A growing body of evidence suggests that metabolic syndrome is associated with endocrine disorders including thyroid dysfunction. Thyroid dysfunction in metabolic syndrome patients may further add to cardiovascular disease risk thereby increasing mortality. This study was done to assess thyroid function in metabolic syndrome patients and evaluate its relationship with the components of metabolic syndrome.A cross sectional study was carried out among 169 metabolic syndrome patients at B P Koirala Institute of Health Sciences, Dharan, Nepal. Anthropometric measurements  and blood pressure were taken. Fasting blood samples were analysed to measure glucose, triglyceride, high density lipoprotein (HDL) cholesterol and thyroid hormones .n = 54) metabolic syndrome patients. Subclinical hypothyroidism (26.6 %) was the major thyroid dysfunction followed by overt hypothyroidism (3.5 %) and subclinical hyperthyroidism (1.7 %). Thyroid dysfunction was much common in females  than males  but not statistically significant (p = 0.068). The relative risk of having thyroid dysfunction in females was 1.525 (CI: 0.983–2.368) as compared to males. Significant differences (p = 0.001) were observed in waist circumference between patients with and without thyroid dysfunction and HDL cholesterol which had significant negative correlation with thyroid stimulating hormone.Thyroid dysfunction was seen in 31.9 % . Metabolic syndrome constitutes a cluster of risk factors characterized by hypertension, atherogenic dyslipidemia, hyperglycemia, prothrombotic and proinflammatory conditions . This clThyroid dysfunction, prominently subclinical hypothyroidism has been observed more frequently in metabolic syndrome patients than general population . Both meThere is evidence that thyroid function may need to be assessed in patients with metabolic syndrome who are also at higher risk for CVD. Thyroid dysfunction is common in Nepal, and the prevalence of diabetes mellitus and metabolic syndrome has been rising steadily. Reports suggest that 20.7 % of the Nepalese population have metabolic syndrome based on National Cholesterol Education Program (NCEP) criteria , 9. HoweIn this study, we assessed thyroid function and examined the association between components of metabolic syndrome and thyroid function in Nepalese population with metabolic syndrome.A cross-sectional study was conducted among metabolic syndrome patients at the department of biochemistry of B P Koirala Institute of Health Sciences (BPKIHS), Dharan, Nepal from September 2013 to September 2014. One hundred sixty nine metabolic syndrome patients aged ≥20 years were selected from the hospital during the study period. The sample size was calculated by taking prevalence of thyroid dysfunction as 15 % (approximate) in this region. Metabolic syndrome was diagnosed based on modified Asian NCEP-ATP III panel criteria . The excMetabolic syndrome patients were considered to have thyroid dysfunction if patients thyroid hormones level fell outside the reference range (free T3 (4.0–8.3 pmol/L), free T4 (9.0–20.0 pmol/L) and TSH level (0.25–5 mIU/L)). Patients were said to be euthyroid if all thyroid hormone levels fell within reference range. Overt hypothyroidism was defined as TSH > 5 mIU/L and free T3 < 4.0 pmol/L and free T4 < 9.0 pmol/L. Subclinical hypothyroidism was considered if TSH > 5 mIU/L and free T3 and free T4 within reference range. Subclinical hyperthyroidism was defined as TSH < 0.25 mIU/L and free T3 and free T4 within reference range. The data generated from study was analyzed using SPSS version 11.0. Continuous variables were expressed as mean ± SD values. Independent sample t test and one way ANOVA was applied for continuous variables and chi square test for categorical variables at 95 % confidence interval. Pearson correlation coefficients were calculated to find relationship between the components of metabolic syndrome and thyroid profile parameters at 95 % confidence interval. Relative risk with 95 % confidence interval (CI) was used to assess the risk factors for thyroid dysfunction in metabolic syndrome.n = 96) males and 43.2 % (n = 73) females, with mean age of 47 ± 12.5 years. Height, weight, body mass index (BMI), waist circumference, systolic BP and diastolic BP were 157.4 ± 8 cm, 70.7 ± 7.9 Kg, 28.6 ± 3.3 Kg/m2, 102.5 ± 6.7 cm, 129.3 ± 13.6 mmHg and 84.9 ± 11.5 mmHg respectively. Similarly, levels of biochemical parameters; fasting blood glucose, triglyceride, HDL cholesterol, free T3, free T4 and TSH were 126.2 ± 50.4 mg/dL, 198.2 ± 90.8 mg/dL, 49.9 ± 15.3 mg/dL, 5.1 ± 1.0 pmol/L, 12.0 ± 2.9 pmol/L and 4.2 ± 3.4 mIU/L respectively. Thyroid dysfunction was found in 31.9 % (n = 54) patients. Subclinical hypothyroidism (26.6 %) was the major thyroid dysfunction followed by overt hypothyroidism (3.5 %) and subclinical hyperthyroidism (1.7 %). Components of metabolic syndrome according to thyroid dysfunction type are shown in Table n = 29) than males 26 % (n = 25) but not statistically significant (p = 0.068). Among males, 23 had subclinical hypothyroidism, 1 had overt hypothyroidism and 1 had subclinical hyperthyroidism. Similarly among females, 22 had subclinical hypothyroidism, 5 had overt hypothyroidism and 2 had subclinical hyperthyroidism. The relative risk of having thyroid dysfunction in females was 1.525  as compared to males. When metabolic syndrome parameters were compared between the patients subgroups (with TSH < 5 mIU/L and TSH ≥ 5 mIU/L), then systolic BP, diastolic BP, waist circumference, blood glucose, triglyceride and HDL cholesterol were 129.7 ± 12.6 mmHg versus 128.4 ± 16 mmHg; p = 0.602, 85.3 ± 11 mmHg versus 84 ± 12.8 mmHg; p = 0.513, 103.4 ± 6.6 cm versus 100.5 ± 6.6 cm; p = 0.008, 125.2 ± 53.2 mg/dL versus 128.6 ± 43.7 mg/dL; p = 0.692, 189.7 ± 75.5 mg/dL versus 217.9 ± 117.6 mg/dL; p = 0.119 and 51 ± 16.8 mg/dL versus 47.4 ± 11 mg/dL; p = 0.103 respectively. Correlation between the components of metabolic syndrome and TSH and free T4 is shown in Table p = 0.037) and free T4  and weak positive correlation with TSH .The study population consisted of 56.8 %  between patients with and without thyroid dysfunction, and HDL cholesterol had significant negative association with TSH level, however, other components of metabolic syndrome had no significant relationships with thyroid dysfunction. Thyroid hormones affect lipid metabolism and thus the components of metabolic syndrome, and there is positive relation between TSH and LDL cholesterol, whereas negative relation between TSH and HDL cholesterol [The correlation between subclinical hypothyroidism and metabolic syndrome and its components varies in different studies and seems to be influenced by age, gender and race of study participants . In the lesterol . Our finlesterol . There alesterol . In a stlesterol . Howeverlesterol .2), had higher triglyceride levels, and an increased likelihood of having metabolic syndrome [In the present study, we observed negative association of BMI with free T3 and free T4 and weak positive correlation with TSH. In a study in Germany, euthyroid subjects with TSH in the upper normal range (2.5–4.5 mU/L) were more obese .


Elaeodendron trichotomum (Turcz.) Lundell is reported.The crystal structure of the triterpene lactone ochraceolide A (3-oxolup-20 (29)-en-30,21α-olide) isolated from  30H44O3 indenofuran-3,11(2H)-dione], is a triterpene lactone, which was isolated from dichloromethane extract of Elaeodendron trichotomum (Turcz.) Lundell (celastraceae) stem bark. The compound has a lupane skeleton and consists of four fused six-membered rings and two five-membered rings. In the crystal, molecules are linked by weak C—H⋯O hydrogen bonds into a three-dimensional network. The configuration of ochraceolide A was proposed based on analogue compounds which belong to the lupane type.The title compound, C Kokoona ochracea (Elm.) Merril stem bark  with an ED50 of 6.0 µM; and hormone-dependent breast cancer with an ED50 of 9.9 µM  isolated from Hippocratea celastroides K.  Lundell stem bark is reported and the crystal structure described.Ochraceolides A–E are a group of cytotoxic lupane γ-lactones isolated from the Celastraceae family. Ochraceolide A was firstly isolated from P212121 with one molecule in the asymmetric unit  and two five-membered rings (E and F). The cyclohexane rings are trans-fused and in standard chair conformations. The cyclopentane (C17–C19/C21/C22) ring is trans-fused to the triterpene D ring and exhibits an envelope conformation [Q = 0.451 (4) Å and θ = 356.7 (5)°] with the puckered C17 atom having the maximum deviation of 0.285 (4) Å. The α-methylene γ-lactone is cis-fused at C19–C21 to the cyclopentane E ring and is essentially planar with a maximum deviation of 0.006 (4) Å for atom C19. The torsion angle C20—C19—C21—O2 is 0.8 (4)° and the weighted average absolute internal torsion angle for the lactone ring is 0.7 (2)°The title compound has a lupane skeleton and crystallizes in the orthorhombic space group it Fig. 1. The triA rings of adjacent molecules interact through two hydrogen bonds (C2—H2A⋯O2 and C24—H24A⋯O3) in a head-to-tail arrangement, forming chains along [001]. These chains are further connected through a weak hydrogen bond between the oxygen of the ketone group (O1) and a methylene group on the C ring (C12), forming an overall three-dimensional network.In the crystal, molecules are linked by weak C—H⋯O hydrogen bonds Table 1. The lacet al., 2016H-cyclopenta[b]furan-2,5(3H)-dione  Lundell was collected from Chunchucmil, Yucatán, México . A voucher specimen (JTun2328) was deposited at the Herbarium Alfredo Barrera Marín, Universidad Autónoma de Yucatán, México. Dried and milled stem bark (2100 g) was exhaustively extracted by dichloromethane using a Soxhlet extraction apparatus to yield 184.2 g of crude extract. A portion of the extract (100 g) was chromatographed on silica gel (40–60 µm) using a gradient elution with n-hexane–ethyl acetate (10–100% ethyl acetate), to obtain 44 fractions. Single crystals suitable for X-ray structure analysis were obtained by slow evaporation of the mixture of solvents present in fractions 7–10 at room temperature.Uiso(H) = 1.2Ueq(C) or 1.5Ueq(methyl C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017012816/lh4023sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017012816/lh4023Isup2.hklStructure factors: contains datablock(s) I. DOI: 1573017CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H-chromen-2-one), the 4-hydroxy-3-methoxyphenyl-substituted derivative of dicoumarol, was deprotonated by the addition of triethylamine, yielding the respective ammonium salt which was crystallized from a methanol solution. The deprotonated dicoumarol derivative exhibits an intramolecular negative charge-assisted hydrogen bond between the deprotonated and non-deprotonated alcohol functions of the coumarol substituents.3,3′-[(3-Methoxy-4-hydroxyphenyl)methanediyl]bis(4-hydroxy-2 H-chromen-2-one) and triethylamine in methanol yielded the title compound triethylammonium 3-[(4-hydroxy-3-methoxyphenyl)(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl]-2-oxo-2H-chromen-4-olate, C6H16N+·C26H17O8− or (NHEt3)+(C26H17O8)−, which crystallized directly from its methanolic mother liquor. The non-deprotonated coumarol substituent shares its H atom with the deprotonated coumarolate substituent in a short negative charge-assisted hydrogen bond in which the freely refined H atom is moved from its parent O atom towards the acceptor O atom, elongating the covalent O—H bond to 1.18 (3) Å. The respective H atom can therefore be described as being shared by two alcohol O atoms, culminating in the formation of an eight-membered ring.The reaction between 3,3′-[(3-methoxy-4-hydroxyphenyl)methanediyl]bis(4-hydroxy-2 The distance of the freely refined hydrogen atom to its parent atom O3 is elongated to 1.18 (3) Å, while the H⋯A hydrogen-bond length to O6 is rather short at only 1.24 (3) Å. This interaction is therefore the second shortest such –CAHB overall and the shortest intramolecular one. In the three related deprotonated dicoumarols, the D⋯A distances range from 2.423 Å . The distances between the alcohol oxygen atoms and bound carbon atoms are 1.3005 (16) Å (O3—C10) and 1.2939 (17) Å (O6—C19); i.e. both very similar and both significantly shorter than those reported for non-deprotonated derivatives, which range from 1.331 to 1.338 Å  with D⋯A = 2.7727 (19) Å and D-–H⋯A = 164.5 (18)°.The ammonium hydrogen atom, which was refined freely, exhibits a hydrogen bond to the carbonyl oxygen atom of the deprotonated coumarol substituent , C11—C1—C20 = 114.44 (12), C2—C1—C20 = 110.79 (11)°] are slightly widened compared to the ideal tetrahedral value. As this is most pronounced for the angle involving the two coumarin substituents, it is most likely based on steric strain. The bond lengths involving the two pyran oxygen atoms  are similar as observed previously, indicating conjugation between the six-membered rings in the two benzopyran systems  in a stacking fashion are observed, O⋯O1(−x + y + z + D⋯A = 2.4139 (15) Å] and as acceptor [O8⋯H27B—C27(−x + y + z + D⋯A = 3.257 (2) Å] in a non-classical hydrogen bond from an amine methyl group  and the non-classical donation towards O8 [C27—H27B⋯O8(−x + y − z + D⋯A = 3.257 (2) (19) Å]. Supported by the hydrogen bond with the carbonyl oxygen atom O1 as acceptor [O1⋯H8O—O8(−x + y − z + D⋯A = 2.6488 (16) Å], these interactions form infinite flat chains with ‘up and down’-pointing benzopyrane moieties protruding along b  bridges the adjacent cation and anion by hydrogen bonding as a classical donor bis(4-hydroxy-2H-chromen-2-one) was carried out by adding 1 mL of triethylamine to its methanolic solution. The resulting