Synthesis, Characterization, and Anti-Algal Activity of Molybdenum-Doped Metal Oxides

: In this study, we attempted to synthesize visible light active nano-sized photocatalysts using metal oxides such as zinc oxide, zirconium oxide, tungsten oxide, and strontium titanium oxide with (MoCl 5 ) 2 as a dopant by the simple ball-milling method. Fourier-transform infrared spectroscopy data conﬁrmed the presence of M-O-Mo linkage (M = Zn, Zr, W, and SrTi) in all the molybdenum-doped metal oxides (MoMOs), but only MoZnO inhibited the growth of the bloom-forming Microcystis aeruginosa under visible light in a concentration-dependent manner up to 10 mg / L. Further, structural characterization of MoZnO using FESEM and XRD exhibited the formation of typical hexagonal wurtzite nanocrystals of approximately 4 nm. Hydroxyl radical ( · OH), reactive oxygen species (ROS), and lipid peroxidation assays revealed · OH generated by MoZnO under the visible light seemed to cause peroxidation of the lipid membrane of M. aeruginosa , which led to an upsurge of intracellular ROS and consequently introduced the agglomeration of cyanobacteria. These results demonstrated that nano-sized MoZnO photocatalyst can be easily synthesized in a cost-e ﬀ ective ball-mill method and utilized for biological applications such as the reduction of harmful algal blooms. Further, our study implies that a simple ball-milling method can provide an easy, green, and scalable route for the synthesis of visible light active doped metal oxides.


Introduction
Photocatalysts are vital photoactive materials that could help eliminate the global energy crisis and mitigate environmental pollution [1]. Photocatalysts generate electrons in the conduction band and holes in the valence band upon illumination with higher light energy compared to its bandgap [2]. These photogenerated electrons and holes can oxidize or reduce surrounding pollutants with suitable redox potentials. Some photocatalysts such as zinc oxide (ZnO), zirconium oxide (ZrO 2 ), and strontium-titanium oxide (SrTiO 3 ) have higher photocatalytic efficiency under ultraviolet (UV) light compared to visible light [3][4][5], meaning an external energy source is needed to activate these photocatalysts. Tungsten oxide (WO 3 ) has also a poor activity under visible light [6]. It is meaningful to synthesize visible light active photocatalysts for practical applications.
Zinc oxide (ZnO), tungsten oxide (WO 3 ), and zirconium oxide (ZrO 2 ), strontium-titanium oxide (SrTiO 3 ) nanoparticles (NPs) are significant semiconductor material and of great interest due to   Atomic% in MoZnO was analyzed using EDX. EDX spectrum analysis was performed to determine the composition of elements present in MoZnO. The EDX spectrum of MoZnO showed the presence of Zn, Mo, O, and Cl elements ( Figure 2). The presence of both Mo and Zn elements was also observed in the same crystallite. The linkage of (MoCl 5 ) 2 to ZnO was confirmed by the coexistence of Mo and Cl on the surface of MoZnO. The weight% and atomic% of the elements present in MoZnO were shown in Table 1. The atomic% of Zn, Mo, O, and Cl elements were 28.25, 3.23, 61.66, and 6.87, respectively.

Structural Analysis of MoZnO
Doping of ZnO with Mo strongly affected the peak intensity and showed the coexistence of two phases in the XRD pattern ( Figure 3). The weak diffraction peaks related to (100)

Structural Analysis of MoZnO
Doping of ZnO with Mo strongly affected the peak intensity and showed the coexistence of two phases in the XRD pattern (  (Table 2), respectively. These results coincided with FESEM and EDX data. MoZnO showed a small alteration in the position of the diffraction peaks towards slightly higher angles, similar to the standard ZnO crystal, indicating that Mo substituted for Zn in the hexagonal lattice [32,33].     The calculated values of TC(hkl) for seven main peaks were shown in Table 3. Both ZnO and MoZnO had TC(hkl) values somewhat different from one. This indicated that both ZnO and MoZnO had preferential orientation along all planes. This was supported by the values of c/a shown in Table 1. The obtained c/a values for ZnO and MoZnO were just as reported standard c/a values for the wurtzite structure, which was about 1.63 [34]. Therefore, it was concluded that crystallites were oriented along all planes. A sharp decline in peak intensity of ZnO was observed upon doping with Mo. This could arise from the different ionic radii of Zn and Mo, which led to lattice disorder and strain that occurred in the ZnO crystal after the Zn exchanged with Mo at the Zn lattice sites [35]. Table 2 shows the measured values of the lattice constant. The values of both a and c were slightly larger compared to bulk ZnO values (0.3242 nm and 0.5194 nm, respectively, for a and c, JCPDS-75-0576) [35]. The calculated values of the lattice strain were shown in Table 2. The lattice strain value of ZnO increased from 0.130 to 4.560 upon doping with Mo, which may be related to the nucleation process of ZnO. Higher Mo concentration enhanced the number of nucleations of ZnO, resulting in limited grain size and greater lattice strain [36].

