar (2), the solvent was evaporated, and aliquots dissolved  for analysis.An initial semi-preparative HPLC fraction (2.2.33CN (B), both containing 2 mM formic acid and 3.6 mM ammonium formate. The gradient (0.2 mL min−1) was: A held at 70% for 10 min, 70–65% A over 8 min, held 65% A for 2 min, 65–30% A over 4 min, 30–70% A over 2 min, and held at 70% A for 4 min. Purity was assessed using HPLC–PDA (200–600 nm) of each standard at ca 10–20 μg mL−1. Quantitation was based on HPLC–UV (λ = 238 nm) peak areas relative to a CRM of MC-LR (9) as MCs purified in this work share identical UV chromophores to MC-LR. Identities were assigned through the comparison of LC–UV–MSn data  to previous work [5]MC-LR (1) at m/z 981.5, [DMAdda5]MC-LHar (2) at m/z 995.5, MC-LR (5) at m/z 1009.5, [ADMAdda5]MC-LR (3) and MC-LHar (6) at m/z 1023.5, and [ADMAdda5]MC-LHar (4) at m/z 1037.6. Aliquots of 1 and 2 (approximately 200 μg each) were dispensed into vials and the solvent evaporated for NMR spectroscopy. Remaining solutions were portioned into 10 μg aliquots, the solvent evaporated, and stored at −20 °C. A set of working stock solutions at 1.0 μg mL−1 in 10 mM phosphate buffer (pH 7) were maintained (−20 °C) for experiments.A Thermo Scientific Surveyor HPLC system coupled to a Surveyor photodiode array (PDA) detector and an LTQ XL Linear Ion Trap Mass Spectrometer were employed as previously described ,30. Brie2.2.41H and 13C NMR spectra were acquired from CD3OH solutions at 300 K using a Bruker AVIIIHD-800 spectrometer  equipped with a 5 mm TCI cryoprobe  with automatic tuning and matching and Z-gradient accessories. 1H and 2D-COSY, TOCSY, DIPSI2 and ROESY spectra were obtained using in-house variants of standard Topspin pulse programmes with excitation sculpted (ES), continuous wave (CW), or combined ES and CW presaturation of the large CD3OH resonance at ca. 4.8 ppm applied on the F1 channel, and CW presaturation of residual CHD2OH resonance applied on the F2 channel. 1D-SELTOCSY and 1D-SELROESY NMR spectra were obtained with on-resonance F1 channel excitation of target signals and F2 channel CW presaturation of CHD2OH lines. Standard HSQC, HMBC, SHSQC and SHMBC spectra were acquired with CW presaturation of the CD3OH signal. 1H and 13C chemical shifts are reported relative to CHD2OH at 3.31 ppm and CD3OH at 49.3 ppm, respectively. Coupling constants are reported to ±0.1–0.2 Hz for CHx signals or 0.3 Hz for NH signals.2.3Nostoc sp. strain 152. The resultant eluates were evaporated, the residues reconstituted in 10% CH3CN (approx. 800 mg biomass mL−1), and further diluted (water) for analysis.Grab-samples of blooms collected from the West Coast , Midwest , and East Coast  with internal standards added (d7-MC-LR and d5-MC-LF), and analyzed using targeted LC-MS/MS (section 1–6) and those listed in section 5]MCs (3–6), [DMAdda5]MCs (1 and 2), and other MCs (7–11) sharing the same precursor ions are shown in Intact MCs were quantitated as diluted aliquots in water  to achieve absorbances within range of the calibration curve (0.15–4.0 ng mL−1). To assess cross-reactivities, 4-parameter logistic curves were constructed from dilutions of a CRM of MC-LR  and compared to curves, generated independently, of the [DMAdda5]MCs and [ADMAdda5]MCs . A SpectraMax M2 microplate reader coupled to a computer running SoftMax Pro 7 Software  was utilized to obtain absorbances. GraphPad Prism 7.4  was used to calculate IC50 values.Extracts of field samples were analyzed using an Adda-ELISA  loaded in duplicate as previously described . Serial-2.5Nostoc sp. strain 152 extract, which was diluted to fit the calibration curve. A CRM of MC-LR (9) and each isolated variant (1–6) were diluted in water  and analyzed in duplicate following the manufacturer’s directions  kit (Eurofins Abraxis) at the same sample concentrations, except for the rections . The sam2.62.6.19), [DMAdda5]MC-LR (1) and [ADMAdda5]MC-LR (3) (10 μg mL−1) were oxidized for characterization of their oxidation products. Standard curves (in duplicate) were also prepared by oxidation of 1, 3 and 9 at concentrations 1, 5, 10, 50, and 100 ng mL−1. The remaining variants isolated in this study (2 and 4–6) were assessed with the oxidative cleavage method as 4-point curves . Each field sample extract was diluted in water (biomass concentrations 0.2–1 mg mL−1) to achieve analyte responses within the range of the standard curves. Each extract was oxidized in triplicate with spikes (n = 2). Spikes were prepared pre-oxidation using standards of MC-LR, [DMAdda5]MC-LR and [ADMAdda5]MC-LR at sufficient levels to double peak areas for quantitation (standard addition).Solutions of MC-LR , for a final reaction volume of 1 mL. Final reagent concentrations were 50 mM KMnO4, 50 mM NaIO4 and 100 mM K2CO3. After 1 h, 40% (w/v) NaHSO3 (75–125 μL) was added until solutions were clear. Solutions were applied to Strata-X SPE columns (60 mg), the column was washed (3 × 1 mL water), eluted , the eluate evaporated to dryness, the residue dissolved , and syringe-filtered prior to analysis.Solutions of 1 M K5]MC-LR (1), [ADMAdda5]MC-LR (3) and MC-LR (9) was conducted (in triplicate) to monitor the formation of targeted oxidation products at ambient temperature. A solution (500 μL) containing 250 ng of each standard in water was oxidized , and sub-sampled  NaHSO3) after 5, 15, 30, 60 and 120 min. The solutions were fractionated by SPE, the residue from the eluate dissolved in water (375 μL), and filtered, as described above.A time-course for oxidative cleavage of MC-LR (3) and [DMAdda5]MC-LR (1) were scanned in negative ionization mode. A list of target molecular ions was generated based on full-scan mass spectra. MS/MS spectra (negative ionization) were obtained using 20% CE for m/z 207 (MMPB), m/z 193 (2R-methyl-3S-hydroxy-4-phenylbutanoic acid (MHPB)) and m/z 291 (2-methyl-3-oxo-4R-methyl-5S-acetyloxy-6-phenylhexanoic acid (MOMAPH)). MS/MS/MS spectra were obtained for additional characterization of m/z 291 (MOMAPH) using 35% CE of the dominant product ion in the MS/MS spectrum (m/z 231). Positive ionization was also used for MS/MS characterization of m/z 293 (MOMAPH).Oxidized standards (10 μg mL2.6.2.2−1) was 45–10% A over 10 min, 10–45% A over 2 min, and held 45% A for 2 min, with 20 μL full-loop injections. Negative ionization MS (m/z 50–300) and MS/MS spectra (10% CE) were obtained for m/z 235 (2R-methyl-3S-acetyloxy-4-phenylbutanoic acid (MAPB)), m/z 193 (MHPB), m/z 207 (MMPB), m/z 291 (MOMAPH), m/z 249 (2-methyl-3-oxo-4R-methyl-5S-hydroxy-6-phenylhexanoic acid (MOMHPH)) and m/z 263 (2-methyl-3-oxo-4R-methyl-5S-methoxy-6-phenylhexanoic acid (MOMMPH)). Positive ionization MS/MS spectra were also obtained for m/z 293 (MOMAPH) using 15% CE and compared to spectra from the ion trap experiments.A TSQ Quantum Access MAX Triple Quadrupole MS system equipped with a Heated Electrospray Ionization (HESI-II) Probe and Surveyor MS Pump Plus (Thermo Scientific) was employed with sheath and auxiliary gas flow at 45 and 15 arbitrary units, respectively; capillary temperature 275 °C; vaporizer temperature 300 °C (HESI-II); isolation width 1.0, and; spray voltage at 3.5 kV (negative ionization) or 4 kV (positive ionization). A Kinetex F5 LC column was used with mobile phases A (0.05% acetic acid in 5% methanol) and B (0.05% acetic acid in 95% methanol). The gradient ; MOMAPH m/z 291 → 231, 131, 119, and 60 (15% CE), and; MHPB m/z 193 → 131, 119, and 73 (15% CE), with quantification ions in bold. Standard curves prepared from MC-LR (9), [DMAdda5]MC-LR (1) and [ADMAdda5]MC-LR (3)  were analyzed and used to determine method detection limits (S/N = 3).Routine analysis of samples included transitions for MMPB 2.6.3Nostoc sp. strain 152 (75 mg) was vortex-mixed in 2.5 mL of oxidant , allowed to react for 1 h, and the reaction stopped by dropwise addition of 40% (w/v) NaHSO3 until the solution turned cloudy white. The sample was centrifuged and the supernatant retained. The pellet was resuspended , centrifuged, and the two supernatants pooled. The solution was applied to a Strata-X SPE column (200 mg), and the column was washed , eluted , and the eluate evaporated to dryness. The residue was dissolved  and injected onto a NovaPak C18  column eluted isocratically with 35% CH3CN containing 2 mM formic acid and 3.6 mM ammonium formate (1 mL min−1) while monitoring the absorbance at 254 nm using the HPLC equipment described in Section Lyophilized 2.6.42O and CH3CN, respectively, each of which contained formic acid (0.1% v/v). A