Both complexes are characterized by a D3 symmetric structure involving three equivalent η6‐bound benzene ligands and a closed‐shell singlet electronic ground state. The analysis of the electronic structure shows that the complexes exhibit metal–ligand bonds that are typical for transition metal compounds. The chemical bonds can be explained in terms of weak donation from the π MOs of benzene ligands into the vacant (n−1)d AOs of M and strong backdonation from the occupied (n−1)d AO of M into vacant π* MOs of benzene ligands. The metals in these 20‐electron complexes have 18 effective valence electrons, and, thus, fulfill the 18‐electron rule if only the metal–ligand bonding electrons are counted. The results suggest that the heavier alkaline earth atoms exhibit the full bonding scenario of transition metals.We report the synthesis and spectroscopic identification of the trisbenzene complexes of strontium and barium M(Bz) Bonding scenarios: The synthesis and spectroscopic identification of the trisbenzene complexes of strontium and barium M(Bz)3  in a low‐temperature Ne matrix is reported. The complexes exhibit metal–ligand bonds that are typical for transition‐metal compounds. The results suggest that the heavier alkaline‐earth atoms exhibit the full bonding scenario of transition metals. These classical s‐block main group elements were for a long time considered to be among the rather uninteresting elements in terms of chemical bonding due to preconceived notions about their valence orbitals, their reactivity and handling.82)8.Oh) adducts possess metal–ligand bonds that are typical for transition‐metal complexes. The participation of the (n−1)d atomic orbitals (AOs) of barium in chemical bonding had been suggested already in earlier theoretical studies3 . The synthesis of the first transition metal benzene complex Cr(Bz)2 by Fischer and Hafner in 1955 was a milestone in transition metal chemistry.3 and Ba(Bz)3 now opens the door to another chapter of metal complexes.The alkali metals and the alkaline earth elements with a (n)s−1 region from co‐deposition of laser‐evaporated strontium atoms with 0.1 % C6H6 are represented in Figure The strontium and barium–benzene complexes were prepared by co‐deposition of laser‐evaporated strontium and barium atoms with benzene molecules in excess neon at 4 K.λ=495–600 nm). Since some bands are partially overlapped by the benzene absorptions, a difference spectrum taken from the spectrum after visible light excitation minus the spectrum after 12 K annealing is obtained, as shown in Figure 4 in the reactant gas to serve as an electron trap.4 captures most of electrons produced by laser ablation during sample deposition, and thus would facilitate the survival of more cation products. The spectra from the experiment with 0.01 % CCl4 added to the neon matrix gas are compared to the spectra from the experiment without CCl4 doping in Figure S1 of Supporting Information. The results show that the observed product absorptions are reduced relative to other absorptions, indicating that these product absorptions are due to a neutral species rather than a cation.A group of metal‐dependent product absorptions are observed after sample deposition at 4 K, remain almost unchanged on sample annealing to 12 K and increase under 20 min of visible light excitation using a high pressure mercury arc lamp with a 495 nm long‐wavelength pass filter  were performed for product identification based on isotopic shifts and absorption splitting. As shown in Figure 13C6H6 and C6D6 samples. The assignment of these product absorptions to the tris(benzene)strontium complex is based on the isotopic splitting of the most intense band at 924.9 cm−1, which is due to the benzene ring breathing mode based on the isotopic data. The spectrum from the experiment using an equimolar mixture of 12C6H6 and 13C6H6 shows that a quartet with two weak intermediates is produced 3, See Figure −1 band is due to a doubly degenerate benzene ring breathing mode of a tris(benzene) complex involving three equivalent benzene ligands. The positions and mode assignment of the observed bands are listed in Table −1 is assigned to the C−C stretching mode. The third strongest band at 1159.1 cm−1 is attributed to the C−H in‐plane bending mode. The remaining bands are weak. The bands at 1332.6 and 1320.1 cm−1 are assigned to mixed modes involving C−C stretching and in‐plane C−H bending. The bands at 933.8 and 810.7 cm−1 are attributed to the out‐of‐plane C−H bending modes.Isotopic substitution experiments (6H6)3 absorptions are the only product absorptions observed in the experiments using different benzene concentrations as well as laser energy. The spectra from experiments using different laser energy are shown in Figure S2. There is no any experimental evidence of formation of the Sr(C6H6) and Sr(C6H6)2 complexes in these experiments.The Sr(Cλ=495–600 nm), but decrease upon UV‐visible light irradiation using the high pressure mercury arc lamp without filters (λ=250–600 nm). The band positions are only slightly blue‐ or red‐shifted with respect to the corresponding modes of tris(benzene)strontium (within 10 cm−1 difference). The spectra from the isotopic‐labeled experiments are shown in Figures S3 and S4. The band positions are listed in Table Similar absorptions are also observed in the barium experiments. The IR spectra are shown in Figure ilters λ=50–600 nm3  and analyzed their electronic structures with quantum chemical methods using density functional theory (DFT) at the M06‐2X(D3)/def2‐TZVPP level of theory, which considers also dispersion interactions. Details of the methods are given in Supporting Information. The calcium complex was also included in the theoretical work in order to provide a comparison with the experimentally observed strontium and barium adducts.We calculated the trisbenzene complexes M(Bz)D3 symmetric M(Bz)3 equilibrium structures with a singlet (1A1) electronic state as energy minima  than in free benzene (1.388 Å), while the other C−C bond lengths are only slightly altered. Calculations with enforced Dh3 symmetry give structures being marginally higher in energy than the D3 form 3 and Sr(Bz)3 have 3 imaginary frequencies, but the Dh3 form of Ba(Bz)3 is calculated as energy minimum, which is only 0.2 kcal mol−1 less stable than the D3 structure. The Dh3 structures of M(Bz)3 have four C−C bonds that are clearly longer (1.411–1.412 Å) than in free benzene while two C−C bonds are a bit shortened (1.382–1.384 Å). The corresponding triplet states of M(Bz)3 posses C1 symmetry for M=Ca and C2 symmetry for M=Sr, Ba and they are 0.6–3.8 kcal mol−1 higher in energy than the singlet D3 structures .The geometry optimizations give a Figure  a. The bm Figure  b. The D3 for the loss of one or all three benzene ligands. The calculations suggest that the BDE of one ligand ranges between De=19.4 kcal mol−1 for M=Ca and De=26.1 kcal mol−1 for M=Sr. The BDEs at room temperature become smaller after correcting for thermodynamic contributions, having values between ΔG298=6.5 kcal mol−1 (Ca) and ΔG298=14.2 kcal mol−1 (Sr). The total BDEs for fragmentation into three benzene molecules are between De=38.6 kcal mol−1 (Ca) and De=46.0 kcal mol−1 (Ba), but the free energies at room temperature are significantly smaller having values between ΔG298=8.7 kcal mol−1 (Ca) and ΔG298=19.5 kcal mol−1 (Ba). Note that the latter process includes the electronic relaxation of the metal atoms to the 1S electronic ground state. The lower BDE of the calcium complex may be the reason why Ca(Bz)3 could not be observed experimentally.Table 3 complexes and their 13C and deuterated isotopes in the region >600 cm−1 as well as the frequency shifts are shown in Tables 3 . The radical cations [M(Bz)3]+  were also calculated. The optimized structures are shown in Figure S6. Both cations are predicted to have D3 symmetric structures with a doublet (2A1) electronic ground state. The calculated vibrational frequencies and intensities are listed in Tables S1 and S2, respectively. Both cations are predicted to have two intense well‐separated C=C stretching modes. Therefore, the assignment of the experimentally observed species to the radical cations can clearly be ruled out, as only one C=C stretching band is observed experimentally.The calculated infrared signals of the M(Bz)3  are formally 20‐electron species possessing a singlet (1A1) electronic ground state. Our previously reported isoelectronic alkaline earth complexes M(CO)82)8Oh) symmetry that possess a triplet (3A1g) ground state. The latter molecules have two singly occupied degenerate orbitals and one lower lying ligand‐based molecular orbital (MO) with a2u symmetry that has zero coefficient at the metal, because it has no valence AO having proper symmetry. We earlier reported the observation of the 20‐electron transition metal octa‐carbonyl anions [TM(CO)8]− .1A1g) ground state, where the degenerate HOMO is fully occupied. The 18‐electron rule first suggested by LangmuirThe complexes M(Bz)Dh3 field of three benzene ligands along with plots of the associated occupied MOs of M(Bz)3. In the Dh3 symmetry field, the five d orbitals of metal split into three irreducible representations (a1′ + e′ + e′′). The a1′ orbital, which is the dz2AO of the metal, provides backdonation to the antibonding π* orbital of the benzene ligands leading to the bonding MO (2a1′). The remaining eight metal AOs serve as acceptor orbitals for the charge donation from the bonding π orbitals of the benzene ligands. There are nine symmetry‐adapted linear combination orbitals from the bonding π orbitals of three benzene ligands. The 1a2′ MO remains as a ligand‐only orbital, because there is no valence AO of the metal possessing this symmetry. All of these bonding and nonbonding orbitals are doubly occupied, resulting in a closed‐shell singlet ground state. Thus, the M(Bz)3 complexes have a total of 20 valence electrons, but the metal centers have only 18 effective valence electrons, and thus still fulfill the 18‐electron rule if only the metal–ligand bonding electrons are considered.Figure 3 in the D3 field of the benzene ligands at the equilibrium geometries and the shape of the occupied MOs of M(Bz)3. Comparing the latter MOs with the Dh3 orbitals shows only negligible differences. The 2a2 MO of the D3 form takes the position of the 2a2′ MO of the Dh3 form and could, in principle, mix with one (n)p AO of the metal. Inspection of the MO coefficients shows that the contribution of the (n)p AO of the metal is nearly zero. Thus, the pattern of the orbital interaction of the D3 structures is very similar to the Dh3 form. We think that the deviation from perfect Dh3 geometry of the M(Bz)3 complexes is due to weak steric