The structure includes what is thought to be an ordered propanol molecule (POL) only in chain D(4) and a solvated acetate molecule (ACT) coordinated to the Zn atom only in chain B(2). Possible origins and consequences of the propanol and acetate molecules are discussed. Three types of amino acid representation in the electron density are examined in detail: (i) sharp with very clearly resolved features; (ii) well resolved but clearly divided into two conformations which are well behaved in the refinement, both having high quality geometry; (iii) poor density and difficult or impossible to model. An example of type (ii) is observed for the intra-chain disulphide bridge in chain C(3) between Sγ6–Sγ11 which has two clear conformations with relative refined occupancies of 0.8 and 0.2, respectively. In contrast the corresponding S–S bridge in chain A(1) shows one clearly defined conformation. A molecular dynamics study has provided a rational explanation of this difference between chains A and C. More generally, differences in the electron density features between corresponding residues in chains A and C and chains B and D is a common observation in the Insugen (I) structure and these effects are discussed in detail. The crystal structure, also at 0.92 Å and 100 K, of a second commercially available form of human recombinant insulin, Intergen (II), deposited in the Protein Data Bank as 3W7Y which remains otherwise unpublished is compared here with the Insugen (I) structure. In the Intergen (II) structure there is no solvated propanol or acetate molecule. The electron density of Intergen (II), however, does also exhibit the three types of amino acid representations as in Insugen (I). These effects do not necessarily correspond between chains A and C or chains B and D in Intergen (II), or between corresponding residues in Insugen (I). The results of this comparison are reported.The crystal structure of a commercially available form of human recombinant (HR) insulin, Insugen (I), used in the treatment of diabetes has been determined to 0.92 Å resolution using low temperature, 100 K, synchrotron X-ray data collected at 16,000 keV (λ = 0.77 Å). Refinement carried out with anisotropic displacement parameters, removal of main-chain stereochemical restraints, inclusion of H atoms in calculated positions, and 220 water molecules, converged to a final value of R = 0.1112 and RThe online version of this article (doi:10.1186/s13065-017-0296-y) contains supplementary material, which is available to authorized users. A definitive account of the 1.5 Å resolution structure (PDB 4INS) of hexagonal porcine insulin, which differs in sequence by only one amino acid at B30 (and D30) from human insulin . Seven of the amino acid side-chains were assigned less ordered conformations, refined with separate atomic coordinate sets and occupancy factors. Commercial human recombinant insulin is now available from several sources. The present study describes the ultra-high resolution (0.92 Å) low temperature structure of Insugen (I) human recombinant insulin, Fig. Success in the use of pig insulin to control diabetes ultimately lies in its ability to mimic the activity of the human form, which is a consequence of near perfect structural isomorphism. However, the use of non-human forms of insulin to control diabetes is known to lead to both allergic reactions and other complications resulting from antibody production in some patients . For thiHR insulin, Fig. See also “Human recombinant insulin (Insugen-30/70) was supplied by Biocon (India) Ltd. See Additional file The crystals were prepared at room temperature by a batch method similar to that of Baker et al. , modifie3.The solution was then warmed to 50 °C by surrounding the test tube with preheated water in a Dewar. This allowed the solution to cool slowly to room temperature. The test tube was lightly sealed with cling film; crystals formed within a few days and were of a suitable size for X-ray diffraction within 2 weeks; the test tube containing crystals was kept at 4 °C prior to data collection. The crystal used for data collection was about 0.2 mmThe following details were supplied by Sakabe . In contCrystals grown at room temperature were passed through a 30% glycerol solution, prepared in mother liquor, prior to cryo cooling in liquid nitrogen. Crystals were screened with three test shots, separated by 45° using 0.5 s exposure and 0.5° oscillation. Data were collected at 16,000 keV (λ = 0.77 Å) and 100 K with the Pilatus 6 M detector as close to the sample as possible 179.5 mm). The EDNA strategy [.5 mm. Thmerge for Iobs = 0.05579 for 57006 hkl’s. The resolution of useful diffraction data achieved and used for structure analysis was 0.92 Å [The following details were supplied by Sakabe . A synchs 0.92 Å –14. The Manual processing of the data was carried out using XDS  to integMolecular replacement was carried out with the published structure 3E7Y as a search model in the program MOLREP , followeFurther details can be found in Additional file A fluorescence mca scan, Fig. free  were 0.108 and 0.146, respectively. The program MolProbity [Model inspection and rebuilding were performed using the program WinCoot 0.7  and furtlProbity  was usedfree were 0.162 and 0.180, respectively. Inspection of the Ramachandran plot revealed that 96.81% of the residues are in the allowed regions. All coordinates and data are deposited in the Protein Data Bank, with identification code 3W7Y.The structure for 3W7Y was determined by molecular replacement and refined using the program REFMAC . Non-hydSuperficially the ultra-high resolution structure of HR insulin (Insugen I), as expected, strongly resembles that of 2Zn porcine insulin see “” having As described below there are significant and interesting differences between the detailed ultra-high resolution structures of Insugen (I) and Intergen (II) and also between the two human recombinant insulin structures and the less detailed porcine insulin . For exaThe ultra-high resolution refinement of HR insulin, Insugen (1) undertaken in the analysis described above has enabled a study of the secondary structure motifs in the insulin molecule to be carried out in detail which exceeds all previous studies.Helix A1  values are indeterminate because it is N-terminal, this residue does seem to be part of the helix. SerA9 is at the C-terminal end of the helix, its side chain H-bonding to the peptide N of IleA10. Details are in Fig. Strand A2 . Note there is only one β-bridge, at CysA11.Helix A3  values of SerA12 suggest it is part of the preceding strand and not this helix. A3 Fig. a, c: thiStrand A4 . The strand extends to SerC12. C2 Fig. a: IleC10Helix C3  structure are listed in Table Figure Of the 102 amino acid residues in Insugen (I) a total of 18:2 in both chains A and C; 8 in chain B; and 6 in chain D do not form any hydrogen bond interactions with solvated water molecules. These residues are as follows:The table above indicates the 18 residues in Insugen (I) which do not form any hydrogen bond interactions with solvated water molecules. There are 2 in both chains A and C; 8 in chain B; and 6 in chain D. Entries in bold are common to two chains, either A and C, or B and D.The residues common to two chains are in bold: Leu16 in both chains A and C are without water interactions as are Leu11, Val12, Ala 14, Leu15 and Cys 19 in both chains B and D. The sequence LeuD11–ValD12–GluD13–AlaD14–LeuD15 is shown in Fig. It is of interest to note that 14 of the 18 residues that do not associate with solvated water molecules are located in α-helical structures. These are: CysA6 (helix A1); LeuA16 (helix A3); LeuB11, ValB12, AlaB14, LeuB15 and CysB19 (helix B2); IleC2 (helix C1); LeuC16 (helix C3); LeuD11, ValD12, AlaD14, LeuD15 and CysD19 (helix D2).It is well known that ultra-high resolution protein structures derived from X-ray diffraction data using cryo cooled crystals often reveal amino acid residues which display more than a single ordered conformation. See for example Smith et al.  and AddlProperties of the electron density for Insugen (I) are summarised in colour code in Fig. The electron density of Insugen (I) chain A is of very high quality (mainly blue) with few problems associated with fitting the amino acid residue structures; only the C-terminal residue N21 exhibits a double conformation with two weak regions of density at the end of the chain.In contrast chain C exhibits the following characteristics: residues Q5, Y14 and Q15 have mainly good density but with some poorly defined regions; residues C6 and C11 participating in an S–S bridge, and L16 demonstrate clear electron density but corresponding to double residue conformations with good geometry (orange). The remaining residues are clearly defined in strong electron density (blue).The electron density of Insugen (I) chain B exhibits the following characteristics: residues L11, V12, and E13 and T27 have clear electron density with two distinct conformations (orange); residues Q4 and L17 show mainly clear double conformations but with some poor density at the extreme end; residue F25 clearly adopts two conformations but both phenyl rings A and B occupy very weak regions of density; residues K29 and T30 are mainly clear single conformations but with some terminal disorder. The remaining residues are clearly defined in strong electron density (blue).In contrast the electron density of Insugen (I) chain D can be described as follows: residues F1, V2, Q4, E21 and K29 have overall poorly defined electron density; residues V12 and V18 have clear double conformations (orange); residue T27 is mainly a clear double conformation but with some missing terminal density. The remaining residues are clearly defined in strong electron density (blue).Why is chain A so well ordered while the related chain C shows a number of double conformations and poorly defined residues?Chains B and D both show a number of double conformations. Double conformations L11, E13 and R22 occur only in chain B; the double conformation V18 occurs only in chain D; double conformations V12 and T27 occur in both chains B and D. L17, R22, F25 and T30 are disordered in chain B alone; F1, V2 and Q4 are disordered in chain D alone; E21, T27 and K29 are disordered in both chain B and D.For the Insugen (I) structure the following points may be considered.It may be possible to rationalise these differences for example via molecular dynamics simulations.The residues most likely to affect biological activity in an adverse way are those which display conformational differences between the corresponding chains A and C, or between chains B and D, particularly with respect to the way the residues have been rated in Fig. being disordered: PheB25, and to a lesser extent GlnC5, AsnA21, LysB29, LysD29 and ThrB30;exhibiting two clear conformations: CysC6–CysC11, LeuB11 and ValB12, and to a lesser extent LeuC16 and GluB13.It follows that the most likely residues are by virtue of:The distribution of these residues in the crystal asymmetric unit is shown in Fig. Properties of the electron