Alcohol dehydrogenase mutants and their use in enzymatic synthesis of loratadine intermediates

Abstract

The application discloses an alcohol dehydrogenase mutant and application thereof in enzymatic synthesis of a loratadine intermediate. A plurality of alcohol dehydrogenase mutants are developed, and a loratadine chiral intermediate is obtained through an enzyme catalytic synthesis method based on the mutants, wherein the reaction condition is mild, the environment is friendly, the reaction has high regional selectivity and stereoselectivity, the reaction conversion rate is high, the chiral purity of the product is high, the enzyme consumption is low, the preparation cost is low, the method is suitable for industrial production, the method avoids the problems of difficult chiral resolution and heavy metal residue in a product in a conventional method, and the method makes up for the deficiencies of a traditional chemical method.

Description

Technical Field

[0001] This invention relates to the field of biosynthesis technology, and in particular to alcohol dehydrogenase mutants and their application in the enzymatic synthesis of lorlatinib intermediates. Background Technology

[0002] Lorlatinib, a third-generation ALK / ROS1 tyrosine kinase inhibitor, is one of the core targeted therapies for the treatment of non-small cell lung cancer. This drug demonstrates good clinical efficacy against mutant cell lines resistant to first- and second-generation ALK inhibitors and exhibits significant blood-brain barrier penetration, effectively controlling brain metastases. The lorlatinib molecule contains a chiral center, the stereochemical purity of which directly affects the binding affinity between the drug and the target, in vivo efficacy, and drug safety. Therefore, developing a synthetic process that can efficiently and selectively construct this chiral center is of great significance for ensuring drug quality, reducing the risk of toxic side effects, and meeting global clinical needs.

[0003] (S)-1-(2-iodo-5-fluorophenyl)ethanol is a key chiral intermediate in the synthesis of lorlatinib. Currently, the preparation of this intermediate mainly relies on traditional chemical synthesis methods, which generally suffer from high energy consumption, poor process safety, and low stereoselectivity. Taking the racemic resolution route as an example, its theoretical maximum yield does not exceed 50%, resulting in poor atom economy, causing not only a large waste of raw materials but also a serious environmental burden. In contrast, bio-enzymatic catalysis is considered a green synthetic alternative strategy, but it still faces challenges in industrial applications: wild-type alcohol dehydrogenases have insufficient catalytic activity and stereoselectivity for the poorly water-soluble and large molecular weight substrate 2-iodo-5-fluoroacetophenone; in addition, traditional enzyme catalysis systems mostly rely on exogenous coenzymes and their complex regeneration systems, resulting in lengthy processes, high costs, and difficulty in meeting the requirements of large-scale production.

[0004] Therefore, there is an urgent need to develop a novel biocatalytic method for 2-iodo-5-fluoroacetophenone that has high catalytic activity and high stereoselectivity and can be produced without relying on an exogenous coenzyme cycle, so as to achieve a green, economical, and industrially suitable synthesis process for (S)-1-(2-iodo-5-fluorophenyl)ethanol. Summary of the Invention

[0005] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide various alcohol dehydrogenase mutants and their applications in the enzymatic synthesis of lorlatinib intermediates. Based on the obtained alcohol dehydrogenase mutants, this invention can obtain lorlatinib chiral intermediates through enzymatic synthesis. The reaction conditions are mild, environmentally friendly, and exhibit high regioselectivity and stereoselectivity. It boasts high conversion rates, high product chiral purity, low enzyme consumption, and low preparation costs, making it suitable for industrial production. It avoids the difficulties in chiral resolution and heavy metal residues in the product found in conventional methods, thus overcoming the shortcomings of traditional chemical methods.

[0006] A first aspect of the present invention provides an alcohol dehydrogenase mutant, said alcohol dehydrogenase mutant comprising: (1) An alcohol dehydrogenase mutant obtained by substituting at least one amino acid from A48, I51, V88, A136, and V183 based on the sequence shown in SEQ ID NO:1; or (2) Based on (1), a mutant of alcohol dehydrogenase with the same function as (1) is further modified by substitution and / or deletion and / or addition of one or more amino acids.

[0007] In some embodiments of the present invention, the number of amino acid residues replaced, deleted, or added is 1-5.

[0008] In some embodiments of the present invention, the number of amino acid residues replaced, deleted, or added is 1, 2, 3, 4, or 5.

[0009] In some embodiments of the present invention, the alcohol dehydrogenase mutant has at least 95%, 96%, 97%, 98%, or 99%, or about 100%, sequence identity with respect to the sequence shown in SEQ ID NO:1.

[0010] In this invention, the phrase "having the same function" refers to protecting the catalytic activity (enzyme activity) of the substrate, in which the substrate is 2-iodo-5-fluoroacetophenone. In this invention, "alcohol dehydrogenase mutant with the same function" means having at least 50%-10000% catalytic efficiency or catalytic ability compared to the alcohol dehydrogenase mutant shown in SEQ ID NO:1 or (1).

[0011] In some embodiments of the present invention, the amino acid residue substitutions include at least one of the following: A48F, A48G, A48Q, I51L, I51C, I51A, V88N, V88K, V88F, A136F, A136I, A136Q, V183A, V183H, and V183K.

[0012] In some embodiments of the present invention, the amino acid residue substitutions include at least two or three of the following: A48F, A48G, A48Q, I51L, I51C, I51A, V88N, V88K, V88F, A136F, A136I, A136Q, V183A, V183H, and V183K.

[0013] In some embodiments of the present invention, the amino acid residue substitution is selected from one of: A48F, A48G, A48Q, I51L, I51C, I51A, V88N, V88K, V88F, A136F, A136I, A136Q, V183A, V183H, V183K; or A48F / I51A, A48F / V88K, A48F / A136I, A48F / V183H, I51L. / V88K、I51L / A136I、I51L / V183H、V88K / A136I、V88K / V183H、A136I / V183H、A48F / I51A / V88K、A4 8F / I51A / A136I, A48F / I51A / V183H, I51A / V88K / A136I, I51A / V88K / V183H, V88K / A136I / V183H.

