A process for the enzymatic preparation of chiral alpha-monofluorocyclopropane compounds with high enantioselectivity

Abstract

The present application relates to the technical field of biopharmacy and bio-chemical engineering, and particularly relates to a method for preparing chiral alpha-monofluorocyclopropane compounds with high enantioselectivity by enzyme method, which adopts a heme iron enzyme Rma NOD single mutant Rma NOD-Q52V or double mutant Rma NOD-Q52V V97I, and successfully realizes high-efficiency bio-catalytic conversion of a fluorobenzene ethylene substrate under the action of diazoacetic acid ethyl ester. The technology has excellent catalytic efficiency, and the substrate conversion rate is as high as 90%; the enantiomeric excess value (e.e.) of the product is more than 80%; and the reaction system is simple, the reaction conditions are mild (normal temperature and normal pressure), and the reaction system meets the requirements of green chemistry. Compared with the traditional chemical synthesis method, the bio-catalytic path of the present application has significant advantages in terms of atom economy, environmental friendliness and optical purity of the product, and provides an innovative solution for the synthesis of alpha-monofluorocyclopropane.

Description

Technical Field

[0001] This invention relates to the fields of biopharmaceutical and biochemical technologies, specifically to a method for the enzymatic preparation of chiral α-monofluorocyclopropane compounds with high enantioselectivity. Background Technology

[0002] Cyclopropane structures are widely found in various important natural products and bioactive molecules. Due to their unique conformational characteristics and electronic effects, they have become core structural units with significant application value in medicinal chemistry research. Furthermore, fluorine substituents are widely present in drug molecules. Introducing fluorine atoms during the structural modification of bioactive molecules can significantly regulate key properties such as hydrogen bonding ability, pharmacokinetics, and cell permeability without altering steric hindrance. Therefore, by integrating the structural advantages of cyclopropane with the special physicochemical properties of fluorine substituents, fluorocyclopropane compounds have become highly attractive pharmacodynamic groups in medicinal chemistry research.

[0003] Currently, the most common method for synthesizing chiral α-monofluorocyclopropanes is metal-asymmetric catalytic cyclopropanation of olefins. Previous studies have used copper and rhodium catalysts to achieve the synthesis of chiral α-monofluorocyclopropanes, but these methods all require heavy metal catalysts, which may pose a problem of heavy metal residues for pharmaceutical production. In recent years, enzyme catalysis has emerged as a highly promising method for achieving olefin cyclopropanation reactions. Previous researchers have used engineered heme iron enzymes to achieve highly stereoselective synthesis of chiral cyclopropane compounds. However, enzyme catalysis has not yet been reported for the reaction of α-monofluoroolefins with ethyl diazonate to generate chiral α-monofluorocyclopropane compounds.

[0004] In view of the above-mentioned defects, the inventors of this invention have finally obtained this invention after a long period of research and practice. Summary of the Invention

[0005] The purpose of this invention is to solve the problem of how to use enzymes as chiral catalysts in the enantioselective enzymatic catalysis of carbene insertion in fluorostyrene, and to provide a method for the enzymatic preparation of chiral α-monofluorocyclopropane compounds with high enantioselectivity.

[0006] To achieve the above objectives, this invention discloses a method for the enzymatic preparation of chiral α-monofluorocyclopropane compounds with high enantioselectivity, comprising the following steps:

[0007] Step S1: Dissolve the fluorinated ethylene substrate in ethanol to obtain a mixed solution;

[0008] Step S2: Add the mixed solution obtained in step S1 to a reaction buffer containing a heme ironase Rma NOD variant. After the reaction, a chiral α-monofluorocyclopropane compound is obtained. The heme ironase Rma NOD variant is either a heme ironase Rma NOD monovariate Rma NOD-Q52V or a heme ironase Rma NOD duplex variant Rma NOD-Q52V V97I.

[0009] The amino acid sequence of the heme ironase Rma NOD monomorph Rma NOD-Q52V is shown in SEQ ID NO.4; the amino acid sequence of the heme ironase Rma NOD duplex variant Rma NOD-Q52V V97I is shown in SEQ ID NO.5.

