Mesoporous cerium – zirconium oxides modi ﬁ ed with gold and copper – synthesis, characterization and performance in selective oxidation of glycerol †

The main goal of this work was the synthesis of mesoporous ceria, zirconia and cerium – zirconium oxides, the application of these supports for the preparation of monometallic (copper or gold) and bimetallic (copper and gold) catalysts and characterisation of their performance in the oxidation of glycerol in basic aqueous solution in the presence of oxygen. The methods applied for the study included: adsorption/ desorption of nitrogen, XRD, ICP-OES, XPS, UV-vis, STEM-EDXS, H 2 -TPR. The presence of the following species Au 0 , Au d + , negatively charged gold Au (cid:1) or Au 0 in bimetallic particles, Cu + , Cu 2+ , Ce 3+ and Ce 4+ , Zr 4+ was con ﬁ rmed and their content was found to depend on the catalyst composition. The interaction between gold and copper species in bimetallic samples caused the formation of the negative charge on gold and the increase of the amount of Cu + cations on the catalyst surface. This feature and the higher gold dispersion gave rise to a signi ﬁ cant increase in the catalytic activity in glycerol oxidation of bimetallic catalysts with respect to that of monometallic samples. The high selectivity to glyceric acid (GLA) is an important advantage of the bimetallic catalysts (copper – gold), especially those loaded on mixed cerium – zirconium oxides. The bimetallic catalysts were stable and in the second run of the reaction they almost did not change glycerol conversion and increased the selectivity to GLA. The texture parameters of the catalysts had also impact on the selectivity of the reaction: the lower the microporosity the higher the selectivity to GLA. The role of the support is the interaction with copper and gold active species and enhancement of the reagents' chemisorption and electron transfers.

Introduction
Bimetallic catalysts supported on metal oxides receive much interest. Bimetallic catalysts containing gold are attractive in different catalytic oxidation processes, e.g. oxidation of carbon monoxide, 1-7 methanol, [8][9][10][11] and other alcohols. [12][13][14][15][16][17][18] In the case of gold catalysts, metallic gold on the surface of catalysts promotes oxidative properties by leading to higher mobility of oxygen and to a much easier reduction of the copper species. 19 The transfer of electrons is difficult on a metallic gold surface; however, copper has a greater electron-donating ability and it can modify the electronic and structural properties of gold. 20 Catalysts containing gold that is modied with copper can be useful for the simultaneous total oxidation of carbon monoxide and methanol from gases emitted by automotive devices and during industrial processes. 11,21,22 The bimetallic copper-gold catalysts promoted with ceria and/or zirconia loaded on mesoporous SBA-15 materials exhibited higher activity in methanol oxidation than analogues monometallic gold or copper catalysts. 11 The chemical composition and structure of the support used for gold-copper play an important role in the activation of bimetallic particles. Recently, it was shown that gold-copper loaded on ceria, zirconia and mixed ceria-zirconia supports have changed electronic state of gold and that the mobility of oxygen from the support plays an important role in methanol oxidation. 23 The activation of the catalysts at higher temperature, which is necessary for oxidation of methanol, strongly affected the surface properties and activity and selectivity in this reaction.
The idea of this work is to apply mesoporous ceria-zirconia mixed oxides with different chemical composition as support for the copper-gold active phase and to investigate the role of chemical composition and structure on the catalytic performance in the liquid phase glycerol oxidation. Ceria-zirconia supports are widely applied in exhaust-gas purication, 24,25 but they are not oen used for ne chemical catalytic syntheses. The addition of zirconia to pure ceria improves not only the resistance against sintering at a high temperature compared to pure ceria, 25,26 but it also affects the oxygen storage capacity. 25 This may be very attractive for the interaction with copper-gold phases and for their application in oxidation processes. In contrast to the earlier study, 23 in this work the catalysts are not activated at higher temperature before the catalytic oxidation of glycerol. Glycerol oxidation over gold catalysts on different supports has been studied by many authors over the last 15 years. [27][28][29][30][31][32][33][34][35] We analyzed the synergistic effect between copper and gold as well as the inuence of the supports.

Experimental
2.1. Preparation of catalysts 2.1.1. Synthesis of zirconia. Zirconia was synthesized by the hydrothermal method using a surfactant as a structure directing agent. [36][37][38][39] A micellar solution of cetyltrimethylammonium bromide (CTMABr, Sigma-Aldrich) was prepared by dissolving the surfactant at room temperature for 3 h in an aqueous acidic solution (pH ¼ 2 controlled by 35-38% HCl, Chempur). Zirconium n-tetrapropanol (Zr(OC 3 H 7 ) 4 , 70% in 1-propanol, Fluka) was added dropwise to the solution of the surfactant to reach the surfactant/precursor molar ratio of 0.33. The gel obtained aer stirring for 1 h at 313 K was sealed into a polypropylene bottle and heated for 24 h at 353 K. The template was removed by extraction in Soxhlet apparatus at 351 K using ethanol for 30 h. Then, the oxide was dried in a vacuum rotator at 343 K (200-300 mbar) and calcined at 673 K for 4 h with a heating rate of 3 K min À1 . The calcination was used to remove the organic template and other carbonates, which are present aer drying.
2.1.2. Preparation of mixed cerium-zirconium oxides, CeZrO x (molar ratio of Ce : Zr ¼ 1 : 2 and 1 : 1). The mixed oxides to be studied were synthesized according to the literature methods. [36][37][38][39] CeZrO x were prepared using an aqueous acidic solution (pH ¼ 2 controlled by 35-38% HCl, Chempur) of 15 wt% cetyltrimethylammonium bromide (CTMABr, Sigma-Aldrich) by dissolving CTMABr at 313 K under stirring for 3 h. The appropriate amounts (depending on the assumed Ce : Zr ratio) of zirconium n-tetrapropanol (Zr(OC 3 H 7 ) 4 , 70% in npropanol, Fluka) and powder of cerium(III) nitrate (Ce(NO 3 ) 3 -$6H 2 O, Sigma-Aldrich) were added into the above-mentioned solution with a surfactant/precursors molar ratio of 0.33. Aer further stirring for 1 h at 313 K, the mixture was heated at 353 K for 24 h. The template was removed by extraction in a Soxhlet apparatus at 351 K using ethanol for 30 h. Then, the oxide was dried in a vacuum rotator at 343 K (200-300 mbar) and calcined at 673 K for 4 h at the heating rate of 3 K min À1 . The nal materials were obtained aer calcination at 673 K for 4 h at the heating rate of 3 K min À1 . The organic template and other carbonates were removed by the calcination.
2.1.3. Preparation of mixed cerium-zirconium oxide CeZrO x (Ce : Zr ¼ 2 : 1). The syntheses of mixed cerium-zirconium oxide, CeZrO x (Ce : Zr ¼ 2 : 1) was carried out using a solution of organic copolymer -Pluronic P-123 (polyethyl glycol-polypropyl glycol-polyethyl glycol, Sigma-Aldrich) in ethanol (POCh). A portion of 1.2 g of Pluronic P-123 was dissolved in 12 cm 3 of ethanol. P123 was mixed with ethanol at room temperature for 1 h, then powders of metals sources -cerium(III) nitrate (Ce(NO 3 ) 3 $6H 2 O, Sigma-Aldrich) and zirconium oxonitrate(V) (ZrO(NO 3 ) 2 , Sigma-Aldrich), were added to ethanol solution of the template and the mixture was stirred for 1 h. In the sample of CeZrO x (2 : 1), the ratio of ceria : zirconia was 2 : 1. The nal material was obtained by heating the mixture at 673 K for 10 h in air atmosphere (a heating rate of 1 K min À1 ). The supports prepared were labelled as XYO x (A : B) where X ¼ Ce; Y ¼ Zr; A : B ¼ ceria : zirconia nominal molar ratio. Calcination removed the organic template and other carbonates.
The main reason of change in the procedure of oxides preparation was the achievement of a high yield of the synthesis. The usage of CTMABr was effective only in the case of small amount of cerium in the synthesis mixture (i.e. the nominal molar ratio CeO 2 : ZrO 2 ¼ 1 : 1 and 1 : 2). For a higher cerium content the change of the template was necessary. Therefore, for the sample with the nominal molar ratio of CeO 2 : ZrO 2 ¼ 2 : 1 and CeO 2 we used Pluronic P-123 as a template.
2.1.4. Preparation of ceria. The synthesis of pure ceria, CeO 2 was carried out using a solution of organic copolymer -Pluronic P-123 (polyethyl glycol-polypropyl glycol-polyethyl glycol, Sigma-Aldrich) in ethanol (POCh). A portion of 1.2 g of Pluronic P-123 was dissolved in 12 cm 3 of ethanol. P123 was mixed with ethanol at room temperature for 1 h, then powders of metal sourcecerium(III) nitrate (Ce(NO 3 ) 3 $6H 2 O, $99.99%, Sigma-Aldrich) was added to ethanol solution of the template and was stirred for 1 h at room temperature. The nal material was obtained by heating the mixture at 673 K for 10 h in air (a heating rate of 1 K min À1 ).
2.1.5. Modication of mesoporous oxides with gold. The modication of mesoporous oxides with 1.5 wt% of Au species was realised as follows. A portion of 4.500 g of dried material (ceria, zirconia or ceria-zirconia oxides) was modied according to the literature method. [40][41][42] Urea was used as precipitation agent to form solid nanoparticles of gold hydroxide obtained by hydrolysis of gold precursor (tetrachloroauric acid (HAuCl 4 -$3H 2 O), Johnson-Matthey). The method of gold deposition using urea and washing with ammonia aqua solution was used according to the procedure described in ref. 40 to obtain small gold particles (below 5 nm). A portion of 137 mg of tetrachloroauric acid was added to 217 cm 3 of distillate water. Then aqua solution of urea (99%, Fluka, 0.16 mol dm À3 ) was added (molar ratio of urea/gold was 100). Finally the support (ceria, zirconia or ceria-zirconia oxides) was included into such obtained solution (pH ¼ 2). The mixture was stirred in a quartz ask for 4 h at 353 K. Then the product was ltered and washed with 100 cm 3 of aqua solution of ammonia (25%, NH 3 $H 2 O, Chempur) and 850 cm 3 of distilled water to obtain pH ¼ 7. The product was dried at room temperature for 24 h and nal material was obtained aer calcination at 623 K for 3 h. Thus, ve gold catalysts: Au/CeO 2 , Au/CeZrO x (2 : 1), Au/CeZrO x (1 : 1), Au/CeZrO x (1 : 2) and Au/ZrO 2 were obtained.
2.1.6. Introduction of copper by incipient wetness impregnation. Copper was introduced by incipient wetness impregnation to get 2.0 wt% of copper. Before impregnation, the support was dried using a vacuum rotator (200-300 mbar) at 343-348 K for 1 h. The source of copper, i.e. 0.146 g of copper(II) nitrate Cu(NO 3 ) 2 $2.5H 2 O (Sigma-Aldrich) was dissolved at room temperature in 2 cm 3 of distilled water and used to ll the oxide pores. The wetted oxide was dried under vacuum at 343 K for 1 h, then at 333 K for 16 h at atmospheric pressure. The materials were calcined in an oven at 623 K for 3 h (a heating rate of 3 K min À1 ). The following catalysts were obtained: Cu/CeO 2 , Cu/ CeZrO x (2 : 1), Cu/CeZrO x (1 : 1), Cu/CeZrO x (1 : 2) and Cu/ZrO 2 .
2.1.7. Modication of gold-containing samples with copper species. To introduce copper into samples by incipient wetness impregnation, portions of 2.000 g of dried Aucontaining catalysts were treated with 2 cm 3 of aqueous solution of Cu(NO 3 ) 2 $2.5H 2 O (0.146 g, Sigma-Aldrich). Before impregnation, the support was dried using a vacuum rotator (200-300 mbar) at 343-348 K for 1 h. The amount of copper nitrate used for the modication was calculated to achieve the loading of copper equal to 2 wt%. The CuAu/CeO 2 , CuAu/ CeZrO x (2 : 1), CuAu/CeZrO x (1 : 1), CuAu/CeZrO x (1 : 2), CuAu/ ZrO 2 materials were dried under vacuum at 343 K for 1 h, then at 333 K for 16 h at atmospheric pressure and then calcined at 623 K for 3 h.

