Application of artificial photoenzyme as catalyst in synthesis of esomeprazole compound

Abstract

The invention belongs to the crossing field of chemical biology and green catalysis, and particularly relates to application of an artificial photoenzyme as a catalyst to synthesis of an esomeprazole compound. The artificial photoenzyme is constructed by inserting non-natural amino acid containing a benzophenone structure into a protein skeleton; the invention aims to realize in-situ generation and chiral control of singlet oxygen by introducing a benzophenone photosensitive group into a protein skeleton at a fixed point, and solves the technical problems of need of expensive renewable coenzyme, poor enzyme stability, high loading capacity and low yield in the existing esomeprazole enzyme method process.

Description

Technical Field

[0001] The present invention belongs to the interdisciplinary field of chemical biology and green catalysis, and specifically relates to the use of an artificial photoenzyme as a catalyst for synthesizing an esomeprazole compound. Background Art

[0002] Esomeprazole is a second-generation proton pump inhibitor pioneered by AstraZeneca in the United States and is the (S)-isomer of omeprazole. Compared to the racemic form of omeprazole, esomeprazole has a low first-pass effect in the liver, high bioavailability, and a prolonged duration of action, thus possessing broad market application prospects. Its chemical name is S-5-methoxy-2-{[(4-methoxy-3,5-dimethyl-2-pyridyl)methyl]sulfinyl}-1H-benzimidazole, and its chemical formula is:

[0003]

[0004] Currently, the preparation of esomeprazole both domestically and internationally is primarily based on chemical asymmetric oxidation synthesis. A commonly used synthetic route uses the inexpensive and readily available pharmaceutical intermediate omeprazole sulfide (2-[(4-methoxy-3,5-dimethylpyridin-2-yl)methyl]-5-methoxythiophene) as the starting material, tetraisopropyloxytitanium as a catalyst, and diethyl tartrate as a chiral ligand to produce esomeprazole via an asymmetric oxidation reaction. This route offers mild reaction conditions and high optical purity, but it is prone to the formation of byproducts such as omeprazole N-oxide and omeprazole sulfone, which compromise product quality and yield, making separation and purification difficult and the process complex. In recent years, patents such as CN106345526A and CN109705091A have attempted to introduce metal-chiral amine systems or oxidant modification strategies to improve selectivity and environmental friendliness, but most still rely on heavy metals or traditional oxidants.

[0005] The bioproduction of esomeprazole, which uses oxygen as an inexpensive, environmentally friendly oxidant, has attracted increasing attention in recent years due to its environmentally friendly, mild conditions, strong specificity, and ease of separation and purification. Existing representative patents, such as CN108251466A and CN108251465A, employ a one-pot monooxygenase synthesis or enzymatic oxidation using omeprazole sulfide as a substrate. However, these catalytic reactions often require expensive renewable coenzymes, and the biocatalysts suffer from poor stability, high loadings, and low yields, limiting their practical application. Summary of the Invention

[0006] In response to the above-mentioned defects or improvement needs of the prior art, the present invention provides an artificial photoenzyme used as a catalyst for the synthesis of esomeprazole compounds. The purpose is to achieve in situ generation and chirality control of singlet oxygen by constructing an artificial photoenzyme by point-introducing a benzophenone photosensitizing group into the protein backbone, thereby solving the technical problems of the existing esomeprazole enzymatic process, such as the need for expensive renewable coenzymes, poor enzyme stability, high loading amount, and low yield.

[0007] To achieve the above objectives, according to one aspect of the present invention, an artificial photoenzyme is provided as a catalyst for the synthesis of esomeprazole compounds. The artificial photoenzyme is constructed by inserting a non-natural amino acid containing a benzophenone structure into a protein backbone.

[0008] Preferably, the non-natural amino acid containing a benzophenone structure is benzophenone alanine or 3-fluoro-substituted benzophenone alanine.

[0009] Preferably, the protein backbone is selected from LmrR or RamR.

[0010] Preferably, the substrate omeprazole sulfide shown in Formula 1 is catalyzed by an artificial photoenzyme to generate an esomeprazole compound shown in Formula 2. The reaction for synthesizing the esomeprazole compound is as follows:

[0011]

[0012] Preferably, the non-natural amino acid containing a benzophenone structure is inserted into an amino acid site of the protein backbone that has a hydrophobic interaction with the substrate; preferably, the site is R75 or M89.

[0013] Preferably, the specific conditions of the reaction are: dissolving the omeprazole sulfide in a mixture of an organic solvent and a buffer solution, then adding an acid solution to adjust the pH of the solution to 6.0-8.0, then adding the artificial photoenzyme, transferring the reaction solution to a sealed container protected by a non-oxidizing gas, maintaining the temperature at 20-35° C., and reacting under 320nm-400nm ultraviolet light for 4-12 hours to produce the esomeprazole.

[0014] Preferably, the mass ratio of the omeprazole sulfide to the organic solvent is 1:(3-20); and the concentration of the artificial photoenzyme in the reaction solution is 10-100 μmol / L.

[0015] Preferably, the method further comprises adding a stabilizer to the reaction solution, wherein the stabilizer is selected from glycerol, dithiothreitol or magnesium chloride; and the buffer solution is selected from 3-(N-morpholino)propanesulfonic acid buffer (MOPS) or 4-hydroxyethylpiperazineethanesulfonic acid buffer (HEPES).

[0016] Preferably, after the esomeprazole is generated, the process further comprises: centrifuging or membrane filtering the product solution, then adding ethyl acetate to extract the organic phase, concentrating the obtained organic phase by rotary evaporation, and purifying the concentrated product by column chromatography.

[0017] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art:

[0018] 1. The present invention realizes the in situ generation and chirality control of singlet oxygen by introducing a benzophenone photosensitive group into the protein skeleton. The system generates singlet oxygen in situ ( 1 O2), induces the selective oxidation of omeprazole sulfide to esomeprazole, and does not rely on renewable coenzymes such as NAD(P)H required for traditional enzyme catalysis, avoiding problems such as poor stability, harsh reaction conditions, large loading amount, and low yield.