Anti-Algal Assay
A preliminary study revealed that only MoZnO inhibited the growth of M. aeruginosa cells as a function of concentration, while others did not (Supplementary Figure S1). MoZnO showed a linear relationship between the initial rate of reaction and the concentration of photocatalyst, which was one of the characteristics of an ideal photocatalyst (Supplementary Figure S2). Based on the preliminary study, MoZnO was used for further anti-algal study. The MoZnO concentration-dependent growth inhibition result was displayed in Figure 5. We also examined the anti-algal activity of metal salts, metal oxides, and their combinations against M. aeruginosa to rule out their contribution to the anti-algal activity of MoZnO (Supplementary Figure S4). As speculated, they did not show any anti-algal activity. As shown in Figure 5A, the pigment of chlorophyll was faded over the time in a concentration dependent manner, indicating that the algal cells gradually died. To present the anti-algal activity quantitatively, the optical density of M. aeruginosa was measured at 680 nm (OD680) ( Figure 5B). The inhibition rate was calculated with OD680 values ( Figure 5C). The inhibition rate of 1 mg/L MoZnO was increased gradually over the time, whereas the inhibition rate of 2.5, 5, and 10 mg/L MoZnO was saturated relatively faster. This result clearly demonstrated that MoZnO was a visible light active photocalyst. The minimal effective concentration (MEC) was found to be 1.0 mg/L in this study.

Anti-algal Assay
A preliminary study revealed that only MoZnO inhibited the growth of M. aeruginosa cells as a function of concentration, while others did not (Supplementary Figure S1). MoZnO showed a linear relationship between the initial rate of reaction and the concentration of photocatalyst, which was one of the characteristics of an ideal photocatalyst (Supplementary Figure S2). Based on the preliminary study, MoZnO was used for further anti-algal study. The MoZnO concentrationdependent growth inhibition result was displayed in Figure 5. We also examined the anti-algal activity of metal salts, metal oxides, and their combinations against M. aeruginosa to rule out their contribution to the anti-algal activity of MoZnO (Supplementary Figure S4). As speculated, they did not show any anti-algal activity. As shown in Figure 5A, the pigment of chlorophyll was faded over the time in a concentration dependent manner, indicating that the algal cells gradually died. To present the anti-algal activity quantitatively, the optical density of M. aeruginosa was measured at 680 nm (OD680) ( Figure 5B). The inhibition rate was calculated with OD680 values ( Figure 5C). The inhibition rate of 1 mg/L MoZnO was increased gradually over the time, whereas the inhibition rate of 2.5, 5, and 10 mg/L MoZnO was saturated relatively faster. This result clearly demonstrated that  aeruginosa. Statistical significance (determined by paired t-test) is shown by * = p < 0.05, ** = p < 0.001, *** = p < 0.0001, when compared to control (0).

Mechanisms of Algae Growth Inhibition
The extracellular hydroxyl free radical, intracellular reactive oxygen species, lipid peroxidation, and agglomeration effect were examined to speculate a mechanism of anti-algal activity of MoZnO.
Extracellular ·OH generated by MoZnO under the illumination of ~2000 lx in the absence (C, EX)