[0014] Conservative substitutions, additions or deletions of one or more amino acids, amino-terminal truncation, and carboxyl-terminal truncation of other amino acid sites of the above-mentioned alcohol dehydrogenases are also included within the scope of this invention.

[0015] In some embodiments of the present invention, the alcohol dehydrogenase mutant further includes: (1) An alcohol dehydrogenase mutant obtained by substituting at least one amino acid from K132, T152, I223, V87, V118, and H62, based on the alcohol dehydrogenase mutant described above; or (2) Based on (1), a mutant of alcohol dehydrogenase with the same function as (1) is further modified by substitution and / or deletion and / or addition of one or more amino acids.

[0016] In some embodiments of the present invention, the amino acid residue substitutions include at least one of K132R, T152E, I223P, V87F, V118P, and H62F.

[0017] In some embodiments of the present invention, the amino acid residue substitutions include at least two of the following: K132R, T152E, I223P, V87F, V118P, and H62F.

[0018] In some embodiments of the present invention, the amino acid residue substitution is selected from one of: K132R, T152E, I223P, V87F, V118P, H62F; or H62F / I223P.

[0019] In some embodiments of the present invention, the amino acid residues are replaced with: I51L / V183H / H62F / I223P.

[0020] In a second aspect, the present invention provides a nucleic acid molecule encoding the alcohol dehydrogenase mutant described above.

[0021] In some embodiments of the present invention, the nucleic acid molecule may further be linked with modifying sequences and / or functional sequences.

[0022] In some embodiments of the present invention, the modified sequence and / or functional sequence includes at least one of the following: signal peptide, promoter, enhancer, terminator, tool enzyme recognition site, ribozyme, self-cleaving intron, miRNA binding site or ribosome binding site (RBS).

[0023] A third aspect of the present invention provides a biomaterial comprising at least one of the following: (1) An expression vector containing the nucleic acid molecules described above; (2) Transformants containing the nucleic acid molecules described above; (3) Transformants containing the expression vector in (1).

[0024] In some embodiments of the present invention, the expression vector includes a plasmid.

[0025] In this invention, the term "expression vector" refers to a vector or expression system used to integrate or insert a targeted exogenous gene.

[0026] In some embodiments of the present invention, the transformant includes bacteria, fungi, viruses, plant cells, or animal cells.

[0027] In this invention, the term "transformant" refers to a recipient cell that acquires a new genetic marker after incorporation or introduction of a foreign gene.

[0028] In some embodiments of the present invention, the transformant does not involve plant or animal reproductive materials.

[0029] In some embodiments of the present invention, the transformants include common cell vectors such as Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and Pichia pastoris.

[0030] In some embodiments of the present invention, the expression vector and transformant can be constructed based on any conventional techniques in the art.

[0031] In this invention, the genetically engineered bacteria expressing alcohol dehydrogenase are obtained by inserting the encoding nucleotide sequence of alcohol dehydrogenase into a plasmid vector to form a recombinant vector, which is then transformed into host cells. The plasmid vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, animal cell viruses, retroviruses, or other vectors well-known in the art. In this invention, the vectors that can be used include, but are not limited to, the pET-28 vector expressed in *E. coli*. Any vector that can stably replicate and exist in the host cell can be used to construct a recombinant expression vector.

[0032] In some embodiments of the present invention, the recombinant vector is obtained by inserting the alcohol dehydrogenase DNA fragment into the cloning site of the pET-28b vector to obtain a recombinant plasmid. Optionally, the cloning site is a DpnI restriction site.

[0033] In some embodiments of the present invention, the host cell includes prokaryotic cells, such as archaea cells, bacterial cells, or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.

[0034] In some embodiments of the present invention, the genetically engineered bacteria are recombinant engineered bacteria constructed from Escherichia coli.

[0035] A fourth aspect of the present invention provides a composition comprising at least one of the alcohol dehydrogenase mutants, nucleic acid molecules, or biological materials described in the above aspects.

[0036] In some embodiments of the present invention, the composition further includes excipients.

[0037] In some embodiments of the present invention, the excipients are rationally selected based on factors such as the product form of the composition, its intended use, and the method of feeding, and include, but are not limited to: diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, dicalcium phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), disintegrants (such as sodium hydroxymethyl starch, crospovidone, etc.), lubricants (such as talc, hydrogenated vegetable oil, polyethylene glycol, etc.), colorants (such as titanium dioxide, methylene blue, etc.), coating materials, solvents, pH adjusters, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), isotonic adjusters (such as glucose, sodium chloride, etc.), and chelating agents (such as disodium EDTA).

[0038] A fifth aspect of the invention provides the use of at least one of the alcohol dehydrogenase mutants, nucleic acid molecules, biomaterials, or compositions described above in biosynthesis.

[0039] In some embodiments of the present invention, the biosynthesis includes at least one of biocatalytic reaction, biofermentation and bioenzymatic hydrolysis.

[0040] In some embodiments of the present invention, the biosynthesis is a biocatalytic reaction.

[0041] In some embodiments of the present invention, at least one of an alcohol dehydrogenase mutant, a nucleic acid molecule, a biomaterial, or a combination thereof is used to prepare the lorlatinib intermediate in the biocatalytic reaction.

[0042] In some embodiments of the present invention, the lorlatinib intermediate comprises (S)-1-(2-iodo-5-fluorophenyl)ethanol.

[0043] A sixth aspect of the present invention provides a method for preparing (S)-1-(2-iodo-5-fluorophenyl)ethanol, comprising the following steps: At least one of the alcohol dehydrogenase mutants, nucleic acid molecules, biological materials, or compositions described above is used to catalyze 2-iodo-5-fluoroacetophenone.

[0044] In some embodiments of the present invention, the catalytic conditions include: a catalytic temperature of 15-50°C and a catalytic time of 2-30 h.