transparent yellowish solution was left standing overnight to grow transparent crystals of triethylammonium 3-[(4-hydroxy-3-methoxyphenyl)(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl]-2-oxo-2H-chromen-4-olate.3,3′-[(3-Methoxy-4-hydroxyphenyl)methanediyl]bis(4-hydroxy-2Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 2PLATON   I. DOI: 10.1107/S2056989018001561/lh5866Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018001561/lh5866Isup3.cmlSupporting information file. DOI: 1818945CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Individual ions and the solvating water molecule assemble into dimeric units located around crystallographic inversion centers  8H13N)(C17H14P)]Cl·H2O, assemble into dimeric units located around crystallographic inversion centers via N—H⋯Cl and O—H⋯Cl hydrogen bonds. These discrete fragments are further interconnected into chains by C—H⋯O interactions. The disubstituted ferrocene core in the {[1′-(diphenylphosphino)ferrocenyl]methyl}dimethylammonium cation has an approximate synclinal eclipsed conformation and is tilted by 3.40 (11)°.Individual ions and the solvating water molecule constituting the structure of the title compound, [Fe(C The phosphine substituent at the other cyclopentadienyl ring is oriented so that one of its pivotal P—C(Ph) bonds lies nearly in the plane of the bonding five-membered ring C6–C10, while the other is roughly parallel with the axis of the ferrocene unit. The angle at which the P—C18 bond intersects the C6–C10 plane is 13.17 (10)°, whereas the angle subtended by the P—C12 bond and the Cg1⋯Cg2 line is only 8.68 (5)°.The protonated aminomethyl chain is directed away from the ferrocene core, with the angle between the C1—N bond and the axis of the ferrocene unit, 2PfcCH2NHMe2]+ cation in the structure of the title compound is involved in an N—H⋯Cl hydrogen bond to a proximal chloride anion 2Cl2(H2O)2} around the crystallographic inversion centers. These discrete units are further interlinked into chains along the a axis via the weaker C—H⋯O and C—H⋯Cl interactions, as shown in Fig. 2Each X, where X = Cl and ClO4 3SO3  was dissolved in acetic acid (10 mL) and the solution was evaporated under reduced pressure. After this procedure was repeated twice using chloroform as a solvent, the residue was dissolved in a minimum amount of hot ethyl acetate. The solution was filtered and layered with hexane. Crystallization by liquid-phase diffusion over several days afforded orange crystals of the title compound. The yield was not determined.The ‘amine’ Ph25H27FeNP]Cl·H2O (481.76 g mol−1): C 62.32, H 6.07, N 2.91%. Found: C 62.23, H 5.91, N 2.79%. ESI MS: m/z 383 ([Ph2PfcCH2]+), 428 ([Ph2PfcCH2NMe2 + H]+)Analysis calculated for [CUiso(H) set to 1.2Ueq of their bonding atom. Hydrogen atoms bonded to carbons were included in their theoretical positions and refined as riding atoms with Uiso(H) = 1.2Ueq(C).Relevant crystallographic data and structure refinement parameters are summarized in Table 210.1107/S2056989017013408/im2484sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989017013408/im2484Isup3.hklStructure factors: contains datablock(s) I. DOI: 1575391CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title compound provides the first crystal structure of an α-alkenyl oxathiazolone ring. 10H7NO2S, provides the first structure of an α-alkenyl oxathiazolone ring. The phenyl ring and the oxathiazolone groups make dihedral angles of 0.3 (3) and −2.8 (3)°, respectively, with the plane of the central alkene group; the dihedral angle between the rings is 2.68 (8)°. A careful consideration of bond lengths provides insight into the electronic structure and reactivity of the title compound. In the crystal, extended π-stacking is observed parallel to the a-axis direction, consisting of cofacial head-to-tail dimeric units [centroid–centroid distance of 3.6191 (11) Å]. These dimeric units are separated by a slightly longer centroid–centroid distance of 3.8383 (12) Å, generating infinite stacks of molecules.The title compound, C In addition, the C1—S1 bond [1.7379 (18) Å] is slightly shorter than the statistical average for molecules of this type (1.75±0.02 Å). Thus the pattern of bonding within the heterocycle is consistent with the Krayuskin conjugation model, leading to the hypothesis that decarbonylation of this derivative should occur with milder conditions than observed for heterocycles substituted with saturated substituents.The title compound Fig. 1 is the fThe atoms in the ring of the oxathiazolone heterocycle form bond angles that sum to 540.0° consistent with a planar ring . The torsion angles O1—C2—C3—C4 [−2.8 (3)°] and C3—C4—C5—C6 [−179.81 (17)°] confirm a near planarity of the molecule favorable for conjugation between the π-systems of the two rings and the central alkene.et al., 2002et al., 1989et al., 1999et al., 2002The planarity, bond lengths and angles in the styryl fragment are comparable with previously reported values .There are eleven crystal structures of oxathiazolone derivatives reported in the literature  λmax (log ∊) : 228 nm (4.21), 307 nm (4.52).The title compound was prepared following literature methods  I. DOI: 10.1107/S2056989017011264/hb7694Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017011264/hb7694Isup3.cmlSupporting information file. DOI: 1565845CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Please note that column heights and error bars in the original figures and data in the ESM tables are correct and statistical tests are valid. These corrections do not affect any results or conclusions in this article.Unfortunately, the original version of Figs. 4, 5 and 6b in the article  containen numbers are as follows:The correct http://media.springernature.com/full/springer-static/image/art%3A10.1186%2Fs13550-016-0218-3/MediaObjects/13550_2016_218_Fig4_HTML.gif): ‘HCT116’ (IC) n = 8; ‘A549’ (WWU) n = 7; ‘HCT116’ (WMIC) n = 3; ‘H1975’ (AZ) n = 6.Figure 4 : ‘ONU (AZ)’ n = 6; ‘H1975 in ONU (AZ)’ n = 6; ‘A431 in ONU (AZ)’ n = 6.Figure 5 : ‘PC9’ n = 13.Figure 6b (


The title compounds are differing only by the position of the chlorine atom in the benzene ring. The molecular structures are very similar, except for the relative position of the hydroxyphenyl rings. 23H21ClN2O2, differ from each other only by the position of the Cl atom on the corresponding benzene ring: meta relative to the central sp3 C atom for (I) and para for (II). In (I), the hydroxyphenyl rings are almost parallel, the dihedral angle between the mean planes being 9.2 (2)°, but in (II), the relative position of the ring is different, characterized by a dihedral angle of 48.5 (1)°. Compound (I) features intramolecular O—H⋯N and intermolecular C—H⋯O hydrogen bonds, while in (II), intramolecular O—H⋯N, C—H⋯N hydrogen bonds and weak intermolecular C—H⋯π interactions are observed. Compound (I) was refined as an inversion twin.The title compounds, C Recent reports on a single-crystal study , C15(S). The chlorophenyl group is almost planar with atom Cl1 deviating by 0.013 (1) Å from the ring in (I)The molecular structure of the title compounds, (I)In compounds (I)C(9) chains propagating along [010]; see Fig. 4C(11) chains propagating along the ab plane of the unit cell; see Fig. 5In the crystal of (I)E)-({1-(3-chlorophenyl)-2-[(E)-(2-hydroxybenzylidene)amino]propyl}imino)methyl]phenol was achieved by the condensation of salicylaldehyde (0.02 mol) and 1-(3-chlorophenyl)propane-1,2-diamine (0.01 mol) in ethanol . The completion of the reaction was monitored by TLC. The obtained yellow solid was purified by recrystallization from ethanol. Single crystals suitable for X-ray analysis were obtained by slow evaporation from ethanol. The above procedure was repeated with 1-(4-chlorophenyl)propane-1,2-diamine (0.01 mol) instead of 1-(3-chlorophenyl)propane-1,2-diamine to synthesise 2-[(1E)-({1-(4-chlorophenyl)-2-[(E)-(2-hydroxybenzylidene)amino]propyl}imino)methyl]phenol.The synthesis of the salen ligand 2-[(1Uiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) for other H atoms. Pairs of O—H bond distances were restrained to 0.82 (1) Å. Compound (I)Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017016292/zq2239sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989017016292/zq2239Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017016292/zq2239IIsup3.hklStructure factors: contains datablock(s) II. DOI: 1412946, 1412945CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


E conformation with respect to the C=N bond, and a dihedral angle of 14.54 (11)° between the benzene ring and the mean plane of the N—N—C(N)=S hydrazinecarbothioamide unit.The molecule of the title Schiff base, has an  8H8BrN3OS·C2H6OS, which crystallizes as a dimethyl sulfoxide (DMSO) monosolvate, displays an E configuration with respect to the C=N bond, with a dihedral angle of 14.54 (11)° between the benzene ring and the mean plane of the N—N—C(N)=S unit. In the crystal, molecules are linked by N—H⋯O hydrogen bonds, forming chains propagating along the b-axis direction. Within the chains there are R23(11) ring motifs, which are reinforced by C—H⋯ODMSO hydrogen bonds enclosing secondary R12(6) and R23(9) loops. The chains are linked by O—Hhydroxyl⋯S hydrogen bonds, forming layers parallel to (011). Inversion-related layers are linked by short Br⋯Br interactions [3.5585 (5) Å], forming slabs parallel to (011). The intermolecular interactions have been investigated using Hirshfeld surface studies and two-dimensional fingerprint plots. The crystal structure of the unsolvated form of the title compound has been reported previously , and its solid-state structure is compared with that of the title solvated form.The molecule of the title Schiff base, C Schiff bases are nitrogen-containing active organic compounds that play a vital role in enzymatic reactions involving interaction of an enzyme with a carbonyl group of a substrate ° between the benzene ring and the mean plane of the N1/N2/C8/N3/S1 unit. The C8—-S1 bond distance of 1.698 (2) Å is close to that expected for a C=S bond -2-thiosemicarbazone and (E)-2-[(1H-indol-3-yl)methylene]thiosemicarbazone -1-[4-(dimethylamino)benzylidene]thiosemicarbazide  ring motif. Comparing the two molecules, as shown in the structural overlay of Fig. 2ca. 180° with respect to that in the unsolvated form of the molecule. The bond lengths and bond angles of the two molecules are similar. In the title compound, the dihedral angle between the benzene ring and the mean plane of the N—N—C(N)=S hydrazinecarbothioamide unit is 14.54 (11)° compared to ca 7.05° in the unsolvated phase. Kargar et al. ; see Fig. 4x, −y + 1, −z + 1) interactions of 3.5585 (5) Å, forming slabs parallel to (011), as illustrated in Fig. 5In the crystal, the Schiff base hydrazone is hydrogen bonded , S⋯H/H⋯S (18.8%), O⋯H/H⋯O (13.3%), Br⋯H/H⋯Br (11.6), C⋯H/H⋯C (8.8%), N⋯H/H⋯N (3.4%), C⋯C (2.8%), Br⋯N/N⋯Br (2.0%), Br⋯Br (1.5%), Br⋯O/O⋯Br (1.1%), Br⋯C/C⋯Br (1.1%), C⋯N/N⋯C (1.0%), S⋯S (0.7%), S⋯N/N⋯S (0.6%) and S⋯C/C⋯S (0.2%), as shown in the two-dimensional fingerprint plots in Fig. 7The three-dimensional et al., 2016viz. 5-bromo-2-hydroxybenzaldehyde thiosemicarbazone -2-(2-hydroxybenzylidene)hydrazinecarbothioamide hydrate] have been reported at 100 K (UJIPIN) and 203 K (UJIPOT and UJIPUZ) by Monfared et al. ° in the title compound.A search of the Cambridge Structural Database  of thiosemicarbazide  and a hot ethanolic solution of 5-bromosalicylaldehyde . The solution was then cooled and kept at room temperature. The precipitate that formed was filtered off and recrystallized from dimethyl sulfoxide. Colourless block-like crystals, suitable for the X-ray analysis, were obtained in a few days on slow evaporation of the solvent.Uiso(H) = 1.5Ueq and 1.2Ueq for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018000233/su5414sup1.cifCrystal structure: contains datablock(s) global, I, 1. DOI: 10.1107/S2056989018000233/su5414Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018000233/su5414Isup3.cmlSupporting information file. DOI: 1587285CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports6: Article number: 37272; 10.1038/srep37272 published online: 11172016; updated: 03072017.In the methods section, under the subheading ‘Human Fetal Brain Atlas Dataset’50”.“Generally, this dataset includes T2 templates and tissue probability maps  for ages between 23–37 weeks of gestation. To create the 4D probabilistic atlas, 142 T2-weighted fast-spin echo images are acquired on 3 T Philips Intera system with MR sequence parameters TR = 1712 ms, TE = 160 ms, flip angle 90° and voxel sizes 0.86 × 0.86 × 1 mm. The atlas construction is described in detail elsewhereshould read:et al.50”.“T2 weighted MR images from 80 fetuses with normal brain appearances were used to create the 4D atlas. The age range at the time of scan was 21.7 to 38.7 weeks gestational age (GA), with mean and standard deviation of 29.6 ± 4.6 weeks. The images were acquired on 1.5 T Philips Achieva system with the following parameters: T2 weighted single shot Fast Spin Echo (ssFSE) TR = 15000 ms, TE = 160 ms, flip angle = 90° and voxel size = 1.25 × 1.25 × 2.5 mm. For each subject multiple stacks of images  were acquired in approximately transverse, sagittal and coronal planes and the data reconstructed into a single 3D brain volume using the slice-to-volume reconstruction method. The reconstruction voxel size was 1.18 × 1.18 × 1.18 mm. More details can be found in studies from Serag In addition, reference 50 was listed incorrectly. The correct reference appears below:et al. Construction of a consistent high-definition spatio-temporal atlas of the developing brain using adaptive kernel regression. NeuroImage59, 2255–2265 (2012).Serag, A.