density for Intergen (II) are summarised in Fig. The electron density of Intergen (II) chain A is of very high quality with no major problems associated with fitting the amino acid residue structures and no multiple conformations or other disorder.In contrast chain C exhibits the following characteristics: residues 1–4, 7, 8, 12, 13, 16,17,19–21, have clear well defined density; residue Q5 has mainly clear density but with missing terminal density; residues S9, I10, N18 and C6–C11 are modelled as single conformations but are probably well ordered double conformations  structure the following points can be made for Intergen (II). Why is chain A so well ordered while chain C shows a number of double conformations and poorly defined residues? Chain B shows one double conformation. There are no double conformations in chain D.Both A-chains have mostly well-defined electron density with very few problems in their interpretation.For the C-chains the only notable difference here lies in the assignment of a double conformation for the C6–C11 disulphide bridge in Insugen (I). As mentioned above the electron density for Intergen (II) in this region, Fig. Comparison of the B-chains of Insugen (I) and Intergen (II): differences here occur for residues L11, V12 and R22 which have double conformations in Insugen (I) and T27 which has a double conformation in Intergen (II). Insugen (I) chain B also has problem residues E13, L17, E21, F25, T27, K29 and T30, which are well behaved in Intergen (II). Intergen (II) chain B has one residue T27 modelled as a double conformation but which is single in Insugen (I).Referring to Fig. There are a number of differences between Insugen (I) chain D and Intergen (II) chain D. In Insugen (I) F1, V2 and T27 all have problem electron density but are well behaved in Intergen (II); Q4, E21 and K29 have weak or poorly defined electron density in both structures; residues S9, V12 and V18 have double conformations in Insugen (I) but not in Intergen (II).The above analysis has indicated that in both the Insugen (I) and Intergen (II) structures the sequence equivalent protein chains A and C, and B and D, respectively exhibit significant differences with respect to their corresponding amino acids such as double conformations and quality of the electron density. It is of interest to note that Baker et al.  in discuThe electron density excerpt below, Fig. 3 = Cβ6–Sγ6–Sγ11A–Cβ11A = 108.23° corresponds to right handed chirality [Figure hirality .Fig. 14left handed chirality [Figure hirality . AdditioFigure right handed chirality [left handed, χ3 = −79.10°.Figure hirality  the minoPossible explanations for the observed bifurcation of chain C(3) Sγ6–Sγ11 disulphide are as follows: CysC6 is hydrogen bonded to a water molecule and there are several other waters modelled in this region which may be associated with greater conformational flexibility compared to CysA6. In addition CysA6 is in a hydrophobic pocket devoid of solvate molecules and consequently the disulphide may be more restricted by this environment. This is supported by the fact that the section of chain D close to chain A(1) Sγ6–Sγ11 disulphide is disordered  whereas the section of chain B close to chain C(3) Sγ6–Sγ11 disulphide is not, Fig. Inspection of the deposited X-ray structure of Intergen (II) (3W7Y), indicates that no attempt was made to model CysC6–CysC11 in chain C in a double conformation. However the superposition of the refined Insugen (I) chain C with the 3W7Y chain C indicates that the alternate conformation of the disulphide from residues CysC6–CysC11 is likely also to be present, but not modelled, in the 3W7Y structure. This is indicated by the presence of negative electron density (green) in the same position as the CysC11 γ sulphur atom in the second (minor) conformation and positive (red) electron density in the over modelled main conformation, Fig. The possibility of this effect being accounted for by radiation damage in the Insugen (I) structure was investigated by closely inspecting the intensity data collected. This led to the conclusion that there is no global suggestion of radiation damage in the data. Next a number of subsets of data were integrated and scaled and the minimum set of data with acceptable completeness was assembled by using images 1–600 (the first third of the data). When solved and initially refined there was still evidence for the second conformation at this disulphide bond. As two clear conformations, rather than complete disorder have been assigned successfully it may be concluded that this is a reflection of the true state in the crystal, rather than radiation damage. Further examination of the difference in the disulphides A6–A11 (single ordered conformation) and C6–C11 (clear ordered double conformation) may be explained by the difference in solvent exposure. C6 is less than 4 Å from the nearest solvent molecule and there are several waters modelled in that area which may give greater conformational flexibility to the region. A6 is in a hydrophobic pocket and consequently the disulphide may be more restricted by that environment. This is supported both by the fact that the section of chain D in this vicinity of the part of the molecule is also disordered (see above).The ultra-high resolution Insugen (I) X-ray structure has been found to include an unexpected solvated propanol molecule (POL5001), Fig. Figure Figure It is interesting to note that Step 12 of US Patent Application Number US 13/032,797  describeThe use of the molecular modelling procedures described in “Molecular dynamics” to investigate reasons for the presence of propanol in the binding site located on chain D of Insugen (I) described here is currently in progress.Insugen (I) and Intergen (II) have been synthesised to include the Zn ions present in naturally occurring insulins. The Zn ions are an essential feature in the formation of the crystal structure and are located on a crystallographic three-fold axis. In porcine insulin 2 Zn crystals , three iThe electron density in the vicinity of Zn2 in molecule 1 is shown in Fig. Figure Additional file Figure As discussed previously in the ultra-high resolution X-ray structure of Insugen (I) in the internal disulphide bridge of chain C (CysC6–CysC11) CysC11 is disordered into two sites: A (80%) and B (20%), Figs. (™) [In order to prepare for the molecular dynamics (MD) simulations, two pdb files, Cys11_80percent.pdb and Cys11_20percent.pdb were generated from the original high resolution crystal structure by editing the atom records for Cys11, generating two separate pdb files, one with the chain C CysC6–CysC11 disulphide in the major (80%) conformation, the other with the minor (20%) conformation. Following this, both structures, including water molecules in the crystal structure were subjected to energy minimisation using HyperChem 8 Professional(™) . Energy (™) , using t(™) , with onAs described below MD simulations were then performed. Two MD simulations were run for each pdb file. The first simulation was run at 310 K for 300 ps, using an initial heat time of 5 ps, with data collected every 0.01 picoseconds, with a time step of 0.002 ps, using NVT dynamics with a Berendsen thermostat . The secThe results of both simulations showed several notable changes regarding torsion angle χ3 of the internal Cys6–Cys11 disulphide bonds, where χ3 is defined by the atoms Cβ6–Sγ6–Sγ11–Cβ11. At the lower temperature (310 K) in the major conformation CysA6–CysA11 of chain A underwent a conformation change around 8 ps, decreasing from about 120° to 50°, and then increased marginally before staying relatively constant between 60° and 80°. Chain C stayed relatively constant between 90° and 120°, Fig. The structure of Intergen (II) was also minimised using the method described above for Insugen (I), but was not split into two pdb files beforehand as no disordering was modelled for this structure, Fig. . FollowiThe results for the simulations run at 310 K showed torsion angles χ3 for both Cys 6–Cys 11 in chains A and C largely remaining relatively steady around 80°–100°, except for some transient spikes above or below this range Fig. a.Fig. 24Upon repeating the simulations at the higher temperature of 320 K, torsion angle χ3 for CysA6–Cysa11 in chain A started off mostly steady around 80°–100° up to about 35–55 ps, and then temporarily sharply decreased before increasing again and then remaining relatively constant and mostly steady around 80°–100° for the rest of the stimulation, except for some transient spikes. CysC6–CysC11 of chain C did not show any noticeable changes at the higher temperature, and remained relatively constant around 80°–100°, except for some transient spikes, which is similar to the result obtained for 310 K Fig. b. The RMFrom the results of both the torsion angle plots and the RMSD kinetics for the Cys6–Cys11 SS bonds for Insugen (I), it is clear that both the Cys6–Cys11 internal disulphide bridges in chains A and C possess flexibility. However the flexibility of Cys6–Cys11 of chain C appears to be much greater, as both the MD simulations for the minor conformation at 310 K and the major conformation show times when the torsion angle of Cys6–Cys11 of chain C shows rapid decreases followed by rapid increases. In contrast, Cys6–Cys11 of chain A only showed one major change, in the major conformation at 310 K, at all other times staying relatively constant. The rapid changes in torsion angles shown by Cys6–Cys11 of chain C of Insugen (I) would appear to explain why it shows disorder in the original crystal structure.From a structural point of view, examination of the secondary structures of chains A and C in the original crystal structure of Insugen (I) may provide an explanation for this increased flexibility. The structure of these chains consists of a single loop between two α-helices. The length of the loop shows differences in chains C and A. In chain C the loop is long enough to contain both Cys residues involved in the internal disulphide bridge, whereas in chain A it is shorter and so one of the Cys residues is located on an α-helix. The longer loop of chain C would be more flexible and therefore may possibly allow for more movement of the Cys residues involved in the disulphide bond. Over the course of the MD simulation changes in secondary structure occur, most noticeably in chain C, with significant portions becoming converted to coils, which may further affect flexibility Fig. . In contFrom the results of both the torsion angle plots and the RMSD kinetics for the Cys6–Cys11 S–S bonds of Insugen (I), it is clear that both the Cys6–Cys11 internal disulphide bridges in chains A and C of possess flexibility. However the flexibility of Cys6–Cys11 of chain C appears to be much greater, as both the MD simulations for the minor conformation at 310 K and the major conformation show times when the torsion angle of Cys6–Cys11 of chain C shows rapid decreases followed by rapid increases. In contrast, Cys6–Cys11 of chain A only showed one major change, in the major conformation at 310 K, at all other