Through the series the electrochemical activity displays a bell shape variation that peaks at 260 mAh/g for the composition x = 0.6 with barely no capacity for the x = 0 and x = 3 end members. We show that this capacity results from cumulated anionic (Se2−/Sen−) and (S2−/Sn−) and cationic Ti3+/Ti4+ redox processes and provide evidence for a metal-ligand charge transfer by temperature-driven electron localization. Moreover, DFT calculations reveal that an anionic redox process cannot take place without the dynamic involvement of the transition metal electronic states. These insights can guide the rational synthesis of other Li-rich chalcogenides that are of interest for the development of solid-state batteries.Expanding the chemical space for designing novel anionic redox materials from oxides to sulfides has enabled to better apprehend fundamental aspects dealing with cationic-anionic relative band positioning. Pursuing with chalcogenides, but deviating from cationic substitution, we here present another twist to our band positioning strategy that relies on mixed ligands with the synthesis of the Li 2TiS3-xSex.Activation of anionic redox in battery materials promises great benefits for battery materials, but remains an elusive phenomenon. Here, the authors present anion-doping as a novel strategy to unlock electrochemical activity in the cobalt/nickel free cathode material, Li TM) oxides, LixTMyO2 (x > y), constitute a promising family for high-capacity cathode materials, relying on cationic and anionic redox processes for charge compensation2. The archetypical Li1.2Ni0.13Mn0.54Co0.13O2 (Li-rich NMC) can deliver specific capacities above 270 mAh/g to reach 1.000 Wh/kg of specific energy at the material level4. As established by extensive theoretical work, the introduction of alkaline ions into the TM layer results in 2p lone pairs on the oxygen7. Their electronic states serve as a reservoir of electrons that can potentially participate in anionic redox processes and liberate additional capacity compared to conventional cathode materials based on cationic redox, provided that they are made accessible through TM(nd)/O(2p) hybridization owing to local distortions n− species9 or the formation of Ir = O double bonds involve the O 2p lone pairs11. Due to difficulties in decoupling the anionic and cationic processes in highly covalent systems, it is likely that both mechanisms contribute almost equally to the electronic structure of the delithiated materials as different mesomeric structures.Layered lithium-rich transition metal -based materials are attractive since anion–cation redox competition was recognized in such materials back to the 1960s by Rouxel12. Ongoing from O to S, Se, or Te, the top of the anionic band is raised and can eventually interact strongly with the d states of TMs. Internal redox chemistry can be triggered by careful selection of the chemical elements and consecutive alignment of the respective bands: TiS2 is a semiconductor (Ti4+), while TiSe2 is a semimetal13, and finally TiTe2 is a metal with a significant transfer of electrons from the anionic Te band into the cationic bands of Ti14, as corroborated by density functional theory (DFT) calculations15. The propensity of stabilizing isolated holes at the top of the anion band increases concomitantly from O to Te: it is not possible to remove copper from Cu0.5Cr3+S2, while the formation of the selenide homolog Cr3+Se2 is feasible16. Equally, this explains why when moving from the left-hand side of the periodic table the disulfides form layered structures with fully reduced S2− anions , while the formation of persulfide type S–S bonds is encountered only on the right-hand side of the periodic table 18. Ultimately, the persulfide TiS3 shows two types of oxidation states for the sulfur (S2− and (S2)2−)20, which suggests that anionic redox can be activated in materials with d0 metals. These mechanisms of hole stabilization constitute a rich playground for the synthesis of materials with reversible anionic redox chemistry21. Within this framework, NaCrS2 is a notable material, as it is one of the first layered chalcogenides to solely utilize the S2−/Sn− redox couple upon electrochemical cycling22. Furthermore, anion doping has been reported for Li2FeSx2 − Sex in an attempt to control the covalency and the corresponding anionic redox potential23. Unfortunately, the overall performance of this material deteriorated with increasing Se content.In this context, chalcogenide 24. The introduction of non-d0 cations 26 associated with the concomitant lowering of the cationic d levels  is a viable strategy to activate Li2TiS3 using either solid-state synthesis in quartz tubes or ball milling27. In this work, we propose an alternative approach to electrochemical activate Li2TiS3: instead of lowering the cationic levels, we adjust the energy and symmetry of the anionic p levels by doping Se2− into Li2TiSx3 − Sex , electron paramagnetic resonance (EPR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy, DFT calculations) is used to understand and explain the underlying mechanisms of anionic redox in these mixed anionic materials. Altogether, these results point towards better exploitation of the ligand chemistry to broaden the class of electrode materials benefiting from anionic redox activity.Pursuing this direction, researchers targeted the electrochemically inactive Li2TiS3 were used as a starting point for preparing members of the Li2TiSx3 − Sex series 24. For compositions x ≤ 2, Li2S, TiS2, and TiSe2 were used as reagents, while for compositions x > 2, Li2S, Li2Se, and TiSe2 were ground and calcined in evacuated quartz tubes (see Supplementary information for details). Inductively coupled plasma analysis of the Li and Ti content of the final products confirmed that the ratio is constant along the full range 0 ≤ x ≤ 3. The powder XRD (PXRD) patterns of the as-synthesized materials  compared to S2− (1.848 Å)28.The synthetic conditions reported for Lials Fig.  can be f3¯m Fig. . The lat2TiSx3 − Sex on the electrochemistry, Swagelok-type cells were assembled and cycled against Li+/Li as shown in Fig. x: while at the end members Li2TiS3 and Li2TiSe3 allow for the removal of only 0.1 and 0.4 Li, respectively; up to 1.7 Li can be extracted at 0.6 < x < 1.5, see Fig. 2TiSx3 − Sex series, the first charging process is characterized by a voltage plateau alike what has been reported in other Li-rich TM chalcogenides utilizing anionic redox30. The potential of this plateau decreases along Li2TiSx3 − Sex from 2.9 V in Li2TiS3 to 2.46 V in Li2TiSe3, as shown in Fig. yTiSx3 − Sex, resulting in S-shaped voltage traces. However, the smaller hysteresis together with the absence of irreversibility during the first cycle suggests much limited irreversible structural damages for the heavier chalcogenides S/Se as compared to oxides. Altogether, the gravimetric discharge capacity peaks at x = 0.6 corresponding to a capacity of 235 mAh/g or 550 Wh/kg  cells over 25 cycles shows sustained capacity retention with, however, a noticeable discharge-voltage fade of 80 mV 25, but dramatically lower than ~200 mV reported for Li-rich oxides31. The differential capacity dQ/dE is given for the 1st, 2nd, and 25th cycles in the inset. After the initial activation plateau at 2.7 V, consecutive oxidation cycles display broad contributions with two maxima at 2.3 and 2.6 V, respectively. This is reminiscent of Li2TiS3, for which an oxidation process at ca. 2.3 V was related to the cationic Ti3+/4+ redox couple and a second process at 2.6 V to an anionic redox process Sn−2−/ (n < 2)24.Galvanostatic cycling of Li/Li mV Fig. . This is2TiS2.4Se0.6 and Li2TiS1.5Se1.5. The voltage gap  after 4 h of relaxation amounts to ΔE = 90 mV for x = 0.6 as compared to 70 mV for x = 1.5. This voltage gap could not be mitigated by applying a very low cycling C/40 rate . These results imply the existence of two electrochemical processes having different kinetics as frequently observed for anionic redox materials, with faster and slower ones being associated with the cationic and anionic redox couples, respectively. Altogether, we note that increasing the selenium content to x = 1.5 (Li2TiS1.5Se1.5) partially mitigates the hysteresis most likely due to the increase of covalence in the Ti–Ch bonds, which enables faster charge transfer. However, this positive effect is counterbalanced at high Se content by a lowering of electrochemical capacity, hence questioning the role of the crystal vs electronic structure in governing the performances of the Li2TiSx3 − Sex members.To check the effect of increasing the Se content on both the polarization and anionic redox response, galvanostatic intermittent titration technique experiments were conducted on Ligap Fig.  measured2TiS2.4Se0.6 was studied in greater detail for its composition, morphology, structure and charge compensating mechanism, and coupling diffraction techniques with microscopy and spectroscopy. EDX compositional maps confirm the homogeneous distribution of Ti, S, and Se, in the atomic ratio 1.05(2):2.34(5):0.62(6), which is in excellent agreement with the nominal composition . Additional sharp diffuse intensity lines clearly visible in the [110] ED pattern point towards a 2D honeycomb Li–Ti ordering and mandate a more rigorous crystallographic treatment in the monoclinic 2TiS3 was used as a starting point for a combined Rietveld refinement of SXRD and neutron powder diffraction data32 in the 2TiS2.4Se0.6 is isostructural to Li2TiS3  image nicely visualizes the O3 stacking of the closely packed layers along the c-axis, while the honeycomb ordering and stacking faults are clearly visible along the [110] direction  Å).Finally, robust structural refinements were carried out on SXRD data of isostructural LiyTiS2.4Se0.6 induced by electrochemical cycling was studied in an operando PXRD experiment. Upon oxidation, a gradual shift of the (104) reflection towards smaller d-spacing  is observed until 1.7 Li are removed at 3.0 V  vs 3.5812(1) Å). Conversely, a nonlinear expansion occurs along c and the inter-slab distance in fully relithiated Li2TiS2.4Se0.6 is slightly larger than that in the pristine material (18.1315(10) vs 18.2638(4) Å). Upon subsequent charge–discharge cycles, a more reversible evolution is witnessed  and STEM imaging was performed on chemically charged Limentclasspt{minimax < 3) and on samples for x = 0.6 at different states of charge. The S 2p, Se 3p, and Ti 2p core spectra were collected at two different photon energies: 1487 eV (home XPS) and 10 keV  to probe the sample surface (7–8 nm) and the bulk (43–44 nm), respectively33.Although PXRD suggests the participation of the chalcogenide anions in the overall redox process, it cannot decouple how such a contribution is parted between S and Se. To answer this question, XPS was carried out on pristine materials (0 < E(Ti–Ch) between the Ti 2p3/2 and the chalcogenide S 2p3/2 or Se 3p3/2 peaks with increasing x , recovered from electrochemical half-cells cycled to different states of charge , and also to access the S 1s core peak, which is free from any overlapping with selenium peak  and partially oxidized Sen− (Se 3p3/2 at 161.6 eV). Upon charge, there is a clear growth of the Sen− (n < 2) component that is accompanied by the gradual disappearance of the signal