Although thorium is traditionally considered to have dominant 6d-orbital contributions to its bonding, contrasting to majority 5f-orbital character for uranium, computational analyses suggests that the bonding of thorium can be more nuanced, in terms of 5f- versus 6d-orbital composition and also significant involvement of the 7s-orbital and how this affects the balance of 5f- versus 6d-orbital bonding character.Despite the burgeoning field of uranium-ligand multiple bonds, analogous complexes involving other actinides remain scarce. For thorium, under ambient conditions only a few multiple bonds to carbon, nitrogen, oxygen, sulfur, selenium and tellurium are reported, and no multiple bonds to phosphorus are known, reflecting a general paucity of synthetic methodologies and also problems associated with stabilising these linkages at the large thorium ion. Here we report structurally authenticated examples of a parent thorium(IV)–phosphanide (Th–PH Despite the burgeoning field of uranium-ligand multiple bond chemistry, analogous thorium compounds remain scarce. Here, the authors synthesize and characterize a series of thorium complexes with multiple bonds to phosphorus, and probe their 5f versus 6d orbital bonding character. In recent years the study of actinide-ligand bonding has flourished due to a desire to better understand the nature of covalency in these linkages in terms of the extent of covalency and levels of metal-derived 7s-, 7p-, 6d- and 5f-orbital character1245671011113141516171820212223242526272930313334353738395-C5Me5)2(OMe)}2(μ-PH)] ] ref.  has beenTIPS)(EH2)] (TrenTIPS=N(CH2CH2NSiPri3)3; E=N, P, As] and parent uranium-imido/-phosphinidene/-arsinidene [U(TrenTIPS)(EH)][K(L)2] (L=a 15-crown-5 ether) complexes2033TIPS)AsK2]4 could be prepared52TIPS)(PH2)] (2), parent thorium(IV)–phosphinidene [Th(TrenTIPS)(PH)][Na(12C4)2] , parent dithorium(IV)–phosphinidiide [{Th(TrenTIPS)}2(μ-PH)] (5) and dithorium(IV)-μ-phosphido [{Th(TrenTIPS)}2(μ-P)][Na(12C4)2] (6) complexes. These complexes can be prepared by more than one route using a new thorium(IV) separated ion pair [Th(TrenTIPS)(DME)][BPh4] (1) or the thorium(IV)-cyclometallate complex [Th{N(CH2CH2NSiPri3)2(CH2CH2NSiPri2C(H)MeCH2)}] (4)  ref. . QuantumTIPS)(Cl)] and MEH2  did not work because these pnictides are too soft to displace the hard chloride, and instead the separated ion pair complex [U(TrenTIPS)(THF)][BPh4] (OCH2CH2CH2CH2NEt3)][BPh4] (1 in 91% isolated yield (In our prior work33h4] ref.  must be f 4 ref.  with Et3)][BPh4]  as the med yield .1 with NaPH2 in DME affords the colourless parent thorium(IV)–phosphanide complex 2 in 39% crystalline yield, 31P{1H} nuclear magnetic resonance (NMR) spectrum exhibits a singlet at −144.1 p.p.m., which resonates as a triplet in the 31P NMR spectrum (JPH=159 Hz) confirming the presence of the two phosphanide hydrogen atoms that resonate as a doublet at 1.4 p.p.m. in the 1H NMR spectrum, as determined by two-dimensional (2D)-correlation . For unequivocal confirmation, when 2 is prepared using NaPD2 to give 2D the two absorptions at 2,276 and 2,251 cm−1 shift to 1,661 and 1,639 cm−1 (P-H/νP-D=1.38)  ref. .2 with benzyl sodium and two equivalents of 12C4 ether in benzene affords the yellow terminal parent thorium(IV)–phosphinidene complex 3, 3 can be prepared from the cyclometallate complex 4 , confirming the presence of the phosphinidene hydrogen atom . When 3 is prepared using NaPD2 to give 3D the intensity of the absorbance at 2,083 cm−1 decreases significantly and a shoulder peak at ∼1,500 cm−1 becomes broader and more ill-defined (P-H/νP-D=1.39)  ref. .3 established, we investigated routes to thorium–phosphido complexes and elucidated two routes to a μ-phosphido {P(H)Na}] complex reacts with NaPH2 to eliminate Na2PH and generate 2, the latter of which reacts further with 4 to give 5. The yellow dithorium(IV)-μ-phosphido complex 6 can be prepared by treating 3 with one equivalent of 4 in benzene2 treated with two equivalents of 4 . A P-H stretching frequency of 2,216 cm−1 is computed for 5, likely reflecting the lack of an anharmonic correction and the ill-defined position of the P-H vibration experimentally. The ATR-IR spectrum of 6 °). The Th1-P1 bond length of 2.982(2) Å is slightly longer than the sum of the single-bond covalent radii2 is at the upper end of thorium–phosphanide bond lengths, a feature noted with the uranium congener, and can be compared with Th–P distances in [Th(η5-C5Me5)2(μ-PPh2)2Ni(CO)2] (av. 2.885 Å), [Th(η5-C5Me5)2(μ-PPh2)2PtPMe3] (av. 2.923 Å), [Th(η5-C5Me5)2(PPh2)2] (av. 2.866 Å), [Th(η5-C5Me5)2{P(SiMe3)2}(Me)] (2.888(4) Å) and [Th(η5-C5Me5)22] (av. 2.879 Å)  Å) (ref. Complex  Å) refs , 58, 59.] av. 2.8 Å,  }2(μ-P)] [TrenPr=N(CH2CH2NC(H)Me2)3] (ref. 5-C5Me5)2}2(μ-P)] (ref. amine-Th–P angles (N4-Th1-P1=178.16(12); N8-Th2-P1=175.78(12)°) are similar to 5 and the Th1-P1-Th2 angle of 176.21(8)° is almost linear. The removal of the phosphinidiide proton to generate the μ-phosphido unit is reflected by the Th1–P1 and Th2–P1 distances of 2.740(2) and 2.735(2) Å, respectively, which are 0.15 Å shorter than the Th–P distances in its formal precursor 5, ∼0.24 Å shorter than the Th–P single-bond distance in 2, and ∼0.01 Å shorter than the formal Th=P double bond in 3. The thorium(IV)-amide and -amine bond lengths (av. 2.366 and 2.818 Å), like anionic 3, are longer than in neutral 2 and 5.The dithorium(IV)-μ-phosphido formulation of )3] ref.  and moreP)] ref. . The Nam2, 3, 5 and 6 we computationally probed these molecules with DFT, NBO and QTAIM topology analyses and salient data are compiled in 2 and 5 and the whole-anion components of 3 (−3) and 6 (−6). Bond lengths and angles of the geometry optimized structures (To gain more insight into the nature of the Th–P bonding in ructures  are compq charges for 2 (2.47), −3 (2.24) and 5 (2.96/3.05) are in-line with thorium(IV)-Tren complexes  are high and suggest ionic bonding between the two thorium ions and the phosphido. The calculated phosphorus MDC-q charges reflect the changes from mono-anionic phosphanide (−0.56) to dianionic terminal phosphinidene (−0.91) to dianionic bridging phosphinidiide (−2.26) and trianionic phosphido (−2.07). The calculated Th–P Mayer bond orders of 0.77 and 1.67 for 2 and −3 are consistent with formal polarized covalent Th–P single and Th=P double bonds, respectively. The analogous bond orders for 5 (0.94 and 0.95) suggest formal Th–P single bonds augmented by donation from the remaining phosphorus lone pair to each thorium, whereas the corresponding bond orders for −6 (1.26 and 1.28) are consistent with the electron-rich phosphido forming Th=P pseudo double-bond interactions with each thorium but because bridging not as strongly as the terminal phosphinidene.The computed thorium MDC-xes ref. , and for2, −3, 5 and −6, 2 and is predominantly a phosphorus 3p-orbital that constitutes a σ-bonding interaction and pseudo lone pair. In 3, the quasi-degenerate HOMO and HOMO-1 orbitals principally represent the thorium(IV)-phosphinidene double bond, with the former of π-symmetry, lying ∼0.02 eV higher in energy than the latter which is the σ-bond. For 5, the HOMO is a phosphorus 3p-orbital lone pair orientated perpendicular to the Th–P–Th and P–H vectors and is partially polarized towards the two thorium centres giving weak π-interactions; the HOMO−1 lies ∼0.67 eV below the HOMO and is a phosphorus 3p-orbital aligned along the Th–P–Th vector to give a 3-centre-2-electron σ-bonding interaction. The HOMO to HOMO−2 of −6 represent the main Th–P interactions in this compound. The HOMO and HOMO−1 of −6 are two quasi-degenerate (∼0.04 eV separation) mutually orthogonal phosphorus 3p orbitals aligned perpendicular to the Th–P–Th axis and polarized to an extent that significant π-interactions to each thorium centre are exhibited. The HOMO−2 of −6 lies ∼0.44 eV below the HOMO−1 and like 5 represents a 3-centre-2-electron Th–P–Th σ-bonding interaction utilizing a phosphorus 3p-orbital. When taken together the two delocalized Th–P π-interactions along with the σ-bonding constitutes pseudo triple bonds to each thorium but equate to overall Th=P double bonds in a Lewis bonding scheme, although each is not as well-developed as the phosphinidene Th=P interaction  with the remainder being 45% 5f and 54% 6d character. For 5 and −6 ionic Th–P interactions are returned. The σ-bonds in 5 and −6 return 6–8% thorium contributions with the remaining 92–94% being phosphorus character. Within the thorium contribution to the Th–P σ-bonds, the 7s contribution returns at 11–14%, but 7p contributions remain negligible at 1% and the 5f and 6d contributions are consistently 35–38 and 50–52%, respectively. For both 5 and −6 the phosphorus 3p orbitals arranged orthogonally to the Th–P–Th bonds are essentially localized at phosphorus and due to the NBO detection threshold we can only say that the thorium contributes <5% to these Th–P bonds.Although the NBO calculations emphasize a more ionic bonding scenario than the DFT picture2, −3, 5 and −6 using the QTAIM approachρ(r), the Laplacian of the electron density ∇2ρ(r), the electronic energy density H(r) and the bond ellipticity parameters ɛ(r). In each case 3,−1 bond critical points (BCPs) were found indicating the presence of Th–P bonds. The BCP data with ρ(r) values of 0.05–0.07 suggest fairly ionic bonding combinations with modest covalent contributions; covalent bonds tend to exhibit ρ(r) values >0.2 but whether this convention, formulated for lighter elements, is appropriate for heavy actinide elements is debatable2 can be considered to present a benchmark and is fairly ionic with a spherical σ-bond (ɛ(r)=0.07). For comparison, a σ-bond or a bond with two π-components presents a cylindrical spread of electron density (ɛ(r) ∼0), whereas a bond with one π-component should be asymmetric (ɛ(r)>0); for example, the ɛ(r) parameters for the C–C bonds in ethane, benzene, and ethene are calculated to be 0.0, 0.23 and 0.45, respectively2, −3 exhibits an ɛ(r) parameter of 0.40 consistent with the presence of a Th=P phosphinidene double bond, and although it is polarized it exhibits a marginally larger ρ(r) value of 0.07 compared with 2. The two ρ(r) values of 0.05 for 5 are the same as for 2, however the Th–P bonds in 5 are asymmetric with ɛ(r) values averaging 0.26, and the QTAIM data suggest that the lone pair on the phosphinidiide may be interacting with the two thorium centres to generate asymmetric Th–P bonding combinations. The ρ(r) values for −6 (0.06) are intermediate to 2 and −3 and the ɛ(r) values are close to zero (0.01 and 0.02), consistent with Th–P bonds with one σ- and two π-bond compositions.We examined the topologies of the Th–P bonds in 1. We have not observed any issues preparing [U(TrenTIPS)(THF)][BPh4] (ref. TIPS)(OCH2CH2CH2CH2NEt3)][BPh4] is the major product of the analogous preparation1 without complication.The difference in the (six-coordinate) effective ionic radiih4] ref.  in neat 2P− parent phosphanide at thorium is relatively straightforward like the uranium analogue3 can be prepared by two routes, either by deprotonation of 2 and abstraction of the sodium ion with 12C4 or by treatment of the thorium(IV)-cyclometallate complex 4 with the sodium phosphanide transfer reagent and 12C4. The latter method has the benefit that it circumvents the requirement to convert 4 to a separated ion pair followed by salt elimination, and instead deprotonation of the phosphanide, protonation of the cyclometallate, formation of a Th=P double bond and abstraction of the sodium ion are all effected in one step. Whilst the preparation of 3 from 4 is notable because it works with so many bond cleavage and formation steps occurring in one reaction, we note that when 3 is prepared from 4 compound 6 is also formed in small but significant amounts (∼15%), presumably due to the large size of thorium coupled to the relative rates of reaction between NaPH2 and 4 and once formed 3 with 4. Germane to this point, we note that 3 is more prone to decomposition than the uranium(IV) analogue, presumably reflecting the larger size and more ionic bonding of thorium(IV) compared with uranium(IV) (see below).Installation of the H5 and 6, apart from the multiple