Design and construction method and application of artificial enzyme containing non-natural tertiary amine catalytic center

Abstract

The invention discloses a design and construction method and application of an artificial enzyme containing a non-natural tertiary amine catalytic center. LmrR protein is taken as a starting material, a tertiary amine cofactor with catalytic activity is introduced into the 89 site of LmrR through covalent linkage, and after the protein is purified, the novel artificial enzyme LmrRM89CTA is successfully constructed. The constructed novel artificial enzyme LmrRM89CTA containing the tertiary amine catalytic center can catalyze a non-natural asymmetric [3 + 2] cycloaddition reaction, and a target chiral spiro product is obtained with the conversion rate of 10% and the enantioselectivity of 84%.

Description

Technical Field

[0001] The invention relates to a design and construction method of an artificial enzyme containing a non-natural tertiary amine catalytic center and application thereof, belonging to the technical field of biocatalysis. Background Art

[0002] In recent years, biocatalytic synthesis technology has been widely used in many fields due to its significant advantages. As a biocatalyst, enzymes are particularly suitable for the development of green chemistry due to their high catalytic efficiency and mild reaction conditions. Compared with traditional chemical methods, enzyme-catalyzed reactions are not only more economical, but also highly specific and selective, which makes them play an increasingly important role in the pharmaceutical, textile, chemical and other industries. Through biosynthesis, various functional molecules can be produced more efficiently and environmentally friendly, promoting the sustainable development of human society. However, enzymes in nature are composed of 20 natural amino acid residues and a limited number of cofactors. The limited number of functional groups in these amino acid residues and cofactors limits the diversity of enzyme-catalyzed reactions. Therefore, by introducing non-natural small molecule catalysts (cofactors) into the protein skeleton, artificial enzymes with higher catalytic activity or new reactivity can be obtained.

[0003] Through the research in recent years, the construction methods of artificial enzymes can be summarized into the following three types: non-natural amino acid insertion, supramolecular assembly technology, and covalent connection. These different construction methods can introduce a large number of non-natural cofactors into different protein skeletons to expand the application of biocatalysis in organic synthesis.

[0004] Organic tertiary amine catalysts TA have been widely used in organic synthesis in recent years and can catalyze various types of addition and cycloaddition reactions. At present, organic tertiary amine catalysts TA have been widely used in the synthesis of fine chemicals, medicines, pesticides, dyes, fragrances and other products. Organic catalysis, as the main tool for asymmetric chemical transformation, has now matured and can use the bioorthogonality of organic catalysis for important chemical and synthetic biology applications.

[0005] In 2010, Chen and colleagues pioneered the use of isatin as an electrophilic reagent for asymmetric allylic alkylation to synthesize MBH carbonates. Since then, isatin-derived MBH carbonates have become particularly useful C3 synthons in the enantioselective cyclization of enantioenriched spiroindoles, including [3+2] cyclization, [3+3] and [4+3] cyclization to synthesize a variety of potentially bioactive chiral spirocyclic compounds. As an effective small molecule catalyst, organic tertiary amine catalysts TA are used in such chemical reactions due to their excellent chemical functions. However, the development and synthesis of chiral organic tertiary amine catalysts TA are difficult, the conditions for chemical catalytic reactions are severe, and the amount of catalyst required is high, which seriously limits their application in asymmetric synthesis. Therefore, based on our previous research on the design of new functional enzyme elements, we sought to design and construct a new artificial enzyme containing a tertiary amine catalytic center to catalyze a variety of asymmetric non-natural cycloaddition reactions, which will effectively expand the diversity and application range of enzymes.

[0006] Lactococcal multidrug resistance regulatory protein (LmrR) is a homodimeric protein with large hydrophobicity and promiscuous pockets at its dimer interface, making it a good carrier for the construction of artificial enzymes. Previously, the Roelfes team at the University of Groningen in the Netherlands had introduced a variety of catalytically active groups into the hydrophobic pocket of LmrR through genetic codon expansion technology and π-π stacking, and achieved non-natural asymmetric catalytic reactions such as cyclopropanation and Friedel-Crafts alkylation. These studies have shown that LmrR has great application potential in the construction of artificial enzymes. However, to date, there have been no reports of using LmrR as a protein skeleton to construct a new type of artificial enzyme containing a tertiary amine catalytic center. Summary of the invention

[0007] To address the deficiencies of the prior art, the present invention prepares a method for designing and constructing an artificial enzyme containing a non-natural tertiary amine catalytic center and its application for catalyzing a non-natural asymmetric [3+2] cycloaddition reaction.

