This study investigated the potential of vitamin K1 as a novel lens aldose reductase inhibitor in a streptozotocin-induced diabetic cataract model. A single, intraperitoneal injection of streptozotocin (STZ) (35 mg/kg) resulted in hyperglycemia, activation of lens aldose reductase 2 (ALR2) and accumulation of sorbitol in eye lens which could have contributed to diabetic cataract formation. However, when diabetic rats were treated with vitamin K1  it resulted in lowering of blood glucose and inhibition of lens aldose reductase activity because of which there was a corresponding decrease in lens sorbitol accumulation. These results suggest that vitamin K1 is a potent inhibitor of lens aldose reductase enzyme and we made an attempt to understand the nature of this inhibition using crude lens homogenate as well as recombinant human aldose reductase enzyme. Our results from protein docking and spectrofluorimetric analyses clearly show that vitamin K1 is a potent inhibitor of ALR2 and this inhibition is primarily mediated by the blockage of DL-glyceraldehyde binding to ALR2. At the same time docking also suggests that vitamin K1 overlaps at the NADPH binding site of ALR2, which probably shows that vitamin K1 could possibly bind both these sites in the enzyme. Another deduction that we can derive from the experiments performed with pure protein is that ALR2 has three levels of affinity, first for NADPH, second for vitamin K1 and third for the substrate DL-glyceraldehyde. This was evident based on the dose-dependency experiments performed with both NADPH and DL-glyceraldehyde. Overall, our study shows the potential of vitamin K1 as an ALR2 inhibitor which primarily blocks enzyme activity by inhibiting substrate interaction of the enzyme. Further structural studies are needed to fully comprehend the exact nature of binding and inhibition of ALR2 by vitamin K1 that could open up possibilities of its therapeutic application. It is thus clear that sorbitol and fructose formation could lead to the breakdown of normal lens physiology, triggering cataract formation. A lot of studies thus have suggested that the inhibitors of aldose reductase could have the ability to limit certain diabetic complications4. Many aldose reductase inhibitors (ARI), both synthetic and natural, have been tested in animal models. Some of the natural ARIs include, ethyl acetate extract from leaf of Aegle marmelos5, glucogallin from Embilica officinalis6, flower bud extract from Magnolia fargesii7, roots of Pueraria lobata8, extracts of Litchi chinensis9, curcumin10, Zingiber officinale12, while some of the synthetic ARIs include zopolrestat16, tolrestat19, sorbinil20 and fidarestat21. However, though synthetic ARIs have gained prominence their efficacy in terms of ALR inhibition in diabetic complications, especially in neuropathy, has not been satisfactory25. Our focus is to identify natural compounds with potential ARI properties. In our recent study, preliminary findings suggested that vitamin K1 could possess potent lens ARI activity and this was also supported by a reduction in lens sorbitol accumulation26. To further explore the potential of vitamin K1 as an effective ARI we compared it with zopolrestat, a standard ARI, and tried to understand the mechanism of action of AR inhibiting activity of vitamin K1.Aldose reductase inhibition is one of the best strategies to inhibit polyol pathway and its associated complications in diabetes. Sorbitol is the first product of the pathway and is further broken down to fructose and fructose by itself leads to production of free radicals. One major consequence of sorbitol formation is that it can accumulate within lens cells resulting in osmotic stress27. Studies with zopolrestat have been performed in normal and diabetic rats28 and healthy male volunteers29 and it has shown 85% absorption30 without major side effects. Though there are reports on increased half-life of zopolrestat in certain tissues like nerve, eye and kidney28, it has been shown to be relatively safe and also partially improved functional abnormalities in left ventricles of diabetic patients31. Nevertheless, a more recent report by Bouguerra et al.32 has demonstrated eryptosis triggered by zopolrestat at relevant in vivo dosages, which could be an important point to consider during its usage.Zopolrestat is an acetic acid derivative and is known to inhibit AR activity, thereby controlling the level of sorbitol, fructose and myo-ionositol in lens, retina, kidney and sciatic nerves of diabetic rats33, insulin sensitivity and modulating glycemic status36, prevention of bone loss in diabetic patients37, blocking insulin resistance38, inhibiting production of inflammatory cytokines associated with insulin response39 and modulating dephospho uncarboxylated protein40.As far as vitamin K is concerned, studies have shown the importance of its members in controlling both diabetes and its associated complications. In diabetes, vitamin K2 has been shown to be important for acute insulin response43. Studies have also shown that when men with lower phylloquinone were tested for oral glucose tolerance there was decreased insulin and an increase in the glucose level when compared to men with higher phylloquinone level that showed an increase in insulin secretion and decreased blood glucose44. More recently, studies on isolated mouse islets have shown that menaquinone-4 potentiated glucose induced insulin secretion suggesting an insulinotrophic potential for vitamin K family45. Indeed, one previous study has also shown that circulating levels of vitamin K1 is inversely proportional to risk of type 2 diabetes in humans46.Even though the exact mechanism as to how vitamin K1 improves insulin sensitivity and reduces glycemic status in type 2 diabetes remains unclear, studies have proposed that osteocalcin plays a crucial role in glucose metabolism by increasing the secretion of insulin, enhancing β islet cell proliferation and by increasing the expression of adiponectin in adipocytes, thereby increasing insulin sensitivity47 and thus inhibit type 1 diabetes-induced cataractogenesis26. In addition to this, if vitamin K1 could prove to be an ARI, then this would be a unique compound unlike other ARI, wherein it has two important activities, (1) anti-hyperglycemic and (2) ARI. Thus, in this study we probed the mechanisms of aldose reductase inhibition by vitamin K1 and compared it with a synthetic aldose reductase inhibitor zopolrestat.In addition to these, our studies with vitamin K1 have shown that vitamin K1 could prevent hyperglycemia by protecting pancreatic isletsStreptozotocin, β-NADH, thiobarbituric acid (TBA), sorbitol dehydrogenase and DL- glyceraldehyde were procured from Sigma Aldrich. Zopolrestat was gifted by Dr. Ravichandran Ramasamy, NYU Langone Medical Center, New York and human recombinant aldose reductase  was a generous gift from Dr. Srinivasan Vedantham, SASTRA University.Male albino Wistar rats (200–225 g) were procured from Central Animal Facility, SASTRA University, Thanjavur. All experiments were approved by Institutional Animal Ethics Committee (IAEC), Central Animal Facility, SASTRA University (125/SASTRA/IAEC/RPP). All experiments were carried out in accordance with CPCSEA  guidelines and regulations. Rats were housed in an air-conditioned room (22 ± 2 °C) with a lighting schedule of 12 h light: 12 h dark and fed with commercial rat diet from Provimi Animal Nutrition, India, Pvt. Ltd.47. After 72 h, blood from tail vein was withdrawn and glucose estimated using Microdot glucometer. Rats with glucose levels < 220 mg/dL were excluded from the experiment and the remaining rats were randomized into two groups of seven animals each.Animals were acclimatized to laboratory conditions for 1 wk. STZ was administered after 4 h of fasting as a single, intraperitoneal injection (35 mg/kg) in sterile citrate buffer Group 1: controlGroup 2: STZ47.Group 3: STZ plus vitamin K1. Vitamin K1  was administered 72 h after STZ injection twice a week for 3 months. Vitamin K1 is not toxic and the dosage used here is based on our previous studies13,) was administered 72 h after STZ injection twice a week for 3 months.Group 4: STZ plus zopolrestat. Zopolrestat  at 4 °C to obtain a 10% homogenate. The lens homogenate was centrifuged at 11,000 × g for 30 min and the supernatant obtained was stored at −70 °C until its use for further biochemical analysis. All the assays were carried out within 48 hours after sample collection.26. Lens aldose reductase (ALR2) activity was estimated according to47. Briefly, in a test tube the following were mixed together; 100 mL each of 50 mM potassium phosphate buffer (pH 6.2), 0.4 M lithium sulphate, 5 mm 2-mercaptoethanol, and 10 mM of DL-glyceraldehyde. After incubation , reaction was initiated with the addition of 100 mL of 0.1 mM NADPH and 100 mL lens tissue homogenate (enzyme source). Optical density was measured at 340 nm and AR activity expressed as nmol/min/mg protein.Sorbitol in lens was estimated as per our previous reportIn this, we made an attempt to understand the nature of ALR2 inhibition by vitamin K1 under various experimental conditions.100 µl of the control eye lens homogenate was incubated first with 1.5 mg of vitamin K1 for 5 min. 1.5 mg is the average dosage of vitamin K1 administered to each rat when measured at a concentration of 5 mg/kg bw. After incubation, 100 µl of 50 mM potassium phosphate buffer (pH 6.2), 100 µl of 0.4 M lithium sulphate, 100 µl of 5 mM 2-mercaptoethanol and 100 µl of 10 mM of DL-glyceraldehyde was added and further incubated at 37 °C for 2 min and the reaction was initiated with the addition of 100 µl of 0.1 mM NADPH. Optical density was measured at 340 nm against a blank containing buffer instead of the homogenate. ALR2 activity was expressed as nmoles of NADPH oxidized/min/mg protein. For co-incubation studies, 100 µl of the control eye lens homogenate was incubated for 5 min with 1.5 mg of vitamin K1 along with reagents for ALR2 measurement as mentioned above. At the end of incubation, the reaction was initiated with the addition of 100 µl of 0.1 mM NADPH and ALR activity measured as described above.100 µl of control eye lens homogenate was pre-incubated with 1.5 mg of vitamin K1 for different time points . At the end of incubation, lens ALR2 activity was measured as mentioned above.100 µl of control eye lens homogenate was pre-incubated with different concentrations of vitamin K1  and incubated for 5 min. At the end of incubation, lens ALR2 activity was measured as mentioned above.1 µg of recombinant human aldose reductase was pre-incubated for 5 min with 0.74 nM vitamin K1. After incubation, 50 µl each of 50 mM potassium phosphate buffer (pH 6.2), 0.4 M lithium sulphate, 5 mM 2-mercaptoethanol and 10 mM DL-glyceraldehyde were added and incubated at 37 °C for 2 min. The reaction was initiated with the addition of 50 µl of NADPH . ALR activity was measured as described above.1 µg of recombinant human aldose reductase was pre-incubated with 0.74 nM of vitamin K1 for 5 min or the recombinant human ALR was added directly to a reaction mixture (without vitamin K1) containing 50 µl each of 50 mM potassium phosphate buffer (pH 6.2), 0.4 M lithium sulphate, 5 mM 2-mercaptoethanol and DL-glyceraldehyde  and incubated at 37 °C for 2 min. ALR activity was measured as mentioned above.1 µg of recombinant human aldose reductase was incubated for 5 min with vitamin K1 . After incubation, ALR activity was measured as mentioned above.48. After incubation, ALR activity was measured as mentioned above.1 µg of recombinant human aldose reductase was incubated for 5 min with zopolrestat . Zopolrestat concentration was based on the work of Nakano and Petrash50. Further, protein preparation wizard from Schrödinger was used to remove non-protein atoms, water molecules from the PDB files and to correct the structural