CN108300744A - Synthetic method, kit and the application of D- heterocyclic amino acids - Google Patents
Synthetic method, kit and the application of D- heterocyclic amino acids Download PDFInfo
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Abstract
The present invention provides a kind of synthetic method, kit and the applications of D heterocyclic amino acids.The synthetic method includes：Using SEQ ID NO:The Mek-Tol Unit compound of D heterocyclics is converted to D heterocyclic amino acids by diaminopimelate dehydrogenase shown in 1.The ketone acid compound of D heterocyclics is converted by D heterocyclic amino acids using the diaminopimelate dehydrogenase of the application, it can obtain the Non-natural chiral amino acid of higher conversion and higher ee, and the stable process conditions used in synthetic method are carried out using the diaminopimelate dehydrogenase, reaction condition is mild, it is easy to operate in entire production process, pollution is relatively low, provide a kind of new idea and method for artificial synthesized D heterocyclic amino acids.
Description
Technical field
The present invention relates to the synthesis fields of non-natural amino acid, in particular to a kind of synthesis of D- heterocyclic amino acids
Method, kit and application.
Background technology
Non-natural chiral amino acid and its derivative are the important composition units of polypeptide, class peptide and many drug molecules,
It is also the key intermediate of synthesis of chiral drug.Currently, the non-natural heterocyclic amino acid of synthesis of chiral, mainly uses chemistry side
Method, including precious metal asymmetric catalytic hydrogenation is used to realize the conversion of single configuration to a certain key intermediate, use hand
Property reagent resolving racemic, using chiral auxiliary asymmetric syntheses, use the methods of the controlled syntheses of chiral raw material.However these
Method has the following defects：1) single configuration is realized to a certain key intermediate using precious metal asymmetric catalytic hydrogenation
Conversion, the disadvantage is that precious metal asymmetric catalyst is expensive, reaction needs a large amount of organic solvents, has heavy metal in product
It remains and there may be the by-product of over reduction, and because containing heterocycle in the raw material of synthesis, can usually interfere valuable gold
The combination of category and ligand, causes catalytic efficiency not high；2) use traditional chiral separation method, one obtained in raceme different
Structure body can cause other wastage of material；3) asymmetric syntheses for utilizing chiral auxiliary or chiral raw material, is related to expensive
Chiral raw material, longer synthetic route and a large amount of organic solvent, and for the synthesis of certain heterocyclic amino acids, obtain
Optical purity of products is not high or product and impurity are not readily separated.
Amino acid is divided into two kinds of D- amino acid and l-amino acid according to chirality.Natural amino acid is existing mainly based on L-type
There is the method there are also document or patent report using biological enzymatic synthesis L- non-natural amino acids in technology.D- heterocyclic amino acids
It is one kind of non-natural amino acid, other than most of function with natural amino acid, also there is non-natural amino acid institute not
The excellent performance having has been widely used in pharmaceutical synthesis (medicine and pesticide), food and feed etc. tool.But by
Special in the Nature comparison of heterocyclic amino acid, the prior art produces chiral alpha-non-natural amino acid by chemical method and is asked there are following
Topic：Generally require that chiral metal catalyst, synthetic route are long, largely cause using organic reagent that environmental pollution is serious, production
Of high cost and product optical purity is relatively inaccessible to require.There is presently no suitably closed by the bioconversion of biological enzyme
At the report of the amino acid of D- heterocycle structures.
Invention content
The main purpose of the present invention is to provide a kind of synthetic method, kit and the applications of D- heterocyclic amino acids, to carry
Selectivity is improved come the methods of biosynthesis D- heterocyclic amino acids using biological enzyme for a kind of.
To achieve the goals above, according to an aspect of the invention, there is provided a kind of synthesis side of D- heterocyclic amino acids
Method, the synthetic method include：Using SEQ ID NO:Diaminopimelate dehydrogenase shown in 1 is by the Mek-Tol Unit of D- heterocyclics
It closes object and is converted to D- heterocyclic amino acids.
