Global minimum searches revealed that the most stable structures of Ln3B18– are hollow cages with Dh3 symmetry. The B18-framework in the Ln3B18– cages can be viewed as consisting of two triangular B6 motifs connected by three B2 units, forming three shared B10 rings which are coordinated to the three Ln atoms on the cage surface. These metallo-borospherenes represent a new class of unusual geometry that has not been observed in chemistry heretofore.The discovery of borospherenes unveiled the capacity of boron to form fullerene-like cage structures. While fullerenes are known to entrap metal atoms to form endohedral metallofullerenes, few metal atoms have been observed to be part of the fullerene cages. Here we report the observation of a class of remarkable metallo-borospherenes, where metal atoms are integral parts of the cage surface. We have produced La 3B18– metallo-borospherenes with unusual spherical trihedron geometry, in which the lanthanide atoms surprisingly form a part of the cage surface.Borospherenes are the boron-based analogs of fullerene cages. Here, the authors report a class of Ln Because of the strong boron–boron bonding, there were speculations about the formation of boron nanotubes composed of a triangular boron lattice5, after the discovery of carbon nanotubes. The triangular boron lattice can be viewed as a graphene-like sheet with the filling of a boron atom in each B6 hexagon. Further theoretical calculations revealed, however, that triangular lattices with hexagonal vacancies were more stable and more suitable to construct boron nanotubes7. In the meantime, combined spectroscopic and theoretical studies have shown that size-selected boron clusters all have 2D structures with delocalized multi-center bonding within the cluster plane11. The discovery of the hexagonal 2D B36 clusters provided the first experimental evidence of the viability of atom-thin boron nanostructures with hexagonal vacancies, named as borophene akin to graphene12. Borophenes have been recently synthesized using atomic vapor deposition on Ag(111) substrates14, forming a new class of synthetic 2D nanomaterials15. The analogy between nanostructures made of boron and carbon has been further extended when the B40 and B39– clusters were found to have global minimum cage structures17, i.e. borospherenes analogous to the fullerenes. Fullerenes are known to form endohedral metallofullerenes for alkali, alkali earth, lanthanide, and actinide elements19, albeit not for transitions metals. Heterofullerenes in which one carbon atom is substituted by a transition metal atom have been observed in the gas phase, but the metal substitution induces large local structural distortions and such heterofullerenes have not been synthesized in the bulk22. The first cage cluster made of multiple metal atoms and carbons was proposed to be Ti8C12+ 23. However, subsequent theoretical calculations showed that the metallocarbohedrene is not stable and the global minimum of Ti8C12+ consisted of a tetrahedral, close-packed Ti8 clusters coordinated by six C2 units on the cluster surface24. In the present article, we report the first observation of a class of metallo-borospherenes, hollow cage clusters consisting of three lanthanide (Ln) atoms and 18 boron atoms (Ln3B18–).The electron deficiency of boron often leads to electron delocalization and the violation of the octet rule in boron compounds and three-dimensional (3D) cage units in different bulk boron allotropesn− (n = 8–10)26, as well as metal-centered nanotubular structures27. More interestingly, it has been shown that transition metals can be an integral part of larger 2D boron clusters28, leading to the possibility of metallo-borophenes29. Lanthanide-doped boron clusters, however, have been found recently to form very different structures, due to both charge transfer interactions and strong (d–p)π bonding30. For example, lanthanide-doped boron clusters do not form similar borometallic molecular wheels as the transition metals. Instead, they form inverse-sandwich-type structures for Ln2Bn– clusters (n = 7–9)32. The most recent study indicates that the inverse-sandwich structure may extend to form lanthanide-boron nanowires33.Transition-metal-doped boron clusters were first found to form aromatic borometallic molecular wheels, M©B18 clusters (La3B18– and Tb3B18–), which are found to possess unprecedented Dh3 cage structures with the Ln atoms being integral parts of the cage surface. These Dh3 metallo-borospherenes belong to an unusual class of geometry known as spherical trihedron. The B18 framework consists of two B6 triangles connected by three B2 units, forming three shared B10 rings. The high stability of the spherical trihedral structures is derived from the strong interactions between the Ln atoms and the B10 rings via charge transfer interactions and d–p covalent bonding. Theoretical calculations show that the entire series of lanthanide elements (Ln = La–Lu) can form spherical trihedral Ln3B18– metallo-borospherenes with tunable magnetic properties, making them a fascinating series of building blocks for new types of magnetic materials.Here we report a joint photoelectron spectroscopy (PES) and quantum chemistry study of two tri-lanthanide-doped B3B18– cluster  for band X was evaluated from its onset to be 2.80 eV, which also represents the electron affinity (EA) of neutral La3B18. The higher binding energy bands  correspond to detachment transitions to the excited states of neutral La3B18. The A band at 3.64 eV was broad and not well resolved at 193 nm, but it was slightly better resolved in the 266 nm spectrum . Band B at 4.28 eV and band C at 4.52 eV were relatively weak and closely spaced, whereas band D at 4.77 eV was much more intense. Beyond ~5 eV, the signal-to-noise ratio was poor and no obvious spectral bands were observed. Band E close to the threshold at a VDE of ~6.2 eV was tentatively labeled. The overall spectral pattern for Tb3B18– exhibits some similarity to that of La3B18–. In particular, the strong X and D bands in Tb3B18– are similar to the strong X and C bands in La3B18–. There is a large energy gap on the high binding energy side in both spectra. Similar spectral patterns could be an indication of similar structures, as have been observed for a series of dilanthanide clusters (Ln2B8–)31.The PE spectrum of Tb, D Fig. . The X b−1 of the global minimum at the levels of PBE/TZP and PBE0/TZP are presented in Supplementary Fig. 3B18– is a hollow cage with a closed-shell ground state (1A1) and Dh3 symmetry. This is a hetero-metallo-borospherene, in which the three La atoms are integral parts of the cage surface, as shown in Fig. Dh3 metallo-borospherene exhibits overwhelming stability relative to the other low-lying isomers: it is more stable than the nearest isomer with Cs symmetry by ~19 kcal mol−1 at the PBE/TZP and PBE0/TZP levels of theory. The B18 framework in the La3B18– cage can be viewed as consisting of two B6 triangles linked together at their three corners by three B2 units, creating three shared B10 rings along the C3 axis. The three La atoms are coordinated by the three B10 rings, giving rise to the closed cage structure. The La3B18– metallo-borospherene has an oblate shape with a diameter of 4.62 Å along the C3 axis (between the two B6 triangles) and 5.09 Å encompassed by the three equatorial La atoms. The relevant bond lengths of the La3B18– metallo-borospherene are shown in Supplementary Fig. The low-lying isomers within 55 kcal mol3B18– was totally unexpected and the geometry is highly unusual. To further examine its stability and robustness, we performed ab initio molecular dynamics (AIMD) simulations at different temperatures, 300, 500, 700, and 1000 K .The similarity in their PE spectra suggested that the global minima of TbDh3 cage structure for La3B18– and Tb3B18–, we calculated their ADEs and VDEs using the ΔSCF–TDDFT formalism. Figures Dh3 global minimum structures, in comparison with the experimental data. The computed ADE/VDE1 at the CCSD(T) level are 2.828/2.972 eV for La3B18–  yields one detachment channel, as shown in Supplementary Table e′′ HOMO (2.972 eV) and 8a1′ HOMO-1 (2.987 eV) are very close to each other, in excellent agreement with the experimental VDE of the X band (2.97 eV). In fact, each of the observed PES band corresponds to two detachment channels, as given in Supplementary Table Dh3 cage global minimum for La3B18–. We have also simulated the PE spectra for the next nine higher-lying isomers of La3B18–, as shown in Supplementary Fig. Dh3 global minimum structure.To validate the 3B18– are 2.901/3.017 eV at the CCSD(T) level  was shown to contain characteristic cages of B20 units, with an octahedral Ni6 cluster nested inside35. Our observation of the Ln3B18– metallo-borospherenes represents the first isolated molecules of Ln–B cages in the gas phase.The observation of the La18 framework in two different pathways and then its bonding with the three La atoms, as schematically shown in Fig. 18 framework by the fusion of three B10 rings. Four of the boron atoms in each B10 ring are shared with the other B10 rings, which each coordinate to a La atom to form three shared La©B10 units. This hypothetical formation pathway of La3B18– can be expressed by the following steps:To understand the stability of these remarkable cage structures, we carried out fragment MO analyses by first considering the construction of the B3B18– cage at the PBE/TZP level of theory. The interactions between the B18 framework and the three La atoms are extremely strong (steps 2 and 3), which underlies the stability of the La3B18– cage. It should be noted that in platonic solids four surfaces are the minimum number to form a 3D object, i.e., the tetrahedron. However, Fig. 10 surfaces are fused together to form the Dh3 La3B18– cage. This is because the La©B10 surface is curved. It turns out that the Ln3B18– metallo-borospherenes belong to a class of geometry mathematically known as n-gonal hosohedron, which is basically a tessellation of lunes on a spherical surface, such that each lune shares the same two vertices. Thus, the Dh3 La3B18– cage is a trigonal hosohedron, also known as spherical trihedron, where the two vertices consist of the triangular B6 units. To the best of our knowledge, such a geometry has not been observed in any cluster or molecular systems heretofore.The energetics were calculated from single-point energy differences of the reactants and products, using the geometries directly taken from the optimized La3B18– cage, in which the B18 framework is formed by two triangular B6 motifs linked by three B2 bridges, such that three B10 rings are created. This hypothetical pathway can be represented by the following steps:Figure 18 framework and the three La atoms to form the La3B18– cage are represented by steps 2/3 or 6/7 with an estimated binding energy of 724.6 kcal mol-1, i.e., 241.5 kcal mol−1 for the binding energy between each La atom and the B10 ring. This huge La-B10 binding energy underlies the extraordinary stability of the La3B18– metallo-borospherene. Compared with the pathway in Fig. 3B18– cage depicted in Fig. The interactions between the B18 is a planar structure36, the stabilization of the 3D B18 framework is entirely due to its strong bonding with the three La atoms, as discussed above. We have analyzed the nature of the La–B10 bonding in the La3B18– metallo-borospherene using several different methods. The MO energy-level diagram and the relevant MOs of La3B18– derived from the La3– and B18 moieties are shown in Fig. e′′, 9e′, and 1a1′′ MOs of La3B18– (red-colored) represent the bonding orbitals between the three La atoms and the B18 moiety, mainly corresponding to the interactions between the irreducible representations, 4e′′, 6e′, and 1a1′′ on the B18 moiety and 6e′′, 12e′, and 2a1′′ on the La3– moiety (the red highlighted MOs). Supplementary Table e′′, 9e′, and 1a1′′ MOs, which are dominated by contributions from the B18 moiety. Hence, there is a strong charge transfer from La to B18, resulting in a closed-shell La3B18– with a large HOMO-LUMO gap of 1.51 eV computed at the PBE/TZP level. The La atoms are in their favorite +III oxidation state in La3B18–, which can be viewed approximately as (La3+)3[B1810–]. As shown in Fig. e′′, 12e′, and 2a1′′ irreducible representations on the La3– moiety are of La 5d characters, while the 4e′′, 6e′, and 1a1′′ irreducible representations on the B18 framework are of B 2p characters. Hence, the 5e′′, 9e′, and 1a1′′ MOs also