Synthesis method of epinose

Abstract

The invention belongs to the technical field of sugar synthesis chemistry, and particularly relates to a synthesis method of epinose. According to the method disclosed by the invention, beta-octa-acetyl lactose is taken as a raw material, brepinose is obtained by modifying and overturning a glucose fragment 2-OH, the raw material is easy to obtain, and the method is simple in process, low in cost and suitable for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of sugar synthesis chemistry technology, specifically relating to a method for synthesizing ipilactose. Background Technology

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Epilactose (I), also known as epilactose, has the chemical structure galactose-β-(1→4)-mannose and is an isomer of lactose (II). Epilactose is a rare reducing disaccharide with important biological functions and beneficial effects, such as promoting intestinal absorption of minerals and reducing arteriosclerosis.

[0004] Due to the important application value of epilactose, its preparation method has always been highly valued. Previous chemical synthesis methods (Wang Jiayao, Jiang Rui, Liang Xiaomei, Jin Shuhui, Wang Daoquan, Zhang Jianjun. Efficient Synthesis of Epilactose[J]. Chin. J. Org. Chem., 2017, 37(2): 375-384.;Huang S, Yu H, Chen X. Disaccharides as Sialic Acid AldolaseSubstrates: Synthesis of Disaccharides Containing a Sialic Acid at the Reducing End [J]. Angewandte Chemie International Edition, 2007, 46(13):2249-2253 DOI:10.1002 / anie.200604799.) adopted the method of coupling galactose and mannose fragments, which generally has a long route, complicated process and low yield. Existing enzymatic methods for synthesizing epimerose (Ito, S., Taguchi, H., Hamada, S. et al. Enzymatic properties of cellobiose 2-epimerase from Ruminococcus albus and the synthesis of rare oligosaccharides by the enzyme. Appl Microbiol Biotechnol 79, 433-441 (2008). https: / / doi.org / 10.1007 / s00253-008-1449-7) suffer from poor reproducibility, difficulties in separation and purification, and challenges in large-scale production. Summary of the Invention

[0005] To address the problems in the prior art, this invention provides a method for synthesizing ipilactose, using readily available raw materials, and suitable for large-scale production.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] A method for synthesizing ipilactose, comprising the following steps: (1) beta-octaacetyl lactose and PCl5 were mixed in an organic solvent and reacted at room temperature. After the reaction was completed, the temperature was lowered and intermediate 1 was obtained by separation. (2) Intermediate 1, N,N-dimethylformamide (DMF), and water were reacted in an organic solvent at room temperature. After the reaction was completed, intermediate 2 was obtained by separation. (3) Intermediate 2 and acetic anhydride react in an organic solvent, and intermediate 3 is obtained after the reaction is completed; (4) Intermediate 3 and sodium borohydride react in an organic solvent, and intermediate 4 is obtained after the reaction is completed; (5) Intermediate 4 and sodium methoxide react in methanol at room temperature. After the reaction is complete, the product is separated and purified.

[0008] In step (1), the molar ratio of beta-octaacetyl lactose to PCl5 is 1:(1~2.5); preferably 1:(1.5~2.5); more preferably 1:(2~2.5).

[0009] In step (1), the mixing and cooling temperature is -10℃ to 0℃; preferably -10℃ to -5℃.

[0010] In step (1), the separation step involves adding a saturated sodium bicarbonate solution at a temperature not higher than 0°C to the reaction system until the pH is greater than 7, allowing it to stand to separate the organic phase, and removing the solvent.

[0011] In step (2), the mass ratio of water to beta-octaacetyl lactose is 2:5. The volume ratio of DMF to water is 1:4; the volume ratio of DMF to organic solvent is 1:100.

[0012] In step (2), the separation step is to remove the solvent from the reaction system, add an equal amount of dichloromethane to the original volume, wash with saturated brine, let stand to separate the organic phase, and remove the solvent.

[0013] In step (3), the molar ratio of acetic anhydride to beta-octaacetyl lactose is (5~10):1; preferably (5~7.5):1.

[0014] In step (3), the separation step involves adding 4 to 5 times the volume of dichloromethane to dilute the reaction system, then adjusting the pH to be greater than 7 with saturated sodium bicarbonate solution, allowing it to stand to separate the organic phase, and removing the solvent.