[0010] In step S1, the fluorostyrene compound substrate is any one of compounds Ia, IIa, IIIa, and IVa, and its structural formula is shown below:

[0011] .

[0012] In step S2, the reaction buffer solution also contains sodium dithionite and ethyl diazonium.

[0013] The concentration of ethyl diazonium acetate is 20 mM, and the concentration of sodium dithionite is 10 mM.

[0014] In step S2, the reaction buffer is a 50 mM phosphate buffer with pH=7.0.

[0015] In step S2, the concentration of the fluorinated ethylene substrate in the reaction system is 10 mM, and the final concentration of the heme ironase Rma NOD variant is OD. 600 = 40, the reaction is carried out under anaerobic conditions.

[0016] In step S2, the reaction conditions are pH 7.0, 20~25℃, and 220 rpm for 16 h.

[0017] In step S2, the chiral α-monofluorocyclopropane compound is any one of compounds Ib, IIb, IIIb, and IVb, and its structural formula is shown below:

[0018] .

[0019] The term "biocatalyst" as used in this invention refers to a reaction catalyzed by using enzymes or microbial cells as biocatalysts. Enzymes can exist both intracellularly and extracellularly, and biological cells can be living or dead cells.

[0020] Heme ironase RmaNOD and its duplex variant RmaNOD-Q52V V97I, derived from *Halophyta rubrum*, efficiently catalyze the carbene insertion reaction of fluorostyrene with ethyl diazonate in aqueous solution, successfully achieving highly enantioselective synthesis of chiral α-monofluorocyclopropane compounds. The enantiomeric excess (ee) of the product can reach over 80%, and the substrate conversion is as high as 90%. Furthermore, optimization of reaction conditions further improves catalytic efficiency and substrate adaptability, expanding the novel function of heme ironase in non-natural carbene transfer reactions. This catalytic system operates under ambient temperature and pressure in an aqueous phase, eliminating the need for an external coenzyme recycling system, simplifying the reaction process and reducing operating costs.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention is the first to apply heme iron enzyme Rma NOD and its dual variant Rma NOD-Q52V V97I to the enzymatic catalytic asymmetric cyclopropanation reaction of fluorostyrene, achieving excellent catalytic effect. It shows good conversion rate and high enantioselectivity for a variety of fluorostyrene substrates, and the product has high optical purity, providing a green and efficient new biocatalytic pathway for the construction of fluorinated chiral cyclopropane structural units.

[0022] The enzymatic method of the present invention for the preparation of chiral α-monofluorocyclopropane with high enantioselectivity has the following advantages compared with other chemical synthesis methods:

[0023] 1. The reaction system is simple, the conditions are mild (room temperature and pressure), the operation is safe, and the product yield is high with excellent enantioselectivity;

[0024] 2. The biocatalyst in this invention has good substrate compatibility and can be applied to a variety of fluorostyrene derivatives;

[0025] 3. The catalytic process does not require a metal catalyst, thus avoiding the problem of heavy metal residues and meeting the requirements of green pharmaceuticals and sustainable development. Attached Figure Description

[0026] Figure 1 This is the synthetic route of the present invention;

[0027] Figure 2 The chiral analysis spectrum of the racemic product of compound Ib;

[0028] Figure 3 The chiral analysis spectrum of compound Ib;

[0029] Figure 4 Chiral analysis spectrum of the racemic product of compound IIb;

[0030] Figure 5 The chiral analysis spectrum of compound IIb;

[0031] Figure 6 Chiral analysis spectrum of the racemic product of compound IIIb;

[0032] Figure 7 The chiral analysis spectrum of compound Ⅲb;

[0033] Figure 8 Chiral analysis spectrum of the racemic product of compound IVb;

[0034] Figure 9 The chiral analysis spectrum of compound IVb;

[0035] Figure 10 NMR of compound Ib 1 H NMR spectrum, the green curve in the figure is 1 The height of the integral curve of the H NMR spectrum represents the integral area of ​​each hydrogen signal (i.e., the proportion of hydrogen atoms).