interactions between the benzene ligands, which have a negligible effect on the orbital interactions. This corresponds to the reduction of the energy difference between the D3 and Dh3 forms of M(Bz)3 having the order of Ca>Sr>Ba, because the metal–ligand bonds and thus the ligand–ligand distances increase d2ns0np0 electron configuration and three benzene ligands in the singlet state. The strength of the pairwise orbital interactions can be estimated with the EDA‐NOCV  method.2 configuration did not converge. Therefore, we used the BP86‐D3(BJ) functional in conjunction with the TZ2P basis set. Our experience in numerous studies has shown that the EDA‐NOCV calculations are not very sensitive to the functional.3  at the D3 equilibrium structures.On the basis of above bonding analysis, it is clear that the M(Bz)3 attractive interactions come mainly from the covalent term ΔEorb, which provides between 72 % (M=Ca) and 54 % (M=Ba) to the total attraction. The breakdown of ΔEorb into pairwise orbital interactions shows that the dominant contribution ΔEorb(1) comes from the backdonation of the occupied (n−1)d AO of the metal into the vacant π* orbitals of the ligands denoted as (Bz)3←M(d). This orbital interaction contributes between 83 % (M=Ca) and 75 % (M=Ba) to ΔEorb. The following terms ΔEorb(2) and ΔEorb(3) also involve the (n−1)d AOs of the metals. They are due to the donation from two sets of degenerate occupied π MOs of the ligands into vacant (n−1)d metal AOs denoted as (Bz)3→M(d). Thus, the valence AOs of the metals that contribute to the metal–ligand bonding are dominated by the (n−1)d orbitals.The data in Table Eorb(1) interaction to (Bz)3←M(d), which has a1 symmetry, may be questioned because the MO diagram shows that the metal (n)s AO correlates in a D3 environment also with a1 symmetric MOs s AO to the 2a1 MO is negligible. Also, the EDA‐NOCV calculation of the Dh3 structures of M(Bz)3 gives values that are very similar to the D3 analysis. The numerical results of the EDA‐NOCV calculation using the Dh3 structures are shown in Table S3. Figure ρ1−Δρ7 of Ba(Bz)3 which nicely illustrate the charge alteration due to the pairwise orbital interactions ΔEorb(1)−ΔEorb(7). The color code of the charge flow is red → blue. The deformation densities Δρ1−Δρ7 of M(Bz)3 calculated with Dh3 symmetry are displayed in Figures S7–S9. The shape of Δρ7 clearly reveals that the small stabilization energy ΔEorb(7) is solely due to the polarization of the ligand orbitals. The deformation densities Δρ1−Δρ7 of Ca(Bz)3 and Sr(Bz)3 closely resemble those of Ba(Bz)3. They are shown in Figures S10 and S11.The assignment of the Δs Figure  b. Inspe2, which transforms to a1′ in Dh3 symmetry. But the shape of the occupied 1a1′ MO of the complex Ba(Bz)3 shown in Figure D3 symmetric structure s2 and (Bz)3 in the doubly excited singlet state with two electrons in the π* MO. It has been shown by us in numerous studies that those fragments, which give the smallest absolute values for the orbital term ΔEorb, are the most suitable species to represent the bonding of the complex, since they change the least in the formation of the bond. This method has proven to be very helpful for a large number of chemical compounds whose nature is not directly apparent.Eorb values using atom M in the electronic ground state and (Bz)3 in the excited state are significantly larger than those in Table One referee pointed out that the choice of the electronic reference states of the fragments is somewhat arbitrary and that the same state can be obtained from a ground state metal atom and excited benzene molecules, which would yield a different pattern of donation and back‐donation. This is a justified objection, which needs to be addressed. The electronic ground state of the metal atoms has the electron configuration (n)se Figure  b. The v3 using the NBO 6.0 method3 follows the Dewar–Chatt–Duncanson (DCD) model for transition metal compounds, which considers the metal atom and the ligands prior to bond formation.However, there is an alternative option for the choice of the interacting fragments. The calculated partial charges of M(Bz)Eorb value is given when the singly charged species M+ in the doublet state with the electron configuration (n−1)d1ns0np0 and doublet (Bz)3− are employed as interacting fragments. Fragments with other charges or electron configurations give larger ΔEorb values . Table +‐(Bz)3− interactions.We carried out EDA‐NOCV calculations using charged fragments as interacting moieties. We found that the lowest ΔEorb is now a bit smaller than the Coulombic attraction ΔEelstat. The strongest pairwise orbital interaction ΔEorb(1) between the fragments in the electronic doublet state comes from the electron‐sharing bond formation between the unpaired electrons of M+ and (Bz)3−. It is interesting to see that the associated deformation density Δρ1 shown in Figure Eorb(1) when neutral fragments are used 3− have a polarity from the metal towards the ligands. As can be expected, the associated eigenvalue υ1, which indicates the magnitude of the net charge flow, is much smaller for the charged fragments than for the neutral fragments. The relative contribution of the 2a1 orbital interaction between the charged fragments is smaller than the dominating 2a1 term of the neutral fragments, whereas the 2e and 3e orbital terms that come from (Bz)3→M(d) π donation are larger when charged fragment are used. But the most relevant results is the finding that the (n−1)d AOs of the metal are still clearly the most important valence orbitals for covalent metal‐(Bz)3 binding even when charged fragments are used for the EDA‐NOCV calculations.The data in Table d Figure . It mean3 gives neutral atoms M and three neutral ligands Bz and the data in Table + and the negatively charged ligand cave (Bz)3−. EDA‐NOCV calculations using the doubly charged fragments M2+ and (Bz)32− give larger ΔEorb values  than singly charged fragments. The results in Tables The two sets of EDA‐NOCV results in Tables 2,6‐bound benzene ligands, are unprecedented. In the literature, only very few metallocene complexes of actinides involving three η5‐bound cyclopendadienyl ligands have been reported.Benzene is an excellent ligand in the organometallic chemistry of low‐valent transition metals. Since the first synthesis of bis(benzene)chromium Cr(Bz)3 is not just a surprising finding of some exotic species. It seems that the bonding situation of the arene complexes plays also an important role in some intermediates of alkaline earth catalyzed reactions. Alonso, Harder and co‐workers recently reported the alkene hydrogenation catalyzed by simple compounds of the alkaline earth elements Ca, Sr, Ba.D3 where Ca is sandwiched between two aromatic rings 3. A similar situation was reported by the group for the alkaline earth catalyzed imine hydrogenation.The observation of the alkaline earth trisbenzene complexes M(Bz)s Figure . The str3  featuring three equivalent η6‐bound benzene ligands in low‐temperature Ne matrix. The analysis of the electronic structure shows that the complexes exhibit metal–ligand bonds that are typical for transition metal compounds. The chemical bonds can be explained in terms of weak donation from the π MOs of benzene ligands into the vacant (n−1)d AOs of M and strong backdonation from the occupied (n−1)d AO of M into vacant π* MOs of benzene ligands. The metals in these 20‐electron complexes have 18 effective valence electrons, and thus still fulfill the 18‐electron rule, because two valence electrons occupy a ligand‐based MO that has zero coefficient at the metal. The results suggest that the heavier earth alkaline atoms Ca, Sr, Ba may exhibit the full bonding scenario of transition metals.In summary, we report the synthesis and spectroscopic identification of the unprecedented trisbenzene complexes M(Bz)The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Mercury adsorption rates of 6–22% are observed within 30 min and after 48 days, the adsorption is greater than 98%. In stark contrast, no adsorption is found during a testing period of 560 days when the solutions are subject to potassium permanganate‐persulfate digestion. New glass flasks and polypropylene bottles are free of mercury contamination but reused flasks are a major source of mercury cross‐contamination. To minimize adsorption and cross‐contamination, standard solutions are treated by potassium permanganate‐persulfate or BrCl digestion, and each individual sample and standard solution should be stored and prepared in single‐use polypropylene bottle, without transference.Adsorption, sample preservation, and cross‐contamination are the major impediments to the accurate and sensitive analysis of low‐level mercury samples. Common measures to deal with this issue are to use Teflon, quartz, or borosilicate glass bottles for sampling, standard solution and sample preservation with oxidative chemicals, to prepare standard solutions daily and to use dedicated glassware. This paper demonstrates that these measures are neither efficient nor effective. Two common laboratory sample containers (borosilicate volumetric glass flasks and polypropylene tubes) are investigated for the preparation and preservation of water samples and standard solutions of 0.2–1 µg L Adsorption and cross‐contamination are the major challenges for accurate analysis of trace mercury. This study demonstrates that adsorption is the root cause for cross‐contamination, and single use of polypropylene plastic bottles for sample preparation and storage is highly efficient to completely solve these notorious problems as long as sample and standard solutions are subject to potassium permanganate‐persulfate digestion. For most elements, standards and samples at µg L−1 and mg L−1 with 2% v/v HNO3 are stable for months. However, mercury standards and samples are an exception. Significant losses of mercury from µg L−1 solutions have been observed regardless of container materials  on to which mercury may be adsorbed.[qv: 6d] The adsorbed Hg2+ can be reduced to Hg+ and/or Hg0  in the presence of a reducing agent occurring either naturally  plastic bottles and 2% v/v HNO−1 in 1% HNO3 in 250 mL glass flasks in 10 days while Heiden and Aikens[qv: 6b] reported 75% losses for similar solutions in 500 mL PE containers in 15 days. We also observed similar rates of mercury losses from µg L−1 solutions preserved in 50 mL volumetric glass flasks and PP centrifuge tubes. Creswell et al. observed positive biases of total mercury analysis from the original collection bottle after a sub‐sample was removed for other analyses and identified that the positive biases were attributed to the adsorption of mercury on the interior wall of the original collection bottle.