represent significant La 5d and B18 2p covalent bonding. It is the strong covalent and ionic bonding between the La atoms and the B10 rings that gives rise to the extraordinary stability of the La3B18– cage structure. These bonding characteristics are found in all lanthanide boride compounds due to the low electronegativity of the lanthanide elements and their diffuse 5d orbitals37.Since the global minimum of B10 interactions can be further characterized using the EDA-NOCV method with B18 (…6a1′21a1′′06e′04e′′0) and La3– (…6e′′42a1′′212e′4) fragments, a powerful energy decomposition tool to give insight into chemical bonding38. We analyzed the B18…La3– interaction by the decomposition of the orbital terms into pairwise contributions, as shown in Supplementary Fig. E1, ΔE2, and ΔE3 associated with the deformation densities Δρ1, Δρ3, and Δρ3, respectively. The remaining terms contribute <10% to the total orbital interactions. The color code of the deformation densities indicates the direction of the charge flow from red → blue. It is interesting to see that the 1a1′′ orbital of La3B18–, which is analogous to the (d–p)δ bonding MO in the Ln2B8– inverse sandwich complexes32, contributes significantly  and Δρ3 (28.9%) deformation densities correspond to the degenerate 9e′ and 5e′′ MOs, respectively. The direction of the charge flow is from the La3– to the B18 moiety, consistent with the fragment MO analyses discussed above  approach39, as shown in Fig. σ bonds formed within the three B2 units and between the B2 units and the three apexes of the two B6 triangles. The second row reveals the delocalized bonds in the B6 triangles, with four three-center two-electron (3c–2e) σ bonds within each B6 unit. The multi-center 12c–2e and 18c–2e delocalized bonds can be viewed as π bonds within the B6 units. The third row represents totally delocalized σ and π bonds within the B18 framework. The last row shows five totally delocalized 21c–2e bonds between the La atoms and the B18 framework, corresponding to the 5e′′, 9e′, and 1a1′′ MOs in Fig. 3B18– metallo-borospherene possesses both 3D aromaticity with calculated nucleus-independent chemical shifts (NICS)40 of −47.87 ppm at the cage center, and planar aromaticity on each B6 triangles with NICS(0) of −31.44 ppm and NICS(1) of −2.16 above the plane center, as shown in Supplementary Table I]8 compound41.We further analyzed the chemical bonding in the La2 bridges have shorter bond lengths and higher bond orders than those of the B6 triangles. In terms of the La–B interactions, the distances and bond order indices are similar to those in the lanthanide–boron complexes reported previously33.We also performed bond-order index analyses for the B–B and La–B interactions, as presented in Supplementary Table 3B18– and Tb3B18– metallo-borospherenes suggest that other lanthanide elements could also form similar structures because of the similarity in the chemical properties of the whole series of lanthanides. We have calculated the Dh3 cage structures for all the lanthanide elements, Ln3B18– (Ln = Ce–Lu). The coordinates obtained at the PBE0 level are given as Supplementary Data 3B18– and La3B18 are provided in Supplementary Table 3B18– metallo-borospherenes. While borospherenes have not been observed beyond the B40 cluster42, the unique bonding characteristics between lanthanide and boron suggest that other lanthanide metallo-borospherenes with different sizes and LnxBy– stoichiometries may exist. Recent studies of transition-metal borides showed that the metal–boron interactions have major influences on their magnetic properties44. Hence, the understanding of Ln–B interactions in the metallo-borospherene systems may provide insights for the design of lanthanide borides with tunable magnetic or catalytic properties.The discoveries of the La3B18– , show similar and relatively simple spectral patterns, suggesting that they have similar highly symmetric structures. Theoretical calculations reveal that the Ln3B18– anions have cage-like structures with Dh3 symmetry: two planar B6 triangular units linked by three B2 bridges to form the B18 framework consisting of three shared B10 rings coordinated to the three Ln atoms. Strong ionic and covalent chemical bonding is found between the Ln atoms and the B18 framework. The extraordinary stabilities of the metallo-borospherenes are understood by various theoretical analyses. La3B18– is found to have a closed-shell electron configuration with a large HOMO-LUMO gap and possesses 3D aromaticity. The Ln3B18– cage complexes are expected to exist for all lanthanide elements, suggesting the possibility that there may exist a large class of lanthanide metallo-borospherenes with different Ln/B stoichiometries and tunable properties.In conclusion, we report the observation of the first tri-lanthanide-doped boron cage clusters , in which the metal atoms are integral parts of the cage surface. Photoelectron spectra of two representative systems, Ln11. The La3B18– and Tb3B18– clusters were produced by laser vaporization of a La/11B or Tb/11B mixed target, respectively. The laser-induced plasma was cooled by a He carrier gas seeded with 5% Ar, initiating nucleation between the boron and lanthanide atoms. The nascent clusters were entrained in the carrier gas and underwent a supersonic expansion. Negatively-charged clusters were extracted from the collimated cluster beam and analyzed by a time-of-flight mass spectrometer. Both pure (Bn–) and mixed (LnxBy–) clusters were produced from the cluster source. The La3B18– and Tb3B18– clusters of current interest were mass-selected and photodetached by the 193 nm (6.424 eV) radiation from an ArF excimer laser or the fourth harmonics from a Nd:YAG laser . Photoelectrons were collected and analyzed in a 3.5-m-long electron flight tube at nearly 100% efficiency. The photoelectron spectra were calibrated by the known transitions of Au– and Bi–. The resolution of the PES apparatus (ΔKE/KE) was around 2.5%, that is, about 25 meV for photoelectrons with 1 eV kinetic energy (KE).The experiments were carried out using a magnetic-bottle PES apparatus equipped with a laser vaporization supersonic cluster source, details of which have been published elsewhere3B18– cluster were performed using the TGMin 2.0 code45. The global minimum structure of Tb3B18– was not searched separately. More than 2000 structures were evaluated for La3B18– using the constrained Basin–Hopping algorithm at the PBE/DZP47 level from the ADF 2017 software48. A Dh3 cage structure was found to be the global minimum, which was significantly lower in energy in comparison to the next lowest-lying isomer  and considering the negligible geometry change due to the occupations of the localized 4f orbitals , we used the 4f-in-core pseudopotentials34 for the lanthanide elements to optimize the geometric parameters in the other Ln3B18– (Ln = Ce–Lu) species.Unbiased global-minimum structural searches for the La52 with the SAOP exchange-correlation functional53 to account for the long-range interactions. The ground state adiabatic and vertical detachment energies were calculated at the DFT levels, as well as the more accurate DLPNO-CCSD(T) level54 with the Def2-TZVP basis sets55 and the Def2-TZVPP pseudopotential for La34, utilizing the AutoAux generation procedure55. We also used the 4f-in-core pseudopotential34 for the simulation of the PE spectrum of Tb3B18– without consideration of the 4f electron detachment channels. Previous studies showed that such detachment channels carried very low detachment cross sections at the low detachment photon energies used and the main PES features of Ln–B binary clusters were dominated by MOs with Ln sd or B sp characters33. Chemical bonding and electronic structure analyses were carried out by canonical molecular orbital (MO) theory and the semi-localized AdNDP method39. We also performed calculations using the energy decomposition analysis–natural orbitals for chemical valence (EDA–NOCV) approach38 to quantitatively elucidate the bonding mechanisms between the B18 and La3– moieties. The bond order indexes of different interatomic interactions were calculated using the Mayer56, Gopinathan–Jug (G–J)57, and Nalewajski–Mrozek schemes58.The simulation of the PE spectra was done using the ΔSCF-TDDFT methodSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1


As a solution to overcome this challenge, a strategy allowing synthesis and preservation of SNPs at high mass loadings within multishell hollow metal–organic frameworks (MOFs) is demonstrated. First, alternating water‐decomposable and water‐stable MOFs are stacked in succession to build multilayer MOFs. Next, using controlled hydrogen bonding affinity, isolated water molecules are selectively sieved through the hydrophobic nanocages of water‐stable MOFs and transferred one by one to water‐decomposable MOFs. The transmission of water molecules via controlled hydrogen bonding affinity through the water‐stable MOF layers is a key step to realize SNPs from various types of alternating water‐decomposable and water‐stable layers. This process transforms multilayer MOFs into SNP‐embedded multishell hollow MOFs. Additionally, the multishell stabilizes SNPs by π‐backbonding allowing high conductivity to be achieved via the hopping mechanism, and hollow interspaces minimize transport resistance. These features, as demonstrated using SNP‐embedded multishell hollow MOFs with up to five shells, lead to high electrochemical performances including high volumetric capacities and low overpotentials in Li–O 2 batteries.Hydrogen affinity‐controlled water molecule transfers throughout multilayer metal–organic frameworks result in sub‐nanometric particle (SNP)‐embedded multishell hollow metal–organic frameworks (MOFs), where the multishell stabilizes SNPs by π‐backbonding allowing high conductivity and hollow interspaces that minimize transport resistance. Moreover, increasing the mass loading of SNPs within multishell hollow MOFs is demonstrated to enable higher capacities and smaller overpotentials in Li–O SNPs are typically stabilized through agglomeration due to their unsaturated surface bonds, even after being synthesized by complex processes, so that the strong interactions with solid supports have been quite successful in stabilizing them. Meanwhile, this method was possible only at low mass loadings where the collision frequency of SNPs can be inhibited. In another way, the pyrolysis of organometallic compounds induces the formation of heterogeneous SNPs at nonuniform particle sizes. However, as agglomeration is driven by surface energy differences attributed to different particle sizes, these heterogeneous SNPs also grow to become larger particles.Despite sub‐nanometric particles (SNPs) having unique abilities such as maximized atom‐utilization efficiency and governable catalytic selectivity,1, 2 such that stable MOFs allow the controlled mass transfer and quantitative stabilization of atomic clusters, while the constituents of decomposable MOFs can work as precursors for SNPs. Accordingly, there were many great efforts to utilize MOFs as materials for preparing various shaped structures with advanced functionalities. In this work, we develop multilayer MOFs with alternating stable and decomposable layers for the autogenous production and stabilization of SNPs in scalable mass loadings. Interestingly, we also show that the single water molecule transfer throughout multilayer MOFs plays a very important role to activate the autogenous synthesis of SNPs. The step‐by‐step deterioration of a decomposable MOF layer by the single water molecule leads to the construction of uniform atomic clusters, thus promoting the synthesis of SNPs in the stable MOF layer. It is also investigated how to introduce the single water molecules in multilayer MOFs and study the interaction between the single water molecule and its environment as a key factor. Finally, the higher mass loading of SNPs in multishell MOFs is realized by the autogenous production and stabilization of dinuclear SNPs with multishell hollow MOFs, and they are applied as electrocatalysts to realize high performance in Li–O2 batteries.We hypothesize that the key requisite to make homogeneous and robust SNPs is a chemically designable system providing manipulative formation and simultaneous stabilization of SNPs. In order that the system is effectively implemented, it should have a high porosity and a large surface area for high SNP loading. Metal–organic frameworks (MOFs) provide