The crystal structures of the 1:1 salts of nicotine with 3,5-dinitrosalicylic acid and with 5-sulfosalicylic acid both show polymeric hydrogen-bonded and π–π-bonded structures but these differ in that in the first example, cations and anions form separate cation chains or anion columns which are unassociated through formal hydrogen bonds while in the second, hydrogen-bonded cation–anion chains are found. R,2S)-1-methyl-2-(pyridin-3-yl)-1H-pyrrolidin-1-ium 2-carboxy-4,6-dinitrophenolate, C10H15N2+·C7H3N2O7−, (I), and -1-methyl-2-(pyridin-3-yl)-1H-pyrrolidin-1-ium 3-carboxy-4-hydroxybenzenesulfonate, C10H15N2+·C7H5O6S−, (II), are reported. The asymmetric units of both (I) and (II) comprise two independent nicotinium cations (C and D) and either two DNSA or two 5-SSA anions (A and B), respectively. One of the DNSA anions shows a 25% rotational disorder in the benzene ring system. In the crystal of (I), inter-unit pyrrolidinium N—H⋯Npyridine hydrogen bonds generate zigzag NIC cation chains which extend along a, while the DNSA anions are not involved in any formal inter-species hydrogen bonding but instead form π–π-associated stacks which are parallel to the NIC cation chains along a [ring-centroid separation = 3.857 (2) Å]. Weak C—H⋯O interactions between chain substructures give an overall three-dimensional structure. In the crystal of (II), A and B anions form independent zigzag chains with C and D cations, respectively, through carboxylic acid O—H⋯Npyridine hydrogen bonds. These chains, which extend along b, are pseudocentrosymmetrically related and give π–π interactions between the benzene rings of anions A and B and the pyridine rings of the NIC cations C and D, respectively [ring centroid separations = 3.6422 (19) and 3.7117 (19) Å]. Also present are weak C—H⋯O hydrogen-bonding interactions between the chains, giving an overall three-dimensional structure.The structures of the 1:1 anhydrous salts of nicotine (NIC) with 3,5-dinitrosalicylic acid (DNSA) and 5-sulfosalicylic acid (5-SSA), namely (1 S-1-methylpyrrolidin-2-yl)pyridine] is well known as a toxic liquid alkaloid which is found in the leaves of the tobacco plants Nicotiana tabacum and N. rustica  and the sulfate, dihydrochloride, bitartrate and bipicrate salts have been reported  nicotinate  and 3-carboxy-4-hydroxybenzenesulfonic acid  (pKa < 1) are capable of forming salts with most Lewis bases and have been used for the formation of crystalline salts suitable for X-ray analysis, e.g. with 5-SSA 10H15N2+ C7H5O6S−, (II)3,5-Dinitrosalicylic acid (DNSA) (pR) chiral centre relative to the known C21(S) centre. Also, in both (I)+ cations (C and D) and either, for (I)A and B) , C21(S) absolute configuration.In both the nicotinium salts of DNSA (I)) Figs. 1, 2 ▶. WiC and D cations are conformationally similar but in (II)C/D—C3C/D—C21C/D—N11C/D are −71.9 (4) (C) and −68.8 (4)° (D) in (I)C) and 125.7 (3)° (D) in (II)]. This conformation with the two rings antiplanar is usual for cationic nicotine structures, e.g. Arnaud et al.  being for the C3B nitro group [173.7 (3)°]. In the B anion, there is 25% rotational disorder about the C1⋯C4 ring vector, which generates a second phenolic O-component (O21B). This phenomenon has precedence in DNSA salt structures, e.g. with the nicotinamide salt  al. 2007. The subphenol hydrogen bond  Å]. The presence of π–π stacking is unusual in DNSA cation structures. In the crystal, there are a number of intermolecular CC/D—H⋯OA/B hydrogen-bonding interactions, which give an overall three-dimensional structure.In the supramolecular structure of (I)s Table 1, giving 1a Fig. 3. With thA and B 5-SSA anions form carboxylic acid O—H⋯Npyridine hydrogen bonds with the D and C NIC cations, respectively  and with anion D, linear (O52Aii). These give independent zigzag chain substructures which extend along b. Although there are no formal hydrogen-bonding links between the two chains, there are π–π interactions between 5-SSA anion A and B benzene rings and C and D NIC cation pyridine rings, respectively [ring-centroid separations = 3.6422 (19) and 3.7117 (19) Å] y Table 2 Uiso(H) = 1.5Ueq(O or methyl-C) or 1.2Ueq.Crystal data, data collection and structure refinement details are summarized in Table 3B) and its other component (O21B) in anion B of (I)The site occupancy factors for the rotationally disordered phenolate components (O2S) absolute configuration was invoked. The Flack parameter for (I) [0.2 (16)] has no physical meaning. The absolute structure of compound (II) was confirmed by resonant scattering [Flack parameter = −0.02 (9)].In both structures, the known C21 global, I, II. DOI: 10.1107/S1600536814023253/lh5736Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536814023253/lh5736IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S1600536814023253/lh5736Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S1600536814023253/lh5736IIsup5.cmlSupporting information file. DOI: 1030394, 1030395CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Anti-tumor responses were observed in some patients, but overall results lack consistency. This might be related to systemic activation of Vγ9Vδ2 T cells in these trials, not providing a specific trigger for these cells to accumulate at the tumor site.Gamma delta T cells expressing the Vγ9Vδ2 T cell receptor (TCR) are the most predominant γδ-T cell subset in peripheral blood accounting for approximately 1-5 % of all T cells. Vγ9Vδ2 T cells recognize phosphoantigens (pAg) such as isopentenyl pyrophosphate (IPP), a naturally occurring pAg that can accumulate in tumor cells, resulting in activation, cytokine release and anti-tumor activity of Vγ9Vδ2 T. The use of Vγ9Vδ2 T cells in clinical trials, either via adoptive transfer of in vitro.In order to improve the efficacy of Vγ9Vδ2 T cell based immunotherapy, we focused on the design of a tumor-targeting construct that binds both the TCR of Vγ9Vδ2 T cells and the Epidermal Growth Factor Receptor (EGFR), which is over-expressed by many tumor types, including Vγ9Vδ2 T cell susceptible tumors like colon carcinoma and head and neck cancer. For this bi-specific construct an antagonistic anti-EGFR single domain antibody fragment (VHH or Nanobody) and an agonistic anti-Vγ9Vδ2 TCR VHH were identified, characterized and constructed into a bi-specific targeting molecule. Only when bound to both EGFR expressing tumor cells and Vγ9Vδ2 T cells, this bi-specific targeting molecule induced Vγ9Vδ2 T cell activation, release of IFN-γ and TNF-α as well as up-regulated expression of cytolytic molecules such as perforin-and granzyme B. Importantly, tumor targeted Vγ9Vδ2 T cells were able to efficiently lyse EGFR expressing tumor cells This study shows that bi-specific anti-Vγ9Vδ-T-anti-EGFR-nanobodies can specifically and efficiently lyse EGFR-expressing tumor cells and are promising candidates for cancer immunotherapy.


Attempts to effect 4″-epimerization of these 5-substituted UDP-glucose with UDP-glucose 4″-epimerase from yeast were unsuccessful, while use of the corresponding enzyme from Erwinia amylovora resulted in efficient epimerization of only 5-iodo-UDP-Glc, but not the corresponding 5-aryl derivatives, to give 5-iodo-UDP-Gal. Given the established potential for Pd-mediated cross-coupling of 5-iodo-UDP-sugars, this provides convenient access to the galacto-configured 5-substituted-UDP-sugars from gluco-configured substrates and 5-iodo-UTP.Glucose-1-phosphate uridylyltransferase in conjunction with UDP-glucose pyrophosphorylase was found to catalyse the conversion of a range of 5-substituted UTP derivatives into the corresponding UDP-galactose derivatives in poor yield. Notably the 5-iodo derivative was not converted to UDP-sugar. In contrast, UDP-glucose pyrophosphorylase in conjunction with inorganic pyrophosphatase was particularly effective at converting 5-substituted UTP derivatives, including the iodo compound, into a range of  In brief, enzymatic synthesis approaches to such compounds may employ a number of different enzymes, either affording the required sugar nucleotide via pyrophosphate bond formation [action of uridylyltransferase  or pyrophosphorylase ], or by epimerization of the C-4″ stereochemistry of the pre-formed sugar nucleotide [action of epimerase ] ] .13 We ha2.12.1.11b–f, a multienzyme approach was assessed (5a) and glucose-1-phosphate with UDP-glucose pyrophosphorylase  to generate UDP-Glc (2a) in situ. Galactose-1-phosphate uridylyltransferase  then catalyses the reaction of UDP-Glc (2a) and α-d-galactose-1-phosphate, giving the corresponding UDP-Gal (1a). UDP-Glc (2a) is only produced in catalytic quantity  as it is continuously recycled, via Glc-1-P, by the action of GalU (1a) sugar nucleotide.In an attempt to generate 5-substituted UDP-Gal derivatives assessed .14,15 Th of GalU . In this1a) using an equimolar quantity of UTP (5a) and a catalytic amount of UDP-Glc (2a). The transformation reached a complete conversion (by SAX HPLC) within 1 h (data not shown). Next, the 5-substituted UTP derivatives 5b–f were used in combination with Gal-1-P in an attempt to generate the corresponding 5-substituted UDP-Gal derivatives 1b–f (1d) (5%) and 5-(2-furyl)-UDP-Gal (1e) (23%) and the products were isolated and characterized. The formation of 5-(4-methoxyphenyl)-UDP-Glc (2d) and 5-(2-furyl)-UDP-Glc (2e) as intermediates in the reaction is implicit, but their presence in the reaction mixture was not detected. The 5-iodo-UTP (5b) and the 5-(5-formyl-2-thienyl)-UTP (5f) derivatives were not converted into the corresponding sugar nucleotides at all; in the case of the 5-phenyl-UTP (5c), the conversion was less than 5% and the product 1c was not isolable. In order to assess which of the enzymes, GalU or GalPUT, is failing to use these latter base-modified compounds as substrates, a series of reverse reactions and inhibition experiments was performed.In a control experiment, Gal-1-P was converted into UDP-Gal  into UDP-Glc (2a). As shown in 1H NMR analysis of the diagnostic anomeric signals (dd) of the sugar phosphates. When a synthetic sample of 5-(5-formyl-2-thienyl)-UDP-Gal (1f) was subjected to equivalent conditions, no conversion was observed after 1 h (data not shown). After extended incubation (24 h) only traces of 5-(5-formyl-2-thienyl)-UDP-Glc (2f) and Gal-1-P were detectable by 1H NMR (1f) either does not bind to the GalPUT active site or that it might bind in a non-productive way. If the latter were true, 1f should act as a GalPUT inhibitor.In the presence of excess Glc-1-P, GalPUT can be used to run the reverse conversion, UDP-Gal (y 1H NMR B. This r2.1.31a), Glc-1-P and 1f were mixed in a molar ratio 1:10:3.5, the conversion to UDP-Glc (2a) after 30 min was the same as in the absence of 1f (not shown), implying that 1f does not compete with UDP-Gal (1a) to bind in the active site of GalPUT. This implies that the formylthienyl substitution of the uracil base prevents the corresponding sugar nucleotides from binding to GalPUT and explains the observed lack of conversion of 2f into 1f in the one-pot GalU-GalPUT protocol. However, the lack of conversion might also be due to a lack of tolerance of GalU for 5-substitution of the uracil ring of UTP.When UDP-Gal (2.1.45a)] showed the rapid conversion of UTP (5a) into UDP-Glc (2a), as