Catalyst characterization
2.2.1. N 2 adsorption and desorption isotherms. The N 2 adsorption/desorption isotherms were obtained using a Quantachrome Instruments autosorb iQ2 at 77 K. The samples were pre-treated in situ under vacuum at 573 K for 4 h.
The nitrogen adsorption isotherms for CeZrO x (1 : 1), Au/ CeZrO x (1 : 1), Cu/CeZrO x (1 : 1) and CuAu/CeZrO x (1 : 1) were obtained using a ASAP 2020 Instruments at 77 K aer pretreated in situ under high vacuum at 573 K for 8 h. The surface area was calculated by the BET method. Mesopores volume and mean pore size were determined from BJH (adsorption branches).
2.2.2. X-ray diffraction (XRD). XRD measurements were carried out on a Bruker AXS D8 Advance diffractometer with Cu K a radiation (l ¼ 0.154 nm), with a step size of 0.5 in the wideangle range . The XRD method was used to estimate the size of gold particles using the Scherrer formula.
2.2.3. Ultra-violet spectroscopy (UV-vis spectroscopy). UVvis spectra were recorded using a Varian-Cary 300 Scan UVvisible spectrophotometer. Powder calcined samples aer drying at 383 K for 13 h were placed in a cell equipped with a quartz window. The spectra were recorded in the range from 800 to 190 nm. Spectralon® was used as the reference material.
2.2.4. STEM-EDXS (scanning-transmission electron microscopy-edge differential X-ray spectroscopy). Metal particles of gold were characterized using scanning transmission electron microscopy (STEM) and estimated using ImageJ™ soware. STEM images were acquired with an aberrationcorrected dedicated STEM microscope HD 2700 CS (Hitachi, acceleration potential of 200 kV) and obtained with a high-angle annular dark-eld (HAADF) detector to produce images with atomic number (Z) contrast. The size distribution and average size of gold particles were estimated from STEM images using ImageJ soware. The precision of particle size determination was ca. AE0.5 nm. Finally, the fractions of gold particles of the same size in all gold particles were calculated to determine the particle size distribution.
2.2.5. H 2 -TPR (temperature programmed reduction by hydrogen). Temperature-programmed reduction (H 2 -TPR) was performed using a CATLAB system connected to a mass spectrometer using a ow of hydrogen as a reducing agent. At the beginning, a calcined catalyst (0.040 g) in the form of powder was packed into the tube quartz reactor and connected to the CATLAB device. Then, the sample was heated in a ow of helium and argon (He/Ar -90/10 vol%, total ow rate of 40 cm 3 min À1 ) for 40 min at 313 K for the stabilization of signals from the mass spectrometer. Aer this time, the mixture of gases was changed into the nal reducing mixture: H 2 /Ar/He (5 vol% H 2 , 10 vol% Ar and 85 vol% He, total ow rate of 40 cm 3 min À1 ). Aer 20 min, the signals of mass ions were stable and the heating started from 313 K to 1173 K at the heating rate of 10 K min À1 . A quadruple mass spectrometer measured the signals of mass ions for m/z ¼ 2.00 (H 2 ), 18.00 (H 2 O), 32.00 (O 2 ) and 36.00 (Ar). The TPR proles presented the correlation between hydrogen consumption and increasing temperature. The TPR prole recorded for the reactor without the catalyst was subtracted from those recorded with the catalysts.
2.2.6. XPS (X-ray photoelectron spectroscopy) study. X-ray photoelectron spectra were recorded on an Ultra High Vacuum (UHV) System. The study was conducted using X-ray Al K a ¼ 1486.6 eV with the following lamp parameters: 14.5 kV, 20 mA. Measurements were carried out in the chamber of the analyser under vacuum of approximately 2.0 Â 10 À8 mbar. The spectra were recorded for the energy range from 1100 eV to 0 eV in increments of 0.5 eV, the energy transition CAE ¼ 100 eV. Different regions were recorded in increments of 0.1 eV, the energy transition CAE ¼ 30 eV and time counts 50 ms. The number of scans in the measured range was tted to the observed signal to noise ratio. The powder sample was put on the conductive tape of carbon (graphite), which was adhered to the carrier surface and placed perpendicular to the axis of the analyser. The X-ray source was set at the angle of 60 to the plane of the surface. The area of the samples analysed corresponded to the size of the aperture of the analyser used (large area), ca. 50 mm 2 . Deconvolution of XP spectra was carried out using the OMNIC 8.0™ soware. Bands intensities were estimated by calculating the integral of each band aer smoothing and baseline correction. Then the deconvolution parameters for the experimental curve, that is the Voigt function, small signal sensitivity and constant base line, were chosen in the OMNIC 8.0™ soware. Atomic ratios were computed from the intensity ratios normalized by the atomic sensitivity factors. An estimated error of AE0.1 eV can be assumed for all measurements. of sodium hydroxide, NaOH (Merck, >99.0%, NaOH : glycerol molar ratio ¼ 2 : 1). 15.00 g of distillate water was put into the reactor, then 0.0015 mol (0.138 g) of glycerol (Sigma-Aldrich, >99.0%), a base -NaOH (0.12 g) and 0.030 g of catalyst were added and the autoclave was closed. Oxygen was introduced to the autoclave at the selected pressure and the mixture was heated to 333 or 363 K (a heating rate 2 K min À1 ) and stirring at 400, 800, 1000 or 1200 rpm. The time of reaction was initiated by stirring at the moment when the mixture achieved the selected temperature. The reaction was conducted for 5 h at a selected temperature and stirring speed before analysis of products. The oxygen content was estimated by the electronic sensor of pressure which measured the pressure of oxygen in the real time of the reaction.
2.3.2. Analyses of products. The quantitative analyses of the reaction mixtures were performed using a high performance liquid chromatograph Merck Hitachi HPLC L-7000 series and the products of the reaction were analysed by a VWR Hitachi Chromaster RI Detector. The reactant and the products were separated on an ion exchange Aminex HPX-87H HPLC column heated at 308 K. The eluent was an aqua solution of H 2 SO 4 (0.005 M) and its ow rate was set at 0.6 cm 3 min À1 . The samples were taken at the end of the reaction: 1 cm 3 of the reactant/products solution was diluted in 10 cm 3 of water and 10 ml of such solution was analysed. The conversion of glycerol and selectivity to selected products were calculated by comparing the area of chromatographic peaks recorded for sample solutions with those of substrate respectively products found in calibration curves. Calibration curves aqua solutions (in the range 0.01-0.10 M) using pure chemical reagents: glycerol (Sigma-Aldrich, 99%), lactic acid (Sigma-Aldrich, 90%), tartronic acid (Fluka, 97%), glycolic acid (Sigma-Aldrich, 99%), oxalic acid (Sigma-Aldrich, 99%), glyceraldehyde (Sigma, 90%), formic acid (Sigma-Aldrich, 98%), glyoxylic acid (Sigma-Aldrich, 95%), 1,3-dihydroxyacetone (Sigma-Aldrich, aqua solution 50%), glyceric acid (Sigma-Aldrich, aqua solution 20%) and NaOH (Merck, >99.0%). Four aqua solutions were prepared for each reagent. The concentrations of these solutions were 0.01, 0.02, 0.05 and 0.10 M and pH was 13.3. Then 1 cm 3 of each solution was diluted in 10 cm 3 of water and 10 ml of such solution was analysed using high performance liquid chromatography. The areas of chromatographic peaks were related to the concentrations of reagents by a straight line, using linear regression analysis. The glycerol conversion and the selectivities to the products of glycerol oxidation were described by the equation: y ¼ ax + b, where y was the instrument response, a represented the sensitivity, and b was a constant that described the background. The concentration (x) of reagent in the solution aer reaction was calculated from this equation.

Structure of the supports
Undoped zirconia has the monoclinic structure, 24 which is a distorted uorite-type or tetragonal structure. Pure ceria is cubic. The structure of mixed ceria-zirconia oxides strongly depends on the composition and the preparation conditions. With increasing cerium content, the monoclinic structure of mixed oxide solid solution changes to a mixture of monoclinic, tetragonal, pure tetragonal and cubic structures. 24 The type of structure and chemical composition are reected by the stability of the system and the isotropic atomic displacement parameter of oxygen atoms, U(O). This parameter is a good indicator of oxygen diffusivity, which is important in catalytic oxidation reactions. High U(O) values indicate a high static positional disorder and a high dynamic thermal vibration, which enhance the movement of oxide ions across the material. Yashima and Wakita 43 found that Ce 0.5 Zr 0.5 O 2 shows the highest value of U(O) among the materials with different ceria to zirconia ratios. We used three different ceria to zirconia ratios: 2 : 1, 1 : 1 and 1 : 2. The XRD pattern ( Fig. 1) of ceria was characteristic of its cubic phase (JCPDS ICDD PDF card -00-043-1002). The pattern of zirconia identied its structure as a mixture of tetragonal (JCPDS ICDD PDF card -00-042-1164) and monoclinic (JCPDS ICDD PDF card -00-007-0343) phases. The XRD patterns of the mixed oxides varied with the chemical composition ( Fig. 1). In the samples with a ceria to zirconia ratio of 2 : 1, the ceria and zirconia phases showed independent XRD patterns. The intensity of the zirconia phase was very low, which is in agreement with the low content of zirconia. Increasing the amount of zirconia to a ceria : zirconia ratio of 1 : 2 induced asymmetry in the peaks in the XRD patterns; peaks characteristic of both zirconia and ceria, with a slight domination of those from zirconia, were present. Contrary to pure zirconia, which contains both monoclinic and tetragonal structures, the mixture of ceria-zirconia with molar ratio of 1 : 2 exhibits the presence of only tetragonal zirconia. At equimolar ceria and zirconia ratio, weak reections of ceria were visible. The structure of zirconia with a uniform distribution of cerium ions within this phase can be assumed.
The differences in the phase compositions also affect the textural parameters determined from nitrogen adsorption/ desorption isotherms (Fig. S1, † Table 1). The isotherms were of type IV indicating mesoporous materials. Ceria, zirconia, and CeZrO x (1 : 2) contained small amounts of micropores. Because of larger microporosity, the surface area of zirconia is much higher (121 m 2 g À1 ) than that of ceria (25 m 2 g À1 ). The inclusion of a small amount of zirconia into ceria (ceria : zirconia ¼ 2 : 1) decreased the surface area to 16 m 2 g À1 . Further increasing the zirconium content led to a slight increase of the surface area to 103 and 108 m 2 g À1 for CeZrO x (1 : 1) and CeZrO x (1 : 2), respectively. Table 2 shows the composition of the catalysts. The values estimated from ICP-MS analyses were similar to those assumed in the preparation, i.e., the molar ceria : zirconia ratio of ca. 2 : 1, 1 : 1 and 1 : 2. The modication of all mixed oxide supports, especially by gold, decreased the molar ratio of ceria : zirconia. Thus, metal addition led to a partial removal of cerium by the interaction of ceria with tetrachloroauric. Notably, a higher percentage of cerium removal was observed for CeZrO x (1 : 1); about 25% compared to 0-14% for the others.