times staying relatively constant. The rapid changes in torsion angles shown by Cys6–Cys11 of chain C of Insugen (I) would appear to explain why it shows disorder in the original crystal structure.From a structural point of view, examination of the secondary structures of chains A and C of Insugen (I) in the original crystal structure may provide an explanation for this increased flexibility. The structure of these chains consists of a single loop between two α-helices. The length of the loop shows differences in chains C and A. In chain C the loop is long enough to contain both Cys residues involved in the internal disulphide bridge, whereas in chain A it is shorter and so one of the Cys residues is located on an α-helix. The longer loop of chain C would be more flexible and therefore may possibly allow for more movement of the Cys residues involved in the disulphide bond. Over the course of the MD simulation changes in secondary structure occur in Insugen (I), most noticeably in chain C, with significant portions becoming converted to coils, which may further affect flexibility.The ultra-high resolution X-ray structures of two forms of human recombinant insulin, Insugen (I) and Intergen (II), has revealed several quite unexpected and previously unpredicted features. Both Insugen (I) and Intergen (II) structures exhibit structural features that can be described as: (a) highly ordered; (b) clear and resolved double conformations; (c) badly disordered. The assembled molecule comprises polypeptide chains A, B, C and D where A and C are sequence equivalent, as are B and D. It is somewhat surprising that the occurrence of structural features (a), (b) and (c) between say Insugen (I) chains A and C is by no means one to one but rather almost lacking in correspondence. This observation applies to all pairs of like polypeptide chains in both Insugen (I) and Intergen (II) and to all pairs of like polypeptide chains one from Insugen (I) and one from Intergen (II). It would be of interest (1) to find explanations for these differences and (2) to know whether they affect the therapeutic properties of these preparations? As described in “6 human insulin (Lilly Research Laboratories) at 120 K and 1.0 Å resolution [GlnB4, ValB12, GluB21, ArgB22, ThrB27 and LysD29; human GlnB4, ValB12, GluB17, GluD21, GluC5, LeuC16, ValD12, ValD18 and GluD21. Only two residues are in common in this list: GlnB4 and ValB12. This result follows the trend reported here for Insugen (I) and Intergen (II) that correspondence between the two structures with respect to multiple or disordered conformations does not follow any fixed pattern. However, interestingly, reference to Fig. GlnB4 and ValB12 presented problems in the interpretation of their electron densities. Of the other residues in the above porcine list some are clear single conformations and others are problematic in either Insugen (I) or Intergen (II). Similar comments apply with respect to the above list for biosynthetic form of T6 human insulin. GlnB4 and ValB12 are the only residues in all four of these insulin structures that presented problems. According to Fig. ValB12 is significantly involved in the interaction of insulin with its receptor.Previous studies: (i) that of Baker et al.  at room solution  have revsolution  list sevA significant example of structural differences in the ultra-high resolution 100 K structures of Insugen (I) and Intergen (II) can be found in the phenylalanine residues PheB24 and B25 see Fig. . As statThe significant observation here is the extremely poor electron density for PheB25 in Insugen (I) which has been modelled as disordered with two distinct conformations, as opposed to PheB24 in Insugen (I) and PheB24 and PheB25 in Intergen (II) which are all excellent examples of strong, clearly resolved single conformation electron density. It is of interest to note that PheB25 in the X-ray structure of porcine insulin  has compThe intra-chain S–S bridge in chain C(3) Cys6–Cys11 has been observed in Insugen (I) to exhibit two ordered conformations. Cys6 occupies a single site while Cys11 occupies two sites with relative occupancies of 0.8 and 0.2, respectively. The geometry and all other refinement characteristics of this bifurcated cysteine bridge are of excellent quality as discussed previously. The corresponding S–S bridge in Insugen (I) chain A(1) is completely ordered which again poses a question about the origin of the distinction between the two molecular dynamics simulations. The ultra-high resolution Insugen (I) X-ray structure was found to include an ordered solvated propanol molecule which forms H-bonds with the prominent OG1A of ThrD27 in chain D(4) and two water molecules. The electron density for this solvate is clear and the geometry of the refined propanol is excellent. There is no solvated propanol in Insugen (I) chain B(2) or in either chain B or D in Intergen (II). These differences again offer a challenge to a rational explanation. The origin of the solvated propanol in Insugen (I) may be questioned. However it is known that propanol is a minor component used in the manufacturing process and is most likely to have been introduced into the protein at some stage of the synthesis procedure. To the best of our knowledge no other insulin structure has been shown to include structurally ordered propanol.Insugen (I) and Intergen (II) have been synthesised to include the essential Zn ions present in naturally occurring insulins. The Zn ions are an essential feature in the formation of the crystal structure and are located on a crystallographic three-fold axis.The electron density in the vicinity of Zn2 in molecule 1 revealed an unexpected feature which was shown to be a solvated acetate molecule. Zn2 is coordinated to both His10B Nε in chain B and an oxygen atom of the acetate. It is most likely that the presence of solvated acetate originated during crystallization. There are no other solvated acetate sites in either Insugen (I) or Intergen (II).


E)-2-(4-(4-(bis(pyridin-2-ylmethyl)amino)styryl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene)malononitrile) with strong donor–π-acceptor structure was synthesized, which showed both colorimetric and fluorescent sensing ability toward Cu2+ with high selectivity and sensitivity. Job plot and mass spectra measurement revealed a 1 : 1 coordination mode between Cu2+ and probe BT in ethanol/HEPES (1 : 4 v/v) buffer (pH 7.2) solution, and the binding constant was calculated to be 3.6 × 104 M–1. The colour of BT solution (10 µM) immediately turned from purple red to yellow and the red fluorescence was quenched obviously when a certain amount of Cu2+ was added, which enabled a dual-channel detection of Cu2+. A paper strip pre-stained with BT solution was further fabricated and it also showed excellent sensing ability toward Cu2+ with a detection limit as low as 10−6 M with the naked eye, which represents better portability and operation simplicity that is favourable for on-site analysis of Cu2+ in water.In this paper, BT (( ESI-HRMS: [M+H]+, [C30H25N6O]+, calcd, m/z = 485.20; found, m/z = 485.23.The synthesis route of BT is shown in 3.3.1.a). The absorption maximum was at 558 nm which made BT solution show light purple red colour. The fluorescence spectrum revealed a maximum emission at 636 nm, which is red fluorescence owing to its low bandgap. And the fluorescence quantum yield of BT in ethanol/HEPES (1 : 4 v/v) buffer (pH 7.2) was calculated to be 0.13 using rhodamine B as a reference. To test the selectivity of BT to Cu2+ ions, 16 kinds of other metal ions including Ag+, Al3+, Ba2+, Ca2+, Cd2+, Cr3+, Fe2+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+ were chosen as contrast. Five equivalents of metal ions were added separately to BT solution, then the absorption and emission spectra were measured immediately and the results are shown in 2+ could change the spectra obviously with absorption maximum blue-shifted by 141 nm to 417 nm with sharp contrast. The colour change could be observed obviously by the naked eye (b inset). The phenomenon was ascribed to the decreased ICT effect between BPA unit and TCF unit, owing to the strongly weakened electron-donating ability of aniline N atom in BPA upon coordination to Cu2+. Although Fe3+ can make the absorption peak blue-shifted by 31 nm, the colour change of solution was not so obvious in that it cannot be clearly distinguished by the naked eye. BT solution with Ni2+ showed a weak shoulder band at 417 nm; however, the absorption maximum was still at 558 nm and the colour of solution was not changed. These results clearly demonstrated that BT was highly selective towards Cu2+ in colorimetric method. The fluorescence changes of BT solution were also determined after addition of metal ions. As shown in figure 2c, only Cu2+ could quench the fluorescence emission of BT efficiently with fluorescence intensity decreased by 15 times. Other metal ions did not affect the fluorescence obviously, except that Ag+ showed certain disturbance. There may be some interactions between pyridine unit and Ag+, thus photon-induced electron transfer may occur and affect the fluorescence emission of BT. The influence of Ag+ or other metal ions except Cu2+ on fluorescence of BT is not so obvious that it cannot be perceived by the naked eye when the solution was irradiated by a UV lamp with light of 365 nm as shown in figure 2d. Therefore, BT could also act as a fluorescence turn-off probe for Cu2+ with good selectivity.Solution of BT in ethanol/HEPES buffer was carefully prepared with a concentration of 10 µM. Owing to the ICT effect from the electron donor (BPA) to the electron acceptor (TCF), BT covered a wide absorption range a. The abaked eye b inset. 