assigned to Se2−  corresponding to oxidized S species (Sn−) emerges at higher binding energy. In the fully charged sample, selenium is predominantly oxidized to Sen−, while 50% of sulfur is found as Sn−, implying that selenium is preferentially oxidized compared to sulfur. It is also important to notice the decrease of the binding energy difference between the Ti 2p3/2 component of Ti4+ and the S 2p3/2 component of sulfide S2−, ΔE(Ti–S), which drops from 295.6 to 295.1 eV, relating to the increasing covalent character of the Ti–S bond upon lithium extraction. This process is partially reversible upon the following discharge (increase up to 295.4 eV). It is also worth noting that the Sen− peak is slightly shifting by −0.25 eV with respect to the Se2− peak upon the charge, and is shifting back upon the following discharge. This shift is rather small but significant, which tends to indicate that the “n” value is not constant.HAXPES and XPS spectra were then collected on ex situ samples of Lieak Fig. . This pee2− Fig. . Concomin−) and selenium (Sen−) species are not fully re-reduced to S2− and Se2− . But signals associated with Ti3+ emerge at temperatures below ~170 K + oxidation state with δ linked to the Se content is consistent with the absence of EPR signal at room temperature and with our inability to detect Ti3+ by XPS.EPR spectra were measured for both pristine materials in Li0 K Fig. . The ina3+ increases owing to the electron localization on Ti, which results in a progressive distortion of the octahedral site. Such a temperature-driven distortion is confirmed by the narrowing of the EPR signal linewidth leading to the appearance of separate and well-defined 3 eigenvalues for the g-factors at 5.2 K 6 octahedra: starting from the TiS6 end-member, the g-factor increases initially due to an increase of local structural disorder, or equivalently due to the widest variety of TiSn6 − Sen environments (n = 1–6). Finally, the g-factor decreases due to the larger spin–orbit coupling of TiSe6 compared to TiS6. It is worth mentioning that pristine Li2TiSe3 shows a Pauli paramagnetic behavior (χ positive and independent of temperature), implying that Ti d and Se p states constitute its Fermi level.Figure yTiS2.4Se0.6  EPR measurements using echo detection reveal a strong isotropic signal centered at g = 1.98 and an anisotropic signal  for both the delithiated and relithiated sample, indicating the presence of Ti3+ . The static 7Li one-pulse NMR spectrum consists of a lineshape typical of quadrupolar nuclei with two sets of satellite transitions  and 1 gaussian peak of width ~50 ppm arising for more mobile or symmetric environments ) using the VASP program package (see Supplementary information for details)36. Having first ensured that the unit-cell parameters obtained from full structural relaxation match the experimental data, we computed the projected density of states  and are therefore typical electrochemical descriptors that allow identifying the redox centers of an electrochemical reaction. Computed for the two Li2TiS3 and Li2TiSe3 end-members, they reveal that S and Se are the redox centers of the oxidation process, as holes are mainly localized on the Ch p lone-pair states , in the absence of an electron mediator , is consistent with the near absence of electrochemical activity for the pure-S and Se end-members.But still, the electrochemical bell-shaped activation in Lites Fig. . Surprissee Fig. . Their nCh6 octahedra. The Fukui function computed for the mixed Li2TiS1.5Se1.5 phases now shows the participation of both the titanium and the chalcogens in the early stage of oxidation /Se(4p)/S(3p) in the mixed phases, which is fully consistent with the evolution of the g-factor deduced from EPR measurements. Hence, despite the apparent similarity of the density of states computed for the whole Li2TiSx3 − Sex series, substantial differences occur in the nature of the electronic states involved in the oxidation process through an internal redox process that is activated by the symmetry lowering induced by the heteroleptic TiCh6 environments. This explains the activation of the electrochemical activity of the S/Se mixed electrodes. The internal redox in pristine Li2TiSx3 − Sex is corroborated by Bader atomic charge analysis performed on the whole Li2TiSx3 − Sex series as a function of x  due to the formation of heteroleptic Tisee Fig. . The natp states in its lower energy part and Se 4p states in its higher energy part, due to the different electronegativity of the two chalcogen atoms  while it is activated preferentially on Se in distorted TiSn6 − Sen environments /Se(p) states). Keeping in mind that the statistical occurrence of the distorted configurations increases with the Se content up to x = 1.5 and then decreases from x = 1.5 to 3, this explanation is consistent with the electrochemical activity of the Li2TiSx3 − Sex phases following the bell-shaped behavior observed experimentally. Finally, the obvious evidence of having two coexisting ligand activities within the same compounds comforts previous theoretical models of Li-rich oxides that rely on nonuniform holes repartitions  and nonoxidized (O2−) oxygen as opposed to uniformly oxidized (O2)n− oxygen).Altogether, these computational results allow rationalizing the observed bell-shaped variation of the electrochemical activity of the electrodes. According to our results, anionic oxidation should be prevented in regular TiS2TiS3 can overcome an unfavorable band positioning to eventually trigger electrochemical activity. With the aim of checking the robustness of this novel strategy to stimulate anionic redox activity, we continued by investigating the Zr and Hf homologs. Compared to titanium, Zr and Hf are more electropositive and show correspondingly d bands positioned at higher energy, hence raising the question of how Se substitution would reshuffle the relative band positions. Here, we synthesized the Li2MSx3 − Sex  series by solid-state reaction (see Supplementary information). Both sulfides, Li2ZrS3 and Li2HfS3, were briefly mentioned before, but in neither case structural or electrochemical characterization was attempted38. As in the case of Li2TiSx3 − Sex, full solid solutions form between the sulfide and selenide end-members  to yellow-off-white (Li2ZrS3) to red-greyish (Li2HfS3). As Zr and Hf are more electropositive than Ti, the conduction band, composed of the 4d0 band (Li2ZrS3) or the 5d0 band (Li2HfS3), is positioned higher in energy compared to that of Li2TiS3  and cationic redox (Ti3+/4+) redox processes. We propose anion substitution as a new strategy to unlock reversible anionic redox in Li-rich materials by changing not only the energy but also the symmetry of the electronic band. This triggers the dynamic involvement of the electronic TM states, without which anionic redox cannot happen. As importantly, our low-temperature EPR/NMR measurements provide experimental evidence of internal ligand to metal charge transfer in this highly covalent system through temperature-driven electron localization, which indicates that band alignment and positioning is a dynamic process escaping simple description and assignment of oxidation states. These compelling findings offer a fertile playground for shedding light on the fundamentals of anionic redox. Performance-wise, both LTFS and Li2TiSx3 − Sex show comparable sustainable specific energy density. So in short, this type of chalcogenide chemistry opens the door to solid-state electrochemists for tuning cationic/anionic band positions  and hence increasing the electrochemical capacities of Li-rich sulfides. Chemical compatibility with S-based ionic conductors in solid-state batteries could be a surplus, rekindling the interest within the battery community.We have shown the feasibility to synthesize the full Li-rich layered chalcogenide LiSupplementary Information


These specific interactions influence the S1 electronic structures along the photoisomerization paths, ultimately leading to internal conversion in Cph1 but intersystem crossing in AnPixJ. This explains why the excited‐state relaxation in AnPixJ is much slower (ca. 100 ns) than in Cph1 (ca. 30 ps). Further, we predict that efficient internal conversion in AnPixJ can be achieved upon protonating the carboxylic group that interacts with PCB.Phytochrome proteins are light receptors that play a pivotal role in regulating the life cycles of plants and microorganisms. Intriguingly, while cyanobacterial phytochrome Cph1 and cyanobacteriochrome AnPixJ use the same phycocyanobilin (PCB) chromophore to absorb light, their excited‐state behavior is very different. We employ multiscale calculations to rationalize the different early photoisomerization mechanisms of PCB in Cph1 and AnPixJ. We found that their electronic S QM/MM calculations reveal that hydrogen‐bond networks interacting with the PCB chromophore govern the distinct excited‐state relaxations of red‐light photoreceptors Cph1 and AnPixJ. The present results contribute to decipher the natural evolution of both cyanobacteriochromes and phytochromes. Z‐E photoisomerization of the C15=C16 double bond in the bilin chromophore, buried within the GAF domain  minimum structures of Cph1 and AnPixJ optimized at the QM(CASSCF)/MM level of theory are depicted in Figure + group should be primarily assigned to the C pyrrole ring of PCB in Cph1 but to the B pyrrole ring in AnPixJ. This is also supported by the largest positive charge found in the C and B pyrrole rings of Cph1 and AnPixJ, see Tables S1 and S2, respectively. This difference is due to different hydrogen‐bonding interactions of PCB with the nearby Asp residue involving the neutral carbonyl group in Cph1 and the negatively charged carboxylic group in AnPixJ . Accordingly, the valence bond pattern of the B and C pyrrole rings differs in Cph1 and AnPixJ, ultimately leading to dissimilar excited‐state structures, isomerization, and decay dynamics, as it will be discussed below .The ground state —in close agreement to the experimental values of 43.3 and 44.1 kcal mol−1, respectively.[1 electronic excited state at the Franck‐Condon region is dominantly caused by a local electronic excitation in both Cph1 and AnPixJ (Table S4). However, the relaxed S1 state geometry of the PCB chromophore changes remarkably with respect to the S0  and in the B, C, and D pyrrole rings of AnPixJ (C6‐NB‐C9‐C10‐C11‐C12‐C13‐C14‐C15‐C16‐ND). As a result, the C14−C15 single bond and C15=C16 double bond in the S0 state change to double and single bonds in the S1 states of both Cph1 and AnPixJ, respectively. The corresponding BLA indexes for C14−C15 and C15−C16 are calculated to be −0.091 Å, and 0.101 Å for Cph1 and −0.075 Å and 0.091 Å for AnPixJ. It is also interesting that compared to the S0 electronic structure at the S0 minima, there is a significant multireference character in the S1 electronic structure at the S1 minima, with two comparable contributions: from both the closed‐shell and charge‐transfer electronic configurations (Table S4).The calculated QM(CASPT2)/MM vertical excitation energies to the S1 state along the decisive C14‐C15‐C16‐C17 dihedral angle (Tables S5–S10). The S1 potential energy surface in Cph1 .1/S0 conical intersection along the photoisomerization reaction is responsible of the excited‐state decay of Cph1. Given the small barrier associated with this process, it is expected to be fast, which is consistent with the experimentally measured time constants of ca. 30 ps.