defects in raw state such as missing atoms and residues51. All the visualizations of molecular structures were performed with PyMOL 52. The chemical structure of vitamin K1 was drawn using ACD/Chemsketch and saved in mol2 file format. LigPrep was used from Schrödinger software to take 2D structures and produce the corresponding low-energy 3D structures with correct chiralities53. Minimization was performed using OPLS_2005 force field and Epik ionizer54 at the standard pH of 7 and with maximum number of conformers per structure as 1000 with RMSD 1.0 Å.To perform molecular docking between aldose reductase (ALR2)/aldehyde reductase (ALR1) and vitamin K1, three dimensional structures were obtained from RCSB Protein Data Bank , which are solved at 0.85 Å and 2.48 Å resolution, respectively55, for the molecular docking studies. Glide uses a hierarchical series of filters to search for possible locations of the ligand in the active-site region of the receptor. Further, it exhibits excellent docking accuracy and high enrichment across a diverse range of receptor types. Since the active site of ALR1/ALR2 are highly conserved, based on various co-factors bound ALR structures, the binding site residues are identified which includes Thr19, Trp20, Lys21, Asp43, Tyr48, His110, Ser159, Asn160, Ser210, Ser214, Lys262 and Asn27256. Further, receptor grid generation program56 was used to define the volume of grid box set to centroid of the active site residues and the number of docking runs was set as 1000. In the present study, we have used the Extra precision (XP) scoring function for molecular docking, as it uses only good ligand poses to perform docking and it gives better result than the SP (standard-precision) scoring function57. The predicted protein ligand complexes were optimized and ranked according to the empirical docking scoring function, calculated using the following equation:In the present study, we have used Glide software (Grid-based Ligand Docking with Energetics), available in Schrödinger suite58 with charmm27 force field59 for all amino acid residues. The ligand topologies were generated using SwissParam online server60. All the MD simulations were done at 300 K for 50 ns at the in-house facility, 11 TF High-Performance Computing cluster. The protein was completely solvated in a cubic box containing TIP3P water model61 and for setting up the MD system for simulation we followed the same protocols as used in our earlier work62. The protein-ligand binding free energy (ΔG) was calculated from the MD trajectories using the software program myPresto version 5 (https://www.mypresto5.jp/en/)63.Molecular dynamics (MD) simulations for the docked complexes of ALR2 and ALR1 bound with Vitamin K1 were performed using GROMACS 5.1.2 packageSpectrofluorimetric analysis was performed to analyze the change in the secondary structure of the recombinant human ALR2 due to binding of vitamin K1, DL-glyceraldehyde, NADPH or zopolrestat. The reaction mixture consisted of varying combinations of 30 µl each of human recombinant ALR2, vitamin K1, DL-glyceraldehyde, NADPH or zopolrestat, made up to 150 µl with 50 mM potassium phosphate buffer (pH 6.2) so that the final concentration was 1 µg/ml of recombinant human ALR2, 0.74 nM of vitamin K1, 10 mM of DL-glyceraldehyde, 0.1 mM of NADPH and 0.74 nM of zopolrestat. The fluorescence was obtained by exciting at 280 nm and scanned between 300–500 nm in a Jasco spectrofluorometer FP- 8200.et al.64.Protein from the eye lens homogenate was estimated according to Lowry p < 0.05) was followed by Tukey’s post hoc test to evaluate the significant difference between STZ + vit K1, STZ and between STZ + zopolrestat, STZ.All enzyme assays and spectrofluorometry studies were performed in duplicates. Results are expressed as mean ± S.D of at least seven determinations. One-way analysis of variance  and till the end of the study at 90 days . Whereas in STZ + vit K1 group, due to vitamin K1 administration there was a gradual decrease in blood glucose level at the end of first month  till the end of the study at 90 days . However, as expected in STZ + zopolrestat group, the blood glucose level at the end of the first month were observed to be increased  when compared to control (92 ± 5.21 mg/dL) and blood glucose levels remained high till the end of the study at 90 days . We would like to point out that the observed glucose levels in the case of zopolrestat treated diabetic rats was nevertheless still lower than STZ alone administered (group II) rats, at the end of 90 days. This has not been demonstrated with previous reports65 on zopolrestat and we do not have a clear explanation other than speculate on the strain of animal used. However we would like to emphasise here that in this study animals with blood glucose more than 220 mg/dL were considered to be diabetic/hyperglycemic and thus this also falls into that category, notwithstanding the lower levels when compared to STZ alone rats. These results suggest that vitamin K1 had more beneficial effect in terms of decrease in blood glucose level in diabetic rats, when compared to zopolrestat and is very effective in inhibiting hyperglycemia, which could contribute to its anti-diabetic function.As shown in Fig. p < 0.05) due to hyperglycemia. As expected treatment of diabetic rats with zopolrestat, a known inhibitor of ALR2, resulted in a significant inhibition of lens ALR2 activity , when compared to untreated diabetic rats. It should be mentioned here that we got an inhibition of around 59% with zopolrestat whereas in one of the earlier studies, the authors have shown greater inhibition at much lower concentrations of zopolrestat66. This could be attributed to differences in experimental conditions. However, when compared to STZ group, there was a significant reduction. Similarly, treatment of diabetic rats with vitamin K1 also resulted in a significant inhibition of lens ALR2 activity  when compared to untreated diabetic rats. As shown in Fig. p < 0.05) in STZ-induced rats as compared with the controls (650.32 ± 136.21 nmoles/ g lens). As expected, treatment of diabetic rats with zopolrestat  or with vitamin K1 (895.23 ± 136.21 nmoles/ g lens) resulted in significant decrease in sorbitol accumulation, when compared to untreated diabetic rats. These results suggest that by inhibiting ALR2, both zopolrestat and vitamin K1 were able to inhibit sorbitol accumulation in diabetic lens.Activation of polyol pathway is a major factor in diabetic complications especially in diabetic cataract. Aldose reductase is the first and rate limiting enzyme of the polyol pathway to be activated by hyperglycemia and we compared the ALR2 inhibiting potential of vitamin K1 with zopolrestat. The activity of aldose reductase .(i) In the first of these experiments, we made an attempt to understand the effect of pre- or co-incubation of vitamin K1 on lens ALR2 activity. As shown in Fig. p < 0.05), when compared to control (9.4 ± 0.04 nM of NADPH oxidized/min/mg protein). At 10 min  and 15 min , the activity was lower than control but higher than that obtained at 5 min pre–incubation. Beyond 15 min, increase in pre-incubation time resulted in rescue of lens ALR2 activity and the activity at 60 min pre-incubation (9.36 ± 0.06 nM NADPH oxidized/min/mg protein) was similar to control lens ALR2 activity.(ii) Next, we analyzed the effect of pre–incubation time of vitamin K1 with lens homogenate on lens ALR2 activity. For this, vitamin K1 was incubated with lens homogenate for 1, 5, 10, 15, 30, 45 and 60 min and then ALR2 activity was assayed. As shown in Fig. p < 0.05) resulted in significant inhibition in lens ALR2 activity when compared to control lens. However, maximum inhibition was observed at 0.74 nM  and this was followed by 33.2 pM  and 0.664 nM . At very high concentrations, vitamin K1 failed to inhibit control lens ALR2 activity . Hence, 0.74 nM appears to be the optimum concentration required to produce maximum inhibition of lens ALR2 activity. Thus, for all further studies the following parameters were used: vitamin K1 concentration = 0.74 nM and incubation time = 5 min.(iii) Next, we determined the effect of increase in vitamin K1 concentration on lens aldose reductase activity. For this, we tested vitamin K1 at the following concentrations: 0.0664 fM, 1.328 pM, 3.32 pM, 33.20 pM, 0.664 nM, 3.32 nM, 3.32 µM, 0.0332 M and 166 M. In addition, we also included 0.74 nM, which is the actual concentration of vitamin K1 measured in two lenses from diabetic animals that were treated with 5 mg/kg bw vitamin K1 twice a week for 90 days. Control lens were pre-incubated with vitamin K1 for 5 min and then ALR2 activity was assayed. As can be seen in Fig. Having established the potential of vitamin K1 in inhibiting ALR2 activity in control lens homogenate, we made an attempt to understand the effectiveness of viatmin K1 in interacting with and inhibiting recombinant human aldose reductase enzyme (ALR2) to validate our findings with lens homogenate.(i) To determine whether an increase in substarte concentration could rescue recombinant human ALR2 activity inhibition by vitamin K1, we analysed ALR2 activity in the presence of increasing concentrations of DL-glyceraldehyde  and in the presence or absence of 0.74 nM of vitamin K1. As shown in Fig. (ii) Next we determined whether increase in the concentration of co-factor NADPH, could rescue recombinant human ALR2 activity. For this, human ALR2 was incubated with different concentrations of NADPH  in the presence or absence of 0.74 nM vitamin K1. As shown in Fig. (iii) To check the effect of increase in vitamin K1 concentration on recombinant human ALR2 activity, the enzyme was incubated for 5 min with different concentrations of vitamin K1 . The reaction contained 10 mM of DL-glyceraldehyde and 0.1 mM of NADPH. As shown in Fig. 29. As shown in Fig. (iv) To further validate the ALR2 inhibitory potential of vitamin K1, we estimated the activity of recombinant human ALR2 in the presence of different concentrations of zopolrestat, a well known ALR inhibitor. The concentrations used were 0, 0.74, 72, 144, 216 and 719 nMTaken together the results on the nature of inhibition of recombinant human ALR2 by vitamin K1, suggests that vitamin K1 has a very good potential in interacting with and subsequently inhibiting the aldose reductase enzyme. Together with the earlier findings on lens ALR2 activity, we show the potential of vitamin K1 as a novel and potent ALR inhibitor.The molecular simulation and docking was performed on human aldose reductase (ALR2) and aldehyde reductase (ALR1) molecules as described in the methods section. Though the amino acid sequence similarity between ALR1 and ALR2 is 50%, the overall three-dimensional structures are highly similar and the R.M.S. deviation between ALR1 and ALR2 was found as 0.76 Å. The binding mode of vitamin K1in the NADPH binding site is highly comparable in both ALR1 and ALR2 structures. Interestingly the present docking study clearly displays the location of vitamin K1, which partially overlaps the NADPH binding site and thus hinders the NADPH availability for ALR. The docking glide XP score for vitamin K1 with ALR1 and ALR2 were found to be −5.013 kcal/mol and −8.869 kcal/mol, respectively with corresponding glide Emodel scores of −50.1 kcal/mol and −75.8 kcal/mol. Emodel scores indicates the estimated conformational energy of the ligand. Further, Molecular dynamics simulation data for the docked conformations of ALR1 and ALR2 with vitamin K1 was generated for 50 ns. The compactness of the protein fold was ensured by monitoring time evolution of radius of gyration of the complexes, root-mean-square deviation (RMSD) and Root Mean Square Fluctuation (RMSF). Interestingly, we observed that the binding mode of vitamin K1 in ALR is consistent during the entire 50 ns simulation  and the side chain conformation of