A method for chemical synthesis of hydroxytyrosol glucoside

Abstract

The application discloses a chemical synthesis method of hydroxytyrosol glucoside. The method uses hydroxytyrosol as a starting material, and acetyl protection, glycosylation, hydrolysis and other steps are performed to prepare the hydroxytyrosol glucoside. The synthesis method of the hydroxytyrosol glucoside has the characteristics of low cost, simple process and industrialization.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis and discloses a method for preparing hydroxytyrosol glucoside. Background Technology

[0002] Hydroxytyrosol is a natural polyphenolic compound derived from olive oil. First discovered in extracts of olive leaves and fruits, it possesses potent antioxidant properties. Furthermore, hydroxytyrosol exhibits various biological activities, including anti-inflammatory, antibacterial, and anticancer effects.

[0003] Glycosides are widely found in various plants, mainly in the form of thioglycosides, cyanoglycosides, phenolic glycosides, and flavonoid glycosides. Glycosylation can alter the activity, stability, and solubility of small molecule compounds. Many glycosides possess diverse biological activities and have important medicinal value.

[0004] Hydroxytyrosol glucoside, also known as hydroxy-β-rhodioloside, is a derivative of hydroxytyrosol and rhodioloside. It also has strong antioxidant activity and good neuroprotective effects, and has extremely high development value in the fields of medicine, health care and cosmetics.

[0005] Currently, there are very few patent reports on hydroxytyrosol glucoside. The preparation method involves bio-fermentation synthesis. For example, CN114317480B discloses a gene-edited glycosyltransferase biocatalytic method for synthesizing hydroxyrhodioloside. Using rhodioloside as a raw material, this method uses in vitro enzymatic catalysis to biosynthesize hydroxyrhodioloside. However, this process uses expensive enzyme catalysts and raw materials, resulting in low product purity, low economic efficiency, and difficulty in large-scale production. CN116478950A discloses a bio-fermentation method for synthesizing hydroxy-β-rhodioloside using gene-edited Saccharomyces cerevisiae cell lines and tyrosine as a raw material. This method involves complex gene editing and strain cultivation processes, resulting in low product purity and difficulty in large-scale production.

[0006] In summary, there is currently no suitable chemical synthesis method for industrial-scale production of hydroxytyrosol glucoside, either domestically or internationally. Based on the glycosylation reaction, this invention develops a method for preparing hydroxytyrosol glucoside using hydroxytyrosol as a raw material through a chemical process. Summary of the Invention

[0007] This invention proposes a synthetic method for preparing hydroxytyrosol glucoside, which features high yield and simple process.

[0008] This invention describes a method for preparing hydroxytyrosol glucoside, which uses hydroxytyrosol as a starting material and involves acetylation, glycosylation, and hydrolysis to obtain hydroxytyrosol glucoside.

[0009]

[0010] In a specific embodiment of the present invention, the method for preparing hydroxytyrosol glucoside of the present invention includes the following steps:

[0011] Step 1: Dissolve hydroxytyrosol in a polar aprotic solvent and react it with an acetylation reagent under the action of a base to generate 3,4-diacetoxyphenethanol;

[0012] Step 2: 3,4-Diacetoxyphenylethanol and β-D-glucopentaacetate undergo a condensation reaction under the action of Lewis acid to obtain 2-(3,4-diacetoxyphenyl)ethyl-(2,3,4,6-O-tetraacetyl)-β-D-glucopyranoside.

[0013] Step 3: 2-(3,4-diacetoxyphenyl)ethyl-(2,3,4,6-O-tetraacetyl)-β-D-glucopyranoside is hydrolyzed under alkaline conditions to remove the acetyl group, yielding hydroxytyrosol glucoside.

[0014] In some specific examples, the polar aprotic solvent mentioned in step 1 is selected from tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, or dimethyl sulfoxide, with N,N-dimethylformamide being the preferred polar aprotic solvent. The amount of polar aprotic solvent used can be in accordance with the conventional usage of solvents in the art, and the amount used has no significant effect on the reaction.

[0015] In some specific examples, the base mentioned in step 1 is selected from: sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium methoxide, sodium tert-butoxide, potassium tert-butoxide, pyridine, triethylamine, or N,N-diisopropylethylamine. Preferred bases are sodium hydroxide, potassium hydroxide, potassium tert-butoxide, or triethylamine. In some embodiments, the molar ratio of potassium hydroxide to hydroxytyrosol is (2.0–2.5):1.0, and the preferred molar ratio is (2.0–2.1):1.0. When the amount of potassium hydroxide added is too low or too high, the reaction yield decreases.