Chemical composition
The efficiency of copper introduction was higher than that of gold. On all the supports, the copper loading was in the range of 1.4-1.8 wt%, introducing 2.0 wt% during the synthesis. Gold introduction ranged between 0.6 and 1.1 wt%, intending 1.5 wt%. More gold was incorporated on pure ceria than on pure zirconia and for copper, this was the similar. If copper was loaded on the supports already modied with gold species, the total amount of copper introduced onto ceria was greater, suggesting interaction between gold and copper during modi-cation. The difference in gold concentration calculated from ICP and XPS, in which XPS is more surface sensitive, showed that gold was preferably located on the external surface of the oxides ( Table 2). The surface concentration of the gold depended on the composition of the supports and was inuenced by the admission of copper. The highest gold surface content was on CeZrO x (1 : 1) in the bimetallic (copper-gold) catalyst (11.4 wt%). The monometallic (gold) catalyst, based on the same support, exhibited a lower content of surface gold species (9.1 wt%). On all other supports, the concentration of surface gold was lower aer copper loading. Loading copper involved the transfer of gold species into the bulk of these supports. In monometallic samples, the surface concentration of gold was higher on ceria than on zirconia. For cerium-zirconium mixed oxides, there was no obvious relationship between the ceria : zirconia ratio and the content of surface gold species. Similarly, copper  content on the surface of the supports did not change linearly with the increase of zirconia. Notably, for monometallic copper on pure zirconia and CeZrO x (1 : 1), the concentration of copper on the surface was lower than the total content of copper, indicating partial incorporation of copper into the support. The same phenomenon was observed for some bimetallic samples. This behavior indicates the mobility of the catalyst components and the dependence of the modiers' location on the chemical composition of the support. Such mobility is enhanced by thermal treatments. 23 The gold : copper molar ratio in most bimetallic catalysts is much higher on the surface of the material than in the bulk ( Table 2). This difference is negligible only for Cu-Au loaded on zirconia suggesting that in this material bimetallic species are distributed more uniformly. The surface and bulk concentrations of cerium and zirconium were estimated on the basis of the ceria : zirconia ratios ( Table  2). The values of this ratio differed from the assumed ones. The higher ratio from the XPS analysis compared to ICP indicated enrichment of the surface in cerium. It was most clearly seen for CeZrO x (2 : 1) and all bimetallic catalysts.

Texture/structure of catalysts
Modication with copper species did not lead to the appearance of new reections in the XRD patterns (Fig. 1). The XRD patterns of ceria-containing with only gold did not exhibit reections from crystalline gold, suggesting the absence of large gold particles. Gold supported together with copper on ceriumzirconium oxides and pure zirconia [CuAu/ZrO 2 ] gave a reection at 2 theta at ca. 38 , which originates from metallic gold. 5,[44][45][46] Using the Scherrer equation, the average size of the particles in CuAu/CeZrO x (1 : 1) and CuAu/ZrO 2 was 7.0 nm and 5.2 nm, respectively. Table 1 presents the textural/structural parameters of the materials modied with gold and/or copper and those of pristine supports. The surface areas of these samples ranged from 13 [Au/CeZrO x (2 : 1)] to 123 m 2 g À1 [Cu/ZrO 2 ]. The samples based on zirconia were characterized by the highest surface area and with increasing cerium content, the surface area decreased. The addition of gold and/or copper slightly modied the surface area of the supports. The largest decrease in the surface area as a result of metal loading was observed on CeZrO x (1 : 1). The removal of cerium from this support during modication with metals may correlate to the decrease in the surface area. STEM-EDXS analysis was used to estimate the relative composition and particle size distribution of supported particles. The bimetallic catalysts CuAu/CeZrO x (1 : 1) and CuAu/ZrO 2 had average sizes of 2.2 nm (and a median particle size of 0.83 nm) and 1.6 nm (with a median particle size of 0.73 nm), respectively (Fig. 2). These values were smaller than those obtained from the XRD patterns using the Scherrer formula, which is dominated by the larger particles (7.0 and 5.2 nm, respectively). The particle-size distribution was wider on zirconia than on the mixed oxides, but on both support types, it also covered the larger particles observed in XRD patterns. The differences in the size of gold particles measured by STEM-EDXS and XRD methods resulted from the differences in the resolution of both methods (XRD analysis does not indentify small metal particles). Notably, the average size of gold particles in the bimetallic sample CuAu/CeO 2 presented in the PSD diagram (Fig. 2) was greater than that noted on CuAu/ZrO 2 and CuAu/CeZrO x (1 : 1). The comparison of the gold particle size distribution on pure zirconia support for monometallic (Au/ZrO 2 ) and bimetallic (CuAu/ZrO 2 ) systems indicated a decrease in the size of gold particles aer the incorporation of copper. Thus, the incorporation of copper enhanced the dispersion of the particles. An important factor that affects the performance of multifunctional catalysts is the location of their components, which was studied for CuAu/CeZrO x (1 : 1) material by STEM-EDXS analysis. Fig. S2 † shows STEM images and EDX results.
The distribution of cerium and zirconium was almost the same, conrming the formation of the mixed oxides. Gold and copper species are located in the same areas, which indicates that they are in close vicinity to each other.
Individual species of gold and copper respectively ceria and zirconia were difficult to identify in the STEM images, because their contrast is too similar (Fig. S2 †). To determine the composition of the white particles with diameters in the range of 10-70 nm, EDXS spot analyses were performed. The EDX spectrum of area 1, (Fig. S2 †), indicated that the main component in the catalysts at this spot is gold. Moreover, a high intensity of gold signals, small intensities of zirconium and copper signals and the lowest intensity of cerium signals revealed that gold, zirconium, and copper were present in the same area. The EDX spectrum of area 2 in Fig. S2 † showed that not all signals were as intense; in particular, the signals assigned to gold were less pronounced. This suggested that the larger white particles in STEM images were clusters of mixed oxides of cerium and zirconium and/or copper oxide clusters, with smaller contributions of gold.

Oxidation state of components
3.4.1. UV-vis spectroscopy. Fig. 3 presents the UV-vis spectra of the catalysts aer drying at 383 K for 13 h. The UVvis spectra of the supports in Fig. 3Aa-Ca showed a band at approximately 225 nm and other broad bands centered at ca. 270-285 nm and 340-350 nm. The lower coordination number of cerium surrounded by oxygen ions requires the higher energy for a charge transfer from oxygen ions to cerium cations. Therefore the rst band was assigned to the charge transfer in crystalline ceria in tetrahedral coordination (O 2À to Ce 4+ ligand to metal charge transfer). The bands at ca. 270-285 nm and 340-350 nm were attributed to the charge transfer from oxygen to cerium cations in hexa-or octacoordinated species or to cerium cations at in lower oxidation states. 47 With increasing zirconium content in the mixed oxides (Fig. 3Da), the band at ca. 350 nm became less resolved than the band at ca. 270 nm. This nding suggested that in the mixed oxides, the content of cerium cations at an oxidation state lower than +4 and/or coordination number higher than four decreased. The band at approximately 213 nm (Fig. 3Ea) is typical of a charge transfer from the O 2À to Zr 4+ ion with tetrahedral conguration in zirconia crystallites. [48][49][50] In the spectra of the mixed oxides, the bands attributed to charge transfers in ceria overlapped with those from the charge transfer in zirconia. Aer copper and gold loading on ceria, CeZrO x (2 : 1) and CeZrO x (1 : 2), the UV-vis bands coming from the charge transfer from oxygen to cerium cations were still present in the spectra, but with lower intensities. This nding indicated that both gold and copper interacted with the cerium cations, which could lead to changes in their oxidation states.
The details on the oxidation states of all components are further considered below in the analyses of the XPS data.
The UV-vis spectra of gold-containing samples showed the characteristic ultraviolet-visible band at ca. 525-555 nm, typical of metallic gold. 51 This plasmon resonance band corresponds to metallic gold nanoparticles of about 2-6 nm and are in accordance with the literature data. 40 The well-resolved metallic gold band was observed for the samples loaded with both copper and gold (spectra d in Fig. 3). The band at ca. 260 nm, which can arise from either the charge-transfer transitions of Au 3+ or Au + ions with ligands or from the absorption caused by the transition of electrons between molecular orbitals of the few-atomic clusters Au n (n < 10), 52 could not be distinguished from the band coming from the charge transfer from oxygen to cerium cations.
The modication of supports with copper species changed the UV-vis spectra (Fig. 3Ac-Ec), which was visible for zirconia because in its spectrum, the region above 230 nm did not contain UV-vis bands characteristic of pristine zirconia. A new band at ca. 265 nm appeared. According to literature data, [53][54][55] bands at 220-260 nm were assigned to the charge-transfer transitions from oxygen in the crystal lattice O 2À to an isolated copper ion, Cu 2+ (O 2À / Cu 2+ ; ligand to metal charge transfer). The changes in the UV-vis spectra of the samples containing cerium resulted from copper loading were less pronounced, because the charge transfer in cerium gave bands in the same region. Fig. 3E showed that aer gold loading, the broadened absorption line covered the whole range between 240 and 640 nm, whereas the copper loaded on zirconia gave rise to two well-distinguished bands at 256 and weak in the region 400-600 nm. For copper species, the rst band was assigned to the charge-transfer transitions from oxygen ions in the crystal lattice of the support to isolated copper ions (O 2À to Cu 2+ ). 54,55 The second band came from the d-d transition of Cu 2+ ions located in an octahedral environment. 56 This band was also observed (although less intense) in the spectra of Cu/ CeZrO x (1 : 2) and Cu/CeZrO x (1 : 1), whereas in the UV-vis spectra of Cu/CeZrO x (2 : 1) and Cu/CeO 2 , the tail of the spectral line could overlap with the band discussed. This band was also assigned in literature 57 to oligonuclear copper oxygen species: Cu d+ /O dÀ /Cu d+ . In the spectra of bimetallic systems (gold-copper), this band coexisted with the band assigned to dd transitions in metallic gold clusters.
3.4.2. X-ray photoelectron spectroscopy. XPS was used to obtain the oxidation states of the gold, copper, cerium, and zirconium. Table 3 presents the binding energies (BE) of gold, copper, and zirconium (Table 4) and their relative distribution. Cerium was present in two oxidation states, +3 and +4, copper in two oxidation states, +1 and +2, and gold in the metallic form, cationic species (+d; d $ 1), and moreover metallic gold species labelled as negatively charged gold Au À or Au 0 in bimetallic particles. The distribution of all these species strongly depended on the chemical composition of the materials. Zirconium was present in one oxidation state, +4, characterized by BE at approximately 182 eV (in the range of 182.1-183.0 eV for the supports and 181.4-183.6 eV for the copper and/or gold modied samples). This indicated the interaction between the elements in mixed oxides and with gold and copper species loaded on the surface of the supports. The highest interaction seems to occur between copper, gold and zirconia as evidenced from the very low BE for the samples based on pure zirconia.
There is a domination of Ce 4+ on the surface of all mixed cerium-zirconium oxides ( Table 4). The increase in zirconium content in the mixed oxides slightly increased the amount of Ce 4+ from 78 to 85%. Modication of the supports with gold affected the distribution of cerium species. A signicant increase in the content of cerium in the lower oxidation state (+3) was noted in Au/CeZrO x (1 : 2) (from 15 to 34%), suggesting the reduction of cerium cations during the modication with auric acid and urea. This reduction of Ce 4+ was paralleled by formation of more oxidized gold species (61% of Au d+ ).
The distribution of cerium cations on the surface of the supports was also inuenced by modication with copper and both copper and gold species. It is difficult to note a simple relationship between the oxidation state of cerium and the chemical composition of the catalysts. However, there is no doubt that copper and gold loading inuence the oxidation state of cerium. The reducing environment of the vacuum may contributed in degree of cerium(IV) reduction.
Gold loaded on ceria, zirconia and cerium-zirconium mixed oxides was characterized in the XP spectra by the two spin-orbit components Au 4f 7/2 and Au 4f 5/2 , separated and composed of two doublets attributed to different gold species (examples in Fig. S3 †), corresponding to Au 0 and Au d+ species (Table 3). [58][59][60][61][62][63] It is difficult to distinguish different oxidation states in cationic   7 eV). These energies are lower than typical of bulk metallic gold (84.0 eV). 64 The lowering of BE can be caused by a decrease in the size of gold particles, as noted in ref.

