Preparation method and application of thiogalactoside

Abstract

The invention provides a preparation method of thiogalactoside. The preparation method comprises the following steps: carrying out reaction on D-galactose and an acylation reagent containing an R1 group under the action of a catalyst to obtain pentaacylgalactose; the preparation method comprises the following steps: mixing and reacting pentaacylgalactose with thioether R2-S-S-R2, ammonium chloride and a metal reducing agent to obtain R2-beta-D-thiotetraacyl galactoside; the R2-beta-D-thiotetraacyl galactoside is subjected to a hydrolysis reaction under the action of a catalyst, and thiogalactoside, namely the R2-beta-D-thiogalactoside, is obtained. According to the method, thioether is adopted as a raw material to form an intermediate in the presence of a catalytic system, so that the intermediate and pentaacylgalactose are subjected to glycosylation reaction, and workers are prevented from being in direct contact with mercaptan with pungent smell; meanwhile, the product is good in stereoselectivity, high in yield and simple and convenient to operate, column chromatography purification is not needed, and high-purity biochemical-grade R2-beta-D-thiogalactoside can be prepared by desalting and removing impurities through a simple refining process.

Description

Technical Field

[0001] The invention belongs to the field of organic synthesis, and specifically relates to a preparation method of thiogalactoside and an application thereof, and in particular to a preparation method of high-purity biochemical-grade thiogalactoside and an application thereof. Background Art

[0002] Since the beginning of the 21st century, with the rapid development of cell biology, a large number of microorganisms have been used as cell factories to produce various proteins, sugars and other metabolites through genetic modification and induction of gene expression, which has generated huge market value. Thiogalactoside has been widely used in detection reagents (CN117924437A) and synthetic biology (CN117929706A).

[0003] Aliphatic thiogalactosides such as thiogalactosides including ethyl-β-D-thiogalactoside are widely used in the field of drug synthesis as galactose donors (Med.Chem.Lett.2014,5,1054-1059). Isopropyl-β-D-thiogalactoside (IPTG) is a commonly used molecular biological reagent among thiogalactosides, which is often used for blue-white screening of cloned bacteria and protein expression in IPTG-induced bacteria (CN117987403A). Aromatic thiogalactosides are generally used as galactose donors in the total synthesis of some biologically active drug molecules or active compounds (Med.Chem.Lett.2017,8,6,642–647).

[0004] Taking the synthesis of IPTG as an example, the most widely used IPTG synthesis method is to use galactose as a raw material, react with acetic anhydride to obtain pentaacetyl galactose; further react with isopropyl mercaptan under the condition of boron trifluoride as Lewis acid to obtain isopropyl-β-D-thioacetyl galactoside, and finally hydrolyze in an alkaline system to obtain IPTG. The defects of this method are: the large amount of acetic anhydride used as both a solvent and a reaction reagent makes post-treatment difficult, produces a large amount of acidic hazardous waste and environmental pressure; in addition, the violent exothermic reaction will produce a large amount of impurities that are difficult to separate and purify. The boron trifluoride ether used in this route is flammable and explosive, and isopropyl mercaptan has an unpleasant odor and is toxic; this method will produce an α isomer impurity while generating the key intermediate isopropyl-β-D-thioacetyl galactoside, which requires silica gel column chromatography purification, resulting in low yield and high cost, and the intermediate obtained in the second step can only be obtained as a slurry due to its low purity, which is not conducive to industrial operation and material transfer. It is only suitable for laboratory synthesis, not for industrial production.

[0005] Another common method (Regis C.Saliba, Organic letters, 2018, 20(3), 800-803) is to first prepare bromotetraacetylgalactoside from β-D-pentaacetylgalactose, acetic anhydride and 30% hydrogen bromide acetic acid solution, and then react with thiourea to obtain 2-S-(2,3,4,6-tetra-O-acetyl-β-D-pyranosylgalactosyl)-2-isothiourea hydrobromide; the acetylated galactosylthiourea hydrobromide is reacted with isopropyl bromide to obtain isopropyl-β-D-thioacetylgalactoside; in the fourth step, an organic base such as triethylamine is used to remove the acetyl group to obtain isopropyl-β-D-thiogalactoside (IPTG). The product prepared by this method has low purity and needs further purification. In addition, the reaction steps are many and the operation is cumbersome, which is not conducive to industrial production.