[0045] In some embodiments of the present invention, the catalytic conditions include: a catalytic temperature of 25-35°C and a catalytic time of 2-24 h.

[0046] In some embodiments of the present invention, the catalytic system further contains a solvent.

[0047] In some embodiments of the present invention, the solvent includes a buffer solution.

[0048] In some embodiments of the present invention, the buffer solution is selected from one of TEA buffer, PB buffer, and Tris-HCl buffer.

[0049] In some embodiments of the present invention, the concentration of the TEA buffer is 0.05-0.20 M and the pH value is 6-8.

[0050] In some embodiments of the present invention, the concentration of the PB buffer solution is 0.05-0.10 M and the pH value is 6-8.

[0051] In some embodiments of the present invention, the concentration of the Tris-HCl buffer is 0.05-0.10 M and the pH value is 6-8.

[0052] In some embodiments of the present invention, the buffer solution is a Tris-HCl buffer solution.

[0053] In some embodiments of the present invention, the concentration of the Tris-HCl buffer is 0.1 M and the pH value is 6.0.

[0054] In some embodiments of the present invention, the catalytic system further contains a co-solvent.

[0055] In some embodiments of the present invention, the co-solvent includes at least one of methyl tert-butyl ether, DMSO (dimethyl sulfoxide), acetonitrile, ethyl acetate, 1,4-dioxane, Tween 20, isobutyl acetate, butyl acetate, methanol, and Span 80.

[0056] In some embodiments of the present invention, the co-solvent is methyl tert-butyl ether.

[0057] In some embodiments of the present invention, the pH of the catalytic system is 6-8.

[0058] In some embodiments of the present invention, the pH of the catalytic system is 6.

[0059] In some embodiments of the present invention, at least one of the alcohol dehydrogenase mutant, nucleic acid molecule, biomaterial, or composition is used as a catalyst.

[0060] In some embodiments of the present invention, the form of the catalyst is not limited, and may include wet bacterial cells, wet bacterial cells immobilized with cells, enzymes extracted from wet bacterial cells after ultrasonic disruption, or immobilized enzymes. When it is a wet bacterial cell, the water content can be 70-90%.

[0061] In some embodiments of the present invention, the culture medium for the bacteria may be any suitable culture medium in the art, including but not limited to LB medium.

[0062] In some embodiments of the present invention, the catalytic reaction is carried out in a single aqueous phase system.

[0063] In some embodiments of the present invention, the substrate dosage is 20-400 g / L.

[0064] In some embodiments of the present invention, the substrate dosage is 50-200 g / L.

[0065] In some embodiments of the present invention, the alcohol dehydrogenase mutant is added in the form of wet cells of genetically engineered bacteria, and the amount of wet cells is 10-200 g / L.

[0066] In this invention, the alcohol dehydrogenase mutant can be a culture of the aforementioned recombinant alcohol dehydrogenase mutant genetically engineered bacteria, or it can be bacterial cells obtained by centrifuging the culture medium or its processed products. The processed products refer to extracts, lysates, or isolated products obtained by separating and / or purifying the nitrile hydrolase from the bacterial cells, or immobilized products obtained by immobilizing the extract or processed products.

[0067] In some embodiments of the present invention, the weight ratio of substrate to catalyst is 10-200:20-400.

[0068] In some embodiments of the present invention, the weight ratio of substrate to catalyst is approximately 10:200.

[0069] In this invention, the term "about" means a deviation of less than or equal to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0070] In some embodiments of the present invention, the method specifically includes: adding recombinant transformant wet cells expressing alcohol dehydrogenase, recombinant transformant wet cells expressing formate dehydrogenase or formate dehydrogenase, buffer, substrate, solubilizer, ammonium formate and coenzyme to a reaction vessel, reacting at 25-35°C for 2-24 hours, and after extraction, separation and rotary evaporation, lorlatinib intermediate is obtained.

[0071] In some embodiments of the present invention, the amount of co-solvent used is 50-500 mL / L.

[0072] In some embodiments of the present invention, the amount of ammonium formate used is 0.2-1 mol / L.

[0073] In some embodiments of the present invention, the amount of coenzyme used is 0.1-2.5 mmol / L.

[0074] In some embodiments of the present invention, based on a 1 L reaction system, 10-200 g of wet recombinant transformants expressing alcohol dehydrogenase, 10-200 g of wet recombinant transformants expressing formate dehydrogenase, 20-100 g of substrate, 50-500 mL of cosolvent, 0.2-1 mol of ammonium formate, 0.1-2.5 mmol of coenzyme, and the remainder of buffer solution.

[0075] In some embodiments of the present invention, the wet cells of the recombinant transformants expressing formate dehydrogenase are 25-75 g.

[0076] In this invention, a coenzyme cycling system is constructed by recombinant transformant wet cells expressing formate dehydrogenase, ammonium formate, and coenzyme.

[0077] In some embodiments of the present invention, the coenzyme is NADP+ or NAD+.

[0078] A seventh aspect of the present invention provides a method for preparing lorlatinib, comprising the following steps: (1) Using at least one of the alcohol dehydrogenase mutants, nucleic acid molecules, biological materials, or compositions described above to catalyze the production of (S)-1-(2-iodo-5-fluorophenyl)ethanol from 2-iodo-5-fluoroacetophenone; (2) Lorlatinib was prepared using (S)-1-(2-iodo-5-fluorophenyl)ethanol.

[0079] In some embodiments of the present invention, the preparation method in step (2) is not limited and can be prepared by any known preparation method in the art.

[0080] In some embodiments of the present invention, the catalytic conditions are limited as defined above.

[0081] In some embodiments of the present invention, the catalytic system is defined as described above.

[0082] In some embodiments of the present invention, the weight ratio of substrate to catalyst is limited as defined above.

[0083] The beneficial effects of this invention are: 1. The method in this invention is a biosynthesis method, which has mild reaction conditions, is environmentally friendly, has high regioselectivity and stereoselectivity, and effectively avoids the difficulties of chiral resolution and heavy metal residues in the products of conventional methods, thus making up for the shortcomings of chemical methods.

