Therapeutic interventions that increase plasma high density lipoprotein (HDL) and apolipoprotein (apo) A-I levels have been reported to reduce plasma glucose levels and attenuate insulin resistance. The present study asks if this is a direct effect of increased glucose uptake by skeletal muscle. Incubation of primary human skeletal muscle cells (HSKMCs) with apoA-I increased insulin-dependent and insulin–independent glucose uptake in a time- and concentration-dependent manner. The increased glucose uptake was accompanied by enhanced phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), the serine/threonine kinase Akt and Akt substrate of 160 kDa (AS160). Cell surface levels of the glucose transporter type 4, GLUT4, were also increased. The apoA-I-mediated increase in glucose uptake by HSKMCs was dependent on phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt, the ATP binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-B1). Taken together, these results establish that apoA-I increases glucose disposal in skeletal muscle by activating the IR/IRS-1/PI3K/Akt/AS160 signal transduction pathway. The findings suggest that therapeutic agents that increase apoA-I levels may improve glycemic control in people with type 2 diabetes. In normal human plasma 5–10% of the total apoA-I exists in a lipid-free or lipid-poor form that accepts cholesterol from peripheral cells expressing the ATP-binding cassette transporter A1 (ABCA1)2. Lipid-free/lipid-poor apoA-I also interacts with scavenger receptor class B type 1 (SR-B1) to activate signal transduction pathways3.Apolipoprotein (apo) A-I is the most abundant apolipoprotein constituent of high density lipoproteins (HDLs)4. We, and others, have reported that apoA-I increases insulin secretion from pancreatic beta-cells in an ABCA1-dependent manner. HDLs also increase insulin secretion from pancreatic beta-cells, but in an ABCG1-dependent manner5. Furthermore, mice that are deficient in apoA-I have impaired glucose tolerance6, while overexpression of apoA-I increases insulin sensitivity7. Similar findings have been reported in people with type 2 diabetes, where infusions of reconstituted HDL consisting of apoA-I complexed with phosphatidylcholine increase plasma insulin levels and reduce plasma glucose levels8. Torcetrapib, a small molecule inhibitor of cholesteryl ester transfer protein activity that increases plasma HDL cholesterol and apoA-I levels by approximately 70%, also improves glycaemic control in people with type 2 diabetes9. There is evidence that HDLs and apoA-I may mediate these effects by increasing glucose disposal in skeletal muscle via activation of the AMP-activated protein kinase (AMPK) signalling pathway8. We have also reported that a single infusion of apoA-I increases glucose disposal and phosphorylation in skeletal muscle in db/db mice, both in the presence and in the absence of insulin10.HDLs and apoA-I have anti-diabetic properties11. This leads to phosphorylation of tyrosine residues in insulin receptor substrate-1 (IRS-1)12, and the p85 subunit of phosphatidylinositol 3-kinase (PI3K)13. Phosphorylated PI3K then activates a downstream signal transduction pathway that phosphorylates serine/threonine kinase Akt (protein kinase B)15 and the Rab GTPase-activating protein, Akt substrate of 160 kDa (AS160)16. This culminates in the translocation of glucose transporter 4 (GLUT4) to the cell surface, and increases glucose uptake17. The present study establishes that apoA-I enhances this signalling pathway and the translocation of GLUT4 to the cell surface.Insulin-dependent glucose uptake into skeletal muscle is initiated by the binding of insulin to the α-subunit of the insulin receptor (IR), and phosphorylation of tyrosine residues in the IR β-subunit (IRβ)p < 0.05)6. Incubation of HSKMCs with lipid-free apoA-I alone increased glucose uptake to 1.26 ± 0.09 nmol/mg/h , while incubation with apoA-I plus insulin increased glucose uptake to 1.46 ± 0.08 nmol/mg/h   with insulin alone increased glucose uptake from 1.0 nmol/mg/h  and 1.11 ± 0.02 nmol/mg/h (p < 0.001), respectively,  . Insulin-independent glucose uptake into HSKMCs was 0.40 ± 0.01 nmol/mg/h at 0.5 h and 1.66 ± 0.14 nmol/mg/h at 5 h .ApoA-I also increased glucose uptake into HSKMCs in a time-dependent manner. When the cells were incubated with PBS in the absence of apoA-I, insulin-dependent glucose uptake into HSKMCs was 0.49 ± 0.01 nmol/mg/h at 0.5 h and 1.84 ± 0.13 nmol/mg/h at 5 h  and 0.72 ± 0.11 (p < 0.05), respectively . Incubation with apoA-I plus insulin increased the pIRS-1/total IRS-1 ratio to 0.62 ± 0.02 . The phosphorylated Akt (pAkt)/total Akt ratio in control HSKMCs was 0.54 ± 0.22  and 0.97 ± 0.04 for cells incubated with apoA-I (p < 0.01). Incubation of HSKMCs with insulin plus apoA-I increased the pAkt/total Akt ratio to 1.52 ± 0.05 . The phosphorylated AS160 (pAS160)/total AS160 ratio, which was 0.57 ± 0.1 in the control HSKMCs  and 1.01 ± 0.006 (p < 0.001), respectively, when the cells were incubated with insulin or apoA-I .To determine if apoA-I increased glucose uptake by activating the IR/IRS-1/PI3K/Akt/AS160 signal transduction pathway, phosphorylation of IRβ, IRS-1, Akt, and AS160 was assessed by western blotting. Incubation with insulin alone or apoA-I alone increased the HSKMC phosphorylated IRβ (pIRβ)/total IRβ ratio from a control value of 0.44 ± 0.03 . When the scrambled siRNA-transfected cells were incubated with apoA-I plus insulin, glucose uptake further increased to 1.31 ± 0.01 nmol/mg/h (p < 0.001 vs. control). Glucose uptake decreased to 0.86 ± 0.06 nmol/mg/h when HSKMCs were transfected with IRS-1 siRNA and incubated in the absence or presence of insulin .Incubation of the scrambled siRNA-transfected HSKMCs with insulin only or apoA-I only increased glucose uptake from a normalized value of 1 nmol/mg/h to 1.17 ± 0.02 and 1.18 ± 0.02 nmol/mg/h, respectively . Incubation with apoA-I plus insulin in the absence of wortmannin further increased glucose uptake to 1.45 ± 0.07 nmol/mg/h (p < 0.001 vs. control).To determine if activation of PI3K/Akt was required for the apoA-I-mediated uptake of glucose into HSKMCs, incubations were also carried out with the PI3K/Akt inhibitor, wortmannin. Akt Fig. , and AS1Glucose uptake was decreased in the wortmannin-treated HSKMCs, irrespective of whether or not insulin was present in the incubation  and 181.8 ± 33 MFI, respectively (p < 0.01 vs. control) . Incubation of HSKMCs with insulin or apoA-I did not affect total cell GLUT4 levels . Glycosylation of GLUT4 can cause heterogeneous bands in western blots. However, GLUT4 western blots can also be well focussed, as is the case in Fig. 21. Incubation of HSKMCs with apoA-I plus insulin increased cellular GLUT4 mRNA levels by 45.3 ± 21% (p < 0.01 vs. control). Incubation of HSKMCs with apoA-I did not alter IR, IRS-1, PI3K and Akt mRNA levels  in untreated HSKMCs . Addition of insulin, apoA-I or insulin plus apoA-I to scrambled siRNA-transfected HSKMCs increased glucose uptake from 1 nmol/mg/h to 1.17 ± 0.02, 1.18 ± 0.02 and 1.31 ± 0.01 nmol/mg/h, respectively . Glucose uptake by HSKMCs that were transfected with ABCA1 siRNA and incubated with PBS was 0.89 ± 0.03 nmol/mg/h . Inclusion of apoA-I in the incubations of ABCA1 siRNA-transfected HSKMC did not increase glucose uptake, irrespective of whether or not insulin was present . ApoA-I, by contrast, did not increase glucose uptake into the SR-B1-transfected HSKMCs in either the presence or absence of insulin  and Coomassie Blue staining.HDLs were isolated from pooled samples of autologously donated human plasma  by sequential ultracentrifugation in the 1.063< d <1.21 g/mL density range and delipidated as described2 (v/v) in Minimum Essential Medium-α  containing 10% (v/v) FBS. When 90% confluent, the cells were plated into 12-well plates (3 × 104 cells/mL) and cultured in MEM-α containing 2% (v/v) horse serum for 4–5 days. Differentiation into myotubes was monitored by phase contrast microscopy.Primary HSKMCs  were cultured at 37 °C in 5% CO3H]2-deoxy-glucose uptake the cells were washed with Krebs-Ringer Bicarbonate (KRB) buffer containing 2 mmol/L sodium pyruvate (pH7.4). Glucose uptake was initiated by incubating the cells for 0.5–5 h with 10 µmol/L [3H]2-deoxy-glucose  in KRB buffer in the presence or absence of insulin . The cells were then rinsed with ice-cold Dulbecco’s PBS, lysed with SDS  and subjected to liquid scintillation counting. Non-carrier-mediated glucose uptake was determined by incubating the cells for 15 min with cytochalasin B  prior to [3H]2-deoxy-glucose uptake34. The results were corrected for cell protein and non-carrier-mediated glucose uptake. The values for the control cells that were incubated in the absence of insulin and apoA-I were normalised to 1 nmol/mg protein/h. All experiments were carried out in triplicate. The results are representative of three or more independent experiments.Fully differentiated HSKMCs were incubated at 37 °C for 16 h in serum-free MEM-α in the presence or absence of lipid-free apoA-I . The apoA-I was removed and the cells were incubated in fresh MEM-α for 1 h with or without insulin . For determination of [3H]2-deoxy-glucose uptake was determined as described above.For incubation with wortmannin, fully differentiated HSKMCs were incubated for 1 h in serum-free MEM-α with wortmannin , then incubated for 16 h with wortmannin in the presence or absence of apoA-I . The apoA-I was removed, and the cells were incubated for 1 h with wortmannin with or without insulin . [4 cells/mL) were plated in 12-well collagen-coated plates. Skeletal muscle differentiation medium was added to the cells the following day. After 3 days (~48 h prior to full differentiation), the cells were transfected for 16 h with scrambled siRNA, IRS-1 siRNA, ABCA1 siRNA or SR-B1 siRNA  using DharmaFECT 4 siRNA transfection reagent .Near-confluent HSKMCs . Primary anti-GLUT4  and secondary DyLight488 donkey anti-goat  antibodies were combined and maintained at room temperature for 10 min. The cells were washed with PBS and incubated for 30 min with the antibody complex in the presence and absence of insulin . After washing and fixing the cells with 2.5% (v/v) neutral buffered formalin for 20 min, GLUT4 translocation to the cell surface was analyzed using a FACSverse flow cytometer .GLUT4 translocation was measured as described previously2, 5 mmol/L EGTA-Na2, 2 mmol/L Na3VO4, 10 mmol/L HEPES (pH 7.4), 0.1% Triton X-100 (v/v), 1 mmol/L PMSF, and a protease inhibitor cocktail (1:1000 (v/v)). The cell lysates (30 µg protein) were subjected to SDS-PAGE, transferred electrophoretically to a nitrocellulose membrane, and incubated with primary antibodies at dilutions of 1:3,000 (v/v) for anti-β-actin , and 1:1,000 (v/v) for all other antibodies. The membrane was then incubated with horseradish peroxidase-conjugated secondary antibodies . Antibodies against phospho-IRβ (tyr1345) (#3026), IRβ (#3020), phospho-Akt (ser473) (#4058), Akt (pan) (#4685), IRS-1 (#2390), phospho-AS160 (ser588) (#8730), and AS160 (#2670) were from Cell Signalling . The phospho-IRS1 antibody  was from Merck Millipore . The ABCA1 antibody (#NB400–105) was from Novus Biologicals , and the SR-B1 antibody (#1971–1) was from Abcam . The blots were developed with ECL and quantified using Image Lab Analysis Software .Fully differentiated HSKMCs were incubated at 37 °C for 16 h in serum-free MEM-α with or without apoA-I . The apoA-I was removed, and the cells were incubated with or without insulin  for 30 min. The cells were then washed with cold PBS and lysed with 50 mmol/L Na pyrophosphate, 50 mmol/L NaF, 50 mmol/L NaCl, 5 mmol/L EDTA-NaTotal RNA was isolated from HSKMCs using an RNeasy Mini Kit  and reverse transcribed (100 ng RNA) using an iScript cDNA Synthesis Kit (Bio-Rad). Real-time PCR was performed with the MyiQ single colour real-time PCR detection system (Bio-Rad) using the iQ SYBR Green Supermix (Bio-Rad) in a BioRad iQ5 thermocycler. Relative changes in mRNA levels were determined by the ΔΔCT method. Results are presented as the ratio of the gene of interest to β-actin. Primer sequences are shown in Supplemental Table I.2  and BHT . Methanol:hexane ) was added to the samples, which were mixed and centrifuged at 2,000 g for 5 min. The hexane layer was collected and dried, dissolved in isopropanol/acetonitrile (70/30 (v/v), 200 µL) and applied to a Supelco ODS LC-18 column  with a 2-cm Pelliguard column (Sigma) attached to a Shimadzu HPLC system . Cholesterol levels were quantified at 205 nm with reference to cholesterol standards (Sigma)36.HSKMCs were lysed in water (500 µL). The lysates (400 µL) were added to water (600 µL) containing EDTA-Nap < 0.05 was considered significant.Results are presented as mean ± SD of at least three independent experiments. Multiple comparisons were analysed by one-way ANOVA with Tukey’s post-test, or two-way ANOVA with Bonferroni post-test . A value of Supplementary Information