[qv: 7b] It is generally recognized that Teflon, quartz, and borosilicate glass have lower tendencies to adsorb mercury than polyethylene (PE) and polypropylene (PP) plastics, although considerable losses of mercury in quartz, Teflon, and glass containers have also been observed[qv: 7a] . However, we often observed mercury contamination for glass flasks after sample digestion even though the flasks had been identified as not contamination by the 24 h acid filling test prior to sample digestion. Parker and Bloom found that new glass flasks were free of mercury contamination.[qv: 6d] However, the use of new volumetric glass flasks for each sample and standard solution is a very costly imposition for routine analysis and testing.FigureThe accurate determination of mercury concentrations at low levels has long been a challenge and the problem is illustrated by an inter‐laboratory proficiency study of trace mercury analysis in water Figure1, whic2−1 solutions (2% HNO3) were observed within 30 min from preparation.[qv: 7a] Indeed, we observed 6–22% losses of mercury from standard solutions of 0.2–1 µg L−1 in 2% HNO3 from 50 mL volumetric glass flasks within 30 min from preparation. The solutions lost 36–43% of mercury from 50 mL PP centrifuge tubes in one day and the losses increased with standing time of the solution, reaching 98% by 48 days  stored in glass flasks and PP tubes while the adsorption rates observed here were similar to the results reported in the literature  with 2% HNO3. In this context, the long‐term stability of mercury solutions after the KMnO4–K2S2O8 digestion was because the digestion deactivates the interior surface of containers, thus preventing adsorption of mercury. To verify this hypothesis, the glass flasks and PP tubes were treated with the KMnO4–K2S2O8 digestion to “deactivate” the interior surface of the flasks and tubes and then mercury solutions were added for the stability test. The solutions prepared with 2% HNO3 in the “deactivated” glass flasks and PP tubes lost greater than 65% of mercury after 48 days whilst the solutions prepared with the “digestion solution” were stable both in “un‐deactivated” and “deactivated” containers  are designed to maintain mercury in the form of Hg2+ in an oxidizing environment. Clearly, mercury in the solutions prepared with “digestion solution” was not in an oxidizing environment because the oxidative chemicals KMnO4 and K2S2O8 are completely reduced by the presence of the excess reducing agent, hydroxylamine hydrochloride  in 50 mL glass flasks and PP tubes were placed on laboratory bench. After 28 days, 25 mL 6% NaCl or 6% NaNO3 was added and then equilibrated for 7 days prior to CVAFS measurement. We found that 94 ± 2% and 74 ± 3% of mercury was recovered from glass by NaCl and NaNO3, respectively and much lower recoveries (54 ± 1% and 53 ± 1%) from the PP tubes. Therefore, the addition of salts  is able to desorb mercury from glass and plastic PP containers. In this context, it would be interesting to see whether the KMnO4–K2S2O8 digestion is able to quantitatively recover mercury from the interior surface of sample containers, in particular from PP containers.To examine the effect of ionic strength on mercury stability, we added NaCl and NaNOns Figure4. The Table4–K2S2O8. The results indicate that the KMnO4–K2S2O8 digestion quantitatively recovered mercury adsorbed on the interior surface of both glass flasks and PP tubes, because at least 60% of the mercury would have been adsorbed from such unpreserved samples in accordance with previous results  prior to total mercury analysis.−1 preserved in HDPE bottle with 4% HNO3. The supplier provided a one‐year guarantee of the stability from the date of receipt. Different batches of this mercury stock solution were tightly capped and stored in laboratory cupboards without any other particular protection. We tested the stability of these stock solutions by instant CVAAS measurement after appropriate dilutions and found that the concentration was within an error of 5% over a period of 5 years from date of receipt. Clearly, the long‐term stability of these mercury stock solutions in the HDPE bottles did not support the loss mechanism of mercury via diffusion through the wall of sample containers and the threads of sample bottle's cap.−1 solutions was most likely that the amount of mercury adsorbed was insignificant relative to the total amount of mercury from such high concentrations.The above results confirm that adsorption of mercury ions on the interior wall of container is the dominant cause for the loss of mercury from standard solutions and samples. Yu and Yan provided a well‐summarized report on the factors affecting the stability of mercury solutions and samples, including concentration of mercury species, sample composition, container material, storage temperature, pH of sample solution and light.[qv: 7a] In general, the relative adsorption rate decreases with increasing concentration of mercury in the solution.[qv: 7a] Toribara et al. observed that the more dilute Hg(II) solutions would be less stable and lose mercury more readily.Table3, placed on a bench for 72 h and then analyzed directly by CVAAS. No mercury contamination was identified , standing for at least 24 h and analyzing the solution,[qv: 15c‐f] while the APHA standard method recommends use of dedicated glassware whenever possible.[qv: 15a] 31)3 soaking overnight is not able to completely remove trace amount of mercury from the interior surface of glass flasks;The typical cleaning procedure using 40% HNO2)Glass flasks and bottles for sample preparation and storage incur high risks of mercury cross‐contamination;3)−1 solutions in 2% HNO3 between volumetric glass flasks and polypropylene tubes;There are no significant differences in the rates of mercury adsorption from µg L4)4–K2S2O8 and BrCl digestion quantitatively desorbs mercury from the interior surface of bottles back into solution;The KMnO5)4–K2S2O8 or BrCl digestion;Mercury samples and standard solutions are extremely stable both in glass flasks and PP tubes after the KMnO6)4–K2S2O8 digestion and prepared by a simple dilution using the KMnO4–K2S2O8 digestion blank solutions.There are no differences in the stability of mercury standards treated by the KMnOFindings of this work are:1)Use new PP bottles for field sampling and storage for trace total mercury analysis and do not add additional “preservative” chemicals;2)Digest, make up and preserve/store the sample in the original sampling bottles;3)Use new PP bottles for standard solutions and prepare the standards as per samples;4)Calibrate the instrument using as‐prepared standard solutions until finished (prepare new after 3 months).The following recommendations are made to eliminate mercury adsorption and cross‐contamination:4Chemicals and Apparatus: High‐purity water used throughout the study was prepared from Millipore Milli‐Q deionized‐water system . Ultrapure concentrated nitric acid , analytical grade sulfuric acid (H2SO4), hydrochloric acid (HCl), stannous chloride (SnCl2), hydroxylamine hydrochloride (HONH3Cl), potassium permanganate (KMnO4), and potassium persulphate (K2S2O8) were obtained from BDH Chemicals, Poole, England. Stock mercury solutions of 20 mg L−1  and 100 µg mL−1  were used for preparation of sample solutions of 0.1–1 µg L−1 by dilution with 2% HNO3. Cold vapor atomic absorption spectrometer (CVAAS), Perkin Elmer FIMS‐400 with an AS‐90 autosampler or cold vapor atomic fluorescence spectrometer (CVAFS), Beijing Titan Instruments AFS‐9130, China, were employed to measure mercury concentrations .acid H2SO, hydroch4–K2S2O8 DigestionKMnO: Mix the sample thoroughly to achieve homogeneity and transfer 25 mL aliquot of the sample into a 50 mL volumetric borosilicate glass flask or 50 mL polypropylene centrifuge tube, add 1 mL of concentrated HNO3 and 10 mL of 0.25 mol L−1 H2SO4. Mix the solution after each addition, then add 7.5 mL of 5% w/v KMnO4 solution and 2.5 mL of 5% w/v K2S2O8 solution to the flask and mix thoroughly. Place the flask in the water bath for 1 h at 95 °C. Cool and add enough 10% hydroxylamine hydrochloride (HONH3Cl) solution (10% w/v) to reduce excess amount of KMnO4 and K2S2O8 in solution until the purple color disappears. Dilute to the mark with water and filter through a 0.22 µm membrane (Whatman Filter No. 541), if necessary. When deionized water was used instead of mercury sample, the resultant solution was termed “Digestion solution.”BrCl Digestion: Transfer 100 mL sample into a 125 mL fluoropolymer bottle. For clear water and filtered samples, add 0.5 mL of BrCl; for brown water and turbid samples, add 1.0 mL; for sewage effluent, add 5 mL. Cap the bottle and digest at room temperature for a 12 h minimum . Add 0.2–0.25 mL of HONH3Cl solution to the BrCl‐oxidized sample, cap the bottle and swirl the sample until the yellow color disappears. Allow the sample to react for 5 min with periodic swirling. Connect a fresh trap to the bubbler, pour the reduced sample into the bubbler, add 0.5 mL of SnCl2 solution, and purge the sample onto a gold trap with N2 at 350 ± 50 mL min−1 for 20 min, prior to CVAFS detection.Analysis with CVAAS or with CVAFS: Mercury was determined by cold vapor atomic absorption spectrometer using tin(II) chloride as reductant at a wavelength of 253.7 nm or cold vapor atomic fluorescence spectrometer using sodium borohydride as reductant. Loss and recovery of mercury from solutions were calculated byC0 is initial concentration of mercury and C is the concentration of mercury after sample processing.Preservation and Storage of Mercury Standard Solutions and Samples: Mercury solutions of 1 µg L−1 were prepared in 50 mL volumetric glass flasks or 50 mL PP centrifuge tubes by dilution from stock mercury solutions with 2% HNO3 or the digestion blank solution (see Section 4–K2S2O8 DigestionKMnO). For comparing stability of mercury solutions, the glass flasks and PP tubes were “de‐activated” by the KMnO4–K2S2O8 digestion and the de‐activated flasks and tubes were used for storing mercury solutions. After preparation, the solutions and samples in glass flasks and PP tubes were capped and placed on the open laboratory bench .Cleaning Procedure for Glass Flasks and Bottles: Glass bottles/flasks were soaked in hot (65–75 °C) concentrated HNO3 for at least 48 h. After cooling, they were rinsed three times with reagent water followed by detergent and deionized water wash. Then, they were rinsed three times with reagent water, and placed in a mercury‐free Class‐100 clean bench until the outside surfaces were dry. The cleaned bottles/flasks were tightly capped and stored in plastic boxes until use.The authors declare no conflict of interest.