Trp 220, which is sterically incompatible to the naphthoquinone moiety Fig. . In contSpectrofluorimetric analysis was perfromed inorder to confirm the binding of vitamin K1 in ALR active site. Spectra for recombinant aldose redcutase was performed by exciting at 280 nm and scanning from 300–500 nm 10, whereas the present study shows the possibility of utilising vitamin K1 as a common inhibitor for both ALR1 and ALR2. Interestingly, the binding site residues are similar for both vitamin K1 and curcumin , except the hydrophobic tail’s conformation of vitamin K1. This clearly indicates the importance of blocking the NADPH binding site to inhibit both ALR1 and ALR2, irrespective of the replacements found at Leu300 and Leu301.This observation is in contrast to the earlier To understand the nature of interaction and inhibition of lens ALR2 by vitamin K1, we performed a series of experiments under different conditions using lens homogenate from non-diabetic healthy animals. Results from vitamin K1 concentration in eye lens of vitamin K1 administered rats and dose-dependency suggested that 0.74 nM could be the optimum dosage for effective ALR2 inhibition by vitamin K1. It should be noted here that increase in vitamin K1 concentration, over and above 0.74 nM, failed to inhibit ALR2 activity over that observed with 0.74 nM. This we speculate could be due to the saturation of binding site between vitamin K1 and ALR2, which could have resulted in no significant change in ALR2 inhibition with increasing concentrations of vitamin K1. Use of vitamin K1 pre-incubation and co-incubation with control lens homogenate showed that vitamin K1 pre-incubation was more effective in inhibiting lens ALR2 activity. This suggests that prior presence of vitamin K1 is essential for it to interact effectively with the enzyme and produce inhibition. However, under co-incubation conditions, it is possible that the active sites are already occupied and vitamin K1 is unable to bind. Similarly, time for pre-incubation suggested that 5 min is the optimum. Even though inhibition of ALR2 was observed at 1, 10 and 15 min, maximum inhibition was observed at 5 min. Beyond 15 min vitamin K1 had no effect on ALR2 inhibition. The reason we speculate for no inhibition of ALR2 activity after 15 min of pre-incubation could be the loss of interaction between vitamin K1 and ALR2 after around 15 min of incubation. This we think is possible since the interaction between vitamin K1 and ALR2 is based on weak bonding. So with time this interaction becomes weaker and weaker resulting in the separation of vitamin K1 from the active site of the enzyme.22. However, when the assay was performed in the absence of vitamin K1, there was an increase in the activity of ALR2 with increase in the concentration of the substrate. Since, there was a contradictory result in the activity of ALR2 with increase in concentration of DL-glyceradehyde, assays were performed by varying the concentration of NADPH which is the cofactor for ALR. The activity of ALR2 increased with increase in concentration of NADPH even when vitamin K1 was present and these results suggests that vitamin K1 probably interferes more with substrate binding at the active site of the enzyme than with co-factor binding.To further validate the inhibitory potential of vitamin K1, recombinant human aldose reductase (ALR2) was used in inhibition assays. The ALR2 assay was performed with various concentration of the substrate, DL-glyceraldehyde, in the presence or absence of 0.74 nM vitamin K1 and with 5 min pre-incubation. The results were surprising as the human ALR2 activity was inhibited with increase in the concentration of DL-glyceraldehyde and in the presence of vitamin K1. This is in contrast to the available literature where the ALR activity was generally found to increase with increasing concentration of the substrate in the presence of an inhibitor49 clearly shows that there is no overlap in their binding sites. Interestingly the docking studies show that the binding site of vitamin K1 overlaps with the binding position of glyceraldehyde. In order to verify this in silico observation, we have performed an ALR inhibitory assay using recombinant ALR2 with 0.1 mM NADPH, 0.74 nM of vitamin K1 and with varying concentration of DL-glyceraldehyde. The assay shows that in the absence of vitamin K1, the ALR2 activity increased with increase in concentration of DL-glyceraldehyde. However, in the presence of vitamin K1, the ALR2 activity showed a decreasing trend with increase in DL-glyceradehyde concentration. This effect may be due to the non-availability of binding site for glyceraldehyde upon binding of vitamin K1.The assay clearly confirms the possibility of having overlapping binding sites of glyceraldehyde and vitamin K1 which may hinder the role of glyceraldehyde.The crystal structure of NADPH and glyceraldehyde bound formet al.10 using curcumin as ARI demonstrated that curcumin at a concentration of 100 µM was effective in inhibiting sorbitol level in RBC under high glucose concentrations. In another study, two compounds orientin and isoorientin isolated from the leaves of Colocasia esculenta were used to explore their inhibitory potential on rat eye lens ALR. Orientin was used at a concentration of 1.65 µM and isoorientin at 1.92 µM81. Of all the concentrations of vitamin K1 assayed, 0.74 nM of vitamin K1 was effective in inhibitng ALR2 activity. Though there is a difference in the nature of these molecules and perhaps on the mechanism of their interaction with ALR, when compared to other studies vitamin K1 was able to inhibit the activity of ALR2 even at very low concentrations and this was further compared with zopolrestat. Zopolrestat dosage was selected based on Nakano and Petrash48. In their work they have shown through fluorescence study that zopolrestat and substrate, DL-glyceradehyde, bind at the same site leading to inhibition of ALR activity. In the present study, inhibition of ALR2 activity was observed with increasing concentartion of zopolrestat and the inhibition pattern was similar to the inhibitory activity of vitamin K1 on human ALR. Thus, these results show that the binding of vitamin K1 in the active site of human ALR is potential enough to inhibit its activity.A study by Muthenna In order to confirm the binding of vitamin K1 in the active site of recombinant ALR2, spectrofluorimetry studies were performed. The complex of protein, substrate and the inhibitor molecules were analysed at the concentrations described in the methods section. The fluorescence intensity maxima of native protein, (without inhibitor or co-factor) and DL-glyceraldehyde was independently observed at 339–341 nm with an intensity of 1397 a.u. and 110 a.u., respectively. Whereas zopolrestat, NADPH and vitamin K1 gave an emission maxima at 356 nm (intensity 6937 a.u.), 462 nm (intensity 2906 a.u.) and 477 nm (intensity 750 a.u.), respectively. Recombinant protein along with cofactor NADPH was scanned for emission maxima. The emission maxima of the protein (339 nm) obtained is the excitation wavelength for NADPH and hence it transfers the fluorescence resonance energy to give an emission maxima at 465 nm with a fluorescence intensity of 3404 a.u. Due to high fluorescence intensity, the protein peak obtained at 339 nm was not significant.Whereas when recombinant protein along with glyceraldehyde and along with vitamin K1 was scanned separately emission maxima was obtained at 339–342 nm, which is consistent with maxima observed when all the three are complexed . Further, we repeated the fluorescence measurements with a standard inhibitor, zopolrestat by replacing vitamin K1 which shows similar fluorescence maxima. When both zopolrestat and vitamin K1 are complexed with recombinant ALR2 along with DL-glyceraldehyde, the fluorescence maxima shifts to 378 nm. This may be due to the overlapping binding site of vitamin K1 and zopolrestat as observed in the molecular docking studies Fig. .Overall the fluorescence studies confirms the binding of vitamin K1 in the active site of recombinant ALR2 which is overlapping with the binding site of zopolrestat. These results also lead us to speculate that viatmin K1 binding to ALR2 active site is more on substarte binding site than co-factor binding site. However, it is interesting to note that vitamin K1 is able to occupy both these sites which are important for the optimal activity of the enzyme. Further, structural studies are needed to fully elucidate the interaction of vitamin K1 with lens ALR2. Nevertheless, these results conclusively show that vitamin K1 indeed interacts with ALR2 and inhibits it, thus demonstrating that vitamin K1 could be a novel and potent ARI with potential clinical applications.Vitamin K1 prevents diabetic cataract by inhibiting lens aldose reductase 2 (ALR2) activity


The codoping lowers the chemical valence of W to ensure the durability of W‐based catalyst, improves the electron‐withdrawing capability of W and O to stabilize the Co and Fe in OER‐favorable high valence state, and enriches the surface hydroxyls, which act as reactive sites. The Co&Fe‐WO shows ultralow overpotential , low Tafel slope (33.7 mV dec−1), and good conductivity. This catalyst is finally applied to a photovoltaic‐water splitting system to stably produce hydrogen for 50 h at a high solar‐to‐hydrogen efficiency of 16.9%. This work highlights the impressive effect of electronic structure modulation on W‐based catalyst, and may inspire the modification of potential but unstable catalyst for solar energy conversion.Oxygen evolution electrode is a crucial component of efficient photovoltaic‐water electrolysis systems. Previous work focuses mainly on the effect of electronic structure modulation on the oxygen evolution reaction (OER) performance of 3d‐transition‐metal‐based electrocatalyst. However, high‐atomic‐number W‐based compound with complex electronic structure for versatile modulation is seldom explored because of its instability in OER‐favorable alkaline solution. Here, codoping induced electronic structure modulation generates a beneficial effect of transforming the alkaline‐labile WO The urchin‐like structure with abundant nanorods grown vertically on the surface can adequately contact with electrolyte, accelerate charge transport, and improve adsorptive separation. The codoping induced notably improved stability, enhanced conductivity, enriched surface hydroxyl groups, and enlarged electrochemical active surface area (ECSA). The reduced chemical valence of W made most sense for the persistent stability meanwhile the enriched surface hydroxyl groups and better conductivity provided the kinetic advantage for low‐overpotential OER process, which were the major points as to why the codoped catalyst showed the attractive performance. Therefore, the Co&Fe‐WO sample exhibited excellent activity  which is, to the best of our knowledge, the top‐two performance among W‐based catalysts and exceeds most of the non‐W based catalysts. This OER catalyst as a core component of the photovoltaic‐electrocatalytic water splitting device cooperated well with the silicon solar cell to operate at high current (≈50 mA) approaching the short‐circuit current. The excellent oxygen evolution ability of Co&Fe‐WO ensured the overall water splitting system to steadily perform at a low voltage (≈1.61 V) and achieved stable hydrogen output for 50 h with an average solar to hydrogen efficiency of 16.9%.In this study, Co and Fe ions were codoped into highly porous urchin‐like WO22.72 (denoted as WO) was easily prepared following a hydrothermal method reported by our group.Figure2 g−1, which is nearly twofold of that of WO (126.0 m2 g−1)  although the morphologies of them are similar. Pore size distribution  measured from BET shows that the pore size is obviously decreased after the doping , indicating that the metal ion doping