indicated by the diagnostic uracil H6 signals in 1H NMR spectra (5a) and 5-iodo-UTP (5b) in molar ratio 1:1:5, UTP (5a) remained almost completely intact (5b) was rapidly converted into 5-iodo-UDP-Glc (2b), as shown by a new H6 signal (2b) ([M−H]−m/z 691) was detected, but one for UDP-Glc (2a) ([M−H]−m/z 565) was absent. These data suggest that, if used in excess, 5-iodo-UTP (5b) can out-compete UTP (5a), the natural substrate of GalU, indicating some degree of relaxed GalU substrate specificity. The fact that no formation of 5-iodo-UDP-Gal (1b) was observed in the multi-enzyme transformation (2b) may have been formed in that reaction, it could not be further processed by GalPUT.A series of experiments were conducted to assess the flexibility of GalU towards 5-substitution of its UTP substrate. A control experiment , within 10 min with 10 mM PPi (1b) was subjected to analogous conditions no conversion was observed even after incubation for 60 min (data not shown). To see whether the lack of conversion of the galacto-configured substrates was down to lack of binding or to non-productive binding of the substrates, inhibition experiments employing 5 equiv of 5-iodo-UDP-Gal (1b), 1 equiv of 5-iodo-UDP-Glc (2b) and excess PPi were conducted. By 1H NMR, 5-iodo-UDP-Glc (2b) was fully converted into Glc-1-P and 5-iodo-UTP (5b) within 10 min as in the no inhibitor control reaction (1b) was detected, which suggests that 5-iodo-UDP-Gal (1b) does not bind to the active site of GalU. A competition experiment was designed to show whether a large excess of Gal-1-P (5 equiv) can outcompete the natural acceptor Glc-1-P (1 equiv) in a GalU mediated conversion of 5-iodo-UTP (5b) (1 equiv) into 5-iodo-UDP-sugar. 1H NMR spectra showed that only 5-iodo-UDP-Glc (2b) was formed and no trace of 5-iodo-UDP-Gal (1b) was detected, even after 120 min (not shown).The lack of GalU-mediated conversion of Gal-1-P with 5-iodo-UTP was somewhat unexpected and warranted further analysis. First, it was shown that in the presence of a high concentration of inorganic pyrophosphate (PPi), GalU can perform the reverse conversion from 5-iodo-UDP-Glc (0 mM PPi . When 5-reaction . No conv5b), although both in their own right are productive substrates in the presence of alternative co-substrates. It may be that a conformational change is required in order to enable co-substrates to bind to GalU in a productive manner, but this is either too slow, or it does not happen at all, when Gal-1-P and 5-iodo-UTP are employed. Further structural analyses are required in order to address this point.From the above data, it is evident that GalU is not able to simultaneously bind both Gal-1-P and 5-iodo-UTP  is an enzyme known to catalyse the conversion of UDP-Gal (1a) into UDP-Glc (2a), with the equilibrium favouring the latter over the former (ca 1:4).1f) is not a substrate for Streptococcus thermophilus GalE.ScGalE)Erwinia amylovora .As noted above, GalU successfully produces a range of base-modified 1a) into UDP-Glc (2a) was achieved using both ScGalE and EaGalE and the progress of the epimerization was followed by 1H NMR. Under the condition employed, the equilibrium reaction mixtures were reached within 10 min and the ratio between galacto-/gluco-configured products were approximately 1:4, as expected (1f) with ScGalE and EaGalE did not show any 4″-OH epimerization by 1H NMR, even after prolonged incubation (120 min). Similarly, when 5-iodo-UDP-Gal (1b) was used as a substrate, ScGalE failed to effect conversion, even after extended incubation (120 min).As control experiments, the conversion of UDP-Gal  into 5-iodo-UDP-Glc (2b) and the transformation reached equilibrium after about 30 min giving mixed galacto-/gluco-configured products in the ratio 3:7. The reverse conversion of 2b into 1b using EaGalE was also shown to achieve a ca 7.5:2.5 equilibrium mixture of gluco-/galacto-configured sugar nucleotides after 30 min  or pyrophosphorylase ] or epimerization of the C-4″ stereochemistry of the pre-formed sugar nucleotide [action of epimerase ]. These studies demonstrate that the one-pot combination of glucose-1-phosphate uridylyltransferase  and UDP-glucose pyrophosphorylase  is able to catalyse the conversion of 5-substituted UTP derivatives into the corresponding 5-substituted UDP-galactose derivatives in a number of instances, albeit in poor yield (<5–23% isolated yield). It appears that the specificity of GalPUT is a limiting factor in the utility of this reaction. In contrast, GalU in conjunction with inorganic pyrophosphatase was able to convert 5-substituted UTP derivatives plus a range of gluco-configured sugar-1-phosphates into the corresponding sugar nucleotides in practical yields (20–98%). Subsequent attempts to convert these gluco-configured compounds to the corresponding galacto-isomers proved problematic, with UDP-glucose 4″-epimerase  from both yeast and Erwinia proving ineffective for bulky 5-aryl derivatives. However, in contrast to the yeast enzyme, the Erwinia GalE proved effective with 5-iodo-UDP-glucose, readily converting it to 5-iodo-UDP-galactose. Given the established potential for Pd-mediated cross-coupling of 5-iodo-UDP-sugars, the enzymatic procedures elaborated in this study provide useful additions to the repertoire of transformation available for the production of novel sugar nucleotides.5-Substituted 44.14.1.11a, 2a and 4a) was confirmed by comparison of 1H NMR spectra and/or HPLC retention times of authentic samples. 5-Substituted UTP derivatives 5b–f1b), 5-iodo-UDP-Glc (2b) and 5-(5-formyl-2-thienyl)-UDP-Gal (1f)1b),1d),1e),2b),2c),2d),2e),3a),4b).All chemicals and reagents were obtained commercially and used as received unless stated otherwise. The identity of products from our control experiments  from galactose-adapted yeast  was purchased from Sigma–Aldrich. Uridine-5′-diphosphogalactose 4″-epimerase  from Erwinia amylovora  was cloned, overexpressed and purified as detailed below.Galactose-1-phosphate uridylyltransferase  from 4.1.4E. amylovora strain Ea273 (ATCC 49946) using the following primers: GalE-F 5′-CGATCACCATGGCTATTTTAGTCACGGGGG and GalE-R 5′-CGATCACTCGAGTCAACTATAGCCTTGGGG. These primers included NcoI and XhoI restriction sites, respectively (underlined). The PCR product was purified from agarose gel using a QIAquick gel extraction kit  and treated for 3 h at 37 °C with NcoI and XhoI  for double digestion. After purification using QIAquick PCR Purification Kit , the digested PCR product was ligated into pETM-30 vector.Escherichia coli NovaBlue cells , purified using a DNA miniprep kit  and sequenced by Microsynth AG (Switzerland) to test the correctness of the gene sequence. E. coli BL21 (DE3) chemically competent cells  were transformed with the pETM-30::GalE construct for expression of the recombinant GST-fusion protein. Cells containing the construct were grown overnight in 10 mL 2 × YT medium containing Kanamycin (30 μg mL−1) at 37 °C. The starter culture was used to seed 1 L of medium (1:100 dilution) and the culture was grown at 37 °C for 3 h (O.D. 0.8). The temperature was then decreased to 18 °C and the culture was left to equilibrate for 1 h before induction with 1 mM IPTG for 16 h. Cells were harvested by centrifugation at 4500g for 15 min at 4 °C, re-suspended in 50 mL ice cold PBS containing 0.2 mg mL−1 lysozyme and protease inhibitors, stirred for 30 min at room temperature and lysed by sonication  on ice for 2 min using 2 s cycles (15.6 MHz). After centrifugation at 18000g for 20 min at 4 °C the supernatant was filtered and loaded onto a GSTrap HP 5 mL column  equilibrated with PBS at a flow rate of 1.5 mL min−1. The column was then washed with PBS until the A280 reached the baseline and the enzyme was eluted with 10 mM reduced glutathione in 50 mM TRIS–HCl buffer at pH 8.0. The eluted protein was dialysed against 50 mM TRIS–HCl buffer at pH 8.0 containing 10% glycerol, concentrated to 0.1 mg mL−1 and stored at −20 °C. Protein purity was confirmed by SDS–PAGE.The GalE gene (ENA accession number FN666575.1) was amplified by PCR from genomic DNA isolated from 4.24.2.1265. After multiple injections, the column was washed with 3 CV of 1 M ammonium bicarbonate followed by 3 CV of MQ water.Strong anion-exchange (SAX) HPLC on Poros HQ 50. An aqueous solution of a sample was applied on a Poros HQ 50 column . The column was first equilibrated with 5 CV of 5 mM ammonium bicarbonate buffer, followed by a linear gradient of ammonium bicarbonate from 5 mM to 250 mM in 15 CV, then hold for 5 CV, and finally back to 5 mM ammonium bicarbonate in 3 CV at a flow rate of 8 mL/min and detection with an on-line detector to monitor A4.2.23NHOAc, pH 6.8 with 1.5% CH3CN in 8 CV at a flow rate of 5 mL/min and detection with on-line UV detector to monitor A265. Fractions containing the sugar nucleotide were pooled and freeze-dried.Reverse phase (RP) C18 purification. The purification was performed on a Dionex Ultimate 3000 instrument equipped with UV/vis detector. A solution of a sample in water was applied on a Phenomenex Luna 5 μm C18(2) column  and eluted isocratically with 50 mM Et4.34.3.15a–f, 0.5 mg, 1 equiv), α-d-galactose-1-phosphate (1 equiv) and UDP-Glc  were dissolved in buffer . A small sample (50 μL) was separated for no enzyme control. Then enzymes were added to give final concentration of GalU (137 μg/mL), IPP (1.4 U/mL) and GalPut (329 μg/mL) in a final volume of 700 μL. The mixture was incubated at 37 °C with gentle shaking. At time points analytical samples were separated (50 μL) and MeOH was added (50 μL) to precipitate the enzymes. The sample was vortexed for 1 min, centrifuged  and the supernatant was filtered through a disc filter (0.45 μm). The filtrate was analysed by SAX HPLC . After 24 h the reaction was quenched by addition of MeOH (the same volume as the sample volume) and processed as indicated for the analytical sample. Products were isolated using SAX HLC (Purification method 1). Pooled fractions containing the sugar nucleotide were freeze-dried. When necessary, Purification method 2 was also applied.UTP analogue  and sugar-1-phosphate (1 equiv) were dissolved in deuterated buffer  and 1H NMR was acquired (100 scans) of the no enzyme control. GalU  was added to total 0.7 mL. Where indicated IPP  was added to this mixture. 1H NMR spectra were acquired at time points 10, 30, 60, 120 min to monitor reaction progress. The reaction was quenched by addition of an equal volume of methanol (0.7 mL), and the resulting solution was filtered through a 0.22 μm disc filter and products were purified using Purification method 1.UTP analogue  was dissolved in deuterated buffer  and 1H NMR of no enzyme control was acquired (100 scans). GalE was added  to give final volume of 0.7 mL and 1H NMR spectra were acquired at time points 10, 30 and 60 min.Appropriate sugar nucleotide  and Glc-1-P  as described in General procedure 2 and the product was isolated using Purification method 1 followed by Purification method 2 with the following modification: isocratic elution for 20 min at flow 5 mL/min with 50 mM Et3N.HOAc, pH 6.8 with 1.5%  and acetonitrile , UV detection at 350 and 265 nm. The title compound 2f eluted at fR = 9.5 min and was obtained after freeze-drying as bistriethylammonium salt .The diagnostic peaks were extracted from a spectrum of the crude mixture purified by SAX only (Purification method 1) giving bisammonium salt of 2f. 1H NMR : δ 9.69 , 8.32 , 7.92 , 7.64 , 6.01 , 5.51 . HRMS, ESI negative: m/z calcd for C20H25N2O18P2S− [M−H]−: 675.0304, found: 675.0304.The title compound 2f was prepared from 5-(5-formyl-2-thienyl)-UTP  and GlcN-1-P  as described in General procedure 2 and the product was isolated using Purification method 1 followed by Purification method 2. The title compound 3b eluted at Rf = 12.9 min and was obtained after freeze-drying as a triethylammonium salt . 1H NMR : δ 8.13 , 5.85 , 5.66–5.60 , 4.31–4.27 , 4.21–4.12 , 3.87–3.66 , 3.45–3.39 , 3.19 3N), 1.17 3N). HRMS, ESI negative: m/z calcd for C15H23IN3O16P2− [M−H]−: 689.9604, found: 689.9596.The title compound


