N-boc-l-alaninol and its preparation method and application

Abstract

The application belongs to the technical field of organic synthesis, and particularly relates to N-Boc-L-propanolamine and a preparation method and application thereof. The preparation method of the N-Boc-L-propanolamine comprises the following steps: (1) preparing N-Boc-propanolamine: reacting raw material 2-amino propanol with Boc anhydride to obtain N-Boc-propanolamine; (2) synthesizing N-Boc-L-propanolamine: dispersively dissolving N-Boc-propanolamine obtained in step (1), adding hydroxyl oxidase and a pH buffer system, and passing in oxygen, so that N-Boc-propanolamine is reacted with the hydroxyl oxidase; after the reaction is completed, extraction, filtration, alkali washing, concentration, crystallization and drying are performed to obtain the N-Boc-L-propanolamine. The preparation method has the advantages of easy availability of raw materials, low price, simple operation, high product yield and easy industrial production.

Description

Technical Field

[0001] This invention belongs to the technical field of organic synthesis, specifically relating to N-Boc-L-propanol, its preparation method, and its application. Background Technology

[0002] Chiral amino alcohols are a class of optically active amino alcohols obtained by the reduction of amino acids using chiral sources. They are widely used in the organic synthesis of pharmaceuticals, fine chemicals, materials, and asymmetric catalysis, especially in the pharmaceutical field where they are key chiral raw materials for peptide and quinolone drugs. N-Boc-L-propanol is a multifunctional chiral amino alcohol. It is a white to off-white solid, readily soluble in organic solvents such as methanol, ethyl acetate, and dichloromethane. In the pharmaceutical field, N-Boc-L-propanol can serve as a key intermediate in the synthesis of chiral compounds such as anticancer drugs, for example, as an intermediate in the synthesis of dalolutamide.

[0003] Darolutamide is an androgen receptor (ARi) inhibitor. Its unique chemical structure binds to the receptor with high affinity, exhibiting strong antagonistic activity, thereby inhibiting receptor function and the growth of prostate cancer cells. Darolutamide is an oral nonsteroidal androgen receptor (AR) inhibitor with a unique chemical structure that binds to the receptor with high affinity, exhibiting strong antagonistic activity, thereby inhibiting receptor function and the growth of prostate cancer cells. Darolutamide is indicated for the treatment of adult patients with high-risk, non-metastatic, castration-resistant prostate cancer.

[0004] There are numerous methods for synthesizing N-Boc-L-propanoamine, including asymmetric and symmetric synthesis. Current techniques generally involve directly reacting L-propanoamine with an N-Boc protecting group (such as Boc anhydride) in an alcohol solvent. The synthetic route is as follows:

[0005]

[0006] However, L-propanol, a commonly used raw material for the synthesis of N-Boc-L-propanol, is not a natural product; it is an optical isomer of 2-aminopropanol. The racemic form of 2-aminopropanol (DL-2-aminopropanol) has two optical isomers: L- and D-. L-propanol requires a complex stereoselective synthetic method to be prepared.

[0007] Patent CN101648879A provides a method for synthesizing L-aminopropanol, summarizing the synthetic pathways of L-propanol: 1) Direct reduction method: L-alanine is reduced to L-propanol using LiAlH4. The solution is heated to reflux in a 5% LiAlH4 THF solution, diluted with dichloromethane, and then treated with a certain amount of water. Filtration is performed to remove inorganic salts. The resulting filter cake is treated twice to achieve satisfactory results, with a yield of 75%. This reduction method is simple, but the reducing agent LiAlH4 is expensive and the operation is dangerous. Furthermore, the subsequent processing is too complex; therefore, this method is rarely used nowadays.

[0008]

[0009] 2) Esterification reduction method: L-alanine is reacted with thionyl chloride and anhydrous ethanol to form an ester. An ethanol solution of L-alanine ethyl ester is continuously added dropwise to an aqueous solution of NaBH4 for 1 hour, and the mixture is stirred at room temperature for 3 hours to reduce L-alanine alcohol. The alcohol is extracted with ethyl acetate and then distilled under reduced pressure, with a yield of 67%.

