Method for synthesizing quinolizinone compound by enzyme method
阅读说明：本技术 酶法合成喹嗪酮化合物的方法 (Method for synthesizing quinolizinone compound by enzyme method ) 是由 史社坡 王娟 王晓晖 刘晓 吴云 丁宁 齐博文 屠鹏飞 于 2019-10-18 设计创作，主要内容包括：本发明公开了一种酶法合成喹嗪酮化合物的方法。该酶法合成喹嗪酮化合物的方法采用Ⅲ型聚酮合酶,通过酶法合成喹嗪酮化合物。本发明的酶法合成喹嗪酮化合物的方法可以实现绿色合成,避免使用价格昂贵的稀有金属催化剂以及反应试剂环境不友好问题。(The invention discloses a method for synthesizing a quinolizinone compound by an enzymatic method. The method for synthesizing the quinolizinone compound by the enzyme method adopts type III polyketide synthase and synthesizes the quinolizinone compound by the enzyme method. The method for synthesizing the quinolizinone compound by the enzyme method can realize green synthesis, and avoids the problems of expensive rare metal catalysts and environment-unfriendly reaction reagents.)
1. A method for synthesizing a quinolizinone compound by an enzyme method is characterized in that a polyketide synthase III is adopted to synthesize the quinolizinone compound by the enzyme method.
2. The method of claim 1, wherein the quinolizinone compound is enzymatically synthesized using polyketide synthase type iii and coenzyme a ligase; the coenzyme A ligase is selected from phenylacetyl coenzyme A ligase PCL or malonyl coenzyme A ligase at.
3. The method of claim 2, wherein the quinolizinone compound is synthesized by an enzymatic method using three enzymes of polyketide synthase type iii, phenylacetyl-coa ligase PCL and malonyl-coa ligase at.matb, using a pyridine acetic acid compound and a malonic acid compound as substrates; wherein the type III polyketide synthase is selected from polyketide synthase HsPKS 3; the pyridine acetic acid compound is selected from pyridine acetic acid or substituted pyridine acetic acid; the malonic acid compound is selected from malonic acid or substituted malonic acid.
4. The method according to claim 3, wherein the pyridine acetic acid compound is at least one selected from pyridine acetic acid, 2- (5-fluoropyridin-3-yl) acetic acid, and 2- (6-fluoropyridin-3-yl) acetic acid; the malonic acid compound is at least one selected from malonic acid, methyl malonic acid, ethyl malonic acid and allyl malonic acid.
5. The method according to claim 4, characterized by comprising the following specific steps:
adding MgCl into phosphate buffer solution2、NaCl、DTT、ATP Na2Reacting CoA, a pyridine acetic acid compound, a malonic acid compound, phenylacetyl coenzyme A ligase PCL and malonyl coenzyme A ligase at.MatB at 25-35 ℃ for 1h, adding polyketide synthase HsPKS3, and continuing to react at 28-45 ℃ for overnight; the next day, the reaction solution is extracted, concentrated and purified to obtain a quinolizinone compound; wherein the molar ratio of the pyridine acetic acid compound to the malonic acid compound is 0.8-1.2: 0.8-1.2; the molar ratio of the pyridine acetic acid compound to the phenylacetyl coenzyme A ligase PCL is 8 multiplied by 105～1.2×1061, preparing a catalyst; MatB is 8 multiplied by 10 in the molar ratio of malonate compound to malonyl-CoA ligase at5～1.2×1061, preparing a catalyst; the molar ratio of phenylacetyl coenzyme A ligase PCL to malonyl coenzyme A ligase at.MatB to polyketide synthase HsPKS3 is 0.8-1.2: 0.8-1.2.
6. The method according to claim 1, characterized in that a quinolizinone compound is enzymatically synthesized by catalyzing pyridine acetyl-type coa and malonyl-type coa using only polyketide synthase type iii as the only enzyme; wherein the type III polyketide synthase is selected from polyketide synthase HsPKS 3; the pyridine acetyl coenzyme A is selected from pyridine acetyl coenzyme A or substituted pyridine acetyl coenzyme A; the malonyl-coenzyme A is selected from malonyl-coenzyme A or substituted malonyl-coenzyme A.