Further, the Mek-Tol Unit compound of D- heterocyclics be selected from it is following any one：
D- heterocyclic amino acids be selected from it is following any one：
Further, above-mentioned synthetic method includes：The Mek-Tol Unit compound of D- heterocyclics is mixed with amino group donor, two
It is reacted under the action of diaminopimelic acid dehydrogenase and coenzyme, obtains D- heterocyclic amino acids.
Further, amino group donor is selected from ammonium formate or ammonium chloride.
Further, coenzyme is selected from β-NADP+Or β-NAD+。
Further, the pH of reaction is 7.8~9.0, and the temperature preferably reacted is 30~40 DEG C.
Further, reaction carries out in buffer solution, and buffer solution is selected from Tris-HCl salt buffers, triethanolamine buffers
Liquid, phosphate buffer or boric acid-sodium hydrate buffer solution.
Further, above-mentioned synthetic method includes：The Mek-Tol Unit compound of D- heterocyclics is mixed with amino group donor, two
It is reacted under the action of diaminopimelic acid dehydrogenase and coenzyme, obtains reaction product；And reaction product is post-processed, it obtains
D- heterocyclic amino acids；Preferably, the step of post-processing includes：Reaction terminating liquid is added into reaction product, obtains termination system；
Termination system is filtered, filtrate is obtained；And the purpose product in filtrate is purified, obtain D- heterocyclic amino acids；
It is further preferred that reaction terminating liquid is hydrochloric acid, the additive amount of hydrochloric acid so that subject to pH≤1 of the system of termination, the step of filtering in use
Celite pad.
To achieve the goals above, according to an aspect of the invention, there is provided a kind of examination of synthesis D- heterocyclic amino acids
Agent box, the kit include biological enzyme, and biological enzyme is SEQ ID NO:Diaminopimelate dehydrogenase shown in 1.
Further, kit further includes the Mek-Tol Unit compound of D- heterocyclics, preferably the Mek-Tol Unit chemical combination of D- heterocyclics
Object be it is following any one：
According to another aspect of the present invention, SEQ ID NO are provided:Diaminopimelate dehydrogenase shown in 1 is screening
Application in D- heterocyclic amino acid synthesis materials.
It applies the technical scheme of the present invention, using the diaminopimelate dehydrogenase of the application by the ketone acid of D- heterocyclics
It closes object and is converted into D- heterocyclic amino acids, higher conversion and the Non-natural chiral amino acid compared with high chiral purity can be obtained,
And the stable process conditions used in synthetic method are carried out using the diaminopimelate dehydrogenase, reaction condition is mild, entirely
It is easy to operate in production process, pollution it is relatively low, provide a kind of new idea and method for artificial synthesized D- heterocyclic amino acids.
Specific implementation mode
It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase
Mutually combination.Below in conjunction with embodiment, the present invention will be described in detail.
As background technology is previously mentioned, there are the defects such as yield is low for the synthesis of D- heterocyclic amino acids in the prior art, are
Improve this present situation, inventor studies the existing method using biological enzymatic synthesis D- heterocyclic amino acids, concurrently
Now the low reason of the synthesis yield of current D- heterocyclic amino acids is, is not directed to specific synthesis material and selects suitable diamino
Base pimelic acid dehydrogenase synthesizes corresponding D- heterocyclic amino acids.In order to find the D- heterocyclic amino acids synthesis material to match and
Diaminopimelate dehydrogenase, inventor have screened from existing numerous Mek-Tol Unit raw materials of compound and have had been commercialized on the market
Raw material or be easy prepare Mek-Tol Unit compound, and be directed to these raw materials, after having screened nearly 100 kinds of enzymes in enzyme library, hair
A kind of existing diaminopimelate dehydrogenase (SEQ ID NO from bacillus stearothermophilus:Sequence shown in 1：
MNKVHKIAVVGYGNIGKYAVQALNRAPDMELAGVVRRARSARDVPPELAGVPIATSIDELEGVEAAILATPTRTTPE
YASDILSKGIHTVDSYDIHGELADVRRKLDDIAKRHGSVAIVSAGWDPGTDSMIRSMLEFMAPGGVTYTNFGPGMSM
GHSVAVKAIDGVKDALSMTIPLGTGVHRRMVYVECEAGADFETVKEKVLADPYFVNDETHVIQVDDVQQLVDVGHGV
SMERKGVSGATHNQLFNFEMRINNPALTSQVLVAAARATFKQQPGAYTMIEVPIID FMYGDREELIKRLV) it is synthesizing
It can reach higher conversion ratio and selectivity on D- heterocyclylamino group acid.