This packing motif still enables significant π–π interactions between two pyridyl groups, and may result from the close proximity of the tetrafluoridoborate ions to the platinum(II) complexes, resulting in intramolecular H⋯F distances between 2.156 and 2.573 Å.The crystal structure of a platinum(II) supramolecular building block, [Pt(dbbpy)(NCCH The solubility and apt geometry of the (dbbpy)platinum(II) complex make it a desirable building block for coordination-driven self-assembly of homo-metallic PtCl2 molecule: one with Pt—N distances of 2.013 (2) and 2.011 (2) Å and a 79.79 (6)° N—Pt—N angle Pt(OH2)2](OTf)2 Pt(NCCH3)(Ph)] [BAr’4], containing a Pt—N distance of 2.000 (4) Å, located trans to the acetonitrile, while the phenyl ligand causes an elongation to 2.092 (4) Å for the other Pt—N bond  and 1.995 (4) Å, respectively, with a bond angle of 80.5 (2)°. These are shorter than those affected by the stronger Day, 2009, and theDay, 2009. The Pt—2 2](OTf)2 (Ph)][BAr′4] phenyl; McKeown et al., 2011et al., 2003tert-butly groups for the (dbbpy)Pt(NCCH3)2 cation and its corresponding dimer  are at an angle of 10.82°], both of which accommodate the bulky tert-butyl groups of the dbbpy ligands. The intramolecular Pt—Pt distance is quite long at 4.5123 (3) Å, yet the pyridyl rings of the dbbpy are positioned for π–π interactions with distances between 3.616 (5) Å (N1⋯N1i) and 4.032 (7) Å (C4⋯C4i)  occurring between the two rings 2](OTf)2 2](ClO4)2  compounds containing the bulky dbbpy ligand pack as head-to-tail dimers, such as the aforementioned (dbbpy)PtClDay, 2009, [Pt(SO3CF3)2 4](BF4)2 Cl3)2](BF4)2[Pt(dbbpy) of Pt(dbbpy)Cl2, and 164 mg (0.8425 mmol) of AgBF4 was refluxed under stirring until a yellow solution formed. The solution was isolated, via cannula, from the AgCl precipitate and condensed under reduced pressure until ∼5 mL of orange solution remained. This was combined with 25 ml of Et2O and the resulting precipitate was washed with 3 × 20 mL Et2O to give 206.9 mg (83.8% yield) of product. UV–vis λmax (∊ Lmol−1cm−1): 211 (4.6 × 104), 249 (4.2 × 104), 306 (2.0 × 104), 319 (2.4 × 104) and 346 (6.0 × 103) nm.Yellow crystals of the title compound were grown from liquid diffusion of hexanes into a dilute acetone solution.Uiso(H) = 1.2Ueq(CH) or Uiso(H) = 1.2Ueq(CH3).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018005923/jj2198sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018005923/jj2198Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018005923/jj2198Isup3.molSupporting information file. DOI: 1837532CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A step-like conformation about the pyranyl ring is found for the molecular structure of the title compound. The three-dimensional packing is sustained by π–π, C—Cl⋯π and C—H⋯O interactions. 19H13ClO3Se {systematic name: 2-[(4-chlorophenyl)selanyl]-2H,3H,4H,5H,6H-naphthopyran-5,6-dione}, has the substituted 2-pyranyl ring in a half-chair conformation with the methylene-C atom bound to the methine-C atom being the flap atom. The dihedral angle between the two aromatic regions of the molecule is 9.96 (9)° and indicates a step-like conformation. An intramolecular Se⋯O interaction of 2.8122 (13) Å is noted. In the crystal, π–π contacts between naphthyl rings [inter-centroid distance = 3.7213 (12) Å] and between naphthyl and chlorobenzene rings [inter-centroid distance = 3.7715 (13) Å], along with C—Cl⋯π(chlorobenzene) contacts, lead to supramolecular layers parallel to the ab plane, which are connected into a three-dimensional architecture via methylene-C—H⋯O(carbonyl) interactions. The contributions of these and other weak contacts to the Hirshfeld surface is described.The title organoselenium compound, C It exhibits biological activities in the context of cancer  can be isolated from the bark of the lapacho tree found in Central and South American countries Compounds of the bio-essential element selenium, found in amino acids such as selenocysteine and selenomethionine, are known to hold potential as pharmaceutical agents i.e. Cg(C2–C4/C9–C11)⋯Cg(C3–C8)i = 3.7213 (12) Å for an angle of inclination = 0.72 (9)° and symmetry operation (i) −x, −y, −z. Two types of interactions connect centrosymmetric aggregates into a supramolecular layer parallel to the ab plane . Thus, π–π interactions between naphthyl and chlorobenzene rings are formed,  along with C—Cl⋯π(chlorobenzene) contacts between centrosymmetrically related rings (Table 1b).In the molecular packing of (I)ne Fig. 2a. Thus,s Table 1. Connects Table 1 to consos Table 1b.et al., 2016dnorm in Fig. 3dnorm-mapped Hirshfeld surface confirms the absence of conventional hydrogen bonds in the structure except for a weak C—H⋯O interaction as given in Table 1The Hirshfeld surfaces calculated on the structure of (I)a) and those delineated into H⋯H, O⋯H/H⋯O, Cl⋯H/H⋯Cl, C⋯C, C⋯H/H⋯C, C⋯Cl/Cl⋯C and Cl⋯O/O⋯Cl contacts de + di ∼2.3 Å in Fig. 6b is the result of a short interatomic H⋯H contact ; the points arising from the short interatomic O⋯H contacts are merged in the plot.The overall two-dimensional fingerprint plot Fig. 6a and tht Table 2. The intot Fig. 6c; the pe, characterizes the two π–π stacking interactions, one between inversion-related naphthyl rings, and the other between the chlorobenzene and (C2–C4/C9–C11) rings as the two overlapping triangular regions at around de = di ∼1.8 and 1.9 Å, respectively, having green points in the overlapping portion. The presence of these two π–π stacking interactions is also seen in the flat regions around the participating rings labelled with 1, 2 and 3 in the Hirshfeld surface mapped over curvedness in Fig. 7The fingerprint plot delineated into C⋯C contacts, Fig. 6i.e. 3.0%, contribution from C⋯Cl/Cl⋯C contacts  to the Hirshfeld surface is the result of its involvement in a C—Cl⋯π contact formed between symmetry-related chlorobenzene atoms . Its presence is also clear from the fingerprint plot delineated into Cl⋯H/H⋯Cl , and Cl⋯O/O⋯Cl contacts . The contribution from C⋯H/H⋯C contacts  and other contacts (Table 3The chlorine atom on the benzene (C14–C19) ring makes a useful contribution to the molecular packing. The small, ts Fig. 6g to thems Fig. 5c. Its pCl Fig. 6d, and Cts Fig. 6h. The cts Fig. 6f and ots Table 3, includiet al., 2016i.e. (II)  as eluent to afford α-lapachone and β-lapachone (I)Referring to the reaction scheme, in a double-necked flask equipped with a magnetic bar and reflux condenser, under a nitrogen atmosphere, lawsone , paraformaldehyde , the vinyl selenide  and the ionic liquid 1-butyl-3-methylimidazolium chloride, [Bmim]Cl  were added over 1,4-dioxane (2 ml). The reaction mixture was heated at 383 K and stirred over 2 h. The reaction mixture was cooled and diluted with dichloromethane (100 ml) and then washed with water (3 × 50 ml). The organic phase was dried over NaUiso(H) set to 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989017007605/wm5392sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017007605/wm5392Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017007605/wm5392Isup3.cmlSupporting information file. DOI: 1551641CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The B—H groups exhibit an intramolecular close contact with a C—H group of the pyridine ring, which may be evidence of electrostatic attraction between the hydridic B—H and the electropositive aromatic C—H.The asymmetric unit contains two independent molecules, which exhibit coplanar, mostly  14H19BN2O, contains two independent molecules in the asymmetric unit. Both molecules exhibit coplanar, mostly sp2-hybridized methoxy and dimethylamino substituents on their respective aromatic rings, consistent with π-donation into the aromatic systems. The B—H groups exhibit an intramolecular close contact with a C—H group of the pyridine ring, which may be evidence of electrostatic attraction between the hydridic B—H and the electropositive aromatic C—H. There appears to be weak C—H⋯π(arene) interactions between two of the H atoms of an aminomethyl group and the methoxy-substituted benzene ring of the other independent molecule, and another C—H⋯π (arene) interaction between one of the pyridine ring H atoms and the same benzene ring.The title compound [systematic name: 4-(dimethylamino)pyridine–4-methoxyphenylborane (1/1)], C RBH2) have been the focus of chemical research for over fifty years, most notably for their use in the indispensable hydroboration reaction, which permits reduction of olefins, carbonyl compounds and others  and C1′—B1′—N1′ = 111.0 (1)°] . The B1—C1 and B1′—C1′ distances  are consistent with a formal C—B single bond. The oxygen atom of both methoxy groups appears to be mostly 2sp hybridized, [C7—O1—C4 = 117.3 (1) and C7′—O1′—C4′ = 117.4 (1)°] and is close to coplanar with the phenyl ring [torsion angles C7—O1—C4—C3 = −7.4 (2) and C7′—O1′—C4′—C3′ = −7.1 (2)°], consistent with π-donation into the phenyl ring.The asymmetric unit contains two independent molecules Figs. 1 and 2 ▸ 2sp hybridized [torsion angles C13—N2—C10 = 121.0 (1)° and C13′—N2′—C10′ = 122.2 (1)°] and is close to coplanar [torsion angles C13—N2—C10—C11 = 2.4 (2) and C13′—N2′—C10′—C11′ = 3.4 (1)°] consistent with π-donation into the pyridine ring.The geometries of the 4-(dimethylamino)pyridine (DMAP) fragment of both molecules is similar to other structures of DMAP–borane adducts. The nitrogen atom of the dimethylamino fragment appears to be Database survey). Interestingly, the B—H atoms exhibit intramolecular close contacts with the C—H atoms of the pyridine ring [H12⋯H2B = 2.26 (3) and H12′⋯H2B′ = 2.27 (3) Å] and are close to coplanar [torsion angles H2B—B1—N1—C12 = 4(1) and H2B—B1—N1—C12 = 16 (1)°], which may be evidence of electrostatic interactions between the hydridic B—H atoms and electropositive aromatic C—H atoms, and is observed in other DMAP–borane adducts (see Database Survey). The planes of the pyridine rings and the benzene rings are almost normal to one another [the dihedral angle between the C1–C6 and C8–C12/N1 rings is 73.14 (7)° and that between the C1′–C6′ and C8′–C12′/N1′ rings is 74.15 (7)°]. Perhaps the most significant difference between the two molecules is the 9.0° difference in the torsion angle about the B—N bond [C1—B1—N1—C8 = −63.9 (2) while C1′—B1′—N1′—C8′ = −72.9 (2)°]  and 1.595 (2) Å, respectively] are consistent with formal N—B single bonds, and are within the range observed for other DMAP–borane adducts  interactions between two of the hydrogen atoms of the aminomethyl group and the methoxyphenyl group of a neighboring molecule  interactions described in the previous section  yielded four structures: UTOZEJ , followed by extraction with anhydrous dichloromethane (4 mL). The extract was filtered through a 0.45 µm PTFE syringe filter. The solvent was again removed in vacuo to afford a white solid . Crystals suitable for X-ray diffraction were grown by diffusion of pentane into a concentrated solution of the title compound in anhydrous dichloromethane.In a nitrogen-filled glove box, sodium 4-methoxyphenylborohydride  and 4-dimethylaminopyridine  were combined in a 20 mL vial containing a stir bar and dissolved in anhydrous THF (4 mL). The solution was cooled to 247 K in the freezer and chlorotrimethylsilane  was added dropwise 1H NMR  δ (ppm): 8.12 , 7.23 , 6.80 , 6.52 , 3.78 , 3.11 . 13C NMR  δ (ppm): 157.3, 154.9, 146.7, 145.0 (br s), 134.5, 122.9, 106.5, 55.0, 39.5. 