Application of transaminase in preparation of Sacubitril intermediate
阅读说明：本技术 转氨酶在制备Sacubitril中间体中的应用 (Application of transaminase in preparation of Sacubitril intermediate ) 是由 孙传民 田振华 于 2019-03-29 设计创作，主要内容包括：本发明提供了转氨酶在制备Sacubitril中间体中的应用,具体地,本发明公开了转氨酶可以用于Sacubitril(AHU-377)中间体的制备,在此基础上获得了一种反应条件温和、收率高、成本低的Sacubitril(AHU-377)中间体(R)-2-(N-叔丁氧羰基氨基)联苯丙醇的制备方法。(The invention provides application of transaminase in preparation of a Sacubitril intermediate, and particularly discloses a preparation method of a Sacubitril (AHU-377) intermediate (R) -2- (N-tert-butoxycarbonylamino) diphenylpropanol, wherein the preparation method has the advantages of mild reaction conditions, high yield and low cost, and the transaminase can be used for preparing the Sacubitril (AHU-377) intermediate.)
1. Application of transaminase in preparing (R) -2- (N-tert-butyloxycarbonylamino) biphenylpropanol is provided.
2. The use of claim 1, wherein said transaminase is selected from the group consisting of:
(A) a polypeptide having an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 2;
(B) a polypeptide having at least 80% homology (preferably at least 90% homology; more preferably at least 95% homology; most preferably at least 97% homology, e.g., at least 99% homology) with the amino acid sequence shown in any one of SEQ ID NO. 3 or SEQ ID NO. 2, and which has catalytic activity;
(C) a derivative polypeptide which is formed by substituting, deleting or adding 1-5 amino acid residues of an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 2 and retains the catalytic activity.
3. The use of claim 2, wherein the amino acid sequence of said transaminase is set forth in SEQ ID NO 3 or SEQ ID NO 2.
4. The use of claim 2, wherein said catalytic activity is that said transaminase catalyzes the reaction of compound 3 as a substrate to compound 2 according to the following formula:
5. a process for preparing (R) -2- (N-t-butoxycarbonylamino) biphenylpropanol comprising the steps of:
(1) preparing reaction system and carrying out enzyme catalysis reaction
The reaction system comprises a compound 3 as a substrate and transaminase; taking the compound 3 as a substrate, and obtaining a compound 2 under the action of transaminase;
the reaction formula is as follows:
6. the method of claim 5, wherein said transaminase is selected from the group consisting of:
(A) a polypeptide having an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 2;
(B) a polypeptide having at least 80% homology (preferably at least 90% homology; more preferably at least 95% homology; most preferably at least 97% homology, e.g., at least 99% homology) with the amino acid sequence shown in any one of SEQ ID NO. 3 or SEQ ID NO. 2, and which has catalytic activity;
(C) a derivative polypeptide which is formed by substituting, deleting or adding 1-5 amino acid residues of an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 2 and retains the catalytic activity.
7. The method of claim 6, wherein the amino acid sequence of said transaminase is set forth in SEQ ID NO 3 or SEQ ID NO 2.
8. The method according to claim 5, wherein in the step (1), the reaction system further comprises pyridoxal phosphate and isopropylamine.
9. The method of claim 5, further comprising step (2): reacting compound 2 with Boc2And (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol is prepared by the O reaction:
10. a method of preparing secubitril (AHU-377), comprising the steps of:
(1) preparing (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol according to the process of claim 5;
(2) and (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol prepared in the step (1) is used as an intermediate to react to obtain Sacubitril (AHU-377).
Technical Field
The invention belongs to the field of biological enzyme catalysis, and particularly relates to application of transaminase in preparation of Sacubitril (AHU-377) intermediate (R) -2- (N-tert-butoxycarbonylamino) diphenylpropanol.
Background
LCZ696 is a new antihypertensive drug developed by Nowa company, Sacubitril (AHU-377) and Valsartan form LCZ696 together, Sacubitril has an action mechanism of blocking 2 polypeptides threatening to lower blood pressure, Valsartan can improve vasodilation and stimulate the body to excrete sodium and water, and the two combine to play pharmacological actions, so that the drug has double inhibition of angiotensin II receptor and enkephalinase, shows a unique action mode clinically, is considered to be capable of reducing strain of failing heart, is superior to antihypertensive action of standard drugs, and obtains rapid channel evaluation qualification of American FDA and European Union EMEA. It is generally accepted that LCA696 will bring innovation to the traditional treatment of heart failure.