Mechanisms of Algae Growth Inhibition
The extracellular hydroxyl free radical, intracellular reactive oxygen species, lipid peroxidation, and agglomeration effect were examined to speculate a mechanism of anti-algal activity of MoZnO.
Extracellular ·OH generated by MoZnO under the illumination of~2000 lx in the absence (C, EX) and presence (SC, SEX) of M. aeruginosa was investigated. Isopropanol was added to S and SC groups to scavenge the ·OH generated by MoZnO, which eventually thus decreased the ·OH fluorescence intensity of the control group [42]. As shown in Figure 6, the MoZnO was effective in generating ·OH radical in solution (EX and SEX). The shading of M. aeruginosa seemed to cause the lower production of ·OH radical by MoZnO (Supplementary Figure S8). Upon shading, the fluorescence intensity of SEX group was decreased by 2.7-fold, compared to the EX group. The irradiation of MoZnO with visible light triggered the generation of a negatively charged conduction band (eCB − ) and positively charged valence band (hVB + ) [43]. The hVB + extracted electrons from nearby water molecules, producing hydroxyl radicals and protons. The high oxidation potential of the generated ·OH can damage harmful cyanobacteria. The intracellular ROS content of M. aeruginosa was also increased in a concentration-dependent manner ( Figure 7). Not surprisingly, the ROS content was shown to decrease 2h after MoZnO treatment. This was in accordance with the anti-algal results, in which algal cells started perishing 2h after treatment, reducing the concentration of live cells. The detection of ROS is impossible in dead cells. The plausible steps for the generation of ·OH free radical by MoZnO were given as follows: The irradiation of MoZnO with visible light triggered the generation of a negatively charged conduction band (e CB − ) and positively charged valence band (h vB + ) [43]. The h vB + extracted electrons from nearby water molecules, producing hydroxyl radicals and protons. The high oxidation potential of the generated ·OH can damage harmful cyanobacteria. The intracellular ROS content of M. aeruginosa was also increased in a concentration-dependent manner (Figure 7). Not surprisingly, the ROS content was shown to decrease 2 h after MoZnO treatment. This was in accordance with the anti-algal results, in which algal cells started perishing 2 h after treatment, reducing the concentration of live cells. The detection of ROS is impossible in dead cells. The intracellular ROS content of M. aeruginosa was also increased in a concentration-dependent manner (Figure 7). Not surprisingly, the ROS content was shown to decrease 2h after MoZnO treatment. This was in accordance with the anti-algal results, in which algal cells started perishing 2h after treatment, reducing the concentration of live cells. The detection of ROS is impossible in dead cells. The membrane integrity was evaluated using lipid peroxidation assay. The MDA content in the treated cells was increased 1.8-2-fold more than in the control group, which indicates that the lipid was more exposed to the ·OH in the treated groups (Supplementary Figure S5). The optical microscope and SEM images of M. aeruginosa showed that algal cells appeared agglomerated in the MoZnO treated samples (Supplementary Figures S6 and S7). In the control group, they did not show any agglomeration. The membrane integrity was evaluated using lipid peroxidation assay. The MDA content in the treated cells was increased 1.8-2-fold more than in the control group, which indicates that the lipid was more exposed to the ·OH in the treated groups (Supplementary Figure S5). The optical microscope and SEM images of M. aeruginosa showed that algal cells appeared agglomerated in the MoZnO treated samples (Supplementary Figures S6 and S7). In the control group, they did not show any agglomeration.