11B NMR  δ (ppm): −5.0 . FTIR : 3012, 2952, 2923, 2853, 2610, 2346, 2288, 2227, 1634, 1548, 1442, 1418, 1392, 1237, 1223, 1161, 1076, 1031, 811, 797, 548, 515.Uiso(H) = 1.5Ueq(C-methyl) and 1.2eq(C) for other H atoms. The B-bound H atoms were located in a difference-Fourier map and freely refined. Methyl H atoms were refined without restrictions on rotation around the C—C bonds, HFIX 138 in SHELXL  I. DOI: 10.1107/S2056989017015171/lh5853Isup2.hklStructure factors: contains datablock(s) I. DOI: 1580559CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The configuration about each double bond in the N—N=C—C=C chain is E; the chain has an all trans conformation. In the crystal, N—H⋯S and C—H⋯π interactions link the molecules into a three-dimensional network.The title dithiocarbazate ester has approximate mirror symmetry with the putative plane bisecting the –CH 18H18N2S2 [systematic name: (E)-4-methylbenzyl 2-[(E)-3-phenylallylidene]hydrazinecarbodithioate, comprises an almost planar central CN2S2 residue [r.m.s. deviation = 0.0131 Å]. The methylene(tolyl-4) group forms a dihedral angle of 72.25 (4)° with the best plane through the remaining non-hydrogen atoms [r.m.s. deviation = 0.0586 Å] so the molecule approximates mirror symmetry with the 4-tolyl group bisected by the plane. The configuration about both double bonds in the N—N=C—C=C chain is E; the chain has an all trans conformation. In the crystal, eight-membered centrosymmetric thioamide synthons, {⋯HNCS}2, are formed via N—H⋯S(thione) hydrogen bonds. Connections between the dimers via C—H⋯π interactions lead to a three-dimensional architecture. A Hirshfeld surface analysis shows that (I) possesses an interaction profile similar to that of a closely related analogue with an S-bound benzyl substituent, (II). Computational chemistry indicates the dimeric species of (II) connected via N—H⋯S hydrogen bonds is about 0.94 kcal mol−1 more stable than that in (I).The title dithiocarbazate ester (I), C The dihedral angles between the central group and the S2- and N2-bound substituents are 71.65 (4) and 7.08 (8)°, respectively. The dihedral angle between the outer groups is 72.33 (4)° and is indicative of an approximately orthogonal relationship. Indeed, the r.m.s. deviation of all non-hydrogen atom in (I)2(tolyl-4) residue is 0.0586 Å, and the angle between this plane and that through the CH2(tolyl-4) residue is 72.25 (4)°. The 1,4-carbon atoms of the 4-tolyl ring lie on the approximate mirror plane defined by the rest of the molecule with the remaining pairs of ring atoms being related across the putative plane.The molecular structure of (I)E in each case. This implies the N1—N2=C2—C3=C4 sequence has an all trans conformation as seen in the N1—N2—C2—C3, N2—C2—C3—C4 and C2—C3—C4—C5 torsion angles of 177.41 (13), −178.70 (15) and 178.23 (15)°, respectively. The C1—S2 [1.7455 (16) Å] and, especially, C11—S2 [1.8233 (16) Å] bond lengths are considerably longer than the C1—S1 bond [1.6752 (16) Å] consistent with considerable thione character in the latter. This is borne out also by the observation that the angles about the C1 atom involving S1 are wider, by over 7°, i.e. S1—C1—S2 = 125.20 (10)° and N1—C1—S1 121.06 (12)°, cf. N1—C1—S2 of 113.74 (11)°.The configuration about the C2=N2 imine [1.284 (2) Å] and C3=C4 ethene [1.339 (2) Å] bonds is Computational chemistry calculations.Further discussion on the molecular geometry of (I)2 mediated by N—H⋯S(thione) hydrogen bonds, Fig. 2a and Table 1b, via methylene-C—H⋯π(tolyl), tolyl-C—H⋯π(phenyl) and phenyl-C—H⋯π(tolyl) interactions, Table 1bc plane and these are linked by the N—H⋯S hydrogen bonds.The most prominent feature of the molecular packing is the formation of an eight-membered, centrosymmetric thioamide synthon, {⋯HNCS}et al., 20082(tolyl-4) group, that might be regarded as the ‘parent’ compound, hereafter referred to as (II). While detailed discussion on the comparison of their molecular geometries and computational modelling are given in Computational chemistry calculations, the present section focuses upon the study of intermolecular interactions formed by (I)et al., 2016The most closely related compound in the crystallographic literature is one with a benzyl substituent at the S2 atom (Tarafder ca 25.4% for (I)ca 17.5 and 16.9%], N⋯H/H⋯N [ca 5.6 and 5.5%] as well as other minor interactions including N⋯C/C⋯N, S⋯C/C⋯S and S⋯N/N⋯S, which constitute less than 5% of the overall contacts.Both (I)divs de at the intervals of 0.01 Å reveals that (I)a,. Specifically, the decomposed fingerprint plot of H⋯H for (I)de + di contact distance of 1.96 Å which is approximately 0.43 Å (17%) shorter cf. 2.36 Å for (II), Fig. 4b. Both (I)c, at approximately 2.7 Å, which is slightly shorter than the van der Waals radii of 2.9 Å. The decomposed fingerprint plots of S⋯H/H⋯S  and N⋯H/H⋯N contacts  for (I)cf. (II), in which the difference is merely 0.04 Å (1.7%). Similarly, the S⋯H/ H⋯S contacts of both (I)cf. the sum of their van der Waals radii by 0.53 and 0.58 Å, respectively (21.5 and 24.0%). As a result, those contacts display intense red spots on their Hirshfeld surface, Fig. 4d.A detailed comparison of the two-dimensional fingerprint plots of ⋯S Fig. 4d and N⋯ts Fig. 4e for , exhibits a greater molecular volume and surface area, and is slightly less globular. This results in a lower surface-to-volume ratio and density for (I)In view of the close structural similarity between (I)et al., 2016As mentioned in the previous section, the ‘parent’ compound represents the most closely related analogue to (I)GaussView5  and pre-optimized using a semi empirical method (PM6) with a precise self-consistent field criterion. Subsequently, the geometries were further optimized at B3LYP/6-311+G without imposing symmetry constraints. A frequency analysis was performed on each optimized structure using the same level of theory and basis set to validate that each structure was indeed the local minimum structure with no imaginary frequency. All calculations were performed using the Gaussian09 software package ca 0.02 and 0.03 Å, respectively. In the chain, the C1—N1 bond lengths have lengthened by ca 0.03 Å, a difference accompanied by a contraction in the N1—N2 bond length by about the same amount. Minor differences are also noted in bond angles with widening of S1—C1—S2 and the angles subtended at the nitrogen atoms by 2–3° with similar contractions in the C1—S1—C11 and S1—C1—N1 angles.The results, as shown from the superposition of the experimental structure and theoretical model of (I)d,p) basis set. It has been demonstrated that the long-range corrected hybrid method can greatly reduce self-interaction errors  was obtained upon the correction of basis set superposition error (BSSE) by counterpoise correction. All calculations were performed in gas phase using Gaussian09 software EBSSEint) of −12.92 and −13.86 kcal mol−1, respectively. The range is approximately 3.89 to 5.23 kcal mol−1 less than the energy computed for a pair of thiourea dimers at the RIMP2/cc-pVDZ and cc-pVTZ levels of theory  is lower in energy  cf. (I)cf. (I)The dimeric species of (I)et al., 2010S-4-methylbenzyldithiocarbazate  was dissolved in hot acetonitrile (100 ml) and added to an equimolar amount of cinnamaldehyde  in absolute ethanol (20 ml). The mixture was heated for about 2 h and was then allowed to stand overnight. The pale-brown crystals that formed were filtered and washed with absolute ethanol at room temperature. Yield: 70%. M.p. 463–466 K. Analysis: Calculated for C18H18N2S2: C, 66.22; H, 5.56; N, 8.58. Found: C, 65.87; H, 5.77; N, 9.00%. FT–IR : 3102, ν(N—H); 1613, ν(C=N); 1021, ν(N—N); 749, ν(CSS).The following procedure was adapted from the literature (Ravoof Uiso(H) set to 1.2–1.5Ueq(C). The nitrogen-bound H atom was located in a difference-Fourier map but was refined with a distance restraint of N—H = 0.88±0.01 Å, and with Uiso(H) set to 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989017003991/hb7666sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017003991/hb7666Isup2.hklStructure factors: contains datablock(s) I. DOI: 1537500CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


III defined by an N4O3 donor set. The packing features supramolecular layers sustained by C—H⋯O, C—H⋯π(aryl) and C—Cl⋯π(aryl) interactions.The title compound features an amine-N-capped octahedral coordination geometry for Yb III atom in the title complex, [Yb(C27H24Cl3N4O3)] }tris(4-chlorophenolato)ytterbium(III)], is coordinated by a trinegative, heptadentate ligand and exists within an N4O3 donor set, which defines a capped octahedral geometry whereby the amine N atom caps the triangular face defined by the three imine N atoms. The packing features supramolecular layers that stack along the a axis, sustained by a combination of aryl-C—H⋯O, imine-C—H⋯O, methylene-C—H⋯π(aryl) and end-on C—Cl⋯π(aryl) interactions. A Hirshfeld surface analysis points to the major contributions of C⋯H/ H⋯C and Cl⋯H/H⋯Cl interactions  to the overall surface but the Cl⋯H contacts are at distances greater than the sum of their van der Waals radii.The Yb Being large and having seven potential donor atoms, these ligands are capable of coordinating lanthanides even if the atomic sizes of lanthanides are greater in comparison to their transition metal counterparts Despite being less studied than transition metal complexes, the crystal chemistry of lanthanides is rich and diverse as their complexes can display various coordination numbers and geometries that are not readily predicted °, indicating a parallel disposition. There is a range in the Yb—O bond lengths, i.e. >0.02 Å, with the shortest Yb—O1 bond being trans to the most loosely bound imine-N4 atom, and the longest Yb—O2 bond being trans to most tightly held imine-N2 atom, Table 1The molecular structure of (I)b axis, Fig. 2a. These are reinforced by methylene-C—H⋯π(aryl) contacts, also shown in Fig. 2a. Chains are connected into supramolecular layers in the bc plane by end-on C—Cl⋯π(aryl) interactions, Fig. 2b. Layers stack along the a axis with no directional interactions between them, Fig. 2c dnorm contact distances within the range of −0.18 to 1.65 Å through calculation of the internal (di) and external (de) distances of a particular Hirshfeld surface point to its nearest nucleus a shows a butterfish-like two-dimensional fingerprint plot for (I)ca 26 and 5%, respectively, of the overall interactions in the crystal, with de + di distances of ∼2.54 Å and ∼2.43 Å, respectively, Fig. 3b and 3c. As seen from the images of Fig. 4d. Finally, the Cl⋯H/H⋯Cl interactions appear as the third most dominant interaction, right after H⋯H and C⋯H/H⋯C, with an overall contribution of ca 24% to the Hirshfeld surface, despite the fact that these interactions are considered weak with contact distances greater than the sum of van der Waals radii. However, as seen from Fig. 3e, these interactions are responsible for the appearance of the tails of the ‘butterfish-shape’.The Hirshfeld surface analysis was performed on (I)III complex has been the subject of two independent determinations  structures are available for comparison where the ligand is identical to that in (I)i.e. P21/n, for Ln = Sm  bond lengths. The only anomalous parameter might be the length of the Gd—N(amine) bond, i.e. 2.737 (8) Å, the relatively high standard uncertainty value notwithstanding.A wider range of lanthanide : 1628 (s) ν(C=N), 1517 (m), 1449 (m), 1392 (m) ν(–O—C=C–), 1158 (m) ν(C—O—C). Analysis calculated for C27H24Cl3N4O3Yb: C, 44.31; H, 3.31; N, 7.66%. Found: C, 44.63; H, 3.12; N, 7.92%.The Schiff base ligand, tris{[amino]ethyl}amine (Kanesato Uiso(H) set to 1.2Ueq(C). Owing to poor agreement, one reflection, i.e. (7 0 4), was omitted from the final cycles of refinement. The maximum and minimum residual electron density peaks of 1.65 and 0.45 e Å−3, respectively, were located 0.84 and 1.51 Å from the Yb and N2 atoms, respectively.Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989016013748/hb7612sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989016013748/hb7612Isup2.hklStructure factors: contains datablock(s) I. DOI: 1501230CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


