CN110643652A

CN110643652A - Method for synthesizing quinolizinone compound by enzyme method

Info

Publication number: CN110643652A
Application number: CN201910992734.5A
Authority: CN
Inventors: 史社坡; 王娟; 王晓晖; 刘晓; 吴云; 丁宁; 齐博文; 屠鹏飞
Original assignee: Beijing University of Chinese Medicine
Current assignee: Beijing University of Chinese Medicine
Priority date: 2019-10-18
Filing date: 2019-10-18
Publication date: 2020-01-03
Anticipated expiration: 2039-10-18
Also published as: CN110643652B

Abstract

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.

Description

Method for synthesizing quinolizinone compound by enzyme method

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.

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).

Example 1 preparation of polyketide synthase HsPKS3 in Huperzia serrata

An expression strain (org.Lett.,2016,18,3550-3553) containing the HsPKS3 gene (SEQ ID NO:1) was added to 10mL of LB liquid medium at a volume ratio of 1:100, and activated at 37 ℃ overnight; among them, LB liquid medium contained 50. mu.g/mL kanamycin. The next day, the LB liquid medium was expanded, and the expression strain containing HsPKS3 gene was added to the LB liquid medium at a volume ratio of 1:100, and cultured with shaking at 37 ℃ to OD₆₀₀And adding 1 volume per thousand of IPTG (isopropyl thiogalactoside) of 1M into the mixture between 0.4 and 0.6, and carrying out low-temperature induced expression for 16 hours at 23 ℃. Centrifugation was carried out at 4 ℃ and 7500 Xg for 5min, and the cells were collected.

And (3) suspending the collected thalli in a precooled lysis buffer, adding 1vol per mill of PMSF, and carrying out ultrasonic crushing in an ice bath. The ultrasonicator was set to: crushing for 2s, stopping crushing for 4s, and crushing for 10 min. Centrifuging the crushed solution at 4 deg.C and 8000 Xg for 40min until thallus is settled to obtain supernatant containing polyketide synthase HsPKS 3; the supernatant containing polyketide synthase HsPKS3 was quickly transferred to a clean 50mL centrifuge tube and placed on ice. Then filtering the supernatant with 0.45 μm microporous membrane, and loading on pre-balanced Ni²⁺Affinity of chelate resinA chromatographic column; removing foreign proteins by KPB washing buffer solution, eluting by KPB elution buffer solution containing 400mM imidazole, ultrafiltering, centrifuging, concentrating, and desalting to obtain polyketide synthase HsPKS3 in Huperzia serrata. The results of SDS-PAGE gel electrophoresis of the polyketide synthase HsPKS3 in Huperzia serrata are shown in FIG. 1.

Example 2a preparation of pyridine acetyl coenzyme A

Cloning and expressing phenylacetyl coenzyme A ligase PCL cloned and expressed in penicillium chrysogenum:

penicillium chrysogenum MT-12 is separated and purified from Huperzia serrata and identified by ITS rDNA sequence sequencing (GenBank accession number MF 765611).