[0084] 2. The mutant in this invention can catalyze the production of (S)-1-(2-iodo-5-fluorophenyl)ethanol from 2-iodo-5-fluoroacetophenone, exhibiting high catalytic efficiency, low dosage, and high substrate concentration. This results in a dual improvement in conversion rate and chiral purity, demonstrating significant advantages for industrial application. The optimal mutant achieves a conversion rate of up to 99% and an optical purity greater than 99%. This method requires only one reaction step to obtain the target product, reducing production costs and possessing broad application prospects and considerable market value.

[0085] 3. The method of this invention allows the use of free alcohol dehydrogenase (e.g., in the form of enzyme powder), or in the form of cells expressing the alcohol dehydrogenase (e.g., wet cells); or in other forms, such as the supernatant of cell lysis expressing the alcohol dehydrogenase or whole-cell immobilization, or immobilization of free enzyme powder. It exhibits excellent compatibility.

[0086] 4. In the method for preparing alcohol dehydrogenase in this invention, the host microorganism for enzyme protein expression can be *Escherichia coli*, and the recombinant plasmid can be the pET28 plasmid; the selection itself is not limited. Furthermore, it can be completed based on conventional experimental procedures, making it simple and easy to implement. Attached Figure Description

[0087] Figure 1 This is a flowchart of the reaction process for synthesizing the chiral intermediate of lorlatinib based on enzyme in this invention. Detailed Implementation

[0088] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0089] In this invention, the LB liquid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, with the remainder being water. The LB solid culture medium is based on the LB liquid culture medium formula, with the addition of 15 g / L agar.

[0090] Example 1 In this embodiment, a method for preparing alcohol dehydrogenase and formate dehydrogenase is provided.

[0091] Specifically, based on the amino acid sequences shown in the table below, various alcohol dehydrogenases and formate dehydrogenases were prepared using a solid-phase synthesis method (commissioned by Shanghai Sangon Biotech). Then, conventional recombinant expression vectors were synthesized. The expression vectors used were commercially available pET28 plasmids. The expression vectors were constructed by inserting the corresponding coding sequences into pET28, resulting in recombinant expression vectors for alcohol dehydrogenase and formate dehydrogenase.

[0092] Table 1. Alcohol dehydrogenase sequence information

[0093] Table 2 Formate dehydrogenase sequence information

[0094] The enzyme protein was expressed using *E. coli* BL21(DE3). Specifically, the obtained recombinant expression vector was sequentially transformed into competent *E. coli* BL21(DE3) cells. First, the obtained alcohol dehydrogenase recombinant expression vector was transformed into competent *E. coli* BL21(DE3) cells, and then plated on LB agar plates (with 50 mg / L kanamycin) and incubated at 37 °C for 12 hours. Then, a single colony was picked and transferred into LB liquid medium (containing 50 mg / L kanamycin), incubated at 37 °C for 12 hours, and the bacterial culture was sequenced for verification. After confirmation, recombinant *E. coli* expressing alcohol dehydrogenase was obtained.

[0095] The alcohol dehydrogenase was screened. Specifically, following the method described in the above examples, recombinant *E. coli* transformed with the recombinant expression vector for alcohol dehydrogenase were prepared. These recombinant *E. coli* (corresponding to ADH1-10, respectively) were inoculated (1 v / v %) in 10 mL LB liquid medium and cultured at 37°C and 180 rpm for 8-10 h. Then, the bacterial culture was inoculated into 100 mL LB liquid medium at 1 v / v % and cultured at 37°C and 180 rpm for 2-2.5 h (equivalent to an OD600 value of 0.6-0.8). Then, IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 24°C and 180 rpm for 12 h. The cultured bacterial culture was poured into a 500 mL centrifuge cup, centrifuged at 8000 rpm for 10 min at 4°C, the supernatant was discarded, and the bacterial cells were collected and weighed.

[0096] Following the same method, wet formate dehydrogenase cells were obtained based on the formate dehydrogenase recombinant expression vector and Escherichia coli BL21(DE3) competent cells.

[0097] 0.05 g of collected recombinant *E. coli* cells transformed with recombinant expression vectors ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, ADH8, ADH9, and ADH10 were added to 2 mL EP tubes, with a blank control group (without bacteria) included. 0.05 g of the formate dehydrogenase cells prepared above were added to each EP tube. Then, 700 µL of PB buffer (0.1 M, pH 7), 100 µL of 7 M ammonium formate solution, 100 µL of 25 mM NADP+ solution, and 100 µL of methyl ether solution containing 2-iodo-5-fluoroacetophenone (final concentration 50 g / L) were added. After thorough mixing, the EP tubes were placed in a constant temperature mixer and reacted at 30°C and 1200 rpm for 4 h with shaking.

[0098] After the reaction was completed, 200 µL of the reaction solution was taken and 200 µL of methyl ether was added. The mixture was shaken and mixed for 2 min, centrifuged at 12000 rpm for 1 min, and the supernatant was collected for HPLC analysis.

[0099] The conversion rates and ee values ​​of each alcohol dehydrogenase were determined by HPLC and are shown in the table below.

[0100] Table 3. Conversion rate and ee value of different alcohol dehydrogenases

[0101] The results showed that ADH6 and ADH9 had the highest conversion rates, with ADH6 producing a product that exhibited better chirality (S-type) and an ee value of 89.92%. Therefore, ADH6 was subsequently used as a template to construct mutants.

[0102] Example 2 In this embodiment, an alcohol dehydrogenase mutant library was further constructed based on ADH6. The amino acid sequence of ADH6 is: MTDRLKGKVAIVTGGTLGIGLAIADKFVEEGAKVVITGRHADVGEKAAKSIGGTDVIRFVQHDASDEAGWTKLFDTTEEAFGPVTTVVNNAGIAVSKSVEDTTTEEWRKLLSVNLDGVFFGTRLGIQRMKNKGLGASIINMSSIEGFVGDPTLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLEGAEEMMSQRTKTPMGHIGEPNDIAWICVYLASDESKFATGAEFVVDGGYTAQ (SEQ ID NO: 1).