O:O′-bridged dinuclear unit which is extended through N-atom donors of the pyrimidine ligand into a two-dimensional layered structureThe coordination polymeric silver(I)–diclofenac complex including pyrimidine is based on a centrosymmetric carboxylate  14H11Cl2NO2) (diclH) and pyrimidine (pym), namely poly[{μ2-2-[2-phenyl]acetato-κ2O:O′}(μ2-pyrimidine-κ2N1:N3)silver(I)], [Ag(C14H10Cl2NO2)(C4H4N2)]n or [Ag(μ-dicl)(μ-pym)]n, the very distorted tetrahedral AgN2O2 coordination centres comprise two N-atom donors from bridging pym ligands [Ag—N = 2.381 (3) and 2.412 (3) Å] and two carboxylate O-atom donors from dicl ligands [Ag—O = 2.279 (2) and 2.280 (2) Å], which bridge Ag atoms, giving a centrosymmetric dinuclear units with a short Ag⋯Ag separation [2.8931 (5) Å]. Within the units are short intraligand C—Cl⋯π(pym) interactions [3.6409 (15) Å]. The units are linked through the bridging N atoms of the pym ligand into a two-dimensional sheet–polymer structure lying parallel to (100) and stabilized by inter-ring π–π interactions between the pym ligands [Cg⋯Cg = 3.4199 (17) Å]. Additional inter-unit C—H⋯O and C—H⋯Cg hydrogen-bonding interactions between the sheets give an overall three-dimensional structure.In the title mixed-ligand silver(I) coordination polymeric complex with the non-steroidal anti-inflammatory drug diclofenac (C These studies have shown that short Ag⋯Ag separations are one of the most important factors for the manifestation of such properties  from separate dicl ligands and two nitrogen atoms /141°}, where α and β are the largest angles around the metal atom) is 0.732 and indicates substantial deviation from ideal tetrahedral geometry 2(μ-pym)2]n n 2] units  Å] is significantly shorter than the sum of the van der Waals radii for two silver atoms (3.44 Å), indicating weak interactions between adjacent AgI ions, forming an [Ag2(COO)2] units. If coexisting strong argentophilic Ag1⋯Ag1i interactions are considered as coordinative, it could be reasoned that the coordination around Ag1 is slightly distorted trigonal–bipyramidal [the structural distortion index tau (τ) was calculated to be 0.06] ds Fig. 1. The dists Fig. 2. Within 2-1N,N-bridging ligand between neighboring [Ag2(COO)2] units, leading to the formation of a two-dimensional coordination polymer, extending along (100) 2] units, which comprise eight-membered rings, can be defined as the nodes of the structure. Connection of the four different pym ligands to these nodes provides continuity of the structure  Fig. 4. In othere Fig. 4.carboxyl hydrogen-bonding interaction [2.971 (3) Å] °, the conformation of the ligand being stabilized by an intramolecular N1—H1⋯O2] Table 2.iii hydrogen-bonding interaction stabilizes the crystal packing (Table 2Cg6iv interaction to a pym ring [3.983 Å] and a strong π–π stacking interaction between aromatic rings of the pym ligands , shown in Fig. 3In the crystal, a C16—H16⋯O1g Table 2. In addi3) of AgNO3  with stirring. A white suspension with a white precipitate formed and the addition of acetonitrile (10 cm3) to this resulted in a clear solution which was left to stand for slow evaporation in darkness at room temperature. Single crystals of (I)All reactions were performed with commercially available reagents and used without further purification. Solid sodium 2-phenylacetate (Nadicl)  and pyrimidine  were added to an aqueous solution  in the frequency range 4000–600 cmas) and symmetric (υs) vibrations of the carboxylate group. The difference between the asymmetric and symmetric carboxylate stretching [Δν = υas(COO−) - υs(COO−)] is often used to correlate the infrared spectra of metal carboxylate structures. When Δν < 200 cm−1, the carboxylate groups of the complexes can be considered bidentate  = 1.2Ueq(C). The N-bound H atom was located in a difference-Fourier map but was also allowed to ride in the refinement with Uiso(H) = 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989016014730/zs2370sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016014730/zs2370Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016014730/zs2370sup3.tifSupporting information file. DOI: 1500646CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports7: Article number: 45206; 10.1038/srep45206 published online: 05302017; updated: 12222017.This Article contains errors. In the legend of Figure 1,a) Spectral flux per bunch through an on-axis 1 mm2 area delivered by Beamline ID19 in the four bunch mode (40 mA storage ring current), including 2.8 mm diamond and 1.4 mm aluminium filtering. The total flux was 1.2 × 109 photons s−1 mm−2 on-axis.”“(should read:a) Spectral flux per bunch through an on-axis 1 mm2 area delivered by Beamline ID19 in the four bunch mode (40 mA storage ring current), including 2.8 mm diamond and 1.4 mm aluminium filtering. The total flux was 4 × 107 photons/bunch/mm2 on-axis.”“ delivered per bunch (150 ps duration23) on Beamline ID19 in the four bunch mode.”“Figure 1(a) shows the calculated on-axis spectral X-ray flux (1.2 × 10should read:7 photons/bunch/mm2 on-axis) delivered per bunch (150 ps duration23) on Beamline ID19 in the four bunch mode.”“Figure 1(a) shows the calculated on-axis spectral X-ray flux (4 × 10In the Discussion section,“The time at which the leading lateral release waves reached the shock front on-axis in the PC-PC and Cu-PC loadings were estimated to be 3.30 μs and 2.53 μs, respectively.”should read:“The times at which the leading lateral release waves reached the shock front on-axis in the PC-PC and Cu-PC loadings were estimated to be 2.3 μs and 1.9 μs, respectively.”


Scientific Reports7: Article number: 42192; 10.1038/srep42192 published online: 02082017; updated: 01292018.This Article contains an error in Figure 2. For CYP3A5 *3 = AG without hypertension,“N = 31 Dose = 3.73”should read:“N = 31 Dose = 4.13”The correct Figure 2 appears below as


The packing features C—H⋯S and C—H⋯π interactions.With both chelating and μ 2(C8H8NS2)4], is a centrosymmetric dimer with both chelating and μ2-tridentate dithiocarbamate ligands. The resulting S5 donor set defines a CdII coordination geometry intermediate between square-pyramidal and trigonal–bipyramidal, but tending towards the former. The packing features C—H⋯S and C—H⋯π interactions, which generate a three-dimensional network. The influence of these interactions, along with intra-dimer π–π interactions between chelate rings, has been investigated by an analysis of the Hirshfeld surface.The title compound, [Cd The S3 dithiocarbamate ligand is strictly chelating, with Δ(Cd—S) = 0.08 Å. Reflecting the symmetric modes of coordination of the dithiocarbamate ligands, the C—S bond lengths are equal within 5σ (Table 1The centrosymmetric binuclear molecule of (I)σ Table 1.5 donor set defines a highly distorted pentacoordinate geometry, with the major distortions due to the disparate Cd—S bond lengths and the acute angles subtended at the CdII atom by the chelating ligands °. A measure of the distortion of a coordination geometry from the ideal square-pyramidal and trigonal–bipyramidal geometries is given by the value of τ or Fig. 2.et al., 2016dnorm in the range −0.055 to 1.371 au  and 5(b), respectively, and again highlight the influence of C—H⋯S interactions, short C10⋯C15 contacts and C—H⋯π interactions involving phenyl rings (atoms C3–C8) as the acceptor. Thus, the C—H⋯S interactions involving the phenyl-ring C4, C5 and H5 atoms with S1 are shown with black dashed lines in Fig. 5a); the red dashed lines indicate short interatomic C⋯C contacts .The Hirshfeld surface analysis for (I)au Fig. 3, the briau Fig. 3. The donau Fig. 3 give risau Fig. 3. The imms Table 3. The C—Het al., 2007a)–(e); their relative contributions to the Hirshfeld surface are summarized quantitatively in Table 4b) that H⋯H contacts do not exert much influence on the molecular packing, as their interatomic distances are greater than the sum of their van der Waals radii, i.e. de + di > 2.8 Å. A pair of peaks appearing in the fingerprint plot delineated into S⋯H/H⋯S contacts at de + di ∼ 2.8 Å  arise from the C5—H5⋯S1 interaction; the weaker C4⋯H4⋯S1 interaction and short interatomic H⋯S/S⋯H contacts involving the S3 atom  indicate the significance of these contacts through the presence of C—H⋯π interactions and short interatomic C⋯H/H⋯C contacts in the crystal. A pair of green lines within the forceps also indicates the influence of these contacts. Finally, an arrow-shaped distribution of green points in the centre in the plot corresponding to S⋯S contacts , together with the contribution from Cd⋯S/S⋯Cd contacts to the Hirshfeld surface (Table 4Cg⋯Cg = 3.6117 (11) Å; symmetry code: −x, 1 − y, 1 − z]. The small contributions from Cd⋯H/H⋯Cd and N⋯H/H⋯N contacts −1. 1H and 13C NMR spectra were recorded at room temperature in DMSO-d6 solution on a Jeol ECA 400 MHz FT–NMR spectrometer.All chemicals and solvents were used as purchased without purification, and all reactions were carried out under ambient conditions. The melting point was determined using an Electrothermal digital melting-point apparatus and was uncorrected. The IR spectrum was obtained on a PerkinElmer Spectrum 400 FT Mid-IR/Far-IR spectrophotometer from 4000 to 400 cm−1): 1491 (m) [ν(C—N)], 1160 (m), 964 (s) [ν(C—S)] cm−1. 1H NMR: δ 7.26–7.42 , 2.05 . 