chemical flexibility and high porosity,10 sucFigure thus providing the manipulative formation of SNP precursors while the water clusters formation is blocked. Owing to their hydrophobicity, the stable MOF layers effectively transferred water molecules to the decomposable MOF layers without adsorption of water molecules. Subsequently, SNPs derived from the decomposable MOFs were stabilized inside the pores of the water‐stable MOFs. We chose zeolitic imidazolate framework‐8 (ZIF‐8) and ZIF‐67 as water‐stable and water‐decomposable MOFs, respectively. ZIF‐8 enabled water molecules to be fed one by one, because the penetration of the isolated water molecule could be manipulated by sieving EG–water complexes through hydrophobic micropores. The X‐ray diffraction (XRD) patterns of ZIF‐67 after hydrolysis  and the unchanged XRD patterns of ZIF‐8  demonstrate that all the metal complexes in ZIF‐67 are disconnected from the organic linkers when exposed to water. ZIF‐67, which is isostructural with ZIF‐8 because both MOFs are composed of 2‐methylimidazole (2‐mim), was seeded for the epitaxial growth of ZIF‐8 to construct multilayer ZIFs  . The disassembly of the bonds between the Co ions and 2‐mim in ZIF‐67 generates SNP precursors and interspaces, as identified by the elemental mapping and HAADF‐STEM images  Figure . The bou) Figure  and by t2. To further clarify the different structures of ML‐ZIFs[2L] and H‐ZIF‐8[1S], the Co K‐edge X‐ray absorption near‐edge structure (XANES) spectra were collected 2 becomes almost flat with the formation of centrosymmetric edge‐shared CoO6 octahedra. Thus, the geometric transformation from ML‐ZIFs[2L] to H‐ZIF‐8[1S] results in a transition of Co coordination from tetrahedral to distorted octahedral, attributable to 3d–4p orbital mixing initiated by the slightly tilted centrosymmetric coordination. Moreover, Co atoms are more oxidized through the formation of Co—O bonds, thereby enhancing the 1s–4p transition on back scattering induced by the structural transformation, resulting in the disappeared shoulder edge and higher energy shift. The red area in the 2D contour map  indicates the formation of a higher oxidation state after the structural transformation of ML‐ZIFs[2L] to H‐ZIF‐8[1S]. The coordination environment of Co was explored using the K‐edge extended X‐ray absorption fine structure (EXAFS) spectroscopy , and to oxygen edge‐sharing Co—Co bonds at 2.90 Å. The changes in the coordination environment during the transformation of ML‐ZIFs[2L] to H‐ZIF‐8[1S] can be confirmed by the disappearance of the 2‐mim signals and the appearance of only two peaks . The Co–Co peak is significantly weaker than the Co–OH peak, which indicates the existence of atomic particles. In the Co K‐edge simulation curves . The XANES and EXAFS spectra  verify that Zn is not affected in the process. These results are consistent with the effective disconnection of all the coordinate bonds of ZIF‐67 and the generation of dinuclear Co(OH)2 SNPs inside the micropores of H‐ZIF‐8[nS]. In addition, the “disordered‐to‐crystalline” phenomenon involves agglomeration of SNPs upon exposure to a highly enhanced electron beam, thereby corroborating the existence of SNPs inside the damaged multishell hollow MOFs .To elucidate the chemical state of Co SNPs in the ZIF‐8 shell, the X‐ray photoelectron spectroscopy (XPS) measurements were conducted. No Co 2p characteristics are observed in ML‐ZIFs[2L] , but the XPS peaks of H‐ZIF‐8[1S] confirm the existence of divalent Co atoms comparable to those in a bulk Co(OH)ed Figure2a, whicy Figure . The mainS] at diffraction angles lower than 20° 2 SNPs in micropores results in a shift to higher angles owing to the slightly reduced pore sizes, with the reduced intensities for the {011} facets. The decreased pore volumes and pore sizes in the Brunauer–Emmett–Teller (BET) isotherms as well as the pore size distribution curves also indicate the micropore filling by the Co(OH)2 SNPs . The near‐edge X‐ray absorption fine structure (NEXAFS) spectra showed a changed dipole transition from core electrons to unoccupied molecular orbitals caused by the micropore filling. The trigonal or tetragonal distortions of octahedral bulk Co(OH)2 lead to t2g and eg orbital splitting, as represented by Co L2,3‐edge multiplets  to the higher energy eg orbital, which is distinguishable from Co2+ . The blue‐shifted IR spectrum  supports the presence of Co3+ in H‐ZIF‐8[1S]. The electron‐deficient Co3+ strongly attracts hydroxyl group lone pair electrons to fill the completely empty eg orbitals, as confirmed by the strong σ* transition at 781.7 eV. The enriched σ bonds also result in shorter bond lengths 2 and ML‐ZIFs[2L]. The N K‐edge NEXAFS spectrum of H‐ZIF‐8[1S] . Imidazole rings are stronger π‐acceptors than hydroxyl groups, so that the −0.7 eV shift of the π* transition is derived from the increased electron density resulting from π‐backbonding by the surrounding Co(OH)2 SNPs. Furthermore, preferential π‐backbonding between Co(OH)2 SNPs and 2‐mim lowers the energy level of the t2g orbital. The NEXAFS spectra show 22.5% Co3+ and 44.6% π‐backbonding, suggesting that Co(OH)2 SNPs are stabilized in H‐ZIF‐8[1S] . The splitting of the π* transition indicates that Co is not substituted at the Zn tetrahedral site in ZIF‐8, as supported by the fingerprint region of the IR spectrum , which shows the N–Zn–N stretching mode at 420 cm−1 without the N–Co–N stretching mode at 424 cm−1. It has been reported that the lower electron density of Co results in a shift of 0.3 eV toward higher energies relative to Zn, which is more electron dense, as observed in the case of ZIF‐67 and ML‐ZIFs[2L]. These observations are also supported by the absence of changes in the C 1s, N 1s, and Zn 2p XPS spectra and the Zn L‐edge NEXAFS spectra .The role of each micropore was also confirmed by the changes in the physical and chemical properties of H‐ZIF‐8[1S]. The XRD patterns of ZIF‐8 agree with the simulated patterns of H‐ZIF‐8 . Then, Co atoms in the clusters are laminated on the inner shell surface and pass through the micropores, suppressing the growth of sheet‐like structures . The limited amount of water prevents complete hydration and regulates Co ion nucleation. Co ions are fed into the micropores prior to bulk Co(OH)2 formation and then form dinuclear Co(OH)2 SNPs as water molecules are supplied one by one into the micropores. To confirm the isolation of water molecules by EG, we used the nuclear magnetic resonance (NMR) spectroscopy. EG has exchangeable hydroxyl groups that are indistinguishable on the 1H‐NMR time scale . However, a strong peak is observed at 5–6 ppm when water molecules are added to EG . The 1H–1H correlation spectroscopy (COSY), which can explain a spin–spin coupling between EG and water molecule, was conducted to find the reason for newly appeared peak by interpretation of the nature of the EG–water complex . The cross‐peaks, denoted as “OH‐H2O”  was also utilized to determine the cluster size. The diffusion coefficient calculated for the intramolecular form indicates minor interactivity, as the value is not affected significantly by the presence of water. In contrast, the value determined for the intermolecular form is approximately 20‐fold higher than that for the intramolecular form, indicating that the intermolecular form behaves like a huge cluster similar to a structure of consecutively bridged water molecules . As a result of this huge EG–water cluster, the isolated water molecules come into contact with the decomposable MOFs one by one, which prevents supersaturation of the reactants and thus avoids rapid nucleation. This approach not only makes hydrolysis controllable by regulating molecular water transfer, but also effectively suppresses nucleation, resulting in the autogenous production of uniform SNPs at high mass loadings. This method can be used to synthesize multishell MOFs via selective detachability, even in the presence of SNPs, which has previously been difficult to implement. After water molecule transfer, the 1H‐NMR and 13C‐NMR spectra show weakening of the hydroxyl group peak and disappearance of the water and intermolecular peaks 2 SNPs, while simultaneously loading more active materials into the multishell structures 2 SNPs and multishell structures. For pristine ZIF‐8, the charging curve shows a high overpotential with a potential gap of 1.494 V at half‐capacity  upon charging. The potential gap of 0.540 V at half‐capacity is approximately 63.9% lower than that of pristine ZIF‐8. Notably, the oxo groups between the Co ions promote the rapid oxidization of Co2+ to Co4+, facilitating the OER. The Co3+ content of 22.5% enhances the OER kinetics by providing a shortcut to Co4+ and more active sites. Although bulk Co(OH)2 exhibits an improved overpotential, the discharge potential gradually decreases, whereas H‐ZIF‐8[1S] sustains a higher voltage . This result indicates that bulk Co(OH)2 is a good electrocatalyst for the OER upon charging, but unsuitable for the oxygen reduction reaction (ORR) upon discharging. Meanwhile, H‐ZIF‐8[1S] not only provides a large surface area for the accumulation of the discharge product Li2O2 during the ORR, but also helps to decompose Li2O2 efficiently during the OER. The specific gravimetric capacity clarifies the importance of Co(OH)2 SNPs, but a smaller amount of H‐ZIF‐8[nS] is loaded as the number of shells increases at the same weight. In this reason, the specific geometric performance was utilized to determine the trend as the number of shell increases. The geometric performance  of H‐ZIF‐8[5s] is significantly smaller than that of pristine ZIF‐8, as is the solution resistance (Rs) . The reduced Rct suggests that Co(OH)2 SNPs enhance the poor electrical conductivity of ZIF‐8 through the hopping transport mechanism. Furthermore, the reduced Rs indicates that the hollow structure minimizes transport resistance, as the diffusion time is inversely proportional to the diffusion length. The π‐backbonding between Co(OH)2 SNPs and micropores enhances stability owing to strong adhesion. Thus, a larger amount of Co(OH)2 SNPs within multishell ZIF‐8 should give excellent cycling stability, as demonstrated by the superiority of H‐ZIF‐8[5S] in Li–O2 batteries . The patterns were well maintained after 20 cycles, while the intensity associated with 011 facets was decreased due to the Li2O2 products of electrochemical reactions. This indicates that the structure of H‐ZIF‐8[1S] is stable during electrochemical reactions. Figure There are two strategies to increase the active sites for electrocatalysis, which include i) exposing more active sites or/and ii) loading more active materials.42 We y Figure . The Co3)2·6H2O, 98+%), zinc(II) nitrate hexahydrate (Zn(NO3)2·6H2O, 98+%), 2‐methyleimidazole , polyvinylpyrrolidone , EG , cobalt(II) hydroxide (95%), methanol (99.9+%), ethanol (99.5+%), poly(tetrafluoroethylene) , lithium perchlorate , and tetraethylene glycol dimethyl ether  were purchased from Sigma‐Aldrich. Deionized (d.i.) water was obtained from a purifying system using UV treatment. A carbon fiber paper  was purchased from Toray Co., Ltd. Ketjen Black  was purchased from Lion Specialty Chemicals Co., Ltd.Cobalt(II) nitrate hexahydrate (Co(NO−3m Co(NO3)2 in d.i. water and 800 × 10−3m 2‐mim, 30 × 10−3m Zn(NO3)2, and 30 × 10−3m 2‐mim in methanol were prepared. PVP (5 g) was mixed with 200 mL of methanol. All solutions were sonicated for 30 min and cooled to room temperature. Then, to prepare the ZIF‐67 seeds, 3.3 mL of the 800 × 10−3m 2‐mim solution and 3.9 mL of the PVP solution were transferred into a 50 mL conical tube and then mixed with 1.5 mL of the 95 × 10−3m Co(NO3)2 solution. The mixture immediately turned purple and was kept for 1 h at room temperature. Subsequently, the purple solution was centrifuged at 8500 rpm for 10 min. To prepare ML‐ZIFs[2L], 7.5 mL of the 30 × 10−3m 2‐mim solution was added to the collected purple powder in a conical tube. The mixture was sonicated for 10 min and then mixed with 7.5 mL of the 30 × 10−3m Zn(NO3)2 solution. After being kept for 3 h at room temperature, the precipitate was collected by centrifugation at 600 rpm for 10 min and then washed with methanol three times. A new conical tube was used for every step. Finally, ML‐ZIFs[nL] was prepared via the following steps. Initially, 3 mL of the 800 × 10−3m 2‐mim solution (4 mL for the subsequent layer‐stacking processes), freshly prepared in methanol, was added to the ML‐ZIFs powder in a conical tube. The mixture