We are thankful to Dr. Deepak Gupta  for bringing to our attention a typographical error in our manuscript .Table 2, ROW 12 was wrongly written as:Number of patients with sore throat (Yes/No)P value).At 0 h 22/48 (Glidescope), 41/48 (Macintosh), 0.001 (It should be read as: Number of patients with sore throat (Yes/No)P value).At 0 h 22/48 (Glidescope), 41/29 (Macintosh), 0.001 (


Evidence was collected for the intermediate formation of thionium ions in Brønsted acid-catalyzed [2 + 2] photocycloaddition and electrophilic addition reactions to enone dithianes and dithiolanes. Low-temperature NMR studies helped to elucidate the structure and configuration of the thionium ions and thus support previous and current results obtained by UV/Vis spectroscopy.The online version of this article (10.1007/s11244-018-0905-6) contains supplementary material, which is available to authorized users. For low temperature irradiation, the reaction vessel was placed in the photoreactor for 20 min prior to irradiation. Flash column chromatography was performed on silica 60  a Bruker DRX400 was used. 1H-NMR spectra were referenced to the residual solvent signal of chloroform-d1 (CHCl3 δ = 7.26 ppm), benzene-d6 (C6HD5 δ = 7.16 ppm) or dichloromethane-d2 (CHDCl2 δ = 5.32 ppm). 13C-NMR spectra were referenced to the 13C-D triplet of CDCl3 (δ = 77.2 ppm) and C6D6 (δ = 128.1 ppm), or to the 13C-D2 quintet of CD2Cl2 (δ = 54.0 ppm). Apparent multiplets which occur as a result of accidental equality of coupling constants to those of magnetically non-equivalent protons are marked as virtual (virt.). The following abbreviations for individual multiplicities were used: br-broad, s-singlet, d-doublet, t-triplet, q-quartet, quint.-quintet. Assignments and the multiplicity of the 13C-NMR signals were determined by two-dimensional NMR experiments . Infrared spectra were recorded by the attenuated total reflection (ATR) technique using a JASCO IR-4100 spectrometer or a Perkin Elmer Frontier IR-FTR spectrometer. The signal intensities are assigned using the following abbreviations: s (strong), m (medium), w (weak). IR signals are reported as wave numbers −1). Mass spectra were carried out on a Agilent MS5977A MSD spectrometer coupled to a Agilent 7890 B gas chromatograph using a HP-5MS UI column . HRMS data were determined at a Thermo Scientific DFS-HRMS spectrometer. UV/Vis Spectroscopy was performed on a Perkin Elmer Lambda 35 UV/Vis spectrometer. Spectra were recorded using a Hellma precision cell made of quartz SUPRASIL® with a pathway of 1 mm in dry CH2Cl2. Concentrations are given for each spectrum.NMR spectra were recorded at room temperature either on a 5): 7-(Pent-4-en-1-yl)-1,4-dithiaspiro[4.5]dec-6-ene  was dissolved in 9.7 mL CH2Cl2 (c = 10 mM) in a Duran tube and the solution was cooled to − 40 °C. After addition of Tf2NH , the solution was irradiated at λ = 366 nm (128 W) for 13.5 h. Irradiation was stopped and NEt3  was added. The solution was warmed to room temperature and solvent was removed in vacuo. After column chromatography , the title compound was obtained as a colourless oil . TLC: Rf = 0.32 (P/Et2O = 99/1) [KMnO4]; IR (ATR): −1) = 2925 (sp3-CH), 2848 , 1448 , 635 ; MS : m/z (%) = 240 (88) [M]+, 212 (99) [M-C2H4]+, 179 (100) [C11H15S]+, 171 (31), 131 (74) [C5H7S2], 91 (41), 79 (34); 1H-NMR : δ (ppm) = 1.19 , 1.37–1.43 , 1.48–1.53 , 1.55–1.62 , 1.65 , 1.72–1.81 , 1.82–1.92 , 1.97 , 2.08–2.14 , 2.21 , 2.33–2.41 , 3.16–3.36 ; 13C-NMR : δ (ppm) = 20.5 , 25.6 , 27.8 , 29.3 , 32.6 , 34.6 , 38.0 , 38.1 , 39.8 , 40.8 , 48.7 , 49.3 , 70.3 ; HRMS : calculated: (C13H20S2): 240.1001; found: 240.1000.Octahydro-6H-spirocyclobuta-benzene-5,2′-dithiolane] (8.36]hexadec-10-ene (12): 8-(4-Methylpent-4-en-1-yl)-1,5-dithiaspiro[5.5]undec-7-ene  was dissolved in dry CH2Cl2 (8.6 mL) and the resulting mixture was cooled to − 78 °C. Following the addition of 1,1,2,2,3,3-hexafluoropropane-1,3-disulfonimide  the solution was stirred for 5.5 h at − 78 °C in the dark. After which the reaction was quenched via the addition of NEt3 . The solution was allowed to warm to room temperature and the solvent was removed in vacuo. After column chromatography , the title compound was obtained as a colourless oil . TLC: Rf = 0.35 (P/Et2O = 98/2) [KMnO4]; IR (ATR): −1) = 2926 , 1444 , 1275 (w), 795 ; MS : m/z (%) = 268 (100) [M]+, 200 (52) [M-C5H8]+, 159 (60), 118 (62), 105 (58), 91 (32); 1H-NMR : δ (ppm) = 1.28 , 1.36–1.46 , 1.53–1.58 , 1.62 , 1.60–1.73 , 1.78 , 1.82–1.95 , 1.96–2.09 , 2.17 , 2.68–2.79 , 2.89–2.99 , 5.32 ; 13C-NMR : δ (ppm) = 19.1 , 22.7 , 24.2 , 25.8 , 26.7 , 26.8 , 33.6 , 35.1 , 36.9 , 38.6 , 42.9 , 47.3 , 49.9 , 119.7 , 132.5 ; HRMS : calculated: (C15H2432S2): 268.1314; found: 268.1309, calculated: (C1413CH2432S2): 269.1348; found: 269.1343.10-Methyl-1,5-dithiadispiroundec-7-ene  was added. After mixing, the orange-yellow solution was warmed to 203 K (− 70 °C) in a Bruker DRX400 spectrometer and spectra were recorded at 203 K. For 13C-NMR and 2D experiments dithiane 9  and Tf2NH  were dissolved in 0.6 mL dry CD2Cl2.Under an argon atmosphere Tf4 were unsuccessful if the irradiation was performed at λ = 398 nm. Gratifyingly, we found that the reaction was feasible once a short-wavelength light source was employed. At λ = 366 nm, a complete conversion to the desired cyclobutane 5 was observed after six hours in the presence of either 10 or one equivalent (equiv) of Brønsted acid (Table 4 was found to be its somewhat lower basicity and/or lower tendency to form a thionium ion. If treated with the Brønsted acid Tf2NH the UV/Vis absorption (c = 0.5 mM in CH2Cl2 solution) did not reach saturation even upon addition of 20 equivalents of the acid . In stark contrast, thionium ion 3 had shown saturation under otherwise identical condition already with 12.5 equiv of Tf2NH and had produced an absorbance of 1.195 [−1 cm−1 was thus calculated according to the Lambert–Beer law.Attempts to induce an intramolecular 2 + 2] photocycloaddition of 1,3-dithiolane  + 2 photof 1.195 . At λ = 7 (c = 1.0 mM) derived from acetophenone could not be converted into a species with a more intense chromophore (20 equiv Tf2NH) and it is thus likely that there is no thionium ion being formed or that its concentration is very low at best. The more rigid 1,3-dithiane 8 (c = 1.0 mM) derived from α-tetralone exhibited a detectable absorption upon acid treatment (20 equiv Tf2NH) although the measured absorbance was not very high (A = 0.1238). Still, it is evident that the rigid ring system facilitates formation of the respective thionium ion by placing the conjugated arene π-system in proper conjugation with the cationic π-system of the cation. Similarly, the 1,3-dithiane 9 (c = 0.5 mM) derived from 2-methyl-2-cyclohexenone is stabilized by the olefinic π-system and the formation of a thionium ion seems to be facile. Indeed, the compound behaved similar to dithiane 1 and produced quantitatively the respective cation upon addition of 12.5 equiv of Tf2NH. The calculated absorption coefficient at λ = 349 nm was ε = 23,940 M−1 cm−1. By comparison with the 1,3-dithiane derived from 2-pentyl-2-cyclohexenone (see Supporting Information) it was shown that the slightly hypsochromic absorption shift as compared with 1 (vide supra) is due to the length of the different alkyl chain but not due to the absence of the olefinic double bond.We further explored the structural parameters which favor the formation of thionium ions. While Satchell and co-workers had already observed colored intermediates if treating 2,2-diaryl-1,3-dithianes with perchloric acid , the 1,39 and given the less complex structure of 9 compared to 1, it was attempted to substantiate the formation of the cation by low-temperature NMR studies. Upon addition of 6.25 equivalents of Tf2NH at − 70 °C, a solution of compound 9 in CD2Cl2 produced a 1H-NMR spectrum  and ● (minor) in the spectrum. A third species was identified based on its signals at high (δ = 1.40 ppm) and low field (δ = 7.35 ppm). All other signals of this species overlap with the signals of the major isomer and they cannot be distinguished at higher concentration which are necessary for a sufficient signal-to-noise ratio in 2D NMR experiments. Upon addition of acid, the structure of 1,3-dithiane 9 changes, indicated by the fact that the two enantiotopic hydrogen atoms in 5-position of the 1,3-dithiane ring are no longer split into two signals (δ = 1.68 and 1.91 ppm). Rather they appear in a single broad peak centered at δ = 2.08 ppm overlapping with signals from other methylene protons.Given the apparently clean formation of a thionium ion from rum Fig.  in which9 is shifted by 1.92–2.03 ppm from δ = 5.32 ppm to δ = 7.35, 7.31, and 7.24 ppm for each species. The strong downfield shift can be explained by an electron withdrawing atom or group, generated by the acid. The chemical shift at δ = 3.61 ppm is attributed to a methylene group next to a thionium ion, as already observed by Lambert et al. [13C-NMR spectrum at δ = 23.3 and 20.5 ppm also indicate the presence of a thiol bound to an alkyl chain [13C-NMR spectrum further exhibits downfield shifted signals between 190.1 and 197.4 ppm which are assigned to the olefinic quartenary carbon atoms in conjugation to the carbocation center. The cation center itself resonates at 218.3–223.1 ppm in the three species mentioned above. The 13C-NMR signals of the sp2 hybridized carbon atoms of all three species appear in three groups of three signals each , suggesting a structural similarity of all three species. In a NOESY experiment cross peaks between the signals of the methyl groups (δ = 2.47 and 2.36 ppm), the olefinic hydrogen atoms (δ = 7.31 and 7.24 ppm) and the endocyclic methylene group in β position to the positive charged sulfur atom (δ = 3.07 and 3.17 ppm) were observed for the major and minor species.1H-NMR timescale [10a was assigned to the major (Z)-diastereoisomer of the thionium ions and structure 10b was assigned to the (E)-diastereoisomer