13C NMR: δ 46.6 (Me) 125.6, 128.4, 129.6, 147.9 (aromatic C), 207.8 (CS2).Sodium methylphenyldithiocarbamate  in methanol (25 ml) was added to cadmium chloride  in methanol (10 ml). The resulting mixture was stirred and refluxed for 2 h. The filtrate was evaporated until an off-white precipitate was obtained, which was recrystallized in methanol. Slow evaporation of the filtrate yielded colourless crystals of the title compound . IR (cmUiso(H) values set at 1.2–1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989017002705/hb7659sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017002705/hb7659Isup2.hklStructure factors: contains datablock(s) I. DOI: 1533246CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Prunus domestica ssp. domestica), six colorless non‐fluorescent chlorophyll catabolites (NCCs) were characterized, named Pd‐NCCs. In addition, several minor NCC fractions were tentatively classified. The structure of the most polar one of the NCCs, named Pd‐NCC‐32, featured an unprecedented twofold glycosidation pattern. Three of the NCCs are also functionalized at their 32‐position by a glucopyranosyl group. In addition, two of these glycosidated NCCs carry a dihydroxyethyl group at their 18‐position. In the polar Pd‐NCC‐32, the latter group is further glycosidated at the terminal 182‐position. Four other major Pd‐NCCs and one minor Pd‐NCC were identified with five NCCs from higher plants known to belong to the ‘epi’‐series. In addition, tentative structures were derived for two minor fractions, classified as yellow chlorophyll catabolites, which represented  oxidation products of two of the observed Pd‐NCCs. The chlorophyll catabolites in leaves of plum feature the same basic structural pattern as those found in leaves of apple and pear trees.In cold extracts of senescent leaves of the plum tree ( Hv‐NCC‐1 from senescent leaves of barley (Hordeum vulgare).Hv‐NCC‐1 as a 1‐formyl‐19‐oxobilin‐type linear tetrapyrrolepFCCs), with species‐dependent configuration of their formation.pFCCs are rapidly hydroxylated to 32‐OH‐pFCC (probably still in the chloroplast).Fig. About 25 years ago, chlorophyll (Chl) breakdown and the appearance of the fall colors were still a stunning mystery.Table In the meantime, NCCs have been found in extracts of senescent leaves of a range of plants,Fig. However, as was recognized recently, Chl‐breakdown ‘branches out’, and furnishes ‘1,19‐dioxobilin‐type’ Chl‐catabolites (DCCs)Prunus domestica ssp. domestica). As shown below, Chl‐breakdown in senescent leaves of this fruit tree follows the ‘PaO/phyllobilin’ pathway of Chl‐breakdown.epi‐type’. In addition, in the extracts several YCCs were also found.In the context of investigations of Chl‐catabolites in domestic agricultural plants, we have studied the nature of such phyllobilins in stone fruit and report here our work on the Chl‐catabolites in leaves of the plum tree (Prunus domestica) and frozen for storage. Five major and nine minor colorless NCCs were provisionally identified in extracts of senescent leaves of plum trees on the basis of their characteristic UV‐absorbance properties, using analytical HPLC , as first reported in the spectrum of Hv‐NCC‐1.Yellow senescent and green leaves were collected from plum trees , analyzed by UV/VIS‐spectroscopy first  and of Hv‐NCC‐1R)‐configuration at the stereogenic C(10).For spectroscopic analysis of the most abundant NCCs in the leaves of Pd‐NCC‐32 (1) displayed a strong signal at m/z 1025.3839, corresponding to C47H62N4NaO20+  and establishing the molecular formula as C47H62N4O20. Likewise, a positive‐ion‐mode ESI‐MS spectrumFig. pseudo‐molecular ion [M + H]+ at m/z 1003.1, also consistent with the molecular formula of C47H62N4O20. Characteristic fragment ions at m/z 841.2, 684.1 and 679.2 were also detected, which indicated the loss of a sugar moiety (C6H10O5) from [M + H]+,D  in CD3OD at 10 °C ) at low field, four Me group singlets at high field and a singlet for the methyl ester group at 3.75 ppm. The typical signals for a peripheral vinyl group were not observed. From 1H,13C‐heteronuclear (HSQC and HMBC) and 1H,1H‐homonuclear NMR‐correlations (COSY and ROESY) of Pd‐NCC‐32 (1) in CD3OD, assignment of the signals of 47 H‐atoms and 45 13C‐nuclei could be achieved  (4.17 ppm) and H–C(1″) (4.33 ppm), as well as H–C(2′) (3.17 ppm) and H–C(2″) (3.21 ppm), the chemical shifts of the pairs of signals differed significantly . Chemical shifts and doublet nature (J = 7.8 Hz) of H–C(1′) and H–C(1″) indicated β‐anomeric attachment of both sugar moieties, as observed earlier for the 32‐glucopyranoside moieties of NCCs.1H‐ and 13C‐chemical shifts of Pd‐NCC‐32 (1) with those of the known NCCs with a peripheral glucopyranosyl group at C(32).1H,13C‐HMBCs from H–C(1′) with C(32) and from H–C(1″) with C(182) established the attachment of one sugar moiety at each one of the terminal C‐atoms of the Et side chain at C(3) (ring A) and of the 1,2‐dihydroxyethyl group at C(18) (ring D). The 1H‐ and 13C‐chemical shifts at the positions C(182) and C(32) were also consistent with an attached peripheral sugar substituent. However, as with other 1,2‐dihydroxyethyl substituted NCCs,1 the configuration at C(182) remains unknown.In a 600 MHz Pd‐NCC fractions , Pd‐NCC‐40 (3), Pd‐NCC‐56 (4), Pd‐NCC‐60 (5), and Pd‐NCC‐71 (6) were also isolated and purified by HPLC. A positive‐ion‐mode ESI‐MS spectrum of Pd‐NCC‐60 (5) showed a pseudo‐molecular ion [M + H]+ at m/z 645.2, consistent with the molecular formula of C35H40N4O8. Characteristic fragment ion peaks were visible at m/z 613.2 and 522.1, corresponding to the loss of MeOH and the loss of ring D from [M + H]+. The same molecular formula and fragmentation is known for the major NCC from Cercidiphyllum japonicum (Cj‐NCC‐1),epi’‐configuration at C(16).Pd‐NCC‐60 (5) and Cj‐NCC‐1 were separately analyzed by analytical HPLC, as well as a 1:1 mixture of both in a co‐injection   of Pd‐NCC‐60 (5). Consistent with their origin from a common ‘primary’ FCC,epi‐pFCC, the other colorless Pd‐NCCs were also deduced to belong to the epi‐series.Five other tion see , Fig. 10Pd‐NCC‐56 (4) was determined as C41H50N4O13 by ESI mass spectrometry, which furnished a base peak [M + H]+ at m/z 807.2. Fragment‐ions at m/z 775.3, 684.2 and 645.2 corresponded to the loss, alternatively, of MeOH, of ring D and of a hexose moiety from [M + H]+. These data indicate the presence of one hexopyranose moiety at the HO–C(32) group of ring A of Pd‐NCC‐56 (4) and a vinyl group at C(18) of ring D. This indicates a common chemical constitution of 4 and of Nr‐NCC‐2Fig. The molecular formula of Pd‐NCC‐40 (3) could be deduced tentatively as C35H42N4O10 by ESI mass spectrometry, which showed the experimental base peak [M + H]+ at m/z 679.2. In the mass spectra, characteristic fragment‐ion peaks at m/z 647.2 and 522.1 were also detected, which corresponded to the loss of MeOH and to the loss of ring D (from [M + H]+). Accordingly, the catabolite Pd‐NCC‐40 (3)  indicated a pseudo‐molecular ion at m/z 841.2, consistent with the molecular formula of C41H52N4O15. The fragments at m/z 809.3, 684.2, 679.2 and 522.1 indicated the loss of MeOH, the loss of ring D, the loss of a sugar moiety and the loss of ring D and a sugar moiety. Thus, the catabolite 2 carries a sugar substituent at the C(3) hydroxyethyl side chain (ring A) and a 1,2‐dihydroxyethyl group at C(18) (ring D). According to their fragmentation pattern,Pd‐NCC‐35 (2)  was determined as C35H40N4O7 with a pseudo‐molecular ion at m/z 629.2. Fragments at m/z 597.2 and 506 indicate the loss of MeOH and ring D. Pseudo‐molecular ion and fragment‐ions are consistent with a chemical constitution of 6, as previously found for Cj‐NCC‐2  and of Cj‐NCC‐2 was supported by a common retention time of 6 and Cj‐NCC‐2 in a HPLC co‐injection experiment.The molecular formula of Pd‐NCC‐54) by LC/ESI‐MS revealed a pseudo‐molecular ion at m/z 661.2 ([M + H]+), consistent with the molecular formula of C35H40N4O9. We suspected Pd‐NCC‐54 as product of the formal addition of an O‐atom to Pd‐NCC‐60 (5) from an endogenous oxidation process. Indeed, as shown recently,2O may eliminate easily, resulting in corresponding YCCs.Pd‐YCC‐67, which showed mass spectral data (pseudo‐molecular ion with m/z 643.2) consistent with its formation as the formal product of an oxidative dehydrogenation of Pd‐NCC‐60 (5). A further minor fraction, classified as YCC from a prominent absorption maximum near 420 nm, was also subjected further to ESI‐MS analysis. The latter data suggested Pd‐YCC‐61 (m/z 805.1) to represent a YCC derived from oxidation of the glucosylated Pd‐NCC‐56 (4). When extracts were prepared after storage of senescent leaves of the plum tree at room temperature for 7 min, an increase of the content of both YCCs (Pd‐YCC‐61 and Pd‐YCC‐67) was observed, as well as the formation of 15‐OH‐Pd‐NCC‐60, identified by comparison with its analogue from the established oxidation of Cj‐NCC‐1.Pd‐NCC‐54. Clearly, work‐up and preparation of extracts of cold senescent leaves need to be done swiftly, in order to avoid oxidation artefacts.Analysis of a minor NCC (tentatively named Prunus domestica ssp. domestica) were shown to contain a range of NCCs, two YCCs, and, in traces, a PiCC, all members of the ‘type I’ phyllobilin family. In spite of the absence of DCCs,Pd‐NCC‐32 (1) showed a previously unknown structure and is functionalized with two glycopyranose moieties on the ‘distant’ pyrrole rings A and D. The structure of Pd‐NCC‐32 (1) also provided the first (indirect) evidence for enzymatic glycosidation of an FCC at the 182‐position .Pd‐NCC‐60 (5) and Pd‐NCC‐71 (6) with corresponding Cj‐NCCs, indicated the plum NCCs to belong to the C(16)‐epi series, as well.Fig. Extracts of naturally senescent leaves of the plum tree , LC/MS gradient grade MeOH from or VWR , and AcONH4, puriss. p.a., from Fluka . KH2PO4, puriss. p.a., K3PO4 dibasic‐anh., puriss. p.a., and hexane were from Sigma–Aldrich . Sand was from J. T. Baker , Sep‐Pak®C18 cartridges (1 and 5 g) were from Waters Associates. pH Values were measured with a WTW SenTix 21 electrode connected to a WTW pH525 digital pH meter.HPLC grade MeOH was purchased from HPLC. Dionex Summit HPLC system with manual sampler, P680 pump, online degasser and diode array detector, 1.35 ml or 200 μl injection loop. Data were collected and processed with Chromeleon V6.70.i) Anal. HPLC. Kinetex 00G‐4601‐E0‐5u‐C18‐100A 250 × 4.6 mm i.d. column at 20 °C protected with a Phenomenex AJ0‐4287 C18 4 × 3.0 mm i.d. pre‐column was used with a flow rate of 0.5 ml min−1. Solvent A: 50mm aq. potassium phosphate buffer (pH 7.0), solvent B: MeOH, solvent C: H2O; solvent composition (A/B/C) as a function of time (0 – 90 min): 0 – 5, 80:20:0; 5 – 60, 80:20:0 to 40:60:0; 60 – 80, 40:60:0 to 0:100:0; 80 – 85, 0:100:0; 85 – 87, 0:100:0 to 0:20:80; 87 – 90, 0:20:80 to 80:20:0.ii) Semi‐prep. HPLC (90 min run). 00G‐4252‐NO Luna 5u C18(2) 100A 250 × 10 mm i.d. column at 20 °C protected with a Phenomenex AJ0‐7220 250 × 10 mm i.d. pre‐column was used with a flow rate as a function of time: 0 – 5 min: 1 – 4 ml min−1; 5 – 90 min: 4 ml min−1. Solvent A: 4mm aq. AcONH4, solvent B: MeOH with AcONH4 (c = 4mm), solvent C: H2O; solvent composition (A/B/C) as a function of time (0 – 90 min): 0 – 5, 80:20:0; 5 – 60, 80:20:0 to 40:60:0; 60 – 80, 40:60:0 to 0:100:0; 80 – 85, 0:100:0; 85 – 87, 0:100:0 to 0:20:80; 87 – 90, 0:20:80 to 80:20:0.iii) Semi‐prep. HPLC (70 min run). 