[0019] 2. The present invention selects R75 or M89 as the insertion site for the non-natural amino acid because R75 and M89 are located at key positions in the protein binding pocket and form a hydrophobic interaction with the substrate. Through precise structural modification, the reaction interface between the photosensitive group and the substrate is optimized, which significantly improves the selectivity and catalytic efficiency of the reaction and realizes the precise spatial positioning of the photosensitive group on the substrate in the hydrophobic binding pocket. DETAILED DESCRIPTION

[0020] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0021] Example 1

[0022] The present invention provides an application of an artificial photoenzyme as a catalyst for synthesizing an esomeprazole compound, which specifically comprises the following steps:

[0023] 1. Preparation of artificial photoenzyme

[0024] (1) Preparation of competent cells: DH5α and BL21(DE3) competent cells were prepared by purchasing E. coli DH5α and BL21(DE3) cells from Takara Biomedical Technology Co., Ltd. E. coli DH5α and BL21(DE3) cells were then treated with calcium chloride to render them susceptible to foreign DNA, i.e., DH5α or BL21 competent cells.

[0025] The specific steps are as follows:

[0026] A single colony of Escherichia coli DH5α or BL21 was picked and inoculated into 10 mL of LB medium, and cultured at 37°C with shaking overnight for 14 h.

[0027] Inoculate into 100 mL of LB medium at a ratio of 1:50, culture with rapid shaking at 37°C for 2 to 3 hours, and stop the culture when the OD600 is approximately 0.5.

[0028] Pour into a sterile 50 mL centrifuge tube, place on ice for 30 min, centrifuge at 8000 rpm for 5 min, and discard the supernatant.

[0029] Add 20 mL of 0.1 mol / L CaCl2 pre-cooled solution to each tube, resuspend the bacterial pellet and transfer it to a tube, and continue to ice bath for 30 min.

[0030] Centrifuge at 8000 rpm for 5 min, discard the supernatant, add 6 mL of 0.1 mol / L CaCl2 pre-cooled solution, and then add 2 mL of 80% glycerol to resuspend the bacterial pellet.

[0031] Aliquot the competent cells into 200 μL cell solution per tube and store in a -80°C refrigerator until use.

[0032] (2) Plasmid construction: Due to the presence of a DNA binding region in the lactococcal multidrug resistance regulator (LmrR) from Lactococcus cremoris subsp. cremoris MG1363, K55 in the sequence was mutated to aspartic acid (D), and K59 was mutated to glutamine (Q), referring to the strategy for constructing artificial metalloenzymes. The codon-optimized LmrR gene sequence (including the C-terminal Strep-tag II purification tag) was synthesized by Genewiz / Azenta and constructed into the expression vector pET17b, obtaining the recombinant vector pET17b_LmrR.

[0033] The codon-optimized nucleic acid sequence of LmrR is:

[0034] ATGGGTGCCGAAATCCCGAAAGAAATGCTGCGTGCTCAAACCAATGTCATCCTGCTGAATGTCCTGAAACAAGGCGATAACTATGTGTATGGCATTATCAAACAGGTGAAAGAAGCGAGCAACGGTGAAATGGAACTGAATGAAGCCACCCTGTATACGATTTTTGATCGTCTGGAACAGGACGGCATTATCAGCTCTTA CTGGGGGTGATGAAAGTCAAGGCGGTCGTCGCAAATATTACCGTCTGACCGAAATCGGCCATGAAAACATGCGCCTGGCGTTCGAATCCTGGAGTCGTGTGGACAAAATCATTGAAAATCTGGAAGCAAACAAAAAATCTGAAGCGATCAAATCTAGAGGTGGCAGCGGTGGCTGGAGCCACCCGCAGTTCGAAAAATAA.

[0035] The amino acid sequence of the codon-optimized LmrR (SEQ ID NO: 1) is: MGAEIPKEMLRAQTNVILLNVLKQGDNYVYGIIKQVKEASNGEMELNEATLYTIFDRLEQDGIISSYWGDESQGGRRKYYRLTEIGHENMRLAFESWSRVDKIIENLEANKKSEAIKSRGGSGGWSHPQFEK.

[0036] (3) PCR to obtain esomeprazole synthase variant plasmid 1: Site-directed mutagenesis primers were designed and codon replacement was achieved at a specific site in the gene sequence by PCR amplification, with site R75 selected. The codons corresponding to the unnatural amino acids were all designed to be TAG, while the codons for the other amino acids were selected from codons commonly used in the LmrR nucleic acid sequence.

[0037] Forward primer F1: CGCGGATCCTTACGCATTAGCCTGCTGTTTGG (SEQ ID NO. 14)

[0038] Reverse primer R1: CCAAACAGCAGGCTAATGCGTAAGGATCCGCG

[0039] The nucleic acid sequence of artificial photoenzyme 1 (protein backbone: LmrR, insertion site: R75) is:

[0040] ATGGGTGCCGAAATCCCGAAAGAAATGCTGCGTGCTCAAACCAATGTCATCCTGCTGAATGTCCTGAAACAAGGCGATAACTATGTGTATGGCATTATCAAACAGGTGAAAGAAGCGAGCAACGGTGAAATGGAACTGAATGAAGCCACCCTGTATACGATTTTTGATCGTCTGGAACAGGACGGCATTATCAGCTCTTA CTGGGGTGATGAAAGTCAAGGCGGTTAGCGCAAATATTACCGTCTGACCGAAATCGGCCATGAAAACATGCGCCTGGCGTTCGAATCCTGGAGTCGTGTGGACAAAATCATTGAAAATCTGGAAGCAAACAAAAAATCTGAAGCGATCAAATCTAGAGGTGGCAGCGGTGGCTGGAGCCACCCGCAGTTCGAAAAATAA.