Discussion
In this study, we synthesized molybdenum-doped metal oxides (MoZnO, MoZrO, MoWO, and MoSrTiO) using the solvent-free ball-milling method. The decrease in the crystallite size of ZnO was observed upon doping with Mo. Swapna et al. [35] obtained similar results for Mo-doped ZnO thin films synthesized by spray pyrolysis method. This indicated that the degradation of the crystallinity of MoZnO was the result of doping with high Mo concentration. The lower product yield of MoZrO, MoWO, and MoSrTiO in this study may indicate that the solvent-free ball milling was not a suitable method for the synthesis of these photocatalysts. The molar ratio of metal oxides was lower in MoZrO, MoWO, and MoSrTiO, but was higher in MoZnO, which could be the plausible reason for their low yield. The higher molar ratio of ZnO may increase the chances of the interaction of ZnO with (MoCl 5 ) 2 and result in higher yield and doping of Mo. Ideal photocatalysts have a linear relationship between the initial rate of reaction and the concentration of photocatalyst [44]. MoZnO had a linear relationship between the initial rate of reaction and the concentration of photocatalyst (Supplementary Figure S2). After achieving the maximum rate, the rate of reaction may remain constant, or in some cases, it may decrease with an increase in the concentration of photocatalyst. The decrease in rate may be due to the increased scattering of the visible light. The rate of reaction of MoZnO was decreased at higher concentrations, which may be due to the increased scattering of the incident light by MoZnO. Unlike MoZnO, MoZrO, MoWO, and MoSrTiO were not active. The reason of inactivity may be due to the low percentage doping of Mo in MoZrO and MoWO, and electron-hole pair recombination in case of MoSrTiO. Previously, it was reported that the catalytic activity of molybdenum-doped metal oxides was dependent on the concentration of the dopant [45,46]. The low percentage doping of Mo in WO 3 increased the bandgap of the resulting MoWO and decreased its reactivity under visible light [45]. On the contrary, Chary et al. [46] showed that the catalytic activity of MoO 3 /ZrO 2 increased with the increase in Mo dopant. Nosaka et al. [47] found that Mo doping in SrTiO 3 increased the recombination of generated electron and hole under visible light. Taken together, precipitation [48], RF magnetron Catalysts 2020, 10, 805 9 of 17 sputtering [49], and hydrothermal [47,50] methods could be more efficient than solvent-free ball mill method for the synthesis of MoZrO, MoWO, and MoSrTiO.
We demonstrated the application of the visible light active MoZnO for the inhibition of algal growth. Fan et al. [51] used 1 mg/L Cu-MOF-74, and Yu et al. [52] used AgBiO 3 , AgNO 3 , and NaNO 3 to inhibit the growth of M. aeruginosa. They noticed that the growth of algal cells was inhibited after 24 and 96 h, respectively. Interestingly, MoZnO synthesized in this study showed rapid growth inhibition of M. aeruginosa at a minimum concentration of 2.5 mg/L after 4 h. ZrO 2 , WO 3 , and SrTiO 3 did not show growth inhibition of M. aeruginosa (Supplementary Figure S1). The component of MoZnO may contribute to the anti-algal activity. Du et al. [21] noticed that 0.71 mg/L Zn 2+ ion and 1 mg/L ZnO NP promoted the growth of M. aeruginosa. Similar results were obtained after incubating M. aeruginosa with 2.5 mg/L ZnO and ZnO + Na 2 MoO 4 ·2H2O for 8 h. However, in this study, Zn 2+ ion from 2.5 mg/L ZnSO 4 treatment did not have any significant effect on M. aeruginosa growth. We performed the ·OH and ROS assay to confirm the generation of extracellular ·OH and intracellular ROS in M. aeruginosa. MoZnO generated extracellular ·OH, which has high oxidation potential. Ding et al. [25] found that nano-ZnS-montmorillonite caused concentration-dependent production of ·OH radicals. Additionally, we found that M. aeruginosa had a shading effect on ·OH production efficiency of MoZnO, decreasing the actual efficiency of MoZnO. Although the shading by M. aeruginosa cells decreased the efficiency of ·OH production, ·OH-mediated growth inhibition of algal cells was considered an important mechanism of inhibition by MoZnO [43]. The intracellular ROS was increased for up to 2 h and then decreased in the MoZnO samples. The decrease of intracellular ROS was likely to the extensive cell death 2 hr after MoZnO treatment, as shown in the anti-algal assay.

Preparation of MoZnO
Molybdenum-doped zinc oxide (MoZnO) photocatalyst was synthesized in the solvent-free medium in a vacuum planetary ball mill (QM-1F, Nanjing University Instrument Plant, Nanjing, China). 1000 mg of ZnO powder was mixed with agate ball in a 1:10 ratio in the agate ball milling tank, and then a 500 mg of (MoCl 5 ) 2 was added. (MoCl 5 ) 2 is a highly unstable metal chloride which can react efficiently with stable metal oxides to produce the molybdenum-doped metal oxide [31]. The mixture was milled for 2 h with the milling rate of 300 rpm. The resulting powder was rinsed four times with deionized water and three times with absolute ethanol before being kept at 353 K up to 6 h in an oven. In addition, we used zirconium oxide (ZrO 2 ), tungsten oxide (WO 3 ), and strontium titanate (SrTiO 3 ) to synthesize respective molybdenum-doped metal oxides (MoZrO, MoWO, and MoSrTiO) using the same reaction condition. The molar ratios of ZnO, ZrO 2 , WO 3 , and SrTiO 3 to (MoCl 5 ) 2 were 6.8, 4.5, 2.4, and 3.2, respectively.