00G‐4252‐NO Luna 5u C18(2) 100A 250 × 10 mm i.d. column at 20 °C protected with a Phenomenex AJ0‐7220/1 C18 250 × 10 mm i.d. pre‐column was used with a flow rate as a function of time: 0 – 5 min: 1 – 4 ml min−1; 5 – 70 min: 4 ml min−1. Solvent A: 50mm aq. potassium phosphate buffer (pH 7.0), solvent B: MeOH, solvent C: H2O; solvent composition (A/B/C) as function of time (0 – 70 min): 0 – 5, 80:20:0; 5 – 50, 80:20:0 to 47.3:52.7:0; 50 – 55, 47.3:52.7:0 to 0:52.7:47.3; 55 – 60, 0:52.7:47.3 to 0:100:0; 60 – 65, 0:100:0; 65 – 67, 0:100:0 to 0:20:80; 67 – 70, 0:20:80 to 80:20:0.LC/MS. i) Pre‐Purifcation of Minor Fractions Pd‐NCC‐54 and Pd‐NCC‐71 on an anal. HPLC. Minor catabolite fractions were first purified by HPLC  according to following procedure: 3 – 4 g of leaf material were ground in mortar and pestle under liquid N2 with addition of ca. 1 g of sand, a tip of a spatula of CaCO3 and 4 – 5 ml of MeOH. The mixture was centrifuged  and the supernatants were stored at −80 °C until use. An aliquot of the supernatant was centrifuged (1 min at 7200 g), diluted (1:1 v/v) with aq. potassium phosphate buffer  and centrifuged again . In total 300 μl (3 × 100 μl) of the supernatant were purified on the anal. HPLC  at a flow rate of 0.5 ml min−1 using 50mm aq. K3PO4 as solvent A and MeOH as solvent B , and desired fractions were collected and combined.ii) LC/MS Analysis of minor fractions of the collected HPLC fractions were analyzed on an LC/MS system  using AcONH4 buffer  and MeOH  as eluents . Twenty microliter of the collected catabolite fraction were injected and analyzed at a flow rate of 0.5 ml min−1 .Spectroscopy. UV/VIS Spectra: Agilent Technologies Cary 60 spectrophotometer; λmax (nm) (rel. ε). CD Spectra: Jasco J715, λmax and λmin (nm), Δε. 1H‐ and 13C‐NMR: Bruker 600 MHz Avance II+ (δ(C1HD2OD) 3.31 ppm, and δ(13CD3OD) 49.0 ppm, δ in ppm,J in Hz. Mass Spectrometry: Finnigan LCQ Classic, electrospray ionization (ESI) source, positive‐ion mode,Senescent plum tree leaves were harvested in November 2013 from a commercial orchard in Aldino (South Tyrol). They were immediately frozen in a freezer (−80 °C) and transported in a cold box (−20 °C) to Innsbruck, where they were stored cold (−80 °C).2) was frozen in liquid N2, grounded in a mortar and extracted with 1 ml of MeOH. The resulting suspension was centrifuged for 3 min at 13,000 g. Five hundred microliter of the MeOH supernatant were diluted with 2 ml of 50mm aq. potassium phosphate buffer (pH 7.0). After centrifugation for 3 min at 13,000 g, 200 μl of the extract was analyzed by HPLC . Yellow‐greenish senescent plum tree leaves (18.7 g) were frozen in liquid N2, pulverized to a fine powder and extracted with 60 ml of MeOH. The suspension was centrifuged for 5 min at 4000 g. Forty‐two milliliter of the supernatant were diluted with 168 ml of 50mm aq. potassium phosphate buffer (pH 7.0). After centrifugation for 5 min at 4000 g, the soln. was extracted two times with hexane. The MeOH extract was diluted with 300 ml of 50mm potassium phosphate buffer (pH 7.0) and applied to a pre‐conditioned 5 g SepPak cartridge. This was washed with 35 ml of H2O and the NCC‐containing fraction was eluted with 30 ml of MeOH. The solvents were removed by using a rotary evaporator. The residue was dissolved in 1 ml of MeOH and 4 ml of 50mm aq. potassium phosphate buffer (pH 7.0) using an ultrasonic bath. After centrifugation for 3 min at 13,000 g, the sample was divided in four aliquots and applied to semi‐prep. HPLC; injection volume, 1.25 ml; flow rate, 0 – 5 min: 1 – 4 ml min−1, 5 – 90 min: 4 ml min−1; solvent A: 4mm aq. AcONH4, solvent B: MeOH with AcONH4 (c = 4mm), solvent C: H2O; solvent composition (A/B/C) as a function of time (0 – 90 min): 0 – 5, 80:20:0; 5 – 60, 80:20:0 to 40:60:0; 60 – 80, 40:60:0 to 0:100:0; 80 – 85, 0:100:0; 85 – 87, 0:100:0 to 0:20:80; 87 – 90, 0:20:80 to 80:20:0. Fractions containing Pd‐NCC‐32 (1) of five consecutive semi‐prep. HPLC runs were collected and dried under reduced pressure. The residue was dissolved in 200 μl of MeOH and 800 μl of 50mm aq. potassium phosphate buffer (pH 7.0) and re‐purified by semi‐prep. HPLC; injection volume, 1.00 ml; flow rate, 0 – 5 min: 1 – 4 ml min−1, 5 – 70 min: 4 ml min−1; solvent A: 50mm aq. potassium phosphate buffer (pH 7.0), solvent B: MeOH, solvent C: H2O; solvent composition (A/B/C) as a function of time (0 – 70 min): 0 – 5, 80:20:0; 5 – 50, 80:20:0 to 47.3:52.7:0; 50 – 55, 47.3:52.7:0 to 0:52.7:47.3; 55 – 60, 0:52.7:47.3 to 0:100:0; 60 – 65, 0:100:0; 65 – 67, 0:100:0 to 0:20:80; 67 – 70, 0:20:80 to 80:20:0. The fraction containing Pd‐NCC‐32 (1) was collected between and diluted with 20 ml of 50mm aq. potassium phosphate buffer (pH 7.0). For de‐salting, the aq. soln. was applied to a pre‐conditioned 5 g SepPak cartridge, washed with 15 ml of H2O and eluted with 5 ml of MeOH. After removal of the solvents using a rotary evaporator, the sample was dried under high vacuum and a uniform sample of 0.29 mg of Pd‐NCC‐32 (1) was obtained.Isolation of Raw Pd‐NCCs for Structural Analysis. 12 anal. extracts were prepared, combined and diluted with 95 ml of 50mm aq. potassium phosphate buffer (pH 7.0). This was applied to a pre‐conditioned 5 g SepPak cartridge, washed with 30 ml of H2O and the NCC‐containing fraction was eluted with 30 ml of MeOH. The fraction was dried under reduced pressure and the precipitate was dissolved in 400 μl of MeOH and 1.6 ml of 4mm aq. AcONH4. After centrifugation for 3 min at 13,000 g, the sample was divided in two aliquots and applied to semi‐prep. HPLC; injection volume, 1.00 ml; flow rate, 0 – 5 min: 1 – 4 ml min−1, 5 – 90 min: 4 ml min−1; solvent A: 4mm aq. AcONH4, solvent B: MeOH with AcONH4 (c = 4mm), solvent C: H2O; solvent composition (A/B/C) as a function of time (0 – 90 min): 0 – 5, 80:20:0; 5 – 60, 80:20:0 to 40:60:0; 60 – 80, 40:60:0 to 0:100:0; 80 – 85, 0:100:0; 85 – 87, 0:100:0 to 0:20:80; 87 – 90, 0:20:80 to 80:20:0. The fractions containing Pd‐NCC‐32 (1), Pd‐NCC‐35 (2), Pd‐NCC‐40 (3), Pd‐NCC‐56 (4) and Pd‐NCC‐60 (5) were collected and to obtain pure samples from all fractions an anal. HPLC run with AcONH4 had to be performed; injection volume, 200 μl; flow rate, 0.5 ml min−1; solvent A: 4mm aq. AcONH4, solvent B: MeOH with AcONH4 (c = 4mm), solvent C: H2O; solvent composition (A/B/C) as a function of time (0 – 90 min): 0 – 5, 80:20:0; 5 – 60, 80:20:0 to 40:60:0; 60 – 80, 40:60:0 to 0:100:0; 80 – 85, 0:100:0; 85 – 87, 0:100:0 to 0:20:80; 87 – 90, 0:20:80 to 80:20:0. In each anal. HPLC run, the desired catabolite was collected.Pd‐NCC‐32 (1). tR = 32.6 min. UV/VIS : 244sh (0.83), 314 (1.00) : 226 (8), 249 (−3), 263 (−3), 283 (−8), 319 (1). 1H‐NMR : 1.92 ); 2.08 ); 2.12 ); 2.23 ); 2.26 – 2.30 ); 2.31 – 2.35 ); 2.37 – 2.41 ); 2.60 – 2.65 , Ha–C(121)); 2.71 – 2.77 ); 2.91 ); 3.17 ); 3.21 ); 3.24 – 3.28 , H–C(4″), H–C(5′), H–C(5″)); 3.33 – 3.39 , H–C(3′), H–C(3″)); 3.62 – 3.69 , Ha–C(6″), Hb–C(32)); 3.71 ); 3.75 ); 3.83 – 3.87 , Hb–C(6″)); 3.96 – 4.01 , Hb–C(182)); 4.02 – 4.09 , Hb–C(5)); 4.17 ); 4.33 ); 4.77 ); 4.87 ); 9.32 ). 13C‐NMR : 8.4 (C(21)); 9.1 (C(71)); 9.1 (C(131)); 12.5 (C(171)); 22.0 (C(121)); 23.6 (C(5)); 24.8 (C(31)); 29.7 (C(15)); 36.9 (C(10)); 39.6 (C(122)); 52.7 (C(85)); 62.1 (C(6′)); 62.1 (C(6″)); 62.5 (C(16)); 66.6 (C(181)); 67.7 (C(82)); 70.3 (C(32)); 71.1 (C(4′)); 71.1 (C(4″)); 73.2 (C(182)); 74.8 (C(2′)); 74.8 (C(2″)); 77.7 (C(3′)); 77.7 (C(3″)); 77.7 (C(5′)); 77.7 (C(5″)); 103.9 (C(1′)); 103.9 (C(1″)); 112.2 (C(7)); 114.8 (C(13)); 120.0 (C(3)); 120.0 (C(12)); 124.2 (C(11)); 124.2 (C(14)); 125.6 (C(8)); 128.5 (C(1)); 133.9 (C(6)); 134.0 (C(2)); 139.6 (C(4)); 159.3 (C(17)); 130.2 (C(18)); 161.5 (C(9)); 171.4 (C(83)); 174.8 (C(19)); 177.2 (C(20)). ESI‐MS: 1079.2 ; 1063.3 ; 1047.3 ; 1041.3 ; 1025.3 ; 1005.1 (20), 1004.1 (53), 1003.1 ; 987.3 (7); 879.3 ; 863.4 ; 841.2 ; 827.3 (4); 684.1 ; 679.2 .see Fig. . CD (MeOPd‐NCC‐35 (2). tR = 35.5 min. UV/VIS (4mm aq. AcONH4/MeOH 63:37): 284 (0.75), 316 (1.00). ESI‐MS: 879.3 ; 863.3 ; 843.2 (15), 842.2 (47), 841.2 ; 825.3 (6); 809.3 ; 684.2 ; 679.2 ; 522.1 .Pd‐NCC‐40 (3). tR = 40.6 min. UV/VIS (4mm aq. AcONH4/MeOH 59:41): 278 (0.78), 316 (1.00). ESI‐MS: 755.1 ; 739.2 ; 717.3 ; 701.3 ; 681.2 (10), 680.1 (40), 679.2 ; 647.2 ; 522.1 .Pd‐NCC‐54. UV/VIS (4mm aq. AcONH4/MeOH 40:60): 316 nm. ESI‐MS: 699.2 ; 678.2 ; 663 (4), 662 (32), 661.2 ; 629.3 .Pd‐NCC‐56 (4). tR = 56.4 min. UV/VIS (4mm aq. AcONH4/MeOH 47:53): 316 nm. ESI‐MS: 845.3 ; 829.3 ; 809.2 (15), 808.2 (46), 807.2 ; 775.3 ; 684.2 ; 645.2 .Pd‐NCC‐60 (5). tR = 60.0 min. UV/VIS (4mm aq. AcONH4/MeOH 44:56): 315 (1.00). ESI‐MS: 683.2 ; 667.3 ; 647.2 (11), 646.2 (39), 645.2 ; 613.2 ; 522.1 .Pd‐NCC‐60 (5) and Cj‐NCC‐1Pd‐NCC‐60 (5), of Cj‐NCC‐1, as well as a 1:1 mixture of both were analyzed by anal. HPLC . tR = 70.7 min. UV/VIS (50mm aq. potassium phosphate buffer (pH 7.0)/MeOH 20:80): 239sh (1.00), 316 (0.87). ESI‐MS: 667.1 ; 631 (2), 630 (35), 629.2 ; 597.2 ; 506 . Provisional identification of Pd‐NCC‐71 (6) with Cj‐NCC‐2Pd‐NCC‐71 (6), a separate sample of purified Cj‐NCC‐2, as well as a mixture of both were analyzed by anal. HPLC.Pd‐YCC‐61. tR = 61.3 min. UV/VIS (50mm aq. potassium phosphate buffer (pH 7.0)/MeOH 40:60): 246 (0.73), 313 (1.00), 429 (1.77). ESI‐MS: 881.1 ; 865.3 ; 843.1 ; 827.3 ; 807.2 (17), 806.1 (48), 805.1 ; 796.6 (16); 774.4 (26); 756.3 (17); 700.3 (15); 643.1 ; 611.3 .Pd‐YCC‐67. tR = 67.1 min. UV/VIS (50mm aq. potassium phosphate buffer (pH 7.0)/MeOH) 25:75): 247 (0.74), 316 (1.00), 428 (1.29). ESI‐MS: 719.1 ; 703.2 ; 681.1 ; 665.3 ; 645.2 (11), 644.2 (40), 643.2 ; 611.2 .Pd‐PiCC‐75. tR = 75.0 min. UV/VIS (50mm aq. potassium phosphate buffer (pH 7.0)/MeOH 1:9): 314 (0.75), 525 (1.00).Pd‐PiCC‐75 (in an extract of a plum tree leaf) and of a purified sample of Cj‐PiCCProvisional identification of


Glycosylation of the immunoglobulin G (IgG)-Fc tail is required for binding to Fc-gamma receptors (FcγRs) and complement-component C1q. A variety of IgG1-glycoforms is detected in human sera. Several groups have found global or antigen-specific skewing of IgG glycosylation, for example in autoimmune diseases, viral infections, and alloimmune reactions. The IgG glycoprofiles seem to correlate with disease outcome. Additionally, IgG-glycan composition contributes significantly to Ig-based therapies, as for example IVIg in autoimmune diseases and therapeutic antibodies for cancer treatment. The effect of the different glycan modifications, especially of fucosylation, has been studied before. However, the contribution of the 20 individual IgG glycoforms, in which the combined effect of all 4 modifications, to the IgG function has never been investigated. Here, we combined six glyco-engineering methods to generate all 20 major human IgG1-glycoforms and screened their functional capacity for FcγR and complement activity. Bisection had no effect on FcγR or C1q-binding, and sialylation had no- or little effect on FcγR binding. We confirmed that hypo-fucosylation of IgG1 increased binding to FcγRIIIa and FcγRIIIb by ~17-fold, but in addition we showed that this effect could be further increased to ~40-fold for FcγRIIIa upon simultaneous hypo-fucosylation and hyper-galactosylation, resulting in enhanced NK cell-mediated antibody-dependent cellular cytotoxicity. Moreover, elevated galactosylation and sialylation significantly increased (independent of fucosylation) C1q-binding, downstream complement deposition, and cytotoxicity. In conclusion, fucosylation and galactosylation are primary mediators of functional changes in IgG for FcγR- and complement-mediated effector functions, respectively, with galactose having an auxiliary role for FcγRIII-mediated functions. This knowledge could be used not only for glycan profiling of clinically important (antigen-specific) IgG but also to optimize therapeutic antibody applications. The importance of the biological properties of antibodies to specifically engage a target of choice and activate complement and Fc gamma receptors (FcγR) on immune cells  is curreThis fucose residue is part of a conserved glycan on asparagine 297 in the Fc domain of immunoglobulin G (IgG). This glycan is important for the quaternary structure of the Fc part, since its removal abrogates binding of FcγR and C1q and hence the antibody’s effector functions , 11. ThiN-acetylglycosamines and mannoses and can be found in human serum with variable levels of core fucose, bisecting N-acetylglycosamine, galactose, and terminal sialic acids  or RBCs. NK cell isolation was only performed with blood from well-genotyped donors who do not express FcγRIIc  to excluEscherichia coli strain DH5α was used for recombinant DNA work. Restriction endonucleases, DNA modification enzymes were obtained from Thermo Fisher Scientific . Oligonucleotides were obtained from Geneart (Thermo Fisher Scientific) or Integrated DNA Technologies .Variable (V) genes for anti-human RhD (anti-D clone 19A10) heavy and light chain were sequenced from a single human B cell from a hyper immunized donor . A singll-fucose (2FF)  or 1 mM 2-deoxy-2-fluoro-d-galactose (2FG) (Carbosynth), respectively, was added to the cell suspension 4 h post transfection. To increase bisecting GlcNAc, 1% pEE6.4 + GNTIII encoding mannosyl -glycoprotein beta-1,4-N-acetylglucosaminyltransferase (GNTIII) enzyme was co-transfected with 99% IgG1-κ HC + LC vector. To increase galactose, 1% pEE6.4 + B4GALT1 encoding β-1,4-galactosyltransferase 1 (B4GALT1) enzyme was co-transfected with 99% IgG1 vector and 5 mM d-galactose  was added to the cell suspension 1 h before transfection. To increase sialylation, the level of galactosylation must also be elevated as sialic acid is the terminal sugar group with galactose residues as substrate. Thus, 1% pEE6.4 + B4GALT1 and 2.5% pEE14.4 + STGALT encoding β-galactoside alpha-2,6-sialyltransferase 1 (ST6GALT) were both co-transfected 96.5% IgG1 vector and 5 mM d-galactose was added to the cell suspension 1 h before transfection. To further increase sialylation, in vitro sialylation (ivs) was performed on the purified in vivo sialylated IgG created using the previous method. Recombinant human α-2,6-sialyltransferase  and cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-NANA) (Roche) were incubated at 37°C for 24 h with purified IgG1 with already in vivo enhanced galactose and sialic acid (as described above), after incubation samples were re-purified with protein A, as described previously (IgG1 production in human embryonic kidney (HEK) F cells and purification using protein A affinity chromatography was performed as described previously by Kruijssen et al.  