An Improved Method for the Synthesis of Butein Using SOCl2/EtOH as Catalyst and Deciphering Its Inhibition Mechanism on Xanthine Oxidase

Butein (3,4,2′,4′-tetrahydroxychalcone) belongs to the chalcone family of flavonoids and possesses various biological activities. In this study, butein was synthesized through aldol condensation catalyzed by thionyl chloride (SOCl2)/ethyl alcohol (EtOH) for the first time. The optimal reaction conditions including the molar ratio of reactants, the dosage of catalyst, and the reaction time on the yield of product were investigated, and the straightforward strategy assembles the yield of butein up to 88%. Butein has been found to inhibit xanthine oxidase (XO) activity. Herein, the inhibitory mechanism of butein against XO was discussed in aspects of inhibition kinetic, fluorescence titration, synchronous fluorescence spectroscopy, and molecular docking. The inhibition kinetic analysis showed that butein possessed a stronger inhibition on XO in an irreversible competitive manner with IC50 value of 2.93 × 10−6 mol L−1. The results of fluorescence titrations and synchronous fluorescence spectroscopy indicated that butein was able to interact with XO at one binding site, and the fluorophores of XO were placed in a more hydrophobic environment with the addition of butein. Subsequently, the result of molecular docking between butein and XO protein revealed that butein formed hydrogen bonding with the amino acid residues located in the hydrophobic cavity of XO. All the results suggested that the inhibitory mechanism of butein on XO may be the insertion of butein into the active site occupying the catalytic center of XO to avoid the entrance of xanthine and inducing conformational changes in XO.


Introduction
Hyperuricemia characterized by an elevated level of uric acid in human blood is a critical cause of gout [1][2][3]. It can result in the deposition of monosodium urate crystals in joint and soft tissue, with accompanying inflammation and degenerative consequences [4,5]. Currently, the increasing clinical reports have demonstrated that hyperuricemia is associated with some other diseases such as cardiovascular disorders, renal dysfunction, obesity, diabetes, hyperlipidemia, cancer, hypertension, and metabolic syndrome [6]. Thus, maintaining uric acid at a normal level becomes an important therapy for prevention of gout and related disorders. Xanthine oxidase (XO), with a function of catalyzing hypoxanthine and xanthine to uric acid, is a pivotal enzyme in purine metabolism. Structurally, XO is a homodimer with each monomer consisted of the molybdopterin (Mo-pt), the flavin adenine dinucleotide

Synthesis of Butein
As outlined in Scheme 1, butein (c) was prepared via aldol condensation catalyzed by SOCl 2 in ethyl alcohol between 2,4-dihydroxyacetophenone (a) and 3,4-dihydroxybenzaldehyde (b). Although the SOCl 2 /EtOH employed to synthesis chalcones has already been described [20,21], butein was synthesized in this study using SOCl 2 /EtOH as a catalyst in one step for the first time and the protection of the hydroxyl groups were avoided. The reaction mixture was extracted by ethyl acetate to obtain the crude product. Pure butein was purified by reversed-phase preparative HPLC and structural elucidation was identified by MS and NMR spectra. In order to further improve the synthetic reaction efficiency of butein, we studied the influence of various parameters of the reaction such as the molar ratio of reactants, the dosage of catalyst, and reaction time. The overall yield of butein was then measured by HPLC. Firstly, the concentration of SOCl 2 in ethyl alcohol was critical in aldol condensation. The reactants a and b (1:1, molar ratio) with different concentrations of SOCl 2 (0.25, 0.5, 1, 1.5, 2.0 mol L −1 ) were evaluated for the synthetic reaction capacity. As depicted in Table 1, the yields increased quickly among the concentrations of SOCl 2 from 0.25 mol L −1 to 1 mol L −1 (entry 1-3), and declined suddenly when the concentrations of SOCl 2 exceeded 1 mol L −1 (entry [4][5]. The yields of high catalyst concentration (2.0 mol L −1 ) and low catalyst concentration (0.25 mol L −1 ) were only 9% and 2%, respectively. The highest yield of butein (85%) was afforded when the concentration of catalyst was 1 mol L −1 , and thus was chosen for further study. Secondly, the molar ratio of two reactants was Molecules 2019, 24,1948 3 of 13 also investigated. As can be seen in Table 1, the molar ratio of a and b increasing from 1:0.6 to 1:1 would lead to the increased of yields (entry 6-7). However, the yields decreased with the molar ratio increasing from 1:1 to 1:1.4 (entry [8][9]. Therefore, the molar ratio of two reactants of 1:1 (entry 3) was recommended in our case. Besides, the yield of butein was not significantly increased at the longer reaction time. To our delight, the yields could reach 88% within 1 h (entry [10][11][12], which extremely saved the preparation time. On the basis of above results, a sets of parameters to synthesize butein was proposed: Reaction was carried out at SOCl 2 1 mol L −1 , under room temperature for 1 h, and the molar ratio of a and b is 1:1 (entry 11).
Molecules 2019, 24, x FOR PEER REVIEW 3 of 13 further study. Secondly, the molar ratio of two reactants was also investigated. As can be seen in Table 1, the molar ratio of a and b increasing from 1:0.6 to 1:1 would lead to the increased of yields (entry 6-7). However, the yields decreased with the molar ratio increasing from 1:1 to 1:1.4 (entry [8][9]. Therefore, the molar ratio of two reactants of 1:1 (entry 3) was recommended in our case. Besides, the yield of butein was not significantly increased at the longer reaction time. To our delight, the yields could reach 88% within 1 h (entry 10-12), which extremely saved the preparation time. On the basis of above results, a sets of parameters to synthesize butein was proposed: Reaction was carried out at SOCl2 1 mol L −1 , under room temperature for 1 h, and the molar ratio of a and b is 1:1 (entry 11).