[0016] In some specific instances, the acetylation agent in step 1 is acetyl chloride. In some embodiments, the molar ratio of acetyl chloride to hydroxytyrosol is (2.0–2.2):1.0, and preferably 2.05:1.0. The inventors have found that using acetyl chloride can significantly improve the product yield.

[0017] In some specific examples, the reaction temperature in step 1 is -5 to 25°C, with a preferred temperature of 20 to 25°C for adding the alkali and a reaction time of 20 to 40 minutes. The acetylation reaction temperature is -5 to 10°C, and the reaction time is 2 to 3 hours. The reaction process does not need to maintain a specific temperature point; fluctuations within this range are acceptable. If the temperature at which the alkali is added is too high, the raw materials will degrade; if the temperature is too low, the reaction time needs to be extended. Excessive acetylation reaction temperature easily generates impurities, while excessively low temperature prolongs the reaction time.

[0018] In some specific instances, the reaction solution from step 1 can be further neutralized, extracted, washed, dried, and concentrated to obtain a colorless oily substance, 3,4-diacetoxyphenylethanol. The neutralization, extraction, washing, drying, and concentration can be performed according to conventional methods in the art.

[0019] In some specific examples, the molar ratio of 3,4-diacetoxyphenylethanol to β-D-glucose pentaacetate in step 2 is 1:(1.0 to 1.4), with a preferred molar ratio of 1:1.2.

[0020] In some specific instances, the Lewis acid described in step 2 is boron trifluoride. The inventors have found that using boron trifluoride as the Lewis acid significantly improves the yield. Boron trifluoride is often sold dissolved in tetrahydrofuran, and this reaction can directly incorporate boron trifluoride dissolved in tetrahydrofuran into the reaction.

[0021] In some specific instances, the solvent in step 2 is DCM (dichloromethane). The amount of solvent used can be in accordance with the conventional usage of solvents in the art, and the amount used has no significant effect on the reaction.

[0022] In some specific examples, the reaction temperature in step 2 is 40-45℃, and the reaction time is 3-5 hours. The reaction process does not need to maintain a certain temperature point; fluctuations within this range are acceptable.

[0023] After the reaction in step 2, the reaction in step 3 can be carried out directly. Alternatively, the reaction solution in step 2 can be washed, dried, and concentrated to obtain a pale yellow oily substance before proceeding with the reaction in step 3.

[0024] In some specific examples, the solvent used in step 3 is selected from methanol, ethanol, isopropanol, tetrahydrofuran, N,N-dimethylformamide, and N,N-dimethylacetamide, with methanol being preferred. The amount of solvent used can be in accordance with the conventional amount of solvent used in the art, and the amount used has no significant effect on the reaction.

[0025] In some specific examples, the base mentioned in step 3 is selected from: sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium methoxide, sodium tert-butoxide, and potassium tert-butoxide. Preferred bases are sodium hydroxide or potassium hydroxide.

[0026] In some specific examples, the amount of alkali used in step 3 is to adjust the pH of the reaction solution to be 9-11, with a preferred pH of 10-11.

[0027] In some specific examples, the reaction temperature in step 3 is -5 to 10°C, and the reaction time is 0.5 to 1 hour.

[0028] In some specific examples, the reaction solution in step 3 can be neutralized, filtered, concentrated, dissolved, and purified with macroporous resin to obtain a white solid, namely hydroxytyrosol glucoside. Neutralization, filtration, concentration, and dissolution can be performed using conventional methods in the art. The macroporous resin is selected from: YKDH-5, YKDH-11, D101, D4008, H107, HP-20, with YKDH-11 being preferred. These macroporous resins are all commercially available.

[0029] In some specific instances, the eluent for the macroporous resin purification in step 3 is selected from methanol, ethanol, and isopropanol, with ethanol being the preferred choice.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The chemical synthesis method of hydroxytyrosol glucoside provided by the present invention prepares hydroxytyrosol glucoside through a three-step reaction using hydroxytyrosol as a raw material. It has the characteristics of high production efficiency, readily available raw materials, high reaction yield, and low cost.

[0032] (2) This invention compares the effects of different protecting groups on the reaction. When acetyl is used as the protecting group, the reaction has three steps, each with high yield and simple operation. The second step does not require purification and can be directly used for the next step, reducing product loss and simplifying the process.

[0033] In summary, the method for synthesizing hydroxytyrosol glucoside provided by this invention has high yields in each step, a simple process, and is suitable for large-scale industrial production, showing great development potential. Attached Figure Description

[0034] Figure 1 : The 1H NMR spectrum of 2-(3,4-diacetoxyphenyl)ethyl-(2,3,4,6-O-tetraacetyl)-β-D-glucopyranoside in Example 1.