Based on the studies above as a result, in a kind of typical embodiment of the application, a kind of D- heterocyclic amino acids are provided
Synthetic method, which includes：Using SEQ ID NO:Diaminopimelate dehydrogenase shown in 1 is by D- heterocyclics
Mek-Tol Unit compound is converted to D- heterocyclic amino acids.Using SEQ ID NO:Diaminopimelate dehydrogenase shown in 1 is synthesizing
It can reach higher conversion ratio and selectivity on D- heterocyclylamino group acid.
It is screened by the Mek-Tol Unit compound to existing a variety of D- heterocyclics, inventor has found to appoint when selection is following
It anticipates a kind of compound： When, carrying out synthesis with above-mentioned diaminopimelate dehydrogenase has chiral purity
High advantage.
Correspondingly, when using the Mek-Tol Unit compound of above-mentioned D- heterocyclics as raw material, the D- heterocyclic amino acids obtained select
From it is following any one：
Above-mentioned synthetic method can on the basis of the method for existing diaminopimelate dehydrogenase synthesizing amino acid, according to
Different rationally adjusted to synthesis condition progress of raw material and biological enzyme obtains.In order to further increase the chiral purity of product,
In a kind of preferred embodiment of the application, above-mentioned synthetic method includes：By the Mek-Tol Unit compound and amino group donor of D- heterocyclics
Mixing, reacts under the action of diaminopimelate dehydrogenase and coenzyme, obtains D- heterocyclic amino acids.
Amino group donor in above preferred embodiment can be existing common amino group donor, in order to make the conversion of substrate
Rate higher, in a kind of preferred embodiment of the application, above-mentioned amino group donor is selected from ammonium formate or ammonium chloride.Different substrates can
To select identical amino group donor that can also select different amino group donors, but in order to make the chiral purity higher of product, for
Different substrate raw materials can carry out above-mentioned synthetic reaction by the most suitable amino group donor of optimum choice.For example, when raw material is When, select ammonium formate to make
For amino group donor, reaction condition is enabled to be more suitable for, so that the chiral purity higher of product.And when raw material isOrWhen, selective chlorination ammonium enables to reaction condition more suitable as amino group donor
It closes, so that the conversion ratio higher of raw material.
In above-mentioned synthetic method, in order to improve the catalytic activity of biological enzyme, coenzyme is usually added to promote the progress of reaction.
In a kind of preferred embodiment of the application, above-mentioned coenzyme is selected from β-NADP+Or β-NAD+。
In above-mentioned synthetic method, the actual conditions of reaction can carry out reasonably optimizing adjustment according to substrate conversion efficiency.At this
Apply in a kind of preferred embodiment, the pH of above-mentioned reaction is 7.8~9.0, and the temperature preferably reacted is 30~40 DEG C.It is above-mentioned anti-
Not only the chiral purity of product is high but also relatively mild under the conditions of answering, convenient for control so that the synthetic method is more simple and easy to do.When
So, for different raw materials, most suitable pH conditions and reaction temperature also slightly have difference, can according to specific raw material into
Row rationally adjustment.Preferably, when raw material is When, the most suitable pH of reaction is 8.5-9.0.And when raw material isWhen, the most suitable pH of reaction is 7.8-8.2.
It, will be above-mentioned anti-in a kind of preferred embodiment of the application in order to make the product chiral purity higher of above-mentioned reaction
It should be carried out in buffer solution, buffer solution is selected from Tris-HCl salt buffers, Triethanolamine buffer, phosphate buffer or boron
Acid-sodium hydrate buffer solution.Different buffer solutions slightly have difference to the effect of optimization of different raw materials.For example, in Tris-HCl salt
In buffer solution,The reaction conversion ratio of raw material is relatively more
It is high.And in Triethanolamine buffer,The reaction conversion ratio of raw material is relatively higher.In phosphoric acid buffer
In liquid, especially sodium phosphate buffer,The reaction conversion ratio of raw material is relatively more
It is high.
