Method for the enzymatic production of (s)-n-tert-butoxycarbonyl-3-hydroxypyrrolidine by co-expression of enzymes

Abstract

The application discloses a method for preparing (S)-N-tert-butyloxycarbonyl-3-hydroxypyrrolidine through co-expression enzyme catalysis, and belongs to the technical field of biochemistry. (S)-N-tert-butyloxycarbonyl-3-hydroxypyrrolidine is prepared through co-expression enzyme catalysis reaction with N-tert-butyloxycarbonyl-3-pyrrolidone as a substrate, and glucose is subjected to coenzyme circulation under the action of glucose dehydrogenase. The conversion rate of enzyme catalysis reaches more than 99%. After decolorization, rotary evaporation and drying, the purity of the product is detected to be greater than 99%, the enantiomeric excess is greater than 99% ee, and the yield reaches 91%. The method has the characteristics of high conversion rate and high yield, and is easy to be industrialized.

Description

Technical Field

[0001] This invention discloses a method for preparing (S)-N-tert-butoxycarbonyl-3-hydroxypyrrolidine by co-expression enzyme catalysis, which belongs to the field of biochemical technology. Background Technology

[0002] (S)-3-hydroxypyrrolidine is an important structural fragment in drug design and is commonly used for structural modification of various drugs. As a key structural fragment, dafinarax is a selective muscarinic M3 receptor antagonist developed by Pfizer. Clinically, it is used to treat overactive bladder symptoms such as urge incontinence, urinary urgency, and urinary frequency. Its selective action on the M3 receptor significantly reduces the incidence of adverse central nervous system and cardiovascular reactions, demonstrating good efficacy, tolerability, and safety.

[0003] Barnidipine is a novel long-acting dihydropyridine calcium channel blocker developed by Yamanouchi Corporation of Japan. It exhibits high vascular selectivity, strong and sustained action, and protective effects on the heart and kidneys, making it a clinically used drug for treating essential hypertension and renal hypertension. Currently, the market supply of (R)-3-hydroxypyrrolidine is relatively abundant, while the supply of (S)-3-hydroxypyrrolidine is extremely scarce. Existing literature mainly describes its synthesis methods, including those described in J. Mol. Catal. A: Chem., 2006, 104 and Tetrahedron, 2006, 62, 5763. These methods involve cyclizing L-malic acid with benzylamine to generate (S)-1-benzylmalimide, followed by reduction with lithium aluminum hydride and then debenzylation under pressure to obtain the target compound. Synthetic Metals, 2006, 156, 671. Using (S)-4-amino-2-hydroxybutyric acid as a starting material, (S)-3-trimethylsiloxy-2-pyrrolidone was obtained via hydroxyl silanization protection and intramolecular acylation cyclization, followed by reduction with lithium aluminum hydride to yield the product. In the above synthetic method, lithium aluminum hydride is used due to its high reactivity, difficult post-processing, the requirement for an absolutely anhydrous solvent in the reaction system, and the partial racemization of the chiral center.

[0004] K. Labelled Compd. Rad., 2001, 44, 31-41 and WO2007050522A1 describe a method using L-hydroxyproline as a starting material, involving decarboxylation, tert-butoxycarbonyl protection of the amino group, Mitsunobu reaction, and hydrolysis of the ester bond to finally obtain (S)-1-N-tert-butoxycarbonyl-3-hydroxypyrrolidine. This method employs column chromatography purification, which is unsuitable for industrial production.

[0005] Therefore, it is necessary to develop an economical synthetic route suitable for the industrial production of (S)-1-N-tert-butoxycarbonyl-3-hydroxypyrrolidine. Summary of the Invention

[0006] To overcome the aforementioned technical deficiencies, this invention provides a method for preparing (S)-N-tert-butoxycarbonyl-3-hydroxypyrrolidine via co-expression enzyme catalysis. Using N-tert-butoxycarbonyl-3-pyrrolidone as a substrate, (S)-N-tert-butoxycarbonyl-3-hydroxypyrrolidine is obtained through a co-expression enzyme catalytic reaction, while glucose undergoes a coenzyme cycle under the action of glucose dehydrogenase. The enzyme-catalyzed conversion rate reaches over 99%. After decolorization, rotary evaporation, and drying, the product purity is >99%, the enantiomeric excess is >99%ee, and the yield reaches 91%. This method features high conversion and high yield, and is easily industrialized.