[0010]

[0011] In the two reduction methods for synthesizing L-propanol mentioned above, although the reaction conditions are relatively mild, the reducing agents NaBH4 and LiAlH4 used in the reduction method are expensive, flammable, highly dangerous, and highly toxic. Moreover, the product separation and purification process is complex, which is not conducive to industrial production. A large amount of organic solvents are used in the synthesis and separation process, and solvent recovery is difficult in the production process. A large amount of pollutants, such as wastewater and solid waste, are generated, which is very detrimental to environmental protection.

[0012] Furthermore, the esterification-reduction method requires an esterification reaction followed by reduction to obtain the target product, which undoubtedly increases the number of reaction steps, prolongs the production cycle, and increases production costs, all of which limit the application of this synthesis method in industrial production. Patent CN101648879A uses L-aminopropionic acid and H2 as the main raw materials and ruthenium carbon as the catalyst for the direct catalytic hydrogenation synthesis of L-propanediol, but it also faces the problem of the high price of the precious metal ruthenium carbon.

[0013] Patent CN106810458A discloses a method for resolving DL-2-aminopropanol to prepare L-2-aminopropanol, which includes the following steps: 1) dissolving L-tartaric acid in water to obtain an aqueous solution of L-tartaric acid; 2) dissolving DL-2-aminopropanol in an alcohol solvent at room temperature, and then adding the aqueous solution of L-tartaric acid dropwise while stirring under cooling conditions. After the addition is complete, the solution is cooled to 0-5°C and kept at that temperature to obtain a cooled solution; 3) adding a small amount of seed crystals to the cooled solution, and allowing it to crystallize at -15°C to 25°C for 16-24 hours to precipitate L-tartaric acid-L-2-aminopropanol acid salt crystals; 4) dissolving the L-tartaric acid-L-2-aminopropanol acid salt crystals in an alcohol solvent, adding an inorganic base in batches, stirring until the amino alcohol is completely released, filtering, and distilling the filtrate under reduced pressure to obtain L-propanol. This patent uses L-tartaric acid as a resolving agent, and the steps are cumbersome and the operation is complicated.

[0014] Therefore, there is an urgent need to develop a new method for preparing N-Boc-L-propanoamine, which uses readily available raw materials, has a simple process route, and is easy to scale up, without the need for additional preparation using expensive reducing agents such as LiAlH4, NaBH4, and precious metal catalysts such as ruthenium carbon. Summary of the Invention

[0015] The purpose of this invention is to address the problems of existing N-Boc-L-propanol preparation processes, which use raw materials that are difficult to obtain and expensive, by providing a method for preparing N-Boc-L-propanol. This method uses readily available and inexpensive raw materials, has simple operation steps, high product yield, and is easy to industrialize.

[0016] The technical solution of this invention is: a method for preparing N-Boc-L-propanol, comprising the following steps:

[0017] (1) Preparation of N-Boc-propanol: The raw material 2-aminopropanol (DL-2-aminopropanol) is reacted with Boc anhydride to obtain N-Boc-propanol. The N-Boc-propanol obtained at this time is a racemic form.

[0018] (2) Synthesis of N-Boc-L-propanol: Disperse and dissolve the N-Boc-propanol obtained in step (1), add hydroxy oxidase and pH buffer system, introduce oxygen, and N-Boc-propanol reacts with hydroxy oxidase.

[0019] After the reaction is complete, the N-Boc-L-propanol is obtained by extraction, filtration, alkali washing, concentration, crystallization and drying.

[0020] The synthetic route for the above reaction steps is as follows:

[0021] (1)

[0022] (2)

[0023] The preparation method of N-Boc-L-propanol in this invention is specifically carried out as follows:

[0024] (1) Preparation of N-Boc-propanol: First, 2-aminopropanol (DL-2-aminopropanol) is added to an organic solvent, and under the condition of controlling the temperature <40℃, Boc anhydride is added to carry out the reaction. The organic solvent can be dichloromethane, methanol, methanol and water. Methanol is preferred.

[0025] After the reaction is complete, the resulting reaction solution is concentrated to remove the organic solvent; then deionized water is added, and the solution is concentrated again to remove the residual organic solvent, yielding a concentrated solution of N-Boc-propanol.