[−1.As suggested experimentally,1 Figure  is quite1 and S0 energies gradually increase, they remain well separated energetically along the photoisomerization path, and there is no hint of an S1/S0 conical intersection that could promote fast S1→S0 internal conversion. Instead, around 100° the T1 energy gets close to the S0 one and the T1 state should thus be able to decay to the S0 state after being formed through an S1→T1 intersystem crossing from the S1 state. However, as mentioned above, this intersystem crossing is not efficient since the SOC values are smaller than 1 cm−1, and thus the entire excited‐state relaxation process becomes very slow compared with that in Cph1. This could explain the experimental observation that excited‐state relaxation in AnPixJ takes ca. 100 ns.In contrast, the excited‐state decay of AnPixJ is experimentally much slower.1 states along the photoisomerization paths (Table S4). We find that the S1 electronic structure of Cph1 is of charge‐transfer character and remains almost constant during the photoisomerization, being essentially the same as at the S1 minimum and at the S1/S0 conical intersection. Both structures exhibit similar valence‐bond patterns . Consequently, the rotation around the C15−C16 single bond is rather straightforward with a small barrier in the S1 state. Noteworthy, similar excited‐state isomerization paths were found in the isolated PΦB chromophore in vacuum (PCB in this work).−1 in PCB versus 0.7 kcal mol−1 in PΦB) and the roles of triplet states and local hydrogen‐bonding network were not studied in the previous work.In order to get insight into the physical origin of the distinct excited‐state decay dynamics of PCB in Cph1 and AnPixJ, we have analyzed the electronic structure of the corresponding S Figures  and 3 c,1 electronic structure at the C14‐C15‐C16‐C17 dihedral angle of −100° is different from that at the S1 minimum. We recall that the S1 state is of charge‐transfer character at its minimum, and the valence bond patterns of the B, C, and D pyrrole rings are different from those at the S0 minimum .Instead, we attribute the different excited‐state decay dynamics of Cph1 and AnPixJ to their distinct Sn Figure . While i1 and S0 electronic structures is the hydrogen‐bonding interaction with the nearby Asp residue involving its neutral carbonyl group in Cph1 and its negatively charged carboxylic group in AnPixJ . However, the computed photoisomerization path  is different and actually resembles the one in Cph1 in terms of the energetics as well as bond lengths and most importantly: there is an energetically accessible S1/S0 conical intersection that controls the excited‐state relaxation dynamics of this mutant form. At the S1/S0 conical intersection, the C14−C15 and C15−C16 bonds are of typical double‐ and single‐bond characters , as in the case of Cph1  as well as the minimum‐energy photoisomerization paths are similar to those in Cph1 and AnPixJ . This reveals that in this case the environmental effects involving other residues play a minor role.The protein environmental effects are further investigated by constructing cluster models including only the PCB chromophore and the nearby Asp residue (see Section S4). Interestingly, the optimized S1 minimum‐energy isomerization paths of Cph1 and AnPixJ at the QM(OM2/MRCI)/MM level using the 100 ns MD snapshots as the initial structures. The photoisomerization paths are the essentially same as those using 1 ns MD snapshot . These results demonstrate that the protein fluctuation will not change the proposal that the hydrogen‐bond networks determine the early photoisomerization processes of Cph1 and AnPixJ phytochromes.Finally, in order to investigate protein fluctuation effects, an extended 200 ns MD simulation was carried out. Figure 1, T1, and S0 minima exhibit distinct geometric and electronic structures because of individual hydrogen‐bonding networks with the nearby Asp residue. These networks also regulate the S1 electronic structures along the photoisomerization paths and ultimately lead to different excited‐state behaviors in Cph1 and AnPixJ. In the former, the S1 charge transfer character is maintained along the entire photoisomerization path, which provides access to an S1/S0 conical intersection that allows an efficient S1→S0 internal conversion to the S0 state. In the latter, the S1 electronic structure along the computed path evolves from initial charge transfer to diradical characters in the later phase, which prevents the formation of an S1/S0 conical intersection so that the excited‐state decay will be less efficient (involving consecutive S1→T1 and T1→S0 intersystem crossings to the S0 state). These different excited‐state dynamics rationalize the experimental observations that excited‐state relaxation is slow (ca. 100 ns) in AnPixJ but fast (ca. 30 ps) in Cph1.1→S0 internal conversion via an S1/S0 conical intersection‐underscoring the decisive influence of the protonation state of nearby residues on the excited‐state dynamics of biological chromophores. In general, our findings demonstrate the feasibility of multi‐scale QM/MM calculations to provide exquisite insight into the photoresponse of red‐light photoreceptors to understand the natural evolution of phytochromes and cyanobacteriochromes.We used accurate multi‐scale QM(CASPT2)/MM calculations to investigate the early PCB photoisomerization in Cph1 and AnPixJ with atomistic detail. We found that the SThe 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.Supporting InformationClick here for additional data file.


E. coli to select variants of the barley chymotrypsin inhibitor 2 with increased stability. We find two variants that are more than 3.8 kJ mol−1 more stable than the wild-type. In one case, the increased stability is the effect of the single substitution D55G. The other case is a double mutant, L49I/I57V, which is 5.1 kJ mol−1 more stable than the sum of the effects of the individual mutations. In addition to demonstrating the strength of our selection system for finding stabilizing mutations, our work also demonstrate how subtle conformational effects may modulate stability.Most single point mutations destabilize folded proteins. Mutations that stabilize a protein typically only have a small effect and multiple mutations are often needed to substantially increase the stability. Multiple point mutations may act synergistically on the stability, and it is often not straightforward to predict their combined effect from the individual contributions. Here, we have applied an efficient in-cell assay in  E. coli to look for mutants of chymotrypsin inhibitor 2 which are either destabilized or stabilized, in an effort to identify a larger library of mutants which might improve stability prediction. Their work demonstrates how subtle conformational effects in proteins may modulate protein stability.Louise Hamborg et al. use a cell-based screen in  For using proteins in biotechnological and biopharmaceutical applications it is often an advantage that the proteins have long shelf-lives and are not degraded too rapidly during the applications2. Our ability to engineer proteins with increased stability or to understand how amino acid changes cause decreased stability has thus been the subject of large number of studies. Although our understanding of the physics and thermodynamics of protein stability is rather advanced and thus to a great extent allows us to calculate structures with amazing precision using either molecular dynamics simulations based on a physical a description of the interactions stabilizing a protein structure3 or by bioinformatics and machine learning4, we are still not able to accurately predict how multiple mutations in a protein act together to change the stability of the protein.Understanding how the stability of a protein changes when an amino acid residue is changed is fundamental for several biological processes and the aetiology of many diseasesE. coli, that can be used to measure both protein translation and folding stability in vivo5. The translation sensor part of the system is based on an RNA hairpin structure inserted into a polycistronic mRNA coding for the protein of interest and for the fluorescent protein mCherry, thus making it possible to read out efficient translation through a red fluorescent signal. The protein folding and stability sensor is based on GFP-ASV through a system engineered to be expressed as a response to protein misfolding. Green fluorescence is thus used as a proxy for low in vivo protein stability. This misfolding response relies on a heat shock promoter, lbpAp, and the E. coli heat shock system. With increasing levels of protein misfolding, more of the chaperone DnaK will bind the misfolded protein instead of the E. coli heat shock sigma factor, RpoH. In the absence of DnaK, RpoH can participate in assembly of the RNA polymerase sigma 32 complex, which can drive transcription from the lbpAp promoter. The presence of misfolded protein that can bind DnaK thus results in the expression of GFP and a green fluorescence signal. By expressing libraries of random mutations in a given protein in this bacterial sensor system and analysing the cells by fluorescence-activated cell sorting (FACS), it is possible to select large sets of protein variants that retain a folded structure, thus avoiding complications from using a functional assay as a proxy for folding stability.Recently, we described a system based on recombinant expression in Gf) between a wild-type protein and a mutant22. In general, the methods perform rather well when predicting the effects of destabilizing mutations but often fail in predicting stabilizing mutations23. A comparison of several stability predictors showed an average correlation of around 0.6 between experimentally determined and computed changes in stability for all types of mutations25. The algorithms are better at predicting deletion mutations in the hydrophobic core, than mutations that increase the size of the side chain, mutations on the protein surface and mutations where electrostatic interactions contribute to the stabilization. This is partly a result of the data available for training the algorithms that mainly consist of deletion mutations in the hydrophobic core26, A particular challenge in predicting stabilizing protein variants is that among the few single substitutions that are actually stabilizing the effects are often small, so that multiple substitutions may be needed to create a substantial stabilizing effect17. As the effects of the mutations are not always independent, and non-additivity may result in both positive or negative epistasis28, it can be difficult to predict the stability of proteins with multiple substitutions. One way to improve the computational methods is to generate stability data on a larger set of protein variants generated to scan sequence space better than the current available datasets and including also stabilizing variants.An alternative to screening mutant libraries for proteins with altered stability is to calculate the effect of substituting amino acids and find variants with the desired properties. Several computational