Entry SOCl2 (mol L −1 ) Molar Ratio (a/b) Time (h) Yield (%)
The inhibitory effect of butein on XO activity was evaluated by the formation of uric acid using xanthine as the substrate. As shown in Figure 1, the activity of XO was effectively inhibited by butein; the inhibition rate increased significantly with increasing concentration of the inhibitor. The IC50 value of butein was determined to be 2.93 × 10 −6 mol L −1 , which was slightly higher than that of allopurinol (1.10 × 10 −6 mol L −1 ). This IC50 value of butein was in keeping with the result of previous report on XO inhibitory activity [16]. The results indicated that butein was an effective XO inhibitor compared to positive control allopurinol, as a result of the presence of carbonyl group and four hydroxyl groups on two phenyl rings [22]. In the previous research, chalcones 3,4,2',5'-tetrahydroxychalcone and 3,4,2',6'-tetrahydroxychalcone with a similar structure as butein have shown weaker inhibition on XO with IC50 values of (17 ± 8) × 10 −6 mol L −1 and (35 ± 10) × 10 −6 mol L −1 , respectively [16]. The presence of hydroxyl groups at C (2′) and C (4′) on ring B showed stronger activity than those with the equivalent substitutes but located at para-position on ring B at C (2′) and C (5′) or meta-position at C (2′) and C (6′) may be contributes to an increment in the stabilization of the aromatic ring due to induce effect [22,23]. Similarly, compound 2,4,2',5'-tetrahydroxychalcone with two hydroxyl groups located at the meta-position on ring A at C (2) and C (4) showed weaker inhibition activity (16.3 × 10 −6 mol L −1 ) than butein [22]. That may be because the higher polarizability enhances the attractive dispersion interactions with an aromatic Scheme 1. Synthesis of butein.