[0026] Concentration can be performed using conventional vacuum concentration, with the temperature controlled between 40-60℃. Concentrate to a syrupy (viscous) consistency. The degree of concentration can be determined by testing the level of loss (LOD). Specifically, the N-Boc-propanol concentrate is dried to remove LOD. Take 1.0g of the N-Boc-propanol concentrate sample and vacuum dry it at 50℃ for 30 minutes, requiring an LOD ≤ 20.0%.

[0027] (2) Synthesis of N-Boc-L-propanol: The concentrated N-Boc-propanol solution obtained in step (1) is first dispersed and dissolved in deionized water, then a pH buffer system and hydroxy oxidase are added. The pH value of the pH buffer system is adjusted to 6-8, oxygen is introduced, and the reaction is carried out under the condition of controlling the temperature <40℃. The role of deionized water is to disperse and dissolve the concentrated N-Boc-propanol solution. The amount of deionized water added is generally 5-7 times the mass of N-Boc-propanol.

[0028] After the reaction is complete, add an extractant, filter to remove the hydroxyl oxidase, and then separate the phases. Dichloromethane can be used as the extractant.

[0029] An alkaline solution was added to the organic phase obtained after phase separation, followed by water washing and phase separation again.

[0030] The organic phase obtained from this phase separation is concentrated, crystallized, filtered, and dried to obtain the N-Boc-L-propanohydrin. Isohexane can be added to induce crystallization. After crystallization, the mixture is cooled and filtered.

[0031] In the present invention, the molar ratio of 2-aminopropanol to Boc anhydride in step (1) of the preparation method of N-Boc-L-propanol is 1:0.95-1.1.

[0032] In the preparation method of N-Boc-L-propanol in this invention, the reaction temperature in step (1) is 20-30℃ and the reaction time is 2-5h.

[0033] In the present invention, in the method for preparing N-Boc-L-propanol, the mass ratio of N-Boc-propanol to hydroxy oxidase in step (2) is 1:0.01-0.03.

[0034] In the preparation method of N-Boc-L-propanol in this invention, the reaction temperature in step (2) is 20-35℃ and the reaction time is 5-7h.

[0035] In the preparation method of N-Boc-L-propanol in this invention, the pH value of the pH buffer system in step (2) is 6.5-7.5.

[0036] The pH buffer system is a phosphate buffer system; this phosphate buffer system consists of the following components: disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, ammonia, and a solvent. The ammonia is used to adjust the pH, and the pH value of the system is mainly adjusted by ammonia during the reaction process.

[0037] Preferably, the mass ratio of disodium hydrogen phosphate dodecahydrate to sodium dihydrogen phosphate dihydrate is 5-7:1; the solvent is deionized water.

[0038] The amino acid sequence of the hydroxy oxidase in step (2) is SEQ ID NO.1.

[0039] Preferably, the nucleotide sequence of the hydroxyoxidase is SEQ ID NO.2.

[0040] In step (2), the concentration of the alkaline solution is 10wt%-20wt%. The alkaline solution can be a 20wt% sodium bicarbonate solution, a 20wt% sodium carbonate solution, or a 10wt% sodium hydroxide solution, etc. Preferably, the alkaline solution is a 20wt% sodium bicarbonate solution.

[0041] An N-Boc-L-propanoamine is prepared using the method described above. The obtained N-Boc-L-propanoamine has an ultra-high performance liquid chromatography purity >99.9% and an ee value of up to 99.9%.

[0042] The application of N-Boc-L-propanol prepared by the above method in the drug dalolutamide.

[0043] An anticancer drug, dalolutamide, has N-Boc-L-propanol as an intermediate in its synthesis.