Hybrid organic–inorganic sol gel inks that can undergo both condensation and radical polymerization are developed, enabling fabrication of complex objects by additive manufacturing technology, yielding 3D objects with superior properties. The 3D objects have very high silica content and are printed by digital light processing commercial printers. The printed lightweight objects are characterized by excellent mechanical strength compared to currently used high‐performance polymers (139 MPa), very high stability at elevated temperatures (heat deflection temperature >270 °C), high transparency (89%), and lack of cracks, with glossiness similar to silica glasses. The new inks fill the gap in additive manufacturing of objects composed of ceramics only and organic materials only, thus enabling harnessing the advantages of both worlds of materials. Recent interest in 3D printing has led to the development of novel ink compositions and printing technologies, such as for printing of polymer‐derived ceramics,Z‐axis, in order to create a 3D structure, which corresponds to a computer‐aided designed (CAD) 3D model.Z‐axis and 20 µm at XY‐axes) composed of various materials, without the need of support materials, and at a high processing speed.All 3D printing techniques are based on sequential deposition of 2D patterns along the For the radical polymerization initiated by a UV light source at the DLP printer, there is a need for organic photopolymerizable groups. Therefore, most of the structures manufactured by stereolithography are mainly composed of organic polymers with a large variety of optical properties, (transparency or colored), mechanical properties, (flexibility or hardness), and biocompatibility. However, due to the organic backbone, such materials suffer from the lack of toughness and low thermal stability.A combination of organic and inorganic components is a perspective route for obtaining materials with improved properties. For example, Kotz et al. have reported on inks containing silica nanoparticles dispersed in organic photopolymerizable resin,The use of hybrid UV‐cured materials in photolithography has been already reported in the literature.Here, we report on new, 3D printable sol‐gel‐based ink compositions, which will enable fabrication of 3D objects, with unique properties, by the DLP additive manufacturing technology. The liquid ink is composed of hybrid UV‐curable metal‐alkoxy oligomers that can undergo both a sol‐gel process and radical polymerization. The synthesis of hybrid photopolymerizable oligomers is performed by a sol‐gel process, and the curing of the 2D layers of the 3D structures is done via photopolymerization during printing. After printing, during aging, a polycondensation process occurs, in which condensation reactions take place between adjacent silanol groups, forming a stronger network.FigureMore specifically, the hybrid ink was prepared via the sol‐gel process by combining a silicon alkoxy precursor with one photopolymerizable acrylate group, 3‐acryloxypropyltrimethoxysilane (APTMS) with conventional sol‐gel monomers, tetramethyl orthosilicate (TMOS), and methyl trimethoxysilane (MTMS), under acidic conditions Figure1a,b. TTo print the sol‐gel‐based ink by the DLP technology, the ink needs to remain in its liquid form and, therefore, it is crucial to increase its gelation time. This can be achieved by keeping a low pH (≈3) during the synthesis, near the isoelectric point of the silica sol,X–Y axes resolution of the printer used in this study was 39 µm, and the used Z‐axis resolution was in the range of 50–350 µm   Figure2. The During printing, localized radical photopolymerization occurs, forming a covalent bond between the acrylic groups in the oligomers, thus enabling initial fixation of the desired structure. The resulting printed structure at this stage is not rigid, and is a gel‐like material. Following the initial formation of the object, it is left for an aging process. During this process a sol‐gel polycondensation reaction occurs, which finally results in a hybrid, ceramic–organic transparent 3D object with a complex structure and superior properties Figure d.FigureWe have evaluated two types of aging processes: (a) aging at room temperature for the duration of up to one month, (b) aging for 7 d at RT, followed by 3 d at 60 °C and then at higher temperatures, ranging from 60 to 400 °C for 2 h. As shown in The mechanical strength of 3D‐printed objects depends on the properties of the constituent material, its porosity and the architecture of the structure itself.[[qv: 1a]] In sol‐gel processes, the pH during the reaction enables control of the properties of the formed silica gel.Figure−3 (1.364 ± 0.006 g cm−3 after 3 d aging at 60 °C). As the aging temperature was increased (up to 200 °C), polycondensation and further solvent evaporation from the structure continued, causing a further increase in shrinkage and mass loss, which resulted in a density decrease. As the temperature increased from 200 to 300 °C, the density, the shrinkage and the mass , hence, it did not result in any deformation of the 3D structure.To evaluate the mechanical strength of the printed structures, rectangular objects were printed, and the maximum compression stress was measured for samples that were aged at the same process as above. As shown in Figure 3 dimensions were printed, and aged as above. FigureTo evaluate the transparency of the printed 3D objects, cubes with 5 × 5 × 5 mmIn conclusion, a new hybrid, organic–inorganic sol‐gel ink that can undergo both condensation and radical polymerization was developed. This composition enables fabrication of 3D objects with very high silica content by DLP technology. The printed objects are characterized by excellent mechanical strength compared to currently used high‐performance polymers (139 MPa). They are also characterized by very high stability at elevated temperatures (HDT > 270 °C), high transparency (89%), and lack of cracks, with glossiness similar to that of silica glass. The formed objects have a density only slightly above that of common plastics. Such inks pave the way for the printing of various complex and lightweight structures and objects which can be used in additive manufacturing processes while using commercially available low‐cost printers for the production of microfluidic reactors, and molds for the plastics and dental industry. These new inks will narrow the gap in additive manufacturing of objects composed of ceramics only and of organic materials only, thus enabling harnessing the advantages of both worlds of materials.Ink Composition: The ink was prepared as follows: First, the monomers TMOS , MTMS , and APTMS  were mixed at a weight ratio of 3:15:2. Then 16 wt% of 0.5 × 10−3m of HCl solution (Sigma Aldrich) and 1 wt% of TPO  were added to the monomers' mixture. The composition that was achieved was sealed in a dark vessel and stirred at 50 °C for 30 min, after which the temperature was increased to 70 °C and the vessel was opened. The composition was kept under stirring for additional 120 min, until the formed oligomers could form a gel matrix upon exposure to UV light (test preformed in a mold). Then the reaction vessel was closed and cooled down to room temperature.3D Printing: A predesigned model was 3D‐printed using a DLP 3D printer . This printer operates by UV‐LED light source (405 nm), with light intensity of 27 mW cm−2. The printer monomer bath was filled with the hybrid ink, and the exposure time which was suitable for obtaining the 3D objects was between 2 s for layer thickness of 50 µm and 5 s for layer thickness of 350 µm. After 3D printing, the structures were kept in a sealed vessel at room temperature for 7 d for further gelation, then, in open vessel, at 60 °C for 3 d to continue the aging process.Characterization: TGA of printed samples was performed with a TGA/DSC1 stare system Mettler–Toledo in the range of 25–1000 °C at a heating rate of 1 °C min−1.3 with Z‐axis resolution of 350 µm. The three‐point bend test was done on printed and aged models with dimensions of 127 mm × 12.7 mm × 3.2 mm that was cured in a mold.Mechanical tests were performed with Instron 4502 Universal Testing Machine. The measurements of the compression strength were done on printed and aged rectangular models with dimensions of 1 × 1 × 2 cm3.Shrinkage was measured by a caliber for printed and aged cube models with a size of 5 × 5 × 5 mmThe light microscope images of lattice shaped structure  were taken by CX41microscope (Olimpus).3 diamond‐like printed structure were taken by high‐resolution scanning electron microscope (HRSEM) Sirion .SEM images of 1 × 1 × 1 cmEDX of the samples after 1000 °C was performed using Environmental SEM Quanta 200  equipped with an EDX spectroscopy probe .The determination of carbon was performed using the Thermo Scientific FLASH 2000 HT Elemental Analyzer (Thermo Scientific).3 and z‐axis resolution of 350 µm were performed with a UV spectrophotometer .The transmittance measurements of a printed cube with dimensions of 5 × 5 × 5 mm−1 to 270 °C for 2 h, under atmospheric environment.HDT measurement was performed according to ASTM D648 standard with load of 1820 kPa and heating speed of 120 °C . Note: the instrument reached its maximal temperature limit (270 °C) for all the duplicates, hence the exact HDT of the material remains unknown. The samples that demonstrated the HDT were cured by UV light in a mold. The dimensions of the samples were 127 mm × 12.7 mm × 3.2 mm , and their thermal stability was tested by placing 50 g weight while heating at 1 °C min−1 at 25 °C. The ink showed Newtonian fluid behavior.The viscosity of the ink was measured using a HAAKE RheoStress 6000 (Thermo Scientific) with a C60/1° Ti polished cone, and at shear rates between 0.1 and 100 sThe authors declare no conflict of interest.SupplementaryClick here for additional data file.


All three Group 11 metals are strongly corroded by the IL at moderate temperatures to give a very high content of dissolved CuI, AgI, and AuI species. The IL–metal solutions are stable against contact with water and air. The replacement of imidazolium with inorganic sodium cations decreased metal corrosion rates by orders of magnitude. Our results clearly indicate metal oxidation by iodide from dibromoiodide anions to form molecular iodine and anionic [Br‐MI‐Br]−  complexes stabilized by imidazolium counterions. From experiments with a trihalide IL with imidazolium methylated at the 2‐position, we ruled out the formation of imidazole–carbene as a cause of the observed corrosion. In contrast to Group 11 metals, molybdenum is inert against the trihalide IL, which is attributed to surface passivation.The reactions of copper, silver, and gold with the imidazolium‐based polyhalide ionic liquid (IL) [C They exhibit unique physicochemical properties, such as extremely low vapor pressures, wide electrochemical windows, and unusual solvation characteristics, to name only a few, which makes them candidates for many applications.3]−/I− redox couple.3]−/Br−‐based redox couples have also come into focus in this context.Polyhalide anions have a long history in aqueous systems, but have only recently been an active area of interest in ILs. New synthesis techniques have allowed for an expansion of the number of known polyhalide anions , and several interesting potential applications for polyhalide ILs have emerged:2I]− and [I3]− ILs, the authors showed that the vapor pressure was low enough to perform XPS and that beam damage effects were negligible when X‐ray exposure was restricted. By analyzing the shifts in binding energy and shake‐up loss features, Men et al. clearly showed that [Br2I]− and [I3]− are weakly coordinating anions with very low basicity. The negative excess charge on the anions is delocalized over the trihalide complexes to form a π system. For [Br2I]−, the authors found the anionic central iodine atom sitting in a linear [Br‐I‐Br]− complex to be less electronegative than its bromine neighbors.One of the key properties of ILs is their low volatility, which allows for investigations under ultrahigh vacuum (UHV) conditions, such as X‐ray photoelectron spectroscopy (XPS). Over the last decade, XPS studies on a large number of nontrihalide IL systems have been successfully carried out, proving the strength of this surface‐sensitive technique  and 1‐butyl‐2,3‐dimethylimidazolium dibromoiodide ([C4C1C1Im][Br2I]). The latter was chosen to elucidate the role of potential metal carbene formation. We demonstrate that our corrosion procedure is a simple way to synthesize IL systems with a high content of gold(I), silver(I), and copper(I) species, which are stable against exposure to ambient conditions and water. In contrast to the extensive studies of