7. The method according to claim 6, wherein the pyridine acetyl-type coenzyme A is selected from at least one of pyridine acetyl-coenzyme A, 2- (5-fluoropyridin-3-yl) acetyl-coenzyme A, 2- (6-fluoropyridin-3-yl) acetyl-coenzyme A; the malonyl-coenzyme A is at least one selected from malonyl-coenzyme A, methylmalonyl-coenzyme A, ethylmalonyl-coenzyme A, and allylmalonyl-coenzyme A.
8. The method according to claim 6, characterized by comprising the following specific steps:
adding pyridine acetyl coenzyme A, malonyl coenzyme A and polyketide synthase HsPKS3 into a phosphate buffer solution, and reacting overnight in a water bath shaking table at the temperature of 28-45 ℃; the next day, the reaction solution is extracted, concentrated and purified to obtain a quinolizinone compound; wherein the molar ratio of the pyridine acetyl coenzyme A to the malonyl coenzyme A to the polyketide synthase HsPKS3 is 800-1200: 6.5-9.5.
9. The method according to any one of claims 6 to 8, further comprising a step of catalyzing the pyridine acetic acid compound to generate pyridine acetyl-type coenzyme A by phenylacetyl-coenzyme A ligase PCL; wherein the pyridine acetic acid compound is selected from pyridine acetic acid or substituted pyridine acetic acid.
10. The method of claim 9, wherein the phenylacetyl-coa ligase PCL catalyzes the production of pyridine acetyl-coa from a pyridine acetic acid compound by: taking Tris-HCl, NaCl and MgCl2、CoA、ATP Na2Pyridine acetic acid compounds and phenylacetyl coenzyme AInoculating enzyme PCL, adding water, and reacting at 25-35 ℃ for 4-8 h to obtain pyridine acetyl coenzyme A; wherein the molar ratio of the pyridine acetic acid compound to the phenylacetyl coenzyme A ligase PCL is 8 multiplied by 105～1.2×106:1。
Technical Field
The invention relates to a method for synthesizing a quinolizinone compound by an enzyme method.
Background
Quinolizinone compounds are a class of heterocyclic compounds which have a bicyclic ring system in structure and contain a nitrogen atom at the ring junction. The quinolizinone compound has polar zwitterion characteristics, shows unique physical and chemical properties such as easy binding with a protein receptor as a ligand molecule, has a moderate LogP value and the like. Quinolizinone compounds also have very broad pharmacological activities, such as antiviral, antitumor, antibacterial, etc., and are commonly used for treating Alzheimer's disease, type II diabetes, HIV, malaria, spinal muscular atrophy, etc.
At present, although a plurality of methods for constructing a quinolizinone structural parent nucleus of a quinolizinone compound are available, the methods are chemical synthesis methods, and most synthesis methods cannot avoid the problems of harsh reaction conditions, use of expensive rare metal catalysts, environment-friendliness of reaction reagents and the like. For example, CN105001216A discloses a preparation method of quinolizinone, which uses substituted azacyclylamine and carbon monoxide as raw materials, and performs carbonylation reaction in the presence or absence of a transition metal catalyst and a ligand to obtain a compound with quinolizinone structure. CN106928215A discloses a method for preparing a quinolizinone compound of formula i, which comprises: oxidizing a compound of a formula III in a solvent under the action of an oxidant, under an electro-redox condition or under a photo-redox condition, and then treating with an acid to obtain a cyclized cis-form compound of a formula II; and step (B) hydrolyzing the compound of formula II to obtain the compound of formula I.
How to synthesize the quinolizinone compound in a green way, and the problems of expensive rare metal catalysts and environment-unfriendly reaction reagents are avoided, and no relevant report exists at present.
Enzymatic synthesis has received increasing attention from scientists as an important component in green chemistry.
In view of the defects of the prior art, a method for enzymatically synthesizing the quinazinone alkaloid and the derivatives thereof is established, so that green synthesis is realized, and the problems of expensive rare metal catalysts and environment-unfriendly reaction reagents are avoided, which is very necessary.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for synthesizing a quinolizinone compound by an enzymatic method, which adopts an enzymatic synthesis method to realize green synthesis and avoids the problems of using expensive rare metal catalysts and using reaction reagents that are not environment-friendly.
The invention adopts the following technical scheme to achieve the purpose.
The invention provides a method for synthesizing a quinolizinone compound by an enzyme method, which adopts III type polyketide synthase to synthesize the quinolizinone compound by the enzyme method.