3.2.2+. As shown in figure 3a, although Fe3+ can make the absorption peak slightly blue-shift and Ni2+ showed some background absorbance, they still did not affect the colorimetric recognition ability of the probe to Cu2+. As shown in figure 3b, Cu2+ can quench the fluorescence of BT solution efficiently even if the other metal ions are present. And it is interesting to note that the coexistence of most of the metal ions except Fe3+ strengthened the recognition ability toward Cu2+, as the change in fluorescence intensity was enhanced. In general, the BT probe demonstrated good anti-interference ability when detecting Cu2+ in both colorimetric and fluorescent modes.Competition experiment was performed to further confirm the selectivity of BT toward Cu3.3.2+ ions, we investigated the coordination mode between them. The stoichiometry for the binding between BT and Cu2+ was studied by Job's plot. We maintained the total concentration (C0) of BT and Cu2+ unchanged, then altered the Cu2+ content  solution. This coordination mode was further confirmed by mass spectrum of the mixed solution of BT and Cu2+ as shown in figure 4b, where the molecular ion peak of [BT–Cu2+] could be found .As the selectivity of the probe has been confirmed, to further understand the recognition nature of BT to Cu3.4.2+ to BT solution, and both the absorption and fluorescence spectra were recorded. As shown in figure 5a, in the UV–visible spectra, the absorbance at 558 nm decreased, while that at 417 nm increased with the addition of Cu2+, revealing the formation of new complex between BT and Cu2+. From the normalized absorption signal response to the concentration of Cu2+ (figure 5b), the detection limit of colorimetric method was calculated to be 2.4 × 10−7 M [figure 5c, the fluorescence intensity decreased gradually with the addition of Cu2+, and the curve became smoother when more than 2 equivalents of Cu2+ was added. The fluorescence intensity was linearly related to the concentration of Cu2+ from 1 to 10 µM, and the fluorescence detection limit was 1.02 × 10−7 M that was calculated on the basis of 3σ/k (σ is the standard deviation of the blank measurement and k is the slope of a plot of the fluorescence intensity versus Cu2+ concentration). The association constant of BT–Cu2+ was calculated to be 3.6 × 104 M−1 using Benesi–Hildebrand analysis [The titration experiment was performed by adding various amounts of Cu× 10−7 M . In the analysis  (see ele3.5.F0) and after (F) addition of two equivalents of Cu2+ was separately collected. As shown in F0/F demonstrated obvious contrast with value more than 5 in the pH range of 5.0–8.5, manifesting that the probe can work well in this range. Too acidic an environment may protonate the aniline N and pyridine moiety which reduces the coordination ability of BPA group, and too basic an environment may reduce the free Cu2+ ions in solution and influence the sensitivity.The performance of probe BT was tested in various pH environments. The fluorescence signal of BT solution before  in acetone for one minute, and then the paper was dried in air. For testing the performance of the strips, they were separately immersed into various concentrations of Cu2+ each for only one second. Obviously, the colour change could be observed by the naked eye immediately even if the concentration of Cu2+ was as low as 1 × 10−5 M (a). Fluorescence signal was also collected with the strip irradiated by a UV lamp, and the detection limit of this method can reach a level of 1 × 10−6 M (b). Different Cu2+ concentrations could give different signal changes in both colorimetric and fluorescence modes using this paper strip, revealing that the Cu2+ strip prepared here could work well.Probe BT showed high selectivity and sensitivity to Cu× 10−5 M a. Fluore× 10−6 M b. Differ4.2+ constructed by BPA and TCF moieties was successfully synthesized with a wide absorption range and red emission. It showed high selectivity and sensitivity toward Cu2+ in ethanol/HEPES (1 : 4 v/v) buffer (pH 7.2) solution in both colorimetric and fluorescent modes. A paper strip was further fabricated easily by dipping common filter paper into BT solution, and the test results also showed good recognition ability to Cu2+, which makes the BT probe more portable and convenient. As a result, real-time and naked eye detection of Cu2+ ion could be realized by using a Cu2+ strip like the pH strip, which is useful in environment monitoring and water analysis.A BT probe for the detection of Cu


Herein, we present a computational study on the [(TPB)FeN2]− complex and describe its conversion into ammonia through the addition of electrons and protons. In particular, we tested the consecutive proton transfer on only the distal nitrogen atom or alternated protonation of the distal/proximal nitrogen. It is found that the lowest energy pathway is consecutive addition of three protons to the same site, which forms ammonia and an iron‐nitrido complex. In addition, the proton transfer step of complexes with the metal in various oxidation and spin states were tested and show that the pKa values of biomimetic mononuclear nitrogenase intermediates vary little with iron oxidation states. As such, the model gives several possible NH3 formation pathways depending on the order of electron/proton transfer, and all should be physically accessible in the natural system. These results may have implications for enzymatic nitrogenases and give insight into the catalytic properties of mononuclear iron centres.Nitrogenases catalyse nitrogen fixation to ammonia on a multinuclear Fe‐Mo centre, but their mechanism and particularly the order of proton and electron transfer processes that happen during the catalytic cycle is still unresolved. Recently, a unique biomimetic mononuclear iron model was developed using tris(phosphine)borate (TPB) ligands that was shown to convert N By using electron paramagnetic resonance (EPR), Mössbauer and extended X‐ray absorption fine structure (EXAFS) spectroscopy, several nitrogen‐based intermediates were trapped and characterised including a doubly protonated hydrazido complex.2O)2H+ as an acid in tetrahydrofuran solvent.Thus, to understand the structural and functional features of enzyme active sites, biomimetic models are often developed that mimic the reactive centre; that is, the metal with its ligand coordination features, but these are then studied in solution.2 , so that it can split off an ammonia molecule and leave an iron‐nitrido complex. Scheme 1. The first step considers single proton transfer to either the distal nitrogen (via TS1d) or the proximal nitrogen atom (via TS1p) to form the singly‐protonated species 2d and 2p, respectively. The next proton transfer onto 2d gives an [FeNHNH] (3dp) or [FeNNH2] (3dd) complex via transition states TS2p or TS2d. Proton transfer towards 3dp and 3dd can give the ammonia complex 4ddd or the [FeNHNH2] complex 4dpd and their mechanistic pathways were studied here as well. Thus, we consider consecutive proton transfer as well as an alternating proton transfer, in which the first proton is donated to the distal nitrogen atom, the second proton to the proximal nitrogen atom (Np), the third proton to the distal nitrogen atom again, etc.Currently, little is known on the proton‐ and electron‐transfer processes in nitrogenases and particularly lacking is the order in which these processes happen. The same is true for the synthetic mononuclear model shown in Scheme 2 on a mononuclear iron centre, we performed a computational study, which may have further relevance to the mechanism of nitrogenase enzymes. Our comprehensive DFT study on the proton‐ and electron‐transfer mechanisms for reduction of N2 to ammonia considers a large variety of possible processes ranging from consecutive proton transfer to alternating proton/electron transfer steps. These studies give valuable insight into the nitrogen fixation reaction using the mononuclear iron model from Scheme In addition, each proton‐transfer step can be followed by electron transfer directly. Clearly, the mechanism of nitrogen reduction to ammonia on a nonheme iron centre can take place through many possible reaction pathways. To gain insight into the mechanism of ammonia production from N2]− complex shown in Scheme −1, respectively. Geometrically, the doublet and quartet spin states have a linear Fe‐N‐N group, whereas it is bent (by 137°) in the sextet spin state. Although we calculated the lowest three spin states for each structure, we will focus on the lowest lying one in the main paper only; the results covering all other spin states are deposited in the Supporting Information.The study presented in this work started from the x+y−1+ complexes with x=0–3 and y=0–3 and assume that only proton transfer reactions take place. We considered complexes with the distal as well as proximal nitrogen atom protonated. However, the structures with a value of x>y, that is, more protons bound to the proximal nitrogen than to the distal nitrogen atom, either failed to converge or, during the geometry optimisation, were reoriented in such a way that the nitrogen atom with the most number of protons became the terminal nitrogen atom (Nd). Therefore, the [(TPB)FeNp(H)Nd]0 structure (2p) is unstable and its geometry optimisation gave [(TBP)FeNNH]0 (2d) instead. Consequently, the first proton transfer can only take place on the distal nitrogen atom. Structure 2d has close lying doublet and quartet spin state structures  and hence will react through two‐state reactivity on competing doublet and quartet spin surfaces, similar to what was seen for mononuclear iron(IV)‐oxo complexes with heme and nonheme ligand environments.2d and located two local minima, namely [(TPB)FeNp(H)Nd(H)]+ (3dp) and [(TPB)FeNpNdH2]+ (3dd), whereby the latter is 2.9 kcal mol−1 lower in energy than the former in the quartet spin state.We first considered the mechanism of three consecutive proton‐transfer reactions starting from the iron‐dinitrogen complex d with EtO2H+ as t2O)2H+ from 22d leading to these structures using isolated species according to reactions 1 and 2 and find values of ΔE+ZPE=−14.6 kcal mol−1 for Equation −1 for Equation −1. Consequently, the kinetics for both pathways was considered here.We then calculated the driving force for proton transfer from ‐tosylimido complexes calculated previously.−1) and the doublet spin state by 30.7 kcal mol−1. The final dissociation of ammonia from the 44ddd complex only costs 5.4 kcal mol−1 at the ΔE+ZPE level of theory through the breaking of the hydrogen bond between ammonia and the iron(IV)‐nitrido group. We also tested the proximal protonation of 3dd (as will be discussed in the next section) and found it to be similar in energy.In the final proton‐transfer step in Scheme 1, 2d, 3dd, 3dp and 4ddd, and included a proton source as used in Ref. 2O)2H+, to the complex. The structures with (Et2O)2H+ included in the model are labelled with RC in subscript after the name and have the same spin state ordering and relative energies as the isolated structures. Thus, 1RC represents [1‐(Et2O)2H+], 2RC is [2‐(Et2O)2H+]+ etc.Subsequently, we investigated the kinetics of the proton‐transfer reactions displayed in Scheme 1 in the absence of an electron donor. For each of the individual reactant complex structures, that is, 1RC, 2d,RC, 3dd,RC, 3dp,RC and 4ddd,RC, on the doublet, quartet and sextet spin states a geometry scan for proton transfer was performed, whereby one degree of freedom (the reaction coordinate) was fixed and all other geometric degrees of freedom minimised. The maxima of these geometry scans were subjected to a transition state search and gave us the proton‐transfer transition states for each of the reaction steps. Thus, 2,4,6TS1d are the transition states for the proton transfer from (Et2O)2H+ to 2,4,61 at the distal nitrogen atom.Figure 2O)2H+ to [(TPB)FeN2]− to give the [(TPB)FeNNH] and two diethyl ether molecules. Optimised geometries starting from the end point of the geometry scans originating from 2,4,61RC resemble the 2,4,62d optimised structures  and confirm the pathway from 1 to 2d. Subsequently, we took the 2,4,62d structures and added the proton donor group (Et2O)2H+ and optimised 2,4,62d,RC. Thereafter, the proton‐transfer pathway was investigated and led to the transition states for distal protonation. The energy landscape described in Figure 21. The first proton transfer to [(TPB)FeN2]− takes