Inhibition of Butein on XO Activity
The inhibitory effect of butein on XO activity was evaluated by the formation of uric acid using xanthine as the substrate. As shown in Figure 1, the activity of XO was effectively inhibited by butein; the inhibition rate increased significantly with increasing concentration of the inhibitor. The IC 50 value of butein was determined to be 2.93 × 10 −6 mol L −1 , which was slightly higher than that of allopurinol (1.10 × 10 −6 mol L −1 ). This IC 50 value of butein was in keeping with the result of previous report on XO inhibitory activity [16]. The results indicated that butein was an effective XO inhibitor compared to positive control allopurinol, as a result of the presence of carbonyl group and four hydroxyl groups on two phenyl rings [22]. In the previous research, chalcones 3,4,2 ,5 -tetrahydroxychalcone and 3,4,2 ,6 -tetrahydroxychalcone with a similar structure as butein have shown weaker inhibition on XO with IC 50 values of (17 ± 8) × 10 −6 mol L −1 and (35 ± 10) × 10 −6 mol L −1 , respectively [16]. The presence of hydroxyl groups at C (2 ) and C (4 ) on ring B showed stronger activity than those with the equivalent substitutes but located at paraposition on ring B at C (2 ) and C (5 ) or metaposition at C (2 ) and C (6 ) may be contributes to an increment in the stabilization of the aromatic ring due to induce effect [22,23]. Similarly, compound 2,4,2 ,5 -tetrahydroxychalcone with two hydroxyl groups located at the metaposition on ring A at C (2) and C (4) showed weaker inhibition activity (16.3 × 10 −6 mol L −1 ) than butein [22]. That may be because the higher polarizability enhances the attractive dispersion interactions with an aromatic residue of enzyme binding site through π-π stacking interactions [24,25]. Thus, the potential inhibitory mechanism of butein on XO is worthy of further investigation. residue of enzyme binding site through π-π stacking interactions [24,25]. Thus, the potential inhibitory mechanism of butein on XO is worthy of further investigation.

Irreversibility
The inhibition of XO by diverse concentrations of butein was illustrated in Figure 2. The plots of the velocity versus at various concentrations of XO were constructed to confirm the irreversibility of butein mediated inhibition. The plots offered a series of parallel straight lines and the slopes of the lines were unchanged with the increasing concentrations of butein, which indicated that butein was an irreversible inhibitor on XO. To the best of our knowledge, most of flavonoids were reported as a reversible inhibitor on XO [26][27][28]. The different inhibition type of butein on XO prompts us to further understand the potential mechanism between butein and XO.

Inhibition Kinetic Analysis
Enzyme inhibition kinetic experiments were carried out to further characterize the binding type of butein against XO by analyzing the data from Lineweaver-Burk plots. As shown in Figure  3a, the vertical axis intercept (1/Vmax) of the double-reciprocal Lineweaver-Burk plots remained the same and horizontal axis intercept (−1/Km) increased with accumulating concentrations of butein, suggesting that butein is a competitive inhibitor. Figure 3b exhibits good linearity between Km app and the relative concentration of butein, signifying that butein bound in a single class of inhibition sites on XO. The inhibition kinetic analysis can be inferred that butein bound to the same active site of XO as xanthine and competed with xanthine during catalysis, just as the allopurinol under the uniform experimental conditions [29]. The value of Ki (inhibition constant) was determined to be 0.46 × 10 −7 mol L −1 .

Irreversibility
The inhibition of XO by diverse concentrations of butein was illustrated in Figure 2. The plots of the velocity versus at various concentrations of XO were constructed to confirm the irreversibility of butein mediated inhibition. The plots offered a series of parallel straight lines and the slopes of the lines were unchanged with the increasing concentrations of butein, which indicated that butein was an irreversible inhibitor on XO. To the best of our knowledge, most of flavonoids were reported as a reversible inhibitor on XO [26][27][28]. The different inhibition type of butein on XO prompts us to further understand the potential mechanism between butein and XO. residue of enzyme binding site through π-π stacking interactions [24,25]. Thus, the potential inhibitory mechanism of butein on XO is worthy of further investigation.

Irreversibility
The inhibition of XO by diverse concentrations of butein was illustrated in Figure 2. The plots of the velocity versus at various concentrations of XO were constructed to confirm the irreversibility of butein mediated inhibition. The plots offered a series of parallel straight lines and the slopes of the lines were unchanged with the increasing concentrations of butein, which indicated that butein was an irreversible inhibitor on XO. To the best of our knowledge, most of flavonoids were reported as a reversible inhibitor on XO [26][27][28]. The different inhibition type of butein on XO prompts us to further understand the potential mechanism between butein and XO.