[0041] The amino acid sequence of artificial photoenzyme 1 (protein backbone: LmrR, insertion site: R75) (SEQ ID NO: 2) is:

[0042] MGAEIPKEMLRAQTNVILLNVLKQGDNYVYGIIKQVKEASNGEMELNEATLYTIFDRLEQDGIISSYWGDESQGGR(BpA)RKYYRLTEIGHENMRLAFESWSRVDKIIENLEANKKSEAIKSRGGSGGWSHPQFEK.

[0043] (4) Transformation of plasmids to obtain recombinant genetically engineered bacteria: After adding plasmid 1 to the EP tube containing competent cells, place on ice for 30 minutes; heat shock at 42°C for 90 seconds. To increase the transformation efficiency, repeat this heat shock step, then quickly place on ice for 3-5 minutes; add 400 μL of fresh LB liquid culture medium, place in a metal bath mixer at 37°C and 700 rpm for 1 hour; centrifuge at 4000 rpm for 5 minutes, discard 200 μL of culture medium, resuspend the bacteria, spread on a plate (containing ampicillin), and incubate at 37°C for 12-16 hours. Recombinant genetically engineered bacterial strain 1 was obtained.

[0044] (5) Preparation of genetically engineered bacteria: The recombinant E. coli genetically engineered strain, strain 1, was inoculated into 3 mL of fresh culture medium containing 100 μg / mL ampicillin resistance (Amp+) and cultured overnight at 37°C and 200 rpm in a shaker. The pre-cultured bacterial solution was then transferred to 1 L of LB culture medium and cultured at 37°C until the OD600 reached 0.8 h. 100 μL of 1 M IPTG inducer was added, and the culture was continued at 30°C for 20-24 h. The bacterial sludge was collected by centrifugation at 8000 rpm and the supernatant was removed and stored in a -80°C freezer.

[0045] (6) Preparation of enzyme solution: Weigh 20 g of the bacterial sludge of strain 1 obtained by fermentation, resuspend the precipitate in buffer W (100 mM Tris-HCl, 150 mM NaCl, 10% glycerol, pH 8.0), and then add DNase I (final concentration 0.1 mg / mL, 10 mM MgCl2) and PMSF (final concentration 1.0 mM). Subsequently, use a cell disruptor to perform ultrasonic disruption for 40 minutes (45-60% amplitude, 5 seconds on, 8 seconds off), and collect the supernatant by centrifugation (8000 rpm, 4°C, 1 hour). The supernatant was collected by centrifugation under low temperature conditions to obtain the enzyme solution of the corresponding strain, which was frozen at -20°C.

[0046] (7) Preparation of enzyme powder:

[0047] Measure 50 ml of the enzyme solution from strain 1 and transfer it to a Strep-Tactin purification column. Incubate it in a 4°C refrigerator or ice box for 1 hour. Then, wash the purification column with 5×1CV (column volume) of Buffer W and elute it with 10×0.5CV of Buffer E (Buffer W containing 2.5 mM desthiobiotin) to obtain the pure LmrR protein with the Strep-tag II purification tag. Then, place it in a drying dish and pre-freeze it in a -20°C refrigerator for 24 hours. The sample is then placed in a freeze dryer for lyophilization. The cold trap temperature of the freeze dryer is -50°C and the drying chamber pressure is 100 Pa. Freeze-dry for 24 hours to obtain a solid enzyme powder, which is stored in a refrigerated container at 4°C.

[0048] 2. Synthesis of esomeprazole catalyzed by artificial photoenzyme

[0049] In a 500 mL three-necked round-bottom flask, add 50 mL of DMSO solution, stir and add 65.8 g of omeprazole sulfide to completely dissolve it, add 250 mL of MOPS buffer, adjust the pH to 8.0 with glacial acetic acid, add 10 g of the above-synthesized artificial photoenzyme powder, transfer the reaction solution to a nitrogen-protected sealed container, maintain the temperature at 20 ° C, and illuminate the UV LED array with a wavelength of 365 nm and 30 mW / cm 2Under the control of irradiation intensity, esomeprazole was generated by light reaction. The reaction was completed after 10 hours. The reaction conversion rate monitored by high performance liquid chromatography was 78.2% and the ee value was 99%.

[0050] Characterization method:

[0051] Chiral ee value detection conditions of product esomeprazole:

[0052] A Shimadzu LC-20AT liquid chromatograph was used, and a Daicel column IC was used. The mobile phase was n-hexane:isopropanol (90:10), with a flow rate of 1 mL / min, a column temperature of 35 °C, and an ultraviolet detection wavelength of 254 nm.

[0053] Peak time: esomeprazole is 15.108 min, and isomer impurity is 16.508 min.

[0054]

[0055] Where: A API is the peak area of ​​product esomeprazole; A IMP is the peak area of ​​the isomeric impurity.

[0056] The omeprazole sulfide prepared in this example was monitored by high performance liquid chromatography. The conversion rate of the reactant omeprazole sulfide was 78.2%, and the ee value of the product esomeprazole was 99%.

[0057] Example 2

[0058] The present invention provides an application of an artificial photoenzyme as a catalyst for synthesizing an esomeprazole compound, which specifically comprises the following steps:

[0059] 1. Preparation of artificial photoenzyme

[0060] (1) Preparation of competent cells: DH5α and BL21(DE3) competent cells: E. coli DH5α and BL21(DE3) cells were purchased from Takara Biomedical Technology. E. coli DH5α and BL21(DE3) cells were then treated with calcium chloride to render them susceptible to foreign DNA, i.e., DH5α or BL21 competent cells.

[0061] The specific steps are as follows:

[0062] A single colony of Escherichia coli DH5α or BL21 was picked and inoculated into 10 mL of LB medium, and cultured with shaking at 37°C overnight for 16 h.

[0063] Inoculate into 100 mL of LB medium at a ratio of 1:50, culture with rapid shaking at 37°C for 2 to 3 hours, and stop the culture when the OD600 is approximately 0.5.