is an effective method to form a highly porous structure for better adsorptive separation and thus the Co&Fe‐WO catalyst possesses large specific surface area. High‐resolution TEM (HRTEM) images show clear lattice fringes with the distance of 0.368 .Classic urchin‐like WOm KOH aqueous solution. Figure−2 and a Tafel slope of 58.5 mV dec−1 . The Tafel slope of codoped Co&Fe‐WO (33.7 mV dec−1) is also notably lower than that of WO. The performances of different ratios of Co/Fe are presented in Figure S6 (Supporting Information), and the optimal Co&Fe‐WO was tested for several times . The OER polarization curves are normalized by the BET surface area  to confirm that the intrinsic activity of Co&Fe‐WO is notably higher than other samples. For better assessment of the OER electrode, the electrochemical performance of Co&Fe‐WO was evaluated in 2‐electrode configuration. A small onset potential of 1.48 V can be observed in the LSV curve . Electrochemical impedance spectra (EIS) test was also introduced to study the electrochemical conductivity of the catalyst. The Nyquist plots , and W6+ (36.3 and 38.4 eV).6+ is gradually reduced to lower valence state during the doping process. The proportions of the peak area for the W5+ were calculated and listed in the Table S3 (Supporting Information). This value of Co&Fe‐WO (66.9%) is obviously higher than that of WO (55.3%), which further proves the decrease in the average valence of the catalyst. Judging from the W 4f spectra, the Co ion brings a stronger effect in reducing the oxidation state of W because of the more negative shift (≈0.54 eV) of the W 4f peak compared with that of the Fe‐WO (≈0.28 eV).X‐ray photoelectron spectroscopy (XPS) can show the electronic structure of the catalysts,3+ which is generally OER favorable.2+,3+ (the binding energy is 780.1 eV) is observed in Co&Fe‐WO sample  of the optimal sample was also measured, and the current density is still very stable. The initial current density is 12.05 mA cm−2, and maintained at 11.92 mA cm−2 after testing for 10 h. To the best of our knowledge, the alkaline resisting WO2 has stable oxygen evolution ability. We therefore turned instead to investigate whether the valence change accounts for the stabilization of the catalyst. On reducing the valence state of W, the WO sample was reduced by the H2 for 2 h and characterized by XRD measurement . The emerging planes of (231), (232), and (113), as well as the shoulder peak (at 23.32°) next to the strongest peak correspond well to the WO2 (JCPDS No. 82‐0728). The color of the sample became darker, indicating the valence of W decreased .2 reduced WO and the most thoroughly reduced Co‐WO (the most negative shift in W 4f XPS spectrum) catalysts do not perform the best stability, a slight increase in the potential happened during the tests. We speculated that the decrease in the oxidation state of W is not the only reason for the improvement in stability.The stability tests were performed by chronopotentiometry at the current density of 10 mA cmh Figure e. AlmostAs shown in Figure Cdl)Cdl values were obtained by calculating the slope of the lines in Figure −2) which is 3.47 times as large as that of WO (4.28 mF cm−2). The Cdls of the Co‐WO and the Fe‐WO are 26.69 and 9.73 mF cm−2, respectively. The sample with the highest Co content possesses the largest active area, but does not show the best OER activity, which may be ascribed to that the insufficient binding capacity to OH and large charge transfer resistance weaken the benefit brought by the high active surface. Subsequently, the polarization curves  normalized by the ECSA show that the Co&Fe‐WO still presents the best performance of oxygen evolution, manifesting that the enhancement in active area was not the exclusive factor for promoting the activity.We also investigated the electrochemical active surface area to further elucidate the origin of the superior performance of the Co&Fe‐WO. Normally, an increase of ECSA provides additional active area and leads to the enhancement of catalytic activity.For the constructing of W‐oxide‐based OER electrocatalyst here, the method of ion doping induced many positive effects Figure g: 1) redFigure−2), and the Co&Fe‐WO was coated on Ni foam (1.0 mg cm−2) as the oxygen evolution anode. The water splitting reactor was connected to a closed gas circulation system equipped with online gas chromatography. External silicon film solar cell was illuminated by a solar simulator  to accomplish the energy input. The light power in the range of 0.70 to 1.24‐fold AM 1.5 was supplied to the device, and the H2 yield  is nearly proportional to the light power under the relative weak light illumination. The solar‐to‐hydrogen efficiency was decreased while stronger light (≥1.12‐fold AM 1.5) was input . Here, the illumination power of 0.87‐fold AM 1.5 was selected in subsequent tests, and the maximum solar photovoltaic conversion efficiency under this power is 24.3%. The operating voltage of about 1.61 V was determined by the J–V curves of the solar cell and the electrodes . As a contrast, LSV curve of a pair of Pt sputtered Ni foam electrodes was tested in the same configuration , which shows that larger voltage is necessary for Pt/Pt electrodes to reach the same current, revealing that the as‐prepared Co&Fe‐WO/Pt electrodes are more energy‐efficient. Remarkably, a phenomenon occurred during the tests of Co&Fe‐WO/Pt electrodes that the voltage between two electrodes gradually increased due to the effect induced by the double‐layer capacitance. Surface charge was constantly accumulated, accompanied by the decreasing of the actual voltage for water splitting. Therefore, two pairs of electrodes were alternately used every 6 h in this experiment. The electrodes could be naturally discharged when not in use so that the activity of them got back close to the optimum value when used again. In this way, the stability test of 100 h was performed and both of the two pairs worked for 50 h. The average yields of H2 and O2 are shown in Figure 2 (885.8 µmol h−1) is nearly twofold of that of O2 (447.5 µmol h−1) with the initial Faradic efficiency of 96.8%. Stable solar to hydrogen (STH) conversion was achieved under the low voltage (≈1.61 V) and high current value (≈51 mA), and finally the average STH conversion efficiency is 16.9%  which was applied to the PV‐water splitting device. The 3d metals exerted an electron interacting with W to lower the valence state of W, which effectively modified the alkaline‐labile WO2.72 to be alkaline solution available. The codoping enabled Co and Fe ions to stabilize in high valence state and planted abundant surface hydroxides to not only act as reactive sites but also protect the catalyst base. The Co&Fe‐WO sample shows very low overpotential of 226 mV (J = 10 mA cm−2), small Tafel slope of 33.7 mV dec−1 and good conductivity. The PV‐water electrolysis device realized stable solar‐to‐hydrogen conversion for 50 h with the average ηSTH of 16.9%. This work propelled the exploration of W‐based OER electrocatalyst, which has complex electronic structure for versatile modulation and paved an avenue to effectively improve the stability of catalyst for efficient energy conversion applications.In summary, Co and Fe ions were homogenously codoped in the porous urchin‐like WO4Preparation of Urchin‐Like Co&Fe‐WO, Co‐WO, Fe‐WO, and WO: Anhydrous WCl6 (1.2 g), CoCl2 (0.3 g), and FeCl3 (0.3 g) were dissolved in 60 mL absolute ethanol and stirred for 20 min to form a homogeneous solution. The solution was then moved into a 100 mL Teflon‐lined stainless steel autoclave and held at 180 °C for 20 h. A yellow powder (Co and Fe codoped WO2.72) was obtained and finally washed with ethanol and distilled water alternately. This product was denoted as Co&Fe‐WO.2.72 (Co‐WO), Fe doped WO2.72 (Fe‐WO), and WO2.72 (WO) were synthesized following the similar method, except without FeCl3, CoCl2 and either of them, respectively.The contrast samples, Co doped WOCharacterizations: X‐ray diffraction patterns were recorded by X‐ray diffractometer . The morphologies of the samples were observed by the transmission electron microscope  and scanning electron microscope . Chemical valence analysis was performed by X‐ray photoelectron spectroscopy . Elemental composition of the catalysts was detected by ICP‐MS . The Brunauer–Emmett–Teller area was measured by nitrogen physisorption . The UV–vis–NIR absorption spectra of the samples were recorded via a UV–visible–NIR spectrophotometer .Electrochemical Measurements: 5 mg of catalyst powder was dispersed in a mixture of water (100 µL) and ethanol (300 µL), and then 20 µL of Nafion solution (5 wt% in water) was added to this suspension. After ultrasonic stirred for 30 min, 80 µL of the homogeneous ink was deposited onto a 1 × 1 cm2 Ni foam substrate . The Ni foam was pretreated with diluted H2SO4 solution and washed by distilled water until the pH of water reached neutral.m KOH aqueous solution. The reference electrode was a Ag/AgCl (3 m KCl) electrode, and the counter electrode was a Pt wire electrode. The linear sweep voltammetry test was performed at a scan rate of 5 mV s−1. All the LSV curves are iR corrected by compensating the series resistance (Rs) values which were extracted from electrochemical impedance Spectroscopy. Double‐layer capacitance (Cdl) values were deduced from cyclic voltammetry measurements which were cycled between 1.15 to 1.25 V (vs RHE) at different scan rates. The electrochemical impedance measurement was evaluated at 1.45 V (vs RHE) from 1 × 106 to 0.1 Hz in the same configuration as LSV measurements.Electrochemical performance was evaluated in a three‐electrode configuration by electrochemical workstation . All the measurements were carried out in 1.0 Photovoltaics‐Water Splitting Evaluation: The PV‐water splitting reaction was performed in a water splitting reactor which was connected to a closed gas‐circulation system . The electrolysis reaction was performed in 1 m KOH solution with the distance of 1 cm between the anode (Co&Fe‐WO/NF) and cathode (Pt coated Ni foam). A solar simulator  was used as the light source to input the solar energy to a crystalline silicon solar cell. The illumination area of the silicon film solar cell is 4.73 cm2. The H2 and O2 evolution was detected by online gas chromatography equipped with thermal conductivity detector .Calculation of Solar‐To‐Hydrogen Energy Conversion Efficiency: The Solar‐to‐Hydrogen conversion efficiency for every hour was calculated by the following two methods:−1)2 was obtained according to the standard molar enthalpy of combustion (−285 kJ mol−1). For the calculation of every hour, the H2 moles represent the H2 evolution amount in 1 h, and the time equals to 3600 s.one is on the basis of the standard molar enthalpy of combustion  of water splitting, and assuming a 100% Faradaic efficiency2 per hour is 0.8858 mmol,The example calculations are as following:the average amount of H1 = 285 kJ mol−1 × 0.8858 mmol/ (0.412 W × 3600 s) = 16.9%η2 = 51.4 mA × 1.23 V × 100%/ 0.412 W = 15.3%ηCalculation of Faradaic Efficiency: Faraday efficiency is defined as the ratio of the actual yield to the theoretical yield. The calculation is as followingm is the actual moles of the product, n is the number of electron transfer, F is the Faraday constant (96 485 C mol−1), J is the current value obtained from the linear sweep voltammetry in the same configuration as PV‐electrocatalytic water splitting, and t is the reaction time.2 evolution is about 51.4 mA, the amount of detected H2 for the first hour is 0.928 mmol, n equals to 2 and time equals to 3600 s. The Faraday Efficiency is calculated as: 0.928 mmol × 2 × 96 485 C mol−1/ (51.4 mA × 3600 s) × 100% = 96.8%An example calculation is given as follows: the current for HThe authors declare no conflict of interest.SupplementaryClick here for additional data file.