In a kind of preferred embodiment of the application, above-mentioned synthetic method includes：By the Mek-Tol Unit compound of D- heterocyclics
It is mixed with amino group donor, is reacted under the action of diaminopimelate dehydrogenase and coenzyme, obtain reaction product；To reaction product
It is post-processed, obtains D- heterocyclic amino acids.The impurity in reaction product can be removed by post-processing, to obtain purity
Higher target product.
Above-mentioned post-processing is common post-processing operation in Amino acid synthesis step, including the operation of reaction terminating and right
The operation that product is purified.In a kind of preferred embodiment of the application, the step of above-mentioned post-processing, includes：To reaction product
Middle addition reaction terminating liquid, obtains termination system；Termination system is filtered, filtrate is obtained；And to the purpose in filtrate
Product is purified, and D- heterocyclic amino acids are obtained.
In a kind of preferred embodiment of the application, above-mentioned reaction terminating liquid is hydrochloric acid (preferably concentrated hydrochloric acid, the amount of addition
Few, the amount of being actually added into is subject to pH≤1), the step of filtering in use Celite pad.It can using the termination that hydrochloric acid is reacted
Fast implement the termination of reaction.And it is filtered with effective, at low cost advantage easy to operate using Celite pad.
In second of typical embodiment of the application, a kind of kit of synthesis D- heterocyclic amino acids is provided, it should
Kit includes biological enzyme, which is SEQ ID NO:Diaminopimelate dehydrogenase shown in 1.Using containing the diamino
The kit of base pimelic acid dehydrogenase carries out the synthesis of D- heterocyclic amino acids, has the advantages that high selectivity.
Further include the ketone compounds of D- heterocyclics in a kind of preferred embodiment, in mentioned reagent box, preferably D- is miscellaneous
The ketone compounds of ring class be it is following any one：
It, can when using the ketone compounds of above-mentioned several D- heterocyclics as substrate
The synthesis of D- heterocyclic amino acids is set to have the advantages that high income and high selectivity.
In the application in the third typical embodiment, SEQ ID NO are provided:Diaminopimelic acid shown in 1 is de-
Application of the hydrogen enzyme in screening D- heterocyclic amino acid synthesis materials.The application new, conversion ratio and/or specificity in capable of screening
Higher substrate, to be formed, type is more, the wider D- heterocyclic amino acids of application range.
Further illustrate the advantageous effect of the application below in conjunction with specific embodiments.
Embodiment 1
(1) it feeds intake：Into tetra- mouthfuls of round-bottomed bottles of 2L, 10g main materials are added250ml Tris-HCl fliud flushings
(100mmol/L, pH=9.0), raw material are dispersed in Tris-HCl salt buffers；PH to 8.5 is adjusted with 10M, NaOH, according to
The secondary ammonium formate that 16g is added, 0.3g β-NAD+, pH to 8.5 is adjusted with 10M, NaOH.Finally the diaminopimelic acid of 80g is taken off
Hydrogen enzyme, hydrogenlyase (the cycle H of 20g+) above-mentioned reaction system is added, and pH is adjusted to 8.5.
(2) it reacts：And 30 DEG C are warming up to, it is reacted.
(3) it post-processes：System is tracked after reaction 16h, detection raw material reaction finishes, is down to room temperature, delays into four-hole boiling flask
The slow concentrated hydrochloric acid that is added dropwise makes the pH value of mixed system after reaction to 1 to terminate reaction, and system crosses diatomite, filtrate 30%NaOH tune
PH to 8.0 is saved, is extracted 1 time, water phase 30%NaOH tune pH to 10.0 with 300ml MTBE, 22g tertbutyloxycarbonyls are added, is added dropwise
30%NaOH, control pH are 10.0, and temperature is that 16h is reacted at 25 DEG C, and 300ml MTBE are added and extract twice.Water phase 10wt%
Hydrochloric acid tune pH to 4.5 is extracted 3 times with 300ml MTBE.Organic phase is washed twice with 300ml saturated common salts, and water phase is abandoned after liquid separation
It, it is organic to be added to the drying of 10g anhydrous magnesium sulfates, it is filtered after dry, filter cake is rinsed with MTBE.Filtrate is contracted again does, and obtains
9.2g solid.