Single-crystal-to-single-crystal (SCSC) reactions are rare for organic solids. We describe the first example of an SCSC [2+2] photodimerization that is mediated by halogen bonding. Halogen bonds have emerged as noncovalent forces that govern the assembly of molecules in organic solids with a degree of reliability akin to hydrogen bonds. Although the structure-directing roles of halogen bonds are often compared to hydrogen bonds, general knowledge concerning the fundamental structural behavior of halogen bonds has had limited opportunity to develop. Following an investigation of solid-state reactions involving organic syntheses and the development of photoresponsive materials, this work demonstrates the ability of the components of intermolecular N⋯I halogen bonding – a ‘workhorse’ interaction for the crystal engineer – to support a single-crystal-to-single-crystal [2+2] photodimerization. A comparison is provided of the geometric changes experienced by the halogen-bonded components in the single-crystal reaction to the current crystal landscape of N⋯I halogen bonds, as derived from the Cambridge Structural Database. Specifically, a linear-to-bent type of deformation of the halogen-bonded components was observed, which is expected to support the development of functional halogen-bonded materials containing molecules that can undergo movements in close-packed crystal environments. Flexibility in the solid state can give rise to atomic and molecular movements that support single-crystal-to-single-crystal (SCSC) transformations   can be considered fundamentally important in the development of machine-like materials   halogen bond has been exploited extensively in the field of crystal engineering to sustain discrete and extended supramolecular frameworks ·2(bpe) , which assemble via N⋯I halogen bonds to form a cocrystal that reacts quantitatively to form tpcb (tpcb = rctt-tetrakis(4-pyridyl)cyclobutane). We demonstrate that the components engaged in halogen bonding exhibit structural flexibility, their geometries undergoing a linear-to-bent type of deformation in the SCSC transformation. The movements span the outer geometries of N⋯I bonds that have been reported to date.In this contribution, we report a halogen-bonded cocrystal with components that support an SCSC [2+2] photodimerization (Scheme 1). We show the components of (I2.2.1.1H NMR spectra were recorded using a Bruker AVANCE-300 NMR spectrometer operating at 300 MHz using DMSO-d6 as solvent. Photoreactions of (I4F16cb)·2(bpe) were conducted on a glass plate using UV radiation from a 450 W medium-pressure mercury lamp inside an ACE Glass photochemistry cabinet. Powder X-ray diffraction data were collected from samples mounted on glass slides by a Siemens D5000 X-ray diffractometer using Cu Kα1 radiation (λ = 1.54056 Å).The bipyridine bpe (SIGMA) and all solvents were commercially available and were used without further purification (FISHER). 2.2.4F16cb was synthesized as reported ·2(bpe) were obtained as colorless plates by combining acetonitrile solutions (1.5 ml each) of I4F16cb  and bpe  (1:2 molar ratio). Single crystals suitable for single-crystal X-ray diffraction studies were obtained after a period of approximately 1 d.I2.3.4F16cb)·2(bpe) were collected with a Bruker APEXII Kappa diffractometer equipped with an Oxford Cryostream low-temperature device using Mo Kα radiation (λ = 0.71073 Å). All calculations dealing with data collection, initial indexing, frame integration, Lorentz polarization corrections and final unit-cell parameters were carried out by APEX2 ·2(tpcb) were collected on a Nonius KappaCCD single-crystal X-ray diffractometer at room temperature using Mo Kα radiation (λ = 0.71073 Å). Data collection, cell refinement and data reduction were performed using COLLECT  = 1.2Ueq(C).Single-crystal data for (I2.3.1.P21/c (No. 14), a = 13.0184 (13), b = 9.7153 (10), c = 19.986 (2) Å, β = 104.649 (5)°, V = 2445.6 (4) Å3, Z = 2, T = 298.15 K, μ(Mo Kα) = 2.653 mm−1, Dcalc = 2.059 g cm−3, 52 897 reflections measured (5.926° ≤ 2θ ≤ 52.87°), 5012 unique  which were used in all calculations. The final R1 was 0.0198 [I > 2σ(I)] and wR2 was 0.0460 .Crystal system: monoclinic, space group: 2.3.2.P21/c (No. 14), a = 12.9647 (13), b = 10.2219 (10), c = 19.519 (10) Å, β = 109.387 (5)°, V = 2440.0 (13) Å3, Z = 2, T = 298.15 K, μ(Mo Kα) = 2.659 mm−1, Dcalc = 2.064 g cm−3, 12 227 reflections measured (5.956° ≤ 2θ ≤ 50.786°), 4464 unique  which were used in all calculations. The final R1 was 0.0330 [I > 2σ(I)] and wR2 was 0.0627 .Crystal system: monoclinic, space group: 3.e.g. ladderanes) and/or of controlling the bulk-physical properties of solids . In this context, a major focus has been the use of noncovalent forces to direct the formation of carbon–carbon single (C—C) bonds in cocrystals via [2+2] photodimerizations  and thus provide insight into the structural behaviors and dynamics of molecular components engaged in intermolecular bonding. Although there have been two recent reports of intermolecular N⋯I halogen bonds that support photocycloaddition reactions in the solid state ·2(bpe). We have reported the synthesis of I4F16cb in the solid state using 1,8-bis(4-pyridyl)naphthalene (dpn) as a template  in acetonitrile afforded colorless plate-shaped crystals after a period of 1 d. The formulation of (I4F16cb)·2(bpe) was confirmed by single-crystal and powder X-ray diffraction (at 298 K), as well as by 1H NMR spectroscopy.The halogen-bonded cocrystal that undergoes SCSC photodimerization is (I4F16cb)·2(bpe) crystallizes in the monoclinic space group P21/c. Half a molecule of I4F16cb and one molecule of bpe lie in the asymmetric unit  ], organizes bpe in a face-to-face π-stacked geometry with two C=C bonds of bpe parallel and separated by 3.83 Å. The olefin lies disordered over two sites [highest occupied site: 0.769 (7)]. The stacking satisfies the criteria for a [2+2] photodimerization : πF–πF = 3.40, πF–πpyr = 3.99 and πpyr–πpyr = 3.84 Å]. Olefins of neighboring chains are separated by 7.89 Å which is outside the limits for a photoreaction.Single-crystal X-ray analysis revealed that ·2(bpe) were exposed to UV irradiation (450 W medium-pressure Hg lamp) for a period of 30 h, tpcb formed quantitatively, indicated by the disappearance of the olefinic peaks (7.54 p.p.m.) and the appearance of cyclobutane peaks (4.66 p.p.m.) in the 1H NMR spectrum (DMSO-d6). Optical microscopy confirmed that the crystals remained intact during the photodimerization.When single crystals of (I4F16cb)·2(bpe) undergoes an SCSC reaction to form (I4F16cb)·2(tbcb). The bpe olefin reacted to give tpcb and, as a result, maintained the structure of the halogen-bonded chains   confirmed that (I2) Fig. 3. The hal3.2.4F16cb)·2(bpe), we constructed a novel IsoStar scatterplot  used to describe halogen bonds. The boundary is rectangular in shape . Given that the separation distance of the C atoms of the C=C bonds decreases to form the C—C bonds, we expected a change in the πcentroid—N⋯I angle to describe the ‘sweep’ of the 4-pyridyl group away from ca 180° in the photodimerization.To gain insight into the geometric changes experienced by the halogen-bonded components of ·2(bpe) and (I4F16cb)·2(tbcb) fall within these ranges. The πcentroid—N⋯I angles from the CSD range from 132.6 to 180.0°. This range is larger than that of the C—I⋯N angles, whereas the corresponding angles for (I4F16cb)·2(bpe) and (I4F16cb)·2(tbcb) fall within the values. Considered together, we believe the IsoStar scatterplot shows that the changes in geometry of the 4-pyridyl groups in the photodimerization correspond to a linear-to-bent type of deformation from the plane of the best-fit p-C6F4-I group.The SD Fig. 4. The disX distances and C—X⋯N angles. Specifically, XB1 underwent N⋯I lengthening from 2.782 (8) to 2.878 (5) Å  to 161.1 (2)° and from 175.8 (3) to 166.4 (2)°, respectively.The changes in geometry of the halogen bonds in the SCSC transformation involve changes to the N⋯B2 Fig. 4, the N⋯I3.3.et al., 20044F16cb itself can be synthesized as a powdered cocrystal using dpn in a cycloaddition sustained by N⋯I halogen bonds ·2(bpe), the SCSC reveals that the components of the N⋯I halogen bonds undergo linear-to-bent bending in order to accommodate the formation of tpcb in the solid state. We note that movements of components in the form of rotations of molecules engaged in N⋯X halogen bonds have been very recently considered as being relevant in the development of molecular-scale machine assemblies  and exhibit SCSC reactivity. We also envisage an ability to construct solids wherein halogen-bonded components exhibit a combination of movements (e.g. bending and rotation). Mechanical properties of the resulting materials will also be studied  publication_text, mcg164, mcg1611a. DOI: 10.1107/S2052252518007583/ed5014sup2.pdfSupporting information. DOI: 1521899, 1521900CCDC references:


Protein ubiquitination plays a key role in the regulation of cellular processes, and misregulation of the ubiquitin system is linked to many diseases. So far, development of tool compounds that target enzymes of the ubiquitin system has been slow and only a few specific inhibitors are available. Here, we report the selection of single-domain antibodies (single-dAbs) based on a human scaffold that recognize the catalytic domain of HOIP, a subunit of the multi-component E3 LUBAC and member of the RBR family of E3 ligases. Some of these dAbs affect ligase activity and provide mechanistic insight into the ubiquitin transfer mechanism of different E2-conjugating enzymes. Furthermore, we show that the co-crystal structure of a HOIP RBR/dAb complex serves as a robust platform for soaking of ligands that target the active site cysteine of HOIP, thereby providing easy access to structure-based ligand design for this important class of E3 ligases.  •Single-domain antibodies (dAbs) modulate the activity of the E3 ligase HOIP•dAbs facilitate HOIP RBR domain crystallization and ligand soaking•HOIP/dAb complex crystals provide access to structure-based inhibitor development Tsai et al. identify single-domain antibodies (dAbs) that modulate the activity of the E3 ligase HOIP and facilitate crystallization of its catalytic RBR domain. HOIP/dAb crystals serve as a robust platform for ligand soaking and provide a novel tool for structure-based inhibitor development of E3 ligases. Ubiquitin modification of protein substrates provides signals for a myriad of cellular responses, from protein degradation to DNA repair or immune signaling. Ubiquitin can be attached to the substrate in the form of a single ubiquitin molecule, or can form polyubiquitin chains via one of its seven Lys residues or the N-terminal methionine (M1) amine group. Substrate modification with ubiquitin requires an enzymatic cascade involving E1-activating enzymes, E2-conjugating enzymes, and E3 ubiquitin ligases. Ubiquitin is first activated in an ATP-dependent manner to form an E1-Ub conjugate and subsequently transferred onto an E2-conjugating enzyme. E3 ligases mediate substrate selection, while specific combinations of E2 and E3 enzymes can determine the type of ubiquitin modification formed . A multiAt present, there is a paucity of validated tool compounds that can be used to study the function of LUBAC and other RBR ligases in a cellular context. BAY11-7082 and gliotoxin both have been described to inhibit LUBAC activity, but lack substrate specificity . More reWe aimed to develop single chain antibody-based crystallization chaperones to assist structural studies on the HOIP RBR domain in a straightforward fashion, at high resolution. Furthermore, we wanted to test if single-chain antibodies could be used as activity modulators to provide mechanistic insight into E3 ligase function. Cameloid-derived nanobodies have proved very useful tools to assist crystallizing complex protein architectures; for example, a ubiquitin-bound kinase PINK  and actiHerein, we report the screening of a large synthetic single-dAb library against the RBR of HOIP to successfully obtain tight-binding dAbs which display different functional effects on E3 ligase activity, depending on the identity of the E2-conjugating enzyme. Moreover, we solved the co-crystal structure of a HOIP RBR/dAb complex and demonstrate that an RBR E3 ligase can serve as a robust platform for soaking of ligands, an expedient approach for structure-based ligand design that should aid the development of modulators of this important class of enzymes.in vitro on the Lys residue within the AviTag peptide. Biotinylated HOIP RBR was immobilized on streptavidin resin and three rounds of phage display