place on the doublet spin state via 2TS1d and has an energy of 4.9 kcal mol−1, which is lower in energy than the nearest quartet and sextet spin states. By contrast, optimised geometries of structures 2 implicate them to be close in energy on the doublet and quartet spin state surfaces. Consequently, during the lifetime of structure 2 it is likely that full equilibration will lead to a mixture of doublet and quartet spin state structures. Geometrically, 21 and 22d structures contain very similar Fe‐N and N‐N distances and hence their electronic configuration is the same.The first pathway investigated relates to the proton transfer from 2H+ by a deuteron may increase the barriers slightly but not by a huge amount.2TS1d is located at a relatively large distance of 2.02 Å, which appears unusually long. However, as can be seen from the structure, 2TS1d reflects the simultaneous breaking of the Et2OH+⋅⋅⋅OEt2 hydrogen bond and the transfer of the proton to Nd. These two simultaneous steps prevent a closer approach of the proton to Nd.The optimised geometry of 2,4,62d can again lead to distal and proximal protonation to form the isomeric complexes 2,4,63dd and 2,4,63dp, respectively. However, despite the fact that the geometries of 2,4,63dp could be optimised, no valid reaction pathways were identified for direct proton transfer from (Et2O)2H+ to the proximal position of 2,4,62d. In particular, all geometry scans for proximal protonation of 2,4,62d failed, and led to distal protonation transition states instead. Clearly, distal protonation of 2 is energetically and kinetically favoured over proximal protonation.The reaction step from 2,42d leads to 2,43dd via a barrier 2,4TS2d with values of 6.4 (doublet) and 5.0 (quartet) kcal mol−1 with respect to their precursor local minima. These structures, similar to 2TS1d, have a small imaginary frequency of i197 (i190) cm−1 for 2TS2d (4TS2d), respectively. Also in these structures the transferring proton is still located at a large distance; hence, the structures are early on the potential energy surface. Previously,Distal protonation of 3dd from 2d leads to significant changes in bond lengths along the Fe−N and N−N interactions. To be specific, the Fe−N bond shortens to 1.718 Å in 23dd as a result of increasing double bond character in the Fe−N interaction. At the same time, the N−N bond elongates to well over 1.32 Å and loses double bond character. We find the quartet spin state to be the ground state of 3dd and 3dp with the doublet and sextet spin states well above the quartet spin state by 17.2 and 14.7 kcal mol−1, respectively. These results appear to contradict experimental observations that implicated a doublet spin ground state.3dd−), which we also calculated has a doublet spin ground state. It could very well be, therefore, that the experimentally characterised EPR spectrum may reflect the doubly reduced species instead. Further work will be necessary to establish this.Formation of intermediate 3dd is on the quartet spin state surface with a barrier (4TS3d) of about 15.0 kcal mol−1. Therefore, the energetic barrier for the third consecutive protonation step is considerably larger than the first two proton transfer barriers. Furthermore, the reaction from 43dd leading to 44ddd is endothermic by 4.0 kcal mol−1. Structurally, it is akin to 43dd with Fe‐N and N‐N distances of 1.819 and 1.325 Å. Similar to the 2TS1d and 2, 4TS2d structures discussed above, the donating proton is at a relatively large distance from Nd of well over 2 Å and has a small imaginary frequency of i148 cm−1. After passing the proton transfer transition states 2,4,6TS3d, the geometry scans fall to complex 4ddd, which is an iron‐nitrido with a weakly bound ammonia molecule. This complex dissociates ammonia to form the final products.The lowest lying transition state for protonation of 2]− in a highly exothermic reaction pathway. Therefore, triple protonation without electron donation is a feasible process for this mononuclear nonheme iron complex. It may very well be, therefore, that in the multinuclear FeMoco cluster, binding of N2 can also be followed by triple protonation without electron transfer. However, reduction steps could speed up the process and hence we investigated that possibility.In summary, the calculations shown in Figure 2,4,62p; however, we did succeed in optimising the structures of 2,4,63dp and 2,4,64dpd . We therefore decided to investigate the proton transfer pathways leading to these intermediates. Unfortunately, in analogy to the problem associated with locating 2TS1p, we also failed the geometry optimisations of 2,4,6TS2p, where the proton during the geometry optimisations moved to the distal nitrogen atom instead. These transition states, therefore, must be considerably higher in energy than those leading to 2,4,6TS2d.As noted above, we failed to optimise 2,4,6TS3′d) for the distal protonation of 2,4,63dp as well as those for proximal protonation from 2,4,63dd FeN(H)NH2]2+ (44dpd) products. Structure 3dp, in analogy to 3dd, has a quartet spin ground state that is separated from the sextet and doublet spin states by 10.8 and 23.1 kcal mol−1, respectively. The proton‐transfer barrier (via 4TS3′d) is about 4.5 kcal mol−1 in energy (ΔE+ZPE) above the value of 43dp and its structure has long Fe‐N and N‐N distances of 1.941 and 1.331 Å, respectively. Similar to the transition states reported above, the imaginary frequency is small (i285 cm−1). Therefore, once the [(TPB)FeN(H)NH]+ structure is formed, it can rapidly pick up a proton to form the [(TPB)FeN(H)NH2]2+ structure along an alternative pathway.Subsequently, we located several transition states (2]+ (3dd)  of only 15.1 kcal mol−1 was found for this process, which is only 0.1 kcal mol−1 higher in energy than distal protonation. Consequently, 43dd can react through either distal or proximal nitrogen protonation with almost equal barriers and hence probabilities. Structurally, the transition states for proximal and distal protonation of 43dd are very similar.The final alternative pathway considered was proximal protonation from  system shows similar trends with larger exothermicity of the proton transfer reaction and lower barrier heights. These studies implicate that although triple protonation is a feasible process without the addition of external electrons, the overall reaction efficiency can be improved by adding electrons to the system. Most probably, the alternating addition of protons and electrons will be the energetically favourable process for nitrogen fixation. However, in the absence of available electron donors, the reaction can still proceed through sequential protonation, albeit considerably slower.As follows from the data shown in Table 21 for alternating and consecutive proton and electron transfer; the results are shown in Figure 3O+/H2O couple or the (Et2O)2H+/2Et2O couple to balance the reaction. However, the H3O+/H2O couple just shifts the relative free energies of proton transfer and makes all reaction pathways much more exergonic. As such, proton transfer will be a very fast process in a water solution. Using the (Et2O)2H+/2Et2O couple the proton transfers are all exergonic but by a mild amount, which means these reaction steps will be kinetically slower and individual intermediates may be trapped accordingly, in agreement with the experimental observations. Nevertheless, the proton transfer trends are the same regardless of the proton donor and the effect is only systematic.To shed more light on the possible reaction pathways for electron‐ and proton‐transfer, we calculated individual reactions starting from 22d0 and the proton transfer pathways are given from left to right and the electron transfer from top to bottom. Thus, proton transfer to form 43dd+ is exergonic by ΔG=−32.8 kcal mol−1 with (Et2O)2H+ as a proton source, whereas electron transfer to give 32d− releases ΔG=−51.2 kcal mol−1. As such, electron transfer is energetically favoured over proton transfer in a diethyl ether solution. The proton transfer energies between 21− and 22d0 using (Et2O)2H+ as proton source matches the value from Figure 43dd+. Overall, the three consecutive proton‐transfer steps from 21− are all exergonic and hence possible at room temperature. However, the proton‐transfer steps are energetically the most favourable from complexes with an overall charge of Q=−1; that is, 21−, 32d− and 23dd−. Most probably the overall charge of −1 gives these complexes high pKa values and make them react fast with external protons. This means that alternating proton and electron transfer should give the most energetically favourable reaction process for conversion of N2 into ammonia and an iron‐nitrido complex.Figure 43dd+ and 54ddd+, which release ΔGET=−80.9 and −96.0 kcal mol−1, respectively. Therefore, the nitrogen fixation reaction is possible through many different reaction pathways of proton and electron donations. We find a thermodynamic viable process of triple proton transfer without electron donation as well as ones where triple proton transfer is coupled to one or two electron transfers.On the other hand, the energetically most favourable reduction steps will be those where a positively charged complex is reduced to a neutral one; that is, 1, 2d, 3dd and 4ddd in detail. Figure 2d and a valence bond description of the electronic configurations of 21 and 2,42d. These valence bond descriptions highlight the molecular orbitals that are formed and broken in each step and the formal charge distributions on the atoms. In particular, in the valence bond drawings, electrons are represented by a dot, and a line between two dots gives a molecular orbital occupied by two electrons. In the past we used these assessments to explain regioselectivities of reaction mechanisms and the origins of bifurcation processes and explained experimental reactivity trends.xy orbital is a non‐bonding orbital in the plane of the phosphorus atoms and has a shape seen before for nonheme iron complexes.xz/πyz, not shown in Figure xz and π*yz) orbitals that cover the Fe, Nd and Np atoms. In blue, we give the four electrons that occupy the πxz and π*xz orbitals and in purple those for the πyz and π*yz orbitals. Even in the reactant complex 21 there is considerable mixing of the iron and N2 π‐type orbitals and, consequently, also charge transfer from nitrogen to metal. In the reactant complex the π*xz and π*yz orbitals are doubly occupied and, as a result, the Fe−N bond length is short and corresponds to a formal single bond with a distance of 1.805 Å. The atomic 3dz2 orbital of iron is involved in a bonding orbital with the 2pz on boron to form the σ*z2 orbital, which also gives considerable charge transfer from iron to boron of Q=−0.42 and a relatively short Fe−B distance of 2.397 Å. The highest lying 3d orbital on iron is singly occupied (σ*x2‐y2) and gives 21 an overall orbital occupation of π*xy2 π*xz2 π*yz2 σ*z22 σ*x2‐y21 and a formal 3d9 configuration. The valence bond structure, therefore, gives eight electrons along the Fe−N bond, representing the occupation of the πxz2 