[0103] In this embodiment, the ADH6 mutant library was prepared by site-directed mutagenesis. Using the ADH6 recombinant expression vector (pET28b(+)-ADH6) prepared in the above embodiment as a template, polymerase chain reaction (PCR) was performed using the primers shown in the table below. Then, after digestion with DpnI, the sample was transformed into Escherichia coli BL21(DE3) competent cells and cultured in 10 mL LB agar plates at 37°C for 12-16 h.

[0104] Table 4. PCR primers used for constructing mutants

[0105] The PCR reaction system (25 µL) consisted of: 1 µL forward primer (100 μM), 1 µL reverse primer (100 μM), 12.5 µL 2×Phanta buffer (purchased from Novizan), 0.5 µL dNTP mixture (10 mM each), 1 µL recombinant expression plasmid template, 0.5 µL DNA polymerase Phanta (purchased from Novizan), and 8.5 µL ultrapure water.

[0106] The PCR program was set according to the Phanta Super-Fidelity DNA Polymerase Manual (Novizan). Specifically, the PCR program was as follows: 95℃ pre-denaturation for 5 min; then 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 4 min, for a total of 30 cycles; 72℃ final extension for 10 min; and incubation at 16℃.

[0107] Positive clones and original ADH6 recombinant Escherichia coli were randomly selected from the plates and inoculated into 10 mL of LB liquid medium (containing 50 mg / L kanamycin) and cultured in a temperature-controlled shaker at 37°C and 200 rpm for 8–10 h. The culture was then inoculated into 100 mL of LB liquid medium (containing 50 mg / L kanamycin) at a 1% (v / v) inoculation rate and cultured in a temperature-controlled shaker at 37°C and 180 rpm for 2–2.5 h (equivalent to an OD600 value of 0.6–0.8). An inducer (IPTG) was added to a final concentration of 0.1 mM and the culture was further incubated in a temperature-controlled shaker at 24°C and 180 rpm for 12 h. The cultured bacterial solution was poured into an 800 mL centrifuge cup and centrifuged at 8000 rpm for 10 min at 4°C. The supernatant was discarded, and the bacterial cells were collected and weighed. Wet cells of original ADH6 recombinant Escherichia coli and ADH6 mutant recombinant Escherichia coli were obtained.

[0108] Based on the single mutant, iterative mutations are performed to obtain multiple mutants. Specifically, the mutant recombinant expression plasmids obtained in the above steps include: pET28b(+)-ADH6-A48F, pET28b(+)-ADH6-A48G, pET28b(+)-ADH6-A48Q, pET28b(+)-ADH6-I51L, pET28b(+)-ADH6-I51C, pET28b(+)-ADH6-I51A, pET28b(+)-ADH6-V88N, pET28b(+)-ADH6-V88 Using the primers pET28b(+)-ADH6-V88F, pET28b(+)-ADH6-A136F, pET28b(+)-ADH6-A136I, pET28b(+)-ADH6-A136Q, pET28b(+)-ADH6-V183A, pET28b(+)-ADH6-V183H, and pET28b(+)-ADH6-V183K as templates, polymerase chain reaction (PCR) was performed using the PCR primers listed in Table 4. Multiple mutants were obtained: ADH6-A48F / I51A, ADH6-A48F / V88K, ADH6-A48F / A136I, ADH6-A48F / V183H, ADH6-I51A / V88K, ADH6-I51A / A136I, ADH6-I51A / V183H, ADH6-V88K / A136I, and ADH6-V88K / V183. H, ADH6-A136I / V183H, ADH6-A48F / I51A / V88K, ADH6-A48F / I51A / A136I, ADH6-A48F / I5 1A / V183H, ADH6-I51A / V88K / A136I, ADH6-I51A / V88K / V183H, ADH6-V88K / A136I / V183H.

[0109] Following the method described in the above embodiments, 2-iodine-5-fluoroacetophenone was used as a substrate, and 2-iodine-5-fluoroacetophenone and (S)-1-(2-iodine-5-fluorophenyl)ethanol were synthesized by catalysis using original ADH6 recombinant Escherichia coli or ADH6 mutant (including single mutant and multiple mutant) recombinant Escherichia coli wet cells. The concentration and chirality of the obtained 2-iodine-5-fluoroacetophenone and (S)-1-(2-iodine-5-fluorophenyl)ethanol were detected by HPLC, and the conversion rate was calculated. Using the conversion rate and chirality as indicators, the dominant enzyme mutant recombinant strains were screened. Specifically, the reaction system consisted of 1 mL of 1 mL of bacterial wet weight 50 g / L, a final substrate concentration of 100 g / L, and 100 mMPB buffer (pH 7.0) as the reaction medium. Additional ingredients included 0.7 M ammonium formate, 50 g / L of the formate dehydrogenase wet cells prepared in the above example, and 2.5 mM NADPH. 0.1 mL of methyl ether was used as a co-solvent. The reaction was carried out at 30°C and 1200 rpm for 1 h. 200 μL of the reaction solution was then added to 50 μL of 6 M hydrochloric acid (to terminate the reaction), followed by extraction twice with 200 μL of ethyl acetate. The ethyl acetate phases were combined. The concentrations and chirality of 2-iodo-5-fluoroacetophenone and (S)-1-(2-iodo-5-fluorophenyl)ethanol were determined by HPLC, and the conversion rate was calculated.

[0110] The reaction flow diagram is as follows Figure 1 As shown in the table below, the reaction results are as follows.