According to the full-length sequence (SEQ ID NO:2) of the phenylacetyl-CoA ligase PCL (AJ001540) gene in Penicillium chrysogenum (Penicillium chrysogenum) recorded in NCBI, forward and reverse specific primers PCL-F-EcoR I and PCL-R-Hind III are designed, and Penicillium chrysogenum genome DNA (number: MT-12) is used as a template to perform PCR amplification through Pyrobest TM DNApolymerase to obtain the target sequence with the full-length 1737bp and encoded 563 amino acids. Construction of recombinant plasmid pMAl-C₂X-PCL and transferred into E.coli expression strain E.coli BL21(DE 3). Single colonies of positive clones were picked by colony PCR and inoculated into 10mL of LB liquid medium containing 100. mu.g/mL of ampicillin, and activated overnight at 37 ℃. The next day, the LB liquid medium was enlarged, the bacterial liquid was added at a volume ratio of 1:100, and the culture was shaken at 37 ℃ to OD₆₀₀0.5 vol% of 1M IPTG is added between 0.4-0.6, and the expression is induced at the low temperature of 17 ℃ for 16 hours. The cells were collected by centrifugation at 7500 Xg for 5min at 4 ℃. Then, the collected thalli are resuspended in precooled TANG Buffer, 1vol per thousand PMSF is added, and ice bath ultrasonication is carried out. The ultrasonicator was set to: crushing for 2s, stopping crushing for 4s, and crushing for 10 min. The disrupted solution was centrifuged at 8000 Xg and 4 ℃ for 40min until the cells settled, to obtain a supernatant containing phenylacetyl-CoA ligase PCL. The supernatant containing phenylacetyl-coa ligase PCL was quickly transferred to a clean 50mL centrifuge tube and placed on ice. Then, the supernatant was filtered through a 0.45 μm microporous membrane and applied to Ni which had been previously balanced with TANG Buffer²⁺On chelating resin affinity column(ii) a Removing impure protein by TANG Buffer, eluting by Washing Buffer containing 500mM imidazole, centrifugally concentrating eluent by Centricon Plus-80 Millipore ultrafiltration centrifugal tube, and removing imidazole by substituting the Washing Buffer by TANG Buffer to obtain the phenylacetyl coenzyme A ligase PCL. The results of gel electrophoresis of the cloned pcl gene are shown in FIG. 2. The results of SDS-PAGE gel electrophoresis of the clonally expressed phenylacetyl-CoA ligase PCL are shown in FIG. 3.

② the pyridine acetyl coenzyme A catalyzed by phenylacetyl coenzyme A ligase PCL

5mL of Tris-HCl (100 mM) and pH 8.5, 0.8mL of NaCl (2M) and 1mL of MgCl (62.5 mM) were taken₂11.5mg of CoA, 18.16mg of ATP Na₂Adding double distilled water to 10mL of phenylacetyl coenzyme A ligase PCL obtained in the step (i) with 10mM pyridine acetic acid and 10nM, and reacting for 6h at 30 ℃ to obtain the pyridine acetyl coenzyme A.

Example 2b preparation of 2- (5-Fluoropyridin-3-yl) acetyl-CoA

(ii) preparation of phenylacetyl-CoA ligase PCL by "cloning and expressing phenylacetyl-CoA ligase PCL in Penicillium chrysogenum" under the same conditions as those in example 2 a.

② the phenylacetyl coenzyme A ligase PCL catalyzed synthesis of 2- (5-fluoropyridine-3-yl) acetyl coenzyme A

5mL of Tris-HCl (100 mM) and pH 8.5, 0.8mL of NaCl (2M) and 1mL of MgCl (62.5 mM) were taken₂11.5mg of CoA, 18.16mg of ATP Na₂10mM 2- (5-fluoropyridin-3-yl) acetic acid and 10nM phenylacetyl coenzyme A ligase PCL obtained in the step (i) are added with double distilled water to 10mL and reacted at 30 ℃ for 6h to obtain 2- (5-fluoropyridin-3-yl) acetyl coenzyme A.

Example 2c preparation of 2- (6-Fluoropyridin-3-yl) acetyl-CoA

② the phenylacetyl coenzyme A ligase PCL catalyzed synthesis of 2- (6-fluoropyridine-3-yl) acetyl coenzyme A

Taking 5mL thickTris-HCl with a pH of 8.5 at a concentration of 100mM, 0.8mL of 2M NaCl, 1mL of 62.5mM MgCl₂11.5mg of CoA, 18.16mg of ATP Na₂10mM 2- (6-fluoropyridin-3-yl) acetic acid and 10nM phenylacetyl coenzyme A ligase PCL obtained in the step (i) are added with double distilled water to 10mL and reacted at 30 ℃ for 6h to obtain 2- (6-fluoropyridin-3-yl) acetyl coenzyme A.