linear gradient (0.3 mL min−1) was used from 20 to 90% B over 18 min, then to 100% B over 0.1 min, followed by a hold at 100% B (2.9 min), then returned to 20% B over 0.1 min with a hold at 20% B (3.9 min) to equilibrate the column. Injection volume was 5 μL. The mass spectrometer was calibrated from m/z 74–1622 and m/z 69–1780 in positive and negative ionization modes, respectively, the spray voltage was 3.7 kV, the capillary temperature was 350 °C, and the sheath and auxiliary gas flow rates were 25 and 8 units, respectively, with MS data acquired from 2 to 20 min. Mass spectral data were collected using full scan mode with alternating positive and negative scans with data collected from m/z 150–500 using the 60,000 resolution setting, an AGC target of 1 × 106 and a max IT of 120 ms. Putative MOMAPH was further probed in a targeted manner in negative ionization mode using the PRM scan mode at m/z 291.1 with a ±0.5 m/z precursor isolation window, the 30,000 resolution setting, an AGC target of 1 × 106 and a max IT of 100 ms, with a stepped collision energy of 15, 20 and 25 eV.LC–HRMS was conducted with a Q Exactive HF Orbitrap mass spectrometer equipped with a HESI-II probe , an Agilent 1200 G1312B binary pump, a G1367C autosampler, and G1316B column oven . Analyses were performed with a 3.5 μm Symmetry Shield C18 column  held at 40 °C with mobile phases A and B of H33.13.1.15]MCs (3–6) were purified from Nostoc sp. strain 152, two of which (3 and 4) were also hydrolysed to [DMAdda5]MCs (1 and 2) in this study  MCs 1 and 2 eluted far earlier than the Adda5-containing MC-LR (9), with [ADMAdda5]MCs 3 and 4 eluting just after 9, followed by MC variants 5 and 6  are acquiring data and that peaks of interest do not co-elute with non-retained matrix. MS/MS fragmentation patterns also allow for some differentiation, as relative fragment ion intensities varied between Adda-, DMAdda- and ADMAdda-containing MCs , but the corresponding dominant fragment ion with m/z 599 for [DMAdda5]MC-LHar (2) was not unique as it was shared with Arg4-Adda5-containing MCs. The origin of the m/z 599 product ion from Arg4-containing ADMAdda-MCs has been reported as being [Arg4-ADMAdda5-Glu6 – CO + H]+ [m/z with [Arg4-Adda5-Glu6 + H]+. LC-MS/MS generated curves exhibited good linearity (All R2 ≥ 0.995) from 1 to 100 ng mL−1 for isolated standards (1–6)  . Stock s3.1.25]MCs 1 and 2 were confirmed through NMR spectroscopy  [5]MC-LHar (4) [1H NMR data for MC-LHar (22) [5]MC-LR (1) MCs (1 and 2) or [DMAdda5]MCs (3–6) at concentrations of 1 or 10 ng mL−1. Therefore, higher concentrations were tested (50–1000 ng mL−1), with IC50 values determined to be > 200 ng mL−1 as compared 0.49 ng mL−1 for MC-LR (9)   and S32,tibodies , indicat1–6) had similar inhibitory potencies (IC50 0.37–0.52 ng mL−1) to MC-LR (9) (0.42 ng mL−1) , retained their hepatotoxicity when administered intraperitoneally (i.p.) to mice [50  was found to be similar to that of MC-LR (9) for [ADMAdda5]MC-LR (3) and [ADMAdda5]MC-LHar (4), at 60 μg kg−1 body weight (bw), with MC-LR (5) being slightly less toxic (LD50 of 160 μg kg−1 bw) [50  for [DMAdda5]MC-LR (1) was also determined to be 97 μg kg−1 bw, only slightly less toxic than for MC-LR (9) [5]MC-LHar (2) has not been previously determined, but PP2A inhibition data reported here suggests a similar toxic potential to that of MC-LR.The PP2A inhibition assay indicated that all of the ADMAdda- and DMAdda-containing MCs isolated in this work (ng mL−1) . Other wof MC-LR . PurifieC-LR (9) . The tox3.35]MC-LR (3) and [DMAdda5]MC-LR (1) (9)) that were initially observed in total ion LC–MS spectra (m/z 180–300) (9 produced prominent peaks with m/z 207 (MMPB) and 263 (MOMMPH). Peaks from oxidized [DMAdda5]MC-LR were observed with m/z 193 (MHPB), 235, and 249 (MOMHPH). Finally, oxidation products from [ADMAdda5]MC-LR resulted in LC–MS peaks at m/z 235, 291 (MOMAPH) and 231. LC–MS/MS experiments were used to establish tentative structural identities and to verify that oxidation products were conserved across congeners containing the same type of Adda variant. Similar to the chromatographic behavior of intact MCs , the oxidized products followed the same order of retention, with the smaller molecules  eluting approximately 1 min prior to their larger counterparts    producedC-LR (1)  analogou180–300) . Oxidati MOMAPH)  and S34.3, the expected smaller compound MAPB, formed through cleavage of the 6,7-ene, was initially targeted. However, although a peak with a pseudomolecular ion corresponding to MAPB (m/z 235 in negative mode) was detected after oxidation, a peak with the same retention time and product ion spectrum was also observed after oxidation of [DMAdda5]MC-LR (1) (9) did not result in the formation of this compound, so m/z 235 was therefore considered to be unique to ADMAdda and DMAdda, but not Adda. Since the m/z 235 peak was not unique to ADMAdda, further investigations using MAPB as a diagnostic compound were abandoned. Rather, the compound formed through cleavage of 4,5-ene, and exhibiting [M−H]− at m/z 291, was assessed as a unique conserved product from oxidative cleavage of ADMAdda-containing MCs.During the oxidative cleavage of the ADMAdda in C-LR (1)  and S35.m/z 291 (MOMAPH) showed a facile neutral loss of acetic acid (60 Da) to give a product ion at m/z 231. Low resolution MS/MS spectra of MOMAPH showed the most intense product ion at m/z 231, with a weaker product ion at m/z 131 (m/z 231 ([M−H–CH3CO2H]−) gave MS3 product ions at m/z 131 (100%), 187 (26%) and 169 (3%) , which was confirmed using both low- and high-resolution mass spectrometry. LC-MS/MS analyses in negative ionization of the peak with  m/z 131 . MS/MS f169 (3%) . Positiv spectra , which w spectra  in the s spectra  followedion mode . LC–HRMS/MS data  was also/MS data .5]MCs purified in this work (3–6) were used to prepare 5-point standard curves ranging from 1 to 100 ng mL−1 and oxidized, and the resultant oxidation product, MOMAPH, was analyzed via LC-MS/MS (2) ≥ 0.992. The differences observed in MOMAPH formation may be attributed to variability introduced during quantification of the original standard (via HPLC–UV relative to MC-LR), variability in oxidation efficiency specific to analyte chemistry, or the competition of oxidant with the two closely located alkenes. Differences in molecular weight of the intact congeners varied less than 3% and could not have significantly contributed to observed differences in MOMAPH production.[ADMAddaLC-MS/MS . The sta5]MCs confirmed the formation of MHPB  via cleavage of the 6,7-ene (m/z 73 (C3H5O2−), 119 (C8H7O−) and 131 (C10H11−) observed at varying relative intensities (m/z 119 (C8H7O−) and 85 (C5H9O−) (5]MCs (1 and 2) show that MHPB formed less efficiently from [DMAdda5]MC-LR (1) than from [DMAdda5]MC-LHar (2). In contrast, the opposite situation was observed for the formation of MOMHPH and MHPB from 1 and 2, indicating that differences in compound chemistry affect the cleavage reaction even with two very closely related alkenes.LC–MS/MS experiments conducted on oxidatively cleaved MC-LR (1) was confirmed, accounting for 1.3% of the total targeted MCs and 1.4% relative to MC-LR. Oxidative cleavage with analysis for MHPB indicated a similar amount of DMAdda (1.8%). The sum of MCs was 1425 μg g−1 by oxidative cleavage, indicating that >80% of identified MCs were accounted for by LC–MS/MS. This was supported by both the PP2A inhibition assay  and Adda-ELISA (1500 μg g−1 Adda-containing MCs).The chniques . A targeMicrocystis bloom showed it was dominated by MC-LA (17) (1420 μg g−1), making up 89% of the total targeted MCs. Only a small amount of [DMAdda5]MC-LR (1) was detected (2 μg g−1), which was 2.8% that of its methylated counterpart, MC-LR (9). However, oxidative cleavage indicated 140 μg g−1 DMAdda  to be present  and Adda-ELISA (1700 μg g−1 Adda-containing MCs).The targeted LC–MS/MS analysis of the Midwest  present , Fig. 10 present , but thid-Leu1]MC-LR together with lower levels of MC-LR, as reported previously MC-LR previously reported in the sample [5]MC-LR would have been 0.9 μg g−1, which is below the method detection limit (1 μg g−1). The Adda-ELISA (350 μg g−1) and PP2A inhibition (400 μg g−1) analyses indicated that most of the MCs were accounted for in the targeted LC–MS/MS analysis (sum 386 μg g−1), with the total by oxidative cleavage (620 μg g−1) possibly representing some decomposed MCs (unrecognized by the ELISA) (stored 7 y).The targeted LC–MS/MS analysis of MCs in the