CN101104862B - Synthesis of D-arylglycine by heterogeneous enzyme-catalyzed hydrolysis of 5-arylhydantoin - Google Patents

Abstract

The invention relates to the field of chemical industry and pharmacy, in particular to a method for synthesizing D-arylglycine by hydrolyzing 5-arylhydantoin with heterogeneous enzyme catalysis. The enzyme preparation prepared by fermentation of the mutant bacteria UV-LZ99 of the formamide hydrolase-producing bacteria Agrobacteriumradiobacter LZ99 is used as a catalyst, and 5-arylhydantoin in an amount 3-10 times higher than that of the homogeneous reaction is put into the heterogeneous catalytic reaction to make the product The D-arylglycine is directly precipitated in a solid form during the hydrolysis process of the substrate, thereby improving the synthesis efficiency, reducing evaporation energy consumption, and stable product quality.

Description

Synthesis of D-arylglycine by heterogeneous enzyme-catalyzed hydrolysis of 5-arylhydantoin

technical field

The invention relates to the field of chemical industry and pharmacy, in particular to a method for synthesizing D-arylglycine by hydrolyzing 5-arylhydantoin with heterogeneous enzyme catalysis.

Background technique

D-arylglycine is a very important class of non-natural amino acids. Because its configuration is just opposite to that of natural amino acids, it is mainly used in the synthesis and preparation of drugs, peptides and sweeteners. In recent years, with the increase in its application value Further development, the market demand is growing rapidly.

The traditional preparation method of D-arylglycine includes chemical synthesis and resolution method and biocatalytic D-arylhydantoin hydrolysis method, wherein all reported technologies are liquid-liquid homogeneous processes, while homogeneous biocatalysis Due to the low solubility of D-arylglycine by hydrolysis, the reaction volume is large, the production capacity is low, and the energy consumption is high.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for preparing D-arylglycine by catalyzing the hydrolysis of 5-arylhydantoin with heterogeneous enzymes, so as to overcome the problems existing in the prior art.

Design of the present invention is such:

On the basis of preparing the substrate 5-arylhydantoin by chemical synthesis, the mutant strain UV-LZ98 of Arthrobacter aurescens LZ98 and the mutant strain of Agrobacterium radiobacter LZ99 produced by hydantoinase were used The enzyme preparation prepared by mixed fermentation of LZ99 is used as a catalyst, and 5-arylhydantoin is put into the heterogeneous catalytic reaction in an amount 3-10 times higher than that of the homogeneous reaction, so that the product D-arylglycine is mixed with the substrate in a solid form. It is directly obtained by hydrolysis to improve production efficiency and reduce evaporation energy consumption.

In the present invention, D-arylglycine includes D-phenylglycine, D-fluorophenylglycine, D-chlorophenylglycine, D-bromophenylglycine, D-iodophenylglycine, D-alkoxyphenylglycine, D-pyridineglycine, D-naphthylglycine, D-quinolineglycine, D-pyrimidineglycine, D-pyrazineglycine, D-pyridazineglycine, D-indoleglycine, D-thiopheneglycine, D-furanglycine, D-pyrroleglycine, D -Pyrazole glycine, D-imidazole glycine, D-thiazole glycine, D-oxazole glycine and D-isoxazole glycine, etc. Most of the D-quinoline glycine series are new compounds, and the α carbon of glycine can be connected to aromatic on different ring carbons of the ring.

According to the enzyme activity of the enzyme preparation, the present invention adjusts the reaction temperature and the reaction pressure, can control the reaction speed and the crystallization speed of the product, and avoid the substrate hydantoin being trapped.

Technical solution of the present invention is:

The method for synthesizing D-arylglycine by heterogeneous enzyme catalyzed hydrolysis of 5-aryl hydantoin of the present invention comprises the following steps:

①In the biocatalytic hydrolysis reactor, add solvent water, 5-aryl hydantoin, enzyme preparation, acid or alkali, etc. in sequence, and stir to form a suspension. 5-21% (w/w), adding acid to adjust the pH value to 6.5-7.8;

② Put 2-8% of the total mass of the reaction liquid into the above-mentioned biocatalytic hydrolysis reactor with an enzyme preparation with an enzyme activity of 0.28 U/mL or more, control the temperature at 25-40°C, and start the stirring reaction;

③ During the reaction process, control the pressure of the reaction system between 0.09 and 0.03Mpa, take samples at intervals of 6 hours, and use liquid chromatography to detect the reaction situation. When the reaction lasts for about 8 hours, D-arylglycine crystals begin to precipitate. When the substrate 5-arylhydantoin cannot be detected in the reaction system, the reaction is basically finished;

④ The coarse crystals obtained by screening and separation can be recrystallized to obtain the product D-arylglycine.

The preparation method of the enzyme preparation solution is as follows: the mutant bacteria UV-LZ98 of Arthrobacter aurescens LZ98 and the mutant bacteria UV-LZ99 of Agrobacterium radiobacter LZ99 are mixed and fermented to obtain an enzyme preparation containing hydantoinase and carbaamide hydrolase, namely "two Bacterial enzymes".

Further, the D-arylglycine is D-phenylglycine, D-fluorophenylglycine, D-chlorophenylglycine, D-bromophenylglycine, D-iodophenylglycine, D-methoxyphenylglycine, D-pyridine Glycine, D-naphthylglycine, D-quinolineglycine, D-pyrimidineglycine, D-pyrazineglycine, D-pyridazineglycine, D-indoleglycine, D-thiopheneglycine, D-furanglycine, D-pyrroleglycine , D-pyrazole glycine, D-imidazole glycine, D-thiazole glycine, D-oxazole glycine and D-isoxazole glycine and other D-amino acids, wherein the α carbon of glycine can be connected to different positions of the aromatic ring on the carbon atom.

Further, the D-arylglycine is D-phenylglycine, and its synthesis method comprises the following steps:

①In a 10L enzyme-catalyzed reaction tank, add 7kg of water, 0.9-1kg of 5-phenylhydantoin, and add hydrochloric acid or sulfuric acid to adjust the pH to 6.8;

②Add wet enzyme preparation solution with 0.62U/ml enzyme activity under stirring condition, then add 0.3L of enzyme activity, control the temperature at 25-38℃, and stir to react;

③ Sampling at intervals of 6 hours, using liquid chromatography to detect the reaction, when the reaction lasted for about 8 hours, D-phenylglycine crystals began to precipitate, and the substrate 5-phenylhydantoin was not detected in the reaction system, and N - when carbamyl phenylglycine is below 0.25%, the reaction is basically finished;

④ The separated coarse crystals can be recrystallized to obtain the qualified product D-phenylglycine;

⑤ Dialyze the feed liquid through the sieve holes, and use membrane filtration, concentration and other refining processes to obtain another part of D-phenylglycine in the solution.

Further, the D-arylglycine is D-pyridineglycine, and its synthesis method comprises the following steps:

①In a 10L enzyme-catalyzed reaction tank, add 6.5kg of water, add 1kg of 5-pyridylhydantoin under stirring, add hydrochloric acid or sulfuric acid to adjust the pH to 6.5;

②Add about 0.4L of wet enzyme preparation solution with an enzyme activity of 0.58U/ml, and stir the reaction at a controlled temperature of 28-36°C;

③ Sampling was taken at intervals of 8 hours, and the reaction was detected by liquid chromatography. When the reaction lasted for about 9 hours, D-pyridine glycine crystals began to precipitate. When the substrate 5-pyridylhydantoin cannot be detected in the reaction system, and the N-carbamoyl-D-pyridine glycine is below 0.20%, the reaction is basically completed;

④ The separated coarse crystals can be recrystallized to obtain the qualified product D-pyridine glycine.

⑤ Dialyze the feed liquid through the sieve holes, and use membrane filtration, concentration and other refining processes to obtain another part of D-pyridine glycine in the solution.

Further, the D-arylglycine is D-naphthylglycine, and its synthesis method comprises the following steps:

①In a 10L enzyme-catalyzed reaction tank, add 7.5kg of water, add 0.8kg of 5-naphthylhydantoin under stirring, add hydrochloric acid or sulfuric acid to adjust the pH to 7.0;

②Add wet enzyme preparation solution with an enzyme activity of 0.62U/ml under the condition of stirring.

③Sampling at intervals of 8 hours, using liquid chromatography to detect the reaction situation, when the reaction proceeds to about 7 hours, D-naphthylglycine crystals begin to precipitate, and the substrate 5-naphthylhydantoin and the corresponding intermediate, the reaction is basically over;

④ The coarse crystals obtained by screening and separation can be recrystallized to obtain the qualified product D-naphthylglycine.

⑤ Dialyze the feed liquid through the sieve holes, and use membrane filtration, concentration and other refining processes to obtain another part of D-naphthylglycine in the solution.

Further, the D-arylglycine is D-quinolineglycine, and its synthesis method comprises the following steps:

①In a 10L enzyme-catalyzed reaction tank, add 8 kg of water, add 0.85 kg of 5-quinoline hydantoin under stirring conditions, and adjust the pH to 6.5;

② Then add about 0.35L of wet enzyme preparation solution with an enzyme activity of 0.68U/ml, and stir and react at 35-38°C;

③Sampling at intervals of 6 hours, using liquid chromatography to detect the reaction situation, when the reaction was carried out to about 8 hours, D-quinoline glycine crystals began to precipitate, and the substrate 5-quinoline hydantoin was not detected in the reaction system, and When N-carbamoyl-D-quinoline glycine is lower than 0.30%, the reaction is basically finished;

④ The coarse crystals obtained by screening and separation can be recrystallized to obtain the qualified product D-2-quinolineglycine.

⑤ Dialyze the feed liquid through the sieve holes, and use membrane filtration, concentration and other refining processes to obtain another part of D-quinoline glycine in the solution.

The heterogeneous catalysis of the present invention is realized on the premise that microorganisms are catalysts, and more importantly, the reactants and most of the products are in the solid phase throughout the process, and the entrapment of solids is avoided through the control of conditions, so that The conversion rate has reached more than 99.5%.

In the present invention, the biocatalytic preparation of D-arylglycine by 5-aryl hydantoin undergoes two-step reactions. The first step is that the substrate 5-arylhydantoin is hydrolyzed to the intermediate product D-N-carbamoyl-arylglycine under the catalysis of hydantoinase; the second step is that the intermediate product is hydrolyzed to The product D-arylglycine.

The catalysis of enzymes is a complex process, which is affected by the structure of enzyme molecules, the interaction between enzymes and substrate molecules, the directional effect of enzymes and substrates, the chiral selective binding of enzymes and substrates, and the combination with reaction transition states. Impact. In practical applications, metal ions play an important role in the catalytic ability of enzymes. Divalent ions such as Ni and Cu have inhibitory effects, while divalent ions such as Mn and Mg have obvious activation effects.

The kinetics study of enzyme-catalyzed hydrolysis of 5-arylhydantoin to D-arylglycine showed that under the premise that the enzyme concentration was fixed and the basic conditions were unchanged, the reaction basically showed a first-order reaction when the substrate concentration was low. When the concentration of reactants increases to saturation, the reaction rate tends to be constant and reaches a maximum. If it can be ensured that the concentration of the substrate 5-arylhydantoin in the system is in a saturated state for a long time, then the enzyme-catalyzed reaction speed can be maintained at the maximum value for a long time. Under the premise that the enzyme activity of the enzyme preparation reaches a high enough level, in the reaction system in the presence of water, the solid state (not completely forming a solution) substrate 5-arylhydantoin is catalyzed and hydrolyzed to produce products while the solute substrate in the solution is hydrolyzed. Rapid dissolution keeps the solution in a saturated state and ensures that the enzymatic reaction proceeds at the highest speed. At the same time, the generated product will crystallize out in the form of crystals after reaching saturation with the increase of concentration, which breaks the dissolution balance and enables the enzymatic reaction to proceed smoothly, thereby realizing the heterogeneous biocatalytic hydrolysis of 5-aryl Hydantoin synthesis of D-arylglycine.

The traditional "one bacterium, two enzymes" method can complete the entire transformation and hydrolysis process in one cell, while the "two bacteria, two enzymes" method is completed in two different cells. Therefore, the mass transfer process of substrates, intermediates and products between different cells is the key to this technology. In order to reduce the influence of mass transfer on the reaction rate, good results can be obtained by adding cationic surfactants to the reaction system.

The heterogeneous biocatalytic process is different from the chemical catalytic process, but the catalytic activity of the biocatalyst also declines with the prolongation of the reaction time. Therefore, it is necessary to control the total feeding amount of the substrate. In the present invention, it is more appropriate to determine that the amount of the substrate 5-arylhydantoin accounts for 5-18% of the total amount of the reactant system, and different aryl groups have different ideal values. The amount ratio here is mass ratio, and the reactant system includes water, enzyme preparation, acid (sulfuric acid or hydrochloric acid, phosphoric acid, etc.), alkali (sodium hydroxide, potassium hydroxide, sodium carbonate or other carbonates, etc.), 5- Aryl hydantoin and other additives, etc.

During the implementation of the two-bacteria and two-enzyme heterogeneous method, the unreacted solid substrate 5-arylhydantoin, the intermediate product N-carbamoyl-arylglycine solid and the product D-arylglycine solid co-exist in the same reactor In , there are multiple phases in the whole process. Moreover, the solid intermediate product D-N-carbamoyl-arylglycine and the solid product D-arylglycine are formed under the conditions of the presence of a large amount of substrates, and the problem of mutual wrapping or trapping is very easy to cause the conversion rate to decrease. Advantageously, however, the solids of the substrates are powders and the D-arylglycamino acids are crystalline. Controlling the precipitation speed of D-arylglycyl amino acid crystals will prevent solids from wrapping each other, and the conversion rate can reach more than 99.6%. The specific method is the optimization and control of the reaction conditions and the adjustment of the enzyme activity of the enzyme preparation.

The effect of pH. According to the principle of catalytic reaction, ammonia, carbon dioxide and D-arylglycine are produced during the conversion process. The generated ammonia easily forms salts with amino acids and accumulates in the reaction system, causing the pH in the later stage of the reaction to rise rapidly to 8.5, thereby strongly inhibiting the catalytic activity of N-aminocarboxamide hydrolase, resulting in a slowdown or even stop of the reaction . Therefore, it is very important to control the acidity and alkalinity in time for the reaction, and 6.5-7.8 is the appropriate pH.

The effect of temperature on the reaction. Under the conditions of fixed concentration and pH, temperature also has a great influence on the rate of enzyme-catalyzed reaction. When the temperature is 25°C, the reaction speed is slow, but the crystallization speed of the product is also slow, which is conducive to the growth of the crystal nucleus, and the encapsulation phenomenon is almost invisible; as the reaction temperature increases, the reaction speed also accelerates, and when it reaches 41°C When , the reaction speed is very fast, and the crystal nucleus formation speed is also accelerated in the supersaturated system, and the encapsulation phenomenon tends to be obvious. A suitable temperature is 25-40°C.

Compared with the prior art, the present invention has the following significant advantages:

1. Select two different strains of bacteria to produce an enzyme each to form a "two bacteria and two enzymes" catalytic system, which can not only speed up the reaction speed to the greatest extent, but also complete the two-step catalytic hydrolysis at one time. Compared with the original "one bacteria, two enzymes" technology, the reaction speed is greatly improved.

2. The input amount of 5-arylhydantoin is 3-10 times that of the traditional process, and it is much higher than its solubility. As the reaction proceeds, the product D-arylglycine will accumulate until the crystals are precipitated, and the yield of crystals at one time can reach 40-75% (w/w) of the total product amount, reducing the consumption of concentrated evaporation steam and saving energy. Above 40%.

3. The product quality is stable, the amount of primary water is reduced, and the yield is higher than the original process by more than 4 percentage points.

Description of drawings

Fig. 1 is the structural diagram of D-amino acid;

Fig. 2 is a schematic diagram of the reaction for preparing D-arylglycine by enzymatic hydrolysis of 5-arylhydantoin.

Explanation: R in Figure 1 is an aryl group; R in Figure 2 is the same as R in Figure 1.

Detailed ways

The present invention will be further described below.

Firstly, the enzyme preparation is prepared by mixed fermentation of hydantoinase-producing bacteria Arthrobacter aurescens LZ98 mutant bacteria UV-LZ98 and carbamidase-producing bacteria Agrobacterium radiobacter LZ99 mutant bacteria UV-LZ99. The process of the invention is then completed.

Embodiment 1, the preparation of D-phenylglycine

In a 10L enzyme-catalyzed reaction tank, first add 7kg of water, then add 1kg of 5-phenylhydantoin, add hydrochloric acid or sulfuric acid to adjust the pH to 6.8, and then add wet enzyme activity of 0.62U/ml under stirring conditions The preparation solution is about 0.3L, the temperature is controlled at 28°C, and the reaction is stirred. Samples were taken at intervals of 6 hours, and the reaction was detected by liquid chromatography. When the reaction lasted for about 8 hours, D-phenylglycine crystals began to precipitate, and the product concentration in the reaction solution was basically constant at 2.45%. When the substrate 5-phenylhydantoin cannot be detected in the reaction system and the N-carbamoylglycine is below 0.25%, the reaction is basically completed, and the conversion rate is above 99.6%. The crude crystals obtained by sieving and separation can be recrystallized to obtain 446.2g of qualified product D-phenylglycine; the material that has passed through the sieve is obtained by membrane filtration, concentration and other refining processes to obtain another part of D-phenylglycine product 240.2g. A total of 686.4 g of the product was obtained with a yield of 80%.

D-p-fluorophenylglycine can be obtained by hydrolysis of 5-p-fluorophenylhydantoin, the preparation method is similar to that of Example 1, and the yield is 77%.

D-p-chlorophenylglycine can be obtained by hydrolysis of 5-p-chlorophenylhydantoin, the preparation method is the same as that of Example 1, and the yield is 81%.

D-p-bromophenylglycine can be obtained by hydrolysis of 5-p-bromophenylhydantoin, the preparation method is similar to that of Example 1, the yield is 73%.

D-p-iodophenylglycine can be obtained by hydrolysis of 5-p-iodophenylhydantoin, the preparation method is similar to that of Example 1, and the yield is 69%.

The preparation method of D-methoxyphenylglycine can be obtained by hydrolyzing 5-p-iodophenylhydantoin, and the preparation method is the same as that of Example 1, and the yield is 75%.

Embodiment 2, preparation of D-3-pyridine glycine

In a 10L enzyme-catalyzed reaction tank, first add 6.5kg of water, add 1kg of 5-(3-pyridyl)hydantoin under stirring conditions, adjust the pH to 6.5, and then add wet enzyme activity of 0.58U/ml Enzyme preparation solution is about 0.4L, stirred and reacted at 32-36°C. Samples were taken at intervals of 8 hours, and the reaction was detected by liquid chromatography. When the reaction lasted for about 9 hours, D-pyridine glycine crystals began to precipitate. When the substrate 5-(3-pyridyl)hydantoin cannot be detected in the reaction system and the N-carbamoylpyridine glycine is below 0.20%, the reaction is basically completed, and the conversion rate is about 99.5%. The coarse crystals obtained by sieving and separation can be recrystallized to obtain 391.6 g of qualified product D-3-pyridine glycine; the material that has passed through the sieve is obtained by membrane filtration, concentration and other refining processes to obtain another part of product 261.1 g. A total of 652.7g of the product, the yield is 76%.

Other D-pyridine glycine preparation methods are basically the same.

Embodiment 3, the preparation of D-2-naphthylglycine

In a 10L enzyme-catalyzed reaction tank, first add 7.5kg of water, add 0.8kg of 5-(2-naphthyl)hydantoin under stirring conditions, adjust the pH to 7.0, and then add wet enzyme activity to 0.65U/ml The enzyme preparation solution is about 0.4L, and the reaction is stirred at 30-40°C. Samples were taken at intervals of 8 hours, and the reaction was detected by liquid chromatography. When the reaction was carried out to about 7 hours, D-2-naphthylglycine crystals began to precipitate. When no 5-(2-naphthyl)hydantoin and corresponding intermediates can be detected in the reaction system, the reaction is basically finished, and the conversion rate is about 99%. The coarse crystals obtained by sieving and separation can be recrystallized to obtain 327.3g of qualified product D-2-naphthylglycine; the material that has passed through the sieve is obtained by membrane filtration, concentration and other refining processes to obtain another part of D-2-naphthylglycine 192.2g . The total product is 519.5g, and the yield is 73%.

Other D-naphthylglycine preparation methods are basically the same.

Embodiment 4, the preparation of D-2-quinoline glycine

In a 10L enzyme-catalyzed reaction tank, first add 8kg of water, add 0.85kg of 5-(2-quinolyl)hydantoin under stirring conditions, adjust the pH to 6.5, and then add wet enzyme activity to 0.68U/ml The enzyme preparation solution is about 0.35L, and the reaction is stirred at 35-38°C. Samples were taken at intervals of 6 hours, and the reaction was detected by liquid chromatography. When the reaction lasted for about 8 hours, D-2-quinoline glycine crystals began to precipitate. When the substrate 5-(2-quinolyl)hydantoin cannot be detected in the reaction system, and N-carbamoyl-D-2-quinoline glycine is lower than 0.30%, the reaction is basically completed, and the conversion rate is 99% %about. The coarse crystals obtained by sieving and separation can be recrystallized to obtain 346.1g of qualified product D-2-quinoline glycine; the material that has passed through the sieve is obtained by membrane filtration, concentration and other refining processes to obtain another part of product D-2-quinoline Glycine 221.2g. The total product is 567.3g, and the yield is 75%.

Other D-quinoline glycine production methods are basically the same.

Claims (1)

1. the method for heterogeneous enzyme catalyzed hydrolysis hydantoin synthetic D-arylglycine, it is characterized in that described D-arylglycine is D-naphthylglycine, and its synthetic method comprises the following steps: ①In the enzyme-catalyzed reaction tank, add 7.5kg of water, add 0.8kg of 5-naphthylhydantoin under stirring, add hydrochloric acid or sulfuric acid to adjust the pH to 7.0; ②Add 0.4L of wet enzyme preparation solution containing hydantoinase and carbamidohydrolase with an enzyme activity of 0.62U/ml under the condition of stirring, control the temperature at 36-40°C and stir for reaction; ③Sampling at intervals of 8 hours, using liquid chromatography to detect the reaction situation, when the reaction proceeds to 7 hours, D-naphthylglycine crystals begin to precipitate, and the substrate 5-naphthylhydantoin and the corresponding intermediate body time, the reaction ends; ④The coarse crystals obtained by sieving and separation can be recrystallized to obtain the qualified product D-naphthylglycine.

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