[0007] The method for preparing (S)-N-tert-butoxycarbonyl-3-hydroxypyrrolidine by co-expression enzyme catalysis according to the present invention comprises the following steps: using N-tert-butoxycarbonyl-3-pyrrolidone as a substrate, a co-expression enzyme catalytic reaction is performed to obtain (S)-N-tert-butoxycarbonyl-3-hydroxypyrrolidine; the reaction equation is shown below:

[0008] Furthermore, in the above technical solution, the co-expressed enzyme is selected from engineered bacterial ketone reductase and glucose dehydrogenase.

[0009] Furthermore, in the above technical solution, the ketone reductase (KRED) expression strain is selected from 21Duet-KG, 21Duet-GK, 21pET-KG, or 21pET-GK.

[0010] Furthermore, in the above technical solution, the ketone reductase (KRED) gene sequence is shown in SEQ ID NO.1.

[0011] Furthermore, in the above technical solution, the glucose dehydrogenase (GDH) gene sequence is shown in SEQ ID NO.3.

[0012] Furthermore, in the above technical solution, the reaction process is as follows: using N-tert-butoxycarbonyl-3-hydroxypyrrolidone as the reaction substrate, glucose and water are added, stirred and dissolved, and then alkali is added to adjust the pH to 6.0-7.5. Then, NADP+ and crude enzyme are added to carry out the reaction.

[0013] Furthermore, in the above technical solution, the reaction process temperature is controlled at 25~35℃, and the alkali is selected from sodium hydroxide aqueous solution.

[0014] Furthermore, in the above technical solution, the product post-processing operation is as follows: after the reaction is completed, ethyl acetate is added for extraction, activated carbon is added to the organic phase for decolorization and then filtered; saturated sodium chloride solution is added for separation, anhydrous magnesium chloride is added to the organic phase for drying, vacuum concentration is carried out, petroleum ether is added for crystallization, and drying is carried out to obtain (R)-N-tert-butoxycarbonyl-3-hydroxypyrrolidine.

[0015] Beneficial effects of the invention A. The target product is prepared by co-expression dual-enzyme coupling catalysis, using whole-cell crude enzymes, which makes the operation simpler, lower in cost, and more environmentally friendly.

[0016] B. The substrate concentration reaches 120 g / L, the overall reaction time is short, completed within 24 hours, with high process efficiency and a yield of over 90%. Attached Figure Description

[0017] Figure 1 shows the enzyme activity detection of ketone reductase and glucose dehydrogenase in the co-expressed engineered bacteria; Figure 2 shows the conversion rate detection graph of the enzyme-catalyzed reaction; Figure 3 shows the purity detection results of the product after extraction and purification. Detailed Implementation

[0018] Construction of engineered bacteria for the basic expression of ketone reductase (KRED) and glucose dehydrogenase (GDH)

[0019] The amino acid sequence of ketone reductase was codon-optimized and synthesized, then directly cloned into the pET-28a(+) expression vector between the BamHI and EcoRI restriction sites to obtain the ketone reductase expression vector pET 28-KRED. The recombinant plasmid was then heat-shock transformed into *E. coli*. E. coli 21-KRED, an engineered strain expressing racemic enzyme, was obtained from BL21 (DE3) competent cells.

[0020] The amino acid sequence of glucose dehydrogenase was codon-optimized and synthesized, then directly cloned into the pET-28a(+) expression vector between the BamHI and EcoRI restriction sites to obtain the glucose dehydrogenase expression vector pET28-GDH. The recombinant plasmid was then heat-shocked into *E. coli*. E. coli BL21 (DE3) competent cells were used to obtain dehydrogenase-expressing engineered bacteria 21-GDH.