was sonicated for 5 min to achieve dispersion, and then 0.75 mL of the 95 × 10−3m Co(NO3)2 solution was added. After 30 min, the solution was centrifuged at 8500 rpm for 10 min, 12.5 mL of the 30 × 10−3m 2‐mim solution was added, and the mixture was sonicated for 5 min. Then, 12.5 mL of the 30 × 10−3m Zn(NO3)2 solution was added and the mixture was kept for 3 h. The above process was repeated every time two new layers of ML‐ZIFs[2L] were stacked.First, precursor solutions of 95 × 10nL] was mixed with the EG–water solution. The mixture was sonicated for 6 h at 5 °C until a transparent purple solution was obtained. The solution was transferred into a 1 L round‐bottom flask and stirred at room temperature for 6 h, and the resulting mixture was filtered using a membrane filter. The powder on the membrane was collected by sonication in methanol. Following centrifugation at 6000 rpm for 10 min, the powder was washed with methanol three times. Finally, the collected powder was dried in a vacuum oven at 60 °C for 24 h.First, 760 mL of pure EG (stored at −15 °C in the refrigerator) was transferred into a 1 L HDPE Nalgene bottle. Then, 40 mL of d.i. water was added and the solution was sonicated for 10 min. Subsequently, 100 mg of as‐synthesized ML‐ZIFs[The powder XRD patterns were obtained using a SmartLab X‐ray diffractometer  with Cu‐Kα radiation at 1200 W . The TEM and SEM images and videos were obtained using a JEM‐ARM200F instrument  operated at 200 kV and a JEM‐7600F instrument  operated at 15 kV, respectively. ICP‐OES data were obtained using an ICP‐MS 7700S instrument . The XPS spectra were collected using a Thermo VG Scientific K‐alpha spectrometer  using Al‐Kα radiation at 350 W (3 mA). The Fourier transform infrared spectra were obtained using an FT/IR‐6100FV spectrometer  via the attenuated total reflectance technique. The X‐ray absorption spectroscopy (XAS) measurements, including the XANES and EXAFS analyses, were conducted at the multipole‐wiggler 10C beamline at the Pohang Accelerator Laboratory . The NEXAFS measurements were performed at the 10D beamline, where the synchrotron radiation was monochromatized using a Si(111) double crystal monochromator and the incident beam was detuned at the appropriate rate for harmonic rejection. All the NMR experiments were performed on a Bruker Avance 400 MHz spectrometer, equipped with a 5 mm BBFO probe with z‐gradients. The Topspin software was used for spectrometer control, and all the pulse sequences were used without modification.2 battery test, a Swagelok‐type cell  was assembled with a Li foil anode, a glass fiber filter  as a separator, 1 m LiClO4 in TEGDME as an electrolyte, and the as‐prepared working electrode. Water in TEGDME was removed using molecular sieves (3 Å) for 10 days. The assembled cell was purged with a 25 cm3 min−1 flow of pure oxygen for 3 h at 1.1 bar. The oxygen purging pressure was maintained during the Li–O2 battery test and confirmed at an outlet valve using an MP112 micromanometer . The EIS measurements were conducted with a PGSTAT302N potentiostat/galvanostat  using an FRA32MBA module.The electrochemical performance measurements were conducted using a WBCS3000L32 instrument  as a galvanostatic cycle tester. The ink used for the working electrode was prepared by mixing H‐ZIF‐8, KB, and PTFE in a weight ratio of 4:5:1 with ethanol. The ink was coated to a thickness of 400 µm on CFP, which was cut into circular pieces with a 10 mm diameter. The electrode was placed in a vacuum oven and dried at 60 °C for 12 h. After weighing, the electrode was rinsed with acetone several times and vacuum dried in a glove box at 60 °C for 12 h. For the Li–OThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Movie 1Click here for additional data file.


The theoretical charge density study for the gas phase of anti-leprosy drug Dapsone has been carried out in the light of the theory of atoms in molecules using density functional theory employing B3LYP hybrid functional completed with dispersion corrections. The Hirshfeld surface analysis as well as fingerprint plots has been utilized to visualize and quantify the intermolecular contacts present in the molecule. The topological properties such as electron density and its Laplacian, delocalization index have been elucidated to throw light into the chemical bonding and atomic and molecular details. The electron localization function has been used to visualize and deduce information on the lone pair and the subshells of the Cl atom. The electrostatic potential visualizes the positive and negative electrostatic potential regions which are susceptible to nucleophilic and electrophilic attack. On the whole, this study provides an exact mechanism, interaction, and topological and electrostatic properties of the drug through theoretical insights which all will be a platform for our further investigation of the interaction between dapsone and dihydropteroate synthase (DHPS). Mycobacterium leprae infections (leprosy)  aniline) molecule using theoretical models and is thoroughly analyzed with the help of Quantum theory of atoms in molecules for the intuitive information on the molecule itself. In a theoretical model such as DFT  , a free The experimentally determined position values of the atoms of dapsone have been given as input for the theoretical optimization in GAUSSIAN09 software package  with DFTThe minimum energy structure of the dapsone molecule has been shown in Fig. The shapes of the two amine groups are found to be a slight tetrahedral with the expected bond angle 113°. The atoms of N1 and N2 make equal dihedral angles with the respective planes C3-C4-C5 and C9-C10-C11 and are found to be 177°.The torsion angles which are formed by the atoms C1 and C7 with O2-S1-O1 plane individually are found to be 123.4° which clearly suggest the symmetry of the molecule.c* axis and the intermolecular interactions are sustained by Van der Waals interactions.In order to better understand the packing and interactions of the molecule with the neighboring molecules, the information from the experimental cif file has been given as the input for CrystalExplorer software 3.0 , 27. TheThe molecular packing of the crystal is stabilized by N–H…O, N–H…N, and π…π types of intermolecular interactions Fig.  and is cThe hydrogen bond parameters of the strongest N1–H9…O2 interaction are: H9…O2; 1.990Å and N1–H9, 1.009Å, and the angle is 159.46°, and another weak N2–H11…O1 interaction are: H11…O1; 2.423Å, N2-H11; 1.009Å, and the angle is 145.29°. The hydrogen bonding parameters are given in Table di and de in the form of a 2D fingerprint plot is used to provide ample information regarding the percentage contribution of various intermolecular contacts present in the molecule. Figure The combination of Investigation on topological properties of electron density in the light of AIM theory allows us to look into the details of atomic, molecular, and chemical bonding. Figure The range of electron density and Laplacian of C–C bonds in both the phenyl rings is 2.05e/Å3 to 2.105e/Å3 and − 20.1e/Å5 to − 20.8e/Å5. The C–S bonds exhibit very poor electron density as well as Laplacian values which shows that the bonds are weak. Both the C–N bonds show the same charge density and Laplacian values and are found to be 2.045e/Å3 and − 21.25e/Å5, respectively, and this might be stabilized with same charge environment. Similarly, the N–H and C–H bonds show good accumulation of charge density between the bonds Fig. . In addiThe very low ellipticity value (ca 0.01) for the bonds S–O and C–H portrays the near cylindrically symmetric nature. The ellipticity values for the C–C bonds are in the range 0.20–0.22, and the increased values show the π character of the C–C bonds.Among the C–C bonds, the bonds C2–C3 and C8–C9 have the highest delocalization index value which shows not only the strength of the bonds but also denotes the occurrence of resonance/delocalization of the bonds in both the phenyl rings. Among the C–H bonds, the C2–H1 bond has the highest delocalization index value which is supported by the high charge density values (1.92e/Å3) as well as high Laplacian values (− 23.9e/Å5). Similarly, the C7–S1 bond exhibits a high value of the delocalization index (1.42) even though the charge density values of both the C–S bonds are the same. This might be because of the influence of the S1 atom that is higher in C7–S1 bond than the C1–S1 bond.As the kinetic energy density dominates, the total energy density becomes positive which shows the instability of the bonds. Among all the bonds, the N–H bonds have high total energy density values which show the low stability of the bonds.The sulfur atom exhibits a very high positive Bader atomic charge 2.36e) as it is attached with the two most electronegative oxygen atoms (− 1.27e) on both sides. The electronegative N1 and N2 atoms exhibit negative charges (− 1.07e), and they are attached with the two positive H atoms each. The group charge of both the primary amine (−NH2) is negative − 0.23e), and they can donate electrons as donors. Both the C–N bonds are well polarized where the carbon atoms  possess positive charges (0.40e) and the nitrogen atoms  possess negative charges (− 1.07e). The most electronegative atoms O1 and O2 exhibit greater volume, and the values are found to be 111.18 Å3 and 111.19 Å3 respectively (Table 6e as it Some of the molecular properties like membrane permeability and bioavailability are straightly connected with some molecular descriptors such as log P (partition coefficient), molecular weight (MW), hydrogen bond acceptors, and donors count in a molecule. Lipinski rule of five  which isThe electrostatic potential is primarily used to trace out the electrostatic positive as well as electrostatic negative regions where the ligand-receptor interactions possibly occur . An asymAs shown in the lock and key shape of the molecular electrostatic potential of dapsone, the negative potential which is in the vicinity of O1 and O2 atoms is susceptible to severe electrophilic attack. When dapsone is taken as an antibacterial, the nucleophilic/electrophilic region of dapsone interacts with the electrophilic/nucleophilic region of the dihydrofolic acid, and the electrophilicity of the new molecule gets equalized by the electronic charge transfer between the ligand and the receptor . In thisMycobacterium leprae was obtained from Uniprot database. The homology modeling of extracted protein sequence was given as an input on the automated SWISS-MODEL server [Mycobacterium tuberculosis was selected as a template (PDB ID: 1EYE). Finally, the refined structure was used for further studies.The crystal structure of DHPS is not available in protein database. Therefore, the proteomic sequence of DHPS (Uniprot ID: P0C0X1) of L server . Based oTwo-dimensional structure of dapsone (CID: 2955) was obtained from PubChem database. The .sdf file converted into .pdb by using Open Babel tool .The molecular docking was performed by using Molecular Graphics Laboratory AutoDock (4.2) tools. The preparation of receptor and ligand, grid, and docking calculation was described in the previous studies .Molecular docking calculation of dapsone with target enzyme dihydropteroate synthase (DHPS) generated six clusters of conformers using a root mean square difference (RMSD) tolerance of 2.0°A. The two non-covalent π-cation interactions estimated on the amino acid residues TRP 132 of DHPS with respective oxygen and sulfur atoms of dapsone Fig. . The resWe have determined the theoretical charge density distribution for the gas phase of dapsone on the basis of the AIM theory. The identification of close contacts and distinguishing them from each other has been accomplished through the Hirshfeld surface analysis and the fingerprint plots. The topological analysis reveals the strength and chemical bonding details of all the covalent bonds in the molecule. The subshells of S atom and lone pairs of O and N atoms are visible in the ELF plots, and the ELF clearly shows the closed-shell interaction of the S–O bonds. In the ESP map, a high electronegative region is found in the vicinity of the O1 and O2 atoms, and this is the active site for electrophilic attack. This theoretical charge density study clearly provides the fine details of structural information, intermolecular interaction, and charge density distribution which are the necessary parameters to interpret the drug-receptor interactions between the dapsone molecule and enzyme of dihydropteroate synthase (DHPS).