The nuclear magnetic data of obtained solid is as follows：1H NMR(400MHz,D2O)：δ 8.43 (d, 2H), 7.87 (d, 1H), 7.50
(t, 1H), 3.97 (t, 1H), 3.24 (dd, 2H).
It is detected through nuclear-magnetism, the content of internal standard (i.e. target product) is 98.5%, and through liquid chromatographic detection, chiral purity is
99.7%.
Embodiment 2
(1) it feeds intake：Into tetra- mouthfuls of round-bottomed bottles of 2L, 10g main materials are added250ml Tris-HCl punchings
Liquid (100mmol/L, pH=9.0), raw material are dispersed in Tris-HCl salt buffers；PH to 9.0 is adjusted with 10M, NaOH,
Sequentially add the ammonium formate of 16.2g, 0.3g β-NAD+, pH to 9.0 is adjusted with 10M, NaOH.Finally by diamino heptan of 40g two
Above-mentioned reaction system is added in the hydrogenlyase of acidohydrogenase, 20g, and adjusts pH to 9.0.
(2) it reacts：And 40 DEG C are warming up to, it is reacted.
(3) it post-processes：System is tracked after reaction 16h, detection raw material reaction finishes, and room temperature is down to, slowly to it under stirring
The middle concentrated hydrochloric acid that is added dropwise makes the pH value of mixed system after reaction to 1 or less；The Celite pad that system after tune acid is crossed to 1~2cm, is used
200ml purified water filter washes cake 2 times aqueous solution filters the mixing between obtained filtrate pH value is adjusted to 5 with sodium hydroxide
Solution obtains filter cake and filtrate, filter cake respectively with 300ml purifying washings twice after, then with 5 times of volume pure water agitator treatings 3 times, pumping
Product 7.4g is obtained after filter cake drying after filter.
The nuclear magnetic data of products obtained therefrom is as follows：1H NMR(400MHz,D2O)：δ 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).
It is detected through nuclear-magnetism, target content is 98.4% in nuclear-magnetism, through liquid chromatographic detection, chiral purity 98.5%.
Embodiment 3
(1) it feeds intake：Into tetra- mouthfuls of round-bottomed bottles of 2L, 10g main materials are added250ml triethanolamines
Buffer solution (100mmol/L, pH=9.0), raw material is dispersed in Triethanolamine buffer；With 10M, NaOH adjust pH to
8.5, sequentially add the ammonium formate of 7.4g, 0.3g β-NAD+, pH to 8.5 is adjusted with 10M, NaOH.Finally by diamino heptan of 20g
Above-mentioned reaction system is added in the hydrogenlyase of two acidohydrogenases, 10g, and adjusts pH to 8.5.
(2) it reacts：And 35 DEG C are warming up to, it is reacted.
(3) it post-processes：System is tracked after reaction 16h, detection raw material reaction finishes, is down to room temperature, delays into four-hole boiling flask
The slow concentrated hydrochloric acid that is added dropwise makes the pH value of mixed system after reaction to 1 to terminate reaction, and system crosses diatomite, obtained filtrate ice-water bath
It is lower to adjust pH value between 6 with solid sodium hydroxide, it is filtered after stirring 30min, obtains solid；Solid is washed with 10mL again, is dried
It is dry to obtain product 8.5g.
The nuclear magnetic data of products obtained therefrom is as follows：1H NMR(400MHz,D2O)：δ 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).
It is detected through nuclear-magnetism, interior target content is 95.0%, through liquid chromatographic detection, chiral purity 99.4%.
