Brain amyloid deposition is one of the main pathological characteristics of Alzheimer’s disease (AD). Soluble oligomers formed during the process that causes β-amyloid (Aβ) to aggregate into plaques are considered to have major neurotoxicity. Currently, drug development for the treatment of Alzheimer’s disease has encountered serious difficulties. Our newly proposed solution is to accelerate the aggregation of Aβ to reduce the amount of cytotoxic Aβ oligomers in brain tissue. This strategy differs from the existing strategy of reducing the total Aβ content and the number of amyloid plaques.In this study, we screened a small library and found that a flavonoid compound (ZGM1) promoted the aggregation of β-amyloid (Aβ). We further verified the binding of ZGM1 to Aβ42 using a microscale thermophoresis (MST) assay. Subsequently, we used dot blotting (DB), transmission electron microscopy (TEM), and thioflavin T fluorescence (ThT) measurements to study the aggregation of Aβ under the influence of ZGM1. By using cell experiments, we determined whether ZGM1 can inhibit the cytotoxicity of Aβ. Finally, we studied the protective effects of ZGM1 on cognitive function in APPswe/PS1 mice via behavioral experiments and measured the number of plaques in the mouse brain by thioflavin staining.ZGM1 can bind with Aβ directly and mediate a new Aβ assembly process to form reticular aggregates and reduce the amount of Aβ oligomers. Animal experiments showed that ZGM1 can significantly improve cognitive dysfunction and that Aβ plaque deposition in the brain tissue of mice in the drug-administered group was significantly increased.Our research suggests that promoting Aβ aggregation is a promising treatment method for AD and deserves further investigation. Amyloid plaque accumulation is one of the two pathological features of Alzheimer’s disease (AD). These plaques are mainly formed by β-amyloid peptide (Aβ), which consists of 39–42 amino acid residues and is produced by the amyloidogenic pathway via the hydrolysis of amyloid precursor protein (APP) by β-secretase and γ-secretase , 2. SincThe aggregation of Aβ involves a complex nucleation-dependent polymerization process with multiple stages and intermediates . AccumulThe earliest evidence for the toxicity of AβOs came from fluid dynamics experiments, which found that AβOs in solution could not be separated by centrifugation . The mosIn summary, because the complicated process of Aβ aggregation is not well understood, it is not clear which form of Aβ causes cell death and eventually leads to AD, so reducing the total Aβ amount in brain tissue has been the main principle used for screening drugs to treat AD. Recent studies have shown that soluble AβOs are a major cause of the loss of synaptic function and neurons compared to mature amyloid fibrils and Aβ monomers . TherefoHere, we discovered a small flavanol compound, 2--5,7-dimethoxychroman-4-one (ZGM1), which promoted the aggregation of Aβ monomers and led to an assembly process that formed reticular aggregate, thereby reducing the amount of toxic Aβ oligomers. In addition, we proved the preventive effect of ZGM1 in AD mice, which provided a new strategy for the prevention and treatment of AD.phenyl)-5,7-dimethoxychroman-4-one (0.2 mmol) was dissolved in 2 M HCl (10 mL), and then the mixture was stirred while refluxing for 2 h. The concentrated mixture was treated with cold water and extracted with ethyl acetate. The ethyl acetate layer was washed with brine, dried over anhydrous magnesium sulfate, and concentrated in vacuo. ZGM1 was finally produced from the residue that was purified on a silica gel column.Synthetic Aβ42 peptides  or synthetic Aβ40 peptides  were dissolved in DMSO to obtain a 5 mM stock solution. Each stock solution was then diluted with D-PBS. ZGM1 was dissolved in DMSO to obtain a 0.1 M stock solution and then diluted with D-PBS to obtain the working concentration. Aβ was incubated with ZGM1 at 37 °C for 12–120 h at specific proportions. Congo red, which has been reported to inhibit Aβ aggregation, was used as a control.2O. The mixture was electrophoresed in a Nu-PAGE 4%~ 12% Bis-Tris premade gel (Invitrogen NP0321BOX) at 200 V for 30 min, and the protein was transferred to a 0.2 μm PVDF membrane. The 6E10 antibody was used for Western blotting to detect Aβ aggregation. Another 2 μl of the sample was incubated for 24–120 h or was not incubated and was spotted on an NC membrane (Sangon) for dot blotting analysis to detect Aβ oligomers.A total of 3 μl of each sample was incubated for 0 h, 12 h, and 24 h with 3 μl Nu-PAGE™ LDS Sample Buffer (Invitrogen) and 6 μl ddHg for 20 min, and then the supernatant was retained for subsequent experiments. These reagents were mixed at a ratio of 1:1:1 so that the final concentration of Aβ was 10 μM. Then, the mixtures were added to a black-walled 96-well plate and incubated at 37 °C, and the fluorescence signals were detected at 0 h, 28 h, 50 h, 72 h, 98 h, 118 h, and 166 h. The excitation wavelength was 440 nm, and the emission wavelength was 476.5 nm.Thioflavin T  was dissolved in DMSO to obtain a 0.1 M stock solution. The ThT, Aβ, and ZGM1 stock solutions were diluted with D-PBS. Aβ (30 μM) was centrifuged at 17,000×The edge of the copper mesh was clamped with tweezers, and 6 μl of the incubated sample was added to the center of the front side of the copper mesh and allowed to remain for 90 s. The sample was gently removed with absorbent paper, and a drop of uranyl acetate was added to the front of the copper mesh and immediately removed. The processed was repeated. After the third drop of uranyl acetate was added, it was allowed to remain on the mesh for 30 s before being removed. The copper mesh was dried and put into the storage box for observation. The images were obtained by transmission electron microscopy .g for 20 min at 4 °C, and then the supernatant was retained. The ZGM1 stock solution was diluted to a concentration of 2 mM with D-PBS. ZGM1 was titrated at a 1:1 dilution 16 times beginning at 2 mM. 5’FAM-Aβ was added to each tube and mixed; the final concentration of 5’FAM-Aβ was 200 nM, and the highest concentration of ZGM1 was 1 mM. A capillary tube  was inserted into each tube to allow the sample to enter the capillary. The capillary was placed in each sample well in order of the ZGM1 concentration (from low to high) and was detected using microscale thermophoresis .Aβ42 linked to a 5-carboxyfluorescein tag at the N-terminus  was dissolved in DMSO to obtain a 5 mM stock solution. Each stock solution was diluted with D-PBS to obtain a concentration of 400 nM and centrifuged at 17,000×Mice at 17–18 days of pregnancy were sacrificed. The abdominal cavity was carefully opened, and the embryos were removed; the whole brain was also removed and placed in DMEM/F12 (1:1) medium. The olfactory bulb and brain stem were removed, and the vascular membrane was peeled off. The remaining tissue was crushed with a yellow pipet tip, transferred into a 15 mL centrifuge tube containing 0.05% Trypsin , placed on ice for 15 min, and then incubated at 37 °C for 10–15 min for digestion. Most of the supernatant was aspirated. Then, 50 μL DNase I  was added, and the tissue was digested at 37 °C for 3 min. A total of 10 mL of DMEM/F12 (1:1) medium containing 10% FBS was added to terminate digestion. The mixture was mixed 20 times by aspiration and then centrifuged at 1200 rpm for 4 min, and the supernatant was discarded. The cells were resuspended gently in 3 ml of neurobasal  medium, and then a cell sieve  was used to filter the sample. The cells were diluted in adherent medium  and then inoculated into 96-well plates at a density of 40,000 cells per well. The growth medium  was prewarmed for fluid exchange after 4 h.2 in a humidified atmosphere.SH-SY5Y cells were grown in 1:1 DMEM/F12 medium  supplemented with 10% fetal bovine serum (Biowest), 0.5% MEM non-essential amino acids 100× solution , and 0.5% sodium pyruvate 100× solution  at 37 °C with 5% COThe experimental groups were treated with ZGM1, Aβ, or both, while the control groups were treated with an equal amount of DMSO; each gradient used three replicates. After 48 h, the medium was changed to a medium containing 5% CCK-8 reagent , and the incubation was continued for 1 h in the dark. Then, the absorbance was measured at a wavelength of 450 nm using a microplate reader .All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals  and Regulations for the Administration of Affairs Concerning Experimental Animals . APP/PS1 mice and 129/C57BL/6 wild type mice were purchased from the Model Animal Research Center of Nanjing University and were raised in the same environment with sufficient food and water and a light to dark cycle of 12:12 h.Four-month-old APP/PS1 TG mice  were divided into three groups: Group T, L-Z, and H-Z, and 4-month-old WT  comprised the W group. The dry ZGM1 powder was resuspended in 0.1% sodium carboxymethylcellulose . The drug was delivered into the stomach by a gavage needle, and the dosage was determined by the body weight. The dosage of the L-D group was 40 mg/kg per day, and the dosage of the H-D group was 120 mg/kg per day. Groups W and T were fed an equal amount of 0.1% SCC as controls for 8 weeks. The mice were administered the drug daily except for the weekends.g for 15 min at 4 °C, and 200 μL of the supernatant was added to a new 1.5 mL EP tube. The 0 h mouse blood sample was used as a blank. ZGM1 powder was dissolved to generate a standard curve for the absolute quantification of ZGM1 in the samples. All samples and standards were tested by the Metabolomics Facility at the Technology Center for Protein Science, Tsinghua University.The mice were orally administered the drug at a dosage of 40 or 120 mg/kg. The mice were sacrificed by perfusion, and the brain tissue was obtained. The brain tissue, an equal mass of glass beads , and 4 volumes of methanol prechilled at − 80 °C were added to the homogenate tubes, and the mixture was homogenized (6000 g) for 30 s and then cooled for 1 min on ice, which was repeated 6 times. The homogenate was placed at − 80 °C for 2 h. The homogenate was centrifuged at 15000×The data analyses, including the recordings of all behavioral responses, were transcribed manually into a computer-acceptable format by researchers blinded to the group assignments.The movement and retention information of the mice over 5 min were recorded by a camera. The next mouse experiment was performed after cleaning the field with 75% alcohol. After all experiments were completed, the total distance, the central