According to the method of the present invention, preferably, the quinolizinone compound is enzymatically synthesized using polyketide synthase type iii and coenzyme a ligase; the coenzyme A ligase is selected from phenylacetyl coenzyme A ligase PCL or malonyl coenzyme A ligase at.
According to the method, preferably, three enzymes of type III polyketide synthase, phenylacetyl coenzyme A ligase PCL and malonyl coenzyme A ligase at.MatB are adopted, pyridine acetic acid compounds and malonic acid compounds are used as substrates, and the quinolizinone compounds are synthesized by an enzyme method; wherein the type III polyketide synthase is selected from polyketide synthase HsPKS 3; the pyridine acetic acid compound is selected from pyridine acetic acid or substituted pyridine acetic acid; the malonic acid compound is selected from malonic acid or substituted malonic acid.
According to the method of the present invention, preferably, the pyridine acetic acid compound is at least one selected from pyridine acetic acid, 2- (5-fluoropyridin-3-yl) acetic acid, and 2- (6-fluoropyridin-3-yl) acetic acid; the malonic acid compound is at least one selected from malonic acid, methyl malonic acid, ethyl malonic acid and allyl malonic acid.
The method according to the invention preferably comprises the following specific steps:
adding MgCl into phosphate buffer solution2、NaCl、DTT、ATP Na2Reacting CoA, a pyridine acetic acid compound, a malonic acid compound, phenylacetyl coenzyme A ligase PCL and malonyl coenzyme A ligase at.MatB at 25-35 ℃ for 1h, adding polyketide synthase HsPKS3, and continuing to react at 28-45 ℃ for overnight; the next day, the reaction solution is extracted, concentrated and purified to obtain a quinolizinone compound; wherein the molar ratio of the pyridine acetic acid compound to the malonic acid compound is 0.8-1.2: 0.8-1.2; the molar ratio of the pyridine acetic acid compound to the phenylacetyl coenzyme A ligase PCL is 8 multiplied by 105～1.2×1061, preparing a catalyst; MatB is 8 multiplied by 10 in the molar ratio of malonate compound to malonyl-CoA ligase at5～1.2×1061, preparing a catalyst; the molar ratio of phenylacetyl coenzyme A ligase PCL to malonyl coenzyme A ligase at.MatB to polyketide synthase HsPKS3 is 0.8-1.2: 0.8-1.2.
According to the process of the present invention, preferably, a quinolizinone compound is enzymatically synthesized by catalyzing pyridine acetyl-type coa and malonyl-type coa using only type iii polyketide synthase as the only enzyme; wherein the type III polyketide synthase is selected from polyketide synthase HsPKS 3; the pyridine acetyl coenzyme A is selected from pyridine acetyl coenzyme A or substituted pyridine acetyl coenzyme A; the malonyl-coenzyme A is selected from malonyl-coenzyme A or substituted malonyl-coenzyme A.
According to the method of the present invention, preferably, the pyridine acetyl-type coenzyme A is selected from at least one of pyridine acetyl-coenzyme A, 2- (5-fluoropyridin-3-yl) acetyl-coenzyme A, 2- (6-fluoropyridin-3-yl) acetyl-coenzyme A; the malonyl-coenzyme A is at least one selected from malonyl-coenzyme A, methylmalonyl-coenzyme A, ethylmalonyl-coenzyme A, and allylmalonyl-coenzyme A.
The method according to the invention preferably comprises the following specific steps:
adding pyridine acetyl coenzyme A, malonyl coenzyme A and polyketide synthase HsPKS3 into a phosphate buffer solution, and reacting overnight in a water bath shaking table at the temperature of 28-45 ℃; the next day, the reaction solution is extracted, concentrated and purified to obtain a quinolizinone compound; wherein the molar ratio of the pyridine acetyl coenzyme A to the malonyl coenzyme A to the polyketide synthase HsPKS3 is 800-1200: 6.5-9.5.
According to the method, preferably, the method further comprises a step of catalyzing a pyridine acetic acid compound to generate pyridine acetyl coenzyme A by using phenylacetyl coenzyme A ligase PCL; wherein the pyridine acetic acid compound is selected from pyridine acetic acid or substituted pyridine acetic acid.