[0006] Therefore, how to provide a method for preparing high-purity biochemical grade thiogalactoside has become an urgent problem to be solved. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for preparing thiogalactoside and its application, and in particular to provide a method for preparing high-purity biochemical grade thiogalactoside and its application. The preparation method provided by the present invention only requires three steps, with short steps and high yield; the purification method is simple and efficient, the product purity can reach more than 99.9%, and the quality can fully meet the high standard requirements of biochemical experiments or synthetic biology.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] The first object of the present invention is to provide a method for preparing thiogalactoside, the preparation method comprising the following steps:

[0010] (1) reacting D-galactose and an acylating agent containing an R1 group under the action of a catalyst to obtain pentaacylgalactose;

[0011] (2) reacting pentaacylgalactose with thioether R2-SS-R2, ammonium chloride and a metal reducing agent to obtain R2-β-D-thiotetraacylgalactoside;

[0012] (3) hydrolyzing R2-β-D-thiotetraacylgalactoside under the action of a catalyst to obtain thiogalactoside, i.e., R2-β-D-thiogalactoside;

[0013] The reaction route is as follows:

[0014]

[0015] Wherein, R1 in the acylating agent is an acetyl group, and R2 in the thioether is an alkyl group, an aryl group or a hydrocarbon group having no more than 10 carbon atoms.

[0016] Specifically, the acylating agent in step (1) is acetic anhydride;

[0017] The catalyst includes any one of calcium acetate, sodium acetate, potassium acetate, and zinc acetate, or a combination of at least two of them.

[0018] Specifically, the molar ratio of D-galactose, acylating agent and catalyst in step (1) is 1:(5-10):(0.1-1);

[0019] Preferably, the reaction temperature in step (1) is 65-110°C, and the reaction time is 1-10h; more preferably, the reaction temperature in step (1) can be 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, 105°C or 110°C, and the reaction time can be 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h or 10h, but are not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are equally applicable.

[0020] Specifically, step (1) further comprises a solvent, wherein the solvent is one or a combination of at least two selected from ethylene glycol diethyl ether, isopropyl acetate, anisole, fluorobenzene, chlorocyclohexane, benzene, toluene and xylene;

[0021] Specifically, in step (2), R2 is at least one selected from methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, n-pentyl, isopentyl, hexyl, heptyl, phenyl, and p-tolyl;

[0022] The metal reducing agent is one selected from aluminum powder, zinc powder and iron powder.

[0023] Specifically, the molar ratio of the pentaacylgalactose, thioether, ammonium chloride and metal reducing agent in step (2) is 1:(0.5-1):(1-3):(1-3);

[0024] Preferably, the reaction temperature in step (2) is 0-35°C, and the reaction time is 2-12h; more preferably, the reaction temperature in step (2) can be 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C or 35°C, and the reaction time can be 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h or 12h, but are not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are equally applicable.

[0025] Specifically, the catalyst in step (3) includes any one of sodium, potassium, calcium, sodium methoxide, NaOH, KOH, Na2S, K2S, Na2CO3, K2CO3, CaO, MgO, and BaO, or a combination of at least two thereof.

[0026] Specifically, step (3) further comprises a solvent, and the solvent is methanol or a mixture of methanol and an alkane reagent.

[0027] Specifically, the molar ratio of the R-β-D-thiotetraacylgalactoside to the catalyst in step (3) is 1:(0.001-0.5);

[0028] Preferably, the reaction temperature in step (3) is -15-25°C, and the reaction time is 0.5-8h; more preferably, the reaction temperature in step (3) can be -15°C, -10°C, -5°C, 0°C, 5°C, 10°C, 15°C, 20°C or 25°C, and the reaction time can be 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h or 8h, but are not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are equally applicable.