[0044] The beneficial effects of this invention are as follows: This invention provides a novel method for preparing N-Boc-L-propanoamine. First, N-Boc-propanoamine is obtained by reacting inexpensive and readily available raw materials 2-aminopropanol and Boc anhydride. The obtained N-Boc-propanoamine is then placed in a buffer system, oxygen is introduced, and hydroxyl oxidase is used to oxidize the D-configuration compounds contained in the racemic N-Boc-propanoamine to carboxylic acids. These are then removed by alkali washing, concentrated, crystallized, and dried to obtain N-Boc-L-propanoamine. This process eliminates the need for flammable and expensive reagents such as LiAlH4, NaBH4, and ruthenium carbon, effectively improving production safety. The raw materials are readily available, the conditions are mild, the operation is simple, and the overall yield is >80%, with a product purity exceeding 99.9%, making it suitable for industrial production. Attached Figure Description

[0045] Figure 1 This is an ultra-high performance liquid chromatogram of the purity of N-Boc-L-propanol in Example 1.

[0046] Figure 2 The image shows the 1H NMR spectrum of N-Boc-L-propanol from Example 1.

[0047] Figure 3 This is a localization diagram of the N-Boc-D-propanol isomer of the product in a specific implementation embodiment.

[0048] Figure 4 This is the spectrum of the isomer detection of N-Boc-L-propanol in Example 1.

[0049] Figure 5 This is an ultra-high performance liquid chromatogram of the purity of N-Boc-L-propanol in Example 2.

[0050] Figure 6 The image shows the 1H NMR spectrum of N-Boc-L-propanol from Example 2.

[0051] Figure 7 This is the spectrum of the isomer detection of N-Boc-L-propanol in Example 2.

[0052] Figure 8 This is an ultra-high performance liquid chromatogram of the purity of N-Boc-L-propanol in Example 3.

[0053] Figure 9 The image shows the 1H NMR spectrum of N-Boc-L-propanol from Example 3.

[0054] Figure 10 This is the spectrum of the N-Boc-L-propanol isomer detection in Example 3.

[0055] Figure 11This is an ultra-high performance liquid chromatogram of the purity of N-Boc-L-propanol in Example 4.

[0056] Figure 12 The image shows the 1H NMR spectrum of N-Boc-L-propanol, the product from Example 4.

[0057] Figure 13 This is the spectrum of the N-Boc-L-propanol isomer detection in Example 4. Detailed Implementation

[0058] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings.

[0059] To facilitate understanding of the present invention, the following embodiments are provided. Those skilled in the art should understand that these embodiments are merely illustrative and should not be construed as limiting the scope of the invention.

[0060] 1. The purity of the products, isomer purity, and NMR of the products obtained in the following examples were determined by Waters AcquityUPLC H-Class ultra-high performance liquid chromatography for product purity, Agilent Technologies 1100 Series liquid chromatograph for isomer testing, and Bruker Magnet System 400'54 Ascend NMR spectrometer for characterization.

[0061] 2. The hydroxy oxidase is obtained through the following steps:

[0062] (1) Cloning of the hydroxy oxidase gene and preparation of expression vector:

[0063] The amino acid sequence of hydroxy oxidase is shown in SEQ ID NO.1. Its encoding gene was obtained through codon optimization, and the full-length sequence was further synthesized by a relevant gene synthesis company. PCR primers were designed as follows:

[0064] Upstream primer: 5'-gtgccgcgcggcagc catatg ATGGAAATTACGAACGTGAATG-3'SEQ ID NO.3

[0065] Downstream primer: 5'-acggagctcgaattc ggatcc TTAATCCCAATCGGCGGCAATG-3'SEQ ID NO.4

[0066] The upstream primer introduces the Nde I restriction site (underlined part), and the downstream primer introduces the Bam HI restriction site (underlined part).

[0067] The synthesized hydroxy oxidase DNA fragment and pET-28a empty plasmid were digested with restriction endonucleases Nde I and Bam HI for 2 h at 37 °C, purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit.

[0068] The target fragment was ligated overnight at 4°C using T4 DNA ligase to obtain the expression plasmid pET28a-hro.

[0069] The above expression plasmid was transformed into Escherichia coli DH5α competent cells. Positive clones were screened on kanamycin-containing resistant plates, and single clones were selected. The positive clones were verified by colony PCR.

