Method for preparing L-galactose

Abstract

The invention discloses a method for preparing L-galactose, which comprises the following steps: mixing pectin and dilute sulphuric acid, and hydrolyzing to generate D-galacturonic acid; adding CaCO3 to neutralize, filtering to remove CaSO4, and neutralizing heat to preheat a cyclization system; lactic acid and acetic acid are added into the hydrolysate, the temperature is raised to 88-92 DEG C at the speed of 1-2 DEG C / min, heat preservation is conducted for 3-4 hours, and L-galactosyl-1, 4-lactone is generated; concentrating the cyclized liquid, adding L-tartaric acid, adjusting the pH value to 8.5-9.0, and reducing lactone by using NaBH4; recrystallizing with ethanol to obtain an L-galactose crystal; according to the method, cyclization is promoted while the pH is stabilized through the lactic acid-acetic acid synergistic effect, and the lactone yield exceeds 75%; l-tartaric acid multiple hydrogen bonds are used for fixing lactone conformation, NaBH4 is forced to attack carbonyl carbon from the Re surface, the L-configuration selectivity reaches 97% ([alpha] D20 = + 72.5 degrees), and a chromatographic separation step is omitted.

Description

Technical Field

[0001] The present invention relates to the technical field of L-galactose preparation, and particularly to a method for preparing L-galactose. Background Art

[0002] L-galactose is a rare monosaccharide with important physiological activities and is widely used in the fields of food, medicine, and cosmetics. However, its efficient preparation still faces many technical bottlenecks, mainly concentrated in problems such as low raw material utilization efficiency, difficult control of side reactions, poor chiral selectivity, and high energy consumption. In the prior art, there are many defects in the processes for pectin hydrolysis, cyclization, and reduction.

[0003] For example, although the traditional acid hydrolysis method catalyzed by sulfuric acid can effectively break the pectin glycosidic bond, when the acid concentration exceeds 1.5%, it is easy to cause the dehydration reaction of D-galacturonic acid to generate carbonized products such as 5-hydroxymethylfurfural (HMF), resulting in the yield of the target product dropping to less than 60%. For example, the patent CN104086667B uses ultrasonic-assisted acid method, although it shortens the extraction time, still needs to strictly control the acid to avoid molecular chain degradation, but does not solve the side reaction problem caused by too high local H⁺ concentration. The conventional sodium borohydride reduction method lacks an effective chiral induction mechanism, and the L / D configurations are mixed in the product, and the ratio of the two configurations is about 1:1. It needs to rely on chromatographic separation and purification, with cumbersome steps and a yield loss of more than 30%. In the prior art, although attempts have been made to use chiral ligands such as binaphthol for directional regulation, the ligand synthesis is complex and difficult to recycle, and it is difficult to be applied industrially. The patented technology represented by CN104086667B improves the pectin extraction efficiency through ultrasonic assistance, but still has the following disadvantages: (1) It does not design a buffer system for the carbonization side reaction, and the HMF content is higher than 10%; (2) The cyclization stage depends on external temperature control, with high equipment investment; (3) It lacks a means of stereoselective regulation, and the optical purity of the product is insufficient. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing L-galactose. The present invention uses the synergistic effect of lactic acid and acetic acid to effectively control the conversion of D-galacturonic acid to cyclize to form L-galactono-1,4-lactone, and then uses the two hydroxyl groups (-OH) and two carboxylic acid groups (-COOH) of L-tartaric acid to form multiple hydrogen bonds with the carbonyl oxygen and adjacent hydroxyl groups of the lactone, fixing the lactone molecular conformation, forcing NaBH4 to attack the carbonyl carbon from the Re face, hindering the formation of the D-configuration transition state, and increasing the yield of the L-configuration.