[0021] Ketoreductase (KRED) gene sequence SEQ ID NO.1 Amino acid SEQ ID NO. 2 of ketoreductase (KRED) MAYTVITGANGFIAQHIKILLSNGHKVIGTVRTQEKADTIANLFDNENLELEIVPDLNAVEAFDPLFKKYNTQIKYHTASPVDSTLTDLQNDFINTAITGTLSVFNAIKKYAADSVESVVYTSSSVAGANFKTFLDPTAVINEESWNPDDREDVVDILSAYSVSKKLAEKAAWNFKEENKDVIKFRLSTVNPFFVTGPQAFDETAKGKLNTTASYFEAILASKPDGNLAPFIGSPAVDVRDYAKAHYLALTEPRFDGQRIFLDGENYTTQQWYDVIHEKFPELNGKIAKGQPGTNKFKEGSMAKMFSKSKKLLGFDQIPLKQTITDGMRQLLRVRGIPQN* Glucose dehydrogenase (GDH) gene sequence SEQ ID NO.3 Glucose dehydrogenase (GDH) amino acid sequence SEQ ID NO.4 MTEQKAIVTDAPKGGVKYTTIDMPEPEHYDAKLSPVYIGICGTDRGEVAGALSFTYNPEGENFLVLGHEALLRVDDARDNGYIKKGDLVLPLVRRPGKCINCRIGRQDNCSIGDPDKHEAGITGLHGFMRDVIYDDIEYLVKVEDPELGRIAVLTEPLKNVMKAFEVFDVVSKRSIFFGD DSTLIGKRMVIIGSGSEAFLYSFAGVDRGFDVTMVNRHDETENKLKIMDEFGVKFANYLKDMPEKIDLLVDTSGDPTTTFKFLRKVNNAGVVILFGTNGKAPGYPVDGEDIDYIVERNITIAGSVDAAKIHYVQALQSLSNWNRVHPDAMKSIITYEAKPETNIFFQKPHGEIKTVIKWQ* Construction of co-expression engineered bacteria

[0022] The PCR primers used for constructing the co-expression vector are shown in Table 1, the PCR system is shown in Table 2, and the PCR conditions are shown in Table 3.

[0023] Table 1 Primers used for PCR

[0024] Table 2 PCR reaction system

[0025] Table 3 PCR reaction parameters

[0026] The GDH gene sequence was amplified from the expression vector pET28-GDH using primers GDH-F1 / GDH-R1, and then cloned into the pET28-KRED vector between the EcoRI and NotI restriction sites to obtain the co-expression vector pET28-KG. The recombinant plasmid was then heat-shocked into *E. coli*. E. coli Racemic enzyme expression engineered strain 21pET-KG was obtained from BL21 (DE3) competent cells.

[0027] The KRED gene sequence was amplified from the expression vector pET28-KRED using primers KRED-F1 / KRED-R1, and then cloned into the pET28-GDH vector between the EcoRI and NotI restriction sites to obtain the co-expression vector pET28-GK. The recombinant plasmid was then heat-shocked into E. coli. E. coli Racemic enzyme expression engineered strain 21pET-GK was obtained from BL21 (DE3) competent cells.

[0028] The KRED gene sequence was amplified from the expression vector pET28-KRED using primers KRED-F2 / KRED-R2 and cloned into the pRSFDuet-1 vector between the NcoⅠ and NotⅠ restriction sites to obtain the expression vector pRSFDuet-KRED. Subsequently, the GDH gene sequence was amplified from the expression vector pET28-GDH using primers GDH-F2 / GDH-R2 and cloned into the pRSFDuet-KRED vector between the NdeⅠ and AvrⅡ restriction sites to obtain the co-expression vector pRSFDuet-KG. The recombinant plasmid was then heat-shock transformed into *E. coli*. E. coli Racemic enzyme expression engineered strain 21Duet-KG was obtained from BL21 (DE3) competent cells.

[0029] The GDH gene sequence was amplified from the expression vector pET28-GDH using primers GDH-F3 / GDH-R3 and cloned into the pRSFDuet-1 vector between the NcoI and NotI restriction sites to obtain the expression vector pRSF Duet-GDH. Subsequently, the KRED gene sequence was amplified from the expression vector pET28-KRED using primers KRED-F3 / KRED-R3 and cloned into the pRSFDuet-GDH vector between the NdeI and AvrII restriction sites to obtain the co-expression vector pRSFDuet-GK. The recombinant plasmid was then heat-shock transformed into *E. coli*. E. coli Racemic enzyme expression engineered strain 21Duet-GK was obtained from BL21(DE3) competent cells. Example 1 I. Cultivation of engineered bacteria that co-express enzymes

[0030] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0031] TB liquid culture medium: tryptone 12 g / L, yeast extract 24 g / L, glycerol 4 mL / L, sodium chloride 10 g / L, dipotassium hydrogen phosphate 12.54 g / L, potassium dihydrogen phosphate 2.313 g / L.