Upon introducing [(PPh3)AuMe] into the 1,2‐diaminoethane (en) solution of (Ge2As2)2−, the heterometallic cluster anion [Au6(Ge3As)(Ge2As2)3]3− is obtained as its salt [K(crypt‐222)]3[Au6(Ge3As)(Ge2As2)3]⋅en⋅2 tol (1). The anion represents a rare example of a superpolyhedral Zintl cluster, and it comprises the largest number of Au atoms relative to main group (semi)metal atoms in such clusters. The overall supertetrahedral structure is based on a (non‐bonding) octahedron of six Au atoms that is face‐capped by four (GexAsx4−)x−  units. The Au atoms bind to four main group atoms in a rectangular manner, and this way hold the four units together to form this unprecedented architecture. The presence of one (Ge3As)3− unit besides three (Ge2As2)2− units as a consequence of an exchange reaction in solution was verified by detailed quantum chemical (DFT) calculations, which ruled out all other compositions besides [Au6(Ge3As)(Ge2As2)3]3−. Reactions of the heavier homologues (Tt2Pn2)2−  did not yield clusters corresponding to that in 1, but dimers of ternary nine‐vertex clusters, {[AuTt5Pn3]2}4− , since the underlying pseudo‐tetrahedral units comprising heavier atoms do not tend to undergo the said exchange reactions as readily as (Ge2As2)2−, according to the DFT calculations.The Zintl anion (Ge2As2)3]3− is obtained from [(PPh3)AuMe] and the binary Zintl anion (Ge2As2)2−, which readily undergoes an in situ atom exchange in solution to form (GeAs3)− and (Ge3As)3−. The latter is required to form the product, which comprises the largest number of Au atoms relative to main group (semi)metal atoms in such clusters. For this, the chemistry of As4 requires even more sophisticated strategies for its preparation, storage and controlled delivery to chemical reactions. These include the reversible coordination of As4 to transition metals4 as guest in porous materials.3 in condensed phase, which was previously studied in gas phase only.As a consequence of its metastable, yet controllable, character under ambient conditions, white phosphorus P has beenpseudo‐)tetrahedral units of main group semi‐metals is the isoelectronic replacement of some or all of the pnictogen atoms in Pn4  with  negatively charged atoms of group 13 or 14 elements. This is realized in salts of the corresponding Zintl anions, which show relatively high  stabilities and solubility in polar solvents. In the past decade, such binary pseudo‐tetrahedral anions were shown to be excellent starting materials to the formation of a large variety of heterometallic and intermetalloid Zintl clusters.Another approach to work with (2Pn2)2− anions , for instance, a variety of ternary clusters were obtained through interaction with d‐ and f‐block organometallic reagents.4 and As4. For instance, (Sn2Sb2)2− was drastically reorganized in a reaction with [LCu(NCMe)] C(Me)}2CH]−) to form a dimer of ternary 9‐vertex units, {[CuSn5Sb3]2−}2.2As2)2− during the stepwise formation of Ta/Ge/As clusters.With . Reactions of these species, all of which needed to be performed in liquid ammonia due to the corresponding salts’ poor solubility (in the absence of organic ligands),+, Au+ or Zn2+ ions.The tendency to fragmentation is in sharp contrast to the behavior of homoatomic Zintl anions Tt10 metals, in particular, have been witnessed of their strong abilities not only to activate P4 and As4, but also the isoelectronic group 14 tetrahedra mentioned above. The anionic clusters [(MesCu)2]4−  were among the first examples for successful solution chemistry with Tt44−.44− tetrahedra were linked through a central gold atom to form the first binary Au/Sn polyanion [Au(η2‐Sn4)2]7−,1∞[Au(η2:η2‐Tt4)]3− 1∞[Au(η2:η2‐TlSn3)]3− 2‐(Sn2Sb2)}2]3−, was obtained in a relatively mild environment due to the reduced overall charge of the binary pseudo‐tetrahedron.3(Ge3P)3]3− was synthesized, comprising binary pseudo‐tetrahedral units.2P2)2− to be a suitable precursor for multinary cluster synthesis, the reaction product indicated an equilibrium of various (GexPx4−)x− species to form in solution, which allowed for a heretofore unknown species (Ge3P)3− to be available for the cluster synthesis. However, to the best of our knowledge, molecular architectures involving more than three anionic ligands or more than three metal atoms have not been known to date. An overview of structural motifs for the coordination of d10 metals by (pseudo‐)tetrahedral anions is provided in Figure Coinage metals and other d2(Ge2As2)3)AuMe] in en, yielding the supertetrahedral Zintl cluster [Au6(Ge3As)(Ge2As2)3]3− as its [K(crypt‐222)]+ salt [K(crypt‐222)]3[Au6(Ge3As)(Ge2As2)3]⋅en⋅2 tol (1). Remarkably, reactions of the heavier homologous Zintl anions (Tt2Pn2)2−  with the gold complex under identical reaction conditions yield yet another type of ternary heterometallic cluster. The anions of the products [K(crypt‐222)]4{[AuTt5Pn3]2}⋅n Sol  are isostructural to a corresponding copper cluster {[CuSn5Sb3]2}4− that were reported only recently.In this work, we describe the reaction of the binary Zintl anion [K(crypt‐222)]en⋅2 tol . Remarka1 was characterized by means of single‐crystal X‐ray diffraction (SC‐XRD)w=2360.01 g mol−1) under ESI‐MS conditions. Therefore, quantum chemical calculations were undertaken to confirm the number of Ge and As atoms in 1. These studies also served to assign Ge and As atoms to their preferred positions within the cluster structure, and to analyze the electronic structure and the bonding situation.Compound 1 x−  pseudo‐tetrahedra in η3 fashion. The fourth apex of each of these units point away from the cluster center, hence giving the whole anion an overall supertetrahedral shape with  dT symmetry.According to SC‐XRD, the structure of the anion in 1 Figure  is basedpseudo‐element concept to rationalize the overall anionic charge of −3 that was unambiguously deduced from the number of three [K(crypt‐222)]+ cations per formula unit. Under the reasonable assumption of  Au+ cations to be present (Au‐Ge/As 2.5369(5)–2.5661(6) Å), the total charge of the four pseudo‐tetrahedral units needs to sum up to −9. This would be achieved if three of the anionic main group units were (Ge2As2)2− while one of them was (Ge3As)3−. Alternatively, the three lower‐charge units could be protonated (Ge3AsH)2−. Several methods were applied to clarify, which of these options would be the correct one. First, we checked the elemental composition by means of EDS. This revealed atom% values of K:Au:Ge:As to be 6.8 %:26.3 %:36.9 %:30.0 % . As a deviation of the K amount is rather common for Zintl cluster compounds, we also compared the Au:Ge:As percentages alone, which are 28.2 %:39.6 %:32.2 % according to the EDS measurement, hence in very good agreement with the theoretical values of Au6Ge9As7 (27.3 %:40.9 %:31.8 %). For comparison, the theoretical values for a protonated cluster K3Au6Ge12As4H3 would be K:Au:Ge:As=12.0 %:24.0 %:48.0 %:16.0 % and Au:Ge:As=27.3 %:54.5 %:18:2 %, which clearly is too far apart from the measured data.Since Ge and As atoms cannot be distinguished by means of common X‐ray diffraction experiments, the number and position of the main group atoms was determined in another way. First, we applied the pseudo‐tetrahedral units are orientated such that one As atom points outwards and is not involved in the coordination. Hence, the (Ge3As)3− unit binds with all three Ge atoms, and the (Ge2As2)2− ligands involve two Ge atoms and one As atom each. In a recent comprehensive study of binary pseudo‐tetrahedral anions,−1), 4:0, 3:1, and 2:2 ratios of coordinating atom types are realized for two Au atoms each, which is a more significant deviation from equality. As can be seen from the energy difference between these isomers, this is a slight, yet noticeable effect, as the total number of coordinating Ge and As atoms are the same in these species.To further corroborate our assumed composition, and to clarify the distribution of the Ge and As atoms on the atomic positions, we additionally applied DFT calculationsnd the GeAs22− ligFor elucidation of the bonding situation, a localization procedure was applied to the 70 valence orbitals, see Figure 2 interaction 3− that has not yet been isolated in a salt on its own. While it is a common observation to find fragmentation and rearrangement of the pseudo‐tetrahedral binary Zintl anions to larger moieties in the presence of transition metal atoms or ions , a reorganization of the 4‐vertex units themselves has only been detected by mass spectrometry in some cases, and in the case of the lightest known binary species, Ge2P2, a corresponding unit was included in the ternary complex [Cd3(Ge3P)3]3− mentioned above.Another issue that required clarification is the observation of the Evidence for this reaction to actually take place is given by ESI mass spectra of the reaction solution, in which we identified all three species with Ge:As ratios of 2:2, 3:1, and 1:3 to coexist .pseudo‐tetrahedra were only observed for the anion in 1 and for [Cd3(Ge3P)3]3−[7c] so far, we calculated reaction energies EXR for the first step of this equilibrium, considering all—known or hypothetical—elemental combinations of Tt and Pn from periods 3–6 . Hence anions with a 3:1 or 1:3 ratio of the involved atom types are stabilized more efficiently in comparison to the anions with a 2:2 atomic composition, if the sizes of the involved atoms are more similar; we attribute this to the fact that the 3:1 and 1:3 anions can be understood as triangles of one atom type with a single atom of the other type capping it, for which the size difference matters more than for two Tt−Tt or Pn−Pn “dumbbells” forming the 2:2 anions. Although these geometric consideration alone do not explain the particular situation of the Ge species (GexPnx4−)x−, we are delighted to find the exchange reactions involving Ge to be distinctly more preferable than calculated for any other Tt/Pn combination—with the (Ge2P2)2− anion being only slightly more prone to follow Equation (1) than (Ge2As2)2−. This finding is in excellent agreement with our experimental observations, in which only the exchange reaction products of (Ge2P2)2− and (Ge2As2)2− seem to form in sufficiently large amounts in solution to be available as ligands for transition metal atoms. According to the calculations, we suggest that the elemental combination Ge/Sb should show a similar behavior—yet, the underlying anion has not yet been explored to date.As discussed recently, the ratio of the atomic radii seems to play a role for the (relative) stabilities of binary 2–4 were also characterized by means of SC‐XRD3)− upon redissolving the single‐crystals in the case of 2. Yet, the sensitivity of the compounds did not allow for their transfer into the gas phase as a whole. The anions in 2–4 are isostructural to each other and with the anion of the above‐mentioned copper homologue of compound 3. They thus represent dimers of trimetallic 9‐vertex cages, which can be understood as heterometallic superatoms, according to comprehensive quantum chemical studies carried out on the anion {[CuSn5Sb3]2}4−.5Sb3]2}4− in compound 4 is shown in Figure Compounds ion {CuSnSb32}4−.5In this case, the assignment of Pb and Sb atoms was unambiguous and not affected by positional disorder. Pb and Sb atoms occupy the