[0005] To solve this technical problem, the technical solution of the present invention is: a method for preparing L-galactose, comprising the following steps: S1. The synergistic catalysis of lactic acid and acetic acid cyclizes D-galacturonic acid to form L-galactono-1,4-lactone; Lactic acid that stabilizes the carboxylic acid form of D-galacturonic acid and acetic acid that promotes the condensation of the C1 carboxylic acid and the C4 hydroxyl group within the D-galacturonic acid molecule through hydrophobic interaction are added to a solution containing D-galacturonic acid; The temperature is raised to 88 °C - 92 °C and kept warm for 3 hours - 4 hours; the reaction ends; S2. Concentrate the reaction solution obtained in S1; S3. Add L-tartaric acid to the concentrated solution obtained in S2. L-tartaric acid fixes the conformation of L-galactonic acid-1,4-lactone through a hydrogen bond network, and NaBH4 is used to directionally reduce L-galactonic acid-1,4-lactone to obtain L-galactose.

[0006] Preferably, the method for catalytic hydrolysis of pectin to produce D-galacturonic acid includes the following steps: S11. Mix pectin and dilute sulfuric acid, raise the temperature to 90 °C - 100 °C, stir, and react for 3 hours - 5 hours; S12. After the HPLC monitors that the yield of D-galacturonic acid exceeds 80%, add CaCO3 in batches to neutralize the sulfuric acid in the reaction solution to a solution pH of 4.8 to 5.2, and release the heat of neutralization. Filter to remove CaSO4.

[0007] In step S11 of the present invention, it is necessary to continuously stir and react at 90 °C - 100 °C for 3 - 5 hours to ensure that pectin is fully hydrolyzed into D-galacturonic acid. The temperature needs to be maintained before the reaction ends and cannot be cooled naturally.

[0008] Preferably, the mass fraction of dilute sulfuric acid in step S11 is 0.8% - 1.2%. The present invention utilizes the dissociation of dilute sulfuric acid into H + and SO4 2- . H + . As a protonic acid, it preferentially attacks the oxygen atom of the α-1,4-glycosidic bond in the pectin molecule, resulting in the cleavage of the glycosidic bond and the formation of D-galacturonic acid and short-chain oligosaccharides; the present invention controls the mass fraction of dilute sulfuric acid to 0.8% - 1.2% to limit the dehydration reaction, i.e., the side reaction, caused by excessive H⁺.

[0009] Preferably, the solid-liquid ratio of pectin to dilute sulfuric acid is 1 g:10 ml.

[0010] Preferably, the lactic acid concentration in step S1 is 0.5 M - 1 M, and the volume ratio of lactic acid to acetic acid is 3:1.

[0011] The present invention strictly controls the volume ratio of lactic acid to acetic acid and strictly maintains the pH at 4.0 - 5.0. It uses lactic acid to stabilize the carboxylic acid form and acetic acid to control the pH and promote cyclization, thereby improving the yield.

[0012] Preferably, in step S1, the initial temperature of the preparation process of the cyclization of D-galacturonic acid catalyzed by lactic acid-acetic acid to produce L-galactonic acid-1,4-lactone increases due to the absorption of the neutralization heat of CaCO3, and then it is raised to 90 °C at a rate of 1-2 °C / min.

[0013] Preferably, the process conditions for the directional reduction of L-galactonic acid-1,4-lactone by NaBH4 to obtain L-galactose in step S3 are as follows: S31. Adjust the pH of the system to 8.5 to 9.0, add NaBH4, and the molar ratio of NaBH4 to L-galactonic acid-1,4-lactone is 1.2:1; S32. Recrystallize with ethanol to obtain L-galactose.

[0014] In the present invention, NaBH4 is in excess to ensure the full reduction of the lactone carbonyl group.

[0015] In the present invention, by the synergistic action of multiple hydrogen bonds and steric hindrance, the ratio of the L-configuration to the D-configuration reaches 88:12, while it is 50:50 without adding an inducer. The optical purity [α]D 20 = +72.5°, approaching the enzymatic method level; and in the present invention, L-tartaric acid is used. L-tartaric acid has high water solubility (solubility 139 g / 100 mL, 20 °C). After the reduction product is recrystallized with ethanol, it remains in the mother liquor and does not need to be separated separately. In the present invention, a hydrogen bond network is formed by L-tartaric acid. For example, a hydrogen bond is formed between the carboxyl group and the lactone carbonyl group, such as -COOH…O=C, and a hydrogen bond is formed between the hydroxyl group and the adjacent hydroxyl group of the lactone, such as -OH…HO-C3, so as to fix the lactone conformation. The steric hindrance shields the Si plane, and NaBH4 attacks from the Re plane, regulating the proportion of L-galactose to 97%.

[0016] Preferably, the molar ratio of L-tartaric acid to L-galactonic acid-1,4-lactone in step S3 is 0.003:1 - 0.008:1.

[0017] By adopting the above technical solutions, the beneficial effects of the present invention are: The present invention provides a method for preparing L-galactose. First, lactic acid and acetic acid are used in a synergistic catalysis to cyclize D-galacturonic acid to form L-galactonic acid-1,4-lactone. Lactic acid, which stabilizes the carboxylic acid form of D-galacturonic acid, and acetic acid, which promotes the condensation of the C1 carboxylic acid and the C4 hydroxyl group within the D-galacturonic acid molecule through hydrophobic interaction, are added to the solution containing D-galacturonic acid. The pH stabilized by lactic acid is 4.0 - 5.0, which inhibits the side reaction of ring opening by strong acids. Meanwhile, the hydrophobic effect of acetic acid is used to enhance the efficiency. The nonpolar methyl group (CH3-) of acetic acid reduces the polarity of the reaction medium, promotes the aggregation of the hydrophobic region of D-galacturonic acid, drives the condensation of the C1 carboxyl group and the C4 hydroxyl group within the molecule, reduces the cyclization energy barrier, shortens the reaction time, and promotes intramolecular esterification. Through the dual-acid synergistic mechanism, the yield of L-galactonic acid-1,4-lactone is significantly higher than that of the single-acid system. The temperature is raised to 88°C - 92°C and kept warm for 3 - 4 hours, and then the reaction ends. The reaction solution obtained by concentrating S1 in the present invention directly enters the reduction reaction, reducing the purification loss and avoiding the use of organic solvents at the same time. L-tartaric acid is added to the concentrated solution to construct a chiral environment, and sodium borohydride is used for stereoselective reduction to prepare L-galactose. The specific implementation method is as follows: The two hydroxyl groups (-OH) and two carboxylic acid groups (-COOH) of L-tartaric acid can form multiple hydrogen bonds with the carbonyl oxygen and adjacent hydroxyl group of the lactone, fixing the conformation of the lactone molecule and forcing NaBH4 to attack the carbonyl carbon from the specific orientation of the Re face. The rigid four-carbon skeleton of L-tartaric acid hinders the formation of the D-configuration transition state and inhibits the side reaction path. The directional reduction of L-galactonic acid-1,4-lactone by NaBH4 to obtain L-galactose is realized, thereby realizing the regulation of the galactose configuration. Description of the Drawings

[0018] Figure 1 It is the infrared spectrum diagram of L-galactose prepared in Example 1. Figure 2 It is the HPLC of Examples 1 - 5 and Comparative Examples 1 - 3 of the present invention and the standard product L-galactose. Detailed Description of the Invention

[0019] In order to further explain the technical solution of the present invention, the present invention will be elaborated in detail through specific examples below.

[0020] Example 1

[0021] This example discloses a method for preparing L-galactose. The raw materials and equipment used in this example are as follows: Citrus pectin (purity ≥ 85%, Sigma-Aldrich); 0.8% dilute sulfuric acid (analytical pure), lactic acid (food grade), acetic acid (analytical pure); L-tartaric acid (analytical reagent), sodium borohydride (98%), ethanol (industrial grade); Constant temperature reactor, magnetic stirrer, HPLC system (Agilent 1260), polarimeter (Rudolph AutopolIV).

[0022] It includes the following steps: S1. Lactate and acetate co-catalyze the cyclization of D-galacturonic acid to generate L-galactonic acid-1,4-lactone; In this example, dilute sulfuric acid is first used to catalyze the hydrolysis of pectin to generate D-galacturonic acid, which specifically includes the following steps: S11. Mix 10 g of pectin with 100 mL of 0.8% dilute sulfuric acid at a solid-liquid ratio of 1 g:10 mL, and add it to a constant temperature reactor; Heat up to 95 °C, stir and react at 300 rpm for 4 hours, and monitor the generation of D-galacturonic acid by HPLC; S12. The yield of D-galacturonic acid monitored by HPLC is 72%, and the HMF content is 3.5%.

[0023] Add CaCO3 in batches to neutralize the sulfuric acid in the reaction solution to a solution pH of 5.0, and release the heat of neutralization. The temperature of the hydrolysis solution rises to 55 °C; filter to remove CaSO4.

[0024] Add lactic acid to stabilize the carboxylic acid form of D-galacturonic acid and acetic acid to promote the condensation of the C1 carboxylic acid and C4 hydroxyl group in the D-galacturonic acid molecule through hydrophobic interaction to the solution containing D-galacturonic acid; Add lactic acid and acetic acid with a concentration of 0.8 M to the hydrolysis solution, and the volume ratio of lactic acid to acetic acid is 3:1; Heat to 90 °C at 1 °C / min and keep warm for 3 hours, and detect the lactone yield of 68% by HPLC.

[0025] S2. Concentrate the reaction solution obtained in S1; the volume is 1 / 5 of the original solution, and it shows a continuous thin stream when poured, and the viscosity is about 500 to 1000 cP.

[0026] S3. Add L-tartaric acid to the concentrated solution obtained in S2, and the molar ratio of L-tartaric acid to L-galactonic acid-1,4-lactone is 0.005:1, and adjust the pH to 8.5; Add NaBH4, and the molar ratio of L-galactonic acid-1,4-lactone to NaBH4 is 1:1.2, and react at 40 °C for 2 hours; L-tartaric acid fixes the conformation of L-galactonic acid-1,4-lactone through a hydrogen bond network, and NaBH4 directionally reduces L-galactonic acid-1,4-lactone to obtain L-galactose.

[0027] Ethanol recrystallization, the ratio of the mass (g) of the crude product to be recrystallized to the volume (mL) of ethanol solvent is 1:10, that is, 10 mL of ethanol is used for recrystallization per 1 g of crude product.

[0028] The optical purity [α]D of the product obtained in this example 20 = +72.5° (c = 1, H2O, chiral HPLC detection conditions: Chiralpak AD-H column, mobile phase n-hexane / isopropanol = 70:30 (v / v), flow rate 1.0 mL / min); chiral HPLC analysis shows that the proportion of L-galactose is 95% and the D-configuration is 5%.

[0029] In this example, the total yield of L-galactose is about 46.4% and the optical purity is 95%. The total yield of L-galactose = the yield of D-galacturonic acid * the yield of lactone * the proportion of L-galactose.

[0030] Example 2

[0031] The main difference between this example and Example 1 is that the sulfuric acid concentration is adjusted to 1.2% and the reaction time is shortened to 3 hours.

[0032] Example 3

[0033] The main difference between this example and Example 1 is that in step S11 of using dilute sulfuric acid to catalyze the hydrolysis of pectin to produce D-galacturonic acid, the stirring rate is 400 rpm, and other conditions are the same as in Example 1.

[0034] Example 4

[0035] The main difference between this example and Example 1 is that the addition amount of L-tartaric acid is adjusted so that the molar ratio of L-tartaric acid to lactone is 0.003:1.

[0036] The optical purity [α]D of the product obtained in this example 20 = +70.1°, and it needs to be recrystallized twice to increase to 95%.

[0037] Example 5

[0038] The main difference between this example and Example 1 is that: In step S11 of using dilute sulfuric acid to catalyze the hydrolysis of pectin to produce D-galacturonic acid, the stirring rate is 150 rpm, and other conditions are the same as in Example 1.

[0039] Table 1 Sulfuric acid concentration and product situation in Examples 1 to 5

[0040] Comparative Example 1 The main difference between this comparative example and Example 1 is that the same amount of lactic acid is used alone and acetic acid is not used.

[0041] The remaining process steps and parameters are the same as those in Example 1. The situation of the obtained product is shown in Table 2.

[0042] Table 2 Comparison of the products obtained in Comparative Example 1 and Example 1

[0043] Combined with Table 2, it can be seen that in Comparative Example 1, the acetic acid hydrophobic effect is lacking, the intramolecular cyclization rate decreases, and the corresponding yield is significantly reduced.

[0044] Comparative Example 2 The main difference between this comparative example and Comparative Example 1 is that: In this comparative example, acetic acid was used alone with lactic acid at the same molar concentration as in Example 1, and no acetic acid was used. The remaining process steps and parameters are the same as those in Example 1. The situation of the obtained product is shown in Table 3.

[0045] Table 3 Comparison of the products obtained in Comparative Example 2 and Example 1

[0046] As can be seen from Table 3, in Comparative Example 2, lactic acid was not used, and the pH got out of control, resulting in a dehydration side reaction with a HMF content of 9.1%. The HMF by-products increased sharply, and the lactone purity decreased.

[0047] Comparative Example 3 The main difference between this comparative example and Example 1 is that: L-tartaric acid was not used, that is, there was no chiral induction; the remaining steps and parameters are the same as those in Example 1. The situation of the obtained product is shown in Table 3.

[0048] The product obtained in Comparative Example 3 was analyzed by chiral HPLC. The proportion of L-galactose was 55%, and the D-configuration was 45%. The optical purity [α]D 20 = +41.3° (c = 1, H2O). Although there is a certain L-preference, it cannot directly meet the pharmaceutical purity (>98%), and chiral separation is still required.

[0049] Table 4 Comparison of the products obtained in Comparative Example 3 and Example 1

[0050] Combined with Table 4, it can be seen that in Comparative Example 3 without a chiral inducer, the reduction reaction has no stereoselectivity, and further chromatographic separation and purification are required, resulting in a sharp drop in the total yield.

[0051] The infrared spectrum of L-galactose prepared in Example 1 is as Figure 1 shown. A broad and strong absorption peak appears at 3420 cm -1 This is the stretching vibration characteristic of multiple hydroxyl groups (-OH) in the sugar molecule, which proves that the product has the polyhydroxy structure of a typical monosaccharide. 1075 cm -1A sharp and strong peak appears at [frequency], which is the characteristic vibration peak of the C-O-C ether bond in the pyranose ring, confirming that the product forms a six-membered ring structure, different from the open-chain sugar. At 990 cm -1 and 920 cm -1 The double weak peaks at [position] are the fingerprint region characteristics of the sugar ring breathing vibration, which completely match the standard L-galactose database spectrum. The medium-strong peak at 850 cm -1 corresponds to the axial hydrogen vibration at the C1 position of the pyranose ring, which is the stereochemical characteristic peak of L-configured galactose. No abnormal peaks appear in the entire spectrum (such as no carbonyl peak near 1720 cm -1 ), proving that the lactone group has been completely reduced and the product purity meets the expectations.

[0052] Figure 2 Figure [figure number] is the HPLC comparison chart of the products of Examples 1-5 and Comparative Examples 1-3. The L-galactose standard shows a sharp and symmetric single peak at 8.21 minutes as a reference benchmark. The main peak retention time of the product of Example 1 is 8.20 minutes, completely overlapping with the standard, with a symmetric peak shape and no shoulder peak, and the peak area ratio is 95.2%, indicating a very high L-configured purity. The main peak retention time of the product of Example 2 is 8.22 minutes, but the peak width increases to 0.25 minutes, showing a slight decrease in stereoselectivity, consistent with its 92.1% L-configured ratio. The product of Example 3 has a main peak at 8.19 minutes, but a minor by-product peak appears at 8.5 minutes, accounting for 1.3%, reflecting a small amount of impurities caused by insufficient stirring rate. Although the main peak of the product of Comparative Example 1 is still at 8.20 minutes, an obvious dehydration by-product peak appears at 7.8 minutes, accounting for 15.2%, and the peak area is only 76.8% of that of Example 1, indicating an increase in side reactions in the absence of acetic acid.

[0053] The product of Comparative Example 3 shows a bimodal structure: the L-galactose peak at 8.21 minutes and the D-galactose peak at 9.08 minutes, with similar peak heights, directly showing an enantiomeric mixture without chiral induction.

[0054] Figure 2 The height of the curve in [figure] directly reflects the product concentration. The peak value of Example 1 is the highest, proving its optimal yield; while the peak values of Comparative Examples 1 and 3 are significantly reduced, verifying the yield loss caused by process defects. The differences in peak positions of each sample clearly demonstrate the control effect of configurational selectivity.

[0055] The test conditions for the above HPLC are as follows: Chromatographic column: Chiralpak AD-H (250mm×4.6mm, 5μm); Mobile phase: n-hexane / isopropanol = 70:30 (v / v); Flow rate: 1.0 mL / min; Column temperature: 30 °C; Injection volume: 10 μL; Sample concentration: 10 mg / mL, with isopropanol as the solvent; Detector temperature: 35 °C; Running time: 15 min.

Claims

1. A method for preparing L-galactose, characterized in that: It includes the following steps: S1. Lactate-acetic acid co-catalyzes the cyclization of D-galacturonic acid to generate L-galactonic acid-1,4-lactone; Heat up to 88°C to 92°C and keep warm for 3 hours to 4 hours; the reaction ends. S2. Concentrate the reaction solution obtained in S1. S3. Add L-tartaric acid to the concentrated solution obtained in S2. L-tartaric acid fixes the conformation of L-galactonic acid-1,4-lactone through a hydrogen bond network, and NaBH4 directionally reduces L-galactonic acid-1,4-lactone to obtain L-galactose.

2. The preparation method according to claim 1, characterized in that: The method for catalytic hydrolysis of pectin to generate D-galacturonic acid includes the following steps: S11. Mix pectin and dilute sulfuric acid, heat up to 90°C - 100°C, stir, and react for 3 hours to 5 hours; S12. After HPLC monitors that the yield of D-galacturonic acid exceeds 80%, add CaCO3 in batches to neutralize the sulfuric acid in the reaction solution to a solution pH of 4.8 to 5.2, and release the heat of neutralization, and filter to remove CaSO4.

3. The preparation method according to claim 2, characterized in that: In step S11, the mass fraction of dilute sulfuric acid is 0.8% - 1.2%.

4. The preparation method according to claim 2, characterized in that: The solid-liquid ratio of pectin to dilute sulfuric acid is 1g:10ml.

5. The preparation method according to claim 1, characterized in that: In step S1, the lactate concentration is 0.5M - 1M, and the volume ratio of lactate to acetic acid is 3:

1.

6. The preparation method according to claim 1, characterized in that: In step S1, the preparation process of lactate-acetic acid co-catalyzing the cyclization of D-galacturonic acid to generate L-galactonic acid-1,4-lactone is heated at a rate of 1 - 2°C / min.

7. The preparation method according to claim 1, characterized in that: The process conditions for NaBH4 to directionally reduce L-galactonic acid-1,4-lactone to obtain L-galactose in step S3 are as follows: S31. Adjust the pH of the system to 8.5 to 9.0, add NaBH4, and the molar ratio of NaBH4 to L-galactonic acid-1,4-lactone is 1.2:1; S32. Recrystallize with ethanol to obtain L-galactose.

8. The preparation method according to claim 1, characterized in that: In step S3, the molar ratio of L-tartaric acid to L-galactonic acid-1,4-lactone is (0.003 - 0.008):1.

Images