Inhibition Kinetic Analysis
Enzyme inhibition kinetic experiments were carried out to further characterize the binding type of butein against XO by analyzing the data from Lineweaver-Burk plots. As shown in Figure  3a, the vertical axis intercept (1/Vmax) of the double-reciprocal Lineweaver-Burk plots remained the same and horizontal axis intercept (−1/Km) increased with accumulating concentrations of butein, suggesting that butein is a competitive inhibitor. Figure 3b exhibits good linearity between Km app and the relative concentration of butein, signifying that butein bound in a single class of inhibition sites on XO. The inhibition kinetic analysis can be inferred that butein bound to the same active site of XO as xanthine and competed with xanthine during catalysis, just as the allopurinol under the uniform experimental conditions [29]. The value of Ki (inhibition constant) was determined to be 0.46 × 10 −7 mol L −1 .

Inhibition Kinetic Analysis
Enzyme inhibition kinetic experiments were carried out to further characterize the binding type of butein against XO by analyzing the data from Lineweaver-Burk plots. As shown in Figure 3a, the vertical axis intercept (1/V max ) of the double-reciprocal Lineweaver-Burk plots remained the same and horizontal axis intercept (−1/K m ) increased with accumulating concentrations of butein, suggesting that butein is a competitive inhibitor. Figure 3b exhibits good linearity between K m app and the relative concentration of butein, signifying that butein bound in a single class of inhibition sites on XO. The inhibition kinetic analysis can be inferred that butein bound to the same active site of XO as xanthine and competed with xanthine during catalysis, just as the allopurinol under the uniform experimental conditions [29]. The value of K i (inhibition constant) was determined to be 0.46 × 10 −7 mol L −1 .

Fluorescence Quenching of XO upon Butein
Fluorescence spectroscopy analysis was further used to research the binding properties of butein with XO on account of its ability to provide information on the molecular level of the binding mode, binding constant, and intermolecular distance. As shown in Figure 4a, under 280 nm excitation wavelength, the fluorescence emission spectra of XO inhibited by different concentrations of butein were excited. XO displayed two strong fluorescence emissions at 340 nm and 405 nm wavelengths due to the presence of three intrinsic fluorophores on XO, including tryptophan, tyrosine, and phenylalanine [30]. Nevertheless, butein has no intrinsic fluorescence under the same experiment conditions. Besides, fluorescence intensity of XO is mainly attributed to tryptophan residues due to the low quantum yield of phenylalanine. The results showed that XO emission intensities decreased dramatically with increasing of butein concentrations, which indicated that butein directly interacted with XO and quenched its intrinsic fluorescence.
Generally, the ground state complex caused the static quenching, while the collision between the fluorophore and the quencher lead to the dynamic quenching. Their excited-state lifetime was used to distinguish two modes of mechanism [31]. The Stern-Volmer equation was applied to calculate the KSV and Kq values with maximal emission at 340 nm in order to ascertain the mechanism of butein binding to XO, as follows [30]: (1)

Fluorescence Quenching of XO upon Butein
Fluorescence spectroscopy analysis was further used to research the binding properties of butein with XO on account of its ability to provide information on the molecular level of the binding mode, binding constant, and intermolecular distance. As shown in Figure 4a, under 280 nm excitation wavelength, the fluorescence emission spectra of XO inhibited by different concentrations of butein were excited. XO displayed two strong fluorescence emissions at 340 nm and 405 nm wavelengths due to the presence of three intrinsic fluorophores on XO, including tryptophan, tyrosine, and phenylalanine [30]. Nevertheless, butein has no intrinsic fluorescence under the same experiment conditions. Besides, fluorescence intensity of XO is mainly attributed to tryptophan residues due to the low quantum yield of phenylalanine. The results showed that XO emission intensities decreased dramatically with increasing of butein concentrations, which indicated that butein directly interacted with XO and quenched its intrinsic fluorescence.  As shown in Figure 4b, the plots of F0/F versus [Q] at three temperatures (298, 304 and 310 K), and the relevant Kq values were given in Table 2. A high linearity suggested that only one type of quenching process occurred, either static or dynamic quenching. When the fluorophore is a biomacromolecule, its maximum scatter collision quenching constant is 2.0 × 10 10 L mol −1 s −1 [33].
The Kq values (at 298, 304 and 310 K, respectively) were 2.09 × 10 12 , 2.55 × 10 12 , 3.10 × 10 12 L mol −1 s −1 , which were considerably greater than the maximum scatter collision quenching constant. The results implied that the fluorescence quenching mechanism of XO by butein is static quenching, and resulted from the formation of a butein-XO complex [34].
Afterwards, the fluorescence titration data were further analyzed to acquire the association constant (Ka) applying the modified Stern-Volmer equation [35]: Generally, the ground state complex caused the static quenching, while the collision between the fluorophore and the quencher lead to the dynamic quenching. Their excited-state lifetime was used to distinguish two modes of mechanism [31]. The Stern-Volmer equation was applied to calculate the Molecules 2019, 24, 1948 6 of 13 K SV and K q values with maximal emission at 340 nm in order to ascertain the mechanism of butein binding to XO, as follows [30]: F 0 and F are steady-state fluorescence intensities of fluorophore in the absence and presence of butein, respectively. K SV and [Q] are Stern-Volmer dynamic quenching constant and quencher concentration, respectively. K q is the quenching rate constant of biomolecule (K q = K SV /τ 0 ). τ 0 is the average biomolecule lifetime, and its value is 10 −8 s [32]. K SV can be obtained from linear regression plot slope of F 0 /F versus [Q].
As shown in Figure 4b, the plots of F 0 /F versus [Q] at three temperatures (298, 304 and 310 K), and the relevant K q values were given in Table 2. A high linearity suggested that only one type of quenching process occurred, either static or dynamic quenching. When the fluorophore is a biomacromolecule, its maximum scatter collision quenching constant is 2.0 × 10 10 L mol −1 s −1 [33]. The K q values (at 298, 304 and 310 K, respectively) were 2.09 × 10 12 , 2.55 × 10 12 , 3.10 × 10 12 L mol −1 s −1 , which were considerably greater than the maximum scatter collision quenching constant. The results implied that the fluorescence quenching mechanism of XO by butein is static quenching, and resulted from the formation of a butein-XO complex [34].
Afterwards, the fluorescence titration data were further analyzed to acquire the association constant (K a ) applying the modified Stern-Volmer equation [35]: where f a is the fraction of accessible fluorescence. The linear regression of F 0 /(F 0 − F) versus 1/[Q] gave the K a values for the butein-XO complex at 298, 301, and 310 K ( Table 2). The K a values were in order of 10 4 L mol −1 and the high linear correlation coefficient R indicated a moderate affinity for the butein-XO interaction [26]. For the static quenching reaction, it was assumed that small molecules bind independently into a class of equivalent sites on a macromolecule. The apparent binding constant K b and the number of binding sites n can be calculated from the following equation [36]: where [Q] is the total concentrations of butein. As shown in Table 2, the values of n were found to be nearly equal to 1, implying the existence of one binding site for butein on XO, which was consistent with the results of inhibition-mode studies.

Thermodynamic Analysis
Generally, hydrogen bonds, van der Waals forces, electrostatic forces and hydrophobic interactions are regarded as the main forces acting small ligands bind to biomolecules. The acting force of the butein-XO complexation could be illustrated by calculating the thermodynamic parameters. The values of ∆H and ∆S were determined from the van't Hoff equation: where K a and R represent the binding constant and the gas constant (8.314 J mol −1 K −1 ). The temperatures were 298, 304 and 310 K. The values of ∆H and ∆S were determined by plotting logK a versus 1/T, and then the free energy change (∆G) was obtained from Equation (5). As shown in Table 2, the negative signal for ∆G meant that the interaction of butein with XO was spontaneous.
The positive values of ∆H (103.45 kJ mol −1 ) and ∆S (427.94 J mol −1 K −1 ) suggested that the binding was an exothermic process, and the complexation was predominately driven by hydrophobic forces [37]. Table 2. Quenching rate constant K q , modified Stern-Volmer association constant K a , and relative thermodynamic parameters for the interaction between butein and XO at different temperatures.

Synchronous Fluorescence
The synchronous fluorescence spectroscopy has been performed to explore the molecular environment around the chromophore groups (tyrosine and tryptophan) of XO [38]. Both of the synchronous fluorescence intensities of tyrosine (∆λ = 15 nm) and tryptophan (∆λ = 60 nm) residues declined obviously with the addition of butein to XO solution. As shown in Figure 5, tyrosine had a visible blue-shift (from 290 nm to 286 nm) and tryptophan had a slight blue-shift (from 276 nm to 275 nm), suggesting that the polarity decreased and the hydrophobicity increased in the vicinity of tyrosine and tryptophan, specifically, that the fluorophores of XO were exposed in a less hydrophilic environment, and more exposed to the solutions with the addition of butein, which was consistent with the result of thermodynamic analysis. where Ka and R represent the binding constant and the gas constant (8.314 J mol −1 K −1 ). The temperatures were 298, 304 and 310 K. The values of ∆H and ∆S were determined by plotting logKa versus 1/T, and then the free energy change (∆G) was obtained from eq (5). As shown in Table 2, the negative signal for ∆G meant that the interaction of butein with XO was spontaneous. The positive values of ∆H (103.45 kJ mol −1 ) and ∆S (427.94 J mol −1 K −1 ) suggested that the binding was an exothermic process, and the complexation was predominately driven by hydrophobic forces [37].

Synchronous Fluorescence
The synchronous fluorescence spectroscopy has been performed to explore the molecular environment around the chromophore groups (tyrosine and tryptophan) of XO [38]. Both of the synchronous fluorescence intensities of tyrosine (∆λ = 15 nm) and tryptophan (∆λ = 60 nm) residues declined obviously with the addition of butein to XO solution. As shown in Figure 5, tyrosine had a visible blue-shift (from 290 nm to 286 nm) and tryptophan had a slight blue-shift (from 276 nm to 275 nm), suggesting that the polarity decreased and the hydrophobicity increased in the vicinity of tyrosine and tryptophan, specifically, that the fluorophores of XO were exposed in a less hydrophilic environment, and more exposed to the solutions with the addition of butein, which was consistent with the result of thermodynamic analysis.

Computational Docking of the Butein-XO Complex
Molecular docking was carried out to improve the understanding of interactions between butein and XO protein. The crystal structure of XO enzyme (PDB ID, 3EUB) in complex with xanthine (Xan) from bovine milk was used for the docking calculations [39]. For validation of the credible docking method, the natural ligand Xan was docked back into the 3EUB protein as a control until the root mean square deviation (RMSD) of best docked ligand conformation was less than 2.0 Å . As shown in Figure 6A, a total of six conformational clusters were obtained from 100 docking runs at an RMSD tolerance of 2.0 Å , and the lowest binding energy with −7.77 kcal mol −1 ,were the most optimal among all the clusters (38 out of 100, red histogram in Figure 6A). In particular, as shown in Figure 6B,C, ligand Xan and potent inhibitor butein were located at the

Computational Docking of the Butein-XO Complex
Molecular docking was carried out to improve the understanding of interactions between butein and XO protein. The crystal structure of XO enzyme (PDB ID, 3EUB) in complex with xanthine (Xan) from bovine milk was used for the docking calculations [39]. For validation of the credible docking method, the natural ligand Xan was docked back into the 3EUB protein as a control until the root mean square deviation (RMSD) of best docked ligand conformation was less than 2.0 Å. As shown in Figure 6A, a total of six conformational clusters were obtained from 100 docking runs at an RMSD tolerance of 2.0 Å, and the lowest binding energy with −7.77 kcal mol −1 ,were the most optimal among all the clusters (38 out of 100, red histogram in Figure 6A). In particular, as shown in Figure 6B,C, ligand Xan and potent inhibitor butein were located at the same hydrophobic pocket of XO, which is the active site with molybdenum atomic (Mo) domain, indicating that butein is a competitive xanthine oxidase inhibitor against xanthine with occupying the catalytic center of XO to avoid the entrance of xanthine and inducing conformational changes in XO. The docking results for butein suggest that three hydrogen bonds were formed between the OH group of butein and the oxygen or hydrogen atom of key amino acids ( Figure 6C,D). The carboxy group of Glu802 was found to form H-bonds with the hydrogen atom of 3-OH and 2 -OH in butein respectively. The carbonyl group in butein was connected with the hydrogen atom of β-OH in Ser876. In addition, key hydrophobic contacts of residues Ala1079, Phe914, Phe1009, Ala1078, Arg880, Val1011, Leu1014, Leu873 and Leu648 were also found in the hydrophobic region of XO. All of these suggest that hydrogen bonds and hydrophobic interaction play an important role in the binding between butein and XO.
Molecules 2019, 24, x FOR PEER REVIEW 8 of 13 same hydrophobic pocket of XO, which is the active site with molybdenum atomic (Mo) domain, indicating that butein is a competitive xanthine oxidase inhibitor against xanthine with occupying the catalytic center of XO to avoid the entrance of xanthine and inducing conformational changes in XO. The docking results for butein suggest that three hydrogen bonds were formed between the OH group of butein and the oxygen or hydrogen atom of key amino acids (Figure 6 C,D). The carboxy group of Glu802 was found to form H-bonds with the hydrogen atom of 3-OH and 2′-OH in butein respectively. The carbonyl group in butein was connected with the hydrogen atom of β-OH in Ser876. In addition, key hydrophobic contacts of residues Ala1079, Phe914, Phe1009, Ala1078, Arg880, Val1011, Leu1014, Leu873 and Leu648 were also found in the hydrophobic region of XO. All of these suggest that hydrogen bonds and hydrophobic interaction play an important role in the binding between butein and XO.

Materials
Mass spectra were obtained with a Bruk micro-TOFQ mass spectrometer. NMR spectra were acquired on a Bruker AV-500 FT-NMR spectrometer (Bruker Daltonics, Bremen, Germany) operating at 500.1 MHz for 1 H and at 125.8 MHz for 13

Materials
Mass spectra were obtained with a Bruk micro-TOFQ mass spectrometer. NMR spectra were acquired on a Bruker AV-500 FT-NMR spectrometer (Bruker Daltonics, Bremen, Germany) operating at 500.

General Procedure for the Synthesis of Butein
SOCl 2 (0.4 mL) was added dropwise to a stirred solution of the 2,4-dihydroxyacetophenone (1 mmol) and 3,4-dihydroxybenzaldehyde (1 mmol) in ethanol (5.1 mL). The reaction mixture was stirred at room temperature for 2 h. At the end of reaction, the mixture was extracted with ethyl acetate by the addition of water. The ethyl acetate layer was washed with water and saturated brine, dried over anhydrous Na 2 SO 4 . After concentration under reduced pressure, the crude product was further purified by reversed-phase preparative HPLC (Agilent Prepstar SD-1 pump connected to a Prostar UV-Vis detector, column Megress ODS-C 18 , 20 × 250 nm, i.d., 10 µm) to give butein. The elution was MeOH-H 2 O (55:45, v/v) at a flow rate 3.0 mL/min, and the sample injection volume was 200 µL. The UV detector was set at 254 and 365 nm.

Enzyme Activity Assay
The XO inhibitory activity assay was performed according to the method modified by our group [29]. The butein was dissolved in dimethyl sulfoxide (DMSO) and subsequently diluted with phosphate buffer (0.07 mol L −1 , pH = 7.5) to a final concentration containing less than 1% DMSO (v/v). In the 0.8 mL reaction system, a series of assay solutions prepared in phosphate buffer (0.07 mol L −1 , pH = 7.5) consisting of a fixed concentration of XO (0.024 U mL −1 ) and various concentrations of inhibitor solutions were incubated at 25 • C for 30 min, and the reaction was initiated by adding the substrate xanthine (300 × 10 −6 mol L −1 ) to the complex solutions. The assay mixture was incubated at 25 • C for 30 min. The reaction was stopped by the addition of HCl (1 mol L −1 ). The absorbance of uric acid level was determined by an HPLC method using a Diamonsil ODS-C 18 column (250 × 4.6 mm i.d., 5 µm, Dikma Technologies, Beijing, China) on an Agilent 1260 system equipped with a G1311C quaternary pump, a G1329B autosampler, a G1316A thermostatted column compartment, and a G1314F variable wavelength detector coupled with an analytical workstation (Agilent Technologies, Inc., Santa Clara, CA, USA). Averages of three replicates are presented and the allopurinol was used as a positive control.

Kinetic Analysis for Inhibitory
The assay was investigated applying the XO activity assay methodology in the absence and presence of butein with a series of concentrations of substrate xanthine (25, 37.5, 50, 75, and 100 × 10 −6 mol L −1 ), where butein was at diverse concentrations (0, 0.2, 0.4, 0.8 × 10 −6 mol L −1 ) and xanthine at a fixed concentration. The involved inhibition mode of the butein on xanthine oxidase was analyzed from Lineweaver-Burk plots, and K i value was identified by the software GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). For competitive inhibition, the Lineweaver-Burk equation can be written in double reciprocal form as follows: A secondary plot can be plotted from: where K i and K m represent the inhibition constant and Michaelis-Menten constant, respectively, and their values can be calculated from Equations (6) and (7). linearly fitted, indicating a single inhibition site or a single class of inhibition site [40].

Fluorescence Titration
Fluorescence titration assay was conducted by a spectrofluorimeter (Model F-4600, Hitachi, Japan) equipped with a thermostat bath and a 150 W xenon lamp to characterize the interaction between butein and XO. Briefly, 2.0 mL sample of XO solution (0.1 U mL −1 ) was titrated with different concentrations of butein from 0 to 36.36 × 10 −6 mol L −1 and then the solutions were incubated 5 min to equilibrate. Afterwards, fluorescence emission spectra of the solutions were measured using a spectrofluorometer at different temperatures (298, 304 and 310 K) in 300-500 nm wavelength range upon excitation 280 nm. The widths of both the excitation and emission slits were set at 5 nm. The background fluorescence of the buffer (0.07 mol L −1 PBS solution, pH = 7.5) was subtracted from butein-XO complexes.
Synchronous fluorescence spectra were performed by setting the excitation and emission wavelength interval (∆λ) at 15 and 60 nm over a wavelength range of 260-330 nm and 250-330 nm, respectively.
All the fluorescence-quenching data were corrected for absorption of excitation light and reabsorption of emitted light by the following relationship [41]: where F c and F m represent the corrected and measured florescence. A 1 and A 2 are the absorbance of butein at excitation and emission wavelengths, respectively.

Molecular Docking
The potential binding site of XO-butein was researched on AutoDock (version 4.2, The Scripps Research Institute, La Jolla, CA, USA). The X-ray crystal structure (PDB ID, 3EUB) of XO used for the docking studies [42] was download from the Protein Data Bank (http://www.rcsb.org/pdb). All water molecules and ligand in XO were removed prior to the docking program running, after which the polar hydrogen atoms and Gasteiger charges were added to the macromolecule file. The 3D structures of butein were obtained in Chem 3D Ultra 8.0. The active site with a fixed grid spacing (0.375 points) was enclosed by a rigid dimension of docking center (100 × 100 × 100 points). The best scoring docked model of the ligand was chosen to represent its most desirable binding mode predicted by AutoDock. The PyMol molecular graphic system was used to visualize the conformations and interactions between the butein and the target proteins.

Conclusions
In summary, a simple and effective method for the synthesis of butein by aldol condensation with the catalytic system SOCl 2 /EtOH was conducted for the first time in the present study. The optimal reaction conditions including the molar ratio of reactants, the dosage of catalyst, and the reaction time were investigated, and its potential inhibition of XO was discussed in aspect of inhibition kinetics, fluorescence titrations, synchronous fluorescence spectroscopy, and molecular docking. The butein can be prepared in good yield from 2 ,4 -dihydroxyacetophenone and 3,4-dihydroxybenzaldehyde based on the catalytic system SOCl 2 /EtOH, without protecting the hydroxyl groups. In addition, the inhibitory activity on XO showed that butein could significantly reduce uric acid concentration and the IC 50 value of restraining XO was found as 2.93 × 10 −6 mol L −1 in vitro. Additionally, an attractive inhibition mechanism of irreversible competition is worth further exploration, as it might be helpful in treating gout. The intrinsic fluorescence of XO was quenched statically by butein, and butein bound spontaneously into the active cavity of XO to form the butein-XO complex with one high-affinity binding sites. Besides, hydrophobic interaction is the main driving force of butein to binding with XO. The molecule docking revealed butein is a competitive XO inhibitor and has a high affinity binding site on XO. The effectiveness of butein in reducing uric acid concentration in the blood is required to evaluate by the further in vivo studies. This study has supplied significant insights into the inhibitory mechanism of butein on XO, which will promote butein to be a leading compound by structural modification on the treatment of gout.

Conflicts of Interest:
The authors declare no conflict of interest.