The conformation of the piperidine ring is modified by the hybridization state of the C atom in the α-position to the piperidinic N atom. N-substituted 4-piperidinone 1-[(1R)-2-hydroxy-1-phenylethyl]piperidin-4-one, C13H17NO2, (I), has a chair conformation, while the piperidine substituted in position 2 with a thiocarbonyl group, 8-[(1S)-1-phenylethyl]-1,4-dioxa-8-azaspiro[4.5]decane-7-thione, C15H19NO2S, (II), features a half-chair conformation. Comparison of the two structures, and data retrieved from the literature, suggests that the conformational flexibility is mainly related to the hybridization state of the C atom α to the piperidinic N atom: a Csp3 atom favours the chair conformer, while a Csp2 atom distorts the ring towards a half-chair conformer. In the crystal structure of (I), weak C—H⋯O hydrogen bonds link the molecules into supramolecular chains propagating along the b-axis direction. In the crystal of (II), the molecules are linked by weak C—H⋯S contacts into supramolecular chains propagating along the b-axis direction.The crystal structures of the two title piperidine derivatives show different conformations for the six-membered heterocycle. The  N-(1-phenethyl-4-piperidyl) propionanilide], a strong agonist of μ-opioid receptors, used for its potent analgesic activity. This industrial synthesis, patented by Janssen Pharmaceutica  Å, and the Cremer parameters are θ = 168.8 (3)° and φ = 171.8 (18)°. The deviation from the ideal conformation, θ = 180°, may be related to the heterocyclic nature of the ring, with short C—N bond lengths and longer C—C bond lengths, as expected. Moreover, atom C4 has a geometry consistent with its sp2 hybridization state, while N1 is essentially tetrahedral, with the lone pair occupying the axial position. The equatorial group substituting this N atom is rigid, as a result of its chiral character. However, the spatial orientation of this group allows the hydroxyl group to interact with the nitrogen lone pair, stabilizing the observed molecular conformation.The first piperidin-4-one derivative .The crystal structure of (II)Compound (I). The synthesis is illustrated in Fig. 3R)-(−)-2-phenylglycinol  with an excess of ethyl acrylate in methanol (60 mL), was stirred overnight at 298 K. The reaction mixture was concentrated, and the crude purified by column chromatography , to afford (2) as a colorless oil (98%). An amount of (2) (40.6 mmol) was added to a mixture of MeONa in anh. benzene. After refluxing the mixture for 5 h, a solid was obtained, which was filtered and dried in air. This solid was treated with AcOH:water  until pH = 1, initiating the decarboxylation process. The mixture was refluxed until gas evolution stopped. After cooling down to 298 K, pH was adjusted to 7 with NaHCO3, and the mixture was washed with CH2Cl2 (3 × 50 ml). The organic phase was dried over Na2SO4, and concentrated. Compound (I)2, CH2Cl2:MeOH, 95:5). Compound (I)n-hexane mixture (1:1).Compound (II). The synthesis is illustrated in Fig. 4S)-(−)-phenylethylpiperi-2,4-dione, has been reported previously . The organic phase was dried over Na2SO4, and then concentrated under reduced pressure. The crude reaction was purified by column chromatography , to afford compound (4) as a white oil, in 95% yield. Next, a suspension of Lawesson’s reagent  in dry toluene (60 mL) was refluxed until complete dissolution of the reagent. The solution was cooled to 313 K, and a solution of (4) in anh. toluene  was added . The product was recrystallized from CH2Cl2:n-hexane (1:1).3), 0.97 (methylene CH2), or 0.98 Å (methine CH). For (I)Uiso(H) = xUeq(carrier atom), where x = 1.5 for methyl and hydroxyl H atoms, and x = 1.2 otherwise. The absolute configuration for chiral centers C7 in (I)et al., 2013Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015017119/xu5868sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989015017119/xu5868Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015017119/xu5868IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989015017119/xu5868Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015017119/xu5868IIsup5.cmlSupporting information file. DOI: 1424094, 1424093CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal of the title molecular salt, the pyridinium cation and the 3-hydroxy-2,4,6-trinitrophenolate anion are linked through bifurcated N—H⋯ hydrogen bonds forming an  5H6N+·C6H2N3O8− , the pyridinium cation and the 3-hydroxy-2,4,6-trinitrophenolate anion are linked through bifurcated N—H⋯ hydrogen bonds, forming an R12(6) ring motif. The nitro group para with respect to phenolate ion forms an intramolecular hydrogen bond with the adjacent phenolic –OH group, which results in an S(6) ring motif. The nitro group flanked by the phenolate ion and the phenolic –OH group deviates noticeably from the benzene ring, subtending a dihedral angle of 89.2 (4)°. The other two nitro groups deviate only slightly from the plane of the benzene ring, making dihedral angles of 2.8 (4) and 3.4 (3)°. In the crystal, the 3-hydroxy-2,4,6-trinitrophenolate anions are linked through O—H⋯O hydrogen bonds, forming chains along [100]. These anionic chains, to which the cations are attached, are linked via C—H⋯O hydrogen bonds, forming a three-dimensional structure. Impact friction sensitivity tests and TGA/DTA studies on the title molecular salt imply that it is an insensitive high-energy-density material.In the crystal structure of the title molecular salt, C The title molecular salt decomposes (70–80%) in two stages. For each stage, the energy of activation was determined employing Kissinger 1957 [stage IStyphnic acid  dissolved in 25 mL of absolute alcohol was mixed with pyridine  and stirred continuously for 6 hrs and then kept aside for 2 h. The yellow-coloured amorphous solid obtained was filtered, washed with 30 ml of dry ether and recrystallized from ethylene glycol. Yellow crystals formed in an ethylene glycol solution after slow evaporation at 298 K over a period of 2 weeks .Uiso(H) = 1.5Ueq(O) for the hydroxyl H atom and = 1.2Ueq(C) for the other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989014027704/su5046sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989014027704/su5046Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989014027704/su5046Isup3.cmlSupporting information file. DOI: 1006492CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Both polyanions present two ·5H2O, A, and bis{[tris(hydroxymethyl)methyl]ammonium} decaaquadi-μ4-oxido-tetra-μ3-oxido-hexadeca-μ2-oxido-hexaoxidodimanganese(II)decavanadate(V) dihydrate, [NH3C(CH2OH)3]2[V10O28{Mn(H2O)5}2]·2H2O, B, have been synthesized under mild reaction conditions in an aqueous medium. Both polyanions present two [Mn(OH2)5]2+ complex units bound to the decavanadate cluster through oxide bridges. In A, the decavanadate unit has 2/m symmetry, whereas in B it has twofold symmetry. Apart from this, the main differences between A and B rest on the organic cations, tetramethylammonium and [tris(hydroxymethyl)methyl]ammonium, respectively, and on the number and arrangement of the water molecules of crystallization. In both compounds, the H atoms from the coordinating water molecules participate in extensive three-dimensional hydrogen-bonding networks, which link the cluster units both directly and through solvent molecules and, in B, through the ‘tris’ cation hydroxyl groups. The cation in B also participates in N—H⋯O hydrogen bonds. A number of C—H⋯O interactions are also observed in both structures.Two heterometallic decavanadate(V) compounds, bis(tetramethylammonium) decaaquadi-μ Product A contains two tetramethylammonium cations and the [V10O28]6– unit is covalently bound to two [Mn(OH2)5]2+ complexes by terminal oxido bridges.While the synthesis of decavanadates with different organic cations as building blocks for supramolecular assemblies is largely explored (da Silva 10O28{Mn(H2O)5}2]2–, in its turn, was achieved by reaction of NH4VO3 with tris(hydroxymethyl)methylamine (‘tris’) and manganese(II) chloride at pH 3 in a 5:3:1 molar proportion. Yellow crystals of [NH3C(CH2OH)3]2[V10O28{Mn(H2O)5}2]·2H2O (B) were isolated in 12% yield, as the only reaction product, after one week at room temperature. X-ray diffraction analyses revealed very similar structures for the heteropolyanions in A and B.The rational synthesis of the heteropolyanion 2– are in the ranges reported in the literature  has the shortest V—O distance, ca 1.6 Å, corresponding to a vanadyl group. The more ‘internal’ vanadium atoms in each structure, viz V3 in A, and V6 and V7 in B, have more uniform V—O distances in more regular octahedral patterns.Bond angles and lengths determined for 2– heteropolyanion, see: Klištincová et al.  water. Commercial reagents were used without purification. The starting materials NH4VO3, MnCl2·4H2O and Mn(OAc)2·4H2O were supplied by Aldrich, while mannitol [C6H8(OH)6] and (Me4N)Cl were purchased from USB and Merck, respectively. Infrared (FTIR) spectra were recorded on a BIORAD FTS-3500GX spectrophotometer from KBr pellets in the 400–4000 cm−1 region.All reactions were performed in air with purified (Milli-Q4)2[V10O28{Mn(H2O)5}2]·5H2O (A)Synthesis of  and [(CH3)4N]Cl  were added to a solution of mannitol  in 60 mL of water to produce a suspension that turned into a deep blue–greenish solution after one hour under reflux. After 24 more hours, a solution of Mn(OAc)2·4H2O  in 10 mL of water was added to this reaction mixture, which remained under reflux for one more day. The solution was concentrated to one third of its initial volume and, after four weeks at room temperature, a mixture of deep-green crystals of (Me4N)6[V15O36(Cl)] . The FTIR spectrum recorded for A shows the characteristic bands of the Me4N+ cation at 3031, 1639, 1485 and 1263 cm−1 and of the inorganic anion at 966, 833, 744, 584 and 455 cm−1.Solid NH3C(CH2OH)3]2[V10O28{Mn(H2O)5}2]·2H2O (B)Synthesis of .In 10.1107/S2056989014028230/hb7337sup1.cifCrystal structure: contains datablock(s) Compound-A, Compound-B, global. DOI: 10.1107/S2056989014028230/hb7337mpf-Asup2.hklStructure factors: contains datablock(s) mpf-A. DOI: Click here for additional data file.10.1107/S2056989014028230/hb7337mpf-Asup4.cdxSupporting information file. DOI: 10.1107/S2056989014028230/hb7337mpf-Bsup3.hklStructure factors: contains datablock(s) mpf-B. DOI: Click here for additional data file.10.1107/S2056989014028230/hb7337mpf-Bsup5.cdxSupporting information file. DOI: 1041495, 