ways that they may be prepared, is the straightforward way the P–H bonds can be sequentially cleaved, leading to increasing formal charge accumulation at phosphorus. Thus, 4 easily removes a proton from 2 to give the dithorium(IV)-phosphinidiide 5, perhaps reflecting high reactivity of the Th–C bond in 4. More surprising, is that the dithorium(IV)-μ-phosphido 6 can be prepared directly from 4 and NaPH2 with elimination of Na2PH, though the reaction 2 NaPH2→Na2PH+PH3 has precedent.A surprising aspect of the synthesis of −1 for 2 compare well with 2,295 and 2,255 cm−1 for [U(TrenTIPS)(PH2)] 2(OMe)}2(μ-PH)] 3 of 2,083 cm−1 is shifted from the P–H stretch of 2,350 cm−1 for the uranium(IV)-phoshinidene analogue31P NMR chemical shifts of 2, 3, 5 and 6 , double-bond deshielded , moderately deshielded  and twice double-bond substantially deshielded , consistent with their phosphanide, phosphinidene, phosphinidiide and phosphido formulations.ATR-IR data are consistent with the proposed formulations, and are generally similar to uranium analogues where comparisons are available. The P–H stretches of 2,276 and 2,251 cm2)] ref. , and the 5 and 6  follow t2 confirms a rare example of a terminal H2P− phosphanide anion2 is in good agreement with the sum of the single-bond covalent radii for thorium and phosphorus5455565758TIPS ligand whereas all other thorium(IV)–phosphanide complexes involve a bis(pentamethylcyclopentadienyl) ligand combination that presents a sterically more accessible wedge for phosphanide ligation5455565758The solid-state structure of 3 confirms the presence of a terminal thorium(IV)–phosphinidene linkage. On the basis of the sum of the covalent double-bond radii of thorium and phosphorus3 can be considered to be long. However, a similar effect was observed in the uranium(IV) analogueTIPS)(PH)]− fragment in 3 and the sterically demanding nature of TrenTIPS. Moreover, unlike in 2 where the Th–P distance can be considered on the basis of additive covalent single-bond radii to be long when compared with the uranium analogue, in 3 the Th=P bond is ∼0.05 Å longer than the corresponding U=P bond3 can thus be considered shorter than might have been expected on the basis of the uranium congener. Germane to this point is the acute Th=P-H bond angle, which is reminiscent of the acute M=C(R)-H angles in some alkylidenesJPH coupling moving from 2 (JPH=159 Hz) to 3 (JPH=107 Hz) as well as the infrared data, and this suggests the thorium is electron deficient and so seeks electron donation from any available electron reservoir, which may also modulate the Th=P distance.The crystal structure of 5 are between the Th–P distances in 2 and 3. This can be explained on the basis that the Th–P distances in 5 should be shorter than in 2 because the HP2− carries a greater charge than the H2P− unit. However, since the dianionic charge of HP2− is spread between two metals in 5 but only one metal in 3 it is logical that the former presents longer Th–P bonds than the latter. The Th–P distances in 5 are slightly longer than in the only other structurally authenticated dithorium(IV)–phosphinidiide complex  (ref. TIPS compared with the bis(pentamethylcyclopentadienyl) ligand combination. The essentially linear Th–P–Th angle in 5 contrasts to the only other structurally characterized actinide parent phosphinidiide complex [{U(η5-C5Me5)2(OMe)}2(μ-PH)]6 are shortened markedly compared with those in 5, and indeed as well as being the first example of an actinide–phosphido complex outside of matrix isolation523 and 6. However, we note that the Th–P distances in 6 are ∼0.29 Å longer than the sum of the double-bond covalent radii3, but this can be justified on basis of the anionic formulation of the dithorium(IV)-μ-phosphido component of 6 and also shorter Th–P distances being precluded by the bulky TrenTIPS ligands that efficiently encapsulate the Th–P–Th unit. Support for these suggestions comes from inspection of the thorium(IV)–amide and –amine bond lengths which are, like for 3, relatively long. Notably, however, even considering the anionic nature of the dithorium(IV)–phosphido unit in 6 the thorium(IV)–amine distances are very long (>2.8 Å), which we suggest results from a strong trans-influence from the trianionic μ-phosphido.The two Th–P bond lengths in 2}] ref. , but thiq charges suggest that the phosphorus ligands in the complexes reported here in terms of donor ability can be ordered: HP2− >H2P− >HP2− (bridging)>P3− (bridging) for −3, 2, 5 and −6. This is consistent with what might be anticipated except for the μ-phosphido, but the bridging nature of this group clearly attenuates its donor capacity. Interestingly, calculated Mayer bond orders arrange the ligands as HP2− >P3− (bridging)>HP2− (bridging)>H2P− , as would be expected, highlighting that for thorium care must be taken when attempting to correlate calculated charges to ligand donor strength. The charge on phosphorus increases from −0.56 to −0.91 commensurate with the formally mono-anionic phosphanide and dianionic phosphinidene formulations in 2 and −3, respectively. As expected the phosphinidiide in 5 carries a charge of −2.26 consistent with its dianionic formulation, and the fact that it is higher than the also dianionic phosphinidene unit in −3 reflects that it is bound more ionically to the two thorium centres in 5 than the phosphinidene to one thorium ion in −3. The charge on the μ-phosphido in −6 is lower than the phosphinidiide in 5 but the μ-phosphido would be expected to be a better donor than the phosphinidiide with formal single Th–P bonds.The MDC-2, −3, 5 and −6 is that where comparisons can be made with analogous uranium(IV) complexes the thorium(IV) bonding is more ionic. For example, in [U(TrenTIPS)(PH2)] and [U(TrenTIPS)(PH)]−  values of 0.05 for both thorium and uranium parent phosphanides, shows a slight difference between thorium and uranium for the corresponding phosphinidenes (0.07 versus 0.08); although the latter data present a small change it is consistent with the overall trend. The most striking difference of the thorium contributions to the thorium–phosphorus bonds compared with the uranium analogues is the far greater involvement of 7s-orbital contributions to the former, as high as 14%, which is in stark contrast to the latter which present at most 2% 7s-orbital contributions. Also, the 5f:6d balance is different for thorium compared with uranium. For the phosphanides the 5f-orbital contributions are essentially the same for thorium and uranium, but for thorium the 6d-orbital contribution is reduced compared with uranium because of significant 7s-orbital involvement. Where the phosphinidenes are concerned, however, the 7s- and 7p-orbital contributions are negligible for both thorium and uranium, but whereas for the former the 5f- and 6d-orbital contributions are comparable  for the latter the 5f-orbital dominates . Interestingly, for 5 and −6, the 5f- and 6d-orbital components are reasonably balanced at ∼36 and 50%, respectively, which is perhaps surprising given that thorium bonding is traditionally considered to have dominant 6d-orbital character whereas uranium is usually considered to have dominant 5f-orbital contributionsThe DFT and NBO analyses return the anticipated Th–P bonding interactions and notably in all cases these bonding interactions return π-bonds higher in energy, even if only slightly, than σ-bonds as is normally the case. This contrasts to uranium-nitrides3133)]− ref.  the bondExperiments were carried out under a dry, oxygen-free dinitrogen atmosphere using Schlenk-line and glove-box techniques. All solvents and reagents were rigorously dried and deoxygenated before use. All compounds were characterized by elemental analyses, NMR, FTIR and UV/Vis/NIR electronic absorption spectroscopies, single-crystal X-ray diffraction studies, and DFT, NBO and QTAIM computational methods. See also 4  and [HNEt3][BPh4)] . The colourless slurry was allowed to warm to room temperature and was stirred at room temperature for 16 h. The solvent and volatiles were removed in vacuo affording a colourless oil. The oil was washed with HMDSO (2 × 10 ml) to afford a colourless powder that was isolated by filtration and dried in vacuo. Yield: 2.12 g, 64%. Crystalline material suitable for X-ray crystallography was obtained from a solution in THF (3 ml) layered with hexanes (1 ml). Anal. Calcd for: C67H118BN5OSi3Th: C, 60.20; H, 8.90; N, 5.24%. Found: C, 59.09; H, 8.69; N, 4.83%. 1H NMR : δ 0.77 3), 1.00 3 ), 1.36 , 2.59 , 3.36 , 3.52 , 7.20 , 7.38 , 8.08  p.p.m. 29Si{1H} NMR : not observed. 13C{1H} NMR : δ 13.37 (CH(CH3)3), 18.81 (NCH2CH3), 19.60 (CH(CH3)3), 25.62 (NCH2CH3), 41.46 (CH2), 45.92 (CH2), 46.77 (CH2), 59.86 (CH2), 63.04 (CH2), 68.92 (CH2), 122.46 (p-Ar-CH), 126.40 (m-Ar-CH), 137.28 (o-Ar-CH) p.p.m. FTIR: ν 3,054 (w), 2,940 (m), 2,861 (m), 1,461 (m), 1,261 (w), 1,101 (m), 1,062 (m), 1,009 (m), 925 (m), 881 (m), 804 (w), 730 (s), 704 (s), 673 (s) 633 (m), 547 (w), 513 (w) cm−1.THF (25 ml) was added to a cold (−78 °C) mixture of 4  and [HNEt3][BPh4)] . The colourless slurry was allowed to warm to room temperature and was stirred at room temperature for 16 h. The solution was filtered and volatiles were removed in vacuo affording 1 as a colourless powder. Yield: 0.85 g, 91%. Crystalline 1 as a DME solvate suitable for X-ray crystallography was obtained from a solution in C6D6 with a few drops of added DME stored at room temperature. Anal. Calcd for C61H105N4BO2Si3Th: C, 58.44; H, 8.44; N, 4.47%. Found: C, 58.09; H, 8.40; N, 4.68%. 1H NMR : δ 0.79 3), 1.05 3), 2.46 , 3.01 , 3.32 , 7.22 , 7.39 , 8.07  p.p.m. 29Si{1H} NMR : not observed. 13C{1H} NMR : δ 11.84 (CH(CH3)3), 19.63 (CH(CH3)3), 41.04 (CH2), 71.66 (CH2), 122.30 , 126.18 (m-Ar-CH), 136.84, (o-Ar-CH) p.p.m. FTIR: ν 3,055 (w), 2,942 (m), 2,863 (m), 1,463 (m) 1,268 (w), 1,024 (m), 1,005 (m), 925 (m), 879 (m), 810 (w), 671 (m), 632 (m), 611 (m), 571 (w), 515 (w) cm−1.DME (25 ml) was added to a cold (−78 °C) mixture of 1  and NaPH2 . The colourless slurry was allowed to warm to room temperature and stirred for 2.5 h to afford a pale orange solution. Solvent was removed in vacuo and the product was extracted into toluene. Filtration and removal of toluene in vacuo afforded a pale yellow solid. Crystalline 2 was obtained from a hexanes (4 ml) solution stored at room temperature. Yield: 0.50 g, 39%. Anal. Calcd for C33H77N4PSi3Th: C, 45.18; H, 8.85; N, 6.39%. Found: C, 45.46; H, 8.81; N, 6.09%. 1H NMR : δ 1.24 3), 1.39 3), 1.44 , 2.48 , 3.60  p.p.m. 31P NMR : δ −144.08  p.p.m. 29Si{1H} NMR : δ 5.65 (Si(CH(CH3)2)3) p.p.m. 13C{1H} NMR : δ 13.82 (CH(CH3)2), 20.26 (CH(CH3)2), 47.25 (CH2), 64.02 (CH2). FTIR: ν 2,939 (m), 2,886 (m), 2,682 (m), 2,276 (w), 2,251 (w), 1,459 (m), 1,260 (m), 1,045 (s), 1,009 (s), 923 (m), 731 (s), 672 (m), 629 (m) cm−1. For isotopomer-shift infrared studies the P–D analogue of 2, 2D, was prepared by replacing NaPH2 with NaPD2.DME (25 ml) was added to a cold (−78 °C) mixture of 2 , 12C4  and NaCH2Ph . The reaction mixture was sonicated for 5 min at room temperature to afford a yellow solution. Method ‘B': a solution of 12C4  in DME (25 ml) was added to a cold (−78 °C) mixture of 4  and NaPH2 . The yellow slurry was allowed to warm to room temperature with stirring for 15 min. Solvent was immediately removed in vacuo to afford a bright yellow solid. The product was extracted into toluene to afford a pale yellow solution and removal of the solvent in vacuo afforded a yellow solid. Recrystallization of the yellow solid from toluene (4 ml) afforded yellow crystals of 3 suitable for X-ray crystallography following storage at −30 °C for 24 h. Yield: 0.38 g, 38%. Anal. Calcd for C49H108N4PSi3ThNaO8· 0.5C7H8: C, 48.59; H, 8.70; N, 4.32%. Found: C, 48.74; H, 8.27; N, 4.64%. 