transition metals, aluminum, and steel compounds corroded by ionic liquids,To elucidate the effect of trihalide ILs on noble metals, we carried out a systematic investigation of Group 11 metals . These three metals from the same group have been chosen because they are relevant electrode‐contact materials in DSSCs and in electronic devices in general. They were exposed to two imidazolium‐based ILs with the trihalide anion dibromoiodide [Br22.16C1Im][Br2I] IL, it was noted that the initially dark red‐brown IL turned black within minutes and became more viscous over time. This darkening process occurred much faster with copper foil than with gold and silver foils. With molybdenum foil, no color change was noted.When the copper, silver, and gold foils were immersed in the [C6C1Im][Br2I] for 6 h at 40 °C  pictures were recorded after the samples were rinsed with acetone . Whereas the unexposed part of the foil revealed a smooth and flat appearance, the immersed part displayed a rough surface with etched grooves and pits. Moreover, the exposed Au wire clearly revealed a 6 % decrease in diameter, and the formerly smooth surface became rough and decorated with etching pits on the μm scale . In contrast to the Group 11 metals, molybdenum exhibited no visual changes in the surface; the negligible mass loss and the unchanged surface appearance both indicate that no corrosion of the molybdenum foil took place.All the Group 11 metal foils  showed considerable mass loss after exposure to [C6C1Im][Br2I], which leads to an IL solution with a metal content well above 20 mol %. For comparison with common dissolution rates, such as metal mass loss per exposed surface area per unit time [mg dm−2 d−1] and changes in foil height per unit time [μ min−1], the corresponding values are also given in Table −1 in aqueous I2/I− or alkaline cyanide etching processes.From the mass losses summarized in Table Due to the high metal contents in the IL solutions obtained after dissolution, metal core‐level signals were expected to be clearly detectable XPS. After the 6 h corrosion experiments, the solutions were placed onto a molybdenum XPS sample holder and transferred to the XPS system. The chemical nature of the dissolved metal species, as deduced by using XPS, will be discussed in detail in Section 2.2. The concentration values derived from XPS intensities were directly compared with the mass‐loss values shown in Table 6C1Im][Br2I] and the solutions after the corrosion experiments will be discussed below in detail.The mass losses of the foils after immersion for 6 h in the polyhalide IL, and the quantification of the XPS signals of the remaining solutions, consistently revealed that, for gold, silver, and copper, a considerable amount of metal was dissolved into the IL, whereas molybdenum was virtually unaffected. The XPS spectra of the neat trihalide IL [C2.26C1Im][Br2I] quickly changed color from dark brown to black on contact with the Group 11 foils in the corrosion experiments. The black solutions were spread onto a flat XPS molybdenum sample holder and placed in the fast entry load lock of a UHV system overnight . During degassing under vacuum, the films lost considerable color to give a more‐or‐less transparent liquid layer on the surface of the sample holder the following day. This change in color from dark black to a fully clear film was most pronounced for the solution from the copper corrosion experiment, in which the most metal was dissolved due to the high etching rate (see Section 2.1.). Survey XPS spectra and IL‐ and metal‐related detailed core‐level scans are shown in Figures 6C1Im][Br2I] trihalide IL and for the IL solutions after corrosion of the gold, silver, copper, and molybdenum foils for 6 h. The Calkyl peak at 284.8 eV served as an internal reference for the binding energies, as previously described.As described above, [C6C1Im]BrI quicklyalkyl) and from the five carbon atoms directly attached to a nitrogen atom at 286.5 eV (Chetero). The integrated areas of the two carbon signals of the neat IL revealed a Calkyl/Chetero ratio of 1.1:1.0, which matches the expected 5:5 ratio to within the accuracy of our method (±10 %). The anion‐related signals of the neat IL showed a single iodide peak in the I 3d5/2 region at 620.7 eV, and in the Br 3d3/2,5/2 region at around 68.5 eV . Notably, the I 3d5/2 and the Br 3d signals of [C6C1Im][Br2I] are considerably shifted to higher binding energies (by +2.4 and +1.1 eV), compared with the analogous monohalide ILs [C6C1Im]I and [C6C1Im]Br, respectively . These shifts to higher binding energy are mainly related to the delocalization of negative charge over the trihalide anion compared with the single halide anions, and have already been discussed by Licence and co‐workers.2I]− anion is an extremely weakly coordinating anion with very low basicity. In our measurements, this is reflected by the larger binding energy separation of the Calkyl and Chetero peaks for [C6C1Im][Br2I] compared with the [C6C1Im]Br and [C6C1Im]I monohalide ILs . Note that we derived the iodine intensity only from the main I 3d5/2 line, which includes the broad shake‐up shoulder around 626 eV that contains some intensity from the trihalide anions due to their delocalized charge distribution.Quantification of the XPS intensities of neat [C6C1Im]BrI led to 6C1Im][Br2I] signals, along with an increase in the Calkyl signal and the appearance of an O 1s peak at 530 eV. These additional Calkyl and O signals are attributed to the appearance of nonvolatile contaminants in the near‐surface region after the corrosion experiment, which possibly originate from residual glassware contamination and/or adsorption from lab air. Such contaminants have been reported to be surface‐activeThe XPS spectra after the 6 h corrosion experiment with Mo foil showed no Mo signals (e.g. Mo 3d at around 230 eV), which corroborates the negligible mass loss found for the IL‐exposed Mo foil , so it is most likely that molecular iodine I2 was pumped away. After testing in a separate UHV system, gas‐phase mass spectra indeed showed the release of I2 from [C6C1Im][Br2I] when the IL was in contact with copper . It is important to note that the Br 3d peaks given in Figure −) during corrosion was ruled out due to the absence of single‐halide Br− signals in the Br 3d region at lower binding energies.Next, we discuss the IL‐derived XPS spectra in Figure  neat C6CImBr2I].I.5/2 reg5/2,7/2 between 92 and 79 eV, Ag 3d3/2,5/2 between 388 and 364 eV, and Cu 2p1/2,3/2 between 960 and 925 eV. For comparison, the XPS spectra of Au0, Ag0, and Cu0 in the form of freshly sputtered metal foils are also depicted  with no loss features was observed, which is indicative of the formation of AgI.3/2 at 932.3 eV) with respect to clean copper foil (Cu 2p3/2 at 932.9 eV). The magnitude of the shift to lower binding energies and the absence of pronounced shake‐up structures—which should be present for a CuII species—are fully in line with the formation of a CuI species.Clean silver foil exhibits a sharp Ag 3d doublet, with the Ag 3d2.3I, AgI, and CuI species was dissolved in the trihalide IL at the expense of iodine. Whereas AgI and CuI species are stable oxidation states in aqueous systems , AuI is metastable and prone to disproportionation to AuIII and Au0 in water without stabilizing ligands.As shown above, a considerable amount of metal in the form of Au6C1Im][Br2I].When the gold‐corrosion experiment was performed under anhydrous and water‐saturated conditions for 6 h at 40 °C, we found no significant differences in the amount of dissolved Au, and also no significant difference in the XPS spectra  compared with the results described above. This indicates that neither water nor oxygen are necessary for (or inhibitory of) the corrosion of gold in contact with [Br2I], that was methylated at the 2‐position to prevent proton abstraction. Due to the higher melting point of this IL, we carried out the corresponding corrosion experiment at 50 °C, and the foil mass loss results are given in Table S1. The mass loss of gold after 6 h was about 30 % less than with the nonmethylated IL, which might be related to the higher viscosity of [C4C1C1Im][Br2I] even at 50 °C; the molar amount of gold dissolved per mole of [C4C1C1Im][Br2I], however, was independent of the methylation. Overall, the efficiency of Au corrosion was found to be very similar for [C6C1Im][Br2I] and [C4C1C1Im][Br2I].Stable Au2I] system was prepared by dissolving NaBr (25 mmol) and IBr (13 mmol) in water . A gold foil (305 mg) was then immersed into this highly concentrated solution for 24 h at room temperature. After rinsing and drying the remaining gold foil, we found a mass loss of (12±1) mg, that is, about 0.06 mmol Au dissolved, which proved that gold is moderately soluble under these circumstances. Note that about 1 mL of [C6C1Im][Br2I] or [C4C1C1Im][Br2I] is able to dissolve at least ten times more gold within 6 h.Finally, we explored whether the trihalide anion was able to dissolve gold without the presence of an imidazolium counterion. For this purpose, a saturated solution of an aqueous Na[Br36C1Im][Br2I] and [C4C1C1Im][Br2I] are able to dissolve the Group 11 metals copper, silver, and gold efficiently by forming +1 metal ions at the expense of iodine; this is in contrast to the situation for molybdenum, which is not attacked. Based on these experimental results, the following reaction mechanism of a Group 11 M0 metal with an [Br2I]− anion is highly plausible:We have consistently shown by using weight loss and XPS experiments that the trihalide ILs [C2 that is later removed under vacuum conditions due to its high vapor pressure. We propose that the MI ion is stabilized by two Br− anions arranged in a linear fashion to form a [Br‐MI‐Br]− anionic complex, which is known, for example, in AuI–halide systems.in which the iodide of the trihalide anion oxidizes the metal atom to oxidation state +1. The reduced iodine radical eventually recombines with another iodine radical, which leads to dissolved iodine I2 that remains dissolved under ambient pressure, but is lost under vacuum. The latter process was revealed by gas‐phase analysis and by the decoloration of the IL film on the sample holder after degassing. According to the proposed reaction mechanism, the amount of Br remains essentially constant, as confirmed by using XPS. The fact that the Br 3d XPS signals from the trihalide IL before and after corrosion do not exhibit new signals at different binding energy—which should be visible through significant peak shape changes—clearly supports the proposed idea that the Br atoms remain in an anionic [Br‐MI‐Br]− complex with the negative charge delocalized. The formation of free bromide Br− and free iodide I− anions can be ruled out by using XPS because of their very different binding energy positions.This reaction mechanism explains the XPS metal signals of oxidation state +1 in solution, the selective decrease in the iodine signals in the XPS spectra and the increase in iodine in the gas phase over the course of the reaction. We attribute the color change of the trihalide ILs (from deep brown to black) upon immersion of the Cu, Ag, and Au foils to the formation of I4C1C1Im][Br2I] compared to the nonmethylated [C6C1Im][Br2I] IL and the lack of significant spectral changes in the cation‐related XPS peaks revealed that the imidazolium C2 proton is not involved. This conclusion was confirmed by the NMR spectra of the [C6C1Im][Br2I] IL after gold corrosion, which showed no indication of an AuI–carbene complex. Finally, the poor ability of highly concentrated aqueous Na[Br2I] to corrode metallic gold indicated that the imidazolium cation is not an innocent counterion, but plays an important role in the dissolution of the anionic [Br‐MI‐Br]− complex. In particular, the fact that the trihalide ILs oxidized gold to a stable +1 state, even in the presence of excess water, is worth noting. AuI is known to be relatively unstable in aqueous solution unless there is a sufficient concentration of halide ions and a low enough pH to maintain a stable complex, and will otherwise disproportionate to Au0 and AuIII.2]− complex, which makes the imidazolium trihalide IL suitable for the corrosion process. Here, further experimental evidence, such as crystal‐structure investigations and tests with other IL cations, is needed, but is, however, out of the scope of this study.For the most noble metal, Au, experiments with both a carefully dried IL and in the presence of water, showed no significant difference in the corrosion efficiency, which ruled out any significant influence of water. Moreover, the similar corrosion rate of gold by [Br2I] was prepared by mixing 1‐hexyl‐3‐methylimidazolium bromide  with iodine monobromide  in a 1:1 molar