same positions as the Sn and Sb atoms, respectively, in the previously reported Cu cluster, which serves to confirm the previous assignment that was made based on perturbation theory for the quoted cluster. The Au atoms are coordinated by two Pb and two Sb atoms each, in a distorted square planar fashion, with the Au–Pb bonds being by 0.12–0.17 Å longer than the Au–Sb bonds. Two additional, yet significantly longer (weaker) Au⋅⋅⋅Pb interactions complement the arrangement into a distorted trigonal prismatic environment.2–4, starting out from the precursor units, as these reactions take place under overall oxidation of the main group elements, we suspect that the lower preference for the reorganizations shown in Equation (1) allows for such alternative fragmentation and rearrangement reactions to take place instead. One should additionally take into consideration that the bonding within the 8‐vertex units has an even more delocalized character than in the pseudo‐tetrahedral anions,Although it is not possible to calculate reaction energies of the reorganization to form the 8‐vertex units that coordinate the Au atoms in compounds 2–4, and also of the yet “missing” Au/Pb/Bi analogue, we can make the following conclusions: First, the atom assignment of tetrel and pentel atoms clearly is preferred in the way shown in Figure 1—but prefer the formation of heterometallic (mainly three‐center) bonds. As the bonding situation in 2–4 is qualitatively the same as in the copper analogue of compound 3, we do not detail it here, but refer to the respective literature.According to DFT calculations of the anions in 2Bi2)2−, or to find any further evidence for the formation of a cluster anion comprising Au besides the said main group metals. As both elemental combinations, Au/Bi and Au/Pb, have been realized (see below), and as other clusters with the elemental combinations Ln/Pb/Bi,2Bi2)2− anion to reorganize seems to be similar to that of (Sn2Bi2)2− 2− and (Ge2As2)2− readily undergo reorganization reactions in solution to provide (Tt3Pn)3− units for the cluster formation, while other elemental combinations prefer other rearrangement pathways.In summary, we presented two new types of intermetalloid cluster architectures comprising gold atoms, resembling a yet unprecedented 22‐atomic supertetrahedral arrangement and a series of dimeric clusters of ternary 9‐vertex cages. Besides experimental work including X‐ray diffraction, EDS/μ‐XFS, and ESI mass spectrometry, quantum chemical studies were applied to confirm the composition and to determine atomic positions in the structures. In addition, the calculations verified that intense Au–Tt or Au–Pn bonding outplays Au⋅⋅⋅Au interactions in these cases, and they served to illustrate that the lightest congeners used in ternary intermetalloid cluster syntheses,  should be addressed to the authors.SupplementaryClick here for additional data file.


Three deactivation mechanisms  in the ratio 3:2:4 were identified. Pathways I and III involve ultrafast intersystem crossing and internal conversion, whereas pathway II involves only internal conversion.The design of photoswitchable transition metal complexes with tailored properties is one of the most important challenges in chemistry. Studies explaining the underlying mechanisms are, however, scarce. Herein, the early relaxation dynamics towards NO photoisomerization in  SHARC attack: The early relaxation dynamics towards NO photoisomerization in trans‐[RuCl(NO)(py)4]2+ was elucidated by means of SHARC non‐adiabatic dynamics, which provided time‐resolved information and branching ratios. Three deactivation mechanisms  in ratio 3:2:4 were identified. Pathways I and III involve ultrafast intersystem crossing and internal conversion, whereas pathway II involves only internal conversion. For simplicity, we carried out non‐adiabatic excited‐state dynamics only in the gas phase to describe the processes occurring in the crystal structure using trajectory surface‐hopping methods.Herein, we report the first study on the relaxation dynamics of 1GS, is an orange crystal that corresponds to an N‐bonded structure with a Ru‐N‐O bond angle of 180°. Upon absorption of the first blue photon, the complex is excited to a metal‐to‐ligand charge‐transfer (MLCT) singlet degenerate (S1 and S2) state, from which relaxation following two different nonradiative pathways is plausible. In the first pathway , together with the close proximity of these excited states, should favour the ISC towards the triplets. Once in the T1 state, the complex relaxes to the N‐bonded triplet minimum, labelled 3GS. From here, the system can either relax back to the 1GS isomer through ISC via the easily accessible MECP1 (orange dotted line), or proceed towards the metastable 1MS22. According to the DFT energy profiles of Figure 3GS isomer and MECP2 is considerably lower (<0.1 eV), and this suggests an efficient route to the 1MS2 intermediate.To facilitate the interpretation of the dynamical simulations it is useful to review here briefly the results of the available stationary calculations. Previous DFT1MS2 through a conical intersection between S1 and S0 near the transition state 1TS1. Both time‐dependent DFT (TD‐DFT) and MS‐CASPT2 calculations1MS2 can absorb a second blue photon and be re‐excited to a 1MLCT singlet state, which again relaxes by following two distinct non‐radiative pathways. In the first, the system undergoes ISC and IC to reach the NO‐bonded 3MS2 isomer, from which the 3MS1 is accessed after overcoming the barrier associated with 3TS2. From 3MS1 an additional ISC through MECP4 leads finally to the O‐bonded 1MS1 isomer (solid black arrows). As in the case of the first photon, the MS‐CASPT2 calculations predict that the last isomer 1MS1 can also be alternatively reached by a pathway involving only singlet states  does not absorb in the blue spectral range and it is observed as a stable green crystal.1GS to 1MS2.The second non‐radiative relaxation pathway  involves exclusively IC among singlet states. This path leads directly to the NO‐bonded singlet intermediate state Ideally, the most appropriate way to describe this complex is with multiconfigurational multistate complete active space second order perturbation theory (MS‐CASPT2).3]2+ complex.1 and S0 brings instabilities, which only a multiconfigurational method can properly account for. However, TD‐DFT is the most accurate method that can be computationally used for the system and we expected it to bring new insights complementary to stationary calculations.To avoid a bias of the presumably complex configurational space, in this work we resorted to full‐dimensional trajectory surface‐hopping methodsAccordingly, the electronic energies, gradients and spin–orbit couplings (SOCs) were obtained on‐the‐fly by using TD‐DFT, for which a new version of the ADF program package1/S2 are populated by a blue photon.1/S2 pair of states and propagated within the lowest three singlet states  and the three lowest triplet states .According to the available experimental data, at the equilibrium geometry only the bright states Strans‐[RuCl(NO)(py)4]2+. This is composed of the two degenerate 1MLCT states, S1 and S2  with ±0.25 eV, as in the photoconversion experiment.1 state and 70 to the S2 state. From them, 47 trajectories (33 %) propagated during 1 ps, while the remaining 97 trajectories (67 %) stopped within the first 200 fs, that is, as soon as they reached the zone near TS1 where S0 and S1 get close in energy, suffer instabilities and the calculation is terminated. Although this is a severe problem of DFT, the behaviour of the trajectories was systematic and allowed us to clearly identify all these “conflicting” trajectories with a particular mechanism.The irradiation wavelength was chosen not necessarily to tune the maximum of the SThe dynamical simulations revealed three distinct relaxation mechanisms, which will be discussed separately. Two sets of trajectories nicely confirm the coexistence of pathways I and II predicted by stationary calculations, while a third pathway (denoted as III) emerged from the simulations.1 and S2 excited states are equally populated (≈50 %) but within few femtoseconds the triplet states start to become populated due to ISC, so that after approximately 100 fs . A hopping‐event analysis between each pair of states reveals that S2 relaxes to the T3 state, whereas S1 relaxes towards the T2 state. Accordingly, the non‐adiabatic transition S2→T3 is more efficient than S1→T2. This large difference between the two pairs of states can be explained by the magnitude of the SOC between the excited states. Near the Franck–Condon region, the S2/T3 pair shows a SOC of approximately 100 cm−1, whereas that of S1/T2 is only approximately 40 cm−1. The analysis of the one‐electron transition density matrix of the excited states in the Franck–Condon region shows that the smaller value of the SOC between S1 and T2 is compatible with the El‐Sayed rule,1 and T2 share the same character in their electronic transitions, whereas the wave‐function character of T3 is different from that of S1 or S2 . Upon relaxation to T3, the system continues to be deactivated non‐radiatively through IC towards T2 with a time constant of 180±20 fs, and eventually to T1 with a fitted constant of 150±20 fs. These initial findings underline the role played by the ISC and triplet states in the NO photoisomerization of trans‐[RuCl(NO)(py)4]2+.A closer look at the individual state populations reveals that the moderate decay rate of the singlets is essentially due to slow depopulation of the SMore insight into the dynamics can be obtained by analyzing the geometrical changes induced by the various relaxation processes discussed above. The time evolution of the Ru‐N‐O angle and Ru−NO distance, which are the most important coordinates related to the N→O linkage isomerization, are shown in Figure 1GS isomer undergoes coherent bending of the nitrosyl ligand, from almost a linear Ru‐N‐O configuration to approximately 142° in about 160 fs. The analysis of the excited‐states character in Figure 3GS isomer, de facto the nearest reachable minimum along the N→O isomerization pathway . Concomitant to the Ru‐N‐O bending, the Ru−NO distance elongates, increasing from approximately 1.76 A  to approximately 2.20 Å during the first 60 fs. This elongation is thus also related to the increasing MLCT character of the excited states. Within 160 fs the Ru−NO bond stabilized at approximately 1.90 Å, consistent with the bond length found for 3GS. Accordingly, from a structural point of view, the trajectories reach the minimum 3GS within 160 fs. However, such a short time is not enough for all the trajectories to relax to the lowest triplet state T1, as the S1 state is still substantially populated at this stage, also due to the weak SOC between S1 with its nearest triplet state T2. Furthermore, inspection of the TD‐DFT single‐point calculations showed that the geometry of 3GS is also a minimum in the S1 PES . Thus, the discussed Ru‐N‐O angle and Ru−NO bond length also correspond to some trajectories remaining in S1.Initially, the geometry of the 3GS, most trajectories fluctuate in energy until the end of the propagation time, as shown by the thickening of the ensemble in Figures 3GS