Synthetic method of epilactose

Abstract

The invention discloses a synthetic method of epilactose, and belongs to the technical field of sugar chemistry. The synthetic method of the epilactose comprises the following steps: beta-octa-acetyl lactose is taken as a raw material, five-step reaction is carried out, and intermediates are directly and continuously fed. The method comprises the following steps: 1, mixing raw materials with PCl5 in dichloromethane at-10 to 0 DEG C, and reacting at normal temperature to obtain an intermediate 1; 2, dissolving the intermediate 1 in acetone, adding 1: 4 DMF-water, and reacting at normal temperature to obtain an intermediate 2; 3, dissolving the intermediate 2 in dichloromethane-pyridine, and dropwise adding trifluoromethanesulfonic anhydride at the temperature of 10 DEG C below zero to 0 DEG C to obtain an intermediate 3; 4, dissolving the intermediate 3 in DMF, adding sodium nitrite, and reacting at normal temperature to obtain an intermediate 4; and 5, dissolving the intermediate 4 in methanol, adding sodium methoxide, reacting at room temperature, and separating and purifying to obtain the epilactose. According to the preparation method, beta-octa-acetyl lactose is taken as a raw material, the epilactose 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 relates to the field of saccharide chemical synthesis technology, and specifically to a method for synthesizing epilactose. Background Technology

[0002] 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. Due to its important applications, its preparation methods have always been of great interest. Previous chemical synthesis methods (Organic Chemistry, 2017, 2, 375;) Angew. Chem., Int. Ed. (2007, 46, 2249.) The method using the coupling of galactose and mannose fragments generally involves a long route, cumbersome process, and low yield. The enzymatic synthesis method for epilactose described in the literature (…) Appl. Microbiol. Biotechnol. (2008, 79, 433.) Poor reproducibility, difficult separation and purification, and difficult to prepare in large quantities. Summary of the Invention

[0003] The purpose of this invention is to provide a method for synthesizing epilactose, which solves the problems of efficient preparation and large-scale production in existing epilactose synthesis methods.

[0004] This invention provides a method for synthesizing epilactose, comprising the following steps: Step 1: At -10℃ to 0℃, beta-octaacetyl lactose and PCl5 are mixed in dichloromethane at a molar ratio of 1:1 to 2.5. The mixture is heated to room temperature to carry out the reaction. The reaction is stopped after the starting material disappears. The reaction system is cooled to -10℃ to 0℃ to separate intermediate 1, which is then directly added to the next reaction step. Step 2: Dissolve intermediate 1 in acetone, add a mixed solution of DMF and water with a volume ratio of 1:4 (1:100), react at room temperature, monitor the disappearance of the raw material, separate to obtain intermediate 2, and directly add to the next reaction step. Step 3: Dissolve intermediate 2 in a mixed solution of dichloromethane and pyridine, and add trifluoromethanesulfonic anhydride dropwise at -10℃ to 0℃. The molar ratio of trifluoromethanesulfonic anhydride to beta-octaacetyl lactose is 4~5:1, and the molar ratio of trifluoromethanesulfonic anhydride to pyridine is 1:3.5~4. After monitoring the disappearance of the starting material, separate intermediate 3 and directly add it to the next reaction step. Step 4: Dissolve intermediate 3 in DMF, add sodium nitrite and react at room temperature. The molar ratio of sodium nitrite to beta-octaacetyl lactose is 2~3:1. After monitoring the disappearance of the raw material, separate to obtain intermediate 4, and directly add to the next reaction step. Step 5: Dissolve intermediate 4 in methanol, add sodium methoxide and react at room temperature. The molar ratio of sodium methoxide to beta-octaacetyl lactose is 1:16~30. After the reaction is completed, separate and purify to obtain epilactose. The reaction route diagram is as follows: .

[0005] The beneficial effects of this application, based on the above technical solutions, are as follows: 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, overcoming the difficulties of traditional methods in preparing ipilactose and promoting the development and application of the functions of epilactose. Attached Figure Description

[0006] Figure 1 Chemical structural diagrams of lactose (I) and lactose (II) provided for embodiments of the present invention; Figure 2 A synthetic route diagram of epilactose provided in an embodiment of the present invention; Figure 3 The 1H NMR spectrum of epilactose provided in this embodiment of the invention; Figure 4 The carbon NMR spectrum of epilactose provided in this embodiment of the invention; Figure 5 The high-performance liquid chromatography (HPLC) spectrum of epilactose (sample number: 25773-19) provided in an embodiment of the present invention; Figure 6 The high-resolution mass spectrometry detection image of epilactose (sample number: 25773-19) provided in the embodiment of the present invention. Detailed Implementation

[0007] To fully illustrate the preparation concept of this invention, the process of this solution is described below with reference to specific embodiments. The embodiments are for illustrative purposes only and should not be interpreted or construed as limiting the protection of this invention. In addition, all experimental materials used in this invention are commercially available products unless otherwise specified.