bloom collected from the Poplar reservoir in the Chesapeake Bay revealed the predominance of MCs targeted by LC–MS/MS (13 μg g−1), although the basic conditions of the oxidation procedure (pH > 10) may have caused partial hydrolysis of the acetate group, which might account for this. Kinetic data from Ballot et al. (2014) [1 of DMAdda-equivalents. The [DMAdda5]MC-LR (1) and [DMAdda5]MC-LHar (2) levels represented 2.3% and 2.5% of their ADMAdda-counterparts, respectively. Total DMAdda was slightly higher at 3.4% of total ADMAdda as determined by oxidative cleavage, but can be accounted for by hydrolytic cleavage of the acetate groups of the [ADMAdda5]MCs and their oxidative cleavage products.MOMAPH (from oxidation of ADMAdda) was not detected in any of the three oxidized planktonic bloom extracts, but was quantitatively measured in the rocedure , Fig. 10. (2014)  indicate5]MCs and [ADMAdda5]MCs, the Adda-ELISA showed very low cross-reactivity with crude extracts Nostoc sp. strain 152. The MCs measured with the other three methods were >250 times the level measured by the Adda-ELISA. While the Adda-ELISA was not representative of the toxic MC content, the PP2A inhibition assay detected 1000 μg g−1 MC-LR equivalents, which was higher than the sum of MCs by oxidative cleavage (609 μg g−1) and the sum of MCs targeted by LC–MS/MS (692 μg g−1).As observed with the purified standards of [DMAdda5]MCs are frequently present when the profile is dominated by Adda-containing MCs. This suggests that DMAdda may be present due to incomplete O-methylation during MC biosynthesis [5]MCs. Due to a lack of commercially available standards and their early elution, the presence of DMAdda-containing MCs is likely underreported. Similarly, [ADMAdda5]MCs in samples may also be underreported due to the apparent absence or low contribution of [Adda5]MCs by cyanobacteria that produce [ADMAdda5]MCs. While commonly employed analysis techniques  might account for the Adda contribution, any [ADMAdda]MCs or [DMAdda]MCs could easily be overlooked.The results of samples analyzed in this work and others ,40,41 su45-and DMAdda5-containing MCs. Furthermore, the lack of commercially available standards hampers targeted analysis approaches. This is problematic, as protein phosphatase inhibition assays (this work and MC-LR, [ADMAdda5]MC-LR). Utilizing standard addition, the oxidative cleavage and analysis procedure applied to field samples was not only helpful in qualifying results, but was also successful in estimating the total Adda-, DMAdda- and ADMAdda-containing MCs in the samples. Data and interpretations were confirmed using LC-MS/MS, PP2A inhibition assay, and the Adda-ELISA.During the investigation of the chemical oxidation of MCs, it was determined there were two sites for the oxidative cleavage of Adda, ADMAdda, and DMAdda, leading to multiple oxidation products that could be targeted for analysis. This observation is important, as the competitive oxidation of the two olefinic sites could impact quantitation if not carefully calibrated using pre-oxidation standard addition with representative congeners .This work was partly supported by the Amanda J. Foss: Conceptualization, Methodology, Investigation, Writing - original draft, Writing - review & editing, , Writing. Christopher O. Miles: Investigation, (LC–HRMS/MS), Writing - original draft, Writing - review & editing. Alistair L. Wilkins: Investigation, (NMR), Writing - original draft, Writing - review & editing. Frode Rise: Investigation, (NMR), Resources. Kristian W. Trovik: Investigation, (NMR). Kamil Cieslik: Investigation, (ELISA). Mark T. Aubel: Resources, Supervision.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


H,3H-dithienopyrazine and 3,4,8,10,11,13-hexahydro-1H,6H-bispyrazine, both form two-dimensional coordination polymers.On reaction with silver nitrate the new pyrazinethiophanes, 5,7-dihydro-1 H,3H-dithienopyrazine, C8H8N2S2, L1, and 3,4,8,10,11,13-hexahydro-1H,6H-bispyrazine, C12H16N2S4, L2, both crystallize with half a molecule in the asymmetric unit; the whole molecules are generated by inversion symmetry. The molecule of L1, which is planar (r.m.s. deviation = 0.008 Å), consists of two sulfur atoms linked by a rigid tetra-2,3,5,6-methylenepyrazine unit, forming planar five-membered rings. The molecule of L2 is step-shaped and consists of two S–CH2–CH2–S chains linked by the central rigid tetra-2,3,5,6-methylenepyrazine unit, forming eight-membered rings that have twist-boat-chair configurations. In the crystals of both compounds, there are no significant intermolecular interactions present. The reaction of L1 with silver nitrate leads to the formation of a two-dimensional coordination polymer, poly[(μ-nitrato-κ2O:O′)silver(I)], [Ag(NO3)(C8H8N2S2)]n, (I), with the nitrato anion bridging two equivalent silver atoms. The central pyrazine ring is situated about an inversion center and the silver atom lies on a twofold rotation axis that bisects the nitrato anion. The silver atom has a fourfold AgO2S2 coordination sphere with a distorted shape. The reaction of L2 with silver nitrate also leads to the formation of a two-dimensional coordination polymer, polypyrazine-κ3S:S′:S′′](nitrato-κO)silver(I)], [Ag(NO3)(C12H16N2S4)]n, (II), with the nitrate anion coordinating in a monodentate manner to the silver atom. The silver atom has a fourfold AgOS3 coordination sphere with a distorted shape. In the crystals of both complexes, the networks are linked by C—H⋯O hydrogen bonds, forming supramolecular frameworks. There are additional C—H⋯S contacts present in the supramolecular framework of II.The two new pyrazineophanes, 5,7-dihydro-1 H,3H-dithienopyrazine (L1), and 3,4,8,10,11,13-hexahydro-1H,6H-bispyrazine (L2), are new N2Sx (x = 2 in L1 and = 4 in L2) ligands designed for the formation of coordination polymers , with the nitrato anion bridging two equivalent silver atoms  analyses  coordination complexes involving various anions, viz. catena-bis(acetonitrile)silver(I) hexafluoridophosphate] benzene-S,S,S′]bis(acetonitrile)silver(I) tetrafluoridoborate] benzene-S,S,S′]benzonitrilosilver tetrafluoridoborate benzonitrile solvate] ](p-tolylsulfonato)silver(I) benzene clathrate] (2)benzothiophene)bis(p-tosyloxy)disilver(I) benzene solvate]  for the silver atom, which has an AgN2S2coordination sphere. NUTBUZ is a two-dimensional coordination polymer. Here, the silver atom has a fivefold AgN2S3 coordination sphere with a distorted shape; the fivefold geometry index τ5 is 0.77  is a two-dimensional coordination polymer, with the silver atom having a fourfold AgOS3 coordination sphere with a trigonal–pyramidal geometry, the fourfold geometry index τ4 being 0.83. The Ag—S bond lengths involving the fourfold coordinated silver atoms vary from 2.4708 (13) Å in NUTCAG to 2.6077 (7) Å in QACYUO/01. The values of the various Ag—S bond lengths in I and II fall within these limits methyl]pyrazine}trisilver(I)], the Ag—O bond lengths vary from 2.567 (5) to 2.752 (5) Å. The values observed for I and II, 2.5849 (15) and 2.492 (3) Å, respectively, are similar to those mentioned above.The nitrate anion can coordinate in at least ten different ways and is extremely useful for designing multi-dimensional coordination polymers, as shown by a search of the CSD. We have previously examined the role of the nitrate anion in the formation of coordination polymers when reporting on the results of the reaction of silver nitrate with some tetrakis-thioether-substituted pyrazine ligands  was first synthesized by Ferigo et al. :L1 was first prepared by the reaction of TBr with Na2S·9H2O, using the procedure of Shimizu et al.  was dissolved in a solution of MeOH/CH2Cl2  in a three-necked flask (500 ml) equipped with a reflux condenser topped by a CaCl2 drying tube, an addition funnel (50 ml) and a magnetic stirring bar. To this mixture was added slowly through the addition funnel a solution of TBr  in CH2Cl2 (25 ml). The reaction mixture was stirred vigorously for 3 h. Removal of the solvent resulted in a brown residue that was extracted into CH2Cl2 (200 ml), washed with water (3 × 30 ml), dried over anhydrous MgSO4 and then, after filtration, evaporated to dryness. The resultant residue was chromatographed over deactivated silica gel using CH2Cl2 as eluent. The main eluted fraction was evaporated to give a white solid that was dried under vacuum yielding pure L1 . Colourless rod-like crystals were formed from a concentrated solution of pure L1 in CH2Cl2, after standing for one week at 