Glyco-eneviously , 34.via a CaptiveSpray source with a NanoBooster . The latter enriched the N2 flow (3 l/min) with CH3CN (pressure 0.2 bar), resulting in increased sensitivity. The samples were ionized in positive ion mode at 1,100 V. The Maxis Impact quadrupole-TOF–MS  was used as detector. MS1 spectra were collected at a frequency of 1 Hz with a scan range of m/z 550–1,800. The mass spectrometric data were calibrated internally in DataAnalysis 4.0  using a list of known IgG glycopeptide masses. MSConvert (Proteowizard 3.0) (m/z window of ±0.2 and within a time window of ±15 s surrounding the retention time) was extracted using the in-house developed 3D Max Xtractor software tool. If above a signal:background ratio of 3, the background-subtracted area of the first three isotopic peaks of each glycopeptide in both 2+, 3+, and 4+ charge state were summed, and this summed value was then divided by the total summed value of all IgG1 glycopeptides to arrive at a percentage for each glycopeptide. From these percentages, we calculated several derived traits using the following formulas: fucosylation (H3N3F1 + H4N3F1 + H5N3F1 + H6N3F1 + G0F + G1F + G2F + H6N4F1 + G0FN + G1FN + G2FN + H6N5F1 + H4N3F1S1 + H5N3F1S1 + H6N3F1S1 + G1FS + G2FS + H6N4F1S1 + G2FS2 + G1FNS + G2FNS + H6N5F1S1 + G2FNS2), bisection (H6N4F1 + G0FN + G1FN + G2FN + H6N5F1 + H6N4F1S1 + G1FNS + G2FNS + H6N5F1S1 + G2FNS2 + H6N4 + G0N + G1N + G2N + H6N5 + H6N4S1 + G1NS + G2NS + H6N5S1 + G2NS2), galactosylation [(H4N3F1 + H5N3F1 + G1F + H6N4F1 + G1FN + H6N5F1 + H4N3F1S1 + H5N3F1S1 + H6N3F1S1 + G1FS + H6N4F1S1 + G1FNS + H6N5F1S1 + H4N3 + H5N3 + H6N3 + G1 + H6N4 + G1N + H6N5 + H4N3S1 + H5N3S1 + H6N3S1 + G1S + H6N4S1 + G1NS + H6N5S1) * 0.5 + G2F + G2FN + G2FS + G2FS2 + G2FNS + G2FNS2 + G2 + G2N + G2S + G2S2 + G2NS + G2NS2], sialylation [(H4N3F1S1 + H5N3F1S1 + H6N3F1S1 + G1FS + G2FS + H6N4F1S1 + G1FNS + G2FNS + H6N5F1S1 + H4N3S1 + H5N3S1 + H6N3S1 + G1S + G2S + H6N4S1 + G1NS + G2NS + H6N5S1) * 0.5 + G2FS2 + G2FNS2 + G2S2 + G2NS2], hybrid-types (H5N3F1 + H6N3F1 + H6N4F1 + H6N5F1 + H5N3F1S1 + H6N3F1S1 + H6N4F1S1 + H6N5F1S1 + H5N3 + H6N3 + H6N4 + H6N5 + H5N3S1 + H6N3S1 + H6N4S1 + H6N5S1), and high-mannose (H5N2 + H6N2 + H7N2 + H8N2 + H9N2). For some of the minor hybrid-type glycans, it could not be determined conclusively whether a galactose or a bisecting N-acetylglucosamine was present, so an educated guess was made based on structural knowledge .Immunoglobulin G-Fc glycan composition of produced IgG1 was determined by mass spectrometry as described previously by Dekkers et al.  Trypsin-ard 3.0)  was usedard 3.0)  was usedProtein A purified IgG was analyzed for monomeric and dimeric IgG on a Superdex 200 10/300 gel filtration column  connected to an Äkta explorer  HPLC system at RT with a flow rate of 0.5 ml/min and PBS as running buffer. Elution profiles were obtained by recording the absorbance at 215 nm.Human FcγR constructs  for surface plasmon resonance (SPR) analysis were obtained from Sino biological . To further include all human FcγRs, a fusion Fc–FcγR construct composed of the extracellular domain of the FcγRIIIb in both allotypes followed by a Fc domain was created. To create the fusion Fc–FcγRIIIb constructs the amino acid code of the extracellular domain of either FcγRIIIb of NA1 allotype or FcγRIIIb NA2 allotype  NCBI re. For bioKD) was done using an equilibrium analysis by linear intrapolation to Rmax = 500  at 109 cells/ml. An amount of 105 erythrocytes were incubated with NK cells for 2 h at 37°C in a 2:1 ratio in Iscove’s modified dulbecco’s medium  supplemented with 10% fetal calf serum  and anti-D IgG1-glycoforms at a total volume of 100 µl. To determine 100% lysis, 2.5% saponine  was added to RBC in control wells and spontaneous lysis (sp) was determined by incubation of RBC without NK cells. Supernatants were collected and released 51Cr was quantified in a Packard Cobra II Auto-Gamma Counter Model D5005 (PerkinElmer). Percentage cytotoxicity was determined by the following formula: NK cells were isolated from Ficoll-Plaque™-Plus  gradient obtained PBMCs by a CD56 magnetic-activated cell separation isolation kit , according to manufacturer’s description. D + RBCs were isolated and labeled with radioactive chromium  and incubated 30 min at RT. To remove unbound TNBS, the solution was dialyzed  using a dialysis cassette  for 1.5 h at RT against PBS and additionally overnight at 4°C to obtain HSA-TNP.A 2.4-mM 2,4,6-trinitrobenzenesulfonic acid (TNBS) (Sigma-Aldrich) solution was added to 20 mg human serum albumin (HSA) diluted to 20 mg/ml  in 0.2 M NA+/+/plx {veronalbuffer  + 10 mM CaCl2 (Merck) + 2 mM MgCl2 (Merck) + 0.1% poloxamer} for 1 h at RT. When C1q was blocked, 10 min prior to addition of serum to the ELISA plate, anti-C1q-85 blocking antibody  and the reaction was stopped with the addition of 100 µl 2 M H2SO4 (Merck). The optical density (OD) was measured at A450 nm using a plate reader .To coat, maxisorp plates  were incubated o/n at RT with 20 µg/ml HSA-TNP in PBS. The plates were washed 5× with PBS + 0.1% tween-20 (Sigma-Aldrich) (wash buffer) using an ELISA washer . All following washing steps were done similarly. The IgG samples were diluted in 100 µl PBS/plx [PBS + 0.1% poloxamer ] per well and incubated for 1.5 h at RT. The plates were washed and incubated with 100 µl 1:35 serum pool in VBantibody  was addeti-C1q-2 , 0.5 µg/ti-C1q-2 , 0.6 µg/ti-C1q-2 , or 1 µgThe results were analyzed with a parallel line assay in Microsoft Office Excel . We asse+ RBCs obtained from heparinized blood were mixed with 350 µl 0.313 mM TNBS in 0.15 M Na2HPO4, pH 8.8 and incubated for 10 min at RT. TNPylated RBCs were centrifuged for 2 min at 350 × g and washed two times with PBS. RBC were resuspended into VBG+/+ [VB+/+ + 0.05% w/v gelatin (Sigma-Aldrich)]. Anti-TNP IgG1 was serially diluted in VBG−/− . In round bottom plates to a final volume of 100 µl we combined the diluted IgG1, 10% serum, ~4.5 × 106 RBC, and a glass bead  to ensure mixing of the solution during incubation . This amount of RBC was taken to ensure the 100% absorbance between 1.8 and 2.2 delta (Δ) A412–A690 nm. The plates were incubated for 90 min at 37°C while shaking at 150 rpm . After incubation, 1.25% w/v saponine was supplemented to the 100% control wells, 100 µl VBG−/− was added to all wells and the plates were centrifuged for 2 min at 350 × g. Subsequently, 150 µl of supernatant was transferred into a separate plate and OD was measured at Δ A412–A690 nm using a plate reader. The percentage of lysed cells was calculated as follows: 50) for each replicate of the different glycoforms using a non-linear fit for normalized response with a variable slope and combined these to an average EC50.Fifty microliters of washed, packed, Dp < 0.05 using two-tailed tests.Statistical analyses were performed using GraphPad Prism version 6.00 for Windows . The level of significance was set at x-axis legend, Figures N-acetylglucosaminyltransferase III (GntIII) to increase bisection —but this was only observed in samples with low starting-levels of galactosylation   Figure , 47, 48.) Figure , with boKD of more than twofold from unmodified IgG to be potentially meaningful changes and within the scope of the SPR method, using a simplified 1:1 Langmuir model that does not fully represent the actual interaction which is more complicated  and subsequent C4b deposition was titrated by serial dilution . All glycovariants of anti-TNP bound TNP-HSA equally well , but C1q binding and C4b deposition differed profoundly for the different glycoforms . The relative C1q binding and C4b deposition were then calculated Figures A and witWe have previously created an orthogonal set of glyco-engineering tools  which weOf the FcγRs, we only observed an effect of glycosylation on binding to the FcγRIII-family of receptors, both FcγRIIIa and FcγRIIIb and their allotypes, which confirms and expands recent studies using a limited set of glycovariants presented here , 30. IncThe enhanced binding of galactosylated and afucosylated IgG was slightly weakened by addition of sialic acid, but only if a bisecting GlcNAc was present. A similar negative effect of sialylation has previously been observed for mouse FcγR by Ravetch and colleagues . ImportaIn vitro, this has been found result in stronger functional efficacy for the V158-variant  . In vitr-variant –57. In vlearance . It shoulearance . We alsolearance , 62, 63.Importantly, the observed changes in FcγRIIIa-binding due to glycosylation reliably translated into functional NK cell-mediated ADCC lysis of RBC. For FcγRIIIa and FcγRIIIb it was known that absence of IgG-Fc core-fucosylation increases the affinity of interaction due to a glycan–glycan interaction between the Fc glycan and the N162-glycan uniquely found in the FcγRIII family . Our appThe possible effect of the Fc-glycans on complement activity, has until now remained enigmatic. It has been proposed for a long time that agalactosylated IgG activates complement more efficiently through the lectin pathway (MBL) . To our Low galactosylation level in total IgG generally correlates with disease severity of several autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis , 14. WhiIn summary, we show here that a set of glyco-engineering techniques we recently developed  can be cPeripheral blood from anonymous, healthy volunteers was obtained with informed, written consent of all subjects, in accordance with Dutch regulations. This study was approved by the Sanquin Ethical Advisory Board in accordance with the Declaration of Helsinki.GD, TK, DW, TR, MW, and GV designed the research. GD, AB, DW, TR, MW, and GV designed the experiments. GD, LT, RP, AB, MdB, CK, SL-T, RV, and YM performed the experiments. GD, RP, AB, MdB, CK, TR, TK, MW, and GV analyzed data, GD and GV wrote the manuscript. All authors contributed to and approved the final manuscript.The authors declare that this study received funding from Sanquin Bloedvoorziening, a not-for-profit organization. The funder was not involved in the study design or collection, analysis, or interpretation of the data.