[0036] Figure 11 NMR of compound IIb 1 H NMR spectrum, the green curve in the figure is 1 The height of the integral curve of the H NMR spectrum represents the integral area of ​​each hydrogen signal (i.e., the proportion of hydrogen atoms).

[0037] Figure 12 NMR of compound IIIb 1 H NMR spectrum, the green curve in the figure represents 1 The height of the integral curve of the H NMR spectrum represents the integral area of ​​each hydrogen signal (i.e., the proportion of hydrogen atoms).

[0038] Figure 13 NMR of compound IVb 1 H-NMR spectrum, the green curve in the figure represents... 1 The height of the integral curve of the H NMR spectrum represents the integral area of ​​each hydrogen signal (i.e., the proportion of hydrogen atoms). Detailed Implementation

[0039] The above-mentioned and other technical features and advantages of the present invention will be described in more detail below with reference to the accompanying drawings.

[0040] The 50 mM Kpi buffer (pH 7.0) in the example was prepared by dissolving 50 mmol of potassium dihydrogen phosphate in 1 L of pure water and adjusting the pH to 7.0 with NaOH.

[0041] Example 1

[0042] Expression of heme ironase Rma NOD using Escherichia coli:

[0043] 1. Preparation of recombinant vectors:

[0044] The preparation method of Rma NOD protein described in this embodiment is as follows:

[0045] (1) The Rma NOD expression vector was constructed by Nanjing Genscript Biotech Co., Ltd. using the pET-22b(+) vector (synthesized by Genscript). Specifically, the Rma NOD gene fragment was used as a template to clone the Rma NOD gene into the pET-22b(+) vector. The corresponding promoter was the T7 promoter, and the corresponding restriction enzyme sites were NdeI and XhoI. The resulting recombinant vector was denoted as RmaNOD_pET-22b(+). The DNA sequence of the Rma NOD gene is as follows:

[0046] ATGGCGCCGACCCTGTCGGAACAGACCCGTCAGTTGGTACGTGCGTCTGTGCCTGCACTGCAGAAACACTCAGTCGCTATTAGCGCCACGATGTATCGGCTGCTTTTCGAACGGTATCCCGAAACGCGGAGCTTGTTTGAACTTCCTGAGAGACAGATACACAAGCTTGCGTCGGCCCTGTTGGCCTACGCCCGTAGTATCGACAACCCATCGGCGTTACAGGCGGCCATC CGCCGCATGGTGCTTTCCCACGCACGCGCAGGAGTGCAGGCCGTCCATTATCCGCTGGTTTGGGAATGTTTGAGAGACGCTATAAAAGAAGTCCTGGGCCCGGATGCCACCGAGACCCTTCTGCAGGCGTGGAAGGAAGCCTATGATTTTTTAGCTCATTTACTGTCTACCAAGGAAGCGCAAGTCTACGCTGTGTTAGCTGAACTCGAGCACCACCACCACCACCAC (SEQ ID NO.1).

[0047] The amino acid sequence of 6X His is shown below: HHHHHH (SEQ ID NO.2).

[0048] 2. Expression of protein Rma NOD:

[0049] Expression of protein Rma NOD: The obtained plasmid Rma NOD was transformed into Escherichia coli BL21(DE3) by heat shock method and cultured overnight on TB plates containing a final concentration of 50 μg / mL ampicillin. Colonies that grew after overnight culture were considered positive colonies.

[0050] Positive single colonies containing recombinant plasmids were inoculated into TB liquid medium containing 50 μg / mL ampicillin and cultured at 37℃ and 200 rpm for 10-12 h with shaking. The cultured bacterial solution was then inoculated into fresh TB liquid medium containing 50 μg / mL ampicillin at a volume ratio of 1:50 and cultured at 37℃ and 200 rpm with shaking until the bacterial concentration OD600 was 0.8-1.2. Then, inducers IPTG (isopropyl-β-D-thiogalactopyranoside) and ALA (5-aminolevulinic acid) were added to a working concentration of 0.2 mM. The culture was continued at 16℃ and 180 rpm for 10-26 h, centrifuged for 20 min, and the bacterial cells were collected and resuspended.