[0029] The second object of the present invention is to provide an application of thiogalactoside. The thiogalactoside prepared by the above preparation method is used as a molecular biological reagent for inducing gene expression and as an important intermediate of candidate drugs containing a galactose molecular skeleton. The thiogalactoside includes ethyl-β-D-thiogalactoside, isopropyl-β-D-thiogalactoside, phenyl-β-D-thiogalactoside, and tolyl-β-D-thiogalactoside.

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

[0031] The invention provides a preparation method of high-purity biochemical grade thiogalactoside and application thereof. By taking thioether as a raw material, an intermediate is formed in the presence of a catalyst system, and then a glycosylation reaction is carried out with pentaacylgalactose, thereby avoiding direct contact of workers with thiol with pungent odor; at the same time, the product has good stereoselectivity, high yield, and simple operation, and does not need to be purified by column chromatography. By removing salt and impurities through a simple refining process, high-purity biochemical grade R2-β-D-thiogalactoside can be prepared. DETAILED DESCRIPTION

[0032] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0033] Example 1

[0034] This embodiment provides a kind of isopropyl-β-D-thiogalactoside (IPTG) The preparation method comprises the following specific steps:

[0035] (1) Add 3.6g sodium acetate (0.044mol), 62.0g acetic anhydride (0.6mol), and 80.0g benzene to a reaction flask, heat to 80°C and stir for 30min, weigh 18.0g D-galactose (0.1mol) and add it to the reaction flask in batches, and continue the reaction for 2h after completion. After the reaction is completed, pour the reaction solution into water and stir, separate the organic phase, dry and concentrate the organic phase, and further recrystallize it in ethanol to obtain 37.6g of white solid, which is pentaacetyl galactose, with a yield of 96% and a HPLC purity of 99%;

[0036] (2) Add 150 ml of toluene, 12.0 g of isopropyl disulfide (0.08 mol), 2.7 g of aluminum powder (0.1 mol), and 10.7 g of ammonium chloride (0.2 mol) to the reaction flask, stir at room temperature for 1 hour, then dissolve 39.0 g of pentaacetyl galactose (0.1 mol) in 100 ml of toluene and dropwise add to the above reaction system, the dropwise addition time is 0.5 hour, and the reaction is continued for 5 hours after the dropwise addition is completed, and the reaction is detected by TLC. After the reaction is completed, the reaction solution is neutralized with a saturated sodium bicarbonate solution, the organic phase is separated, dried, and concentrated to obtain a colorless slurry, which is isopropyl-β-D-thiotetraacetyl galactoside, which is recrystallized from ethanol to obtain 35.4 g of a white solid, with a yield of 87% and a HPLC purity of 98%;

[0037] (3) Dissolve 20.3 g of isopropyl-β-D-thiotetraacetyl galactoside (0.05 mol) in 100 ml of methanol, add 0.15 g of sodium methoxide (0.0025 mol), and react at 0°C for 3 h. After the reaction is completed, neutralize the reaction solution with 10% hydrochloric acid to pH = 7, dry and concentrate, and recrystallize from methanol to obtain 10.7 g of biochemical grade IPTG with a purity of 99.9% and a yield of 90%.

[0038] The characterization data are as follows: 1 H-NMR (500MHz, CD3OD) δ4.39(d,J=9.4Hz,1H),3.87(dd,J=3.3,1.1Hz,1H),3.72(dd,J=11.4,6.8Hz,1H),3.68(dd,J=11.4,5. 5Hz, 1H), 3.53–3.48 (2H), 3.45 (dd, J = 9.3, 3.3Hz, 1H), 3.23 (hept, J = 6.8Hz, 1H), 1.31 (d, J = 7.1Hz, 3H), 1.30 (d, J = 7.2Hz, 3H).