ratio. After mixing both components, we obtained a deep red‐brown liquid with a considerably lower viscosity than [C6C1Im]Br. We avoided exposure of the IL to daylight to prevent any photochemical reactions that might occur.The RT trihalide IL [C4C1C1Im][Br2I], methylated at the 2‐position of the imidazolium ring. This IL was prepared by mixing dry 1‐butyl‐2,3‐dimethylimidazolium bromide  with IBr , that is, a 1:1 molar ratio. When the two ingredients were mixed in a mortar, the IL initially liquefied and a deep red‐brown liquid was obtained. The mixture was then placed in an evacuated bell jar to remove adsorbed water from the hydrophilic compound, which led to solidification after several minutes under vacuum. After regrinding, the obtained deep‐orange powder exhibited a melting point in the range of 40–50 °C.To investigate whether carbene formation played a role in the reaction, we studied a noncarbene‐forming trihalide counterpart, [C2C1Im][AuBr4]) was synthesized as an IL‐analogous AuIII compound, which served as an XPS binding energy reference: AuBr3  and [C2C1Im]Br  were placed in a 50 mL 2‐neck flask fitted with a septum and a reflux condenser. Dry CH3CN (20 mL) was added through the septum using a syringe, and the initial suspension was heated at reflux overnight. After cooling, the deep‐red solution was syringe‐filtered  and transferred to a 50 mL flask. The solvent was evaporated by using a rotary evaporator to give a dark‐red solid . The material was analyzed using inductively coupled plasma optical emission spectrometry (ICP‐OES), which revealed an Au content of 30.3 wt % .1‐Ethyl‐3‐methylimidazolium tetrabromoaurate ([C6C1Im][Br2I] (≈1 mL) in a glass tube under air. The tubes were closed and then heated to 40 °C for 6 h in a water bath. A second gold foil (619.8 mg) was also corroded in [C4C1C1Im][Br2I] by using the same procedure, except that the reaction was carried out at 50 °C due to the higher melting point of the C2‐methylated IL. For each corrosion experiment, the boiling tubes were gently shaken occasionally. After 6 h, a few drops of the liquid were removed from the tubes and placed on a molybdenum sample holder to form a thin layer for XPS analysis. The metal foils were removed from the tubes, rinsed with acetone, dried, and reweighed. In addition, a 0.1 mm thick gold foil and a 1 mm thick gold wire were half‐immersed into [C6C1Im][Br2I] at RT for about 17 h. These samples were also studied by using SEM.To investigate the effect of the trihalide ILs on Group 11 metals, 15×20 mm foils of polycrystalline copper , silver , gold , and molybdenum  were rinsed with acetone, dried, weighed, and then immersed in [C6C1Im][Br2I], and “wet” [C6C1Im][Br2I] to elucidate the role of water in the gold corrosion process: In the dry IL experiment, [C6C1Im][Br2I]  was dried by heating to 50 °C in a continuously evacuated Schlenk flask for 24 h. A gold foil (609 mg) was added and the vessel was purged with nitrogen  before being sealed against air contact. For the wet IL experiment, the IL  was added to a Schlenk flask, followed by deionized water . The two liquids were not miscible even after several minutes of agitation, and the water formed a layer above the IL. This water layer initially had a slight red‐brown color. A gold foil (526 mg) was then placed into the bilayered system and fully immersed in the IL phase. Both Schlenk flasks were held in a water bath at 40 °C for 6 h. Samples of the ILs were taken for XPS analysis after the reaction, and the masses of the remaining gold foils were measured.Two further corrosion experiments were performed with “dry” [C−7 mbar) and pumped down overnight before being transferred to the main UHV system for XPS analysis (base pressure better than 1×10−10 mbar).Before and after the reactions, XPS analysis was performed for all IL samples. They were placed on a precleaned molybdenum sample holder by spreading a thin liquid film (≈0.1 mm thick) at the bottom of a reservoir (dimensions 14×20×0.5 mm) milled into the sample holder. The sample holder was introduced into a fast‐entry load lock . Metal peaks were fitted similarly, except the pseudo‐Voigt profiles had an 80 % Lorentzian contribution. Shirley backgrounds were used. Carbon 1s spectra were fitted with a two‐peak model, Calkyl and Chetero. The Chetero peak was constrained to a FWHM of 1.1 times the width of the Calkyl peak.Peaks in the XPS spectra associated with nonmetal elements were fitted with a pseudo‐Voigt profile with a 30 % Lorentzian contribution. Doublets due to spin‐orbit splitting (such as I 3d) were fitted with two peaks constrained to have equal full width at half maxima (FWHM) and the expected peak‐area ratio  should be addressed to the authors.SupplementaryClick here for additional data file.


Heterochromatin protein 1α (HP1α) is a protein that mediates cancer‐associated processes in the cell nucleus. Proteomic experiments, reported here, demonstrate that HP1α complexes with importin α (IMPα), a protein necessary for its nuclear transport. This data is congruent with Simple Linear Motif (SLiM) analyses that identify an IMPα‐binding motif within the linker that joins the two globular domains of this protein. Using molecular modeling and dynamics simulations, we develop a model of the IMPα‐HP1α complex and investigate the impact of phosphorylation and genomic variants on their interaction. We demonstrate that phosphorylation of the HP1α linker likely regulates its association with IMPα, which has implications for HP1α access to the nucleus, where it functions. Cancer‐associated genomic variants do not abolish the interaction of HP1α but instead lead to rearrangements where the variant proteins maintain interaction with IMPα, but with less specificity. Combined, this new mechanistic insight bears biochemical, cell biological, and biomedical relevance. Alterations in HP1 proteins range from overexpression, somatic mutations, CNVs, and abnormalities in the function of their upstream regulators. Early studies performed by our group and others have demonstrated that signaling‐mediated mechanisms, yet to be fully understood, lead to the extensive post‐translational modifications of HP1 proteins. Some of these modifications fall in the globular domains of these proteins; the globular domains read histone marks and are responsible for homodimerization and heterodimerization. These domains are joined by an instrinsically disordered region known as the linker, which serves as an integrator of phosphorylation‐mediated signaling events. However, some of the detailed structural‐functional relationships of the linker domain are by far less understood than those of the globular domains. Thus, the current study uses the HP1α linker domain, as a model for extending our understanding of how these proteins can be regulated via the linker domain through heterodimerization events that ultimately lead to the translocation of this protein to the nucleus, the cellular region where it is functionally needed. Moreover, we model how post‐translational modifications, deposited in the linker region in response to upstream regulators such as cancer‐associated mutations, alter linker structure and thereby bonding patterns during interphase, likely impacting import and the downstream pathway.Three known HP1 isoforms, HP1α CBX5), HP1β (CBX1), and HP1γ CBX3), are critical for regulating gene expression networks that are crucial for normal embryonic development, are cri, HP1β , HP1γ (CBX3), and HP1β (CBX1) were downloaded from UniProt2.2The canonical binding mode of the nuclear importin receptor protein, IMPα, and nuclear localization signal (NLS) containing proteins was explored in the PDB and the literature.2.3The crystal structure of the linker peptide which connects the chromodomain and chromoshadow domain has not been solved, likely due to its flexibility. Therefore, we built the model of the peptide using the sequence of residues 87‐109 and the Builder Function in Discovery StudioXenopus laevisThe binding mode of a bipartite NLS to IMPα was previously demonstrated for the histone‐binding protein N1N2 in 2.4HP1α immunoprecipitation was performed from HeLa cells. An antibody to HP1α  or control IgG was conjugated to Protein A/G Magnetic Beads (ThermoFisher Scientific) through disuccinimidyl suberate (DSS) crosslinking. Cells were lysed with immunoprecipitation Lysis/Wash Buffer (Thermo Scientific‐Pierce), and lysates were incubated with the conjugated antibodies overnight at 4°C. Immunoprecipitated complexes were washed, eluted and run on a 4%‐15% Criterion Tris‐HCl Protein Gel (Bio‐Rad), which was subsequently visualized with BioSafe Coomassie Stain (Bio‐Rad). Each gel lane was divided into eight sections, destained, dehydrated, dried, and subjected to trypsin digestion. Liquid chromatography (LC)‐ESI‐MS/MS analysis was then performed on a Thermo Scientific LTQ Orbitrap mass spectrometer at the Mayo Clinic Proteomics Core.2.5−4) were mined from The Cancer Genome Atlas (TCGA),We modeled experimentally relevant phosphorylated, nonphosphorylatable mutant, or phosphomimetic mutant forms of S92, S95, S97, and S103 within the HP1α linker separately or in combination. As controls for binding, HP1α NLS binding mutants were also generated by mutating each cluster of basic residues within the bipartite sequence to acidic residues. Additionally, rare single nucleotide variants in HP1α that are rare in the currently healthy adult population  implicit solvent model in Discovery StudioMolecular dynamics (MD) simulations were executed as in our previous studies.2.7α atoms of IMPα. We calculated root‐mean‐square deviation (RMSD) and root‐mean‐square‐fluctuation (RMSF) values to infer the flexibility and mobility of the peptides throughout the simulation and using Cα atoms. The surface area of the IMPα receptor alone and the peptide alone were measured to uncover any differences in conformation or folding, and then buried solvent accessibility surface area (SASA) of the peptide in each condition was measured and plotted. Hydrogen bonds and salt bridges between the linker peptide and IMPα were calculated across the conditions and visualized as heat maps. We analyzed hydrogen bonds that had a frequency ≥ 0.5 in at least three different conditions. The energy of interaction, encompassing van der Waals and electrostatic forces, was calculated to survey the interaction strength of each complex. The Cα atoms of residue pairs involved in hydrogen bonding or salt bridge interactions between the peptide and IMPα were selected for distance metrics as simplified measures of interaction. Contact maps defining the intermolecular contacts between the HP1α peptide across the trajectory were generated. Atoms were considered contacting if they were within 3.9 Å and the fraction of simulation time spent in contact was recorded. Principle component analysis (PCA) of the Cα atoms was calculated in Cartesian space. All distributions were compared using permutation and t tests, as previously described.Prior to calculations, all trajectories were superimposed using C2.8Initial protein models were constructed in Discovery Studio v201733.1The translocation of HP1α to the nucleus is a critical step for realizing its functional role in epigenetic inheritance of cancer; however, mechanisms that can regulate this critical process remained poorly studied. Notably, SLiM analysis identified a bipartite NLS within the HP1α linker (IDR2)  and adjacent residues in IMPα. Monitoring salt‐bridge formation revealed that many interprotein interactions are common to all conditions, but some are variant specific Figure ; specifiThe nonphosphorylatable variants  formed new salt bridges through K105 and K107 that contributed to the stabilization of the interaction with IMPα. Phosphorylation at S92 only or S92‐95‐97 resulted in the formation of a salt‐bridge between R90  in HP1α and D208 (major groove) in IMPα, which is congruent with the occurrence of a conformational change that causes the N‐terminal part of the HP1α NLS (KRK) to lose contact with the minor groove and make new contacts with the major groove to preserve the interaction. Only one new salt‐bridge was formed upon phosphorylation of S92‐95‐97‐103, and this intrachain interaction occurred at the end of the peptide between E108 and R107. The phosphomimetic mutants again displayed different patterns of interactions from that of the phosphorylated protein. New salt‐bridges were formed between the mutated residues  and nearby residues in IMPα suggesting a closer affinity of the two proteins. This observation is important as experimentalists typically consider phosphomimetic mutants to be similar to a phosphorylation event, but our data revealed differences.Next, we computed the total interaction energy between each HP1α