[0064] Pour into a sterile 50 mL centrifuge tube, place on ice for 30 min, centrifuge at 8000 rpm for 5 min, and discard the supernatant.

[0065] Add 20 mL of 0.1 mol / L CaCl2 pre-cooled solution to each tube, resuspend the bacterial pellet and transfer it to a tube, and continue to ice bath for 30 min.

[0066] Centrifuge at 8000 rpm for 5 min, discard the supernatant, add 6 mL of 0.1 mol / L CaCl2 pre-cooled solution, and then add 2 mL of 80% glycerol to resuspend the bacterial pellet.

[0067] Aliquot the competent cells into 200 μL cell solution per tube and store in a -80°C refrigerator until use.

[0068] (2) Plasmid construction: The lactococcal multidrug resistance regulator (LmrR) from Lactococcus cremoris subsp. cremoris MG1363 has a DNA binding region. Based on the artificial metalloenzyme construction strategy, K55 in the sequence was mutated to aspartic acid (D), and K59 was mutated to glutamine (Q). The codon-optimized LmrR gene sequence (including the C-terminal Strep-tag II purification tag) was synthesized by Genewiz / Azenta and constructed into the expression vector pET17b, obtaining the recombinant vector pET17b_LmrR.

[0069] (3) PCR to obtain esomeprazole synthase variant plasmid 2: Site-directed mutagenesis primers were designed and PCR amplification was performed to achieve codon substitution at a specific site in the gene sequence, with site M89 selected. The codons corresponding to the unnatural amino acids were all designed to be TAG, while the codons for the other amino acids were selected from codons commonly used in the LmrR nucleic acid sequence.

[0070] Forward primer F1: GGAATTCCATATGAGTACCAAGATGGATTTTGATGC (SEQ ID NO. 13).

[0071] Reverse primer R1: CGCGGATCCTTACGCATTAGCCTGCTGTTTGG (SEQ ID NO. 14).

[0072] The nucleic acid sequence of artificial photoenzyme 2 (protein backbone: LmrR, insertion site: R89) is:

[0073] ATGGGTGCCGAAATCCCGAAAGAAATGCTGCGTGCTCAAACCAATGTCATCCTGCTGAATGTCCTGAAACAAGGCGATAACTATGTGTATGGCATTATCAAACAGGTGAAAGAAGCGAGCAACGGTGAAATGGAACTGAATGAAGCCACCCTGTATACGATTTTTGATCGTCTGGAACAGGACGGCATTATCAGCTCTTA CTGGGGGTGATGAAAGTCAAGGCGGTCGTCGCAAATATTACCGTCTGACCGAAATCGGCCATGAAAACTAGCGCCTGGCGTTCGAATCCTGGAGTCGTGTGGACAAAATCATTGAAAATCTGGAAGCAAACAAAAAATCTGAAGCGATCAAATCTAGAGGTGGCAGCGGTGGCTGGAGCCACCCGCAGTTCGAAAAATAA.

[0074] The amino acid sequence of artificial photoenzyme 2 (protein backbone: LmrR, insertion site: R89) (SEQ ID NO: 3) is:

[0075] MGAEIPKEMLRAQTNVILLNVLKQGDNYVYGIIKQVKEASNGEME LNEATLYTIFDRLEQDGIISSYWGDESQGGRRKYYRLTEIGHENM(BpA)RLAFESWSRV DKIIENLEAN KKSEAIKSRG GSGGWSHPQF EK.

[0076] (4) Transformation of plasmid to obtain recombinant genetically engineered bacteria: After adding plasmid 2 to the EP tube containing competent cells, place on ice for 30 minutes; heat shock at 42°C for 90 seconds (this heat shock step can be repeated to increase transformation efficiency), then quickly place on ice for 3-5 minutes; add 400 μL of fresh LB liquid culture medium, place in a metal bath mixer at 37°C and 700 rpm for 1 hour; centrifuge at 4000 rpm for 5 minutes, discard 200 μL of culture medium, resuspend the bacteria, spread on a plate (containing ampicillin), and incubate at 37°C for 12-16 hours. Obtain recombinant genetically engineered bacterial strain 2.

[0077] (5) Preparation of genetically engineered bacteria: The recombinant E. coli genetically engineered strain 2 was inoculated into 3 mL of fresh culture medium containing 100 μg / mL Amp+ and cultured overnight at 37°C and 200 rpm in a shaker. The pre-cultured bacterial solution was then transferred to 1 L of LB culture medium and cultured at 37°C until the OD600 reached 0.8 h. 100 μL of 1 M IPTG inducer was added, and the culture was continued at 30°C for 20-24 h. The bacterial slurry was collected by centrifugation at 8000 rpm and the supernatant was removed and stored in a -80°C freezer.

[0078] (6) Preparation of enzyme solution: Weigh 20 g of the bacterial sludge of strain 2 obtained by fermentation, resuspend the precipitate in buffer W (100 mM Tris-HCl, 150 mM NaCl, 10% glycerol, pH 8.0), and then add DNase I (final concentration 0.1 mg / mL, 10 mM MgCl2) and PMSF (final concentration 1.0 mM). Subsequently, use a cell disruptor to perform ultrasonic disruption for 40 minutes (45-60% amplitude, on 5 seconds, off 8 seconds), and collect the supernatant by centrifugation (8000 rpm, 4°C, 1 hour). The supernatant was collected by centrifugation under low temperature conditions to obtain the enzyme solution of the corresponding strain, which was frozen at -20°C.

[0079] (7) Preparation of enzyme powder:

[0080] Measure 50 ml of the enzyme solution from strain 2 and transfer it to a Strep-Tactin purification column. Incubate it in a 4°C refrigerator or ice box for 1 hour. Then, wash the purification column with 5×1CV (column volume) of Buffer W and elute it with 10×0.5CV of Buffer E (Buffer W buffer containing 2.5 mM desthiobiotin) to obtain the pure LmrR protein with the Strep-tag II purification tag. Then, place it in a drying dish and pre-freeze it in a -20°C refrigerator for 24 hours. The sample is then placed in a freeze dryer for freeze-drying. The cold trap temperature of the freeze dryer is -50°C and the drying chamber pressure is 100 Pa. Freeze-dry for 24 hours to obtain a solid powder of the enzyme, which is stored in a refrigerated container at 4°C.