equilibrium value. A few other trajectories deviate from this minimum and relax back to the original 1GS geometry through ISC between T1 and S0. A look at the T1→S0 hopping geometries  indicates that the ISC occurs near the easily accessible MECP1. With TD‐BP86 this crossing point is located in between the 3GS and 1MS2 intermediate, 0.17 eV above 3GS . The trace of the T1→S0 hopping is also apparent from Figure 0 state recovers starting from 250 fs, while T1 starts to become depopulated after 750 fs. The depletion of the norm of the one‐electron transition density matrix . This relaxation pathway represents a major quenching funnel that could make the entire photoisomerization process quite inefficient, as it reverts the system back to the original isomer 1GS. Given the small number of events (only 8 (5 %) trajectories out of 144) that undergo T1→S0 ISC within 1 ps, the calculated time constant has a large error (1600±600 fs) and should also be considered only qualitatively.Upon relaxation to 1MS2 due to the short propagation times and the systematic underestimation of the triplet excitation energies by TD‐BP86 with respect to MS‐CASPT2,1/S0 MECPs. In particular, the position of MECP2 is crucial, as it allows for the 3GS→1MS2 passage . Because, according to TD‐BP86, MECP2 is located 0.62 eV above the minimum 3GS (see Table S7 in the Supporting Information), a large barrier must be surmounted to reach 1MS2. Thus, the 3GS→1GS pathway through the MECP1 is boosted instead, in accordance with the more affordable (0.17 eV) barrier between 3GS and MECP1. These facts indicate that TD‐BP86 artificially hinders the 1GS→1MS2 isomerization, which otherwise should be favourable, according to MS‐CASPT2 . This hand‐in‐hand analysis of the dynamical simulations with the quantum chemical calculations is thus critical to reach a comprehensive interpretation of the complex photochemistry of trans‐[RuCl(NO)(py)4]2+.The dynamical events related to pathway I are summarized on the right‐hand side of Figure 1 and S0 state are less than approximately 0.2 eV around TS1 and the multireference character of the ground state wave function becomes significant. Figure 3GS and TS1. The singlet trajectories undergo a series of IC transitions bringing population to S1 and S0 in the region around TS1 . None of these trajectories stopped on S2, and this suggests ultrafast radiationless decay from this state, as was seen in the trajectories pertaining to pathway I. However, in this case depopulation is also due to the rapid energy increase of S2 in the region between 3GS and TS1 . Indeed, at 3GS the stationary BP86 calculations predict an S1–S2 energy gap of 0.32 eV, whereas at TS1 this gap becomes 0.7 eV. On the other hand, in the same region the S0 energy increases with increasing Ru‐N‐O bending angle, whereas the S1 energy decreases  and, as a result, the S0–S1 energy gap reduces. Although near the transition state TS1 the gap was calculated to be 0.24 eV by stationary calculations , the dynamical simulations demonstrate that these two states can get closer, supporting the presence of a S1/S0 conical intersection near TS1 that can promote efficient formation of 1MS2 through the singlet manifold, as suggested by the MS‐CASPT2 calculations.1 state will undergo IC through this conical intersection, with the two main ground‐state relaxation pathways leading to 1GS and 1MS2. Because the momentum associated with the Ru‐N‐O angle is mainly decreasing at the time when the trajectories crash , we expect the relaxation path to 1MS2 to be favoured over the backward return to 1GS for inertial reasons , which confirms this hypothesis. The remaining trajectories that halted on a triplet state underwent efficient ISC and IC transitions . Reaching this crossing point involves no energy barrier and little structural change. Pathway III is thus a hypothesis that relies on the assumption that the T1 population can be transferred to S0 by ISC via the accessible MECP2.We now briefly discuss the mechanisms behind the trajectories related to pathways II and III, which account for 64 % of the trajectories. These trajectories are terminated within 30–200 fs, as soon as the Strans‐[RuCl(NO)(py)4]2+ can proceed by three mechanisms coexisting with a ratio of about 3:2:4 during the first 200 fs. Crucial to each pathway is the presence of non‐radiative ISC and IC, which compete on pathways I and III, whereas IC alone is present on pathway II. Pathway I is followed by 33 % of the trajectories, which undergo IC and ISC towards the triplet minimum 3GS in the T1 PES. ISC occurs with a time constant of 160±30 fs. Within the 1 ps simulation time, a small amount (5 %) of trajectories come back to the starting isomer 1GS, and this highlights one of the possible quenching funnels that can slow down the entire photoisomerization process. 24 % of the trajectories belong to pathway II and halt in the region near TS1 in singlet state S1 or S0 within the first 200 fs. This mechanism does not involve ISC, but only IC relaxation towards TS1 or the nearby conical intersection to eventually reach 1MS2 by another IC. Finally, 43 % of the trajectories halted on a triplet state during the first 200 fs in the region near TS1 (pathway III), which similarly to pathway I, involves ISC and IC, but has a faster ISC process with a time constant of 80 fs. Once in the triplet state these trajectories could either reach 1MS2 by ISC through the nearby MECP2 or undergo barrierless relaxation back to the minimum 3GS.We propose that NO photoisomerization in trans‐[RuCl(NO)(py)4]2+ as a photoswitching agent, and evidence the complexity of ruthenium nitrosyl photochemistry. The present study also illustrates the complementarity of stationary quantum chemical calculations and dynamical simulations, as high‐level quantum chemical calculations are indispensable to assess the validity of the different regions of the PES and critically interpret the outcome of the dynamics.The three mechanisms highlight the versatility of 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.


P = .025). p16 expression in a biopsy is related both to viral production and transformation, while decreased E4 expression relates to methylation, indicating advanced HSIL. p16 expression in ≥2/3 of the epithelium and absent E4 indicate likely HSIL on a subsequent LEEP specimen.The decision to treat a cervical squamous intraepithelial lesion (SIL) by loop electrosurgical excision procedure (LEEP) relies heavily on a colposcopy‐directed biopsy showing high‐grade (H)SIL. Diagnosis is often supported by p16, an immunohistochemical (IHC) biomarker of high‐risk (hr)HPV E7 gene activity. Additional potential markers include methylation of tumor suppressor genes FAM19A4/miR124‐2 in cervical cytology for advanced transforming HSIL and the IHC marker HPV E4 for productive, potentially regressing lesions. In 318 women referred for colposcopy, we investigated the relationship between staining patterns of p16 and E4 IHC in the worst biopsy, and the relation of these to FAM19A4/miR124‐2 methylation status in cytology. E4‐positive staining decreased with increasing SIL/CIN grade from 41% in LSIL to 3% in HSIL/CIN3. E4 positivity increased with grade of p16 when p16 expression was limited to the lower two third of the epithelium (r = 0.378), but fell with expression over. Loss of E4 expression in the worst lesion was associated with the methylation of FAM19A4/miR124‐2. We also examined whether these biomarkers can predict the histological outcome of the LEEP biopsy in a subgroup of 119 who underwent LEEP treatment. About 85% of women with ≥lower two third p16 staining/E4‐negative HSIL biopsies and 65% with limited p16 staining/E4‐positive HSIL biopsies had ≥HSIL in the LEEP specimen ( We found an inverse relation between HPV E4 expression on biopsy and hypermethylation of FAM19A4/miR124‐2 on cytology sample. p16 and hypermethylation increased with the severity of the lesion and may be used to identify premalignant transformation. The use of p16/E4 immunostaining and methylation could offer valuable information beyond current grading practice. Since histological diagnosis of HSIL/CIN2‐3 is the basis for surgical treatment, accuracy of diagnosis is key. However, hematoxylin and eosin stain (H&E) diagnosis is subjective and substantial interobserver and intra‐observer variability has been reported.The use of biomarkers, whose results are unambiguous and reproducible, may provide a solution. Immunohistochemical (IHC) staining with p16, single or in combination with the proliferation marker Ki‐67, has been identified as a valuable marker in diagnosis of ≥HSIL/CIN2.Hypermethylation of the CpG islands in promotor regions of several tumor suppressor genes is extremely high in CC This study aims to describe the relationships between the immunohistochemical expression patterns of markers p16 and HPV E4 in hrHPV‐positive colposcopy‐directed cervical biopsy, and also the relationship of these to methylation markers FAM19A4/miR124‐2 in cervical cytology samples of women referred to a colposcopy due to an abnormal screening test. In addition, we studied if biomarkers p16/E4 on biopsy and methylation on cytology can predict the histological outcome on loop electrosurgical excision procedure (LEEP) in a subgroup of 119 who underwent LEEP treatment.22.1The EVAH is a follow‐up study of women referred to the colposcopy clinic of Hospital Clínic, Barcelona, Spain because of abnormal cytology (≥ASC‐US).2.2Physician‐taken cervical cytology samples were collected using a Cervex‐Brush  and were rinsed in 20 mL of Thinprep medium (Hologic), before colposcopy. Samples were stored at room temperature for up to 6 months before DNA was isolated for hrHPV testing. An input volume of 250 μL was used to obtain 100 μL of eluate with the QIAamp MinElute Virus Spin kit (QIAgen Inc). hrHPV detection was performed using the GP5+/6+‐PCR‐EIA . The EIA‐positive GP5+/6+ amplimers were genotyped using a strip‐based test by the Genotyping kit HPV GP , genotyping hrHPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.Residual material after HPV testing and cytology was concentrated and sent to DDL Diagnostic Laboratory for methylation testing.2.3Histologic samples were fixed in 10% neutral buffered formalin and embedded in paraffin following routine procedures. Four‐micrometre‐sections were obtained for H&E and p16 staining and these were assessed by an expert local pathologist following morphologic criteria supported by p16 staining according to the LAST recommendations.Biopsies were recut at DDL Diagnostic Laboratory, Rijswijk, The Netherlands according to the sandwich methods, producing one 4‐µm‐thick H&E before slide, two tubes with three 8‐µm‐thick sections for HPV testing, three 4‐µm‐thick slides for immunohistochemistry, a 4‐µm‐thick H&E after slide, and up to three 4‐µm‐thick back‐up slides. Diagnoses based on H&E and IHC recut slides were considered as the definitive diagnosis. DNA for HPV detection and genotyping was isolated using proteinase K isolation and SPF10‐PCR‐DEIA‐LiPA25 . Women for whom the worst diagnosis on the local histology slide was more than one SIL/CIN grade different from the recut histology slides were excluded. This was done as the study histology could not be considered