selection were carried out. Phage ELISAs were evaluated following second and third rounds of selection to determine the enrichment of antigen-specific phage. Sequences of randomly selected clones from second and third rounds were analyzed to confirm binder diversity. After obtaining several hundreds of unique dAb candidates, biolayer interferometry (BLI) was employed to evaluate dAb binding to HOIP RBR. Full-length or truncated versions of RBR were included in the evaluation process to eliminate dAbs that only bind RING1 or RING2-LDD domains, because those dAbs are less likely to contribute toward stabilization of the flexible linkers that connect RBR subdomains. Other selection criteria included fast-association and slow-dissociation rates to identify tight and stable binders. More than 80 binders were selected and purified in soluble form for further assessment, including association/dissociation rate evaluation by BLI, dAb oligomerization state evaluation by SEC-MALLS, and dAb and dAb/RBR thermal stability evaluation by differential scanning fluorimetry. Finally, 10 dAbs were selected to be taken forward and their interaction with HOIP RBR was quantified by BLI, which showed that most of the binding affinities (KD) of the selected dAbs are in the nanomolar range   and lysine-reactive E2s, especially members of the UbcH5 family, which are highly promiscuous and active with most E3 ligases. To investigate functional effects of the selected dAbs on the activity of HOIP with UbcH7 and UbcH5C, respectively, we carried out n assays A, and adgle-dAb)  had no egle-dAb) B.Figure D = 320 nM; and dAb 2, KD = 7.5 nM) that have only a minor effect on activity with either E2   F and 1G  3.2 nM) H–1M. Thi 3.2 nM) C.D = 1.7 μM)  A–S2C. ToHDX-MS is useful for monitoring the exchange of peptide backbone amide protons. The technique was used here to map changes in solvent accessibility and hydrogen bonding in HOIP RBR upon dAb complexation, as determined by differential rates of deuterium incorporation of pepsin-derived peptides from HOIP. In simple cases, binding epitopes are revealed by appearance of “protected” patches of surface amides that exchange more slowly as they are shielded from solvent  on antibody binding. Interestingly, this was not what we saw. Firstly, the pattern of changes we observed was varied among the dAbs, suggesting we had identified a range of binding sites and binding modes. Secondly, antibodies induced not only regions of HDX-MS protection, but also showed significant regions of deprotection, suggesting that some dAbs may lock HOIP in a more open conformation. To more easily visualize and compare the behavior of these dAbs, the differential deuteration (as a proportion of the maximum deuteration) was plotted versus the pepsin-derived HOIP peptides for each dAb complex and clustered using a Euclidean distance approach A.Figure donor) site on IBR, and, secondly, sterically interfering with the domain movement necessary for catalysis  all strongly reduce amide exchange in the linker connecting RING1 and IBR, here referred to as the helix-turn-helix (HTH) region of HOIP, suggesting that this is likely to be an interaction site for all these proteins B–2D. Howatalysis C and 2D.atalysis .The three antibodies in class 4  have non-classical protection patterns with a very small region of protection in IBR and large regions of deprotection in RING1, HTH, and RING2, especially for dAb25 and dAb34. We hypothesize that these dAbs may bind to IBR of HOIP stabilizing a distinct conformation, more open than its average solution conformation, such that many of the backbone amides are more solvent exposed than normal.H and VK of a conventional antibody  B. InteredAb3 and RBRUbcH5B−Ub are tilted at different angles with respect to the IBR  through fragment-based covalent ligand screening and reported a ligand-bound RING2-LDD structure solved at 2.15 Å resolution (referred to as compound (5) in -fibrinopeptide B was used for internal calibration. To identify HOIP peptides, additional non-deuterated, uncomplexed samples were processed identically except that an MSe fragmentation method was used. The output was processed using ProteinLynx Global Server v3.0.2 (Waters), using low- and high-energy thresholds of 250 and 100 respectively. Identified ion peaks were searched against the HOIP construct sequence used, using non-specific peptide cleavage and methionine oxidation as a variable modification. Results were analysed using DynamX v3.0 (Waters). PLGS-identified peptides were filtered to those of length 7-to 25 with a PLGS peptide score > 7.0. A low energy signal threshold of 500 was applied. Peptide and ion assignments were manually checked and refined where necessary.Sample handling and mixing were performed using a LEAP H/D-X PAL robot  and liquid chromatography was performed using an Acquity M class UPLC . Samples were subjected to a standard deuteration method using 20-fold dilution into deuteration buffer . Similarity clustering of this data across dAbs and peptides was performed using UPGMA (unweighted pair group method with arithmetic mean) clustering with a Euclidean distance measure, as implemented in Spotfire.A complex of HOIP RBR and dAb3 was prepared by mixing RBR:dAb3 at 1:3 molar ratio. The mixture was loaded onto a Superdex 75 10/300 GL column  with 50 mM HEPES pH 7.5, 150 mM NaCl and purified at a flow rate of 0.8 mL/min. Sample purity was evaluated by SDS-PAGE B and S4CInhibitor compounds were stored at 10-20 mM in 100% DMSO. Prior to soaking, compounds were diluted by 100-fold in crystallization reservoir solution to achieve final concentration of 1% DMSO. Trehalose was supplemented in the reservoir for a final concentration of 30% in the drop. Compound-soaked protein crystals were harvest after 24 h with MicroLoops LD and flash-frozen with liquid nitrogen.3 or 4 were collected on MX beamline IO4 (wavelength 0.9795 Å); crystals soaked with 2 were collected on MX beamline IO4-1 (wavelength 0.9159 Å) while crystals soaked with 5 were collected on MX beamline IO3 (wavelength 0.9763 Å) at Diamond Light Source (UK) at 100K. Data were processed with autoPROC (1OHQ) (4LJQ)   as the s) (4LJQ)  as searc) (4LJQ) , REFMAC5) (4LJQ)  and PHEN) (4LJQ) . PHENIX ) (4LJQ)  in CCP4 ) (4LJQ) . FurtherAnhydrous solvents were commercially acquired in 100 mL bottles and used as received. Commercially acquired chemicals were used without further purification. Chemical names were generated in the ChemDraw Professional . Microwave reactions were carried out in sealed Biotage microwave vials in a Biotage Initiator+ microwave equipped with IR temperature control. For working up reaction mixtures, hydrophobic filters were generally used to remove traces of water from the extraction solvent.2Cl2 and eluted with mixtures of petroleum ether (bp 40-60), ethyl acetate and MeOH typically over 12 column volumes (CV).Column chromatography was carried out on a Biotage Isolera One automated equipment. Biotage SNAP (Ultra) KP-Sil cartridges were used for normal phase. The samples were generally loaded dissolved in CHtr) are reported in minutes. The solvents employed were A  and B  in a gradient of 5→97% B in A (v/v) over 4 min. Flow rate 0.5 mL/min.UPLC-MS analysis was conducted on an Acquity UPLC CSH C18 column  at 40°C. Injection volume: 1 μL. The UV detection was a summed-up signal from wavelengths between 210 to 400 nm. UPLC retention times (Preparative HPLC purification was conducted on an Agilent 5 C18 column (50 mm × 21.2 mm) at ambient temperature. The solvents employed for purification were A  and B . Flow rate: 25 mL/min. Injection volume: <1 mL. The DAD UV detection was a summed-up signal from wavelengths between 210 to 360 nm, and the HPLC was coupled to an Agilent Infinitylab LC/MSD unit with positive/negative electrospray ionization (scan range 100 to 1000 AMU) and mass- and UV-based automated fraction collection.1H, 13C and 2D NMR spectra were recorded on a Bruker 400 MHz instrument equipped with a 5 mm probe. The obtained FID-files were processed with MestReNova v.12.0.3-21384 software (Mestrelab Research). Signals are reported in ppm (δ) using the solvent as reference (DMSO-d6: 2.50 ppm (1H), 39.52 ppm (13C); CDCl3: 7.27 (1H), 77.16 (13C)) (J) are given in Hertz (Hz), and JHH were rounded to the nearest 0.1 Hz. Multiplet patterns were assigned the following abbreviations or combinations of these: br – broad, m – multiplet, s – singlet, d – doublet, t – triplet. Signal assignment was made from unambiguous chemical shifts and COSY, HSQC and HMBC experiments. Unassigned aromatic signals were denoted Ar-C and Ar-CH respectively.6 (13C)) . CouplinLow resolution mass spectrometry (LRMS) was recorded on a Waters ZQ MS unit with alternate scan positive/negative electrospray ionization (scan range 100 to 1000 AMU).High resolution mass spectrometry (HRMS) was recorded on a Waters XEVO G2-XS MS unit with positive electrospray ionization (scan range 100 to 1200 AMU) after sample elution from an Acquity UPLC equipped with an CSH C18 column , held at 50°C, and using a solvent gradient of 97% A in B, v/v → 100% B over 8.5 min. The solvents employed were A  and B . Flow rate: 0.8 mL/min. Injection volume: 0.2 μL. Molecular masses were calculated in the ChemDraw Professional .Rf) values were rounded to the nearest 0.05. The yields reported are from single reaction runs.Thin layer chromatography (TLC) was carried out using pre-coated silica gel 60 plates that were eluted using mixtures of solvents as indicated. The plates were visualized using UV light (254 nm) or potassium permanganate stain, and retention factor (2 and 3 have previously been reported (4 is described below. The synthesis of inhibitor 5 (HOIPIN-8) was carried out largely according to the procedure previously published by Katsuya et al. -4-but-2-enoic acid SI-1  was stirred with 4-nitrophenyl 2-iodoacetate  and NaHCO3  in DMSO (3.5 mL) at ambient temperature for 24 h. The reaction mixture was filtered and adsorbed onto a Biotage SNAP cartridge, dried, and then purified by column chromatography (Biotage Ultra 25 g KP-Sil column) using a mixture of cyclohexane and EtOAc  to afford the activated ester SI-2 (130 mg), which was used without further purification.reported , (E)-4- was stirred with N,N-diisopropylethylamine  and methylamine  in anhydrous DMF (1 mL) at ambient temperature under N2 for 1 h. The reaction mixture was filtered and purified by preparative HPLC to afford the product amide 4  as a white solid. UPLC: tr = 1.75 min; 1H NMR : δ 12.28 , 10.07 , 8.07 , 7.99-7.93 , 7.01 , 5.91 , 4.48 , 4.16 , 2.62-2.56 , 1.68 . One set of Ar-CH2 signals overlaps with the solvent peak; 13C NMR : δ 166.9 (CH2C=O), 164.8 (ester-C=O), 163.7 ((C=O)NHCH2), 162.0 (pyridone-C=O), 148.3 (pyridone-C6), 147.1 (CH2CH), 145.3 (pyridone-C4), 119.4 (CHCOO), 116.9 (pyridone-C3), 113.9 (pyridone-C5), 62.4 (OCH2), 26.3, 25.3 (CH2CH2CH2CH2), 25.2 (NHCH3), 21.8, 20.9 (CH2CH2CH2CH2). The peak corresponding to CH2CH overlaps with the solvent signal; HRMS: HRMS (ESI+) m/z [M + H]+ Calcd for C17H22N3O5+ 348.1554, found 348.1569.NBS bromination: Similar to the method of Katsuya et al.  and dibenzoyl peroxide  were dissolved in anhydrous CHCl3 in a 100 mL round-bottom flask. The flask was fitted with a condenser and the reaction mixture was refluxed at 70°C for 4 h, then allowed to cool. CH2Cl2 (20 mL) was added and the combined organic fractions were washed with saturated aqueous NaHCO3, water and brine (40 mL each). The organic solution was filtered through a hydrophobic frit and concentrated in vacuo to afford the crude product as a yellow oil that solidified upon standing. Purification by column chromatography , v/v, over 12 column volumes) gave 3,5-dibromoisobenzofuran-1(3H)-one SI-3-1 (2.94 g) as a white solid. TLC: Rf = 0.45 ; 1H NMR : δ 7.83-7.74 , 7.35 ; 13C NMR : δ 166.5 (C=O), 150.6 (Ar-C), 