CN104177361B - An ASA-type enzyme inhibitor based on auxin-regulated gene GH3 and preparation method thereof - Google Patents

Abstract

The invention discloses an ASA-type enzyme inhibitor based on the auxin-regulated gene GH3 and a preparation method thereof, wherein the structural formula of the enzyme inhibitor is: R is phenethyl or naphthyl. The IC ₅₀ of the enzyme inhibitor of the present invention for GH3-6 is 16.00±1.80 and 0.45±0.02 μM respectively, has good enzyme inhibitory activity, and shows potential application prospects in fields such as pesticides. The preparation method of the enzyme inhibitor of the present invention is simple, does not need special reaction conditions, reacts at normal temperature and pressure, has simple post-treatment and high product yield.

Description

An ASA-type enzyme inhibitor based on auxin-regulated gene GH3 and preparation method thereof

1. Technical field

The invention relates to an enzyme inhibitor and a preparation method thereof, in particular to an ASA-type enzyme inhibitor based on the auxin regulating gene GH3 and a preparation method thereof.

2. Background technology

Auxin is the earliest discovered plant hormone. Its chemical essence is a class of organic small molecules without structural characteristics, including salicylic acid (SA), jasmonic acid (JA) and the most representative 2-indoleacetic acid. (IAA), etc., are mainly concentrated in the plant's shoots, tops, root tips and other parts that have growth advantages. They play an important role in physiological regulation of cell division and proliferation, tissue and organ differentiation, flowering and fruit ripening, seed germination and dormancy. Auxin has a dual regulatory effect on the physiology of plants: at low concentrations, it can promote the growth of shoots and develop root tips; at high concentrations, it can inhibit the growth of plant tops and roots, and even cause plant death.

At present, the GH3 gene family is the most thorough research on auxin-regulated genes. In the Arabidopsis genome, 19 gene members have been found so far, named GH3-1, GH3-2, GH3-3, ... GH3-19 and so on. It has been shown that the protein encoded by the GH3 gene can catalyze the adenylation of salicylic acid (SA), indoleacetic acid (IAA), and jasmonic acid (JA), thereby regulating the levels of these plant hormones in vivo. In addition, studies have also found that they can also catalyze the connection reaction between IAA and various amino acids, and store them in the body in the form of inactive forms of IAA-X (X=Ala, Asp, Glu, Leu, etc.), so as to achieve the regulation of IAA and related amino acids. purpose at the in vivo level. The specific process is shown in the following formula:

The physiological adenylation process of IAA is: under the action of adenosine triphosphate (ATP), 2-indoleacetic acid (IAA) generates its activated intermediate - adenosine monophosphate (IAA-AMP), and at the same time removes a molecule of pyrophosphate (PPi); then various amino acids react with IAA-AMP to form a complex (IAA-aa) of indole acetic acid and amino acid (aa), which is the inactive storage form of IAA and can regulate the hormone of IAA in plants level, which plays an important role in the regulation of plant growth.

3. Contents of the invention

The present invention aims to provide an ASA-type enzyme inhibitor based on auxin-regulating gene GH3 and a preparation method thereof. The technical problem to be solved is to select a compound with better enzyme-inhibiting activity on GH3 gene through molecular design.

The present invention prepares a series of various ASA-carboxylic acid complexes (ASA-ca) based on ASA intermediates through molecular design, and they have an adenosine structure similar to IAA-AMP.

The present invention is based on the structural formula of the ASA-type enzyme inhibitor of the auxin-regulated gene GH3:

R is phenethyl or naphthyl, the same below.

The preparation method of the ASA type enzyme inhibitor based on the auxin regulating gene GH3 of the present invention is as follows:

1) Activation of the carboxyl group

Under the protection of nitrogen, dissolve the carboxylic acid and the carboxyl activator in an organic solvent, the solution is clear, and react in a closed manner at room temperature for 1-2 hours to obtain intermediate 1, which can be directly used in the next step without separation .

The molar ratio between the carboxylic acid and the carboxyl activator is 1:1.1-1.3;

Described carboxylic acid is phenylpropionic acid or naphthoic acid etc.;

The carboxyl activator is carbonyldiimidazole (CDI), N-hydroxysuccinimide (NHS), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC .HCl) or dicyclohexylcarbodiimide (DCC), etc.;

The organic solvent is anhydrous acetonitrile, N,N-diformamide (DMF), dimethyl sulfoxide (DMSO) or dry dichloromethane, etc.;

The structural formula of the intermediate 1 is:

2) Coupling

Add ASA intermediate to the reaction solution prepared in step 1), the solution becomes turbid, and add 1,8-diazacyclo[5.4.0]undec-7-ene to the reaction solution under the condition of ice-water bath (DBU), the solution becomes clear, and reacts for 8-12h under airtight conditions at room temperature. After the reaction finishes, the solvent is distilled off under reduced pressure, then CHCl is added for dissolving, then the citric acid solution of mass concentration ₃ % is added for washing, and the organic phase is separated and collected. The organic phase was washed with water and saturated brine successively, dried over anhydrous sodium sulfate, and CHCl ₃ was removed by distillation under reduced pressure, and then separated on a silica gel column (with a volume ratio of CHCl ₃ /MeOH=10-12:1 as the mobile phase) to obtain intermediate body 2;

The molar ratio of intermediate 1, ASA intermediate and DBU in the reaction liquid is 1:1:1.8-2.5.

The structural formula of the ASA intermediate is:

The structural formula of the intermediate 2 is:

3) deprotection group

Add intermediate 2 into the deprotecting group reagent solution, react at room temperature for 1.5-2.5 hours, remove the solvent under reduced pressure after the reaction, and obtain the target product after recrystallization with acetone;

During the deprotection reaction process, the addition amount of the deprotection reagent is greatly excessive, and generally the molar ratio of the deprotection reagent to the intermediate 2 is controlled at 150-300:1.

The deprotection group reagent solution is 80% HCOOH aqueous solution, 5-10% hydrochloric acid solution or trifluoroacetic acid (TFA) and the like.

Described concrete process with acetone recrystallization is as follows:

Put the white solid obtained after removing the solvent under reduced pressure into a flask, add acetone, heat to reflux and continuously add new acetone until the white solid is no longer dissolved, filter while it is hot, and the filtrate crystallizes in an ice-water bath , filtered, washed and dried to obtain the target product.

Reaction scheme of the present invention is shown in following formula:

The ASA intermediate used in the present invention has a molecular structure similar to that of the IAA-AMP complex, and the adenosine structure in it can be strongly and specifically recognized by the enzyme encoded by the GH3 gene.

【Determination of enzyme inhibitory activity】

After confirming the purity of the product by 1HNMR and elemental analysis, the inventor tested the inhibitory effect of the prepared target product on GH3-6 enzyme activity. The specific test steps are as follows:

1) Enzyme-catalyzed reaction: Add Tris-HCl (pH8.0), MgCl ₂ , ATP, IAA, aspartic acid, enzyme inhibitors and GH3-6 to a 2mL centrifuge tube in order to control the total volume of the liquid in the centrifuge tube The concentration of Tris-HCl (pH8.0) in the centrifuge tube is 100mM, the concentration of MgCl ₂ is 5mM, the concentration of ATP is 500μM, the concentration of IAA is 100μM, the concentration of aspartic acid is 1mM, GH3-6 The concentration was 14 μg/mL, and cultured at 30°C for 10 minutes;

2) Color reaction: Add 50 μL of 21 mM ammonium molybdate reagent, 200 μL of the enzyme-catalyzed reaction solution obtained in step 1), 50 μL of 0.5M 2-mercaptoethanol, and 20 μL of Eikonogen reagent (containing 21 mM sodium sulfite, 0.77 M sodium metabisulfite) into a 2 mL centrifuge tube in sequence. , 10.4M 1-amino-2-naphthol-4-sulfonic acid), after equilibrating at room temperature for 30 minutes, measure its absorbance at 580 nm.

The IC ₅₀ of the enzyme inhibitor of the present invention for GH3-6 is 16.00±1.80 and 0.45±0.02 μM respectively, has good enzyme inhibitory activity, and shows potential application prospects in fields such as pesticides. The preparation method of the enzyme inhibitor of the present invention is simple, does not need special reaction conditions, reacts at normal temperature and pressure, has simple post-treatment and high product yield.

4. Specific implementation

Example 1:

1) Activation of the carboxyl group

Under nitrogen protection, add phenylpropionic acid (0.6g, 4.0mmol) and CDI (0.72g, 4.45mmol) in the round bottom flask of 200mL, inject about 50mL dry acetonitrile in the flask with needle then, nitrogen protection about 20min, React at room temperature for 1-2 h under airtight conditions to obtain intermediate 1, which can be directly used in the next reaction without isolation.

2) Coupling

Add ASA intermediate (1.4g, 3.64mmol) to the reaction solution prepared in step 1), the solution becomes turbid, and 1mL DBU (about 6.58mmol) is added to the reaction solution under the condition of ice-water bath, the solution becomes clear and sealed at room temperature The reaction was carried out under the conditions for 8-12 hours, and thin layer chromatography (TLC) detection showed that after the reaction was completed, the solvent was distilled off under reduced pressure to obtain a white solid. Add 150mL CHCl to the obtained white solid to dissolve, then add 150mL of ₃ % citric acid solution to wash, separate and collect the organic phase, repeat this process 2-3 times, combine the organic phase; wash the organic phase with water and saturated brine in turn After drying over anhydrous sodium sulfate, the organic solvent was removed by distillation under reduced pressure, and then separated by silica gel column (using volume ratio CHCl ₃ /MeOH=10:1 as mobile phase) to obtain intermediate 2 (1.69 g, yield 81.8%) .

3) deprotection group

Intermediate 2 (0.78g, 1.52mmol) was added to 20mL of HCOOH aqueous solution with a mass concentration of 80%. The reaction solution was transparent and reacted at room temperature until the reaction was detected by TLC. The solvent was removed under reduced pressure to obtain a white solid, which was recrystallized from acetone. Obtain target product (0.54g, yield 75.9%);

Described concrete process with acetone recrystallization is as follows:

Put the white solid obtained after removing the solvent under reduced pressure into a flask, add acetone, heat to reflux and continuously add new acetone until the white solid is no longer dissolved, filter while it is hot, and the filtrate crystallizes in an ice-water bath , filtered, washed and dried to obtain the target product.

The structural formula of the target product is:

The enzyme inhibitors prepared in this example have the following steps for testing the inhibitory effect on the enzyme activity of GH3-6:

1) Enzyme-catalyzed reaction: Add Tris-HCl (pH8.0), MgCl ₂ , ATP, IAA, aspartic acid, enzyme inhibitors and GH3-6 to a 2mL centrifuge tube in order to control the total volume of the liquid in the centrifuge tube The concentration of Tris-HCl (pH8.0) in the centrifuge tube is 100mM, the concentration of MgCl ₂ is 5mM, the concentration of ATP is 500μM, the concentration of IAA is 100μM, the concentration of aspartic acid is 1mM, GH3-6 The concentration was 14 μg/mL, and cultured at 30°C for 10 minutes;

2) Color reaction: Add 50 μL of 21 mM ammonium molybdate reagent, 200 μL of the enzyme-catalyzed reaction solution obtained in step 1), 50 μL of 0.5M 2-mercaptoethanol, and 20 μL of ikonogen reagent (containing 21 mM sodium sulfite, 0.77 M sodium metabisulfite) into a 2 mL centrifuge tube successively. , 10.4M 1-amino-2-naphthol-4-sulfonic acid), after equilibrating at room temperature for 30 minutes, measure its absorbance at 580 nm.

It was determined that the IC ₅₀ of the enzyme inhibitor prepared in this example for GH3-6 was 16.00±1.80 μM, showing good enzyme inhibitory activity.

Example 2:

1) Activation of the carboxyl group

Under nitrogen protection, add naphthoic acid (0.50g, 2.90mmol) and CDI (0.56g, 3.43mmol) into a 200mL round bottom flask, then inject about 50mL of dry acetonitrile into the flask with a needle, nitrogen protection for about 20min, airtight Under the condition of room temperature reaction for 1-2h, the intermediate 1 is obtained, and the prepared intermediate 1 can be directly used in the next reaction without isolation.

2) Coupling

Add ASA intermediate (1.02g, 2.64mmol) to the reaction solution prepared in step 1), the solution becomes turbid, and 0.68mLDBU (about 4.47mmol) is added to the reaction solution under the condition of an ice-water bath, the solution becomes clear, room temperature The reaction was carried out under airtight conditions for 8-12 hours. Thin layer chromatography (TLC) detection showed that after the reaction was completed, the solvent was distilled off under reduced pressure to obtain a white solid. Add 150mL CHCl to the obtained white solid to dissolve, then add 150mL of ₃ % citric acid solution to wash, separate and collect the organic phase, repeat this process 2-3 times, combine the organic phase; wash the organic phase with water and saturated brine in turn After drying over anhydrous sodium sulfate, the organic solvent was removed by distillation under reduced pressure, and then separated by silica gel column (using volume ratio CHCl ₃ /MeOH=12:1 as mobile phase) to obtain intermediate 2 (1.07g, yield 75.4%) .

3) deprotection group

Intermediate 2 (1.07g, 1.98mmol) was added to 30mL of HCOOH aqueous solution with a mass concentration of 80%, the reaction solution was transparent, and reacted at room temperature until the reaction was detected by TLC. After the reaction was completed, the solvent was removed under reduced pressure to obtain a white solid. The target product (0.82g, yield 82.5%) was obtained after recrystallization;

Described concrete process with acetone recrystallization is as follows:

Put the white solid obtained after removing the solvent under reduced pressure into the flask, add acetone, heat to reflux and add new acetone until the white solid is completely dissolved, filter while it is hot, and the filtrate is left to crystallize under the condition of an ice-water bath. The target product was obtained after filtration, washing and drying.

The structural formula of the target product is:

The enzyme inhibitors prepared in this example have the following steps for testing the inhibitory effect on the enzyme activity of GH3-6:

1) Enzyme-catalyzed reaction: Add Tris-HCl (pH8.0), MgCl ₂ , ATP, IAA, aspartic acid, enzyme inhibitors and GH3-6 to a 2mL centrifuge tube in order to control the total volume of the liquid in the centrifuge tube The concentration of Tris-HCl (pH8.0) in the centrifuge tube is 100mM, the concentration of MgCl ₂ is 5mM, the concentration of ATP is 500μM, the concentration of IAA is 100μM, the concentration of aspartic acid is 1mM, GH3-6 The concentration was 14 μg/mL, and cultured at 30°C for 10 minutes;

2) Color reaction: Add 50 μL of 21 mM ammonium molybdate reagent, 200 μL of the enzyme-catalyzed reaction solution obtained in step 1), 50 μL of 0.5M 2-mercaptoethanol, and 20 μL of ikonogen reagent (containing 21 mM sodium sulfite, 0.77 M sodium metabisulfite) into a 2 mL centrifuge tube successively. , 10.4M 1-amino-2-naphthol-4-sulfonic acid), after equilibrating at room temperature for 30 minutes, measure its absorbance at 580 nm.

After determination, the enzyme inhibitor prepared in this example has an IC ₅₀ of 0.45±0.02 μM for GH3-6, showing a good enzyme inhibitory activity.

Claims (7)

1., based on an ASA type enzyme inhibitors of plant hormone regulatory gene GH3, it is characterized in that its structural formula is:

Wherein R is styroyl or naphthyl.

2. a preparation method for the ASA type enzyme inhibitors based on plant hormone regulatory gene GH3 according to claim 1, is characterized in that comprising the following steps:

1) activation of carboxyl

Under nitrogen protection, be dissolved in organic solvent by carboxylic acid and carboxyl activator, confined reaction 1-2h under room temperature, obtains intermediate 1;

Mol ratio between described carboxylic acid and described carboxyl activator is 1:1.1-1.3;

The structural formula of described intermediate 1 is:

R is styroyl or naphthyl;

2) coupling

To step 1) add ASA intermediate in obtained reaction solution, in the condition downhill reaction liquid of ice-water bath, add 1,8-diazacyclo [5.4.0] 11 carbon-7-alkene, react 8-12h under room temperature air tight condition, after separation and purification, obtain intermediate 2;

Mol ratio in reaction solution between intermediate 1, ASA intermediate and 1,8-diazacyclo [5.4.0] 11 carbon-7-alkene is: 1:1:1.8-2.5;

The structural formula of described ASA intermediate is:

The structural formula of described intermediate 2 is:

R is styroyl or naphthyl;

3) Deprotection

Added by intermediate 2 in Deprotection reagent solution, react 1.5-2.5h under room temperature, reaction terminates rear removed under reduced pressure solvent, to obtain target product after acetone recrystallization.

3. preparation method according to claim 2, is characterized in that:

Step 1) described in carboxylic acid be phenylpropionic acid or naphthoic acid; Described carboxyl activator is carbonyl dimidazoles.

4. preparation method according to claim 2, is characterized in that:

Step 1) described in organic solvent be the methylene dichloride of anhydrous acetonitrile, DMF, methyl-sulphoxide or drying.

5. preparation method according to claim 2, is characterized in that:

Step 2) described in the detailed process of separation and purification as follows:

By reaction solution underpressure distillation desolvation after reaction terminates, then add CHCl ₃dissolve, then add the citric acid solution washing of mass concentration 3%, separated and collected organic phase, through anhydrous sodium sulfate drying after organic phase washed with water and saturated common salt water washing, underpressure distillation removes CHCl ₃, cross silicagel column separation subsequently and obtain intermediate 2.

6. preparation method according to claim 2, is characterized in that:

Step 3) described in Deprotection reagent solution be the HCOOH aqueous solution of mass concentration 80%, the 5-10% hydrochloric acid soln of mass concentration or trifluoroacetic acid.

7. preparation method according to claim 2, is characterized in that:

Step 3) described in as follows with the detailed process of acetone recrystallization:

The white solid obtained after removed under reduced pressure solvent is added in flask, adds acetone, be heated to reflux state and constantly add new acetone, until white solid no longer dissolves, filtered while hot, filtrate leaves standstill crystallization under the condition of ice-water bath, filters, washs and namely obtain target product after drying.