tools have been developed that predict the change in free energy for folding  to broadly cover sequence and stability space. CI2 is a small single domain protein of 64 residues, which has been extensively used as a model to understand key concepts of protein folding and stability−1 more than the sum of the individual effects of the two single variants. A detailed analysis of the structural changes in L49I/I57V suggests that several subtle long-range effects underlie the high stability gain.In the current work we show that our sensor system can indeed be used to select for proteins with increased stability, even in an already highly stable protein. Many of the 25 variants for which we measured the stability destabilize CI2. Still, we found two variants, L49I/I57V and D55G, that are significantly stabilized relative to wild-type CI2. For L49I/I57V we find that there is a strong positive synergistic effect between the two substitutions and this variant is stabilized by 5.1 kJ mol5. It will thus not be possible to separate stabilized variants from the wild-type or from variants with stabilities close to that of the wild-type. A library of random mutations in wild-type CI2 will therefore be most suited for selecting variants with stabilities that are lower than that of the wild-type protein but that are still able to fold. To select variants of CI2 that are more stable than the starting point we opted to use a destabilized background as starting point. The I57A variant of CI2 is significantly destabilized (ΔGf = 14 kJ mol−1) and results in a high GFP signal in the sensor system . In total we found 71 unique sequences without stop codons, deletions or insertions in the region encoding the 64 amino acid residues of CI2. 41 of the sequences were from the wild-type library and 30 sequences were from the I57A library .To express and purify the 71 CI2 variants, they were subcloned into pET11a without the hexa-His tag, which is part of the folding sensor system. The presence of this C-terminal His-tag interferes with key interactions of the CI2 C-terminal carboxylate and compromises the stability of the protein during purification. However, the destabilization gives just the right stability window for finding variants with altered stability in the sensor system. Thus, the FACS selection was done on CI2 libraries with the His-tag, whereas the in vitro stability measurements were performed on CI2 variants without the His-tag. Although we selected for protein variants that do not activate the misfolding sensor, some of the variants still did not behave well in the expression system and did not result in pure and stable protein or resulted in protein with multistate folding behaviour. We thus ended with 13 unique variants in the wild-type background and 12 unique variants in the I57A background that could be used for stability measurements. In the set of variants that we have analysed, we also included L49I and D55G . From previous work, it is known that I57V is slightly more stable than the wild-type protein32, and valine at position 57 is also preferred in CI2 from many other plant species35. Most other mutations that occur together with I57V are less stable than I57V alone, but still more stable than the I57A background. There are however two exceptions. Both the L49I/I57V and D55G/I57V are even further stabilized than I57V. Both I at position 49 and G at position 55 are often seen in CI2 from other species35. The L49I and D55G variants have not previously been characterized so we also included these single variants in our analysis.To measure the in vitro stability for folding we used our recently described combined two-dimensional thermal and chemical protein unfolding assay, where the unfolding of the protein is followed by the change in intrinsic Trp fluorescence as the temperature is increased at multiple concentrations of denaturant Fig. 34. The sin Table . We findGf for each variant  of the experimentally determined values with those calculated using FoldX, Rosetta and SEQ are 0.81, 0.74 and 0.72, respectively. In general, the three stability predictors agree in the overall effect of mutations, but they are inconsistent in the exact value. Importantly, while the methods are relatively good at predicting destabilizing effects, they are generally not able to predict stabilized variants, though FoldX does predict D55G to be highly stabilizing.To compare how well the stability of the selected set of CI2 variants can be predicted computationally we used FoldX, Rosetta and a sequence-based method that analyses variability in a multiple sequence alignment of homologous sequences (hereafter referred to as SEQ), to calculate ΔΔm-value of 8.4 ± 0.3 kJ/mol/M corresponding to the average of the m-values for wild-type, L49I, D55G, I57V, D55G/I57V and L49I/I57V . As the m-value is not a fitting parameter in this analysis the standard errors of the other fitting parameters decreases and cannot be directly compared to results presented in Fig. Gf = −2.2 ± 0.1 kJ mol−1 . Indeed, the two mutations have an unfavourable synergistic effect, ΔΔΔGf, of 2.6 ± 0.4 kJ mol−1. In contrast, introducing L49I, which on its own destabilizes by ΔΔGf = 3.4 ± 0.1 kJ mol−1, together with I57V results in a total stabilization of the L49I/I57V double mutant of ΔΔGf = −3.8 ± 0.1 kJ mol−1. In this case the synergistic effect of introducing both L49I and I57V is ΔΔΔGf = −5.1 ± 0.2 kJ mol−1.In an attempt to understand better the origin of the increased stability of the two double mutants (D55G/I57V and L49I/I57V), we performed a more detailed analysis of the double mutation cycles from the wild-type through the single mutants to the double mutants. To compare the variants in the two double mutation cycles we re-analysed the stability data assuming a common 7V Table . As longl−1 Fig. . For D55Gf from FoldX, Rosetta and SEQ. Particularly, FoldX does a good job in predicting the effects of the L49I and D55G mutations  following the manufacturer’s instructions and everything was plated on LB agar plates with 100 µg mL−1 ampicillin.The wild-type CI2 sequence used here is UniProt: P01053, residues 22–84 with an additional N-terminal Met. The numbering we use, start at this Met, which is the numbering system commonly used in the literature. cDNA encoding this sequence was inserted into a pET22-mCherry vector between the NdeI and HindIII restriction sites to preserve the translation coupling using Gibson assembly. The QuickChange Lightning II mutagenesis kit (Agilent) was used to create the I57A variant in the CI2_WT_pET22_mCherry vector. Mutant libraries of CI2 WT and CI2 I57A were generated using the GeneMorph II Random mutagenesis kit (Agilent). The mutation frequency was aimed at 0-4 amino acid substitutions per gene by adjusting the initial target DNA and the number of amplification cycles. The PCR product from the random mutagenesis was used as a MEGAprimer for the insertion of the mutants into the CI2 WT or CI2 I57A backgrounds. The PCR products were transformed into MegaX DH10B T15. After recovery, the transformants were directly inoculated in 3 mL LB medium containing 100 µg mL−1 ampicillin, 25 µg mL−1 chloramphenicol and 50 µg mL−1 spectinomycin and grown overnight at 37 °C and 180 rpm. Cells were transferred into fresh medium and grown at 30 °C and 250 rpm to an OD600 of 0.5–0.7. Expression was induced by addition of 0.5 mM IPTG and the growth temperature of the culture was shifted to 30 °C.The CI2 libraries were transformed into electrocompetent Rosetta2(DE3)pLysS pSEVA631-IBpAP-GFP-ASV cellsE. coli fraction harboring proteins with altered protein stability, protein expression was induced again and cells  were sorted as described above. The following day, the sorted cell population was analysed 1 h after induction of protein expression by flow cytometry . Single cells were sorted directly into 100 µl LB supplemented with antibiotics in 96 well culture plates and grown overnight at 37 °C and 300 rpm. The single cells were characterized by Sanger sequencing.One hour after induction cells were analysed by flow cytometry . 100,000 cells expressing a CI2 WT mutant protein with increased GFP signal, and 100,000 cells expressing a CI2 I57A mutant protein with decreased GFP signal were sorted . The CI2 genes were amplified using the Phusion Hot Start II DNA Polymerase (Thermo Scientific) and region of interest-specific primers with overhang adapters. The PCR amplicons were purified using AMPure XP beads (Beckman Coulter) following the manufacturer’s instructions. Dual indices and Illumina sequencing adapters (Nexteras XT Index kit (Nextera v2 D)) were attached using the KAPA HiFi HotStart DNA polymerase. The amplicon libraries were purified using AMPure XP beads (Beckman Coulter) following the manufacturer’s instructions. Concentrations of the purified amplicon libraries were quantified using a Qubit 2.0 Fluorometer and the dsDNA broad range kit. To determine the average bp length, a bioanalyzer and the DNA 1000 kit (Agilent) was used following the manufacturer’s instructions. The libraries were normalized to 10 nM and all libraries were pooled. The samples were spiked with 5% Phi-X control DNA (Illumina) and loaded onto the flow cell and sequenced on and then applied onto an Illumina MiSeq instrument.Samples from the CI2 WT library were extracted from each step of FACS selection for NGS. 2 mL culture was centrifuged for 10,000 × NdeI and BamHI restriction sites. All clones were verified using Sanger sequencing. All protein expression were performed in BL21(DE3)pLysS. For small scale protein expression, the bacteria were grown in 2 ml ZYM5052 autoinduction media in a 24 well cell culture plate with incubation at 37 °C for 4 h followed by 20 h at 20 °C. Low expression level plasmids were expressed in 50 mL ZYM5052 autoinduction media. Cells were harvested by centrifugation at 5000 × g for 15 min. The pellet frozen at −20 °C and resuspended in 10 mM Na-acetate, pH 4.4 before centrifugation at 20,000 × g for 30 min. The supernatant was further diluted in the same buffer. The samples were applied onto a 1 ml Resource S column equilibrated with 20 mM Na-acetate, pH 4.4 and step eluted with 20 mM Na-acetate, pH 4.4, 1 M NaCl. The peak fraction was applied onto a Superdex 75 16/85 column equilibrated with 50 mM NH4HCO3, and the fractions containing CI2 were collected and lyophilized before dissolving, in 50 mM MES, pH 6.25. For expression of protein for structure determination the bacteria were grown in LB media. The expression was induced by 0.4 mM IPTG at OD600 0.6–0.8. Cells were harvested by centrifugation at 5000 × g for 15 min and resuspended in 25 mM Tris-HCl, pH 8, 1 mM EDTA before lysis by two freeze-thaw cycles. The sample was cleared by centrifugation at 20,000 × g at 4 °C. Polyetylenimine was added to a concentration of 1% and the sample centrifuged for 15 min at 20,000×g. Ammonium sulphate was added to the supernatant to 70% saturation, and left for 30 min at 4 °C before centrifugation at 20,000 × g. The pellet was resuspended in 25 mM Tris-HCl, pH 8 and heated at 40 °C until all precipitate was solubilized. The samples were centrifuged at 20,000 × g for 10 min before size exclusion chromatography in 10 mM NH4HCO3. Peak fractions were pooled and lyophilized and finally resuspended in MilliQ water and dialysed