阅读说明：本技术 一种d-泛解酸的制备方法 (Preparation method of D-pantoic acid ) 是由 陈本顺 江涛 何伟 杨涛 于 2020-06-19 设计创作，主要内容包括：本发明提供一种D-泛解酸的制备方法,具体涉及药物化学技术领域,以化合物II(3-羟基-4,4-二甲基二氢呋喃-2-酮)为原料,在臭氧的作用下发生氧化反应得到化合物III(4,4-二甲基二氢呋喃-2,3-二酮),随后发生水解反应,并在酮还原酶的作用下发生不对称还原得到含有化合物I((R)-2,4-二羟基-3,3-二甲基丁酸)的混旋粗品,含有化合物I的混旋粗品重结晶步骤后得到高纯度的D-泛解酸。本方法合成步骤少,反应条件温和,工艺稳定性高,产物对映体过量值达99%以上,具有广阔的市场前景,更易于化合物I的工业化生产。(The invention provides a preparation method of D-pantoic acid, and particularly relates to the technical field of pharmaceutical chemistry, wherein a compound II (3-hydroxy-4, 4-dimethyldihydrofuran-2-ketone) is used as a raw material, an oxidation reaction is carried out under the action of ozone to obtain a compound III (4, 4-dimethyldihydrofuran-2, 3-dione), a hydrolysis reaction is carried out subsequently, asymmetric reduction is carried out under the action of ketoreductase to obtain a racemic crude product containing a compound I ((R) -2, 4-dihydroxy-3, 3-dimethylbutyric acid), and the racemic crude product containing the compound I is recrystallized to obtain high-purity D-pantoic acid. The method has the advantages of few synthesis steps, mild reaction conditions, high process stability, over 99 percent of enantiomeric excess value of the product, wide market prospect and easy industrial production of the compound I.)

1. A preparation method of D-pantoic acid is characterized in that a compound III (4, 4-dimethyldihydrofuran-2, 3-dione) is used as a raw material, hydrolysis reaction is carried out firstly, then asymmetric reduction reaction is carried out under the action of ketoreductase, a racemic crude product containing a compound I ((R) -2, 4-dihydroxy-3, 3-dimethylbutyric acid) is obtained, and high-purity D-pantoic acid ((R) -2, 4-dihydroxy-3, 3-dimethylbutyric acid) is obtained after a recrystallization step of the racemic crude product containing the compound I, and the synthetic route of the reaction is as follows:

2. the method for preparing D-pantoic acid according to claim 1, wherein the compound III is synthesized by using compound II (3-hydroxy-4, 4-dimethyldihydrofuran-2-one) as raw material and performing oxidation reaction under the action of ozone, and the specific synthetic route is as follows:

3. the preparation method of D-pantoic acid according to claim 2, characterized in that the specific method of synthesizing compound III is to use compound II as raw material, add solvent A and alkali, inject ozone at low temperature for reaction, after GC (gas chromatography) detects that the raw material has reacted completely, add acid to adjust pH to 7, and obtain compound III through extraction, suction filtration, and concentration under reduced pressure.

4. The process according to claim 3, wherein the base is selected from sodium methoxide, potassium tert-butoxide, sodium tert-butoxide, potassium hydroxide and sodium hydroxide, and the solvent A is selected from ethyl acetate, dichloromethane, ethanol, isopropanol and tetrahydrofuran.

5. The process according to claim 3, wherein the temperature is controlled to a value below-10 ℃ and O is introduced subsequently₃Solvent A, base and O₃The reaction temperature is-20 ℃ to 20 ℃.

6. The process for preparing D-pantoic acid according to claim 3, characterized in that compound II, base and O₃1: (1-5): (1-5).

7. The process for the preparation of D-pantoic acid according to any one of claims 1 to 6, characterized by the specific steps of:

1) adding hydrolysis solvent, compound III, ketoreductase, glucose dehydrogenase and NADP⁺Reacting with formic acid, performing suction filtration after reacting for a period of time, and concentrating the filtrate to obtain a crude product containing the compound I;

2) adding dichloromethane into the crude racemic mixture containing the compound I obtained in the step 1) to dissolve, adding a proper amount of NaOH solution to adjust the pH value to be 5, drying, removing dichloromethane under reduced pressure, and recrystallizing in a solvent B to obtain high-purity D-pantoic acid.

8. The preparation method of D-pantoic acid according to claim 7, wherein in step 1), the reaction temperature is 10 to 30 ℃ and the reaction time is 24 hours; the mass ratio of the compound III to the ketoreductase is (10:1) - (20: 1); the mass ratio of the compound III to the glucose dehydrogenase is (10:1) - (50: 1); compound III and NADP⁺The mass ratio is (50:1) - (200: 1); the molar ratio of the compound III to the formic acid is (10:1) - (1: 1).

9. The process for preparing D-pantoic acid according to claim 7, wherein the DNA sequence of the ketoreductase is represented by SEQ ID No.1, and the amino acid sequence is represented by SEQ ID No. 2; the DNA sequence of the glucose dehydrogenase is shown as SEQ ID No. 3.

10. The method for preparing D-pantoic acid according to claim 7, wherein the hydrolysis solvent in step 1) comprises a hydrolysis solvent A and a hydrolysis solvent B, the hydrolysis solvent A comprising one of sulfuric acid, hydrochloric acid or sodium hydroxide; the hydrolysis solvent B comprises one of ethyl acetate, dichloromethane, ethanol, isopropanol or tetrahydrofuran, the solvent B used for recrystallization in the step 2) is a mixture of isopropanol and acetone, and the mass ratio of the isopropanol to the acetone is 2: 1.

Technical Field

The invention belongs to the technical field of medicinal chemistry, and particularly relates to a preparation method of D-pantoic acid.

Background

Pantothenic acid, a B vitamin (vitamin B5), is a key precursor for the biosynthesis of coenzyme a (coa) and Acyl Carrier Protein (ACP). Both CoA and ACP are essential cofactors for cell growth and are involved in many metabolic reactions in key biosynthetic pathways. Pantothenic acid is synthesized by both microorganisms and plants, and must be obtained from the diet of mammals. Therefore, it is necessary to synthesize D-pantoic acid by a simple and efficient method.

There are related documents (Si D, Urano N, Nozaki S, et al, l-Pantoyl lactosucrose dehydrogenase from Rhodococcus erythropolis: genetic analytes and applications of the steric oxidation of l-Pantoyl lactone [ J ]. Applied microbiology and Biotechnology,2012,95(2):431 and 440.) reporting the synthesis of D-pantoic acid starting from 3-hydroxy-4, 4-dimethyldihydrofuran-2-one, the specific route is as follows:

the route involves the production of 4, 4-dimethyldihydrofuran-2, 3-dione by the enzyme LPLDH dehydrogenase, followed by hydrolysis and finally by the action of KPA reductase to produce D-pantoic acid. The hydrolysis step of the route can not realize direct synthesis from the compound III (4, 4-dimethyldihydrofuran-2, 3-dione) to the compound I ((R) -2, 4-dihydroxy-3, 3-dimethylbutyric acid), and the method has the disadvantages of high consumption of reaction substrates and high cost, and greatly limits the industrial production of the compound I.

In addition, by consulting the literature, it was found that there is no report on the realization of the one-step synthesis of compound III (4, 4-dimethyldihydrofuran-2, 3-dione) to compound I ((R) -2, 4-dihydroxy-3, 3-dimethylbutyric acid), i.e., the synthesis of compound I directly from compound III. In view of the fact that D-pantoic acid is an important vitamin and is essential in biosynthetic applications, it is essential to develop a new scheme route which is more economical and more favorable for industrial production, and we developed a class of routes for preparing the target product D-pantoic acid from 4, 4-dimethyldihydrofuran-2, 3-dione by a one-step reaction.

It is known from the above literature that the compound III (4, 4-dimethyldihydrofuran-2, 3-dione) is an important intermediate for the preparation of D-pantoic acid. Japanese patent JP05306276 provides a route for the preparation of compound III, which is as follows:

the above route uses 3-hydroxy-4, 4-dimethyldihydrofuran-2 (3H) as the starting material, DMSO as the solvent, and Cl (O ═ CC (═ O) Cl as the oxidant to synthesize 4, 4-dimethyldihydrofuran-2, 3-dione compounds, which involves the use of toxic reagents and the reaction conditions are carried out at-60 ℃, and clearly this scheme is not a desirable oxidation process. Similarly, Chang et al (Chang H S, Woo J C, Lee K M, et al, FACILE SYNTHESIS OF α -KETOCARBONYL COMPOUNDSFROM α -HYDROXYARBONYL COMPOUNDS [1] [ J ]. Synthetic Communications,2002,32(1):31-35) reported that the target product (4, 4-dimethyldihydrofuran-2, 3-dione) was obtained by oxidation reaction under the action of sodium hypobromite and hydrochloric acid. The reaction is complicated in operation and complicated in post-treatment. Furthermore, the documents relating to such reactions reported earlier that, with the rapid progress of organic synthesis, a novel research method was necessary for the synthesis of 4, 4-dimethyldihydrofuran-2, 3-dione compounds, that is, a method for synthesizing 4, 4-dimethyldihydrofuran-2, 3-dione under mild reaction conditions, which is advantageous for the preparation of D-pantoic acid.

Disclosure of Invention

In order to solve the problems of improper preparation conditions of D-pantoic acid, complex operation of a synthetic route, higher cost and the like in the prior art, the invention provides a more economic preparation method of D-pantoic acid, and a scheme for synthesizing a compound III (4, 4-dimethyldihydrofuran-2, 3-dione) to a compound I ((R) -2, 4-dihydroxy-3, 3-dimethylbutyric acid) in one step is realized, so that the utilization rate of a substrate is improved, the enantiomeric excess value of a product can reach more than 99 percent, and the production cost is greatly reduced.

The purpose of the invention can be realized by the following technical scheme:

a preparation method of D-pantoic acid comprises the steps of taking a compound III (4, 4-dimethyldihydrofuran-2, 3-dione) as a raw material, firstly carrying out hydrolysis reaction, then carrying out asymmetric reduction reaction under the action of ketoreductase to obtain a racemic crude product containing a compound I ((R) -2, 4-dihydroxy-3, 3-dimethylbutyric acid), and recrystallizing the racemic crude product containing the compound I to obtain high-purity D-pantoic acid ((R) -2, 4-dihydroxy-3, 3-dimethylbutyric acid), wherein the synthetic route of the reaction is as follows:

preferably, the compound III is synthesized by using a compound II (3-hydroxy-4, 4-dimethyldihydrofuran-2-one) as a raw material and performing an oxidation reaction under the action of ozone, wherein the specific synthetic route is as follows:

the reaction condition of the route is mild, and the use of polluting heavy metals and toxic reagents is avoided.

Preferably, the compound III is synthesized by a specific method, in which the compound II is used as a raw material, the solvent a and the base are added, ozone is introduced at a low temperature for reaction, GC (gas chromatography) is used to detect that the raw material has completely reacted, acid is added to adjust pH to 7, and the compound III is obtained by extraction, suction filtration, and concentration under reduced pressure.

Preferably, the base used is any one of sodium methoxide, potassium tert-butoxide, sodium tert-butoxide, potassium hydroxide or sodium hydroxide, preferably sodium hydroxide. The solvent A used comprises one or a mixture of more than two of ethyl acetate, dichloromethane, ethanol, isopropanol or tetrahydrofuran in any proportion.

Preferably, the temperature is controlled below-10 ℃, and then O is introduced₃Solvent A, base and O₃The reaction temperature is-20 ℃ to 20 ℃.

Preferably, compound II, base and O₃1: (1-5): (1-5). More preferably, the molar ratio of the compound II to the alkali and the ozone is 1: (1-3): (1-3).

Preferably, the specific steps are as follows:

1) adding hydrolysis solvent, compound III, ketoreductase, and NADP⁺Reacting with formic acid and glucose dehydrogenase, filtering after a period of reaction, and concentrating the filtrate to obtain a crude product containing the compound I;

2) adding dichloromethane into the crude racemic mixture containing the compound I obtained in the step 1) to dissolve, adding a proper amount of NaOH solution to adjust the pH value to be 5, drying, removing dichloromethane under reduced pressure, and recrystallizing in a solvent B to obtain high-purity D-pantoic acid.

Preferably, in the step 1), the reaction temperature is 10-30 ℃, and the reaction time is 24 hours; the mass ratio of the compound III to the ketoreductase is (10:1) - (20: 1).

Preferably, in step 1), the mass ratio of the compound III to the glucose dehydrogenase is (10:1) to (50: 1).

Preferably, in step 1), the mass ratio of the compound III to the NADP + is (50:1) - (200:1)

Preferably, in step 1), the molar ratio of the compound III to formic acid is (10:1) to (1: 1).

Preferably, the ketoreductase has a DNA sequence as set forth in SEQ ID No.1 and an amino acid sequence as set forth in SEQ ID No. 2; the DNA sequence of the glucose dehydrogenase is shown as SEQ ID No. 3.

Preferably, the hydrolysis solvent in step 1) comprises a hydrolysis solvent A and a hydrolysis solvent B, and the hydrolysis solvent A comprises one of sulfuric acid, hydrochloric acid or sodium hydroxide; the hydrolysis solvent B comprises one of ethyl acetate, dichloromethane, ethanol, isopropanol or tetrahydrofuran.

Preferably, the solvent used for recrystallization in step 2) is a mixture of isopropanol and acetone, and the mass ratio of isopropanol to acetone is 2: 1.

the invention has the beneficial effects that:

1. according to the invention, 4-dimethyldihydrofuran-2, 3-dione (compound III) is used as a raw material, and then the raw material is further hydrolyzed under the action of ketoreductase and is asymmetrically reduced to obtain (R) -2, 4-dihydroxy-3, 3-dimethylbutyric acid (compound I, namely D-pantoic acid) through one-step synthesis, so that the yield and the purity are high, the product purity reaches more than 99%, and the structure is consistent with the structure reported by literatures. The method has the advantages of few synthesis steps, higher process stability, suitability for large-scale production, simple operation of the synthesis route, low cost, suitability for industrial production and wide market prospect.

2. Taking 3-hydroxy-4, 4-dimethyl dihydrofuran-2-ketone (compound II) as a raw material, and reacting the raw material with O₃Oxidation to give 4, 4-dimethylbisThe hydrofuran-2, 3-diketone (compound III) has mild reaction conditions, and avoids the use of polluting heavy metals and toxic reagents.

Detailed Description

These two enzymes were used in all examples and are reported below:

glucose dehydrogenase: 3.6U/g, the DNA sequence is shown in SEQ ID No.3, and since the DNA sequence is known and the preparation method of glucose dehydrogenase is known, the preparation method is not written in detail, and the sequence is written to better distinguish the type of glucose dehydrogenase used.

Ketoreductase enzymes: 5.6U/g, the DNA sequence is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No. 2; since both the DNA sequence and the amino acid sequence are known and the method for preparing the ketoreductase is well known, the preparation method is not described in detail and the sequence is written to better distinguish the type of ketoreductase used.