1H NMR : δ 1.57 3), 2.04 3), 2.61 , 3.30 , 3.72 , 9.44  p.p.m. 31P NMR : δ 150.79  p.p.m. 29Si{1H} NMR : δ 2.48 (Si(CH(CH3)2)3) p.p.m. 13C{1H} NMR : δ 14.69 (CH(CH3)2), 21.11 (CH(CH3)2), 46.01 (CH2), 65.26 (CH2), 68.08 (OCH2) p.p.m. FTIR: ν 2,911 (m), 2,857 (m), 2,087 (w), 1,461, (w), 1,246 (m), 1,135 (s), 1,095 (s), 932 (m), 917 (m), 735 (s), 669 (m), 626 (m), 553 (m) cm−1. For isotopomer-shift infrared studies the P–D analogue of 3, 3D, was prepared by method ‘B' replacing NaPH2 with NaPD2.Method ‘A': deuterated benzene (0.5 ml) was added to an NMR tube containing 2  and 4 . The reaction mixture was sonicated for 5 min at room temperature to afford a yellow solution. Method ‘B': DME (25 ml) was added to a cold (−78 °C) mixture of 4  and NaPH2 . The yellow slurry was stirred at −78 °C for 35 min, allowed to warm to room temperature and stirred at room temperature for a further 1.5 h to afford a bright yellow solution. The solvent was removed in vacuo to afford a bright yellow solid. The product was extracted into toluene to afford a pale orange solution. Removal of the solvent in vacuo afforded a yellow solid. Recrystallization of the yellow solid from toluene (4 ml) afforded yellow crystals of 5 on storage at −30 °C for 24 h. The crystals were isolated and washed with pentane (3 × 3 ml) and dried in vacuo for 30 mins. Yield: 0.35 g, 40%. Anal. Calcd for C66H151N8PSi6Th2: C, 46.07; H, 8.85; N, 6.51%. Found: C, 46.57; H, 8.91; N, 5.85%. 1H NMR : δ 1.42 3), 1.75 3), 2.54 , 3.60  p.p.m. 31P NMR : δ 24.46  p.p.m. 29Si{1H} NMR : δ 3.17 (Si(CH(CH3)2)3) p.p.m. 13C{1H} NMR : δ 14.41 (CH(CH3)2), 20.76 (CH(CH3)2), 46.09 (CH2), 65.03 (CH2) p.p.m. FTIR: ν 2,938 (m), 2,885 (m). 2,860 (m), 2,164 (w), 1,458 (m), 1,237 (m), 104 5 (m), 930 (s), 880 (s), 861 (m), 734 (s), 671 (s), 629 (m) cm−1. For isotopomer-shift infrared studies the P–D analogue of 5, 5D, was prepared by method ‘B' replacing NaPH2 with NaPD2.Method ‘A': deuterated benzene (0.5 ml) was added to an NMR tube containing 3  and 4 . The reaction mixture was sonicated for 5 min at room temperature and yellow precipitate was observed. The solvent was removed in vacuo to afford a bright yellow solid. Method ‘B': a solution of 12C4  and NaPH2  in DME (20 ml) was added to a solution of 4  in DME (15 ml) dropwise over 15 min. The resulting yellow solution was stirred for a further 15 min and then the solvent was removed in vacuo as the resulting solid was washed with toluene to afford 6 as an analytically pure bright yellow solid. Yield: 0.48 g, 57%. Crystalline 6 suitable for X-ray crystallography was obtained by dissolution in DME (5 ml) and stored at −30 °C for 24 h. Anal. Calcd for C82H182N8PSi6Th2NaO8 · 0.8C7H8: C, 48.61; H, 8.81; N, 5.06%. Found: C, 49.05; H, 9.11; N, 5.55%. 1H NMR : δ 1.27 3), 1.57 3), 2.53 , 3.68  p.p.m. 31P NMR : δ 553.50  p.p.m. 29Si{1H} NMR : not observed. 13C{1H} NMR : δ 15.13 (CH(CH3)2), 21.62 (CH(CH3)2), 46.23 (CH2), 59.08 (CH2), 72.94 (OCH2) p.p.m. FTIR: ν 2,913 (m), 2,855.63 (m), 2,818 (m), 1,446 (w), 1,288 (w), 1,244 (w), 994 (s), 882 (m), 796 (m), 742 (s), 655 (m), 626 (m), 556 (w), 475 (w) cm−1.Method ‘A': deuterated benzene (0.5 ml) was added to an NMR tube containing 3  in Et2O (10 ml) was slowly added to a cold (−90 °C) solution of LiAlD4  in Et2O (40 ml) and stirred at this temperature for 30 min. The reaction vessel was connected to a flask containing a solution of sodium anthracenide (6.40 mmol in 50 ml THF) and cooled with liquid nitrogen. The temperature of the first reaction vessel was raised to room temperature and all volatiles condensed into the flask cooled with liquid nitrogen. The liquid nitrogen bath was exchanged with a bath at −90 °C (EtOH/liquid nitrogen) and the reaction mixture stirred overnight while the temperature was allowed to rise to room temperature. The reaction mixture was concentrated to ca. 10 ml and toluene (50 ml) was added. The suspension was centrifuged and the resulting yellowish solid washed twice with warm toluene (2 × 40 ml) each time. Drying the remaining solid in vacuo afforded a yellowish solid. Yield: 0.3 g, 78% (based on sodium anthracenide). FTIR: ν 1,622 , 775  cm−1 (A solution of PClPD) cm−1 .www.ccdc.cam.ac.uk/data_request/cif. All other data are available from the corresponding authors on request.The X-ray crystallographic coordinates for structures reported in this article have been deposited at the Cambridge Crystallographic Data Centre (CCDC), under deposition number CCDC 1476593-1476598. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via How to cite this article: Wildman, E. P. et al. Thorium–phosphorus triamidoamine complexes containing Th–P single- and multiple-bond interactions. Nat. Commun. 7:12884 doi: 10.1038/ncomms12884 (2016).Supplementary Figures 1-25, Supplementary Tables 1-4, Supplementary Methods and Supplementary References1CIF data for 2CIF data for 3CIF data for 5CIF data for 6CIF data for CIF data for ThOR


In the crystal, weak 4-tolyl-C—H⋯π°. Overall, the molecule has the shape of the letter L. In the crystal, supramolecular layers in the ab plane are formed via weak 4-tolyl-C—H⋯π(C6-ring of indole) and S—O⋯π contacts. The aforementioned interactions along with interatomic H⋯H and H⋯O contacts are all shown to make significant contributions to the calculated Hirshfeld surfaces.In the title indole derivative, C Herein, the crystal and molecular structures of (I)Nitrogen-based heterocycles comprise a class of compounds with significant biological importance that are crucial in organic synthesis  and 0.021 (14) Å for the C1 and O2 atoms, respectively. This planarity extends over the entire dioxolo-indole residue, which exhibits an r.m.s. deviation of 0.0249 Å for the 12 constituent atoms with maximum deviations of 0.058 (2) and 0.0284 (14) Å for the C1 and C8 atoms, respectively. The dihedral angle between the residues linked at the S atom is 89.95 (6)°, i.e. indicating a perpendicular relationship consistent with the shape of the letter L. The CNO2 atoms about the S atom define a tetrahedron with widest angle being subtended by the doubly bonded O3 and O4 atoms, i.e. O3—S—O4, is 120.32 (10)°.The molecular structure of (I)Analysis of the Hirshfeld surface (§4). Three specific points of contact between molecules are highlighted here, i.e. within the standard distance criteria in PLATON . These are: a 4-tolyl-C11—H11⋯π contact and a pair of S—O⋯π contacts, Table 1ab plane as shown in Fig. 2a. Layers stack along the c axis with no directional interactions between them, Fig. 2b.The molecular packing of (I)et al., 2017C and dioxole-O1 on the Hirshfeld surfaces mapped over dnorm in Fig. 3c axis. On the Hirshfeld surfaces mapped over the electrostatic potential, Fig. 4The Hirshfeld surfaces calculated for (I)a and those delineated into H⋯H, O⋯H/H⋯O and C⋯H/H⋯C contacts  ring is viewed as the pair of characteristic wings in the fingerprint plot delineated into C⋯H/H⋯C contact, Fig. 6d. The presence of a pair of intermolecular S—O⋯π contacts in the crystal is also indicated by small but significant contributions from C⋯O/O⋯C and O⋯O contacts to the Hirshfeld surface, Table 3The overall two-dimensional fingerprint plot for (I)i.e. 5-benzyloxy-3-methyl-1-tosyl-1H-indole —C(=O) bridge and with each nitrogen bound to a benzyl group, i.e. 1,2-bis[5-benzyl-5H-dioxoloindole-6-yl]ethane et al., 2015The compound was prepared and characterized as described in the literature (da Silva Uiso(H) set to 1.2–1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989018000889/hb7727sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989018000889/hb7727Isup2.hklStructure factors: contains datablock(s) I. DOI: 1816872CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


We recently developed a polyethylenimine (PEI) and polyethylene glycol (PEG) dual-functionalized reduced graphene oxide (GO)  for high-efficient gene delivery in HepG2 and Hela cell lines. To evaluate the feasibility and applicability of RGPP as a gene delivery carrier, we here assessed the transfection efficiency of RGPP on gene plasmids and siRNA in 11 different cell lines. Commercial polyalkyleneimine cation transfection reagent (TR) was used as comparison. In HepG2 cells, RGPP exhibited much stronger delivery ability for siRNA and large size plasmids than TR. For green fluorescent protein (GFP) plasmid, RGPP showed about 47.1% of transfection efficiency in primary rabbit articular chondrocytes, and about 27% of transfection efficiency in both SH-SY5Y and A549 cell lines. RGPP exhibited about 37.2% of GFP plasmid transfection efficiency in EMT6 cells and about 26.0% of GFP plasmid transfection efficiency in LO2 cells, but induced about 33% of cytotoxicity in both cell lines. In 4T1 and H9C2 cell lines, RGPP had less than 10% of GFP plasmid transfection efficiency. Collectively, RGPP is a potential nano-carrier for high-efficiency gene delivery, and needs to be further optimized for different cell lines. A varSafe and efficient carrier is critical for gene delivery . PolyethIn this report, we evaluate the delivery ability of RGPP for siRNA and plasmids in 11 kinds of cell lines. Commercial polyalkyleneimine cation transfection reagent (TR) was used as comparison. In HepG2 cells, RGPP exhibited better gene delivery ability than TR for siRNA, large size for plasmids such as GFP-Bcl-xL (9.8 kb) and other functional gene plasmids including GFP-Bak, GFP-Puma, GFP-Bax 1–2/L-6 and GFP-NPM NLS1/2D. RGPP could efficiently deliver GFP plasmid into primary rabbit articular chondrocyte, SH-SY5Y, A549, EMT6 and LO2 cell lines. However, RGPP inefficiently delivered GFP plasmid into H9C2 and 4T1 cell lines.2.2.1.N-(3-dimethylaminopropyl-N′-ethylcarbodiimide) hydrochloride (EDC) were purchased from Sigma-Aldrich . 8-arm-polyethylene glycol amine  was purchased from Shanghai Seebio Biotech, Inc. . Turbofect™ transfection regent  was purchased from Thermo Fisher Scientific . Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories . Dulbecco's modified Eagle's medium (DMEM) and RPMI 1640 medium were purchased from Gibco . Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing Biological Engineering Materials Co. Ltd .GO was purchased from Nanjing XFNano Materials Tech Co. Ltd . Branched PEI (25 kDa) and Human hepatoma cell line HepG2 was purchased from the Experimental Animal Center of Sun Yat-Sen University . Human neuroblastoma cell line SH-SY5Y, human breast cancer cell line MCF-7, mouse melanoma cell line B16 and high metastatic mouse melanoma cell line B16F10 were obtained from American Type Culture Collection. Human normal hepatocyte cell line LO2, human lung cancer cell line A549, mouse breast cancer cell line EMT6 and high metastatic mouse breast cancer cell line 4T1 were obtained from Jinan University . Rat myocardial cell line H9C2 was obtained from Guangdong Provincial People's Hospital . Primary rabbit articular chondrocytes were prepared according to our previously reported method and identified by using toluidine blue staining .Venus , C32 V , VCV , VCVV , EGFP-Bak (#32564), EGFP-Puma (#16590), EGFP-Bax 1-2/L-6 (#30533), EGFP-Bcl-xL , GFP-Bcl2 (#17999), GFP-Bcl2-Cb5 (#18000), GFP-Bcl2-Maob (#18001), GFP-NPM WT (#17578), GFP-NPM NESD (#13283), GFP-NPM NLS1/2D (#13287) and EGFP (#74165) plasmids were obtained from Addgene . FAM-labelled siRNA was purchased from GenePharma .2.2.2COOH (1.0 g) were added to the nGO suspension and sonicated for 30 min in ice bath to obtain carboxylation of nGO (nGO−COOH). The resulting nGO−COOH suspension was washed three to five times with deionized water by using 30 kDa molecular weight cut off (MWCO) ultrafiltration device  to remove ions.GO was prepared by a modified Hummers method using expandable graphite flake ,52. GO (−1) was added to 10 ml of nGO suspension (1.0 mg ml−1), and pH of the mixture was adjusted to 8.0 by 5 mM NaOH and then sonicated for another 10 min. Next, 30 mg of 8-arm PEG-NH2 was added to the above suspension, and the mixture was sonicated for 10 min and then stirred for 12 h at room temperature to obtain nGO–PEG. The resulting nGO–PEG suspension was washed three to five times with deionized water by using ultrafiltration device (30 kDa MWCO) to remove the unreacted PEG and ions.PEG−nGO was obtained just as described previously . Briefly2.3.