peptide and IMPα. The interaction energy is influenced by all other interactions quantified above, and thus the combination of metrics is more interpretable than each alone. The wild‐type and nonphosphorylatable variants showed lower interaction energies , compared to phosphorylated HP1α peptides and phosphomimetic mutants  Figure C. The siWe also monitored the distance between residues participating in hydrogen bonds as a simplified geometric measure to facilitate interpretation Figure A. The fi3.4We considered the specific patterns of residue‐level rearrangements across conditions. First, we visualized the final conformation between the two members of the complex, as a visual guide to how the interactions changed during simulation Figure . We quanSubsequently, we analyzed conformational dynamics of the bound linker peptide using PCA. Comparing the mean values for each trajectory in PC1‐PC2 space shows variation among technical replicates Figure , but wit3.5To gain more insights into how the regulation of HP1α, through its linker domain, may be altered by cancer‐associated mutations, we evaluated six additional genomic variants, applying the same approaches used above to assess phosphorylation. Peptide RMSD measures global changes from the initial contacts; S92Y and Q109H were associated with smaller deviations, while F94L and D99A were associated with larger departures Figure . RMSF vaWe next analyzed interacting residues at the binding interface between both proteins. Certain hydrogen bonds between the peptide and IMPα were stable across all variants. For example, the hydrogen bond between R90 in HP1α and E324 in IMPα was stable in all but D99A. However, all the variants formed new bonds with IMPα . Interes4Our laboratory has studied HP1 proteins for several years in relationship to their function as epigenomic effectors of growth signaling pathways. Interestingly, our own work and that of others has been, throughout the years, biased to studying the structural‐functional relationship of the globular domain of these proteins. Thus, the current study, which is focused on using molecular modeling and dynamic stimulations to interpret HP1α linker phosphorylation and cancer‐associated genomic variants bears novelty and biomedical relevance. In particular, we specifically investigate how these regulatory and pathological events alter interactions with the nuclear receptor, IMPα. Linear motif analyses identify, with a high level of confidence, an NLS consensus site within this intrinsically disordered region of the protein. This data is congruent with our immunopurification experiments that reveal the existence of a HP1α‐IMPα complex. Linear Motif analyses for post‐translational modifications combined with data from large scale mass spectrometry experiments demonstrate that the linker is amenable to phosphorylation at different sites. Thus, the HP1α linker appears to function as a binding interface for IMPα and in the integration for upstream regulatory cell signaling pathways. More importantly, this complex is predicted to be responsible of transporting HP1α into the nucleus, where it becomes functional. We find that several cancer‐associated mutations affect the sequence of this domain in a manner that we predicted may disrupt these functions. Indeed, molecular modeling and dynamic simulations, combined with an analytical approach that uses different metrics, provide evidence to support this prediction. Briefly, we find that the unmodified linker peptide binds to IMPα via hydrogen bonds and salt‐bridges formed between the bipartite NLS of HP1α and acidic patches within the major and minor grooves of IMPα. The selected variants studied here were rare missense mutations reported in various cancers but not in the general population. Our modeling experiments show that the interaction between these proteins is inhibited by phosphorylation of the four serine residues within the linker, which leads to a more extended HP1α conformation that is not compatible with the canonical binding mode for IMPα. Missense mutations in the HP1α linker rearranged interactions with IMPα, likely leading to loss of binding or nonspecific binding. The changes in linker conformation were mediated by modification of intramolecular and intermolecular contacts that reflect the dynamic property of the linker peptide. Interestingly, phosphorylation of S92 might be sufficient to disrupt the association of the HP1α linker with IMPα; however, the phosphomimetic mutant of the same residue was more potent at shifting the peptide out of the IMPα binding pocket. Furthermore, the phosphorylation of S92 only or even phosphorylation of S92‐95‐S97 did not achieve the full effect in the form of reduced binding affinity for IMPα, as did phosphorylation of all four serine residues , suggesting that all four residues may affect the mechanism of IMPα interaction. These data support our own and previous findings that each serine residue within the NLS is phosphorylated during mitosis.The authors declare no potential conflict of interest.Table S1. Multiple Sequence Alignment (MSA) of human HP1α, HP1γ, and HP1β. The MSA was used for comparison of shared sequence and structure among the HP1 isoforms. Protein sequences were acquired from UniProt. Reported SNVs in disease and normal populations are shown in the first row. MESSA predictions of secondary structures  and disorder predictions (shown by asterisks highlighted in gray) are shown in the second and third rows, respectively.Table S2 HP1α is phosphorylated at serine residues within the linker by numerous kinases.CBX5)Table S3 Genomic variants reported in HP1α  Total surface area of the HP1α linker peptide is reduced only with mutation of the basic residues in the NLS binding motifs (KRK or KKK or both) to acidic residues.Figure S2 Visualization of superimposed final conformations of the HP1α linker peptide bound to IMPα in related conditions. Conditions are grouped based on the residue (s) of interest in comparison to wild‐type. (A) S92 conditions (B) S92‐95‐97 conditions (C) S92‐95‐97‐103 conditions (D) NLS binding mutant conditions.Figure S3 Genomic variants strengthen and increase the number of Intermolecular contacts between HP1α and IMPα. Residue‐residue contacts between HP1α and IMPα were calculated and plotted in a contact map matrix. A) Points indicate residue pairs that are within 6 Å. B) A grayscale gradient indicates average proximity.Figure S4 Genomic variants in the HP1α linker promote reorganizing of the intermolecular contacts to stabilize the complex with IMPα. (A) Hydrogen bond matrix of hydrogen bond donor and acceptor pairs in all conditions. The constant presence (probability ≥0.5) or absence (probability ≤0.5) of a hydrogen bond is defined by the black to white color scale. (B) The salt‐bridge matrix uses the distribution value of 7.5 as a cut‐off for the presence of a salt‐bridge between the donor and acceptor pair. Colors in the range of light purple to black indicate the presence of a salt‐bridge. Chain A refers to IMPα and chain B represents the HP1α peptide. (C) Total interaction energy of the HP1α‐IMPα complex across all conditions. Negative kcal/mol values represent the energy of unbinding and destabilization of the complex. Values closer to or greater than zero indicate a stable interaction. (D‐F) Distance metrics of each residue pair as outlined in Figure Figure S5 Phosphorylation and phosphomimetic mutation of all serine residues in the HP1α linker increases deviation from the wild‐type conformation. Principle component analysis of all frames of the trajectories from each experimental condition demonstrates an overall divergence of the fully phosphorylated or phosphomimetic HP1α peptide from wild‐type and all other conditions. (A) Comparison of PC1 and PC2 shows clustering of the wild‐type, non‐phosphorylatable, and partially phosphorylated or phosphomimetic peptides. (B) Comparison of PC1 and PC3 illustrates the similarity via clustering together of the phosphorylation or phosphomimetic mutation of S92 through S103. (C) Comparison of PC2 and PC3 shows that phosphorylation of S92 through S103 diverged from all the conditions except the NLS binding mutants. HP1α linker genomic variants are comparable to the wild‐type protein. Principle component analysis of the trajectories demonstrates clustering of the variants with wild‐type with the exception of two replicate outliers from wild‐type and the I101M variant. (D) PC1 vs PC2 (E) PC1 vs PC3 (F) PC2 vs PC3.Figure S6 Genomic variants in the HP1α linker do not drastically affect the overall mobility or conformation of the peptide. (A) RMSD calculation illustrates little deviation of the variants from wild‐type but increased variability similar to wild‐type in the I101M and Q109H variants. (B) RMSF calculation shows the F94 L variant diverges most from wild‐type. (C) Buried SASA of the HP1α linker peptide in all conditions. (D) Total surface area of the HP1α linker peptide in all conditions.Figure S7 Visualization of the final conformation of the HP1α linker variant peptides bound to IMPα. (A) Wild‐type (B) S92Y (C) F94 L (D) S95 L (E) D99A (F) I101M (G) Q109H (H) Superimposition of all peptides bound to IMPα.Click here for additional data file.


Importantly, the lead fragment only bound at the interface and was not observed in the ATP binding site of the protein when co-crystallised with CK2α. The fragment-like molecules discovered in this study represent unique scaffolds to CK2 inhibition and leave room for further optimisation.Increased CK2 levels are prevalent in many cancers. Combined with the critical role CK2 plays in many cell-signaling pathways, this makes it a prime target for down regulation to fight tumour growth. Herein, we report a fragment-based approach to inhibiting the interaction between CK2α and CK2β at the α-β interface of the holoenzyme. A fragment, CAM187, with an IC If inhibited below the high critical threshold, the cancerous cell would no longer be malignant as there are no alternative signaling pathways which could by-pass the need for CK2.1.250. Additionally, in one study CX4945 was shown to inhibit CLK2 more potently than CK2 (3.8 nM compared to 14.7 nM).Previous research into CK2 inhibition has focused on small molecules that inhibit the kinase activity at the ATP-binding site.Alternative strategies to selectively inhibit CK2 have recently been explored; these exploit other aspects of the protein unique to CK2. The αD pocket, which is unique to CK2, was discovered near the ATP site. It has been exploited by linking a low affinity ATP site fragment to an elaborated fragment in the αD site. This led to a novel and selective CK2 inhibitor.15Pc, was found to inhibit the interaction between CK2α and CK2β.One of the other areas to probe for non-ATP site inhibition is the protein-protein interaction (PPI) interface between the catalytic and regulatory subunits of CK2: CK2α and CK2β. To date three small molecules and a peptide have been shown to bind in the interface pocket on CK2α. The fragment, DRB, binds at both the ATP and the α/β interface site .16 W16 a1.3−1) to identify starting points against any target of interest.The fragment-based approach to drug discovery utilises small compounds  conducted initial high throughput screening of fragments from their fragment library against CK2 using an FP (fluorescence polarization) assay. This library consisted of over 400 compounds from the Abell Group. The most promising fragment from this screen was NMR154 .24 This A crystal structure was obtained of NMR154L, the ethyl analogue of NMR154, a showed With this crystal structure in hand, it was hoped that the binding could be improved by modifying NMR154 along a series of vectors, utilizing anchoring in the pocket and increasing the interactions outside the pocket with the protein residues .3.2The first iteration explored was substituting the methylamine group 50 ≈ 0.4 μM)ture see . W16  giving an IC50 of 150 μM. Realising this trend additional biaryl compounds were screened against CK2 to give the lead compound, CAM187 (7). These compounds affect their binding by the biaryl part of the molecule filling the PPI pocket on the protein and the pendent ammonium group forming an H-bond with an Asp37 above the pocket.By screening the NMR compound library an initial hit was found for an inhibitor of the PPI between the α-β subunits of CK2 with an ICCAM187 is the first small molecule fragment to inhibit the α-β interface of CK2 without also binding in the ATP site. Due to its fragment-like properties, CAM187 leaves room for further development. These developments could lead to drug-like molecules with high potency and selectivity against CK2 as well as deeper understanding between the structure activity relationship.