According to the method of the invention, preferably, the phenylacetyl-CoA ligase PCL catalyzes the pyridine acetic acid compound to generate the pyridine acetyl-CoA by the following steps: taking Tris-HCl, NaCl and MgCl2、CoA、ATP Na2Adding water into a pyridine acetic acid compound and phenylacetyl coenzyme A ligase PCL, and reacting for 4-8 h at 25-35 ℃ to obtain pyridine acetyl coenzyme A; wherein the molar ratio of the pyridine acetic acid compound to the phenylacetyl coenzyme A ligase PCL is 8 multiplied by 105～1.2×106:1。
The method for synthesizing the quinolizinone compound by the enzyme method adopts the type III polyketone synthase, and synthesizes the quinolizinone compound by the enzyme method, so that green synthesis is realized, and the problems of expensive rare metal catalysts and environment-unfriendly reaction reagents are avoided.
Drawings
FIG. 1 shows the results of SDS-PAGE gel electrophoresis of HsPKS3, a polyketide synthase, in Huperzia serrata.
FIG. 2 shows the results of gel electrophoresis of the cloned pcl gene.
FIG. 3 shows the result of SDS-PAGE gel electrophoresis of the clonally expressed phenylacetyl-CoA ligase PCL.
FIG. 4 shows the results of SDS-PAGE gel electrophoresis of malonyl-CoA ligase at.
FIG. 5 is a scheme showing the synthesis of quinolizinone compounds using three enzymes, polyketide synthase type III (HsPKS 3), phenylacetyl-CoA ligase PCL and malonyl-CoA ligase at.
FIG. 6 is a scheme showing the synthesis scheme for the synthesis of quinolizinone compounds using only polyketide synthase type III (polyketide synthase HsPKS3) as the sole enzyme catalyzing pyridine acetyl-type CoA and malonyl-type CoA.
FIG. 7 is a synthesis scheme of phenylacetyl-CoA ligase PCL catalyzing pyridine acetic acid compounds to generate pyridine acetyl-CoA.
Fig. 8 is a scheme showing the synthesis of malonyl-coa from malonyl-coa ligase at.matb catalyzing malonates.
Figure 9 is a synthetic scheme for a quinolizinone compound.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the scope of the present invention is not limited thereto.
The traditional preparation methods of the quinolizinone compounds all adopt chemical synthesis methods, and most of the synthesis methods cannot avoid the problems of harsh reaction conditions, use of expensive rare metal catalysts, environment-friendly reaction reagents and the like. The application unexpectedly finds that the quinolizinone compound is synthesized by adopting the III type polyketide synthase through an enzyme method, green synthesis can be realized, and the problems of expensive rare metal catalysts and environment-unfriendly reaction reagents are avoided. Furthermore, the reconstitution of the synthetic pathway of a quinolizinone compound in a microorganism can be achieved by enzymatically synthesizing the quinolizinone compound using a type iii polyketide synthase to provide necessary genetic elements.
The method for synthesizing the quinolizinone compound by the enzyme method comprises the following steps: the quinolizinone compound is synthesized by enzyme method by using type III polyketide synthase. The method adopts III type polyketone synthase to synthesize the quinolizinone compound by an enzyme method, realizes green synthesis, and avoids the problems of expensive rare metal catalysts and environment-unfriendly reaction reagents.
In the present invention, type III polyketide synthase is a reused subunit molecule of 40X 10 in size3～47×103The homodimer autonomous synthase can directly catalyze the condensation between pantephthalein coenzyme A to form monocyclic or bicyclic aromatic polyketides. The polyketide synthase type III is preferably polyketide synthase HsPKS3, more preferably polyketide synthase HsPKS3 clonally expressed from Huperzia serrata.
In the present invention, the quinolizinone compound can be enzymatically synthesized using polyketide synthase type III and coenzyme A ligase. The coenzyme a ligase is preferably selected from the group consisting of phenylacetyl-coa ligase PCL or malonyl-coa ligase at.matb, more preferably phenylacetyl-coa ligase PCL and malonyl-coa ligase at.matb. Preferably, the quinolizinone compound is enzymatically synthesized using a type iii polyketide synthase in combination with three enzymes, phenylacetyl-coa ligase PCL and malonyl-coa ligase at. The phenylacetyl-coa ligase PCL is preferably a phenylacetyl-coa ligase (PCL) which is clonally expressed from Penicillium chrysogenum. Malonyl-coa ligase (at.matb) is preferably malonyl-coa ligase (at.matb) which is clonally expressed from arabidopsis thaliana (a.thaliana).
