阅读说明：本技术 一种埃博霉素b的高效提取纯化方法 (Efficient extraction and purification method of epothilone B ) 是由 徐有安 付光明 龚美芳 于 2020-11-30 设计创作，主要内容包括：本发明涉及化学工业领域,特别涉及一种埃博霉素B的高效提取纯化方法,将富含埃博霉素B的发酵液过滤后得到湿树脂,该湿树脂先水洗再用一定质量分数的碳酸氢钠溶液洗涤2次之后再水洗、提取、浓缩至一定体积,再用一定质量分数的碳酸氢钠溶液洗涤2次,以彻底除去绝大部分的蛋白类物质,消除了其对后面两次结晶、溶解和正相制备色谱操作的影响,提高了产品质量同时节约了成本,能得到纯度≥99.0％的埃博霉素B的白色固体。(The invention relates to the field of chemical industry, in particular to a high-efficiency extraction and purification method of epothilone B, which is characterized in that fermentation liquor rich in epothilone B is filtered to obtain wet resin, the wet resin is washed by water firstly, then washed by sodium bicarbonate solution with a certain mass fraction for 2 times, then washed by water, extracted and concentrated to a certain volume, and then washed by sodium bicarbonate solution with a certain mass fraction for 2 times, so that most protein substances are thoroughly removed, the influence of the wet resin on the subsequent crystallization, dissolution and normal-phase preparation chromatographic operation of the two times is eliminated, the product quality is improved, the cost is saved, and white solid of epothilone B with the purity of more than or equal to 99.0% can be obtained.)

1. The efficient extraction and purification method of the epothilone B is characterized by comprising the following steps:

s1, collecting and washing resin: filtering the washed fermentation liquor of the epothilone B to obtain wet resin, washing the wet resin with a sodium bicarbonate solution, and then washing the wet resin with water;

s2, concentrating and extracting: filtering the wet resin, eluting the resin by using an eluent EA solution to obtain an ethyl acetate elution collected liquid, standing the obtained ethyl acetate elution collected liquid, separating a small amount of water in the lower layer, carrying out reduced pressure concentration on the ethyl acetate phase in the upper layer until the volume of the ethyl acetate phase is 10-20% of the volume before concentration, washing the obtained concentrated liquid by using a sodium bicarbonate solution, and concentrating the concentrated liquid again until the volume of the ethyl acetate phase is 10-20% of the volume before concentration;

and S3, carrying out crystallization twice, dissolution, normal-phase preparative chromatography and final crystallization on the solution obtained in the step S2 to obtain the epothilone B.

2. The method for efficiently extracting and purifying epothilone B according to claim 1, wherein the method comprises the following steps: the washing with the sodium bicarbonate solution in the S1 and the S2 means that the sodium bicarbonate solution with the mass fraction of 6% is used for washing twice.

3. The method for efficiently extracting and purifying epothilone B according to claim 1, wherein the method comprises the following steps: the two-time crystallization in the S3 comprises primary crystallization and secondary crystallization, wherein the primary crystallization is to add toluene with the same volume under stirring at 20-30 ℃, keep the temperature at 20-30 ℃ after the addition is finished, stir for 30 minutes, then uniformly heat to 40 ℃ I, uniformly cool to 0 ℃ and keep the temperature at 0 ℃ for stirring for 1 hour, and leach a filter cake obtained by filtration with toluene to obtain a primary crude product; and the secondary crystallization is to transfer the filter cake obtained by the primary crystallization to a container by using an EA solution, and repeat the primary crystallization again to obtain a secondary crude product.

4. The method for efficiently extracting and purifying epothilone B according to claim 1, wherein the method comprises the following steps: the dissolving in S3 means dissolving with dichloromethane.