against water. We note that while we attempted to express 71 variants of CI2, a number of them displayed did not yield soluble protein or give useful data in the equilibrium unfolding experiments. We hypothesise that this could be caused by a subset of the variants bypassing the folding sensor by weakening the interaction with DnaK as suggested by predicting DnaK binding site with the Limbo algorithm42 . CI2 genes were amplified and ligated into a pET11a vector using the 34. The degree of unfolding was followed by fluorescence measurements on a Prometheus NT.48 (NanoTemper) using Prometheus NT.48 high sensitivity capillaries. The temperature was ramped from 15 to 95 °C with a temperature increment of 1 °C min−1. Global analysis of temperature and solvent denaturation was performed as described34.Protein concentrations were determined by absorbance at 280 nm measured on a NanoDrop 1000 due to the low volume samples. Equilibrium stability in GuHCl was measured with a final protein concentration of 10 µM at 6–16 concentrations of GuHCl evenly distributed in the range from 0 to 5, 6 or 7 M depending on the stability of the variant19 was used to estimate the free-energy change upon mutations of CI2, using the coordinates of the PDB entry 2CI237. The RepairPDB function of FoldX was first applied to the wild type structure. The resulting structure was used as input to the BuildModel function to generate the models of the investigated mutants and to evaluate their ΔΔGf.Version 4 of the FoldX energy function15 in its cartesian version43 was also used to estimate ΔΔGf, using the coordinates of PDB entry 2CI2. The wild-type structure was first relaxed in cartesian space with restrained backbone and sidechain coordinates. The resulting coordinates were then used to build the model of the investigated mutants and to evaluate their ΔΔGf by means of the Cartesian_ddg function. The calculations were repeated on five independent runs, whose results were then averaged to obtain the final values reported in the manuscript. The resulting difference in stability was multiplied by 1.44 to bring the ΔΔG values from Rosetta energy units onto a scale corresponding to kJ mol−1 (ref. 44).The Rosetta energy function45, which takes into account not only single-site conservation, but also correlated mutations between site pairs. This approach aims at exploiting the structural and functional constraints encoded in the family evolution46, assigning to each specific sequence a score related to the probability of being a good representative of that family47. Even if this measure is more related to the general fitness of the sequence, it can also be used bona fide to judge the effect of a specific mutation on protein stability48. To have a variation model which is statistically significant, we obtained a larger multiple sequence alignment containing CI2 homologues by building a hidden Markov model of the protein family, based on 4 iterations of Jackhmmer49 and extracting the sequences from the Uniprot Uniref100 database50. Sequences containing more than 50% of gaps with respect to the wild-type sequence were excluded, together with the sequences sharing more than 90% of sequence identity, resulting in an alignment of 1198 sequences, corresponding to 942 independent sequences at the 95% identity level. We then used the asymmetric plmDCA algorithm52 to calculate the parameters of the sequence model. The score of the wild-type sequence was then subtracted to the one of each analysed sequence to obtain the final values reported in the manuscript. The scripts and sequence alignment used in these analyses are available from https://github.com/KULL-Centre/papers/tree/master/2021/CI2-Hamborg-et-al.Looking at the mutational pattern observed in a multiple sequence alignment of homologous sequence, it is possible to build a global statistical model of the relative protein family variability4)2SO4, 50 mM Tris-HCl, pH 8.0 at a protein concentration of 75 mg ml−1. CI2 I57V were crystalized in 0.1 M Tris-HCl, 8% PEG 8000, pH 8.5. Data for WT were collected on an inhouse setup with an Agilent SuperNova diffraction source (1.5406 Å) and an Atlas CCD detector. Data for L49I, I57V and L49I/I57V were collected at DESY, Hamburg, beamline P13 (0.9763 Å) equipped with a Pilatus 6M-F, S/N 60-0117-F detector.CI2 WT, L49I and L49I/I57V were crystalized at 293 K in 40% . The latter program was employed because of pronounced anisotropic distribution of reflections. The structures were solved by molecular replacement using the CCP4 program Phaser55. The initial search model was wild-type CI2 (PDB code 2CI2)37. Later molecular replacement solutions were obtained using the higher resolution structures described in the present paper. The structures were carefully examined, adjusted and refined with Coot and Refmac5, respectively57. To make sure that the structures were not in a domain swapped configuration58, molecular replacement solutions were also sought using the domain swapped structure with PDB accession code 6QIZ. These consistently yielded significantly worse statistics.The reflections were collected using autoPROC34. For most variants two denaturant series were fitted (n = 2), except for L49I (n = 4), I57V (n = 3) and L49I/I57V (n = 6). The reproducibility of the fitted ΔGf at 25 °C is estimated to 1.2 kJ mol−1 from the pooled standard deviation59. No additional statistical analysis was used in this work.Each stability titration curve was made from at least 6 samples at different denaturant concentrations. We previously performed a systematic analysis of the minimal number of samples required for a robust analysis and found that five samples were sufficient for getting reliable resultsFurther information on research design is available in the Peer Review FileSupplemental InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Reporting Summary


Sputtered Fe72Ga28 layers have their magnetization in the sample plane with a magnetic fluctuation that gives rise to magnetic ripple. In order to turn its magnetization into the out of plane (OOP) direction, it has been magnetically coupled with Cr2O3 that has magnetic moments along the c-axis, that is the perpendicular direction when properly aligned. Cr2O3 has been obtained from Cr oxidation, whereas Fe72Ga28 has been deposited on top of it by sputtering in the ballistic regime. Although a uniaxial in-plane magnetic anisotropy is expected for Fe72Ga28 thickness above 100 nm, the interfacial coupling with Cr2O3 prevents this anisotropy. The formation of stripe domains in Fe72Ga28 above a critical thickness reveals the enhancement of the out of plane component of the Fe72Ga28 magnetization with respect to uncoupled layers. Due to the interface coupling, the Fe72Ga28 magnetization turns into the out-of-plane direction as its thickness is gradually reduced, and a perpendicular magnetic anisotropy of 3·106 erg·cm−3 is inferred from experimental results. Eventually, the coupling between Cr2O3 and Fe72Ga28 promotes an exchange-bias effect that has been well fitted by means of the random field model.Here it is investigated the effect of the antiferromagnet Cr However, in many applications as for example: high density magnetic storage, spintronic devices, non-volatile random access memories, logic devices, skyrmions or sensors, materials with OOP magnetization are desirable6.Control of the magnetization is an important issue for the development of pioneering magnetic devices. Typically, thin films have the magnetization in the sample plane in order to reduce the energy of the system0 FePt and CoPt thin films are extensively investigated but, their large coercivity and switching fields can represent a drawback for their integration in devices8. Therefore, it is of interest the investigation of other materials with PMA, or the possibility of turning the magnetization into the OOP direction. Stripe domains appear above a critical thickness when a moderate PMA is present9. In permalloy films, stripes have been observed because of columnar growth10, but they have also been promoted when coupled with NdCo11. Recently, the magnetization direction has been tuned in Fe–N layers by ion implantation and heat treatment conditions9, and by annealing in Fe87Si9B1312. Even more notably, the control of the magnetic anisotropy by means of voltage has been observed at magnetic transition metal/oxide interfaces2.Systems with large perpendicular magnetic anisotropy (PMA) such as L116. Also interesting is the possibility in sputtered Fe72Ga28 layers of controlling the in-plane magnetic anisotropy by growth conditions19 or by thermal treatments combined with mechanical stress20. Molecular beam epitaxy (MBE) FeGa can exhibit domain stripes due to a low PMA23, but the general behavior observed by magnetic force microscopy (MFM) in FeGa deposited either by electrodeposition24, sputtering25 or even MBE26 is a magnetic ripple due to magnetic fluctuations in layers with magnetization in the sample plane.FeGa alloys are extensively studied because of their large magnetostriction constant and low coercivity72Ga28 magnetization into the OOP direction when appropriately coupled with Cr2O3, an antiferromagnet that has its magnetic moments along the hexagonal c-axis being possible to have moments in the perpendicular direction when properly aligned29. Although Cr2O3 has already been coupled with ferromagnetic metals32, the interaction with Fe72Ga28 seems not a trivial problem. Fe72Ga28 has already been coupled with TbFe2 in [Fe72Ga28/TbFe2] multilayers to turn its magnetization into the OOP direction34. However, only a tilt of the magnetization of around 25° with respect to the sample plane was achieved despite the large PMA of TbFe234. Here we show that by means of a suitable experiment design in terms of layer thickness and materials, it is possible to reach an effective interfacial interaction that enables to turn the Fe72Ga28 magnetization into the OOP direction. In addition, because of the interfacial interaction we have observed exchange-bias effect in the perpendicular direction being possible to fit the experimental exchange-bias fields (HE) by means of the random field model37.In this work, we have explored the possibility of turning the Fe2O3 was synthetized from the evaporation of Cr on glass substrates that was subsequently oxidized in oxygen atmosphere at 750 °C during 3 h. Fe72Ga28 layers with a thickness ranging from 240 to 20 nm were grown by the DC magnetron sputtering technique in the ballistic regime at room temperature on top of the Cr2O3. The sputtering deposition was carried out in oblique incidence with an angle between the vapor beam and the perpendicular to the sample of about 25° and a distance of 9 cm between target and substrate. This direction of the vapor beam within the sample plane is taken as the reference direction to control the direction of the in-plane uniaxial anisotropy axis when created38. Fe72Ga28 films were deposited from a target with a composition of Fe72Ga28 with a diameter of 5 cm and a thickness of 2 mm using an Ar pressure of 3·10−3 mbar and a growth power of 90 W in all cases. A 20-nm thick Mo layer was used as a capping layer to avoid FeGa oxidation. Mo was also deposited with a power of 90 W and with an Ar pressure of 3·10−3 mbar. Therefore, the structure of the studied samples