In the present invention, the quinolizinone compound may be enzymatically synthesized using only type III polyketide synthase as the only enzyme and pyridine acetyl coenzyme A and malonyl coenzyme A as substrates. Pyridine acetyl coenzyme A can be obtained by a commercially available method, a conventional chemical synthesis method, or a biological method such as an enzymatic synthesis method. Preferably, the pyridine acetyl-type coenzyme A is obtained by biological methods such as enzymatic synthesis. Malonyl-coa can be obtained commercially, by conventional chemical synthesis, or by biological methods such as enzymatic synthesis. Preferably, malonyl-coa is obtained by biological methods, such as enzymatic synthesis.
According to one embodiment of the invention, three enzymes of type III polyketide synthase, phenylacetyl coenzyme A ligase PCL and malonyl coenzyme A ligase at.MatB are adopted, pyridine acetic acid compounds and malonic acid compounds are taken as substrates, and then the enzyme method is adopted to synthesize the quinolizinone compounds; wherein the type III polyketide synthase is selected from polyketide synthase HsPKS 3; the pyridine acetic acid compound is selected from pyridine acetic acid or substituted pyridine acetic acid; the malonic acid compound is selected from malonic acid or substituted malonic acid. Wherein, the synthetic route of the quinolizinone compound is shown in figure 5.
In the present invention, the substituent of the substituted pyridine acetic acid may be selected from any one of hydroxyl, C1-C9 alkyl, C1-C9 alkoxy, halo and aryl, preferably any one of hydroxyl, C1-C4 alkyl, C1-C4 alkoxy, halo, phenyl and benzyl, more preferably any one of hydroxyl, methyl, ethyl, methoxy, ethoxy and halo. Examples of C1-C4 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. The halo group is preferably selected from fluoro, chloro, bromo.
In the present invention, the substituent of the substituted malonic acid may be selected from any one of hydroxy, allyl, C1-C9 alkyl, C1-C9 alkoxy, halo, and aryl, preferably any one of hydroxy, allyl, C1-C4 alkyl, C1-C4 alkoxy, halo, phenyl, and benzyl, and more preferably any one of hydroxy, allyl, methyl, ethyl, methoxy, ethoxy, and halo. Examples of C1-C4 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. The halo group is preferably selected from fluoro, chloro, bromo.
In the present invention, the pyridine acetic acid compound is preferably at least one selected from the group consisting of pyridine acetic acid, 5-F-pyridine acetic acid (2- (5-fluoropyridin-3-yl) acetic acid), and 6-F-pyridine acetic acid (2- (6-fluoropyridin-3-yl) acetic acid). The malonic acid-based compound is preferably at least one selected from malonic acid, methylmalonic acid, ethylmalonic acid and allylmalonic acid.
In certain embodiments, the enzymatic synthesis of a quinolizinone compound comprises the following specific steps: adding MgCl into phosphate buffer solution2、NaCl、DTT、ATP Na2Reacting CoA, a pyridine acetic acid compound, a malonic acid compound, phenylacetyl coenzyme A ligase PCL and malonyl coenzyme A ligase at.MatB at 25-35 ℃ for 1h, adding polyketide synthase HsPKS3, and continuing to react at 28-45 ℃ for overnight; the next day, the reaction solution is extracted, concentrated and purified to obtain the quinolizinone compound. The molar ratio of the pyridine acetic acid compound to the malonic acid compound may be 0.8 to 1.2:0.8 to 1.2, preferably 0.9 to 1.1:0.9 to 1.1, and more preferably 0.95 to 1.05:0.95 to 1.05. The molar ratio of the pyridine acetic acid compound to the phenylacetyl coenzyme A ligase PCL can be 8 × 105～1.2×1061, preferably 9X 105～1.1×1061, more preferably 9.5X 105～1.05×106:1. MatB may be 8X 10 in the molar ratio of malonates to malonyl-CoA ligase at5～1.2×1061, preferably 9X 105～1.1×1061, more preferably 9.5X 105～1.05×106:1. The molar ratio of phenylacetyl coenzyme A ligase PCL, malonyl coenzyme A ligase at.MatB and polyketide synthase HsPKS3 can be 0.8-1.2: 0.8-1.2, preferably 0.9-1.1: 0.9-1.1, and more preferably 0.95-1.05: 0.95-1.05.