[0081] 2. Synthesis of esomeprazole catalyzed by artificial photoenzyme

[0082] In a 500 mL three-necked round-bottom flask, add 50 mL of DMSO solution, stir and add 65.8 g of omeprazole sulfide to completely dissolve it, add 250 mL of MOPS buffer, adjust the pH to 7.0 with glacial acetic acid, add 10 g of the synthesized artificial photoenzyme, transfer the reaction solution to a nitrogen-protected sealed container, maintain the temperature at 30 ° C, and illuminate the UV LED array with a wavelength of 365 nm and 30 mW / cm 2Under the control of irradiation intensity, esomeprazole was generated by light reaction. The reaction was completed after 10 hours. High performance liquid chromatography monitoring showed that the conversion rate of omeprazole sulfide was 85.7%, and the ee value of the product esomeprazole was 99%.

[0083] Example 3

[0084] The present invention provides an application of an artificial photoenzyme as a catalyst for synthesizing an esomeprazole compound, which specifically comprises the following steps:

[0085] 1. Preparation of artificial photoenzyme

[0086] (1) Preparation of competent cells: DH5α and BL21(DE3) competent cells: E. coli DH5α and BL21(DE3) cells were purchased from Takara Biomedical Technology. E. coli DH5α and BL21(DE3) cells were then treated with calcium chloride to render them susceptible to foreign DNA, i.e., DH5α or BL21 competent cells.

[0087] The specific steps are as follows:

[0088] A single colony of Escherichia coli DH5α or BL21 was picked and inoculated into 10 mL of LB medium, and cultured at 37°C with shaking overnight (14-16 h).

[0089] Inoculate into 100 mL of LB medium at a ratio of 1:50, culture with rapid shaking at 37°C for 2 to 3 hours, and stop the culture when the OD600 is approximately 0.5.

[0090] Pour into a sterile 50 mL centrifuge tube, place on ice for 30 min, centrifuge at 8000 rpm for 5 min, and discard the supernatant.

[0091] Add 20 mL of 0.1 mol / L CaCl2 pre-cooled solution to each tube, resuspend the bacterial pellet and transfer it to a tube, and continue to ice bath for 30 min.

[0092] Centrifuge at 8000 rpm for 5 min, discard the supernatant, add 6 mL of 0.1 mol / L CaCl2 pre-cooled solution, and then add 2 mL of 80% glycerol to resuspend the bacterial pellet.

[0093] Aliquot the competent cells into 200 μL cell solution per tube and store in a -80°C refrigerator until use.

[0094] (2) Plasmid construction: The lactococcal multidrug resistance regulator (LmrR) from Lactococcus cremoris subsp. cremoris MG1363 has a DNA binding region. Based on the artificial metalloenzyme construction strategy, K55 in the sequence was mutated to aspartic acid (D), and K59 was mutated to glutamine (Q). The codon-optimized LmrR gene sequence (including the C-terminal Strep-tag II purification tag) was synthesized by Genewiz / Azenta and constructed into the expression vector pET17b, obtaining the recombinant vector pET17b_LmrR.

[0095] (3) PCR to obtain esomeprazole synthase variant plasmid 3

[0096] Site-directed mutagenesis primers were designed and PCR amplification was used to replace codons at specific sites in the gene sequence, with the selected site being R75. The codons corresponding to the unnatural amino acids were all designed to be TAG, while the codons for the other amino acids were selected from codons commonly used in the LmrR nucleic acid sequence.

[0097] Forward primer F1: GGAATTCCATATGAGTACCAAGATGGATTTTGATGC (SEQ ID NO. 13).

[0098] Reverse primer R1: CGCGGATCCTTACGCATTAGCCTGCTGTTTGG (SEQ ID NO. 14)

[0099] (4) Transformation of plasmids to obtain recombinant genetically engineered bacteria: After plasmid 3 was added to the EP tube containing competent cells, the tube was placed on ice for 30 minutes; heat-shocked at 42°C for 90 seconds, and then quickly placed on ice for 3-5 minutes; 400 μL of fresh LB liquid medium was added, and the tube was cultured in a metal bath mixer at 37°C and 700 rpm for 1 hour; centrifuged at 4000 rpm for 5 minutes, 200 μL of the culture medium was discarded, the bacteria were resuspended, plated (containing ampicillin), and incubated at 37°C for 12-16 hours. Recombinant genetically engineered bacterial strain 3 was obtained.

[0100] (5) Preparation of genetically engineered bacteria: The recombinant E. coli genetically engineered strain, strain 3, was inoculated into 3 mL of fresh culture medium containing 100 μg / mL Amp+ and cultured overnight at 37°C and 200 rpm in a shaker. The pre-cultured bacterial solution was then transferred to 1 L of LB culture medium and cultured at 37°C until the OD600 reached 0.8 h. 100 μL of 1 M IPTG inducer was added, and the culture was continued at 30°C for 20-24 h. The bacterial slurry was collected by centrifugation at 8000 rpm and the supernatant was removed and stored in a -80°C freezer.

[0101] (6) Preparation of enzyme solution: Weigh 20 g of the bacterial sludge of strain 3 obtained by fermentation, resuspend the precipitate in buffer W (100 mM Tris-HCl, 150 mM NaCl, 10% glycerol, pH 8.0), and then add DNase I (final concentration 0.1 mg / mL, 10 mM MgCl2) and PMSF (final concentration 1.0 mM). Subsequently, use a cell disruptor to perform ultrasonic disruption for 40 minutes (45-60% amplitude, on 5 seconds, off 8 seconds), and collect the supernatant by centrifugation (8000 rpm, 4°C, 1 hour). The supernatant was collected by centrifugation under low temperature conditions to obtain the enzyme solution of the corresponding strain, which was frozen at -20°C.