π*xz2 πyz2 π*yz2 orbitals. The Fe−B interaction is formally a single bond through occupation of the σ*z2 orbital with two electrons , whereas the unpaired electron in σ*x2‐y2 is given in red.To understand the electronic changes during the proton‐transfer reaction, we analysed the electronic configurations and orbital descriptions of complexes 2,42, the degeneracy of the π*xz/π*yz couple is lost. Specifically, the 2py orbital on the terminal nitrogen atom splits off from the molecular πyz/π*yz interaction and forms a σNH bond with the 1s orbital of the incoming proton. Electronically, the two electrons that form the σNH orbital both originate from the transfer of one electron from the π*xz and π*yz orbitals into σNH. As a result of this, the quartet spin state drops in energy and becomes the most favourable configuration, with orbital occupation σNH2 π*xy2 π*xz1 π*yz1 σ*z22 σ*x2‐y21. The loss of π*xz/π*yz electrons in 2d leads to elongation of the N−N distance to 1.25 Å in both the quartet and doublet spin states. However, the Fe−B interaction stays intact at a distance of 2.405 (2.534) Å in 42d (22d), respectively, due to the still doubly occupied σ*z2 orbital.Upon protonation of the terminal nitrogen atom, as in 2,42d to form 43dd is electronically depicted in Figure 43dd the N=N double bond is broken and charge density migrates from nitrogen to iron strengthening the Fe−Np bond. As a consequence of strengthening of the Fe−N bond, the Fe−B bond is weakened or broken and the σ*z2 orbital converts into an atomic orbital. It is seen that the Fe−B distance elongates to well over 3 Å in both structures 3 and 4. At the same time, one electron from σ*z2 is promoted into the π*yz orbital to get the overall orbital occupation π*xy2 π*xz1 π*yz2 σ*z21 σ*x2‐y21. In the final proton transfer to the terminal nitrogen atom, an ammonia molecule is formed and the Fe−N distance reduces to 1.688 Å, but the electronic configuration stays the same.The second proton transfer from We present a series of density functional theory calculations on the electron‐ and proton‐transfer processes in a mononuclear iron model of nitrogenase. We calculate barrier heights for proton transfer in the absence of an electron donor and show a feasible process with three consecutive proton transfer barriers in an overall exothermic process. Protonation is favoured on the distal nitrogen atom so that triple proton transfer forms one ammonia molecule and an iron‐nitrido complex. Tests on proximal protonation find barriers that are higher in energy due to stereochemical interactions and shielding of the proximal site. We finally tested proton transfer from complexes with different overall charge and show that the energetically favourable process is consecutive proton and electron transfer. However, alternative pathways are energetically accessible and cannot be ruled out. The calculations point out that proton and electron transfer do not necessarily need to happen consecutively because the energetics are also favourable for multiple proton transfer without electron transfer. As such, multiple pathways for the formation of ammonia from molecular nitrogen are possible, which give the enzyme sufficient flexibility to operate under low acid or low reduction conditions.2]− structure as described by Peters et al.,2O)2H+. As the overall charge of our chemical system ranges from −1 to +2 we did full geometry optimisations with a solvent model included; namely, the polarised continuum model (CPCM),E+ZPE data with energies at BS2 and zero point corrections with BS1.All computational studies presented here used density functional theory methods as implemented in the Gaussian 09 program package,−6 hartrees and neither did the optimised geometries change significantly. The reproducibility of the calculations was investigated by reoptimising the proton‐transfer reaction from 21 to 22 with the PBE0 density functional method.To test the accuracy of our optimisation algorithm, we repeated the geometry optimisations of local minima and some of the transition states using tight convergence criteria in Gaussian. In none of these cases; however, did the energy change by more than 10The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The single crystalline structures were obtained both in case of 0D  SnO2 powders and in case of 0D nanofibers, as confirmed by electron diffraction of TEM. It was found that SnO2 synthesis parameters significantly affect materials’ properties by contributing to the difference in morphology, texture formation, changes in IR spectra of 1D structure as compared to 0D powders, increases in the specific surface area of nanofibers, and the alteration of current-voltage characteristics 0D and 1D SnO2 nanostructures. It was established that gas sensors utilizing of 1D nanofibers significantly outperform those based on 0D powders by providing higher specific surface area and ohmic I–V characteristics.Zero- and 1D  tin (IV) oxide nanostructures have been synthesized by thermal evaporation method, and a comparison of their morphology, crystal structure, sorption properties, specific surface area, as well as electrical characteristics has been performed. Synthesized SnO For the synthesis of 0D and 1D SnO2 nanostructured materials, 2 g of SnC2O4 powder was loaded into 50 × 10 × 10 mm alumina boat each, which were placed inside of a quartz tube in two different horizontal-type furnaces. Two furnaces had identical settings with the only exception that one furnace provided faster heating rate of 80 K/min, and a second furnace provided only 20 K/min heating rate. The N2 gas with 0.005% O2 impurity content was used as an inert atmosphere that was supplied to the quartz tube before heating began [2 was obtained due to following reactions [Tin (II) oxalate (SnC4) as in , was useng began . Both fueactions :\documen2 sample with the fast heating rate was marked as TO1, and the SnO2 sample with slow heating rate was named TO2.The SnO2 samples. The powdered samples were scanned from 20 to 80 2θ at 1°/min with a scanning step of 0.0001°. XRD patterns were analyzed by the PDXL software package using database ICDD/PDF-2 and COD. The crystalline size and lattice parameters of the materials were calculated automatically by the software.In X-ray diffractometer Ultima IV  with CuКα radiation at 40 kV, 30 mA was used to collect diffraction patterns of the SnO2 samples.Both Transmission Electron Microscopy PEM 100–01  and Scanning Electron Microscopy REM 106I  were used for characterization of particle’s size and morphology of the obtained SnOSpecific surface area of the samples was studied by adsorption/desorption of nitrogen  using Langmuir isotherm and Brunauer-Emmett-Teller (BET)-based software.−1 wavenumber spectra of SnO2 were collected using FTIR spectrometer . For spectra collection, SnO2 samples were mixed with pre-dried KBr  at 1:30 SnO2/KBr ratio.IR 4000–400 cm2 nanostructures were carried by using gas sensors developed utilizing a special test structures. A 6 × 10 × 2 mm crystalline glass ceramic material  [2 material was deposited on the top of the Ni electrodes. The deposition of sensitive layer was performed by the sedimentation on the surface of electrodes. For this purpose, SnO2 1 g/cm3 suspension in ethanol was prepared and stirred in ultrasound bath for 90 s, after which the complete test structure with deposited interdigitated Ni electrodes, contact pads and parts of Cu wires was fully immersed into the suspension and left overnight to achieve a complete liquid evaporation. After the deposition of SnO2 layer on Ni-interdigitated electrodes of the test structure, the gas sensor was produced.Measurements of current-voltage characteristics of SnOUkraine)  was usedThe block diagram of the electrical circuit with resistance connected in parallel used to measure the electrical properties of gas sensors are shown in Fig. 2 nanostructured materials are shown in Fig. 2 synthesized at 80 K/min faster heating rate  and (101) peaks of SnO2 [2 synthesized at faster heating rate of 80 K/min shows 100/95 ratio of two major hkl peaks; however, for SnO2 synthesized at slower heating rate of 20 K/min, the (110)/(101) peak intensity significantly reversed to 100/125, thus this nanostructure has a strong preferred orientation of growth in <110> crystallographic direction. Therefore, even based on the analysis of X-ray diffraction patterns of SnO2 synthesized at different heating rates, one can conclude that the preferred orientation of the material is different and this will certainly affect the morphology of the SnO2.X-ray diffraction patterns for two SnOate Fig. a, while ate Fig. b. As it  41-1445 . The ave of SnO2 , the SnO2 samples processed at two different heating rates are shown in Fig. 2 sample synthesized at 80 K/min fast heating rate  and SnO2 synthesized at slower heating rate of 20 K/min (TO2). Pursuant to these data, the 1D SnO2 has specific surface area five times higher than 0D SnO2. Thus, the ratio of surface to volume really increases in the 1D nanostructures as compared with 0D particles.Table 2 samples are non-porous; they are consistent with the results of electron diffraction and microscopy. The revealed negligible porosity was caused by gaps between the primary particles  oxide sample is characterized by linear (ohmic) current-voltage dependences. The various nature of curves for 0D and 1D nanostructures related to the different surface to volume ratios. Change in this ratio leads to a change in the I–V behavior of the material. It is known that both surface and bulk conductivities of the SnO2 contribute to the overall conductivity.As seen on Fig. 2 are significantly improved if the material is showing ohmic type semiconducting behavior [In addition, it is known that the ohmic behavior of current-voltage characteristics is very important for the sensing properties of the material, as the sensing properties of SnObehavior . Therefo2 of different morphology  and one-dimensional (1D) nanostructures) were obtained by thermal evaporation method. Such significant difference in the morphology of the SnO2 nanostructures were achieved due to their different synthesis conditions, as it was found that slower heating rate during the thermal evaporation brings changes to the SnO2 morphology allowing to receive 1D nanofibers. The comparison of different properties of 0D and 1D SnO2 nanostructures is presented. It was determined that the morphology has significant impact on the structural and functional properties of SnO2 as it is reflected in changes in crystal structure where texture formation was recorded, variation of IR spectra, as well as different I–V characteristics of gas sensors based on 0D and 1D SnO2 structures. It was also established that considerable changes in behavior of SnO2 depends also on surface to volume ratios of nanostructures.The single crystalline particles of SnOBased on the experimental data, 1D nanostructures are more desirable for use in gas sensors. Further comparative research of 0D and 1D nanostructures will be carried out regarding sensory properties.