[0008] In this embodiment of the invention, "continuous addition" means that the intermediate does not need to be purified and can be directly used in the next reaction step.

[0009] In the embodiments of this invention, both room temperature and ambient temperature refer to 20~25℃.

[0010] Example 1: Synthesis of lactose Step 1: Add 10.0 g (14.7 mmol) of beta-octaacetyl lactose to 100 mL of dichloromethane with stirring. Stir until homogeneous and cool to below -5°C. Then, add 6.76 g (32.5 mmol) of PCl5 in portions, maintaining the system temperature below -5°C throughout the addition process. After the addition is complete, raise the system temperature to 25°C and continue stirring for 6 hours. After the starting material spot disappears as determined by TLC (electrolyte 1 / 1 ethyl acetate / petroleum ether), cool the system to below -5°C and slowly pour it into a vigorously stirred saturated sodium bicarbonate ice-water solution. Stir for 30 minutes. The system should be weakly alkaline (pH = 7.1–8.5). Allow to stand to separate the organic phase. Concentrate under reduced pressure to remove the solvent, then add the resulting oily intermediate 1 to the next step.

[0011] Step 2: Dissolve the oily intermediate 1 from Step 1 in 100 mL of acetone at room temperature. Add 4 mL of water and 1 mL of DMF to the system, and then stir the reaction at room temperature. After about 6 hours, TLC detection (eluent 1 / 1 ethyl acetate / petroleum ether) showed that the starting material spot had disappeared. Then, concentrate the system under reduced pressure to remove the solvent. Dissolve the resulting oily substance in 100 mL of dichloromethane, wash twice with saturated brine, separate the organic phase, concentrate under reduced pressure to remove the solvent, and then add the oily intermediate 2 to the next step.

[0012] Step 3: Dissolve the oily intermediate 2 from Step 2 in 100 mL of dichloromethane and 20 mL (19.6 g, 248 mmol) of pyridine. Cool to below -5°C, then slowly add 16.6 g (64.7 mmol) of trifluoromethanesulfonic anhydride dropwise under controlled temperature. After the addition is complete, maintain the system temperature below -5°C and stir for approximately 2 hours. After the starting material spot disappears according to TLC (electrolyte 1 / 1 ethyl acetate / petroleum ether), pour the system into a saturated sodium bicarbonate ice-water solution and stir to neutralize until it becomes weakly alkaline (pH = 7.1~8.5). Allow to stand to separate the dichloromethane phase, concentrate under reduced pressure to remove the solvent, and add the oily intermediate 3 to the next step.

[0013] Step 4: Dissolve oily intermediate 3 from Step 3 in 100 mL of DMF. Add 2.02 g (29.4 mmol) of sodium nitrite while stirring at room temperature. Stir and react for about 6 hours. After the starting spot disappears according to TLC (eluent 1 / 1 ethyl acetate / petroleum ether), pour the system into a 1N hydrochloric acid ice-water solution and stir thoroughly for 30 minutes. Extract the aqueous phase three times with 100 mL of ethyl acetate each time. Combine the three ethyl acetate phases, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent, obtaining oily intermediate 4, which is then added to the next step.

[0014] Step 5: Dissolve the oily intermediate 4 from Step 4 in 50 mL of methanol, add 50 mg (0.92 mmol) of sodium methoxide, stir at room temperature for 1 hour, and monitor by TLC (electrolyte 10 / 1 dichloromethane / methanol) until no raw material or intermediate remains. Then, add Dowex 50W-X8 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, resulting in the formation of a white insoluble substance. Cool the system to 0°C and continue stirring for 1 hour, then filter to obtain a white powdery epilactose product. Dry the product with hot air at 40-50°C until no weight loss occurs (i.e., constant mass), and weigh to obtain 1.91 g of product (sample number: 25773-19), with an overall yield of 38%. The product has an HPLC purity of 100%.

[0015] The above products 1 H NMR, 13 The C NMR and HRMS spectra are as follows: Figure 3-5 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).

[0016] 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).

[0017] The proton and carbon spectral data are consistent with those in the literature. Angew. Chem. Int. Ed. 2007, 46, 2249-2253; Chin. J. Org. Chem . 2017, 37, 375-384.).

[0018] HRMS(ESI, m / z) calculated for C 12 H 22 O 11Cl [M+H] + : 377.0851, found377.0856; Molecular formula is C 12 H 22 O 11 .

[0019] The lactose sample above 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).

[0020] The HPLC chromatogram of the ipilactose sample is shown below. Figure 5 As shown, the peak elution time is approximately 14.83 min, and the content is 100% according to the area normalization method.

[0021] Example 2 Synthesis of lactose Step 1: Add 500 g (735 mmol) of beta-octaacetyl lactose to 5 L of dichloromethane with stirring. Stir until homogeneous and cool to below -5°C. Then, add 338 g (1.625 mol) of PCl5 in batches, maintaining the system temperature below -5°C throughout the addition process. After the addition is complete, raise the system temperature to 25°C and continue stirring for 6 hours. After the starting material spot disappears as determined by TLC (electrolyte 1 / 1 ethyl acetate / petroleum ether), cool the system to below -5°C and slowly pour it into a vigorously stirred saturated sodium bicarbonate ice-water solution. Stir for 30 minutes. The system should be weakly alkaline (pH = 7.1–8.5). Allow to stand to separate the organic phase. Concentrate under reduced pressure to remove the solvent, then add the resulting oily intermediate 1 to the next step.

[0022] Step 2: Dissolve the oily intermediate 1 from Step 1 in 5 liters of acetone at room temperature. Add 200 ml of water and 50 ml of DMF to the system, and then stir the reaction at room temperature. After about 6 hours, TLC detection (eluent 1 / 1 ethyl acetate / petroleum ether) showed that the starting material spot had disappeared. Then, concentrate the system under reduced pressure to remove the solvent. Dissolve the resulting oily substance in 5 liters of dichloromethane, wash twice with saturated brine, separate the organic phase, concentrate under reduced pressure to remove the solvent, and then add the oily intermediate 2 to the next step.

[0023] Step 3: Dissolve the oily intermediate 2 from Step 2 in 5 L of dichloromethane and 1 L (980 g, 12.4 mol) of pyridine. Cool to below -5°C, then slowly add 830 g (3.24 mol) of trifluoromethanesulfonic anhydride dropwise under controlled temperature. After the addition is complete, maintain the system temperature below -5°C and stir for about 2 hours. After the starting material spot disappears according to TLC (electrolyte 1 / 1 ethyl acetate / petroleum ether), pour the system into a saturated sodium bicarbonate ice-water solution and stir to neutralize until it becomes weakly alkaline (pH = 7.1~8.5). Allow to stand to separate the dichloromethane phase, concentrate under reduced pressure to remove the solvent, and add the oily intermediate 3 to the next step.

[0024] Step 4: Dissolve the oily intermediate 3 from Step 3 in 5 liters of DMF. Add 101 g (1.47 mol) of sodium nitrite while stirring at room temperature, and then stir the reaction for about 6 hours. After the starting material spot disappears according to TLC (eluent 1 / 1 ethyl acetate / petroleum ether), pour the system into a 1N hydrochloric acid ice-water solution and stir thoroughly for 30 minutes. Extract the aqueous phase three times with ethyl acetate, using 5 liters of ethyl acetate each time. Combine the three ethyl acetate phases, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent, obtaining oily intermediate 4, which is then added to the next step.

[0025] Step 5: Dissolve the oily intermediate 4 from Step 4 in 2.5 L of methanol, add 2.5 g (46 mmol) of sodium methoxide, stir at room temperature for 1 hour, and monitor by TLC (electrolyte 10 / 1 dichloromethane / methanol) until no raw material or intermediate remains. Then, add Dowex 50W-X8 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 500 mL of methanol and 1000 mL of methyl ether to the concentrated residue and stir for 1 hour, resulting in a white insoluble substance. Cool the system to 0 °C and continue stirring for 1 hour, then filter to obtain a white powdery epilactose product. Dry with hot air at 40-50 °C until no weight loss occurs (i.e., constant mass), and weigh to obtain 95.5 g of product, with a total yield of 38%. The product has 100% HPLC purity.

[0026] Example 3 Synthesis of lactose Step 1: Add 1000 g (1.47 mol) of beta-octaacetyl lactose to 10 L of dichloromethane with stirring. Stir the system until homogeneous and cool to below -5°C. Then, add 676 g (3.25 mol) of PCl5 in batches, maintaining 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 starting material spot disappears as detected by TLC (electrolyte 1 / 1 ethyl acetate / petroleum ether), cool the system to below -5°C and slowly pour it into a vigorously stirred saturated sodium bicarbonate ice-water solution. Stir for 30 minutes. The system is weakly alkaline (pH = 7-8). Allow to stand to separate the organic phase. Concentrate under reduced pressure to remove the solvent, and add the resulting oily intermediate 1 to the next step.

[0027] Step 2: Dissolve the oily intermediate 1 from Step 1 in 10 liters of acetone at room temperature. Add 400 ml of water and 100 ml of DMF to the system, and then stir the reaction at room temperature. After about 6 hours, TLC detection (eluent 1 / 1 ethyl acetate / petroleum ether) showed that the starting material spot had disappeared. Then, concentrate the system under reduced pressure to remove the solvent. Dissolve the resulting oily substance in 10 liters of dichloromethane, wash twice with saturated brine, separate the organic phase, concentrate under reduced pressure to remove the solvent, and then add the oily intermediate 2 to the next step.

[0028] Step 3: Dissolve the oily intermediate 2 from Step 2 in 10 L of dichloromethane and 2 L (1960 g, 24.8 mol) of pyridine. Cool to below -5°C, then slowly add 1.66 kg (6.47 mol) of trifluoromethanesulfonic anhydride dropwise under controlled temperature. After the addition is complete, maintain the system temperature below -5°C and stir for about 2 hours. After the starting material spot disappears according to TLC (electrolyte 1 / 1 ethyl acetate / petroleum ether), pour the system into a saturated sodium bicarbonate ice-water solution and stir to neutralize until it becomes weakly alkaline (pH = 7~8). Allow to stand to separate the dichloromethane phase, concentrate under reduced pressure to remove the solvent, and add the oily intermediate 3 to the next step.

[0029] Step 4: Dissolve oily intermediate 3 from Step 3 in 10 L of DMF. Add 202 g (2.94 mol) of sodium nitrite while stirring at room temperature. Stir for approximately 6 hours. After the starting material spot disappears according to TLC (eluent 1 / 1 ethyl acetate / petroleum ether), pour the system into a 1N hydrochloric acid ice-water solution and stir thoroughly for 30 minutes. Extract the aqueous phase three times with ethyl acetate, using 10 L of ethyl acetate each time. Combine the three ethyl acetate phases, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to remove the solvent, yielding oily intermediate 4, which is then added to the next step.

[0030] Step 5: Dissolve the oily intermediate 4 from Step 4 in 5 liters of methanol, add 5 g (92 mmol) of sodium methoxide, stir at room temperature for 1 hour, and monitor by TLC (electrolyte 10 / 1 dichloromethane / methanol) until no raw material or intermediate remains. Then, add Dowex 50W-X8 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 1 liter of methanol and 2 liters of methyl ether to the concentrated residue and stir for 1 hour, resulting in the formation of a white insoluble substance. Cool the system to 0°C and continue stirring for 1 hour. Filter to obtain a white powdery epilactose product. Dry with hot air at 40-50°C until no weight loss occurs (i.e., constant mass), and weigh to obtain 191 g of product, with a total yield of 38%. The product has 100% HPLC purity.

[0031] In Examples 1-3 of this invention, when the starting material beta-octaacetyl lactose is dissolved in dichloromethane in step 1, its molar concentration is not higher than 0.5 mol / L; When intermediate 1 is dissolved in acetone in step 2, its molar concentration shall not exceed 0.5 mol / L; When intermediate 2 is dissolved in the dichloromethane and pyridine mixture of step 3, its molar concentration shall not exceed 0.5 mol / L; When intermediate 3 is dissolved in DMF in step 4, its molar concentration is not higher than 0.5 mol / L.

[0032] Detection method: (HPLC) Table 1: Mobile phase: Table 2: Chromatographic conditions: Table 3: Mass Spectrometry Report of Sample 25773-19

Claims

1. A method for the synthesis of epilactose, characterized in that, Comprising the following steps: Step 1, beta-octacetyl lactose is mixed with PCl5 in dichloromethane at a molar ratio of 1:1-2.5 at -10-0°C, the reaction is carried out by warming to room temperature, and the reaction is stopped after monitoring the disappearance of the raw material, the reaction system is cooled to -10-0°C, and the intermediate 1 is separated and directly connected to the next step; Step 2, the intermediate 1 is dissolved in acetone, a mixed solution of DMF and water with a volume ratio of 1:4 is added, the volume ratio of DMF to acetone is 1:100, and the reaction is carried out at room temperature, and the intermediate 2 is separated after monitoring the disappearance of the raw material, and directly connected to the next step; Step 3, the intermediate 2 is dissolved in a mixed solution of dichloromethane and pyridine, triflic anhydride is added dropwise at -10-0°C, the molar ratio of triflic anhydride to beta-octacetyl lactose is 4-5:1, and the molar ratio of triflic anhydride to pyridine is 1:3.5-4, and the intermediate 3 is separated after monitoring the disappearance of the raw material, and directly connected to the next step; Step 4, the intermediate 3 is dissolved in DMF, sodium nitrite is added and reacted at room temperature, the molar ratio of sodium nitrite to beta-octacetyl lactose is 2-3:1, and the intermediate 4 is separated after monitoring the disappearance of the raw material, and directly connected to the next step; Step 5, the intermediate 4 is dissolved in methanol, sodium methoxide is added and reacted at room temperature, the molar ratio of sodium methoxide to beta-octacetyl lactose is 1:16-30; after monitoring the end of the reaction, the epilactose is obtained by separation and purification; The reaction route is as follows: 。 2. The method of synthesis of claim 1, wherein, The separation step in step 1 is to add saturated sodium bicarbonate ice water solution to the reaction system to adjust the pH to 7.1-8.5, separate the organic phase, evaporate the solvent under reduced pressure to obtain the intermediate 1.

3. The method of synthesis of claim 1, wherein, The separation step in step 2 is to remove the solvent under reduced pressure to obtain an oil, dissolve the oil in dichloromethane with the same volume as the reaction solvent, wash with saturated brine, separate the organic phase, repeat the washing, and evaporate the solvent under reduced pressure to obtain the intermediate 2.

4. The method of synthesis of claim 1, wherein, The separation step in step 3 is to add the reaction system to saturated sodium bicarbonate solution with a temperature ≤0°C to adjust the pH of the system to 7.1-8.5, separate the organic phase, and evaporate the solvent under reduced pressure to obtain the intermediate 3.

5. The method of synthesis of claim 1, wherein, The separation step in step 4 is to pour the reaction system into 1N hydrochloric acid solution with a temperature of 0-4°C, extract with ethyl acetate for 3 times, combine the obtained ethyl acetate phase, dry with anhydrous sodium sulfate, and evaporate the solvent under reduced pressure to obtain the oil intermediate 4.

6. The method of synthesis of claim 5, wherein, The amount of hydrochloric acid solution is 2mL:1g of beta-octacetyl lactose.

7. The method of synthesis according to claim 5 or 6, wherein, The single amount of ethyl acetate is 5-10mL:1g of beta-octacetyl lactose.

8. The method of synthesis of claim 1, wherein, The separation and purification step in step 5 is: adding an acidic resin to neutralize the system to neutral, filtering out the acidic resin, and then concentrating under reduced pressure to obtain a residue, adding methanol and methyl tert-butyl ether in a volume ratio of 1:2 to the residue, the total volume of the methanol and methyl tert-butyl ether being 0.5-1 times the volume of the reaction system, stirring, cooling to 0°C, continuing to stir, and then filtering, and drying the filtrate to obtain lacto-N-tetraose.

9. The method of synthesis of claim 1, wherein, The molar ratio of beta-octacetyl lactose to PCl5 is 1:2.21; the molar ratio of trifluoromethanesulfonic anhydride to beta-octacetyl lactose is 4.4:1; the molar ratio of trifluoromethanesulfonic anhydride to pyridine is 1:3.8; the molar ratio of sodium nitrite to beta-octacetyl lactose is 2:1; and the molar ratio of the amount of sodium methoxide added to beta-octacetyl lactose is 1:16.

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