阅读说明：本技术 一种2-氯-4-硝基苯-α-L-岩藻糖苷的制备方法 (Preparation method of 2-chloro-4-nitrobenzene-alpha-L-fucoside ) 是由 潘得成 徐文明 祝长斌 马迪 钟佩岑 于 2021-07-02 设计创作，主要内容包括：本发明公开了一种2-氯-4-硝基苯-α-L-岩藻糖苷的制备方法,属于体外诊断领域。本发明以L-岩藻糖为起始原料,通过乙酰化反应,制得1,2,3,4-四乙酰基-α-L-岩藻糖,再与2-氯-4-硝基苯酚发生糖苷化反应,制得2-氯-4-硝基苯-2,3,4-三乙酰基-α-L-岩藻糖苷,其中,所述催化剂选择三氟甲磺酸三甲基硅酯或三氟化硼乙醚；最后进行脱乙酰基反应,制得2-氯-4-硝基苯-α-L-岩藻糖苷。本发明方法反应路线较短,通过三步反应制得最终产品；且以L-岩藻糖为原料,起始原料价格相对便宜,2-氯-4-硝基苯酚用量明显减少；后处理过程简单且反应重现性好,可实现工业化生产。(The invention discloses a preparation method of 2-chloro-4-nitrobenzene-alpha-L-fucoside, belonging to the field of in vitro diagnosis. The method comprises the steps of taking L-fucose as an initial raw material, preparing 1,2,3, 4-tetraacetyl-alpha-L-fucose through acetylation, and then carrying out glycosylation reaction with 2-chloro-4-nitrophenol to prepare 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside, wherein the catalyst is selected from trimethylsilyl trifluoromethanesulfonate or boron trifluoride diethyl etherate; finally, performing deacetylation reaction to prepare the 2-chloro-4-nitrobenzene-alpha-L-fucoside. The method has a short reaction route, and a final product is prepared through three steps of reactions; the L-fucose is used as a raw material, the price of the initial raw material is relatively cheap, and the dosage of the 2-chloro-4-nitrophenol is obviously reduced; the post-treatment process is simple, the reaction reproducibility is good, and the industrial production can be realized.)

1. A method for preparing 2-chloro-4-nitrobenzene-alpha-L-fucoside, which is characterized by comprising the following steps:

(1) taking L-fucose as a starting material, and preparing 1,2,3, 4-tetraacetyl-alpha-L-fucose through acetylation reaction;

(2)1,2,3, 4-tetraacetyl-alpha-L-fucose and 2-chloro-4-nitrophenol undergo glycosylation reaction to prepare 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside, wherein a reaction solvent is dichloromethane, triethylamine is used as an acid-binding agent, and the catalyst is one of trimethylsilyl trifluoromethanesulfonate or boron trifluoride diethyl etherate;

(3) performing deacetylation reaction on the 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside to prepare the 2-chloro-4-nitrobenzene-alpha-L-fucoside.

2. The preparation method according to claim 1, wherein in the step (1), the acetylation reaction is carried out with a base as a catalyst, wherein the base is selected from one of pyridine and sodium acetate; the acetylation reagent is one or more selected from acetic anhydride, acetic acid and acetyl chloride.

3. The process according to claim 1, wherein in the step (1), the acetylation reaction is carried out at a temperature of 25 to 120 ℃ for 4 to 12 hours.

4. The production method according to any one of claims 1 to 3, wherein in the step (2), the catalyst is boron trifluoride diethyl etherate; 4-10 equivalent of catalyst; the dosage of the 2-chloro-4-nitrophenol is 1 to 4 equivalents.

5. The method according to any one of claims 1 to 4, wherein the reaction temperature of the glycosidation reaction in step (2) is 25 to 50 ℃, preferably 40 to 50 ℃, and the reaction time is 48 to 72 hours.

6. The preparation method according to any one of claims 1 to 5, wherein in the step (2), the 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside is obtained by recrystallization after the reaction, and the recrystallization solvent is one or more selected from methanol, ethanol and isopropanol.

7. The preparation method according to any one of claims 1 to 6, wherein in the step (3), the deacetylation reaction is carried out by using a base as a catalyst, wherein the base is selected from one or more of sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium tert-butoxide, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide and lithium hydroxide, and is preferably sodium methoxide, sodium ethoxide and sodium tert-butoxide.

8. The method according to any one of claims 1 to 7, wherein in the step (3), the reaction temperature of the deacetylation reaction is 0 to 50 ℃ and the reaction time is 0.5 to 2 hours.