[0015] In step (4), the molar ratio of sodium borohydride to beta-octaacetyl lactose is (1~5):1; preferably (3~5):1.

[0016] In step (4), the separation step is to pour the reaction system into a 1N hydrochloric acid solution at 0℃~4℃, and then extract it with dichloromethane. The extraction is performed 1-3 times. The dichloromethane phase obtained by separation is dried to remove the solvent. The ratio of hydrochloric acid solution to beta-octaacetyl lactose is 2mL:1g, and the ratio of dichloromethane to beta-octaacetyl lactose is 5mL:1g.

[0017] In step (5), the molar ratio of beta-octaacetyl lactose to sodium methoxide is (10~20):1.

[0018] In step (5), the separation and purification steps are as follows: after neutralizing the reaction system, the solvent is removed, and methanol and methyl ether with a volume ratio of 1:2 are added to the residue; the product is obtained by filtration and drying; the volume of methanol and methyl ether added is 0.5 to 1 times that of the reaction system.

[0019] In the above steps, the concentration of beta-octaacetyl lactose and each intermediate in the organic solvent is not higher than 0.5 mol / L; preferably 0.05 mol / L to 0.5 mol / L; more preferably 0.05 mol / L to 0.15 mol / L. The organic solvent is selected from at least one of dichloromethane, acetone, methanol, and dimethyl sulfoxide (DMSO).

[0020] The present invention has the following advantages: This invention uses readily available commercially available beta-octaacetyllactose as a starting material. Through a five-step continuous conversion process, the 2-OH of the glucose fragment is modified and inverted to efficiently obtain ipilactose. This overcomes the difficulties in preparing ipilactose using traditional methods and promotes the development and application of ipilactose's functions. Attached Figure Description

[0021] Figure 1 It is lactose. 1 H NMR spectrum; Figure 2 It is lactose. 13 C NMR spectrum; Figure 3 This is a high-resolution mass spectrometry (HRMS) image of ipilactose; Figure 4 This is an HPLC chromatogram of ipilactose. Detailed Implementation

[0022] Unless otherwise specified, all materials used in the experiments of this invention are commercially available products. Unless otherwise specified, room temperature refers to 10℃~30℃; low temperature refers to no higher than 5℃, especially -10℃~5℃; ice water is 0℃~4℃; weakly alkaline pH is 7.1~8.5.

[0023] A method for preparing ipilactose, the reaction route is as follows: .

[0024] Specifically, the above preparation method includes the following steps: (1) At -10℃~0℃, beta-octaacetyl lactose and PCl5 were mixed in dichloromethane at a molar ratio of 1:(1~2.5), and the mixture was heated to room temperature for reaction. The reaction was stopped after the raw materials disappeared. The reaction system was cooled to -10℃~0℃, and intermediate 1 was obtained by separation and directly added to the next reaction. Specifically, the separation steps are as follows: saturated sodium bicarbonate ice water solution is added to the reaction system until it is weakly alkaline (pH>7), the organic phase is separated by standing, and the solvent is evaporated from the organic phase under reduced pressure to obtain oily intermediate 1.

[0025] (2) Intermediate 1 is dissolved in acetone, and DMF and water are added in a volume ratio of 1:4. The volume ratio of DMF to acetone is 1:100. The reaction is carried out at room temperature. After the raw material disappears, intermediate 2 is separated and directly added to the next reaction. Specifically, the separation steps are as follows: the solvent is removed from the reaction system under reduced pressure to obtain an oily substance, which is then dissolved in an equal volume of dichloromethane, washed with saturated brine, allowed to stand to separate the organic phase, and the washing is repeated once. The solvent is evaporated from the organic phase under reduced pressure to obtain oily intermediate 2.

[0026] (3) Intermediate 2 and acetic anhydride react in DMSO. The molar amount of acetic anhydride added is 5 to 10 times that of beta-octaacetyl lactose. After monitoring the disappearance of the raw material, intermediate 3 is separated and directly added to the next reaction. Specifically, the separation steps are as follows: the reaction system is diluted with 4 to 5 times the volume of dichloromethane, then the pH is adjusted to be greater than 7 with saturated sodium bicarbonate solution, the organic phase is separated by standing, and the solvent is removed.