Embodiment 4
(1) it feeds intake：Into tetra- mouthfuls of round-bottomed bottles of 2L, 10g main materials are added250ml Tris-HCl punchings
Liquid (200mmol/L, pH=9.0), raw material are dispersed in Tris-HCl salt buffers；PH to 8.5 is adjusted with 10M, NaOH,
Sequentially add the ammonium formate of 9.8g, 0.3g β-NAD+, pH to 8.5 is adjusted with 10M, NaOH.Finally by diamino heptan of 100g two
Above-mentioned reaction system is added in the hydrogenlyase of acidohydrogenase, 10g, and adjusts pH to 8.5.
(2) it reacts：And 35 DEG C are warming up to, so that reaction system is reacted according to following chemical formula.
(3) it post-processes：System is tracked after reaction 16h, detection raw material reaction finishes, and room temperature is down to, slowly to it under stirring
The middle concentrated hydrochloric acid that is added dropwise makes the pH value of mixed system after reaction to 1 or less；The Celite pad that system after tune acid is crossed to 1~2cm, is used
Between obtained filtrate pH value is adjusted to 6 by sodium hydroxide, crude product aqueous solution is obtained.Crude product aqueous solution is crossed into model 001 × 7
Storng-acid cation exchange resin resin column purification, gained crude product are filtered after being washed with absolute ethyl alcohol, and filter cake is dried solid
Body product；The storng-acid cation exchange resin resin column purification that filtrate crosses model 001 × 7 obtains solid product 5.1g.
The nuclear magnetic data of products obtained therefrom is as follows：1H NMR(400MHz,D2O)：δ 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).
It is detected through nuclear-magnetism, target content is 98.2% in nuclear-magnetism, through liquid chromatographic detection, chiral purity 99.1%.
Embodiment 5
(1) it feeds intake：Into tetra- mouthfuls of round-bottomed bottles of 2L, 10g main materials are added250ml sodium phosphate fliud flushings
(200mmol/L, pH=8.0), raw material is dispersed in sodium phosphate buffer；PH to 7.8 is adjusted with 10M, NaOH, is added successively
Enter the ammonium chloride of 29g, the glucose of 29.3g, 0.33g β-NADP+, pH to 7.8 is adjusted with 10M, NaOH.Finally by the two of 50g
Above-mentioned reaction system is added in the glucose dehydrogenase of diaminopimelic acid dehydrogenase, 10g, and adjusts pH to 7.8.
(2) it reacts：And 35 DEG C are warming up to, so that reaction system is reacted according to following chemical formula.
(3) it post-processes：System is tracked after reaction 16h, detection raw material reaction finishes, and room temperature is down to, slowly to it under stirring
The middle concentrated hydrochloric acid that is added dropwise makes the pH value of mixed system after reaction to 1 or less；The Celite pad that system after tune acid is crossed to 1~2cm, is used
200ml purified water filter washes cake 2 times aqueous solution filters the mixing between obtained filtrate pH value is adjusted to 6 with sodium hydroxide
Solution obtains filter cake and filtrate, filter cake respectively with 300ml purifying washings twice after, then with 5 times of volume pure water agitator treatings 3 times, pumping
Product 8.2g is obtained after filter cake drying after filter.
The nuclear magnetic data of products obtained therefrom is as follows：1H NMR(400MHz,D2O)：δ 7.30 (d, 1H), 7.02 (t, 1H), 6.93
(s, 1H), 3.50 (t, 1H), 3.15 (d, 2H).
It is detected through nuclear-magnetism, interior target content is 98.0%, through liquid chromatographic detection, chiral purity 98.5%.
Embodiment 6
(1) it feeds intake：Into tetra- mouthfuls of round-bottomed bottles of 2L, 10g main materials are added250ml boric acid-hydroxide
Sodium fliud flushing (200mmol/L, pH=9.0), raw material is dispersed in sodium phosphate buffer；PH to 8.5 is adjusted with 10M, NaOH,
Sequentially add the ammonium chloride of 15g, the glucose of 15.5g, 0.33g β-NADP+, pH to 8.5 is adjusted with 10M, NaOH.Finally will
Above-mentioned reaction system is added in the glucose dehydrogenase of the diaminopimelate dehydrogenase of 60g, 20g, and adjusts pH to 8.5.