[0035] Figure 2 : The 1H NMR spectrum of 2-(3,4-dihydroxyphenyl)ethyl-β-D-glucopyranoside in Example 1. Detailed Implementation

[0036] The following description, in conjunction with several preferred embodiments, further illustrates the technical solution of the present invention in a non-limiting manner. Reagents or instruments used without a specified manufacturer are considered to be conventional products that can be purchased on the market.

[0037] Example 1: Preparation of hydroxytyrosol glucoside by acetyl protection method

[0038]

[0039] Step 1: Preparation of 3,4-diacetoxyphenylethanol

[0040] 20 g (0.13 mol) of hydroxytyrosol was dissolved in 80 ml of N,N-dimethylformamide. 15.3 g (0.272 mol) of KOH was added in portions under an ice-water bath, maintaining an internal temperature of 20–25 °C. After the addition was complete, stirring was continued at 20–25 °C for 0.5 h. The temperature was lowered to -5–10 °C, and 20.9 g (0.266 mol) of acetyl chloride was added dropwise. After the addition was complete, the reaction mixture was kept at this temperature for 2–3 h. After the reaction was complete, the reaction solution was poured into 800 ml of water and extracted with DCM. The DCM layer was washed successively with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution, and water. The DCM layer was dried, filtered, and concentrated to obtain 26.7 g of a colorless viscous liquid. Yield: 86.5%, HPLC purity: 98.57%.

[0041] Step 2: Preparation of 2-(3,4-diacetoxyphenyl)ethyl-(2,3,4,6-O-tetraacetyl)-β-D-glucopyranoside

[0042] Dissolve 20 g (0.083 mol) of 3,4-diacetoxyphenylethanol in 100 ml of DCM, add 38.8 g (0.099 mol) of β-D-glucose pentaacetate and 2.9 g (0.021 mol) of 48% boron trifluoride in tetrahydrofuran solution, heat to 40–45 °C and reflux for 3–5 h. After the reaction is complete, filter the reaction solution and wash the filtrate successively with saturated sodium bicarbonate and water. Dry the DCM layer, filter, concentrate, and purify by column chromatography (200–300 mesh silica gel column), eluting with PE / EA = 2 / 1, to obtain 29.8 g of colorless viscous liquid, yield: 63.3%, HPLC purity: 98.64%. ¹H-NMR (500 MHz, DMSO-D6): see [link to relevant data]. Figure 1 .

[0043] Step 3: Preparation of 2-(3,4-dihydroxyphenyl)ethyl-β-D-glucopyranoside

[0044] 25 g (0.043 mol) of 2-(3,4-diacetoxyphenyl)ethyl-(2,3,4,6-O-tetraacetyl)-β-D-glucopyranoside was dissolved in 125 ml of methanol. The solution was cooled to -5 to 10 °C, and NaOH was added in batches until the pH reached 10 to 11. After the addition was complete, stirring was continued for 0.5 to 1 h. The pH of the reaction solution was adjusted to neutral, and the mixture was filtered. The filtrate was concentrated to a solid-liquid mixture, and 100 ml of ethanol was added to adjust the pH to 2 to 4. The mixture was stirred at room temperature for 0.5 h and then filtered. The filtrate was concentrated, and the concentrate was subjected to column chromatography with DCM / methanol = 5 / 1 elution to give 12.3 g of off-white hydroxytyrosol glucoside solid. Yield: 89.6%, HPLC purity: 98.73%. Melting point: H-NMR (500 MHz, DMSO-D6): see Figure 2 .

[0045] Example 2: Preparation of hydroxytyrosol glucose by acetyl protection method

[0046] Step 1: Preparation of 3,4-diacetoxyphenylethanol

[0047] 100 g (0.649 mol) of hydroxytyrosol was dissolved in 400 ml of N,N-dimethylformamide. 76.4 g (1.362 mol) of KOH was added in portions under an ice-water bath, maintaining an internal temperature of 20–25 °C. After the addition was complete, stirring was continued at 20–25 °C for 0.5 h. The temperature was lowered to -5–10 °C, and 104.4 g (1.33 mol) of acetyl chloride was added dropwise. After the addition was complete, the reaction mixture was kept at this temperature for 2–3 h. After the reaction was complete, the reaction solution was poured into 4 L of water and extracted with DCM. The DCM layer was washed successively with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution, and water. The DCM layer was dried, filtered, and concentrated to obtain 135.4 g of a colorless viscous liquid. Yield: 87.6%, HPLC purity: 97.96%.

[0048] Step 2: Preparation of 2-(3,4-diacetoxyphenyl)ethyl-(2,3,4,6-O-tetraacetyl)-β-D-glucopyranoside

[0049] Dissolve 130 g (0.538 mol) of 3,4-diacetoxyphenylethanol in 650 ml of DCM, add 252 g (0.645 mol) of β-D-glucose pentaacetate and 19.0 g (0.134 mol) of 48% boron trifluoride tetrahydrofuran solution, heat to 40–45 °C and reflux for 3–5 h. After the reaction is complete, filter the reaction solution and wash the filtrate successively with saturated sodium bicarbonate and water. Dry the DCM layer, filter, and concentrate to obtain 363 g of a pale yellow oily substance, which can be used directly in the next step.

[0050] Step 3: Preparation of 2-(3,4-dihydroxyphenyl)ethyl-β-D-glucopyranoside

[0051] Dissolve 363g of the concentrate from step 2 in 1.5L of methanol, cool to -5 to 10℃, and add NaOH in batches until the pH reaches 10 to 11. Continue stirring for 0.5 to 1 hour after the addition is complete. Adjust the pH of the reaction solution to neutral, filter, concentrate the filtrate to a solid-liquid mixture, add 1.2L of ethanol, adjust the pH to 2 to 4, stir at room temperature for 0.5 hours, and filter. Concentrate the filtrate, add 600ml of water to dissolve the concentrate, adsorb with YKDH-11, elute with an ethanol gradient, collect the eluent, concentrate, and dry to obtain 95.2g of off-white hydroxytyrosol glucoside solid, yield: 57.3% (combined yield of steps 2 and 3), HPLC purity: 98.34%.

[0052] Example 3:

[0053] Step 1 of Example 3 is as follows: The key difference between Example 3 and Example 1 is that potassium tert-butoxide is used instead of KOH:

[0054] 20 g (0.13 mol) of hydroxytyrosol was dissolved in 80 ml of N,N-dimethylformamide. 30.6 g (0.272 mol) of potassium tert-butoxide was added in portions under an ice-water bath, maintaining an internal temperature of 20–25 °C. After the addition was complete, stirring was continued at 20–25 °C for 0.5 h. The temperature was lowered to -5–10 °C, and 20.9 g (0.266 mol) of acetyl chloride was added dropwise. After the addition was complete, the reaction mixture was kept at this temperature for 2–3 h. After the reaction was complete, the reaction solution was poured into 800 ml of water and extracted with DCM. The DCM layer was washed successively with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution, and water. The DCM layer was dried, filtered, and concentrated to obtain 25.5 g of a colorless viscous liquid. Yield: 82.5%, HPLC purity: 95.112%.

[0055] Steps 2 and 3 are the same as in Example 1.

[0056] Example 4:

[0057] Step 1 of Example 4 is as follows: The key difference between Example 4 and Example 1 is that triethylamine is used instead of KOH:

[0058] 20 g (0.13 mol) of hydroxytyrosol was dissolved in 80 ml of N,N-dimethylformamide. 27.6 g (0.272 mol) of triethylamine was added dropwise under an ice-water bath, maintaining an internal temperature of 20–25 °C. After the addition was complete, stirring was continued at 20–25 °C for 0.5 h. The temperature was lowered to -5–10 °C, and 20.9 g (0.266 mol) of acetyl chloride was added dropwise. After the addition was complete, the reaction mixture was kept at this temperature for 2–3 h. After the reaction was complete, the reaction solution was poured into 800 ml of water and extracted with DCM. The DCM layer was washed successively with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution, and water. The DCM layer was dried, filtered, and concentrated to obtain 48.6 g of a colorless viscous liquid. Yield: 82.5%, HPLC purity: 91.529%.

[0059] Steps 2 and 3 are the same as in Example 1.

[0060] As can be seen from Examples 3 and 4, the alkalis described in this invention can all achieve high yields.

[0061] Example 5:

[0062] Step 1 of Example 5 is as follows: The key difference from Example 1 is that the molar ratio of KOH to hydroxytyrosol is reduced from 2.1:1 to 2.0:1.

[0063] 20 g (0.13 mol) of hydroxytyrosol was dissolved in 80 ml of N,N-dimethylformamide. 14.6 g (0.259 mol) of KOH was added dropwise under an ice-water bath, maintaining an internal temperature of 20–25 °C. After the addition was complete, stirring was continued at 20–25 °C for 0.5 h. The temperature was lowered to -5–10 °C, and 20.9 g (0.266 mol) of acetyl chloride was added dropwise. After the addition was complete, the reaction mixture was kept at this temperature for 2–3 h. After the reaction was complete, the reaction solution was poured into 800 ml of water and extracted with DCM. The DCM layer was washed successively with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution, and water. The DCM layer was dried, filtered, and concentrated to obtain 24.1 g of a colorless viscous liquid. Yield: 77.9%, HPLC purity: 97.152%.

[0064] Steps 2 and 3 are the same as in Example 1.

[0065] Example 6:

[0066] Step 1 of Example 6 is as follows: The key difference from Example 1 is that the molar ratio of KOH to hydroxytyrosol is increased from 2.1:1 to 2.3:1.

[0067] 20 g (0.13 mol) of hydroxytyrosol was dissolved in 80 ml of N,N-dimethylformamide. 16.7 g (0.298 mol) of triethylamine was added dropwise under an ice-water bath, maintaining an internal temperature of 20–25 °C. After the addition was complete, stirring was continued at 20–25 °C for 0.5 h. The temperature was lowered to -5–10 °C, and 20.9 g (0.266 mol) of acetyl chloride was added dropwise. After the addition was complete, the reaction mixture was kept at this temperature for 2–3 h. After the reaction was complete, the reaction solution was poured into 800 ml of water and extracted with DCM. The DCM layer was washed successively with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution, and water. The DCM layer was dried, filtered, and concentrated to obtain 19.0 g of a colorless viscous liquid, yield: 61.3%, HPLC purity: 97.187%.

[0068] Steps 2 and 3 are the same as in Example 1.

[0069] Examples 5 and 6 show that both excessively high and low KOH feed ratios affect the yield. Reaction tests revealed that when the KOH feed ratio is too low, the raw material hydroxytyrosol fails to fully participate in the reaction, leading to a reduced yield. When the KOH feed ratio is too high, excess KOH reacts with acetyl chloride, consuming the acetylation reagent and further reducing the yield. Therefore, the range described in this invention can effectively guarantee the yield.

[0070] Comparative Example 1: Preparation of Hydroxytyrosol Glucose by Benzoyl Protection Method

[0071]

[0072] Step 1: Preparation of 3,4-dibenzoyloxyphenylethanol

[0073] 20 g (0.13 mol) of hydroxytyrosol was dissolved in 80 ml of N,N-dimethylformamide. 15.3 g (0.272 mol) of KOH was added in portions under an ice-water bath, maintaining an internal temperature of 20–25 °C. After the addition was complete, stirring was continued at 20–25 °C for 0.5 h. The temperature was lowered to -5–10 °C, and 37.4 g (0.266 mol) of benzoyl chloride was added dropwise. After the addition was complete, the reaction mixture was kept at this temperature for 2–3 h. After the reaction was complete, the reaction solution was poured into 800 ml of water and extracted with DCM. The DCM layer was washed successively with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution, and water. The DCM layer was dried, filtered, concentrated, and purified by column chromatography (200–300 mesh silica gel column) with PE / EA = 5 / 1 elution, yielding 15.6 g of a colorless viscous liquid. Yield: 33.2%, HPLC purity: 98.31%.

[0074] Step 2: Preparation of 2-(3,4-benzoyloxyphenyl)ethyl-(2,3,4,6-O-tetraacetyl)-β-D-glucopyranoside

[0075] 15 g (0.041 mol) of 3,4-dibenzoyloxyphenylethanol was dissolved in 75 ml of DCM, and 19 g (0.049 mol) of β-D-glucose pentaacetate and 1.4 g (0.01 mol) of 48% boron trifluoride / tetrahydrofuran were added. The mixture was heated to 40–45 °C and refluxed for 3–5 h. After the reaction was completed, the reaction solution was filtered, and the filtrate was washed successively with saturated sodium bicarbonate and water. The DCM layer was dried, filtered, concentrated, and purified by column chromatography (200–300 mesh silica gel column) with PE / EA = 2 / 1 elution to give 15.3 g of a pale yellow viscous liquid. Yield: 54.4%, HPLC purity: 98.52%.

[0076] Step 3: Preparation of 2-(3,4-dihydroxyphenyl)ethyl-β-D-glucopyranoside

[0077] 14 g (0.02 mol) of 2-(3,4-benzoyloxyphenyl)ethyl-(2,3,4,6-O-tetraacetyl)-β-D-glucopyranoside was dissolved in 70 mL of methanol. The solution was cooled to -5 to 10 °C, and NaOH was added in batches until the pH reached 10 to 11. After the addition was complete, stirring was continued for 0.5 to 1 h. The pH of the reaction solution was adjusted to neutral, and the mixture was filtered. The filtrate was concentrated to a solid-liquid mixture, and 140 mL of ethanol was added to adjust the pH to 2 to 4. The mixture was stirred at room temperature for 0.5 h and then filtered. The filtrate was concentrated, and the concentrate was eluted by column chromatography with a DCM / methanol ratio of 5 / 1 to give 4.9 g of off-white hydroxytyrosol glucoside solid. Yield: 79.0%, HPLC purity: 99.41%.

[0078] It can be observed that when protected with benzoyl, the reaction consists of three steps, with low yields in each step. The introduction of benzoic acid structure into the process makes the first step reaction and the purification of the final product difficult.

[0079] Comparative Example 2: Preparation of Hydroxytyrosol Glucoside by Benzyl Protection Method

[0080]

[0081] Step 1: Preparation of 3,4-dibenzyloxyphenylethanol

[0082] 20 g (0.13 mol) of hydroxytyrosol was dissolved in 80 ml of N,N-dimethylformamide. 15.3 g (0.272 mol) of KOH was added in portions under an ice-water bath, maintaining an internal temperature of 20–25 °C. After the addition was complete, stirring was continued at 20–25 °C for 0.5 h. The temperature was lowered to -5–10 °C, and 33.7 g (0.266 mol) of benzyl chloride was added dropwise. After the addition was complete, the reaction mixture was kept at this temperature for 3–5 h. After the reaction was complete, the reaction solution was poured into 800 ml of water and extracted with DCM. The DCM layer was washed successively with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution, and water. The DCM layer was dried, filtered, concentrated, and purified by column chromatography (200–300 mesh silica gel column) with PE / EA = 5 / 1 elution, yielding 18.9 g of a colorless viscous liquid. Yield: 43.5%, HPLC purity: 98.71%.

[0083] Step 2: Preparation of 2-(3,4-dibenzyloxyphenyl)ethyl-(2,3,4,6-O-tetraacetyl)-β-D-glucopyranoside

[0084] 15 g (0.044 mol) of 3,4-dibenzyloxyphenylethanol was dissolved in 75 ml of DCM, and 20.6 g (0.053 mol) of β-D-glucose pentaacetate and 1.6 g (0.011 mol) of 48% boron trifluoride / tetrahydrofuran were added. The mixture was heated to 40–45 °C and refluxed for 3–5 h. After the reaction was completed, the reaction solution was filtered, and the filtrate was washed successively with saturated sodium bicarbonate and water. The DCM layer was dried, filtered, concentrated, and purified by column chromatography (200–300 mesh silica gel column) with PE / EA = 2 / 1 elution to give 14.1 g of colorless viscous liquid. Yield: 48.2%, HPLC purity: 98.65%.

[0085] Step 3: Preparation of 2-(3,4-dibenzyloxyphenyl)ethyl-β-D-glucopyranose

[0086] 13 g (0.019 mol) of concentrated 2-(3,4-dibenzyloxyphenyl)ethyl-(2,3,4,6-O-tetraacetyl)-β-D-glucopyranoside was dissolved in 65 mL of methanol. The solution was cooled to -5 to 10 °C, and NaOH was added in batches until the pH reached 10 to 11. After the addition was complete, stirring was continued for 0.5 to 1 h. The pH of the reaction solution was adjusted to neutral, and the solution was filtered. The filtrate was concentrated, and the concentrate was subjected to column chromatography with DCM / methanol = 10 / 1 elution to give 9.1 g of a pale yellow viscous liquid. Yield: 95.8%, HPLC purity: 99.05%.

[0087] Step 4: Preparation of 2-(3,4-dihydroxyphenyl)ethyl-β-D-glucopyranoside

[0088] 9 g of 2-(3,4-dibenzyloxyphenyl)ethyl-β-D-glucopyranoside was dissolved in 100 ml of methanol, and 0.9 g of palladium on carbon was added. Hydrogen gas was bubbled through the solution, and the reaction was carried out at 40–50 °C for 8–12 h. After the reaction was completed, the solution was filtered, the filtrate was concentrated, and the concentrate was subjected to column chromatography with a DCM / methanol ratio of 5 / 1 to elute, yielding 5.5 g of off-white hydroxytyrosol glucoside. Yield: 96.8%, HPLC purity: 98.16%.

[0089] It can be observed that when benzyl protection is used, the reaction involves four steps. The first step has a low yield and is difficult to purify. The reaction process is lengthy, resulting in a low overall yield and requiring dangerous operations such as hydrogenation reduction.

[0090] Comparative Example 3: The effect of different Lewis acid catalysts on glycosylation reaction.

[0091] 1. Stannzyl tetrachloride-catalyzed glycosylation reaction

[0092] 10 g (0.041 mol) of 3,4-diacetoxyphenylethanol was dissolved in 50 ml of DCM, and 19.3 g (0.049 mol) of β-D-glucose pentaacetate and 2.7 g (0.01 mol) of tin tetrachloride were added. The reaction was carried out at 20–25 °C for 2–3 h. After the reaction was completed, the reaction solution was washed twice with 1 M hydrochloric acid, twice with saturated sodium bicarbonate, and once with water. The DCM layer was dried, filtered, concentrated, and purified by column chromatography (200–300 mesh silica gel column) with PE / EA = 2 / 1 elution to give 9.0 g of a pale yellow oily substance. Yield: 38.6%, HPLC purity: 98.84%.

[0093] 2. Ferric chloride-catalyzed glycosylation reaction

[0094] 10 g (0.041 mol) of 3,4-diacetoxyphenylethanol was dissolved in 50 ml of DCM, and 19.3 g (0.049 mol) of β-D-glucose pentaacetate and 1.7 g (0.01 mol) of ferric chloride were added. The mixture was reacted at 20–25 °C for 3 h. After the reaction was completed, the reaction solution was washed twice with 1 M hydrochloric acid, twice with saturated sodium bicarbonate, and once with water. The DCM layer was dried, filtered, concentrated, and purified by column chromatography (200–300 mesh silica gel column) with PE / EA = 2 / 1 elution, yielding 7.6 g of a pale yellow oily substance, yield: 32.6%, HPLC purity: 99.59%.

[0095] 3. Glycosylation reaction catalyzed by aluminum trichloride

[0096] 10 g (0.041 mol) of 3,4-diacetoxyphenylethanol was dissolved in 50 ml of DCM, and 19.3 g (0.049 mol) of β-D-glucose pentaacetate and 1.4 g (0.01 mol) of aluminum trichloride were added. The reaction was carried out at -5 to 10 °C for 1 to 2 h. After the reaction was completed, the reaction solution was washed twice with 1 M hydrochloric acid, twice with saturated sodium bicarbonate, and once with water. The DCM layer was dried, filtered, concentrated, and purified by column chromatography (200-300 mesh silica gel column) with PE / EA = 2 / 1 elution to give 5.10 g of pale yellow oil, yield: 27.3%, HPLC purity: 99.74%.

[0097] 4. Magnesium chloride-catalyzed glycosylation reaction

[0098] 10 g (0.041 mol) of 3,4-diacetoxyphenylethanol was dissolved in 50 mL of toluene. 19.3 g (0.049 mol) of β-D-glucose pentaacetate, 1.0 g (0.01 mol) of magnesium chloride, and 4 g of anhydrous calcium sulfate were added. The mixture was heated to 80 °C and reacted for 3–5 h. After the reaction was complete, the reaction solution was filtered. The filtrate was washed twice with 1 M hydrochloric acid, twice with saturated sodium bicarbonate, and once with water. The toluene layer was dried, filtered, concentrated, and purified by column chromatography (200–300 mesh silica gel column) with PE / EA = 2 / 1 elution, yielding 1.5 g of a pale yellow oily substance. Yield: 6.5%, HPLC purity: 99.15%.

[0099] It is evident that using a Lewis acid different from that of the present invention leads to a decrease in yield.

[0100] Comparative Example 4: Effect of different acetylation reagents on the reaction in step 1 of the acetyl protection method

[0101] 20 g (0.13 mol) of hydroxytyrosol was dissolved in 80 ml of N,N-dimethylformamide. 15.3 g (0.272 mol) of potassium hydroxide was added dropwise under an ice-water bath, maintaining an internal temperature of 20–25 °C. After the addition was complete, stirring was continued at 20–25 °C for 0.5 h. The temperature was lowered to -5–10 °C, and 27.1 g (0.266 mol) of acetic anhydride was added dropwise. After the addition was complete, the reaction mixture was kept at this temperature for 2–3 h. After the reaction was complete, the reaction solution was poured into 800 ml of water and extracted with DCM. The DCM layer was washed successively with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution, and water. The DCM layer was dried, filtered, and concentrated to obtain 11.7 g of a pale yellow viscous liquid. Yield: 37.9%, HPLC purity: 67.674%.

[0102] When acetic anhydride was used instead of acetyl chloride, the yield and purity decreased significantly. Monitoring of the reaction revealed that acetic anhydride byproduct acetic acid reacted further with the product to generate impurities.

[0103] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A method for preparing hydroxytyrosol glucoside, characterized in that, This method uses hydroxytyrosol as a starting material and involves acetylation, glycosylation, and hydrolysis to prepare hydroxytyrosol glucoside. Step 1: Dissolve hydroxytyrosol in a polar aprotic solvent, and react it with an acetylation reagent under the action of a base to generate 3,4-diacetoxyphenethanol; Step 2: 3,4-Diacetoxyphenylethanol and β-D-glucopentaacetate undergo a condensation reaction under the action of Lewis acid to obtain 2-(3,4-diacetoxyphenyl)ethyl-(2,3,4,6-O-tetraacetyl)-β-D-glucopyranoside. Step 3: 2-(3,4-diacetoxyphenyl)ethyl-(2,3,4,6-O-tetraacetyl)-β-D-glucopyranoside is hydrolyzed under alkaline conditions to remove the acetyl group, yielding hydroxytyrosol glucoside.

2. The method for preparing hydroxytyrosol glucoside according to claim 1, characterized in that, The polar aprotic solvent mentioned in step 1 is selected from: tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide or dimethyl sulfoxide, with N,N-dimethylformamide being the preferred polar aprotic solvent.

3. The method for preparing hydroxytyrosol glucoside according to claim 1, characterized in that, The base mentioned in step 1 is selected from: sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium methoxide, sodium tert-butoxide, potassium tert-butoxide, pyridine, triethylamine or N,N-diisopropylethylamine; the preferred base is sodium hydroxide, potassium hydroxide, potassium tert-butoxide or triethylamine; preferably, the molar ratio of potassium hydroxide to hydroxytyrosol is (2.0-2.5):1.0, and the preferred molar ratio of potassium hydroxide to hydroxytyrosol is (2.0-2.1):1.

0.

4. The method for preparing hydroxytyrosol glucoside according to claim 1, characterized in that, The acetylation reagent mentioned in step 1 is acetyl chloride; preferably, the molar ratio of acetyl chloride to hydroxytyrosol is (2.0~2.2):1.0, and more preferably, the molar ratio of acetyl chloride to hydroxytyrosol is 2.05:1.

0.

5. The method for preparing hydroxytyrosol glucoside according to claim 1, characterized in that, In step 1, the reaction temperature is -5 to 25°C, and the preferred reaction temperature for adding alkali is 20 to 25°C, with a reaction time of 20 to 40 minutes; the acetylation reaction temperature is -5 to 10°C, with a reaction time of 2 to 3 hours.

6. The method for preparing hydroxytyrosol glucoside according to claim 1, characterized in that, The reaction solution from step 1 was further neutralized, extracted, washed, dried, and concentrated to obtain a colorless oily substance, 3,4-diacetoxyphenylethanol.

7. The method for preparing hydroxytyrosol glucoside according to claim 1, characterized in that, In step 2, the molar ratio of 3,4-diacetoxyphenylethanol to β-D-glucose pentaacetate is 1:(1.0-1.4), preferably 1:1.2; preferably, the Lewis acid in step 2 is boron trifluoride; preferably, the solvent in step 2 is DCM; preferably, the reaction temperature in step 2 is 40-45°C, and the reaction time is 3-5 h; preferably, step 3 is carried out directly after step 2, or the reaction solution in step 2 is washed, dried, concentrated to obtain a pale yellow oil before step 3 is carried out.

8. The method for preparing hydroxytyrosol glucoside according to claim 1, characterized in that, The solvent used in step 3 is selected from methanol, ethanol, isopropanol, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, with methanol being preferred; preferably, the reaction temperature in step 3 is -5 to 10°C, and the reaction time is 0.5 to 1 h.

9. The method for preparing hydroxytyrosol glucoside according to claim 1, characterized in that, The alkali used in step 3 is selected from: sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium methoxide, sodium tert-butoxide, and potassium tert-butoxide; the preferred alkali is sodium hydroxide or potassium hydroxide; preferably, the amount of alkali used in step 3 is to adjust the pH of the reaction solution to maintain 9-11, and the preferred pH is 10-11.

10. The method for preparing hydroxytyrosol glucoside according to claim 1, characterized in that, The reaction solution in step 3 is neutralized, filtered, concentrated, dissolved, and purified by macroporous resin to obtain a white solid, which is hydroxytyrosol glucoside. Preferably, the macroporous resin is selected from: YKDH-5, YKDH-11, D101, D4008, H107, HP-20, with YKDH-11 being the most preferred. Preferably, the eluent for the macroporous resin purification in step 3 is selected from: methanol, ethanol, isopropanol, with ethanol being the most preferred.

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