EXAMPLE 3a preparation of malonyl-CoA

Preparation of malonyl coenzyme A ligase at

Malonyl-coa ligase at.matb was successfully cloned from arabidopsis thaliana (a.thaliana). Adding an expression strain containing the at.MatB gene into 10mL of LB liquid culture medium (containing 50 mu g/mL kanamycin) at a volume ratio of 1:100 for activation at 37 ℃ overnight; among them, LB liquid medium contained 50. mu.g/mL kanamycin. The next day, the LB liquid medium was enlarged, the bacterial liquid was still added at a volume ratio of 1:100, and the mixture was shake-cultured at 37 ℃ to OD₆₀₀And adding 1 volume per thousand of IPTG (isopropyl thiogalactoside) of 1M into the mixture between 0.4 and 0.6, and carrying out low-temperature induced expression for 16 hours at 23 ℃. The cells were collected by centrifugation at 7500 Xg for 5min at 4 ℃. Then, the collected thalli are resuspended in precooled lysis buffer, 1vol per thousand PMSF is added, and ice bath ultrasonication is carried out. The ultrasonicator was set to: crushing for 2s, stopping crushing for 4s, and crushing for 10 min. The disrupted solution was centrifuged at 8000 Xg at 4 ℃ for 40min until the cells settled, to obtain a supernatant containing malonyl-CoA ligase at. The supernatant containing malonyl-coa ligase at.matb was quickly transferred to a clean 50mL centrifuge tube and placed on ice. Then filtering the supernatant with 0.45 μm microporous membrane, and loading on pre-balanced Ni²⁺A chelating resin affinity chromatography column; firstly, removing impure protein by using a washing buffer solution, then eluting by using an elution buffer solution containing 400mM imidazole, centrifugally concentrating the eluent by using a Centricon Plus-80 Millipore ultrafiltration centrifugal tube, and then removing imidazole by using a desalting buffer solution to obtain malonyl coenzyme A ligase at. The concentration of malonyl-CoA ligase at.MatB was 1.2mg/mL as determined by the BCA method. The results of SDS-PAGE gel electrophoresis of malonyl-CoA ligase at.MatB are shown in FIG. 4.

Catalytic synthesis of malonyl-coenzyme A by malonyl-coenzyme A ligase at. MatB

3.9mL of 100mM potassium phosphate buffer solution at pH 7.0, 1.6mL of 62.5mM MgCl₂、23.02mg CoA、36.31mg ATP Na₂And (3) adding 3.10mg of DTT, 6mM of malonic acid and 10nM of malonyl-CoA ligase at.MatB obtained in the step (i) to 10mL of double distilled water, and reacting at 30 ℃ for 12 hours to obtain malonyl-CoA.

EXAMPLE 3b preparation of methylmalonyl-CoA

(ii) preparation of malonyl-CoA ligase at. MatB using exactly the same conditions as in (i) in example 3 a.

Catalytic synthesis of methylmalonyl-coenzyme A by malonyl-coenzyme A ligase at. MatB

3.9mL of 100mM potassium phosphate buffer solution at pH 7.0, 1.6mL of 62.5mM MgCl₂、23.02mg CoA、36.31mg ATP Na₂3.10mg of DTT, 6mM of methylmalonic acid and 10nM of malonyl-CoA ligase (at. MatB) obtained in step (i), adding distilled water to 10mL, and reacting at 30 ℃ for 12 hours to obtain methylmalonyl-CoA.

Example 3c preparation of ethylmalonyl-coenzyme A

Catalytic synthesis of ethylmalonyl-coenzyme A by malonyl-coenzyme A ligase at. MatB

3.9mL of 100mM potassium phosphate buffer solution at pH 7.0, 1.6mL of 62.5mM MgCl₂、23.02mg CoA、36.31mg ATP Na₂3.10mg of DTT, 6mM of ethylmalonic acid and 10nM of malonyl-CoA ligase At.MatB obtained in the above step (i), adding double distilled water to 10mL, and reacting at 30 ℃ for 12 hours to obtain ethylmalonyl-CoA.

EXAMPLE 3d preparation of allylmalonyl-CoA

Catalytic synthesis of allylmalonyl-coenzyme A by malonyl-coenzyme A ligase at. MatB

3.9mL of 100mM solution at pH 70 potassium phosphate buffer, 1.6mL MgCl at a concentration of 62.5mM₂、23.02mg CoA、36.31mg ATP Na₂3.10mg of DTT, 6mM of allylmalonic acid and 10nM of malonyl-CoA ligase (at. MatB) obtained in step (i) were added with distilled water to 10mL and reacted at 30 ℃ for 12 hours to obtain allylmalonyl-CoA.