−1) was mixed with 8 ml PEI (25 kDa) solution (0.75 mg ml−1) and then bathed at 80°C for 2 h to obtain RGPP solution. The resulting RGPP solution was dialysed against deionized water in a 30 kDa MWCO dialysis membrane for 2 days. The 630 nm ultraviolet–visible (UV–vis) absorbance of cuprammonium complex formed between PEI and copper(II) ion was measured by UV–vis spectrometer  for determining the amount of modified PEI in RGPP [RGPP was prepared as described previously . Briefly in RGPP .2.4.2 at 37°C.HepG2, SH-SY5Y, MCF-7, B16, B16F10, A549 cells and primary rabbit articular chondrocytes were cultured in high-glucose DMEM supplemented with 10% FBS. H9C2 cells were cultured in low-glucose DMEM containing 10% FBS. LO2, EMT6 and 4T1 cells were cultured in RPMI 1640 containing 10% FBS. All cells were maintained in a humidified incubator with 5% CO4 cells well−1 and incubated for 24 h. Plasmid (0.2 µg) and different amount of RGPP or TR were mixed in 20 µl serum-free media and incubated at room temperature for 20 min. Plasmid/RGPP or plasmid/TR complexes were incubated with cells for 4 h in 100 µl serum-free media. Then, serum-free media were replaced by fresh media containing 10% FBS and the cells were incubated for another 44 h before CCK-8 assay.Cytotoxicity of RGPP and TR as well as plasmid was assessed by CCK-8 assay according to the manufacturer's protocol just as described previously . Cells w2.5.5 cells/well and incubated for 24 h before transfection. For siRNA transfection, 0.369 µg of FAM-labelled siRNA and different amount of RGPP or TR were mixed in 100 µl serum-free media and incubated at room temperature for 20 min. Complexes of RGPP/siRNA or TR/siRNA were incubated with cells for 4 h in 500 µl serum-free media, and then siRNA transfection efficiency was determined by fluorescence microscope imaging  and flow cytometry analysis . For plasmid transfection, 1 µg of plasmid and different amount of RGPP or TR were mixed in 100 µl serum-free media and incubated at room temperature for 20 min. After the complexes of RGPP/plasmid or TR/plasmid were incubated with cells for 4 h in 500 µl serum-free media, the serum-free media were replaced by fresh media containing 10% FBS and the cells were cultured for another 44 h before fluorescence microscope imaging or FCM analysis for transfection efficiency.Cells were seeded in 24-well plates with a density of 1 × 102.6.t-test was used to evaluate the significance of difference between two groups. Statistical and graphic analyses were done using the software SPSS 19.0  and Origin 8.5 (OriginLab Corporation). p < 0.05 was defined as statistically significant difference.Data were presented as mean ± s.d. from at least three independent experiments. The Student's 3.3.1.a,b). RGPP at N/P ratio of 60 and 0.4% of TR were used for plasmid transfection without indication.After exposure of cells to RGPP or TR for 48 h, CCK-8 assay showed that both RGPP and TR exhibited dose-dependent cytotoxicity, and RGPP at N/P ratio of 60 and 0.4% (TR volume/medium volume) of TR had similar cytotoxicity a,b. RGPPc). Fluorescence microscopic images of cells exclusively transfected GFP-Bak, GFP-Puma, GFP-Bax 1-2/L-6, GFP-Bcl-xL and GFP-NPM NLS1/2D plasmids by using RGPP and TR for 48 h also demonstrated that RGPP showed higher delivery ability for these five plasmids than TR (d). We also determined the transfection ability of both RGPP and TR for V, C32 V, VCV and VCVV plasmids, and found that both RGPP and TR efficiently delivered these plasmids into cells, and the transfection ability of TR was slightly better than that of RGPP (c). After the complexes of RGPP/siRNA or TR/siRNA were incubated with cells for 4 h, FCM analysis showed that RGPP at N/P ratios of 10 and 15 exhibited 43.4 ± 2.1% and 62.8 ± 2.4% of siRNA transfection efficiency, much higher than that of 0.1% and 0.2% of TR (e). Fluorescence microscopic images also showed that RGPP at N/P ratio of 15 delivered more siRNA into cells than 0.2% of TR (f).We evaluated the transfection ability of RGPP/TR for 10 functional gene plasmids by using FCM analysis. FCM analysis showed that RGPP had higher transfection efficiency than TR for GFP-Bak , GFP-Puma , GFP-Bax 1-2/L-6 , GFP-Bcl-xL  and GFP-NPM NLS1/2D  plasmids c. Fluore than TR d. We als of RGPP c. After 2% of TR e. Fluore2% of TR f.c), suggesting that RGPP exhibited better delivery ability for large-size plasmid than TR. It was reported that huge aggregates (greater than 1000 nm) of DNA/transferrin-PEI exhibited much higher transfection efficiency than small aggregates in in vitro transfection assay [figure 1e,f), which might benefit from the inhibitory effect of RGPP on gene degradation [Size of plasmid is one of the elements influencing transfection efficiency during transient transfection assay ,57. Tranfigure 1on assay . Diameteon assay , which mradation .3.2.a,b shows the cytotoxicity of different doses of RGPP or TR in SH-SY5Y cells, and RGPP at N/P ratios of 15, 30, 60 and 90, respectively, exhibited 2.1 ± 0.3%, 5.1 ± 0.6%, 14.0 ± 1.2% and 27.2 ± 1.5% of transfection efficiency, while 0.2%, 0.4% and 0.6% of TR, respectively, showed 9.3 ± 1.7%, 15.0 ± 0.6% and 12.3 ± 0.5% of transfection efficiency (c). RGPP at N/P ratio of 60 and 0.4% of TR exhibited similar cytotoxicity (about 4.5%) and transfection efficiency (about 14.0%) in SH-SY5Y cells (a–c). RGPP at N/P ratio of 90 showed the highest transfection efficiency (about 27.2%), about 1.8-fold that of 0.4% of TR , suggesting that RGPP at N/P ratio of 90 was superior to 0.4% of TR as a gene delivery carrier for SH-SY5Y cells. Fluorescence microscopic images also showed that RGPP at N/P ratio of 90 delivered more GFP plasmid into SH-SY5Y cells than 0.4% of TR (figure 2d).We evaluated the delivery ability of RGPP on GFP plasmid by using FCM analysis in three human cancer cell lines: human neuroblastoma cell line (SH-SY5Y), human lung cancer cell line (A549) and human breast cancer cell line (MCF-7). Figure 2ficiency c. RGPP a5Y cells a–c. RGPPThe cytotoxicity and transfection efficiency of RGPP and TR in A549 and MCF-7 cells are listed in 3.3.a,b, both RGPP and TR exhibited dose-dependent cytotoxicity in EMT6 cells, and RGPP at N/P ratios of 15, 30 and 60, respectively, had 2.4 ± 0.8%, 5.5 ± 1.1% and 37.2 ± 3.2% of transfection efficiency, and 0.2%, 0.4% and 0.6% of TR, respectively, had 2.0 ± 0.3%, 6.5 ± 1.5% and 7.8 ± 1.1% of transfection efficiency (figure 3c). In EMT6 cells, RGPP at N/P ratio of 60 and 0.6% of TR exhibited about 35% of cytotoxicity, but transfection efficiency of RGPP at N/P ratio of 60 (about 37.2%) was about fourfold higher than that of 0.6% of TR . Similarly in 4T1 cells, both RGPP and TR also exhibited dose-dependent cytotoxicity , and RGPP at N/P ratios of 15, 30, 60 and 90, respectively, exhibited 7.9 ± 2.7%, 10.2 ± 0.8%, 9.9 ± 1.0% and 9.9 ± 1.2% of transfection efficiency, and 0.2%, 0.4% and 0.6% of TR, respectively, had 1.5 ± 0.6%, 3.4 ± 2.0% and 4.3 ± 2.7% of transfection efficiency (figure 3g). Both RGPP and TR showed less than 10% of transfection efficiency indicating that both RGPP and TR were inefficient in delivering plasmid into 4T1 cells. Fluorescence microscopic images also showed that both RGPP and TR were inefficient in delivering plasmid into 4T1 cells (figure 3h).We next evaluated the delivery ability of RGPP on GFP plasmid in four mouse cancer cell lines: mouse breast cancer cell line (EMT6), high metastatic mouse breast cancer cell line (4T1), mouse melanoma cell line (B16) and high metastatic mouse melanoma cell line (B16F10). As shown in figure 3The cytotoxicity and transfection efficiency of RGPP and TR in B16F10 and B16 cells are listed in 3.4.a–c). Both RGPP at N/P ratio of 45 and 0.4% of TR had about 32.6% of cytotoxicity, but the transfection efficiency of RGPP at N/P ratio of 45 was about 26.1%, about twofold that of 0.4% of TR (about 12.6%) in LO2 cells (a–c). In H9C2 cells, RGPP at N/P ratios of 15 and 30, respectively, exhibited only 1.4 ± 0.4% and 2.0 ± 1.0% of transfection efficiency with 7.5 ± 2.4% and 18.7 ± 0.6% of cytotoxicity, and 0.2% and 0.4% of TR exhibited 0.8 ± 0.3% and 3.2 ± 0.4% of transfection efficiency without cytotoxicity, and 0.6% of TR had 2.8 ± 0.8% of transfection efficiency with 7.9 ± 8.4% of cytotoxicity (d–f). Transfection efficiencies of RGPP and TR were less than 4% (f), indicating that both RGPP and TR were inefficient in delivering plasmid into H9C2 cells. In primary rabbit articular chondrocytes, RGPP at N/P ratios of 15, 30, 60 and 90, respectively, had 1.2 ± 0.2%, 3.4 ± 0.7%, 47.1 ± 2.0% and 15.1 ± 3.3% of transfection efficiency, and 0.2%, 0.4%, 0.6% and 0.8% of TR, respectively, exhibited 13.3 ± 0.1%, 10.5 ± 1.4%, 39.0 ± 1.5% and 48.0 ± 0.5% of transfection efficiency (c). RGPP at N/P ratio of 60 and 0.8% of TR exhibited similar cytotoxicity (about 8.5%) and transfection efficiency  in primary rabbit articular chondrocytes (a–c). Therefore, both RGPP at N/P ratio of 60 and 0.8% of TR were excellent gene delivery carriers for primary rabbit articular chondrocytes.Finally, we evaluated the delivery ability of RGPP on GFP plasmid in normal cells including human normal hepatocyte cell line (LO2), rat myocardial cell line (H9C2) and primary rabbit articular chondrocytes. In LO2 cells, RGPP at N/P ratios of 15, 30 and 45, respectively, exhibited 1.6 ± 0.4%, 3.6 ± 0.3% and 26.1 ± 2.8% of transfection efficiency with 14.2 ± 7.1%, 27.0 ± 6.7% and 32.6 ± 0.3% of cytotoxicity, and 0.2%, 0.4% and 0.6% of TR, respectively, showed 6.2 ± 0.9%, 12.6 ± 2.9% and 16.3 ± 3.0% of transfection efficiency with 7.3 ± 6.3%, 32.6 ± 12.5% and 54.2 ± 4.6% of cytotoxicity a–c. BothO2 cells a–c. In Htoxicity d–f. Tran than 4% f, indicaficiency c. RGPP adrocytes a–c. Ther4.The transfection efficiency of RGPP for GFP plasmid in 10 different cell lines summarized in


Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. Previous studies have shown that the Sonic hedgehog (Shh) signaling pathway may reduce cell apoptosis by inhibiting oxidative stress in A. cantonensis infection. In this study, we investigated the relationship between cytokine secretion and Shh pathway activation after treatment with excretory/secretory products (ESP) of fifth-stage larval A. cantonensis (L5).The results showed that IL-1β and IL-6 levels in mouse astrocytes were increased. Moreover, ESP stimulated the protein expression of Shh pathway molecules, including Shh, Ptch, Smo and Gli-1, and induced IL-1β and IL-6 secretion. The transcription factor nuclear factor-κB (NF-κB) plays an important role in inflammation, and it regulates the expression of proinflammatory genes, including cytokines and chemokines, such as IL-1β and TNF-α. After ESP treatment, NF-κB induced IL-1β and IL-6 secretion in astrocytes by activating the Shh signaling pathway.A. cantonensis stimulates astrocyte activation and cytokine generation through NF-κB and the Shh signaling pathway.Overall, the data presented in this study showed that ESP of fifth-stage larval The online version of this article (10.1186/s13071-017-2385-0) contains supplementary material, which is available to authorized users. These cells can protect neurons and induce inflammatory responses by releasing anti-apoptotic proteins or cytokines in response to pathogen infection , 2. Durineration . Astrocyneration . This ban tissue . Severaltrocytes .via a series of inhibitory steps, and it can trigger a common signaling pathway. In the absence of Shh, transmembrane Patched (Ptc) receptors block the function of another transmembrane protein, Smoothened (Smo). In contrast, following Shh interaction with Ptc, Smo can be activated via inhibition of Ptc. These changes initiate a signaling cascade that activates the Glioma-associated oncogene (Gli) family of transcription factors (Gli1-Gli3) [Helicobacter pylori infection, the Shh pathway can positively regulate the expression of Interleukin-1β (IL-1β), IL-10, IL-12, IFNγ and MIP-2 in mouse stomach tissues [The Hedgehog (Hh) signaling pathway and secreted proteins play important roles in animal development. This pathway regulates morphogenesis of a variety of tissues and organs . Hh has i1-Gli3) , 14. Sev tissues . IL-1β i tissues , 17. IL- tissues –20.Angiostrongylus cantonensis, the rat lungworm, was found in the hearts and pulmonary arteries of Rattus rattus and Rattus norvegicus in Guangzhou (Canton), China, by Chen in 1935 [A. cantonensis infection, the fifth-stage larvae (L5) induce a wide range of immune responses, including eosinophil recruitment and cytokine release  in the CNS of humans [ in 1935 , 22. Thi in 1935 –28. By 2 in 1935 , 30. In f humans , 