is: glass/ Cr2O3/Fe72Ga28/Mo. For further comparisons, we have also deposited single Fe72Ga28 layers with thickness of 20 and 150 nm between Mo buffer and capping layers to avoid oxidation.30 nm-thick Crα wavelength (1.54056 Å). A Digital Instruments Nanoscope IIIa instrument was used to obtain MFM images. We monitored the cantilever’s phase of oscillation while the magnetic tip was scanning the sample surface to work in the phase detection mode. The distance between sample and surface was 40 nm on average (lift mode)33. The MFM measurements were performed without magnetic field after an OOP magnetic field of 10 kOe was applied. The topography of the samples has also been obtained in this microscope working as an Atomic Force Microscope (AFM).We have used X-ray diffractometry (XRD) in the Bragg–Brentano configuration to study the structural properties. Measurements were performed in a Philips X’Pert MPD using the Cu K38. Hysteresis loops at 5 K were measured in a SQUID EverCool MPMS SQUID magnetometer from Quantum Design after field-cooling (FC) at 1 kOe from 360 K and zero-field-cooling (ZFC) at 0 kOe from 360 K. To reduce any hypothetical systematic error from the SQUID, a field step of 10 Oe has been used in the low field region of the hysteresis loops. Also, the comparison between FC and ZFC hysteresis loops has been used to cross-check the shift in the field axis. At low temperature, only OOP loops were recorded. Magnetic moment versus temperature was also measured with the magnetic field in the perpendicular direction.In-plane and OOP hysteresis loops were performed in a vibrating sample magnetometer (VSM) from LakeShore 7304 at room temperature. In the sample plane, we measured loops at different angles between the applied magnetic field and the in-plane reference direction. As a reference direction, we considered as 0° the beam incidence direction in the sample plane2O3, FeGa or Mo  the main diffraction peak19. For Fe72Ga28 we can determine the lattice parameter (a) thanks to Bragg’s law:α in this case). In the cubic system, we can obtain a from dhkl since:h, k, and l are the Miller indexes of the family of planes. From experimental results for the (110) peak we have determined a lattice parameter of 2.90 Å, in agreement with previous works18.In the XRD measurements we have only observed diffractions peaks related to either Cr Mo Fig. a. We hav2O3 show no variations upon the deposition of Fe72Ga28 on top of it as it can be inferred from the comparison with a single layer of Cr2O3 deposited in the same growth conditions  and therefore, it has a rhombohedral structure with lattice parameters a = b = 4.95 Å and c = 13.56 Å. Cr2O3 layers are not fully c-oriented since (012), (104) and (116) diffraction peaks for Cr2O3 have been detected by XRD. However, it is expected a magnetic contribution along the perpendicular direction due to these family of planes. The Nèel temperature (TN) of the Cr2O3 has been obtained from the measurement of the magnetization as a function temperature  defined as the ratio between the remanence (Mr) and the maximum magnetization (Mmax) obtained in the perpendicular hysteresis loops. In Table 72Ga28 thickness is reduced, mostly for a thickness below 80 nm. The OOP squareness and hysteresis loops of coupled layers can be compared with those of a Fe72Ga28 thin film grown in similar conditions  sample to show the magnetization direction has been turned into the perpendicular direction becoming the OOP direction an easy axis.The VSM hysteresis loops reveal the change of the magnetization direction from in-plane to OOP as the Feced Fig. b. We canns Table ; Fig. 3cKFM, can be experimentally inferred taking into account that:MFM the saturation magnetization of the Fe72Ga28, and HK the anisotropy field in the hard direction. For the samples with Fe72Ga28 thickness of 20 nm and 40 nm the in-plane is a hard axis, and we have obtained an average value of KFM of 3·106 erg cm−3 :Aex is the exchange energy per unit length. When Q > 0.1, stripe domains are wider than the layer thickness, whereas they exhibit a periodicity equals to the layer thickness if Q < 0.1.For materials with moderate or low PMA, KFM = 3·106 erg/cm3, and MFM = 1100 emu cm−326, it is inferred a Q of 0.3 in our samples and therefore, stripes are expected above a critical thickness. Considering Aex = 1.7·10−6 erg cm−1 from the literature40, and using Eq.  at 5 K after a FC process at 1 kOe  is higher than the theoretical value of Cr2O3 (KAF = 2·105 erg·cm−3)42, and some chemical roughness is expected at the Fe72Ga28/Cr2O3 interface, we have used the random field model proposed by Malozemoff37 to calculate the theoretical HE values. In this random field model, the AF layer breaks into domains, and the exchange-bias field is obtained by means of the expression:z is generally taken as the unity, and AAF is the exchange stiffness of the antiferromagnet that takes a value of 4·10−7 erg cm−142. With this expression  and a ferromagnet (Fe72Ga28) with the magnetization in the sample plane but with a fluctuation that produces magnetic ripple. First of all, the in-plane magnetic anisotropy of Fe72Ga28 is completely vanished due to the coupling. Secondly, stripe domains are promoted due to the enhancement of the OOP component of the Fe72Ga28 magnetization. We have observed that the magnetization direction of Fe72Ga28 is gradually turned from in-plane to the OOP direction as the Fe72Ga28 thickness is reduced A perpendicular magnetic anisotropy of 3·106 erg·cm−3 has been inferred from experimental results in the Fe72Ga28 layers. It has also been observed exchange-bias phenomena in the perpendicular direction fitting the experimental data to the random field model.In summary, we have studied the effect of the interfacial coupling between an antiferromagnet with magnetic moments along the Supplementary Figure 1.


The formation of this BST2‐orf7a complex destabilizes BST2 dimerization, thus impairing the antiviral activity of the latter.We present in this work a first X‐ray Absorption Spectroscopy study of the interactions of Zn with human BST2/tetherin and SARS‐CoV‐2 orf7a proteins as well as with some of their complexes. The analysis of the XANES region of the measured spectra shows that Zn binds to BST2, as well as to orf7a, thus resulting in the formation of BST2‐orf7a complexes. This structural information confirms the the conjecture, recently put forward by some of the present Authors, according to which the accessory orf7a  viral protein are capable of interfering with the BST2 antiviral activity. Our explanation for this behavior is that, when BST2 gets in contact with Zn bound to the orf7a Cys Think zinc! We conjecture that in the case of SARS‐CoV‐2, the orf7a accessory protein acts as a BST2 antagonist. We provide evidence, based on sequence analysis, Molecular Dynamics simulations as well as XAS experiments, that Zn ions play a key role in the SARS‐CoV‐2 virus strategy to escape the immune response mediated by the BST2 host protein. In this situation, it is of the utmost importance to also continue looking for effective antiviral drugs.One important step in this direction is the identification and characterization of the human proteomes interacting with the SARS‐CoV‐2 proteins. Insert the note „For a review describing the whole set of SARS‐CoV‐2 coded proteins see Ref. [5].” A rather complete and accurate study in this direction has been recently reportedIt is found that the percentages of Zn, Mg, Fe, Cu, and Mn metalloproteins belonging to the human proteome interacting with the SARS‐CoV‐2 proteins  are found to be 12.3 %, 3 %, 1.8 %, 0.3 % and 0.3 %, respectively, with the majority of them (about 17 %) interacting with the accessory protein SARS‐CoV‐2 orf8.In this context an interesting observationThe orf7a and orf8 proteins (of yet not fully understood function) are coded by the SARS‐CoV‐2 open reading frames ORF7a and ORF8, respectively. The orf7a protein is common to all SARS‐CoV type coronaviruses and is highly conserved,The mechanism of immobilization is mainly carried out by proteins of the tetherin family, also known as BST2 or cluster of differentiation 317 (CD317).53, Cys63, and Cys91 in human BST2) stabilize BST2 homodimerization through intermolecular disulfide bonds.Three invariant cysteine residues  is used. On the other hand, X‐ray Absorption Spectroscopy (XAS) measurements at the Zn K‐edge represents a powerful tool for investigating the near atomic environment of Zn that may allow identifying possible specific Zn‐protein binding sites.With this in mind, we have performed the first measurements of the entire XAS spectrum of the BST2 protein and the N‐terminal ectodomain of orf7a (PDB 7CI3) at the Zn K‐edge, covering both the X‐ray Absorption Near Edge Structure (XANES) and Extended X‐Ray Absorption Fine Structure (EXAFS) regions, although in the present paper we limit ourselves to comparison, analysis and theoretical calculations of the XANES region of the measured spectra, postponing the study of the EXAFS data to a forthcoming work.15 of orf7a in the Zn mediated interactions with BST2. For this reason we designed and expressed two orf7a constructs, that we call orf7aL1 (aa. 16–82) and orf7aL2 (aa. 15–82), differing for the absence and the presence of the initial Cys15. Measurements were performed on the BST2 protein and the two orf7a constructs separately as well as on samples where BST2 and orf7a are simultaneously present.Since Cys is a common Zn ligand, we considered especially important to provide experimental evidence for the role played by CysOf course it would be of the utmost importance to perform the same kind of experiments and analysis by replacing orf7a with orf8. Unfortunately, at the moment we have been unable to produce a sufficiently large amount of the purified orf8 protein, adequate for XAS measurements.The analysis of the extensive XAS measurements we have performed shows that both BST2 and orf7a are able to bind Zn. We also have indications that the BST2/orf7a complex get formed in the presence of Zn ions, supporting the hypothesis, put forward in Ref. [9], according to which Zn plays a pivotal role in helping to make BST2 available for orf7a binding and inactivation.These results, besides shedding some light on the selective role of orf7a in the development of the disease could open the way for possible therapeutic strategies against SARS‐CoV‐2 based on the modulation of cellular Zn homeostasis.7, 9Zn is an irreplaceable element in the biochemistry of living cells because of its essential role as a catalytic and structural cofactor.As we said, in this context XAS is an ideal tool to study the local Zn environment which is capable of clarifying whether and how Zn can bind to BST2 and/or orf7a.L1 (aa. 16–82) and orf7aL2 (aa. 15–82) that differ for the absence and presence of Cys15, respectively. Samples were prepared according to the following protocol.For the purpose of our XAS experiments we have prepared a number of samples containing either BST2 or the orf7a N‐terminal ectodomain, or the two together in the presence of Zn ions in different solution conditions. For the reasons explained above, we designed and expressed two orf7a constructs, orf7aE. coli strain BL21 (DE3) pLySS and expressed overnight at 18 °C after induction with 0.5 mm IPTG  in LB