[0032] The engineered bacteria were inoculated into 5 mL of LB kanamycin-resistant (50 μg / mL) liquid medium and cultured overnight at 37°C and 220 rpm (12–16 h). Subsequently, they were transferred to 1 L of fresh TB liquid medium with the same resistance and cultured at 37°C and 220 rpm with shaking until the OD600 reached 0.6–0.8. IPTG inducer was then added to a final concentration of 0.2 mM, and the culture was induced at 28°C for 20 h. After the culture was completed, the fermentation broth was centrifuged at 8000 rpm for 20 min, the supernatant was discarded, and the bacterial cells were collected and stored at -80°C for later use. The frozen whole-cell culture was used as crude enzyme for enzymatic reactions. II. Determination of Ketoreductase Activity

[0033] At 30°C and pH 7.0, the substrate 1-tert-butoxycarbonyl-3-pyrrolidone is converted to (R)-1-tert-butoxycarbonyl-3-hydroxymethylpyrrolidone under the catalysis of ketoreductase (KRED) and coenzyme NADPH. One enzyme activity unit (U) is defined as the number of micromoles of NADP+ generated from NADPH per gram of enzyme per minute. 3 g of frozen whole bacterial cells were taken, resuspended in 15 mL of PBS buffer (20 mM, pH 7.5), sonicated for 20 min, centrifuged at 8000 rpm for 10 min, and the supernatant was collected to obtain the crude enzyme solution. In a 1 cm cuvette with a total volume of 4 mL, add 3.6 mL of 20 mM PBS buffer and 200 μL of 100 mM substrate solution, then add 100 μL of diluted enzyme solution. Mix well and zero the sample at 340 nm. Add 100 μL of 20 mM NADPH solution, mix well, and then measure at 340 nm. Measure the absorbance every 30 s and calculate the ketone reductase activity according to the NADPH standard curve. III. Determination of glucose dehydrogenase activity

[0034] At 30℃ and pH 7.0, glucose was converted to gluconic acid by glucose dehydrogenase using NADP as a coenzyme. In this process, the number of micromoles of NADPH generated from NADP per minute by each gram of glucose dehydrogenase is defined as one enzyme activity unit (U). 3 g of frozen whole bacterial cells were taken, resuspended in 15 mL of PBS buffer (20 mM, pH 7.5), sonicated for 20 min, centrifuged at 8000 rpm for 10 min, and the supernatant was collected to obtain the crude enzyme solution. In a 1 cm cuvette with a total volume of 4 mL, add 3.6 mL of 20 mM PBS buffer and 200 μL of 2 M substrate solution, then add 100 μL of diluted enzyme solution. After mixing thoroughly, zero the sample at 340 nm. Add 100 μL of 20 mM NADP solution, mix well, and then detect the absorbance at 340 nm. Measure the absorbance every 30 s and calculate the ketone reductase activity according to the NADPH standard curve. IV. Enzyme Activity Detection of Different Co-expressed Engineered Bacteria

[0035] Following the above method, the enzyme activities of engineered bacteria expressing ketone reductase 21-KRED and glucose dehydrogenase 21-GDH were detected, respectively. Subsequently, the enzyme activities expressed individually were used as controls (100%), and the activities were further measured. The activities of ketone reductase (KRED) and glucose dehydrogenase (GDH) in co-expressed strains 21Duet-KG, 21Duet-GK, 21pET-KG, and 21pET-GK were measured. As shown in Figure 1, the engineered strain 21pET-GK showed the highest KRED and GDH activities. V. Optimization of Enzyme Catalysis Process

[0036] In a 500 mL reaction system, N-tert-butoxycarbonyl-3-pyrrolidone was added as the substrate, along with the corresponding mass of glucose and 450 mL of water. Stirring was started until the glucose dissolved. The mixture was then adjusted with NaOH solution. Adjust the pH to 6.0–7.5, add 0.3% NADP+ (substrate mass), and finally add a certain amount of 21pET-GK crude enzyme. Make up the volume to 500 mL with water and begin the reaction. Control the reaction temperature at 25–35℃ and maintain the pH at 6.0–7.5 using 2.5 M NaOH solution. After 20 h, take samples for HPLC analysis. The conversion rate is expressed as the peak area ratio of product to substrate (Table 4).

[0037] Table 4 Optimization of enzyme-catalyzed reaction parameters

[0038] VI. Enzyme-catalyzed preparation of (S)-1-N-tert-butoxycarbonyl-3-hydroxypyrrolidine Enzyme-catalyzed reaction: 120 g of N-tert-butoxycarbonyl-3-pyrrolidone was weighed and added to a reaction flask, followed by 140 g of glucose and 950 mL of water. Stirring was started until the glucose dissolved. The pH was adjusted to 7.0 with NaOH solution, 0.4 g of NADP+ was added, followed by 20 g of 21pET-GK crude enzyme. The volume was brought to 1 L with water, and the reaction was initiated. The reaction temperature was controlled at 30℃, and the pH was maintained at 6.5-7.0 with NaOH solution. After 20 h of reaction, the conversion rate reached over 99% (Figure 2).

[0039] Product extraction and purification: Add half the volume of ethyl acetate to the reaction solution and stir for 30 min. Collect the organic phase and repeat the extraction twice. Combine the organic phases. Add 10 g of activated carbon to the organic phase, stir at 40℃ for decolorization, and then filter. Add 500 L of saturated sodium chloride solution and stir to remove water. Separate the liquid and collect the organic phase. Add 20 g of anhydrous magnesium chloride to the organic phase to remove water, stir for 3 h, and then filter. Rotary evaporate under reduced pressure at 45℃. After rotary evaporation, add 300 mL of petroleum ether, stir at -5℃ to crystallize, and filter. Repeat the rotary evaporation process for the uncrystallized portion, stir to crystallize, combine the crystals, and dry to obtain the product (S)-1-N-tert-butoxycarbonyl-3-hydroxypyrrolidine. Sample testing showed a purity >99% (Figure 3), chirality >99%ee, and a molar yield of 91%.

[0040] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing (S)-N-tert-butoxycarbonyl-3-hydroxypyrrolidine via co-expression enzyme catalysis, characterized in that, The process includes the following steps: using N-tert-butoxycarbonyl-3-pyrrolidone as a substrate, a co-expression enzyme catalyzed reaction is used to obtain (S)-N-tert-butoxycarbonyl-3-hydroxypyrrolidone.

2. The method for preparing (S)-N-tert-butoxycarbonyl-3-hydroxypyrrolidine by co-expression enzyme catalysis according to claim 1, characterized in that: The co-expressed enzymes are selected from engineered bacterial ketone reductase and glucose dehydrogenase.

3. The method for preparing (S)-N-tert-butoxycarbonyl-3-hydroxypyrrolidine by co-expression enzyme catalysis according to claim 2, characterized in that: The ketone reductase expression strains are selected from 21Duet-KG, 21Duet-GK, 21pET-KG, or 21pET-GK.

4. The method for preparing (S)-N-tert-butoxycarbonyl-3-hydroxypyrrolidine by co-expression enzyme catalysis according to claim 3, characterized in that: The ketone reductase gene sequence is shown in SEQ ID NO.

1.

5. The method for preparing (S)-N-tert-butoxycarbonyl-3-hydroxypyrrolidine by co-expression enzyme catalysis according to claim 2, characterized in that: The glucose dehydrogenase gene sequence is shown in SEQ ID NO.

3.

6. The method for preparing (S)-N-tert-butoxycarbonyl-3-hydroxypyrrolidine catalyzed by the co-expression enzyme according to claim 1, characterized in that, The reaction procedure was as follows: using N-tert-butoxycarbonyl-3-hydroxypyrrolidone as the substrate, glucose and water were added, stirred to dissolve, and then the pH was adjusted to 6.0-7.5 with alkali. NADP was then added. + It reacts with the crude enzyme.

7. The method for preparing (S)-N-tert-butoxycarbonyl-3-hydroxypyrrolidine catalyzed by the co-expression enzyme according to claim 1, characterized in that, The post-processing of the product was as follows: after the reaction was completed, ethyl acetate was added for extraction, the organic phase was decolorized with activated carbon and then filtered; saturated sodium chloride solution was added for separation, the organic phase was dried with anhydrous magnesium chloride, concentrated under reduced pressure, petroleum ether was added for crystallization, and dried to obtain (S)-1-N-tert-butoxycarbonyl-3-hydroxypyrrolidine.

8. The method for preparing (S)-1-N-tert-butoxycarbonyl-3-hydroxypyrrolidine catalyzed by the co-expression enzyme according to claim 1, characterized in that: The reaction process is controlled at a temperature of 25~35℃, and the alkali is selected from an aqueous solution of sodium hydroxide.

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