[0111] Table 5. Conversion rate and ee value of alcohol dehydrogenase mutants

[0112] For single mutants, ADH6-A48F, ADH6-A48G, ADH6-A48Q, ADH6-I51L, ADH6-I51C, ADH6-I51A, ADH6-V88N, ADH6-V88K, ADH6-V88F, ADH6-A136F, ADH6-A136I, ADH6-A136Q, ADH6-V183A, ADH6-V183H, and ADH6-V183K all showed superior mutant performance. Among multiple mutants, ADH6-I51L / V183H exhibited the best results. ADH6-I51L / V183H will be the primary target for future testing.

[0113] Example 3 In this embodiment, the stability of the alcohol dehydrogenase mutant was tested using ADH6-I51L / V183H as an example.

[0114] Specifically, wet WT and ADH6-I51L / V183H bacterial cells were stored at 25°C in the dark. Samples were taken at 12 h and 24 h, and enzyme activity was measured using the method described in Example 1 above. The percentage of residual activity at different times was calculated with the initial activity as 100%.

[0115] The results are shown in the table below.

[0116] Table 5. Stability of alcohol dehydrogenase mutants

[0117] The results showed that the stability of the ADH6-I51L / V183K mutant was slightly lower than that of the WT mutant.

[0118] To address this, the inventors started with the ADH6-I51L / V183K mutant and performed iterative saturation mutations on sites that might affect structural stability in order to obtain an enzyme mutant that combines high catalytic activity with excellent stability.

[0119] Specifically, the inventors analyzed the homologous three-dimensional structure of the ADH6-I51L / V183K mutant and selected the following sites that may enhance structural rigidity, hydrophobic stacking, or surface salt bridges for iterative mutation: K132R, T152E, V87F, H62F, V118P, and I223P.

[0120] Specifically, using the pET28b(+)-ADH6-I51L / V183H recombinant expression plasmid as a template, iterative mutations were performed according to the method described in the above examples to obtain new multiple mutants. The PCR primer information used is shown in the table below.

[0121] Table 6. Primer information for stable iterative mutation PCR

[0122] Single clones were randomly picked from the culture plate, with ADH6-I51L / V183K mutant and WT(ADH6) as controls. Protein expression was induced and bacterial cells were collected according to the method described in the above examples. The collected wet bacterial cells were resuspended in an equal volume of pH 7.0 phosphate buffer and vortexed to mix. 100 μL of the bacterial suspension was placed in a PCR tube and heat-treated in a 55°C metal bath for 30 minutes, followed immediately by cooling in an ice bath for 5 minutes. Using the untreated bacterial suspension under the same conditions as the activity benchmark (100%), enzyme activity was measured according to the method described in the above examples (where the reaction time was shortened to 30 minutes) to obtain its conversion rate. The residual activity (%) of thermal stability was calculated to determine its stability.

[0123] Wherein, residual thermal stability activity (%) = × 100%.

[0124] The results are shown in the table below.

[0125] Table 7 Initial screening data of stable iterative mutants

[0126] In the table, M2 represents the ADH6-I51L / V183K mutant. For example, M2-K132R means that the ADH6-I51L / V183K mutant has been further mutated with K132R.

[0127] The results showed that the thermal stability of most of the mutants obtained in this embodiment was improved compared to M2, but the initial catalytic activity of some mutants decreased. Considering both thermal stability, residual activity, and initial conversion rate, the inventors screened out two mutation sites, I223P and H62F, which can significantly improve the thermal stability of the ADH6-I51L / V183K mutant while maintaining its high level of catalytic activity.

[0128] Furthermore, using the positive multimutant plasmid pET28b(+)-ADH6-M2-H62F constructed in the above steps as a template, iterative mutagenesis was performed using the primers shown in Table 6 to construct the two-point iterative mutant ADH6-M2-I51L / V183H / H62F / I223P, which was named M3. Stability testing was performed according to the above steps. The results are shown in the table below.

[0129] Table 8 Initial screening data of stable iterative mutants

[0130] The results showed that while inheriting the ultra-high catalytic performance of M2 (conversion rate >99%, ee value >99.5%), the thermal stability, solvent tolerance and storage stability of the M3 mutant were comprehensively and significantly improved, successfully solving the problem of decreased stability.

[0131] Example 4 In this embodiment, the effects of each mutant obtained in the above embodiments are further tested.

[0132] (1) Thermal stability test: In this embodiment, the thermal stability test includes three aspects: melting temperature (Tm) determination, thermal deactivation half-life determination, and residual activity determination after thermal shock.

[0133] The melting temperature (Tm) was determined using differential scanning fluorometry (DSF). Specifically, the WT and M3 mutants were diluted separately in the same reaction buffer (50 mM sodium phosphate buffer, pH 7.0) to a final enzyme concentration of 0.1 mg / mL. A fluorescent dye (SYPRO Orange) was added. The temperature was increased from 25°C to 95°C at a rate of 1°C / min on a real-time quantitative PCR instrument, and the fluorescence signal was monitored. The melting temperature (Tm) of the protein was determined by derivative curve analysis. Each sample was tested in triplicate.

[0134] The determination of the heat inactivation half-life involved incubating WT and M3 mutants in a 55°C constant temperature water bath. Samples were taken at 0, 5, 15, 30, 60, 120, and 240 minutes, and immediately cooled in an ice bath. Residual enzyme activity was then measured under standard reaction conditions (30°C) following the method described in the previous examples. The relative activity at each time point was calculated with the initial activity as 100%. The time required for half of the enzyme activity to be lost was calculated by fitting a first-order inactivation kinetic equation; this is the half-life (t1 / 2).

[0135] The determination of residual activity after heat shock included: incubating WT and M3 mutants in water baths at 50°C and 60°C, respectively; sampling at 30 minutes and 1 hour; cooling in an ice bath; and determining residual enzyme activity under standard reaction conditions (30°C) according to the method described in the above examples. A control group without heat treatment was also included, and the percentage of residual activity was calculated.