278 K.The yield could be increased by as much as 11% using a method similar to that described by Boekelheide  al. 1973. Well-gr1H NMR : δ 4.22  ppm. 13C NMR : δ 152.30, 34.44 ppm. Analysis for C8H8N2S2 (Mr = 196.30 g mol−1). Calculated (%): C 48.95, H 4.11, N 14.27, S 32.67. Found (%): C 49.02, H 4.23, N 14.04, S 32.60. MS , m/z (%): 196 .Synthesis of 3,4,8,10,11,13-hexahydro-1H,6H-bispyrazine (L2):2CO3  was suspended in DMF (250 ml) in the flask. To this well-stirred suspension was added dropwise through the addition funnel a solution of TBr  and 1,2-ethanedithiol  dissolved in DMF (50 ml), at a rate of about 10 ml h−1. The mixture was stirred for a further 20 h and then filtered. The orange filtrate was evaporated under reduced pressure. The residue was extracted into CH2Cl2 (300 ml) then washed with water (3 × 30 ml), dried over anhydrous MgSO4 and then, after filtration, evaporated to dryness. The resultant residue was chromatographed over deactivated silica gel using CH2Cl2 as eluent. The main eluted fraction was evaporated to give a white solid that was dried under vacuum to obtain 0.35 g (50% yield) of pure L2 . Slow evaporation at room temperature of a solution of L2 in CHCl3 in a 5 mm diameter glass tube gave colourless plate-like crystals.A 500 ml three-necked flask was equipped with a reflux condenser, a 50 ml addition funnel, and a magnetic stirring bar. The entire system was purged and kept under a nitrogen atmosphere using vacuum line techniques. Then well-ground Cs1H NMR : δ 4.08 , 2.92  ppm. 13C NMR : δ 151.15, 34.40, 34.09 ppm. Analysis for C12H16N2S4 (Mr = 316.54 g mol−1). Calculated (%): C 45.53, H 5.09, N 8.85, S 40.52. Found (%): C 45.34, H 5.30, N 8.68, S 40.33. MS , m/z (%): 316 .Synthesis of complex I:L1  in THF (5 ml) was introduced into a 16 mm diameter glass tube and layered with MeCN (2 ml) as a buffer zone. Then a solution of AgNO3  in MeCN (5 ml) was added very gently to avoid possible mixing. The glass tube was sealed and left in the dark at room temperature for at least two weeks, whereupon colourless needle-like crystals of complex I were isolated in the buffer zone.A solution of 8H8N3O3S2Ag (Mr = 366.18 g mol−1). Calculated (%): C 26.24, H 2.21, N 11.48, S 17.51. Found (%): C 26.27, H 2.10, N 11.29, S 17.19.Analysis for CSynthesis of complex II:L2  in CH2Cl2 (10 ml) was introduced into a 16 mm diameter glass tube and layered with MeCN (2 ml) as a buffer zone. Then a solution of AgNO3  in MeCN (5 ml) was added very gently to avoid possible mixing. The glass tube was sealed and left in the dark at room temperature for at least three weeks, whereupon thin, colourless plate-like crystals of complex II were isolated at the interface between the two solutions. No analytical data are available for this complex.A solution of Uiso(H) = 1.2Ueq(C). For L1, the rather high Rint value of 0.159 is due to the poor quality, viz. large mosaic spread, of the crystal.Crystal data, data collection and structure refinement details are summarized in Table 610.1107/S205698902000362X/lh5951sup1.cifCrystal structure: contains datablock(s) L1, L2, I, II, global. DOI: 10.1107/S205698902000362X/lh5951L1sup2.hklStructure factors: contains datablock(s) L1. DOI: 10.1107/S205698902000362X/lh5951L2sup3.hklStructure factors: contains datablock(s) L2. DOI: 10.1107/S205698902000362X/lh5951Isup4.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S205698902000362X/lh5951IIsup5.hklStructure factors: contains datablock(s) II. DOI: 1989541, 1989540, 1989539, 1989538CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A new form of the synthetic psychedelic miprocin, a psilocin derivative, is reported. The title compound has a single protonated tryptammonium cation and one half of a fumarate in the asymmetric unit. The ions are held together in an elaborate set of rings and chains along (110). N-isopropyl-N-methyltryptammonium) (4-HO-MiPT) fumarate (systematic name: bis{[2-(4-hydroxy-1H-indol-3-yl)ethyl](methyl)propan-2-ylazanium} but-2-enedioate), 2C14H21N2O+·C4H2O42−, has a singly protonated tryptammonium cation and one half of a fumarate dianion in the asymmetric unit. The tryptammonium and fumarate ions are held together in one-dimensional chains by N—H⋯O and O—H⋯O hydrogen bonds. These chains are a combination of R42(20) rings, and C22(15) and C44(30) parallel chains along (110). They are further consolidated by N—H⋯π interactions. There are two two-component types of disorder impacting the tryptammonium fragment with a 0.753 (7):0.247 (7) occupancy ratio and one of the fumarate oxygen atoms with a 0.73 (8):0.27 (8) ratio.The title compound, bis(4-hydroxy- A wide variety of naturally occurring organisms, including over 200 species of ‘magic’ mushrooms, contain psychoactive tryptamine compounds  fumarate, which is a synthetic analogue of psilocybin. The compound crystallized as a one-to-one tryptammonium/hydrofumarate salt fumarate by treating 4-AcO-DMT fumarate with one half equivalent of lead(II) acetate, precipitating half of the fumarate dianions as lead(II) fumarate , aka ‘miprocin’, is a psilocybin analogue. Its synthesis was first reported in 1981 by Repke and co-workers  fumarate contains one tryptammonium cation and one half of a fumarate dianion :0.247 (7) ratio, with the two moieties related to each other by a pseudo-mirror operation. In solution, the two conformations are most likely interconverting into each other by rapid de- and reprotonation. One oxygen atom of the half fumarate anion is also disordered over two positions in a 0.73 (8):0.27 (8) ratio. Half of the fumarate dianion is present in the asymmetric unit, with the other half generated by inversion; it is slightly distorted from planarity with r.m.s. deviations of 0.020 and 0.070 Å for the two components. The carboxylate unit is fairly delocalized, with C—O distances ranging from 1.251 (10) to 1.284 (2) Å.The asymmetri unit of bis acetate was dissolved in 2 mL of deionized water and then added to the tryptamine solution. After sonication, a white precipitate formed. The powder was removed A, H2 and H2A were found from a difference- Fourier map and were refined isotropically, using DFIX restraints with N—H distances of 0.87 (1) Å and an O—H distance of 0.88 (1) Å. Isotropic displacement parameters were set to 1.2Ueq of the parent indolic nitrogen atom and 1.5Ueq of the parent oxygen atom and the parent ammonium nitrogen atoms. All other hydrogen atoms were placed in calculated positions with appropriate carbon–hydrogen bond lengths: (2sp) 0.95 Å, (CH3) 0.98 Å, (CH2) 0.99 Å and (CH) 1.00 Å. Isotropic displacement parameters were set to 1.2Ueq(C) for 2sp, CH and CH2 parent carbon atoms and 1.5Ueq(C-methyl). Atoms N2 and C11–C14 were modeled as being disordered over two sets of sites [0.753 (7):0.247 (7)] and refined with SADI (0.03) restraints on C—C(methyl) and N—C(methyl) bonds to maintain consistent bond lengths in the disorder. Oxygen atom O3 was also modeled as disordered over two sites [0.73 (8):0.27 (8)].Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989020002923/zl2774sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020002923/zl2774Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020002923/zl2774Isup3.cmlSupporting information file. DOI: 1987588CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The clinical features of COVID-19 have a considerable range from a mild illness to severe disease. Underlying pathophysiological mechanisms of the rapidly progressive, and often fatal, pulmonary disease frequently observed in COVID-19 need to be elucidated, in order to develop new treatment strategies for different disease endotypes. Fatal cases can display features of a cytokine storm, which may be related to hemophagocytic lymphohistiocytosis. Also, a spectrum of vascular changes, including microvascular damage, is known to accompany severe COVID-19. In this paper, we describe the co-occurrence of hemophagocytic lymphohistiocytosis and extensive pulmonary microvascular damage with thrombosis and its sequelae in a patient with fatal COVID-19. We believe these response patterns may be linked by common mechanisms involving hypercytokinemia and require further investigation as a fatal constellation in COVID-19, to generate appropriate treatment in patients who display these combined features. The clinical features of a patient with COVID-19 range from a mild illness with slight complaints such as sore throat and headache to patients with a very severe illness who have acute hypoxemic respiratory failure and