1041494CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The structures of these hybrids were elucidated by extensive analyses of spectra and calculated electronic circular dichroism (ECD) method.The basic substances of life include various amino acids and sugars. To search such molecules is the precondition to understand the essential nature. Here we reported four unprecedented hybrids of  The plant is an annual herb belonging to the Ranunculaceae family, and its root is used in traditional Chinese Medicine as a treatment for faucitis. Additionally, the ethanol extract of the plant has clearly exhibited antitumor and antituberculosis activities3R. ternatus, which other researchers have not exploited, four novel compounds (561–4 possess a relative integrated γ-aminobutyric acid (GABA) moiety and monosaccharide or disaccharide moieties. More intriguingly, all of the compounds have potential tail to tail ether-connected  bonds. This represents an unprecedented structural phenomenon for the connections of sugars with the participation of γ-amino acids. Considering the significant roles of sugars and γ-amino acids, these novel compounds could provide opportunities for expanding our understanding of various natural chemical and biosynthetic phenomena.Amino acids and sugars perform vital roles in all living organisms. They are involved in complex biological processes such as catalysis and highly selective molecular recognition. Their hybrids, sugar amino acids (SAAs), which feature both a sugar framework and amino acid functional groups, have attracted substantial interest for their roles in peptidomimetic studies1ompounds  were iso1 was obtained as a yellow amorphous powder. Its molecular formula was determined to be C16H23NO9 based on the high resolution electrospray ionization mass spectroscopy (HRESIMS) quasi-molecular ion at m/z 396.1266 [M + Na]+ , corresponding to 6 degrees of unsaturation. The infrared radiation (IR) spectrum of 1 revealed the presence of hydroxyl (3388 cm−1), carbonyl (1712 cm−1), and aldehyde (1649 cm−1) groups. The 1H NMR spectrum , a system of sp2 aromatic resonances at δ 6.98  and 6.27 , and four methylenes at δ 4.50 , 4.25 , 2.20 , and 1.85 . Interestingly, instead of one remaining sugar moiety, which would be expected considering the molecular weight of the compound, two sets of similar sugar resonances were observed; additionally, the 13C NMR sugar resonances were presented in pairs, implying that two anomers of the sugar were in equilibrium. In the 13C NMR spectrum  experiment (δ 4.25) correlated with C-2′ (δ 132.0), C-5′ (δ 139.2), C-3 (δ 26.3) and C-2 (δ 30.7); H-3 (δ 1.85) correlated with C-4 (δ 44.2), C-2, and C-1 (δ 173.9); H-2 (δ 2.20) correlated with C-1 and C-3, indicating the existence of a 4-(2-formyl-1H-pyrrol-1-yl) butanoic acid moiety. Furthermore, an oxygenated methylene hydrogen signal at H-6′ (δ 4.50) correlated with C-4′ (δ 111.2) on the pyrrol ring, as well as C-6″α (δ 70.8) and C-6″β (δ 72.4) on the fructose moiety. Additionally, correlations between H-6″β  and C-6′β (δ 63.3), and H-6″α  and C-6′α (δ 63.6) were observed, which indicated that the oxygenated methylene of the 4-(2-formyl-1H-pyrrol-1-yl) butanoic acid moiety was connected with position C-6 on the fructose moiety through an ether linkage. Thus, compound 1 was determined and named ranunculin A.Compound spectrum  of 1 disspectrum , the resperiment , the pro2 was also obtained as a yellow amorphous powder. The HRESIMS gave a quasi-molecular ion at m/z 396.1266 [M + Na]+ , which corresponded to the molecular formula C16H23NO9. The IR, UV, and NMR data  butanoic acid moiety. However, NMR resonances for a fructose moiety in 1 were absent and were replaced by signals attributed to the glucose moiety in 2. Furthermore, correlations between H-6″ and C-6′ (δ 63.5 and 63.4) of the 4-(2-formyl-1H-pyrrol-1-yl) butanoic acid moiety and H-6′ (δ 4.50) and C-6″  of the glucose moiety were observed in the HMBC spectra . Comparisons of the molecular weight and sites of unsaturation of compound 3 with those of 2 suggested that an additional hexose moiety existed in compound 3. Further analysis of the NMR spectra showed that most of the data were similar, with the exception of a set of fructose signals  of the glucose moiety and C-2″′(δ 104.1) of the fructose moiety and H-6′ (δ 4.50) and C-6″ (δ 71.9) indicated that the oxygenated methylenes of 4-(2-formyl-1H- pyrrol-1-yl) butanoic acid moiety were connected with the C-6 position of the glucose moiety through an ether linkage, while the C-1 position of β-fructose was connected with the anomeric proton of the glucose moiety through an α-linkage. Thus, compound 3 was determined and named ranunculin C.The molecular formula of  signals . By contmpound 1 . In the mpound 1 , the cor4 was obtained as a yellow amorphous powder. An HRESIMS analysis provided an [M + Na]+ ion at m/z 510.1587, appropriate for a molecular formula of C21H29NO12  requiring 8 sites of unsaturation. Based on the NMR information, a substructure similar to that of compound 2 could be readily deduced, considering the aforementioned structural analysis. However, in contrast to the latter, the NMR signals of 4 were not in pairs, which suggested that the anomeric proton of the glucoside was substituted. In addition to these similar resonances, the 1H NMR  and 3.68 , two methylenes at δ 2.82  and 2.25  and two oxygenated methines at 4.52  and 4.39 . In the 13C NMR spectrum, an ester carbonyl carbon at δ 175.9, one methylene carbon at δ  38.8, and three oxygenated carbons at δ 83.2, 67.6, and 67.1 were observed. The 1H-1H correlated spectroscopy (1H-1H COSY) spectrum  and H-2″′ (δ 4.52), between H-2″′ and H-3″′ (δ 4.39), and between H-3″′ and H-4″′ , suggesting the existence of an additional four-chain carbon moiety. Furthermore, in the HMBC spectra (δ 175.9) were observed; thus, 4 should contain a five-membered lactone with a methylene substitute combing the remaining 2 degrees of unsaturation. Moreover, the HMBC correlations between the methylene proton H-1″′(δ 4.00 and 3.68) and the glucoside anomeric carbon C-1″ (δ 103.0), and between the glucoside anomeric proton H-1″ (δ 4.23) and the C-1″′ indicated that the five-membered lactone was connected with an anomeric carbon of the glucoside moiety through a methylene bond and that the β-configuration of the glucoside was determined based on the coupling constant, J = 8.0, of the anomeric proton. The configurations of C-2″′ and C-3″′ were deduced to be 2″′S, 3″′S or 2″′R, 3″′R on the basis of related literature84a and 4b (4aC1 (92.00%) were obtained by a systematic conformational analysis, which was performed for 4a using an MMFF94 molecular mechanics force field calculation 1011121314head to head , head to tail  and head to body  connections. Coyolosa, a so-called 6,6′-ether-connected sugar, was reported to have been isolated from the root of Acrocomia Mexicana in one study171813C NMR data of C-6 were still at δC 61.1 when the 6,6′-ether-connection was formed. Nevertheless, inspired by the proposed coyolosa, Takahashi et al.18tail to tail connected sugars have been found from natural products, not to mention the participation of γ-amino acid moieties. Thus, compounds 1–4 represent a new connective sample between a 6,6′-ether-connected sugar and γ-amino acid. Subsequently, different synthetic routes were designed to obtain such compounds. For example, the reactions between 4-(2-formyl-5-hydroxymethyl-1H-pyrrol-1-yl) butanoic acid esters and 6-iodo- or 6-trifluoromethanesulfonate- substituted sugars with the remaining hydroxyls by acetonide protection or between 4-(2-formyl-5-chloro-1H-pyrrol-1-yl) butanoic acid esters and the aforementioned sugars with the remaining hydroxyls by acetonide protection, among others. Unfortunately, all of these attempts failed. Thus, these unusual molecules could challenge the synthetic skills of curious investigators. For living organisms in nature, γ-aminobutyric acid is the major inhibitory neurotransmitter in the mammalian central nervous system, whereas D-glucose is one of the main products of photosynthesis and fuel sources for cellular respiration. What factors will play a role in the sustainability of such molecules when they form conjugates by such connection modes? Perhaps only when large amounts of synthetic products are obtained will this exploration be successful.Structurally, for ranunculins A-D , their p1H NMR (500 MHz), 13C NMR (125 MHz), and 2D-NMR spectra were recorded with an INOVA-500 spectrometer with TMS as an internal standard. Values were given in ppm. HRESIMS were obtained using an Agilent 1100 series LC/MSD ion trap mass spectrometer. The optical rotations were measured with a Jasco P-2000 polarimeter. Column chromatography was performed on macroporous resin , Rp-18 , and Sephadex LH-20 . Preparative HPLC separation was performed using a Shimadzu LC-10AT instrument with an SPD-10A detector using a YMC-Pack ODS-A column .The IR spectra were obtained using an IMPACT 400 (KBr) spectrometer. Ranunculus ternatus Thunb were purchased from Beijing Pu Sheng Lin Pharmaceutical Co., Ltd. in Beijing, China, in Feb 2010. The plant material was identified by Prof. Ma Lin . A specimen was deposited at the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Beijing, China.The roots of Ranunculus ternatus Thunb (20.0 kg) were extracted with 95% EtOH twice and then extracted with 70% EtOH once. The residue (5.1 kg) obtained by concentrating the EtOH extract in vacuo was suspended in water and extracted with petroleum ether, ethyl acetate and n-butanol. The remaining water portion after extraction was subjected to HP-20 macroporous resin using an H2O-EtOH gradient to give six fractions. The 30% ethanol elutant from the macroporous resin column was separated into two subfractions by Sephadex LH-20 using an H2O-MeOH gradient. Subsequently, subfraction 1 was processed by a combination of reversed phase silica gel, Sephadex LH-20 column chromatography and preparative HPLC to yield compounds 1 (15 mg), 2 (12 mg), and 3 (10 mg) and subfraction 2 to give 4 (8 mg). The purity of the isolated species were shown by HPLC (μm) maintained at 40 °C under a flow rate of 1 mL/min. The chromatograms of these compounds were achieved under 295 nm with a programmed gradient of methanol (A) and water-acetic acid : 0–30 min, 2–60% A; 30–40 min, 60–100% A.Air-dried powder roots of  by HPLC . The exp1, yellow amorphous powder, max (MeOH): 295 nm. IR: 1712 cm−1, 1649 cm−1. ESI-MS m/z: 374.4 [M + H]+, HRESIMS: m/z 396.1266 [M + Na]+ . 1H, 13C NMR data , see Compound 2, yellow amorphous powder, max (MeOH): 295 nm. IR: 1708 cm−1, 1647 cm−1. ESI-MS m/z: 374.5 [M + H]+, HRESIMS: m/z 396.1266 [M + Na]+ . 1H, 13C NMR data , see Compound 3, yellow amorphous powder, max (MeOH): 295 nm. IR: 1706 cm−1, 1647 cm−1. ESI-MS m/z: 536.1. [M + H]+, HRESIMS: m/z 558.1791. [M + Na]+ . 1H, 13C NMR data , see Compound 4, yellow amorphous powder, max (MeOH): 298 nm. IR: 1776 cm−1, 1712 cm−1, 1650 cm−1. ESI-MS m/z: [M + H]+ 488.1, HRESIMS: m/z 510.1587 [M + Na]+ . 1H, 13C NMR data , see Compound How to cite this article: Feng, Z.-m. et al. Naturally occurring hybrids derived from γ-amino acids and sugars with potential tail to tail ether-bonds. Sci. Rep.6, 25443; doi: 10.1038/srep25443 (2016).