[0008] The present invention is achieved through the following technical solutions: The first object of the present invention is to provide an artificial enzyme containing a non-natural tertiary amine catalytic center, wherein the artificial enzyme is obtained by introducing a cysteine ​​mutation into the hydrophobic cavity of the lactococcal multidrug resistance regulatory protein and connecting a cofactor containing a tertiary amine catalytic group to the cysteine.

[0009] In one embodiment of the present invention, the structural formula of the cofactor is: .

[0010] In one embodiment of the present invention, the amino acid sequence of the lactococcal multidrug resistance regulatory protein is shown as SEQ ID NO.1.

[0011] In one embodiment of the present invention, the cysteine ​​mutation is to mutate the 89th methionine of the lactococcal multidrug resistance regulatory protein with an amino acid sequence as shown in SEQ ID NO.1 to cysteine.

[0012] In the present invention, this cofactor acts as an enzyme catalytic center and can catalyze an asymmetric [3+2] cycloaddition reaction and achieve a certain conversion rate and stereoselectivity.

[0013] A second object of the present invention is to provide a method for preparing the artificial enzyme, comprising the steps of: (1) reacting mercapto tetrahydropyrrole with 2,2'-dithiodipyridine under protective gas conditions, and then reacting the obtained product with 4-chloropyridine to obtain a cofactor; (2) performing site-directed mutagenesis on the 89th amino acid in the hydrophobic cavity of the lactococcal multidrug resistance regulatory protein whose amino acid sequence is shown in SEQ ID NO.1, thereby mutating methionine to cysteine; (3) Covalently linking the cofactor prepared in step (1) to the mutated lactococcal multidrug resistance regulatory protein in step (1) to obtain the artificial enzyme.

[0014] In one embodiment of the present invention, the covalent linking in step (3) is performed by adding the cofactor to the purified protein and reacting at 0-8°C for 20-30h.

[0015] In one embodiment of the present invention, the reaction of mercaptopyrrole and 2,2'-dithiodipyridine is carried out under argon protection for 0.5 to 1 h.

[0016] In one embodiment of the present invention, the reaction of thiopyrrole tetrahydropyrrole and 2,2'-dithiodipyridine is carried out at 20-30°C.

[0017] In one embodiment of the present invention, the reaction product of mercaptopyrrole and 2,2'-disulfide dipyridine and 4-chloropyridine is reacted at 70-90° C. for 10-15 hours.

[0018] The third object of the present invention is to provide the use of the artificial enzyme in catalyzing asymmetric [3+2] cycloaddition reaction.

[0019] In one embodiment of the present invention, the artificial enzyme can catalyze the following reaction: .

[0020] In one embodiment of the present invention, the catalytic reaction conditions are 25-350 C for 40-60h.

[0021] Beneficial effects of the present invention: Starting from the LmrR protein, the present application introduces a tertiary amine cofactor with catalytic activity into the 89th position of LmrR through covalent linkage. After purifying the protein, a new artificial enzyme LmrR_M89C_TA is successfully constructed. The new artificial enzyme LmrR_M89C_TA containing a tertiary amine catalytic center constructed by the present invention can catalyze a non-natural asymmetric [3+2] cycloaddition reaction and obtain the target chiral spirocyclic product with a conversion rate of 10% and an enantioselectivity of 84%. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 C-H spectra of the synthesized cofactor precursor compounds; Figure 2 is the reaction formula for the covalent linkage between the LmrR protein and the cofactor precursor compound; Figure 3 The protein spectrum of the artificial enzyme LmrR_M89C_TA; Figure 4 This is the chiral HPLC spectrum of the asymmetric [3+2] cycloaddition reaction catalyzed by the artificial enzyme LmrR_M89C_TA. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with specific examples. These implementation cases are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the claims attached to the application.

[0025] Culture medium and reagents: LB medium: 1% peptone, 1% sodium chloride and 0.5% yeast extract, pH about 7.0, sterilized at 121°C for 20 min, for the culture of E. coli. Add 20 g / L agar powder to prepare LB solid medium.

[0026] Washing buffer: 50 mM disodium hydrogen phosphate, 150 mM sodium chloride, pH = 8.0. The number of washes and collections in the protein purification step is represented by W1-W3.