need to be admitted to an intensive care unit. Risk factors for severe and fatal disease include elevated serum levels of D-dimer, ferritin, and interleukin (IL)-6 , 3. HoweA 66-year-old male with a history of hypertension, peripheral arterial disease, transient ischemic attacks, and polycythemia vera presented at the emergency department with fever and shortness of breath. The patient used ruxolitinib 25 mg twice daily, acetyl salicylic acid 80 mg, atorvastatin 20 mg, calcium carbonate/colecalciferol 1.25 g/400 IE, irbesartan 300 mg, and metoprolol 200 mg once daily. On examination, he had a fever of 39.1 °C with a blood pressure of 120/90, a pulse rate of 90/min, and an oxygen saturation of 96% without a need for supplemental oxygen. His body mass index (BMI) was 36. Chest X-ray demonstrated bilateral consolidations, mostly on the right side. Polymerase chain reaction (PCR) of a nasopharyngeal swab confirmed infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Laboratory results showed progressive hyperinflammation, with a high probability of having secondary hemophagocytic lymphohistiocytosis using an online HScore calculator (Hscore 180) [IL-1 beta/IL-1F2, below threshold of detection; IL-1ra/IL-1F3, 306.52 ± 225.23 (pg/ml); IL-6, 1.36 ± 0.38 (pg/ml); IL-7, 5.22 ± 1.81 (pg/ml); IL-8/CXCL8, 6.60 ± 3.98 (pg/ml); IL-10, below threshold of detection; IL-12 p70, below threshold of detection; IL-18/IL-1F4, 181.36 ± 83.43 (pg/ml); IL-6R alpha, 30,010.60 ± 6822.03 (pg/ml); TNF-alpha, 2.51 ± 0.71 (pg/ml)Immunohistochemistry was performed with an automated, validated, and accredited staining system  using optiview (OV) or ultraview (UV) universal DAB detection Kit (#760-700). In brief, following deparaffinization and heat-induced antigen retrieval, the tissue samples were incubated according to their optimized time with the antibody of interest Table . IncubatBriefly after deparaffinization, CC1  antigen retrieval was performed for 64 min at 95 °C, followed by incubation for 8 min with the Discovery inhibitor . Primary antibody CD163  was incubated for 32 min at 37 °C followed by detection with Ultramap goat-anti-mouse HRP  and visualization using purple kit for 32 min. CC2 . A stripping step at 100 °C for 8 min was performed, followed by incubation with either CD3 , CD20 , MPO , or glycophorin-C  at 37 °C for 32 min followed by secondary antibody and Ultramap goat-anti-mouse HRP or Ultramap goat-anti-rabbit HRP  at 37 °C for 24 min, and visualization with Teal for 32/16 min  for 32 min  pattern Fig. , relatedThis case demonstrates histopathological evidence of HLH in fatal COVID-19 pneumonia with a concurrent severe microvascular damage pattern in both lungs, with capillary thrombosis and extensive intra-alveolar fibrin exudation with an AFOP pattern. This type of microvascular damage is a distinguishing feature of COVID-19 pneumonia , and the


The apical positions around the paddle-wheel copper centers are occupied by the N atoms of the 4-methylpyridine ligands. Parts of the decanoate chains are disordered with occupancy ratios of 0.817 (9):0.183 (9) and 0.65 (5):0.35 (5).The 4-methylpyridine (4-Mepy) based dimeric copper(II) carboxylate complex [Cu 2(C10H19O2)4(C6H7N)2] or [Cu2(μ-O2CC9H19)4(4-Mepy)2] crystallizes with triclinic  Å] via four bridging carboxylate ligands arranged in the syn–syn coordination mode. The apical positions around the paddle-wheel copper centers are occupied by the N atoms of the 4-methylpyridine ligands. The structure exhibits disorder of the terminal alkyl carbon atoms in the decanoate chains.The 4-methylpyridine (4-Mepy) based dimeric copper(II) carboxylate complex [Cu A dinuclear complex, [Cu2(μ-O2CC5H11)4(C6N2H4)2] 4(4-Mepy)2] crystallizes in the triclinic system, space group PII ion coordinated by the N atom of 4-methylpyridine and by two deprotonated O-monodentate decanoate ligands. The two CuII ions are bridged by four carboxylate ligands in the syn–syn coordination mode, resulting in a distorted square-pyramidal environment with the four O atoms forming the square basal plane and the two pyridyl-N atoms of the 4-Mepy ligands occupying the apical positions. The molecular structure of the complex is shown in Fig. 1The title compound [CuII dimers with a [Cu2(μ-O2CR)4L2]-type structure, [Cu2(μ-O2CCMe3)4(NC5H3(2-NH2)(6-CH3))2] 4(py)2] 4(4-NCpy)2]  , Cu—O (average) [1.9740 (12)], and Cu—N [2.1680 (14) Å] distances are comparable to those observed for structurally similar Cu al. 2000 and Petr al. 1993. The dif al. 1993.gauche conformation at the C18—C19 bond  of a neighbouring 4-Mepy unit with an H⋯centroid distance of 2.94 Å and C—H⋯centroid angle of 134° bis(nicotinamide)dicopper(II) bisdicopper(II) bis(pyridine)dicopper(II)  and sodium decanoate  were stirred in 25 mL of methanol. After 30 minutes, 4-methyl pyridine  was added to the reaction mixture, and stirring was continued for 3 h. The resulting green product was filtered off, washed repeatedly with small volumes of methanol and dried in a vacuum desiccator over fused CaCl2 . The product was dissolved in acetonitrile to give a greenish homogeneous solution, which was allowed to concentrate by evaporation at room temperature. Single crystals suitable for X-ray diffraction analysis were obtained from this solution after one day and were collected by filtration. The compound is insoluble in water but soluble in methanol and acetonitrile.All reagents were purchased from E. Merck and used as received without further purification. CuSO−1): νasym (COO−) 1580, νsym (COO−) 1381, νstretch (C—H) 2800–2950, νstretch (py) 1682, 1489, 1445.IR spectroscopic data  = 1.2Ueq(C) or 1.5Ueq(C-methyl). The twofold disordered parts of the decanoate chains  have been completed through successive electron density difference-Fourier maps and were refined with a sum of their occupancies restrained to unity using geometry (SAME) and Uij restraints (SIMU and RIGU) implemented in SHELXL. The refinement converged with the relative occupancies of 0.817 (9) and 0.183 (9) for the C15–C16 section and 0.65 (5) and 0.35 (5) for the C24–C26 section.C-bound hydrogen atoms were placed in idealized positions with C—H = 0.95–0.99 Å, and refined as riding with 10.1107/S2056989020014103/jq2001sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020014103/jq2001Isup2.hklStructure factors: contains datablock(s) I. DOI: 2039945CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Furthermore, Taurisolo® was able to induce autophagic process via increasing the expression of LC3II, a protein marker involved in formation of autophagosome and ex novo synthesis of sphingomyelin, ceramides, and their metabolites both in HG- and THG-H9c2 cells. Finally, Taurisolo® reduced hypertrophy and induced differentiation of HG-H9C2 cells into cardiomyocyte-like cells. These data suggest that Taurisolo® counteracts the toxicity induced by TMAO and HG concentrations increasing autophagic process and activating de novo sphingolipid synthesis, resulting in a morphological cell remodeling. In conclusion, our results allow speculating that Taurisolo®, combined with energy restriction, may represent a useful nutraceutical approach for prevention of cardiomyopathy in obese subjects.In addition to high plasma glucose, increased levels of trimethylamine N-oxide (TMAO) have been found in obese subjects, where are considered as a novel risk factor for cardiovascular diseases. The present study aimed to investigate the effect of a novel nutraceutical formulation based on grape polyphenols (registered as Taurisolo®) in counteracting TMAO- and high glucose (HG)-induced cytotoxicity in cardiomyoblast H9c2 cells. Cell damage was induced with HG (HG-H9c2) and HG+TMAO (THG-H9c2); both experimental cell models were, thus, incubated for 72 h in the presence or absence of Taurisolo®. It was observed that Taurisolo® significantly increased the cell viability and reduced lactate dehydrogenase and aspartate transaminase release in both HG- and THG-H9c2 cells. Additionally, through its antioxidant activity, Taurisolo® modulated cell proliferation  Obesity is a medical condition mainly characterized by increased body fat accumulation, which negative effects on health are well-known. Excessive adipose tissue is among the leading causes of chronic inflammation that, in turn, plays a pivotal role in developing or worsening the outcome of various pathologies such as metabolic, cardiovascular, respiratory, viral, and tumoral . Obesityin vitro effect and signaling mechanisms of Taurisolo® on proliferation, OS, morphological remodeling, and autophagy, in trimethylamine TMAO and hyperglycemia damaged embryonic rat cardiomyoblast H9c2 cells.Circulating trimethylamine N-oxide (TMAO) levels has been recently highlighted as a potential prognostic marker for cardiovascular diseases (CVD). Increased TMAO concentrations have also been associated with impaired glucose tolerance and diabetes and were accompanied by other metabolic conditions such as low HDL cholesterol and phospholipids and hypomethylation . RecentlAll chemicals, standards, and reagents used were either analytical or mass grade reagents. The water was treated in a Milli-Q water purification system  before use. Bovine serum albumin (BSA) and 3--2,5-diphenyl tetrazolium bromide (MTT) and trimethylamine N-oxide (TMAO) were purchased from Sigma-Aldrich . Phosphate-buffered saline (PBS) and trypsin-EDTA were from Lonza . Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Gibco . Western blot analysis was performed using the following primary antibodies: polyclonal antibody (polyAb) p-ERK 44/42 and polyAb ERK 44/42 from Cell Signaling Technology ; monoclonal Ab Mn-SOD was from Santa Cruz Biotechnology . Blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit or HRP-conjugated goat anti-mouse  secondary antibodies. Anti-mAb was purchased from Santa Cruz Biotechnology and secondary antibodies conjugated to Alexafluor 488 from Life Science, Portland, OR, USA, and 4′,6-diamidino-2-phenylindole (DAPI) from Sigma-Aldrich.Aglianico cultivar grape, collected during the autumn 2016 harvest. Firstly, the Department of Pharmacy, University of Naples Federico II , provided the supplement formulation; then, the large-scale production was accomplished by MBMed Company . For the polyphenol extract production, grapes were extracted with water (50°C), and the solution was filtrated and concentrated and underwent a spray-drying process with maltodextrins as support (40-70%) to obtain a fine microencapsulated powder. As previously reported, the High-Performance Liquid Chromatography-diodearray detector  polyphenol profile of Taurisolo® revealed the presence of: Ferulic acid 10.5 ± 0.70 μg/g, Resveratrol 13.6 ± 0.64 μg/g, Caffeic acid 20.7 ± 0.76 μg/g, Procyanidin B3 dimer 22.05 ± 6.61 μg/g, p-coumaric acid 27.9 ± 0.66 μg/g, Rutin 28.4 ± 0.70 μg/g, Quercetin 40.22 ± 7.11 μg/g, Procyanidin C2 trimer 44.6 ± 0.66 μg/g, Procyanidin B4 dimer 56.6 ± 0.88 μg/g, Procyanidin B1 dimer 62.8 ± 0.59 μg/g, Procyanidin B2 dimer 426.5 ± 5.92 μg/g, Syringic acid 539.2 ± 6.02 μg/g, Epicatechin 886.0 ± 7.82 μg/g, Gallic acid 1463.4 ± 65.5 μg/g, and Catechin 4087.0 ± 64.5 μg/g  MIPC (d20 : 0/26 : 0) (1111 m/z). We observed in the lipid fingerprint profile of Tau-treated HG- and THG-H9c2 cells that the metabolite peaks were centered between 200 and 1500 m/z as sphingosine, C16-C18 sphinganine-1-phosphate, PE-Cer (d16 : 2/20 : 1), SM (d18 : 0/16 : 0), PI-Cer (d18 : 0/16 : 0), 1-O-stearoyl-Cer (d18 : 1/18 : 0)/Or 1-O-eicosanoyl-Cer (d18 : 1/16 : 0), MIPC (18 : /20), PIM2 (16 : 0/18 : 1), and Glu/Gal-ceramide. Cer plays a central role in sphingolipid metabolism. Cer consists of sphingoid long-chain base linked to an acyl chain via an amide bond and synthesized de novo in the endoplasmatic reticulum (ER); it can be modified into Golgi in sphingomyelin (SM), sphingosine, and glycosphingolipids  and are transported to the plasma membrane (PM). Cer can then be metabolized into ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) or be resynthesized back into SM. Sphingosine was associated with growth arrest . Here, we demonstrated that Taurisolo® improves in vitro HG- and TMAO-induced H9c2 dysfunction, suggesting that Taurisolo®, combined with energy restriction, may represent a useful nutraceutical approach for the prevention of cardiomyopathy in obese subjects.Annunziata and colleagues , 17 have


The anti-tubercular activity of the target hybrids was evaluated against drug-susceptible M. tuberculosis strain (ATCC 27294) where hybrids 5d, 5g and 5h were found to be as potent as INH with MIC = 0.24 µg/mL, also the activity was evaluated against Isoniazid/Streptomycin resistant M. tuberculosis (ATCC 35823) where compounds 5g and 5h showed excellent activity (MIC = 3.9 µg/mL). Moreover, the target hybrids were examined against six bronchitis causing-bacteria. Most derivatives exhibited excellent antibacterial activity. K. pneumonia emerged as the most sensitive strain with MIC range: 0.49–7.81 µg/mL. Furthermore, a molecular docking study has proposed DprE1 as a probable enzymatic target for herein reported isatin-nicotinohydrazide hybrids, and explored the binding interactions within the vicinity of DprE1 active site.Joining the global fight against Tuberculosis, the world's most deadly infectious disease, herein we present the design and synthesis of novel isatin-nicotinohydrazide hybrids (  On the other hand, methyl nicotinate 1a and methyl 6-methoxynicotinate 1b was refluxed with hydrazine hydrate in methanol to produce the key intermediate nicotinohydrazides 2a–b which subsequently reacted with N-substituted indoline-2,3-dione derivatives 4a–i in absolute ethanol containing a catalytic amount of glacial acetic acid to get the final compounds 5a–m -1-phenylprop-2-en-1-one 1b was refluxed with NaOH in MeOH to get the free 6-methoxynicotinic acid 10, which then undergone to chlorination via SOCl2 to furnish 6-methoxynicotinoyl chloride 11 where the chloride was subsequently substituted with an azide functionality to produce 6-methoxynicotinoyl azide 12. The azid derivative 12 was subjected to Curtius Rearrangement to get 5-isocyanato-2-methoxypyridine 13, that refluxed with 5-bromoindoline-2,3-dione 3c in dry toluene to furnish the final compound 14.In the last scheme, methyl 6-methoxynicotinate 1H NMR spectra of the final compounds 5a–m and 9a–c were confirmed with the presence of a D2O exchangeable singlet signal attributable to the proton of the hydrazide linker at δ (13.31–13.86) ppm. Moreover, 1H NMR spectra for compounds 9a–c confirmed the presence of singlet signal in the aliphatic region for the CH3 group around δ 2.71 ppm, in addition to the extra aromatic protons for the 6-phenyl ring at a range of δ 7.44–7.57 ppm, whereas, spectra for compounds 5j–m and 14 confirmed the appearance of singlet signal for OCH3 groups at δ (3.82–3.97) ppm.5a, e, g, j, and 9a) was confirmed with the appearance of two triplet signals for (–CH2C3H) and (N-CH2) at δ (0.89-0.91) and (3.70–3.75) ppm, respectively. Also, 1H NMR spectra for compounds  displayed two doublet signals for (–CH(C3H)2) and (N-CH2) in the isobutyl moiety at δ (0.90-0.93) and (3.56-3.74) ppm, respectively, whereas, the ethyl acetate group of compounds  was represented by a triplet signal for (CO-CH2C3H), quartette signal for (CO–C2HCH3) as well as singlet signal for (N-CH2) at δ (1.20–1.22), (4.16–4.17) and (4.72–7.74) ppm, respectively. Furthermore, the N-benzyl substituted counterparts (5d and 5m) showed singlet signals around δ 5.02 ppm attributed to the benzylic (–C2H–C6H5) protons.In addition, the propyl moiety in compounds  and (162.90–166.52) ppm attributable for the carbonyl carbons of the nicotinic hydrazide and indoline-2-one moieties, respectively. Moreover, compounds 5c, l, and 9c showed an extra signal for C = O group for the ester moiety at δ 167.86 ppm, whereas, compound 14 displayed the C = O signal for urea linker at δ 153.69 ppm, and two signals for indoline-2,3-dione moiety at δ 159.57 and 183.65 ppm. Additionally, 13 C NMR spectra for compounds 9a–c showed a signal corresponding to CH3 carbon at δ 23.88–27.24 ppm, while, spectra for compounds 5j–m and 14 displayed a signal corresponding to OCH3 carbon at δ (53.53–54.44) ppm.On the other hand, N-propyl group in compounds  appeared as three signals resonating at δ (11.60–11.65 ppm), (20.81–20.87 ppm), and (41.28–41.48 ppm) for –CH2CH3, N-CH2CH2, and N-CH2, respectively, and the carbons of the N-isobutyl group in compounds  appeared as three signals at a range of δ (20.36–20.45 ppm), (27.22–23.88 ppm) and (47.10–56.51 ppm) due to –CH(CH3)2–, –CH(CH3)2– and –N-CH2–, respectively. Finally, a signal attributable to the benzylic (–CH2–) carbon in the N-benzyl derivatives (5d and 5m) appeared around δ 43.10 ppm.Furthermore, the carbons of the 2.2.2.2.1.M. tuberculosis (ATCC 27294) using the Microplate Alamar Blue Assay (MABA)All of the newly synthesised hybrids were evaluated for their anti-tubercular action towards 5d, 5g, and 5h were found to be as