Example 4a Synthesis of Quinazinone Compound P1 catalyzed by polyketide synthase HsPKS3 in Huperzia serrata

The quinolizinone compound P1 is synthesized by an enzyme method by adopting polyketide synthase HsPKS3, pyridine acetyl coenzyme A and malonyl coenzyme A in huperzia serrata.

Mu. mol of pyridine acetyl-CoA of example 2a, 1. mu. mol of malonyl-CoA of example 3a and 8.2nmol of polyketide synthase HsPKS3 in huperzia serrata of example 1 were added to 10ml of 0.1M potassium phosphate buffer solution having a pH of 7.0, and reacted overnight in a 37 ℃ water bath shaker. The next day, the reaction solution was extracted with 2 times of ethyl acetate for 3 times, and the ethyl acetate layers were combined, spin-dried with a rotary evaporator, and purified by liquid chromatography to obtain quinolizinone compound P1.

Example 4b Synthesis of quinolizinone Compound P1 catalyzed by polyketide synthase HsPKS3 in Huperzia serrata

Polyketide synthase HsPKS3, phenylacetyl coenzyme A ligase PCL and malonyl coenzyme A ligase at.MatB in huperzia serrata are adopted, pyridine acetic acid and malonic acid are taken as substrates, and the quinolizinone compound P1 is synthesized by an enzyme method.

10mM MgCl was added to 10ml of 100mM potassium phosphate buffer solution at pH 7.5₂、200mM NaCl、2mM DTT、6mM ATP Na₂After 1h reaction at 30 ℃ with 4.5mM CoA, 10mM pyridine acetic acid, 10mM malonic acid, 10nM phenylacetyl-CoA ligase PCL of example 2a and 10nM malonyl-CoA ligase at. MatB of example 3a, 10nM polyketide synthase HsPKS3 in Huperzia serrata of example 1 was added and the reaction was continued overnight at 33 ℃. The next day, the reaction solution was extracted with 2 volumes of ethyl acetate for 3 times, and the ethyl acetate layers were combined, spun-dried in a vacuum centrifugal concentrator, and purified by liquid chromatography to obtain quinolizinone compound P1.

Example 5a Synthesis of quinazolinone Compounds by polyketide synthase HsPKS3 catalysis in Huperzia serrataObject P2

The quinolizinone compound P2 is synthesized by an enzyme method by adopting polyketide synthase HsPKS3, pyridine acetyl coenzyme A and methylmalonyl coenzyme A in huperzia serrata.

Mu. mol of pyridine acetyl coenzyme A of example 2a, 1. mu. mol of methylmalonyl coenzyme A of example 3b and 8.2nmol of polyketide synthase HsPKS3 of huperzia serrata of example 1 were added to 10ml of 0.1M potassium phosphate buffer solution having a pH of 7.0, and reacted overnight in a 37 ℃ water bath shaker. The next day, the reaction solution was extracted with 2 times of ethyl acetate for 3 times, and the ethyl acetate layers were combined, spin-dried with a rotary evaporator, and purified by liquid chromatography to obtain quinolizinone compound P2.

Example 5b Synthesis of quinolizinone Compound P2 catalyzed by polyketide synthase HsPKS3 in Huperzia serrata

Polyketide synthase HsPKS3, phenylacetyl coenzyme A ligase PCL and malonyl coenzyme A ligase at.MatB in huperzia serrata are adopted, and pyridylacetic acid and methylmalonic acid are taken as substrates to synthesize the quinolizinone compound P2 by an enzyme method.

10mM MgCl was added to 10ml of 100mM potassium phosphate buffer solution at pH 7.5₂、200mM NaCl、2mM DTT、6mM ATP Na₂4.5mM CoA, 10mM pyridylacetic acid, 10mM methylmalonic acid, 10nM of the phenylacetyl-CoA ligase PCL of example 2a and 10nM of the malonyl-CoA ligase at. MatB of example 3a, after reaction for 1h at 30 ℃ 10nM of the polyketide synthase HsPKS3 in Huperzia serrata of example 1 was added and the reaction was continued overnight at 33 ℃. The next day, the reaction solution was extracted with 2 volumes of ethyl acetate for 3 times, and the ethyl acetate layers were combined, spun-dried in a vacuum centrifugal concentrator, and purified by liquid chromatography to obtain quinolizinone compound P2.

Example 6a Synthesis of Quinazinone Compound P3 catalyzed by polyketide synthase HsPKS3 in Huperzia serrata

The quinolizinone compound P3 is synthesized by an enzyme method by adopting polyketide synthase HsPKS3, pyridine acetyl coenzyme A and ethyl malonyl coenzyme A in huperzia serrata.

Mu. mol of pyridine acetyl coenzyme A of example 2a, 1. mu. mol of ethylmalonyl coenzyme A of example 3c and 8.2nmol of polyketide synthase HsPKS3 of huperzia serrata of example 1 were added to 10ml of 0.1M potassium phosphate buffer solution having a pH of 7.0, and reacted overnight in a 37 ℃ water bath shaker. The next day, the reaction solution was extracted with 2 times of ethyl acetate for 3 times, and the ethyl acetate layers were combined, spin-dried with a rotary evaporator, and purified by liquid chromatography to obtain quinolizinone compound P3.

Example 6b Synthesis of quinolizinone Compound P3 catalyzed by polyketide synthase HsPKS3 in Huperzia serrata

Polyketide synthase HsPKS3, phenylacetyl coenzyme A ligase PCL and malonyl coenzyme A ligase at.MatB in huperzia serrata are adopted, and pyridine acetic acid and ethylmalonic acid are taken as substrates, and the quinolizinone compound P3 is synthesized by an enzyme method.

10mM MgCl was added to 10ml of 100mM potassium phosphate buffer solution at pH 7.5₂、200mM NaCl、2mM DTT、6mM ATP Na₂4.5mM CoA, 10mM pyridylacetic acid, 10mM ethylmalonic acid, 10nM of the phenylacetyl-CoA ligase PCL of example 2a and 10nM of the malonyl-CoA ligase at. MatB of example 3a, after reaction for 1h at 30 ℃ 10nM of the polyketide synthase HsPKS3 in Huperzia serrata of example 1 was added and the reaction was continued overnight at 33 ℃. The next day, the reaction solution was extracted with 2 volumes of ethyl acetate for 3 times, and the ethyl acetate layers were combined, spun-dried in a vacuum centrifugal concentrator, and purified by liquid chromatography to obtain quinolizinone compound P3.

Example 7a Synthesis of Quinazinone Compound P4 catalyzed by polyketide synthase HsPKS3 in Huperzia serrata

The quinolizinone compound P4 is synthesized by an enzyme method by adopting polyketide synthase HsPKS3, pyridine acetyl coenzyme A and allyl malonyl coenzyme A in huperzia serrata.

Mu. mol of pyridine acetyl coenzyme A from example 2a, 1. mu. mol of allyl malonyl coenzyme A from example 3d and 8.2nmol of polyketide synthase HsPKS3 from Huperzia serrata from example 1 were added to 10ml of 0.1M potassium phosphate buffer solution at pH 7.0 and reacted overnight in a 37 ℃ water bath shaker. The next day, the reaction solution was extracted with 2 times of ethyl acetate for 3 times, and the ethyl acetate layers were combined, spin-dried with a rotary evaporator, and purified by liquid chromatography to obtain quinolizinone compound P4.

Example 7b Synthesis of quinazolinone Compounds by polyketide synthase HsPKS3 in Huperzia serrataObject P4

Polyketide synthase HsPKS3, phenylacetyl coenzyme A ligase PCL and malonyl coenzyme A ligase at.MatB in huperzia serrata are adopted, pyridine acetic acid and allylmalonic acid are taken as substrates, and the quinolizinone compound P4 is synthesized by an enzyme method.

10mM MgCl was added to 10ml of 100mM potassium phosphate buffer solution at pH 7.5₂、200mM NaCl、2mM DTT、6mM ATP Na₂4.5mM CoA, 10mM pyridylacetic acid, 10mM allylmalonic acid, 10nM of the phenylacetyl-CoA ligase PCL of example 2a and 10nM of the malonyl-CoA ligase at. MatB of example 3a, after reaction for 1h at 30 ℃ 10nM of the polyketide synthase HsPKS3 in Huperzia serrata of example 1 was added and the reaction was continued overnight at 33 ℃. The next day, the reaction solution was extracted with 2 volumes of ethyl acetate for 3 times, and the ethyl acetate layers were combined, spun-dried in a vacuum centrifugal concentrator, and purified by liquid chromatography to obtain quinolizinone compound P4.

Example 8a Synthesis of Quinazinone Compound P5 catalyzed by polyketide synthase HsPKS3 in Huperzia serrata

The quinolizinone compound P5 is synthesized by an enzyme method by adopting polyketide synthase HsPKS3, 2- (6-fluoropyridin-3-yl) acetyl coenzyme A and allylmalonyl coenzyme A in huperzia serrata.

Mu. mol of 2- (6-fluoropyridin-3-yl) acetyl-CoA of example 2c, 1. mu. mol of allylmalonyl-CoA of example 3d and 8.2nmol of polyketide synthase HsPKS3 from Huperzia serrata of example 1 were added to 10ml of 0.1M potassium phosphate buffer solution at pH 7.0 and reacted overnight in a 37 ℃ water bath shaker. The next day, the reaction solution was extracted with 2 times of ethyl acetate for 3 times, and the ethyl acetate layers were combined, spin-dried with a rotary evaporator, and purified by liquid chromatography to obtain quinolizinone compound P5.

Example 8b Synthesis of quinolizinone Compound P5 catalyzed by polyketide synthase HsPKS3 in Huperzia serrata

Polyketide synthase HsPKS3, phenylacetyl coenzyme A ligase PCL and malonyl coenzyme A ligase at.MatB in huperzia serrata are adopted, 2- (6-fluoropyridin-3-yl) acetic acid and allylmalonic acid are taken as substrates, and the quinolizinone compound P5 is synthesized by an enzyme method.

At a concentration of 100mM in 10ml and a pH of 7.5 potassium phosphate buffer solution 10mM MgCl was added₂、200mM NaCl、2mM DTT、6mM ATP Na₂After 1h reaction at 30 ℃ with 4.5mM CoA, 10mM 2- (6-fluoropyridin-3-yl) acetic acid, 10mM allylmalonic acid, 10nM phenylacetyl-CoA ligase PCL of example 2a and 10nM malonyl-CoA ligase at. MatB of example 3a, 10nM polyketide synthase HsPKS3 in Huperzia serrata of example 1 was added and the reaction was continued overnight at 33 ℃. The next day, the reaction solution was extracted with 2 volumes of ethyl acetate for 3 times, and the ethyl acetate layers were combined, spun-dried in a vacuum centrifugal concentrator, and purified by liquid chromatography to obtain quinolizinone compound P5.

Experimental example 1

The quinolizinone compounds P1 obtained in examples 4a and 4b were subjected to structural identification.

Mass spectrum showed, [ M-H ]]^-Peak m/z: 160.0404, calculated as: peak m/z: 160.0406[ M-H]^-Molecular formula is C₉H₇NO₂。

Hydrogen spectrum of nuclear magnetic resonance¹H NMR(500MHz，CD₃OD):δ_H 6.01(1H,s,H-3),6.39(1H,s,H-1),6.92(1H,t,J＝6.0Hz,H-7),7.34(1H,t,J＝8.0Hz,H-6),7.44(1H,d,J＝9.0Hz,H-8),8.81(1H,d,J＝7.0Hz,H-5)。

Carbon spectrum of nuclear magnetic resonance¹³C NMR(125MHz，CD₃OD):δ_C 96.4(C-1),168.8(C-2)，95.1(C-3),161.7,(C-4),127.3(C-5),131.2(C-6),114.5(C-7),125.5(C-8),144.8(C-9)。

From this, it is presumed that the quinolizinone compound P1 is 2-hydroxyquinolizinone. The chemical structural formula of the quinolizinone compound P1 is shown as follows:

experimental example 2

The quinolizinone compounds P2 obtained in examples 5a and 5b were subjected to structural identification.

Mass spectrum showed, [ M-H ]]^-Peak m/z: 174.0563, calculated as: peak m/z: 174.0561[ M-H]^-Molecular formula is C₉H₁₀NO₂。

Hydrogen spectrum of nuclear magnetic resonance¹H NMR(600MHz,DMSO-d₆):δ_H 2.01(3H,s),6.37(1H,s,H-1),6.86(1H,t,J＝6.5Hz,H-7),7.26(1H,t,J＝6.5Hz,H-6),7.46(1H,d,J＝7.5Hz,H-8),8.74(1H,d,J＝6.0Hz,H-5)。

Carbon spectrum of nuclear magnetic resonance¹³C NMR(150MHz,DMSO-d₆):δ_C 158.5(C-4),92.8(C-1),100.8(C-3),112.8(C-7),124.2(C-8),126.0(C-5),128.7(C-6),139.9(C-9),162.2(C-2),9.7(C-10)。

The quinolizinone compound P2 is thus presumed to be 2-hydroxy-3-methyl quinolizinone. The chemical structural formula of the quinolizinone compound P2 is shown as follows:

experimental example 3

The quinolizinone compounds P3 obtained in examples 6a and 6b were subjected to structural identification.

Mass spectrum showed, [ M + H ]]⁺Peak m/z: 190.0861, calculated as: peak m/z: 190.0861[ M + H]⁺Predicted molecular formula is C₁₁H₁₁NO₂。

The molecular weight of the quinolizinone compound P3 is 28 more than that of the quinolizinone compound P1 with a determined structure, and the molecular weight of the ethyl group is 28, so that the quinolizinone compound P3 is determined to be 2-hydroxy-3-ethyl-quinolinone. The chemical structural formula of the quinolizinone compound P3 is shown as follows:

experimental example 4

The quinolizinone compounds P4 obtained in examples 7a and 7b were subjected to structural identification.

Mass spectrum showed, [ M + H ]]⁺Peak m/z: 202.0863, calculated as: peak m/z: 202.0863[ M + H]⁺Predicted molecular formula is C₁₂H₁₁NO₂。

The molecular weight of the quinolizinone compound P4 is 40 more than that of the quinolizinone compound P1 with a determined structure, and the molecular weight of allyl is 40, so that the quinolizinone compound P4 is determined to be 2-hydroxy-3-allyl-quinolinone. The chemical structural formula of the quinolizinone compound P4 is shown as follows:

experimental example 5

The quinolizinone compounds P5 obtained in examples 8a and 8b were subjected to structural identification.

Mass spectrum showed, [ M-H ]]^-Peak m/z: 178.0337, calculated as: peak m/z: 178.0310[ M-H]^-Predicted molecular formula is C₉H₆FNO₂。

The molecular weight of the quinolizinone compound P5 is 18 more than that of the quinolizinone compound P1 with a determined structure, and meanwhile, the molecular weight of the F atom is 18, so that the quinolizinone compound P5 is determined to be 2-hydroxy-6-F-quinolizinone. The chemical structural formula of the quinolizinone compound P5 is shown as follows:

the present invention is not limited to the above-described embodiments, and any variations, modifications, and substitutions which may occur to those skilled in the art may be made without departing from the spirit of the invention.

Sequence listing

<110> Beijing university of traditional Chinese medicine

<120> method for synthesizing quinolizinone compound by enzyme method

<130> YC12019060030-A

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ctatcacaac tgatttcagc taacattgag acgatcctct cggaggcatt gcggaaagca 900

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ctaggctatt tgaacaacga aaaagccacc gcagagacat ttgtggacgg atggatgcgt 1320

acgggagacg aggctgttat ccgtaaaagc ccgaagggca tcgagcacgt gtttattgtc 1380

gatcggatta aggagttgat caaggtcaag ggtctgcaag tcgcgcctgc cgagctcgaa 1440

gcccatatcc tcgcccaccc ggatgtttcg gactgtgctg tcatcgctat cccggatgat 1500

cgtgcaggag aagtacctaa ggccattgtt gtgaagtccg ccagcgcagg atcggacgaa 1560

tctgtctccc aggctctcgt gaagtatgtt gaggaccaca aggctcgtca caagtggttg 1620

aagggaggta ttagatttgt ggatgccatc cccaagagcc cgagtggtaa gattcttcgt 1680

cggttgatcc gtgaccaaga gaaggaggca cggagaaagg ctggtagcaa gatctaa 1737

Claims

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:

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:

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。