[0027] Elution buffer: 50 mM disodium hydrogen phosphate, 150 mM sodium chloride, 5 mM desthiobiotin, pH = 8.0. The elution collection times in the protein purification step are represented by E1-E6.

[0028] Example 1: Synthesis of artificial cofactor precursor compounds Add 203 mg (1 mmol) of Boc-protected thiotetrahydropyrrole to 10 mL of TFA / DCM mixed solvent, stir at 0°C for 2 hours and vacuum dry. Add 10 mmol of 2,2'-disulfide dipyridine to the product and react at room temperature for 0.5 h under argon protection. After the reaction is completed, dry the mixture and purify it by column chromatography. After adding 4-chloropyridine to the purified product and reacting at 80°C for 12 hours, vacuum evaporate the reaction solution and purify it by column chromatography to obtain cofactor precursor 1. The reaction formula of the synthesis steps is shown below. The carbon-hydrogen spectrum of the cofactor precursor compound 1 is shown below. Figure 1 shown.

[0029]

[0030]

[0031] Example 2: Site-directed mutagenesis and purification and expression of LmrR protein Based on the crystal structure of LmrR protein, we selected 10 sites in the LmrR cavity, namely E7C, M8C, N14C, V15C, D60C, M89C, F93C, V99C, I103C, and E107C, and performed PCR site-directed mutagenesis using LmrR as template. PCR conditions: (1) 98 0 Initial denaturation at 98 °C for 2 min, (2) 25 cycles, 0 Denature at 55-65 °C for 10 s. 0 30 s at 72 °C (depending on the Tm of the primer) and incubate at 72 0 C for 2 min; (3) 72 0 The final extension was performed at 37 0 The cells were digested with DpnI for 1 h at 37 °C and then transformed into competent E. coli DH5-α cells. Single clones were picked from LB solid plates containing ampicillin (100 μg / mL) and inoculated into 3 mL LB medium containing the same concentration of ampicillin. The culture medium was incubated at 37 0C, 220 rpm overnight culture, then extract plasmids for Sanger sequencing of mutants. For protein expression, the extracted plasmids were transformed into E. coli BL21, and a single colony was used to inoculate overnight culture for protein expression and purification.

[0032] The recombinant bacteria were inoculated into 3 mL LB liquid medium, cultured at 37°C, 220 r / min for 14 h, and then 2.5 mL was inoculated into 200 mL fresh sterilized LB medium, cultured at 37°C, 220 r / min for 3-5 h, and when the OD600 reached 0.6-0.8, IPTG inducer was added to a final concentration of 1 mmol / L, and induced culture was carried out at 20°C, 160 r / min for 20 h. The bacterial liquid was collected, centrifuged at 4°C, 6 000 r / min for 20 min to collect the bacterial precipitate, resuspended in 10 mL PBS (pH=8.0), and then ultrasonically disrupted, with the disruption parameters of power 175 W, on 3 s, off 6 s, and 20 min. After disruption, DNA enzyme was added and incubated on ice for 30 min. Centrifuged at 4°C, 12,000 r / min for 40 min. The supernatant was filtered with a 0.45 μm filter membrane and loaded into a Streptactin column and incubated at 4°C, 20 r / min for 1 h. The column was removed, the flow was collected, and the protein was washed with 2 column volumes of washing buffer, repeated 3 times, and the washing solution (W1-W3) was collected. The protein was eluted with 1 column volume of elution buffer, repeated 6 times, and the eluate (E1-E6) was collected. The obtained proteins were LmrR_E7C, LmrR_M8C, LmrR_N14C, LmrR_V15C, LmrR_D60C, LmrR_M89C, LmrR_F93C, LmrR_V99C, LmrR_I103C and LmrR_E107C. The fractionated collection solution in the protein purification process was analyzed by SDS-PAGE, and finally E2-E5 was ultrafiltered and concentrated. The concentrated protein was dialyzed in PBS (pH=6.5) and stored for later use.