[0051] The collected bacterial cells were resuspended in 50 mM Kpi buffer (pH=7.0) to obtain bacterial sludge containing Rma NOD protein.

[0052] The amino acid sequence of heme ironase Rma NOD:

[0053] MAPTLSEQTRQLVRASVPALQKHSVAISATMYRLLFERYPETRSLFELPERQIHKLASALLAYARSIDNPSALQAAIRRMVLSHARAGVQAVHYPLVWECLRDAIKEVLGPDATETLLQAWKEAYDFLAHLLSTKEAQVYAVLAELEHHHHHH (SEQ ID NO. 3).

[0054] The optimal reaction conditions were determined by measuring the yield of the heme ironase Rma NOD-catalyzed reaction under different pH conditions in the buffer solution. The reaction results are shown in Table 1 below.

[0055] Table 1. Synthesis of chiral α-monofluorocyclopropane under different conditions

[0056]

[0057] The optimal reaction buffer solution was determined to be KPI (pH=7), and the reaction temperature was 25 °C. Under these conditions, the initial yield could reach 64%, and the ee value was as high as 77%.

[0058] One round of evolution was carried out through site-specific semi-rational iterative mutation. The resulting heme ironase Rma NOD dual variant was used as a biocatalyst for the reaction. The reaction conditions were: buffer solution of KPI (pH=7) and reaction temperature of 25℃. The reaction results are shown in Table 2 below.

[0059] Table 2. Synthesis of chiral α-monofluorocyclopropane under different biocatalyst conditions in a single evolutionary cycle.

[0060]

[0061] The first round of screening yielded two heme ferase Rma NOD single variants, Rma NOD-Q52V and Rma NOD double variant Rma NOD-Q52V V97I. The amino acid sequence of the heme ferase Rma NOD single variant Rma NOD-Q52V is shown in SEQ ID NO.4, and the amino acid sequence of the heme ferase Rma NOD double variant Rma NOD-Q52V V97I is shown in SEQ ID NO.5.

[0062] The amino acid sequence of the heme ironase monoclonal variant Rma NOD-Q52V is as follows:

[0063] MAPTLSEQTRQLVRASVPALQKHSVAISATMYRLLFERYPETRSLFELPERVIHKLASALLAYARSIDNPSALQAAIRRMVLSHARAGVQAVHYPLVWECLRDAIKEVLGPDATETLLQAWKEAYDFLAHLLSTKEAQVYAVLAELEHHHHHH (SEQ ID NO. 4).

[0064] The amino acid sequence of the heme ironase dual variant Rma NOD-Q52V V97I is as follows:

[0065] MAPTLSEQTRQLVRASVPALQKHSVAISATMYRLLFERYPETRSLFELPERVIHKLASALLAYARSIDNPSALQAAIRRMVLSHARAGVQAVHYPLIWECLRDAIKEVLGPDATETLLQAWKEAYDFLAHLLSTKEAQVYAVLAELEHHHHHH (SEQ ID NO. 5).

[0066] Example 2

[0067] Enzymatic preparation of α-monofluorocyclopropane:

[0068] 24 µmol of fluorostyrene compound was dissolved in 60 µL of ethanol to prepare a fluorostyrene compound stock solution; 48 µmol of ethyl diazonite was dissolved in 60 µL of ethanol to prepare an ethyl diazonite stock solution; 24 µmol of sodium dithionite was dissolved in 60 µL of phosphate buffer to prepare a sodium dithionite stock solution. Under anaerobic conditions, 170 µL of phosphate buffer, 10 µL of sodium dithionite stock solution, 200 µL of bacterial sludge containing Rma NOD-Q52V V97I protein (final concentration OD 40), 10 µL of ethyl diazonite stock solution, and 10 µL of fluorostyrene compound stock solution were added to a reaction flask. After sealing, the mixture was stirred at room temperature for 16 h, and then samples were taken for chiral column analysis.

[0069] The detection method for the product is as follows (the detection method for the product in subsequent embodiments is the same as the detection method in this embodiment):

[0070] After the reaction was complete, an equal volume of dichloromethane was added, the mixture was vigorously shaken for 10 min, then allowed to stand for two minutes. The organic and aqueous layers were then separated by centrifugation at 8000 rpm for 10 min. The lower dichloromethane layer was carefully pipetted through an organic membrane, evaporated to dryness, and dissolved in 300 µL of n-hexane for sample preservation.

[0071] The optical rotation of chiral trifluoromethyl nitro groups was determined using Shimadzu LC columns, specifically CHIRALCEL® OJ-H solvent-resistant bonded chiral columns with silica gel coated with cellulose-tris(4-chlorophenylcarbamate). The mobile phase used was normal phase: n-hexane and isopropanol. The program was as follows: column temperature 40 °C, detector absorption wavelengths: 214 nm and 254 nm, flow rate 1 mL / min, and isocratic elution with 1% isopropanol.

[0072] The enantiomeric excess (ee %) of the product chiral monofluorocyclopropane is calculated by the following formula:

[0073] Enantiomer excess (ee%) ,

[0074] In the formula, S is the peak area of ​​(S)-monofluorocyclopropane, and R is the peak area of ​​(R)-monofluorocyclopropane.

[0075] The retention time of (S)-monofluorocyclopropane was 10.7 min, and the retention time of (R)-monofluorocyclopropane was 12.6 min.

[0076] Example 3

[0077] 24 µmol of fluorostyrene substrate Ia was dissolved in 60 µL of ethanol to prepare a fluorostyrene stock solution; 48 µmol of ethyl diazonite was dissolved in 60 µL of ethanol to prepare an ethyl diazonite stock solution; and 24 µmol of sodium dithionite was dissolved in 60 µL of phosphate buffer to prepare a sodium dithionite stock solution. Under anaerobic conditions, 170 µL of phosphate buffer, 10 µL of sodium dithionite stock solution, 200 µL of bacterial sludge containing Rma NOD-Q52V V97I protein (final concentration OD 40), 10 µL of ethyl diazonite stock solution, and 10 µL of fluorostyrene stock solution were added to a reaction flask. After sealing, the mixture was stirred at room temperature for 16 h, and then samples were taken for chiral column analysis. The chromatogram is shown in [Figure number missing]. Figure 3 (Sample reacted for 16 h, retention time 11.6 min). The chiral analysis chromatogram of the racemic product of compound Ib is shown below. Figure 2 The yield of compound Ib was 90%, with an optical purity of ee% of 80.7%, and NMR... 1 H NMR spectrum as follows Figure 10 As shown.

[0078] Example 4

[0079] 24 µmol of fluorostyrene substrate IIa was dissolved in 60 µL of ethanol to prepare a fluorostyrene stock solution; 48 µmol of ethyl diazonite was dissolved in 60 µL of ethanol to prepare an ethyl diazonite stock solution; and 24 µmol of sodium dithionite was dissolved in 60 µL of phosphate buffer to prepare a sodium dithionite stock solution. Under anaerobic conditions, 170 µL of phosphate buffer, 10 µL of sodium dithionite stock solution, 200 µL of bacterial sludge containing Rma NOD-Q52V V97I protein (final concentration OD 40), 10 µL of ethyl diazonite stock solution, and 10 µL of fluorostyrene stock solution were added to a reaction flask. After sealing, the mixture was stirred at room temperature for 16 h, and then samples were taken for chiral column analysis. The chromatogram is shown in [Figure number missing]. Figure 5 (Sample reacted for 16 h, retention time 11.9 min). The chiral analysis chromatogram of the racemic product of compound IIb is shown below. Figure 4 The yield of compound IIb was 90%, with an optical purity of ee% of 97.0%, and NMR... 1 H NMR spectrum as follows Figure 11 As shown.

[0080] Example 5

[0081] 24 µmol of fluorostyrene substrate IIIa was dissolved in 60 µL of ethanol to prepare a fluorostyrene stock solution; 48 µmol of ethyl diazonite was dissolved in 60 µL of ethanol to prepare an ethyl diazonite stock solution; and 24 µmol of sodium dithionite was dissolved in 60 µL of phosphate buffer to prepare a sodium dithionite stock solution. Under anaerobic conditions, 170 µL of phosphate buffer, 10 µL of sodium dithionite stock solution, 200 µL of bacterial sludge containing Rma NOD-Q52V V97I protein (final concentration OD 40), 10 µL of ethyl diazonite stock solution, and 10 µL of fluorostyrene stock solution were added to a reaction flask. After sealing, the mixture was stirred at room temperature for 16 h, and then samples were taken for chiral column analysis. The chromatogram is shown in [Figure number missing]. Figure 7 (Sample reacted for 16 h, retention time 14.0 min). The chiral analysis chromatogram of the racemic product of compound IIIb is shown below. Figure 6 The yield of compound IIIb was 90%, with an optical purity of ee% of 96.4%, and NMR... 1 H NMR spectrum as follows Figure 12 As shown.

[0082] Example 6

[0083] 24 µmol of fluorostyrene substrate IVa was dissolved in 60 µL of ethanol to prepare a fluorostyrene stock solution; 48 µmol of ethyl diazonite was dissolved in 60 µL of ethanol to prepare an ethyl diazonite stock solution; and 24 µmol of sodium dithionite was dissolved in 60 µL of phosphate buffer to prepare a sodium dithionite stock solution. Under anaerobic conditions, 170 µL of phosphate buffer, 10 µL of sodium dithionite stock solution, 200 µL of bacterial sludge containing Rma NOD-Q52V V97I protein (final concentration OD 40), 10 µL of ethyl diazonite stock solution, and 10 µL of fluorostyrene stock solution were added to a reaction flask. After sealing, the mixture was stirred at room temperature for 16 h, and then samples were taken for chiral column analysis. The chromatogram is shown in [Figure number missing]. Figure 9 (Sample reacted for 16 h, retention time 30.9 min). The chiral analysis chromatogram of the racemic product of compound IVb is shown below. Figure 8 The yield of compound IVb was 90%, with an optical purity of ee% of 92.5%, and NMR... 1 H NMR spectrum as follows Figure 13 As shown.

[0084] The above description is merely a preferred embodiment of the present invention and is illustrative rather than restrictive. Those skilled in the art will understand that many changes, modifications, and even equivalents can be made within the spirit and scope defined by the claims of the present invention, all of which will fall within the protection scope of the present invention.

Claims

1. A method for the enzymatic preparation of chiral α-monofluorocyclopropane compounds with high enantioselectivity, characterized in that, Includes the following steps: Step S1: Dissolve the fluorinated ethylene substrate in ethanol to obtain a mixed solution; Step S2: Add the mixed solution obtained in step S1 to a reaction buffer containing a heme ironase Rma NOD variant. After the reaction, a chiral α-monofluorocyclopropane compound is obtained. The heme ironase Rma NOD variant is either a heme ironase RmaNOD monovariate Rma NOD-Q52V or a heme ironase Rma NOD duplex variant Rma NOD-Q52V V97I. The amino acid sequence of the heme ironase Rma NOD monoclonal variant Rma NOD-Q52V is shown in SEQ ID NO.4; the amino acid sequence of the heme ironase Rma NOD duplex variant Rma NOD-Q52V V97I is shown in SEQ ID NO.

5. In step S1, the fluorostyrene compound substrate is any one of compounds Ia, IIa, IIIa, and IVa, and its structural formula is shown below: ； In step S2, the reaction buffer is a 50 mM phosphate buffer with pH=7.

0. The reaction buffer solution also contains sodium dithionite and ethyl diazonite, wherein the concentration of ethyl diazonite is 20 mM and the concentration of sodium dithionite is 10 mM. In the reaction system, the concentration of the fluorinated vinyl substrate was 10 mM, and the final concentration of the heme ironase Rma NOD variant was OD. 600 = 40, the reaction was carried out under anaerobic conditions; the reaction conditions were pH 7.0, 20~25℃, 220 rpm for 16 h.

2. The method for preparing chiral α-monofluorocyclopropane compounds with high enantioselectivity by enzymatic method as described in claim 1, characterized in that, In step S2, the chiral α-monofluorocyclopropane compound is any one of compounds Ib, IIb, IIIb, and IVb, and its structural formula is shown below: 。

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