[0102] (7) Preparation of enzyme powder:

[0103] Measure 50 ml of the enzyme solution from strain 3 and transfer it to a Strep-Tactin purification column. Incubate it in a 4°C refrigerator or ice box for 1 hour. Then, wash the purification column with 5×1CV (column volume) of Buffer W and elute it with 10×0.5CV of Buffer E (Buffer W buffer containing 2.5mM desthiobiotin) to obtain the pure LmrR protein with the Strep-tag II purification tag. Then place it in a drying dish and pre-freeze it in a -20°C refrigerator for 24-48 hours. Then, place the sample in a freeze dryer for freeze-drying. The cold trap temperature of the freeze dryer is -50°C and the drying chamber pressure is 100Pa. Freeze-dry for 24 hours to obtain a solid powder of the enzyme, which is stored in a refrigerator at 4°C.

[0104] 2. Synthesis of esomeprazole catalyzed by artificial photoenzyme

[0105] In a 500 mL three-necked round-bottom flask, add 50 mL of DMSO solution, stir and add 65.8 g of omeprazole sulfide to completely dissolve it, add 250 mL of MOPS buffer, adjust the pH to 7.0 with glacial acetic acid, add 10 g of the synthesized artificial photoenzyme, transfer the reaction solution to a sealed container protected by nitrogen, maintain the temperature at 30 ° C, and illuminate the UV LED array with a wavelength of 365 nm and 20 mW / cm 2 Under the control of irradiation intensity, esomeprazole was generated by light reaction. The reaction was completed after 10 hours. The conversion rate of omeprazole sulfide monitored by high performance liquid chromatography was 80.3%, and the ee value of the product esomeprazole was 99%.

[0106] Example 4

[0107] 1. Preparation of artificial photoenzyme

[0108] (1) Preparation of competent cells: DH5α and BL21(DE3) competent cells: E. coli DH5α and BL21(DE3) cells were purchased from Takara Biomedical Technology. E. coli DH5α and BL21(DE3) cells were then treated with calcium chloride to render them susceptible to foreign DNA, i.e., DH5α or BL21 competent cells.

[0109] The specific steps are as follows:

[0110] A single colony of Escherichia coli DH5α or BL21 was picked and inoculated into 10 mL of LB medium, and cultured at 37°C with shaking overnight (14-16 h).

[0111] Inoculate into 100 mL of LB medium at a ratio of 1:50, culture with rapid shaking at 37°C for 2 to 3 hours, and stop the culture when the OD600 is approximately 0.5.

[0112] Pour into a sterile 50 mL centrifuge tube, place on ice for 30 min, centrifuge at 8000 rpm for 5 min, and discard the supernatant.

[0113] Add 20 mL of 0.1 mol / L CaCl2 pre-cooled solution to each tube, resuspend the bacterial pellet and transfer it to a tube, and continue to ice bath for 30 min.

[0114] Centrifuge at 8000 rpm for 5 min, discard the supernatant, add 6 mL of 0.1 mol / L CaCl2 pre-cooled solution, and then add 2 mL of 80% glycerol to resuspend the bacterial pellet.

[0115] Aliquot the competent cells into 200 μL cell solution per tube and store in a -80°C refrigerator until use.

[0116] (2) Plasmid construction: The lactococcal multidrug resistance regulator (LmrR) from Lactococcus cremoris subsp. cremoris MG1363 has a DNA binding region. Based on the artificial metalloenzyme construction strategy, K55 in the sequence was mutated to aspartic acid (D), and K59 was mutated to glutamine (Q). The codon-optimized LmrR gene sequence (including the C-terminal Strep-tag II purification tag) was synthesized by Genewiz / Azenta and constructed into the expression vector pET17b, obtaining the recombinant vector pET17b_LmrR.

[0117] (3) PCR to obtain esomeprazole synthase variant plasmid 4: Site-directed mutagenesis primers were designed and PCR amplification was performed to achieve codon substitution at a specific site in the gene sequence, with position M89 selected. The codons corresponding to the unnatural amino acids were all designed to be TAG, while the codons for the other amino acids were selected from codons commonly used in the LmrR nucleic acid sequence.

[0118] Forward primer F1: GGAATTCCATATGAGTACCAAGATGGATTTTGATGC (SEQ ID NO. 13)

[0119] Reverse primer R1: CGCGGATCCTTACGCATTAGCCTGCTGTTTGG (SEQ ID NO. 14)

[0120] (4) Transformation of plasmids to obtain recombinant genetically engineered bacteria: After plasmid 4 is added to the EP tube containing competent cells, place it on ice for 30 minutes; heat shock at 42°C for 90 seconds (this heat shock step can be repeated to increase transformation efficiency), then quickly place it on ice for 3-5 minutes; add 400 μL of fresh LB liquid culture medium, place it in a metal bath mixer and culture it at 37°C and 700 rpm for 1 hour; centrifuge it at 4000 rpm for 5 minutes, discard 200 μL of the culture medium, resuspend the bacteria, spread it on a plate (containing ampicillin), and culture it upside down at 37°C for 12-16 hours. Recombinant genetically engineered bacterial strain 4 is obtained.

[0121] (5) Preparation of genetically engineered bacteria: The recombinant E. coli genetically engineered strain 4 was inoculated into 3 mL of fresh culture medium containing 100 μg / mL Amp+ and cultured overnight at 37°C and 200 rpm in a shaker. The pre-cultured bacterial solution was then transferred to 1 L of LB culture medium and cultured at 37°C until the OD600 reached 0.8 h. 100 μL of 1 M IPTG inducer was added, and the culture was continued at 30°C for 24 h. The bacterial slurry was collected by centrifugation at 8000 rpm and the supernatant was removed and stored in a -80°C freezer.

[0122] (6) Preparation of enzyme solution: Weigh 20 g of the bacterial sludge of strain 4 obtained by fermentation, resuspend the precipitate in buffer W (100 mM Tris-HCl, 150 mM NaCl, 10% glycerol, pH 8.0), and then add DNase I (final concentration 0.1 mg / mL, 10 mM MgCl2) and PMSF (final concentration 1.0 mM). Subsequently, use a cell disruptor to perform ultrasonic disruption for 40 minutes (45-60% amplitude, on 5 seconds, off 8 seconds), and collect the supernatant by centrifugation (8000 rpm, 4°C, 1 hour). The supernatant was collected by centrifugation under low temperature conditions to obtain the enzyme solution of the corresponding strain, which was frozen at -20°C.

[0123] (10) Preparation of enzyme powder:

[0124] Measure 50 ml of the enzyme solution from strain 4 and transfer it to a Strep-Tactin purification column. Incubate it in a 4°C refrigerator or ice box for 1 hour. Then, wash the purification column with 5×1CV (column volume) of Buffer W and elute it with 10×0.5CV of Buffer E (Buffer W containing 2.5 mM desthiobiotin) to obtain the pure LmrR protein with the Strep-tag II purification tag. Then, place it in a drying dish and pre-freeze it in a -20°C refrigerator for 48 hours. The sample is then placed in a freeze dryer for lyophilization. The cold trap temperature of the freeze dryer is -50°C and the drying chamber pressure is 100 Pa. Freeze-dry for 24 hours to obtain a solid powder of the enzyme, which is stored in a refrigerated container at 4°C.

[0125] In a 500 mL three-necked round-bottom flask, add 50 mL of DMSO solution, stir and add 43.9 g of omeprazole sulfide to completely dissolve it, add 250 mL of MOPS buffer, adjust the pH to 8.0 with glacial acetic acid, add 10 g of the synthesized artificial photoenzyme, transfer the reaction solution to a nitrogen-protected sealed container, maintain the temperature at 30 ° C, and illuminate under the UV LED array with a wavelength of 365 nm and 30 mW / cm 2 Under the control of irradiation intensity, esomeprazole was generated by light reaction. The reaction was completed after 10 hours. The conversion rate of omeprazole sulfide monitored by high performance liquid chromatography was 89.7%, and the ee value of the product esomeprazole was 99%.

[0126] Example 5

[0127] The present invention provides an application of an artificial photoenzyme as a catalyst for synthesizing an esomeprazole compound, which specifically comprises the following steps:

[0128] 1. Preparation of artificial photoenzyme

[0129] (1) Preparation of competent cells: DH5α and BL21(DE3) competent cells: E. coli DH5α and BL21(DE3) cells were purchased from Takara Biomedical Technology. E. coli DH5α and BL21(DE3) cells were then treated with calcium chloride to render them susceptible to foreign DNA, i.e., DH5α or BL21 competent cells.

[0130] The specific steps are as follows:

[0131] A single colony of Escherichia coli DH5α or BL21 was picked and inoculated into 10 mL of LB medium, and cultured at 37°C with shaking overnight for 14 h.

[0132] Inoculate into 100 mL of LB medium at a ratio of 1:50, culture with rapid shaking at 37°C for 2 to 3 hours, and stop the culture when the OD600 is approximately 0.5.

[0133] Pour into a sterile 50 mL centrifuge tube, place on ice for 30 min, centrifuge at 8000 rpm for 5 min, and discard the supernatant.

[0134] Add 20 mL of 0.1 mol / L CaCl2 pre-cooled solution to each tube, resuspend the bacterial pellet and transfer it to a tube, and continue to ice bath for 30 min.

[0135] Centrifuge at 8000 rpm for 5 min, discard the supernatant, add 6 mL of 0.1 mol / L CaCl2 pre-cooled solution, and then add 2 mL of 80% glycerol to resuspend the bacterial pellet.

[0136] Aliquot the competent cells into 200 μL cell solution per tube and store in a -80°C refrigerator until use.

[0137] (2) Plasmid construction: The lactococcal multidrug resistance regulator (LmrR) from Lactococcus cremoris subsp. cremoris MG1363 has a DNA binding region. Based on the artificial metalloenzyme construction strategy, K55 in the sequence was mutated to aspartic acid (D), and K59 was mutated to glutamine (Q). The codon-optimized LmrR gene sequence (including the C-terminal Strep-tag II purification tag) was synthesized by Genewiz / Azenta and constructed into the expression vector pET17b, obtaining the recombinant vector pET17b_LmrR.

[0138] (3) PCR to obtain esomeprazole synthase variant plasmid 1: Site-directed mutagenesis primers were designed and codon replacement was achieved at a specific site in the gene sequence by PCR amplification, with site R75 selected. The codons corresponding to the unnatural amino acids were all designed to be TAG, while the codons for the other amino acids were selected from codons commonly used in the LmrR nucleic acid sequence.

[0139] Forward primer F1: CGCGGATCCTTACGCATTAGCCTGCTGTTTGG (SEQ ID NO. 14)

[0140] Reverse primer R1: CCAAACAGCAGGCTAATGCGTAAGGATCCGCG

[0141] (4) Transformation of plasmids to obtain recombinant genetically engineered bacteria: After adding plasmid 1 to the EP tube containing competent cells, place on ice for 30 minutes; heat shock at 42°C for 90 seconds. To increase the transformation efficiency, repeat this heat shock step, then quickly place on ice for 3-5 minutes; add 400 μL of fresh LB liquid culture medium, place in a metal bath mixer at 37°C and 700 rpm for 1 hour; centrifuge at 4000 rpm for 5 minutes, discard 200 μL of culture medium, resuspend the bacteria, spread on a plate (containing ampicillin), and incubate at 37°C for 12-16 hours. Recombinant genetically engineered bacterial strain 1 was obtained.

[0142] (5) Preparation of genetically engineered bacteria: The recombinant E. coli genetically engineered strain, strain 1, was inoculated into 3 mL of fresh culture medium containing 100 μg / mL Amp+ and cultured overnight at 37°C and 200 rpm in a shaker. The pre-cultured bacterial solution was then transferred to 1 L of LB culture medium and cultured at 37°C until the OD600 reached 0.8 h. 100 μL of 1 M IPTG inducer was added, and the culture was continued at 30°C for 24 h. The bacterial slurry was collected by centrifugation at 8000 rpm and the supernatant was removed and stored in a -80°C freezer.

[0143] (6) Preparation of enzyme solution: Weigh 20 g of the bacterial sludge of strain 1 obtained by fermentation, resuspend the precipitate in buffer W (100 mM Tris-HCl, 150 mM NaCl, 10% glycerol, pH 8.0), and then add DNase I (final concentration 0.1 mg / mL, 10 mM MgCl2) and PMSF (final concentration 1.0 mM). Subsequently, use a cell disruptor to perform ultrasonic disruption for 40 minutes (45-60% amplitude, 5 seconds on, 8 seconds off), and collect the supernatant by centrifugation (8000 rpm, 4°C, 1 hour). The supernatant was collected by centrifugation under low temperature conditions to obtain the enzyme solution of the corresponding strain, which was frozen at -20°C.

[0144] (7) Preparation of enzyme powder:

[0145] Measure 50 ml of the enzyme solution from strain 1 and transfer it to a Strep-Tactin purification column. Incubate it in a 4°C refrigerator or ice box for 1 hour. Then, wash the purification column with 5×1CV (column volume) of Buffer W and elute it with 10×0.5CV of Buffer E (Buffer W containing 2.5 mM desthiobiotin) to obtain the pure LmrR protein with the Strep-tag II purification tag. Then, place it in a drying dish and pre-freeze it in a -20°C refrigerator for 30 hours. The sample is then placed in a freeze dryer for lyophilization. The freeze dryer has a cold trap temperature of -50°C and a drying chamber pressure of 100 Pa. Freeze-dry for 24 hours to obtain a solid enzyme powder, which is stored in a refrigerated container at 4°C.

[0146] 2. Synthesis of esomeprazole catalyzed by artificial photoenzyme

[0147] In a 500 mL three-necked round-bottom flask, add 50 mL of DMSO solution, add 15 mL of glycerol, stir and add 43.9 g of omeprazole sulfide to completely dissolve it, add 250 mL of MOPS buffer, adjust the pH to 7.5 with glacial acetic acid, add 10 g of the synthesized artificial photoenzyme, transfer the reaction solution to a sealed container protected by nitrogen, maintain the temperature at 30 ° C, and illuminate the UV LED array with a wavelength of 365 nm and 30 mW / cm 2 Under the control of irradiation intensity, esomeprazole was generated by light reaction. The reaction was completed after 10 hours. The conversion rate of omeprazole sulfide monitored by high performance liquid chromatography was 92.3%, and the ee value of the product esomeprazole was 99%.

[0148] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An application of an artificial photoenzyme as a catalyst for synthesizing an esomeprazole compound, characterized in that: The artificial photoenzyme is constructed by inserting a non-natural amino acid containing a benzophenone structure into a protein skeleton.

2. The use of an artificial photoenzyme as a catalyst for synthesizing an esomeprazole compound according to claim 1, characterized in that: The non-natural amino acid containing a benzophenone structure is benzophenone alanine or 3-fluoro substituted benzophenone alanine.

3. The use of an artificial photoenzyme as a catalyst for synthesizing an esomeprazole compound according to claim 1, characterized in that: The protein backbone is selected from LmrR or RamR.

4. The use of an artificial photoenzyme as a catalyst for synthesizing an esomeprazole compound according to claim 1, characterized in that: The substrate omeprazole sulfide shown in Formula 1 is catalyzed by an artificial photoenzyme to generate the esomeprazole compound shown in Formula 2. The reaction for synthesizing the esomeprazole compound is as follows:

5. The use of an artificial photoenzyme as a catalyst for synthesizing an esomeprazole compound according to claim 4, characterized in that: The non-natural amino acid containing a benzophenone structure is inserted into a specific site on the protein backbone, and the specific site is an amino acid site having a hydrophobic interaction with the substrate; preferably, the site is R75 or M89.

6. Use of an artificial photoenzyme as a catalyst for synthesizing an esomeprazole compound according to claim 4, characterized in that: The specific conditions of the reaction are: dissolving the omeprazole sulfide in a mixture of an organic solvent and a buffer solution, then adding an acid solution to adjust the pH of the solution to 6.0-8.0, then adding the artificial photoenzyme, transferring the reaction solution to a sealed container protected by a non-oxidizing gas, maintaining the temperature at 20-35°C, and reacting under 320nm-400nm ultraviolet light for 4-12 hours to produce the esomeprazole.

7. Use of an artificial photoenzyme as a catalyst for synthesizing an esomeprazole compound according to claim 6, characterized in that: The mass ratio of the omeprazole sulfide to the organic solvent is 1:(3-20); the concentration of the artificial photoenzyme in the reaction solution is 10-100 μmol / L.

8. The use of an artificial photoenzyme as a catalyst for synthesizing an esomeprazole compound according to claim 6, characterized in that: The method further comprises adding a stabilizer into the reaction solution, wherein the stabilizer is selected from glycerol, dithiothreitol or magnesium chloride; and the buffer solution is selected from 3-(N-morpholino)propanesulfonic acid buffer or 4-hydroxyethylpiperazineethanesulfonic acid buffer.

9. Use of an artificial photoenzyme as a catalyst for synthesizing an esomeprazole compound according to claim 6, characterized in that: After the esomeprazole is generated, the process further includes: centrifuging or membrane filtering the product solution, then adding ethyl acetate to extract the organic phase, concentrating the obtained organic phase by rotary evaporation, and purifying the concentrated product by column chromatography.

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