Broth containing kanamycin at a final concentration of 30 μg/mL. Cells were harvested by centrifugation.The pET28a(+) plasmid harbouring the BST2 wild type gene and SARS‐CoV‐2 ORF7a 15–82 or 16–82 genes were synthesized by GenScriptm Tris HCl, pH 8.0, 0.25 m NaCl, 5 mm imidazole and 1 mm Tris (2‐carboxyethyl)phosphine (TCEP)) supplemented with a cocktail of ethylene‐diaminetetraacetic acid (EDTA)‐free protease inhibitors (Sigma‐Aldrich) and lysed by sonication. The lysate was cleared by centrifugation and the soluble fraction was loaded onto a 2 mL prepacked His trap column  pre‐equilibrated with the binding buffer. The column was washed with the binding buffer to elute weakly bound contaminants and the recombinant protein was eluted by passing over the column binding buffer solutions containing 250 mm imidazole.Cells derived from BST2 expression were resuspended in a binding buffer  in presence of a cocktail of ethylenediaminetetraacetic acid (EDTA)‐free protease inhibitors (Sigma‐Aldrich). Both orf7a protein constructs were purified according to the protocol of Ref. [36] with minor modifications.Cells derived from SARS‐CoV‐2 ORF7a with 15–82 or 16–82 aa. expressions were resuspended in lysis buffer (50 mm NaCl using a molar absorptivity of 7700 m−1 cm−1 (for SARS‐CoV‐2 orf7aL1 and orf7aL2) and of 1615 m−1 cm−1 (for BST2) at 280 nm based on a molecular mass of 10051.22 Da, 9948.08 Da and 14866.61 Da, respectively.Protein concentration was determined spectrophotometrically in the buffer 20 mM Tris HCl, pH 8.0, 0.15 Purity and size of the proteins were checked by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) on a pre‐casted NuPage 4–12 % Bis‐Tris polyacrylamide gel .The purified proteins were identified by mass‐spectrometric analysis. After the denaturing SDS‐PAGE run, the proteins of interest were extracted from the gel and analyzed on an Orbitrap FusionTM TribridTM Mass Spectrometer (Thermo) following a tryptic proteolysis (data not shown).4 stock solution in the buffer 20 mm Tris HCl, pH 8.0, 0.15 M NaCl with 20 % glycerol. The resulting solutions were mixed by pipetting, injected in the sample‐holder used for XAS measurements and immediately frozen in liquid N2. Between the preparation and the analysis, samples in their sample‐holders were kept at a temperature close to 77 K, in a dry‐shipper for the shippingSamples for XAS measurements were prepared by adding Zn to each protein solution from the concentrated stock solutions prepared by diluting the ZnSOIn Table L1 and L2 appended to the name of the orf7a protein (rows 2 and 3) have been introduced to distinguish between the orf7a constructs differing by the absence (orf7aL1) or the presence (orf7aL2) of Cys15 at the beginning of the amino acid sequence. The subscript first appearing next to BST2 and orf7aL2 in the rows 4 and 5, respectively, is there to recall that in the case of the sample BST2first+orf7aL1, the orf7aL1 protein was added to an already Zn‐loaded BST2 solution, while, vice versa in the case of the sample BST2+orf7aL2 first, BST2 was added to an already Zn‐loaded orf7aL2 solution. Note that the Zn concentration has been always taken to be substoichiometric in order to minimize possible spurious XAS signals from free Zn in solution. In the row 6 the concentration of the Zn in buffer is reported.As we already said, the labels m2 . Spectra were recorded in fluorescence mode using a 13‐element solid state Ge detector. To avoid photo‐degradation and spectra evolution during XAS measurements, all the samples, held at 77 K since their preparation, were cooled down and kept at 13 K in a helium cryostat during the whole XAS measurement. For the same reason in the process of data acquisition, samples were systematically moved to a different position after each scan to prevent the beam hitting a previously irradiated spot. Scans never lasted more than 40 minutes. For each sample from 13 to 23 scans, depending on the protein concentration, were merged to obtain average spectra with an acceptable signal‐to‐noise ratio. In the case of the Zn‐buffer, owing to the much higher Zn concentration, average of only 4 scans was enough. XAS spectra were averaged and normalized using the Athena software.XAS measurements on our samples have been carried out at the BM30 beamline of the European Synchrotron Radiation Facility .Although we have measured the whole XAS spectrum (XANES and EXAFS regions) of the samples in Table 2In this section we will discuss the features of the measured XANES spectra and will present some theoretical calculations in support of our analysis.2.1first+orf7aL1 (purple curve) and BST2+orf7aL2 first (orange curve). From this comparison we can make the following observations.A) In the top panel of Figure first+orf7aL1 and BST2+orf7aL2 first. This means that BST2 as well as the other two compounds are able to bind Zn. Furthermore, we observe that in the Zn‐buffer spectrum, where the metal is in an octahedral configurationfirst+orf7aL1 and BST2+orf7aL2 first samples is in a tetrahedral geometry.The XANES spectrum of the Zn‐buffer is significantly different from the spectra of BST2, BST2first+orf7aL1 (purple curve) samples. Such a structural feature is typically attributed to the presence of Cys (and possibly His) ligands around Zn in a tetrahedral configuration.ab initio XANES calculations we present in the dedicated section confirm the occurrence of this peculiar behaviour in the spectrum of trial structures where Zn is coordinated to ligand motifs involving Cys and His residues.There is a double peak at the top of the XANES white‐line of both the BST2 (blue curve) and BST2L2 first sample (orange curve), rather a single peak is visible which, however, has a height definitely smaller than that of the Zn‐buffer white‐line again hinting to a tetracoordinated Zn.No double peak is featured in the spectrum of the BST2+orf7aL1 (red curve) and orf7aL2 (green curve) samples. From this comparison we can make the following observations.B) In the bottom panel of Figure L1 and orf7aL2 samples, indicating that Zn is bound to both orf7a constructs.The XANES spectrum of the Zn‐buffer is significantly different from the spectra of both the orf7aL2 sample shows a sort of two‐fold (or maybe three‐fold) structure which is absent in the case of orf7aL1. Consistently with what we noticed above we interpret this difference as due to the presence of the extra Cys15 as a Zn ligand in the orf7aL2 sequence which is absent in orf7aL1 .The shape of the XANES white‐line of the orf7aC) The picture emerging from the analysis described in A) and B) above is quite interesting.first+orf7aL1 (purple curve) samples should be interpreted by saying that, once BST2 is loaded with Zn, the addition of orf7aL1 does not significantly affect the Zn binding modeFirst of all, as seen from the top panel of Figure L2 first (orange curve) sample is instead appreciably• The XAS spectrum of the BST2+orf7adifferent from that of BST2 (blue curve) and, as one can see in the bottom panel ofL2 (green curve).Figure L2 XANES white‐line disappears when BST2 is added. It is tempting to interpret this behaviour by saying that, upon BST2 addition, the Cys15 residue of orf7aL2, to which Zn is supposed to be bound, is made available for the creation of a new disulfide bond with one of the unpaired Cys of the BST2 monomer.From this last comparison we conclude that the structure visible in the orf7aThe process illustrated above may be the mechanism behind the formation of the BST2/orf7a complex which, preventing the correct BST2 dimerization, impairs the BST2 antiviral ability.2.2In order to get some insight as well as quantitative information on the specific way Zn can bind to the BST2 and orf7a proteins, we have performed calculations of the XANES region of the spectrum of models of BST2‐Zn and orf7a‐Zn complexes, assuming that Zn is bound to either Cys's or His's or both in some effective Zn‐finger motif.For the purpose of this analysis we have made a search in the PDB data bankXANES calculations were performed using the FDMNES code.The theoretical spectra are expressed as function of the photo‐electron energy. To compare them with the experimental spectra and convert photo‐electron energy in incident photon energy, a constant energy value of 9659.3 eV was accounted for in defining the photo‐electron energy. This value was optimized to allow the best energy alignment of the Zn‐buffer white‐line position of both the calculated and experimental spectra. This correction was made in all the spectra.i. e. structures that have the same number of Zn‐coordinated Cys and His) do not allow to push further the interpretation of the calculations and determine exactly what is the Zn local structure.The results collected in Figure first+orf7aL1 and orf7aL1 samples  and orf7aL2 (green curve). The comparison shows that orf7a, in both its constructs (either 16–81 or 15–81), is able to bind Zn. Moreover one should notice the significant difference in the shape of the white‐line around its maximum between the green and the red curve. This difference, according to the results of our XANES calculations , should be ascribed to the presence of the extra Cys15 Zn‐ligand in orf7aL2.In the bottom panel of Figure L2  is added to an already Zn‐loaded BST2 solution (purple curve in the top panel of Figure L1 alone, as seen in the smoother shape of the red curve in the bottom panel of Figure L1 is unable to modify the Zn‐BST2 coordination mode and bind the metal, at least at the concentrations we are working at. This is consistent with the pivotal role of Cys15 as a Zn‐ligand that the interpretation of our data implies.On the contrary, in the situation where orf7aab initio XANES calculations confirm that the structure visible at the top of the white‐line, with its peaks and dips, occurs if Zn in a tetrahedral configuration is coordinated to some Cys/His motif where, together with His's, at least one Cys is present (see Figure Extensive 15 amino acid of orf7a with some of the available Cys along the BST2 chain.This work is a first step in the direction of understanding the role of Zn in the interactions between BST2 and some of the accessory SARS‐CoV‐2 proteins. We have shown that both the host BST2 protein and the viral orf7a protein are able to bind Zn. Our analysis supports the hypothesis that BST2/orf7a complexes get formed with the creation of a disulfide bond pairing the Cysab initio DFT calculations which both will be addressed.To obtain a more accurate picture of where Zn can possibly bind, one needs to extend the present study to include the structural information that can be extracted from the analysis of the EXAFS region of the spectrum. An investigation where the BST2 and orf7a EXAFS data will be employed to identify and more precisely characterize possible Zn binding sites along the two protein sequences is currently in progress.Naturally, a crucial step in the direction of elucidating the alleged inhibition of the BST2 virion immobilization ability operated by accessory proteins is to provide good XAS data also for the orf8 protein. Until today this has not been possible owing to the difficulty of producing a sufficiently large amount of purified orf8 protein. We are confident, however, that we will soon be able to overcome the problem.The authors declare no conflict of interest.