The author wishes to make the following corrections to this paper .The published The order of sub-graph in On page 27, line 5, “Figure 2a” should be “Figure 3a”.On page 32, the caption for Figure 17 “V101 (red) and R15 (blue)” should be “V101 (blue) and R15 (red)”.The authors regret any inconvenience or misunderstanding caused by these errors. The manuscript will be updated and the original will remain available on the article webpage.


The synthesis of acridanes and related compounds through a Cu‐catalysed radical cross‐dehydrogenative coupling of simple 2‐[2‐(arylamino)aryl]malonates is reported. This method can be further streamlined to a one‐pot protocol involving the in situ fomation of the 2‐[2‐(arylamino)aryl]malonate by α‐arylation of diethyl malonate with 2‐bromodiarylamines under Pd catalysis, followed by Cu‐catalysed cyclisation. Within the wider pantheon of C–H activation, cross‐dehydrogenative couplings (CDC) have proved to be versatile procedures for the selective formation of C–C bonds from two different C–H systems under oxidative conditions.2P potassium channels1, Figure 2), as NADH analogues in hydride‐transfer reactions,3), as photoswitchesH‐dibenzoazepines or 5,11‐dihydro‐10H‐dibenzodiazepines by ring expansion.9,10‐Dihydroacridines (acridanes) have garnered much attention4 or acridinium salts 5 aryl]malonates 9 .In the event, l Scheme . First, 15a was then examined. Pleasingly, treatment of 15a under the conditions previously reported delivered the desired acridane 16a in 87 % yield without the need for further optimisation  were prepared, which opens the possibility for further manipulations of the cyclised products (vide infra). The Fmoc‐protected ester 16e was crystalline, which allowed unambiguous confirmation of its structure through X‐ray crystallographic analysis (see the Supporting Information).By following this successful initial result, the generality of this new Cu‐catalysed approach to acridanes was explored Scheme . First, 16g–j in good to excellent yields. Incorporation of a nitrogen atom into one of the aromatic rings was also possible; isolation of the corresponding aza‐acridane 16k was achieved in 66 % yield.Next, the introduction of substituents at various positions on either aromatic ring of the acridane skeleton was explored, which gave 16a–k, we extended this method to the synthesis of other related heterocycles of interest, such as xanthenes and thioxanthenes (Scheme 16l–m). However, cyclisation of a linear diaryl ether in the presence of 10 mol‐% Cu(2‐ethylhexanoate)2 under the standard reaction conditions delivered xanthene 16l in a disappointing yield of 33 %. Also isolated was an equal amount of a by‐product, identified as ethyl 2‐oxo‐2‐(2‐phenoxyphenyl)acetate, which arises from competing decarboxylation and aerial oxidation of the starting material. This problem was exacerbated in the case of thioxanthene 16m, which was isolated in only 20 % yield along with 53 % of the undesired by‐product. Further optimisation of these reactions is clearly required but fortunately, in both cases, the yield of the oxidation by‐products could be minimised by performing the cyclisation under an atmosphere of argon, which leads to formation of xanthene 16l and thioxanthene 16m in 47 and 44 % yields, respectively. While formation of the desired products 16l–m is enhanced under these conditions, performing the reaction under argon necessitates the use of 2.5 equiv. of Cu salt to allow the reaction to proceed to completion 4 to give 19 and thereby generate a new quaternary carbon centre, again in excellent yield. Finally, hydrogenolysis of the benzyl protecting group in 16b delivered acridane 20 bearing a free N–H group in reasonable, un‐optimised yield.In order to demonstrate the utility of the acridanes derived from the Cu‐catalysed cyclisation, a brief study on their further functionalisation was carried out Scheme . For exa16 based on the α‐arylation of 1,3‐dicarbonyl compounds with haloarenes 21 to generate intermediates 15 in situ, which would then undergo the oxidative coupling reaction phenyl triflates 23 in the presence of CsF, and subsequent N‐alkylation  as the catalyst with the bench stable ligand tBu3P·HBF4 (4 mol‐%) in toluene at 70 °C (Table 15n in 61 % (from 21a) and 72 % (from 21b) yields, respectively.No α‐arylation was observed when using the Pd(OAc)N‐methyl‐N‐phenylaniline 21b and diethyl malonate was first carried out by using the optimised conditions described above under an atmosphere of argon. Subsequently, the reaction flask was simply opened to the air, Cu(2‐ethylhexanoate)2 (15 mol‐%) was added and heating continued for a further 24 h to facilitate the cyclisation. In this manner, the target acridane 16n was isolated in a pleasing yield of 74 % over the 2 steps. Furthermore, substituents could be readily introduced on one or both of the aromatic rings, which allows access to more highly substituted acridanes such as 16o and 16p.With conditions for the malonate coupling in hand, our attention then turned to establishing the one‐pot α‐arylation–cyclisation protocol to prepare acridanes directly Scheme . To this2 as the catalyst under mild conditions and thus avoids many of the problems associated with classical strategies for the synthesis of acridanes. The diester moiety resulting from the oxidative coupling reaction serves as a useful handle for further functionalisation. In addition, we have established a streamlined protocol involving the in situ formation of the cyclisation precursor by the α‐arylation of diethyl malonate with a 2‐bromodiarylamine under Pd catalysis, followed by subsequent Cu‐catalysed cyclisation to give the acridanes in a one‐pot fashion. Further studies will be carried out to utilise this new method in target synthesis.In summary, we report a Cu‐catalysed radical cross‐dehydrogenative‐coupling approach to acridanes and related heterocycles from readily available 2‐[2‐(arylamino)aryl]malonates. This highly atom‐economical method uses inexpensive Cu(2‐ethylhexanoate)Representative Procedure for the Cu‐catalysed Synthesis of Acridanes: To a solution of the cyclisation precursor 15 (1.00 mmol) in toluene (10 mL) was added Cu(2‐ethylhexanoate)2 . The reaction mixture was heated to reflux (oil bath at 120 °C) for 17 h with the condenser left open to the air. After cooling to room temp., saturated NH4Cl soln (25 mL) was added, and the aqueous phase extracted with EtOAc (3 × 25 mL). The combined organics were washed with NH4OH soln , dried with MgSO4, filtered and concentrated in vacuo. Purification by flash column chromatography with EtOAc/hexane as eluent afforded the title compound 16 (see the Supporting Information for details).CCDC 1498037 (for 16e) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre.Supporting InformationClick here for additional data file.