The cloning, expression, purification and crystallization of the CH domain of the plant-specific kinesin GhKCH2 is reported. Kd = 0.42 ± 0.02 µM) than most other CH-domain-containing proteins. To understand the underlying mechanism, prokaryotically expressed GhKCH2-CH (amino acids 30–166) was purified and crystallized. Crystals were grown by the sitting-drop vapour-diffusion method using 0.1 M Tris–HCl pH 7.0, 20%(w/v) PEG 8000 as a precipitant. The crystals diffracted to a resolution of 2.5 Å and belonged to space group P21, with unit-cell parameters a = 41.57, b = 81.92, c = 83.00 Å, α = 90.00, β = 97.31, γ = 90.00°. Four molecules were found in the asymmetric unit with a Matthews coefficient of 2.22 Å3 Da−1, corresponding to a solvent content of 44.8%.GhKCH2 belongs to a group of plant-specific kinesins (KCHs) containing an actin-binding calponin homology (CH) domain in the N-terminus. Previous studies revealed that the GhKCH2 CH domain (GhKCH2-CH) had a higher affinity for F-actin ( In comparison, knowledge of plant CH-domain crystal structures remains limited, with only one structure available  fibres in our laboratory, has a motor domain with microtubule-stimulated ATPase activity and a CH domain that strongly interacts with microfilaments, suggesting it to be a candidate for a linker between microfilaments and microtubules , containing the CH domain, had a higher affinity for F-actin (Kd = 0.42 ± 0.02 µM) than most other CH-domain-containing proteins (Kd = ∼5–50 µM) , a KCH previously cloned from cotton (2.2.1.CA TAT GGA TTT GGA ATC TAG AAA AGC TG-3′ (NdeI site in bold) and the reverse primer 5′-CAA CTC GAG TTA CGA GAG CCT CCA CTC GTT ATA G-3′ (XhoI site in bold). The PCR product was digested and inserted into a modified pGEX-4T-2 vector  at the NdeI and XhoI restriction sites with a TEV protease cleavage site between GST and the target gene (Table 1Escherichia coli strain BL21(DE3). Cells were grown at 37°C in LB medium containing 100 mg ml−1 ampicillin to an A600 of 0.6–0.8 and were induced with 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 22°C overnight. The cells were harvested by centrifugation and lysed by gentle sonication in lysis buffer . After high-speed centrifugation, the supernatant was loaded onto a home-made Glutathione Sepharose 4B column  and incubated for 1 h. After washing with wash buffer , TEV protease was added to cleave the fusion protein overnight. The collected flowthrough was purified by Mono Q chromatography and further polished by gel filtration on a HiLoad 16/60 Superdex 75 pg column . The purified proteins were pooled, concentrated to 10–15 mg ml−1, flash-cooled in liquid nitrogen and stored at 80°C. Protein purity and identity were assessed by SDS–PAGE with Coomassie Bright Blue staining  and 1 µl precipitatant solution equilibrated against 100 µl reservoir solution at 277 K.Initial screening for crystallization conditions was carried out in 48-well sitting-drop plates using the commercially available kits Crystal Screen, Crystal Screen 2 and Index  at 4°C. Crystals of GhKCH2-CH were initially grown from a mixture of 1 µl protein solution (10–15 mg mla and 2b). Detailed information on GhKCH2-CH crystallization is given in Table 2Subsequent optimizations were performed using 24-well sitting-drop plates, and the size of the crystals was enlarged by streak-seeding using a cat whisker  PEG 8000, 10%(v/v) DMSO for 5 min, transferred to a solution consisting of 0.1 M Tris–HCl pH 7.0, 20%(w/v) PEG 8000, 20%(v/v) DMSO for a further 5 min and finally flash-cooled in liquid nitrogen. The diffraction data set was collected at 100 K on BL17U1 at Shanghai Synchrotron Radiation Facility (SSRF) using an ADSC Q315 CCD. A total of 360 images with an oscillation angle of 1° each were collected using a 250 mm crystal-to-detector distance and an exposure time of 1 s per frame  = 23.6] and weak reflections with indices of l = 2n + 1   with the highest scores were used as search models. When determining the crystal structure of GhKCH2-CH using Phaser  gave the best results after further refinement among these models. Four molecules were found in the asymmetric unit with a Matthews coefficient of 2.22 Å3 Da−1, corresponding to a solvent content of 44.8%.Indexing with l = 2n + 1) and even components (l = 2n) using MTZUTILS from the CCP4 package (Winn et al., 2011REFMAC or phenix.refine with even reflections (Murshudov et al., 2011et al., 2006et al., 2010Rfree of about 35%. Owing to the low amino-acid sequence identity of the GhKCH2 CH domain to other CH domain-containing proteins, crystallization of selenomethionine-labelled protein is in progress.The entire reflection data set was split into odd (


The title molecule possesses ten-, five- (lactone) and three-membered (epoxide) rings with a pyrimidine group as a substituent. The ten-membered ring displays an approximate chair–chair conformation, while the lactone ring shows a flattened envelope-type conformation. 19H22N2O3, {systematic name -1a,5-dimethyl-8-[(pyrimidin-5-yl)methylidene]-2,3,6,7,7a,8,10a,10b-octahydrooxirenocyclodecafuran-9(1aH)-one} was obtained from the reaction of parthenolide cyclodecafuran-9(1aH)-one] with 5-bromopyrimidine under Heck reaction conditions, and was identified as an E isomer. The molecule possesses ten-, five- (lactone) and three-membered (epoxide) rings with a pyrimidine group as a substituent. The ten-membered ring displays an approximate chair–chair conformation, while the lactone ring shows a flattened envelope-type conformation. The dihedral angle between the pyrimidine moiety and the lactone ring system is 29.43 (7)°.The title compound, C The interesting biological properties of PTL directed our attention to design and synthesize additional bioactive derivatives. In order to obtain detailed information on the structural conformation of the current molecule, including assignment of the absolute configuration of the four stereocentres, and to establish the geometry of the exocyclic double bond, a single crystal X-ray structure determination has been carried out.Parthenolide (PTL) is a sesquiterpene lactone known to significantly target cancer stem cells, which are the putative roots of all types of cancer °. The C11=C13—C16 bond angle of 127.89 (16)° deviates from the ideal value of 120°, but other bond lengths and angles are largely unremarkable. The four chiral carbon atoms in PTL were determined using 1354 quotients ,C5(R),C6(S),C7(S) for the arbitrary atom-numbering scheme used, and is consistent with previous studies , 0.98 Å (RCH3), 0.99 Å (R2CH2) and 1.00 Å (R3CH). Uiso(H) parameters were set to values of either 1.2Ueq or 1.5Ueq (RCH3 only) of the attached atom. The absolute structure parameter [−0.04 (3)] was determined directly from the diffraction data using 1354 Parsons quotients  I, global. DOI: 10.1107/S2056989015021507/zs2350Isup2.hklStructure factors: contains datablock(s) I. DOI: 1436825CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Aging is linked to decrease of the body cell use of growth hormone (GH) and thyroxine, whereas the decrease is via “death hormones” inhibition? This study proposes different viewpoints. Since interleukin 17 receptor C (IL17RC) is highly expressed in tissues from age-related macular degeneration (AMD) patients, IL17RC signaling pathways are explored to evaluate Wnts/vascular endothelial growth factor (VEGF) expression and complement activity, which are pathological factors in AMD. IL17RC overexpression or VEGF treatment was performed in two cell lines for up to two-day. Real-time Quantitative PCR, confocal microscopy, immune-blot, MTT assay, etc. measured downstream effects. IL17RC overexpression increases Wnts and VEGF that forms complexes with Wnt-signaling components. VEGF or the Wnt-signaling components interacting with C3 suggests alternative complement pathway activation. Moreover, IL17RC-overexpressed cells or VEGF-treated cells for two-day, which is overstimulation, increase PI3K/Akt/GSK3 insensitivity and GSK3 activity, and decrease growth/survival. High GSK3 activity associates with many chronic diseases including type II Diabetes. This study shows high GSK3 activity can result from PI3K/Akt overstimulation. Type II Diabetes shows insulin resistance that the body cells decrease insulin use. Possessing little sensitive PI3K/Akt for receptor activation, cells after overstimulation, although live, hardly respond to PI3K/Akt activators including GH, thyroxine and insulin. These results suggest an alternative explanation of the body cells declining hormone use since various kinds of cell signaling-induced overstimulation events almost always linked to PI3K/Akt, increase with age. Playing pathological roles in senescence and diseases, overstimulation eventually generates health problems.The online version of this article (doi:10.1186/2193-1801-3-356) contains supplementary material, which is available to authorized users. In 1974, Dr. W. Donner Denckla proposed Death Hormone or DECO (decreasing oxygen consumption hormone) Theory Denckla . Age-relAMD is the leading cause of irreversible blindness in the elderly population worldwide. The disease causes progressive loss of central vision because of macular geographic atrophy (GA) (dry AMD) or choroidal neovascularization (CNV) (wet AMD) Sunness . AMD is ARPE (RPE from ATCC) and THP-1 cultured monocytes) were obtained from ATCC, and the cells were cultured according to the protocols from ATCC. Transfection (2 μg of empty vector (EV) or 2 μg of IL17RC) was performed using Nucleofector II purchased from Lonza according to the protocols from the manufacturer.Anti (α)-Akt, α-pAkt, α-Axin, α-β-catenin, α-C3, α-c-Myc, α-cyclin D1, α-GSK3, α-GSK3β, α-pGSK3, α-IL17, α-IL17RC, α-IRS1, α-PI3K, α-pPI3K, α-TAU, α-VEGF, α-Wnt-3a, and α-Wnt-10b were purchased from Santa Cruz. α-c/EBPα, α-pc/EBPα, α-ERK1/2, α-pERK1/2, and α-pIRS1 were bought from Cell Signaling Technology. α-GSK3β was obtained from BD Transduction Lab. GAPDH was purchased from ImmunoChemical. IL17A, IL17F and sFRP2 were bought from R and D. VEGF was obtained from Sigma-Aldrich. IL17A or IL17F (500 ng/mL), sFRP2 (250 ng/mL) or VEGF (10 ng/mL) was applied to cell culture. An IL17RC expression vector was purchased from OriGene.EV- or IL17RC-transfected ARPE cells were plated onto sterile cover slips. Cells were left untreated or treated with sFRP2 for two-day. The fixing, blocking and staining procedures have been described  was directly applied to cells in the 96-well plates according to the protocol from the manufacturer to generate RT-PCR results, or RNA was extracted from cells in 24-well plates using a Qiagen kit. Primers to amplify VEGF, Wnt-3a, Wnt-10 and RPLP0 are described as follows: VEGF forward 5′ctacctccaccatgccaagt, reverse 5′tggtgatgttggactcctca; Wnt-3a forward 5′caagattggcatccaggagt, reverse 5′atgagcgtgtcactgcaaag; Wnt-10b forward 5′tgctttttccttctccatgc, reverse 5′tccaagaaatcccgagagaa; RPLP0 forward 5′ ggcgacctggaagtccaact, reverse 5′ ccatcagcaccacagccttc. RT-PCR procedures from total RNA have been described  statistical package based on t-test. An adjusted P-value of < 0.05 was considered significant.Because high expression of IL17RC protein in peripheral blood and chorioretinal tissues with AMD lesions  resulting in increased GSK3 activity Figure . Both paa Figure ; therefoBecause GSK3 is constitutively activated , D  or Figure , represented as mean ± SD. A, * versus EV of the same groups: p < 0.05. B, * versus control in the same groups: p <0.05; In c-Myc and cyclin D1 columns: there is no statistic difference between column 3 and column 1 or 2; # or ## versus any other treatment in the cognate groups: p < 0.05 or 0.01. C. *, ** or *** versus any other treatment in the same groups: p < 0.05, 0.01 or 0.001. (PDF 963 KB)Additional file 1: Figure S1: Statistic results of some blots in Figures  or 5A , represented as mean ± SD. A, * or ** versus any other treatment in the same groups: p < 0.05 or 0.01. B and C, * or ** versus control in the same groups: p < 0.05 or 0.01. (PDF 1 MB)Additional file 2: Figure S2: Statistic results of some blots in Figures Additional file 3: Figure S3: Statistic results of some blots in Figures