5. The method for efficiently extracting and purifying epothilone B according to claim 1, wherein the method comprises the following steps: the normal phase preparative chromatography in the S3 is to fill 10 micron silica gel, sample injection is carried out by 10 times, and the eluent is a mixture of the following materials in a volume ratio of 2: 3, and detecting the detector condition at 290nm, and then eluting and collecting to obtain a collected liquid.

6. The method for efficiently extracting and purifying epothilone B according to claim 1, wherein the method comprises the following steps: and the final crystallization in the S3 means concentration, adding n-heptane, stirring at 20-25 ℃, filtering and drying.

Technical Field

The invention relates to the field of chemical industry, in particular to a high-efficiency extraction and purification method of epothilone B.

Background

In the prior art, the methods for preparing, extracting and purifying the epothilone B fermentation liquid are disclosed in Chinese invention patents, namely an epothilone B purification method (CN 106905338B) and an epothilone B production method (CN 106834377B), but the method for finally obtaining the product by filtering the epothilone B-rich fermentation liquid to obtain wet resin, washing, decompressing and concentrating, extracting, decompressing and concentrating, crystallizing, dissolving and secondarily crystallizing the wet resin still has the following defects in actual operation: the collected liquid obtained after fermentation of epothilone B contains more protein substances, so that a large amount of white protein solids are separated out in the subsequent concentration operation, the crystallization operation is influenced twice, the content of the crude product obtained after crystallization twice is low, more viscous substances are generated when the crude product is dissolved by dichloromethane, the operation is inconvenient, the content of the product obtained by final crystallization is slightly reduced, and more troublesome is that the protein substances can also reduce the column efficiency of a normal-phase preparation chromatographic column, so that the cost is increased, and the product quality is influenced.

Disclosure of Invention

In order to solve the problems, the invention provides a method for efficiently extracting and purifying epothilone B, which is characterized by comprising the following steps:

s1, collecting and washing resin: filtering the washed fermentation liquor of the epothilone B to obtain wet resin, washing the wet resin with a sodium bicarbonate solution, and then washing the wet resin with water;

s2, concentrating and extracting: filtering the wet resin, eluting the resin by using an eluent EA solution to obtain an ethyl acetate elution collected liquid, standing the obtained ethyl acetate elution collected liquid, separating a small amount of water in the lower layer, carrying out reduced pressure concentration on the ethyl acetate phase in the upper layer until the volume of the ethyl acetate phase is 10-20% of the volume before concentration, washing the obtained concentrated liquid by using a sodium bicarbonate solution, and concentrating the concentrated liquid again until the volume of the ethyl acetate phase is 10-20% of the volume before concentration;

and S3, carrying out crystallization twice, dissolution, normal-phase preparative chromatography and final crystallization on the solution obtained in the step S2 to obtain the epothilone B.

Moreover, the washing with the sodium bicarbonate solution in S1 and S2 means washing twice with a sodium bicarbonate solution with a mass fraction of 6%.

In addition, the two-time crystallization in the S3 comprises primary crystallization and secondary crystallization, wherein the primary crystallization is to add toluene with the same volume under stirring at 20-30 ℃, keep the temperature at 20-30 ℃ after the addition is finished, stir for 30 minutes, then uniformly heat up to 40 ℃ I, uniformly cool to 0 ℃ and keep the temperature at 0 ℃ and stir for 1 hour, and filter cakes obtained by filtration are leached by toluene to obtain a primary crude product; and the secondary crystallization is to transfer the filter cake obtained by the primary crystallization to a container by using an EA solution, and repeat the primary crystallization again to obtain a secondary crude product.

The dissolving in S3 means dissolving with methylene chloride.

And the normal phase preparative chromatography in the S3 is to fill 10 micron silica gel, sample injection is carried out by 10 times, and the eluent is a mixture of eluent with the volume ratio of 2: 3, and detecting the detector condition at 290nm, and then eluting and collecting to obtain a collected liquid.

And the final crystallization in S3 is concentration, adding n-heptane, stirring at 20-25 deg.C, filtering, and drying.

Compared with the prior art, the technical scheme has the beneficial effects that: the washing of the sodium bicarbonate aqueous solution is added in the steps of water washing and extraction, and after extraction, the sodium bicarbonate aqueous solution is concentrated and washed by the sodium bicarbonate aqueous solution again, so that most of protein substances are thoroughly removed, the influence of the protein substances on the subsequent two crystallization, dissolution and normal phase preparation chromatographic operation is eliminated, the product quality is improved, the cost is saved, and the white solid of the epothilone B with the purity of more than or equal to 99.0 percent can be obtained.

Drawings

FIG. 1 is an HPLC chromatogram of epothilone B obtained in example 1;

FIG. 2 is an HPLC chromatogram of epothilone B obtained in example 2.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings and examples, and the present invention is not limited to the examples.

Example 1:

1. resin collection and water washing:

(1)100L fermentation broth was filtered to yield 1.7kg of wet resin.

(2) The resin titer was measured as: 66mg B/L. (80mg A/L)

(3) Calculate approximate amount of epothilone B: (66mg B/L) 100L-6.6 g. (8g of A)

(4) Washing with 3L of water 2 times, then with 500ml of 6% by weight sodium bicarbonate solution 2 times, and finally with 3L of water 3 times, filtering off with a Buchner funnel (300mm and 10L filter flask), and then loading 2L of EA (i.e. ethyl acetate) into a resin column (diameter: 18cm and height: 10 cm).

2. Extraction:

(1) and (3) eluting the resin by using 6-8L of ethyl acetate to obtain 6-8L of ethyl acetate elution collecting liquid.

(2) Standing for more than 1 hour to separate a small amount of water from the lower layer.

(3) Concentrating the ethyl acetate phase at the upper layer under reduced pressure to 1.5L, separating out a small amount of solid, and filtering.

(4) The filtrate was washed 2 times with 200ml x 2 of 6% by weight sodium bicarbonate solution.

(5) Continuously concentrating to 200-300 ml.

3. Primary crystallization:

(1) under magnetic stirring at 20-30 deg.C, adding equal volume of toluene, and maintaining the temperature at 20-30 deg.C after about 30 min. After the addition is complete, the temperature is maintained at 20-30 ℃ and stirring is carried out for about 30 minutes.

(2) Raising the temperature to 40 ℃ within 3 hours at a constant speed, then lowering the temperature to 0 ℃ within 2 hours at a constant speed, keeping the temperature at 0 ℃ and stirring for 1 hour.

(3) Filter with a buchner funnel. The filter cake was rinsed with 50ml of room temperature toluene to give crude 1: 6 g.

4. And (3) recrystallizing:

(1) and transferring and washing the filter cake into a single-mouth bottle by using 200-300 ml of ethyl acetate.

(2) Under magnetic stirring at 20-30 deg.C, adding equal volume of toluene, and maintaining the temperature at 20-30 deg.C after about 30 min. After the addition is complete, the temperature is maintained at 20-30 ℃ and stirring is carried out for about 30 minutes.

(3) Raising the temperature to 40 ℃ within 3 hours at a constant speed, then lowering the temperature to 0 ℃ within 2 hours at a constant speed, keeping the temperature at 0 ℃ and stirring for 1 hour.

(4) Filter with a buchner funnel. The filter cake was rinsed with 50ml of room temperature toluene to give crude 2: 5g of the total weight.

DCM dissolution:

(1) dissolved in 50ml of dichloromethane.

(2) Filtering to obtain dichloromethane dissolved solution rich in active ingredients.

6. Normal phase preparative chromatography:

(1) filling 10 micron silica gel, injecting samples in 5 times, and eluting with ethyl acetate (volume ratio): n-heptane 2: 3, under a detector condition of 290 nm.

(2) Eluting and collecting to obtain 5L of collected liquid (purity of epothilone B is more than or equal to 99.0%)

7. And (3) final crystallization:

(1) concentrating to 200-300 ml, and adding 600-900 ml of n-heptane at room temperature.

Keeping the temperature of 20-25 ℃, stirring for 2 hours, filtering and drying to obtain epothilone B: 4.3g, Total yield: 65%, purity 99.41%, batch number: 202008026 and 109, the HPLC profile is shown in FIG. 1.

Example 2:

1. resin collection and water washing:

(1)300L of the fermentation broth was filtered to obtain 5kg of wet resin.

(2) The resin titer was measured as: 70mg B/L. (88mg A/L)

(3) Calculate approximate amount of epothilone B: (70mg B/L) 300L-21 g. (26.4g of A)

(4) Washing was carried out 2 times with 8 L.times.2 of water, 2 times with 1 L.times.2 of a 6% sodium bicarbonate solution and finally 3 times with 8 L.times.3 of water, filtration dried with a Buchner funnel (300mm and 10L filter flask) and then 3L of EA was packed into a resin column (diameter: 18cm and height: 10 cm).

2. Extraction:

(1) and eluting the resin by using 18-24L of ethyl acetate to obtain 18-24L of ethyl acetate elution collecting liquid.

(2) Standing for more than 1 hour to separate a small amount of water from the lower layer.

(3) Concentrating the ethyl acetate phase at the upper layer under reduced pressure to 4L, separating out a small amount of solid, and filtering.

(4) The filtrate was washed 2 times with 500ml x 2 of 6% by weight sodium bicarbonate solution.

(5) Continuously concentrating to 600-900 ml.

3. Primary crystallization:

(1) under magnetic stirring at 20-30 deg.C, adding equal volume of toluene, and maintaining the temperature at 20-30 deg.C after about 30 min. After the addition is complete, the temperature is maintained at 20-30 ℃ and stirring is carried out for about 30 minutes.

(2) Raising the temperature to 40 ℃ within 3 hours at a constant speed, then lowering the temperature to 0 ℃ within 2 hours at a constant speed, keeping the temperature at 0 ℃ and stirring for 1 hour.

(3) Filter with a buchner funnel. The filter cake was rinsed with 150ml of room temperature toluene to give crude 1: 20 g.

4. And (3) recrystallizing:

(1) transferring and washing the filter cake into a single-mouth bottle by using 600-900 ml of ethyl acetate.

(2) Under magnetic stirring at 20-30 deg.C, adding equal volume of toluene, and maintaining the temperature at 20-30 deg.C after about 30 min. After the addition is complete, the temperature is maintained at 20-30 ℃ and stirring is carried out for about 30 minutes.

(3) Raising the temperature to 40 ℃ within 3 hours at a constant speed, then lowering the temperature to 0 ℃ within 2 hours at a constant speed, keeping the temperature at 0 ℃ and stirring for 1 hour.

(4) Filter with a buchner funnel. The filter cake was rinsed with 150ml of room temperature toluene to give crude 2: 18 g.

DCM dissolution:

(1) dissolved in 150ml of dichloromethane.

(2) Filtering to obtain dichloromethane dissolved solution rich in active ingredients.

6. Normal phase preparative chromatography:

(1) filling 10 micron silica gel, injecting sample by 10 times, and eluting with ethyl acetate (volume ratio): n-heptane 2: 3, under a detector condition of 290 nm.

(2) Eluting and collecting to obtain 15L of collected liquid (purity of epothilone B is more than or equal to 99.0%)

7. And (3) final crystallization:

(1) concentrating to 600-900 ml, and adding 1800-2700 ml of n-heptane at room temperature.

Keeping the temperature of 20-25 ℃, stirring for 2 hours, filtering and drying to obtain epothilone B: 15.6g, overall yield: 74%, purity 99.55%, batch number: 202008026-95, the HPLC chromatogram is shown in FIG. 2.