62.
All monometallic (gold) catalysts presented the domination of metallic gold. The distribution of metallic and cationic gold species was determined by the chemical composition of the supports. The higher amount of metallic gold was noted on pure zirconia and ceria (Table 4). More cationic gold species (Au d+ ) were formed on mixed ceria-zirconia supports. Generally, the Au d+ species were present mainly on the surface of monometallic (Au/support) samples. In bimetallic (Cu-Au/support) materials, cationic gold was observed only on pure ceria but in a very small quantities. The introduction of copper not only decreased or removed cationic gold species, it also increased the amount of metallic gold on pure ceria and zirconia and on mixed ceria-zirconia supports, the latter with enhanced shielding of the core hole. This nding indicates that the existence of reducible copper species in copper-gold catalysts is responsible for enhanced core-hole shielding. 12 The species characterized by BE below 83 eV were interpreted by some authors as negatively charged gold on different supports, e.g. ref. 46, 52 and 58. The XP spectra of bimetallic samples in the Au 4f range were characterized by two doublets (with the exception of CuAu/ZrO 2 ), which origin depends on the nature of the supports. In the case of copper-gold loaded on ceria, the species characterized by BE 4f 7/2 of 83.6 and 85.4 eV were attributed to Au 0 and Au d+ , respectively.
The XP spectra of bimetallic samples in the Au 4f range were characterized by two doublets (with the exception of CuAu/ ZrO 2 ), which origin depends on the nature of the supports. In the case of copper-gold loaded on ceria, the species characterized by BE 4f 7/2 of 83.6 and 85.4 eV were attributed to Au 0 and Au d+ , respectively.
On the mixed cerium-zirconium oxides, besides pure metallic gold (BE in the range of 83.6-84.7 eV) a Au 4f 7/2 peak at lower BE (the range of $82-83 eV) was observed. CuAu/ZrO 2 contained only metallic gold species (the peak at 83.4 eV).
The XPS bands covering the range of 83-85 eV is consistent with a Au 0 oxidation state. 62,63 The lower binding energy observed for Au 0 4f 7/2 in the copper-gold catalysts than in monometallic gold catalysts can be attributed to the smaller particle size. 63 The lower coordination of gold atoms causes this band to shi, 65,66 in agreement with the STEM-EDXS results (Fig. S2 †). As concerns the appearance of XP bands in the range of $82-83 eV they should be attributed to the changes in electronic structure of the system and core-hole shielding.
The BE of Cu 2p peaks were assigned based on the literature, 67,68 to Cu + (in the range of 932.0-933.0 eV) and Cu 2+ (934.1-935.5 eV). All samples containing copper (monometallic and bimetallic), except for Cu/CeZrO x (1 : 2) and CuAu/CeZrO x (1 : 2), exhibited the domination of surface Cu + cations. Notably, the Cu + content increased in the presence of gold species (except for CuAu/CeZrO x (1 : 2)). The lower amount of surface Cu + in CuAu/ CeZrO x (1 : 2) compared to monometallic copper catalyst was accompanied by the growth of gold peaks with BE between 82 and 83 eV indicative of direct interaction between gold and copper.

Temperature-programmed reduction by hydrogen (H 2 -TPR)
All components of the catalysts can be reduced by hydrogen. An analysis of gold and copper reduction was made in the temperature range between 373 and 723 K (Fig. 4). In this temperature range the peaks coming from the reduction of cationic gold respectively cationic copper in the 400 to 480 K range are well distinguished. The reduction of cationic gold and copper in bimetallic samples could not be strictly distinguished because the range of the reduction temperatures for both metals is similar and the two metals may affect each other's reduction behavior. Fig. 4A-E shows the TPR proles of monometallic gold, copper and bimetallic copper-gold systems loaded on different supports. For simplicity, only the most intense peak, from the reduction of cationic gold and/or copper in the range at ca. 400-480 K, is considered. The position of this peak was almost independent of the composition of modiers (only copper, only gold or both copper and gold) on ceria (Fig. 4A) or zirconia (Fig. 4E). For mixed cerium-zirconium oxide supports, the reduction temperatures of cationic gold and copper species were determined by the composition of the supports. With increasing zirconium content in the support, the reduction temperatures of copper and gold species were more separated, and generally, they decreased with the increasing content of zirconia in the mixed oxides used. The lowest reduction temperatures were characteristic of metals loaded on CeZrO x (1 : 2) (Fig. 4F).
This phenomenon indicated a stronger interaction of cationic gold and copper with mixed cerium-zirconium oxides, particularly if the zirconium content was higher. The promoting effect of zirconium was shown in the literature. 69,70 Notably, the loading of both copper and gold on the mixed oxides caused a signicant decrease in the reduction temperature, particularly evident for CeZrO x (1 : 2) (a decrease to 398 K). Such a high decrease in the reduction temperature must be inuenced not only by the interaction of modiers with mixed ceria-zirconium oxides but also by the interaction between gold and copper. In the catalysts modied with copper, Cu 2+ loaded on oxides of transition metals (ceria, zirconia or mixed cerium-zirconium oxides) was reduced to metallic copper Cu 0 below 473 K (in the case of smaller particles of copper(II) oxide) and at approximately 523 K (in the case of larger particles of copper(II) oxide). 71 The peaks around 450-475 K can be ascribed mainly to the reduction of copper(II) oxide particles having little or no interaction with the support. This assignment is in agreement with literature data. 72,73 copper(II) oxide clusters. The rst peak, at a lower temperature, was assigned to nanoclusters strongly interacting with ceria. The second peak, at a higher temperature, was attributed to copper(II) oxide species which were more like bulk oxide, less experiencing with ceria. The different interaction can be explained by the different particle size, the smaller the particle diameter, the higher the extent of interaction and therefore the easier reducibility. 81 It is noteworthy that TPR proles of CuAu/CeO 2 and ceria samples (Fig. 4A) show a reduction peak at around 573 K. This indicates that in the case of mesoporous ceria, pure ceria particles can be reduced at around 573 K. In the case of Ce-Zr oxides and pure zirconia, this peak is observed in the samples modied with copper and gold (except CuAu/CeZrO x (1 : 2), Fig. 4D). It is not observed on the TPR proles of supports (oxides) (Fig. 4B, C and E). The synergistic interaction between cerium (in the mixed oxides), zirconium (in the case of pure zirconia as support), gold (as Au d+ ) and copper (as Cu 2+ ) species leads to lower a reduction temperature of copper. 83 Hydrogen consumption above 573 K results from reduction of bulk copper(II) oxide. The temperatures of maximum hydrogen consumption observed in Fig. 4 were signicantly lower than that of bulk copper(II) oxide. If small copper(II) oxide particles were loaded on ceria, 76,[79][80][81] this support could promote their reduction below 573 K. The reduction of copper oxide clusters, strongly interacting with ceria, occurred in the range 398-448 K, and larger copper(II) oxide particles, non-associated with ceria, were reduced at approximately 473 K. 78 Copper(II) oxide-ceria catalysts revealed two maxima of hydrogen consumption: in the range of 423-441 K, assigned to small copper(II) oxide crystallites in contact with ceria, and in the range of 453-498 K, attributed to larger copper(II) oxide particles not in close contact with ceria. 76,77 A comparison of temperatures of maximum hydrogen consumption over coppergold and copper catalysts led to a conclusion that in bimetallic copper-gold samples based on the mixed cerium-zirconium oxides, copper or gold were reduced at lower temperatures than over the analogous monometallic catalysts. This nding conrmed the interaction between copper and gold.