potent as INH with MIC = 0.24 µg/mL. Compounds 5f, 5j, and 5k revealed potent activity with MIC range = 0.48–0.98 µg/mL, whereas derivatives 5a–5c, 5e, 5i, 5l, 5m, and 14 exhibited a moderate activity with MIC range = 1.95–7.81 µg/mL.The results revealed that most tested compounds exerted potent to moderate anti-tubercular action with MIC range = 0.24-7.81 µg/mL. Compounds 5a–5i was affected by two main factors; N-substitution and incorporation of halogen at position 5 of the oxindole ring. Regarding N-substitution it was shown that, compounds bearing isobutyl group: 5b, 5f, and 5h exhibited remarkable activity with MIC values = 1.95, 0.48, and 0.24 µg/mL, respectively. Similarly, compound 5d with N-benzyl moiety revealed a potent activity with MIC = 0.24 µg/mL. In contrast, compounds 5c and 5i carrying the N-ethylcarboxylate group were found to be the least active derivatives (MIC = 7.81 µg/mL). Moreover, the contribution of substitution at position 5 of oxindole ring on the activity was quite important. Incorporating 5-Br group in compounds 5g and 5h (MIC = 0.24 µg/mL) showed better activity than 5-Cl substituted compounds 5e and 5f  and the unsubstituted congeners 5a–5c (MIC range = 3.9–7.81 µg/mL).Concerning the structure–activity relationship, it was observed that the anti-tubercular activity of compounds 5j–5m against the unsubstituted counterparts 5a–5d, it was found that the introduction of 6-methoxy group in compounds 5j–5l (MIC = 0.98-3.9 µg/mL) enhanced the anti-tubercular activity by 2-4 fold compared to unsubstituted analogues 5a–5c (MIC range: 1.95–7.81 µg/mL) except for the benzyl derivatives 5d and 5m. Conversely, the introduction of 6-phenyl moiety to the nicotinohydrazide moiety as in compounds 9a–c (MIC = 15.63–31.25 µg/mL) led to a reduced anti-tubercular activity that may be due to steric factors.Then we turned our attention to study the impact of substitution at position 6 on the nicotinohydrazide moiety of the new hybrids. Comparing the anti-tubercular activity of 6-methoxy nicotinohydrazide derivatives In conclusion, increasing the lipophilicity of the isatin scaffold via C-5 substitution with halogens (Br > Cl) was more advantageous for activity. Also, the anti-mycobacterial action of the target hybrids was decreased in the following order; 6-methoxy nicotinohydrazide > unsubstituted nicotinohydrazide > 2-methyl-6-phenyl nicotinohydrazide .2.2.2.M. tuberculosis (ATCC 35823) was evaluated using MABA5a–m, 9a–c, and 14 (5g and 5h with the best anti-tubercular effect against M. tuberculosis (ATCC 27294), showed excellent activity (MIC = 3.9 µg/mL) resistant M. tuberculosis strain (ATCC 35823), with more than 32 fold increase in the activity compared to INH and Streptomycin (MIC > 125 µg/mL). Moreover, compounds 5d, 5f and 5j elicited a good activity with MIC = 15.63 µg/mL, whereas derivatives 5b, 5k, and 14 produced a moderate activity with MIC = 62.5 µg/mL. Finally, compounds 5a and 5l were found to be least active showing MIC value = 125 µg/mL, while the 6-phenyl pyridine derivatives 9a–c were inactive with MIC >125 µg/mL.The anti-tubercular activity against Isoniazid and Streptomycin resistant , and 14 , it was 2.2.3.5a–m, 9a–c, and 14 was evaluated against bronchitis causing-bacteria; Gram-negative Mycoplasma pneumoniae (ATCC 15531), K. pneumoniae (ATCC 43816), H. influenzae (ATCC 10211), M. catarrhalis (ATCC 25238), B. pertussis (ATCC 9340) and Gram-positive S. pneumoniae (ATCC 1659) using XTT Susceptibility Assay  with MIC range = 0.49–7.81 µg/mL, while M. pneumoniae (ATCC 15531) and M. catarrhalis (ATCC 25238) were the least sensitive strains MIC range = 0.98–125 µg/mL and MIC range = 3.9 to >125 µg/mL, respectively.The antibacterial activity for the synthesised compounds ty Assay 33,34. Th5g and 5h showing best anti-tubercular activity with the minimal bacterial resistance against resistant-TB, revealed a broad-spectrum antibacterial action against most of the tested bronchitis causing-bacteria. They were found to be more potent than the reference drug against H. influenzae, M. catarrhalis, and B. pertussis . Additionally, they were equipotent to Azithromycin against M. pneumoniae, S. pneumoniae and K. pneumoniae .Interestingly, compounds 5k showed MIC values (0.98-3.9 µg/mL) less than that expressed by Azithromycin (MIC = 1.95–7.81 µg/mL) against four bacterial strains: M. pneumoniae (ATCC 15531), H. influenza, M. catarrhalis (ATCC 25238) and B. pertussis (ATCC 9340). Also, compound 5j was as potent as the reference drug against H. influenzae, K. pneumoniae (ATCC 43816), M. catarrhalis (ATCC 25238), and B. pertussis (ATCC 9340). Furthermore, compounds 5f and 14 were two-fold more potent (with MIC = 1.95 µg/mL) than Azithromycin (MIC = 3.9 µg/mL) against H. influenza (ATCC 10211) and B. pertussis (ATCC 9340) and equipotent to Azithromycin against K. pneumoniae (ATCC 43816) with MIC = 0.49 µg/mL. Finally, derivatives 5a, 5b, and 5d produced the same MIC values = 0.49 µg/mL as that expressed by the standard drug against K. pneumoniae (ATCC 43816).Moreover, compound 2.2.4.5g and 5h on normal human cells, an MTT cytotoxicity assay was carried out against non-tumorigenic WI-38 cells. The obtained results ascribed to hybrids 5g and 5h non-significant cytotoxic action with IC50 value = 60.95 and 54.01, respectively , Pantothenate synthetase (PS), Mtb ATP synthase subunit C (AtpE), Flavin-dependent thymidylate synthase (TS or ThyA), and flavoenzyme Decaprenylphosphoryl-β-D-Ribose 2′-Epimerase (DprE1)Over the past decade, several druggable targets have been utilised in TB-drug discovery to develop new therapies in order to fight the global epidemic of TB. Most of these enzyme targets have an essential role in energy production, cell wall biosynthesis, and DNA replication, repair, and transcription. Examples of these enzymes are; Alanine racemase (Alr), Maltosyltransferase (GlgE), 5g and 5h were selected for further in silico investigation to explore their possible molecular target. Two promising drug enzymes in M. tuberculosis were suggested as potential targets for the target compounds in this study; the flavoenzyme Decaprenylphosphoryl-β-D-Ribose 2′-Epimerase (DprE1) and the enoyl-acyl carrier protein reductase (InhA) enzymes. Both enzymes play an essential role in the metabolism of the cell wall and the synthesis of mycolic acid, respectively,On account of their superior anti-mycobacterial activity, compounds 5h has achieved −10.1 and −7.9 kcal/mole energy score for DprE1 and InhA, respectively, whereas, compound 5g has achieved −9.7 and −7.3 kcal/mole for DprE1 and InhA, respectively. Interestingly, both compounds 5g and 5h achieved a higher energy score than the co-crystalized ligand  in DprE1. These findings suggested that DprE1 may be a plausible target for the herein reported compounds.The crystal structures of the two enzymes were downloaded from the protein data bank PDB ID: 4NCR for DprE1 and 2B35 for InhA. Despite, the importance of docking in providing a predicated binding mode and an account for a ligand strength, it remains a theoretical technique that needs proper validation by comparison with an experimental reference. Therefore, both co-crystallised ligands were re-docked into their corresponding enzymes. The calculated RMSD values between the co-crystallised and docked pose were 0.52 and 0.71 for DprE1 and InhA, orderly which indicates a valid and reliable docking protocol. Compound 5g and 5h was engaged in two H-bond interactions via its carbonyl oxygen (C = O) functionality with His-132 and Tyr-415 amino acids. Furthermore, the NH of the hydrazide linker and the nitrogen atom of the pyridine ring were involved in H-bond interactions with His-132 and Asn-385, respectively. Moreover, the nitrogen atom of the pyridine ring in compound 5h achieved an extra hydrogen bond interaction with Cys-387 which may explain its higher energy score than 5g. Worthy of note, the N-isobutyl group of compound 5h established several hydrophobic interactions with non-polar residues lining a hydrophobic pocket, such as Pro-116, Val-121, Ile-131, and Ala-417.Inspection of the docking poses for the two examined compounds revealed their ability to fit perfectly in the active site of the DprE1 enzyme establishing different types of interactions  and 5. T5g and 5h in InhA active site , and the 3 D illustrations of the superimposition for the docking poses and the co-crystalized ligands in DprE1 and InhA active sites , as well as the detailed interactions achieved by compounds 5g