Fe@2 an unprecedentedly detailed structural model complex for this kind of non‐heme iron oxygenases. As, as confirmed by the crystal structure, sufficient space for other organic ligands is available, the biologically relevant ligand α‐ketoglutarate is implemented. We observe biomimetic reaction behavior towards dioxygen that opens studies investigating Fe@2 as a functional model complex.A conceptual approach for the synthesis of quasi‐heteroleptic complexes with properly endo‐functionalized cages as ligands is presented. The cage ligand reported here is of a covalent organic nature, it has been synthesized via a dynamic combinatorial chemistry approach, making use of a masked amine. Inspired by enzymatic active sites, the described system bears one carboxylate and two imidazole moieties as independent ligating units through which it is able to coordinate to transition metals. Analysis of the iron(II) complex in solution and the solid state validates the structure and shows that no undesired but commonly observed dimerization process takes place. The solid‐state structure shows a five‐coordinate metal center with the carboxylate bidentately bound to iron, which makes  endo‐functionalized with one carboxylic acid and two imidazole moieties. These groups can cooperate with each other to coordinate to Zn(II) and Fe(II) resulting in biomimetic quasi‐heteroleptic transition‐metal‐cage complexes. Coordination of α‐ketoglutarate to the iron complex leads to an increase of its reactivity towards dioxygen.A cage compound is synthesized that is  Synthetic supramolecular chemistry received wide attention during the past decades. Beside the diverse applications of supramolecular systems, such as new catalysts or as molecular machines, the synthesis of new motifs and topologies continuously steers the field towards new endeavors.(SbF6)2 and [Fe(MeCN)6](SbF6)2 in [D8]THF and subsequent treatment with triethylamine. To ensure absence of metal salt traces in the follow up chemistry, a modified procedure with understoichiometric use and separation of Fe@2 from the free ligand is employed in the case of iron. The IR absorption at 1710 cm−1 that can be assigned to the carbonyl stretching of the carboxylate of 2 is not found anymore in any case and is presumably shifted into the finger print area of the spectrum (see ESI). Such behavior is indicative for carboxylate coordination to a metal. By high resolution ESI‐MS, [C102H109N10O2Zn] (Zn@2) and [C102H109N10O2Fe] (Fe@2) could be confirmed (see figures S27/S35).With cage 1H, 1H DOSY and 13C NMR spectroscopy in [D8]THF. Comparison of the 1H and 13C NMR spectra of 2 and Zn@2 confirms coordination of the functional residues as we observe a shift for both the imidazole signals and the propylene linker (see ESI for NMR spectra). In the 13C NMR of Zn@2 we see broadening of the propylene linker signals, the carboxyl carbon resonance could not be detected. We attribute this behavior to a limited flexibility upon zinc coordination. 1H DOSY NMR reveals an enhancement of the hydrodynamic radius to 10.6 Å, which is expected due to the ionic nature of Zn@2.The complexes were analyzed by 1H NMR spectrum of Fe@2  is found via DOSY NMR @2Fe@2Fe. Instead, oxygen activation in iron oxygenases only takes place after cofactor and/or substrate binding, for instance after binding of α‐ketoglutarate (αKG) in α‐ketoglutarate‐dependent iron oxygenases. Inspired by this, we examined the reactivity of Fe@2 towards dioxygen in the presence of αKG. Coordination to Fe@2 is confirmed by a spectral change in NMR and via UV/Vis spectroscopy, where upon addition of αKG we observe a feature at 485 nm that is in the range of FeII t2g to αKG π* MLCT transitions observed for αKG‐bound enzymatic FeII sites.In agreement with the reaction mechanisms found for substrate oxidation in iron oxygenases, where O102H109N10O2Fe(C5H5O5)] is found in high resolution ESI‐MS, but besides further adducts.As an additional indication of the αKG complex being formed, [C2 leads to a direct spectral change in UV/Vis even at low temperatures  and iron(II), resulting in the formation of the quasi‐heteroleptic cage‐complexes Zn@2 and Fe@2. Solution and solid‐state investigation show that these are monomeric structures and no undesired dimers are obtained. X‐ray analysis shows that additional coordination of NEt3 to Fe@2 is possible and that the carboxylate unit coordinates in an unsymmetrically bidentate fashion reminiscent of for example the active site of naphthalene dioxygenase. Finally, we found Fe@2 not only being a structural but also a functional model of enzymatic active sites as its reactivity towards oxygen is strongly enhanced in the presence of bound α‐ketoglutarate. Overall, the reported concept of applying cages of lowered symmetry as ligands may lead to huge varieties of this class of metal complexes, whose properties could selectively be tuned by derivatization of the used building blocks. The accessibility of the introduced compound class can especially be of interest for the design of biomimetic complexes as many metalloproteins feature a heteroleptic coordination environment within the active site pocket.In summary, we have reported the stepwise synthesis of the endo‐functionalized cage ligand 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.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


The Y2O2SO4 powder was successfully synthesized by decomposition of Y2(SO4)3 at high temperature. According to the electronic band structure calculations, yttrium oxysulfate is a dielectric material. The difference between the oxygen–sulfur and oxygen–yttrium bond nature in Y2O2OS4 was shown based on partial density of states calculations. Vibrational modes of sulfur ions and [Y2O22+] chains were obtained theoretically and corresponding spectral lines observed in experimental Infrared and Raman spectra.The traditional way for determination of molecular groups structure in crystals is the X-Ray diffraction analysis and it is based on an estimation of the interatomic distances. Here, we report the analysis of structural units in Y The rising in experimental and theoretical studies of rare-earth-activated phosphors over the past decades is primarily associated with their applications in lighting, electronic displays, temperature sensing, etc. . A largeRe3+) ions, the common way for doping is a partial substitution of Re3+ with Ln3+  ions. Recently, Re2O2SO4 oxysulfate was studied as a host for optical materials and it has been shown that the luminescent efficiency of Re2O2SO4:Ln3+ phosphorous depends on the size and shape of particles 2− and [Y2O22+] structural units.In this paper, we report the synthesis of Y2(SO4)3  in an argon atmosphere at a temperature of 700 °C. A schematic of an installation for carrying out high temperature decomposition processes is shown in 2(SO4)3 was placed in a quartz reactor and purged with argon for 30 min at a rate of 6 L/h. After that, the reactor was placed in a heated vertical furnace and held for 10 h. After the completion of the reduction process, the reactor was removed from the furnace and cooled to room temperature. The decomposition recovery process is described by the equation:Yttrium oxysulfate was obtained by decomposition of yttrium sulfate Y−1. Raman spectrum was recorded using an i-Raman Plus spectrometer at a laser excitation wavelength of 785 nm and the spectral resolution was about 4 cm−1. The Infrared as well as the Raman spectrum was obtained at room temperature.Fourier-transformed Infrared spectroscopy (IR) was carried out with the use of a Fourier Transform Infrared Spectrometer FSM 1201, . The sample for the investigation was prepared in a tablet form with addition of annealed KBr. IR spectrum was recorded with spectral resolution 4 cm24p64d15s2, 3s23p4 and 2s22p4 valence electron configurations were used for Y, S and O, respectively. The local density approximation (LDA) based on the Perdew and Zunger parametrization 2− ion with dT symmetry, its site symmetry and the factor group symmetry of the monoclinic cell are presented in 4 should be observed in Raman as in Infrared spectra. The Infrared and Raman spectra of Y2O2SO4 are presented in The Y−1 in the Infrared spectrum and strongest band at 1009 cm−1 in the Raman spectrum are associated with ν1 symmetric stretching vibrations of SO4 groups. Spectral bands above 1000 cm−1 are antisymmetric stretching vibrations (ν3). Bands at 650 and 604 cm−1 in Raman spectrum are ν4 antisymmetric bending of SO4 tetrahedra. The ν4 modes appeared in the Infrared spectrum at 666, 621 and 608 cm−1. Spectral lines in Raman spectrum at 448 and 432 cm−1 are defined as ν2 symmetric bending of sulfur ions. The rotational vibration of SO4 appeared in Raman spectrum as the peak at 195 cm−1. The Raman peak at 168 cm−1 is explained as translations of SO4 in the SO4 structural layer.The weak spectral band at 969 cm4 tetrahedra  with a calculated wavenumber value equal to 546.7 cm−1 is a combination of ν2 vibrations of SO4 and O-O stretching, as shown in uB mode at 499.1 cm−1 is a translation of O, as shown in −1 in Infrared spectra is devoted to oxygen vibration in [Y2O22+] chains, but not to Y-O vibrations as was stated earlier  structural units. Calculated partial density of states shows different electron distribution for s and p orbitals in case of oxygen atoms in [SO4]2− and in case of [Y2O22+]. The formation of hybrid sp orbital in yttrium–oxygen chains is supposed. The electronic structure and band gap value of yttrium oxysulfate was presented for the first time.In summary, we have demonstrated that the lines in vibrational spectra of Y