According to another embodiment of the invention, a quinolizinone compound is enzymatically synthesized using only polyketide synthase type iii as the sole enzyme catalyzing pyridine acetyl-type coa and malonyl-type coa; wherein the type III polyketide synthase is selected from polyketide synthase HsPKS 3; the pyridine acetyl coenzyme A is selected from pyridine acetyl coenzyme A or substituted pyridine acetyl coenzyme A; the malonyl-coenzyme A is selected from malonyl-coenzyme A or substituted malonyl-coenzyme A. The synthetic route of the quinolizinone compound is shown in figure 6.
In the present invention, the substituent of the substituted pyridine acetyl coenzyme A can be selected from any one of hydroxyl, C1-C9 alkyl, C1-C9 alkoxy, halo and aryl, preferably any one of hydroxyl, C1-C4 alkyl, C1-C4 alkoxy, halo, phenyl and benzyl, and more preferably any one of hydroxyl, methyl, ethyl, methoxy, ethoxy and halo. Examples of C1-C4 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. The halo group is preferably selected from fluoro, chloro, bromo.
In the present invention, the substituent of the substituted malonyl-coenzyme A may be selected from any one of hydroxyl, allyl, C1-C9 alkyl, C1-C9 alkoxy, halo and aryl, preferably any one of hydroxyl, allyl, C1-C4 alkyl, C1-C4 alkoxy, halo, phenyl and benzyl, more preferably any one of hydroxyl, allyl, methyl, ethyl, methoxy, ethoxy and halo. Examples of C1-C4 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. The halo group is preferably selected from fluoro, chloro, bromo.
In the present invention, the pyridine acetyl-type coenzyme A is preferably at least one member selected from the group consisting of pyridine acetyl coenzyme A, 5-F-pyridine acetyl coenzyme A (2- (5-fluoropyridin-3-yl) acetyl coenzyme A), and 6-F-pyridine acetyl coenzyme A (2- (6-fluoropyridin-3-yl) acetyl coenzyme A). The malonyl-coenzyme A is preferably at least one member selected from the group consisting of malonyl-coenzyme A, methylmalonyl-coenzyme A, ethylmalonyl-coenzyme A, and allylmalonyl-coenzyme A.
In certain embodiments, the enzymatic synthesis of a quinolizinone compound comprises the following specific steps: adding pyridine acetyl coenzyme A, malonyl coenzyme A and polyketide synthase HsPKS3 into a phosphate buffer solution, and reacting overnight in a water bath shaking table at the temperature of 28-45 ℃; the next day, the reaction solution is extracted, concentrated and purified to obtain a quinolizinone compound; wherein the molar ratio of the pyridine acetyl coenzyme A to the malonyl coenzyme A to the polyketide synthase HsPKS3 is 800-1200: 6.5-9.5. The molar ratio of the pyridine acetyl coenzyme A to the malonyl coenzyme A to the polyketide synthase HsPKS3 is preferably 900-1100: 7-9; more preferably 950 to 1050:7.8 to 8.5. The phosphate buffer solution is preferably selected from potassium phosphate buffer solution or sodium phosphate buffer solution, more preferably potassium phosphate buffer solution. According to a specific embodiment of the invention, pyridine acetyl coenzyme A, malonyl coenzyme A and polyketide synthase HsPKS3 are added into potassium phosphate buffer solution, and the mixture reacts in a water bath shaking table at the temperature of 28-45 ℃ overnight; the next day, the reaction solution is extracted, concentrated and purified to obtain a quinolizinone compound; wherein the molar ratio of the pyridine acetyl coenzyme A to the malonyl coenzyme A to the polyketide synthase HsPKS3 is 950-1050: 7.8-8.5.
In the invention, the method for synthesizing the quinolizinone compound by the enzyme method can also comprise the step of catalyzing a pyridine acetic acid compound to generate pyridine acetyl coenzyme A by phenylacetyl coenzyme A ligase PCL; wherein the pyridine acetic acid compound is selected from pyridine acetic acid or substituted pyridine acetic acid. Wherein, the synthetic route of the pyridine acetyl coenzyme A is shown in figure 7. The substituent of the substituted pyridine acetic acid can be selected from any one of hydroxyl, C1-C9 alkyl, C1-C9 alkoxy, halo and aryl, preferably any one of hydroxyl, C1-C4 alkyl, C1-C4 alkoxy, halo, phenyl and benzyl, and more preferably any one of hydroxyl, methyl, ethyl, methoxy, ethoxy and halo. Examples of C1-C4 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. The halo group is preferably selected from fluoro, chloro, bromo.
In the invention, the phenylacetyl-CoA ligase PCL catalyzes the step of generating the pyridine acetyl-CoA from the pyridine acetic acid compoundThe following are preferred: taking Tris-HCl, NaCl and MgCl2、CoA、ATP Na2Adding water into a pyridine acetic acid compound and phenylacetyl coenzyme A ligase PCL, and reacting for 4-8 h at 25-35 ℃ to obtain pyridine acetyl coenzyme A; wherein the molar ratio of the pyridine acetic acid compound to the phenylacetyl coenzyme A ligase PCL is 8 multiplied by 105～1.2×106:1. The molar ratio of the pyridine acetic acid compound to the phenylacetyl coenzyme A ligase PCL is preferably 9X 105～1.1×1061, more preferably 9.5X 105～1.05×106:1。
In the present invention, the method for synthesizing a quinolizinone compound by an enzymatic method may further comprise a step of malonyl-coa ligase at.matb catalyzing the malonate compound to generate malonyl-coa; wherein the malonic acid compounds are selected from malonic acid or substituted malonic acid. Wherein, the synthetic route of the malonyl-coenzyme A is shown in figure 8. The substituent of the substituted malonic acid can be selected from any one of hydroxyl, allyl, C1-C9 alkyl, C1-C9 alkoxy, halo and aryl, preferably any one of hydroxyl, allyl, C1-C4 alkyl, C1-C4 alkoxy, halo, phenyl and benzyl, and more preferably any one of hydroxyl, allyl, methyl, ethyl, methoxy, ethoxy and halo. Examples of C1-C4 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl. The halo group is preferably selected from fluoro, chloro, bromo.
In the present invention, the step of malonyl-coa ligase at.matb catalyzing the production of malonyl-coa from a malonate-type compound is preferably as follows: taking phosphate buffer solution and MgCl2、CoA、ATP Na2DTT, malonic acid compounds and malonyl coenzyme A ligase at.MatB, adding water, and reacting at 25-35 ℃ for 8-15 h to obtain malonyl coenzyme A; wherein the molar ratio of the malonic acid compound to the malonyl-CoA ligase at.MatB is 4 × 105～9×105:1. The molar ratio of malonates to malonyl-coa ligase at.matb is preferably 5 × 105～8×1051, more preferably 5.5X 105～7×105:1. Phosphate buffer solutionPreferably selected from potassium phosphate buffer or sodium phosphate buffer, more preferably potassium phosphate buffer. According to one embodiment of the invention, phosphate buffer solution, MgCl, is taken2、CoA、ATP Na2DTT, malonic acid compounds and malonyl coenzyme A ligase at.MatB, adding water, and reacting at 25-35 ℃ for 8-15 h to obtain malonyl coenzyme A; wherein the molar ratio of the malonic acid compound to the malonyl-CoA ligase at.MatB is 5.5 × 105～6.5×105:1。
According to one embodiment of the present invention, a method for enzymatically synthesizing a quinolizinone compound comprises the steps of: (1) phenylacetyl coenzyme A ligase PCL catalyzes a pyridine acetic acid compound to generate pyridine acetyl coenzyme A; wherein the pyridine acetic acid compound is selected from pyridine acetic acid or substituted pyridine acetic acid; (2) malonyl-coa ligase at.matb catalyzes the production of malonyl-coa from malonate-like compounds; wherein the malonic acid compounds are selected from malonic acid or substituted malonic acid; (3) synthesizing a quinolizinone compound by an enzymatic method using only type III polyketide synthase as a sole enzyme to catalyze pyridine acetyl coenzyme A and malonyl coenzyme A; wherein said type III polyketide synthase is selected from the group consisting of polyketide synthase HsPKS 3. The synthetic route of the quinolizinone compound is shown in figure 9.
The experimental reagents, instruments and detection indexes adopted in the following examples and experimental examples are as follows:
LB liquid medium: dissolving 10g tryptone, 5g yeast extract, 10g NaCl and distilled water, adjusting pH to 7.0 with NaOH, adding distilled water to constant volume of 1L, and sterilizing with high pressure steam at 121 deg.C for 15 min.
IPTG (isopropyl-. beta. -D-thiogalactoside), PMSF (phenylmethylsulfonyl fluoride) were purchased from Beijing Byeldi Biometrics, Inc. CoA (coenzyme A), ATP Na2Disodium adenosine triphosphate, DTT (dithiothreitol), pyridine acetic acid, 2- (5-fluoropyridin-3-yl) acetic acid, 2- (6-fluoropyridin-3-yl) acetic acid, malonic acid, methylmalonic acid, ethylmalonic acid, and allylmalonic acid were all purchased from Shanghai-derived PhylloBiotech Ltd.
Preparing a buffer solution related to protein crushing, extraction and purification:
lysis buffer: weighing 4.145g K2HPO4，0.25g KH2PO42.92g of NaCl and 0.17g of imidazole are dissolved in 300mL of double distilled water, the pH value is adjusted to 7.9, and the volume is adjusted to 500 mL;
binding buffer: weighing 2.072g K2HPO4，0.125g KH2PO414.61g of NaCl is dissolved in 300mL of double distilled water, the pH value is adjusted to 7.9, and the volume is adjusted to 500 mL;
KPB wash buffer: weighing 2.072g K2HPO40.125g of KH2PO4, 14.61g of NaCl and 1.362g of imidazole are dissolved in 300mL of double distilled water, the pH value is adjusted to 7.9, and the volume is adjusted to 500 mL;
KPB elution buffer: weigh 1.554g K2HPO4，0.096g KH2PO4Dissolving 50mL of glycerol and 13.62g of imidazole in 300mL of double distilled water, adjusting the pH value to 7.9, and metering the volume to 500 mL;
desalting buffer used for desalting: weighing 2.145g K2HPO4，0.0817g KH2PO450mL of glycerol and 1mL of 0.5M EDTA were dissolved in 300mL of double distilled water, the pH was adjusted to 7.9, and the volume was adjusted to 500 mL.
TANG Buffer: 3.028g Tris is weighed, adjusted to pH 7.5 with HCl, 5.844g NaCl, 50mL glycerol, 0.01g sodium azide, and dissolved in 500mL double distilled water.
Washing Buffer: 3.028g Tris was weighed, adjusted to pH 7.5 with HCl, 5.844g NaCl, 50mL glycerol, 0.01g sodium azide, 17.02g imidazole, and dissolved in 500mL double distilled water.
Preparing SDS-PAGE gel electrophoresis related reagents:
protein loading buffer: 40mmol/L Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% beta-mercaptoethanol (now available), 0.1% bromophenol blue.
Gel buffer (0.5M Tris-HCl): 30.285g Tris was weighed, dissolved in 50mL double distilled water, adjusted to pH 6.8 with HCl, and added with 2g SDS to a volume of 500 mL.
Lower layer gel buffer (0.5M Tris-HCl): 90.885g Tris was weighed, dissolved in 50mL double distilled water, adjusted to pH 8.8 with HCl, and then diluted to 500mL with 2g SDS.
Ammonium persulfate (Ammonium persulfate, 10% AP) (100 mL): 10g of ammonium persulfate powder, adding double distilled water to a constant volume of 100mL, subpackaging and storing at-20 ℃ in a dark place.
5 × protein electrophoresis buffer: 15.1g of Tris-HCl, 72g of glycine and 5g of SDS are weighed, and double distilled water is added to the solution to be constant volume of 1L.
Coomassie brilliant blue staining solution: 0.1% Coomassie Brilliant blue R250, 25% methanol, 7% glacial acetic acid, and filter paper. Note that: coomassie Brilliant blue R250 was first dissolved thoroughly in methanol and the other ingredients were added.
Decoloring liquid: 50mL of glacial acetic acid, 100mL of methanol and distilled water are added to a constant volume of 500 mL.
Electrophoresis apparatus (Bio-Rad, USA), PCR amplification apparatus (Eppendorf, Hamburg, Germany), mass spectrometer (Shimadzu, Japan), nuclear magnetic resonance apparatus (Varian, USA), ultrasonication apparatus (Cole Parmer, USA), Centricon Plus-80 Millipore ultrafiltration centrifugal tube (Millipore corporation).
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