134.7 (Ar-CH), 130.6 (Ar-C), 127.3, 127.1 (2 × Ar-CH), 123.1 (Ar-C), 73.4 (CHBr).a et al.  5-BromoiNucleophilic substitution: The intermediate 3,5-dibromoisobenzofuran-1(3H)-one SI-3-1  was transferred to a 20 mL microwave vial and water (20 mL) was added. The vial was capped and the mixture was heated by microwave irradiation at 120°C for 3 h, then allowed to cool. The afforded solid was filtered off, washed with water (2 × 20 mL) and dried in vacuo to afford alcohol SI-3  as a white solid. TLC: Rf = 0.25 ; UPLC: tr = 1.90 min; 1H NMR : δ 8.28 , 7.93 , 7.86 , 7.77  , 6.65 ; 13C NMR : δ 167.5 (C=O), 149.4 (Ar-C), 133.8 (Ar-CH), 128.5 (Ar-C), 127.0, 126.5 (2 × Ar-CH), 125.8 (Ar-C), 97.7 (OCH); LRMS: 229 [M+H]+.SI-3 , 1-methyl-4--1H-pyrazole , Pd(dppf)Cl2 , and K2CO3 . The vial was pump-filled with nitrogen and de-gassed 1,4-dioxane (18 mL) and H2O (2 mL) was added. The mixture was heated in the microwave at 100°C for 2 h. The reaction mixture was quenched with 1 M aqueous HCl (13 mL) and filtered through a plug of Celite. The solids were washed with EtOAc (2 × 20 mL). The phases were separated, and the aqueous phase extracted with EtOAc (3 × 15 mL). The combined organic fractions were filtered through a hydrophobic frit and concentrated in vacuo to afford the crude product as a red solid. Purification by column chromatography  afforded a solid that was washed with Et2O (10 mL) and dried to give biaryl product SI-4  as a pink solid. TLC: Rf = 0.40 ; UPLC: tr = 1.62 min; 1H NMR : δ 8.39 , 8.16 , 8.08 , 7.87-7.83 , 7.81-7.76  (3 × Ar-H), 6.65 , 3.88 ; 13C NMR : δ 168.2 (C=O), 148.6 (Ar-C), 139.0 (Ar-C), 137.0, 129.3 (2 × pyrazol-CH), 126.9, 125.2 (2 × Ar-CH), 123.7, 120.7 (Ar-C), 119.2 (Ar-CH), 98.1 (OCH), 38.8 (CH3); LRMS: 231 [M+H]+.According to the method of Katsuya et al.  a 20 mL Alkylation:SI-4  and 1-ethan-1-one  were dissolved in EtOH (15 mL) and 6 M aqueous NaOH was added . The mixture was stirred at ambient temperature for 30 min. The reaction was quenched with 1 M aqueous HCl  and the mixture was stirred for 10 min. Water (10 mL) was added and the EtOH was removed in vacuo. The residue was washed with cold water and EtOAc then dried to afford the product mixture of SI-5a and SI-5b (0.43 g) as a white solid.Suzuki reaction: A 20 mL microwave vial was charged with SI-5a and SI-5b , tert-butyl 4--1H-pyrazole-1-carboxylate , Pd(dppf)Cl2 , and K2CO3 , capped and pump-filled with nitrogen (3×). De-gassed THF (15 mL) and H2O (1.5 mL) was added and the mixture was purged with nitrogen, then heated in the microwave at 90°C for 2 h. The reaction mixture was quenched on ice with 1 M HCl (7 mL), filtered through a plug of Celite and the solids were washed with EtOAc (3 × 10 mL). The phases were separated, and the aqueous phase was extracted with EtOAc (2 × 10 mL). The combined organic fractions were filtered through a hydrophobic frit and concentrated in vacuo to afford the crude biaryl product as a brown film, which was used in the next step without further purification.Boc-deprotection: 4M HCl in 1,4-dioxane  was added and the crude mixture was stirred at ambient temperature under nitrogen 1.5 h. The solvent was removed in vacuo to give an orange solid. Purification by preparative HPLC gave SI-6 (0.19 g) as a white solid.Ring-opening: SI-6  was dispersed in EtOH (10 mL) and H2O (2 mL) and cooled to 0°C in an ice-water bath. 6 M aqueous NaOH  was then added, and the mixture was stirred at 0°C for 15 min and at ambient temperature for 45 min. The mixture was cooled to 0°C and ice-cold Et2O (40 mL) was added. The mixture was stirred at 0°C for 1 h to give a precipitate, which was filtered off and washed with Et2O (10 mL). Carboxylate 5  sodium salt was isolated as a brown solid. UPLC: tr = 2.20 min (C); 1H NMR : δ 8.80 , 8.25 , 8.18 , 7.96  (4 × pyrazole-CH), 7.91 , 7.62 , 7.52 , 7.45  (5 × Ar-H), 7.15 , 3.86 . The pyrazole-H was not detected. 13C NMR : δ 187.9 (ketone-C=O), 170.0 (carboxylate-C=O), 160.9, 158.5 (2 × Ar-CF), 148.9 , 142.4 (Ar-C), 136.2 (pyrazole-CH), 132.5 , 132.3, 131.6 (2 × Ar-C), 130.2 (Ar-CH), 128.1 (pyrazole-CH), 126.5 (Alkene-CH), 126.3, 122.1 (2 × Ar-CH), 121.4, 118.7, 113.9 (3 × Ar-C), 107.9, 107.7 (2 × Ar-CH), 38.6 (CH3). One Ar-C signal was not detected. HRMS (ESI+) m/z [M+H]+ Calcd for C23H17F2N4O3+ 435.1263, found 435.1273.4 or 5 (20 μM), 1% v/v final DMSO concentration, in 50 mM HEPES pH 7.5, 150 mM NaCl buffer at 23°C. Final volume 75 μL. Aliquots (25 μL) were withdrawn after 0.5, 4 and 24 h incubation, flash frozen in liquid nitrogen and stored at -80°C. Each sample were individually defrosted immediately before injection into the LCMS. Acquisition of mass spectrometry data was carried out using positive electrospray ionisation (ESI) on an Agilent 6224 TOF LCMS system equipped with a Diode Array Detector (DAD), a Wellplate sampler (held at 10°C) and an Agilent PLRP-S reverse phase column  held at 70°C. The following mobile phases were used for the LC: Mobile phase A (MP A) = 0.2% Formic acid in water, v/v; Mobile phase B (MP B) = 0.2% Formic acid in MeCN, v/v. The following LC method was employed: 0-0.5 min , 0.5-5 min , 5-5.7 min , 5.7-5.71 min , 5.71-6.2 min (100% B), 6.2-6.21 min , 6.21-8 min . Flow rate: 0.5 mL/min. Injection volume: 10 μL. The run length was 8 min, and equilibration time 1 min after each sample. DAD absorption was an average of wavelengths between 210 and 280 nm. The TOF mass spectrometer recorded 1 spectrum per second with a scan range between 700 and 2200 AMU. Proteins were deconvoluted in the Agilent MassHunter Workstation Software version B.06.00 build 6.0.633.10 using maximum entropy deconvolution with 1 Da mass step and baseline factor 7 subtractions.HOIP RBR (2 μM) was incubated with compound on) and error values were obtained by using the LINEST function in MS Excel, which uses the least squares method to calculate the statistics for a straight line. The error values of koff were obtained from the standard deviation of independent analysis of the rate of dissociation from at least 4 different protein concentrations. The error values of the equilibrium dissociation constant (KD) were obtained using the following equation in Excel:Data were analysed using the Octet Data software  and Microsoft Excel. The association rate constant  are available at RCSB Protein Data Bank (https://www.rcsb.org/).Crystal structures generated in this study (PDB codes


There has been little previous work directly measuring whether the trafficking of calcium channels requires their ability to bind Ca2+ in the selectivity filter or to conduct Ca2+. Here, we examine trafficking of neuronal CaV2.1 and 2.2 channels with mutations in their selectivity filter and find reduced trafficking to the cell surface in cell lines. Furthermore, in hippocampal neurons, there is reduced trafficking to the somatic plasma membrane, into neurites, and to presynaptic terminals. However, the CaV2.2 selectivity filter mutants are still influenced by auxiliary α2δ subunits and, albeit to a reduced extent, by β subunits, indicating the channels are not grossly misfolded. Our results indicate that Ca2+ binding in the pore of CaV2 channels may promote their correct trafficking, in combination with auxiliary subunits. Furthermore, physiological studies utilizing selectivity filter mutant CaV channels should be interpreted with caution.Voltage-gated calcium channels are exquisitely Ca  •V2 channels block inward Ba2+ currentsSelectivity filter mutations in Ca•V2 channelsSurprisingly, these mutations severely reduce trafficking of the Ca•Pore mutant N-type channels show reduced expression in presynaptic terminals•2δ and thus are not grossly misfoldedPore mutant channels still require β and α V2 channels, preventing inward Ba2+ currents, would reduce trafficking. Pore-mutant channels show strongly reduced cell-surface expression in cell lines and neurons, but still require β and α2δ subunits and thus are not grossly misfolded.Meyer et al. examine whether selectivity filtering mutations in Ca V) are exquisitely Ca2+ selective  for divalent cation binding in the channel pore and for permeation -affinity binding site that is also occupied by blocking cations such as Cd2+  on the intracellular I-II linker  domain, as also elucidated functionally . Our results suggest that a combination of interaction with the auxiliary subunits and Ca2+ binding in the pore promotes the trafficking competency of CaV2 channels.Our main finding is that there are substantial defects in trafficking to the cell surface and into neuronal processes and presynaptic terminals of the divalent cation-impermeant CaV2 channel selectivity filter for channel trafficking, taking advantage of our previously described extracellular hemagglutinin (HA)-tagged CaV2.2 construct, in which the tag does not affect function , either alone or concurrently  retains a small inward Ba2+ current at +5 mV  in the presence of a large driving force. In order to reduce outward currents through these channels, in case these might be masking residual inward current, we then utilized an N-methyl-D-glucamine (NMDG)-based internal solution , like the WT CaV2.2 current, was lost when CaV2.2 channels were blocked using ω-conotoxin GVIA  cells. In our initial experiments, we found that the selectivity filter mutant channels, co-expressed with α2δ-1 and β1b, were present in the plasma membrane to a much lower extent than WT CaV2.2: a 53.5% reduction for CaV2.2 EIA and a 67.8% reduction for CaV2.2 EI, II, III, IVA . The fact that the selectivity filter mutant channels still responded significantly to both β-subunit co-expression and reduced temperature indicates that the CaV2.2 EI, II, III, IVA channels are not grossly misfolded and thus aggregated in the ER. This is supported by the absence of increased co-localization of CaV2.2 EI, II, III, IVA with an ER marker protein disulphide isomerase  exhibited almost no inward or outward current  was also examined by cell-surface biotinylation in tsA-201 cells  is a charge reversal mutation of the selectivity filter E in domain IV of Cafunction . Indeed, current . In agreobserved D and S4E01 cells E and 4F.2.2 EIVK E and 4F.ate WCL; G.Figure V2.2 into hippocampal neurites is highly dependent on the presence of an α2δ subunit. We first examined the effect of α2δ-1 on trafficking of the CaV2.2 selectivity filter mutants in undifferentiated N2A cells. Similar to previous results, cell-surface expression was reduced by 53.5% for CaV2.2 EIA and by 67.8% for CaV2.2 EI, II, III, IVA -mCherry as a marker for presynaptic terminals. Intracellular CaV2.2 expression was monitored by the fluorescence of the GFP tag, and HA immunostaining in non-permeabilized conditions was used to assess its cell-surface expression . This construct showed robust expression in the plasma membrane when co-expressed with β1b and α2δ-1 in tsA-201 cells, although only when using 30°C culture conditions , whereas clear cell-surface expression was observed for CaV2.1-HA EIA, although it was 33.5% less than for WT CaV2.1-HA  remained able to restore both off-gating currents and excitation-contraction coupling when expressed in dysgenic myotubes, whereas the equivalent CaV1.2 mutant did not .