Morphological and Microstructural Analysis
The shape and size of MoZnO were analyzed using field emission scanning electron microscopy (FESEM). FESEM was performed at an accelerating voltage of 0.5~30 KV (JSM-6700F, JEOL, Tokyo, Japan). The elemental proportion of MoZnO was analyzed using an energy dispersive X-ray spectrometer (EDX, INCAx-sight, Oxford).

XRD Analysis
The crystallinity of MoZnO was analyzed using an X-ray diffractometer (Rigaku Ultima IV, Rigaku, Japan), with Cu Kα radiation (wavelength = 15.4 nm) operated at 40 kV and 40 mA. The crystallite size D of ZnO and MoZnO was calculated using the Scherrer equation [56]: where D is the crystalline size, k is the shape factor (0.9), λ is the wavelength of X-ray (1.54056 Å), β is the full width at half maximum (FWHM) of the diffraction peak (in radians), and θ is the diffraction angle of the reflection. Texture coefficients (TC) were calculated to understand the orientation of doped Mo in MoZnO using the 36-1451 JCPDS-ICDD card for all the peaks except (200) peak [57]. The equation of texture coefficient is as follows [57]: where I(hkl) is the measured relative intensity of (hkl) plane, I 0 (hkl) is the theoretical intensity of the same plane taken from the 36-1451 JCPDS-ICDD card, and N is the number of reflections. The random orientation of doped material has TC(hkl) value equal to 1, whereas a value other than 1 indicates the preferred orientation. The lattice constants a and c were measured by applying the following equation [35]: The lattice strain (ε) was measured using the tangent formula [36]:

Algae Culture
A strain of M. aeruginosa (No. FBCC000002) was received from the Nakdonggang National Institute of Biological Resources (Sangju, Korea). The MA was cultured in a 2-L conical flask having BG-11 medium (BGM) (pH 7.0) at 298 ± 1 K [58], exposed to~2000 lx of incandescent lamp with light-dark cycle of 12:12 h. The conical flask was shaken every hour to prevent deposition of the cells.

Anti-Algal Activity
A preliminary study was performed to determine the anti-algal activity of synthesized molybdenum-doped metal oxides (MoZnO, MoZrO, MoWO, and MoSrTiO). Different concentrations of molybdenum-doped metal oxides were used for growth inhibition of M. aeruginosa. Based on the result, MoZnO was used for further study (Supplementary Figure S1). The stock of MoZnO (1000 mg/L) was prepared in a new BG-11 medium. The resulting solution was ultrasonicated for 15 min to homogenize the MoZnO. M. aeruginosa cultures were washed three times and resuspended in fresh culture medium to obtain an eventual concentration of 6.48 × 10 3 cell/mL (OD680 ≈ 0.43), which was nearly equal to the number of toxic concentration of MA blooms occurred in the freshwater bodies [59]. The MoZnO stock solution was mixed with the M. aeruginosa culture to achieve the eventual concentration of 0, 1, 2.5, 5, and 10 mg/L. The resulting solution was incubated according to the conditions given in Section 4.3.1. Next, samples were drawn after successive 1-h time-intervals (1-8 h), and the number of algal cells per milliliter was calculated from OD680 using a UV-Vis spectrophotometer (Bio Drop). The growth inhibition rate of algae was calculated using an inhibitory rate equation [51].
Algae inhibition rate was calculated as follows: where µ a-b is the specific growth rate from t a (h) to t b (h), X a is the OD680 value at t a (h), X b is the OD680 value at t b (h), I r (%) is the inhibition rate based on the specific growth rate, µ 0 is the average of the specific growth rate of the control group, µ e is the specific growth rate of the experimental group, and t a and t b are the initial and final time measured at 1-h intervals.

Effect of Metal Salts, Metal Oxides and Their Combinations
The

Hydroxyl Radical (·OH) Assay
Terephthalic acid (TA) is a nonfluorescent compound, which upon oxidation by hydroxyl radical converted into fluorescent compound 2-hydroxyterephthalic acid. Therefore, the fluorescence intensity is directly proportional to the concentration of ·OH. TA (0.5 mM) was dissolved with excess NaOH (2 g/L) in a 100 mL BGM. Then, the BGM was neutralized by adding HCl, and the BGM was equally distributed into two conical flasks (Supplementary Table S1). One group was labelled as control (C) and the other as experimental group (EX). The final concentration of 10 mg/L MoZnO was mixed with both C and Ex groups, respectively. The C and Ex groups were exposed to~2000 lx of incandescent lamps for 4 h. 1 mM isopropyl alcohol was added to the C group as a free radical scavenger. The solutions were centrifuged at 4500 rpm for 10 min. Finally, the fluorescence spectrum of supernatant was analyzed at excitation and emission wavelengths of 350 and 500 nm. The obtained fluorescence intensity was used to compare the ·OH free radicals formed in C and Ex groups. The same experiment was performed under the M. aeruginosa to study the effect of the shading of algal cells on the efficiency of MoZnO (Supplementary Figure S8). The control (SC) and experimental (SEX) groups were illuminated with 2000 lx of incandescent lamps up to 4 h. The rest of the procedure was the same.

ROS Assay
The intracellular ROS produced in M. aeruginosa incubated with MoZnO was evaluated employing 2 ,7 -Dichlorofluorescein diacetate (DCFH-DA, Sigma-Aldrich, St. Louis, MO, USA), which is a nonfluorescent dye. In the cell, esterase catalyzed DCFH-DA to another nonfluorescent 2 ,7 -dichlorodihydrofluorescein (DCFH) dye. DCFH further oxidized by ·OH to the extremely fluorescent 2 ,7 -dichlorofluorescein (DCF). In detail, 5 mL of M. aeruginosa culture was drawn in 15 mL falcon tube from all flasks, at successive time-interval of 1 h. The DCFH-DA solution was mixed with the M. aeruginosa culture to make an eventual concentration of 5 µM. Next, wrapped all the tubes with aluminum foil and incubated at 30 • C up to 1 h. Finally, the fluorescence spectrum of M. aeruginosa culture was analyzed at excitation and emission wavelengths of 488 and 525 nm, respectively, by making use of a fluorescence spectrometer (F-7000, HITACHI, Japan). The obtained fluorescence intensity was used to calculate the relative ROS content.

Lipid Peroxidation Assay
Lipid peroxidation assay was performed according to Metzler's malondialdehyde (MDA) method [60]. Sample and 10% trichloroacetic acid (TCA) were mixed in the volume ratio of 1:2. The resulting mixture was centrifuged at 11,000 g for 45 min. The supernatant was collected and mixed with 3 mL of 6.7 g/L 2-thiobarbituric acid (TBA). Next, the resulting solution was boiled on the water bath up to 10 min. After the temperature of the solution came to room temperature, the absorbance was measured at the wavelengths of 532 and 600 nm. The value obtained at 600 nm was subtracted from the value at 532 nm. Lipid peroxidation was expressed in terms of mg MDA/kg sample, using the molar extinction coefficient for MDA of 1.56 × 10 5 M −1 cm −1 [61].

Effect of Agglomeration
Optical Microscope Analysis A standard experiment was performed to examine the consequence of agglomeration on the multiplication of M. aeruginosa. The M. aeruginosa culture was incubated with different MoZnO concentrations (0, 1, 2.5, 5, and 10 mg/L) up to 4 h. The incubated M. aeruginosa was analyzed using an optical microscope to detect the aggregated algal cells.

SEM Analysis
SEM analysis was performed to investigate the aggregation of M. aeruginosa incubated with 0, 5, and 10 mg/L of MoZnO up to 4 h. M. aeruginosa cultures were washed 3-times with double-distilled water and pelleted by centrifugation at 3000 rpm for 10 min. Obtained pellets were fixed with 4% formaldehyde for 1 h at room temperature. Fixed pellets were serially dehydrated with 30%-100% ethanol. Subsequently, 30 µL of each sample was put on a glass slide and dried overnight in the open air at room temperature. The SEM analysis of the prepared samples was performed using a (JSM-6490LV, JEOL, Tokyo, Japan) at an accelerating voltage of 0.2-30 KV.

Statistical Analysis
The experiments were carried out in triplicate, and the data are presented as mean and standard deviation (SD). Significant differences between means were identified using the paired t-test. Statistical significance was evaluated using significance levels at 0.05.

Conclusions
MoZnO nanoparticles were successfully synthesized by a facile surfactant-free ball-milling method. This method seemed not to be suitable for the synthesis of