Both enantiomers occur as two crystallographically independent molecules in the asymmetric unit. In the crystal, molecules are linked by N—H⋯O hydrogen bonds forming β-sheet structures. The β-sheets are linked by weaker C—H⋯O hydrogen bonds, forming a supramolecular three-dimensional structure.The title compound, glycidamide, was synthesized  3H5NO2, is the mutagenic and genotoxic metabolite of acrylamide, a food contaminant and industrial chemical that has been classified as being probably carcinogenic to humans. Synthesized via the reaction of acrylonitrile and hydrogen peroxide, it crystallizes with both enantiomers occurring as two crystallographically independent molecules (A and B) in the asymmetric unit. They have similar conformations with an r.m.s. deviation of 0.0809 Å for molecule B inverted on molecule A. In the crystal, molecules are linked by N—H⋯O hydrogen bonds, which lead to the formation of β-sheet structures enclosing R22(8) and R42(8) loops. The β-sheets are linked by weaker C—H⋯O hydrogen bonds, forming a supramolecular three-dimensional structure.The title compound, glycidamide (systematic name: oxirane-2-carboxamide), C Both enantiomers occur as two crystallographically independent molecules  to acrylonitrile  in water (500 ml) and 30% H2O2 . The pH was kept at 7.3–7.5 and the temperature was maintained at 308–310 K. After the reaction was completed (about 12 h), the mixture was treated with 5% palladium on charcoal, stored overnight in a refrigerator and then filtered. The solvent was evaporated and the crude product  was recrystallized from dry acetone at low temperature. Colourless crystals formed after 3–5 days at 243 K. GA is very hygroscopic, so purification of the raw product was carried out in an inert atmosphere. The compound was stored in dry argon at 243 K. Identity and purity were checked by NMR spectroscopic methods and elemental analysis. 1H-NMR : δ 3,49 ; 3,02 ; 2,87 . 13C-{1H}-NMR : δ 170.1 (C1), 48.5 (C2), 45.6 (C3). Elemental analysis for C3H5NO2. Required: C 41.36%; H 5.79%; N 16.09%; found: C 41.41%; H 5.47%; N 16.27%.The synthesis of the title compound (GA) was performed according to a published method with modifications  = 1.2Ueq(N). The C-bound H were placed in calculated positions and refined using a riding model: C—H = 0.99–1.00 Å with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989016010859/su5309sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989016010859/su5309Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016010859/su5309Isup3.cmlSupporting information file. DOI: 1490160CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Two isotypic title compounds comprise a 2-iminopyridine ring fused with a cyclooctane ring. In one compound, the cyclooctane ring adopts a twisted chair–chair conformation, while in the second, this ring adopts a twisted boat–chair conformation. 25H24BrN3, (I), and C25H24FN3, (II), comprise a 2-iminopyridine ring fused with a cyclooctane ring. In (I), the cyclooctane ring adopts a twisted chair–chair conformation, while in (II), this ring adopts a twisted boat–chair conformation. The dihedral angles between the planes of the pyridine ring and the bromobenzene and phenyl rings are 80.14 (12) and 71.72 (13)°, respectively, in (I). The equivalent angles in (II) are 75.25 (8) and 68.34 (9)°, respectively. In both crystals, inversion dimers linked by pairs of C—H⋯N interactions generate R22(14) loops, which are further connected by weak C—H⋯π interactions, generating (110) sheets.The molecules of the two isotypic title compounds, C Its derivatives are known to possess antimicrobial (Jo The structures of compounds (I)ortho position C5. The endocyclic angles of the pyridine ring cover the range 114.29 (18)–123.02 (2)° and 118.86 (13)–123.11 (12)° for compounds (I)et al., 2005 In both compounds, the imino group is nearly coplanar with the pyridine ring, as indicated by the N1=C1—N3—C5 torsion angle pyridine-3-carbonitrile -5,6,7,8,9,10-hexahydrocycloocta[p-toluenesulfonic acid (pTSA) (0.5 mmol) was added. The reaction mixture was heated under reflux for 2–3 h. After completion of the reaction (TLC), the reaction mixture was poured into crushed ice and extracted with ethyl acetate. The excess solvent was removed under vacuum and the residue was subjected to column chromatography using petroleum ether/ethyl acetate mixture (97:3 v/v) as eluent to afford pure product. The product was recrystallized from ethyl acetate, affording colourless crystals of compounds (I)The two compounds were prepared in a similar manner using 4-fluoro aldehyde (1 mmol) for compound (I)2). Imine atom H1 was found in a difference map and refined with a distance restraint in both compounds of N—H = 0.86 (10) Å. Isotropic displacement parameters for H atoms were calculated as Uiso = 1.5Ueq(C) for CH3 groups and Uiso = 1.2Ueq(carrier atom) for all other H atoms. The DELU restraint was applied in compound (II)Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S1600536814022016/hb7284sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S1600536814022016/hb7284Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536814022016/hb7284IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S1600536814022016/hb7284Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S1600536814022016/hb7284IIsup5.cmlSupporting information file. DOI: 1027782, 1027783CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Washout of the CDR antibodies partially recovered CB immunoreactivity, suggesting a transient structural change in CB calcium-binding site. We discovered that CDR2 and CB co-immunoprecipitate. Furthermore, the expression levels of voltage-gated calcium channel Cav2.1, protein kinase C gamma and calcium-dependent protease, calpain-2, were increased after CDR antibody internalization. Inhibition of these signaling pathways prevented or attenuated CDR antibody-induced CB and L7/Pcp-2 immunoreactivity loss, morphological changes and increased protein expression. These results signify that CDR antibody internalization causes dysregulation of cell calcium homeostasis. Hence, drugs that modulate these events may represent novel neuroprotective therapies that limit the damaging effects of CDR antibodies and prevent PC neurodegeneration.Paraneoplastic cerebellar degeneration (PCD) is characterized by loss of Purkinje cells (PCs) associated with progressive pancerebellar dysfunction in the presence of onconeural Yo antibodies. These antibodies recognize the cerebellar degeneration-related antigens CDR2 and CDR2L. Response to PCD therapy is disappointing due to limited understanding of the neuropathological mechanisms. Here, we report the pathological role of CDR antibodies on the calcium homeostasis in PCs. We developed an antibody-mediated PCD model based on co-incubation of cerebellar organotypic slice culture with human patient serum or rabbit CDR2 and CDR2L antibodies. The CDR antibody-induced pathology was investigated by high-resolution multiphoton imaging and biochemical analysis. Both human and rabbit CDR antibodies were rapidly internalized by PCs and led to reduced immunoreactivity of calbindin DThe online version of this article (doi:10.1007/s00401-014-1351-6) contains supplementary material, which is available to authorized users. Paraneoplastic cerebellar degeneration (PCD), caused by Purkinje cell (PC) death in the cerebellum, is a paraneoplastic neurological disorder with severe pancerebellar symptoms, such as ataxia, nystagmus, and dysarthria . Cross-rBrain, normal ovary tissue, and tumor tissue express CDR2 protein , 30, 70.2+ levels and Ca2+ flux via cytoplasmic Ca2+-binding protein calbindin D28K (CB) and the GoLoco domain protein, Purkinje cell-specific protein-2 (L7/Pcp-2), are essential for cellular and molecular mechanisms involved in neurotransmitter release, ion channel permeability, enzyme activity, and gene transcription ; 6 days; ***p < 0.0001; nE = 4; and rCDR [125 ng/mL]; ***p < 0.0001; 6 days; nE = 4; Fig. rCDR2 and rCDR2/2L internalization . rCDR2L internalization had no effect on PKCγ protein expression, while CB+ - and L7/Pcp-2+-PC loss was rescued by K252a co-treatment in the rCDR2L group. The antagonist had no effect on the CB+- or L7/Pcp-2+ count or PKCγ protein expression in the rIgG and untreated naive control.The postsynaptic Carization , 73. PKCrization . Missensrization , 60. We The results show that the CDR antibody-mediated pathology can be attenuated in the presence of VGCC, AMPA receptor, and PKC signaling inhibition.2+ levels and induce Ca2+-dependent proteases activity. To examine whether CDR internalization affects Ca2+-dependent proteases activity of calpain-1 and calpain-2, we performed Western blot analysis of rCDR-treated cOTSC lysates. Calpain-1 expression was not affected, but calpain-2 expression was significantly increased  (6 days) reduced the CB+-PC loss to ~45 %, as well as the CDR-induced pathology on the dendritic arborizations in the remaining PCs  treatment  (18 %) and hCDR2/2L+(PS3) (48 %) -induced L7/Pcp-2+-PC loss was partially rescued by 10 μM MDL28170  showed an improvement of the dendritic morphology and significant reduction of CB+-PC loss .Changes in VGCC, AMPA receptor, and PKC signaling can lead to elevated intracellular Ca2+ to the millimolar range, which thereby activate the Ca2+-dependent protease calpain 2.These data show that CDR internalization leads to an increase in intracellular unbound CahCDR2/2L+  and MAP kinase inhibitor U0126 (5 μM) did not prevent the CB+- or L7/Pcp-2+-PC immunoreactivity loss (CB: hCDR2/2L+(PS3): 70 ± 10 vs. 68 ± 7 % (5 μM U0126); p = 0.4268; L7/Pcp-2: hCDR2/2L+(PS4): 65 ± 3 vs. 64 ± 2 % (5 μM U0126), p = 0.5249; nE = 4; Fig. Activation of mitogen-activated protein (MAP) kinase signaling cascades are associated with PKC-dependent synaptic depression/declustering of glutamate receptors, inflammation, death receptor activation, apoptosis, and oxidative stress , 23, 35.hCDR2/2L.These data suggest that the MAP kinase signaling cascade is not involved in the CB or L7/Pcp-2 immunoreactivity reduction induced by CDR antibodies, also known as onconeuronal Yo antibodies, have attracted increasing interest due to their suggested pathological role in the progressive loss of PCs seen in PCD after binding to CDR2 and CDR2L antigens , 33, 70.OTSC can be used to study neurochemical, structural, and physiological changes linked to diseased in vivo brains , 44. HenhCDR and rCDR can cause similar PC pathology. By using rCDR we exclude cytotoxic T cell involvement, since the cOTSC was not exposed to related peptides which could activate brain-naive resident T cells [Here, we demonstrate that both  T cells . Therefo T cells . However T cells . Our CDR T cells , but the T cells  demonstr T cells .h/rCDR internalization changes PC physiology by targeting postsynaptic signaling factors and thereby modifies Ca2+ homeostasis. We described these factors along with related neurological diseases in Table S1. We found that the immunoreactivity of Ca2+ homeostasis regulator CB and VGCC modulator L7/Pcp-2 was reduced. These proteins are functionally linked, since CB shapes the post-tetanic potentiation induced by the Ca2+ influx through AMPAR and VGCC and can act as an activity-dependent sensor [2+-binding mechanism or protein structure, as seen in other neurodegenerative diseases [In Fig. t sensor , 57. Whet sensor , 37, 74.diseases , 50.Fig.2+-transient elevation which greatly influences Ca2+ homeostasis [2+ microdomain variations due to AMPAR, VGCC, or PKC activity play a crucial role [2+ influx [2+ influx in synaptic Ca2+ microdomains [2+ influx into PCs, which can activate cell death pathways by enhancing PKC and Ca2+-dependent protease activity [CB dysfunction or deletion can result in drastic fast intracellular Caeostasis . In PC sial role , 57, 68.+ influx , 57, 73.odomains . Inhibitactivity .rCDR-treated cOTSCs. We therefore hypothesize that CDR internalization enhances PKC activity, which: first, reduces L7/Pcp-2 expression; second, increases Cav2.1 expression; third, increases intracellular Ca2+, which activates Ca2+-dependent proteases such as calpain-1 and calpain-2 and thereby induces neuronal death.PKC is associated with apoptosis in ischemia and stroke , 29, 60.2+ concentrations. In Alzheimer’s, Huntington’s, and Parkinson’s disease, calpain over-activation or dysregulation triggers neuronal death by truncation of important synaptic substrates [2+ level above 0.250 mM [2+ transient by mediating over-activation of calpain-2 [Calpain-1 and calpain-2 are activated by different cellular Cabstrates , 53, 59.0.250 mM . Inhibit0.250 mM . We hypoalpain-2 . As calpalpain-2 , 23, 35.2+ homeostasis, which lead to the silencing of the PCs. Second, cytototoxic T cells and microglia mediate the clearing of diseased cells, as known from autopsy studies [PCD pathogenesis is largely unknown. We propose a two-step pathology mechanism. First, internalization of CDR antibodies modifies important regulatory factors of Ca studies .2+ homeostasis due to alterations of CB, VGCC, or PKC [2+ levels by inhibition, the VGCC–AMPAR–PKC signaling pathway  There is no pathology seen in hematoxylin and eosin stained, paraffin-embedded ultra thin sections of cOTSC after 6 days of hCDR2/2L+(PS3) serum treatment. Purkinje (PCL), molecular (MCL) and granule (GCL) cell layer, scale bars 50 μm. (b) Double immunostaining of CB (magenta) and cleaved “active” caspase-3 (green) in rIgG and rCDR2/2L-treated cOTSC after 6 days. Orthogonal view shows that cleaved caspase-3 positive apoptotic cells were scattered in the cerebellum in both rIgG control and rCDR2/2L treated group. All of these cells were small and labeling was not co-localized with CB+ PCs; scale bars 15 μm. Supplementary material 1 (TIFF 4816 kb)Fig. S2hCDR and rCDR internalization induces GoLoco domain protein L7/Pcp-2 immunoreactivity loss. (a) L7/Pcp-2 (green) positive PCs are reduced under hCDR2/2L serum from PCD patient 3 and 4 at day 6 (D6); scale bars 40 μm. (b) Internalization of hCDR2/2L+(PS3) and hCDR2/2L+(PS4) (1 μL/mL) reduced the L7/Pcp-2 positive PCs (L7/Pcp-2+) to two-third over time (samples taken: day 2 (D2), day 4 (D4), day 6 (D6); (nE = 3)). (c) rCDR internalization caused similar reduction pattern of L7/Pcp-2 (green) positive PCs after 6 days as seen for hCDR in (a), scale bars 40 μm. (d) rCDR2, rCDR2L and rCDR2/2L internalization led to ~70 % L7/Pcp-2+ PC loss with no difference between the groups (nE = 6). Data in mean ± SEM. Non-parametric two-tailed paired Mann–Whitney’s U test. *p < 0.05; **p < 0.01; ***p < 0.001. The percentage changes to the controls are summarized in Table 1. Supplementary material 2 (TIFF 5778 kb)Supplementary material 3 (DOC 59 kb)Supplementary material 4 (DOC 29 kb)Supplementary material 5 (DOC 51 kb)Below is the link to the electronic supplementary material.