32.A. cantonensis infection in mice increased reactive oxygen species (ROS) and antioxidants in the astrocytes, and activation of the Shh signaling pathway inhibited cell death through the GRP78/Bcl-2-dependent pathway [A. cantonensis L5 induced oxidative stress and cell apoptosis in astrocytes, but Shh pathway activation could reduce cell injury [In our previous studies,  pathway . Moreovel injury . In the Angiostrongylus cantonensis (Taipei strain) was maintained in our laboratory through cycling in Biomphalaria glabrata snails and Sprague-Dawley (SD) rats [w/v) pepsin-HCl (pH 2–3) for 1 h. Each BALB/c mouse was inoculated with 25 L3 via stomach intubation. In this study, SD rats and BALB/c mice were purchased from the National Laboratory Animal Center, Taipei. All procedures involving animals and their care were reviewed and approved by the Chang Gung University Institutional Animal Care and Use Committee.SD) rats . On day A. cantonensis were isolated from the brain tissues of rats by anesthetizing with 30 μl Zoletil 50 (Virbac) after 21 days post infection. After the worms were washed with saline, phosphate-buffered saline, distilled water and RPMI , they were incubated in RPMI without foetal bovine serum (FBS) for 24, 48 and 72 h. The ESP of L5 were collected and concentrated by Amicon Ultra-15 10 K centrifugal filter devices  from the culture medium. The ESP concentration in the medium was determined using a Bio-Rad Protein Assay Kit , according to the manufacturer’s instructions. The ESP were used to treat the astrocytes, and cellular changes were observed [Live L5 of observed .2. Cells were plated onto 10 cm culture dish, incubated in serum-free DMEM/F-12 for 24 h, pretreated with the the recombinant Shh (r-Shh) , Shh agonist (SAG) , Shh pathway inhibitor (Cyclopamine) (Sigma-Aldrich) and NF-Kb inhibitor (JSH-23) (Sigma-Aldrich) for 1 h and then treated with L5-ESP.A mouse brain astrocytic cell line (CRL2535) from ATCC was used in this study . Cells wThe proteins of astrocytes were separated by 12% SDS-PAGE. The separated proteins were transferred to nitrocellulose membrane and incubated with antibodies against Shh (Sigma-Aldrich), Ptch (Sigma-Aldrich), Smo (Sigma-Aldrich), Gli-1 (Sigma-Aldrich), IL-1β , IL-6 (RayBiotech), NF-κB (Sigma-Aldrich) and β-actin (Sigma-Aldrich). The membrane was washed with TBS/T three times and then incubated with a 1:10,000 dilution of anti-rabbit or mouse horseradish peroxidase antibody (Sigma-Aldrich). The reactive bands were detected by ECL reagents  and captured by a UVP BioSpectrum 600 Imaging System . ImageJ software analysis was used to detect the image densitometry of target proteins.The cultured supernatants were collected from astrocytes treated with ESP at 2 h intervals, for up to 8 h. These samples were used to detect the concentration of IL-1β or IL-6 by a mouse-specific ELISA kit (RayBiotech).Total RNA was extracted from the astrocytes by using GENEzol TriRNA Pure Kit . First -strand cDNA was synthesized using SuperScript III reverse transcriptase  with oligo d(T) primer, according to the manufacturer’s instructions. Quantitative real-time PCR was performed using the qPCR Master Mix  on the ABI 700 qPCR System . A β-actin internal control was used. The expression level was detected with specific primers, targeting the Shh , Ptch , Gli-1  and β-actin cDNAs .The astrocytes were seeded into 6 well plates and grown to 70–80% confluence for transfection. X-tremeGene siRNA transfection reagent  was mixed with 40 pmoles Shh siRNA (UCUGAAACGCAGGACAAGG and CCUUGUCCUGCGUUUCAGA) (Sigma-Aldrich) within 5 min. Cells were incubated with the reagent containing siRNA for 8 h and then replaced the fetal bovine serum containing medium without siRNA. After 48 h, cells were collected to detect protein expression.w/v) paraformaldehyde (PFA) and 0.5% (v/v) Triton X-100 in PBS before incubation. Sections were washed in PBS/T (pH 7.4) and blocked for 30 min in PBS containing 2% BSA. Then, brain sections were incubated with primary antibody, including chicken anti-Glial fibrillary acidic protein(GFAP)  and rabbit anti-NF-κB, , for 24 h at 4 °C. Finally, the sections were incubated with the secondary antibodies  for 50 min at room temperature. DAPI was used to detect cell nuclei.The frozen mouse brain tissue sections were fixed and permeabilized with 2% .Student’s t-tests were used to compare the OD values and expression levels using GraphPad Prism 5 software. Data were presented as means ± SD. Differences were considered statistically significant through the A. cantonensis L5 ESP in cytokine expression, we treated astrocytes with 500 μg/ml ESP for 0–8 h. Western blot analysis showed that the protein levels of IL-1β and IL-6 increased in a time-dependent manner in astrocytes, and significant increases (IL-1β: t(4) = 5.368, P < 0.01; IL-6: t(4) = 147.8, P < 0.001) were detected at 2 h  = 3.978, P < 0.05) at 2 h and significantly increased at 6 h (t(4) = 8.725, P < 0.01), and IL-6 was significantly increased (t(4) = 4.893, P < 0.01) at 2 h  members by quantitative real-time PCR  = 16.68, P < 0.001), and Ptch and Gli-1 were significantly elevated for 6 h after ESP treatment (t(4) = 13.24, P < 0.001). Moreover, the protein expression of Shh was significantly elevated for 4 h, and Ptch, Smo, and Gli-1 were significantly elevated for 2 h after ESP treatment (Shh: t(4) = 6.187, P < 0.01; Ptch: t(4) = 8.711, P < 0.01; Smo: t(4) = 5.254, P < 0.01; Gli-1: t(4) = 4.625, P < 0.01)  were not significantly changed for 0–8 h in astrocytes , Shh agonist (SAG), and Shh pathway inhibitor (Cyclopamine). These data showed that r-Shh and SAG stimulated IL-1β and IL-6 generation (IL-1β(r-Shh treatment): t(4) = 4.884, P < 0.01; IL-1β(SAG treatment): t(4) = 8.56, P < 0.01; IL-6(r-Shh treatment): t(4) = 123.3, P < 0.001; IL-6(SAG treatment): t(4) = 66.2, P < 0.001)  = 5.948, P < 0.01; IL-1β(SAG treatment): t(4) = 5.058, P < 0.01; IL-6(r-Shh treatment): t(4) = 7.679, P < 0.01; IL-6(SAG treatment): t(4) = 3.975, P < 0.01)  = 7.447, P < 0.01; IL-6: t(4) = 5.912, P < 0.01). Alternatively, the results showed that the expressions of IL-1β and IL-6 were significantly decreased in Shh siRNA treatment (IL-1β: t(4) = 11.71, P < 0.001; IL-6: t(4) = 5.88, P < 0.01), and the expression levels could be restored after r-Shh treatment (IL-1β: t(4) = 11.83, P < 0.001; IL-6: t(4) = 7.435, P < 0.01)  Fig.  and secr01) Fig.  by activA. cantonensis-infected mice. Immunofluorescence staining with antibodies against GFAP and NF-κB showed that NF-κB was significantly increased in activated astrocytes from A. cantonensis-infected mice after 21 days  = 5.786, P < 0.01)  Fig. . These dt(4) = 9.855, P < 0.001; Shh: t(4) = 6.192, P < 0.01; Ptch: t(4) = 4.454, P < 0.01; Smo: t(4) = 5.853, P < 0.01)  = 5.133, P < 0.01; IL-6: t(4) = 11.28, P < 0.001)  = 4.915, P < 0.01; IL-6: t(4) = 7.22, P < 0.01)  (NF-κB: 01) Fig. . Immunof01) Fig. . These d01) Fig.  and secr01) Fig.  were sigA. cantonensis infection, reactive stress and injury are elevated in the parenchyma, meninges, and cerebrospinal fluid of the mouse brain [A. cantonensis could induce eosinophil recruitment in the brain, but the function and effects of the ESP molecules are poorly understood [In se brain , 38. Morse brain . The ESPderstood . The ESPderstood . HoweverToxoplasma gondii infection, stimulated murine astrocytes can inhibit parasite infection and replication by the generation of cytokines [T. gondii tachyzoites or bradyzoites in astrocytes induce inflammatory cytokine  generation in vitro [Astrocytes are important to brain cells that regulate immunocyte recruitment through the release of cytokines or chemokines, such as IL-1β and IL-6, in response to injuries or pathogen infections . In Toxoytokines , 44. Morin vitro . IL-1β iin vitro .2O2 treatment, and Shh expression in astrocytes is higher than that in fibroblasts and neurons. Following H2O2 treatment, the astrocytes protect cortical neurons by secretion of Shh protein [A. cantonensis soluble antigen treatment [A. cantonensis ESP treatment.The Shh signaling pathway plays an important role in human development; it regulates the morphogenesis of tissues and organs . In the reatment . In thisvia the expressions of cytokines (IL-1β) and chemokines (MCP-1) [Neuroinflammation plays a major role in parasite infection, Parkinson’s disease and Alzheimer’s disease –53. Seve (MCP-1) . In vira (MCP-1) .A. cantonensis ESP-stimulated expression of cytokines, including IL-1β and IL-6, in cultured mouse astrocytes. In conclusion, the present work showed that NF-κB induces the expressions of IL-1β and IL-6 through the Shh signaling pathway in ESP-treated astrocytes.Here, we investigated the molecular mechanisms of


SAMHD1 is an intracellular enzyme that specifically degrades deoxynucleoside triphosphates into component nucleoside and inorganic triphosphate. In myeloid-derived dendritic cells and macrophages as well as resting T-cells, SAMHD1 blocks HIV-1 infection through this dNTP triphosphohydrolase activity by reducing the cellular dNTP pool to a level that cannot support productive reverse transcription. We now show that, in addition to this direct effect on virus replication, manipulating cellular SAMHD1 activity can significantly enhance or decrease the anti-HIV-1 efficacy of nucleotide analogue reverse transcription inhibitors presumably as a result of modulating dNTP pools that compete for recruitment by viral polymerases. Further, a variety of other nucleotide-based analogues, not normally considered antiretrovirals, such as the anti-herpes drugs Aciclovir and Ganciclovir and the anti-cancer drug Clofarabine are now revealed as potent anti-HIV-1 agents, under conditions of low dNTPs. This in turn suggests novel uses for nucleotide analogues to inhibit HIV-1 in differentiated cells low in dNTPs. In cycling THP_1 cells that are relatively permissive to HIV-1 infection, SAMHD1 is largely phosphorylated by cyclin A2/CDK1 at Threonine 592. By contrast, T592 phosphorylation is reduced in differentiated THP-1 cells that are restrictive to HIV-1 infection23242628The rate of HIV-1 proviral synthesis is limited by the intracellular dNTP concentration783435in vitro and cell-based assays to study the mutual effects of nucleotide analogues and SAMHD1 activity on HIV-1 replication.Agents that modulate SAMHD1 function would have great value for studies of its anti-HIV effects. Since the triphosphohydrolase activity of SAMHD1 is regulated allosterically by nucleotide analogues whilst the efficacy of nucleotide analogues can be affected simultaneously by SAMHD1 activity, we used a combination of in vitro enzyme-coupled assay to test the effect on SAMHD1 activity of the triphosphate derivatives of a panel of FDA-approved nucleoside analogues widely used in antiviral and anticancer therapy, detailed in 3738404246We first used an in vitro assay to assess their role as activators, substrates or inhibitors of SAMHD1. We evaluated the triphosphate forms of the panel of nucleoside analogues detailed in 4850in vitro assays, demonstrated that Aciclovir triphosphate (ACV-TP) was a non-hydrolysable allosteric activator of SAMHD1 with comparable efficiency to GTP, Ganciclovir triphosphate (GCV-TP) was also non-hydrolysable but displayed no activation of SAMHD1 and Clofarabine triphosphate (CFB-TP) was a substrate hydrolysed by SAMHD1 at a rate comparable to natural dNTP substrates in the presence of the activator GTP35KS = 226 ± 12 μM, n = 1.7 ± 0.1 and kcat = 0.24 ± 0.04 s−1. ddI-TP only weakly activates SAMHD1 and analysis of the ddI-TP concentration dependence of TTP hydrolysis  showed anti-HIV-1 activities with EC50 values below 10 μM; NVP and RAL inhibited HIV-1 at nanomolar concentrations  and integrase inhibitor (RAL) controls were evaluated in proliferating H9 and Jurkat T cell lines . As woultrations .−) and PMA-differentiated (PMA+) U937 and THP-1 lines a different pattern emerged (− cells (EC50 > 200 μM), ACV showed an increased activity in PMA+ U937 cells (EC50 = 52.2 μM), and both ACV and GCV had extremely significant activities in PMA+ THP-1 cells     when comparing PMA+ to PMA− THP-1 cells  in their activities in PMA− and PMA+ cells  . The ant.003 μM) . Measurectively) . By cont-1 cells . As mighA+ cells .