Catalytic activityglycerol oxidation in the liquid phase
The challenge in the development of efficient oxidation catalysts is to obtain high activity, selectivity, and stability. Activity and selectivity of gold catalysts in glycerol oxidation are inuenced by the nature of the support, which determines the species formed on the surface 30,84,85 and the presence of other catalytically active components (e.g. palladium-gold, platinumgold). 13,86 Catalytic glycerol oxidation was performed by stirring at different rates to nd the conditions in which diffusion had the least inuence on the catalytic reaction (Tables S1 and S2 †). The conversion of glycerol increased up to 1000 rpm. Therefore, further experiments were carried out using this stirring speed. Table 5 shows the results of the catalytic oxidation of glycerol at 333 K in a basic aqueous solution under pure oxygen for monometallic and bimetallic catalysts. Copper-gold catalysts were denitely more active than monometallic catalysts under the same reaction conditions. The monometallic catalysts yielded primarily gaseous products. Changes in the copper and gold dispersion and electronic states, relative to those in monometallic ones, play a crucial role in increasing the catalyst activity and selectivity to glyceric acid. 87,88 Increase of glycerol conversion with increase of gold dispersion and decrease in selectivity to glyceric acid for small mean gold particle size (e.g. 2.7 nm in ref. 88) were observed. Here, the dispersion of gold and glycerol conversion were higher for bimetallic (gold- copper) catalysts than for monometallic ones. However, contrary to the ndings described in literature 87,88 for gold catalysts, bimetallic catalysts in this work presented the higher selectivity to glyceric acid despite the small mean gold particle size (<2.7 nm). All copper-gold catalysts were selective primarily towards glyceric acid. Low selectivities to other oxidation products, such as lactic, oxalic, tartronic, glycolic, and formic acids were observed. The oxidation of glycerol to glyceric acid under basic conditions on copper-gold catalysts proceeds via the initial formation of glyceraldehyde, which is rapidly oxidized to glyceric acid. 14 Tartronic, C2, and C1 acids indicate over-oxidation or transformation of glyceric acid. In the bimetallic system, gold and copper were in close proximity, therefore, the interaction between them made the interaction with reagents more effective. The activity and selectivity of copper-gold catalysts were also inuenced by the chemical composition of the supports. The highest selectivity to glyceric acid was observed for CuAu/CeZrO x (1 : 1). In this catalyst, the microporosity was negligible, and therefore, the diffusion of the primary product, glyceric acid, was easy, preventing capturing the intermediates and preventing over-oxidation.
Among bimetallic catalysts TOF was the highest on CuAu/ ZrO 2 than on the rest of bimetallic catalysts (Table 5). This catalyst had the smallest gold particles (average size 1.6 nm), exclusively metallic gold species and a domination of Cu + cations. Thus, such metallic species will be considered in the drawing of the reaction pathway at the end of this section. The best gold catalysts based on carbon or different metal oxides described in the literature as catalysts attractive for glycerol oxidation exhibited similar or lower TOF than CuAu/ZrO 2 . Table S3 † collects the best TOF data from the literature 28,62,89,90 in which the conditions of glycerol oxidation were similar to those applied in this work. The bimetallic catalysts achieved higher TOFs than monometallic gold catalysts (Table 5), explained by the crucial role of the interaction between gold and copper in the activation of glycerol adsorption and then its oxidation.
3.6.1. Effect of the reaction temperature. To obtain further information on the performance of the catalysts, oxidation experiments were conducted at different temperatures. The increase in the glycerol conversion was signicant for all copper-gold catalysts when the reaction temperature increased from 333 to 363 K. At both reaction temperatures glyceric acid was the main product. The increase in the reaction temperature led to higher yields of lactic acid, as a result of the dehydration of glyceric acid molecules (Table S4 †). At 333 K, glyceric acid transformed to formic acid and glycolic acid (Scheme 1).
At 363 K, the monometallic gold catalysts showed high glycerol conversion on Au/CeO 2 , but much lower on Au/ZrO 2 , yielding selectivity to glyceric acid and lactic acid and to a relatively small amount of formic acid (Table S1 †). On copper catalysts (Table S2 †), in addition to glyceric acid, glycolic and formic acids were formed in high yields at 363 K. This behavior indicated that copper species were responsible for the dehydration of the primary product, glyceric acid, whereas in the presence of gold-active centers, glyceric acid was formed and transformed to lactic acid at a higher temperature.
3.6.2. Changes in activity and selectivity and catalytic stability. A comparison of glycerol conversion (Fig. 5) and the changes in oxygen consumption during the reaction of glycerol oxidation (Fig. S4 †) showed that CuAu/CeZrO x (1 : 2) exhibited the highest conversion in glycerol oxidation at 333 K aer 5 h, at 1000 rpm. Over this catalyst, the oxygen consumption in the reaction was the smallest. Aer 150 min of the reaction over cerium-zirconium oxides modied with copper and gold, the oxygen consumption was smaller than over CuAu/CeO 2 and CuAu/ZrO 2 .
Between 30 and 300 min reaction, the molar ratio oxygen-: glycerol was always higher than 3.5 for all copper-gold catalysts, but this parameter changed linearly from 7 aer 30 min to 21 aer 300 min (Fig. S4 †). At the beginning, the molar ratio oxygen : glycerol increased over all bimetallic catalysts, but aer 180 min, for CuAu/CeO 2 and CuAu/CeZrO x (2 : 1), this value decreased from 6 to 4 and from 16 to 14, respectively.
The reaction proles (Fig. 5) indicated that aer 2 h of the reaction, ca. 60-80% of glycerol was converted; then, the conversion leveled off. The high selectivity to glyceric acid in the initial stages of the reaction indicated that it was formed in the rst step of the reaction. Formic and glycolic acid were also detected, but the selectivity to these compounds increased slowly during the reaction and decreased when the concentration of carbon dioxide increased.
The most active bimetallic catalysts were stable and glycerol conversion in the second cycle of the reaction was not considerably changed. It is characteristic that at constant activity, the selectivity to glyceric acid increased in the second reaction cycle performed over bimetallic catalysts based on zirconia and cerium-zirconium mixed oxides rich in zirconium. This phenomenon suggested changes in the catalyst surface during reaction.
The possibility of reusing catalysts without the loss of activity is very important. Fig. 6 shows the results of the experiments in which the catalysts were reused in the reaction at 333 K and a stirring speed of 1000 rpm. The catalyst used in the rst reaction cycle was recovered by decantation of the solvent aer the reaction and was reused without any washing or drying.
The increase in glyceric acid selectivity in the second cycle was highly desirable. The highest glycerol conversion was achieved by CuAu/CeZrO x (1 : 2) in the rst cycle (87%), whereas the highest selectivity to glyceric acid was reached on CuAu/ ZrO 2 in the second cycle (83%). Notably, CuAu/CeZrO x (1 : 2) material contained the lowest content of Cu + species and the highest amount of bimetallic particles. The increase of GLA selectivity was considered in the context of metal leaching during the rst reaction. Table S5 † shows the amount of copper and gold in the catalysts aer the second reaction. It is clear that zirconium strongly hold copper species and therefore leaching of copper did not occur for CuAu/CeZrO x (1 : 2). In contrast, for this sample gold was partially leached. This behaviour led to the increase of Cu : Au molar ratio which caused the increase of selectivity to GLA. On the basis of the results obtained within this work the reaction pathway of the glycerol oxidation to glyceric acid performed on bimetallic (Cu-Au) catalysts based on metal oxides used in this work was proposed (Scheme 2). Only the initial stages of the reaction are documented in the results obtained within this work. The following steps are hypothetic. On the basis of the highest TOF obtained on CuAu/ ZrO 2 which contained only metallic gold and almost exclusively Cu + cations, Au 0 particles and Cu + are proposed as active centres at the beginning of the reaction.
In the proposed reaction cycle the different surface gold and copper species are involved. Moreover, the role of the hydroxyl groups from the reaction medium are pointed out.
Hydroxide anions from sodium hydroxide initiate the dehydrogenation via H-abstraction of one of the primary OH groups of glycerol according to the literature data. 33 This is enhanced by the interaction of negative charge on gold formed by an electron transfer from Cu + species. The  following electron transfers from the aldehyde ion to Au 0 and from gold containing negative charge to Cu 2+ result in the rebuilding the primary metal species on the surface. In the next step oxygen is dissociatively chemisorbed on metallic gold particle, hydrogen from aldehyde group is abstracted by hydroxyls from the reaction medium and glyceric acid ion is formed. Scheme 2 indicates the crucial role of the electron transfer between the following pairs of copper and gold species: (i) Cu + and Au 0 or (ii) Cu 2+ and Au À . The catalysts exhibiting the highest TOF values, i.e. CuAu/ZrO 2 (TOF ¼ 1236 h À1 ) and CuAu/CeZrO x (1 : 2) (TOF ¼ 901 h À1 ), contain the highest amount of Cu + (86%) -Au 0 (100%) and Cu 2+ (66%) -Au À (67%), respectively. In the other bimetallic catalysts the dominant species of copper and gold are Cu + and Au 0 ( Table  4) between which the electron transfer does not occur. Such catalysts reveal lower TOF values.

Conclusions
All catalysts obtained within this work were mesoporous oxides containing also micropores and the microporosity increases with increasing zirconium content in the supports. It was evidenced the presence of gold species in the metallic form Au 0 , cationic Au d+ and negatively charged gold, the latter only in bimetallic copper-gold, catalysts. Moreover, the presence of cationic copper species, Cu + and Cu 2+ were evidenced. The interaction between all the components of the catalysts was evidenced and its role in the initial step of the catalytic oxidation of glycerol was discussed. Bimetallic gold-copper catalysts were signicantly more active in glycerol oxidation than the respective monometallic samples and they were highly selective to glyceric acid. High gold dispersion in bimetallic catalysts was supposed to be responsible for high glycerol conversion but negative charge on gold seemed to enhance the activity. Cu + species played an important role in the achievement of high selectivity to glyceric acid. The bimetallic catalysts were chemically stable. They were proved to be active in reuse and to show higher selectivity to glyceric acid than in the rst run.
On the basis of the characterization of the catalysts and the results of glycerol oxidation, one can postulate that metallic gold was the active phase in the oxidation of glycerol, whereas copper species drive the selectivity of the reaction. The presence of copper also increased the rate of the rst reaction step, i.e. the H-abstraction from glycerol molecules. Moreover, gold interacted with copper located in vicinity by electron transfer from Cu + to metallic gold or from Au À to Cu 2+ . The electron transfer between the aforementioned metal species is crucial in the reaction steps of glycerol oxidation (Scheme 2). Therefore, for high reaction rate the high content of above indicated pairs of metal species (Cu + -Au 0 and Cu 2+ -Au À ) is required. The interaction of gold and copper species with the supports also inuenced the types of metallic species formed and therefore the chemical composition of the supports played an important role in the catalytic activity and selectivity.


Another application of newly prepared Brønsted-acidic ionic liquids as highly efficient reusable catalysts for neat synthesis of amidoalkyl naphthols

In this work, two newly prepared Brønsted-acidic ionic liquids, [MPyrrSO3H] Cl (IL1) and [MMorSO3H]Cl (IL2), were efficiently used as catalysts for the synthesis of amidoalkyl naphthols through the one‐pot, three‐component reaction of β‐naphthol, aryl aldehydes, and acetamide under neat conditions. High activity of the catalysts, excellent yields, short reaction times, simple procedure with an easy work-up, and the absence of any volatile and hazardous organic solvents are some advantages of the present methodology. Moreover, the catalysts are simply prepared and can be recovered conveniently and reused such that considerable catalytic activity can still be achieved after the fifth run. Subjects: Organic Chemistry; Catalysis; Environmental

Introduction
A major challenge in modern chemistry is the design of highly efficient chemical reaction sequences that provide maximum structural complexity with a minimum number of synthetic steps in short reaction times (Dömling, 2006;Schreiber, 2000). Multicomponent reactions (MCRs) have gained considerable attention as a powerful method in organic synthesis and medicinal chemistry because they involve simultaneous reaction of more than two starting materials to yield a single product through one-pot reaction (Gore & Rajput, 2013;Slobbe, Ruijter, & Orru, 2012;