[0033] LmrR protein amino acid sequence SEQ ID NO.1: GAEIPKEMLRAQTNVILLNVLKQGDNYVYGIIKQVKEASNGEMELNEATLYTIFDRLEQD GIISSYWGDESQGGRRKYYRLTEIGHENMRLAFESWSRVDKIIENLEANKKSEAIKSRGG SGGWSHPQFEK Example 3: Covalent Linking of LmrR Protein to Cofactors After purification, 10 times the equivalent of small molecule cofactor precursor compound (DMF solution) was added to the protein, and the reaction was carried out at 4°C for 24 hours for covalent linking. The reaction solution was ultrafiltered three times using an ultrafiltration tube and the buffer was replaced with PBS at pH = 7.4, and then stored at 4°C for subsequent catalysis. The reaction formula for the connection is as follows: Figure 2 The constructed artificial enzymes are LmrR_E7C_ TA, LmrR_M8C_ TA, LmrR_N14C_ TA, LmrR_V15C_ TA, LmrR_D60C_ TA, LmrR_M89C_ TA, LmrR_F93C_ TA, LmrR_V99C_ TA, LmrR_I103C_ TA and LmrR_E107C_ TA.

[0034] Example 4: Catalytic reaction In a 2 mL centrifuge tube, add PBS buffer (50 mM NaCl, 150 mM NaHCO) at a total volume of 300 μL. 2 PO 4 ) Dilute the protein solution of the artificial enzyme to 275 μL, with a final concentration of 30 μM. Add 10 μL of the stock solution of reactant 2 (prepare a 30 mM DMF solution, and add 10 μL, with a final concentration of 1 mM) and reactant 3 (prepare an 80 mM DMF solution, and add 15 μL, with a final concentration of 4 mM). 0 C. After 48 hours of reaction, ethyl acetate was added for extraction, the extract was dried over anhydrous sodium sulfate and filtered, and the filtrate was spin-dried. The concentrated product 4 was dissolved in 200 μL of chromatographic grade solvent isopropanol, and the conversion analysis and enantioselectivity were detected by HPLC. The reaction formula of the catalytic reaction is shown below.

[0035]

[0036] The catalytic reaction data of different enzyme mutants are shown in Table 1: The new artificial enzyme LmrR_M89C_TA can catalyze a non-natural asymmetric [3+2] cycloaddition reaction and obtain the target chiral spirocyclic product 4 with a conversion rate of 10% and an enantioselectivity of 84%.

[0037] Enzyme catalytic reaction conversion rate (%) = amount of reacted raw material 1 / amount of original raw material 1 * 100% Table 1 Effects of different artificial enzyme mutants on catalytic reactions

[0038] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. An artificial enzyme containing a non-natural tertiary amine catalytic center, characterized in that: The artificial enzyme is obtained by introducing cysteine ​​mutation into the hydrophobic cavity of the lactococcal multidrug resistance regulatory protein and connecting a cofactor containing a tertiary amine catalytic group through cysteine.

2. The artificial enzyme according to claim 1, characterized in that The structural formula of the cofactor is: .

3. The artificial enzyme according to any one of claims 1 or 2, characterized in that The amino acid sequence of the lactococcal multidrug resistance regulatory protein is shown in SEQ ID NO.

1.

4. The artificial enzyme according to claim 3, characterized in that The cysteine ​​mutation is to mutate the 89th methionine of the lactococcal multidrug resistance regulatory protein with an amino acid sequence as shown in SEQ ID NO.1 to cysteine.

5. A method for preparing the artificial enzyme according to any one of claims 1 to 4, characterized in that: The steps include: (1) reacting mercapto tetrahydropyrrole with 2,2'-dithiodipyridine under protective gas conditions, and then reacting the obtained product with 4-chloropyridine to obtain a cofactor; (2) performing site-directed mutagenesis on the 89th amino acid in the hydrophobic cavity of the lactococcal multidrug resistance regulatory protein whose amino acid sequence is shown in SEQ ID NO.1, thereby mutating methionine to cysteine; (3) Covalently linking the cofactor prepared in step (1) to the mutated lactococcal multidrug resistance regulatory protein in step (1) to obtain the artificial enzyme.

6. The preparation method according to claim 5, characterized in that: In step (3), the covalent linking step is to add the cofactor to the purified protein and react at 0-8°C for 20-30h.

7. The preparation method according to claim 5, characterized in that The reaction of mercaptotetrahydropyrrole with 2,2'-dithiodipyridine is carried out under argon protection for 0.5 to 1 h.

8. The preparation method according to claim 5, characterized in that: The reaction product of mercaptopyrrole and 2,2'-dithiodipyridine is reacted with 4-chloropyridine at 70-90° C. for 10-15 hours.

9. Use of the artificial enzyme according to any one of claims 1 to 4 in catalyzing an asymmetric [3+2] cycloaddition reaction.

10. The use according to claim 9, characterized in that: The artificial enzyme can catalyze the following reaction: 。

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