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Annette Dolphin , coated with poly-L-lysine, and cultured in a 5% COThe tsA-201 cells  were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 unit/ml penicillin, 1 μg/ml streptomycin and 1% GlutaMAX .Mouse neuroblastoma N2A cells  were obtained from Professor Roger Morris, Kings College London . They we3 cells in 100 μL of plating solution , HEPES (10 mM), horse serum (5%), glutamine (0.5 mM), and 1 unit/ml penicillin, 1 μg/ml streptomycin) were seeded onto sterile poly-lysine-coated glass coverslips and incubated at 37°C. After 2 h, the plating solution was replaced with 1 mL of growth medium , 2-mercaptoethanol (25 μM), glutamine (0.5 mM), and 1 unit/ml penicillin, 1 μg/ml streptomycin), half of which was replaced every 3-4 days.All experiments were performed using a Schedule 1 method in accordance with the Home Office Animals (Scientific Procedures) Act 1986, UK. Hippocampal neurons were obtained from male P0 Sprague Dawley rat pups. After cervical dislocation and decapitation, brains were excised and rapidly placed in ice-cold Hanks buffered salt solution  and hemisected. After dissecting out the mid brain from each hemisphere, hippocampi were collected and cleaned from the meninges. Hippocampi were chopped into small pieces and incubated in a shaker (200 rpm) for 40 min at 37°C in a HBSS-based dissociation solution containing: papain , bovine serum albumin , L-Cysteine (0.2 mg/ml), glucose (5 mg/ml) and DNase I . The digested tissues were then gently triturated with glass Pasteur pipettes and the cells were counted. Approximately 75 × 101-subunit cDNAs were used: CaV2.2 , always containing an extracellular HA-tag (v2.1 (GenBank: M64373 with E1686R)   , with an M86621) , mCherry M86621)  and VAMP M86621) . The cDN M86621)  was inclV2.2 II-III loop Ab  , anti- α2δ-1 Ab , anti-HA , anti-HA , anti-PDI , anti-Akt Ab  and anti-GAPDH Ab . For immunocytochemistry, secondary Abs (1:500) used were anti-rabbit-Alexa Fluor 594, anti-rat- Alexa Fluor 488, anti-rat-Alexa Fluor 594 and 647 and anti-mouse-Alexa Fluor 488 (ThermoFisher). For immunoblotting, secondary Abs (1:2000) were anti-rabbit-Horseradish Peroxidase (HRP), and anti-mouse HRP (Biorad).Antibodies (Abs) used were: anti-Cayclonal) , anti-CaV2.1-HA or CaV2.2-HA together with α2δ-1 and β1b  in a ratio 3:2:2 (plus 0.5 for mCherry as a transfection marker when included). The transfection reagent used was PolyJet (Tebu-bio Ltd), used in a ratio of 3:1 to DNA mix, according to the manufacturer’s protocol. Culture medium was changed 6 h after transfection and cells were either incubated at 37°C for a further 42 h or transferred to 30°C for 66 h. For electrophysiological studies, tsA-201 cells were transfected as above, except using Fugene6 , according to the manufacturer’s protocol. β1b-GFP or CD8 were used as transfection markers. Similar data were obtained using the two different methods to identify transfected cells for recording . After 2 h, the transfection mixes were replaced with growth medium consisting of 50% conditioned and 50% fresh medium. The DNA mix (in pCAGGS) consisted of CaV2.2 , α2δ-1, β1b and mCherry or VAMP-mCherry, at a ratio of 3:2:2:1. α2δ-1 was replaced by empty vector when appropriate. The cultures were used for immunostaining experiments at 14 days in vitro, as described below.At 7 days V2.2-HA or the EI, II, III, IVA and EIVK pore mutants together with α2δ-1 and β1b. At 48 h after transfection, cells were rinsed with phosphate-buffered saline (PBS) and then incubated for 30 min at room temperature (RT) with 0.5 mg/ml Premium Grade EZ-link Sulfo-NHS-LC-Biotin (Thermo Scientific) in PBS. The reaction was quenched by removing the biotin solution and replacing with PBS containing 200 mM glycine for 2 min at RT. The cells were rinsed with PBS before being resuspended in PBS containing 1% Igepal; 0.1% SDS and protease inhibitors , pH 7.4, for 30 min on ice to allow cell lysis, cleared by centrifugation at 13,000 × g and assayed for total protein . Cleared WCLs corresponding to 20 - 40 μg total protein were mixed with 5 x Laemmli sample buffer to a final dilution of 1 x  together with α2δ-1 and β1b. After transfection, cells were incubated at either 30°C or 37°C, as described, for 48-72 h before being fixed with 4% paraformaldehyde (PFA) in PBS, pH7.4 at RT for 5 min. When permeabilization was required, cells were then incubated in PBS containing 0.2% Triton X-100 for 5 min at RT. Blocking was performed for 30 min at RT in PBS containing 20% goat serum and 5% BSA. For staining of extracellular epitopes, blocking, and primary Ab incubation steps were carried out prior to permeabilization. The indicated primary Abs were applied (diluted in PBS with 10% goat serum and 2.5% BSA) overnight at 4°C or for 1 h at RT. The indicated secondary Abs were applied  at RT for 1 h. Cell nuclei were stained with 0.5 μM 4’,6’-diamidino-2-phenylindole (DAPI) in PBS for 5 min. For surface/intracellular HA staining of CaV2.2-HA as shown in Immunocytochemistry was carried out on N2A cells expressing either Cain vitro, in PBS containing 4% PFA / 4% sucrose for 5 min at RT, and then blocked in PBS containing 20% goat serum and 5% BSA for 30 min at RT. Primary Ab  was applied overnight at 4°C. Coverslips were washed and cells were then permeabilized by incubating with 0.2% Triton X-100 in PBS for 5 min at RT. When required, the second primary Ab  was incubated overnight at 4°C. The appropriate secondary Abs were applied for 1 h at RT. The coverslips were mounted onto glass slides using VECTASHIELD® mounting medium .Cultures of transfected hippocampal neurons were fixed, after 14 days Imaging was performed on Zeiss LSM 780 confocal microscope, at fixed microscope settings for all experimental conditions of each experiment. Images of N2A and tsA-201 cells were obtained using a 63 x oil objective at a resolution of 1,024 × 1,024 pixels and an optical section of 0.5 μm. After choosing a region of interest containing transfected cells, the 3 × 3 tile function of the microscope allowed imaging of a larger area selected without bias. Every cell identified as transfected was included in the measurements, to ensure lack of bias.imagej.net). Surface labeling in non-permeabilized cell bodies was measured using the freehand line tool (5 pixels) to measure the mean intensity of cell-surface staining. Intracellular staining was measured using the freehand selection tool, excluding the nucleus and the plasma membrane. The value of the mean pixel intensity in different channels was measured separately and background was subtracted by measuring the intensity of an imaged area without transfected cells. All data were then normalized to the appropriate positive control for each experiment before combining experiments.Images of tsA-201 and N2A cells were analyzed using imageJ -based internal solution containing in mM: NMDG, 140; EGTA, 5; MgCl2, 2; CaCl2, 0.1; K2ATP, 2; HEPES, 20; pH 7.2, 310 mOsm. The external solution for recording Ba2+ currents contained in mM: tetraethylammonium (TEA) Br, 160; KCl, 3; NaHCO3, 1.0; MgCl2, 1.0; HEPES, 10; glucose, 4; BaCl2, 1,or 2 as indicated, pH 7.4, 320 mosM with sucrose. 1 mM extracellular Ba2+ was the charge carrier. When used, 1 μM ω-CTX was applied by local perfusion. An Axopatch 1D or Axon 200B amplifier was used, and whole cell voltage-clamp recordings were sampled at 10 kHz frequency, filtered at 2 kHz and digitized at 1 kHz. 70%–80% series resistance compensation was applied and all recorded currents were leak subtracted using P/8 protocol. Membrane potential was held at – 80 mV, unless stated. Analysis was performed using Pclamp 9 (Molecular Devices) and Origin 7 . Current-voltage (I-V) relationships were fit by a modified Boltzmann equation as follows: I = Gmax∗(V-Vrev)//k)), where I is the current density (in pA/pF), Gmax is the maximum conductance (in nS/pF), Vrev is the apparent reversal potential, V50, act is the midpoint voltage for current activation, and k is the slope factor. The amplitude of tail currents was measured from the current present upon repolarization to −50 mV from a 20 ms test pulse between −50mV and +70 mV from −100mV holding potential. The activation curve obtained from tail currents was fitted with Boltzmann function.Calcium channel currents in transfected tsA-201 cells were investigated by whole cell patch-clamp recording, essentially as described previously . The patData were analyzed with GraphPad Prism 7  or Origin-Pro 2015 . All data are shown as mean ± SEM; “n” refers to number of cells, unless indicated otherwise, and is given in the figure legends, together with details of statistical tests used. Experiments where representative data are shown were repeated at least 3 times, unless otherwise stated. Graphpad Prism 7 was used for statistical analysis. Statistical significance between two groups was assessed by Student’s t test, as stated. One-way ANOVA and the stated post hoc analysis was used for comparison of means between three or more groups.This study did not generate/analyze datasets or code.


AbstractBrevishieldophyes Chakrabarti & Pandit, gen. n. and Mesoshieldophyes Chakrabarti & Pandit, gen. n., and a new species Mesoshieldophyesvarecae Chakrabarti & Pandit, sp. n. are described. These mites are leaf vagrants. The morphological characters of the afore-mentioned genera and those of Ashieldophyes Mohanasundaram are compared. A key for separating the genera within the subfamily Ashieldophyinae is provided. The diagnostic characters of the subfamily Ashieldophyinae are also revised.Two new genera,  Ashieldophyespennadamensis Mohanasundaram, 1984, infesting Caseariatomentosa Roxb.  from near the Pennadam Sugar Factory, Arcot district, Tamil Nadu, south India, was the type species for the genus Ashieldophyes Mohanasundaram within the new family Ashieldophyidae Mohanasundaram. Later, the family Ashieldophyidae was made one of the subfamilies (Ashieldophyinae) of the EriophyidaeAshieldophyesglochidionae Chakrabarti & Pandit, 2009, infesting Glochidionmultiloculare (Rottler ex Willd.) Voigt (Phyllanthaceae) from Lataguri Forest, Jalpaiguri, West Bengal, was described in this taxon.Caseariavareca Roxb. and C.glomerata Roxb. were collected. Examination of those specimens allowed establishing two new genera, Mesoshieldophyes Chakrabarti & Pandit, gen. n. for accommodating Mesoshieldophyesvarecae Chakrabarti & Pandit, sp. n. and Brevishieldophyes Chakrabarti & Pandit, gen. n. for reassigning A.glochidionae in the Ashieldophyinae.During periodical samplings for exploration of eriophyoid mite diversity in West Bengal & Assam, further samples of eriophyoids infesting Eriophyoid mites were collected and studied as described by Taxon classificationAnimaliaTrombidiformesEriophyidaeMohanasundaram, 1984http://zoobank.org/9A73DBEB-3C29-44D3-89DF-B349D0C8F895sc, placed on lateral margins, directed laterally; pedipalp genual setae d present and simple; femoral setae bv of leg I present; genual setae l″ of leg II present; coxae with setae 1b; female genitalia located between coxae II; genital cover flap lacks ridges.Prodorsal shield small and oval shaped; scapular tubercles absent but with very short scapular setae Ashieldophyespennadamensis Mohanasundaram, 1984.Ashieldophyes Mohanasundaram, 1984, Brevishieldophyes Chakrabarti & Pandit, gen. n., and Mesoshieldophyes Chakrabarti & Pandit, gen. n. belong to the subfamily Ashieldophyinae of family Eriophyidae in having small or moderately developed shield, lacking opisthosomal setae d and e, coxae widely separated anteriorly, female genitalia appressed to the coxae and with a triangular cover flap. These three genera can easily be separated by the characters given in Table Taxon classificationAnimaliaTrombidiformesEriophyidaeMohanasundaram, 1984http://zoobank.org/D84AC07F-CD6A-42E1-9E42-EA6782545674Ashieldophyespennadamensis Mohanasundaram, 1984, Oriental Insects, 18: 251–252.d present; prodorsal shield small and oval; scapular tubercles absent but with very short setae sc; legs with all usual setae; solenidion ω blunt; opisthosoma with equal number of smooth dorsal and ventral semiannuli; setae 1b present.Body vermiform; pedipalp genual seta Gnathosoma 15 (14–15) projecting obliquely down-curved, dorsal pedipalp genual setae d 1 (1–2); chelicerae 13 (13–15). Prodorsal shield small, oval-shaped, without lobe, 9 (9–10) and 23 (22–23) wide, lacking scapular tubercles but with very short scapular setae sc, placed on lateral margin and directed laterally. Leg I from base of trochanter 