PUBLIC INTEREST STATEMENT
Application of new ionic Liquids in organic transformations is of great interest in recent years. Therefore, in this paper, two newly prepared Brønsted-acidic ionic liquids were efficiently used as catalysts for the synthesis of amidoalkyl naphthols through the one-pot, three-component reaction of β-naphthol, aryl aldehydes, and acetamide under neat conditions. Some advantages of this procedure are high yields, short reaction times, easy work-up, absence of volatile and hazardous solvents, and reusability of catalysts for a number of times without appreciable loss of activity. Davoodnia, 2011). High atom economy, good selectivity, time and energy saving, low cost, minimum waste production, and short reaction time make MCRs suitable for the synthesis of complex molecules with potential biological activity (Chebanov & Desenko, 2012;Manjappa, Peng, Jhang, & Yang, 2016;Zang, Zhang, Zang, & Cheng, 2010). On the other hand, the nature of the catalyst plays a crucial role in the determination of the product and selectivity (Khan, Khan, & Bannuru, 2010;Mirzaei & Davoodnia, 2012;Shaterian & Mohammadnia, 2012). Therefore, development of inexpensive, mild, and reusable catalysts for MCRs such as the synthesis of amidoalkyl naphthols remains of interest to the synthetic organic chemists. It has been reported that amidoalkyl naphthols can convert to important biologically active aminoalkyl naphthol derivatives by amide hydrolysis. Later compounds have been evaluated for the hypotensive and bradycardiac effects (Dingermann, Steinhilber, & Folkers, 2004;Shen, Tsai, & Chen, 1999). Amidoalkyl naphthols are generally synthesized via the three-component reaction of β-naphthol, an aldehyde, and an amide in the presence of various catalysts, such as Sb(OAc) 3 (Hakimi, 2016), zirconocene dichloride (Cp 2 ZrCl 2 ) ( (Kiasat, Hemat-Alian, & Saghanezhad, 2016), carbon-based solid acid (Davoodnia, Mahjoobin, & Tavakoli-Hoseini, 2014), H 3 BO 3 (Shahrisa, Esmati, & Nazari, 2012), iodine (Nagawade & Shinde, 2007), nano-sulfated zirconia (Zali & Shokrolahi, 2012) (Kumar, Rao, Ahmad, & Khungar, 2009), and nano silica phosphoric acid (Bamoniri, Mirjalili, & Nazemian, 2014). Although each of these individual methods has its own merits, many suffer from limitations such as long reaction times, unsatisfactory yields, and the use of relatively expensive catalysts. Thus, the exploration of novel methodologies using new efficient and reusable catalysts is still ongoing.
In recent years, ionic Liquids (ILs) have attracted rising interest as eco-friendly solvents, catalysts and reagents in organic transformations due to their advantageous properties, such as non-flammability, negligible vapor pressure, high thermal and chemical stability, and ability to dissolve a wide range of materials (Chowdhury, Mohan, & Scott, 2007;Olivier-Bourbigou, Magna, & Morvan, 2010;Pârvulescu & Hardacre, 2007). ILs are miscible with materials having very wide range of polarities and are simultaneously able to dissolve a wide range of organic, inorganic and organometallic substances. These features offer numerous opportunities for the improvement of organic reactions using ILs as solvents and catalysts. Moreover, their ionic character enhances the reaction rates to a great extent in many reactions. Among them, Brønsted acidic ILs, especially the SO 3 H-functionalized ones, have designed as environmentally friendly catalysts to replace the traditional mineral liquid acids like sulfuric acid and hydrochloric acid in chemical processes (Greaves & Drummond, 2008;Qiu et al., 2016;Shirole, Kadnor, Tambe, & Shelke, 2017;Vafaeezadeh & Alinezhad, 2016;Zolfigol, Khazaei, Moosavi-Zare, & Zare, 2010).

Results and discussion
As a preliminary, we directed our studies toward examination of the effect of various parameters like catalyst composition, effect of solvent, and influence of temperature on the reaction of β-naphthol (1) (1.0 mmol), 4-chlorobenzaldehyde (2d) (1.0 mmol), and acetamide (3) (1.0 mmol) for the synthesis of compound 4d as the model reaction in the absence or presence of IL 1 and IL 2 as catalysts. A summary of the optimization experiments is provided in Table 1. First, to illustrate the need for catalyst in the reaction, the model reaction was studied in the absence of catalyst under solvent-free condition. The yield of the product was trace at 90°C after 60 min (Table 1, entry 1). Next, the reaction was performed in the presence of IL 1 or IL 2 in different solvents as well as under solvent-free conditions. Among the solvents tested, those being EtOH, MeOH, CH 2 Cl 2 , and MeCN, the reaction proceeded most readily to give the highest yield of the product 4d under solvent-free conditions. It was observed that the yield of the final product 4d increased with increasing amount of catalyst in the reaction mixture. The best result was obtained with 10 mol% of the catalyst under solvent-free conditions, which gave the desired product in 95 and 98% yields after 3 and 2 min at 90°C, respectively, for IL 1 and IL 2 (Table 1, entry 12). Further increase in temperature and IL 1 or IL 2 amount were found to have an inhibitory effect on formation of the product (Table 1, entries 13, 16, 17).
With the optimized conditions in hand, β-naphthol was reacted with acetamide and a wide variety of aromatic aldehydes using IL 1 or IL 2 ( Table 2). As it can be seen, the reaction is effective with a variety of aromatic aldehydes with electron-donating or withdrawing substituents. Although the kind of aromatic aldehyde had no significant effect on the reaction, in most cases, but not all, aromatic aldehydes substituted with electron-withdrawing group or none reacted slightly faster than those with electron-donating groups and gave the higher yields of the products. Furthermore, both catalysts were highly efficient, and gave the desired amidoalkyl naphthols in high yields and short reaction times. However, as depicted, IL 2 proved to be the better catalyst than IL 1 in terms of yield and reaction time.
We also investigated recycling of the catalysts under solvent-free conditions using the model reaction. After completion of the reaction, the reaction mixture was cooled to room temperature, and warm distilled water was added. The product was collected by filtration, and washed repeatedly with warm distilled water. The combined filtrate was evaporated to dryness under reduced pressure. The residual ionic liquid was repeatedly washed with diethyl ether, dried under vacuum at 60°C, and used for the subsequent catalytic runs. The recovered catalyst worked well for up to five catalytic runs without any significant loss of its activity (95/98, 95/96, 93/95, 92/93, and 91/93% yields for IL 1 /IL 2 catalysts in first to fifth use, respectively).

Entry
Catalyst ( 190-192 192-194 (Bamoniri et al., 2014) In accordance with the literature (Kiasat et al., 2016;Shahrisa et al., 2012), the suggested mechanism is described in Scheme 2. We believe that these ILs can act as Brønsted acids and therefore promotes the reactions by increasing the electrophilic character of the electrophiles in the reaction.

Conclusion
In conclusion, we showed that two newly synthesized Brønsted-acidic ILs, IL 1 and IL 2 , efficiently catalyze the synthesis of amidoalkyl naphthols by increasing the electrophilic character of the electrophiles in the reaction β-naphthol, aryl aldehydes, and acetamide under solvent-free reactions. The kind of aldehyde had no significant effect on the reaction rates and products' yields. However, in general, electron-poor aldehydes reacted slightly faster than electron-rich ones and gave the higher yields of the products. Also, IL 2 proved to be the better catalyst than IL 1 in terms of yield and reaction time. Some advantages of this procedure are high yields, short reaction times, easy work-up, absence of volatile and hazardous solvents, and reusability of catalysts for a number of times without appreciable loss of activity.

Experimental
The IL 1 and IL 2 were synthesized according to the our previous report (Dehghan et al., 2016). All chemicals were available commercially and used without additional purification. Melting points were recorded on a Stuart SMP3 melting point apparatus. The 1 H NMR spectra were recorded with a Bruker 300 FT spectrometer.

General procedure for the synthesis of amidoalkyl naphthols (4a-k) catalyzed by IL 1 or IL 2
A mixture of β-naphthol (1) (1.0 mmol), an aromatic aldehyde (2a-k) (1.0 mmol), acetamide (3) (1.0 mmol), and IL 1 or IL 2 (0.1 mmol, 10 mol %) was heated in an oil bath at 90°C for 2-6 min. After completion of the reaction, monitored by TLC, the mixture was cooled to room temperature and warm distilled water was added. This resulted in the precipitation of the product, which was collected by filtration. The crude product was washed repeatedly with warm distilled water and then cold ethanol, and subsequently recrystallized from ethanol to give the pure products 4a-k in high yields. The products were characterized according to comparison of their melting points with those of authentic samples and for some of them by their 1 H NMR spectral data.


extfieldsofstudy:
Physics
Materials Science

text:
Mechanistic reaction pathway for hexafluoropropylene oxide pulsed plasma deposition of PTFE-like films

The pulsed plasma deposition of PTFE-like thin films using hexafluoropropylene oxide (HFPO) precursor has been investigated by time-resolved in situ mass spectrometry. Electron-impact and electron-attachment ionisation modes have been employed to monitor neutral plasma species; whilst electrical discharge negative and positive ions have been detected by switching-off the mass spectrometer electron-impact ionisation source. The relative concentrations of neutral versus ionic plasma species were found to rise for shorter pulse duty cycles. Mechanistically, HFPO molecules undergo pulsed plasma-initiated decomposition to form difluorocarbene (:CF2) radical species, and these can combine to form tetrafluoroethylene (CF2 = CF2) molecules, which subsequently polymerise leading to the growth of PTFE-like ([–CF2–]n) perfluoroalkyl chains. Shorter pulsed plasma duty cycles enhance the growth of linear PTFE-like perfluoroalkyl chains.

Introduction
Hexafluoropropylene oxide (HFPO) is a stable fluorine-containing cyclic molecule [1,2]. Thermal or pulsed plasma excitation of HFPO is employed for the deposition of PTFE-like ([-CF 2 -] n ) thin films for a variety of technological applications; these include liquid repellency, low dielectric constant electronic materials, encapsulation barrier layers, and biologically implantable devices [3][4][5]. The HFPO pyrolysis mechanism is understood to proceed via the formation of either fluoro(trifluoromethyl) carbene (II) and carbonyl difluoride (III), or difluorocarbene (VI) and trifluoroacetyl fluoride (VII) species, scheme 1 [6][7][8][9][10][11]. Under these conditions, it is the polymerisation of difluorocarbene (:CF 2 ) radicals which is postulated to give rise to the growth of PTFElike films [12][13][14][15]. Whilst in the case of pulsed plasma excitation of HFPO gas, such intermediate difluorocarbene species, have been detected by time-resolved ultraviolet absorption spectroscopy within the duty cycle on-period (t on ); followed by recombination processes predominating during each subsequent offperiod (t off ) [13]. However, the direct growth of perfluorocarbon chains within the electrical discharge has not previously been validated. Rather, x-ray photoelectron spectroscopy (XPS) and infrared analysis of the pulsed plasma deposited films have shown an enhancement in -CF 2group content with decreasing pulsed plasma duty cycle (i.e. greater structural retention to form PTFE-like films) [4,16]. In this article, the pulsed plasma polymerisation mechanism for HFPO precursor is investigated by time-resolved in situ mass spectrometry, with a particular emphasis on the role played by difluorocarbene species during the growth of PTFE-like perfluoroalkyl chains ([-CF 2 -] n ).