[0027] (4) Intermediate 3 and sodium borohydride react in methanol. The molar amount of sodium borohydride added is 1 to 5 times that of beta-octaacetyl lactose. After monitoring the disappearance of the raw material, intermediate 4 is separated and directly added to the next reaction. Specifically, the separation steps are as follows: the reaction system is poured into a 0℃~4℃, 1N hydrochloric acid solution, and then extracted with dichloromethane. The dichloromethane phase obtained by separation is dried to remove the solvent. The ratio of hydrochloric acid solution to beta-octaacetyl lactose is 2mL:1g, and the ratio of dichloromethane to beta-octaacetyl lactose is 5mL:1g.

[0028] (5) Intermediate 4 and sodium methoxide react in methanol at room temperature. The amount of sodium methoxide added is 1:(10~20) of the molar amount of beta-octaacetyl lactose. After the reaction is completed, the product ipilactose is separated and purified. Specifically, the separation and purification steps are as follows: acidic resin is added to the reaction system to neutralize the system to neutrality, the acidic resin is removed by filtration, and the mixture is concentrated under reduced pressure until no fraction is distilled off. Then, 0.5 to 1 times the volume of methanol and methyl ether in a volume ratio of 1:2 is added to the residue, the mixture is stirred, cooled to 0°C, stirred again, and then filtered. After drying the filtrate, a white powdery product is obtained.

[0029] In the above steps, the concentration of beta-octaacetyl lactose and each intermediate in the organic solvent is no higher than 0.5 mol / L.

[0030] The present invention will be further described below with reference to embodiments and accompanying drawings, but the present invention is not limited to the embodiments described below. Unless otherwise specified, room temperature refers to 10℃~30℃; low temperature refers to no higher than 5℃, especially -10℃~5℃; ice water is 0℃~4℃; weakly alkaline pH is 7.1~8.5.

[0031] Example 1 Synthesis of Ipilactose (1) Add 10.0 g (14.7 mmol) of beta-octaacetyl lactose to 100 mL of dichloromethane under stirring. Stir the system until it is uniform and cool it to below -5°C. Then add 6.76 g (32.5 mmol) of PCl5 in batches, keeping the system temperature below -5°C during the addition process. After the addition is complete, raise the system temperature to 25°C and continue stirring for 6 hours. After the raw material disappears as detected by TLC, cool the system to below -5°C and slowly pour it into a saturated sodium bicarbonate ice water solution that is stirred vigorously. Stir for 30 minutes and detect that the system is weakly alkaline. Then let it stand to separate the organic phase. The oily intermediate 1 obtained by vacuum concentration of the organic phase is added to the next step.

[0032] (2) At room temperature, intermediate 1 was dissolved in 100 mL of acetone. 4 mL of water and 1 mL of DMF were added to the system. The mixture was stirred at room temperature and reacted. After about 6 hours, the TLC showed that the raw material had disappeared. The system was concentrated under reduced pressure. The resulting oily substance was dissolved in 100 mL of dichloromethane. After washing twice with saturated brine, the organic phase was separated. The oily intermediate 2 was concentrated under reduced pressure and added to the next step.

[0033] (3) Dissolve intermediate 2 in 10 mL of DMSO and 10 mL of acetic anhydride, stir at room temperature until the starting material disappears as detected by TLC, then dilute the system with 100 mL of dichloromethane, pour in a saturated sodium bicarbonate aqueous solution and stir to neutralize until it is weakly alkaline. Allow to stand to separate the dichloromethane phase, concentrate under reduced pressure to obtain oily intermediate 3, and add it to the next step.

[0034] (4) Dissolve intermediate 3 in 100 mL of methanol, stir and cool to below -5 °C, then add 2.23 g (58.9 mmol) of sodium borohydride powder in batches, stir and react for 30 minutes below -5 °C, then react at room temperature for about 6 hours. When the starting material disappears as detected by TLC, pour the system into 20 mL of 1N hydrochloric acid ice water solution, stir thoroughly for 30 minutes, and then extract the aqueous phase three times with dichloromethane, using 50 mL of dichloromethane each time. Combine the three dichloromethane phases and concentrate under reduced pressure to obtain oily intermediate 4, which is then added to the next step.

[0035] (5) Dissolve intermediate 4 in 50 mL of methanol, add 50 mg (0.92 mmol) of sodium methoxide, stir at room temperature for 1 hour, and detect no raw material or intermediate by HPLC. Add acidic resin to neutralize the system. After filtering to remove the acidic resin, concentrate the system under reduced pressure until no fraction remains. Then add 10 mL of methanol and 20 mL of methyl ether to the concentrated residue and stir for 1 hour. Then cool the system to 0 °C and continue stirring for 1 hour. Filter the system and dry the filtrate with hot air to obtain 1.76 g of white powder product with a total yield of 35% and a purity of 100% (HPLC method).

[0036] The above products 1 H NMR, 13 The C NMR and HRMS spectra are as follows: Figure 1-3 As shown: 1 H NMR (400 MHz, D2O) δ: 5.21 (d, 0.61 H, J = 1.6 Hz), 4.95 (s, 0.39H), 4.45-4.50 (dd, 1H, J1 = 7.6 Hz, J2 = 2.4Hz), 3.99-4.05 (m, 1.37H), 3.93-3.98(m, 2H), 3.74-3.87(m, 3H), 3.67-3.73(m, 1H), 3.53-3.62(m, 1.33H).

[0037] 13 C NMR (100 MHz, D2O) δ: 103.14, 93.88, 76.66, 75.46, 72.60, 71.12, 71.05, 70.27, 70.21, 69.11, 68.69, 61.20, 60.43; (93.72, 76.25, 75.04, 71.90, 70.72 are peaks of isomers).

[0038] The proton and carbon spectral data are consistent with those in the literature (Disaccharides as Sialic Acid AldolaseSubstrates: Synthesis of Disaccharides Containing a Sialic Acid at the Reducing End [J]. Angewandte Chemie International Edition, 2007, 46(13):2249-2253; Efficient Synthesis of Epilactose[J]. Chin. J. Org. Chem. , 2017, 37(2): 375-384.).

[0039] HRMS(ESI, m / z) calculated for C 12 H 22 O 11 Cl [M+H] + : 377.0851, found377.0856; Molecular formula is C 12 H 22 O 11 .

[0040] The above-mentioned ipilactose sample was prepared into a 50 mg / mL solution using acetonitrile / water at a volume ratio of 80:20 for HPLC detection. The chromatographic conditions are as follows: Column: Durashell NH2, φ4.6×250mm×5μm; Column temperature: 40℃; Mobile phase: Acetonitrile / water = 80:20; Flow rate: 1.0 mL / min; Detector: Evaporative light scattering detector (ELSD).

[0041] The HPLC chromatogram of the ipilactose sample is shown below. Figure 4 As shown, the peak elution time is approximately 14.83 min, and the content is 100% according to the area normalization method.

[0042] Example 2 Synthesis of Ipilactose (1) Add 100.0 g of beta-octaacetyl lactose to 1 L of dichloromethane under stirring. Stir the system evenly and cool it to below -5°C. Then add 67.6 g of PCl5 in batches, keeping the system temperature below -5°C during the feeding process. After the feeding is completed, the system temperature rises to 25°C and the reaction continues to be stirred for 6 hours. After the raw material disappears as detected by TLC, cool the system to below -5°C and slowly pour it into a saturated sodium bicarbonate ice water solution that is stirred vigorously. Stir for 30 minutes and the system is detected to be weakly alkaline. Then let it stand to separate the organic phase. The oily intermediate 1 obtained by vacuum concentration of the organic phase is added to the next step.

[0043] (2) At room temperature, intermediate 1 was dissolved in 1 liter of acetone. 40 ml of water and 10 ml of DMF were added to the system. The mixture was stirred at room temperature and reacted. After about 6 hours, the TLC showed that the raw material had disappeared. The system was concentrated under reduced pressure. The resulting oily substance was dissolved in 1 liter of dichloromethane. After washing twice with saturated brine, the organic phase was separated. The oily intermediate 2 was concentrated under reduced pressure and added to the next step.

[0044] (3) Dissolve intermediate 2 in 100 mL of DMSO and 100 mL of acetic anhydride, stir at room temperature until the starting material disappears as detected by TLC, then dilute the system with 1 L of dichloromethane, pour in saturated sodium bicarbonate aqueous solution and stir to neutralize until it is weakly alkaline. Allow to stand to separate the dichloromethane phase, concentrate under reduced pressure to obtain oily intermediate 3 and add it to the next step.

[0045] (4) Dissolve intermediate 3 in 1 liter of methanol, stir and cool down to below -5°C, then add 22.3 g of sodium borohydride powder in batches, stir and react for 30 minutes below -5°C, then react at room temperature until the raw material disappears as detected by TLC. Pour the system into 200 mL of 1N hydrochloric acid ice water solution, stir thoroughly for 30 minutes, and then extract the aqueous phase three times with dichloromethane, each time using 500 mL of dichloromethane. Combine the three dichloromethane phases and concentrate under reduced pressure to obtain oily intermediate 4, which is then added to the next step.

[0046] (5) Dissolve intermediate 4 in 500 mL of methanol, add 0.5 g of sodium methoxide, stir at room temperature for 1 hour, and monitor with HPLC until no raw material or intermediate is detected. Add acidic resin to neutralize the system. After filtering to remove the acidic resin, concentrate the system under reduced pressure until no fraction remains. Then add 100 mL of methanol and 200 mL of methyl ether to the concentrated residue and stir for 1 hour. A white powder is produced. Cool the system to 0 °C and continue stirring for 1 hour. Filter to obtain a white powder product. After hot air drying, weigh 18.6 g of product, with a total yield of 37% and a purity of 100% (HPLC method).

[0047] Example 3 Synthesis of Ipilactose (1) Add 1 kg of beta-octaacetyl lactose to 10 L of dichloromethane under stirring. Stir the system evenly and cool it to about -10°C. Then add 600 g of PCl5 in batches, keeping the system temperature below -5°C during the feeding process. After the feeding is completed, the system temperature rises to 25°C and the reaction continues for 6 hours. After the raw material disappears as detected by TLC, cool the system to below -5°C and slowly pour it into a saturated sodium bicarbonate ice water solution that is stirred vigorously. Stir until the system is weakly alkaline (pH=7.5) and then let it stand to separate the organic phase. The oily intermediate 1 obtained by vacuum concentration of the organic phase is added to the next step.

[0048] (2) At room temperature, intermediate 1 was dissolved in 10 liters of acetone. 400 ml of water and 100 ml of DMF were added to the system. The mixture was stirred at room temperature until the raw material disappeared as detected by TLC. The system was concentrated under reduced pressure. The resulting oily substance was dissolved in 10 liters of dichloromethane. After washing twice with an equal volume of saturated brine, the organic phase was separated. The oily intermediate 2 was concentrated under reduced pressure and added to the next step.

[0049] (3) Dissolve intermediate 2 in 1 L of DMSO and 1 L of acetic anhydride, stir at room temperature until the starting material disappears as detected by TLC, then dilute the system with 10 L of dichloromethane, pour in saturated sodium bicarbonate aqueous solution and stir to neutralize until it is weakly alkaline (pH=7.5). Allow to stand to separate the dichloromethane phase, concentrate under reduced pressure to obtain oily intermediate 3 and add it to the next step.

[0050] (4) Dissolve intermediate 3 in 10 liters of methanol, stir and cool to about -5°C, then add 200 g of sodium borohydride powder in batches, stir and react for 30 minutes below -5°C, then react at room temperature until the starting material disappears as detected by TLC. Pour the system into 2 liters of 1N hydrochloric acid ice water solution, stir thoroughly for 30 minutes, and then extract the aqueous phase three times with dichloromethane, using 5 liters of dichloromethane each time. Combine the three dichloromethane phases and concentrate under reduced pressure to obtain oily intermediate 4, which is then added to the next step.

[0051] (5) Dissolve intermediate 4 in 5 liters of methanol, add 5 g of sodium methoxide, stir at room temperature for 1 hour, and check by HPLC until no raw material or intermediate is detected. Add acidic resin to neutralize the system to neutral. After filtering to remove the acidic resin, concentrate the system under reduced pressure until no fraction is distilled. Then add 1 liter of methanol and 2 liters of methyl ether to the concentrated residue and stir for 1 hour. Cool the system to 0°C and continue stirring until a large amount of insoluble matter is present. Filter and dry the filtrate to obtain 191 g of white powder product with a total yield of 38% and a purity of 100% (HPLC method).

Claims

1. A method for synthesizing ipilactose, characterized in that, Includes the following steps: (1) beta-octaacetyl lactose and PCl5 were mixed in an organic solvent and reacted at room temperature. After the reaction was completed, the temperature was lowered and intermediate 1 was obtained by separation. (2) Intermediate 1 reacts with DMF and water in an organic solvent at room temperature, and intermediate 2 is obtained after the reaction is completed; (3) Intermediate 2 and acetic anhydride react in an organic solvent, and intermediate 3 is obtained after the reaction is completed; (4) Intermediate 3 and sodium borohydride react in an organic solvent, and intermediate 4 is obtained after the reaction is completed; (5) Intermediate 4 and sodium methoxide react in methanol at room temperature. After the reaction is complete, the product is separated and purified.

2. The synthesis method according to claim 1, characterized in that, In step (1), the molar ratio of beta-octaacetyl lactose to PCl5 is 1:(1~2.5); In step (2), the mass ratio of water to beta-octaacetyl lactose in step (1) is 2:5; the volume ratio of DMF to water is 1:4; and the volume ratio of DMF to organic solvent is 1:

100. In step (3), the molar ratio of acetic anhydride to beta-octaacetyl lactose in step (1) is (5~10):1; In step (4), the molar ratio of sodium borohydride to beta-octaacetyl lactose in step (1) is (1~5):1; In step (5), the molar ratio of sodium methoxide to beta-octaacetyl lactose in step (1) is 1:(10~20); The concentration of beta-octaacetyl lactose and its intermediates in organic solvents shall not exceed 0.5 mol / L; In step (1), the mixing and cooling temperatures are -10℃ to 0℃; The organic solvent is selected from at least one of dichloromethane, acetone, methanol, and DMSO.

3. The synthesis method according to claim 1, characterized in that, In step (1), the molar ratio of beta-octaacetyl lactose to PCl5 is 1:(1.5~2.5); In step (3), the molar ratio of acetic anhydride to beta-octaacetyl lactose in step (1) is (5~7.5):1; In step (4), the molar ratio of sodium borohydride to beta-octaacetyl lactose in step (1) is (3~5):1; The concentration of beta-octaacetyl lactose and its intermediates in organic solvents is 0.05 mol / L to 0.5 mol / L; In step (1), the mixing and cooling temperatures are -10℃ to -5℃; In step (1), the organic solvent is dichloromethane; in step (2), the organic solvent is acetone; in step (3), the organic solvent is DMSO; and in step (4), the organic solvent is methanol.

4. The synthesis method according to claim 1, characterized in that, In step (1), the molar ratio of beta-octaacetyl lactose to PCl5 is 1:(2~2.5); The concentrations of beta-octaacetyl lactose and its intermediates in organic solvents range from 0.05 mol / L to 0.15 mol / L.

5. The synthesis method according to claim 1, characterized in that, In step (1), the separation step is to add a saturated sodium bicarbonate solution at a temperature not higher than 0°C to the reaction system until the pH is greater than 7, let it stand to separate the organic phase, and remove the solvent; In step (2), the separation step is to remove the solvent from the reaction system, add an equal amount of dichloromethane to the original volume, wash with saturated brine, let stand to separate the organic phase, and remove the solvent; In step (3), the separation step involves adding 4 to 5 times the volume of dichloromethane to dilute the reaction system, then adjusting the pH to be greater than 7 with saturated sodium bicarbonate solution, allowing it to stand to separate the organic phase, and removing the solvent. In step (4), the separation step is to pour the reaction system into a 1N hydrochloric acid solution at 0℃~4℃, and then extract it with dichloromethane. The extraction is repeated 1-3 times. The dichloromethane phase obtained by separation is dried to remove the solvent. The ratio of hydrochloric acid solution to beta-octaacetyl lactose in step (1) is 2mL:1g, and the ratio of dichloromethane to beta-octaacetyl lactose in step (1) is 5mL:1g. In step (5), the separation and purification steps are as follows: after neutralizing the system, the solvent is removed, and methanol and methyl ether with a volume ratio of 1:2 are added to the residue. The mixture is then filtered and dried to obtain the product. The volume of methanol and methyl ether added is 0.5 to 1 times that of the reaction system.

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