The glucose consumption of MDA-MB-231 and MCF-7 breast cancer cells and human mammary epithelial cells (HMEpiCs) was assessed. Cells were incubated with γ-Fe2O3@DMSA or γ-Fe2O3@DMSA-DG, and MDA-MB-231 cells which exhibited the highest glucose consumption were used in breast cancer xenografts. Tumor targeting was studied by magnetic resonance imaging and Prussian blue staining in vivo.γ-FeIn vitro, there was significant uptake of γ-Fe2O3@DMSA-DG by MDA-MB-231 and MCF-7 cells within 2 h and this was inhibited by glucose. Uptake of γ-Fe2O3@DMSA-DG was significantly higher in MDA-MB-231 compared with MCF-7 cells, and there was no obvious uptake of γ-Fe2O3@DMSA in either cell line. In vivo, γ-Fe2O3@DMSA-DG could be detected in the liver and in tumors post-injection, while γ-Fe2O3@DMSA was nearly undetectable in tumors.Glucose consumption was highest in MDA-MB-231 and lowest in HMEpiCs. 2O3@DMSA improved tumor targeting of γ-Fe2O3@DMSA which can be assessed by magnetic resonance imaging.2-DG-coated γ-Fe This glucose analog, which permits the quantitative analysis of glycolysis, is by far the most widely used radiolabeling marker in the diagnostic work-up of primary tumors, locoregional recurrence, and distant metastases [18F]FDG emits small amounts of radiation and CT scanners use X-rays to scan, with radiation being potentially harmful to humans, we evaluated the effectiveness of using a magnetic resonance imaging (MRI) contrast agent for tumor metabolic imaging.In the 1920s, Warburg discovered that even in the presence of ample oxygen, cancer cells prefer to metabolize glucose by glycolysis. This is seemingly a paradox, since glycolysis, when compared with oxidative phosphorylation, is a less-efficient pathway for producing ATP  / N, where G0 is the glucose concentration of the primary culture medium, G24 is the glucose concentration of the medium after cells were cultured for 24 h, l is the quantity of culture medium, and N is the number of seeded cells.The glucose metabolic rate of MDA-MB-231 and MCF-7 breast cancer cells and human mammary epithelial cells (HMEpiCs) was assessed using the glucose oxidase-peroxidase (GLU-GOD) assay. Cells were seeded at a density of 5 × 102 at 37 °C. HMEpiCs were cultured in MED-0001 supplemented with SUP-0001. Cells were incubated with 5 ml of culture medium containing γ-Fe2O3@DMSA-DG NPs or γ-Fe2O3@DMSA NPs at an iron concentration of 0.3 μmol/ml for different time intervals . Competition experiments were performed by adding 25 mmol/l d-glucose to γ-Fe2O3@DMSA-DG NPs non-glucose DMEM or MED-0001 growth medium. After incubation, the culture medium was removed. Adherent cells were washed three times with PBS , trypsinized, and centrifuged for 5 min at 2000×g. The number of cells was determined using a Neubauer counting chamber. For Prussian blue staining, cells were seeded in six-well plates on glass coverslips. A total of 1 × 106 cells were embedded in gelatin  at room temperature for MRI measurement; 150 μl of the cell suspension was transferred into a 96-well plate, and the absorbance was read at 480 nm using a microplate reader .MDA-MB-231 and MCF-7 cells were purchased from the cell bank of the Chinese Academy of Sciences , and HMEpiCs and culture medium  were purchased from PriCells . MDA-MB-231 and MCF-7 cells were cultured in DMEM supplemented with 10 % fetal bovine serum in a humidified atmosphere of 5 % COCell monolayers grown on glass coverslips were washed three times with PBS and subsequently fixed with methanol and acetone (−20 °C). The fixed cells were incubated with 10 % potassium ferrocyanide for 5 min, incubated with 10 % potassium ferrocyanide in 20 % hydrochloric acid for 30 min, and counterstained with nuclear fast red.v/v HCl at 60 °C for 2 h. Any ferrous chloride present was oxidized to ferric chloride using 0.1 mg/ml ammonium persulfate (10 ml). Then, the K4[Fe(CN)6]·3H2O solution (20 ml) was added for 10 min to form the iron-thiocyanate complex. An aliquot of this mixture (150 μl) was transferred into a 96-well plate, and the absorbance was read at 480 nm using a microplate reader. A standard curve was generated using FeCl3·6H2O solutions of different concentrations under the same conditions. Each experiment was repeated five times.An ultraviolet colorimetric assay was performed as previously described . Briefly3]. T2 relaxometry was tested using a T2 map. The T2 map calculation can be found under “dynamic analysis,” a mono-exponential fit without signal offset. The detailed formula is as follows:i is the echo time of image I and wi is the weight of image i with T2 MR relaxometry of cells in gelatin was performed using a multi-echo spin echo pulse sequence test / [A]control × 100 %).Cell viability was measured using the 3--2,5-diphenyltetrazolium bromide (MTT) assay. MDA-MB-231 cells (approximately 105 cells/mouse) in the right neck dorsal region of female nude (BALB/c) mice (n = 24) . Animals were injected with a contrast agent formulation for imaging when the tumors were approximately 1 cm in diameter, which occurred approximately 4 weeks after injection of tumor cells. Animals were anesthetized by intraperitoneal injection of diazepam (8 μg/g)  and ketamine hydrochloride (120 μg/g) .MDA-MB-231 cells were selected for xenograft experiments as these cells consume more glucose than MCF-7 cells. All experiments were approved by the governmental review committee on animal care. Human MDA-MB-231 breast cancer xenografts were generated by subcutaneous injection of MDA-MB-231 cells (2 × 10n = 6) were slowly injected with γ-Fe2O3@DMSA-DG NPs (300 μmol Fe/kg) in 1000 μl sterilisata . MRI was performed at six intervals . Control animals (n = 6) were injected with γ-Fe2O3@DMSA NPs at the same dose of Fe, using the same method.Mice with MDA-MB-231 tumors  within a wrist coil. MRI was performed in a coronal plane with a T2W turbo-spin-echo sequence .n = 12) were anesthetized and euthanized by cervical dislocation 24 h after injection. Frozen sections of the tumor, brain, liver, and muscle were fixed in paraformaldehyde (−20 °C). For Prussian blue staining, the fixed tissues were incubated in 10 % potassium ferrocyanide for 5 min and then incubated in 10 % potassium ferrocyanide in 20 % hydrochloric acid for 30 min, followed by counterstaining with nuclear fast red.Mice  of the brain, liver, muscle, and tumor after contrast medium injection within the different groups of mice. p values less than 0.05 were considered statistically significant. All statistical analyses were performed with SPSS software, version 17.0 .All numeric data are expressed as mean ± standard error of the mean. A standard unpaired 2O3@DMSA NPs and γ-Fe2O3@DMSA-DG NPs in water produced nanoparticle aggregates. We previously reported  × 10−4 μmol/cell) after 24 h, whereas HMEpiCs exhibited the lowest levels of glucose consumption ([7.13 ± 0.08] × 10−5 μmol/cell). The difference in glucose consumption was significant between the three cell lines (p < 0.01).The level of glucose consumption by MDA-MB-231 cells . However, there were no significant differences between the cellular iron content of γ-Fe2O3@DMSA NPs and γ-Fe2O3@DMSA-DG NPs in HMEpiCs (p > 0.05). The ultraviolet colorimetric assay also revealed higher uptake of γ-Fe2O3@DMSA-DG NPs in MDA-MB-231 cells compared with MCF-7 cells, while HMEpiCs had the lowest uptake value after the same incubation time , while there were no significant difference in the mean T2 relaxation time change in the different groups of HMEpiCs (p > 0.05). After blocking the binding site with free glucose and subsequent incubation with γ-Fe2O3@DMSA-DG NPs, the T2 relaxation times of MDA-MB-231 and MCF-7 cells were significantly higher than those of MDA-MB-231 and MCF-7 cells incubated with γ-Fe2O3@DMSA-DG NPs in non-glucose medium .These changes were also reflected in the T2 relaxation times. After 10 min of incubation, the mean T2 relaxation time of MDA-MB-231 and MCF-7 cells exposed to γ-Fe2O3@DMSA-DG NPs were 100.23 ± 2.26 (0.3 μmol/ml), 100.19 ± 2.43 (0.6 μmol/ml), 100.11 ± 1.69 (1.2 μmol/ml), 99.48 ± 2.54 (2.4 μmol/ml), and 99.75 ± 2.73 (4.8 μmol/ml). The viability of cells treated with γ-Fe2O3@DMSA NPs was 99.64 ± 1.73 (0.3 μmol/ml), 99.76 ± 2.72 (0.6 μmol/ml), 99.61 ± 1.98 (1.2 μmol/ml), 99.18 ± 2.21 (2.4 μmol/ml), and 97.31 ± 4.02 (4.8 μmol/ml). One-way analysis of variance revealed no significant differences in cell viability between the two groups of treated cells (p > 0.05). The MTT assay did not demonstrate reduced viability of MDA-MB-231 cells after incubation with γ-Fe2O3@DMSA NPs or γ-Fe2O3@DMSA-DG NPs, at variable iron concentrations for 24 h. These results indicate that γ-Fe2O3@DMSA NPs and γ-Fe2O3@DMSA-DG NPs do not inhibit cell growth at Fe concentrations between 0.3 and 2.4 μmol/ml and therefore caused no significant cytotoxicity.The viability values of MDA-MB-231 cells treated with γ-Fe2O3@DMSA NPs group, MRI T2-weighted images showed a decrease in tumor signal intensity 12 h after injection, which returned to basal levels before injection after 24 h  revealed no significant uptake of γ-Fe2O3@DMSA NPs in tumor cells   -DTPA-DG –22 and h9mTc-DTPAosamine) . Glucoseosamine) , 23. In 2O3@DMSA-DG NPs in mice, 1.5-T MRI revealed that the T2 signal intensity in tumors decreased from 12 to 48 h. This was significantly different from the results observed after administration of γ-Fe2O3@DMSA NPs, where the T2 signal intensity in tumors decreased at 12 h but returned to basal levels prior to 24 h. Notably, there were no significant differences in T2 signal intensity between the two groups in the brain, liver, and muscle. Prussian blue staining of tumor tissues revealed iron particle uptake in some tumor cells 24 h after injection of γ-Fe2O3@DMSA-DG NPs, whereas no significant iron particle uptake was observed in tumor cells 24 h after injection of γ-Fe2O3@DMSA NPs. This demonstrates that the targeting NPs prepared in this study had tumor-specific targeting aggregation. Thus, the addition of a targeting molecule onto the surface of NPs increased the selective cellular binding and internalization through receptor-mediated active uptake and endocytosis. Without the incorporation of targeting ligands or ligand analogs, NPs only rely on nonspecific interactions with cell membranes.According to other reports, expression of GLUT1 occurs predominantly around the area of tumor necrosis , 25. Ourin vitro experiments may differ significantly to those obtained in vivo. In vitro, a number of particles may surround the tumor cells; however, in vivo, after intravenous injection, numerous opsonin proteins, cells, and salts bind to the surface of the nanoparticles. This is known as the opsonization process and is responsible for easier recognition by the mononuclear phagocytic system leading to rapid clearance from the circulation. Nanoparticles are usually taken up by macrophages in the liver . After uptake in specialized macrophages, SPIOs are degraded lysosomally. The nanoparticles internalized by tumor cells cannot be metabolized and eliminated quickly. The coating material is eliminated via other decomposition and elimination pathways. For example, the coating of ferumoxtran-10 is degraded via intracellular dextranases and is primarily eliminated renally (89 % in 8 weeks) and the core material is supplied to the iron storage pool of the body.The results of 2O3@DMSA-DG NPs or γ-Fe2O3@DMSA NPs used were non-monodispersed SPIOs, as they were not connected to active surface molecular polyethylene glycol and, as such, could not avoid phagocytosis by the reticuloendothelial system (RES) in the body. They do not have identical physical properties for uncontrolled bio-distribution and bio-elimination. The average hydrodynamic diameter of the nanoparticle was approximately 150 nm, and a large number of these particles were taken up by the RES. In our in vivo study, we observed that the T2-weighted signal in the liver decreased immediately after injection of γ-Fe2O3@DMSA-DG NPs or γ-Fe2O3@DMSA NPs, indicating that most particles were phagocytized by the RES. If the 2-DG-modified SPIO decreases the both signals of liver metastasis and the health parts, the liver tumor may not be easily detected.The γ-FeHowever, the biocompatibility of such particles is not only related to their nano-size but is particularly related to their surface properties, which are the determining factors for cellular uptake and cytotoxicity . In addid-glucosamine hydrochloride and X-ray spectroscopy demonstrated that DG was successfully functionalized onto the surface of γ-Fe2O3-DMSA NPs. The glucose analog, 2-DG, can bind to glucose transporters overexpressed in tumor cells. Our results demonstrated the uptake of γ-Fe2O3@DMSA-DG NPs by breast cancer cells exhibiting high-glucose consumption, but no uptake of γ-Fe2O3-DMSA NPs. These findings indicate that it is possible to non-invasively identify high-glucose metabolic tumors by MRI after injection of γ-Fe2O3@DMSA-DG NPs. However, compared with 18F-FDG PET-CT, one drawback of this approach is that it requires two MRI scans (before injection and 24 h after injection).In this study, DMSA-coated naked iron oxide supplied carboxyl groups, which were linked with