Materials and methods
In situ mass spectrometry analysis was undertaken during pulsed plasma polymerisation inside a cylindrical glass reactor (8.5 cm diameter and 2943 cm 3 volume), figure 1. This chamber was pumped down to a base pressure of 8×10 −3 mbar, via a liquid nitrogen cold trap attached to a turbomolecular drag pump (model TPD 022, Pfeiffer Vacuum Technology AG) backed by a diaphragm pump (model N813.4 ANE, KNF Neuberger GmbH). A copper coil (7 mm diameter, 4 turns, spanning 10.5 cm) wrapped around the glass chamber was connected to Original content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence.
Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. a 13.56 MHz radio frequency (RF) power supply (model RFG 600SE, Coaxial Power Systems Ltd) in conjunction with a LC circuit matching unit (to minimise reflected power). A pulse signal generator (model TG503 5 MHz pulse, Thurlby Thandar Instruments Ltd) was used to trigger the RF power supply and monitored using an oscilloscope (model OX 530 20 MHz, Metrix Ltd). For pulsed plasmas, a fixed 500 ms off-period (t off ) and variable on-periods (t on ) of 10, 5 and 2 ms (corresponding to duty cycles of 2, 1 and 0.4% approximately) were employed in conjunction with 50 W power input during the on-period (P on ).
An electrostatic quadrupole mass spectrometer probe (model HAL EQP 2500 Hiden Analytical Ltd), with a grounded 50 μm orifice was positioned inside the plasma chamber along the central axis (this acted as an earthed substrate), figure 1. The instrument comprised an ion extractor, electron-impact ionisation source, a 45°sector field energy filter, and a triple-filter quadrupole mass filter with secondary electron multiplier detection. The mass spectrometer analyser was pumped by a turbomolecular pump (model TMU 260, Pfeiffer Vacuum Technology AG) backed by a rotary vane pump (model DUO 2.5, Pfeiffer Vacuum Technology AG) to give a mass spectrometer base pressure of better than 2×10 −9 mbar. The electron-impact/attachment ionisation source of the mass spectrometer was operated at 70 eV to allow for the detection of neutral and radical plasma species. Ionised gaseous species (plasma ions) were sampled directly from the electrical discharge through the 50 μm end cap orifice with the mass spectrometer ionisation filament switched off. For time-resolved pulsed plasma measurements, signal gating of the mass spectrometer detector allowed for any specified time window (t on or t off ) to be monitored. This was achieved by using an additional pulse signal generator (model TGP110 Scheme 1. Mechanistic reaction pathways for the fragmentation of hexafluoropropylene oxide (HFPO) and subsequent difluorocarbene polymerisation. The initial fragmentation steps (Route A and Route B) are analogous to reported HFPO thermal decomposition mechanisms [6,8,9]. 10 MHz, Thurlby Thandar Instruments Ltd) connected to the mass spectrometer interface unit through a transistor-transistor logic (TTL) level acquisition input. One pulse signal generator triggered both the plasma duty cycle ignition and the other pulse signal generator; where the latter was set to trigger the mass spectrometer ion counting detector during the whole duty cycle, or either the t on or t off (afterglow) time windows. Therefore, the mass spectra scans sampled the whole duty cycle, or either the t on or t off (afterglow) time frames, and were averaged over multiple pulse cycles to improve signal-to-noise (typically 2 min total scan time for 0-400 m/z range).
Prior to each experiment, the chamber was cleaned with a 50 W oxygen plasma (0.2 mbar pressure) and then purged with HFPO gas precursor (HFPO, +98%, Aldrich Chemical Company Inc.) at a pressure of 0.2 mbar via a fine control needle valve (model 145-217-4P4PC, Meggitt Avionics Ltd) for 20 min, prior to electrical discharge ignition. A range of standard perfluorocarbon molecules were also analysed in the gas phase (no plasma) using the same instrument in order to provide reference electron-impact ionisation mass fragmentation patterns and cross-checked against the American NIST database [17,18]. These included perfluoro-n-pentane (+99%, Fluorochem Ltd) and perfluoro(methylcyclohexane) (+90%, Fluorochem Ltd).

Time-averaged mode detection of plasma species
The parent fragment ion (molecular mass of HFPO=166 amu), was absent during mass spectrometric analysis of the precursor molecule (t on =0 ms, i.e. no plasma), with CO + (28 m/z), CF + (31 m/z), and CF 3 + (69 m/z) being the predominant peaks, figure 2 [19]. Plasma ignition leads to an order of magnitude increase in concentration of neutral species together with the appearance of ionic fragments. In time-averaged mass spectrometry detection mode, the relative concentration of neutral species is several orders of magnitude greater than positive ions, and was found to rise dramatically as the t on period was shortened. Conversely, the number of positive plasma ions detected increased for longer t on values, figure 2. The most intense peaks for electronimpact of neutral plasma species were CO + (28 m/z), and CF 3 + (69 m/z); whilst the most intense peak for positive plasma ions was CF 3 + (69 m/z). At longer t on values, the mass spectra approached those observed for continuous wave (non-pulsed) HFPO plasmas [20]. Whilst for decreasing t on values, the electron-impact of neutral plasma species CO + (28 m/z) : CF 3 + (69 m/z) ratio approaches that observed for the HFPO precursor molecule (t on =0 ms).
In the absence of electron-impact/attachment ionisation, no negative ion species (including Fions) were detected. Whilst for electron-attachment negative-mode mass spectrometry detection of plasma species, only fluorine atoms (19 m/z) were measured in all time frames (time-averaged, resolved on-period and resolved offperiod), figure 3.

Time-resolved electron-impact ionisation positive-mode mass spectrometry of neutral plasma species
In the case of reference mass spectra taken for standard linear C n F n+2 perfluoroalkane molecules, the CF 2 + /CF 3 + ion signal ratio remains fairly constant for n>3. This CF 2 + /CF 3 + ion ratio was also found to be steady for the analysis of HFPO pulsed plasma neutral species, which therefore supports the growth of linear C n F n+2 perfluoroalkyl chains (species (XII) in scheme 1), figure 4. Other evidence for the formation of neutral linear chain perfluorocarbon species included the detection of mass spectrometric electron-impact ionisation mass fragments: (C 2 F 4 + (100 m/z), C 2 F 5 + (119 m/z), C 3 F 6 + (150 m/z), C 3 F 7 + (169 m/z), C 4 F 7 + (181 m/z), C 4 F 9 + (219 m/z) and C 5 F 11 + (269 m/z)), figure 4. The signal intensity of these perfluoroalkyl chain fragments was found to increase by an order of magnitude (in both the t on and t off windows) with decreasing t on period, which supports there being less plasma-induced fragmentation of growing polymer chains for shorter t on values [21].
On the other hand, electrical discharge generated fragments arising from HFPO decomposition byproducts become relatively more predominant at longer t on values; for instance the CO + (28 m/z) signal is greater compared to the CF 3 + (69 m/z) intensity at t on =10 ms and lower for t on =2 ms. These observations are also consistent with the aforementioned attenuation of extended plasma-induced HFPO molecule breakdown at shorter t on periods (less CO by-product formation). The relative signal intensity of neutral species measured during the t off window was greater compared to either time-averaged or t on period detection modes; which indicates that radical concentrations are highest during the t off window (a prerequisite for polymer chain propagation mechanism involving either difluorocarbene (VI) or tetrafluoroethylene (V) intermediate species, scheme 1), figures 2 and 4.

Time-resolved positive-mode mass spectrometry of positive ion plasma species
In contrast to 70 eV electron-impact mass spectrometry of the HFPO precursor molecule (t on =0 ms, figure 4), the HFPO parent molecular positive ion (166 m/z) was detected in the pulsed plasmas suggesting that the HFPO molecule undergoes much more gentle ionisation within the electrical discharge due to lower energy plasma electron-impact collisions [22], allowing the formed HFPO molecule positive ion to remain intact, Figure 5 Linear C n F n+2 perfluorocarbon chain positive ion plasma fragments were also detected (C 2 F 4 + (100 m/z), C 2 F 5 + (119 m/z), C 3 F 6 + (150 m/z), C 3 F 7 + (169 m/z), C 4 F 7 + (181 m/z), C 4 F 8 + (200 m/z), C 4 F 9 + (219 m/ z), C 5 F 8 + (231 m/z), C 5 F 11 + (269 m/z)). The greater visibility of higher mass range fragments in positive-ion detection mode relative to fragments at lower masses is again due to the lower energy plasma electron-impact ionisation process (compared to the higher 70 eV energy electron-impact ionisation employed for neutral species detection mode, figure 4) [22]. The relative intensity of these perfluoroalkyl chain positive ion fragments rises with decreasing t on period, which supports there being less plasma-induced fragmentation/damage of growing polymer chains at shorter t on values. Interactions between ions and HFPO is observed with the exchange of an F − ion with an O 2− ion derived from HFPO giving rise to an intense C 3 F 7 + peak, figure 5 [23].
The previously described enhancement of neutral plasma species concentration during the t off period at shorter t on values detected in mass spectrometry electron-impact ionisation mode ( figure 4) is mirrored by a corresponding drop in intensity of positive-ion plasma species detected during the off-period. Furthermore, the positive-ion signal intensities are several orders of magnitude greater during the t on period (electrical discharge) compared to the t off period, figure 5.

Conclusions
Pulsed plasma polymerisation of HFPO proceeds via electrical discharge initiated decomposition of precursor molecules to yield difluorocarbene (:CF 2 ) radicals during the pulse t on period. These then readily combine to form tetrafluoroethylene (CF 2 =CF 2 ) intermediate species, which are able to subsequently undergo polymerisation to create PTFE-like perfluoroalkyl chains. Shorter pulsed plasma duty cycles (t on ) give rise to a greater concentration of neutral species (including polymerisable tetrafluoroethylene (CF 2 =CF 2 ) molecules), in conjunction with an attenuation of plasma ion concentrations-leading to the growth of longer perfluoroalkyl PTFE-like chains.  . Time-resolved electron-impact ionisation positive-mode mass spectra of HFPO pulsed plasma neutral species as a function of duty cycle t on period (t off =500 ms, P on =50 W, and 0.2 mbar). The detector was triggered for the whole duration of t on and t off time windows respectively. C n F 2n+1 + fragments are labelled as: C 2 =C 2 F 5 + (119 m/z), C 3 =C 3 F 7 + (169 m/z), C 4 =C 4 F 9 + (219 m/z), and C 5 =C 5 F 11 + (269 m/z). t on =0 ms corresponds to the fragmentation pattern of HFPO precursor molecule (absence of plasma ignition). by JPSB and AC Both authors gave final approval for publication.
Data accessibility Supporting data can be accessed at http://collections.durham.ac.uk

Conflicts of interest
There are no conflicts of interest to declare. Figure 5. Time-resolved positive-mode mass spectra of HFPO pulsed plasma positive ions species as a function of duty cycle t on period (t off =500 ms, P on =50 W, and 0.2 mbar). For each duty cycle, the detector was triggered for the whole duration of t on and t off time windows respectively. C n F 2n+1 + fragments are labelled as: C 2 =C 2 F 5 + (119 m/z), C 3 =C 3 F 7 + (169 m/z), C 4 =C 4 F 9 + (219 m/z), and C 5 =C 5 F 11 + (269 m/z).


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Chemistry Biology

text:
Synthesis, molecular modeling and anticancer activity of new coumarin containing compounds

A series of new coumarin containing compounds were synthesized from 4-bromomethylcoumarin derivatives 2a, b and different heteroaromatic systems 4a-e, 6a-d, 8, 10via methylene thiolinker. Twenty-four compounds were screened biologically against two human tumor cell lines, breast carcinoma MCF-7 and hepatocellular carcinoma HePG-2, at the national cancer institute, Cairo, Egypt using 5-fluorouracil as standard drug. Compounds 5h, 7d, 7h, 9a, 13a and 13d showed strong activity against both MCF-7 and HepG-2 cell lines with being compound 13a is the most active with IC50 values of 5.5 µg/ml and 6.9 µg/ml respectively. Docking was performed with protein 1KE9 to study the binding mode of the designed compounds.