+ treated THP-1 cells and monocyte-derived macrophages (MDM), where dNTP levels are reduced3435Increased anti-HIV-1 activities of NRTIs have been reported previously in PMA50) by flow cytometry and calculated the selectivity index (SI) for each compound (50 > 200 μM). In T cells, where ACV and GCV showed no anti-HIV-1 activity they also did not exhibit any cytotoxic effect (CC50 > 200 μM). The anti-HIV-1 activity of CFB in T cells was only observed at greater than cytotoxic concentrations (SI < 1). In contrast, NRTIs  showed SIs > 30 . However, upon differentiation cytotoxicity was greatly decreased (CC50 > 1.6 μM) resulting in SI values of >43 and >533 in PMA+ U937 and THP-1 cells, respectively. The NRTIs  did not show cytotoxicity (CC50 > 200 μM) thus increasing the SIs, particularly in PMA+ THP-1 cells  and differentiated (PMA+) U937 cells transduced with catalytically active SAMHD1 (wt) or an inactive mutant (HD206-7AA). Comparative EC50 data and statistical significance test results from these assays are summarised in To test whether the anti-HIV-1 activities of the selected nucleoside analogues could be further increased through dNTP depletion by SAMHD1, we evaluated antiviral activities in undifferentiated  (+ cells expressing HD206-7AA (EC50 > 200 μM) but in cells expressing wt SAMHD1 anti-HIV-1 activity of GCV was also greatly enhanced, >200 fold  (Inspection of these data showed that ACV and GCV had no anti-HIV-1 activity in PMA 50.9 μM  comparab(+ cells . However 0.0001) . Similar 0.0001) . As was wt SAMHD1 or HD206-7AA did not affect the EC50 values of CFB in PMA− cells, which remained higher than the cytotoxic concentrations  and ABC showed no significant difference  were increased but only to EC50 values comparable with those observed in untransduced cells  were increased significantly  compared to cells expressing HD206-7AA  and differentiated (PMA+) THP-1 cells expressing endogenous SAMHD1 after transduction with the HIV-2 accessory viral protein Vpx, which reduces SAMHD1 levels by targeting it for proteasomal degradation125Since we found significant differences in the anti-HIV-1 activities of nucleoside analogues in U937 cells expressing exogenous − THP-1 cells upon Vpx transduction  (50 > 200 μM), (p < 0.0001)   and GCV  0.0001) . The antpression . HoweverCFB CC50 . In PMA+y 4-fold . This ma− cells resulted in only small effects on the anti-HIV-1 activities of d4T and ddI (2–3 fold lower) and no difference with ABC  and finally once with CH2Cl2. The crude product was purified by flash column chromatography  to give 490 mg of the cycloSaligenyl-triester with minor impurities. Then 47.5 mg (97.2 μmol) of this phosphate triester were dissolved in 1 mL anhydrous DMF. A solution of 96.3 mg (146 μmol) bis-tetrabutylammonium hydrogen pyrophosphate in 0.7 mL DMF, which had been stirred for 1 h over 4 Å molecular sieves, was added in one aliquot. After 72 h the solvent was removed under reduced pressure and the residue was purified by reverse phase flash column chromatography , ion exchange (NH4+) on Dowex (50WX8) and again reversed phase flash column chromatography  to give 20.5 mg (34.5 μmol) of the triphosphate as a colourless solid after freeze drying. 1H NMR: : δ [ppm] = 7.99 , 6.19–6.26 , 5.93–5.89 , 5.52–5.48 , 4.10–4.06 , 4.03–3.98 , 3.23–3.17 , 2.90–2.76 , 1.78–1.71 , 0.98–0.93 , 0.76–0.72 . 13C NMR: : δ [ppm] = 148.9, 139.3, 138.5, 129.2, 68.3, 59.9, 45.5, 33.6, 22.9, 6.7, 6.7. 31P NMR: : −10.73, −10.91, −23.27. HRMS : calcd. for C14H21N6O10P3: 525.0459 [M-H]−, found 525.0360 [M-H]−. IR (ATR): wavenumber [cm−1] = 2988, 2884, 1644, 1445, 1407, 1210, 1118, 891, 832, 484.The synthesis of the ammonium salt of abacavir-5′-triphosphate (ABC-TP) was performed as describedcycloSaligenyl-phosphorcloridate solution (THF). The crude product was purified by flash column chromatography  to give 219 mg of the cycloSaligenyl-triester with minor impurities. In the second step 47.7 mg (106 μmol) cycloSal-triester of carbovir in 1 mL dry DMF and 105 mg (159 μmol) of bis-tetrabutylammonium hydrogen pyrophosphate in 0.8 mL DMF were reacted. After purification and freeze-drying 12.3 mg (22.1 μmol) of the triphosphate was obtained as a colorless solid. 1H NMR: : δ [ppm] = 8.00 , 6.29–6.26 , 5.94–5.92 , 5.54–5.49 , 4.08–3.98 , 3.23–3.17 , 2.85–2.77 , 1.78–1.71 . 13C NMR: : δ [ppm] = 158.6, 153.7, 151.0, 138.4, 138.3, 129.3, 115.2, 68.6, 60.1, 45.4, 33.8. 31P NMR: : −10.87, −10.98, −23.31. HRMS : calcd. for C11H16N5O11P3: 485.9986 [M-H]−, found 486.0020 [M-H]−. IR (ATR): wavenumber [cm−1] = 2988, 1686, 1610 1407, 1214, 1056, 1004, 948, 893, 473.Carbovir 5′-triphosphate (CBV-TP) was prepared similarly. Amounts used were: 308 mg (1.25 mmol) carbovir, 9.5 mL anhydrous pyridine and 1.62 mL  of 1 M 5-chloro-S. cerevisiae Ppx1 exopolyphosphatase activity, described previously192 and 2 mM TCEP) at 25 °C before the reaction was initiated by the addition of substrate . The final concentrations were 100 nM SAMHD1(115–626), 10 nM Ppx1, 2.5 μM BSA, 40 μM MDCC-PBP (80 μM in ABC-TP and CBV-TP assays). Activator, substrate and inhibitor were added at concentrations described below. The fluorescence intensity increase resulting from the phosphate-product binding to MDCC-PBP was recorded at 430 nm excitation and 465 nm emission over 10–30 min in a Clariostar multiwell plate reader (BMG). Steady-state rates were obtained from the fluorescence time courses by fitting a linear function with maximal slope using the Mars software (BMG). Apparent dissociation constant of ddI-TP binding (KS), catalytic constant (kcat), maximal rate (kmax) and the ddI-TP concentration at half maximal activation (Ka) were determined by nonlinear least squares fitting using a Hill-function in the software package Grafit 7.0.3 . All measurements were performed at least in triplicate and data are represented as mean ± SEM.SAMHD1 hydrolysis, activation and inhibition by nucleotide analogues was quantified using a continuous coupled assay employing the fluorescent phosphate biosensor MDCC-PBP to measure phosphate release from combined SAMHD1 triphosphohydrolase and Cell lines were obtained from the Programme EVA Centre for AIDS Reagents and tested free for mycoplasma contamination. JurkatSAMHD1 and HIV-2 Vpx DNA sequences were inserted into pLGatewayIeYFP−) cells (6 × 105 cells/mL) were treated with increasing concentrations of nucleoside analogues  and incubated at 37 °C in 5% CO2 for 48 h. Medium was then replaced and cells were further incubated for 72 h. Non-dividing U937 and THP-1 (PMA+) cells (6 × 105 cells/mL) were differentiated by addition of 100 nM phorbol-12-myristate-13-acetate (PMA+) for 48 h and then treated with increasing concentrations of nucleoside analogues as described above. The percentage of live cells was determined by flow cytometry using Live/Dead fixable blue-fluorescent dyes (Invitrogen) and a Fortessa X20 analyser (BD Biosciences). Data were analysed using the FlowJo software suite.H9, Jurkat and undifferentiated U937 and THP-1 (PMA5 cells/mL) were treated with increasing concentrations of nucleoside analogues  and incubated for 4 h at 37 °C in 5% CO2. Cells were then infected with HIV-1-GFP by spinoculation at 800 × g for 90 min in the presence of 1 μg/mL polybrene, and further incubated at 37 °C in 5% CO2 for 72 h. Undifferentiated U937 and THP-1 (PMA−) cells (6 × 105 cells/mL) were treated with increasing concentrations of nucleoside analogues  and incubated at 37 °C in 5% CO2 for 48 h. Cells were then infected with HIV-1-GFP by spinoculation at 800 × g for 90 min in the presence of 1 μg/mL polybrene, and further incubated at 37 °C in 5% CO2 for 72 h. Non-dividing U937 and THP-1 (PMA+) cells (6 × 105 cells/mL) were differentiated by addition of 100 nM PMA (PMA+) for 48 h and treated with increasing concentrations of nucleoside analogues as described above. Cells were then infected with HIV-1-GFP in the presence of 1 μg/mL polybrene and further incubated at 37 °C in 5% CO2 for 72 h. The percentage of GFP+cells was determined by one-colour flow cytometry using a Fortessa X20 analyser (BD Biosciences) and the data analysed using the FlowJo software suite.H9 and Jurkat cells (6 × 105 cells/mL) were transduced with WT SAMHD1-YFP or HD206-7AA-YFP VLPs by spinoculation at 800 × g for 90 min in the presence of 1 μg/mL polybrene. Similarly, THP-1 cells (6 × 105 cells/mL) were transduced with Vpx-YFP VLPs by spinoculation at 800 × g for 90 min in the presence of 1 μg/mL polybrene. Cells were incubated at 37 °C in 5% CO2 and after 72 h, PMA+ cells (6 × 105 cells/mL) were treated with 100 nM PMA and PMA− cells (7.5 × 104 cells/mL) were left untreated. Cells were then incubated at 37 °C in 5% CO2 for 48 h. Nucleoside analogues were added at increasing concentrations  and cells were further incubated for 48 h. PMA− cells were then infected with HIV-1-GFP by spinoculation at 800 × g for 90 min in the presence of 1 μg/mL polybrene, PMA+ cells were infected with HIV-1-GFP in the presence of 1 μg/mL polybrene only. Cells were incubated for 72 h at 37 °C in 5% CO2 and then analysed by two-colour flow cytometry using a Fortessa X20 analyser (BD Biosciences). Data were analysed using the FlowJo software suite.Antiviral assays were further performed in monocytoid cells transduced with YFP-expressing VLPs containing WT SAMHD1, HD206-7AA or Vpx. Briefly, U937 cells (6 × 1050) and the effective concentration of nucleoside analogue that inhibits infection of cells by 50% (EC50) were calculated for each independent experiment by non-linear regression using GraphPad Prism v6.00 for Windows . Non-ambiguous values were subjected to a replicate test to calculate confidence intervals of 95% (95% CI). CC50 and EC50 values were determined for at least two independent experiments for each nucleoside analogue and a selectivity index (SI) also calculated, (SI = CC50/EC50). EC50 values for each replicate were used to perform an unpaired two-tailed t-test to determine significant differences between U937 cells expressing SAMHD1 wt or HD206-7AA mutant and THP-1 cells expressing Vpx or untransduced cells. P values of <0.005 were considered statistically significant.The cytotoxic concentration of nucleoside analogue that inhibits cell viability by 50% .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The hydrophilic glucose residues lie in the regions z ≃ 0.25 and 0.75. Four classical hydrogen bonds link the molecules to form layers parallel to the ab plane, from which the pyridyl rings project; pyridyl ring stacking parallel to the a axis links adjacent layers.In the title compound, C Thus, (1) reacted with (2) in KOH/acetone to give a product for which two isomeric structures, (3) and (4), seemed possible, corresponding to two possible modes of glucosylation. After deprotection of the product (see Scheme), the final free sugar pyridinethione N-glucoside (5) or its regioisomer pyridine-2-thioglucoside (6) was obtained. Spectroscopic data cannot differentiate between these structures.We report here a novel one-step synthesis of a pyridine-2-thioglucoside derivative by reaction of the pyridine-2(16) as the product in the solid state. The molecule is shown in Fig. 1glucose 1.8016 (15), S—Cpyridyl 1.7723 (13) Å. The main torsional degrees of freedom are between the rings, as defined by the torsion angles N11—C12—S1—C1 = −2.08 (14) and C2—C1—S1—C12 = 152.98 (9)°.The X-ray structure determination indicated unambiguously the formation of the pyridine-2-thioglucoside (6) occupy the regions at z ≃ 0.25 and 0.75. The layer structure of (6) is shown in Fig. 2X interactions also lie within the layer, but are not drawn explicitly in Fig. 2x, y, z) = 3.1417 (10) Å.The glucose moieties of . The interplanar angles are 4.33 (5)°, the centroid-to-centroid distances are 3.96 and 3.72 Å, and the ring offsets are ca 1.26 and 0.94 Å; these cannot be expressed exactly because neighbouring rings are not exactly parallel.Adjacent layers are connected cf. Table 1glucose is 1.793 (3), S—Cpyridyl is 1.759 (3) Å.Perhaps surprisingly, a database search revealed only one other example of a pyridine ring with a thioglucose substituent at the 2-position, namely pyridyl thioglucose monohydrate -thione (1)  in aqueous potassium hydroxide  was added a solution of 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide (2)  in acetone (30 ml). The reaction mixture was stirred at room temperature until the reaction was judged complete by TLC (30 min to 2 h). The mixture was evaporated under reduced pressure at 313 K and the residue was washed with distilled water to remove the potassium bromide. The solid was collected by filtration and crystallized from ethanol to give compound (3) in 85% yield (m.p. 468 K). Dry gaseous ammonia was then passed through a solution of the protected thioglycoside (3) (0.5 g) in dry methanol (20 ml) at 273 K for 0.5 h, then the mixture was stirred at 273 K until completion of the reaction . The mixture was evaporated at 313 K to give a solid residue, which was recrystallized from ethanol to give compound (6) in 85% yield (m.p. 482–483 K).To a solution of the pyridine-2-(1−1. 