Full exploitation of this isotope proxy and thermometer is limited due to time consuming and costly analysis using mass spectrometric instrumentation. Here, we present an all optical clumped CO2 isotopologue thermometer with capability for rapid analysis and simplified sample preparation. The current development also provides the option for analysis of additional multiply-substituted isotopologues, such as 12C18O2. Since the instrument unambiguously measures all isotopologues of the 12C16O2 + 13C16O18O 13C16O2 + 12C16O18O exchange, its equilibrium constant and the corresponding temperature are measured directly. Being essentially independent of the isotope composition of the calibration gas, an uncalibrated working reference is sufficient and usage of international calibration standards is obsolete. Other isotopologues and molecules can be accessed using the methodology, opening up new avenues in isotope research. Here we demonstrate the high-precision performance of the instrument with first gas temperature measurements of carbon dioxide samples from geothermal sources.Simultaneous analysis of carbon dioxide isotopologues involved in the isotope exchange between the doubly substituted  The most prominent uses are linked to the oxygen isotope exchange reaction between the main isotopologue and the 13C-18O containing species of CO2Mass spectrometry of multiply substituted isotopologues or clumped isotopes has become an extremely powerful tool in the natural sciences. Demonstrated applications which investigated carbon dioxide, methane, nitrous oxide, molecular hydrogen and oxygen range from tectonic history and evolution, geobiology and atmospheric chemistry over the investigation of non-equilibrium processes with correction procedures, diagenesis studies, the investigation of mineral formation conditions, the assessment of hydrothermal flow systems and paleo-evolution to paleothermometry; and there are many more potential applications13C16O18O, containing two or more rare isotopes (e.g. 13C and 18O) simultaneously. When compared to the main isotopologue 12C16O2, these are well below 10−4  still suffered from uncertainties in the 20‰ range which exceeds natural 13CH3D variability of about 8‰4. Nevertheless, a more recent diode laser study on doubly substituted methane5 has successfully demonstrated that optical systems can well approach the necessary requirements. The achieved precision level of 200 ppm, however, remains still well above the commonly accepted threshold of 100 ppm (or 0.1‰) required for the study of clumped isotope fractionation in non-hydrogenated molecules.Reaction  involves7, the instruments have to cope with inherent drawbacks. Not only are they relatively costly and heavy, thus not permitting in-field operation; they also require time consuming measurements and careful sample preparation in order to avoid contamination of the measurement signal. With current mass spectrometric procedures, preparation and analysis of a carbonate sample take about 3 to 6 h8. Importantly, only the largest and most sophisticated instruments can reach the mass resolution required to resolve isobaric interferences in CO29. Typical operation conditions are around M/ΔM ~ 40000 or lower, which is insufficient to separate 13C16O2 from 12C16O17O at m/z = 45 or 13C16O18O from 12C17O18O at m/z = 47, for example. In order to resolve these masses, a resolving power of above 52000 is needed – so far only accessible for large radius instruments in ‘non-normal operation’ mode9. This makes multiply substituted isotopologue analysis by mass spectrometry a very exclusive technology that will remain limited to only a handful of highly specialised laboratories worldwide, likely also constraining industrial or commercial use. Note that only two (12C16O2 and 13C18O2) out of twelve stable CO2 isotopologues can be detected entirely free from isobaric interference using a mass spectrometer  a cardinal mass signal. They can thus well be assessed by the same technology except 12C18O2, whose quantification suffers from distorting background signals. For minor contributions to a cardinal mass, such as 12C16O17O however, more advanced sample preparation or conversion technologies12 must be employed at the cost of prolonged measurement time and reduced precision.While mass spectrometers excel in the achieved precision of about 10 to 20 ppm2 isotopologues below the relative abundance level of 10−5, even if these provide the main contribution to a cardinal mass. Assuming the measurement uncertainty being limited by Poisson statistics, the 10 ppm precision is reached after about 3 or 4 h of measurement on m/z = 47 (13C16O18O)13. In order to obtain the same precision for the 12C18O2 isotopologue on m/z = 48, a (n(1613C16O18O)/n(12C18O2))2 ~ 100 times longer analysis time would be required, thus about two weeks. Even measurement times of two days are impractical and contrary to common practice. Demonstrations of ppm level instrument stabilities over such long time periods are lacking too. The m/z = 48 and 49 signals can therefore only be used as an indicator for sample contamination 15 and must remain useless in exploiting 12C18O2 or 13C18O2 as isotopic tracers with mass spectrometry.Simple counting statistics prevent using current mass spectrometer technology for the analysis of CO2 that is not influenced by most of these limitations, most notably the isobaric interference problem. The instrument achieves a measurement accuracy well below the 100 ppm level in the measurement of 13C16O18O and the technique has the capacity of assessing new tracers such as 12C18O2 that likely provide new and complementary information. In principle the developed method is calibration free and has strong potential of becoming a breakthrough technology, because it provides great isotopologue selectivity at reduced time, size and cost factors, which makes it well suited for widespread scientific, laboratory and commercial applications. The paper focusses on the measurement of carbon dioxide and the isotope exchange in the gas phase and we present the analysis of CO2 from thermal sources in the Upper Rhine Valley. The results will be compared to duplicate mass spectrometer measurements of CO2 from the same source. Apart from direct studies of gaseous carbon dioxide, current applications are concerned with multiply-substituted isotopologues in carbonates. Because carbonate isotopologues are obtained from measurements of gaseous CO2 released during the acid digestion of the carbonates, they can be investigated using the same analysis systems. The direct application of carbon dioxide isotopologue analysis to carbonates is further facilitated by the fact that the carbonate clumped isotope scale has been directly tied into the equilibrium CO2 gas scale6.Despite the pioneering achievements of mass spectrometry in rare multi-isotope research, it is evident that alternative technologies are needed to overcome several of the aforementioned limitations. In this paper, we will present the first optical multi-isotopologue analyser for CO2 isotopologues is based on absorption of isotopologue dependent ro-vibrational transitions in the ν3 fundamental band around 4 µm  at 4.3 and 4.4 µm and absorption-balanced light paths of 9.2 cm and 10 m length, respectively, absorption signals of several ten percent of the five most abundant isotopologues can be obtained when a few mbar of pure CO2 are analysed in a cylindrical 0.8 L thermostated stainless steel absorption cell. A Herriott cell configuration made out of two concave mirrors at a distance of about 17.6 cm has been realised in order to achieve the long light path with 58 reflections. The short path traverses the cell just once in the perpendicular direction. More information on the absorption lines and a more detailed description of the set up displayed in Fig. 16.The optical measurement of CO µm Fig. , where i2 at 297 K and 3 to 4 mbar are recorded at a rate of 1.56 kHz by driving laser currents at the same pace. A non-linear current ramp has been chosen for minimising the non-linearity of the laser frequency response over time17. Individual spectra were then averaged over 1 s time interval and analysed using a home-built fitting routine employing Rautian line profiles18. Required line parameters have been taken either from the HITRAN database (position) or were determined in separate experiments (self broadening coefficient and frequency of velocity changing collisions)16. The particle number density n of an isotopologue is obtained from applying the Beer-Lambert law to one of its transitions  = [CO2]) is linked to the optical measurement via19L is the path length, Tr(ν) the transmittance, S the line strength and g(ν − νc) the molecular line shape function of the particular transition. Isotopologue concentrations can thus be obtained from the extinction coefficient α = −ln(Tr(νc)) and the absorption cross section σ = S ⋅ g(0) at peak centre, which remain constant under fixed experimental conditions. The path length cancels in the measurement of an isotopologue ratio when both isotopologues are detected using the same path, e.g.20. [CO2]1/[CO2]2 = α1/α2⋅(σ1/σ2)−1, making the method immune against eventual slight path length changes. Note that in spite of using the simplifying notation on the right hand side of Eq.  measurements have been performed using the ThermoFischer MAT253Plus instrument at IUP Heidelberg, that has been equipped with an additional m/z = 47.5 cup and 10K1 of the isotope exchange reaction 21 or multiply substituted isotopologues23:The equilibrium constant reaction  is stricK1) is completely independent of the bulk isotope composition. Consequently, the 17O isotopic composition does not at all affect the determination of the equilibrium constant. Bulk isotopic compositions are only introduced when isotopologue concentrations are replaced by Δ values as defined in Eq.  and Δ(46CO2) for CO2 equilibrated at 300 K. The reason for the conflicting results is that in the one case approximate but precisely measurable and in the other case exact but only approximatively accessible atomic isotope ratios are used in the calculations of the statistical abundances. Nonetheless, the so defined Δ47 is overwhelmingly influenced by the first of the three terms, which in turn is to a large extent  according to Eq. , necessitating that international standard substances are used and that assumptions on the 17O isotope content are made. In this way systematic biases of up to 40 ppm are introduced24. Equally important, mass spectrometers can only approximately access the clumped 13C16O18O isotopologue  using the m/z = 47 signal and a corresponding scaling factor must be applied14.Unlike the direct measurement of ln ) 27, where k is the Boltzmann constant and where we have adopted a per molecule rather than a per mole definition of energies. The free energy