Introduction
Uncontrolled cellular proliferation, mediated by dysregulation of the cell-cycle machinery and activation of cyclin-dependent kinases (CDKs) to promote cell-cycle progression, lies at the heart of cancer as a pathological process (O'Leary et al., 2016) CDKs are key regulatory kinases of the cell cycle (Morgan, 1997). They regulate different phases of cell cycle by binding to distinct regulatory subunit called a cyclin (Abdel Latif et al., 2016). The importance of CDKs in cell division process directed the attention of the medicinal chemists towards the use of them as a potential targets in the treatment of human cancer (Meijer et al., 1999).
Many molecules were found to be CDK specific ATP-competitive inhibitors and few of them have progressed into human clinical trials as flavopiridol compound I (Fig. 1) which is a coumarin derivative (Senderowicz et al., 1998 andSedlacek et al., 1996).
Coumarins are bioactive compounds of both nature and synthetic origin and there has been a growing interest in their synthe-sis due to their useful and diverse pharmaceutical and biological activities (Salem et al., 2016).
Based on the previous information we designed and synthesized a series of new coumarin containing compounds where the coumarin ring is substituted at position 4 with different five and six member heterocycles via a methylene thiolinker to test the anticancer effect of these new structure combinations against MCF-7 and HepG-2 cell lines (Fig. 2).

Chemistry
Scheme 1 illustrates the synthesis of the final compounds 5a-j, 7a-h, 9a, b. Ethyl 4-bromoacetoacetate 1 was obtained via bromination of ethyl acetoacetate using molecular bromine in ether at room temperature (Sousa et al., 2012). This bromo compound was allowed to go through Pechmann condensation with the phenol derivatives 2a, b in concentrated sulfuric acid to afford the intermediates 4-bromomethylcoumarin derivatives 3a, b Basanagouda et al., 2014). The triazole derivatives 4a-e (Kusmeierza et al., 2014;Malbec et al., 1984;Sandstrom and Wennerbeck, 1966) and oxadiazole derivatives 6a-d (Charistos et al., 1994) were synthesized using the appropriate substituted phenyl hydrazide and phenylisothiocyanate or carbon disulfide, respectively while compound 8  was prepared applying the same procedure using hydrazine hydrate and carbon disulfide. The final compounds 5a-j, 7a-h, 9a, b were obtained in good yield after the reaction of the 4-bromomethylcoumarin derivatives 3a, b with various thiol containing heteroaromatics 4a-e, 6a-d and 8 in acetone utilizing potassium carbonate as a base The synthesis of different pyrimidine-5-carbonitril derivatives 13a-d was described in Scheme 2. The first part of this scheme involved the preparation of the pyrimidine thiol 10 through condensation of benzaldehyde with thiourea and ethyl cyanoacetate according to the reported procedure (Shaquiquzzaman et al., 2012). Alkylation of compound 10 with the bromomethylcoumarin derivative 3a in tetrahydrofuran using triethylamine as a base afforded the intermediate alkylsulfanyl pyrimidine carbonitrile 11, which was subsequently halogenated by the reaction with phosphorous oxychloride to give the reactive chloro intermediate 12. The chloro group in compound 12 was replaced with different aliphatic and aromatic amines in ethanol to afford the final compounds 13a-d in good yield.

Antitumor activity
Twenty-four newly synthesized compounds were tested for their cytotoxic activity against human breast cancer cell line (MCF-7) and hepatocellular carcinoma cell line (HepG-2) using MTT assay according to the method of Mosmann (Mosmann, 1983). The results are presented in Table 1.
From the above data we conclude that the substitution of the nitro group of 5h alters the activity either with methyl group leads to almost inactive compound 5g, 5j, while replacing it with hydrogen decreases the activity as in compounds 5f and 5i. Moreover replacing the benzocoumarine ring of 5h with methylcoumarine ring reduces the activity as in compound 5c. Moving to the oxadiazole series 7a-7h we realized that having fluorine substituted phenyl ring attached to the oxadiazole moiety leads to the most active compounds in these series either with methylcoumarine moiety attached to the oxadiazole ring 7d or with benzocoumarine ring attached to the oxadiazole ring 7h. Replacing this fluorine atom in this series with any other substituents either reduce (7b, 7c, and 7e) or kills the activity (7a, 7f and 7g). Having disubstituted thiadiazole with methylcoumarine compound gives strong active compound 9a, while replacing these two methylcoumarine rings with benzocoumarine ones leads to inactive compound 9b.
The replacement of the hydrazinyl moiety or the piperazinyl one on the pyrimidine ring in 13a and 13d by aniline in 13b or morpholine in 13c respectively resulted in reducing or killing the activity concluding that the most active compound should bear a hydrogen bond donor moiety like in 13 a, 13d.

Molecular docking
Molecular docking is a computational procedure performed on structure-based rational drug design to identify correct conformations of small molecule ligands and also to estimate the strength of the protein-ligand interaction, usually one receptor and one ligand. Docking of ligands that are bound to a receptor through noncovalent interactions is relatively conventional nowadays. The majority of docking methods development research has been focused on the effective prediction of the binding modes of noncovalent inhibitors (Kumalo et al., 2015).
CDKs have a critical role in the control of cell division so deregulation of CDKs can lead to abnormal processes and numerous human diseases, most notably cancer and tumors. Therefore, suppression of CDKs activity serves as an ideal therapeutic strategy for cancer and tumor by interrupting aberrant cell proliferation (Lu et al., 2016). A new class of drugs, termed CDK inhibitors, has been studied in preclinical and now clinical trials. These inhibitors are believed to act as an anti-cancer drug by blocking CDKs to block the uncontrolled cellular proliferation that is hallmark of cancers (Balakrishnan et al., 2016) Docking Studies for the target compounds 5h, 9a and 13a, which showed the highest in vitro activity, were carried out using the molecular operating environment (MOE) to show their binding mode and suggest the proposed mechanism of their antiproliferative activity. Crystallographic structure of cyclin dependent kinase 2 (CDK2) was obtained from the protein data bank (code, 1KE9. pdb) (Bramson et al., 2001) with the help of Pharm Mapper software (Liu et al., 2010). Upon inspection of the protein embedded ligand interaction for the protein molecule 1KE9, it was found that the embedded ligand occupied the ATP-binding site of CDK2 with the formation of two hydrogen bonds, one with the backbone NH of Leu-83 and the other with the backbone carbonyl of Glu-81 as shown in (Fig. 3).
When examining the interaction between the protein and our target compounds, it was found that the target compounds occupied the same pocket of the active site of the protein and formed one hydrogen bond with backbone NH of Leu-83 as shown in . These interactions assume the importance of Leu-83 residue for the activity. Also it was observed that compound 5h showed an additional binding to Lys-89 through arene cation interaction, but this interaction does not seem to have any relation to activity.

Conclusion
The target compounds showed good fit within the active site of the docked CDK2. There is a strong correlation between molecular modeling and biological screening results and it is expected that the target compounds antiproliferative activity may be due to inhibition of CDK2 activity.

Chemistry
All commercial reagents were used without purification. Melting points were determined on a Mel-Temp 3.0 melting point apparatus, and are uncorrected. TLC analysis was carried out on silica gel 60 F254 precoated aluminum sheets using UV light for detection. 1 H and 13 C NMR spectra were recorded on a Bruker 400 MHz spectrom-eter and Bruker 100 MHz spectrometer, respectively using the indicated solvents. Mass spectra were obtained from the Cairo University Mass Spectrometry Laboratory, Cairo, Egypt. Elemental analysis was performed at the Microanalysis Centre, Cairo University.

Synthesis of ethyl 4-bromoacetoacetate (1)
The required ethyl 4-bromoacetoacetate was synthesized by bromination of ethyl acetoacetate with molecular bromine in dry ether according to the published method (Sousa et al., 2012).
4.1.32. Synthesis of 4-hydroxy-2-{[(6-methyl-2-oxo-2H-chromen-4yl)methyl]sulfanyl}-6-phenylpyrimidine-5-carbonitrile (11) Triethylamine (1 ml, 0.01 mol) was added to a solution of 10 (2.29 g, 0.01 mol) in dry tetrahydrofuran. The mixture was stirred at room temperature for 1 h then solution of 3a (2.52 g, 0.01 mol) in dry tetrahydrofuran was added portionwise and the reaction mixture was stirred for additional 24 h at room temperature then the solvent was concentrated under reduced pressure and the residue was poured onto crushed ice with stirring. The precipitate was filtered off and recrystallized from ethanol to give compound 11.
Brownish white solid ( 3.77;N,10.47. Found: C,65.69;H,3.67;N, methyl]sulfanyl}-6-phenylpyrimidine-5-carbonitrile (12) Phosphorous oxychloride (10 ml) was added dropwise with stirring to a flask charged with compound 11 (4.4 g, 0.011 mol) at 0°C. The mixture was stirred at room temperature for 0.5 h then refluxed on water bath at 70°C for 6 h. The reaction mixture was cooled, and poured onto crushed ice. The formed solid was filtered and recrystallized from methylene chloride to give compound 12.
Light brown solid, (3 gm 3.36;N,10.01. Found: C,63.12;H,3.30;N,methyl]sulfanyl}-6-phenylpyrimidine-5-carbonitrile (13a and 13b) Solution of compound 12 (2 gm, 0.005 mol) in absolute ethanol was added portionwise to a well stirred solution of the appropriate amine (0.005 mol) and triethyl amine (0.5 ml, 0.005 mol) in absolute ethanol (10 ml). The mixture was refluxed for 5 h then the solvent was removed under reduced pressure and the remained solid was washed with cold water and purified by recrystallization from ethanol.
Compound 13d was prepared using the same procedure but the filtrate was stirred with 2N hydrochloric acid before filtration,

Biology
All final compounds were evaluated for their antitumor activity against human breast cancer cell line (MCF-7) and hepatocellular carcinoma cell line (HepG-2) adopting MTT assay [1]. All materials were obtained from Sigma Chemical Co (USA). The cell lines were obtained from ATCC via Holding company for biological products and vaccines (VACSERA), Cairo, Egypt. 5-Fluorouracil was used as a standard anticancer drug for comparison. MTT assay is a colorimetric assay based on the reduction of the 3-(4,5-dimethyl-2-thia zolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) to a purple formazan derivative by mitochondrial succinate dehydrogenase in viable cells. Cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum. Antibiotics added were 100 units/ml penicillin and 100 mg/ml streptomycin at 37°C in a 5% CO 2 incubator. The cell lines were seeded in a 96-well plate at a density of 1 Â 10 4 cells/well at 37°C for 48 h under 5% CO 2 . After incubation the cells were treated with different concentration of the tested compounds and incubated for 24 h, then 20 ml of MTT solution at 5 mg/ml was added and incubated for 4 h. 100 ml of dimethyl sulfoxide (DMSO) is added into each well to dissolve the purple formazan formed. The absorbance of each well was measured by a microplate reader (EXL 800 USA) using a test wavelength of 570 nm. The results was expressed as IC 50 , which is the concentration of the drugs inducing a 50% inhibition of cell growth of treated cells when compared to the growth of control cells. The IC 50 values were calculated using different concentrations of the tested compounds (Mosmann, 1983).

Molecular docking
The docking studies and modeling calculations were done using 'MOE version 2009.10 release of Chemical Computing Group's' which was operated under Windows XP operating system installed on an Intel Pentium IV PC with a 2.8 MHz processor and 512 RAM. The tested compounds were built in 2D using ChemBiooffice suite and geometric optimization was done using Hyperchem, then subjected to docking simulation. The X-ray crystallographic structure of CDK2 protein enzyme was obtained from the Protein Data Bank; code '1KE9.pdb'.