area, and the number of crossings in the central area were analyzed.After placing the mice in the center of the elevated plus maze (80 cm × 80 cm), the movement and retention information of the mice over 5 min were recorded by a camera located at the top of the room. The next mouse experiment was performed after cleaning the maze with 75% alcohol. After all experiments were finished, the open arm entry and time in the open arm were statistically analyzed, and the differences between the groups were compared.On the first day, objects A (circles) and B (squares) of the same color were placed in the open field. The mouse was free to explore in this environment for 10 min. The movement and retention information of the mice were recorded. The next mouse experiment was performed after cleaning the field with 75% alcohol. The next day, object B was replaced in the open field by a circular object C, which was slightly larger than object A. The position of object A did not change. After the mice were placed in the field, the movement and retention information of the mice were recorded over 5 min. The number of times and the timing of the sniffing of objects A and C by the mice were statistically analyzed, and the differences between the groups were compared.A platform was placed 1.5 cm below the water surface in the second quadrant of a circular water tank . Titanium dioxide was added to whiten the water. The test was performed for 6 days. Mice were trained for 6 consecutive days with 3 trials per day as acquisition trials. Each trial began with placing the mouse into a different quadrant and allowing it to swim freely for 60 s. After each mouse reached the platform (or were guided to the platform if the mice were unable to locate the platform after 60 s), they were returned to their cage to dry for 20 min. The time that each mouse took to reach the platform was regarded as the escape latency. On the 7th day, the mice were rested for a day. On the 8th day, a probe test was performed without the platform for 60 s. Each mouse was placed into the opposite quadrant of the target zone, from which the platform was removed. The time in the target zone and the number of crossings that occurred in the probe were recorded.After the behavioral studies, the mice were deeply anesthetized with 7% chloral hydrate  and perfused. For ThS staining, excised brains were fixed overnight in 4% paraformaldehyde at 4 °C and immersed in 30% sucrose for 24 h twice for dehydration. Brain slices were cut in the coronal plane (30 μm per section) at − 26 °C. Aβ plaques in brains were visualized using ThS staining. ThS was dissolved in 50% ethanol at 0.4 M (filtered by a 0.22-μm filter), and brain sections were stained for 8 min and then washed with 50% ethanol for 3 min, which was repeated 3 times. The slices were finally sealed with 20% glycerin (dissolved in PBS). The images of the plaques were obtained with an Olympus fluorescence microscope. The number and areas of the plaques were determined using the ImageJ program (NIH). The analyses of the plaque distributions were transcribed manually into a computer-acceptable format by researchers blinded to the group assignments.Brain tissues were homogenized in ice-cold RIPA containing a 1* proteinase inhibitor. The homogenized tissue was ultrasonically crushed on ice for 20 min at 5% of the maximum power. The supernatant of the brain lysate was used for ELISA of Aβ40 or Aβ42 and Western blotting and dot blotting to detect biochemical changes and soluble Aβ. The concentrations of the soluble fractions of brain lysates were determined by using a BCA protein assay kit . Protein samples (20 μg) were loaded in each lane of SDS–PAGE gels for Western blot analyses. GluR1, GluR2, PSD95, synaptotagmin, and App695 were measured. The measurements of Aβ40 and Aβ42 content were completed with an ELISA kit (Invitrogen) according to the manufacturer’s instructions with fivefold diluted soluble fraction samples. Detailed information about the antibodies used is included in the Additional files.P < 0.05, **P < 0.01, ***P < 0.001, other comparisons were not significant).Graphs were obtained with GraphPad Prism 6, and statistical analyses were performed with one-way analysis of variance or two factors analysis of variance  to verify binding by using microscale thermophoresis (MST). The quantitative analysis of the data revealed that the half-maximal binding (EC50) value was 85.8 ± 18.2 μM , as a potent fluorescent marker of amyloid proteins, binds fibrils , 18. TheThe diversity in the ZGM1-generated amyloid fibrils indicated that the presence of ZGM1 may lead to the formation of different types of aggregates. To further explore this, we incubated Aβ42 with different concentrations of ZGM1 and observed the morphology of the ZGM1-generated aggregates by transmission electron microscopy (TEM). After incubation for 24 h at a ratio of 1:50 (ZGM1 to Aβ), the morphology of the Aβ aggregates was significantly different from that of the control. More protofibrils and early amyloid fibrils were produced, which showed more branches  to APPswe/PS1-dE9 mice  revealed that ZGM1 can penetrate the blood-brain barrier and become enriched in the brain , 795 nM (120 mg kg− 1), or 1319 nM (250 mg kg− 1), and the concentration remained relatively stable until 6 h after administration .Mass spectrometry analysis of the mouse tissue after ZGM1 administration , the total distance the mice traveled in the open field test was significantly higher than that of the other groups at the same age    Fig. d, while 01) Fig. d, which Decreased learning and memory ability, along with cognitive dysfunction, are the most important clinical symptoms of AD patients, especially the decline in short-term memory ability, which is often regarded as the earliest symptom , 24. TheFinally, to detect changes in the spatial learning and memory ability of TG mice after ZGM1 administration, we conducted Morris water maze tests. In the Morris water maze test, there was no significant difference in the swimming speed of the mice in the administration group and untreated TG mice Fig. i, which However, the Western blot results showed that there was no significant difference in the levels of learning- and memory-related biomarkers in the mouse brain homogenates between groups  to observe Aβ plaques in the hippocampus and cortical tissue. Compared with WT mice, a smaller number of plaques appeared in the brains of TG mice Fig. a, c. In In addition, the results of ELISA showed that compared to that of TG (319 ng/g protein), the content of Aβ42 increased by 47% after the administration of a low dose of ZGM1, although the content of Aβ40 in the brain tissue of mice was not significantly changed Fig. e, f. WesClinical data have shown that the number of Aβ plaques in the brain is not directly related to the severity of cognitive impairment in patients , 26. TheStudies have mainly focused on finding small molecule compounds to inhibit Aβ aggregation or to promote the depolymerization of amyloid fibrils. Many compounds have been found to have both effects. For example, the Wanker group reported that epigallocatechin gallate (EGCG) can inhibit the formation of Aβ fibrils when incubated with monomers . When EGIn addition, 2,8-bis--7-hydroxy-phenoxazin-3-one (O4) was reported to promote the aggregation of Aβ monomers and the formation of fibrils . Since sHere, we report that  a small However, the high-dose group did not show better results Fig. b–h. We sBased on the above studies, we speculated that ZGM1 can mediate the aggregation of Aβ42 monomers into globulomers, which can be used as seeds in fibril formation. The number of amyloid fibrils can be elevated by increasing the ZGM1 concentration. Amplification of the ThT fluorescence signal Fig. b. In addAlthough the specific metabolic processes and kinetic parameters of ZGM1 remain to be further elucidated, the available results indicated that it may be feasible to accelerate the rapid aggregation of Aβ during the toxic oligomer phase and delay the progression of AD. The rate of Aβ aggregation can be greatly accelerated by a small number of seeds, so ZGM1 can function at concentrations well below the EC50 value. The results of ThT have also demonstrated that ZGM1 can significantly accelerate Aβ aggregation at a concentration of 200 nM. Since mass spectrometry can only detect the abundances of unmetabolized and unmodified ZGM1 prototypes in tissues, it is also possible that other forms of metabolites of ZGM1 may play a role in the pharmacological processes. We also observed that ZGM1 increased the number of Aβ plaques in the brains of mice, which suggested that ZGM1 promoted the formation of amorphous Aβ aggregates. Although these Aβ aggregates are likely to be of low toxicity, the long-term safety of the use of ZGM1 for the early prevention of AD still needs to be further evaluated.Currently, drug development for the treatment of Alzheimer’s disease has encountered serious difficulties. The failure of drugs such as BACE and γ-secretase inhibitors, immunotherapeutics against Aβ, and inhibitors of Aβ aggregation suggests that we need to try new strategies. After analyzing the current status of AD drug development, our proposed solution is to accelerate the aggregation of Aβ to reduce the amount of cytotoxic Aβ oligomers in brain tissue. This strategy differs from the existing idea of reducing the total Aβ content and the number of amyloid plaques. The main goal is to reduce the amount of cytotoxic Aβ oligomers in brain tissue. In this study, we tested a small molecule compound (ZGM1) to determine its ability to promote the aggregation of Aβ monomers, mediate a new Aβ assembly process and reduce the amount of Aβ oligomers. Animal experiments showed that ZGM1 can significantly improve cognitive dysfunction in AD model mice, and we found that Aβ plaque deposition in the brain tissue of the mice in the drug-administered group was significantly increased.Additional file 1:Figure S1. Two-dimensional 1H,15 N correlation spectrum obtained for ZGM1. . Figure S2. ZGM1 concentration in mouse tissue over time after administration. (a) ZGM1 concentration in blood plasma or brain homogenate after administration at a dose of 250 mg/kg. (b) ZGM1 concentration in brain homogenate after administration at a dose of 40 mg/kg, 120 mg/kg and 250 mg/kg. Figure S3. Western blotting analyses of learning- and memory-related proteins in mouse brains. Table S1. NMR data of the ZGM series. Table S2. Escape latency and significance  of the hidden platform test in the Morris water maze.


In the case of H2O ice, H2O2 is formed at the temperatures below 60 К. In the case of H2O:O2 ice, H2O2 is formed in the 20–140 К range. For H2O:O2 = 9:1 ice, we derived H2O2 photochemical quantum yield as a function of sample irradiation temperature. The obtained data can be used for evaluation of H2O2 photoproduction at the surface of astrophysical water ice bodies and inside the particles of Noctilucent Clouds in the Earth’s atmosphere.We report the results of laboratory measurements of H The characteristics of these processes greatly depend on the characteristics of irradiation, ice type, its thickness, temperature, additives, and others. The end products such as H2O2, O2, O3, etc., both in solid and gas phase are of considerable interest for astrophysics as they are all oxidizing agents and may provide a source of chemical energy as a fuel for extraterrestrial life5.It is well known that surfaces of most icy bodies in the outer Solar System and interstellar space consist mainly of water and are regularly bombarded with energetic particles and photons. This irradiation triggers a spectrum of physicochemical processes inside solid phase2O2 on Europa’s surface6 and the discussed existence or absence of H2O2 on Enceladus, Ganymede, and Callisto7 stimulated extensive laboratory studies of the mechanisms of formation and measurement of the parameters of producing concentrations of this component in high-purity H2O ice and H2O ice with different additives irradiated by energetic particles14. In particular, it was shown that, as compared to H2O ice, the presence of О2 greatly increases the production of H2O2 and other products , especially at relatively high irradiation temperatures of 80–120 К. At the same time, except for a few works, significantly less attention was paid to investigation of H2O2 production by VUV photons. In particular, Gerakines et al.15 and Schriver et al.16 reported about H2O2 formation in H2O ice at 10 K irradiated by microwave discharge hydrogen flow lamps. Yabushita et al.17 found out H2O2 presence in H2O ice after its irradiation by 157 nm photons at 90 K. Shi et al.18 measured H2O2 in porous H2O + O2 ice irradiated by 193 nm photons at 40–78 K. Thus, many details of H2O2 production by VUV photons, the temperature dependence in the first place, are still understood poorly.The established detection of H2O2 production inside thin H2O and H2O:O2 ice samples irradiated by 121.6 nm photons in the temperature range of 20–140 K. We discuss possible implications of the obtained results at the surface of astrophysical water ice bodies and inside the particles of Noctilucent Clouds in the Earth’s atmosphere.This work reports the first results of laboratory measurements of H21. Since the clouds discovery22, there were many discussions about their nature (see reviews by Gadsden & Schröder19 and by Thomas20). Only recently, the infrared spectra of clouds showed23 that NLCs consisted mainly of water ice. Thus, it is not doubt now that clouds form by condensation of water vapour and can influence on gas-phase chemistry of this region due to water vapour is its key parameter.There exists at least one analog of such water icy bodies regularly irradiated by VUV photons in the Earth’s atmosphere. Each summer at polar and middle latitudes, one can observe the highest atmospheric clouds called Noctilucent Clouds (NLCs). They appear in mesopause region  at the temperatures of 120–150 Kα line) and the reaction H2O + hv → H + OH provides the main chemical source of the family of odd hydrogen 24. In turn, the reactions with participation of HOx components  remove the components of the odd oxygen family (Ox: O(1D), O(3P), and O3). Therefore, the appearance of NLCs is expected to reduce the concentrations of water vapour and HOx and to increase Ox. However, rocket measurements of the concentration of atomic oxygen made during several campaigns demonstrated unpredicted O depletion around NLCs25. A possible explanation of the revealed effect was proposed by Murray and Plane26 who noticed that there also occurs photolysis of H2O molecules inside NLCs particles. The photoproducts (H and OH) may release into gas phase and additional source of HOx leads to increase of Ox removal. In the work by Kulikov et al.27 this hypothesis was verified by laboratory measurements of the photodesorption rate from thin water ice samples irradiated by 121.6 nm photons in the temperature range of 120–150 K. It was found that most photoproducts did not leave the solid phase and tended to recombine in water molecules back. Basing on the results of ice irradiation by energetic particles at relatively high temperatures of 80–120 К14, we can assume that H2O2 is the principal photoproduct that may accumulate in NLCs.In the conditions of daytime mesopause, water vapour is subjected to intensive solar VUV radiation  taken in different seasons. The model estimates of the concentration of those ions formed as a result of the H2O2 + O2+ → H2O2+ + O2 reaction agreed well with the results of measurements made in different seasons, except summer. The authors conjectured that the discrepancy was caused by the increased H2O2 concentration in the summer time. Later measurements29 revealed positively charged clusters containing H2O2 in the presence of NLCs particles. The researchers arrived at the conclusion that for such clusters to be formed, the concentration of H2O2 must be much higher than the expected level.Note that the experimental evidence of H−1 appeared in FTIR spectra of H2O and H2O:O2 ice samples as the result of their irradiation by calibrated source of 121.6 nm photons at high vacuum conditions. For more details please see Methods.Hydrogen peroxide was found by detecting the IR absorption band of 2850–2860 cmα photons at the photon flux intensity Iα = 5·1014 photons/(cm2·s) are presented in Fig. 2O ice  ≤ 60 К, and the temperature rise from 20 to 60 К results in a monotonic decrease of the integrated area of the 2850–2860 cm−1 band  cm−1. Forth, H2O2 peak position lays at 2850 cm−1 in the range of 20–100 K and shifts to 2852–2854 cm−1 at the temperatures 120–140 K. Note also, in both cases, there are no new features at 1039, 1142, and 1259 cm−1 . Note firstly, the present results are in a qualitative agreement with H2O2 temporal evolution obtained in earlier laboratory studies of the formation of this component in H2O ice irradiated by energetic particles14 and photons15. The temporal evolution of 2O ice, the growth stage continues for ~20–30 min and can be described by a quadratic function of irradiation time (VUV fluence). This corresponds to the results of irradiation of H2O ice by Lyman-α photons at 10 K obtained by Gerakines et al.15. In the case of H2O:O2 ice, the growth stage continues for ~10 min and can be described by a linear function of irradiation time (VUV fluence). Such behavior of 2O:O2 ice was found at other photon flux intensities. It means that, in the case of H2O:O2 ice, the H2O2 production during the growth stage can be fit successfully to a (pseudo) first-order reaction. We can conclude that the rate of H2O2 production is proportional to Iα and can determine the H2O2 photochemical quantum yield (2O2 generated per a Lyman-α photon absorbed by ice) as a function of Tir following, for example, Cooper et al.12 and Hand and Carlson14. For this, we carried out special experiments with H2O:O2 ice at lowIα = 3·1013 photons/(cm2·s) at which the growth stage continued more than 100 min. The Fig. Tir with corresponding linear fits whose values of slope are presented on Fig. Figure 2O2 production after irradiation of different ices36 used the values of −1 band absorption) from two sources10. Moore & Hudson8 measured −17 cm·molecule−1 and this value used for all temperatures. Loeffler et al.10 found out that −17 cm·molecule−1 at 20 K, 5.2·10−17 cm molecule−1 at 80 K, and 4.9·10−17 cm·molecule−1 at 110 K. Thus, for estimation of H2O2 column density (relative concentration) and H2O2 photochemical quantum yield from the results presented in Figs et al.8 data were interpolated into the temperature regions of 20–80 K and 80–110 K, and extrapolated to the temperature region of 110–140 K.All known studies of H2O ice, the maximum of relative concentration H2O2/H2O at 20 K is ~2% at 8 and ~1% at et al.10. In the case of H2O:O2 ice, the maximum of H2O2/H2O at 100 K is ~9% at 8 and ~4.5% at et al.10. In that time, the maximum of 8 and ~(0.0053–0.0175) molecules/photon at et al.10.One can see from Fig. see Fig. . Note al2O2 formation during VUV irradiation of H2O ice was discussed by Gerakines et al.15 and pointed by Loeffler et al.10 in comparison with H2O2 formation by ions. This component is formed due to reaction OH + OH → H2O2. The quadratic character of 2O2 molecule. These photons should produce two OH close to each other due to extremely low mobility of OH in water ice below 80 K38. The saturation of 2O2 + hv → 2OH and H2O2 + OH → H2O + HO2.The possible mechanism of Hsee Fig.  indicate2O:O2 ice, the linear character of 2O2 formation differs from the previous case. Note, Loeffler et al.10 and Hand and Carlson14 found out the same behavior of 2O ice irradiated by high-energy ions and electrons correspondingly. It was proposed for explanation of this, in particular, that two OH could be produced in an ion track caused by an ion10. Evidently, this mechanism cannot be transferred on our situation. But, following Loeffler et al.10 and Hand and Carlson14, we can speculate that the rate of H2O2 formation in H2O:O2 ice irradiated by VUV photons is proportional to VUV intensity and the concentrations of H2O and O2. In other words, one photon, one H2O molecule and one O2 molecule are needed for appearance of one H2O2 molecule. On the other hand, O2 is impurity of H2O ice and typically two O2 molecules are separated from each other by a “paling” of H2O molecules. So, it is hard to imagine that these O2 molecules can participate in formation of one H2O2 molecule due to extremely low mobility of intermediates (such as OH and HO2) in water ice below 80 K as above mentioned. Thus, we can conclude as a first approximation that H2O2 photochemical quantum yield inside VUV irradiated H2O:O2 ice is proportional to the relative concentration of O2 (O2/H2O), when O2/H2O In the case of Hsee Fig.  shows us2O:O2 ice, temperature dependences of H2O2 column density  H2O2 + hv → 2OH and (2) H2O2 + OH → H2O + HO2. So, R1,2 are the corresponding reaction rate coefficients. Note, Loeffler et al.39 measured the loss of H2O2 in H2O:H2O2 at temperatures between 21 and 145 K initiated by UV photons (193 nm). They obtained that the temperature dependences of H2O2 photodestruction cross section  varied in the range of 0.2–0.4 molecules/100 eV at 50–100 K, that is close to G-value of Lyman-α photons at 50–100 K obtained in our paper. Thus, contribution of Lyman-α photons to H2O2 production is defined by the ratio between energy fluxes of photons (EFph) and energetic particles (EFep). In the case of EFph~EFep, photons produce approximately the same amount of H2O2 inside ice as particles. At that, as it was noted by Gerakines et al.40, the penetration depth for Lyman-α photons (~45 nm41) in water ice is essentially less, than for protons which depends on its energy . So, one would expect the high relative H2O2 concentration in the top few tens of nm caused by Lyman-α photons. It means that H2O2 production by VUV photons can be important at EFph/EFep ≥ 10−2.In astrophysics, VUV irradiation competes with energetic particles bombardmentEFph EFep. But, at this moment, there is no information about containing of O2 inside water ice of Noctilucent Clouds. It is well-known that NLCs are formed as a result of gas-kinetic collision of H2O molecules with the surface of mesospheric aerosols, including adsorption and desorption properties. It is, generally, a relatively slow process, with the characteristic time (2–20 hours) depending on temperature43. In the real conditions of a summer mesopause, H2O concentration in gas phase is more than 4 orders of magnitude less than the concentration of O2 in ground (triplet) state and less than daytime concentration of O2 in singlet state ((2–4)·109 cm−3 at 80–85 km44). The molecules of O2 in ground and excited states, like those of H2O, continually bombard the surface of the forming particles of clouds, adsorb on their surface and can be trapped inside the ice matrix as the NCL particles are being covered by new ice layers. This suggests that a small part of O2 molecules from gas phase may be uptaken by the forming matrix of the cloud. It is certainly hard to believe that the relative concentration of O2 inside NCL particles  measurements with the ALOMAR RMR-lidar in Northern Norway , at the altitudes of 81–86 km SNLC varies within the (3–6)·10−8 cm2/cm3 range45. Taking into consideration that at these heights in the conditions of average solar activity Iα~3 · 1011 photons/(cm2·s)24, we obtain 3·3·s). In the real conditions of the mesopause, the NLCs particles are also irradiated by UV solar photons, which leads to H2O2 photodissociation in solid phase with the efficiency close to this process in gas phase39. For the considered range of altitudes of 81–86 km, the photodissociation constant of H2O2 in gas phase −4 s−1. Thus, the equilibrium concentration of H2O2 that may be accumulated in cloud particles irradiated by Lyman-α photons is 7·−3. Gumbel et al.25 reported the maximum of SNLC~10−7 cm2/cm3, which gives the estimate for the maximum values of 2O2 to be 7.5 · 103·3·s) and 5 · 107·−3, correspondingly. Under the conditions of NLCs existence, typical value of gas-phase concentration of H2O2 is ~2 · 105 cm−3 at 81 km and decreases with increasing height down to the values less than 104 cm−3 at 86 km26. Thus, the minimum of 2O2 concentration is equal to 104 cm−3, corresponds to 2 · 10−4 molecules/photon. Following our above conclusions, H2O2 photochemical quantum yield inside H2O:O2 ice is proportional to the relative concentration of O2. Taking into consideration the measured values of 2O:O2 = 9:1 ice at the temperatures of 120–140 K, we estimate that the minimum of −4 molecules/photon corresponds to 2 in NLCs ~(1.4–4.9)·105 cm−3) at 8 and ~(0.11–0.38)% (the absolute concentration ~(2.5–8.3)·105 cm−3) at et al.10. Note, that the estimated values of the O2 absolute concentration in NLCs are several orders of magnitude less than typical gas-phase concentrations of this component in ground and singlet states at the altitudes of 81–86 km.Let us assume that 2, the physicochemical processes occurring in them may remarkably affect the chemical composition of the mesopause region. On the one hand, it may be a possible explanation of the results of early rocket mass-spectrometer measurements29 indicating increased H2O2 concentration in the clouds. On the other hand, H2O2, product of its UV photodissociation (OH), H2O, O2 and other impurities  can participate in subsequent reactions producing more complex chemical compounds inside NLCs as it takes place, for example, in the bulk of supercooled water particles46. We hope that this research stimulates further experimental and theoretical investigations of the chemical composition of cloud particles. Note also that the obtained results are interesting for astrophysical applications, for example, for assessing the contribution of VUV irradiation to H2O2 production in the outer Solar System and interstellar space depending on temperature.To conclude, we have demonstrated for the first time that, if NLCs particles contain ≥0.1% Oet al.27, the apparatus consisted of a Fourier Transform Infrared Spectrometer (Bruker IFS 66 v)), a closed-cycle He refrigerator (Leybold ROK 10–300), a gas preparation and inlet system (further briefly GPIS), and a high-vacuum chamber with a volume of about 1000 cm3 pumped continuously by a turbomolecular pump system (Leybold-Heraeus) securing a high vacuum in the chamber down to the 10−8 mbar range. Inside the chamber, at the cold end of the cryostat there was a vertically mounted aluminium mirror (2.5 × 4 cm in size) as a substrate whose temperature was precisely regulated by a temperature controller . The mirror temperature could be selected in the 10–300 K range. The GPIS was equipped with baratrons and needle valves. The upper part of the high vacuum chamber had two ports, one of which was equipped with a MgF2 (5 mm thick) input window for a VUV lamp. The second port had a KBr window for the IR beam of the FTIR spectrometer. The input for the VUV lamp made an angle of incidence of ~450 to the mirror surface and, according to the estimates of the manufacturer, MgF2 transmitted about 60% of the quantum flux at the wavelength of 121.6 nm. As a VUV source (Lyman–α) we used a resonance hydrogen lamp (Opthos Instruments) containing a mixture of 10% H2 and 90% Ar excited by a microwave generator  with a frequency of 2450 MHz. The lamp intensity was determined by the power supplied by the microwave generator . The FTIR spectrometer was placed on rails allowing precise positioning of the instrument with respect to the cryostat with the sample. This was important for achieving a good overlap of the areas of the light spots from both, infrared (from spectrometer light source) and vacuum ultraviolet irradiation (from VUV lamp) of the ice film sample on the substrate. The operation of the FTIR spectrometer was PC controlled by means of software (OPUS) that permitted scanning spectra over a wide range (from 6000 to 500 cm−1) and analyzing the obtained spectra. The spectra were recorded with a spectral resolution of 0.2–2 cm−1 in the RAIRS mode (reflection absorption infrared spectroscopy) where the IR beam passes through the sample twice.The experimental set-up was the same as we used under laboratory measurements of the photodesorption rate from water ice. As described by Kulikov et al.27, each experiment with a particular sample of ice was conducted in two stages. At the first stage, two background spectra with different resolution were recorded at a mirror temperature of 20 K and at a temperature of subsequent irradiation (Tir). At that time, H2O or H2O + O2 gas was got ready in GPIS. We used the oxygen (Air Liquide 5.5) with purity better than 99.9995 Vol% and triply distilled water with resistivity better than 107 ohm cm, additionally degassed by freeze/thaw cycles in vacuum conditions. As described in our previous study27, then, an ice film was prepared by depositing H2O or H2O + O2 vapour onto the mirror at 20 K. The deposition speed and sample thickness were controlled by a needle valve and internal baratron of GPIS and by tracking the evolution of absorption bands in the FTIR spectra. More specifically, a sample was prepared by successive slow depositions of small vapour portion which were accompanied by the integrated area measurements of the water ice 3275 cm−1 band  of 10.2 eV photons in water ice and close to typical radii of NLC particles48. After the sample preparation, the mirror temperature was set at Tir (in the 20–140 K range) and several IR spectra of unirradiated ice were recorded. At Tir = 120 K and above, the IR spectra showed crystalline features of all ice samples that was in accordance with composition of NLCs49. Note also, Bartels-Rausch et al.50 discussed recently the simulations of disorder on pure ice at different temperatures below melting point (Tm) and showed that, at the temperatures 10 K below Tm, disorder affected the first molecular layer of ice only. In current study, the samples consisted of more than 100 layers of water and the highest temperature in our experiments (140 K) was about 20 K below than characteristic Tm of water ice in our vacuum chamber. Thus, we can conclude that interface processes could not influence essentially on the studied processes inside ice samples.The experimental procedures were almost the same as we used under laboratory measurements of the photodesorption rate from water ice. As described by Kulikov 27, at the second stage, the vacuum ultraviolet lamp was switched on and the ice films were exposed to VUV radiation with intensity set by microwave generator. After each photolysis period, IR spectra of the irradiated ice films were recorded. For improving the signal-to-noise ratio we used the spectral resolution of 2 cm−1 and a large amount of scans (2000). Hydrogen peroxide was found by detecting the IR absorption band of 2850–2860 cm−1 in difference spectra (before and after irradiation). The band was exuded by subtracting the baseline from the spectrum in manner described, for example, in Hand & Carlson14. Values of H2O2 column densities were determined as −1 band, As described in our previous studyet al.27. Before an experiment with specific H2O or H2O + O2 ice sample, we performed series of measurements of the absolute magnitude of the flux of Lyman-α photons that reach the ice sample at different adjustments of the microwave generator power. The widely used “ozone method”51 was applied for the procedure. The intensity of the lamp was determined by measuring the O2 → O3 conversion rate in a VUV photolyzed sample of solid O2 at 16 K. The ozone formation as a function of photolysis time was monitored with the FTIR spectrometer via the O3 absorption band at about 1040 cm−1. More specifically, for finding the VUV intensity at the mirror for a specific generator output power (GOP) we made successive measurements of the integrated area of the 1040 cm−1 absorption band . After that, the lamp intensity at this GOP was determined as Y was the quantum yield for the formation of O3 from O2, and et al.52 and was equal to 8.4 · 10−18 cm·photon−1. We obtained that, depending on GOP varied within the range of 4–120 W, the photon flux intensity varied within the range 5 · 1012–1015 photons/(cm2•s). The stability of lamp intensity at the fixed GOP was checked by means of photodiode SXUV300 .The calibration was carried out in the same manner as it was described by Kulikov


The chemical shift changes were similar in the two titrations, consistent with sugars binding to the cleft between the upper and lower domains; the NMR data show no evidence for simultaneous binding of a NAG6 to two λ lysozyme molecules. Six 150 ns MD simulations of λ lysozyme in complex with NAG4 or NAG6 were performed starting from different conformations. The simulations with both NAG4 and NAG6 show stable binding of sugars across the D/E active site providing low energy models for the enzyme‐inhibitor complexes. The MD simulations identify different binding subsites for the 5th and 6th sugars consistent with the NMR data. The structural information gained from the NMR experiments and MD simulations have been used to model the enzyme‐peptidoglycan complex.The X‐ray structure of lysozyme from bacteriophage lambda (λ lysozyme) in complex with the inhibitor hexa‐N‐acetylchitohexaose (NAG6) (PDB: 3D3D) has been reported previously showing sugar units from two molecules of NAG6 bound in the active site. One NAG6 is bound with four sugar units in the ABCD sites and the other with two sugar units in the E′F′ sites potentially representing the cleavage reaction products; each NAG6 cross links two neighboring λ lysozyme molecules. Here we use NMR and MD simulations to study the interaction of λ lysozyme with the inhibitors NAG4 and NAG6 in solution. This allows us to study the interactions within the complex prior to cleavage of the polysaccharide.  BMRBBiological Magnetic Resonance Data BankHSQCheteronuclear single quantum correlationMDmolecular dynamicsNAGN‐acetylglucosamineNAG4tetra‐N‐acetylchitohexaoseNAG6hexa‐N‐acetylchitohexaoseNAMN‐acetylmuramic acidNMRnuclear magnetic resonancePDBProtein Data BankSPCsimple point chargeT4Llysozyme from T4 bacteriophage1Lysozymes are very widespread throughout nature, being found in phage, bacteria, plants, and animals.The active site of λ lysozyme is located between an upper and a lower domain Figure . The uppA crystal structure of λ lysozyme in complex with hexa‐N‐acetylchitohexaose (NAG6), an inhibitor, has been determined  was calculated for each residue using the standard equation (δ=12δH2+αδN2); the 15N shift changes were scaled by a factor of α = 0.15 compared to the 1HN chemical shift changes.LT) byPT is the total protein concentration, Kd is the dissociation constant and Δδmax is the maximum observed chemical shift difference. In the Kd calculations, the average of the combined chemical shift changes from the residues showing evidence of hydrogen bonding interactions detected from both the simulations and the NMR experiments were used. Neighboring residues that showed significant changes through inductive effects were also included. When all the subsites were considered, the residues used in the Kd calculations were 19, 20, 68, 69, 73, 77, 98, 101, and 102. To decide which combined chemical shift changes are large enough to be considered significant indicators of the binding site, the method from Schumann et alThe combined uation δ=δH2+αδN2;2.3http://www.gromos.net)φ, ψ angles of −84°, 102° which have been identified previously.MD simulations of λ lysozyme were performed using the GROMOS11 biomolecular simulation package  and NAG4 (0‐26 mM); a region of the HSQC spectra is shown in Figure 1HN and 15N chemical shifts and peak intensities were followed through the titrations  and hexa‐NAG (NAG6) to λ lysozyme in solution was characterized, at a residue‐specific level, using NMR methods. 2D s Figure . Residue1HN and 15N chemical shift changes for λ lysozyme in the presence of ~ 6 mM NAG4 and NAG6 are shown as a function of sequence in Figure Kd value.The spectrum of λ lysozyme in the presence of NAG6 shows peak broadening of specific residues in addition to changes in chemical shifts Figures  and 3; a3.2N of Lys138, Ala139, and Asp140 are hydrogen bonded to the CO of Gln134, Phe135, and Glu136, respectively, in the open structure but not in the closed one. Thus, some of the observed peak intensity perturbations for C‐terminal residues may indicate a change in the population of open and closed structures, or in the dynamics of their interconversion, as NAG4/NAG6 bind to λ lysozyme.The most significant broadening of peaks is observed for residues at the C‐terminus of λ lysozyme Figure C. Additi3.3u and Fu. This change is not observed when the protein adopts the closed conformation and so different interactions with the protein are seen for sugars 5 and 6 in the simulations starting from the open and closed states of λ lysozyme.MD simulations of λ lysozyme in complex with NAG4 and NAG6 were used to provide structural models for the complex and to characterize the dynamics of the protein complex. Different conformations of λ lysozyme seen in crystallographic studies were used as starting structures for the simulations to capture a dynamic picture of the enzyme. Two 150 ns simulations of λ lysozyme in complex with NAG6 were performed starting from the open and closed conformations of λ lysozyme  between the individual sugars and the protein were compared in the four NAG4 simulations . For theu Figure . In simuIn both the NAG6_1 and the NAG4_3 simulations there is a similar change in orientation of the D‐E linkage between the sugar units populating the D and E subsites. There is considerable interest in the ligand‐binding modes in subsites D and E since the catalysis of the cleavage of the β(1 → 4) glycosidic bond happens between these two subsites. We identify here changes at the glycosidic linkage at these subsites which enable the adoption of other energetically favorable states during the MD simulation in Figure λ lysozyme showed different conformational states in the MD simulations, especially in the upper and lower lip regions. To characterize this, the distance between the upper lip (Tyr132: Cα) and lower lip (Lys58: Cα) and their distances to the catalytic residue Glu19, which is located between them, was calculated to assess the contribution of each lip region to the opening and closing mechanism of the protein . In addi3.41HN and 15N chemical shift changes in the NAG4 and NAG6 NMR titrations were mapped on to the λ lysozyme complex conformations populated during the different simulations  as illustrated in Figure As discussed above, flexibility of the linkage between the sugars occupying subsites D and E was identified in some of the simulations causing the final two sugar units to adopt alternative conformations populating subsites EWhen a carbonyl group forms a hydrogen bond to the sugar, the largest effect is often seen for the amide group of the following residue rather than the amide group of the same residue. This is seen, for example, for residue Gln68 where its backbone oxygen is involved in hydrogen bonding in the simulations. Its overall backbone shifts were 0.079 and 0.096 ppm while the following residue, Leu69, showed larger shifts of 0.114 and 0.161 ppm in the NAG4 and NAG6 titrations, respectively. Interestingly, in the crystal structure Gln68 hydrogen bonds to the sugar using its side chain oxygen, not its backbone carbonyl oxygen.The region with the most significant broadening during the titrations is the upper lip region (residues 128‐141). Some of the residues in this region, Tyr132 and Phe135, interact with the ligand in both the MD simulations and the crystal structure of the complex. It was noted earlier that some of the broadened residues do not make contact with the sugar in the crystal structure; contacts between these residues and the sugar are also not observed in the MD simulations. The broadening and disappearance of resonances for these residues in the upper lip region may therefore reflect changes in the population and dynamics of the multiple conformations this region samples in solution in the presence of inhibitor.The chemical shift changes observed for the NAG4 and NAG6 titrations are generally similar although there are some interesting differences Figure . Many of3.51‐γ‐D‐Glu2‐L‐Lys3‐D‐Ala4‐D‐Ala5. However, the residue at position 3 is replaced by diaminopimelic acid in Dap‐type peptidoglycan which is mostly seen in Gram‐negative bacteria. Here, we modeled a peptidoglycan octamer with a NAG‐NAM(‐L‐Ala‐γ‐D‐Glu‐L‐Dap‐D‐Ala‐D‐Ala) disaccharide repeating unit since λ lysozyme has been shown to be more active for gram‐negative bacterial cell wall lysis.φ, ψ:−84°, 102° glycosidic angles for the NAM‐NAG disaccharide, which have been previously determined from free‐energy calculations.φ, ψ:−62°, 118° where φ is the O5‐C1‐O4‐C4 dihedral and ψ is the C1‐O4‐C4‐C3 dihedral). The pentapeptide conformation seen in the NMR structures of the unbound peptidoglycan monomer, dimer, and protein‐bound peptidoglycan polymers show that it is not well‐defined.Although there have been many studies of the chemical structure of peptidoglycans, there is no consensus on the preferred 3D molecular structure adopted or its arrangement in the cell wall.u and Fu sugar binding sites when the structure was open. Our λ lysozyme‐peptidoglycan model suggests that in the presence of the natural substrate this EuFu pocket will be occupied by the peptide moiety and the sugars occupy the EF or E′F′ subsites. In addition to that, our model suggests that the peptide moiety P4 binds adjacent to the λ lysozyme lower lip region. Here there was a significant loss of secondary structure in the β3‐β4 strands (residues 52‐56 and 59‐61) during the simulations. However, the model suggests that hydrogen bonding interactions between the peptide moiety P4 and Lys60 and Ser61 in λ lysozyme may stabilize the secondary structure in the lower lip in the complex with peptidoglycan. The additional interactions between these peptide moieties and λ lysozyme are likely to be responsible for the higher affinity of the enzyme for its natural substrate.As can be seen from the molecular surface representation Figure A, after Notably, a previous structural alignment of lysozyme from T4 bacteriophage (T4L) with a dimer portion of peptidoglycan4uFu subsites. The D‐E linkage is in the vicinity of the catalytic residue Glu19 and this is the site where cleavage of the glycosidic bond occurs during catalysis. The combination of persistent hydrogen bonding between the sugar and protein, but with flexibility around the D‐E linkage, may be essential features for the activity of the enzyme. Our λ lysozyme‐peptidoglycan model suggests that in the presence of the natural substrate the Eu‐Fu pocket will be occupied by the peptide chains while the sugars occupy the E‐F or E′‐F′ subsites. This would hold the substrate in place while allowing a conformational change at the active site. In light of this behavior it is interesting that in the crystal structure of the complex of λ lysozyme with NAG6, a single sugar molecule does not occupy all six subsites in a single molecule of λ lysozyme. In molecule A of λ lysozyme subsites A‐D are populated by four of the NAG6 units and subsites E'F’ are populated by a second NAG6 molecule. Therefore, the D‐E linkage, which is flexible in the simulations, is missing in both the molecules in the crystal structure.NMR studies and MD simulations have given a consistent picture of the binding of NAG4 and NAG6 to λ lysozyme in solution. Residues particularly in subsites B, C, and D of λ lysozyme make persistent hydrogen bonds to the sugar units, while flexibility in the D‐E linkage enables the final two sugar units to explore a number of binding modes including the EF, E′F′, and EPrevious NMR studies and MD simulations of λ lysozyme have identified that in the apo state in solution the upper and lower lip regions of the protein are flexible. In the crystal structure of the complex of λ lysozyme with NAG6 both molecules adopt a closed conformation. However, in all the MD simulations of the λ lysozyme complexes reported here, which were started from different conformations of the enzyme and with different sugar binding modes, the upper and lower lip regions are dynamic allowing the enzyme to adopt both open and closed conformations. In addition, in the NMR titrations of λ lysozyme with NAG4 and NAG6 significant broadening of the resonances of residues within the upper lip region is observed although some of these residues are not close to the ligand binding site. This suggests that the flexibility of the lip regions seen for the apo protein in solution is maintained, to some extent, in the complex although there may be changes to the timescale or nature of the dynamics. Our results suggest that an open form is needed for the entry of the substrate and upon ligand binding the upper and lower lips retain flexibility but can close to create a cage around the catalytic Glu19 as required for the activity of the enzyme. This study demonstrates that NMR spectroscopy combined with molecular dynamics simulations can provide information that is complementary to the static crystal structure by highlighting the dynamic nature of the enzyme‐inhibitor complex.Appendix S1: Supplementary MaterialClick here for additional data file.


COX7A1 is a subunit of cytochrome c oxidase, and plays an important role in the super‐assembly that integrates peripherally into multi‐unit heteromeric complexes in the mitochondrial respiratory chain. In recent years, some researchers have identified that COX7A1 is implicated in human cancer cell metabolism and therapy. In this study, we mainly explored the effect of COX7A1 on the cell viability of lung cancer cells. COX7A1 overexpression was induced by vector transfection in NCI‐H838 cells. Cell proliferation, colony formation and cell apoptosis were evaluated in different groups. In addition, autophagy was analyzed by detecting the expression level of p62 and LC3, as well as the tandem mRFP‐GFP‐LC3 reporter assay respectively. Our results indicated that the overexpression of COX7A1 suppressed cell proliferation and colony formation ability, and promoted cell apoptosis in human non‐small cell lung cancer cells. Besides, the overexpression of COX7A1 blocked autophagic flux and resulted in the accumulation of autophagosome via downregulation of PGC‐1α and upregulation of NOX2. Further analysis showed that the effect of COX7A1 overexpression on cell viability was partly dependent of the inhibition of autophagy. Herein, we identified that COX7A1 holds a key position in regulating the development and progression of lung cancer by affecting autophagy. Although the crosstalk among COX7A1, PGC‐1α and NOX2 needs further investigation, our study provides a novel insight into the therapeutic action of COX7A1 against human non‐small cell lung cancer. COX7A1 could inhibit cell proliferation and colony formation ability, as well as promote cancer cell apoptosis. COX7A1 overexpression blocked the autophagic flux via downregulation of PGC‐1α and upregulation of NOX2. To catalyze the reduction of molecular oxygen to water, the cytochrome c is used as a substrate by COX. This is the terminal and rate‐limiting step of mitochondrial respiration, which contributes to the energy stored in the electrochemical gradient.In recent years, scientists have noticed that mitochondrial dysfunction is implicated in multiple diseases, especially for cancer.Autophagy is a type of bulk degradation process, as well as a mechanism of intracellular protein and organelle recycling. In this process, the cell engulfs proteins, protein aggregates, organelles or lipids go into a double‐membrane vesicle, autophagosome, which will further fuses with the lysosome to form the autolysosome finally.NOX2 is a superoxide generating enzyme, which forms ROS.In this study, we mainly explored the effect of COX7A1 on cell viability and autophagy in human non‐small cell lung cancer cells. Our results indicated that COX7A1 suppressed cell proliferation capacity and colony formation ability, and promoted cell apoptosis. In addition, the overexpression of COX7A1 blocked autophagic flux via downregulation of PGC‐1α and upregulation of NOX2, and further analysis showed that the effect of COX7A1 on cell viability was partly dependent on the inhibition of autophagy. Therefore, our study identified that COX7A1 plays a crucial role in the treatment of human non‐small cell lung cancer.22.12. The culture medium was replaced every other day, and the H838 cells were passaged  every 5 or 6 days.Human non‐small cell lung cancer cell lines, NCI‐H838 and NCI‐H1703, were purchased from the American Type Culture Collection (ATCC). The cells were cultured with RPMI‐1640 (Gibco) supplemented with 10% Fetal Bovine Serum (Gibco), 100 U/mL penicillin and 0.1 g/mL streptomycin (Sigma) in a humidified 37°C incubator with 5% CO2.2The coding sequence of human COX7A1 was amplified using PCR  and subsequently cloned into a pCI vector (Promega Corporation). To overexpress COX7A1 in H838 cells, the cells were transfected with the pCI‐COX7A1 vector  using Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol. The control group for COX7A1 overexpression was performed with the transfection of empty vector. Following transfection for 24 hours, the H838 cells in different groups were harvested for subsequent experimentation. The overexpression efficiency was examined using western blot.2.3To evaluate cell proliferation ability, the proliferation index was measured in different groups using CCK‐8 method  as the references.2.4Total mRNA in each group was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. Then, cDNA was synthesized using 2 μg of mRNA and a Transcriptor first‐strand cDNA synthesis kit (Promega). Real‐time qPCR was then performed as previously described.p62‐ Forward 5′‐GACTACGACTTGTGTAGCGTC‐3′.p62‐ Reverse 5′‐AGTGTCCGTGTTTCACCTTCC‐3′.β‐actin‐ Forward 5′‐CCCAGAGCAAGAGAGG‐3′.β‐actin‐ Reverse 5′ ‐GTCCAGACGCAGGATG‐3′.2.5In our study, radioimmunoprecipitation assay lysis buffer  was used to extract the total protein. The protein concentration in different groups was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Herein, the western blot assay was performed as previously described.2.62. Then, the cells were fixed for 20 minutes at room temperature using 4% paraformaldehyde, and stained with crystal violet (5 mg/mL) for 20 minutes at room temperature. Finally, the cell colonies were imaged using an Epson Perfection V600 scanner (Seiko Epson Corporation).In the colony formation assay, 500 cells were seeded into 12‐well plates. The cells were incubated for 7 days in the incubator at 37°C with 5% CO2.7The expression levels of apoptotic genes (Bax and Caspase 3) in different groups were measured using western blot. In addition, cell apoptosis was further analyzed using Annexin V/PI Cell Apoptosis Assay kit (Thermo Fisher Scientific) and Tunel‐FITC Assay Kit (Abcam) according to the manufacturer's protocol. In brief, the cells in each group were dissociated into single cells using trypsin, and washed with PBS. Then, the cell samples were incubated with different staining solutions. Finally, cell samples were detected using a FACSCalibur flow cytometer and FlowJo 7.6.1 software.2.8To knockdown PGC‐1α and NOX2, siRNAs for different genes  were purchased from QIAGEN. All of the siRNAs were transfected into cancer cells using Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol. The cells in the negative control group were transfected with a scrambled sequence of the siRNA. The knockdown effect of siRNA was evaluated using western blot.2.9In our study, ptf‐LC3 vector (mRFP‐GFP‐LC3 reporter construct) was purchased from Addgene, USA. H838 cells were grown in 35 mm glass‐bottom dishes. On reaching 50% confluence, the cells were transfected with different vectors using Lipofectamine 3000 for 24 hours. Then, the cells in different groups were imaged in phenol red‐free medium using a Zeiss Observer Fluorescence Microscope.2.10t tests were applied to compare the means of two groups, and one‐way ANOVA with Bonferroni's correction was used to compare the means of three or more groups. One‐tailed test was used in the Student's t test. P < .05 was considered statistically significant.The results are presented as mean ± SD. All statistical analysis was performed using SPSS 17.0. Herein, unpaired Student's 33.1In this study, human H838 lung cancer cells were tranfected with pCI‐COX7A1 to induce COX7A1 overexpression, and western blot results showed that the expression level of COX7A1 in the Overexpression group was much higher (about 8‐fold) than the Control group  was increased in the COX7A1 Overexpression group (group II) compared with the Control group (group I), and PGC‐1α knockdown abolished the difference between the COX7A1 Overexpression group and Control group. However, the percentage of autolysosome and autophagosome did not show significant any difference among each group . It could be possible that the blockage of autophagy in H838 cells is hard to further downregulate the ration of autolysosomes. To confirm this hypothesis, we also performed a positive control group treated with Bafilomycin A, which is a autophagy inhibitor and has been demonstrated to block the autophagic flux. In this group, the amount of autophagosomes was increased. However, the ratio of autolysosome also did not show significant difference compared with the control group without any treatment, which was similar to the results of the COX7A1 overexpression group  is a type of superoxide generating enzyme complex, which forms ROS and is expressed in myeloid cells, and some cancer cells.3.4The influence of COX7A1 on cell viability was further evaluated in NOX2‐knockdown cells. The results indicated that the inhibition of cell proliferation induced by COX7A1 overexpression could be rescued by NOX2 knockdown in some degree Figure A. Cell a4In recent years, scientists have demonstrated that the regulation of energy generation and cell cycle progression in cancer cells are different from normal cells, and the energy homeostasis also varies in different types of cancers.Autophagy is considered as a survival‐promoting pathway. In this process, the intracellular proteins and organelles can be captured, degraded and recycled in lysosomes, which release the toxic buildup of cellular waste products, and provide substrates to support the metabolism in starvation. During the process of cancer development, autophagy is up‐regulated to make cancer cells survive the microenvironmental stress. In addition, the upregulation of autophagy also promotes the growth and aggressiveness of cancer cells.Some researchers have indicated the effect of COX7A1 on PGC‐1 in skeletal muscle cells, and their results showed that the COX7A1 knockout increased the expression level of PGC‐1β.NOX2 is a superoxide generating enzyme, which forms ROS.However, the detailed mechanism of the effect of COX7A1 on PGC‐1 and NOX2 is still unclear for us. Similar to COX7A1, NOX2 also holds an important role in the process of mitochondrial energy metabolism.In addition, our results indicated that the overexpression of COX7A1 decreased the expression of PGC‐1 and increased the expression of NOX2. However, the relationship between PGC and NOX2 is unclear. We noticed that the knockdown of NOX2 did not affect the expression level of PGC‐1, while the downregulation of PGC‐1 expression induced by COX7A1 overexpression did not show any effect on autophagy in NOX2 knockdown cells. These results suggested that the effect of PGC‐1 on autophagy might depend on the normal expression of NOX2, and NOX2 could be the protein downstream of PGC‐1 in COX7A1‐induced autophagy regulation Figure . NeedlesIn this study, our results indicated that the inhibition of COX7A1 overexpression on cell viability was rescued in some degree by NOX2 knockdown, indicating that the influence of COX7A1 depends on the regulation of autophagy partly. As COX7A1 is a subunit in mitochondrial respiratory chain, it could be possible that COX7A1 regulates cell viability by affecting the mitochondrial respiratory chain and energy generation directly. For example, Mishra et al found that the overexpression of COX7A1 in human lung cancer cells A549) could lead to the inhibition of cell proliferation and the increase in cell apoptosis. To evaluate whether autophagy could be involved in COX7A1‐mediated cell death, they further detected the expression level of BECN1, a gene which holds a key role in autophagy. However, the results showed that no change in the expression level of BECN1 could be observed after COX7A1 overexpression, suggesting that the cell death induced by COX7A1 overexpression might not be due to autophagy regulation in A549 cells. could le5In conclusion, our study mainly addressed the link between COX7A1 and hallmarks of human non‐small cell lung cancer cells. The results indicated that the overexpression of COX7A1 could inhibit cell proliferation and colony formation ability, as well as promote cancer cell apoptosis. In addition, COX7A1 overexpression blocked the autophagic flux via downregulation of PGC‐1α and upregulation of NOX2, and the influence of COX7A1 on cell viability depends on the regulation of autophagy partly. Although the crosstalk among COX7A1, PGC‐1α and NOX2 needs our further investigation, our study provides a novel insight into the therapeutic action of COX7A1 against human human non‐small cell lung cancer.The authors declare that there are no conflicts of interest. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


At room temperature, FeC4H+ is formed as the main ionic product, and the experimental findings are substantiated by labeling experiments. According to extensive quantum chemical calculations, the C−H bond activation step proceeds through a radical‐based hydrogen‐atom transfer (HAT) mechanism. This finding is quite unexpected because the initial spin density at the terminal carbon atom of FeC4+, which serves as the hydrogen acceptor site, is low. However, in the course of forming an encounter complex, an electron from the doubly occupied sp‐orbital of the terminal carbon atom of FeC4+ migrates to the singly occupied π*‐orbital; the latter is delocalized over the entire carbon chain. Thus, a highly localized spin density is generated in situ at the terminal carbon atom. Consequently, homolytic C−H bond activation occurs without the obligation to pay a considerable energy penalty that is usually required for HAT involving closed‐shell acceptor sites. The mechanistic insights provided by this combined experimental/computational study extend the understanding of methane activation by transition‐metal carbides and add a new facet to the dizzying mechanistic landscape of hydrogen‐atom transfer.The reactivity of the cationic metal‐carbon cluster FeC Getting together: Radical‐type hydrogen‐atom transfer transpires by localized spin density generated in situ once the reaction partners approach each other. Transfer of a hydrogen atom constitutes a key step in a broad range of chemical, environmental, and biological processes.δ+−Oδ‐] unit serves as an active site,+ .+ (M=Sc‐Zn),Previous investigations, in which metal oxides were often used as prototypical model systems, suggested that the presence of a pronounced and spatially confined high spin density at the reaction‐initiating center constitutes a prerequisite for an efficient, radical‐based HAT process.As to the role of the spin density in HAT, Ye and Neese demonstrated that HAT from ethane to an active site with depleted spin density is associated with a distinct energy barrier; this is due to the absence of a “prepared” acceptor site of the metal‐oxo group.4+ cluster with methane as revealed in a combined experimental/computational approach. The gas‐phase experiments were performed by using Fourier‐transform ion cyclotron resonance mass spectrometry (FT‐ICR MS) under thermal, single‐collision conditions. Mechanistic aspects were elucidated by high‐level quantum mechanical calculations, which enabled a detailed analysis of the changes of the electronic structure along the reaction coordinate. As will be shown, this study adds another facet to the rich landscape of HAT mechanisms by demonstrating that, in the course of a reaction, a poorly reactive site with a low spin density can be transformed into a highly reactive one carrying high spin density. The knowledge gained from this exercise may open up a new perspective for methane activation through the use of carbon‐based materials in heterogeneous catalysis.Herein, we report an entirely unexpected finding on the reaction of the pristine FeC4+ ions (m/z=104) with CH4, CD4, 13CH4, and a 1:2 mixture of CH4 and CD4 . The FT‐ICR mass spectra, Figure ions m/z=04 with CB with Δm=−48 relative to the precursor ion FeC4+ appears; this signal is assigned to the product ion Fe+, indicating that FeC4+ reacts by carbon‐atom transfer with background gases. Upon leaking CH4 into the ICR cell at a stationary pressure of 2.0×10−8 mbar, a new signal C with Δm=+1 relative to FeC4+ is identified as FeC4H+; clearly, hydrogen‐atom transfer from methane to the precursor ion is accompanied by the release of CH3.  and F (FeC3D2+) appear  and D (FeC3H2+) in Figure G (FeC213CH2+), is identified, .By using CDr Figure  c. If Fe4+ activates methane at ambient temperature. The rate constant k(FeC4+/CH4) is estimated to 5.7×10−10 cm3 molecule−1 s−1. This corresponds to a collision efficiency of ϕ=53 %.4+/CH4/CD4 couples, corrected for background contributions, amounts to 1.4. In addition to the labelling experiments, the elementary compositions of the charged species have been confirmed by exact mass measurements.Clearly, the experimental findings reveal that FeC4+ consists of a linear arrangement of the five atoms having an iron atom at one end of the rod. Other isomers like the spoke and hub (Fe+) arrangement or an Fe+ atom decorated by two C2 ligands can be ruled out; they are much higher in energy  and the other to a quartet state (4[FeC4]+).−1), which is within the error bar of the applied QC method, we are reluctant to assign which of the two forms the ground state; moreover, at room temperature FeC4+ may exist as a mixture of the two states.According to our quite elaborate calculations, performed at the NEVPT217e,15o)/QZ//CASSCF/TZ level of theory, the structure of the most stable isomer of FeC7e,15o/QZ4+, the following was noted: the individual bond length distances of FeC4+ are similar, irrespective of the electronic state. The same holds true for the atomic charge distributions. For both states of FeC4+ almost all of the positive charge is located at the terminal iron atom; Cδ carries less than 0.13|e|, whereas minor negative charges are built up at the remaining three carbon atoms Cα, Cβ, and Cγ. Only the distribution of the spin density exhibits some differences. However, for either spin state, most of the unpaired electrons are located at the iron atom; except for Cβ all carbon atoms of the carbide chain carry some spin density but never more than 0.35 μB.As to the possible effects of the spin states on various molecular features of FeC4+‐mediated C−H bond activation of methane, QC calculations based on the density functional theory (DFT)4 almost always corresponds to the cleavage of the first C−H bond of the substrate and not so much to the subsequent coupling reactions. In addition, our extensive DFT‐based calculations demonstrate the extreme complexity of the follow‐up processes. Therefore, the present study primarily addresses four representative pathways for the initial phase of methane activation by FeC4+; for the sake of clarity, details of all other remaining pathways, considered in this study, are transferred to the Supporting Information .To obtain mechanistic insight into the FeC4, 6[FeC4]+ preferentially from the side of the terminal iron atom. This reflects the fact that most of the positive charge is concentrated at the metal  and B (red). Although 4,6EC1 correspond to the energetically preferred arrangements, these very intermediates prove to be a dead end in the further course of a hydrogen‐atom transfer to Cα of the FeC4 moiety. Both transition states 6TS1 and 4TS1 to bring about HAT are with 70 and 93 kJ mol−1, respectively, located well above the entrance asymptote and without external energy supply not accessible. It was noted in passing that the geometries of the transition states 4,6TS1 are surprisingly similar to those of the FeC+/CH4 system.4,6EC1 only have the choices either to be stabilized by collisional cooling or by the emission of IR‐photons; however, these processes are quite inefficient under the present conditions. Thus, 4,6EC1 can only revert to the reactants.Although neutral and possessing only a negligibly permanent electric dipole moment,δ) of FeC4+, in a side‐on fashion, leads to the formation of the rather flexible and loosely bound encounter complexes 6EC2 and 4EC2 through paths C (black) and D (blue), respectively. In both cases, the intermediates 4,6EC2 are converted barrier‐free to 4,6I2 through 4,6TS2. As 4, 6TS2 are located below the entrance asymptote, FeC4+ is able to activate methane at ambient temperature. In any case, the driving force to generate the rather stable insertion species 4, 6I2 is mostly due to a favorable thermochemistry: The energetic requirements in weakening the Cβ−Cγ bond of the carbon chain as indicated by the change of the bond lengths from 1.29 to 1.36 Å and the cleavage of the C−H bond of methane are overcompensated by the formation of a rather strong C−H bond with an sp‐hybridized carbon atom at the end of the carbon chain.On the other hand, a van der Waals interaction of methane with the terminal carbon‐atom  for 6TS2 indicate that the interaction between the CH3 moiety and FeC4 is negligible. Thus, for thermodynamic reasons, the reaction through a PCET channel does not appear to be feasible.i) Hydride transfer: A closer look at the frontier orbitals of 6[FeC4]+ reveals that all suitable π‐orbitals of the carbon atoms to accept an electron pair, which are sufficiently low in energy, are already doubly or at least singly occupied. Therefore, 6[FeC4]+ does not meet the prerequisites to activate methane through a HT channel.ii) Hydrogen‐atom transfer: Numerous studies have demonstrated that a significant spin density at the hydrogen‐acceptor site plays a crucial role in the thermal, single‐collision activation of methane.2O2]⋅+,4 are located well above the entrance channel. As listed in Table δ atom owns a low spin density of only 0.35 μB. The spin density is not only locally depleted, it also obstructs the HAT process further by being distributed over two different π*‐orbitals.iii) 4+ (Equation 1 a).At a first glance, it seems that all of the established mechanisms cannot explain what happens in the course of hydrogen transfer from methane to FeCδ, HT, and CCH4. This 3c/3e bond is comprised by a doubly occupied σC‐H‐C‐orbital and a singly occupied σ*C‐H‐C‐orbital.Figure δ and CCH4for the stationary points during the C−H bond activation process. It clearly demonstrates that when the two reactants encounter, the approaching methane molecule induces a significant increase in spin density at Cδ, especially at the transit from the encounter complex 6EC2 to the transition state 6TS2. When 6TS2 is converted to the intermediate 6I2, the electron of the moving H‐atom pairs with the single electron at the Cδ‐atom to finally form a σC‐H‐bond. As a consequence, the spin‐density at Cδ almost completely disappears and shifts to the ensuing carbon atom of the CH3‐fragment .Next, Figure t Figure . This fiWhy can the reaction proceed through a radical pathway under thermal, single‐collision conditions even though the initial spin density is low at the reactive site?oxo bond in the quintet oxo‐iron(IV) intermediate from its equilibrium geometry to the transition state amounts to about 39 kJ mol−1.As proposed by Shaikβ−Cγ bond and some bending of the Cβ‐Cγ‐Cδ moiety, the associated contraction of the Cδ−Cγ and Cα−Cβ bonds efficiently compensates the energy requirement for the “preparatory step” . In addition, as proposed by Nørskov,A closer inspection of the structures shown in Figure δ of FeC4+?Finally, the crucial question centers on how methane induces the localization of spin density at the terminal Cδ‐H‐CCH4should be close to 90°. But this is not the case. In fact, the respective transition states are much higher for the reactions involving the other carbon sites . Furthermore, the angle Cδ‐H‐CCH4in 6TS2 is with 114° much larger than the optimum value.If methane were to interact only with the singly occupied π*‐orbital, which is delocalized over the entire carbon chain, a similar reactivity of the individual carbon atoms of the rod should be expected. In addition, the optimum angle Csp‐orbital at Cδ is not innocent. The localization of spin density is ascribed to the electronic reorganization between the doubly occupied sp‐orbital at Cδ and the singly occupied delocalized π*‐orbital along the carbon chain. When methane approaches the terminal Cδ‐atom, a slight bending occurs at the terminal carbon chain (<Cβ‐Cγ‐Cδ=166°). This eases the migration of one of the electrons of the doubly occupied sp‐orbital at Cδ to the delocalized π*‐orbital. As a result, a radical site with high spin density is generated in situ at the terminal carbon atom Cδ, which eventually leads to the radical‐like transition state 6TS2.A closer inspection of the frontier orbital diagram shown in Figure 4+‐mediated activation of methane have been obtained by means of Fourier‐transform ion cyclotron resonance mass spectrometry in combination with high‐level quantum chemical calculations. As shown experimentally, FeC4+ activates the C−H bond of methane at ambient temperature. The reaction is very efficient and proceeds by a radical‐based, classical hydrogen‐atom transfer. This mechanistic scenario is quite surprising as the initial spin density is depleted at the terminal carbon atom. The root causes why nevertheless HAT occurs were addressed by a detailed frontier orbital analysis. As a complement to Neese's and Shaik's hypothesis on the electronic requirements of metal‐oxo intermediates in C−H bond activation, the present study demonstrates on how an active radical center can be generated in situ along the reaction coordinate without a significant energy penalty to pay. The mechanistic insights gained from this study may aid the rational design of carbon‐based catalysts for C−H bond activation.Novel and unprecedented mechanistic insights into the FeCThe 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.


A rich variety of products composed of molybdenum, sulfur, carbon and hydrogen was observed. MoxSy+ species are formed in the first reaction step, together with products containing carbon and hydrogen. The calculations indicate that the strong Mo-S bonds are formed preferentially, followed by Mo–C bonds. Hydrogen is exclusively bound to carbon atoms, i.e. no insertion of a molybdenum atom into a C–H bond is observed. The reactions are efficient and highly exothermic, explaining the rich chemistry observed in the experiment.Molybdenum sulfide is a potent hydrogen evolution catalyst, and is discussed as a replacement of platinum in large-scale electrochemical hydrogen production. To learn more about the elementary steps of MoSThe online version of this article (10.1007/s11244-017-0864-3) contains supplementary material, which is available to authorized users. For this task an FT-ICR mass spectrometer is used, as it is an excellent tool for the examination of ion–molecule reactions in the gas-phase [In this work, the formation of Moas-phase –36, inclas-phase –39. Expe−6–10−4 mbar) by a piezoelectric valve with a backing pressure of about 20 bar. During thermalization cluster formation occurs. The produced (cluster-)ions are entrained by the helium gas carrier and are accelerated and guided by several electrostatic lenses towards the ultrahigh vacuum (UHV) region, with a pressure in the lower 10−10 mbar range without reaction gas. The source gas pulses have no measurable effect on the UHV pressure. The ICR cell is located in the center of the superconducting magnet, where the ions are trapped, mass selected, reacted and detected.The reactivity measurements are performed on a modified Bruker/Spectrospin CMS47X FT-ICR mass spectrometer, equipped with a 4.7 T superconducting magnet and an Apex III data station , 41. The−9 mbar. The liquid sample is degassed by several freeze–pump–thaw cycles. Broadband and single frequency resonant excitation are used to isolate the species of interest. Stable ion signal conditions were obtained for Mon+, n = 1–3, and their reactions with DMDS were monitored by taking mass spectra at delays from 0.0 to 25.0 s. Since ion accumulation in the cell takes 2 s, some reaction products are already present at 0 s reaction delay. Typically, 20 experiment cycles are run at each reaction delay for signal averaging. Kinetic analysis is performed assuming pseudo-first order kinetics. Due to the large number of intermediates and products, only peaks that exceeded a relative intensity of 2% at any time during the first 4 s were taken into account for data evaluation, to keep the fits manageable. After calibration of the measured pressures and correcting for ion-gauge sensitivity [DMDS vapor is introduced into the UHV via a leak valve at a constant background pressure in the range of (6–8) × 10sitivity , pressursitivity . The unc92Mo (STB Isotope Germany GmbH) was used. Metallic powder of 700 mg 92Mo with ≥ 99.9% enrichment was pressed and sintered into a solid target disk. DMDS  was used without further purification.Since natural molybdenum has a broad isotopic distribution, isotopically enriched Structure and energetics of relevant species along the reaction path were calculated using density functional theory (DFT), employing the M06-L functional  along wiSee Supporting Information (SI) for the complete parameter matrices containing all calculated absolute reaction rates, as well as optimized structures and benchmarking of the computational method with respect to other DFT functionals and higher-level methods.+) and its product ions is depicted in Fig. + and one DMDS molecule.Due to the vast variety of products being formed in competing reactions, the reaction kinetics were fitted only for the first 4.0 s. The kinetic diagram showing the time dependent relative intensities of the parent ion . The data at longer times suggest that charge transfer products, in particular C2H6S2+ and C2H5S+, are efficiently formed throughout the reaction sequence, most likely from multiple precursors.At the early stage of the reaction, the primary products MoS+ and CH3SSCH3 interact, there is up to 4.7 eV of energy released . This energy is redistributed among the internal degrees of freedom of the cluster and leads to dissociation of various bonds, leading to the observed reaction products. The high reaction energy also explains why the association product is not observed among the reactions products, since a large number of strongly exothermic reaction channels make radiative association [3· instead of C2H6, leading to less exothermic reactions. There is one reaction that is slightly endothermic  where we might expect that the excess energy is supplied by the kinetic energy of the trapped ions.Structures of possible association and reaction products are shown in Fig. ociation  impossibxSy clusters [+, where only endothermic reactions were observed [From a structural perspective, molybdenum tends to form as many bonds with heavy atoms as possible, as shown also in previous studies on charged and neutral Moclusters , 19, 51.observed –54.tr = 20.0 s reveals that these products continue to react with DMDS until the final products are formed. The molybdenum containing products are shown in Fig. 5C2H6+, MoS4C3H9+ and MoS5C3H9+. It is worth mentioning that neither pure MoSx+ nor MoCxHy+ species were present as final products. Combining the general reaction pattern with theoretical calculations on geometry optimization at the M06-L/def2TZVP level suggests the formation of up to five Mo–S bonds together with Mo–C bonds, see Fig. 2H5+. Other intense charge transfer products are S2C2H6+, S3C3H9+, SC2H3+ and S2C3H7+; their intensities are 88, 53, 41 and 20%, respectively, relative to SC2H5+ with 100%.A qualitative analysis of the mass spectra for reaction delay until 2+ + CH3SSCH3, is discussed in more detail. The reactions 2.1, 2.4 and 2.6 are dominant, consistent with the tendency of molybdenum to directly extract sulfur from DMDS. All reactions included in Table Again, due to the vast variety of simultaneous reactions with different intermediates reacting through competing reaction channels, only the first 4.0 s of the reaction were analyzed with a kinetics fit. The kinetic model was developed iteratively by choosing stoichiometrically allowed and chemically reasonable reaction pathways and testing the quality of the fit. In Fig. 3SSCH3 to Mo2+ is again considerably exothermic  and S3C3H9+; their intensities relative to the most intensive Mo2S8C3H9+ peak are 29, 19 and 11%, respectively.For reaction delays higher than 4.0 s, the acquired mass spectra were analyzed qualitatively, revealing that after a reaction time of approximately 2+ is a product of the reaction, as shown in the mass spectrum in Fig S2, a large number of species can be observed as secondary products. Interestingly, two oxide species Mo2O+ and Mo3S2O+ are observed, suggesting that some of the species present might be capable of dehydrogenating water, which is likely to be present in traces in the vacuum system. The kinetic fit, Fig. 3+ + CH3SSCH3. The first step of the reaction kinetics was modelled as shown in Table For the first 4.0 s, the reaction proceeds smoothly, as shown in Fig. 3+ is predicted to be cyclic, in agreement with previous calculations on neutral systems [3S2 core and two CH3 groups attached to Mo atoms, analogous to the association product of Mo2+ and DMDS CH3, is with ΔE = − 0.98 eV only mildly exothermic, but has almost 50% branching ratio. This indicates that during the formation of the association product, the initial insertion of a Mo center into the S–S bond releases sufficient energy to liberate Mo2+.Molybdenum trimer Mo systems . The tri3+ experiment, which leads to small intensities of secondary products of Mo2+ in the first 4 s of the reaction, below the noise level.Interestingly, no charge transfer products were found in the acquired mass spectra. This might be due to the relatively high noise level in the Mo−10 cm3 s−1 were found. Charge transfer reactions forming mixed SxCyHz+ clusters were observed only for the monomer and dimer in later stages of the reaction; in fact, charge transfer product ions became the dominant products in the monomer kinetics after longer times. At higher reaction delays, the dominant molybdenum containing product ions had the form MoxSy(CH3)z+ with x = 1, 2, y > x and z = 2, 3. Supported by theoretical calculations, it can be assumed that these products consist of two or three methyl groups binding on the sulfided molybdenum cluster. The trimer was found to be relatively unstable, since a dissociative reaction channel was observed producing the fragment ion Mo2+ at a high rate of (5.3 ± 1.5) × 10−10 cm3 s−1. Albeit not being final products, pure MoxSy+ product ions were observed for all examined species, illustrating the strong tendency of molybdenum to form sulfides by direct extraction of sulfur from DMDS.Despite the vast variety of product ions forming, the early stage of each reaction was analyzed for all molybdenum cluster cations examined. Reasonable reaction schemes with realistic reaction rates in the order of 10xHy species incorporated in the product clusters is observed. According to the calculated energetics from the literature [y species incorporated in MoS2 increases from C via CH and CH2 to CH3. This trend is qualitatively reflected in the product distribution, where MopSqCx+ species are very rare.In accordance with literature , the higterature , the binBelow is the link to the electronic supplementary material.Supplementary material 1 (DOCX 741 KB)