of the gas is linked to the gas’ molecular partition function, which sums over all energy states εi taking degeneracies di into account and counting internal energy states from the lowest or zero-point energy (ZPE)  ε0 state of the moleculeUsing the equilibrium constant of an isotope exchange (or isomerisation) reaction with a particular working gas as a thermometer, temperature is directly measured as a thermodynamic variable. The equilibrium constant of an isotope exchange reaction is linked to the reaction free enthalpy Δtrans) from the molecular internal degrees of freedom (int). Since the equilibrium constant is given as a product of partition functions of reactant (react) and product (prod) molecules27M26. The only non-trivial factors are the total internal partition functions that need to be evaluated separately. If one is mainly interested in isotope fractionation effects, it is convenient to normalise the equilibrium constant by dividing through its classical high temperature limiting value K*, which is given as the product of the classical symmetry numbers of product and reactant molecules:In the Eq.  we have Solving for H = ΔU = Δε0, the reaction free enthalpy (TΔS), which is given by the two remaining terms on the right hand side. Eq.  methods33. If highly accurate potential surfaces with spectroscopic quality are available or global effective Hamiltonians have been determined, such as for CO2 34, the total internal partition function in Eq. (2) due to neglecting the quantisation of rotational states, which is only taken into account approximately. In such a case the sum in Eq.  and Aref indicate the measured product of absorbances in Eq.  required isotopologues of a homogenous CO(see Eqs ), which d by Eq. ). Since 2 serves as calibration point, that determines the origin of the optical ln K1 measurements. The gas has been equilibrated at 1000 °C for about 5 h. We use the calculated value of Aref in Eq.  values of ln (K1) show a larger spread between −951 and −968 ppm, giving an order of ±10 ppm agreement (see Table δ13CVPDB = −(40.0 ± 0.3)‰, A natural candidate for the calibration of the optical method using Eq.  are measentclass1pt{minimaentclass1pt{minimaIn the second step, individual samples are measured by alternating acquisition sequences of sample and working gas. Each sequence starts by filling the spectrometer with the working reference gas and acquiring spectra for about 30 s with 1 s integration time. Then fast (~2 min) removal of the working reference occurs and the sample gas is analysed following the same acquisition procedure. Pressures of sample and reference gases are matched to agree within 0.01%. For repeated analysis, the sample is recovered using cryogenic trapping for about 5 minutes. At the end of the evacuation period the optical base line is determined. These repeated sample-reference comparisons, where Eq.  is appli2 isotopologue thermometer using ln K1 as directly observable temperature proxy. Four different samples of equilibrated CO2 have been prepared, with equilibration temperatures at 1 °C, 21 °C, 131 °C, and 1000 °C. For measurements at 1000 °C, pure CO2 gas was filled into quartz vials and kept in a lab oven for about 5 h. For the lower temperatures, droplets of liquid water were added to facilitate isotope exchange between isotopologues of CO2. At 1 °C equilibration times were about one month, and they were about a week for the intermediate temperature at 131 °C. Figure K1 from partition functions determined as direct sums and from the BMU method with harmonic frequencies and ZPE values given by Wang et al.22  and IRMS measurements of the 13C16O18O isotopologue in a case study of natural carbon dioxide extracted from an operating hydrothermal power plant (Soultz) and two shallow wells (Landgrafenbrunnen and Stahlbrunnen), all located in the Upper Rhine Valley. The geothermal reservoir in Soultz  has a temperature of about 200 °C at a depth of 5000 m37. During power plant operation, the water cools down to ~150 °C at the surface. Carbon dioxide for the analysis has been sampled from a separate sampling line, where the water has been rapidly cooled down to 38.5 °C. The mass spectrometric and laser measurements show clumped isotope temperatures between 92 and 108 °C and the two methods agree well within the respective uncertainties of 8 °C for the mass spectrometer determinations and 11 °C for the laser measurements  °C and (15 ± 6) °C, respectively, which is in good agreement with the temperature of the well’s water, Tw = 13.5 °C. A slight deviation from the expected water-CO2 equilibrium towards higher temperature has been observed for Stahlbrunnen, Tw = 12.4 °C versus TIRMS = (20 ± 5) °C and TTLDAS = (28 ± 7) °C. The interpretation of eventual discrepancies between measured apparent equilibration temperatures and parent water temperatures requires further investigation and is beyond the scope of this paper.Clumped isotope thermometry of gas phase CO40 that provides a similar absorption length. Such small volumes imply reduced sample sizes on the order of 10 μmol or below and lead to faster evacuation times due to much simpler geometry without dead volumes. This shortens the time lapse between sample and reference measurement, thus limiting the impact of instrument drift and reducing the overall measurement uncertainty. We further anticipate that the introduction of an automated pressure balance system will allow for more reproducible conditions that further improve the measurement uncertainty.The standard uncertainty of the laser measurements in the 50 ppm range is obtained with samples of about 100 µmol and about 10 sample reference comparisons, which take between 1.5 and 2 h. This is still slightly larger than what can be obtained by mass spectrometry. However, this type of optical measurements is still in its infancy and is expected to improve soon. Already, our 0.8 L Herriott cell can be replaced with a very compact 40 to 140 mL multi-pass cellν3 fundamental region of CO2 around 4.4 µm are not exclusively limited to the detection of the 13C16O18O clumped isotopologue. Figure 13C16O17O can be measured as well. Inspection of spectral data42 further indicates that the P(12)e transition at 2305.365327 cm−1 provides a well isolated absorption line of 12C18O2. Its reported line strength (S = 1.11 ⋅ 10−23 cm molecule−1) is similar to the intensities of 12C16O17O and 13C16O2 used in this work clumping49. The similar magnitude of isotope fractionation in both reactions It is also worth noting that optical measurements in the ν0 and the energies of the lowest states ).).ν0 and 2 at the accuracy level of better than 100 ppm. New advances in laser absorption spectroscopy, such as evidenced by the recent measurement of 12C16O17O at the precision level of 10 ppm within a time frame of 10 min52, indicate that laser instruments will favourably compete with mass spectrometer technology very soon. The comparatively high selectivity of laser-based instruments and their large potential of assessing new tracers, such as 12C18O2 for the homogeneous isotope exchange with 12C16O2, will open up new horizons in clumped isotope science and thermometry. The most important advantage of the technology is that the temperature can be obtained easily and directly via an unambiguous measurement of the equilibrium constant of the isotope exchange reaction. The optical CO2 isotopologue thermometer is a strong example showcasing the full potential for this and other molecules in the future. We have shown how the technology can be used for the thermometry of gaseous CO2 and that new areas related to molecular chemistry and physics are opened up for clumped isotope research. The relatively low cost and size factors of the technology will be additional parameters for developing and easing the spread of this exciting frontier science technology to more laboratories and technological applications.We provide the first optical measurement of multiply-substituted isotopologues of CO


However, luminescence shows a lower local symmetry, as confirmed by the extended X‐ray absorption fine structure. By considering the reduced symmetry of the local structure, this work opens the door to a new class of understanding of the properties of materials.Research on lithium compounds has attracted much attention nowadays. However, to elucidate the precise structure of lithium compounds is a challenge, especially when considering the small ions that may be transferred between the interstitial voids. Here, the discovery of reduced local symmetry (symmetry breaking) in small domains of Li For that lithium compounds have never attracted so much attention in the past until recently, driven by the requirements of energy‐storage and energy‐saving applications, such as Li‐ion batteries (LBs) and light‐emitting diodes (LEDs). Aiming at these applications, lithium‐containing silicates with the general formula ABC2EuSiO4 and considered it isostructural with Li2SrSiO4.2SrSiO4 by taking Li2EuSiO4 as a starting model, confirming that Li2SrSiO4 crystallizes into the trigonal space group P3121.8] polyhedron presents an extremely distorted structure with a much larger eccentric distance and sphericity than expected.P3121. Nevertheless, the structural model of space group P3121 (noted as the P3121 model hereafter) possesses one Sr2+ site and thus cannot interpret the luminescence properties of Eu2+ and Ce3+ in Li2SrSiO4 in a straightforward way.In 1998, Haferkorn first determined the crystal structure of Li+ is the smallest and lightest alkali metal ion and can easily be incorporated into the interstitial sites in crystal lattices. The mobility and static displacement of small atoms may result in characteristic local symmetry breaking due to tiny distortions in the structure that do not propagate over long‐ranges.Structures are always fundamental to understanding material properties.Symmetry is a beautiful and harmonic natural concept, but much of the changes in the world have been caused by symmetry breaking,According to Lee (the Nobel Laureate in Physics in 1957), “the root of all symmetry principles lies in the assumption that it is impossible to observe certain basic quantities.”7FJ splitting and the 5D0‐7FJ transitions of the f6 configuration in 32 point groups, with consideration of the odd crystal field in Judd–Ofelt theory, and reported that the relationship between luminescence spectra and crystal structures could be used to investigate the local environments in crystal structures by doping trace amounts of Eu3+ (or Sm2+) into compounds as a probe.3+, other rare‐earth ions, such as Dy3+ and Sm3+, have also been used as structural probes to study local site symmetry, but comparatively, Eu3+ is the best probe due to the large splitting of the 7F1 or 7F2 levels of Eu3+ in the visible wavelength region, which results in easily identified emission