The structural formula of Sacubitril (AHU-377) is as follows:
WO2008031567B1 reports a preparation method of Sacubitril, which is characterized in that (R) -2- (N-tert-butoxycarbonylamino) biphenyl propanol is used as a raw material, is oxidized into aldehyde by TEMPO, and is synthesized into the Sacubitril by Witting reaction, hydrolysis, asymmetric hydrogenation, deprotection, esterification and amidation, and the specific reaction steps are as follows:
wherein, (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol is a key raw material, so that the preparation method of (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol is important for the production of Sacubitril.
The preparation method of (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol mainly relates to the construction of chiral center, and the main method is a method using chiral compound as raw material, such as J.Med.chem.,1995,38,1689-1700, using D-tyrosine as raw material, and generating D- (N-tert-butoxycarbonylamino) biphenylalanine methyl ester through amino protection, carboxylic acid esterification, hydroxy trifluoromethanesulfonic acid activation and Suzuki coupling reaction, wherein the specific reaction steps are shown as the following formula. The (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol can be prepared by reducing D- (N-tert-butoxycarbonyl) biphenylamino methyl ester
In addition, there is also a method for constructing a chiral center by asymmetric hydrogenation, for example, WO2011035569a1, CN105330569A reports that biphenylcarboxaldehyde is used as a raw material, and cyclizes with N-t-butoxycarbonylglycine in the presence of acid anhydride and alkali to generate an oxazolone compound, the oxazolone compound is hydrolyzed to obtain N-t-butoxycarbonyl bisanilic acid or ester, and the N-t-butoxycarbonyl bisanilic acid or ester is further asymmetrically hydrogenated and reduced under a metal catalyst to obtain (R) -2- (N-t-butoxycarbonylamino) biphenylpropanol. However, the method needs expensive metal catalysts, has high cost and serious heavy metal pollution, and is not suitable for industrial mass production.
Therefore, a method for preparing (R) -2- (N-tert-butoxycarbonylamino) diphenylpropanol with low production cost, environmental friendliness and high stereoselectivity is needed to be found.
Disclosure of Invention
The invention aims to provide a preparation method of Sacubitril (AHU-377) intermediate (R) -2- (N-tert-butoxycarbonylamino) diphenylpropanol, which has the advantages of higher yield, environmental friendliness, low production cost and higher chiral purity.
In a first aspect of the invention, there is provided the use of a transaminase for the preparation of (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol.
In another preferred embodiment, the transaminase is selected from the group consisting of:
(A) a polypeptide having an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 2;
(B) a polypeptide having at least 80% homology (preferably at least 90% homology; more preferably at least 95% homology; most preferably at least 97% homology, e.g., at least 99% homology) with the amino acid sequence shown in any one of SEQ ID NO. 3 or SEQ ID NO. 2, and which has catalytic activity;
(C) a derivative polypeptide which is formed by substituting, deleting or adding 1-5 amino acid residues of an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 2 and retains the catalytic activity.
In another preferred embodiment, the amino acid sequence of said transaminase is shown in SEQ ID NO 3, or SEQ ID NO 2.
In another preferred embodiment, the amino acid sequence of said transaminase is set forth in SEQ ID NO 3.
In another preferred embodiment, the catalytic activity means that the transaminase catalyzes the reaction of compound 3 as a substrate to compound 2, which has the following formula:
in a second aspect of the present invention, there is provided a process for preparing (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol, comprising the steps of:
(1) preparing reaction system and carrying out enzyme catalysis reaction
The reaction system comprises a compound 3 as a substrate and transaminase; taking the compound 3 as a substrate, and obtaining a compound 2 under the action of transaminase;
the reaction formula is as follows:
in another preferred embodiment, the transaminase is selected from the group consisting of:
(A) a polypeptide having an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 2;
(B) a polypeptide having at least 80% homology (preferably at least 90% homology; more preferably at least 95% homology; most preferably at least 97% homology, e.g., at least 99% homology) with the amino acid sequence shown in any one of SEQ ID NO. 3 or SEQ ID NO. 2, and which has catalytic activity;
(C) a derivative polypeptide which is formed by substituting, deleting or adding 1-5 amino acid residues of an amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 2 and retains the catalytic activity.
In another preferred embodiment, the amino acid sequence of said transaminase is shown in SEQ ID NO 3, or SEQ ID NO 2.
In another preferred embodiment, the amino acid sequence of said transaminase is set forth in SEQ ID NO 3.
In another preferred example, in the step (1), the reaction system further comprises pyridoxal phosphate and isopropylamine.