1H NMR (DMSO-d6): δ 2.22 , 2.31 , 3.15–3.80 , 4.40 , 4.90 , 5.30 , 5.59 , 7.19  ppm. 13C NMR: δ 20.7 (CH3), 22.9 (CH3), 61.0 (C6′), 68.9 (C4′), 72.7 (C2′), 75.9 (C3′), 80.7 (C5′), 83.7 (C1′), 103.2 (C3), 116.0 (CN), 119.7 (C5), 149.2 (C4), 159.0 (C6), 164.0 (C2) ppm.IR (KBr): 3600–3258 (OH), 2222 (CN) cmP21, with two independent but virtually identical molecules in the asymmetric unit. It became apparent that the two molecules formed layer structures independent of each other, and were related by a translation vector . The checkCIF program also indicated that the true space group should be centred, with a 100% fit and a small deviation. The same cell was retained for ease of checking, and the structure determination and refinement repeated in space group I2. The refinement was entirely satisfactory, and corresponds to the structure presented here. However, the reflections with (h + k + l) odd, which are required to be systematically absent in I2, seemed to be quite definitely present. We are unable to explain this anomaly. The HKL file appended to the CIF contains these reflections.Crystal data, data collection and structure refinement details are summarized in Table 3aromatic 0.95, C—Hmethylene 0.99, C—Hmethine 1.00 Å).Crystal data, data collection and structure refinement details are summarized in Table 2et al., 2013The absolute configuration was determined by the unambiguous Flack parameter of −0.008 (8)  I, global. DOI: 10.1107/S2056989017015213/qm2120Isup2.hklStructure factors: contains datablock(s) I. DOI: 1580696CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The distorted coordination geometry tends towards square-pyramidal with the pyridyl-N atom occupying the apical position. In the crystal, hydroxyl-O—H⋯O(hydroxyl) and hydroxyl-O—H⋯N(pyridyl) hydrogen-bonding give rise to a supramolecular double-chain along [1-10]; methyl-C—H⋯π(chelate ring) interactions help to consolidate the chain. The chains are connected into a three-dimensional architecture via pyridyl-C—H⋯O(hydroxyl) interactions. In addition to the contacts mentioned above, the Hirshfeld surface analysis points to the significance of relatively weak π–π interactions between pyridyl rings [inter-centroid distance = 3.901 (3) Å].In the title compound, [Zn(C Thus, one-dimensional polymers have been found in the crystals of [Cd(S2CNR2)2(NN)]n in cases where R = Et and NN = 1,2-bis(4-pyridyl)ethylene ethane 2. It was of some surprise that coordination polymers based on Cd←N dative bonds were not formed as the putative bridging NN ligand was terminally bound. The first example of this phenomenon was noted in a compound closely related to the title compound, i.e. Cd[S2CN(n-Pr)CH2CH2OH)]22 CH2CH2OH]2 and 1,2-bis(4-pyridyl)ethylene, i.e. 1:2, 1:1 and 2:1, only the binuclear compound {Cd[S2CN(i-Pr)CH2CH2OH)]2}23, featuring one bridging and two terminally bound 1,2-bis(4-pyridyl)ethylene ligands, could be isolated CH2CH2OH]2}2 aggregate was retained in the structure of [{Cd[S2CN(i-Pr)CH2CH2OH]2}2]2 with two terminally bound 3-pyridinealdazine ligands  dithiocarbamates, Cd(S2CNRR′)2]2(NN), i.e. R = CH2CH2OH and R′ = Me, Et or CH2CH2OH for NN = 4,4′-bipyridyl CH2N(H)C(=Y)C(=Y)N(H)CH2(3-pyridyl) where Y = O 2]2 with an excess of 1,2-bis(4-pyridyl)ethylene in which the binuclear species co-crystallized with an uncoordinated molecule of 1,2-bis(4-pyridyl)ethylene CH2CH2OH)]2, (I)By contrast to the chemistry described above for cadmium dithiocarbamates, no polymeric structures have been observed for zinc analogues with potentially bridging bipyridyl molecules. Instead, only binuclear compounds of the general formula . This observation is also viewed as the flat region around these rings in the Hirshfeld surfaces mapped over curvedness in Fig. 7h, with their tips at de + di ∼ 3.2 Å, and wings in Fig. 6i. The low contribution from other contacts summarized in Table 4The overall two dimensional fingerprint plot, Fig. 6et al., 2016e.g. Berdugo & Tiekink, 2009−S2P(OR)2] derivatives, indicating bridging modes are possible in the presence of 1,1-dithiolate co-ligands. There were six examples of structures where 4-pyridinealdazine was present in the crystal but was non-coordinating, and two where the ligand was terminally bound as in (I)i.e. the cadmium analogue of (I)2)2[O(H)Me]22](ClO4)2·4-pyridinealdazine, 1.72MeOH, 1.28H2O 2]2 2]2}2, was characterized in the same study.A search of the Cambridge Structural Database CH2CH2OH]2 Uiso(H) set to 1.2–1.5Ueq(C). The O-bound H atoms were located in a difference-Fourier map but were refined with distance restraint of O—H = 0.84±0.01 Å, and with Uiso(H) set to 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989017012725/hb7702sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989017012725/hb7702Isup2.hklStructure factors: contains datablock(s) I. DOI: 1572824CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Arabidopsis after γ-ray treatment. The aim of this study was to investigate the role of PLD-mediated glycerolipid metabolism in γ-ray-induced membrane injury.γ-rays are high-energy radiation that cause a range of random injuries to plant cells. Most studies on this issue have focused on γ-ray-induced nucleotide damage and the production of reactive oxygen species in cells, so little is known about the glycerolipid metabolism during γ-rays induced membrane injury. Using an ESI-MS/MS-based lipidomic method, we analysed the lipidome changes in wild-type and phospholipase D (PLD)δ- and α1-deficient Arabidopsis leaves after 2885-Gy γ-ray treatment was less than 10%. High does γ-ray treatment could induce the accumulation of intracellular reactive oxygen species (ROS). Inhibition of PLDα1 caused severe lipid degradation under γ-ray treatment. γ-ray-induced glycerolipid degradation mostly happened in chloroplastidic lipids, rather than extraplastidic ones. The levels of lysophosphatidylcholine (lysoPC) and lysophosphatidylethanolamine (lysoPE) were maintained in the WS ecotypes during γ-ray treatments, while increased significantly in the Col ecotype treated with 1100 Gy. After 210- and 1100-Gy γ-ray treatments, the level of lysophosphatidylglycerol (lysoPG) decreased significantly in the four genotypes of Arabidopsis.The ion leakage of γ-ray-induced membrane injury may occur via an indirect mechanism. The degradation of distinct lipids is not synchronous, and that interconversions among lipids can occur. During γ-ray-induced membrane injury, the degradation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) may be mediated by PLDζ1 or phospholipase A1. The degradation of phosphatidylglycerol was not mediated by PLA, PLDδ or PLDα1, but by phospholipase C or other PLDs. γ-rays can decrease the double-bond index and increase the acyl chain length in membrane lipids, which may make membranes more rigid and further cause injury in membranes.The online version of this article (10.1186/s12870-017-1153-9) contains supplementary material, which is available to authorized users. PLDα1-deficient mutant (PLDα1-def) was generated by antisense suppression from Arabidopsis [Columbia ecotype (Col)]. The mutants were confirmed by the absence of the transcript, protein, and activity of PLDα1 or PLDδ .Measurement of ion leakage was performed as described previously, with minor modifications . Twenty-2DCFDA) which purchased from Sigma (D6883). Leaves of 7-day-old Arabidopsis were treated by various doses of gamma irradiation, and stained with 5 μg ml−1 H2DCFDA immediately after treatment, then rinsed three times with MS liquid medium. After staining and rinsing, the leaves were observed under a confocal laser scanning microscope . Images were processed with Adobe Photoshop CS.Intracellular ROS was measured using the ROS-sensitive dye 2′,7′-dichlorofluorescin diacetate (Hw/v) trichloroacetic acid (TCA) reagent, then centrifuged at 12,000 g for 10 min. After that, 2 ml of supernatant was added to 2 ml 0.6% (w/v) thiobarbituric acid (TBA) and incubated in boiling water for 30 min. After cooling and centrifugation, the absorption of the supernantant was determined at 450, 532 and 600 nm. The content of MDA was calculated using the following formula: C (nmom ml-1) = 6.45 × (A532-A600)-0.56 × A540.The level of lipid peroxidation was estimated by content of malondialdehyde (MDA) according to Duan et al. . After ghttp://www.k-state.edu/lipid/lipidomics). Fresh leaves of Arabidopsis were put into 3 ml of pre-heated (75 °C) isopropanol (0.01% butylated hydroxytoluene) to inhibit lipolytic activity. Then the leaves were extracted with chloroform/methanol (2:1) three times (each time for 12 h). After extraction, the leaves were dried at 105 °C overnight and weighed for dry mass. The lipid samples were dried under N2 gas at room temperature for analysis. Data processing was performed as described previously )/100, where N and n is the number of double bonds and acyl carbons in each lipid molecule respectively.Data from discordant samples were removed according to Q-test . SPSS 13Additional file 1:Figure S1. Growth condition of Arabidopsis after different doses of γ-rays treatment for 10 days. WS, Wassilewskija ecotype; PLDδ-def, PLDδ-knockout mutant with WS background; Col, Columbia ecotype; PLDα1-def, PLDα1-deficient mutant with Col background (JPEG 915 kb)Additional file 2:Figure S2. The subcellular distribution of ROS after different doses of γ-rays treatment. Arabidopsis seedlings were stained with 5 μg ml−1 H2DCFDA, and observed under a confocal laser scanning microscope. Bars = 50 μm. AF, autofluorescence; WS, Wassilewskija ecotype; PLDδ-def, PLDδ-knockout mutant with WS background; Col, Columbia ecotype; PLDα1-def, e mutant with Col background (JPEG 3420 kb)Additional file 3:Figure S3. Levels of MDA after various doses of gamma irradiation treatment. Blank bars represent control, black bars represent 2060 Gy gamma ray treated plants, and light grey bars represent 2786 Gy gamma ray treated plants (TIFF 769 kb)