(2) it reacts：And 35 DEG C are warming up to, so that reaction system is reacted according to following chemical formula.
(3) it post-processes：System is tracked after reaction 16h, detection raw material reaction finishes, is down to room temperature, delays into four-hole boiling flask
The slow concentrated hydrochloric acid that is added dropwise makes the pH value of mixed system after reaction to 1 to terminate reaction, and system crosses diatomite, obtained filtrate ice-water bath
It is lower to adjust pH value between 6 with solid sodium hydroxide, it is filtered after stirring 30min, obtains solid；Solid is washed with 10mL again, is dried
It is dry to obtain 9.0g products.
The nuclear magnetic data of products obtained therefrom is as follows：1H NMR(400MHz,D2O)：δ 7.18,6.82 (2d, 2H, arom.-H),
3.78 (dd, 1H, H α), 3.23 (2dd, 2H, H β), 2.10 (s, 2HCH3)。
It is detected through nuclear-magnetism, interior target content is 99.0%, through liquid chromatographic detection, chiral purity 99.0%.
Comparative example 1
According to document " Transition-Metal-Assisted Asymmetric Synthesis of Amino Acid
Change described in Analogues.A NewSynthesis of Optically Pure D-and L-Pyridylalanines "
It learns synthetic method and synthesizes D- methyl 2- acetylaminohydroxyphenylarsonic acids 3- (4- pyridyl groups)-methyl propionate (product same as Example 1).It will
380mg methyl 2- acetylaminohydroxyphenylarsonic acids 3- (4- pyridyl groups)-methyl acrylate, 46mg (S, S)-[Rh (DIPAMP) (COD)]+
(BF4-) it is added in autoclave with 10ml methanol, leads to H2So that pressure is reached 65psi, tracks to after having reacted, column chromatography for separation
Go out product 349mg, is 92% by gas chromatographic detection chiral purity.
Comparative example 2
Using L-Leu dehydrogenase and hydrogenlyase corresponding amino acid is converted to come the raw material in Catalysis Examples 1-6
Reaction, tracked using liquid chromatographic detection, the results are shown in Table 2.
The chiral purity of the D- heterocyclic amino acids of embodiment 1 to 6 and comparative example 1 is obtained using liquid chromatographic detection, and
It is recorded in table 1.
Table 1：
The D- that embodiment 1 is obtained to 6 preparation method using the present invention of embodiment it can be seen from the data in table 1 is miscellaneous
The chiral purity of cyclic amino acids is all higher than 98%.Moreover, by the comparison of embodiment 1 and comparative example 1 it can be found that using the application
Diaminopimelate dehydrogenase is higher than chemically synthesized method to the selectivity of specific D- heterocyclic amino acids.
In addition, using the L-Leu dehydrogenase of comparative example 2 and formate dehydrogenase enzymatic original identical with Examples 1 to 6
Expect, to attempt to synthesize product identical with Examples 1 to 6, to track using liquid chromatographic detection, be as a result reported in Table 2 below.
Table 2：
Using the L-Leu dehydrogenase of comparative example 2 and formate dehydrogenase enzymatic and reality it can be seen from the data in table 2
1~6 identical raw material of example is applied, the chiral purity of synthetic product is respectively less than 10%.It can be seen that the application diaminopimelic acid is de-
Hydrogen enzyme has hardly possible when being catalyzed specified raw material synthesis D- heterocyclic amino acids relative to other existing diaminopimelate dehydrogenases
With the highly selective of expectation.
It can be seen from the above description that the above embodiments of the present invention realize following technique effect：The present invention's
Heterocycle ketone acid is converted into D- heterocyclic amino acids by diaminopimelate dehydrogenase, obtains higher conversion and the non-day of higher ee
Right chiral amino acid, the stable process conditions that synthetic method uses, reaction condition is mild, easy to operate in entire production process,
It pollutes relatively low, a kind of new idea and method is provided for artificial synthesized D- heterocyclic amino acids.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, any made by repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Sequence table
<110>Kai Laiying Pharmaceutical Groups（Tianjin）Limited liability company
<120>Synthetic method, kit and the application of D- heterocyclic amino acids
<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
1 5 10 15
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 (11)
1. a kind of synthetic method of D- heterocyclic amino acids, which is characterized in that the synthetic method includes：
Using SEQ ID NO:It is miscellaneous that the Mek-Tol Unit compound of D- heterocyclics is converted to D- by diaminopimelate dehydrogenase shown in 1
Cyclic amino acids.