In another preferred example, in the step (1), the compound 3 is dissolved in toluene, and then transaminase liquid, pyridoxal phosphate and isopropylamine are added for reaction.
In another preferred embodiment, in the step (1), the pH of the reaction system is controlled to be 7.0 to 10.0, preferably 8.5 to 9.0.
In another preferred example, the method further comprises the step (2): reacting compound 2 with Boc2And (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol is prepared by the O reaction:
in another preferred embodiment, in the step (1), the temperature of the enzyme-catalyzed reaction is 20-40 ℃.
In a third aspect of the present invention, there is provided a method of preparing secubitril (AHU-377) comprising the steps of:
(1) according to the process of the second aspect of the present invention, (R) -2- (N-t-butoxycarbonylamino) biphenylpropanol is obtained;
(2) and (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol prepared in the step (1) is used as an intermediate to react to obtain Sacubitril (AHU-377).
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Detailed Description
The inventors of the present invention have made extensive and intensive studies and have unexpectedly found that transaminase can be used for producing (R) -2- (N-t-butoxycarbonylamino) biphenylpropanol, and have obtained a method for producing (R) -2- (N-t-butoxycarbonylamino) biphenylpropanol which is mild in reaction conditions, high in yield and low in cost on the basis of the above.
The prior preparation method of (R) -2- (N-tert-butoxycarbonylamino) diphenylpropanol has the defects of low yield, serious environmental hazard and the like, so the invention provides the application of transaminase in the preparation of Sacubitril (AHU-377) intermediate (R) -2- (N-tert-butoxycarbonylamino) diphenylpropanol. The (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol obtained by the method has high chiral purity, is environment-friendly, has low production cost and is beneficial to industrial production.
Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.
Transaminase enzymes
The invention provides application of transaminase in preparation of an intermediate (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol (compound 1) of Sacubitril (AHU-377).
In a preferred embodiment of the present invention, compound 3 and isopropylamine are reacted to compound 2, under transaminase catalysis:
then compound 2 and Boc2And (3) performing O reaction to obtain a target compound 1:
in a preferred embodiment of the invention, the transaminase is selected from the group consisting of: enzyme 3 shown in SEQ ID No. 3, enzyme 2 shown in SEQ ID No. 2, and enzyme 1 shown in SEQ ID No. 1.
The amino acid sequence of enzyme 1 is as follows:
the amino acid sequence of enzyme 2 is as follows:
the amino acid sequence of enzyme 3 is as follows:
the enzyme gene sequences involved in the present invention can be obtained by conventional methods, such as total artificial synthesis or PCR synthesis, which can be used by those skilled in the art. One preferred synthesis method is the asymmetric PCR method. The asymmetric PCR method uses a pair of primers with different amounts to generate a large amount of single-stranded DNA (ssDNA) after PCR amplification. The pair of primers are referred to as non-limiting and limiting primers, respectively, and are typically in a ratio of 50-100: 1. In the first 10-15 cycles of the PCR reaction, the amplification product is mainly double-stranded DNA, but when the restriction primers (low concentration primers) are consumed, PCR using non-restriction primers (high concentration primers) will produce a large amount of single-stranded DNA. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.
The enzyme of the invention may be expressed or produced by conventional recombinant DNA techniques, comprising the steps of:
(1) transforming or transducing a suitable host cell with a polynucleotide encoding a protein of the invention, or with a recombinant expression vector containing the polynucleotide;
(2) culturing the host cell in a suitable medium;
(3) separating and purifying the target protein from the culture medium or the cells to obtain the target enzyme.
Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences encoding the relevant enzymes of the invention and suitable transcription/translation control signals, preferably commercially available vectors: pET28 a. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In addition, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.
The recombinant vector comprises in the 5 'to 3' direction: a promoter, a gene of interest, and a terminator. If desired, the recombinant vector may further comprise the following elements: a protein purification tag; a 3' polyadenylation signal; an untranslated nucleic acid sequence; transport and targeting nucleic acid sequences; selection markers (antibiotic resistance genes, fluorescent proteins, etc.); an enhancer; or operator.
Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. The expression vector may be a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In general, any plasmid and vector may be used as long as it can replicate and is stable in the host.
One of ordinary skill in the art can construct vectors containing the promoter and/or gene sequence of interest of the present invention using well known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
The expression vector of the present invention can be used for transformationSuitable host cells are those that allow the host to transcribe the RNA of interest or to express the protein of interest. The host cell may be a prokaryotic cell, such as E.coli, C.glutamicum, Brevibacterium flavum, Streptomyces, Agrobacterium: or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells. It will be clear to one of ordinary skill in the art how to select an appropriate vector and host cell. Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote (e.g., Escherichia coli), CaCl may be used2The treatment can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods (e.g., microinjection, electroporation, liposome encapsulation, etc.). The transformed plant may be transformed by methods such as Agrobacterium transformation or biolistic transformation, for example, leaf disc method, immature embryo transformation, flower bud soaking method, etc. The transformed plant cells, tissues or organs can be regenerated into plants by conventional methods to obtain transgenic plants.
The term "operably linked" means that the gene of interest to be expressed transcriptionally is linked to its control sequences in a manner conventional in the art to be expressed.
Culture of engineering bacteria and fermentation production of target protein
After obtaining the engineered cells, the engineered cells can be cultured under suitable conditions to express the protein encoded by the gene sequence of the invention. The medium used in the culture may be selected from various conventional media, depending on the host cell, and the culture is carried out under conditions suitable for the growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.
In the present invention, conventional fermentation conditions may be employed. Representative conditions include (but are not limited to):
(a) the fermentation and induction temperature of the enzyme is kept at 25-37 ℃ in terms of temperature;
(b) the pH value of the induction phase is controlled to be 3-9;
(c) in the case of Dissolved Oxygen (DO), DO is controlled at 10-90%, and the maintenance of dissolved oxygen can be solved by introducing oxygen/air mixed gas;
(d) for feeding, the feeding type preferably comprises carbon sources such as glycerol, methanol, glucose and the like, and the feeding can be carried out independently or in a mixed manner;
(e) as for the IPTG concentration during induction, conventional induction concentrations can be used in the present invention, and usually the IPTG concentration is controlled at 0.1-1.5 mM;
(f) the induction time is not particularly limited, and is usually 2 to 20 hours, preferably 5 to 15 hours.
The target enzyme of the present invention exists in Escherichia coli cells, the host cells are collected by a centrifuge, and then the host cells are disrupted by high pressure, mechanical force, enzymatic cell disruption or other cell disruption methods, and the recombinant protein is released, preferably by high pressure method. The host cell lysate can be primarily purified by flocculation, salting out, ultrafiltration, etc., and then purified by chromatography, ultrafiltration, etc., or directly purified by chromatography.
The chromatography includes cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, and affinity chromatography. Common chromatographic methods include:
1. anion exchange chromatography:
anion exchange chromatography media include (but are not limited to): Q-Sepharose, DEAE-Sepharose. If the salt concentration of the fermentation sample is higher, affecting binding to the ion exchange medium, the salt concentration needs to be reduced before ion exchange chromatography is performed. The sample can be replaced by means of dilution, ultrafiltration, dialysis, gel filtration chromatography and the like until the sample is similar to a corresponding ion exchange column equilibrium liquid system, and then the sample is loaded and subjected to gradient elution of salt concentration or pH.
2. Hydrophobic chromatography:
hydrophobic chromatographic media include (but are not limited to): Phenyl-Sepharose, Butyl-Sepharose, octyl-Sepharose. Samples were prepared by adding NaCl, (NH)4)2SO4And increasing the salt concentration, loading, and eluting by decreasing the salt concentration. Removing by hydrophobic chromatographyHetero-proteins with large differences in hydrophobicity.
3. Gel filtration chromatography
Hydrophobic chromatographic media include (but are not limited to): sephacryl, Superdex, Sephadex types. The buffer system is replaced by gel filtration chromatography or further purified.
4. Affinity chromatography
Affinity chromatography media include (but are not limited to): HiTrapTMHeparinHPColumns。
5. Membrane filtration
The ultrafiltration medium comprises: organic membranes such as polysulfone membranes, inorganic membranes such as ceramic membranes, metal membranes. The purification and concentration can be achieved by membrane filtration.
The main advantages of the invention are:
(1) the application of the transaminase in the preparation of (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol is provided for the first time, and a good transaminase with high conversion rate and ee value is obtained through a large number of experiments and screens;
(2) provides a method for preparing (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol, which uses transaminase for catalysis and has mild reaction conditions.
(3) The transaminase with higher activity is screened to be used for preparing (R) -2- (N-tert-butoxycarbonylamino) biphenylpropanol, and experimental results show that the ee value of the obtained target product can reach 99 percent, and the conversion rate can reach about 98 percent.
The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：一种D-泛解酸内酯的生产方法