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

Abstract

The application discloses a design construction method of an artificial enzyme containing a non-natural tertiary amine catalytic center and application thereof, and is characterized in that the application introduces a tertiary amine cofactor with catalytic activity into the 89th position of LmrR by covalent linkage, and successfully constructs a new artificial enzyme LmrR_M89C_TA after protein purification. The artificial enzyme LmrR_M89C_TA containing the tertiary amine catalytic center constructed by the application can catalyze a non-natural asymmetric [3+2] cycloaddition reaction, and a chiral spirocyclic product is obtained at a conversion rate of 10% and an enantioselectivity of 84%.

Description

Technical Field

[0001] The present invention relates to a method for designing and constructing 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 applied in various fields due to its significant advantages. Enzymes, as biocatalysts, are particularly well-suited 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, making them increasingly important in industries such as medicine, textiles, and chemicals. Biosynthesis enables the efficient and environmentally friendly production of various functional molecules, promoting the sustainable development of human society. However, enzymes in nature are composed of 20 naturally occurring 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 reactions catalyzed by enzymes. Therefore, by introducing non-natural small molecule catalysts (cofactors) into the protein backbone, artificial enzymes with higher catalytic activity or novel reactivities can be obtained.

[0003] Recent research has categorized the construction methods of artificial enzymes into three main approaches: unnatural amino acid insertion, supramolecular assembly, and covalent ligation. These diverse approaches allow the introduction of a wide range of unnatural cofactors into diverse protein backbones, expanding the application of biocatalysis in organic synthesis.

[0004] Tertiary amine catalysts (TA) have been widely used in organic synthesis in recent years, catalyzing a variety of addition and cycloaddition reactions. Currently, tertiary amine catalysts (TA) are widely used in the synthesis of fine chemicals, pharmaceuticals, pesticides, dyes, fragrances, and other products. As a primary tool for asymmetric chemical transformations, organocatalysis has matured, and its bioorthogonality can be exploited for important chemical and synthetic biology applications.

[0005] In 2010, Chen and colleagues pioneered the use of isatin as an electrophile for asymmetric allylic alkylation to synthesize MBH carbonates. Since then, isatin-derived MBH carbonates have become particularly useful C3 synthons for enantioselective cyclization of enantiomerically enriched spiroindoles, primarily through [3+2], [3+3], and [4+3] cyclizations to synthesize a variety of potentially bioactive chiral spirocyclic compounds. Tertiary amine catalysts (TAs), as a class of effective small molecule catalysts, have been exploited in these reactions due to their excellent chemical functionality. However, the difficulty in developing and synthesizing chiral tertiary amine catalysts, the harsh reaction conditions required for chemical catalysis, and the high catalyst requirements have severely limited their application in asymmetric synthesis. Therefore, building on our previous research in the design of novel functional enzyme elements, we sought to design and construct a novel artificial enzyme containing a tertiary amine catalytic center to catalyze a variety of asymmetric, non-natural cycloaddition reactions, which would significantly expand the diversity and application of enzymes.

[0006] The 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 on the construction of new artificial enzymes containing tertiary amine catalytic centers using LmrR as a protein skeleton. 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, which is used to catalyze a non-natural asymmetric [3+2] cycloaddition reaction.

[0008] The present invention is achieved through the following technical solutions:

[0009] 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 through cysteine.

[0010] In one embodiment of the present invention, the structural formula of the cofactor is: .

[0011] In one embodiment of the present invention, the amino acid sequence of the Lactococcus multidrug resistance regulatory protein is shown as SEQ ID NO.1.

[0012] 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.

[0013] In the present invention, this cofactor serves as the enzyme catalytic center, which can catalyze an asymmetric [3+2] cycloaddition reaction and achieve a certain conversion rate and stereoselectivity.

[0014] A second object of the present invention is to provide a method for preparing the artificial enzyme, comprising the steps of:

[0015] (1) reacting mercaptotetrahydropyrrole with 2,2'-dithiodipyridine under protective gas conditions, and then reacting the obtained product with 4-chloropyridine to obtain a cofactor;

[0016] (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, and mutating methionine to cysteine;

[0017] (3) Covalently linking the cofactor prepared in step (1) to the mutated lactococcal multidrug resistance regulatory protein to obtain the artificial enzyme.

[0018] 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.