peaks.According to symmetry breaking, forbidden transitions of electrons may occur due to a perturbation in the Hamiltonian. Therefore, fluorescence spectra originating from parity violation promisingly provides a probe for hyperfine interactions. In 1984, Zhang calculated the values of the Stark energy levels for 2SrSiO4. Two Sr2+ sites in the Li2SrSiO4 structure with P3121 symmetry are broken into C2 subgroups, as distinguished by employing Eu3+ as a spectroscopic probe. The difference between the two sites is ≈0.2 nm for the Eu3+ 5D0‐7F0 transition, which is out of the discernable range of X‐ray, electron and neutron diffraction, and nuclear magnetic resonance measurements but is sensitive to Eu3+ fluorescence spectra. This work demonstrates a facile, but powerful, optical tool to probe hyperfine structures and opens a door to identify new material properties and mechanisms by considering their reduced symmetry. Justifiably, symmetry breaking will not only exist in Li2SrSiO4 but also in other lithium compounds. Thereby, this discovery represents a landmark in exploring hyperfine structures with subgroups, which will improve our comprehension of modern physics and existing philosophy.Society longs to find the beauty of a structure. Therefore, in this work, we report the discovery of symmetry breaking in small domains of Li3+, Eu2+, and Eu3+‐doped LiSrSiO4, shown in Figure S1 in the Supporting Information, match well with those of Li2EuSiO4 (JCPDS 47‐0120).3+ (103 pm) and Eu2+ (109 pm) should replace Sr2+ (112 pm) in the blue‐emitting Li2SrSiO4:Ce3+ and yellow‐emitting Li2SrSiO4:Eu2+ phosphors. Eu3+ should also occupy the Sr2+ site in Li2SrSiO4 despite its smaller radius (95 pm), as is known, e.g., from Eu3+‐doped Zn2SiO4 or SrSnO3 perovskites.3+ may be used as a spectroscopic probe for the symmetry of the initial coordination of Sr2+ in Li2SrSiO4.The XRD patterns of CeO4 JCPDS ‐0120.3+ 2SrSiO4:Ce3+ and Li2SrSiO4:Eu2+ comprise more than one peak each, as can be observed in previous reports.3+, the doublet peaks can be attributed to transitions from the lowest 5d excited state to the ground substates of 2F7/2 and 2F5/2 (degenerate). However, this conclusion is not possible for Eu2+ because there is no spin‐orbit splitting for the 8S7/2 ground state of Eu2+. Nevertheless, emission peaks originating from different environments around Eu2+ cannot be excluded. However, there is only one Sr site in the P3121 model of Li2SrSiO4.3+ and Eu2+ in Li2SrSiO4, an accurate crystal structure should be confirmed first.The asymmetrical shapes of the spectra in Figure S2a,b in the Supporting Information suggest that the emission peaks of LiFigure5D0‐7FJ  emission spectra of Li2SrSiO4:Eu3+ with a nominal composition of Li2Sr0.995Eu0.005SiO4. The electric dipole transition of 5D0‐7F2 is parity‐forbidden, but it can potentially occur in a noninversion site by mixing with the opposite component. If allowed, its intensity would be far stronger than the intensity of the magnetic 5D0‐7F1 transition. Therefore, the strong 5D0‐7F1 emission in Figure 3+ that occupies an inversion or approximate inversion site. However, the Wyckoff positions 3abP3121 model have noninversion symmetry.5D0‐7F0 transition as measured with an optical parametric oscillator (OPO) at 20 K. One peak at 579.2 nm can be observed by monitoring the 5D0‐7F2 emission at 611 nm, while two peaks at 579.2 and 579.4 nm can be observed by monitoring the 5D0‐7F1 emission at 586 nm. Theoretically, the number of peaks for the Eu3+ 5D0‐7FJ transition will be no more than 2J + 1, where J is the total angular momentum. Thus, the number of peaks for the 5D0‐7F0 transition at each site, which is strictly forbidden by parity and spin rules due to ΔJ = 0 for the transition from J = 0 to J = 0, should be no more than 1. Two peaks for the 5D0‐7F0 transition can be observed in Figure 3+ in Li2SrSiO4.Figure 5D0‐7F1 transition, for which the splitting strongly depends on the symmetry. In the P3121 model, the Sr2+ at the Wyckoff 3ab2 symmetry. Thus, the 5D0‐7F1 emission should have 3 peaks if only one Sr2+ site with C2 symmetry exists in Li2SrSiO4.5D0‐7F1 emission and the 7F1‐5D0 excitation, are observed in Figure 1 and S2), each with triplet emission. More than 3 peaks for the 5D0‐7F1 emission are observed upon exciting the 5D2 level at 464.3, 464.7, and 446.0 nm, as shown in Figure 5D2 to 5D1 levels. From the above results, we can conclude that two different environments for Eu3+ exist in the crystal lattice of Li2SrSiO4.In addition to detection the number of sites, site symmetry could be probed by the 3+. To analyze this apparent disparity, the long‐range structure of Li2SrSiO4 was studied with X‐ray diffraction (XRD), synchrotron X‐ray diffraction (SXRD), and neutron diffraction (ND). By decreasing the symmetry of the P3121 model, a hypothetical model with the translationengleiche subgroup C2 was derived and tentatively refined. No significant differences between the refined structures in the C121 and P3121 space groups were found, either in the XRD or in the ND data , which can be attributed to regular thermal expansion effects, no significant changes in the crystal structure were detected. Therefore, we can conclude that no phase transition occurs down to 12 K and that the crystal structure is best described by the model of space group P3121 for all investigated temperatures. Details for the methodology of the XRD, SXRD, and ND Rietveld refinement are given in Figures S3–S5 and Table S2 in the Supporting Information.Systematically absent Bragg reflections of [031¯] zone axis, which can be perfectly simulated with both the C2 and the P3121 models . For space group P3121, kinematic theory only allows for 00l reflections with l = 3n, but these reflection may increase in intensity via dynamic electron diffraction. Nevertheless, the 003 peak intensity is much stronger than that of the other peaks in the [350] pattern . However, only one type of chemical environment for Li and Si can be clearly discriminated in FigureP3121 model. The chemical shifts of the 7Li and 29Si resonances are 0.5842 and −68 ppm, respectively, and the twin peaks at −94.2838 and 98.3730 ppm and −191.1380 and 194.2925 ppm are the first two and the second two spinning sidebands of 7Li. Since different sites can have the same chemical shift, the Li‐ and Si‐NMR results are inconclusive. Therefore, the NMR data are consistent with the higher symmetry, but they cannot exclude the lower symmetry.Solid‐state nuclear magnetic resonance (NMR) is sensitive to the local chemical environments of Li and Si atoms. In the FiguresC2 and P3121 models that were hypothesized above and their 2 and 1 Sr‐atom sites, respectively, all 8 Sr–O bonds of the first coordination sphere of Sr cannot be fit at one time due to the large variations in the bond lengths. Instead, fitting was performed by classifying the 8 Sr–O bonds into two groups: a shorter group and a longer group. As shown in Figure C2 model than by the P3121 model. In Figure P3121 model are close to those in the C2 model, including both of the shorter Sr–O1 bonds  and the longer Sr–O2 bonds ; the disorder factors of these bonds are within a reasonable range. When fit with the C2 model, the coordination number for the shorter and longer groups are both 4.0, and the total sum, 8.0, is consistent with the above results from XRD and SXRD; when fit with the P3121 model, however, the coordination number for the shorter and longer groups are both 3.7, and the total sum, 7.4, is far below the coordination number, 8.0, resolved from the XRD and SXRD results. Therefore, the fit from C2 symmetry gives a more reasonable coordination number of N = 8.0, which is consistent with the crystal structure. Based on the coordination numbers , the analyses of the EXFAS spectra indicates a structure with two different environments of Sr in Li2SrSiO4 .Next, the local structure was examined with extended X‐ray absorption fine structure spectroscopy (EXAFS) to obtain information on the local coordination numbers, interatomic distances and possibly structural disorder. The Sr K‐edge EXAFS spectrum and its transformation in K‐space are shown in 2SrSiO4 does not deviate significantly from that of the P3121 model, whereas the cryogenic spectra of Eu3+ demonstrate that more than one Eu3+ site has to be taken into account. The diffraction data give an average structure model that projects the structure into a single unit cell, which requires a periodicity over an area larger than the coherence length of the radiation used. If local distortions are not long‐range ordered, they do not influence the space group assignment.XRD, SXRD, ND, and SAED data indicate that the long‐range average structure of Li5D0‐7FJ transitions of Eu3+, either allowed or forbidden, are determined by the local symmetry. The cryogenic spectra of Eu3+ suggest that more than one Eu3+ site has to be considered. To explore the reason for the electronic structure, the charge deformation density of Li2SrSiO4 was obtained from first‐principles calculation as shown in FigureC2 model, whereas these patterns merge  in Figure P3121. It seems that the spectroscopic probe of Eu3+ also interacts with the shape of the electron density, a further aspect of the local structure in addition to its symmetry. The ab initio geometry optimization shows that the final enthalpies for C2 (−9251.08 eV) and P3121 (−9250.79 eV) are very close to each other. Because the lower symmetry C2 model has a lower energy, local symmetry breaking seems to be possible due to the small energy barrier.The multiple 2SrSiO4. Its crystal structure consists of [LiO4] and [SiO4] tetrahedrons. The periodical arrangement of three [SiO4] and six [LiO4] tetrahedrons forms a channel. Thus, the crystal structure of Li2SrSiO4 may be described by incorporating Sr atoms into the framework channels. There is one type of channel in P3121 SiO4 changes from trigonal to hexagonal,2SrSiO4 has a nonrigid structure. The tiny differences between the channels may lead to symmetry breaking from P3121 into C2  since the superposition of local deviations results in P3121 symmetry, described in Figure C2 or the P3121 model, all the Sr atoms are coordinated by 8 neighboring atoms, as shown in Figure C2 and P3121 models, based on XRD data, the bond lengths of Sr1–O range from 2.553 to 2.691 Å, and Sr2–O range from 2.567 to 2.757 Å in the C2 model, wherein the variation amplitudes of the Sr1–O and Sr2–O bonds are 0.138 and 0.190 Å, respectively. However, the variation range of the Sr–O bond length in the P3121 model is as small as 0.077 Å, changing from 2.576 to 2.653 Å. Because the effects of the surface, interface, crystal defects, etc., are always unavoidable, they cause a wide variation in the Sr–O bond lengths, which breaks the local symmetry of the Sr site in the P3121 model, resulting in the appearance of two Sr sites in the C2 