This may be attributed to increased basal expression of Cyp24a1 in C57BL/6 mice, which promotes inactivation of 1,25(OH)2D3. Decreased expression of Cyp24a1 in susceptible strains was associated with genetic alterations in noncoding regions of Cyp24a1 gene, which were strongly reminiscent of super‐enhancers that regulate gene expression. These observations suggest that higher basal expression of an enzyme required for catabolizing vitamin D renders B6‐kl/kl mice less susceptible to changes in Klotho expression, providing a plausible explanation for the lack of aging phenotypes on C57BL/6 strain.In mutant mice, reduced levels of Klotho promoted high levels of active vitamin D in the serum. Genetic or dietary manipulations that diminished active vitamin D alleviated aging‐related phenotypes caused by Klotho down‐regulation. The hypomorphic Klotho [ By contrast, the kl/kl mutation on the C57BL/6 genetic background displayed normal lifespan with reduced aging‐related phenotypes 2D3] 2D3  specific to kidney samples and a fifth site that was shared with liver  and histone H3 mono‐methylated at lysine 4 (H3K4me1) only in kidney samples. The broad distribution of these marks was reminiscent of super‐enhancers (Whyte et al., Cyp24a1 and presence of H3K27ac‐ and H3K4me1‐marked modified DHS suggests that this cluster of DHS regulates kidney‐specific expression of Cyp24a1 gene.To uncover regulatory mechanisms that alter expression of Cyp24a1 super‐enhancer lead to reduced basal expression of Cyp24a1 in susceptible strains making them more reliant on Klotho for regulation of Cyp24a1 to maintain healthy levels of serum vitamin D3.Sequences surrounding these regions of chromatin modifications and DHS were also found to be conserved in other mammalian species, including humans (Figure None declared.Cyp24a1 gene. JMS and RS wrote the paper and all authors read and commented on the manuscript.JMS designed and supervised the study; AV and MS performed the experiments; AS and RS did the genetic analysis of kidney‐specific variations in  Click here for additional data file. Click here for additional data file.


One phase is charge, orbital and trimeron ordered similar to the ground state of magnetite, Fe3O4, while the other has Fe2+/Fe3+charge averaging. Lattice symmetry is unchanged but differing strains from the electronic orders probably drive the phase separation. Complex low symmetry materials like CaFe3O5 where charge can be redistributed between distinct cation sites offer possibilities for the generation and control of electronic phase separated nanostructures.Incomplete transformations from ferromagnetic to charge ordered states in manganite perovskites lead to phase-separated microstructures showing colossal magnetoresistances. However, it is unclear whether electronic matter can show spontaneous separation into multiple phases distinct from the high temperature state. Here we show that paramagnetic CaFe 3O5.Electronic phase separation is an important feature of many correlated perovskite compounds but hasn’t been seen in other complex oxides with similar physical behaviour such as magnetite. Hong et al. find phase separation between a magnetite-like charge ordered phase and a charge averaged phase in CaFe Both undergo charge ordering on cooling, which for magnetite is accompanied by a lattice distortion below the much-studied Verwey transition at 125 K1. This was recently found to result from a complex ordering of Fe2+/Fe3+ charge states, Fe2+ orbitals, and three-Fe trimeron groups2. However, long range phase segregation is observed below charge ordering transitions in many manganites5 and related perovskites but has not been found in magnetite or related ferrite spinels.Manganites such as LaMFen–1On+1 (n ≥ 4) ferrites with M = Fe7, Mn9 and Ca11, we investigate n = 4 CaFe3O5 and discover long range electronic phase separation between a magnetite-like charge ordered phase and a charge averaged phase.Here, following recent studies of spin and charge ordering in non-spinel 3O5 was synthesised and characterised as described in Methods. The orthorhombic crystal structure consists of FeO6 octahedra sharing edges to form infinite chains parallel to the a-axis and ribbons of three octahedra parallel to the bc-plane as shown in Fig. TM = 302 K and a small net magnetisation of 0.05 µB per CaFe3O5 unit with a moderate coercivity of 0.3 T at 2 K. Ceramic pellets of CaFe3O5 are semiconducting with a small magnetoresistance of −1% at 200 K  phase has ferromagnetic (FM) alignment of spins within the three-atom ribbons parallel to the bc-plane, while the (0 0 0) has FM spin chains parallel to the a-axis. Although both orders are antiferromagnetic, the observation of a small net magnetisation below TM suggests that one or both of the spin structures are canted. The fractions of the two phases are found to be different in the synchrotron and neutron diffraction experiments  crystalline phase of CaFeray Fig.  and neutray Fig.  reveal lnts Fig. , consist3O5, and in the HT phase they have respective Bond Valence Sums (BVSs) of 2.43 and 2.22, equivalent to formal charges of +2.75 and +2.50 when renormalized to the average of +2.67. Differing charge redistributions occur below the 302 K magnetic and phase segregation transition  magnetic order shows the opposite behaviour as the Fe1 and Fe2 BVSs converge to similar values of 2.44(8) and 2.29(7) at 4 K showing that the electronic states at the two sites are not significantly different, hence this phase is charge averaged (CA).Thermal evolution of lattice parameters from neutron and X-ray fits are, respectively, shown in Fig. ion Fig. . In the 6 Fe2+ states are subject to Jahn–Teller distortion which leads to tetragonal octahedral compression in magnetite2, corresponding to negative values of the QJT distortion parameter. The Fe2 site in CaFe3O5 has a large negative QJT value due to intrinsic distortions within the HT structure  and this is the likely driver for the observed averaging of charge states between the two sites in the CA phase so that their minority spin populations become more equal.Degenerate 3dure Fig. , but belase Fig. , consist3O5 do not change the structural symmetry, but they lead to different cell distortions  phases below 220 K, and finally to undergo a further phase separation into a mixture of FM, AFM-A and charge ordered AFM-CE phases below 150 K, with all three phases extant down to the lowest measured temperature of 15 K17. CaFe3O5 undergoes a genuine electronic phase separation in the sense that both of the two low temperature CA and CO phases are electronically and magnetically distinct from the HT paramagnetic state, and so is analogous to e.g. the separation of a fluid into a liquid and gas below a critical transition. Both low temperature CaFe3O5 phases are magnetically ordered and it is remarkable that they appear to share a common magnetic ordering temperature as shown in Fig. Charge ordering in one of the two low temperature phases is clearly a key driver for the long range electronic phase separation in CaFey1−Pry)1−xCaxMnO3 suppresses long range phase separation18. CaFe3O5 is in principle a stoichiometric material although small strain variations due to the 4% substitution of Fe for Ca observed in our polycrystalline sample may tip the local balance between the energies of the two ground states leading to phase coexistence. The resulting disorder in magnetic interactions could also be important in stabilising the phase coexistence in a Griffiths-type model19. All Mn sites are electronically equivalent in the HT aristotype manganite perovskite structure but CaFe3O5 has a further electronic degree of freedom as charge can be redistributed between structurally inequivalent Fe1 and Fe2 sites, leading to the observed extremes of charge ordering (CO) in one phase and charge averaging (CA) in the other. This charge redistribution mechanism is akin to electronic separation models originally proposed for manganites21, although couplings of both the charge redistribution degree of freedom and trimeron formation associated with charge and orbital ordering to the lattice appear to be important factors that drive phase segregation in CaFe3O5.Another important difference is that phase separation in manganites is associated with substantial intrinsic disorder due to cation mixing, and a recent study has demonstrated that chemical ordering of cations in  Thermogravimetric analysis heating the sample in air at 10 °C min−1 to 900 °C, as shown in Supplementary Fig. 3O5. Powder X-ray diffraction confirmed that CaFe3O5 adopts an orthorhombic Sr2Tl2O5 type structure with space group Cmcm as reported previously10.Polycrystalline CaFeMagnetic measurements were carried out with a Quantum Design MPMS XL SQUID magnetometer. Magnetic susceptibility was recorded in zero field cooled (ZFC) and field cooled (FC) conditions between 2 and 400 K with an applied magnetic field of 1000 Oe. Hysteresis loops were also measured at 2, 230 and 300 K. Electrical resistivity measurements were carried out with a Quantum Design PPMS, between 180 and 400 K. Magnetoresistance hysteresis loops were also measured at 200 K.3O5 were Rietveld-fitted using the FullProf Suite24. A small amount of Fe at the Ca site was found from both synchrotron X-ray [4.4(3)% Fe] and neutron [4.0(8)% Fe] refinements of cation site occupancies at 500 K. Crystal structure refinements of the two phases formed below 302 K in CaFe3O5 was possible with both the synchrotron X-ray and neutron powder diffraction data except at temperatures just below the transition (275 and 285 K). Magnetic irreducible representation analysis was carried out using BasIrReps. Bond valence sums for each iron site were calculated using a standard method with linear interpolation to estimate mixed charge states between Fe2+ and Fe3+26. The crystal structure projection in Fig. 27 and the magnetic structures in Fig. High resolution powder X-ray diffraction data were collected at the ID22 beamline of the ESRF with incident wavelength 0.3999 Å. The powder was packed into a glass capillary with an outer diameter of 0.7 mm and spun during data acquisition with temperatures from 80 to 500 K controlled using an Oxford Cryostream system. High resolution neutron diffraction data were collected at the D2B beamline of the ILL with incident wavelength 1.5940 Å. 5 g of powder samples were packed into a vanadium can and diffraction patterns were collected at temperatures from 4 to 500 K. Crystal and magnetic structures of CaFe28 (v16 and 17) utilising planewaves (650 eV cutoff) and on-the-fly pseudopotentials, within the PBE approximation to exchange and correlation. Band structures (shown in Supplementary Fig. U energy, and insulating charge ordered states for both phases at large U. However, values in the range 2.0 < U < 4.0 eV simultaneously predict the charge averaged state for the CA structure and the charge ordered state for the CO structure, and hence confirm that this range is realistic for Fe oxides.DFT+U electronic structure calculations were performed using CASTEP28 v1 and 17 uData that support the findings of this study have been deposited at 10.7488/ds/2378.Supplementary Information