a reliable indicator of the lesion on which treatment was based. Those whose biopsy was hrHPV‐negative by SPF10‐PCR‐DEIA‐LiPA25v1 were also excluded.2.4INK4a antibody  and panHPVE4 mAb , respectively, after heat‐induced epitope retrieval with citrate buffer. Reactivity was visualized using the EnVision Detection System.The biopsy with the worst diagnosis was then stained for p16, using p16Two expert pathologists, blinded to the local histological diagnosis, were asked to score the worst lesion for E4 and p16. Biomarker p16 was scored negative (0) when no staining or patchy staining was observed, diffuse staining restricted to the lower third of the epithelium was scored as 1, staining restricted to the lower two third of the epithelium was scored as 2 and staining above the lower third of the epithelium, including full thickness staining, was scored as 3. E4 staining was scored negative (0), focal when limited positive staining was seen in the superficial layer of the epithelium (1) or extensive when there was widespread‐positive staining in the superficial layer of the epithelium and or below (2). This grading system has been previously validated as reproducible.To investigate further the relation between the expression of p16 and E4 and outcome we combined scores of p16 and E4, and used this to predict the presence of ≥HSIL/CIN2 and ≥HSIL/CIN3 on LEEP treatment. Lesions with p16 expression restricted to the lower third of the epithelium, or less, and all lesions with E4 positivity regardless of p16 expression were classified as combined IHC negative. In line with the LAST recommendations, all biopsies expressing p16 above the lower third of the epithelium,2.5P ≤ .05 for all tests. Agreement between the diagnosis on the locally produced slide and the consensus diagnosis on the final selection of samples was calculated using Cohen's kappa. Correlation coefficients for HPV E4 positivity and methylation positivity were calculated using Spearman's correlation test and differences in positivity percentages were calculated using the Chi‐squared test. The authors confirm that the data supporting the findings of this study are available within the article.Results were analyzed using IBM SPSS version 22.0 for Windows. The level of statistical significance was set at 3A total of 538 women had a smear which was eligible for methylation testing and interpretation of the result.The definitive diagnoses on the recut biopsy used for new H&E and IHC slides were as follows: 90 no CIN (28.3%), 67 LSIL/CIN1 (21.1%), 95 HSIL/CIN2 (29.9%), 62 HSIL/CIN3 (19.5%), 4 cervical carcinomas (CC) (1.2%). The agreement between the diagnosis on the locally produced slide and the consensus diagnosis on the newly sectioned slide was substantial (Cohen's kappa = 0.747).3.1The methylation positivity for markers FAM19A4 individually and combined with miR124‐2, in the cytology samples of these women according to the definitive histological diagnosis are shown in Table P < .001), and in ≥ HSIL/CIN2 .FAM19A4 was more frequently positive than miR124‐2 in both ≤LSIL/CIN1 . In biopsies showing p16 up to two third of the epithelium (n = 189), there was a significant correlation between p16 expression and E4 expression .Expression of E4 increased with increasing p16 expression with the highest percentage of E4 positivity seen in lesions with p16 in the lower two third of the epithelium (37.3%). However, in lesions that express p16 above the lower two third of the epithelium, E4 positivity dropped to 10.1% (P < .001).Of the women with lesions showing no or patchy p16 expression, 26.2% (27/103) had a FAM19A4 and/or miR124‐2 methylation‐positive cytology sample. The frequency dropped to 18.5% methylation‐positive cytology samples in women with lesions showing p16 in the lower third of the epithelium but increased to 42.4% methylation positivity in women with p16 in the lower two third of the lesion and 54.3% in women with lesions showing p16 above the lower two third of the epithelium. There was a significant correlation between grade of p16 expression on biopsy and methylation positivity on cytology sample . The same pattern was observed for the combination of FAM19A4 and/or miR124‐2 methylation markers, which was positive in 52.1% of the women with E4‐negative biopsies and 30.2% of the women with E4‐positive biopsies (P = .010).The relation between hypermethylation of markers FAM19A4 and/or miR124‐2 on cytology sampling and E4/p16 on the worst histological lesion on biopsy was explored by selecting all LSIL/CIN1‐3 lesions and ICCs with diffuse p16 expression in at least the lower third of the epithelium (n = 210). In this group, an inverse relation was found between E4 and FAM19A4/miR124‐2. Figure 3.5To find out the relation between p16/E4 marker patterns on colposcopy‐directed biopsy or FAM/miR on the cytology sample and a ≥HSIL/CIN2 diagnosis on LEEP, 119 women with HSIL/CIN2‐3 on biopsy treated by LEEP were investigated. Of these women, 22 (18.5%) with a mean age of 40.1y had a worst diagnosis of ≤LSIL/CIN1 on LEEP, and 97 (81.5%) women with a mean age of 37.5y had a worst diagnosis of ≥HSIL/CIN2 on LEEP.P = .025). The same was done for the outcome of ≥HSIL/CIN3 on LEEP, showing that 26.1% (6/23) of women with combined IHC‐negative biopsies and 49.0% (47/96) of women with combined IHC‐positive biopsies had ≥HSIL/CIN3 on LEEP (P = .047).Table P = .264). Among the methylation‐negative women there were 34.0% (16/47) with ≥HSIL/CIN3 on LEEP , and among women with methylation‐positive samples there were 51.4% (37/72) with ≥HSIL/CIN3 .Of the 119 women who were treated after a ≥HSIL/CIN2 lesion on biopsy, 60.5% (72/119) had a FAM and/or miR methylation‐positive sample and 39.5% (47/119) had a methylation‐negative sample. ≥HSIL/CIN2 on LEEP occurred in 84.7% (61/72) methylation‐positive women and 76.6% (36/47) methylation‐negative women (4P = .010).This study shows that in women who have hrHPV‐positive SIL/CIN there are complex relationships between expression patterns of the widely used immunohistochemical marker p16 and novel immunohistochemical marker E4 on biopsy. They also show relationships between these biopsy markers and the methylation markers FAM19A4/miR124‐2 on cervical cytology sample. Firstly, there is an inverse relation between productive infection as shown by HPV E4 positivity in the worst lesion present on biopsy, and methylation of markers FAM19A4 and miR124‐2 on cytology which are associated with transforming HPV infection. In women with diffusely p16 positive ≥LSIL/CIN1, a difference in FAM19A4 and/or miR124‐2 positivity rates was found between women with lesions expressing E4 and those who did not: 52.1% of women with E4‐negative and 30.2% with E4‐positive biopsies were positive for the methylation markers  up to 37% in lesions with p16 in the lower two third of the epithelium, but fell to 10% when p16 expression was above two third of the epithelium.Hypermethylation of FAM19A4 and/or miR124‐2 on cervical cytology sample increased with the grade of SIL/CIN, but there was some positivity (24%) in ≤LSIL/CIN1, rising to 66% in HSIL/CIN3 and was seen in all cancers. Methylation positivity progressively increased with grade of diffuse p16 positivity (18.5% in lesions with p16 in the lower third to 54.3% in lesions with p16 up to full thickness). There was also some (26.2%) methylation in samples of women with lesions negative or patchy for p16 IHC.p16 is widely used as marker of transformation in hrHPV‐associated CC and precancer, but this study shows that it is also expressed in up to the lower two third of cervical epithelium in lesions showing completion of the HPV life cycle and HPV production by E4 expression, and that increasing extent of p16 expression up to two third of the epithelium is associated with more frequent E4 expression. This is consistent with the expression of p16 as a surrogate for hrHPV E7 expression and the important role of E7 in driving the increased epithelial proliferation above the basal layer necessary for viral reproduction, as well as playing a part in driving neoplastic transformation. This finding explains the difficulty of using p16 on its own as a reliable transformation marker in SIL/CIN diagnosis. Only expression of p16 above two third of the epithelium is associated with infrequent E4 expression showing loss of viral life cycle completion with virus production and increased frequency of methylation of tumor suppressor genes.We also found evidence that p16 expression and E4 positivity on the worst colposcopy‐directed biopsy related to the frequency of finding HSIL/CIN2 and HSIL/CIN3 on LEEP in women treated for ≥HSIL/CIN2. Our results support the view that lesions with diffuse p16 expression limited to the basal third and E4 positivity are likely to be smaller and/or more regressive lesions. Luttmer et al have previously shown an increasing positivity rate for FAM19A4 analysis performed on cervicovaginal self‐samples and cervical cytology samples with the severity and volume of the lesion.This study was performed in a selected group of women who were referred for colposcopic examination after an abnormal Pap‐smear result, who had a hrHPV‐positive cervical biopsy and had cervical cytology samples suitable for methylation testing. Methylation status was only determined in the cervical cytology samples and not on biopsy. However, a previous study from van Baars et al confirmed that the methylation status of the cervical cytology sample represents the methylation status of the worst lesion present on the cervix when taking biopsies of all lesions detected during colposcopy together with a random biopsy of normal appearing tissue.We used a reference standard diagnosis in which the opinion of at least three pathologists was reflected, and which assured that the worst lesion was present in all tested material. It is known that reproducibility of diagnosis of LSIL/CIN1 and HSIL/CIN2 is limited and intra‐ and interobserver agreement is moderate.Large prospective studies are needed to investigate the outcome of E4‐positive and negative LSIL/CIN1 and HSIL/CIN2. Currently available methylation markers have shown good clinical performance in the detection of advanced transforming precursor lesions and CC in hrHPV‐positive women. In addition, results from a large follow‐up study have shown a low CC risk for hrHPV‐positive women with a negative FAM19A4/miR124‐2 triage test result, offering an objective triage test in hrHPV‐based screening programs.INK4A. Lesions with diffuse, but limited p16 positivity and E4 positivity most likely represent early transforming and productive lesions with variable HPV life cycle completion and might have a higher probability of spontaneous regression. Extensively p16‐positive, E4‐negative lesions in patients, and those with a methylation positive cervical cytology sample are more often ≥HSIL/CIN3 lesions and most likely represent advanced transforming lesions that require LEEP treatment. Further studies are required to establish the progression risk of different biomarker expression patterns in women not treated by LEEP.Our results show that there is a possibility of more specific diagnosis of CIN when using grading of patterns of immunohistochemical expression of HPV E4 in combination with p16