model.Finally, we discuss the origin of symmetry breaking in Li21 Figure6a but C2 structural model of Li2SrSiO4 can be approximated as the P3121 model, which follows the same principle for ignoring higher order terms in the Taylor series expansion in mathematics. However, the results achieved in this work by employing Eu3+ as a spectroscopic probe suggest that the difference between Sr1 and Sr2 in Li2SrSiO4 is non‐negligible, as discussed above, regarding perturbations to the Hamiltonian of the system energy. This work not only distinguishes the hyperfine structures of Li2SrSiO4 but also presents a facile optical tool for detecting local structures and, more importantly, helps to explain crystal structures at the subgroup symmetry level. These discoveries will produce far‐reaching implications for understanding the properties and mechanisms of materials.By neglecting the minor differences in the local environments of the same types of atoms, including Li, Sr, Si, and O, the 2SrSiO4 by employing Eu3+ as a spectroscopic probe. The X‐ray, synchrotron X‐ray, and neutron diffraction results show that the long‐range average structure of Li2SrSiO4 is consistent with the symmetry of space group P3121. However, the cryogenic spectra of Eu3+ suggest a lower local symmetry with the C2 space group. A comparison of the electron diffraction and NMR data suggest the presence of the P3121 model but cannot exclude the possibility of the C2 model. Nevertheless, the Sr K‐edge EXAFS confirms that the local environment deviates from that of the P3121 model but is similar to that of the C2 model. All of these evidences indicate that Li2SrSiO4 exists in a medium possessing characteristic symmetry in space group P3121 for its long‐range structure and a lower symmetry local structure in space group C2. One site of Sr exists in P3121, while two sites of Sr exist in the hypothetical C2 model. All the Sr atoms are eightfold coordinated irrespective of the symmetry model. Two sites of Sr could rationally explain the photoluminescent properties of Eu2+ and Eu3+ in Li2SrSiO4. The resolution of the Eu3+ 5D0‐7F0 emission for distinguishing the two sites of Sr2+ is 0.2 nm. Therefore, the powerful ability of a Eu3+ spectroscopic probe for detecting local hyperfine structures has been demonstrated; moreover, this work opens the door to elucidating the properties of lithium compounds by considering the reduced symmetry of the local structures. These results will produce far‐reaching implications for characterizing material structures and properties.In summary, the phenomenon of a local structure with reduced symmetry, i.e., symmetry breaking, was discovered in Li2SrSiO4 was synthesized from SrCO3 (AR), Li2CO3 (AR), and SiO2 (99%). The residual SrCO3 in the final compound that had not been fully reacted was identified from ND in Figure 2O (AR), SiO2 (99%), Eu2O3 (99.99%), and CeO2 (99.99%) sources via the following process. First, stoichiometric amounts of the raw materials with a 3% excess of Li were thoroughly ground, and the mixture was heated at 500 °C for 2 h in air. Then, the mixture was ground again and sintered at 850 °C for 8 h. The blue‐emitting Li2SrSiO4:Ce3+ and yellow‐emitting Li2SrSiO4:Eu2+ phosphors were reacted under a 90% N2 + 10% H2 reductive atmosphere, and the red‐emitting Li2SrSiO4:Eu3+ phosphor was obtained in air. Conventional XRD was measured using a Rigaku D/max‐IIIA diffractometer with an X‐ray wavelength of 1.5418 Å. SXRD was recorded on the high‐energy X‐ray diffraction beamline SP12B1 of a Spring‐8 in Japan with an X‐ray wavelength of 0.6888 Å. The neutron diffraction was collected on a D20 diffractometer at the Institute Laue‐Langevin in Grenoble, France. The programs of GSAS7Li and 29Si were collected on a Bruker Avance‐III, 400 MHz NMR instrument. The Sr K‐edge EXAFS was collected at the 01C1 beamline of the National Synchrotron Radiation Research Center (NSRRC) in Taiwan. The EXAFS data were fit using the FEFF8‐lite program. The ab initio geometry optimization and first‐principles calculations of the charge deformation density were performed using the CASTEP module of Materials Studio 7.0.Samples were synthesized via a two‐step solid‐state reaction. Initially, LiThe authors declare no conflict of interest.SupplementaryClick here for additional data file.


The interactions of the responsive hydrogen bonding motif with complementary partners were examined through a series of 1H NMR experiments, revealing that the recognition preference of the responsive hydrogen bonding motif in a mixture can be switched between two states.A stimuli responsive linear hydrogen bonding motif, capable of  A pH responsive linear triple hydrogen bonding motif capable of protonation and deprotonation, has been investigated. A series of 1H NMR experiments, revealed that the preference of this responsive hydrogen bonding motif in a mixture of hydrogen bonding motifs, can be switched between two self‐sorted states. D) and acceptors (A) in hydrogen‐bonding motifs (HBMs),1H NMR study of a responsive HBM capable of switching recognition preference towards complementary HBMs, in response to protons.Significant effort in supramolecular chemistry is directed towards use of narcissistic (self‐loving) and/or social (self‐loathing) self‐sortingI) revealed protonation of the imidazole resulting in a donor‐ donor (DD) array . We reasoned that protonation and deprotonation of UIM 1 would result in switching between the donor‐donor‐acceptor (DDA) array of neutral UIM 1 and the donor‐donor‐donor (DDD) array of protonated UIM‐H++1‐H  had in prior work been shown to interact with AIC 2 (AAD array)++1‐H (DDD array) was expected to interact with a complementary AAA array such as BB1 3  was identified as a potential responsive motif; the crystal structure of a synthetic intermediate  distinct changes in chemical shifts were observed to the spectrum of UIM 1 . Resonances Ha, Hb, Hc and Hg (between 6 and 8 ppm) were observed to shift downfield on the addition of hydrochloric acid . In addition, the NH resonances Hd, He, Hf and Hh (between 9 and 13 ppm), which are broadened for UIM 1 , can be seen in the 1H NMR spectra of +1‐H Figure ESI 1c). The changes in chemical shift and sharpening of these specific resonances, relative to UIM 1, indicate a change in chemical environment and protonation of the nitrogen atom in the aminoimidazole ring (at position Hf), in a similar manner to that observed in the crystal structure of intermediate I, to generate UIM‐HCl +1‐H. 1H NMR titration was carried out to determine the equivalents of acid required to protonate UIM 1. At 5 mM of UIM 1 in chloroform 0.25, 0.5, 0.75, 1 and 3 equivalents of 4 M HCl in 1,4‐dioxane were added . This demonstrated that significant changes in chemical shift of the aromatic resonances  occurred even at 0.5 equivalents of hydrochloric acid. With 1 equivalent of hydrochloric acid the broad NH resonances of UIM sharpened, indicating complete protonation of the imidazole. Protonation was reversed by addition of sodium hydrogen carbonate to regenerate neutral UIM 1 see Figure ESI 1b. After filtration of the salts, the 1H NMR spectrum of the resulting solution revealed broadening of the NH resonances  and an upfield change in chemical shift of the aromatic resonances  matching that observed for the neutral species . The switching process can be cycled by the addition of acid and base respectively . After four cycles, the resolution of the NH resonances in the 1H NMR spectrum of +1‐H was diminished, likely due to the concentration of the sample decreasing slightly with each cycle in part through loss of sample during filtration. To further prolong the recyclability of switching, and identify conditions where intermediate filtration can be avoided, a further set of reagents‐trifluoroacetic acid (TFA) and 1,4‐diazabicyclo[2.2.2]octane (DABCO) were considered (condition B). Under these conditions, however, it was necessary to add excess base to ensure complete deprotonation, which made spectral interpretation more challenging . Moreover, the presence of TFA appeared to subtly influence the spectra at higher concentrations, possibly due to interaction of the anion with the HBM. We explored several additional weaker acids and bases e. g. camphor sulfonic acid and 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (not shown); whilst similar spectral changes were observed, the quality and practicality of handling, led us to use conditions A and B in subsequent studies.1 and UIM +1‐H to interact with complementary HBMs was tested by the addition of AIC 2, bearing an AAD array. The heterodimerisation of UIM 1 and AIC 2 through triple hydrogen bonding has been well studied,a, Hb and Hc resonances together with an upfield shift and broadening of resonance Hg of UIM 1 in the 1H NMR and downfield shift of resonance H4 of AIC 2 .The capacity of UIM 1, a complementary HBM 3 (BB1 3 with AAA character) was added to create a three‐component system. On the addition of BB1 3 to 1 ⋅ 2, the heterodimer remained intact and BB1 3 showed no interaction‐imposed perturbations in the 1H NMR spectra . Overall these experiments demonstrated the ability of UIM 1 to undergo proton responsive switching in a mixture and hence switch recognition preference between UIM⋅AIC 1 ⋅ 2 and UIM‐H+⋅BB1 +1‐H ⋅ 3 heterodimers. The observed proton dependent behavior in the system is thus fully consistent with the binding behavior and complexation affinities.To further explore the recognition behavior of the switchable HBM +1‐H may compete for the hydrogen‐bond donor groups, complicating further the equilibria and thus spectral interpretation. Consequently, the chloride ion of +1‐H was exchanged for a non‐competitive anion (hexafluorophosphate (PF6)) and the molecular recognition of UIM‐HPF6 with complementary and competing HBMs (2 and 3) was examined . Overall the recognition behaviour exhibited by UIM‐HPF6 closely mimics the preferences exhibited by HBM UIM‐HCl +1‐H (full discussion can be found in the supporting information). Finally, it should be noted that there is potential for AIC 2 and BB1 3 to become protonated; indeed spectral changes are observed upon addition of HCl to either component , however for AIC 2, in the presence of other components i. e. UIM 1, AIC 2 is not protonated whilst for BB1 3, it is more instructive to consider the 1 ⋅ 3 complex as the preferred site of protonation.In the studies described above, the chloride ion used in generating 1 ⋅ 2 and UIM‐H+⋅BB1 +1‐H ⋅ 3 heterodimers are the preferred complexes in three component mixtures. This stimuli dependent behavior is distinct from prior studies on stimuli dependent H‐bondingIn summary we have used acid‐base chemistry to modulate the recognition preference of a HBM to demonstrate that UIM⋅AIC See supporting information for full experimental details.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.