In the crystal of (II), molecules are linked via O—H⋯O hydrogen bonds, forming inversion dimers with an The title compounds, (I) and (II), differ only in the substituent on the N atom of the central piperidine ring; methyl in (I) and benzyl in (II). In each molecule, the 4,11-dihydroxy groups are involved in intramolecular O—H⋯O hydrogen bonds. In the crystal of (I), molecules are linked  p-tolyl-2,9-dithia-13- azadispiro[4.1.47.35]tetradecan-6-one, C26H31NO3S2, (I), and 13-benzyl-4,11-dihydroxy-1,8-bis(4-methylphenyl)-2,9-dithia-13-azadispiro[4.1.47.35]tetradecan-6-one, C32H35NO3S2, (II), the piperidine rings adopt distorted chair conformations. The thiophene rings in (I) have envelope conformations, with the spiro C atoms as the flaps. In (II), one thiophene ring (D) has an envelope conformation, with the hydroxy-substituted C atom as the flap, while the other thiophene ring (E) has a twisted conformation on the C—C bond involving the spiro C atom and the toluyl-substituted C atom. In (I), the mean plane of the piperidine ring makes dihedral angles of 75.16 (9) and 73.33 (8)° with the mean planes of the thiophene rings (D and E), respectively. In (II), the corresponding dihedral angles are 70.95 (11) and 77.43 (12)°. In both compounds, there is an intramolecular O—H⋯O hydrogen bond forming an S(6) ring motif. In the crystal of (I), molecules are linked via O—H⋯N and C—H⋯O hydrogen bonds, forming chains along [010]. There are also π–π interactions present involving inversion-related benzene rings, linking the chains to form slabs parallel to (100). In the crystal of (II), molecules are linked via O—H⋯O hydrogen bonds, forming inversion dimers with an R44(8) ring motif. The dimers are linked by C—H⋯π interactions, forming slabs parallel to (001).In the title compounds 4,11-dihydroxy-13-methyl-1,8-di- Most piperidine derivatives are known to have chair conformations  adopt distorted chair conformations in both compounds. In compound (I)--C13), by 0.412 (2) and −0.7543 (18) Å, respectively.In both molecules there is an intramolecular O—H⋯O hydrogen bond present forming an D (C7–C10/S1) and E (C14/C16–C18/S2) have envelope conformations with atoms C10 and C14, respectively, as the flaps. They deviate from the mean plane through the four other atoms in the ring by 0.6277 (15) Å for C10 and 0.6494 (15) Å for C14. The mean plane of the piperidine ring A makes dihedral angles of 75.16 (9) and 73.33 (8)° with the mean planes of the thiophene rings D and E, respectively. The mean plane of thiophene ring D makes a dihedral angle of 60.10 (1)° with toluyl ring B (C1–C6), and the mean plane of thiophene ring D make a dihedral angle of 58.14 (1)° with toluyl ring C (C19–C24). Rings B and C are inclined to one another by 66.39 (13)°.In compound (I)D (C7–C10/S1) has an envelope conformation with atom C9 as the flap. It deviates from the mean plane through the other four atoms by 0.621 (2) Å. Thiophene ring E (C13/C15–C17/S2) has a twisted conformation on the C13—C17 bond. These two atoms deviate from the plane (C15/C16/S2) by 0.291 (2) and −0.490 (2) Å, respectively. The piperidine ring A mean plane makes dihedral angles of 70.95 (11) and 77.43 (12)° with the mean planes of thiophene rings D and E, respectively. The mean plane of thiophene ring D make a dihedral angle of 52.42 (1)° with toluyl ring B (C1–C6), and the mean plane of thiophene ring D make a dihedral angle of 65.71 (1)° with toluyl ring C (C18–C23). Benzyl ring F (C25–C30) makes a dihedral angle of 75.09 (1)° with the mean plane of piperidine ring A. Rings B and C are inclined to one another by 74.33 (12)°.In compound (II)via O—H⋯N and C—H⋯O hydrogen bonds, forming chains along the b-axis direction (Table 1via weak π–π stacking interactions involving inversion-related C toluyl rings tetradecan-6-one gave zero hits.A search of the Cambridge Structural Database : A mixture of -1-methyl-3,5-bis(4-methylbenzylidene)piperidin-4-one (1 mmol) 1, 1,4-dithiane-2,5-diol (1 mmol) 2 and triethylamine (0.25 eq) in dichloromethane (6 ml) was heated under reflux for 3 h. After completion of the reaction (TLC), the solvent was removed and the product was purified by flash column chromatography using a petroleum ether–ethyl acetate mixture (4:1 v/v) as eluent to afford pure state of the title compound. After purification the compound was recrystallized in CHCl3 by slow evaporation.Compound (II): A mixture of -1-benzyl-3,5-bis(4-methylbenzylidene)piperidin-4-one (1 mmol) 1, 1,4-dithiane-2,5-diol (1 mmol) 2 and triethylamine (0.25 eq) in dichloromethane (6 ml) was heated under reflux for 3 h. After completion of the reaction (TLC), the solvent was removed and the product was purified by flash column chromatography using a petroleum ether–ethyl acetate mixture (4:1 v/v) as eluent to afford pure state of the title compound. After purification the compound was recrystallized in CHCl3 by slow evaporation.A, was freely refined. Those of compound (I)Uiso(H) = 1.5Ueq(O). The C-bound hydrogen atoms were placed in calculated positions and refined as riding: C—H = 0.93–0.98 Å with Uiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989015020885/su5199sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989015020885/su5199Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015020885/su5199IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989015020885/su5199Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989015020885/su5199IIsup5.cmlSupporting information file. DOI: 1435024, 1435023CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


This compound is of interest as the first to possess cycloheptatrienyl and cycloheptadienyl ligands in an anionic metal complex.The reaction of bis(anthracene)cobaltate(−I) with excess cycloheptatriene, C 7H8, resulted in a new 18-electron cobaltate containing two different seven-membered ring ligands, based on single-crystal X-ray diffraction. The asymmetric unit of this structure contains two independent cation–anion pairs of the title complex, [K(18-crown-6)][Co(η3-C7H7)(η5-C7H9)], where 18-crown-6 stands for 1,4,7,10,13,16-hexaoxacyclooctadecane (C12H24O6), in general positions and well separated. Each (18-crown-6)potassium cation is in contact with the η3-coordinating ligand of one cobaltate complex. Each η3-coordinating ligand behaves as an allylic anion whose exo-diene moiety is bent away from the allylic plane, and thus is not involved directly in the bonding. The metal-coordinating portions of the anionic η5 ligands are planar and one of these ligands is modeled as disordered over two positions, with occupancy ratio 0.699 (5):0.301 (5), such that one orientation is rotated by one carbon atom with respect to the other. The diffraction intensities were integrated according to non-merohedral twin law [-1 0 0/0 -1 0/0.064 0 1], a 180° rotation about reciprocal lattice axis [001], and the masses of the twin domains refined to equal amounts. As both ligands are formally coordinated as anions, the cobalt atom is best considered to be CoI. This compound is of interest as the first to possess cycloheptatrienyl and cycloheptadienyl ligands in an anionic metal complex.The reaction of bis(anthracene)cobaltate(−I) with excess cycloheptatriene, C For the titanium analog, although the homoleptic CHT complex was initially observed by NMR, no crystals were obtained, and it readily isomerized. Metal vapor co-condensation reactions of titanium and iron with CHT also led to the isomerized forms 2, which could be isolated at room temperature, but was observed to isomerize to Mo(η7-C7H7)(η5-C7H9) with a half-life of ca 200 h  transition metal complex, Zr(η5-C7H9)]−, which confirmed that isomerization had occurred and that both anthracene ligands had been displaced. As no spectroscopy had been performed, it is unknown if an anionic intermediate like ‘[Co(η-C7H8)2]−’ was initially formed, and if formed, whether it had any lifetime in cold and/or room-temperature solutions.Given the tendency for homoleptic CHT complexes to isomerize, we decided to investigate whether this would occur in the late transition metal low-valent cobalt system. The 18-electron anionic precursor bis(anthracene)cobaltate(−I) was chosen because it had been demonstrated that the anthracene ligands are quite labile (Brennessel 3-C7H7)(η5-C7H9)], (I)3-coordinating ligands of the cobalt anions, with K⋯C distances ranging from 3.207 (3) to 3.538 (4) Å. The longest K⋯C distance is well within the sum of the van der Waals radii for potassium and carbon of 4.45 Å  and 27.2 (4)°, for anions containing Co1 and Co2, respectively. Interestingly, the tropylium cation (CHT+) also has the formula C7H7; however, tropylium as a ligand is aromatic, and thus planar and with similar C C bond lengths. The η5-coordinating ligands are essentially planar in their cobalt-bonded regions with r.m.s. deviations from planarity of 0.050 and 0.051 Å for planes C8–C12 and C22–C26, respectively ][Co(CO)3]−  and five (‘pentadienyl’) carbon atoms of the seven-membered rings as described above, it is thought best to consider the cobalt atom as formally Coet al., 2004et al., 1989via a ‘[CoH(η-C7H7)(η-C7H8)]−’ intermediate.The exact mechanism of isomerization has not been determined for (I)6-C7H8)2 3(η3-C7H7)], M = Fe  and glass-covered magnetic stir bars were employed. Cycloheptatriene was distilled from Na/K alloy.All operations were performed under an atmosphere of 99.5% argon further purified by passage through columns of activated BASF catalyst and molecular sieves. Standard Schlenk techniques were employed for all reactions with a double manifold vacuum (0.01 Torr) line. Solutions were transferred 2][Co(η4-C14H10)2]  was added, the slurry was filtered. The product was washed with pentane (20 ml) and dried in vacuo, yielding a blackish-gray solid . This product was only characterized by single-crystal X-ray diffraction. Red–brown blocks were grown from a pentane-layered THF solution at 273 K.Excess cycloheptatriene was added dropwise to a deep pinkish-red solution of [K(18-crown-6)(THF)R1 = 0.19, at which point the structure was examined for twinning :0.301 (5), such that the ethyl linkage is shifted by one carbon atom  = 1.2Ueq(C).H-atom positions of ring-ligand carbon atoms, except those in the minor component of the disorder, were located in a difference map and refined freely. All other H atoms were placed geometrically and treated as riding atoms: methine and 10.1107/S2056989015003151/bh2504sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989015003151/bh2504Isup2.hklStructure factors: contains datablock(s) I. DOI: 1049452CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: