A method for preparing rebaudioside B using a mild hydrolysis system

Abstract

The present application relates to the technical fields of natural product chemical synthesis, and particularly relates to a method for preparing rebaudioside B by using a mild hydrolysis system, taking rebaudioside A as raw material, taking tetrahydrofuran (THF) as solvent, adding deionized water, triethylamine (Et3N) and lithium bromide (LiBr) to form a mild hydrolysis system, stirring and reacting at 25-40 DEG C for 4-8 h, and then performing steps such as decompression desolventization, pH adjustment, extraction and recrystallization to obtain high-purity rebaudioside B; the method solves the problems of the existing technology, such as many side reactions, low yield, poor purity, serious equipment corrosion in the strong alkali hydrolysis method, and high cost, long reaction period and difficulty in industrial amplification in the enzyme catalytic hydrolysis method.

Description

Technical Field

[0001] This invention relates to the field of natural product chemical synthesis technology, and specifically to a method for preparing ribobadiol B using a mild hydrolysis system. Background Technology

[0002] Reb A and Reb B are both core diterpenoid sweet glycosides extracted from Stevia rebaudiana. Both are natural, low-calorie, and high-sweetness, with broad application prospects in the food, beverage, and pharmaceutical industries. Reb A is approximately 350–450 times sweeter than sucrose, with a pure sweetness and very weak aftertaste, making it the most widely used high-intensity natural sweetener in industry. Reb B is approximately 150–300 times sweeter than sucrose, with a rich taste and long finish, offering a superior taste compared to Reb A. It has unique application value in functional foods, sugar-free beverages, and pharmaceutical flavoring.

[0003] However, the content of ribobandi glycoside B in natural stevia is extremely low (usually below 2%), far lower than that of ribobandi glycoside A (approximately 50–70%), which cannot meet the needs of industrial production and the market. Therefore, the selective hydrolysis of ribobandi glycoside A to break its specific ester bonds and prepare high-purity ribobandi glycoside B has become the mainstream technical route for the industrial production of ribobandi glycoside B.

[0004] Currently, the existing methods for preparing rebaudioside B by hydrolysis of rebaudioside A are mainly divided into strong alkaline hydrolysis and enzyme-catalyzed hydrolysis. Both methods have obvious technical defects, as follows:

[0005] 1. Strong Alkali Hydrolysis Method: This method uses strong alkalis such as sodium hydroxide and potassium hydroxide as hydrolysis reagents. The reaction conditions are harsh, typically requiring pH 9-12 and 80-100℃. These harsh conditions easily lead to excessive glycosidic bond breakage of ribavirin A and destruction of the steviol nucleocyclic structure, generating a large number of byproducts (such as steviol and stevia glycosides), which can account for 15-30%. Simultaneously, the product purity is low (usually below 85%), requiring multiple recrystallizations to meet industrial application requirements, resulting in a low product yield of only 50-65%. Furthermore, strong alkalis are highly corrosive, severely damaging production equipment, and the resulting waste alkaline liquid has high environmental treatment costs, failing to meet the requirements of green and clean production.

[0006] 2. Enzymatic hydrolysis: This method uses specific glycosidases as catalysts. Although the reaction conditions are relatively mild, it has many limitations: glycosidases are expensive, have poor stability, are easily inactivated during the reaction, and have a long reaction cycle (usually 24-48 hours). Bacterial contamination is also common during the reaction, affecting product quality. Furthermore, enzyme-catalyzed reactions have strict requirements on substrate concentration, which is usually below 5%, leading to low industrial scale-up efficiency, long production cycles, and high costs. In addition, glycosidases have limited selectivity for hydrolysis sites, easily generating isomeric impurities that affect product purity.

[0007] The LiBr / Et3N / THF system is a recognized ultra-mild selective hydrolysis system for esters / glycosides in organic synthesis. Its core advantage lies in its ability to generate low concentrations of LiOH (triethylamine removes protons from trace amounts of water in the system, Li...) in situ. + With OH - This system forms a weakly bound state, avoiding direct attack of the substrate by strong bases. The reaction conditions are mild (room temperature -40℃, near-neutral pH 7–8), and it does not damage sensitive structures such as chiral centers, polyether bonds, and glycosidic rings. It also exhibits extremely high selectivity for ester / glycosidic esters, without hydrolyzing ordinary glycosidic bonds and amide bonds. The system uses inexpensive reagents, has a fast reaction rate, and requires simple post-processing, making it highly promising for industrial applications.

[0008] In view of the above-mentioned defects, the inventors of this invention have finally obtained this invention after a long period of research and practice. Summary of the Invention

[0009] The purpose of this invention is to solve the problems of strong alkaline hydrolysis method having many side reactions, low yield, poor purity, and serious equipment corrosion, as well as enzyme-catalyzed hydrolysis method having high cost, long reaction cycle, and difficulty in industrial scale-up. The invention provides a method for preparing ribobadiol B using a mild hydrolysis system.

[0010] To achieve the above objectives, this invention discloses a method for preparing riboflavin B using a mild hydrolysis system, comprising the following steps:

[0011] S1, using riboboroside A as raw material and tetrahydrofuran as solvent, is composed of deionized water, triethylamine and lithium bromide to form a mild hydrolysis system;

[0012] S2, after stirring the mild hydrolysis system obtained in step S1, ribaodi glycoside B is obtained.

[0013] In step S1, the substrate concentration of ribobandi glycoside A in tetrahydrofuran is 0.1–0.3 mmol / mL.

[0014] In step S1, the volume of deionized water is 1.0% to 3.0% of the volume of tetrahydrofuran.

[0015] In step S1, the molar ratio of triethylamine to ribobandiidine A is 3-5:1.

[0016] In step S1, the molar ratio of lithium bromide to rebaudioside A is 8-12:1.

[0017] In step S2, the reaction temperature is 25~40 ℃ and the reaction time is 4~8 h.

[0018] In step S2, after the reaction is completed, desolvation under reduced pressure, pH adjustment, extraction, and recrystallization are required to obtain high-purity riboside B.

[0019] The structural features of Rebaudioside A are as follows: a β-D-glucopyranosyl-1-O- ester bond (an easily hydrolyzed site) is attached to the C-19 position of the stevioside core, and a trisaccharide chain (β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl) is attached to the C-13 position. The target reaction of this invention is to selectively hydrolyze the glucopyranosyl ester bond at the C-19 position of Rebaudioside A, releasing one molecule of D-glucose while preserving the integrity of the trisaccharide chain at the C-13 position, ultimately generating Rebaudioside B.

[0020] The reaction formula is as follows:

[0021] Reb A + H2O —(LiBr, Et3N / THF, 25–40 ℃)→ Reb B + D-glucose.

[0022] The reaction mechanism of this invention is as follows:

[0023] 1. Triethylamine (Et3N), as a weak base, activates trace amounts of deionized water in the system, removing protons from water molecules to generate Et3NH. + With OH - ;

[0024] 2. Li in the system + With the generated OH - The formation of a weakly bound state of low-concentration active LiOH avoids direct attack of strong bases on the sensitive structure of riboboroside A;

[0025] 3. Li + It chelates with the carbonyl oxygen and ortho heteroatom of the ester group in the riboside A molecule to form a five-membered ring intermediate, thereby activating the carbonyl carbon of the ester group and increasing its electrophilicity;

[0026] 4. Low concentration of OH in the system - It selectively attacks the activated C-19 ester carbonyl group, undergoing a nucleophilic addition-elimination reaction, breaking the ester bond, releasing one molecule of D-glucose, and simultaneously generating riboside B.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] 1. Extremely mild reaction conditions: The present invention adopts a mild hydrolysis system of LiBr / Et3N / THF, with a reaction temperature of 25–40℃ (preferably 30℃) and a system pH of 7–8 (near neutral). It does not require the use of strong acids or bases, and can completely protect the glycosidic bond, chiral center and steviol core structure of ribobadiol B. There is no excessive hydrolysis byproduct generated, and the purity of the product is significantly improved.

[0029] 2. High selectivity and product purity: The hydrolysis system of this invention has a selectivity of over 99% for the C-19 glucosyl ester bond of riboboroside A, which can accurately break the target ester bond and completely preserve the integrity of the C-13 trisaccharide chain; the crude purity of riboboroside B can reach over 95%, and the purity after purification is ≥99% (HPLC detection), which meets the requirements of industrial-grade high purity.

[0030] 3. Significantly improved product yield: The total yield of the method of the present invention can reach 88-90%, which is significantly improved compared with the traditional strong alkali hydrolysis method (50-65%) and enzyme-catalyzed hydrolysis method (65-75%). This effectively reduces raw material loss and improves production economic efficiency.

[0031] 4. High efficiency and low cost: The reaction cycle is only 4–8 hours (preferably 6 hours), which is significantly more efficient than enzyme-catalyzed hydrolysis (24–48 hours); the reagents used (LiBr, Et3N, THF) are all inexpensive industrial-grade reagents, and the THF solvent can be recycled (recovery rate >90%); the reaction does not require special equipment and does not corrode equipment, which greatly reduces the equipment investment and operating costs for industrial production. According to calculations, the cost of industrial production can be reduced by more than 40%.

[0032] 5. Green and environmentally friendly, meeting clean production requirements: No high-salt or high-alkali waste liquid is generated during the reaction process, and the post-processing steps are simple. High-purity products can be obtained simply through extraction, concentration, and recrystallization. The THF solvent can be recycled and reused, resulting in low emissions of waste gas, wastewater, and solid waste. This is environmentally friendly and in line with the development trend of modern industrial green and clean production. Attached Figure Description

[0033] Figure 1 A comparative diagram of the chemical structures of Reb A and Reb B;

[0034] Figure 2 This is the HPLC chromatogram of the refined rabodiin B obtained in Example 1. Detailed Implementation

[0035] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0036] The specific specifications of the raw materials used in the following examples are as follows to ensure the stability, selectivity, and product quality of the reaction:

[0037] 1. Rebadinoside A: Industrial grade, purity ≥90% (HPLC detection);

[0038] 2. Lithium bromide (LiBr): Anhydrous grade, purity ≥99%;

[0039] 3. Triethylamine (Et3N): Analytical grade (AR grade), purity ≥99%, anhydrous treatment before use;

[0040] 4. Tetrahydrofuran (THF): Analytical grade (AR grade), moisture content <0.05%;

[0041] 5. n-Butanol, methanol, and acetone: all were analytical grade (AR grade);

[0042] 6. Deionized water: conductivity ≤10μS / cm;

[0043] 7. Dilute hydrochloric acid: 1 mol / L, prepared by mixing analytical grade concentrated hydrochloric acid with deionized water.

[0044] (1) Limitation of the range of key process parameters

[0045] Rebaudioside A substrate concentration in tetrahydrofuran: 0.1–0.3 mmol / mL;

[0046] The volume of deionized water is 1.0% to 3.0% of the volume of tetrahydrofuran;

[0047] The molar ratio of triethylamine to ribobandiidine A was 3.0–5.0 eq.

[0048] Anhydrous lithium bromide to rebaudioside A molar ratio: 8.0–12.0 eq;

[0049] Reaction temperature: 25–40 °C;

[0050] Reaction time: 4–8 h.

[0051] (2) Optimize process parameters

[0052] The substrate concentration was 0.2 mmol / mL, the deionized water content was 2.0%, the triethylamine was 4.0 eq, the lithium bromide was 10.0 eq, the reaction temperature was 30 ℃, and the reaction time was 6 h.

[0053] Example 1

[0054] 1. Preparation of reaction solution: In a 5L reactor, add 300g of riboboroside A (92% purity, 0.28mol) and 1.4L of anhydrous THF. Stir at room temperature for 30min until riboboroside A is completely dissolved (concentration 0.2 mmol / mL). Add 28mL of deionized water (2.0% by volume) to the above solution and stir until homogeneous. Then add 113g of triethylamine (1.12mol, 4.0 eq) and 244g of anhydrous LiBr (2.8mol, 10.0 eq) in sequence, and continue stirring for 20min until the reagents are completely dissolved to obtain the reaction solution.

[0055] 2. Hydrolysis reaction: Nitrogen gas (N2) was introduced into the reaction vessel for protection, and the reaction temperature was controlled at 30℃. The reaction was stirred at a constant temperature for 6 hours. The reaction progress was monitored by HPLC. The HPLC detection conditions were: C18 column (4.6×250mm, 5μm), mobile phase methanol-water (65:35, v / v), flow rate 1.0mL / min, and detection wavelength 210nm. The reaction was terminated when the residual amount of riboflavin A was detected to be 0.6%.

[0056] 3. Post-treatment and crude product enrichment: The reaction solution was placed in a vacuum distillation apparatus and the THF solvent was removed under reduced pressure at 40℃ and -0.08 MPa. 2.5L of pure water was added to the residue and stirred to disperse evenly. The pH of the system was adjusted to 5.5 with 1mol / L dilute hydrochloric acid. 2L of n-butanol was added for extraction. The extraction was repeated 3 times, and the n-butanol organic phases from the 3 extractions were combined. The organic phase was washed once with 1L of saturated brine. After standing and separating the layers, the aqueous phase was discarded. The organic phase was placed in a vacuum concentration apparatus and concentrated to dryness at 45℃ to obtain 215g of crude riboside B in the form of a light yellow powder with a purity of 96.5%.

[0057] 4. Purification: Add 1.2 L of methanol / acetone mixed solvent (1:1, v / v) to the crude product, heat to 40 °C and stir until completely dissolved; place the solution in a 0 °C refrigerator and crystallize for 8 h; after crystallization, filter, wash the filter cake twice with 50 mL of cold methanol (0–5 °C) each time; place the filter cake in a 45 °C vacuum drying oven and dry for 4 h until constant weight, to obtain 204 g of refined Rebaudioside B, with a yield of 89% and a purity of 99%.

[0058] Example 2

[0059] Parameter optimization experiment: Effect of LiBr dosage:

[0060] Using the same process steps as in Example 1, only the amount of LiBr was changed to 8.0 eq, 10.0 eq, and 12.0 eq, respectively, while other parameters remained unchanged. The experimental results are shown in Table 1 below:

[0061] Table 1. Experimental results obtained with different amounts of LiBr.

[0062]

[0063] Conclusion: As shown in Table 1, the reaction efficiency, product purity and yield are all optimal when the amount of LiBr is 10.0 eq. Too much or too little LiBr will result in a slight decrease in yield or purity.

[0064] Example 3

[0065] Experiment on the effect of substrate concentration on ribobandinoside A:

[0066] Using the same process steps as in Example 1, only the amount of LiBr was changed to 0.1 mmol / mL, 0.2 mmol / mL, and 0.3 mmol / mL, respectively, while keeping other parameters unchanged. The experimental results are shown in Table 2 below:

[0067] Table 2 Effect of different substrate concentrations of ribobandiidine A on preparation efficiency

[0068]

[0069] As shown in Table 2, the substrate concentration of riboside A is limited to 0.1–0.3 mmol / mL, preferably 0.2 mmol / mL; too high a concentration results in poor solubility and incomplete reaction; too low a concentration results in low production capacity.

[0070] Example 4

[0071] Experiment on the effect of deionized water dosage:

[0072] Using the same process steps as in Example 1, only the volume percentage of deionized water was changed to 1.0%, 2.0%, and 3.0%, respectively, while other parameters remained unchanged. The experimental results are shown in Table 3 below:

[0073] Table 3. Effect of different deionized water volume ratios on preparation results

[0074]

[0075] As shown in Table 3, the volume percentage of deionized water is limited to 1.0% to 3.0%, with 2.0% being preferred; insufficient water volume results in weak hydrolysis activity, while excessive water volume can easily trigger secondary hydrolysis.

[0076] Example 5

[0077] Experiment on the effect of triethylamine dosage:

[0078] Using the same process steps as in Example 1, only the amount of triethylamine was changed to 3.0 eq, 4.0 eq, and 5.0 eq, respectively, while other parameters remained unchanged. The experimental results are shown in Table 4 below:

[0079] Table 4 Effect of different amounts of triethylamine on the preparation effect

[0080]

[0081] As shown in Table 4, the amount of triethylamine is limited to 3.0–5.0 eq, with 4.0 eq being preferred; too low a concentration results in insufficient activation, while too high a concentration leads to excessive alkalinity and the generation of secondary impurities.

[0082] Example 6

[0083] Experiment on the effect of reaction temperature:

[0084] Using the same process steps as in Example 1, only the reaction temperature was changed to 25 ℃, 30 ℃, and 40 ℃, while other parameters remained unchanged. The experimental results are shown in Table 5 below:

[0085] Table 5 Effect of different reaction temperatures on the preparation effect

[0086]

[0087] As shown in Table 5, the reaction temperature is limited to 25–40 °C, with 30 °C being preferred; the reaction rate is slow at low temperatures, while high temperatures can easily damage the glycoside structure.

[0088] Example 7

[0089] Effect of reaction time on the experiment:

[0090] Using the same process steps as in Example 1, only the reaction time was changed to 4 h, 6 h, and 8 h, respectively, while other parameters remained unchanged. The experimental results are shown in Table 6 below:

[0091] Table 6 Effect of different reaction times on preparation results

[0092]

[0093] As shown in Table 6, the reaction time is limited to 4 to 8 hours, with 6 hours being preferred. Shorter times result in incomplete reactions, while longer times increase side reactions and decrease yield.

[0094] Comparative Example

[0095] Traditional strong alkali hydrolysis method:

[0096] 1. Preparation of reaction solution: Take 300g of Rebaudioside A raw material (purity 92%, 0.28mol), add 3L of deionized water, stir to dissolve; add sodium hydroxide solid, adjust the pH of the system to 13, stir evenly to obtain the reaction solution.

[0097] 2. Hydrolysis reaction: The reaction temperature was controlled at 60℃ and the reaction was stirred at a constant temperature for 3 hours. The reaction was terminated when the residual amount of riboflavin A was less than 1.0% by HPLC monitoring.

[0098] 3. Post-treatment and purification: Adjust the pH of the system to 7 with 1 mol / L dilute hydrochloric acid, and let it stand to precipitate; collect the precipitate by suction filtration, wash it 3 times with pure water; add the precipitate to methanol, heat to dissolve, recrystallize, refrigerate at 0℃ for 8 hours to crystallize, filter and dry to obtain 126g of ribobadiin B product.

[0099] Results: The purity of the obtained riboside B was 82.3% as determined by HPLC; the total yield was 54.9%; the product contained 12.7% byproducts (stevioside, steviol glycoside, etc.), and some products showed glycosidic bond breakage.

[0100] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. A method for preparing ribobandibidine B using a mild hydrolysis system, characterized in that, Includes the following steps: S1, using riboboroside A as raw material and tetrahydrofuran as solvent, is composed of deionized water, triethylamine and lithium bromide to form a mild hydrolysis system; S2, after stirring the mild hydrolysis system obtained in step S1, ribaodi glycoside B is obtained.

2. The method for preparing ribobandibidine B using a mild hydrolysis system as described in claim 1, characterized in that, In step S1, the substrate concentration of ribobandi glycoside A in tetrahydrofuran is 0.1–0.3 mmol / mL.

3. The method for preparing ribobandibidine B using a mild hydrolysis system as described in claim 1, characterized in that, In step S1, the volume of deionized water is 1.0% to 3.0% of the volume of tetrahydrofuran.

4. The method for preparing ribobandibidine B using a mild hydrolysis system as described in claim 1, characterized in that, In step S1, the molar ratio of triethylamine to ribobandiidine A is 3-5:

1.

5. The method for preparing ribobandibidine B using a mild hydrolysis system as described in claim 1, characterized in that, In step S1, the molar ratio of lithium bromide to rebaudioside A is 8-12:

1.

6. The method for preparing ribobandibidine B using a mild hydrolysis system as described in claim 1, characterized in that, In step S2, the reaction temperature is 25~40℃ and the reaction time is 4~8h.

7. The method for preparing ribobandibidine B using a mild hydrolysis system as described in claim 1, characterized in that, In step S2, after the reaction is completed, desolvation under reduced pressure, pH adjustment, extraction, and recrystallization are required to obtain high-purity riboside B.

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