[0019] In one embodiment of the present invention, the reaction of mercaptopyrrole and 2,2'-disulfide dipyridine is carried out under argon protection for 0.5 to 1 hour.

[0020] In one embodiment of the present invention, the reaction of mercaptopyrrole and 2,2'-disulfide dipyridine is carried out at 20-30°C.

[0021] In one embodiment of the present invention, the reaction product of mercaptopyrrole and 2,2'-disulfide dipyridine with 4-chloropyridine is reacted at 70-90° C. for 10-15 hours.

[0022] The third object of the present invention is to provide the use of the artificial enzyme in catalyzing asymmetric [3+2] cycloaddition reactions.

[0023] In one embodiment of the present invention, the artificial enzyme can catalyze the following reaction:

[0024] .

[0025] In one embodiment of the present invention, the catalytic reaction conditions are 25-35 0 C for 40 to 60 hours.

[0026] Beneficial effects of the present invention:

[0027] Starting with the LmrR protein, this application successfully constructed a novel artificial enzyme, LmrR_M89C_TA, by covalently linking a catalytically active tertiary amine cofactor to position 89 of LmrR. After protein purification, the enzyme, LmrR_M89C_TA, was successfully constructed. This novel artificial enzyme, LmrR_M89C_TA, containing a tertiary amine catalytic center, catalyzed a non-natural asymmetric [3+2] cycloaddition reaction, affording the target chiral spirocyclic product with a conversion of 10% and an enantioselectivity of 84%. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any creative work.

[0029] Figure 1 C-H spectra of the synthesized cofactor precursor compounds;

[0030] Figure 2 is the reaction formula for the covalent linkage of LmrR protein and cofactor precursor compound;

[0031] Figure 3 This is the protein spectrum of the artificial enzyme LmrR_M89C_TA;

[0032] 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

[0033] 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 intended to limit the scope of the present invention. In addition, after reading the content taught by the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims appended hereto.

[0034] Culture medium and reagents:

[0035] LB medium: 1% peptone, 1% sodium chloride, and 0.5% yeast extract, pH approximately 7.0, sterilized at 121°C for 20 min. For culturing E. coli, add 20 g / L agar powder to prepare LB solid medium.

[0036] Washing buffer: 50 mM sodium 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.

[0037] Elution buffer: 50 mM sodium 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.

[0038] Example 1: Synthesis of artificial cofactor precursor compounds

[0039] 203 mg (1 mmol) of Boc-protected mercapto tetrahydropyrrole was added to 10 mL of TFA / DCM mixed solvent, stirred at 0°C for 2 hours and dried under vacuum. The product was added with 10 mmol of 2,2'-disulfide dipyridine and reacted at room temperature for 0.5 hours under argon protection. After the reaction was completed, the mixture was dried and purified by column chromatography. After the purified product was added with 4-chloropyridine and reacted at 80°C for 12 hours, the reaction solution was evaporated under vacuum and purified by column chromatography to obtain cofactor precursor 1. The reaction formula of the synthesis step is shown below. The carbon-hydrogen spectrum of the cofactor precursor compound 1 is shown below. Figure 1 shown.

[0040]

[0041]

[0042] Example 2: Site-directed mutagenesis and purification of LmrR protein

[0043] 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 a 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 Anneal at 72°C for 30 s (depending on the Tm of the primer) and incubate at 72 0 Extension under C for 2 min; (3) 72 0 The final extension was performed at 37°C for 8 min. 0The cells were digested with DpnI for 1 h at 37 °C and then transformed into competent E. coli DH5-α cells. Single colonies were picked from LB solid plates containing ampicillin (100 μg / mL) and inoculated into 3 mL of LB medium containing the same concentration of ampicillin. The culture medium was incubated at 37 0 C, 220 rpm overnight culture, followed by plasmid extraction and Sanger sequencing of mutants. For protein expression, the extracted plasmid was transformed into E. coli BL21, and a single colony was used to inoculate an overnight culture for protein expression and purification.

[0044] The recombinant bacteria were inoculated into 3 mL of LB liquid medium and cultured at 37°C, 220 rpm, for 14 h. After that, 2.5 mL was inoculated into 200 mL of fresh sterile LB medium and cultured at 37°C, 220 rpm, for 3-5 h. When the OD600 reached 0.6-0.8, IPTG was added to a final concentration of 1 mmol / L and cultured at 20°C, 160 rpm, for 20 h. The bacterial suspension was collected and centrifuged at 4°C, 6000 rpm, for 20 min to collect the pellet. The pellet was resuspended in 10 mL of PBS (pH 8.0) and then ultrasonically disrupted at 175 W power, 3 s on, 6 s off, for 20 min. After disruption, DNA enzyme was added and the cells were incubated on ice for 30 min. The cells were centrifuged at 4°C, 12,000 rpm, and 40 min. The supernatant was filtered through a 0.45 μm filter membrane and loaded into a Strep tactin column. The cells were incubated at 4°C, 20 rpm, and 1 h. The column was removed and the flow-through was collected. The protein was washed with 2 column volumes of washing buffer three times, and the washes (W1-W3) were collected. The protein was eluted with 1 column volume of elution buffer six times, and the eluates (E1-E6) were collected. The proteins obtained were LmrR_E7C, LmrR_M8C, LmrR_N14C, LmrR_V15C, LmrR_D60C, LmrR_M89C, LmrR_F93C, LmrR_V99C, LmrR_I103C, and LmrR_E107C, respectively. The fractions collected during the protein purification process were analyzed by SDS-PAGE. Finally, fractions E2-E5 were concentrated by ultrafiltration, dialyzed against PBS (pH = 6.5), and stored for later use.

[0045] LmrR protein amino acid sequence

[0046] SEQ ID NO.1:

[0047] GAEIPKEMLRAQTNVILLNVLKQGDNYVYGIIKQVKEASNGEMELNEATLYTIFDRLEQD GIISSYWGDESQGGRRKYYRLTEIGHENMRLAFESWSRVDKIIENLEANKKSEAIKSRGG SGGWSHPQFEK

[0048] Example 3: Covalent linkage of LmrR protein with co-factor

[0049] The purified protein was added with 10 times equivalent of small molecule co-factor precursor compound (dissolved in DMF) for covalent linkage at 4°C for 24 h. The reaction solution was ultrafiltrated for three times and replaced with buffer to PBS (pH = 7.4) and stored at 4°C for later catalysis. The linkage reaction is shown in 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, respectively.

[0050] Example 4: Catalytic reaction

[0051] The total volume of the reaction in 2 mL centrifuge tube was 300 μL. The protein solution of artificial enzyme was diluted with PBS buffer (50 mM NaCl, 150 mM NaH2PO4, pH 7.4) and the volume was made up to 275 μL with a final concentration of 30 μM. The reagent 2 stock solution (30 mM DMF solution was prepared and 10 μL was added with a final concentration of 1 mM) and reagent 3 (80 mM DMF solution was prepared and 15 μL was added with a final concentration of 4 mM). The reaction system was shaken at 30 0 C. After 48 h of reaction, ethyl acetate was added for extraction. The extract was dried with anhydrous sodium sulfate and filtered. The obtained product 4 was dissolved in 200 μL of chromatographic grade isopropanol and analyzed by HPLC for conversion and enantioselectivity. The reaction equation of catalytic reaction is shown below.

[0052]

[0053] The catalytic reaction data of different enzyme mutants are shown in Table 1: the novel 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%.

[0054] Enzyme catalytic reaction conversion rate (%) = amount of reacted raw material 1 / amount of original raw material 1 * 100%

[0055] Table 1 Effects of different artificial enzyme mutants on catalytic reactions

[0056]

[0057] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall 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 a cysteine ​​mutation into the hydrophobic cavity of the lactococcal multidrug resistance regulatory protein and connecting a cofactor containing a tertiary amine catalytic group through the cysteine; The structural formula of the cofactor is: ; The amino acid sequence of the lactococcal multidrug resistance regulatory protein is shown in SEQ ID NO.1; 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.

2. A method for preparing the artificial enzyme according to claim 1, characterized in that: The steps include: (1) reacting mercaptotetrahydropyrrole 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, and mutating methionine to cysteine; (3) Covalently linking the cofactor prepared in step (1) to the mutated lactococcal multidrug resistance regulatory protein to obtain the artificial enzyme.

3. The preparation method according to claim 2, 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.

4. The preparation method according to claim 2, wherein The reaction of mercaptotetrahydropyrrole with 2,2'-disulfide dipyridine is carried out under argon protection for 0.5 to 1 h.

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

6. Use of the artificial enzyme according to claim 1 in catalyzing an asymmetric [3+2] cycloaddition reaction; The artificial enzyme catalyzes the following reaction: 。

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