9. The preparation method according to claim 8, wherein in the step (3), the post-reaction treatment is a recrystallization treatment, and the final product is obtained, wherein the solvent used for recrystallization is one or more selected from ethanol and isopropanol.

10. Use of the preparation method according to any one of claims 1 to 9 in the field of biological detection.

Technical Field

The invention relates to a preparation method of 2-chloro-4-nitrobenzene-alpha-L-fucoside, belonging to the field of in vitro diagnosis.

Background

alpha-L-fucosidase (AFU) is a lysosomal acid hydrolase that is widely distributed in various tissues, cells, and body fluids in the human body. The liver, kidney and other tissues have high activity, the basic function of the liver, kidney and other tissues is to catalyze the hydrolysis metabolism of fucose-containing oligosaccharide, glycoprotein and glucoside, the essence of the liver, kidney and other tissues is glycoprotein, and the liver, kidney and other tissues play an important role in various aspects including immune reaction, signal transduction and the like. The research finds that the determination of the alpha-L-fucosidase has positive effects in the diagnosis and treatment of various diseases. Wherein, AFU in serum catalyzes 2-chloro-p-nitrophenol alpha-L-fucoside (CNP-AFU) to hydrolyze to generate 2-chloro-4-nitrophenol (CNP) by a rate method (continuous monitoring method), the generation rate of CNP is monitored at the wavelength of 405nm or 410nm, and AFU activity is calculated. The method has no obvious interference on bilirubin 250mg/L, hemoglobin 230mg/L and ascorbic acid 6 g/L. The method has the advantages of simple operation, short determination time and improved sensitivity and anti-interference performance to a certain extent.

At present, the market demand of 2-chloro-4-nitrobenzene-alpha-L-fucoside is large, but the synthesis has great difficulty. The synthesis method of the product comprises the following steps: 1) koichi et al published a Japanese patent in 1994 (JP 06-179690) disclosing for the first time a method for the synthesis of 2-chloro-4-nitrophenyl-alpha-L-fucoside; however, this method is poor in reproducibility, and requires a large amount of 2-chloro-4-nitrophenol to be used, and the post-treatment of the reaction is very difficult. 2) Duyu et al reported a new synthetic route in 2004; however, the starting material of the method, namely ethyl-1-mercapto-beta-L-fucoside and the protecting group material, namely tert-butyldimethylsilyl chloride, are expensive, the reaction yield is low, and the method is not suitable for industrial production. 3) In 2013, a new synthetic route was reported by Beijing Lidman Biochemical GmbH; the alpha configuration product obtained by the method has high proportion, but the synthetic route has longer steps, and ethanethiol is needed, so the odor is large. Therefore, a simple and practical method for preparing 2-chloro-4-nitrobenzene-alpha-L-fucoside is needed.

Disclosure of Invention

[ problem ] to

The existing preparation method has poor reproducibility, difficult post-treatment, or high cost, low yield, complex synthesis steps, use of substances such as ethanethiol and the like with large odor.

[ solution ]

In order to solve the above problems, the present invention provides a novel method for preparing 2-chloro-4-nitrobenzene-alpha-L-fucoside. The method has a short reaction route, and a final product is prepared through three steps of reactions; the L-fucose is used as a raw material, the price of the initial raw material is relatively cheap, and the dosage of the 2-chloro-4-nitrophenol is obviously reduced; the post-treatment process is simple, the reaction reproducibility is good, and the industrial production can be realized.

Specifically, the invention provides a preparation method of 2-chloro-4-nitrobenzene-alpha-L-fucoside, and the reaction route of the method is as follows:

the method comprises the following steps:

(1) taking L-fucose as a starting material, and preparing 1,2,3, 4-tetraacetyl-alpha-L-fucose through acetylation reaction;

(2)1,2,3, 4-tetraacetyl-alpha-L-fucose and 2-chloro-4-nitrophenol are subjected to glycosylation reaction to prepare 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside, wherein the reaction solvent is dichloromethane, triethylamine is used as an acid-binding agent, and the catalyst is selected from trimethylsilyl trifluoromethanesulfonate (TMSOTf) or boron trifluoride diethyl etherate (BF)₃·Et₂O) is selected;

(3) performing deacetylation reaction on the 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside to prepare the 2-chloro-4-nitrobenzene-alpha-L-fucoside.

In one embodiment of the present invention, in step (1), the acetylation reaction is carried out with a base as a catalyst, wherein the base is selected from one of pyridine and sodium acetate, preferably sodium acetate; the acetylation reagent is selected from one or more of acetic anhydride, acetic acid and acetyl chloride, and acetic anhydride is preferred.

In one embodiment of the present invention, in step (1), the amount of the catalyst is 1 to 1.2 equivalents, and the amount of the acetylation reagent is 4 to 5 equivalents (the amount of each step is not specifically indicated, and the default is the starting material for the reaction of the step).

In one embodiment of the present invention, in step (1), the reaction temperature of the acetylation reaction is 25-120 ℃, preferably 60-100 ℃, and the reaction time is 4-12 h.

In one embodiment of the present invention, in step (1), after the acetylation reaction is completed, dichloromethane is added, washing is performed several times, then washing is performed with a saturated sodium chloride solution, and then drying and solvent removal are performed to obtain 1,2,3, 4-tetraacetyl- α -L-fucose.

In one embodiment of the present invention, in step (2), the catalyst is preferably boron trifluoride diethyl etherate; the amount of catalyst used is 4 to 10 equivalents, preferably 5 to 8 equivalents.

In one embodiment of the present invention, in step (2), the 2-chloro-4-nitrophenol is used in an amount of 1 to 4 equivalents, preferably 1.5 to 2.5 equivalents.

In one embodiment of the present invention, in step (2), the reaction temperature of the glycosidation reaction is 25-50 ℃, preferably 40-50 ℃, and the reaction time is 48-72 h.

In one embodiment of the present invention, the reaction of step (2) is carried out under an inert atmosphere, which is nitrogen or argon.

In one embodiment of the invention, in the step (2), the 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside is obtained by recrystallization in the post-reaction treatment, wherein the recrystallization solvent is one or more selected from methanol, ethanol and isopropanol, so that column chromatography is avoided, and the post-treatment difficulty is greatly reduced.

In one embodiment of the invention, in the step (3), the deacetylation reaction uses a base as a catalyst, wherein the base is selected from one or more of sodium methoxide, sodium ethoxide, sodium tert-butoxide, potassium tert-butoxide, sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide and lithium hydroxide, and preferably sodium methoxide, sodium ethoxide and sodium tert-butoxide; the dosage of the catalyst is 0.5-5 mol%, preferably 1-2 mol% of 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside.

In one embodiment of the present invention, in the step (3), the reaction temperature of the deacetylation reaction is 0 to 50 ℃, preferably 20 to 25 ℃, and the reaction time is 0.5 to 2 hours.

In one embodiment of the present invention, in the step (3), the solvent used for the deacetylation reaction is methanol.

In one embodiment of the present invention, in the step (3), the post-reaction treatment is a recrystallization treatment, and the final product can be obtained, wherein the solvent used for recrystallization is one or more selected from ethanol and isopropanol.

The invention also provides the application of the method in the field of biological detection.

Compared with the prior art, the invention has the following advantages and effects:

compared with the prior synthesis method, the starting material L-fucose adopted by the invention has relatively low price, and the glycosylation reaction in the second step is catalyzed by boron trifluoride diethyl etherate, so that compared with the prior art, the use amount of 2-chloro-4-nitrophenol is greatly reduced, the reaction temperature is reduced, the cost is saved, and the difficulty of post-treatment is also reduced; the reaction steps are relatively short, the reaction yield of each step is considerable, column chromatography is not used in the post-treatment process, and industrialization can be realized; (ii) a The final product 2-chloro-4-nitrobenzene-alpha-L-fucoside has better quality; the cost of the total process route is reduced; therefore, the preparation method has good prospect of industrial production.

Drawings

FIG. 12-chloro-4-nitrophenyl-alpha-L-fucoside¹H NMR。

FIG. 22-chloro-4-nitrobenzene- α -L-fucoside HPLC.

Detailed Description

The present invention is further described below with reference to examples, but the embodiments of the present invention are not limited thereto.

Example 1:

(1) synthesis of 1,2,3, 4-tetraacetyl-alpha-L-fucose

Adding 100g L-fucose and 1L pyridine into a reactor, and stirring; 746g of acetic anhydride was added in portions at 25 ℃ to prevent a violent exotherm; after the addition was complete, the reaction was carried out at 25 ℃ for 12 h. After the reaction is finished, removing pyridine by rotation to obtain viscous liquid; and the viscous liquid is poured into 1L dichloromethane, washed by water (5x 500mL), then washed by 500mL saturated sodium chloride solution, dried by anhydrous sodium sulfate, and the solvent is removed by rotation to obtain 200g of 1,2,3, 4-tetraacetyl-alpha-L-fucose, wherein the yield is 99%.

(2) Synthesis of 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside

Into the reactor, 200g of 1,2,3, 4-tetraacetyl- α -L-fucose was added, dissolved in 1L of dichloromethane, and stirred. 232g of 2-chloro-4-nitrophenol and 60g of triethylamine are added, stirred under nitrogen. 426g BF was added dropwise at 25 deg.C₃·Et₂And O, after finishing dropping, continuing to react for 48 hours and stopping the reaction. After the reaction, 1L of water was added to quench the reaction, and 1L of dichloromethane was added to separate the reaction solution. The organic phase was washed with 3X 1L of saturated aqueous sodium hydroxide solution, filtered and separated. The aqueous phases were combined and back-washed with 1L dichloromethane. And (3) combining organic phases, rotatably removing the solvent to obtain a crude product 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside, and recrystallizing the crude product with ethanol to obtain 110g of 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside with the yield of 41%.

(3) Synthesis of 2-chloro-4-nitrobenzene-alpha-L-fucoside

Into the reactor, 100g of 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl- α -L-fucoside and 500mL of methanol were added and stirred at room temperature. 125mg of sodium methoxide was dissolved in 5mL of methanol, and the solution was added dropwise to the reaction system and stirred. TLC monitored the completion of the reaction of the starting materials and stopped the reaction. After the reaction, 30g of + H ion exchange resin was added, and the reaction was quenched with stirring. Filtering and removing the solvent to obtain a crude product 5. Adding 100mL of ethanol, pulping, and filtering to obtain 60g of a final product with the yield of 84%.

The quality (purity) of the final product 2-chloro-4-nitrobenzene-alpha-L-fucoside is determined to be 99%.

In the embodiment, the experiment result is not greatly fluctuated through repeated experiments; in contrast, in the prior art (the synthesis method disclosed in Japanese patent (JP 06-179690)), repeated experiments show that the experimental results fluctuate greatly.

Example 2

Synthesis of 1,2,3, 4-tetraacetyl-alpha-L-fucose

(1) To the reactor, 3kg of acetic anhydride and 600g of anhydrous sodium acetate were added in this order, heated to 80 ℃ and stirred. Adding 1kg of L-fucose in batches to prevent violent heat release; the reaction was exothermic and the internal temperature was controlled not to exceed 120 ℃. After the feeding is finished, the internal temperature is stabilized at 110 ℃ of 100-. The reaction system was cooled, 5L of ice water was added, and stirred overnight. Extracting the product with dichloromethane 3 × 3L, washing with water 2 × 2L, washing with 2L saturated sodium chloride solution once, drying the organic phase with anhydrous sodium sulfate, and removing solvent to obtain 1,2,3, 4-tetraacetyl-alpha-L-fucose 2kg with yield of 99%;

(2) synthesis of 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside

To the reactor, 2kg of 1,2,3, 4-tetraacetyl- α -L-fucose was added, dissolved in 10L of dichloromethane, and stirred. 1160g of 2-chloro-4-nitrophenol and 600g of triethylamine are added, and stirring is carried out under the protection of nitrogen. 4.26kg of BF was added dropwise at 25 deg.C₃·Et₂And O, after finishing dropping, continuing to react for 48 hours and stopping the reaction. After the reaction, 10L of water was added to quench the reaction, and 10L of dichloromethane was added to separate the reaction solution. The organic phase was washed with 3X 10L of saturated aqueous sodium hydroxide solution, filtered and separated. The aqueous phases were combined and back-washed with 1L dichloromethane. And (3) combining organic phases, rotatably removing the solvent to obtain a crude product 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside, and recrystallizing the crude product with ethanol to obtain 1060g of 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside with the yield of 40%.

(3) Synthesis of 2-chloro-4-nitrobenzene-alpha-L-fucoside

Into the reactor, 1000g of 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl- α -L-fucoside and 5000mL of methanol were added and stirred at room temperature. 1250mg of sodium methoxide was dissolved in 50mL of methanol, and the solution was added dropwise to the reaction system with stirring. TLC monitored the completion of the reaction of the starting materials and stopped the reaction. After the reaction, 300g of + H ion exchange resin was added, and the reaction was quenched with stirring. Filtering and removing the solvent to obtain a crude product 5. 1000mL of ethanol is added, and the mixture is pulped and filtered to obtain 580g of a final product with the yield of 81 percent.

The quality (purity) of the final product 2-chloro-4-nitrobenzene-alpha-L-fucoside is determined to be 99%.

Example 3

(1) Synthesis of 1,2,3, 4-tetraacetyl-alpha-L-fucose

Adding 100g L-fucose and 1L pyridine into a reactor, and stirring; 746g of acetic anhydride was added in portions at 25 ℃ to prevent a violent exotherm; after the addition was complete, the reaction was carried out at 60 ℃ for 4 h. After the reaction is finished, cooling, and removing pyridine by rotation to obtain a viscous liquid; pouring the viscous liquid into 1L dichloromethane, washing with water (5x 500mL), washing with 500mL saturated sodium chloride solution, drying with anhydrous sodium sulfate, and removing the solvent to obtain 1,2,3, 4-tetraacetyl-alpha-L-fucose;

(2) synthesis of 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside

Into the reactor, 200g of 1,2,3, 4-tetraacetyl- α -L-fucose was added, dissolved in 1L of dichloromethane, stirred, added 232g of 2-chloro-4-nitrophenol and 60g of triethylamine, protected with nitrogen, and stirred. At 40 ℃, 450g of TMSOTf is added dropwise, and the reaction is continued for 48 hours after the addition is finished, and the reaction is stopped. After the reaction, 1L of water was added to quench the reaction, and 1L of dichloromethane was added to separate the reaction solution. The organic phase was washed with 3X 1L of saturated aqueous sodium hydroxide solution, filtered and separated. The aqueous phases were combined and back-washed with 1L dichloromethane. Combining organic phases, rotatably removing a solvent to obtain a crude product 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside, and recrystallizing isopropanol to obtain 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl-alpha-L-fucoside;

(3) synthesis of 2-chloro-4-nitrobenzene-alpha-L-fucoside

Into the reactor, 100g of 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl- α -L-fucoside and 500mL of methanol were added and stirred at room temperature. 150mg of sodium tert-butoxide was dissolved in 5mL of methanol, and the solution was added dropwise to the reaction system and stirred. TLC monitored the completion of the reaction of the starting materials and stopped the reaction. After the reaction, 30g of + H ion exchange resin was added, and the reaction was quenched with stirring. Filtering and removing the solvent to obtain a crude product 5. Adding 100mL of isopropanol, pulping, and filtering to obtain the final product.

The quality (purity) of the final product 2-chloro-4-nitrobenzene-alpha-L-fucoside is determined to be 99%.

Comparative example 1

Step (1) same as example 1;

step (2): in a 200mL three-necked flask with a stirrer, 4.2g of anhydrous zinc chloride (catalyst), 10g of 1,2,3, 4-tetraacetyl-alpha-L-fucose and 52g of 2-chloro-4-nitrophenol were added, and after the addition was completed, the temperature was raised to 120 ℃ with stirring, and when the reaction solution was clear, the progress of the reaction was monitored by TLC. The reaction of the raw materials is completed, the reaction is stopped, the temperature is reduced, and 250mL of dichloromethane is added to dissolve the reaction solution. Washing with 0.1mol/L sodium hydroxide aqueous solution (3X 500mL), washing with saturated sodium chloride solution (3X 500mL), drying with anhydrous sodium sulfate, and removing solvent; the crude product was obtained by column chromatography (EA/PE ═ 1:5), and recrystallized from ethanol to give 3g of 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl- α -L-fucoside in 23% yield.

Step (3) same as example 1;

comparative example 2

Step (1) same as example 1;

step (2): 83g of anhydrous zinc chloride, 200g of 1,2,3, 4-tetraacetyl-alpha-L-fucose and 520g of 2-chloro-4-nitrophenol are added into a 2L three-neck flask with a stirrer, after the addition is finished, the temperature is raised to 120 ℃ by stirring, and the reaction progress is monitored by TLC after the reaction solution is clear. The reaction of the raw materials is completed, the reaction is stopped, the temperature is reduced, and 5L of dichloromethane is added to dissolve the reaction solution. Washing with 0.1mol/L sodium hydroxide aqueous solution (3X 1L), washing with saturated sodium chloride solution (3X2L), drying with anhydrous sodium sulfate, and removing solvent; the crude product was obtained by column chromatography (EA/PE ═ 1:5), and recrystallized from ethanol to give 48g of 2-chloro-4-nitrobenzene-2, 3, 4-triacetyl- α -L-fucoside in 18% yield.

Step (3) was the same as in example 1.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.