and 5h within the DprE1 binding site (Table S3) were provided in the Supporting Materials.The 2 D diagrams for interactions of compounds 4.5a–m, 9a–c) and isatin-3-pyridylamine hybrid 14 were designed and synthesised. The anti-mycobacterial activity towards M. tuberculosis (ATCC 27294), as well as towards the INH and Streptomycin resistant M. tuberculosis (ATCC 35823), were evaluated for the seventeen newly synthesised 2-oxindolin-3-ylidene-nicotinohydrazide derivatives. The obtained results identified compounds 5g and 5h as the most potent anti-tubercular agents in this study (MIC = 0.24 µg/mL), with minimal mycobacterial resistance (MIC = 3.9 µg/mL). Furthermore, all herein reported hybrids were examined for their antibacterial activity against six bronchitis causing-bacteria; M. pneumoniae, H. influenzae, K. pneumoniae, M. catarrhalis, B. pertussis, and S. pneumonia. Most hybrids exerted significant antibacterial activity in comparison to the reference drug Azithromycin against the tested bacterial strains. The most sensitive strain was K. pneumoniae with MIC range = 0.49–7.81 µg/mL. Interestingly, compounds 5g and 5h displayed a broad-spectrum antibacterial activity against most of the tested bronchitis causing-bacteria. Moreover, an in silico molecular modelling study has proposed DprE1 as a potential enzyme target for herein reported isatin-nicotinohydrazide hybrids. Compounds 5g and 5h were able to fit perfectly in the active site of the DprE1 enzyme achieving a higher energy score  than the co-crystalized ligand . The oxindole ring was engaged in two H-bond interactions via its carbonyl oxygen (C = O) functionality with His-132 and Tyr-415 amino acids, whereas, the NH of the hydrazide linker and the nitrogen atom of the pyridine ring were involved in H-bond interactions with His-132 and Asn-385, respectively. In addition, the N-isobutyl group of compound 5h established several hydrophobic interactions with non-polar residues lining a hydrophobic pocket.In this work, different sets of isatin-nicotinohydrazide hybrids (5.5.1.5.1.1.13 C NMR spectra were run at 100 MHz in deuterated dimethylsulphoxide (DMSO-d6). Chemical shifts (Hδ) are reported relative to the solvent (DMSO-d6). NMR analyses were carried out at the NMR Unit, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt, whereas IR analyses were carried out at the Faculty of Science, Mansoura University, Mansoura, Egypt. Elemental analyses were carried out at the Regional Centre for Microbiology and Biotechnology, Al-Azhar University, Cairo, Egypt. Compounds 2a,b4a–i,811I.R. spectra have been recorded on Schimadzu FT-IR 8400S spectrophotometer. NMR spectra were recorded by Bruker-Avance 400 spectrometer. 5.1.2.2a–b or 8 (2.5 mmol) were added to an equivalent amount of N-substituted indoline-2,3-dione derivatives 4a–i in 15 ml of glacial acetic acid. The previous mixture was heated under reflux with TLC monitoring, after complete consummation of starting materials, the reaction flask was cooled to room temperature. The formed precipitate was collected under vacuum, washed with hexane, cold water and recrystallized from dioxane/methanol mixture to furnish final compounds 5a–m and 9a–c, respectively.In a round-bottom flask, the appropriate nicotinohydrazide derivative 5.1.3.12  was dissolved in anhydrous toluene (20 ml) which then heated under reflux for 1 h. To the previous solution, an equivalent amount of 5-bromoisatin was added and the reaction mixture was refluxed for 5 h. The produced precipitate was filtrated while hot washed with hexane and recrystallized from acetonitrile to furnish compound 14.6-Methoxynicotinoyl azide 5a–m, 9a–c, and 14) have been provided in the Supporting Materials.Full characterisation details  for the target hybrids  against M. tuberculosis ATCC 27294 (Isoniazid-susceptible strain) and Mycobacterium tuberculosis ATCC 35823 (Isoniazid and Streptomycin resistant strain). The detailed procedures were presented in the supporting materials.Microplate Alamar blue assay (MABA)5.2.2.,H. influenzae ATCC 10211, M. pneumoniae ATCC 15531, K. pneumoniae ATCC 43816, M. catarrhalis ATCC 25238, and Bordetella pertussis ATCC 9340, as well as S. pneumoniae ATCC 1659, representing Gram-positive bacterium, that obtained from the American type culture collection. The detailed experimental procedures have been added in the supporting materials.A colorimetric broth microdilution method was utilised to determine the MIC for the tested hybrids following the XTT -reduction assay5.2.3.5g and 5h towards the normal human lung WI-38 cells as reported earlierThe MTT assay5.3.,Aiming to define the molecular target for the best two lead compounds, the crystal structures of both DprE1 and InhA enzymes of TB were downloaded from the Protein data bank ID: 4NCR and 2B35, respectivelyClick here for additional data file.


Scientific Reports 10.1038/s41598-020-70090-2, published online 11 August 2020.Correction to: This Article contains a repeated error in the legends for Tables 3, 5 and 6 where,a95% CI”“should read:a: 95% CI”“


In the 1H NMR spectra of this compound, the NH iminium protons were observed at δ 10.35 p.p.m., which confirms the strong charge-assisted hydrogen bonding (CAHB) in the =HN+—H⋯Br− synthon.In the crystal, the cations and anions of the title salt are linked  16H16N3S+·Br−, adopts an envelope conformation, with the C atom bearing the phenyl ring as the flap atom. In the crystal, the cations and anions are linked by N—H⋯Br hydrogen bonds, forming chains parallel to the b-axis direction. Hirshfeld surface analysis and two-dimensional fingerprint plots indicate that the most important contributions to the crystal packing are from H⋯H (46.4%), C⋯H/H⋯C (18.6%) and H⋯Br/Br⋯H (17.5%) interactions.The central thiazolidine ring of the title salt, C The mean plane of the thiazolidine ring makes dihedral angles of 18.28 (15) and 83.19 (15)°, respectively, with the C5–C10 and C11–C16 phenyl rings of the 3-(benzylideneamino) and 5-phenylthiazolidin groups, while the dihedral angle between them is 82.54 (15)°. The torsion angle of the N2—N1—C4—C5 bridge that links the thiazolidine and 3-(benzylideneamino) units is −175.7 (3)°.The thiazolidine ring (S1/N2/C1–C3) in the cation of the title salt Fig. 1 adopts ab-axis direction. C—H⋯π interactions or π–π stacking interactions contributing to the stabilization of the crystal packing are not observed.In the crystal, adjacent cations and anions are linked by pairs of N—H⋯Br hydrogen bonds Table 1, formingCrystalExplorer 3.1  to 1.3503 a.u. (blue) is shown in Fig. 3dnorm surface arise as a result of short interatomic contacts  shows the two-dimensional fingerprint of the sum of the contacts contributing to the Hirshfeld surface represented in normal mode . All of the contributions to the Hirshfeld surface are given in Table 3et al., 2015The most significant intermolecular interactions are the H⋯H interactions (46.4%) Fig. 5b. All oet al., 2016viz. MOJGUQ  Å] between the major components of the disordered phenyl ring contribute to the stabilization of the molecular packing. In the crystal of XOWXAL  contacts. In the other structures, the 3-N atom carries a C–substituent instead of an N–substituent as found in the title compound. Three of them were determined to be racemic :0.179 (3). The system of hydrogen bonds connecting two cations and two anions into 12-membered rings is identical in the racemic and in the optically active crystals. YITCEJ : C, 53.04; H, 4.45; N, 11.60. Found: C, 53.01; H, 4.42; N, 11.56%. 1H NMR  : 4.58 ; 4,89 ; 5.60 ; 7.37–8.07 ; 8.44 , 10.35 . 13C NMR : 45.36, 55.91, 127.76, 128.65, 128.82, 128.86, 129.09, 131.54, 132.85, 137.48, 151.11, 167.84. MS (ESI), m/z: 282.30 [C16H16N3S]+ and 79.88 Br−.To the solution of 3-amino-5-phenylthiazolidin-2-iminium bromide (1 mmol) in 20 mL of ethanol was added benzaldehyde (1 mmol) and the mixture was refluxed for 2 h. After cooling down to room temperature, the reaction product precipitated as colourless single crystals, which were collected by filtration and washed with cold acetone (yield 76%), m.p. 519 K. Analysis calculated for CUiso(H) = 1.2Ueq. The distances between the carbon atoms of two phenyl groups were constrained with a DFIX instruction [DFIX 1.40 0.02 C C].Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989020001899/rz5269sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020001899/rz5269Isup2.hklStructure factors: contains datablock(s) I. DOI: 1837123CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: