Many drugs currently used are covalent inhibitors and irreversibly inhibit their targets. Most of these were discovered through serendipity. Covalent inhibitions can have many advantages from a pharmacokinetic perspective. However, until recently most organisations have shied away from covalent compound design due to fears of non-specific inhibition of off-target proteins leading to toxicity risks. However, there has been a renewed interest in covalent modifiers as potential drugs, as it possible to get highly selective compounds. It is therefore important to know how reactive a warhead is and to be able to select the least reactive warhead possible to avoid toxicity. A robust NMR based assay was developed and used to measure the reactivity of a variety of covalent warheads against serine and cysteine – the two most common targets for covalent drugs. A selection of these warheads also had their reactivity measured against threonine, tyrosine, lysine, histidine and arginine to better understand our ability to target non-traditional residues. The reactivity was also measured at various pHs to assess what effect the environment in the active site would have on these reactions. The reactivity of a covalent modifier was found to be very dependent on the amino acid residue. When designing a covalent drug it may be useful to consider binding as occurring in two distinct stages to avoid toxicity.inact/KI, where: kinact is the maximum rate at which the reversible complex forms the covalent bond (k3); and KI is defined as (k2 + k3)/k1 and is the concentration of inhibitor which gives half the maximum rate of covalent bond formation (E–I).Kinetic models have been introduced to explain covalent inhibition as shown in However, other factors may affect the process. For example, it is possible in some cases that there is a direct reaction between the covalent inhibitor and the protein without a prior molecular recognition event. In other cases the protein may have a substantial role in covalent inhibitor bond formation.The main concern with covalent modifiers is their ability to bind irreversibly to off target proteins and result in toxicological effects such as immune responses.It should be noted that the reactivity of the covalent warheads measured here was carried out in solution. However in the context of an enzyme, molecular recognition events will constrain the warhead to a limited set of orientations within the active site. The orientation of the warhead will affect the rate at which it reacts with the protein. In an ideal scenario, the warhead should be constrained in orientation to give a favourable trajectory for reacting with the appropriate residue, which should result in the covalent bond formation reaction occurring faster and help reduce off target effects.22.1An NMR assay which can track the amount of the covalent warhead in solution with an amino acid was designed. A typical assay setup is shown in The amino acid is used at 10x the concentration of the warhead to give the reaction pseudo first order kinetics, as the concentration of the amino acid remains in large excess of the warhead throughout the reaction and therefore can be considered to be constant. To ensure that the covalent warhead was reacting with the sulfur of the cysteine or the hydroxyl of the serine the amine group was protected with a BOC group and the carboxylic acid was protected as a methyl ester.As the reaction proceeds the amount of the covalent warhead is reduced and this can be measured as the decrease in the integral of the NMR peaks corresponding to the warhead as seen in By then plotting the natural log of the integral of these peaks against time  a straig2.2The reactivity of a range of covalent warheads measured against cysteine and serine are presented in 15) and the vinyl sulfonamide (16) was measured three times and an average taken. In each case the average was found to be within 7% of the measured rates suggesting that the assay is extremely reproducible , the oxaborole (3) and the nitriles , where a reversible reaction could potentially regenerate the inhibitor. The Michael acceptors  are likely also reversible, albeit the rate of the reverse reaction is likely to be very slow and in many cases essentially irreversible. The irreversible warheads are the β-lactams , where hydrolysis of the inhibitor from the amino acid residue could potentially happen, but would produce a modified and unreactive version of the inhibitor.During this study we have looked at both potentially “reversible” and “irreversible” covalent modifiers. A “reversible” covalent inhibitor would be for example the trifluoroketone (3) and the sulfonyl fluoride (13) are “harder” electrophiles). In contrast, the Michael acceptors 12, 15 and 16 are softer electrophiles and react more rapidly with the “softer” cysteine. The cysteine reacts with these Michael acceptors at least 2 orders of magnitude more rapidly than serine does. The reactions of cysteine with 15 and 16 are significantly faster than any of the other reactions that we investigated. Compound 16 was found to react so quickly with cysteine that a rate constant could not be measured for this reaction. The “hard” or “soft” natures of the electrophiles was suitable for prediction of the relative reactivity between cysteine and serine in the majority of cases, but in some cases, particularly in cases where the electrophile was of moderate “hardness” or “softness” the relative reactivity was more difficult to predict and other factors may be important.It is instructive to compare reactions with cysteine and serine. There is a very different order in reactivity of the different warheads with the two residues. This can be largely explained as serine is a much “harder” nucleophile than cysteine and tends to react faster with the “harder” electrophiles (for example the benzoxaborole (12) is currently under investigation in a range of ongoing covalent modifier drug discovery projects,10 which was found to have very similar reactivity between cysteine and serine. This may be because the nitrogen in compound 10 alters the reactivity of the double bond or it may be that the nitrogen is able to act as a general base and deprotonate the serine to make it more reactive. Compound 11 also had similar reactivity with both cysteine and serine.The acrylamide warhead  appear to react similarly with both cysteine and serine, but have relatively slow rates. The sulfonyl fluoride (13) was quite reactive to both cysteine and serine. The carbamate (14) was significantly more reactive to cysteine than serine.The nitriles (2 (a penicillin) was found to react with serine too slowly for a rate to be measured. This is interesting because penicillins, like compound 2, are known to react with a serine residue in vivo to have an effect. This shows how important the non-covalent binding and orientation of the warhead in active site plays in the ability of covalent drugs to form a covalent bond to their target. The β-lactams 4 and 6 were more reactive than the penicillin with both cysteine and serine. Interestingly compound 6 was moderately reactive with serine. This may be due to the nitrogen lone pair being less available due to delocalisation into the phenyl ring, increasing the reactivity of the carbonyl.Compound A wide range of different covalent warheads, with different reactivities, are found in clinically used drugs. This shows that the reactivity of the warheads depends on a number of factors, including the amino acid residues found in the target and the recognition motif that gives rise to the initial non-covalent binding interaction and selectivity with the target.2.3Tyrosine, arginine, lysine, threonine and histidine were identified as amino acids that might also be targetable using covalent inhibitors. To investigate this, three covalent warheads which displayed the overall trend of being more reactive against cysteine than serine in Amino acids where it is conceivable that there could be significantly different protonation states in proteins were also investigated at other pHs. Histidine (pka = 6) was investigated at pH 5 and pH 9.8. Tyrosine (pKa = 10) and cysteine (pKa = 8) were also investigated at pH 9.8. As was expected where the amino acids were more deprotonated, and therefore more reactive, the reactions proceeded faster.2.4Ultimately, the ability to target a particular amino acid will depend on both the warhead being used and on the environment in which the residue exists.Overall these results provide an indication of how a drug containing a covalent warhead may be tuned to a particular project. If a covalent compound is too reactive and is found to have toxic side effects a less reactive warhead can be selected. Conversely, if the covalent warhead is not reactive enough to form a bond to the target then a more reactive warhead can be selected. Also we have derived information in understanding the differences between the different amino acid residues. The relative orientation of the warhead to the nucleophile in the binding site will also be an important factor in the success of a covalent drug, which has not been assessed here. The advantages of this approach is that it is simple to carry out and gives an indication of the relative reactivity of different warheads with different amino acid side chains. However, it does not take account of the molecular recognition events within an enzyme active site.2.5(12) was synthesised from aniline using acryloyl chloride and triethylamine in dichloromethane at 0 °C. The methyl carbamate (14) was obtained under the same conditions using methyl chloroformate, as was the vinyl sulfonamide (15) using the sulfonyl chloride. The substituted acrylamide (10) was synthesised from aniline and the appropriate carboxylic acid using propylphosphonic anhydride as the coupling reagent in tetrahydrofuran. The 4-β-lactam (4) was synthesised from β-phenylalanine using mesyl chloride and sodium bicarbonate in acetonitrile at 60 °C. The 1-β-lactam (6) was obtained from aniline using 3-bromopropionyl chloride and potassium carbonate in dichloromethane at 0 °C to give the amide which was then cyclised using sodium tert-butoxide in dimethylformamide at 0 °C. The ketones  were synthesised from benzaldehyde using the appropriate Grignard reagent in tetrahydrofuran at 0 °C. These were then oxidised to the ketone using Dess-Martin periodinane. Finally, the sulfonyl fluoride (13) was obtained by treating the sulfonyl chloride with TBAF in tetrahydrofuran.Some of the covalent warheads of interest were commercially available and these were purchased. Those which were not available were synthesised as shown in The amino acids of interest were commercially available with the exception of Boc-Arg-OMe. This was synthesised as shown in 33.11H (400 MHz or 500 MHz), 13C (100 MHz or 125 MHz), and 2D NMR spectra were recorded in CDCl3, MeOD or DMSO‑d6 using a Bruker Avance spectrometer. Proton chemical shifts are reported in ppm relative to the residual chloroform peak (δ = 7.26 ppm), methanol peak (δ = 3.31 ppm) or DMSO peak (δ = 2.50 ppm). Multiplicities are given as s (singlet), d (doublet), t (triplet), q (quartet), qui (quintet), m (multiplet), brs (broad singlet), dd (doublet of doublets), td (triplet of doublets), dt (doublet of triplets) or as a combination of these. Coupling constants (J) are quoted to the nearest 0.1 Hz. 13C chemical shifts are reported in ppm relative to the residual chloroform peak (δ = 77.16 ppm), methanol peak (δ = 49.00 ppm) or DMSO peak (δ = 39.51 ppm). Assignment of proton and carbon signals was achieved using COSY, HSQC and HMBC experiments. LCMS analysis was performed with either an Agilent HPLC 1100 series connected to a Bruker Daltonics MicrOTOF or an Agilent Technologies 1200 series HPLC connected to an Agilent Technologies 6130 quadrupole spectrometer, where both instruments were connected to an Agilent diode array detector. High resolution electrospray measurements were performed on a Bruker Daltonics MicrOTOF mass spectrometer. Preparative HPLC was performed on a Gilson HPLC  connected to a Gilson 155 UV/vis detector using Waters XBridge C18 columns  eluting with 0.1% formic acid in water and acetonitrile (95:5 -> 5:95) as the mobile phase.Chemicals and solvents were purchased from commercial sources and were used without any further purification. Air and water sensitive reactions were carried out under an inert nitrogen atmosphere in oven dried glassware. Analytical thin-layer chromatography (TLC) was performed on precoated TLC plates . Developed plates were air-dried and analysed under a UV lamp (UV254/365 nm) and by staining with permanganate or ninhydrin. Flash column chromatography was performed on prepacked silica gel cartridges  using a Teledyne ISCO Combiflash Rf or Combiflash Rf 200i.3.24, passed through a phase separator and evaporated to dryness. The residue was purified by flash chromatography eluting with a gradient of 0–100% ethyl acetate in heptane to give 4-phenylazetidin-2-one  as a white solid.3-amino-3-phenyl-propanoic acid  was dissolved in MeCN (60 ml) and methanesulfonyl chloride  and sodium hydrogen carbonate  were added. The mixture was heated to 60 °C and stirred overnight. Water (10 ml) was added and the mixture extracted 3x with 30 ml ethyl acetate. The combined organics were dried over MgSO1H : δ 7.39–7.28 , 6.71 , 4.70 , 3.41 , 2.84 ;13C : δ 168.46 (C7), 140.33 (C4), 128.87 (C2), 128.21 (C1), 125.69 (C3), 50.42 (C5), 47.94 (C6).Analysis is in agreement with the literature.3.34, passed through a phase separator and evaporated to dryness. The residue was recrystallised from 5 ml 1:1 heptane: ethyl acetate to give the intermediate  as a white powder.To a suspension of potassium carbonate  in DCM (10 ml) was added aniline . The mixture was cooled to 0 °C and 3-bromopropanoyl chloride  added dropwise. The reaction was stirred at 0 °C for 15 min then allowed to warm up to room temperature. After three hours LCMS showed complete conversion of aniline to the intermediate so 5 ml water was added and the layers separated. The aqueous layer was extracted 3× with 15 ml ethyl acetate and the combined organic layers were dried over MgSOm/z 228.1 [M+H]+MS (ESI) tert-butoxide  was added and the reaction was allowed to slowly warm up to room temperature for three hours. The solvent was removed in vacuo and the residue was dissolved in 10 ml DCM, washed with 10 ml water and the aqueous layer extracted 2× with 15 ml DCM. The combined organics were dried over MgSO4, passed through a phase separator and evaporated to dryness. The residue was purified by flash chromatography eluting with a gradient of 0–80% ethyl acetate in heptane to give 1-phenylazetidin-2-one  as a white powder.This intermediate was dissolved in DMF (10 ml) and cooled to 0 °C. Sodium 1H : δ 7.31 , 7.06 , 3.56 , 3.05 ;13C : δ 164.54 (C7), 138.51 (C4), 129.12 (C2), 123.81 (C1), 116.12 (C3), 37.99 (C5), 36.02 (C6);m/z 148.1 [M+H]+MS (ESI) m/z (ESI+) calcd for C9H10NO [M+H]+: 148.0757, found 148.0464 (6.5 ppm).HRMS Analysis is in agreement with the literature.3.44, passed through a phase separator and evaporated to dryness. The residue was purified by flash chromatography eluting with a gradient of 0–9% methanol in DCM to give a mixture containing (E)-4-(dimethylamino)-N-phenyl-but-2-enamide (46 mg) as a pale yellow powder. This was purified by HPLC eluting with 5–95% acetonitrile in water (0.1% NH4) to give (E)-4-(dimethylamino)-N-phenyl-but-2-enamide  as a white powder.(E)-4-(dimethylamino)but-2-enoic acid hydrochloride  was dissolved in THF (5 ml) with triethylamine  and T3P  (50% in Ethyl acetate (0.6400 ml)) was added. After 15 min aniline  was added and the reaction stirred overnight. Then 10 ml saturated sodium hydrogen carbonate solution was added and the solution extracted 3× with 10 ml DCM. The combined organic layers were dried over MgSO1H : δ 7.92 , 7.57 , 7.28 , 7.08 , 6.94 , 6.14 , 3.04 , 2.23 ;13C : δ 142.83 (C8), 129.20 (C2), 125.76 (C7), 124.56 (C1), 119.95 (C3), 60.53 (C9), 45.71 (C10)m/z (ESI+) calcd for C12H17N2O [M+H]+: 205.1335, found 205.1350 (4.6 ppm).HRMS 3.54 and evaporated to dryness. The residue was purified by flash chromatography with a gradient of 0–40% ethyl acetate in heptane to give 1-phenylprop-2-yn-1-ol  as a colourless oil.Benzaldehyde  was dissolved in THF (20 ml) and bromo(ethynyl)magnesium  (0.5 M in THF) was added dropwise over 10 min and the reaction stirred at 0 °C for 30 min. The reaction was then allowed to warm up to room temperature and stirred for three hours. 10 ml saturated ammonium chloride solution was added and stirred vigorously. 10 ml Ethyl acetate was added and the layers separated, the aqueous layer was extracted 2× with 10 ml ethyl acetate. The combined organic layers were washed with 10 ml water, 10 ml brine, dried over MgSO1H : δ 7.55 , 7.38 , 5.43 , 3.42 , 2.68 .Analysis is in agreement with the literature.3.6(17)  was dissolved in DCM (15 ml) and cooled to 0 °C. Dess-Martin  was added and the reaction stirred while slowly warming up to room temperature. When TLC indicated the reaction was complete 20 ml saturated sodium thiosulfate solution was added, the layers separated and the organic layer washed with 20 ml saturated sodium thiosulfate solution. The aqueous layers were combined and extracted 2× with 15 ml DCM. The combined organics were washed with saturated sodium hydrogen carbonate solution, brine, dried over MgSO4, passed through a phase separator and evaporated to dryness. The residue was purified by flash chromatography eluting with a gradient of 0–50% ethyl acetate in heptane to give 1-phenylprop-2-yn-1-one  as a colourless oil which crystallised on standing.1-phenylprop-2-yn-1-ol 1H : δ 8.17 , 7.64 , 7.50 , 3.43 ;13C : δ 177.54 (C5), 136.29 (C4), 134.67 (C1), 129.85 (C3), 128.83 (C2), 80.87 (C7), 80.41 (C6).Analysis is in agreement with the literature.3.7N-phenylprop-2-enamide  as a white powder.Aniline  and triethylamine  were dissolved in DCM (10 ml) and cooled to 0 °C. Prop-2-enoyl chloride  was added and the reaction allowed to warm up to room temperature overnight. Methanol (10 ml) was added and stirred for 30 min and the reaction was evaporated to dryness. The residue was purified by flash chromatography eluting with a gradient of 0–50% ethyl acetate in heptane to give 1H : δ 10.09 , 7.66 , 7.32 , 7.06 , 6.44 , 6.25 , 5.75 ;13C : δ 163.06 (C6), 138.95 (C4), 131.86 (C7), 128.68 (C2), 126.69 (C8), 123.40 (C1), 119.30 (C3);m/z 148.1 [M+H]+MS (ESI) m/z (ESI+) calcd for C9H10NO [M+H]+: 148.0757, found 148.0743 (12.8 ppm).HRMS Analysis is in agreement with the literature.3.84 and evaporated to dryness. The residue was purified by flash chromatography eluting with a gradient of 0–80% ethyl acetate in heptane to give benzenesulfonyl fluoride  as a colourless oil.Benzenesulfonyl chloride  was dissolved in THF (10 ml) and tetrabutylammonium fluoride  was added. The reaction was stirred at room temperature overnight. 5 ml saturated sodium hydrogen carbonate solution and 10 ml water was added and the reaction extracted 3× with 20 ml DCM. The combined organics were dried over MgSO1H : δ 8.01 , 7.79 , 7.64 ;13C : δ 135.71 (C1), 133.24 , 129.80 (C2), 128.52 (C3).Analysis is in agreement with the literature.3.9N-phenylcarbamate  as a pale yellow oil.Aniline  was dissolved in DCM (25 ml) and cooled to 0 °C. Pyridine  was added followed by methyl chloroformate  and the reaction stirred while slowly warming up to room temperature. When TLC indicated the reaction was complete 10 ml water was added, the layers separated and the aqueous layer extracted 2× with 15 ml DCM. The combined organics were passed through a phase separator and evaporated to dryness. The residue was purified by flash chromatography eluting with a gradient of 0–50% ethyl acetate in heptane to give methyl 1H : δ 7.38 , 7.31 , 7.07 , 6.63 , 3.78 ;13C : δ 154.19 (C6), 138.00 (C4), 129.20 (C2), 123.64 (C1), 118.92 (C3), 52.45 (C7);m/z (ESI+) calcd for C8H07NO2 [M+H]+: 152.0706, found 152.0716 (5.9 ppm).HRMS Analysis is in agreement with the literature.3.104 and evaporated to dryness. The residue was purified by flash chromatography eluting with a gradient of 0–50% ethyl acetate in heptane to give a colourless oil. This was dissolved in 5 ml DCM and 5 ml heptane was added, the DCM was removed under reduced pressure. A precipitate formed and was isolated by filtration to give N-phenylethenesulfonamide  as off white crystals.Aniline  and 2-chloroethanesulfonyl chloride  were dissolved in DCM (10 ml) and cooled to 0 °C. Triethylamine  was added and the reaction was stirred for 10 min then allowed to warm up to room temperature. After 2 h TLC suggested no starting material remained so the reaction was quenched with 20 ml water and the layers separated. The aqueous layer was extracted 2× with 20 ml DCM, the combined organics washed with 20 ml 1 N HCl, 20 ml brine, dried over MgSO1H : δ 7.45 , 7.30 , 7.21 , 7.14 , 6.58 , 6.24 , 5.91 ;13C : δ 137.76 (C4), 136.25 (C6), 129.14 (C2), 127.55 (C7), 123.80 (C1), 119.70 (C3);m/z 184.1 [M+H]+MS (ESI) m/z (ESI+) calcd for C8H10NO2S [M+H]+: 184.0427, found 184.0430 (5.3 ppm).HRMS Analysis is in agreement with the literature.3.114 and evaporated to dryness. The residue was purified by flash chromatography eluting with a gradient of 0–80% ethyl acetate in heptane to give 1-phenylprop-2-en-1-ol .Benzaldehyde  was dissolved in THF (10 ml) and bromo(vinyl)magnesium  (1 M in THF) was added dropwise over 10 min and the reaction stirred at 0 °C for 30 min. The reaction was then allowed to warm up to room temperature and stirred overnight. Then 10 ml saturated ammonium chloride solution was added and stirred vigorously. 10 ml ethyl acetate was added and the layers separated, the aqueous layer was extracted 2× with 10 ml ethyl acetate. The combined organic layers were washed with 10 ml water, 10 ml brine, dried over MgSO1H : δ 7.23 , 7.17 , 5.90 , 5.20 , 5.05 , 5.02 .Analysis is in agreement with the literature.3.12(18)  was dissolved in DCM (15 ml) and cooled to 0 °C. Dess-Martin  was added and the reaction stirred while slowly warming up to room temperature.1-phenylprop-2-en-1-ol 4, passed through a phase separator and evaporated to dryness.When TLC indicated the reaction was complete 20 ml saturated sodium thiosulfate solution was added, the layers separated and the organic layer washed with 20 ml saturated sodium thiosulfate solution. The aqueous layers were combined and extracted 2× with 15 ml DCM. The combined organics were washed with saturated sodium hydrogen carbonate solution, brine, dried over MgSOThe residue was purified by flash chromatography eluting with a gradient of 0–50% ethyl acetate in heptane to give 1-phenylprop-2-en-1-one  as a colourless oil. Despite the product being stored in the freezer it was observed to have turned brown when needed so was repurified by flash chromatography (12 g column) eluting with a gradient of 0–2% methanol in DCM to give 1-phenylprop-2-en-1-one  yield as a colourless oil.1H : δ 7.93 , 7.55 , 7.45 , 7.14 , 6.42 , 5.90 ;13C : δ 191.07 (C5), 137.27 (C4), 133.03 (C1), 132.38 (C6), 130.22 (C7), 128.72 (C2), 128.65 (C3).Analysis is in agreement with the literature.3.13tert-butoxycarbonylamino)-5-guanidino-pentanoic acid  and N,N′-dicyclohexylmethanediimine  were dissolved in DCM (4 ml) with N,N-dimethylpyridin-4-amine . Methanol  was added and the reaction stirred for five days. LCMS indicated the reaction was complete so it was filtered and evaporated to dryness. The residue was purified by flash chromatography eluting with a gradient of 0–10% methanol in DCM followed by HPLC 5–95% acetonitrile in water (0.1% HCOOH) to give methyl 2-(tert-butoxycarbonylamino)-5-guanidino-pentanoate  as a colourless oil.2-: δ 8.19 , 7.18 , 4.21 , 3.73 , 3.30 , 3.19 , 1.84 , 1.69 , 1.41 ;13C : δ 169.64 (C2), 157.75 (C8), 156.37 (C12), 80.39 (H13), 52.99 (C3), 52.69 (C1), 40.93 (C6), 29.92 (C4), 28.49 (C14), 25.07 (C5);m/z (ESI+) calcd for C12H25N4O4 [M+H]+: 289.1870, found 289.1874 (1.3 ppm).HRMS 3.142O:10% D2O) was added 5 μl of the covalent warhead (0.2 M in DMSO‑d6), in an NMR tube and a 1H NMR spectra was acquired. To 0.5 ml of the same buffer was added 5 μl of the amino acid (2 M in DMSO‑d6). This was added to the NMR tube and the solution mixed by inverting the tube several times. If necessary, sonication was used to ensure complete dissolution. This gave a final solution of 1 mM covalent warhead, 10 mM amino acid in 100 mM PBS. A 1H NMR was typically recorded on this sample every 10 min for 6 h. In the experiment, there was a ∼4 s data acquisition time, followed by a 4 s relaxation delay to minimise peak saturation. Each data point required approximately 2 min (16 scans per data point).To 0.5 ml of the assay buffer (100 mM PBS in 90% H


X-ray diffraction (XRD), scanning electronic microscopy (SEM) and transmission electron microscopy (TEM) measurements revealed that the optimal α-LiFeO2 sample was composed of extremely small nanoparticles. The electrochemical properties were tested at 0.1 C in the cut-off voltage of 1.5–4.8 V. The sample obtained at 150 °C for 6 h exhibited the best cycling stability with high initial discharge capacity of 223.2 mAh/g, which was extremely high for pristine α-LiFeO2 without any modification process. After 50 cycles, the discharge capacity could still maintain 194.5 mAh/g with good capacity retention. When the charge–discharge rate increased to 0.2 C and 0.5 C, the initial discharge capacities were 216.6 mAh/g and 171.5 mAh/g, respectively. Furthermore, the optimal sample showed low charge transfer resistance and high lithium-ion diffusion coefficients, which facilitated the excellent electrochemical performance.Nanosized α-LiFeO Sakurai et al. synthesized α-LiFeO2 sample by high temperature solid-state method, but the sample was proven to be electrochemically inactive [2 sample at low temperature of less than 250 °C via H+/Li+ ionic exchange reaction. Although this sample was electrochemically active, it exhibited only reversible capacity of 80 mAh/g and poor cycle performance at 0.1 C [2 synthesized by Li et al. via solvothermal methods exhibited higher initial capacity of 135 mAh/g, but the cycling performance was still poor [2-MWCNT compounds via radio frequency oxygen plasma method [2 with excellent electrochemical performance.Lithium-ion batteries have been widely applied to laptop computers, electric vehicles, hybrid vehicles, and other portable electronic products . As an inontoxic . Accordied forms ,5. Amongoperties . Many reinactive . Then, till poor . To furta method . The opt+ transportation distance. For α-LiFeO2, the electrochemical performance is significantly impaired by the poor conductivity. Thus, reducing the particle size is a valid approach to promote the ionic transportation and the electronic conductivity. Morales et al. [2 nanoparticles with smaller average diameter of about 50 nm. This material could exhibit high initial specific capacity of 150 mAh/g. Hirayama et al. further confirmed the improvement effect of smaller particle size on the electrochemical performance of LiFeO2 [2 nanoparticles reported in the above literature still cannot meet the demands of high reversible capacity of cathode materials. To further enhance the reversible capacity, Wang et al. synthesized α-LiFeO2 nanoparticles with particle size under 10 nm. It showed extremely high initial specific capacity of 290.6 mAh/g at 0.1 C, but the cycling performance was not very satisfactory for cathode materials [According to the existing literature , nanomats et al.  synthesif LiFeO2 . Howeveraterials . Based o2 samples via a simple, quick and eco-friendly strategy at ambient temperature followed by a low temperature calcine process. Compared with the reported methods [2 with high performance, but also alleviates the environment pollution. More importantly, the optimal α-LiFeO2 sample with small particle size showed excellent electrochemical properties at high voltage.We successfully synthesized nanosized α-LiFeO methods ,17,18,192 nanoparticles were prepared by a simple, quick and eco-friendly strategy followed by a low temperature calcined process. Firstly, 2.73 g LiOH·H2O and 3 g Fe(NO)3·9H2O was dissolved in 20 mL ethanol. The suspension was intensely stirred under magnetic stirring at ambient temperature for 5 h. After being washed with distilled water and ethanol repeatedly to eliminate redundant lithium hydrate, the brown product was dried at 80 °C for 12 h in an air oven. The brown particles were obtained by hand-grinding in an agate mortar. Finally, the product was sintered at different temperatures  for 6 h in tube furnace under argon atmosphere.The α-LiFeO2 powders was examined with scanning electron microscope  and transmission electron microscopy . The specific surface area and pore volume were obtained by Brunauer–Emmett–Teller (BET) N2 adsorption–desorption measurement . X-ray photoelectron spectroscopy  was used to estimate the Valence states of α-LiFeO2 electrodes during different process.The crystalline phase was characterized by X-ray diffraction . The particle morphology of the α-LiFeO2 were evaluated at ambient temperature. The positive electrode was prepared with 85% synthesized product, 10% acetylene black, and 5% polyvinylidene fluoride binder in N-methyl-2-pyrrolidone solvent. The slurry was pressed onto Al foil and dried at 100 °C for 4 h and then punched into round positive slices with 12 mm diameter. The lithium foil was the anode electrode, ehilr celgard microporous polypropylene membrane was the diaphragm. The electrolyte solution was 1 M lithium hexafluorophosphate (LiPF6) solution in a mixture of the ethylene carbonate (EC) and diethl carbonate (DEC) in a 1:1 volume ratio. The charge–discharge profiles were investigated by Land CT2001A battery testing system with a voltage window of 1.5–4.8 V. The EIS was studied by CS-350 electrochemical workstation with frequency ranging from 0.1 Hz to 100 kHz. The CV was also tested by CS-350 electrochemical workstation, with scan rate of 0.1 mV/s.The electrochemical performance of α-LiFeO2, where the Li precursor and Fe precursor dissolved in ethanol solution and reacted together under ambient temperature. Herein, the formation of α-LiFeO2 includes two stages, the formation of nucleuses and the growth of α-LiFeO2 crystals. Meanwhile, the low temperature calcination process further improved the crystallinity of α-LiFeO2.2 samples obtained under various temperatures for 6 h in argon atmosphere is presented in 2 well . When the temperature rises to 350 °C, there is an impurity phase, which can be attributed to spinel LiFe5O8 [5O8 impure phase appears after high temperature sinter process [2 would still be the main phase, and the (110), (200) and (220) diffraction peaks gradually become thinner upon increasing temperature.The XRD patterns of the α-LiFeO LiFe5O8 ,21. This2 nanoparticles obtained at various temperatures. As shown in 2 [2 particles tended to agglomerate a bunch-like morphology. Morales et al. synthesized 50 nm α-LiFeO2 nanoparticles via molten-salt way that also agglomerated with a bunch-like morphology, and they believed such particular architecture can provide an uninterrupted pathway for lithium ion diffusion, which enhances the electrochemical properties [own in 2 . Furtheroperties .2 adsorption–desorption measurements were introduced to obtain the surface information of α-LiFeO2 calcined at different temperatures. In 2·g−1, 194.48 m2·g−1, 159.30 m2·g−1 and 103.42 m2·g−1, respectively. Furthermore, the corresponding desorption average pore diameter of these samples are 8.46 nm, 3.27 nm, 3.61 nm and 8.28 nm, respectively. Clearly, α-LiFeO2 calcined at 150 °C possesses the largest specific surface area and smallest pore diameter; these surface structures supply a large specific surface area to enhance the contact area between electrolyte and electrode, and shorten the transportation distance of Li+ ions between the nanoparticles [The Narticles .2 [5O8, which is considered responsible for the sharp decrease of initial discharge capacity [2 synthesized at high temperature was electrochemically inactive, as the higher temperature may increase the disorder degree of iron ions on lithium sites and block lithium diffusion pathways [The cycling performance of the electrodes obtained at different temperature are shown in 2 ,23,24. Tcapacity ,26. Furtpathways ,28.Among these samples, the sample obtained at 150 °C exhibits the highest initial specific capacity of 223.3 mAh/g. More importantly, the discharge capacity retains 194.5 mAh/g after 50 cycles. Although there is a sharp capacity decline in the first five cycles, this sample can show outstanding cycling performance with high capacity retention of 96.9% in subsequent charge–discharge processes. The sample calcined at 150 °C exhibited such excellent electrochemical performance may attributed to small average particle size of below 10 nm and lower calcined temperature.2 electrodes obtained at 150 °C. It can be seen from 2 samples at different rates are very stable and close to 100%, indicating the α-LiFeO2 obtained in this work can show excellent reversible electrochemical performance.To further evaluate the electrochemical performance, the optimal sample sintered at 150 °C was cycled between 1.5–4.8 V at different rates.2, which was also found in orthorhombic, corrugated layer, and tetragonal phase LiFeO2 [4+ atoms, which originally located on octahedral 4a sites might move to tetrahedral 8c position during reaction. This is the main conduction pathway of lithium ions during charging; the displace position of Fe4+ may account for the voltage hysteresis and a continuous decrease of plateau at 4.5–4.8 V. Another explanation is that unstable Fe4+ may react with the electrolyte during charging process. Wu et al. suggested that the reversible electrochemical reaction occurring in the a-LiFeO2 electrode during charge–discharge are Fe3+/Fe2+, the corresponding reaction formation is:2 polymorphs [e LiFeO2 ,29,30. Dlymorphs .2 at pristine state (OCV), charged to 4.8 V and discharged to 1.5 V. The X-ray photoelectron spectroscopy (XPS) was employed to indicate the chemical state of α-LiFeO+ ions are extraction from α-LiFeO2 to form α-Li1+xFeO2 [+ de-intercalated from α-Li1+xFeO2 [2. Evidently, among these electrodes, the electrode calcined at 100 °C demonstrates the lowest current densities and smallest peak areas, exposing that the electrode possesses higher inner resistance and poor electrochemical performance [The cyclic voltammetry measurements shown in i1+xFeO2 ,34. Two i1+xFeO2 . A sharpformance .The lithium-ion diffusion coefficient can be calculated by the following equation .R is the gas constant, T is the absolute temperature, A is the surface area of the electrode, n is the number of electrons per-molecule, F is he Faraday constant, C is the molar concentration of Li+ ions, ω is the angular frequency, and σ could been obtained by liner fitting the relationship curve between 2 calcined at 100 °C, 150 °C, 250 °C and 350 °C were calculated as 2.98 × 10−13, 1.3 × 10−10, 8.08 × 10−11, and 9.11 × 10−12, respectively. The results of calculations are also listed in Li+, which may positively affect the electrochemical performance.2 nanoparticles with average diameter below 10 nm were successfully prepared via a simple, quick and eco-friendly strategy followed by a low temperature calcine process. The optimal α-LiFeO2 calcined at 150 °C for 6 h exhibited excellent electrochemical performance. The initial discharge capacity could reach up to 223.3 mAh/g at 0.1 C, which is close to the theoretical value, and retained 194.5 mAh/g after 50 cycles. It also exhibited high specific capacities of 216.6 mAh/g and 171.5 mAh/g with capacity retention of 77.3% and 84.5% after 50 cycles at 0.2 C and 0.5 C, respectively. Such high electrochemical performance can be attributed to the ultra-small particle size and small charge transfer resistance. The present work may provide a promising strategy to promote the practical application of α-LiFeO2.Ultra-small α-LiFeO


This paper describes the improved performance from an electrochemically treated composite TiO2 electrode for use in aqueous Al-ion batteries. After application of the cathodic electrochemical treatment in 1 mol/dm3 KOH, Mott–Schottky analysis showed the treated electrode as having an increased electron density and an altered open circuit potential, which remained stable throughout cycling. The cathodic treatment also resulted in a change in colour of TiO2. Treated-TiO2 demonstrated improved capacity, coulombic efficiency and stability when galvanostatically cycled in 1 mol·dm−3AlCl3/1 mol·dm−3 KCl. A treated-TiO2 electrode produced a capacity of 15.3 mA·h·g−1 with 99.95% coulombic efficiency at the high specific current of 10 A/g. Additionally, X-ray diffraction, scanning electron microscopy and X-ray photoelectron spectroscopy were employed to elucidate the origin of this improved performance.The potential for low cost, environmentally friendly and high rate energy storage has led to the study of anatase-TiO A capacity of 60 mA·h·g−1 at 2.66 A/g was measured by Hang et al. [2 in aqueous Al3+-containing electrolytes provides an opportunity for creating aqueous cells with high working voltages. However, to date there has been limited discussion of the low coulombic efficiency of anatase TiO2 in aqueous aluminium salt electrolytes, especially during initial cycles. This is an important point for the electrode to be considered for use in full, two electrode battery cells. Furthermore, methods to improve the performance of TiO2 in aqueous aluminium electrolytes have generally focused on nanostructures such as nanospheres, nanotube arrays or the addition of graphene flakes [−1 was measured from TiO2 nanospheres at the low specific current of 50.25 mA/g, while a graphene-TiO2 electrode produced a discharge capacity of ca. 20 mA·h·g−1 at a high current of 6.25 A/g. However, the coulombic efficiency of these TiO2 structures is low, with the coulombic efficiency of graphene incorporated TiO2 being approximately 50%. Aqueous intercalation batteries are being explored as potentially cheap, safe, non-toxic and high power energy storage devices ,2,3,4. Atrolytes ,9,10,11. of Al3+ ,7,9,10, trolytes ,11. Othetrolytes ,13,14,15g et al. . The posg et al. ,18,19. T2 could provide a method to incorporate Ti3+ to improve the electrodes’ ionic and electronic conductivity whilst potentially improving insertion kinetics of Al3+. However, only He et al. have described the potential of doped TiO2 via their synthesis of black nanoleaves [2 electrodes and its application in aqueous batteries [Doped TiOnoleaves . Their matteries ,21. 2 doping have been widely explored for use in other applications such as photocatalytic hydrogen production, environmental pollutant removal or supercapacitor electrodes [2 nanostructures have been prepared via high temperature (> 500 °C) treatments in H2, Ar and N2 atmospheres [2. Electrochemically doped TiO2 nanotube arrays has shown improved electrochemical performance as aqueous supercapacitor current collectors, while self-doped TiO2 nano structures have demonstrated improved photocatalytic activity for water splitting [2 electrodes in order to improve their performance as a negative electrode in aqueous AlCl3 electrolyte, demonstrating high rate capability and stability during cycling. Significantly, doping of TiO2 is demonstrated to be an important factor for improving coulombic efficiency and initial cycling stability in aqueous electrolyte.Other methods of TiOectrodes . It is wospheres ,24,25,26plitting ,28. Here2 electrodes were manufactured through combining TiO2 nanopowder (87.5 wt%), Vulcan XC-72 carbon black (5 wt%), Nafion binder (7.5 wt%) and additional propanol (approximately 3 g for every 1 g of powder). The resulting ink was mixed using a Silverson high speed shear mixer for 30 min at 5000 rpm, then sonicated for a further 20 min. TiO2 was purchased from US-nano and had a nominal particle size of 5 nm. The resulting ink was then coated onto a carbon polymer current collector, which was used because of its electrochemical stability in acidic electrolytes and for being more cost effective than other metal current collectors, such as titanium or platinum foils and meshes. Electrodes were allowed to dry in ambient conditions overnight, resulting in 23–24 mg of active electrode covering an area of 7 cm−2, where the electrode covered 1 cm × 3.5 cm of both sides of the current collector. Composite TiO−3 AlCl3/1 mol·dm−3 KCl. The electrochemical treatment process consisted of holding electrodes at −1.40 V vs. SCE in 1 mol·dm−3 KOH for 15 min, which was repeated ten times with a 3 min open circuit potential (OCP) between repeats. No obvious bubbling or H2 evolution was visible during chronoamperometry. Electrodes were left in the KOH electrolyte for several hours before being washed, in de-ionised water, and transferred into a 3-electrode cell with a AlCl3/KCl electrolyte. Galvanostatic cycling and electrochemical impedance spectroscopy were performed on a Solartron 1470E battery analyser. Electrochemical impedance spectroscopy (EIS) was performed to produce both Mott–Schottky and Nyquist plots. For Mott–Schottky analysis, EIS was performed using a 10 mV perturbation between 10 kHz and 10 Hz at DC bias potentials between 0.4 V and −0.7 V vs. SCE. For the Nyquist plots, EIS was performed on electrodes in partially charged states, achieved by applying a 100 mA/g charge current to −0.8 V and −0.9 V, holding the electrodes at these potentials for 120 s, then performing measurements at OCP. These measurements were performed between 0.1–10 kHz using a 10 mV perturbation around the OCP. Electrochemical experiments were carried out in 3-electrode cells using a saturated calomel reference (SCE) electrode and copper-hexacyanoferrate as a reversible counter electrode. The electrolyte used was 1 mol·dmX-ray diffraction (XRD) was carried out using a Bruker D2 phaser with Cu Kα radiation (average wavelength = 1.5418 Å). Data points were collected in the 2θ range 15° to 75° with a 0.0202572° increment and 0.4 s time step. Scanning electron microscope (SEM) images were obtained using a JSM 6500F field emission electron microscope, which was operated at 15.0 kV and a working distance of 13.7 mm. 2 electrodes. A potential of −1.40 V vs. SCE was applied to the electrode in 1 mol·dm−3 KOH in an attempt to partially reduce the TiO2 active material. The potential was stepped from 0.0 V vs. SCE to −1.40 V vs. SCE and held for 15 min. 2 and treated-TiO2 electrode. Both show the characteristic anatase pattern with no difference between the two, suggesting a lack of bulk structural change to the TiO2 electrodes.The cathodic electrochemical treatment was carried out via a chronoamperometry performed on composite TiO2 electrode, at 1000× and 40000× magnification, with 2. At 1000× magnification, both electrodes show an even coating of active material, though larger agglomerations can also be seen. At 40000× magnification, individual particle agglomerations can be observed, though resolution of individual particles cannot be seen due to the small nominal TiO2 particle size of approximately 5 nm. No significant change can be observed in the structure of the electrodes, suggesting that any change in electrode performance was not due to particle flocculation or minor electrode cracking, which could have increased surface area or improved electrode wettability. 2 (right) electrode following the chronoamperometry shown in 2 electrode can be seen to change colour to pale-yellow. This would be consistent with the introduction of Ti3+ and a change in the band gap of TiO2 [2 nanotube arrays developing a blue colour [2 has undergone hydrogenation [2 doping [ of TiO2 ,29. Despe colour , a yellogenation ,32, hydrgenation  or N2 do2 doping .2) and electrochemically treated TiO2 (treated-TiO2) electrodes. 2 (a) and treated-TiO2 (b) electrodes. No detectable difference can be observed between the samples. This could indicate that Ti3+ states exist within the bulk rather than the surface. However, given that measurements were performed ex-situ, it is more likely that any Ti3+ formation, from the electrochemical reduction treatment, is followed by re-oxidation in air. Ti 2p3/2 peaks are located at 459.40 and 458.71 eV for TiO2 and treated-TiO2 respectively, while Ti 2p1/2 peaks are located at 465.15 and 464.46 eV. Unexpectedly, the presence of inorganic Ti-oxyfluoride < 1%, was detected in both samples, which may arise during ink preparation, but could alternatively arise from low level X-ray degradation of the binder within the ink.X-ray photoelectron spectroscopy was performed on the samples shown in −3 AlCl3/1 mol·dm−3 KCl. 2 and treated-TiO2 electrode at a frequency of 5 kHz. Capacitance values were calculated via Equation (1), where C = capacitance, f = frequency and Z″ = imaginary impedance part. The positive and linear gradient produced by the as-manufactured TiO2 electrode, between approximately −0.45 V and −0.3 V vs. SCE, is indicative of an n-type semi-conductor and has an approximate gradient of 0.0748 [−2 from the treated-TiO2 electrode suggests an increase in the number of donors  according to Equation 1, where C = capacitance, A = area, dN = number of donors, e = electronic charge, V = applied potential, FBV = flat band potential, k = Boltzmann’s constant and T = absolute temperature [f 0.0748 ,36. The perature .(1)C=−i2 and also shows the response at frequencies of 2 kHz and 10 kHz. The Mott–Schottky plot is again indicative of an n-type semi-conductor. The gradient of the linear portion of the plot, between −0.46 and −0.56 V vs. SCE, is approximately 6.25 × 10−5. This is 3 orders of magnitude lower than the approximate gradient found in the as-manufactured TiO2 electrode at the same frequency, providing clearer evidence that the electrochemical treatment resulted in an increase in the electron donor number. The number of donors, dN, was not estimated since the electrochemically active surface area was not known. Nevertheless, the cathodic electrochemical treatment resulted in an increase in the number of donors (electrons) and a shift of the FBV to a more negative potential. Both these criteria would be expected from an introduction of Ti3+ or oxygen vacancies into TiO2, as expected from the cathodic treatment at −1.4 V vs. SCE in 1 mol·dm−3 KOH.2 was then also cycled at 10.0 A/g. 2 becomes most obvious at higher specific currents. 2, at 0.2 A·g−1 to be 25.3 mA·h·g−1, which is actually marginally higher than the 23.8 mA·h·g−1 measured from the treated-TiO2. However, the improved performance of treated-TiO2 becomes obvious at higher specific currents. At 2 A/g, the discharge capacities of TiO2 and treated-TiO2 are 15.9 mA·h·g−1 and 21.3 mA·h·g−1 respectively. At 8 A/g, the TiO2 electrode can produce a capacity of only 3.2 mA·h·g−1 while a capacity of 15.3 mA·h·g−1 was measured from treated-TiO2 at the higher specific current of 10 A/g, clearly showing superior rate capability. The coulombic efficiency of treated-TiO2 was measured to be 81.4% and 91.8% at 0.2 A/g and 0.5 A/g, respectively. This is higher than a coulombic efficiency of 75.9% and 85.0% for the TiO2 electrode. Above specific currents of 1.0 A/g, the coulombic efficiency of both electrodes are roughly similar, increasing from approximately 98% to > 99.95% at 8.0 A/g. To determine variations in electrode performance, 10 galvanostatic cycles were performed at each of the specific currents of 0.2, 0.5, 1.0, 2.0, 4.0, 6.0 and 8.0 A/g. Treated-TiO2 (a) and TiO2 (b) electrodes during the 1st, 10th and 60th cycle. For treated-TiO2, a 1st cycle discharge capacity of 22.5 mA·h·g−1 was measured, corresponding to a coulombic efficiency of 81.7%. This increased to 24.2 mA·h·g−1 at a coulombic efficiency of 94.44% by the 10th cycle with a discharge capacity and coulombic efficiency of 23.1 mA·h·g−1 and 96.2% measured during the 60th cycle. For the untreated TiO2 electrode, an initial discharge capacity of 16.6 mA·h·g−1 was measured with a coulombic efficiency of 70.30%. Discharge capacity and coulombic efficiency were measured as 20.1 mA·h·g−1 and 83.4% during the 10th cycle and 21.4 mA·h·g−1 and 94.0% by the 60th cycle. The increased discharge capacity and coulombic efficiency from the treated-TiO2 electrode during initial cycling would be a particularly important consideration for full cell construction.2 electrode throughout the cycling regime, resulting in a lower equivalent series resistance (ESR) for the electrochemically treated electrode. This can be seen in 2 and TiO2 electrodes is 11.7 mV at 0.2 A/g, increasing linearly to 116.5 mV at 2 A/g. At 10.0 A/g, the IR-drop from treated-TiO2 is 135.2 mV, while the IR-drop from TiO2 at 8.0 A/g is 539.3 mV. The gradient of the linear fits were used to determine an ESR of 0.562 Ω for treated-TiO2 and 2.843 Ω for untreated TiO2. This suggests a significant improvement in the conductivity of TiO2, a reasonable conclusion from the introduction of Ti3+ expected from the electrochemical reduction process, which would decrease the band gap and thus improve conductivity. Electrodes were 23 and 24 mg for the treated and pristine electrodes respectively, both covering an area of 7.0 cm−2 such that differences in electrode impedance arising from differences in mass loading or electrode thickness can be reasonably neglected.The IR-drop, between charge and discharge, was also observed to be lower for the treated-TiO2 and treated-TiO2 electrodes were left at open circuit potential (OCP) for 1 h before being subject to EIS protocols. Measurements were performed between 0.1 Hz–10 kHz with a 20 mV perturbation. Electrodes were analysed in partially charged states by applying a 100 mA/g charge current to −0.8 V and −0.9 V, holding the electrodes at these potentials for 120 s then performing measurements at OCP. Potentials of 0.8 V and 0.9 V vs. SCE correspond to states-of-charge of approximately 20% and 50%, respectively. 2 electrode at OCP after charging to −0.8 V or −0.9 V For both electrodes, the Nyquist plots follow a ca. 45° impedance, starting at 0.13 Ω on the real x-axis for treated-TiO2 and 0.17 Ω for TiO2 due to the series resistance arising from the electrolyte and electrode. This 45° line curves up to > 45° at a frequency of approximately 2 Hz for the treated electrode and at approximately 1 Hz for the as-manufactured electrode. The characteristic semi-circle present for many insertion battery electrodes is not immediately obvious, suggesting either a rapid charge-transfer or a very small charge-transfer resistance; this suggests that charge storage is diffusion limited. The > 45° slope present at mid to low frequencies further suggest the possibility of capacitive behaviour from the electrodes [2 compared to untreated TiO2, suggests a lower impedance. This could be assigned to a lower charge transfer resistance if the high frequency region is represented by a depressed semi-circle with a smaller radius. Alternatively, the ca. 45° line may represent a Warburg impedance, which can describe the transport of ions through electrode pores [The effect of the treatment was further characterised with the use of electrochemical impedance spectroscopy (EIS). TiOectrodes , though de pores .2 and treated-TiO2 were cycled to −1.1 V vs SCE. 2 and treated-TiO2 cycled at 1 A/g to −1.1 V. An increase in discharge capacity is measured from both electrodes: 34.6 mA·h·g−1 and 37.2 mA·h·g−1 for TiO2 and treated-TiO2 respectively. However, there is a considerable decrease in coulombic efficiency from TiO2, measured at only 58.0% compared to 85.0% when cycled to −1.0 V at 1 A/g. In contrast, coulombic efficiency is only marginally lower for treated-TiO2 at 90.3% compared to 90.9% when cycled to −1.0 V. This further demonstrates the drastic improvement provided by the electrochemical reduction treatment.Furthermore, TiO2 was altered compared to TiO2, both before and after cycling. 2 has an OCP of approximately −0.425 V vs. SCE with the OCP of TiO2 being approximately 0.58 V. Post-cycling, the OCP of treated-TiO2 after 1 h is relatively stable at approximately −0.38 V, with TiO2 being at 0.2 V and still increasing after 1 h at OCP. This implies a change in the equilibrium potential and therefore a change in the concentration of some species within the electrode, which could be the incorporation of Ti3+ due to the electrochemical treatment. It cannot be ruled out that the electrochemical reduction process improves performance through factors such as improved electrode wetting or enhanced surface area, though the lack of structural changes observed via SEM imaging and XRD suggest otherwise. However, while XPS showed that there was no measurable change in oxidation state, the observed colour change, different OCP values and Mott-Schottky plots suggest a change in the band gap and charge carrier density of TiO2, which would be consistent with the introduction of Ti3+. This change in OCP also remains stable in the electrolyte and during cycling. It was also observed that the OCP of treated-TiO2 electrodes were prepared through a potentiostatic hold at −1.4 V vs. SCE, in 1 mol·dm−3 KOH. The colour change of a TiO2 electrode from white to pale yellow suggests the introduction of Ti3+, though this could not be confirmed with XPS measurements. However, a Mott–Schottky plot showed treated-TiO2 to have a greater electron donor number, which would be the expected result from the introduction of Ti3+ or oxygen vacancies. The electrochemical reduction protocol was shown to improve capacity, coulombic efficiency and stability during the first 60 galvanostatic cycles. Conductivity also drastically improved, observed via EIS and analysis of the IR-drop between charge and discharge cycles. It is demonstrated that the electrochemical treatment described can improve the performance of composite TiO2 electrodes for use in aqueous Al-ion batteries and could also be applied to titanium oxide electrodes in other metal-ion batteries. A discharge capacity of 15.3 mA·h·g−1 was possible from treated-TiO2 at 10 A/g—the highest specific current recorded from TiO2 in aqueous Al3+ electrolyte and a higher specific current than recorded from NaTi2(PO4)3 in aqueous Na-ion cells. The observed improvements to coulombic efficiency and stability during the first 10 cycles are particularly important for use in full cells, but still requires further improvements and understanding. Cycling a separate treated-TiO2 electrode at 1 A/g allowed for a capacity of 37.2 mA·h·g−1 at 90.3% coulombic efficiency, where an as-manufactured TiO2 electrode produced 34.6 mA·h·g−1 at a much lower coulombic efficiency of 58.0%, further demonstrating the improved stability from the electrochemically treated TiO2 electrodes. This work also highlights the importance of TiO2 doping for improved electrode performance in high-rate, aqueous electrolyte batteries.Electrochemically treated TiO


Catalpa ovata (Bignoniaceae) is widely distributed throughout Korea, China, and Japan. This study investigated the anti-inflammatory effects of catalpalactone isolated from C. ovata in lipopolysaccharide (LPS)-induced RAW264.7 cells. Catalpalactone significantly inhibited nitric oxide (NO) production and inducible NO synthase (iNOS) expression in LPS-induced RAW264.7 cells. The levels of cytokines such as interleukin-6 and tumor necrosis factor-α were reduced under catalpalactone exposure in LPS-induced RAW264.7 cells. Additionally, catalpalactone suppressed signal transducer and activator of transcription 1 (STAT-1) protein expression and interferon-β (IFN-β) production. Treatment with catalpalactone prevented interferon regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB) activation. Taken together, these results suggest that the anti-inflammatory effects of catalpalactone are associated with the suppression of NO production and iNOS expression through the inhibition of IRF3, NF-κB, and IFN-β/STAT-1 activation. Inflammation is a protective immune response that occurs when tissues are damaged by infection. Tissue damage is accompanied by pathological responses such as pyrexia, edema, and pain . LipopolLPS is recognized by toll-like receptor 4 (TLR4), and it promotes the activation of two different signal pathways: myeloid differentiation factor MyD88)- and toll/IL-1R domain-containing adaptor inducing interferon-β (TRIF)-dependent pathways . Stimula8- and toCatalpa ovata G. Don. (Bignoniaceae) is an ornamental tree that is widely distributed across Korea, China, and Japan ..C. ovata2.RAW264.7 macrophages were purchased from the American Type Culture Collection . Cells were cultured in DMEM containing 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen) at 37 °C in a humidified atmosphere with 5% CO5 cells/mL for 24 h. The cells were treated with various concentrations of catalpalactone . After 24 h of incubation, cell viability was analyzed according to the manufacturer’s instructions using an EZ-Cytox cell viability assay kit. Briefly, 10 μL of the kit solution was added to each well and incubated for 4 h at 37 °C under 5% CO2. The cell viability was determined by measuring formazan production using an ELISA reader  based on absorbance at 480 nm. The reference wavelength was 650 nm.Cells were cultured on a 96-well plate at a density of 2 × 105 cells/mL for 24 h. The cells were treated with various concentrations of catalpalactone  and incubated for 2 h. Cells were incubated with LPS (1 μg/mL) for 18 h at 37 °C under 5% CO2. The culture supernatant (100 μL) was reacted with Griess reagent (100 μL) in a 96-well plate and incubated at room temperature for 15 min. The absorbance was measured at 540 nm using an ELISA reader. Nitrite concentrations were calculated using a standard sodium nitrite curve. The results are presented as the mean ± SD of six replicates for one representative experiment.Cells were cultured on a 96-well plate at a density of 2 × 105 cells/mL for 24 h. The cells were treated with various concentrations of catalpalactone  and incubated for 2 h. Cells were then incubated with LPS (1 μg/mL) for 18 h at 37 °C under 5% CO2. The cells were harvested and lysed using NP40 cell lysis buffer (containing 1 mM phenylmethylsulfonyl fluoride and 1× protease inhibitor cocktail) for 30 min on ice. The protein concentration of the cell lysate was determined using a Bio-Rad Protein Assay . Aliquots of 30 μg of protein were loaded and electrophoresed on 10% SDS–polyacrylamide gels and then transferred to nitrocellulose membranes . The membranes were washed once using phosphate-buffered saline (PBS) containing 0.05% Tween 20 and blocked using PBS containing 5% skim milk and 0.05% Tween 20 for 1 h. After blocking, the membranes were incubated with target antibodies  at 4 °C overnight. After incubation, the membranes were washed and incubated for 2 h at room temperature with HRP-conjugated secondary antibodies diluted 1:2000 in blocking buffer. The protein bands were detected using a chemiluminescence detection system .Cells were cultured on 100 mm culture dishes at a density of 2 × 105 cells/mL for 24 h. The cells were pretreated with various concentrations of catalpalactone  for 2 h and then stimulated with or without LPS (1 μg/mL) for 18 h. The culture supernatant was measured using ELISA kits for IL-6, TNF-α, and IFN-β according to the manufacturer’s protocol. The results are presented as the mean ± SD of three replicates for one representative experiment.Cells were cultured in a 6-well plate at a density of 2 × 105 cells/mL for 24 h. pNF-κB-Luc , pIRF3-Luc , and pRL-TK control reporter vectors  were transfected using Lipofectamine 2000 according to the manufacturer’s protocol. After transfection, the cells were pretreated with various concentrations of 30 or 50 µM catalpalactone for 2 h and then stimulated with or without LPS (1 μg/mL) for 24 h. Luciferase activity was measured using the Dual-luciferase Reporter Assay System . The results are presented as the mean ± SD of three replicates for one representative experiment.Cells were cultured in a 6-well plate at a density of 2 × 105 cells/mL for 24 h. The cells were pretreated with various concentrations of catalpalactone  for 2 h and then stimulated with or without LPS (1 μg/mL) for 18 h. Total RNA was isolated from the cells using an RNeasy kit . A PrimeScript 1st strand cDNA synthesis kit was used for reverse transcription (RT) according to the manufacturer’s protocol. A Chromo4 real-time PCR detection system  and SYBR Green were used for RT-PCR amplification for iNOS, IL-6, TNF-α, and β-actin under the following conditions: 50 cycles of 94 °C for 20 s, 60 °C for 20 s, and 72 °C for 30 s. The primer sequences are listed in Cells were cultured in a 6-well plate at a density of 2 × 10T threshold method using the BioRad software tool Genex-Gene Expression Macro [The expression of target genes in comparison with β-actin was evaluated via the comparative Con Macro . The resp < 0.05 was considered to be statistically significant.The differences between datasets were assessed using one-way analysis of variance followed by Tukey’s multiple comparison test. C. ovata in LPS-induced RAW264.7 cells. Catalpalactone was not cytotoxic in RAW264.7 cells at concentrations up to 50 µM. Moreover, our data indicated that catalpalactone inhibited NO and pro-inflammatory cytokine production and suppressed iNOS mRNA and protein expression in LPS-induced RAW264.7 cells in a concentration-dependent manner. In addition, catalpalactone suppressed STAT1 expression and IFN-β production and significantly decreased IRF3 and NF-κB activation in LPS-induced RAW264.7 cells. Taken together, catalpalactone has anti-inflammatory activity via both MyD88-dependent and independent pathway in LPS-stimulated RAW264.7 cells.We observed the anti-inflammatory activities of catalpalactone isolated from On the basis of its low cytotoxicity and potent anti-inflammatory effects, catalpalactone is suggested to be a superior medicine plant resource that can be developed to prevent and treat inflammatory diseases.


The CYP2C11 assay showed good linearity for all components (R2 > 0.999). Substrates and metabolites were found to be stable for up to 72 h. Additionally, the method demonstrated good reproducibility, intra- and inter-day precision (<15%), acceptable recovery and accuracy (80%–120%), and low detection (1.3501 µM and 3.2757 µM) and quantitation limit values (4.914 µM and 9.927 µM) for 16α-hydroxytestosterone and testosterone, respectively. Salicylic acid acts reversibly as a noncompetitive (weak) inhibitor with Ki = 84.582 ± 2.67 µM  = 82.70 ± 2.67 µM) for CYP2C11 enzyme activity. This indicates a low potential to cause toxicity and drug–drug interactions.The inhibitory effect of new chemical entities on rat liver P450 marker activities was investigated in a functional approach towards drug development. Treatment of colorectal cancer (CRC) and chemoprevention using salicylic acid has gained a lot of attention, mainly in the prevention of the onset of colon cancer. Thus, an in vitro inhibitory effect of salicylic acid on rat CYP2C11 activity was examined by using high performance liquid chromatography (HPLC). High performance liquid chromatography analysis of a CYP2C11 assay was developed on a reversed phase C Cytochrome P450s are known as Phase 1 mono-oxygenase drug-mediated enzymes that play a vital role as a body defence in the drug pharmacokinetic field . CYP450 Nowadays, toxicity and bioavailability issues are the most common obstacles in the drug development field . TherefoThe CYP2C11 isoform is the most dominant isoform in male-specific rat liver microsomes and accounts for 50% of the total male-specific male rat hepatocytes . Wojciko2 from fatty acid and increasing the amount of uncoupled fatty acid in the plasma. Until now, various papers on the pharmacokinetics and metabolism of salicylic acid have been reported, but only one paper [Salicylic acid  belongs ne paper  has showConsequently, an analytical HPLC method was validated, which studies the in vitro inhibitory effect of salicylic acid on the activities of major cytochrome CYP450s (CYP2C11) as an example for promoting the safety and efficacy of salicylic acid in clinics. UV-VIS spectrophotometry analysis for CYP2C11 assay was performed by dissolving salicylic acid (100 µM), testosterone (200 µM) and 16-α hydroxytestosterone (50 µM) powder in pure methanol (wavelength cut-off of methanol was 210 nm).The following graph illustrates the measurements of maximum wavelength of each component in the CYP2C11 assay. From overlain spectra of CYP2C11 assay compounds  and Fig, it is sr2 values of 0.9999 and 0.9998, respectively. The linear regression coefficient was within the acceptable range (r2 > 0.99), according to ICH  guidelines. Relative standard deviations (%RSD) at each different concentration (<5%) met the ICH guidelines [Different concentrations of testosterone and its metabolite (16α-hydroxytestosterone) solutions  + 70% of methanol) to evaluate that the method outcomes were not affected by the impurities. Specificity was achieved by choosing the right mobile phase composition (phosphate buffer at pH 3.36 (A) + methanol (B)) using low pressure isocratic elution programming: 68% methanol + 32% phosphate buffer at pH = 3.36. This results in good separation of the enzyme peak (NADPH (Nicotinamide Adenine Dinucleotide Phosphate Hydrogen)-regenerating system) from the CYP2C11 metabolite (16α-hydroxytestosterone), salicylic acid, phenacetin and testosterone peaks at T = 25 °C, using a C18  column at 0.8 mL/min flow rate and a wavelength of λ = 243 nm .Intra-assay variation of testosterone and 16α-hydroxytestosterone was determined by measuring three testosterone concentration levels  and three 16α-hydroxytestosterone concentration levels  3 times in a single batch (n = 3). The outcomes, summarised in Inter-assay variation was determined by measuring testosterone standards of three concentrations levels  and 16α-hydroxytestosterone  for three consecutive days, in separate batches. The results, summarised in m [The stability of testosterone was investigated at three different concentrations , which were chosen as an approximation of its Km , and sto2 = 0.9997), where r2 met ICH guidelines. LOD and LOQ values were 5.4408 and 16.4874 µM, respectively. Percentage recovery for testosterone at concentrations of 25, 100 and 200 µM was found to be within an acceptable range (80%–120%), according to ICH guidelines, and thus the results show high and acceptable accuracy (80%–120%) for testosterone concentrations of 25 and 100 µM. On the other hand, at 200 µM concentration, the accuracy was just beyond the acceptable value of 120.5% for 0 and 24 h. The results indicate that testosterone solution was relatively stable for 72 h at ambient temperature, which is consistent with the literature [The outcomes revealed that there was no variation in the concentration or changes in chromatographic behavior of testosterone at 0, 24, 48, and 72 h, compared to actual concentrations. Calibration curves were plotted for days 1−4 and all four calibration curves were averaged, hence the average calibration curve equation was: y = 0.0236x − 0.0406  was investigated for three different concentrations  stored for 72 h and kept at room temperature in natural light conditions. Three different concentration ranges of 16-alfa hydroxytestosterone were chosen, since the amount of metabolite formed at 200 µM of testosterone was 35 µM, meaning it was necessary to investigate the stability of the metabolite at low, medium, and high concentrations. Phenacetin  was added to each batch. The sample was analysed in triplicate (n = 3) for each batch. The stability test results are summarised in 2 = 0.9992), where r2 met the ICH guidelines. Percentage recovery for 16-alfa hydroxytestosterone at concentrations of 10, 40 and 80 µM was found to be within acceptable ranges (80%–120%), according to ICH guidelines. Thus, the results show high and acceptable accuracy (80%–120%) for 16-alfa hydroxytestosterone concentrations of 10, 40 and 80 µM compared with the standard known concentration. Therefore, the results indicate that 16-alfa hydroxytestosterone solution was stable for 72 h at room temperature, which is consistent with the literature [The results presented in A robustness study was performed by changing the percentage of the mobile phase (increasing the percentage of methanol by 2%) using HPLC, and evaluating the effect on retention time and peak area of each compound. R = 12.359 min) when the concentration of methanol decreased by 2%. This means that testosterone is more soluble in organic solvent than aqueous medium. A mobile phase consisting of 68% methanol with 32% phosphate buffer at pH 3.36 was chosen because the resolution between phenacetin and 16-alfa hydroxytestosterone peaks (1.083 min) was greater than the resolution between phenacetin and 16-alfa hydroxytestosterone peaks (0.842 min) when using 70% methanol with 30% phosphate buffer at pH 3.36. This implies that the CYP2C11 assay method is robust, considering the change in mobile phase ratio.Overall, retention time, peak area and resolution for salicylic acid, phenacetin, testosterone and 16α-hydroxytestosterone did not vary when changing the composition of methanol by 2%, except that testosterone eluted later  at 25 °C was greater than the resolution between salicylic acid and the phenacetin peak (1.26 min) at 30 °C. Overall, this method is robust, considering the change in column temperature.Different concentrations of testosterone  were incubated in the presence of 0, 50, 100 and 200 µM salicylic acid using 0.5 mg/mL of rat liver microsome with 1.0 mM NADPH (Nicotinamide Adenine Dinucleotide Phosphate Hydrogen), 5.0 mM G6P (Glucose-6-Phosphate), 1.7 units/mL G6PDH (Glucose-6-Phosphate Dehydrogenase), 1.0 mM EDTA (Ethylenediamine tetraacetic acid) and 3.0 mM magnesium chloride and the reaction was terminated at different time intervals. The effect of salicylic acid on CYP2C11 activity is shown in The experimental method for a CYP2C11 assay has been validated in this study for testosterone and 16α-hydroxytestosterone. All the analytical parameters  were in line with ICH guidelines.Km  remains unaffected at three salicylic acid concentrations . However, the Vmax  for the inhibition studies  decreased compared to the Vmax of the negative control assay, as shown in i = 84.58 ± 2.67 µM  = 82.70 ± 2.67 µM). A higher value of Ki for salicylic acid in rat liver microsomes has a potentially negligible effect in causing drug interactions with other co-administrated drugs, which are substrates of the CYP2C11 enzyme. Outcomes from this in vitro study looking at the inhibitory effect of salicylic acid on CYP2C11 enzyme activity will be beneficial for a future in vivo study for healthcare screening for the effective use of salicylic acid in clinics and for the safe administration of salicylic acid with other drugs. As salicylic acid metabolism is mediated by two CYP450 enzymes (CYP2C11 and CYP2E1) in rat liver microsomes, so it will be necessary to evaluate the in vitro and in vivo inhibitory effects of salicylic acid on CYP2E1 enzyme activity in humans in order to assess the full effects.Our in vitro results demonstrate clearly that salicylic acid, at therapeutically relevant concentrations (0–200 µM) , acts po+ (Nicotinamide Adenine Dinucleotide Phosphate), magnesium chloride (MgCl2), testosterone, microsomes from liver pooled from male rats (Sprague-Dawley)  and 16-alfa hydroxytestosterone were purchased from Sigma Aldrich, Co . HPLC analytical reagent grade methanol and acetonitrile were purchased from Sigma Aldrich, Co. . Salicylic acid, potassium phosphate monobasic, potassium phosphate dibasic, phenacetin with purity greater than 98%, phosphoric acid (85% w/w), glucose-6-phosphate (G-6-P), glucose-6-phosphate dehydrogenase (G-6-PDH), EDTA (Ethylenediamine tetraacetic acid), NADPA 570 pH Meter purchased from JENWAY Limited  was used. A UV-VIS spectrometry instrument was purchased from VWR International Ltd.  and UV spectra were obtained using a Bio 100 Cary software from Aglient Technologies LDA  and 1 cm length of quartz cuvette. A Shimadzu LC-2010A HT Module liquid chromatographic system was used , combined with a low pressure pump quaternary gradient (series 200 LC (Liquid Chromatography) pump), a degasser, a model series 200 UV-detector, a series 200 Peltier LC column oven for chromatographing the analysed solutions, and a series 200 autosampler. A SUPELCO  C18 column  was used. The data were processed using Shimadzu HPLC 2 data lab solutions software processing system.Validation studies were carried out using a LC-2010A HT Module HPLC system . Chromatographic experiments were assessed in a low pressure gradient mode. The separation of the four target components  was conducted on a SUPELCO C18 column . The mobile phase for chromatographic separation of the four compounds consisted of 32% of phosphate buffer solution (pH = 3.36) and 68% of methanol. The flow rate was set to 0.8 mL/min, and the oven temperature was 30 °C. A wavelength detection of 243 nm was used and 10 µL solution volume was injected for HPLC analysis. The mobile phase consisted of methanol/phosphate buffer (pH 3.36) (68%/32% v/v), which provided good separation and resolution for the investigated compounds.+ (Nicotinamide Adenine Dinucleotide Phosphate) was added to the mixture after pre-incubation of all components for 10 min in a water bath at T = 37 °C [g (37 °C). The reaction was terminated after 65 min by the addition of ice-cold grade acetonitrile containing 50 µM of phenacetin . Tubes were centrifuged in a microcentrifuge  for 12 min to precipitate protein. Then, the supernatant was collected and dissolved in a mobile phase (30% phosphate buffer at pH 3.36 and 70% methanol) and made up to 1000 µL volume. A volume of 10 µL of dissolved supernatant was injected into the instrument for HPLC analysis. CYP2C11 activity was determined using testosterone (substrate) via the formation of 16α-hydroxytestosterone (metabolite for CYP2C11 enzyme). Oxidative metabolism of testosterone was measured using a NADPH-regenerating system consisting of: 1.0 mM NADPH (Nicotinamide Adenine Dinucleotide Phosphate Hydrogen), 5 mM G6P (Glucose-6-Phosphate), 1.7 units/mL G6PDH (Glucose-6-Phospahe Dehydrogenase), 1.0 mM EDTA (Ethylenediamine tetraacetic acid), and 3.0 mM magnesium chloride. Mixtures were incubated in a final volume of 500 µL of NADPH-regenerating system and and a final concentration of 0.067 M potassium phosphate buffer at pH 7.4 using a serial range of testosterone  and a serial range of salicylic acid solutions , which were added to the incubation mixture in triplicate. The reaction was initiated by adding 0.5 mg/mL of pooled liver microsomes to each tube. NADP = 37 °C . The finPhenacetin (50 µM), salicylic acid (100 µM), testosterone (200 µM), and 16α-hydroxytestosterone (50 µM) standard solutions were recorded in the UV region of 200–350 nm using methanol as a blank, and 243 nm absorption wavelength.Different mobile phases for the CYP2C11 assay were used. Thus, the most suitable mobile phase was as follows: HPLC grade methanol (low UV cut-off of 205 nm) as mobile phase (A), and phosphate buffer at pH = 3.36 as mobile phase (B) . Salicylic acid (SA) (1.38 mg) (C = 200 µM) was weighed accurately and dissolved in a 50 mL volumetric flask in a mobile phase (70% methanol + 30% phosphate buffer at pH = 3.36). Serial dilutions were performed, yielding final concentrations of 150, 100, 75, 50, 25, and 10 µM. Testosterone (5.76 mg) (C = 400 µM) was weighed accurately and added to a 50 mL volumetric flask before being dissolved in mobile phase. A serial dilution of testosterone stock solution was made, yielding final concentrations of 300, 200, 150, 100, 50, and 25 µM. Phenacetin was used as an internal standard for the CYP2C11 enzyme assay by dissolving 0.0009 g of the powder in a mobile phase (70% methanol + 30% phosphate buffer at pH = 3.36) and a 100 mL volumetric flask.The metabolite for the CYP2C11 enzyme (16α-hydroxytestosterone) stock solution of 100 µM (in a 50 mL volumetric flask) was prepared, followed by serial dilutions to 80, 60, 40, 20 and 10 µM respectively.The regression equation  consisted of different ranges of testosterone and 16α-hydroxytestosterone concentrations using 50 µM of phenacetin as an internal standard, which was calculated by a weighted least-squares linear regression analysis of mean peak area ratio  versus standard concentrations. Validation parameters were calculated using Microsoft Excel 2010 software .Vm, Km, Clint, α,, Ki) values were obtained from secondary Lineweaver–Burk and Michaelis–Menten plots. Inhibition data of CYP2C11 assays were assumed as non-competitive inhibition based on the shape of Lineweaver–Burk plots, and the standard error. AIC (Akaike information criterion) and SC (Schwarz criterion) were from obtained nonlinear regression analysis. The concentration of inhibitor to cause 50% inhibition of original enzyme activity (IC50) was determined by nonlinear regression using Graphpad Prism software . The percentage inhibition was calculated from Vm values.The CYP inhibition analysis was assessed by measuring the formation of 16α-hydroxytestosterone metabolite of the tested CYP2C11 substrate (testosterone). The peak area ratios of both the metabolite and internal standard were acquired using Microsoft Excel 2010 software. Pharmacokinetic parameter (In conclusion, an HPLC method was developed and validated for high throughput screening of compounds mediated by the CYP2C11 enzyme using salicylic acid as a tested inhibitor. Optimisation for in vitro CYP2C11 enzymatic reaction was achieved with regard to enzyme concentration and incubation time of the reaction. The amount of marker metabolite was quantified by a highly accurate and sensitive HPLC method. In this study, the in vitro CYP2C11 inhibition assay data demonstrated that Salicylic acid acts reversibly as a noncompetitive inhibitor, which may inhibit cytochrome P450 2C11 enzyme activity. The outcomes obtained provide useful tips for the effective use of salicylic acid in clinics. However, further in vitro and in vivo investigational studies are needed to determine the effect of salicylic acid administration in humans for full healthcare screening, regarding the safety of this drug when administrated concomitantly with regular medicines.


OsMDHAR4 in resistance against heat stress remains unclear.Monodehydroascorbate reductase (MDAR or MDHAR), which is responsible for growth, development and stress response in plants, is a key enzyme in the maintenance of the ascorbate acid (AsA) pool through the AsA–glutathione (AsA–GSH) cycle. High temperature affects a broad spectrum of cellular components and metabolism including AsA–GSH cycle in plants. In rice, however, the detailed roles of OsMDHAR4 expression was detected in all tissues surveyed and peaked in leaf blade. OsMDHAR4 was responsive to multiple stresses and was relatively strongly induced by heat treatment. In comparison with wild type, the osmdhar4 mutant exhibited improved tolerance to heat stress, whereas OsMDHAR4 overexpression lines exhibited enhanced sensitivity to heat stress. Moreover, we found that suppression of OsMDHAR4 promoted stomatal closure and hydrogen peroxide accumulation, and overexpression of OsMDHAR4 increased stomatal opening and decreased hydrogen peroxide content in rice leaves.Here, we report that OsMDHAR4 protein was localized to the chloroplasts. OsMDHAR4 negatively regulates tolerance to heat stress by mediating H2O2-induced stomatal closure in rice.Taken together, these results indicated that The online version of this article (10.1186/s12284-018-0230-5) contains supplementary material, which is available to authorized users. The monodehydroascorbate reductase (MDAR or MDHAR), well-known as flavin adenine dinucleotide (FAD) enzyme, is involved in the ascorbate–glutathione cycle, and plays an important role in directly reducing monodehydroascorbate (oxidized ascorbate) to ascorbate using NAD(P)H as an electron donor  enhanced tolerance to temperature and methylviologen-mediated oxidative stresses  enhanced the wheat resistance to Puccinia striiformis f. sp. tritici (Pst)  from the rice plant was cloned, and its function was analyzed. The osmdhar4 mutant displayed significantly improved heat tolerance with increased stomatal closure and reduced water loss by promoting H2O2 accumulation. OsMDHAR4-overexpressing transgenic plants showed obviously enhanced sensitivity to heat stress with decreased stomatal closure and accelerated water loss speed. Our results suggest that the OsMDHAR4 gene negatively regulates resistance to heat stress in rice.In this study, a new gene encoding the chloroplastic MDHAR (OsMDHAR4 gene spans 1431 bp and encodes a protein of 476 amino acids and has a predicted molecular weight of 51.86 kDa. The prediction of protein domains using NCBI and InterProScan databases indicated that OsMDHAR4 protein contains pyridine nucleotide-disulfide oxidoreductase domains (Pyr_redox_2 and NAD(P)-binding domain) and a FAD/NAD-linked reductase, dimerisation domain. The multiple amino acid sequence alignment of OsMDHAR4 and other plant MDHARs was performed using the BLAST tool at the NCBI database. OsMDHAR4 showed 87, 88, 86, 43, 69, 43, 70 and 53% identity and 92, 93, 93, 61, 80, 60, 82 and 69% similarity to ZmMDAR (Zea mays), BdMDAR (Brachypodium distachyon), TaMDHAR4, TaMDAR6 (Triticum aestivum), AtMDAR4, AtMDAR6 , GmMDAR (Glycine max), and SlMDAR (Solanum lycopersicum), respectively  of OsMDHAR4 to the N-terminal of yellow fluorescent protein (YFP) driven by the cauliflower mosaic virus 35S promoter. The empty and recombinant vectors were transiently transformed into the epidermis of Nicotiana benthamiana by agroinfiltration. Fluorescence microscopic analysis revealed that OsMDHAR4-YFP was mainly detected in chloroplast, partially colocalized with the red autofluorescence of chlorophyll. On the contrary, 35S-YFP was widely distributed in the nucleus and cytoplasm and was not colocalized with the red autofluorescence of chloroplasts . The mRNA abundance of OsMDHAR4 was detected using quantitative RT-PCR (qRT-PCR). OsMDHAR4 transcripts were detected in all of the tissues surveyed, and the expression level was relatively lowest in root and peaked in the blade of the flag leaf , abscisic acid (ABA), H2O2, salt and cold, at the seedling stage. It was found that, after heat treatment, the OsMDHAR4 transcripts increased rapidly at 0.5 h (1.8-fold), after which a slight decrease occurred at 3 h, and then they continued to rise and peaked at 24 h (2.8-fold). Under the PEG treatment, the transcript abundance of OsMDHAR4 was nearly unchanged at most time points except for 3 h and 24 h, where two induced peaks (1.7 and 2.2-fold) appeared. In the ABA treatment, OsMDHAR4 was induced quickly at 0.5 h and 3 h, after which a dramatic decrease appeared at 6 h and 12 h, and recovered to initial level until 24 h. When treated with H2O2, the OsMDHAR4 expression showed a significant trend of rising followed by falling. The OsMDHAR4 transcript level was slightly suppressed by salt and cold treatments at 6 h and 12 h . Then, the osmdhar4 mutant (RMD_03Z11DA36), a transfer DNA (T-DNA) insertion line in the japonica rice ZH11 background was obtained   were treated at high temperature (45 °C) for 72 h and subsequently returned to 26 °C for recovery. Survival rates were used as a measure of heat tolerance. After the heat treatment and recovery, 80.5% of the osmdhar4 seedlings survived compared with 45.6% of the WT seedlings  Fig. , left. ongs Fig. . Subsequves Fig. . This deOsMDHAR4 in rice, we constructed an overexpression vector of OsMDHAR4 driven by 35S promoter. The OsMDHAR4-overexpressing vector was transformed into rice ZH11. PCR analysis using OsMDHAR4-specific primers (OsMDHAR4-OE-F and R) and vector-specific primers (Hyg-F and R) confirmed the presence of the transgene in the two independent overexpression lines OsMDHAR4-OE-1 and OsMDHAR4-OE-5 , the results showed that 56.8% of stomata were completely closed in the osmdhar4 mutant, whereas only 25.0% were completely closed in the WT plants. On the other hand, only 10.5% of stomata were completely open in the osmdhar4 mutant, but 46% were completely open in WT. The percentage of partially open stomata didn’t differ significantly from each other , while the percentages of completely open were higher than the WT  . Under both ABA and H2O2 treatment, the OsMDHAR4 expression levels were increased in first and decreased at last, but salt and cold treatments had almost no effect on these  Fig. , the rests) Fig. . We founese Fig. . So, we MDAR genes. Overexpression of LeMDAR gene alleviated photoinhibition of PSI and PSII and enhanced the tolerance to various abiotic stresses by elevating AsA level -like pool , might lead to the accumulation of H2O2 in guard cells and trigger stomatal closure, and enhances drought and salt tolerance , which were responsible for H2O2 removal, were found to be more tolerant to salinity stress  enhanced the sensitivity to drought and salt stress in rice  showed a strongly enhanced tolerance to heat stress with lower water loss rates, more closed stomata and significantly higher H2O2 contents after the treatment as compared with the WT. Our data also showed that the osmdhar4 mutant had more closed stomata and significantly reduced stomatal conductance after heat treatment. Meanwhile, the osmdhar4 mutant accumulated more H2O2 as compared with the WT before or after heat treatment. All of the above was found to be opposite in the OsMDHAR4 overexpression line , simulated drought stress (treated with 20% [w/v] PEG 6000), ABA treatment (100 μM ABA), oxidative stress (treated with 100 mM H2O2), salt stress (treated with 250 mM NaCl), and cold stress (seedlings were transferred to a growth chamber at 4 °C), followed by sampling at the designated time points . We prepared each RNA sample with shoots from at least four seedlings and ensured that every sampled seedling was of the same growth stage.To check the expression levels of the OsMDHAR4-overexpressing transgenic plants were firstly selected by germinated seeds on 1/2 MS medium containing 50 mg L− 1 hygromycin, WT and the osmdhar4 mutant were grown on normal 1/2 MS medium 1 d later. For heat stress testing, the uniformly germinated seeds were sown in the same barrels filled with a well-mixed soil, which was then cultured in a growth chamber with a 13-h-light (28 °C)/11-h-dark (25 °C) photoperiod, 70% humidity. The 5.5- to 6.5-leaf stage seedlings were transferred to a growth chamber with a 13-h-light (45 °C)/11-h-dark (45 °C) photoperiod, 70% humidity for 48 h or 72 h. The treated seedlings were transferred back into the previous growth conditions for recovery, and seedlings with newly growing leaf blades were then counted as surviving plants.Before treatment, OsMDHAR4 was amplified from the cDNA of Zhonghua 11 by PCR. The PCR fragment was inserted into vector pEXT06/g (target gene driven by 35S promoter). The resulting construct was then confirmed by sequencing and transformed into Zhonghua 11, a japonica rice that can be easily transformed, by the Agrobacterium tumefaciens-mediated co-cultivation method. Transformed calli were selected on hygromycin medium. The primers used in this work are listed in Additional file To generate the overexpression construct, the full-length CDS of OsMDHAR4 gene was amplified by PCR and directionally inserted into pCAMBIA1300-35S:YFP-NOS vector. The cultures of the Agrobacterium tumefaciens strain EHA105 harboring empty and recombinant vector were used to infect the healthy leaves of Nicotiana benthamiana (4 weeks old). The fluorescence signals were observed using a confocal microscope (Leica TCS SP5 or Zeiss LSM710). The primers used in this work are listed in Additional file Coding sequence of the OsMDHAR4 were designed using the Roche Web site (Roche Applied Science). The rice Actin1 was used as the internal reference  was used according to the manufacturer’s instructions to extract total RNA. Before reverse transcription, total RNA was treated with gDNA Eraser  for 5 min at 42 °C to degrade possibly contaminated residual genomic DNA. The cDNA templates were synthesized using PrimeScript™ reagent Kit  according to the manufacturer’s instructions. Quantitative real-time PCR was performed on an optical 96-well plate with a CFX96 Real-time PCR Detection System  using SYBR Premix Ex Taq . The PCR thermal cycling protocol was as follows: 95 °C for 10 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Gene-specific primers for 2O2 contents. The contents were measured spectrophotometrically after reaction with potassium iodide (KI). The reaction mixture consisted of 0.5 ml of 0.1% trichloroacetic acid (TCA), leaf extract supernatant, 0.5 ml of 100 mM potassium phosphate buffer (pH 7.8) and 1 ml reagent . The blank control consisted of 1 ml 0.1% TCA and 1 ml KI in the absence of leaf extract. After 1 h of reaction in darkness, the absorbance was measured at 390 nm. The amount of H2O2 was calculated using a standard curve prepared with known concentrations of H2O2.Leaves harvested from 3.5- to 4.5-leaf stage seedlings, with or without heat treatment, were used to measure H2O2. The samples were observed and imaged under a scanner.3,3′-Diaminobenzidine (DAB) staining was performed following a published method with some modifications  as described previously  cellulase RS  for 5 h at 40 °C without shaking to facilitate peeling off the epidermal strips. The epidermal strips were peeled off from leaves using tissue forceps. After washing with loading buffer , the epidermal strips were incubated in staining buffer (loading buffer containing 50 mM H2DCFDA) for 10 min at room temperature in the dark. The epidermal strips were washed with distilled water to remove the excess dye. The fluorescence was examined using a confocal laser-scanning microscope . All confocal images were taken under identical conditions and the guard cell region was selected to quantify the mean grey value of guard cells.Hv/v) glutaraldehyde, and stomatal images  were obtained using scanning electron microscopy (KYKY-EM3200).Leaves of 3.5- to 4.5-leaf stage plants with or without heat treatment for 24 h were fixed with 2.5%  containing 5 mM EDTA and 10 mM dithiothreitol (DTT) for the reduction of DHA to AsA. The reaction mixtures were incubated at room temperature for 10 min and 0.5% N-ethylmaleimide (NEM) was added. AsA was assayed in a similar manner, except that deionized H2O was substituted for DTT and NEM. The color was developed in both the reaction mixtures by the addition of 10% TCA, 44% o-phosphoric acid, α,ά-dipyridyl in 70% ethanol, and 30% FeCl3. The reaction mixtures were incubated at 37 °C for 1 h and quantified spectrophotometrically at 525 nm.Leaves harvested from 3.5- to 4.5-leaf stage seedlings, with or without heat treatment, were used to measure the total AsA (tAsA), AsA and DHA contents determined by the spectrophotometric method described previously , Brachypodium distachyon (BdMDAR), Triticum aestivum , Arabidopsis thaliana , Glycine max (GmMDAR), and Solanum lycopersicum (SlMDAR). Red line: FAD/NAD-binding domain, blue line: Pyr_redox_2 (pyridine nucleotide-disulfide oxidoreductase 2) domain, and green line: FAD/NAD-linked reductase, domain. Dark blue shadow: amino acids are conserved among all 9 sequences, pink shadow: amino acids are conserved among 7 or 8 sequences, light blue shadow: amino acids are conserved among 5 or 6 sequences Sequence alignment was performed using the DNAMAN6 software. (JPG 9854 kb)Additional file 2:Figure S2. Genotyping of osmdhar4 T-DNA insertion mutant. F1, R1 and L4 stand for the primers. W, wild type. M, homozygous mutant. H, heterozygous mutant. (JPG 479 kb)Additional file 3:Figure S3. aOsMDHAR4-overexpressing construct. b PCR identification of transgenic plants from two overexpression lines (OsMDHAR4-OE-1 and OE-5). Positive transgenic plants have a PCR band about 1471 bp or 675 bp when using different primers combinations (OsMDHAR4-OE-F + R or Hyg-F + R). P, positive control. N, negative control. (JPG 420 kb)Additional file 4:Figure S4. a Detection of H2O2 production in guard cells of WT and osmdhar4 mutant plants with H2DCFDA. Scale bars = 10 μm. b Quantitative analysis of H2O2 production in guard cells of WT and osmdhar4 mutant . Error bars indicate the SE based on three biological replicates. *, P < 0.05, by Student’s t-test. (JPG 2714 kb)Additional file 5:Figure S5. a Detection of the AsA content of the WT, the osmdhar4 mutant and OsMDHAR4-OE-1 plants under normal or heat stress conditions. b Detection of the DHA content of the WT, the osmdhar4 mutant and OsMDHAR4-OE-1 plants under normal or heat stress conditions. c AsA/DHA ratio of WT, the osmdhar4 mutant and OsMDHAR4-OE-1 plants under normal or heat stress conditions. Error bars indicate the SE based on three biological replicates. *, P < 0.05, **, P < 0.01, by Student’s t-test. ns, no significant. FW, Fresh Weight. (JPG 480 kb)Additional file 6:Table S1 List of primers used in this study . (DOCX 23 kb)