[0039] Example 2

[0040] This embodiment provides a kind of isopropyl-β-D-thiogalactoside (IPTG) The preparation method comprises the following specific steps:

[0041] (1) Add 3.6g potassium acetate (0.037mol), 62.0g acetic anhydride (0.6mol), and 80.0g ethylene glycol dimethyl ether to a 500ml three-necked flask, heat to 80°C and stir for 30min, weigh 18.0g D-galactose (0.1mol) and add it to the reaction flask in batches, and continue to react for 6h after completion. After the reaction is completed, pour the reaction solution into water and stir, separate the organic phase, dry and concentrate the organic phase, and further recrystallize it in ethanol to obtain 38.3g of white solid, which is intermediate 1 pentaacetylgalactose, with a yield of 98% and a purity of 98%.

[0042] (2) Add 150 ml toluene, 12.0 g isopropyl disulfide (0.08 mol), 5.6 g iron powder (0.1 mol), and 10.7 g ammonium chloride (0.2 mol) to a 500 ml three-necked flask, stir at 60 degrees for 1 hour, and then cool to room temperature. Dissolve 39 g pentaacetyl galactose (0.1 mol) in 100 ml toluene and dropwise add it to the above reaction system at 25 degrees Celsius for 1 hour. After the dropwise addition is completed, continue the reaction at room temperature for 7 hours, and detect the reaction by TLC. After the reaction is completed, neutralize the reaction solution with saturated sodium bicarbonate solution, separate the organic phase, dry and concentrate to obtain a colorless slurry, which is the intermediate 2-isopropyl-β-D-thiotetraacetyl galactoside, and recrystallize it from ethanol to obtain 36.1 g of a white solid with a yield of 89% and a HPLC purity of 98%.

[0043] (3) Dissolve 20.3 g of isopropyl-β-D-thiotetraacetyl galactoside (0.05 mol) in 100 ml of methanol, add 0.35 g (0.0025 mol) of potassium carbonate, and react at 10°C for 1.5 h. After the reaction is completed, neutralize the reaction solution with dilute hydrochloric acid (1 mol / L), adjust the pH of the reaction solution to 5-6, concentrate the filtrate, and dry the solid to obtain a crude off-white solid powder. Recrystallize from methanol to obtain biochemical grade IPTG with an HPLC purity of 99.9% and a yield of 90%.

[0044] Example 3

[0045] This embodiment provides a kind of isopropyl-β-D-thiogalactoside (IPTG) The preparation method comprises the following specific steps:

[0046] (1) Add 3.6g of calcium acetate (0.023mol), 62.0g of acetic anhydride (0.6mol), and 80.0g of benzene to a 500ml three-necked flask, heat to 80°C and stir for 30min, weigh 18.0g of D-galactose (0.1mol) and add it to the reaction flask in batches, and continue to react for 6h after completion. After the reaction is completed, pour the reaction solution into water and stir, separate the organic phase, dry and concentrate the organic phase, and recrystallize it from ethanol to obtain 37.1g of intermediate 1 pentaacetylgalactose, with a yield of 95% and a purity of 98%.

[0047] (2) Add 150 ml of toluene, 12.0 g of isopropyl disulfide (0.08 mol), 6.5 g of zinc powder (0.1 mol), and 10.7 g of ammonium chloride (0.2 mol) to a 500 ml three-necked flask, stir at room temperature for 0.5 h, then dissolve 39.0 g of pentaacetyl galactose (0.1 mol) in 100 ml of toluene and dropwise add to the above reaction system for 1 h. After the addition is complete, continue the reaction for 5 h, and monitor the reaction by TLC. After the reaction is complete, neutralize the reaction solution with a saturated sodium bicarbonate solution, separate the organic phase, dry and concentrate to obtain a colorless slurry, which is the intermediate 2-isopropyl-β-D-thiotetraacetyl galactoside, and recrystallize from ethanol to obtain 36.5 g of a white solid with a yield of 90% and a purity of 98% by HPLC.

[0048] (3) 20.3 g of isopropyl-β-D-thiotetraacetyl galactoside (0.05 mol) was dissolved in 100 ml of methanol, and 0.38 g of barium oxide (0.0025 mol) was added. The mixture was reacted at 15° C. for 8 h. After the reaction was completed, the reaction solution was neutralized with 10% dilute sulfuric acid to pH = 7, dried, concentrated, and recrystallized from methanol to obtain 10.9 g of isopropyl-β-D-thiogalactoside (IPTG), with a yield of 92% and a purity of 99%.

[0049] Example 4

[0050] This embodiment provides a kind of isopropyl-β-D-thiogalactoside (IPTG) The other conditions are the same as those in Example 1, except that step (1) is changed. The detailed steps are as follows:

[0051] Add 36.5g sodium acetate (0.08mol), 103.3g acetic anhydride (1.0mol), and 100.0g benzene to the reaction flask, heat to 80°C and stir for 30min, weigh 18.0g D-galactose (0.1mol) and add it to the reaction flask in batches, and continue to react for 4h after completion. After the reaction is completed, pour the reaction solution into water and stir, separate the organic phase, dry and concentrate the organic phase, and further recrystallize it in ethanol to obtain 36.3g of white solid, which is pentaacetyl galactose, with a yield of 93% and a HPLC purity of 99%.

[0052] Example 5

[0053] The embodiment provides an isopropyl-β-D-thiogalactoside (IPTG) The other conditions are the same as those in Example 1, except that the metal reducing agent in step (2) is changed. The detailed steps are as follows:

[0054] Add 150ml toluene, 12g isopropyl disulfide (0.08mol), 1.35g aluminum powder (0.05mol), 3.25g zinc powder (0.05mol), 10.7g ammonium chloride (0.2mol) to the reaction bottle, stir at room temperature for 1h, dissolve 39.0g pentaacetyl galactose (0.1mol) in 100ml toluene and add dropwise to the above reaction system, the addition time is 0.5h, and the reaction is continued for 5h after the addition is complete, and the reaction is detected by TLC. After the reaction is completed, the reaction solution is neutralized with a saturated sodium bicarbonate solution, the organic phase is separated, dried and concentrated to obtain a colorless slurry, which is isopropyl-β-D-thiotetraacetyl galactoside, and ethanol is recrystallized to obtain 36.6g of a white solid, with a yield of 90% and a HPLC purity of 97%.

[0055] Example 6

[0056] The embodiment provides an isopropyl-β-D-thiogalactoside (IPTG) The other conditions are the same as those in Example 1, except that step (3) is changed. The detailed steps are as follows:

[0057] 20.3g of isopropyl-β-D-thiotetraacetyl galactoside (0.05mol) was dissolved in 70ml of methanol and 30ml of dichloromethane, and 27.6g of potassium carbonate (0.2mol) was added. The reaction was carried out at 0°C for 3h. After the reaction was completed, the reaction solution was neutralized with 10% hydrochloric acid to pH = 7, dried and concentrated, and recrystallized from methanol to obtain 11.3g of biochemical grade IPTG with a purity of 99.9%, with a yield of 95%.

[0058] Example 7

[0059] This embodiment provides a kind of ethyl-β-D-thiogalactoside The preparation method comprises the following specific steps:

[0060] (1) Add 3.6 g sodium acetate (0.044 mol), 103.3 g acetic anhydride (1.0 mol), and 100.0 g anisole to a reaction flask, heat to 110° C. and stir for 30 min, weigh 18.0 g D-galactose (0.1 mol) and add it to the reaction flask in batches, and continue the reaction for 4 h after completion. After the reaction is completed, pour the reaction solution into water and stir, separate the organic phase, dry and concentrate the organic phase, and further recrystallize it from ethanol to obtain 37.6 g (0.96 mol) of white solid, which is pentaacetyl galactose, with a yield of 96% and a HPLC purity of 99%;

[0061] (2) Add 150 ml of toluene, 9.8 g of diethyl disulfide (122.25 g / mol, 0.08 mol), 5.4 g of aluminum powder (0.2 mol), and 16.1 g of ammonium chloride (0.3 mol) to the reaction flask, stir at room temperature for 1 hour, then dissolve 39.0 g of pentaacetyl galactose (0.1 mol) in 100 ml of toluene and dropwise add to the above reaction system, the dropwise addition time is 0.5 hour, and the reaction is continued for 10 hours after the dropwise addition is completed, and the reaction is detected by TLC. After the reaction is completed, the reaction solution is neutralized with a saturated sodium bicarbonate solution, the organic phase is separated, dried, and concentrated to obtain a colorless slurry, which is ethyl-β-D-thiotetraacetyl galactoside, and 35.3 g (0.09 mol) of a white solid is obtained by recrystallization from ethanol, with a yield of 90% and a HPLC purity of 99%;

[0062] (3) 19.6 g of ethyl-β-D-thiotetraacetyl galactoside (0.05 mol) was dissolved in 100 ml of methanol, and 0.15 g of sodium methoxide (0.0025 mol) was added. The mixture was reacted at 0° C. for 3 h. After the reaction was completed, the reaction solution was neutralized with 10% hydrochloric acid to pH = 7, and then dried, concentrated, and recrystallized from methanol to obtain 11.1 g (0.0495) of biochemical grade ethyl-β-D-thiogalactoside with a purity of 99.9%, with a yield of 99%.

[0063] The characterization data are as follows: 1 H NMR (500MHz, CD3OD) δ4.34(d,J=9.6Hz,1H),3.90(d,J=2.5Hz,1H),3.76(dd,J=11.4,6.8Hz,1H),3.70(dd,J=1 1.4,5.3Hz,1H),3.59–3.52(m,1H),3.48(dd,J=9.2,3.3Hz,1H),2.84–2.69(m,2H),1.31(t,J=7.4Hz,3H)ppm.

[0064] Example 8

[0065] This embodiment provides a phenyl-β-D-thiogalactoside The preparation method comprises the following specific steps:

[0066] (1) Add 3.6g sodium acetate (0.044mol), 51.7g acetic anhydride (0.5mol), and 50.0g xylene to a reaction flask, heat to 65°C and stir for 30min, weigh 18g D-galactose (0.1mol) and add it to the reaction flask in batches, and continue the reaction for 10h after completion. After the reaction is completed, pour the reaction solution into water and stir, separate the organic phase, dry and concentrate the organic phase, and further recrystallize it from ethanol to obtain 37.6g (0.96mol) of white solid, which is pentaacetyl galactose, with a yield of 96% and a HPLC purity of 99%;

[0067] (2) Add 150 ml of toluene, 17.5 g (0.08 mol) of diphenyl disulfide, 8.1 g of aluminum powder (0.3 mol), and 5.4 g of ammonium chloride (0.1 mol) to the reaction flask, stir at room temperature for 1 hour, then dissolve 39 g of pentaacetyl galactose (0.1 mol) in 100 ml of toluene and dropwise add to the above reaction system for 0.5 hour. After the addition is complete, continue the reaction for 5 hours, and detect the reaction by TLC. After the reaction is complete, neutralize the reaction solution with saturated sodium bicarbonate solution, separate the organic phase, dry and concentrate to obtain a colorless slurry, which is 44.0 g (0.1 mol) of phenyl-β-D-thiotetraacetyl galactoside, with a yield of 80% and a purity of 99% by HPLC;

[0068] (3) 44.0 g of phenyl-β-D-thiotetraacetyl galactoside (0.1 mol) was dissolved in 100 ml of methanol, and 1.4 g of calcium oxide (0.025 mol) was added. The mixture was reacted at 0°C for 3 h. After the reaction was completed, the reaction solution was neutralized with 10% dilute hydrochloric acid, dried and concentrated to obtain a white solid. Recrystallization from methanol gave 26.7 g of 99.9% biochemical grade phenyl-β-D-thiogalactoside, with a yield of 98%.

[0069] The characterization data are as follows: 1 H NMR (500MHz, MeOD) δ7.58-7.52(m,2H),7.32–7.25(m,2H),7.25–7.19(m,1H),4.60(d,J=9.7Hz,1H),3.91(d,J=3.2Hz,1H),3.77 (dd, J = 11.4, 6.9 Hz, 1H), 3.71 (dd, J = 11.5, 5.2 Hz, 1H), 3.62 (t, J = 9.4 Hz, 1H), 3.57 (t, J = 6.1 Hz, 1H), 3.51 (dd, J = 9.2, 3.3 Hz, 1H).

[0070] Example 9

[0071] This embodiment provides a tolyl-β-D-thiogalactoside The preparation method comprises the following specific steps:

[0072] (1) Add 3.6g sodium acetate (0.044mol), 62.0g acetic anhydride (0.6mol), and 80.0g benzene to a reaction flask, heat to 90°C and stir for 30min, weigh 18.0g D-galactose (0.1mol) and add it to the reaction flask in batches, and continue to react for 8h after completion. After the reaction is completed, pour the reaction solution into water and stir, separate the organic phase, dry and concentrate the organic phase, and further recrystallize it from ethanol to obtain 37.6g (0.96mol) of white solid, which is pentaacetyl galactose, with a yield of 96% and a HPLC purity of 99%;

[0073] (2) Add 150 ml of toluene, 19.7 g (246.39 g / mol, 0.08 mol) of bis(4-tolyl) disulfide, 11.2 g of iron powder (0.2 mol), and 16.1 g of ammonium chloride (0.2 mol) to the reaction flask, stir at room temperature for 1 hour, then dissolve 39.0 g of pentaacetyl galactose (0.1 mol) in 100 ml of toluene and dropwise add to the above reaction system, the dropwise addition time is 0.5 hour, and the reaction is continued for 5 hours after the dropwise addition is completed, and the reaction is detected by TLC. After the reaction is completed, the reaction solution is neutralized with a saturated sodium bicarbonate solution, the organic phase is separated, dried, and concentrated to obtain a colorless slurry, namely 39.4 g (0.09 mol) of tolyl-β-D-thiotetraacetyl galactoside, with a yield of 90% and a purity of 99% by HPLC;

[0074] (3) Dissolve 22.7 g of tolyl-β-D-thiotetraacetyl galactoside (0.05 mol) in 100 ml of methanol, add 1.4 g of potassium hydroxide (0.025 mol), react at 0°C for 3 h, neutralize the reaction solution with 10% dilute hydrochloric acid to pH = 7 after the reaction, dry and concentrate to obtain a white solid. Recrystallize from methanol to obtain 13.7 g of biochemical grade tolyl-β-D-thiogalactoside with a purity of 99.9%, with a yield of 96%.

[0075] The characterization data are as follows: 1 H NMR (MeOD, 600MHz): δ7.45 (d, 2H, J = 8.1Hz), 7.12 (d, 2H, J = 8.1Hz), 4.50 (d, 1H, J = 9.6Hz), 3.89 (d, 1H, J = 3.3Hz), 3.75 (dd, 1H, J = 1 1.4, 6.0Hz), 3.70 (dd, 1H, J = 11.4, 6.0Hz), 3.57 (t, 1H, J = 9.6Hz), 3.53 (t, 1H, J = 6.0Hz), 3.48 (dd, 1H, J = 9.6, 3.3Hz), 2.31 (s, 3H).

[0076] Comparative Example 1

[0077] This comparative example provides a method for preparing isopropyl-β-D-thiogalactoside, which is basically the same as Example 1, except that ammonium chloride is not added in step (2) and the yield is 0.

[0078] Comparative Example 2

[0079] This comparative example provides a method for preparing isopropyl-β-D-thiogalactoside, which is basically the same as Example 1, except that in step (2), the aluminum powder is replaced by 2.4 g of magnesium powder (0.1 mol), and the yield is 0.

[0080] Comparative Example 3

[0081] This comparative example provides a method for preparing isopropyl-β-D-thiogalactoside, which is basically the same as Example 1, except that, in step (2), 150 ml of toluene, 12.0 g of isopropyl disulfide (0.08 mol), 1.9 g of aluminum powder (0.07 mol), and 2.7 g of ammonium chloride (0.05 mol) are added to the reaction bottle, and after stirring at room temperature for 1 h, 39.0 g of pentaacetyl galactose (0.1 mol) is dissolved in 100 ml of toluene and added dropwise to the above reaction system, the dropping time is 0.5 h, and the reaction is continued for 5 h after the dropwise addition is completed, and the reaction is detected by TLC. After the reaction is completed, the reaction solution is neutralized with a saturated sodium bicarbonate solution, the organic phase is separated, dried, and concentrated to obtain a colorless slurry, i.e., isopropyl-β-D-thiotetraacetyl galactoside, and 17.1 g of a white solid is obtained by recrystallization from ethanol, with a yield of 42% and a purity of 98% by HPLC.

[0082] The applicant declares that the present invention illustrates the preparation method and application of thiogalactoside of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0083] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0084] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing thiogalactoside, characterized in that: The preparation method comprises the following steps: (1) reacting D-galactose and an acylating agent containing an R1 group under the action of a catalyst to obtain pentaacylgalactose; (2) reacting pentaacylgalactose with thioether R2-SS-R2, ammonium chloride and a metal reducing agent to obtain R2-β-D-thiotetraacylgalactoside; (3) hydrolyzing R2-β-D-thiotetraacylgalactoside under the action of a catalyst to obtain thiogalactoside, i.e., R2-β-D-thiogalactoside; The reaction route is as follows: Wherein, R1 in the acylating agent is an acetyl group, and R2 in the thioether is an alkyl group, an aryl group or a hydrocarbon group having no more than 10 carbon atoms.

2. The preparation method according to claim 1, characterized in that: The acylating agent in step (1) is acetic anhydride; The catalyst includes any one of calcium acetate, sodium acetate, potassium acetate, and zinc acetate, or a combination of at least two of them.

3. The preparation method according to claim 1 or 2, characterized in that: The molar ratio of D-galactose, acylating agent and catalyst in step (1) is 1:(5-10):(0.1-1); Preferably, the reaction temperature in step (1) is 65-110° C. and the reaction time is 1-10 h.

4. The preparation method according to any one of claims 1 to 3, characterized in that Step (1) further comprises a solvent, wherein the solvent is one or a combination of at least two selected from ethylene glycol diethyl ether, isopropyl acetate, anisole, fluorobenzene, chlorocyclohexane, benzene, toluene and xylene.

5. The preparation method according to any one of claims 1 to 4, characterized in that: In step (2), R2 is at least one selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, n-pentyl, isopentyl, hexyl, heptyl, phenyl, and p-tolyl; The metal reducing agent is one selected from aluminum powder, zinc powder and iron powder.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The molar ratio of the pentaacylgalactose, thioether, ammonium chloride and metal reducing agent in step (2) is 1:(0.5-1):(1-3):(1-3); Preferably, the reaction temperature in step (2) is 0-35°C and the reaction time is 2-12h.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The catalyst in step (3) includes any one of sodium, potassium, calcium, sodium methoxide, NaOH, KOH, Na2S, K2S, Na2CO3, K2CO3, CaO, MgO, and BaO, or a combination of at least two thereof.

8. The preparation method according to any one of claims 1 to 7, characterized in that: Step (3) further comprises a solvent, wherein the solvent is methanol or a mixture of methanol and an alkane reagent.

9. The preparation method according to any one of claims 1 to 8, characterized in that: The molar ratio of the R-β-D-thiotetraacylgalactoside to the catalyst in step (3) is 1:(0.001-0.5); Preferably, the reaction temperature in step (3) is -15-25°C and the reaction time is 0.5-8h.

10. An application of thiogalactoside, characterized in that: The thiogalactoside prepared by the preparation method according to any one of claims 1 to 9 is used as a molecular biological reagent for inducing gene expression and as an important intermediate of a candidate drug containing a galactose molecular skeleton. The thiogalactoside includes ethyl-β-D-thiogalactoside, isopropyl-β-D-thiogalactoside, phenyl-β-D-thiogalactoside, and tolyl-β-D-thiogalactoside.

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