However, the lack of fundamental understanding of the reaction mechanism renders designing a selective catalyst inefficient. Here we show the correlation between the kinetics of product formation and those of surface species conversion during CO2 reduction over Pd/Al2O3 catalysts. The operando transmission FTIR/SSITKA  experiments demonstrates that the rate-determining step for CO formation is the conversion of adsorbed formate, whereas that for CH4 formation is the hydrogenation of adsorbed carbonyl. The balance of the hydrogenation kinetics between adsorbed formates and carbonyls governs the selectivities to CH4 and CO. We apply this knowledge to the catalyst design and achieve high selectivities to desired products.Catalytic CO 2 reduction on a catalyst surface is essential for achieving the desired product selectivity. Here, the authors show an operando kinetic analysis of CO2 hydrogenation over a palladium catalyst in order to address the factors governing the selectivity of the process.Understanding the mechanism of CO Under atmospheric pressure, methanation and reverse water gas shift (RWGS) reaction are the two pathways for thermocatalytic CO2 reduction on Group VIII metals2. Although methanation produces synthetic natural gas, RWGS generates CO, an important component of syngas. Considerable efforts have been devoted to developing catalysts to achieve high selectivity towards either CH4 or CO9. However, for designing a selective catalyst, it is very important to first have a fundamental understanding of the site requirements, elementary steps and intermediates, which can ultimately lead to the construction of viable mechanisms for both methanation and RWGS. Although intricate interplay between various surface species at different catalyst components that determine product selectivity has been recognized, controversies still exist regarding the detailed mechanism and the roles of surface species15. Some issues may originate from the different interrogation methods used; for example, some prior conclusions were based on kinetic and spectroscopic information acquired under transient conditions12. Under transient conditions, the perturbation of the chemical environment around the adsorbates and catalyst surface causes characteristically divergent behaviors from steady state.Heterogeneous catalytic CO17. However, SSITKA itself cannot identify the chemical nature of surface species, a task that can be accomplished by operando spectroscopic measurements, e.g., Fourier transform infrared spectroscopy (FTIR). Correlating the reaction kinetics with the transformations of spectroscopically observable surface species, both simultaneously measured under steady-state reaction conditions, is vital for the complete elucidation of the complex mechanistic framework21. Pd-based catalysts are able to catalyze both CO2 methanation and RWGS23. Our previous work on Pd/Al2O3 bifunctional catalysts has proposed the plausible pathways for both CO2 methanation and RWGS24. However, the factors that govern product selectivity (to CH4/CO) under steady-state CO2 reduction conditions are still not clear.Steady-state isotopic transient kinetic analysis (SSITKA) has evolved as one of the most powerful techniques allowing measurements at steady state to determine the mean surface lifetimes and abundances of intermediates leading to products4 and CO selectivities are governed by the balance of the hydrogenation kinetics between adsorbed formates and carbonyls over Pd/Al2O3 catalysts, which was demonstrated by a combined operando transmission FTIR spectroscopy/SSITKA investigation. Using this knowledge, we show that tailor-made catalysts can be prepared to achieve high selectivity to either of the two desired products.Here we show that CH12CO2/H2/Ar to 13CO2/H2 at 533 K. The fast disappearance of Ar gas in <4 s indicates a short gas hold-up time for the system. The disappearance of 12CO2 was almost as fast as Ar, indicating weak interaction between 12CO2 and the catalyst and the reactor walls. In the meantime, the concentration of 13CO2 signal increased accordingly, with the concentration and the conversion (2.5%) of carbon dioxide (12CO2 + 13CO2) constant during the switch. The decaying signals of 12CH4 and 12CO and the rising signals of 13CH4 and 13CO crossed at y = 0.5, indicating constant rates for methane and carbon monoxide formation regardless of isotopic substitution. These symmetrical responses indicate that the steady state of the catalyst surface was not perturbed during the switch. The concentrations of products 12CH4 and 12CO decreased more slowly than that of 12CO2 and disappeared at ~600 s. This indicates large mean residence times for C-containing surface intermediates leading to the products, and that intermediates which equilibrate with CO2 are not involved in the rate-determining steps for CO2 methanation and RWGS.Figure operando FTIR contour plot. Before the switch, the absorption band between 2,300 and 2,400 cm−1 (red range) is attributed to gaseous 12CO2. The spectral range between 1,800 and 2,100 cm−1 (blue and green range) is assigned to chemisorbed carbonyls (∗12CO) on the surface of Pd metal particles22. The absorption bands around 1,400 (yellow range) and 1,600 cm−1 (red range) arise from adsorbed formates (H12COO∗) on the surface of the Al2O3 support22. After the switch, the bands due to 12CO2 quickly disappeared and a new band developed at lower wavenumbers  attributable to 13CO2, consistent with their rapid responses in MS. The IR signal decay of surface species might be slightly affected by the location of IR beam on the sample wafer25. However, the shift of the IR features of 12CO∗ and H12COO∗ to those of 13CO∗ and H13COO∗ at lower wavenumbers were much slower compared with the shift of 12CO2 to 13CO2 bands. This observation suggests that neither H12COO∗ nor 12CO∗ undergoes reverse reactions to form 12CO2. The disappearance trends of the IR signatures of H12COO∗ and 12CO∗ are similar to those of the MS signals of 12CH4 and 12CO products, suggesting that both H12COO∗ and 12CO∗ are reactive intermediates rather than spectators and, the rate-determining steps for CH4 and CO formation are related to CO∗ and HCOO∗. It is noteworthy that no other species  were observed under steady-state reaction conditions and no carbon deposition was identified in our previous study on Pd/Al2O3 during CO2 reduction24. Therefore, CO∗ and HCOO∗ are the two most abundant surface species and will be the focus of the following discussion.Figure ∗CO and Pd on the 5% Pd/Al2O3, room temperature 12CO adsorption followed by temperature-programed ∗12CO desorption were studied by FTIR  and strongly (∗12COs). The stability of ∗COs at high temperatures is due to the dissociated ∗H, which adsorbs near to ∗COs on the Pd surface and transfers electron density to Pd to strengthen the Pd-CO bond26. The FTIR spectrum of ∗12COs is very similar to that of ∗12CO obtained in CO2 reduction at 533 K, indicating that all ∗12CO observed under reaction conditions were ∗12COs. However, ∗12COw could not be observed during CO2 reduction at ≥ 533 K due to its rapid desorption. As we observe no ∗12COw under steady-state reaction conditions, the 12CO product generated after the switch was mainly from the conversion of the other abundant species, H12COO∗.To gain insight into the interaction between 12CH4 and 12CO with increasing temperature are shown in Supplementary Fig. 13CH4 and 13CO are not displayed. The faster decay in both 12CH4 and 12CO signals at higher temperatures indicates the higher reactivities of intermediates. In contrast, the decay curves of 12CO2 and Ar did not change with temperature, further indicating that any elementary step that directly involves CO2 is not the rate-limiting step for the formation of CH4 and CO. The real mean surface residence times 12CH4 and 12CO (denoted as ICH and ICO) together with the E for 12CH4 and 12CO formation , the excess ∗CO can only occupy the weak adsorption sites of Pd particles to form ∗COw. The ∗COs will be further hydrogenated to CH4 while the ∗COw will desorb.Based on above results and SSITKA theoryrCO) is determined and equal to the rate of HCOO∗ conversion (4 formation (rCH4):The scheme shows that the rate of CO formation , which is proportional to the concentration of ∗COs. The completely filled pool of ∗COs in the 533–573 K temperature range suggests that ∗ reduction to ∗CO  involved24, rCH4 (∗COs ([∗COs]) and ∗H ([∗H]), as well as the rate constant of ∗COs conversion (k1):∗ ([HCOO∗]) and ∗H ([∗H]), and rate constant of HCOO∗ reduction (k2):As the conversions of both HCOO∗COs and HCOO∗ do not share and compete for active sites, as they are located on Pd metal and Al2O3 support, respectively22. Therefore, [∗COs] and [HCOO∗] can be independently changed to tune the rate of CO formation, rCO, as well as the reaction product distribution.It is known that ∗COs  to the empty sites of Rh. The synergistic effect at lower catalyst temperature (423 K) was found to be negligible. Our study showed that RWGS cannot occur over Pd/Al2O3 at 423 K31, so the added Pd/Al2O3 cannot supply additional CO to Rh/Al2O3 catalyst for the further ∗CO hydrogenation to CH4 on Rh/Al2O3. Therefore, the above analysis of their results show that for an incompletely filled pool of ∗COs with 100% CH4 selectivity (∗CO) is a strategy for increasing the rate of CO2 methanation.In the case of an incompletely filled pool of 2 methanation and RWGS are not two parallel reactions during the CO2 reduction over Pd/Al2O3 catalysts. Instead, they share the initial steps and intermediates from bicarbonates to formates until after formate decomposition. The rate of formate decomposition to CO∗ is larger than the rate of ∗CO hydrogenation to CH4 and the excess CO∗ desorbs. The rate-determining step for CO2 reduction and for RWGS is the conversion of HCOO∗, whereas that for CH4 formation is the hydrogenation of ∗CO. The balance of the hydrogenation kinetics between HCOO∗ and ∗CO governs the selectivities to CH4 and CO. Given that ∗CO and HCOO∗ are mainly on metal (Pd) and support (Al2O3), respectively, the balance could be tuned to achieve the desired CH4 and CO selectivities by optimizing the loading of the metal and the surface area of the support. This work has important implications for other bifunctional systems where the balance between different catalytic functions determines the rates and product distribution.In conclusion, CO2O3 catalysts were prepared on a γ-Al2O3 powder  by the incipient wetness method using Pd(NH3)4(NO3)2 as the precursor. After impregnation, the samples were dried at 373 K for 24 h and then calcined at 773 K for 2 h in air (flow rate = 60 mL min−1) and followed by reduction at 773 K for 1 h in 10% H2/He (flow rate = 60 mL min−1) to obtain the Pd/Al2O3 catalysts31. Forty-one milligrams of 5 wt% Pd/Al2O3 was pressed onto a tungsten mesh and loaded into a home-made operando transmission IR cell32. The cell has a very small internal dead volume , resulting in a short gas hold-up time. This renders the system suitable for obtaining accurate kinetic information about intermediates and products during the SSITKA experiments. Before experiments, the catalyst was pretreated by calcination at 673 K for 1 h under air with a flow rate of 10 mL min−1, followed by reduction at 673 K for 1 h under 20% H2 in He with a flow rate of 10 mL min−1. The reactant gas mixture was composed of 4 mL min−1 H2, 1 mL min−1 12CO2 or 13CO2, 1 mL min−1 Ar and He as the diluent with a total gas flow of 10 mL min−1 at atmospheric pressure. Ar gas was used as an inert tracer to correct for gas hold-up of the system and for gas re-adsorption. After the reaction reached steady state at the reaction temperature of 533 K, the reactant was switched from H2/12CO2/Ar to H2/13CO2. During the switch, the gaseous effluent from the cell and the species on the catalyst surface were monitored by MS and FTIR, respectively. The switch was performed in the temperature range of 533–573 K, where the CO2 conversion was <8%. In order to obtain the real mean surface residence times . The number of adsorbed species r) of CH4 and CO products 2 reduction over 41 mg of 5% Pd/Al2O3, the 12COO∗, which was demonstrated to be located on the Al2O3 support. As not all H12COO∗ was converted to 12CO, the amount of sites for formates on Al2O3 is estimated to be at least 1.86 μmol, i.e., 4 formation via ∗12COs and a portion of H12COO∗, the upper limit of the amount of active Pd sites for ∗COs (forming CH4) is 1.95 μmol, i.e., 12COO∗ on the surface shown in Supplementary Fig. The SSITKA theory has earlier been described extensivelyThe data that support the findings of this study are available from the corresponding author on request.Supplementary Information