20 (20–21), femur 7 (7–8), femoral setae bv 7 (7–8), genu 3 (2–3), genual setae l″ 20 (21–23), tibia 5 (3–5), tibial setae l′ 15 (12–15), tarsus 5 (3–5), tarsal setae ft′ and ft″ both 12 (10–12), solenidion ω 4 (3–4), straight and blunt; empodium em simple, 4-rayed; setae u′ 2 (2–3). Leg II from base of trochanter 18 (18–20); femur 6 (5–6), femoral setae bv 5 (5–6), genu 2 (2–3), genual setae lʺ 23 (20–23), tibia 3 (3–4), tibial setae lʹ absent, tarsus 4 (3–4), solenidion ω 8 (7–8), straight and blunt; empodium em simple, 4-rayed; tarsal setae ftʹ 8 (8–10) and ftʺ 12 (10–12), setae uʹ 2 (2–3). Coxigenital area smooth; broadly joined, sternal line absent, coxa I widely separate, setae 1b 2 (2–3) and 5 (5–6) apart, setae 1a 8 (8–9) and 7 (7–8) apart, setae 2a 13 (13–15) and 18 (18–20) apart. Opisthosoma dorsally flat, smooth, with equal number of dorsal and ventral semiannuli, 21 (20–21); setae c2 10 (7–11) on ventral semiannulus 2 (2–3), setae d and e absent, setae f 14 (12–15) on ventral semiannulus 7 (6–7) from rear margin; setae h1 absent, setae h2 12 (12–14). Genital cover flap 10 (9–11) and 16 (17–18) wide, triangular and smooth; setae 3a 8 (6–8). Internal genitalia apodeme short, spermathecae rounded with short funnel-like spermathecal tubes.Female (n = 20). Body vermiform, brown colour in life, dorso-ventrally flattened; 250 (175–250) and 40 (39–46) wide. C.tomentosa, coll. M. Mohanasundaram, coll. no. 427 (TNAU); West Bengal: North 24-Parganas, Madral, 12.VIII.2005, many females and nymphs from C.tomentosa, coll. R Pandit, coll. nos. 1182–1184/19/2005.India: Tamil Nadu: South Arcot District, near Pennadam Sugar Factory, 16.VIII.1981, 2 females from India: Tamil Nadu & West Bengal.The mites inhabit the under surface of leaves as vagrants without showing symptoms of damage to the host plant.This species is so far known only from its type locality and here reported for the first time from West Bengal.Taxon classificationAnimaliaTrombidiformesEriophyidaeChakrabarti & Panditgen. n.http://zoobank.org/16667767-867C-4329-A2F9-CF791F511F0Bsc lacking; pedipalp genual setae d present and simple; coxae without setae 1b; femoral setae bv of leg I present; genual setae l″ of leg II present; empodium simple; female genitalia appressed to the coxae, genital cover flap smooth, triangular, located between coxae II; apodeme normal in length.Body fusiform, dorso-ventrally flattened. Gnathosoma short, downwardly curved, cheliceral stylet short; prodorsal shield small and sickle-shaped; scapular tubercles and scapular setae Ashieldophyesglochidionae Chakrabarti & Pandit, 2009.The genus is monotypic.Brevi derived from the adjective Latin word brevis meaning short  and ophyes derived from eriophyes meaning erion = wool + phyes = a grower/maker.Masculine.Taxon classificationAnimaliaTrombidiformesEriophyidaecomb. n.http://zoobank.org/33D1FFB7-997E-495E-AD21-EF1EDB27CB2BAshieldophyesglochidionae Chakrabarti & Pandit, 2009, International Journal of Acarology, 15:163–164.d present; prodorsal shield small and sickle shaped; scapular setae sc absent; legs with all usual setae; solenidion ω knobbed; opisthosoma with equal number of smooth dorsal and ventral semiannuli; setae 1b absent.Body fusiform; pedipalp genual seta G.multiloculare, coll. R Pandit. coll. nos. 1275–1279/45/2004. Type material of A.glochidionae Meghalaya: Burnihat, 18.X.1985, many females and nymphs, from C.glomerata, coll. B Das, coll. nos. 967–971/61/1985.India: West Bengal: Jalpaiguri, Lataguri forest, 15.X.2004, many females and nymphs from India: West Bengal & Meghalaya.The mites inhabit the under surface of leaves as vagrants without showing symptoms of damage to the host plant.B.glochidionae from G.multiloculare needs further confirmation because this mite species and other two Ashieldophyinae have been collected subsequently from plants of the genus Casearia .The original report of Taxon classificationAnimaliaTrombidiformesEriophyidaeChakrabarti & Panditgen. n.http://zoobank.org/7227C05D-E910-44EE-AC65-49CACF572032d absent; prodorsal shield semi-circular without any lobe, lacking scapular tubercles and scapular setae sc; femoral setae bv of leg I and genual setae l″ of leg II absent; coxae with setae 1b; dorsal and ventral semiannuli with granules; female genitalia appressed to the coxae; genital cover flap triangular and smooth; empodium simple; apodeme short in length.Body fusiform, dorso-ventrally flattened. Gnathosoma short, obliquely down-curved, cheliceral stylet short; pedipalp genual setae Mesoshieldophyesvarecae Chakrabarti & Pandit, sp. n.This genus is monotypic.Mesoshieldophyes is derived from meso = middle, referring to the medium size of prodorsal shield and phyes derived from eriophyes meaning erion = wool + phyes, a grower/maker.The genus name Masculine.The size of the prodorsal shield in this genus is larger than that in the other two genera of this subfamily.Taxon classificationAnimaliaTrombidiformesEriophyidaeChakrabarti & Panditsp. n.http://zoobank.org/E19A73E2-8A4C-4ABD-8C2F-4FC2DDFBC135d absent; prodorsal shield semi-circular; scapular setae sc absent; femoral setae bv on leg I absent; genual setae lʺ on leg II absent; solenidion ω knobbed; opisthosoma with equal number of granulated dorsal and ventral semiannuli; setae 1b present.Body fusiform; pedipalp genual seta Gnathosoma 15 (14–15) projecting obliquely down-wards, dorsal pedipalp genual setae d absent, setae ep 1 (1–2); chelicerae 13 (13–15). Prodorsal shield semicircular, without lobe, 14 (18–20) and 43 (40–43) wide with granules, lacking scapular tubercles and setae sc.Leg I from base of trochanter 20 (20–21), femur 7 (7–8), femoral setae bv absent, genu 3 (2–3), genual setae l″ 20 (21–23), tibia 4 (3–4), tibial setae l′ 10 (10–12), tarsus 5 (3–5), tarsal setae ft′ and ft″ both 12 (10–12), solenidion ω 4 (3–4), straight and knobbed; empodium em 4 (4–5), simple, 4-rayed; setae u′ 2 (2–3). Leg II from base of trochanter 18 (18–20); femur 6 (5–6), femoral setae bv 5 (5–6), genu 2 (2–3), genual setae l″ absent, tibia 3 (3–4), tibial setae l′ absent, tarsus 4 (3–4), tarsal setae ft′ 8 (8–10), ft″ 12 (10–12); solenidion ω 8 (7–8), straight and knobbed; empodium em 4 (4–5), simple, 4-rayed; setae u′ 2 (2–3). Coxigenital area smooth; sternal line absent, coxae widely separated, setae 1b 2 (2–3) and 5 (5–6) apart, setae 1a 8 (8–9) and 7 (7–8) apart, setae 2a 13 (13–15) and 18 (18–20) apart. Opisthosoma dorsally flat, with equal number of dorsal and ventral semiannuli, 21 (20–21), both dorsal and ventral semiannuli ornamented with fine granules; setae c2 10 (7–11) on ventral semiannulus 2 (2–3), setae d and e absent, setae f 14 (12–15) on ventral semiannulus 7 (6–7) from rear margin; setae h1 absent, setae h2 12 (12–14). Genital cover flap 6 (5–6) and 16 (17–18) wide, triangular and smooth; setae 3a 7 (6–7). Internal genitalia apodeme short, spermathecae globose with short, funnel-like spermathecal tubes.Female (n=12). Body fusiform, yellow colour in life, dorso-ventrally flattened; 140 (120–145) and 50 (45–50) wide. Male. Not observed.Caseariavareca Roxb. .The mites inhabit the under surface of leaves as vagrants without showing symptoms of damage to the host plant.26°42′30.1″N, 88°25′36.7″E), 163 m above sea level, 03.II.2015, coll. S Chakrabarti, R Pandit, S Sarkar.India: West Bengal: Darjeeling, Bengdubi Forest ; paratypes: 2 females on slide bearing holotype and 36 females, larvae and nymphs on 10 slides (nos. 1295–1304/N11/2015).varecae is from the specific designation of the host plant in the genitive case.The species name Diagnosis. Prodorsal shield poorly developed to moderately developed, lacking scapular tubercles, scapular setae sc absent but if present very short; sternal line absent; coxae widely separated anteriorly; legs with all segments, setae bv on leg I and genual setae lʺ on leg II may or may not be present; opisthosoma lacking setae d and e but c2 and f present; genitalia appressed to the coxae, genital cover flap triangular; genital apodeme curved and abbreviated and spermathecae globose with short spermathecal tubes.


Both the thiophene and benzene rings of adjacent molecules interact attractively in a face-to-face manner, forming zigzag sheets lying parallel to the (011) plane.In the title compound, the 2,5-dichlorothiophene and 2,4-dichlorophenyl rings, linked  13H6Cl4OS, consists of a 2,5-dichlorothiophene ring and a 2,4-dichlorophenyl ring linked via a prop-2-en-1-one spacer. The dihedral angle between the 2,5-dichlorothiophene ring and the 2,4-dichlorophenyl ring is 12.24 (15)°. The molecule has an E configuration about the C=C bond and the carbonyl group is syn with respect to the C=C bond. The molecular conformation is stabilized by intramolecular C—H⋯Cl contacts, producing S(6) and S(5) ring motifs. In the crystal, the molecules are linked along the a-axis direction through face-to-face π-stacking between the thiophene rings and the benzene rings of the molecules in zigzag sheets lying parallel to the bc plane along the c axis. The intermolecular interactions in the crystal packing were further analysed using Hirshfield surface analysis, which indicates that the most significant contacts are Cl⋯H/ H⋯Cl (20.8%), followed by Cl⋯Cl (18.7%), C⋯C (11.9%), Cl⋯S/S⋯Cl (10.9%), H⋯H (10.1%), C⋯H/H⋯C (9.3%) and O⋯H/H⋯O (7.6%).The molecular structure of the title compound, C S)-flavanones in a stereospecific reaction catalysed by the enzyme chalcone isomerase. The chemistry of chalcones remains a fascination among researchers because of the large number of replaceable hydrogen atoms that allows a number of derivatives with a variety of promising biological activities. They are found in fruits and vegetables, which attracted attention because of their pharmacological activities such as anti-inflamatory -3--1-prop-2-en-1-one -1-(3-bromophenyl)-3-prop-2-en-1-one -3-(2-bromophenyl)-1-prop-2-en-1-one  and S(5) ring motifs.The title compound, Fig. 1Cg1) of adjacent molecules in the alternating sheets along the [100] direction  and between the benzene rings  of the same molecules . As shown Fig. 2c-axis direction through face-to-face π-stacking between the thiophene and benzene rings of pairs of adjacent molecules along the [100] direction  of the title compound, where the sum of Cl⋯H/H⋯Cl interactions comprises 20.8% of the total Hirshfeld surface area of the molecule. The Cl⋯H/H⋯Cl interactions represented by the spikes in the bottom right and left region (de + di ≃ 2.83 Å) indicate that the hydrogen atoms are in contact with the Cl atoms to build the two-dimensional supramolecular framework . Cl⋯Cl contacts  are disfavoured when the number of H atoms on the molecular surface is large because of competition with the more attractive H⋯Cl contacts. Cl⋯Cl contacts from a parallel alignment of C—Cl bonds  −x, y, 1 − z] may be indicated. They are known in the literature as type-I halogen–halogen interactions ; 11.9%) reflect π–π interactions between the above-mentioned aromatic rings. The S⋯Cl contacts  contracted to a much lesser degree. The C⋯H/H⋯C interactions  account for 9.3% of the total Hirshfeld surface of the molecules. The scattered points in the breakdown of the fingerprint plot show the π–π stacking interactions. In the fingerprint plot delineated into H⋯O/O⋯H contacts , the 7.6% contribution to the Hirshfeld surface arises from intermolecular C=O⋯H hydrogen bonding and is viewed as pair of spikes with the tip at de + di ∼ 2.9 Å.The C—H⋯Cl interactions appear as two distinct spikes in the fingerprint plot Fig. 3b of thets Fig. 3c; 18.7%ts Fig. 3e; 10.9%ns Fig. 3g accounts Fig. 3h, the 7et al., 2015The large number of Cl⋯H/ H⋯Cl, Cl⋯Cl, C⋯C, Cl⋯S/S⋯Cl, H⋯H, C⋯H/H⋯C and O⋯H/H⋯O interactions suggest that van der Waals interactions and hydrogen bonding play the major roles in the crystal packing (Hathwar E)-1-(5-chlorothiophen-2-yl)-3-(4-ethylphenyl)prop-2-en-1-one -3-(thiophen-2-yl)prop-2-en-1-one [(X); Yesilyurt et al., 2018E)-1-(2-aminophenyl)-3-(thiophen-2-yl)prop-2-en-1-one [(XI); Chantrapromma et al., 2013The closest related compounds with the same skeleton and containing a similar bis-chalcone moiety to the title compound but with different substituents on the aromatic rings are:  and (IV) are isostructural in space group P21/c. There are no hydrogen bonds of any kind in the structures of compounds (I)C(7) chains by means of C—H⋯O hydrogen bonds. In (V), there are again no hydrogen bonds nor π–π stacking interactions but in (VI), the molecules are linked into C(5) chains by C—H⋯O hydrogen bonds. In each of compounds (I)–(VI), the molecular skeletons are close to planarity, and there are short halogen–halogen contacts in the structures of compounds (II) and (V) and a short Br⋯O contact in the structure of compound (VI).In (I)via weak C—H⋯O and C—H⋯S hydrogen bonds, forming chains propagating along the c-axis direction. In (IX), the thienyl ring is not coplanar with the benzene ring, their planes forming a dihedral angle of 13.2 (4)°. In the crystal, molecules stack along the a-axis direction, with the interplanar separation between the thienyl rings and between the benzene rings being 3.925 (6) Å. In (X), the thiophene ring forms a dihedral angle of 26.04 (9)° with the benzene ring. The molecular conformation is stabilized by an O—H⋯N hydrogen bond. The molecules are connected through C—H⋯O hydrogen bonds, forming wave-like layers parallel to the ab plane, which are further linked into a three-dimensional network by C—H⋯π interactions. In (XI), the molecule is almost planar with a dihedral angle of 3.73 (8)° between the phenyl and thiophene rings. An intramolecular N—H⋯O hydrogen bond generates an S(6) ring motif. Adjacent molecules are linked into dimers in an anti-parallel face-to-face manner by pairs of C—H⋯O interactions. Neighboring dimers are further linked into chains along the c-axis direction by N—H⋯N hydrogen bonds.In (VII), the molecule is non-planar, with a dihedral angle of 22.6 (2)° between the aromatic rings. The molecules are linked by pairs of C—H⋯π interactions, forming inversion dimers. There are no other significant intermolecular interactions present. In (VIII), the molecule is nearly planar, the dihedral angle between the thiophene and phenyl rings being 9.07 (8)°. The molecules are linked et al., 2013abet al., 2014et al., 2010The title compound was synthesized as per the procedure reported earlier (Kumar Uiso(H) = 1.2Ueq(C) for C—H. Owing to poor agreement between observed and calculated intensities, twelve outliers  were omitted in the final cycles of refinement.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018010976/dx2006sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989018010976/dx2006Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018010976/dx2006Isup3.cmlSupporting information file. DOI: 1036797CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Supramolecular chains arise in the crystal as a result of charge-assisted O—H⋯O and N—H⋯O hydrogen bonding.As the piperidin-1-ium group is nearly orthogonal to the quinolinyl residue in each of the two independent cations of the title salt solvate, these cations are  17H17F6N2O+· C4H4O42−·CH3CH2OH, quinolin-4-yl](hydroxy)methyl}piperidin-1-ium butanedioate ethanol monosolvate) comprises two independent cations, with almost superimposable conformations and each approximating the shape of the letter L, a butanedioate dianion with an all-trans conformation and an ethanol solvent molecule. In the crystal, supramolecular chains along the a-axis direction are sustained by charge-assisted hydroxy-O—H⋯O(carboxylate) and ammonium-N—H⋯O(carboxylate) hydrogen bonds. These are connected into a layer via C—F⋯π(pyridyl) contacts and π–π stacking interactions between quinolinyl-C6 and –NC5 rings of the independent cations of the asymmetric unit [inter-centroid separations = 3.6784 (17) and 3.6866 (17) Å]. Layers stack along the c-axis direction with no directional interactions between them. The analysis of the calculated Hirshfeld surface reveals the significance of the fluorine atoms in surface contacts. Thus, by far the greatest contribution to the surface contacts, i.e. 41.2%, are of the type F⋯H/H⋯F and many of these occur in the inter-layer region. However, these contacts occur at separations beyond the sum of the van der Waals radii for these atoms. It is noted that H⋯H contacts contribute 29.8% to the overall surface, with smaller contributions from O⋯H/H⋯O (14.0%) and F⋯F (5.7%) contacts.The asymmetric unit of the centrosymmetric title salt solvate, 2C Plasmodium falciparum -quinolin-4-yl](hydroxymethyl)piperidin-1-ium chloride and its  enantiomer. While the former is effective against malaria, the latter has an affinity for the adenosine acceptors in the brain, inducing serious psychiatric and neurologic side-effects  and 1.285 (3) Å, and C38—O5, O6 = 1.255 (4) and 1.271 (4) Å. While normally these bond lengths might be expected to be closer to equivalent, as noted below, each of the O4 and O6 atoms participate in two strong charge-assisted hydrogen bonds, see Supramolecular features, which explains the slightly longer C O bond lengths formed by these atoms. Further support for proton transfer leading to the formation of piperidin-1-ium cations is supported by the pattern of hydrogen bonding involving the ammonium-N—H hydrogen atoms, as discussed below in Supramolecular features.The asymmetric unit of the salt solvate, (I)R at C12 and S at C13 for the N1-cation, and R at C29 and S at C30 for the N3-cation, i.e. each conforms to the [(+)-erythro-mefloquinium] isomer; space-group symmetry indicates that the unit cell contains equal numbers of both enantiomers. The r.m.s. deviation for the ten atoms comprising the N1-quinolinyl residue is 0.0254 Å [0.0256 Å for the N3-quinolinyl residue], with the hydroxy-O1 and ammonium-N2 atoms lying to either side of the plane, i.e. −0.323 (4) and 1.302 (6) Å, respectively [0.255 (4) Å for O2 and −1.348 (6) Å for N4]. The dihedral angle of 72.55 (9)° [71.48 (9)°] formed between the fused ring system and the least-squares plane through the piperinium ring indicates that, to a first approximation, the molecule has the shape of the letter L. Referring to Table 1+—H⋯O(hydroxy) hydrogen-bond is formed as the hydroxyl-O1 and ammonium-N2 atoms lie to the same side of the cation with the O1—C12—C13—N2 torsion angle of −63.4 (3)° indicating a + syn-clinal relationship [O2—C29—C30—N4 = −68.4 (3)°].The cations exhibit very similar molecular geometries, as highlighted in the overlay diagram of Fig. 2trans conformation (+ anti-periplanar). The dihedral angle formed between the terminal carboxylate residues is 51.0 (2)°, indicating that the dianion is considerably twisted.In the butanedioate dianion, the C35—C36—C38—C39 torsion angle of 175.4 (3)° indicates an all-a-axis direction, Table 1a. Each of the cation-bound hydroxy groups forms a charge-assisted hydroxy-O—H⋯O(carboxylate) hydrogen bond to a carboxylate-O atom, at opposite ends of the butanedioate dianion. In addition, each of the four ammonium-N—H hydrogen atoms connects to a carboxylate-O atom, each derived from a different carboxylate residue, via a charge-assisted ammonium-N—H⋯O(carboxylate) hydrogen bond. Thus, each of the O4 and O6 atoms accept two charge-assisted hydrogen bonds. The carboxylate-O5 atom accepts a hydrogen bond from the solvent ethanol molecule, while ethanol-O7 participates in a methine-C—H⋯O interaction, Table 1e.g. the C35—O3 < C38—O5 < C38—O6 C35—O4 .The most prominent feature of the molecular packing is the formation of twisted supramolecular chains propagating parallel to the c-axis direction are of the type C—F⋯π(pyridyl), Table 16 ring of the N1-quinolinyl residue (C4–C9) and each of the N3-quinolinyl-bound pyridyl   and C6 (C21–C26)  rings. A view of the unit-cell contents is shown in Fig. 3b.The connections between the chains leading to supramolecular layers that stack along the et al., 2019Crystal Explorer 17 dnorm for the constituents of (I)dnorm for the dianion, Fig. 4a and b, and near the respective donors on the Hirshfeld surfaces of the ethanol molecule, Fig. 4c, and mefloquinium cations in Fig. 4d and e. The effects of the short inter-atomic contacts on the packing of (I)It is clearly evident from the numerous characteristic red spots on the Hirshfeld surfaces mapped over a, and those delineated into specific H⋯H, O⋯H/H⋯O, F⋯H/H⋯F, C⋯F/⋯C and C⋯O/O⋯C contacts de + di 2.2 Å in the fingerprint plot delineated into H⋯H contacts in Fig. 9b, is an indication of the short interatomic H⋯H contact between symmetry-related piperidinium-H34A and ethanol-H39A atoms, Table 2c, the pair of long spikes with their tips at de + di ∼1.8 Å are due to the O—H⋯O and N—H⋯O hydrogen bonds involving the carboxylate-O4 atom of the dianion whereas the points corresponding to N—H⋯O hydrogen bonds involving the O3 and O5 atoms of the dianion and those involved in short interatomic O⋯H contacts, Table 2de + di ∼2.5 Å in the fingerprint plot delineated into F⋯H/H⋯F contacts, Fig. 9d, represent the presence of these short contacts. The effect of intermolecular C—F⋯π/π⋯F—C and short interatomic C⋯F/F⋯C contacts on the molecular packing, Table 3de + di ∼3.1 Å in Fig. 9e. The presence of short interatomic C⋯O/O⋯C contacts involving the hydroxyl-O1 and -O2 atoms are characterized as a pair of leaf-like tips at de + di ∼3.0 Å in Fig. 9f. Finally, the presence of π–π stacking interactions between the (C4–C9) ring of the O1-cation and the (C21–C26) and N3-pyridyl rings of the O2-cation are reflected in the 3.4 and 3.3% contributions from C⋯C contacts to the Hirshfeld surfaces of the individual cations; although the contribution from these contacts to the surfaces in the overall structure of (I)The presence of a cone-shaped tip at Chemical context, the specific enantiomer of Lariam is important in terms of pharmacological activity. Hence, considerable investment has been made in attempting to resolve the enantiomers by salt formation. During such studies, a seemingly high propensity towards kryptoracemic behaviour has been revealed. Kryptoracemic behaviour is related to the rare phenomenon where enantiomeric molecules crystallize in one of the 65 Sohncke space groups, i.e. space groups which lack an inversion centre, a rotatory inversion axis, a glide plane or a mirror plane. In these circumstances, the enantiomeric molecules are related by non-crystallographic symmetry, e.g. a non-crystallographic centre of inversion. A review of this phenomenon has appeared for organic compounds  and sodium succinate (2 mmol) in ethanol (15 ml) was refluxed for 30 min. The reaction mixture was left at room temperature and after three days, colourless platy crystals of (I)1H NMR (DMSO-d6): δ: 1.15–1.27 , 1.32–1.47 , 1.48–1.57 , 1.65–1.74 , 2.33 , 2.58–2.67 , 3.00–3.11 , 5.58 , 7.95 , 8.10 , 8.37 , 8.75 , resonances due to OH and NH were not observed. Resonances due to ethanol solvate were also present: 3.45  and 1.07 . 13C NMR (DMSO-d6): δ: 22.92, 24.28, 24.71, 31.76, 45.55, 60.57, 70.33, 115.58, 119.89 , 122.34, 122.64 , 127.77 , 127.76, 129.40, 129.9 , 142.74, 146.56 , 153.13, 175.32). 19F NMR (DMSO-d6); δ: −58.83, −66.63. IR (cm−1) 3500–2100 (br), 1589 (br), 1514, 1454, 1430, 1371, 1312, 1267, 1217, 1182, 1111, 1053, 1018, 986, 941,910, 837, 777, 546, 445.Uiso(H) set to 1.2–1.5Ueq(C). The O- and N-bound H atoms were refined with distance restraints 0.84±0.01 and 0.88±0.01 Å, respectively, and refined with Uiso(H) = 1.5Ueq(O) and 1.2Ueq(N), respectively. Owing to poor agreement, the  I, global. DOI: 10.1107/S2056989019009654/hb7838Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019009654/hb7838Isup3.cmlSupporting information file. DOI: 1938793CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: