CN114350737B - Preparation method of 5 alpha, 6 alpha-epoxy-11 beta, 17 alpha-dihydroxypregna-3, 20-diethylene glycol ketal - Google Patents
Preparation method of 5 alpha, 6 alpha-epoxy-11 beta, 17 alpha-dihydroxypregna-3, 20-diethylene glycol ketal Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims abstract description 58
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- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 claims description 4
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- AWQSAIIDOMEEOD-UHFFFAOYSA-N 5,5-Dimethyl-4-(3-oxobutyl)dihydro-2(3H)-furanone Chemical compound CC(=O)CCC1CC(=O)OC1(C)C AWQSAIIDOMEEOD-UHFFFAOYSA-N 0.000 claims description 2
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- VHRSUDSXCMQTMA-DZDRLISQSA-N (6R,8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-17-(2-hydroxyacetyl)-6,10,13-trimethyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthren-3-one Chemical compound C[C@@H]1C[C@H]2[C@@H]3CC[C@](O)(C(=O)CO)[C@@]3(C)C[C@H](O)[C@@H]2[C@@]2(C)C=CC(=O)C=C12 VHRSUDSXCMQTMA-DZDRLISQSA-N 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a preparation method of 5 alpha, 6 alpha-epoxy-11 beta, 17 alpha-dihydroxypregna-3, 20-diethylene glycol ketal, which takes buffer solution or pure water as a reaction system, adds substrate 6-methyl reducing substance, oxygen donor and oxygen carrier, and uses lipase to carry out biocatalysis synthesis reaction; after the reaction is finished, separating and purifying reactants to obtain a product 6-methyl epoxy compound, namely 5 alpha, 6 alpha-epoxy-11 beta, 17 alpha-dihydroxypregna-3, 20-diethylene glycol ketal, wherein the reaction formula is as followsThe invention uses enzyme method to biologically catalyze and synthesize the 6-methyl epoxy compound, which can avoid adverse effect on environment caused by chemical synthesis, and the enzyme method can synthesize peroxy acid for epoxy reaction in situ, which can avoid the risk of hydrogen peroxide addition in the chemical synthesis process.
Description
Technical Field
The invention relates to a preparation method of 5 alpha, 6 alpha-epoxy-11 beta, 17 alpha-dihydroxypregna-3, 20-diethylene glycol ketal, and belongs to the field of biocatalysis.
Background
Methylprednisone is a medium-effect synthetic product, belongs to adrenocortical hormone medicines in medicines of endocrine systems, is used for emergency treatment of critical diseases, and can also be used for treating endocrine dyscrasia, rheumatic diseases, collagenous diseases, skin diseases, anaphylactic reactions, ophthalmic diseases, gastrointestinal diseases, blood diseases, leukemia, shock, cerebral edema, polyneuritis, myelitis, emesis caused by cancer chemotherapy and the like. Patent CN 108912192 discloses a method for synthesizing 6 beta-methylprednisolone, wherein 5 alpha, 6 alpha-epoxy-11 beta, 17 alpha-dihydroxypregna-3, 20-diethylene glycol ketal is an intermediate for synthesizing methylprednisolone, and 5 alpha, 6 alpha-epoxy-11 beta, 17 alpha-dihydroxypregna-3, 20-diethylene glycol ketal is a 6-methyl epoxy compound with the following structural formula
Currently, the synthesis of 6-methyl epoxy compounds is chemical only and see in particular patent CN 107602652. The method can generate a large amount of toxic byproducts in the reaction process, has toxic effects on the environment and has a certain risk in the production process.
Disclosure of Invention
The invention aims to solve the defects in the background art, and the oxidation reaction is carried out by using a biocatalysis method, so that the required oxidation product is prepared under relatively mild reaction conditions, and the oxidation product does not cause great toxic effect on the environment. The lipase catalysis process does not need to rely on cofactors to catalyze oxidation reactions, and the in-situ production of peroxyacids can solve the risk in the peracid storage process.
In order to achieve the aim of the invention, the invention provides a preparation method of 5 alpha, 6 alpha-epoxy-11 beta, 17 alpha-dihydroxypregna-3, 20-diethylene glycol ketal, which takes buffer solution or pure water as a reaction system, adds substrate 6-methyl reducing substance, oxygen donor and oxygen carrier, and uses lipase to carry out biological catalytic synthesis reaction; after the reaction is finished, separating and purifying reactants to obtain a product 6-methyl epoxy compound, namely 5 alpha, 6 alpha-epoxy-11 beta, 17 alpha-dihydroxypregna-3, 20-diethylene glycol ketal, wherein the reaction formula is as follows
Further, after the reaction is finished, the reaction solution is centrifuged at 4 ℃ and 4000rpm, a filter cake is obtained by suction filtration of a sand core funnel, the filter cake is washed and purified by an organic solvent ethyl acetate until the detection purity is 97%, and the filter cake is dried to constant weight at 60 ℃ in vacuum, wherein the purification method can adopt the organic solvent for extraction.
Further, the buffer solution is one of a sodium phosphate buffer solution, a sodium acetate buffer solution and a sodium citrate buffer solution. Sodium acetate buffer is preferred.
Further, the lipase is one of immobilized lipase IM, biodiesel NS81006 and lipase LIPF. Preferably the lipase LIPF.
Further, the amount of the lipase is 2.5% -17.5% of the substrate mass percentage. Preferably 7.5%.
Further, the reaction temperature is 15 ℃ to 45 ℃. Preferably 30 ℃.
Further, the oxygen donor is H 2 O 2 The molar ratio of oxygen donor to substrate was 1:2-1:8, preferably 1:8.
further, the oxygen carrier is one or more of ethyl acetate, acetone, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, heptanoic acid, caprylic acid, capric acid, citric acid and palmitic acid; preferably octanoic acid; the molar ratio of the oxygen carrier to the substrate is 1:2-1:5, preferably 1:5.
further, a cosolvent is added into the reaction system, wherein the cosolvent is a hydrophobic solvent and/or a hydrophilic solvent; the hydrophobic solvent is one or more of n-hexane, tetrahydrofuran, acetone, toluene and ethyl acetate, and the hydrophilic solvent is one or more of ethanol, isopropanol, acetonitrile and methanol. The cosolvent is preferably isopropanol (2.5% -10%, v/v).
Compared with the prior art, the invention has the beneficial effects that:
the enzyme method is used for biocatalysis synthesis of the 6-methyl epoxy compound, so that adverse effects on the environment caused by chemical synthesis can be avoided, and the enzyme method can be used for in-situ synthesis of peroxyacid for epoxy reaction, so that the risk of hydrogen peroxide addition in the chemical synthesis process can be avoided.
Drawings
FIG. 1 is a control chromatogram of 5α,6α -epoxy-11β,17α -dihydroxypregna-3, 20-diethylene glycol ketal;
FIG. 2 is a chromatogram of the reaction process according to one embodiment of the invention.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings and specific embodiments.
Example 1:
(1) And (3) catalyst screening: the immobilized lipases NOV 40086, NS81006, vlan 8340, IM and LIP F were selected and catalyzed to epoxide reactions.
(2) Bioconversion scheme: the catalytic epoxy reaction system contained 50mM substrate 6-methyl reduction, 1M acetic acid/sodium acetate buffer (pH 5.8), ethyl acetate 7.5% (v/v), substrate: octanoic acid: hydrogen peroxide is 1:5:8, hydrogen peroxide (30% H) 2 O 2 Aqueous solution) was added at once, and then different catalysts (w/w, enzyme solution based on system volume ratio, immobilized enzyme or enzyme powder based on mass ratio, compared with mass percentage of the substrate) were added, and the reaction was started at 30℃and 220 rpm.
(3) The liquid phase detection flow comprises the following steps: after the conversion, the yield or purity was measured by liquid phase under conditions of ODS column (35 μm, 4.6X1250 mm) of a differential detector, acetonitrile: water (60:40), flow rate: 1ml/min.
Yield calculation:
yield = C a /C b *100％
C a 、C b Respectively the mass concentration of the initial addition substrate after 100% conversion compared with the mass concentration of the product at the end of the reaction
The liquid phase was examined for the yields, wherein the enzymes produced by the products were biodiesel NS81006, lipase LIP F, etc. Two enzymes with optimal catalytic efficiency were selected: the optimal addition amounts of immobilized lipase IM and lipase LIP F are optimized respectively, and the results are shown in Table 1, and the preferable result is that the lipase LIP F is used as a catalyst for epoxy reaction to carry out subsequent reactions.
TABLE 1 comparison of catalytic efficiencies of different catalysts
Example 2:
1M acetic acid/sodium acetate buffer (pH 5.8) containing 200g/L substrate 6-methyl reduction, ethyl acetate 7.5% (v/v), substrate: octanoic acid: hydrogen peroxide is 1:5:8, hydrogen peroxide (30% H) 2 O 2 Aqueous solution) was added to the reaction system after the completion of the one-time addition, 65% (w/w) of immobilized lipase LIP F was added to the reaction system, and reacted at different reaction temperatures. After the completion of the reaction, the yield was measured by the liquid phase detection method of example 1, and the results are shown in Table 2, with a reaction temperature of preferably 30℃being the optimum reaction temperature.
TABLE 2 comparison of different reaction temperatures
Example 3:
to reduce costs, enzyme powder is used instead of immobilized lipase LIP F and the appropriate catalyst addition is optimized. The optimized system was 1M acetic acid/sodium acetate buffer (pH 5.8) containing 200g/L substrate 6-methyl reduction, ethyl acetate 7.5% (v/v), substrate: octanoic acid: hydrogen peroxide is 1:5:8, hydrogen peroxide (30% H) 2 O 2 Aqueous solution) was added, different amounts of LIP F enzyme powder were added to the reaction system, and the reaction was started at 30℃and 220 rpm. After the completion of the reaction, the purity of the product in the reaction solution was measured by the liquid phase detection method of example 1, and the result is shown in Table 3, and the LIP F enzyme powder addition amount of 7.5% (w/w) was preferable in consideration of the cost and the impurity content, at this time, the yield was 75.62%.
TABLE 3 influence of catalyst addition on the epoxy reaction
Example 4:
1M acetic acid/sodium acetate buffer (pH 5.8) containing 200g/L substrate 6-methyl reduction, ethyl acetate 7.5% (v/v), substrate: different kinds of oxygen carriers: hydrogen peroxide is 1:5:8, hydrogen peroxide (30% H) 2 O 2 Aqueous solution) was added at a time, 7.5% (w/w) LIP F enzyme powder was added to the reaction system, and the reaction was started at 30℃and 220 rpm. After the reaction was completed, the yield was measured by the liquid phase detection method in example 1, and the results are shown in Table 4, and octanoic acid was preferable as an oxygen carrier for the epoxy reaction as compared with other oxygen carriers.
TABLE 4 influence of oxygen carrier types on epoxy reactions
Example 5:
1M acetic acid/sodium acetate buffer (pH 5.8) containing 200g/L substrate 6-methyl reduction, ethyl acetate 7.5% (v/v), using different substrates: octanoic acid: molar ratio of hydrogen peroxide, hydrogen peroxide (30% H) 2 O 2 Aqueous solution) was added at a time, 7.5% (w/w) LIP F enzyme powder was added to the reaction system, and the reaction was started at 30℃and 220 rpm. After the completion of the reaction, the yield was measured by the liquid phase detection method of example 1, and the results are shown in Table 5, octanoic acid and H 2 O 2 The ratio has obvious influence on the catalytic efficiency, and auxiliary material substrates are preferred: octanoic acid: h 2 O 2 The addition ratio is 1:5:8.
TABLE 5 Effect of adjuvant ratios on epoxy reactions
Example 6:
1M acetic acid/sodium acetate buffer (pH 5.8) contains 200g/L substrate 6-methyl reducing agent, helpingSolvent addition hydrophobic solvent 7.5% (v/v), hydrophilic solvent 5% (v/v), substrate: octanoic acid: the hydrogen peroxide is added in a proportion of 1:5:8, hydrogen peroxide (30% H) 2 O 2 Aqueous solution) was added at a time, and 7.5% (w/w) LIP F enzyme powder was added to the reaction system to start the reaction. After the completion of the reaction, the yield was measured by the liquid phase detection method of example 1, and as shown in Table 6, isopropyl alcohol was preferably used as a cosolvent in the present study, and the addition ratio of isopropyl alcohol was preferably (as shown in Table 7), and 7.5% was selected as the optimum addition amount of the reaction solvent.
TABLE 6 effect of cosolvent types on epoxy reactions
TABLE 7 effect of cosolvent addition on epoxy reaction
Example 7:
adding raw materials (200 g/L substrate 6-methyl reducing substance, substrate: octanoic acid: hydrogen peroxide (30% H2O2 water solution) in a molar ratio of 1:5:8, 7.5% isopropyl alcohol (v/v), 7.5% catalyst lipase LIP F enzyme powder (compared with the mass percentage of the substrate, w/w), after all auxiliary materials are added at one time, reacting at 30 ℃ and 245rpm, centrifuging after the reaction is finished, taking a precipitate after centrifuging, carrying out suction filtration on the precipitate by a sand core funnel to obtain a filter cake, washing the filter cake by using ethyl acetate with a proper proportion until the liquid phase detection purity of the product is 97%, and carrying out vacuum drying at 60 ℃ until the weight yield is 75%.
Comparing the liquid chromatograph of the reference substance of 5 alpha, 6 alpha-epoxy-11 beta, 17 alpha-dihydroxypregna-3, 20-diethylene glycol ketal shown in fig. 1 with the liquid chromatograph of the reaction process of the embodiment of the invention shown in fig. 2, and comparing the liquid chromatograph with the peak time consistent, and proving that the product prepared by the embodiment of the invention is the target product of 5 alpha, 6 alpha-epoxy-11 beta, 17 alpha-dihydroxypregna-3, 20-diethylene glycol ketal.
Finally, it should be noted that: the above embodiments are only for illustrating the technical aspects of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those of ordinary skill in the art that: modifications and equivalents may be made to the specific embodiments of the invention without departing from the spirit and scope of the invention, which is intended to be covered by the claims.
Claims (7)
1. The preparation method of 5 alpha, 6 alpha-epoxy-11 beta, 17 alpha-dihydroxypregna-3, 20-diethylene glycol ketal is characterized in that buffer solution or pure water is used as a reaction system, substrate 6-methyl reducing substance, oxygen donor and oxygen carrier are added, and lipase is used for carrying out biocatalytic synthesis reaction, wherein the use amount of the lipase is 2.5-17.5% in percentage by mass of substrate, the reaction temperature is 15-45 ℃, and the lipase is one of biodiesel NS81006 and lipase LIPF; after the reaction is finished, separating and purifying reactants to obtain a product 6-methyl epoxy compound, namely 5 alpha, 6 alpha-epoxy-11 beta, 17 alpha-dihydroxypregna-3, 20-diethylene glycol ketal, wherein the reaction formula is as follows
2. The method for preparing 5α,6α -epoxy-11β,17α -dihydroxypregna-3, 20-diethylene glycol ketal according to claim 1, wherein after the reaction, the reaction solution is centrifuged at 4 ℃ and 4000rpm, the sand core funnel is filtered to obtain a filter cake, and the filter cake is washed and purified by ethyl acetate as an organic solvent until the detection purity is 97%, and is dried under vacuum at 60 ℃ until the weight is constant.
3. The method of preparing 5α,6α -epoxy-11β,17α -dihydroxypregna-3, 20-diglycol ketal of claim 2, wherein the buffer is one of sodium phosphate buffer, sodium acetate buffer, and sodium citrate buffer.
4. A process for the preparation of 5α,6α -epoxy-11β,17α -dihydroxypregna-3, 20-diglycol ketal according to claim 3 wherein the lipase is LIPF.
5. The method for producing 5α,6α -epoxy-11β,17α -dihydroxypregna-3, 20-diglycol ketal according to claim 1, wherein the oxygen donor is H 2 O 2 The molar ratio of oxygen donor to substrate was 1:2-1:8.
6. the method for preparing 5α,6α -epoxy-11β,17α -dihydroxypregna-3, 20-diglycol ketal according to claim 1, wherein the oxygen carrier is one or more of ethyl acetate, acetone, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, heptanoic acid, caprylic acid, capric acid, citric acid and palmitic acid; the molar ratio of the oxygen carrier to the substrate is 1:2-1:5.
7. the method for preparing 5α,6α -epoxy-11β,17α -dihydroxypregna-3, 20-diglycol ketal according to claim 1, wherein a cosolvent is added in the reaction system, wherein the cosolvent is a hydrophobic solvent and/or a hydrophilic solvent; the hydrophobic solvent is one or more of n-hexane, tetrahydrofuran, acetone, toluene and ethyl acetate, and the hydrophilic solvent is one or more of ethanol, isopropanol, acetonitrile and methanol.
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