2. synthetic method according to claim 1, which is characterized in that the Mek-Tol Unit compound of the D- heterocyclics is selected from such as
Descend any one：
The D- heterocyclic amino acids be selected from it is following any one：
3. synthetic method according to claim 1, which is characterized in that the synthetic method includes：
The Mek-Tol Unit compound of the D- heterocyclics is mixed with amino group donor, in the diaminopimelate dehydrogenase and coenzyme
Under the action of react, obtain the D- heterocyclic amino acids.
4. synthetic method according to claim 3, which is characterized in that the amino group donor is selected from ammonium formate or ammonium chloride.
5. synthetic method according to claim 3, which is characterized in that the coenzyme is selected from β-NADP+ or β-NAD+.
6. synthetic method according to claim 3, which is characterized in that the pH of the reaction is 7.8~9.0, preferably described
The temperature of reaction is 30~40 DEG C.
7. synthetic method according to claim 3, which is characterized in that the reaction carries out in buffer solution, the buffering
Liquid is selected from Tris-HCl salt buffers, Triethanolamine buffer, phosphate buffer or boric acid-sodium hydrate buffer solution.
8. the synthetic method according to any one of claim 3 to 7, which is characterized in that the synthetic method includes：
The Mek-Tol Unit compound of the D- heterocyclics is mixed with the amino group donor, in the diaminopimelate dehydrogenase and
It is reacted under the action of the coenzyme, obtains reaction product；
The reaction product is post-processed, the D- heterocyclic amino acids are obtained；
Preferably, the step of post-processing includes：
Reaction terminating liquid is added into the reaction product, obtains termination system；
The termination system is filtered, filtrate is obtained；And
Purpose product in the filtrate is purified, the D- heterocyclic amino acids are obtained；
It is further preferred that the reaction terminating liquid is hydrochloric acid, the additive amount of the hydrochloric acid so that subject to pH≤1 of the termination system,
Celite pad is used in the step of filtering.
9. a kind of kit of synthesis D- heterocyclic amino acids, the kit includes biological enzyme, which is characterized in that the biological enzyme
For SEQ ID NO:Diaminopimelate dehydrogenase shown in 1.
10. kit according to claim 9, which is characterized in that the kit further includes the Mek-Tol Unit of D- heterocyclics
Compound, preferably the Mek-Tol Unit compound of D- heterocyclics be it is following any one：
11.SEQ ID NO:Application of the diaminopimelate dehydrogenase shown in 1 in screening D- heterocyclic amino acid synthesis materials.
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| US20090117627A1 (en) * | 2007-09-07 | 2009-05-07 | Evonik Degussa Gmbh | Process for preparing enantiomerically enriched amines |
| CN103667380A (en) * | 2012-09-12 | 2014-03-26 | 天津工业生物技术研究所 | Novel method for synthesizing D-amino acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20090117627A1 (en) * | 2007-09-07 | 2009-05-07 | Evonik Degussa Gmbh | Process for preparing enantiomerically enriched amines |
| CN103667380A (en) * | 2012-09-12 | 2014-03-26 | 天津工业生物技术研究所 | Novel method for synthesizing D-amino acid |
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| CN108048317A (en) * | 2017-12-12 | 2018-05-18 | 凯莱英医药集团（天津）股份有限公司 | The continuous synthesis system and method for continuously synthesizing of a kind of alpha-non-natural amino acid |
| CN108048317B (en) * | 2017-12-12 | 2023-08-15 | 凯莱英医药集团（天津）股份有限公司 | Continuous synthesis system and continuous synthesis method of unnatural amino acid |
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