[0136] (2) Organic solvent resistance test: The WT and M3 mutants were mixed separately with buffer solutions containing different volume fractions of organic solvent (20% DMSO or 30% methanol). The mixtures were incubated with gentle shaking at 25°C for 2 hours. After incubation, samples were taken, and enzyme activity was measured under standard reaction conditions according to the method described in the previous examples. The percentage of residual activity after organic solvent treatment was calculated, with the activity of the group incubated in the same buffer solution without the addition of organic solvent as 100%.

[0137] (3) pH stability test: A series of buffer solutions with different pH values ​​(Britton-Robinson buffers, pH 5.0-10.0) were prepared. WT and M3 mutants were mixed with equal volumes of the prepared buffer solutions at different pH values ​​to place the enzymes in the target test pH environment. The mixtures were incubated at 4°C for 24 hours. After incubation, the pH of each group was adjusted back to the standard reaction pH (7.0), and enzyme activity was immediately measured. The residual activity under each pH condition was calculated with the enzyme activity incubated in the optimal pH (7.0) buffer as 100% to assess its pH stability range and tolerance limit.

[0138] (4) Storage stability test: The WT and M3 mutants were placed in standard storage buffer and stored at 4°C and 25°C, respectively, in the dark. Samples were taken on days 0, 7, and 30, and enzyme activity was measured under standard reaction conditions according to the method described in the above examples. The percentage of residual activity at different storage times and temperatures was calculated, with the activity at the beginning of storage as 100%.

[0139] The test results are shown in the table below.

[0140] Table 8 Results of the Effect Test

[0141] The above results indicate that the M3 mutant not only greatly improves the enzyme's catalytic efficiency and enantioselectivity, but also significantly strengthens its three-dimensional structure through synergistic effects, making it exhibit far greater resilience than the wild type when facing harsh industrial conditions such as high temperature, organic solvents, and extreme pH.

[0142] Example 5 In this embodiment, the effect of environmental conditions on the catalytic reaction was tested.

[0143] (1) Selection of cosolvent: Following the method described in the above examples, 0.5 g each of M3 mutant wet cells and formate dehydrogenase wet cells, 7 mL of PB buffer (0.1 M, pH 7), 1 mL of 7 M ammonium formate solution, 1 mL of 10 mM NADP+ solution, and 1 mL of 1000 g / L 2-iodo-5-fluoroacetophenone co-solvent solution were added to the reaction flask. The co-solvents selected for each group were methyl tert-butyl ether, DMSO (dimethyl sulfoxide), acetonitrile, ethyl acetate, 1,4-dioxane, Tween 20, isobutyl acetate, butyl acetate, methanol, and Span 80, respectively. The reaction was carried out at 30°C and 800 rpm. After 1 h of reaction, 1 mL of the reaction solution was taken, extracted with an equal volume of ethyl acetate, centrifuged, and the upper organic phase was analyzed by HPLC.

[0144] The results are shown in the table below.

[0145] Table 9 Test results for different co-solvents

[0146] The results showed that the highest conversion rate (>99%) was achieved when methyl tert-butyl ether was used as the co-solvent, and its chirality was S-type with an ee value of 99.82%.

[0147] (2) Selection of buffer solution: Following the method described in the above embodiments, 0.5 g each of M3 mutant wet cells and formate dehydrogenase wet cells, along with 7 mL of different buffer solutions (0.1 M), 1 mL of 7 M ammonium formate solution, 1 mL of 10 mM NADP+ solution, and 1 mL of 1000 g / L methyl tert-butyl ether solution of 2-iodo-5-fluoroacetophenone, were added to the reaction flask. The buffer solutions included PB, Tris-HCl, or TEA buffer at pH 7.0. The reaction was carried out at 30 °C and 800 rpm. After 0.5 h of reaction, 1 mL of the reaction solution was taken, extracted with an equal volume of ethyl acetate, centrifuged, and the upper organic phase was analyzed by HPLC.

[0148] The results are shown in the table below.

[0149] Table 10 Test results for different buffer solutions

[0150] The results showed that the highest conversion rate was achieved when the buffer solution was Tris-HCl buffer at pH 7.0.

[0151] (3) pH selection: Following the method described in the above embodiments, 0.5 g each of M3 mutant wet cells and formate dehydrogenase wet cells, 7 mL of Tris-HCl buffer (0.1 M), 1 mL of 7 M ammonium formate solution, 1 mL of 10 mM NADP+ solution, and 1 mL of 1000 g / L methyl tert-butyl ether solution of 2-iodo-5-fluoroacetophenone were added to the reaction flask. The pH of the buffer solutions was set to 6.0, 6.5, 7.0, 7.5, and 8.0. The reaction was carried out at 30 °C and 800 rpm. After 0.5 h of reaction, 1 mL of the reaction solution was taken, extracted with an equal volume of ethyl acetate, centrifuged, and the upper organic phase was analyzed by HPLC.

[0152] The results are shown in the table below.

[0153] Table 11 Test results at different pH values

[0154] The results showed that the highest conversion rate was achieved when the buffer solution was Tris-HCl at pH 6.0, with a conversion rate of 80.33% in just 0.5 h.

[0155] (4) Selection of other factors: Following the method described in the above embodiments, 0.5 g each of M3 mutant wet cells and formate dehydrogenase wet cells, 7 mL of PB buffer (0.1 M, pH 7), 1 mL of 25 M ammonium formate solution, 1 mL of 10 mM NADP+ solution, and 1 mL of 2000 g / L methyl tert-butyl ether solution containing the substrate (system concentration 200 g / L) were added to the reaction flask, for a total system volume of 10 mL. The substrate was set as 2-iodo-5-fluoroacetophenone, and the reaction was carried out at 30 °C and 800 rpm. After 6 h of reaction, 1 mL of the reaction solution was taken, extracted with an equal volume of ethyl acetate, centrifuged, and the upper organic phase was analyzed by HPLC.

[0156] In single-factor testing, only the concentration of the target factor is adjusted; the concentrations of other factors are selected in the same manner as in this step.

[0157] The single-factor adjustments and corresponding results are shown in the table below.

[0158] Table 12 Single-factor adjustment details and corresponding results

[0159] Example 6 In this embodiment, the catalytic synthesis of a small-scale system (1 L) was verified using the M3 mutant under the optimal conditions explored in the above embodiments.

[0160] The specific reaction was as follows: Take a 2000 mL three-necked flask, add 50 g each of M3 mutant wet cells and formate dehydrogenase wet cells, then add 500 mL of Tris-HCl buffer (0.1 M, pH 6.0), 100 mL of 25 M ammonium formate solution, 100 mL of 2.5 mM NADP+ solution, and 300 mL of 1000 g / L 2-iodo-5-fluoroacetophenone (containing methyl tert-butyl ether) solution. Add the substrate dropwise using a constant pressure funnel, ensuring complete addition within 2 hours. Place the flask in a 30 ℃ water bath and stir at 300 rpm. At 3 h, 6 h, and 10 h of reaction time, take 500 µL of the reaction solution, extract with an equal volume of ethyl acetate, and then perform TLC analysis.

[0161] The results showed that the conversion was complete after 10 hours of reaction. The reaction solution was extracted with ethyl acetate. For the first extraction, an equal volume of ethyl acetate was added; for the second and third extractions, half a volume of ethyl acetate was added. The specific extraction procedure was as follows: after adding ethyl acetate, the mixture was shaken and centrifuged at 8000 rpm for 5 min. The supernatant was collected and dehydrated using anhydrous Na₂SO₄. After extraction, the extract was rotary evaporated. Specifically, a vacuum pump was used first, followed by an oil pump for rotary evaporation. After rotary evaporation, a small amount of ethyl acetate was used to dissolve the crude product until only a few particles remained, followed by sonication to ensure complete dissolution. Petroleum ether was placed in a beaker on a magnetic stirrer. The dissolved crude product solution was slowly added dropwise to the petroleum ether (while the rotor was continuously stirring) until a large amount of solid precipitated. Stirring was stopped, and the mixture was allowed to stand for 10 min. The solid was then removed using a vacuum filter and dried by rotary evaporation using an oil pump to obtain the final product (lorlatinib chiral intermediate).

[0162] The purity of the crude and final products is shown in the table below.

[0163] Table 13 Test results of crude and final products

[0164] The results showed that the M3 mutant could catalyze the conversion of 300 g / L 2-iodo-5-fluoroacetophenone to (S)-1-(2-iodo-5-fluorophenyl)ethanol, with a conversion rate >99% and an ee value greater than 99%.

[0165] The final product obtained can be synthesized into lorlatinib using conventional methods currently available in the field.

[0166] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An alcohol dehydrogenase mutant, characterized in that, The alcohol dehydrogenase mutant includes: (1) An alcohol dehydrogenase mutant obtained by substituting at least one amino acid from A48, I51, V88, A136, and V183 based on the sequence shown in SEQ ID NO:1; or (2) Based on (1), a mutant of alcohol dehydrogenase with the same function as (1) is further modified by substitution and / or deletion and / or addition of one or more amino acids.

2. The alcohol dehydrogenase mutant according to claim 1, characterized in that, The alcohol dehydrogenase mutant also includes: (1) An alcohol dehydrogenase mutant obtained by substituting at least one amino acid from K132, T152, I223, V87, V118, and H62, based on the alcohol dehydrogenase mutant described in claim 1; or (2) Based on (1), a mutant of alcohol dehydrogenase with the same function as (1) is further modified by substitution and / or deletion and / or addition of one or more amino acids.

3. The alcohol dehydrogenase mutant according to claim 1 or 2, characterized in that, The substitution of the amino acids includes: At least one of A48F, A48G, A48Q, I51L, I51C, I51A, V88N, V88K, V88F, A136F, A136I, A136Q, V183A, V183H, V183K, K132R, T152E, I223P, V87F, V118P, and H62F.

4. A nucleic acid molecule encoding the alcohol dehydrogenase mutant according to any one of claims 1-3.

5. A biomaterial, characterized in that, The biomaterial includes at least one of the following: (1) An expression vector containing the nucleic acid molecule of claim 4; (2) A transformant containing the nucleic acid molecule of claim 4; (3) Transformants containing the expression vector in (1).

6. A composition, characterized in that, The composition contains at least one of the alcohol dehydrogenase mutant according to any one of claims 1-3, the nucleic acid molecule according to claim 4, or the biological material according to claim 5; Preferably, the composition further includes excipients.

7. The use of at least one of the alcohol dehydrogenase mutants of any one of claims 1-3, the nucleic acid molecule of claim 4, the biomaterial of claim 5, or the composition of claim 6 in biosynthesis; Preferably, the biosynthesis includes at least one of biocatalytic reaction, biofermentation, and bioenzymatic hydrolysis.

8. The use of at least one of the alcohol dehydrogenase mutant of any one of claims 1-3, the nucleic acid molecule of claim 4, the biomaterial of claim 5, or the composition of claim 6 in the preparation of lorlatinib intermediate; Preferably, the lorlatinib intermediate comprises (S)-1-(2-iodo-5-fluorophenyl)ethanol.

9. A method for preparing (S)-1-(2-iodo-5-fluorophenyl)ethanol, comprising the following steps: Catalysis of 2-iodo-5-fluoroacetophenone is performed using at least one of the alcohol dehydrogenase mutants of any one of claims 1-3, the nucleic acid molecule of claim 4, the biomaterial of claim 5, or the composition of claim 6.

10. A method for preparing lorlatinib, comprising the following steps: (1) Using at least one of the alcohol dehydrogenase mutants of any one of claims 1-3, the nucleic acid molecule of claim 4, the biomaterial of claim 5, or the composition of claim 6 to catalyze the production of (S)-1-(2-iodo-5-fluorophenyl)ethanol from 2-iodo-5-fluoroacetophenone; (2) Lorlatinib was prepared using (S)-1-(2-iodo-5-fluorophenyl)ethanol.

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