Albeit remotely located, the substituents contort the heptazethrene plane. The phenyl substituents stand approximately perpendicular to the core plane and prevent direct intermolecular contacts of the heptazethrene cores.The title compound was synthesized as a derivative of heptazethrene bearing two methyl and two  50H44, 1, was synthesized as a derivative of heptazethrene bearing two methyl and two tert-butylphenyl substituents, respectively, at the 1,9- and 7,15-positions. The asymmetric unit consists of one half of the molecule, which lies about an inversion centre. Albeit remotely located, the substituents contort the heptazethrene plane. The tert-butylphenyl substituents stand approximately perpendicular to the core plane, with a dihedral angle of 79.09 (5)° between the phenalene ring system and the substituted benzene ring, and prevent direct intermolecular contacts of the heptazethrene cores.The title compound, C As is the case with 2, a typical bond-length alternation in the central hexagon is observed, indicating a quinoidal character for 1  Å is recorded (by using OLEX2) for the carbon atoms at the 7- and 15-positions. The same analysis was applied to 2 , and the maximum distance from the mean plane is 0.103 (3) Å for the carbon atoms at the 8- and 16-positions. The contorted structure of 1 is also evidenced by the torsion angle at the 1–16b–16a–16 °, which indicates that steric interactions between the 1-methyl and 7-phenyl groups may result in the contorted structure.The molecular structure of 1 Fig. 1 consistsnt Fig. 2a. As is 2 Fig. 2c, and t2 -7,15-dimesityl-heptazethrene, and the 1–16b–16a–16 torsion angles are 2.55 (19) and 13.94 (19)°. One molecule is observed in 1,9-bis(hexyloxy)-7,15-bis(pentafluorophenyl)-heptazethrene, and the corresponding torsion angle is 6.44 (18)°.A search of the Cambridge Structural Database , 2-methylnaphthylboronic acid 4 , Pd2(dba)3·CHCl3 , SPhos  and K2CO3  was stirred in a deaerated solvent composed of toluene (70.8 ml), ethanol (17.2 ml) and water (19.0 ml) at 363 K for 24 h. The reaction was quenched by addition of saturated aqueous NH4Cl (50 ml). Organic materials were extracted with CHCl3 (30 ml × 4), and the combined organic phase was washed with brine, dried over MgSO4 and concentrated in vacuo. Crude materials were purified by silica gel column chromatography (eluent: 30% CHCl3/hexane) to afford the coupling product 5 in 2.02 g  as a yellow powder. The compound 5  was dissolved in THF (80.0 mL), and to the solution was added 4-tert-butylphenylmagnesium bromide  at 273 K. The mixture was stirred for 2 h, and saturated aqueous NH4Cl (20 ml) was added. Organic materials were extracted with ethyl acetate (50 ml × 3), and the combined organic phase was washed with brine, dried over Na2SO4 and concentrated in vacuo to give a yellow oil containing diol 6. Without purification, the crude material was dissolved in CH2Cl2 (200 ml), and BF3·Et2O  was added at ambient temperature. After 10 min, methanol (10 ml) was added, and volatile materials were removed in vacuo. The crude material was washed with methanol (50 ml), and a purple solid containing the cyclized compound 7 was obtained. Without purification, the crude material was dissolved in toluene (400 ml), and to the solution was added a solution of 2,3-dichloro-5,6-dicyano-para-benzoquinone  at ambient temperature. After 1 h, the mixture was poured onto a pad of silica gel (250 g) and eluted with toluene to afford the title compound 1. A small amount of contaminants was noted and was removed by washing the compound with methanol (50 ml) to afford the title compound 1  as a purple solid. Suitable single crystals were grown from slow liquid–liquid diffusion of acetonitrile into a toluene solution of 1.The title compound ca 643 K (decomposed); IR : 567, 587, 764, 803, 825, 837, 1017, 1108, 1269, 1362, 1447, 1461, 2864, 2901, 2951 cm−1; 1H NMR  δ 1.32 , 2.36 , 7.03 , 7.09 , 7.25 , 7.28 , 7.36 , 7.37 , 7.38 , 7.72 ; 13C NMR  δ 26.0 (CH3), 31.5 (CH3), 34.6, 125.6 (CH), 126.1* (CH), 127.1 (CH), 127.4 (CH), 129.2 (CH), 129.7, 130.3, 131.0* (CH), 131.7, 132.6 (CH), 133.2, 133.6, 134.7, 136.0, 136.7, 150.3 (Resonances with * appeared with twofold intensities and should contain two overlapping resonances.); HR–MS  calculated for C50H44 [M+H]+ 645.3521, found 645.3545.Physical data: m.p. Uiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) for aromatic H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016020247/is5468sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989016020247/is5468Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016020247/is5468Isup3.molSupporting information file. DOI: 1523781CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Using a carbon microfibers technique, axial stretch was applied to mimic effects of physiological conditions of ventricular filling. A study of cation entry with the same experimental model and the manganese quenching method reported (i) a constitutive cation entry in mdx cardiomyocytes and (ii) the involvement of TRPV2 channels in axial-stretch dependant cation entry, “Axial stretch-dependent cation entry in dystrophic cardiomyopathy: involvement of several TRPs channels”  [1].In this data article, intracellular Ca2+ dye fluo-8 was used for [Ca2+]i measurement, in both resting and stretching conditions, using a perfusion protocol starting initially with a calcium free Tyrode solution followed by the perfusion of 1.8 mM Ca2+ Tyrode solution. The variation of [Ca2+]i was found higher in mdx cardiomyocytes.Here, the Ca Specifications TableValue of the data•Protocol used in these experiments could activate Store-operated calcium channels (SOCs) and stretch-activated calcium channels (SACs).•This protocol can be used to investigate stretch-dependent calcium increases in cardiomyocytes and other cells.•The effects of SOCs and SACs inhibitors and activators may help to understand stretch-dependent calcium increases.12+]i) measured in Wild Type (WT) and mdx cardiomyocytes in two different conditions: at rest and during the application of an axial stretch. Calcium measurement was performed using the Ca2+ dye fluo-8 and during a perfusion protocol starting initially with a calcium free Tyrode solution followed by the perfusion of 1.8 mM Ca2+ Tyrode solution. In these conditions, [Ca2+]i kinetics are described . During experiments, cardiomyocytes were superfused with Tyrode solution containing 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1.8 mM MgCl2, 10 mM Hepes and 11 mM glucose or with calcium-free Tyrode solution containing 140 mM NaCl, 5.4 mM KCl, 1.8 mM MgCl2, 10 mM Hepes, 11 mM glucose, 1 g/L BSA and 20 mM Taurine.Before experiments, cells were stored in culture medium containing Dulbecco׳s modified Eagle׳s medium (DMEM – Lonza: 12-604F), complemented with 10 µg/mL insulin, 10 µg/mL gentamycin, 4 mM NaHCOTranilast (Trn) was purchased from Calbiochem (53902-12-8), GsMTx-4 from Abcam (ab141871), probenecid , Streptomycin sulfate  and 4-methyl-4′-[3.5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-1.2.3-thiadiazole-5-carboxanilide  from Sigma, ryanodine from Merck (15662-33-6), fura-2-AM (108964-32-5) and fluo-8-AM (sc-362561) from Santa-Cruz.2.1.22+-free Tyrode solution. Cells were stretched at 10% of the initial sarcomere length before recording fluorescence.Cells were loaded at room temperature during 20 min with the single-wavelength calcium sensitive probe fluo-8-AM (6 µM) in CaFluorescence images were recorded by Confocal Laser Scanning Microscopy (CLSM) using a Bio-Rad MRC 1024 (Bio-Rad Laboratories) equipped with a 15 mW Ar–Kr gas laser. Confocal unit was attached to an inverted microscope (Olympus IX70) with a 60× water objective and fluorescence signal collection was performed through the control software Lasersharp 3.2 (Bio-Rad Laboratories). Fluorophore was excited with the 488 nm line and the emitted fluorescence was detected at 522 nm (green fluorescence). For analysis, fluorescence was normalized (DF/F0) and calculated through IDL software routines 2.1.3n is the number of cells. Differences were tested with t-test. P<0.05 indicates a statistical significant difference.Data are presented as means±SEM,


The reaction of cadmium dichloride with the ligand 5,6-bis(pyridin-2-yl)pyrazine-2,3-dicarboxylic acid leads to the formation of a two-dimensional coordination polymer. 16H8N4O4)(H2O)2]n. The metal atom is sevenfold coordinated by one pyrazine and one pyridine N atom, two water O atoms, and by two carboxylate O atoms, one of which bridges two CdII atoms to form a Cd2O2 unit situated about a centre of inversion. Hence, the ligand coordinates to the cadmium atom in an N,N′,O-tridentate and an O-monodentate manner. Within the polymer network, there are a number of O—H⋯O hydrogen bonds present, involving the water molecules and the carboxylate O atoms. There are also C—H⋯N and C—H⋯O hydrogen bonds present. In the crystal, the polymer networks lie parallel to the bc plane. They are aligned back-to-back along the a axis with the non-coordinating pyridine rings directed into the space between the networks.The reaction of 5,6-bis(pyridin-2-yl)pyrazine-2,3-dicarboxylic acid with cadmium dichloride leads to the formation of the title two-dimensional coordination polymer, [Cd(C On exposure to air, the compound loses the solvent of crystallization and four water molecules, transforming into a two-dimensional coordination polymer pyrazine-2,3-dicarboxylic acid (W and O2W), and by two carboxylate O atoms (O1 and O3). Atom O1 bridges two cadmium atoms to form a Cd2O2 unit situated about a centre of inversion; the Cd1⋯Cd1ii distance is 3.8753 (8) Å, while the Cd—O1 and Cd—O1ii bonds are, respectively, 2.371 (4) and 2.427 (4) Å, and the Cd1—O1⋯Cd1ii and O1—Cd⋯O1ii bond angles are 107.74 (13) and 72.26 (13)°, respectively. As can be seen in Fig. 1N,N,O) and a monodentate manner (O). It can be seen from the carboxylate C—O bond lengths  that the negative charge is distributed over the O–C–O group for the first, but located on atom O3 for the second.The reaction of 5,6-bis(pyridin-2-yl)pyrazine-2,3-dicarboxylic acid with cadmium dichloride leads to the formation of the title two-dimensional coordination polymer Fig. 1. Here thpyrazine (Cd1—N1) and the Cd—Npyridine (Cd1—N3) bond lengths are the same within 3 s.u.s. [2.418 (4) cf. 2.430 (4) Å]. The Cd—Owater bond lengths [2.301 (4) and 2.317 (3) Å] are shorter than the Cd—Ocarboxylate bond lengths [2.371 (4) and 2.377 (4) Å], while the bridging Cd1⋯O1ii distance is the longest at 2.427 (4) Å. The geometry of the sevenfold-coordinated cadmium atom can best be described as a distorted pentagonal bipyramid, with atoms O1,N1,N3,O2W,O1ii in the basal plane and atoms O1W,O3i in the apical positions with an O1W—Cd1—O3i bond angle of 157.41 (15)° (Table 1Selected bond lengths and angles involving atom Cd1 are given in Table 1° Table 1.The coordinated pyridine ring (N3/C5-C9) and the carboxylate group (O1/O2/C15) are inclined to the pyrazine ring (r.m.s. deviation = 0.03 Å) by 16.9 (2) and 1.9 (6)°, respectively. The non-coordinating pyridine ring (N4/C10–C14) and the second coordinating carboxylate group (O3/O4/C16) are inclined to the pyrazine ring by 60.2 (3) and 89.1 (11)°, respectively. The two pyridine rings are inclined to one another by 75.4 (3) °.bc plane, as illustrated in Figs. 2a axis, with the non-coordinating pyridine rings directed into the space between the networks bis]bistetraaquadicadmium dihydrate) [CSD refcode: FAVHIV; Jiao et al., 20122O2 unit formed about an inversion centre as in the title compound. In FAVHIV, the Cd⋯Cd distance and the angles Cd—O⋯Cd and O—Cd⋯O are, respectively, 4.0408 (5) Å, and 111.05 (8) and 68.95 (7)°, compared to 3.8753 (8) Å, and 107.74 (13) and 72.26 (13) °, respectively, in the title compound. However, such an arrangement is extremely common for cadmium(II) complexes (over 600 hits in the CSD) and the bond lengths and angles vary enormously; for example the Cd⋯Cd distance varies from ca 3.0 to 4.3 Å, the Cd—O⋯Cd angle varies from ca 82 to 119° and the O—Cd⋯O angle from ca 60 to 90°.A search of the Cambridge Structural Database  has been reported previously pyrazine-2,3-dicarboxylic acid  was added to an aqueous solution (25 ml) of CdCl2·2H2O . The colourless solution immediately obtained was stirred for 1 h at room temperature. The reaction mixture was then filtered and the filtrate allowed to evaporate slowly at room temperature. After two weeks, small colourless plate-like crystals of the title compound were obtained, separated by filtration and dried in air . Selected IR bands : ν 1630(m), 1598(vs), 1533(m), 1469(m), 1442(m), 1414(m), 1362(s), 1301(m), 1273(m), 1176(m), 1165(m), 1119(m), 1043(w), 992(w), 829(m), 789(m), 759(m), 675(m), 562(m), 513(m). Analysis for C16H12N4O6Cd (468.71): calculated: C 41.00, H 2.58, N 11.95%; found: C 40.70, H 2.43, N 11.80%.Uiso(H) = 1.5Ueq(O). The C-bound H atoms were included in calculated positions and treated as riding atoms: C—H = 0.94 Å with Uiso(H) = 1.2Ueq(C). The best crystal available was extremely thin (0.01 mm) and as the shape of the crystal was irregular it was not possible to carry out a numerical absorption correction. The displacement ellipsoids for two carboxylate O atoms (O2 and O4) and a water O atom (OW1) are large but attempts to split these atoms were not successful.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989016012858/pk2589sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989016012858/pk2589Isup2.hklStructure factors: contains datablock(s) I. DOI: 1498382CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information: