CN108300744B - Synthesis method, kit and application of D-heterocyclic amino acid - Google Patents
Synthesis method, kit and application of D-heterocyclic amino acid Download PDFInfo
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Abstract
The invention provides a synthetic method, a kit and application of D-heterocyclic amino acid. The synthesis method comprises the following steps: the diaminopimelate dehydrogenase shown in SEQ ID NO:1 is used to convert D-heterocyclic keto acids into D-heterocyclic amino acids. The diaminopimelate dehydrogenase is adopted to convert D-heterocyclic ketonic acid compounds into D-heterocyclic amino acids, the unnatural chiral amino acids with high conversion rate and high ee can be obtained, the technological conditions used in the method for synthesizing the diaminopimelate dehydrogenase are stable, the reaction conditions are mild, the operation is simple in the whole production process, the pollution is low, and a new thought method is provided for artificially synthesizing the D-heterocyclic amino acids.
Description
Technical Field
The invention relates to the field of synthesis of unnatural amino acids, and in particular relates to a synthetic method, a kit and application of D-heterocyclic amino acids.
Background
The non-natural chiral amino acid and the derivatives thereof are important constituent units of polypeptides, peptoids and a plurality of drug molecules and are key intermediates for synthesizing chiral drugs. At present, the synthesis of chiral unnatural heterocyclic amino acids mainly adopts chemical methods, including methods of using noble metal asymmetric catalytic hydrogenation to realize single configuration transformation on a certain key intermediate, using chiral reagents to split racemates, using chiral prosthetic group asymmetric synthesis, using directional synthesis of chiral raw materials, and the like. However, these methods have the following drawbacks: 1) the method has the advantages that the precious metal asymmetric catalyst is expensive, a large amount of organic solvent is needed for reaction, heavy metal residues exist in the product, and a byproduct of over reduction possibly exists, and moreover, the raw material for synthesis contains heterocyclic rings, so that the combination of the precious metal and a ligand is often interfered, and the catalytic efficiency is not high; 2) one isomer in the racemate is obtained by adopting a traditional chiral resolution method, so that additional raw material waste is caused; 3) the asymmetric synthesis of chiral auxiliary group or chiral raw material relates to chiral raw material with high price, longer synthetic route and a large amount of organic solvent, and for the synthesis of some heterocyclic amino acid, the obtained product has low optical purity or the product and impurities are not easy to separate.
The amino acid is divided into two kinds of D-amino acid and L-amino acid according to chirality, natural amino acid is mainly L type, some documents or patent reports adopt a method for synthesizing L-unnatural amino acid by using biological enzyme, D-heterocyclic amino acid is one kind of unnatural amino acid, has excellent performance which is not possessed by unnatural amino acid besides most functions of natural amino acid, and has wide application in the aspects of drug synthesis (medicine and pesticide), food, feed and the like.
Disclosure of Invention
The invention mainly aims to provide a synthetic method, a kit and application of D-heterocyclic amino acid, and provides a method for biologically synthesizing D-heterocyclic amino acid by adopting biological enzyme to improve selectivity.
In order to achieve the above object, according to one aspect of the present invention, there is provided a method for synthesizing a D-heterocyclic amino acid, the method comprising: the diaminopimelate dehydrogenase shown in SEQ ID NO:1 is used to convert D-heterocyclic keto acids into D-heterocyclic amino acids.
Further, the D-heterocyclic ketonic acid compound is selected from any one of the following compounds:
further, the synthesis method comprises the following steps: mixing the D-heterocyclic ketonic acid compound with an amino donor, and reacting under the action of diaminopimelate dehydrogenase and coenzyme to obtain the D-heterocyclic amino acid.
Further, the amino donor is selected from ammonium formate or ammonium chloride.
Further, the coenzyme is selected from β -NADP+Or β -NAD+。
Further, the pH value of the reaction is 7.8-9.0, and the preferable temperature of the reaction is 30-40 ℃.
Further, the reaction is carried out in a buffer selected from Tris-HCl salt buffer, triethanolamine buffer, phosphate buffer or boric acid-sodium hydroxide buffer.
Further, the synthesis method comprises the following steps: mixing D-heterocyclic ketonic acid compounds with an amino donor, and reacting under the action of diaminopimelate dehydrogenase and coenzyme to obtain a reaction product; and carrying out post-treatment on the reaction product to obtain D-heterocyclic amino acid; preferably, the step of post-processing comprises: adding a reaction termination solution into the reaction product to obtain a termination system; filtering the termination system to obtain a filtrate; purifying the target product in the filtrate to obtain D-heterocyclic amino acid; more preferably, the reaction terminating solution is hydrochloric acid, the amount of hydrochloric acid added is such that the pH of the terminating system is not more than 1, and a diatomaceous earth pad is used in the filtration step.
In order to accomplish the above object, according to one aspect of the present invention, there is provided a kit for synthesizing D-heterocyclic amino acids, the kit comprising a biological enzyme which is diaminopimelate dehydrogenase represented by SEQ ID NO: 1.
Further, the kit also comprises D-heterocyclic ketoacids, preferably any one of the following D-heterocyclic ketoacids:
according to another aspect of the present invention, there is provided use of the diaminopimelate dehydrogenase represented by SEQ ID NO:1 for screening D-heterocyclic amino acid synthesis raw materials.
By applying the technical scheme of the invention, the diaminopimelate dehydrogenase is adopted to convert the D-heterocyclic ketonic acid compound into the D-heterocyclic amino acid, the unnatural chiral amino acid with higher conversion rate and higher chiral purity can be obtained, the method for synthesizing the diaminopimelate dehydrogenase has the advantages of stable process conditions, mild reaction conditions, simple operation and lower pollution in the whole production process, and a new thinking method is provided for artificially synthesizing the D-heterocyclic amino acid.
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.
As mentioned in the background art, the synthesis of D-heterocyclic amino acids in the prior art has a defect of low yield, etc., and in order to improve the current situation, the inventors have studied the existing method for synthesizing D-heterocyclic amino acids by using biological enzymes, and found that the reason of the low synthesis yield of D-heterocyclic amino acids is that a suitable diaminopimelate dehydrogenase is not selected for a specific synthesis raw material to synthesize the corresponding D-heterocyclic amino acids, and in order to find out a D-heterocyclic amino acid synthesis raw material and a diaminopimelate dehydrogenase which are matched, the inventors screened commercially available raw materials or easily prepared ketoacids from a large number of existing ketoacid compound raw materials, screened nearly 100 enzymes from an enzyme library for these raw materials, and found that a diaminopimelate dehydrogenase derived from Bacillus stearothermophilus (sequence shown in SEQ ID NO: MNKVHKIAVVGYGNIGKYAVQA L NRAP L AGVVRRARSARDVPPE L0 AGVPIATSIDE L1 EGVEAAI 5639 2ATPTRTTPEYASDI L SKGIHTVDSYDIHGE L ADVRRK DDIAKRHGSVAIVSAGWDPGTDSMIRSM L SMT 36 EFMAPGGVTYTNFGPGMSMGHSVAVKAIDGVKDA L Q7V 3L GTGVHRRMVYVECEAGADFETVKEKV L ADPYFVNDETHVIQVDDVQQ L VDVGHGVSMERKGVSGATHNQ L FNFEMRINNPA L) can achieve high selectivity for synthesizing heterocyclic amino acids and for heterocyclic DME-8292.
Based on the above research results, in one exemplary embodiment of the present application, there is provided a method for synthesizing a D-heterocyclic amino acid, the method comprising: the diaminopimelate dehydrogenase shown in SEQ ID NO:1 is used to convert D-heterocyclic keto acids into D-heterocyclic amino acids. The diaminopimelate dehydrogenase shown in SEQ ID NO. 1 can achieve high conversion rate and selectivity in the synthesis of D-heterocyclic amino acids.
By screening a plurality of existing D-heterocyclic ketoacids, the inventor finds that any one of the following compounds can be selected:
when the diaminopimelate dehydrogenase is synthesized, the chiral purity of the diaminopimelate dehydrogenase is high.
Accordingly, theWhen the D-heterocyclic keto acid compound is used as a raw material, the obtained D-heterocyclic amino acid is selected from any one of the following groups:
the synthesis method can be obtained by reasonably adjusting synthesis conditions according to different raw materials and biological enzymes on the basis of the existing method for synthesizing amino acid by using diaminopimelate dehydrogenase. In order to further improve the chiral purity of the product, in a preferred embodiment of the present application, the above synthesis method comprises: mixing the D-heterocyclic ketonic acid compound with an amino donor, and reacting under the action of diaminopimelate dehydrogenase and coenzyme to obtain the D-heterocyclic amino acid.
The amino donor in the above preferred embodiment may be a conventional amino donor, and in order to achieve a higher conversion of the substrate, in a preferred embodiment of the present application, the amino donor is selected from ammonium formate or ammonium chloride. The same amino donor or different amino donors can be selected for different substrates, but the synthesis reaction can be carried out by optimally selecting the most suitable amino donor for different substrate raw materials in order to ensure higher chiral purity of the product. For example, when the raw material is
In the process, the ammonium formate is selected as the amino donor, so that the reaction conditions are more suitable, and the chiral purity of the product is higher. When the raw material is
Or
When ammonium chloride is selected as the amino groupThe reaction conditions can be more suitable, and the conversion rate of raw materials is higher.
In the above synthesis method, in order to improve the biological enzyme catalytic activity, usually add coenzyme to promote the reaction, in the application of a preferred embodiment, the coenzyme selected from β -NADP+Or β -NAD+。
In the synthesis method, the specific reaction conditions can be reasonably optimized and adjusted according to the conversion rate of the substrate. In a preferred embodiment of the present application, the pH of the reaction is 7.8 to 9.0, and the reaction temperature is preferably 30 to 40 ℃. Under the reaction conditions, the chiral purity of the product is high, the product is relatively mild, and the synthesis method is simple and easy to operate due to convenient control. Naturally, the optimum pH conditions and reaction temperature for different starting materials will also vary slightly and can be adjusted appropriately for the particular starting material. Preferably, when the starting material is
When the reaction is carried out, the optimum pH for the reaction is from 8.5 to 9.0. When the raw material is
When the reaction is carried out, the optimum pH for the reaction is 7.8 to 8.2.
In order to achieve higher chiral purity of the product of the above reaction, in a preferred embodiment of the present application, the above reaction is performed in a buffer selected from Tris-HCl salt buffer, triethanolamine buffer, phosphate buffer, or boric acid-sodium hydroxide buffer. The optimization effect of different buffers on different raw materials is slightly different. For example, in Tris-HCl salt buffer,
the reaction conversion of the starting material is relatively higher. In the triethanolamine buffer solution, however,
the reaction conversion of the starting material is relatively higher. In a phosphate buffer, especially a sodium phosphate buffer,
the reaction conversion of the starting material is relatively higher.
In a preferred embodiment of the present application, the above synthesis method comprises: mixing D-heterocyclic ketonic acid compounds with an amino donor, and reacting under the action of diaminopimelate dehydrogenase and coenzyme to obtain a reaction product; and carrying out post-treatment on the reaction product to obtain the D-heterocyclic amino acid. Impurities in the reaction product can be removed through post-treatment, so that the target product with high purity is obtained.
The post-treatment is a post-treatment operation which is commonly used in the amino acid synthesis step and comprises a reaction termination operation and a product purification operation. In a preferred embodiment of the present application, the post-processing step includes: adding a reaction termination solution into the reaction product to obtain a termination system; filtering the termination system to obtain a filtrate; and purifying the target product in the filtrate to obtain the D-heterocyclic amino acid.
In a preferred embodiment of the present application, the reaction stop solution is hydrochloric acid (preferably concentrated hydrochloric acid, which is added in a small amount, and the actual amount is based on pH less than or equal to 1), and a diatomite pad is used in the filtration step. Termination of the reaction with hydrochloric acid enables rapid completion of the reaction. And the diatomite pad has the advantages of simple and effective operation and low cost.
In a second exemplary embodiment of the present application, a kit for synthesizing a D-heterocyclic amino acid is provided, which includes a biological enzyme that is diaminopimelate dehydrogenase represented by SEQ ID NO: 1. The kit containing the diaminopimelate dehydrogenase is used for synthesizing the D-heterocyclic amino acid, and has the advantage of high selectivity.
In a preferred embodiment, the kit further comprises D-heterocyclic ketone compounds, preferably any one of the following D-heterocyclic ketone compounds:
In a third exemplary embodiment of the present application, there is provided the use of a diaminopimelate dehydrogenase as shown in SEQ ID NO 1 for screening D-heterocyclic amino acid synthesis starting materials. The application can screen novel substrates with higher conversion rate and/or specificity so as to form D-heterocyclic amino acids with more varieties and wider application range.
The advantageous effects of the present application will be further described with reference to specific examples.
Example 1
(1) Charging, 10g of main raw material is added into a 2L four-mouth round-bottom bottle
250ml Tris-HCl buffer (100 mmol/L, pH 9.0), raw material was homogeneously dispersed in Tris-HCl buffer, pH was adjusted to 8.5 with 10M NaOH, 16g ammonium formate, 0.3g β -NAD were added+The pH was adjusted to 8.5 with 10M, NaOH. 80g of diaminopimelate dehydrogenase, 20g of formate dehydrogenase (cycle H)+) The above reaction system was added and the pH was adjusted to 8.5.
(2) Reaction: and the temperature was raised to 30 ℃ to carry out the reaction.
(3) And (3) post-treatment: after 16h of reaction, the system is followed, the reaction of the raw materials is detected to be finished, the temperature is reduced to room temperature, concentrated hydrochloric acid is slowly dripped into a four-neck flask to enable the pH value of the mixed system after the reaction to be 1 so as to stop the reaction, the system is covered by diatomite, the pH value of filtrate is adjusted to 8.0 by 30% NaOH, 300ml of MTBE is used for extracting for 1 time, the pH value of water phase is adjusted to 10.0 by 30% of NaOH, 22g of tert-butyloxycarbonyl group is added, 30% of NaOH is dripped, the pH value is controlled to be 10.0, the reaction is carried out for 16h at the temperature of 25 ℃, and 300 ml. The aqueous phase is adjusted to pH 4.5 with 10% by weight hydrochloric acid and extracted 3 times with 300ml of MTBE. The organic phase was washed twice with 300ml of saturated saline, the aqueous phase was discarded after separation, the organic phase was dried over 10g of anhydrous magnesium sulfate, filtered after drying, and the filter cake was washed with MTBE. The filtrate was again dried to give 9.2g of a solid.
The nuclear magnetic data of the obtained solid are as follows:1H NMR(400MHz,D2O)：8.43(d，2H)，7.87(d，1H)，7.50(t，1H)，3.97(t，1H)，3.24(dd，2H)。
the content of the internal standard (namely the target product) is 98.5 percent through nuclear magnetic detection, and the chiral purity is 99.7 percent through liquid chromatography detection.
Example 2
(1) Charging, 10g of main raw material is added into a 2L four-mouth round-bottom bottle
250ml Tris-HCl buffer (100 mmol/L, pH 9.0), raw material was homogeneously dispersed in Tris-HCl buffer, pH was adjusted to 9.0 with 10M NaOH, 16.2g ammonium formate, 0.3g β -NAD were added in sequence+The pH was adjusted to 9.0 with 10M, NaOH. Finally, 40g of diaminopimelate dehydrogenase and 20g of formate dehydrogenase were added to the above reaction system, and the pH was adjusted to 9.0.
(2) Reaction: and the temperature was raised to 40 ℃ to carry out the reaction.
(3) And (3) post-treatment: after reacting for 16h, a tracking system is added, the reaction of the raw materials is detected, the temperature is reduced to room temperature, concentrated hydrochloric acid is slowly dripped into the system under stirring, and the pH value of the mixed system after the reaction is reduced to below 1; and (3) passing the acid-adjusted system through a 1-2 cm diatomite pad, washing the filter cake for 2 times by using 200ml of purified water to obtain an aqueous solution, adjusting the pH value of the obtained filtrate to be between 5 by using sodium hydroxide, carrying out suction filtration on the mixed solution to obtain a filter cake and a filtrate, washing the filter cake twice by using 300ml of purified water, stirring and washing for 3 times by using 5 times of volume of pure water, and drying the filter cake after suction filtration to obtain 7.4g of a product.
The nuclear magnetic data of the obtained product are as follows:1H NMR(400MHz,D2O)：7.74(d，1H)，7.17(d，1H)，4.20(s，1H)，4.07(t，1H)，3.38(s，1H),3.34(dd，1H),3.10(dd，1H)。
the content of the nuclear magnetic internal standard is 98.4% by nuclear magnetic detection, and the chiral purity is 98.5% by liquid chromatography detection.
Example 3
(1) Charging, 10g of main raw material is added into a 2L four-mouth round-bottom bottle
250ml triethanolamine buffer (100 mmol/L, pH 9.0), uniformly dispersing the raw materials in triethanolamine buffer, adjusting pH to 8.5 with 10M NaOH, sequentially adding 7.4g ammonium formate, 0.3g β -NAD+The pH was adjusted to 8.5 with 10M, NaOH. Finally, 20g of diaminopimelate dehydrogenase and 10g of formate dehydrogenase were added to the above reaction system, and the pH was adjusted to 8.5.
(2) Reaction: and the temperature was raised to 35 ℃ to carry out the reaction.
(3) And (3) post-treatment, namely reacting for 16 hours, then tracing the system, detecting that the raw materials are reacted, cooling to room temperature, slowly dropwise adding concentrated hydrochloric acid into a four-neck flask to ensure that the pH value of the mixed system after the reaction is 1 to stop the reaction, allowing the system to pass through diatomite, adjusting the pH value of the obtained filtrate to be 6 by using solid sodium hydroxide in an ice water bath, stirring for 30min, performing suction filtration to obtain a solid, washing the solid by using 10m L, and drying to obtain 8.5g of a product.
The nuclear magnetic data of the obtained product are as follows:1H NMR(400MHz,D2O)：6.08(q，2H)，4.12(t，1H)，3.41(s，1H)，3.28(dd，1H)，3.16(s，1H),2.94(dd，1H),2.17(s，3H)。
the content of the internal standard is 95.0% by nuclear magnetic detection, and the chiral purity is 99.4% by liquid chromatography detection.
Example 4
(1) Charging, 10g of main raw material is added into a 2L four-mouth round-bottom bottle
250ml Tris-HCl buffer (200 mmol/L, pH 9.0), raw material was homogeneously dispersed in Tris-HCl buffer, pH was adjusted to 8.5 with 10M NaOH, 9.8g ammonium formate, 0.3g β -NAD were added+The pH was adjusted to 8.5 with 10M, NaOH. Finally, 100g of diaminopimelate dehydrogenase and 10g of formate dehydrogenase were added to the above reactionSystem, and adjust pH to 8.5.
(2) Reaction: and the temperature was raised to 35 ℃ to cause the reaction system to react according to the following chemical formula.
(3) And (2) post-treatment, namely reacting for 16 hours, cooling to room temperature after detecting that the raw materials react, slowly dropwise adding concentrated hydrochloric acid into the system while stirring to ensure that the pH value of the mixed system after reaction is below 1, passing the system after acid adjustment through a 1-2 cm diatomite pad, adjusting the pH value of the obtained filtrate to be between 6 by using sodium hydroxide to obtain a crude product aqueous solution, purifying the crude product aqueous solution through a strong acid cation exchange resin column with the model of 001 × 7, washing the obtained crude product with absolute ethyl alcohol, performing suction filtration, drying a filter cake to obtain a solid product, and purifying the filtrate through a strong acid cation exchange resin column with the model of 001 × 7 to obtain 5.1g of the solid product.
The nuclear magnetic data of the obtained product are as follows:1H NMR(400MHz,D2O)：4.90(t，1H)，3.92(m，4H)，3.45(t，1H)，3.40(s，1H)，3.35(s，1H),1.94(dd，1H),1.74(dd，1H),1.54(td，2H)。
the content of the nuclear magnetic internal standard is 98.2% by nuclear magnetic detection, and the chiral purity is 99.1% by liquid chromatography detection.
Example 5
(1) Charging, 10g of main raw material is added into a 2L four-mouth round-bottom bottle
250ml of sodium phosphate buffer (200 mmol/L, pH 8.0), the starting material was homogeneously dispersed in sodium phosphate buffer, the pH was adjusted to 7.8 with 10M NaOH, 29g of ammonium chloride, 29.3g of glucose and 0.33g of β -NADP were added in succession+The pH was adjusted to 7.8 with 10M, NaOH. Finally, 50g of diaminopimelate dehydrogenase and 10g of glucose dehydrogenase were added to the above reaction system, and the pH was adjusted to 7.8.
(2) Reaction: and the temperature was raised to 35 ℃ to cause the reaction system to react according to the following chemical formula.
(3) And (3) post-treatment: after reacting for 16h, a tracking system is added, the reaction of the raw materials is detected, the temperature is reduced to room temperature, concentrated hydrochloric acid is slowly dripped into the system under stirring, and the pH value of the mixed system after the reaction is reduced to below 1; and (3) passing the acid-adjusted system through a 1-2 cm diatomite pad, washing the filter cake for 2 times by using 200ml of purified water to obtain an aqueous solution, adjusting the pH value of the obtained filtrate to be between 6 by using sodium hydroxide, carrying out suction filtration on the mixed solution to obtain a filter cake and a filtrate, washing the filter cake twice by using 300ml of purified water, stirring and washing for 3 times by using 5 times of volume of pure water, and drying the filter cake after suction filtration to obtain 8.2g of a product.
The nuclear magnetic data of the obtained product are as follows:1H NMR(400MHz,D2O)：7.30(d，1H)，7.02(t，1H)，6.93(s，1H)，3.50(t，1H)，3.15(d，2H)。
the content of the internal standard is 98.0% by nuclear magnetic detection, and the chiral purity is 98.5% by liquid chromatography detection.
Example 6
(1) Charging, 10g of main raw material is added into a 2L four-mouth round-bottom bottle
250ml of boric acid-sodium hydroxide (200 mmol/L, pH 9.0), uniformly dispersing the raw materials in sodium phosphate buffer, adjusting pH to 8.5 with 10M NaOH, sequentially adding 15g of ammonium chloride, 15.5g of glucose and 0.33g of β -NADP+The pH was adjusted to 8.5 with 10M, NaOH. Finally, 60g of diaminopimelate dehydrogenase and 20g of glucose dehydrogenase were added to the above reaction system, and the pH was adjusted to 8.5.
(2) Reaction: and the temperature was raised to 35 ℃ to cause the reaction system to react according to the following chemical formula.
(3) And (3) post-treatment, namely reacting for 16 hours, then tracing the system, detecting that the raw materials are reacted, cooling to room temperature, slowly dropwise adding concentrated hydrochloric acid into a four-neck flask to ensure that the pH value of the mixed system after the reaction is 1 to stop the reaction, passing the system through diatomite, adjusting the pH value of the obtained filtrate to be 6 by using solid sodium hydroxide in an ice water bath, stirring for 30 minutes, carrying out suction filtration to obtain a solid, washing the solid by using 10m L water, and drying to obtain 9.0g of a product.
The nuclear magnetic data of the obtained product are as follows:1H NMR(400MHz,D2O)：7.18，6.82(2d，2H，arom.-H)，3.78(dd，1H,Hα)，3.23(2dd，2H，Hβ)，2.10(s，2HCH3)。
the content of the internal standard is 99.0% by nuclear magnetic detection, and the chiral purity is 99.0% by liquid chromatography detection.
Comparative example 1
Synthesis of D-methyl-2-acetylamino-3- (4-pyridyl) -propionic acid methyl ester (same product as in example 1) according to the chemical Synthesis method described in "Transmission-Metal-Assisted Synthesis of Amino acids analogs. A New Synthesis of synthetic Pure D-and L-Pyridylalanines". The Synthesis of D-methyl-2-acetylamino-3- (4-pyridyl) -propionic acid methyl ester (same product as in example 1) 380mg of methyl-2-acetylamino-3- (4-pyridyl) -acrylic acid methyl ester, 46mg of (S, S) - [ Rh (DIPAMP) (COD)]+ (BF4-) and 10ml of methanol were charged into the autoclave and charged with H2The pressure is allowed to reach 65psi, and after the reaction is completed, 349mg of product is separated by column chromatography, and the chiral purity is 92% by gas chromatography detection.
Comparative example 2
The conversion of the starting materials to the corresponding amino acids in examples 1-6 was catalyzed by L-leucine dehydrogenase and formate dehydrogenase and followed by liquid chromatography detection, the results of which are shown in Table 2.
The chiral purity of the D-heterocyclic amino acids of examples 1 to 6 and comparative example 1 was determined by liquid chromatography and is reported in table 1.
Table 1:
as can be seen from the data in Table 1, the chiral purity of the D-heterocyclic amino acids obtained by the preparation method of the invention in examples 1 to 6 is more than 98%. Furthermore, from a comparison of example 1 with comparative example 1, it can be found that the selectivity for a specific D-heterocyclic amino acid using the diaminopimelate dehydrogenase of the present application is higher than that of the chemical synthesis method.
In addition, L-leucine dehydrogenase and formate dehydrogenase of comparative example 2 were used to catalyze the same raw materials as in examples 1-6 to attempt to synthesize the same products as in examples 1-6, followed by liquid chromatography detection, and the results are reported in Table 2.
Table 2:
as can be seen from the data in Table 2, the L-leucine dehydrogenase and formate dehydrogenase of comparative example 2 are used to catalyze the same raw materials as those in examples 1-6, and the chiral purity of the synthesized product is less than 10%.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: the diaminopimelate dehydrogenase provided by the invention converts heterocyclic ketonic acid into D-heterocyclic amino acid to obtain unnatural chiral amino acid with higher conversion rate and higher ee, the synthetic method adopts stable technological conditions, the reaction conditions are mild, the operation is simple in the whole production process, the pollution is low, and a new thought method is provided for artificially synthesizing D-heterocyclic amino acid.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Kai Lai Ying pharmaceutical group (Tianjin) Ltd
Synthesis method, kit and application of <120> D-heterocyclic amino acid
<130>PN82310KLY
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>301
<212>PRT
<213>Novibacillus thermophilus
<400>1
Met Asn Lys Val His Lys Ile Ala Val Val Gly Tyr Gly Asn Ile Gly
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Lys Tyr Ala Val Gln Ala Leu Asn Arg Ala Pro Asp Met Glu Leu Ala
20 25 30
Gly Val Val Arg Arg Ala Arg Ser Ala Arg Asp Val Pro Pro Glu Leu
35 40 45
Ala Gly Val Pro Ile Ala Thr Ser Ile Asp Glu Leu Glu Gly Val Glu
50 55 60
Ala Ala Ile Leu Ala Thr Pro Thr Arg Thr Thr Pro Glu Tyr Ala Ser
65 70 75 80
Asp Ile Leu Ser Lys Gly Ile His Thr Val Asp Ser Tyr Asp Ile His
85 90 95
Gly Glu Leu Ala Asp Val Arg Arg Lys Leu Asp Asp Ile Ala Lys Arg
100 105 110
His Gly Ser Val Ala Ile Val Ser Ala Gly Trp Asp Pro Gly Thr Asp
115 120 125
Ser Met Ile Arg Ser Met Leu Glu Phe Met Ala Pro Gly Gly Val Thr
130 135 140
Tyr Thr Asn Phe Gly Pro Gly Met Ser Met Gly His Ser Val Ala Val
145 150 155 160
Lys Ala Ile Asp Gly Val Lys Asp Ala Leu Ser Met Thr Ile Pro Leu
165 170 175
Gly Thr Gly Val His Arg Arg Met Val Tyr Val Glu Cys Glu Ala Gly
180 185 190
Ala Asp Phe Glu Thr Val Lys Glu Lys Val Leu Ala Asp Pro Tyr Phe
195 200 205
Val Asn Asp Glu Thr His Val Ile Gln Val Asp Asp Val Gln Gln Leu
210 215 220
Val Asp Val Gly His Gly Val Ser Met Glu Arg Lys Gly Val Ser Gly
225 230 235 240
Ala Thr His Asn Gln Leu Phe Asn Phe Glu Met Arg Ile Asn Asn Pro
245 250 255
Ala Leu Thr Ser Gln Val Leu Val Ala Ala Ala Arg Ala Thr Phe Lys
260 265 270
Gln Gln Pro Gly Ala Tyr Thr Met Ile Glu Val Pro Ile Ile Asp Phe
275 280 285
Met Tyr Gly Asp Arg Glu Glu Leu Ile Lys Arg Leu Val
290 295 300
Claims (14)
1. A method for synthesizing a D-heterocyclic amino acid, comprising:
the diaminopimelate dehydrogenase shown in SEQ ID NO:1 is used to convert D-heterocyclic keto acids into D-heterocyclic amino acids.
2. The method of claim 1, wherein the D-heterocyclic ketonic acid compound is selected from any one of the following:
the D-heterocyclic amino acid is selected from any one of the following:
3. the method of synthesis of claim 1, comprising:
mixing the D-heterocyclic ketonic acid compound with an amino donor, and reacting under the action of the diaminopimelate dehydrogenase and a coenzyme to obtain the D-heterocyclic amino acid.
4. The synthesis process according to claim 3, characterized in that the amino donor is chosen from ammonium formate or ammonium chloride.
5. The method of synthesis according to claim 3, wherein the coenzyme is selected from β -NADP + or β -NAD +.
6. The synthesis method according to claim 3, wherein the reaction has a pH of 7.8 to 9.0.
7. The synthesis method according to claim 3, wherein the reaction temperature is 30-40 ℃.
8. The method of synthesis according to claim 3, wherein the reaction is carried out in a buffer selected from Tris-HCl salt buffer, triethanolamine buffer, phosphate buffer or boric acid-sodium hydroxide buffer.
9. The synthesis method according to any one of claims 3 to 8, characterized in that it comprises:
mixing the D-heterocyclic ketonic acid compound with the amino donor, and reacting under the action of the diaminopimelate dehydrogenase and the coenzyme to obtain a reaction product;
and carrying out post-treatment on the reaction product to obtain the D-heterocyclic amino acid.
10. The method of synthesis according to claim 9, wherein the step of post-processing comprises:
adding a reaction termination solution into the reaction product to obtain a termination system;
filtering the termination system to obtain a filtrate; and
and purifying the target product in the filtrate to obtain the D-heterocyclic amino acid.
11. The synthesis method according to claim 10, wherein the reaction terminating solution is hydrochloric acid, the hydrochloric acid is added in an amount such that the pH of the terminating system is not more than 1, and a diatomaceous earth pad is used in the filtering step.
12. A kit for synthesizing D-heterocyclic amino acid comprises a biological enzyme, and is characterized in that the biological enzyme is diaminopimelate dehydrogenase shown as SEQ ID NO. 1, and the kit also comprises D-heterocyclic ketoacids.
13. The kit according to claim 12, wherein the D-heterocyclic ketonic acid compound is any one of:
the application of diaminopimelate dehydrogenase shown in SEQ ID NO. 1 in screening D-heterocyclic amino acid synthetic raw materials.
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