[0070] The bacterial cells were cultured, and after plasmid amplification, the plasmid was extracted and re-transformed into Escherichia coli BL21(DE3) competent cells. The transformation solution was plated on LB agar plates containing kanamycin and incubated overnight at 37°C inverted to obtain positive transformants Escherichia coli BL21(DE3) / pET28a-hro. The positive clones were verified by colony PCR and gene sequencing.

[0071] (2) Expression of oxidase: The Escherichia coli obtained in step (1) was inoculated into LB medium containing kanamycin (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0) and cultured overnight at 37°C with shaking. The inoculation was then carried out at a rate of 1% (v / v) into a 500 mL Erlenmeyer flask containing 100 mL of LB medium and cultured on a shaker at 37°C and 200 rpm.

[0072] When the OD600 of the culture medium reaches 0.6, IPTG with a final concentration of 0.4 mmol / L is added as an inducer. After induction at 26℃ for 12 h, the culture medium is centrifuged, the bacterial cells are collected, and washed twice with physiological saline to obtain wet bacterial cells.

[0073] Weigh the collected wet bacterial cells by centrifugation, and resuspend the cells in 20 mmol / L phosphate buffer (pH = 7.0) at a concentration of 10 mg / mL to obtain a bacterial suspension.

[0074] The bacterial suspension was disrupted using a cell sonicator, and the supernatant was collected after centrifugation to obtain the crude enzyme solution. The crude enzyme solution was purified using existing conventional methods and freeze-dried for 24 hours to obtain the hydroxy oxidase. After collection, it was stored at 4°C.

[0075] Note: hro -- Hydroxyl oxidase

[0076] 3. Calculation method of ee value in each embodiment and comparative example: ee = purity of product in isomer detection spectrum - purity of isomer in detection spectrum.

[0077] Example 1

[0078] The specific steps for preparing N-Boc-L-propanol are as follows:

[0079] (1) Preparation of N-Boc-propanol: First, add 100g of 2-aminopropanol (1.33mol) to 500g of methanol, and slowly add 275g of Boc anhydride (1.26mol) under the condition of controlling the temperature at 20℃, and react at 20℃ for 3h.

[0080] Gas phase detection showed that the remaining raw material was less than 0.1%, indicating the reaction was complete. The resulting reaction solution was concentrated to remove methanol; then 100g of deionized water was added, and the solution was concentrated again to remove residual methanol, yielding a concentrated N-Boc-propanol solution, which was directly used in the next reaction step.

[0081] (2) Synthesis of N-Boc-L-propanol: The concentrated N-Boc-propanol obtained in step (1) was first dispersed and dissolved in 1.0 L of deionized water, and then 1.0 L of phosphate buffer system (the system composition is: 30 g / L disodium hydrogen phosphate dodecahydrate, 6 g / L sodium dihydrogen phosphate dihydrate, 4 wt% ammonia water) and 2.4 g of hydroxy oxidase (the mass ratio of N-Boc-propanol to hydroxy oxidase is 1:0.01) were added. The pH value of the pH buffer system was adjusted to 7.0 by adding 4 wt% ammonia water dropwise. Oxygen was introduced and the temperature was controlled at 23℃ for 7 h.

[0082] After the reaction is complete, add 600 mL of dichloromethane, filter to remove hydroxy oxidase, and then separate the phases to obtain the organic phase.

[0083] Add 300g of a 20wt% sodium bicarbonate solution to the organic phase obtained by phase separation, stir for 30 minutes, wash with water, and separate the phases again.

[0084] The organic phase obtained from the phase separation was concentrated to dryness; 200g of isohexane was added, and the mixture was stirred at room temperature to crystallize; the mixture was filtered and dried to obtain 96.1g of white solid product, with a total product yield of 82.4% and an ee value of 99.95% for the obtained N-Boc-L-propanol.

[0085] Figure 1 The specific parameters for liquid chromatography characterization of N-Boc-L-propanol are shown in Table 1 below.

[0086] Table 1. Liquid Chromatography Characterization Parameters of N-Boc-L-Propanol

[0087] peak Retention time (min) Peak area (mAU*s) Peak height (mAU) Peak area (%) 1 3.520 628 498 0.0198 2 4.636 3175767 1233940 99.9802 Total / 3176395.2 / /

[0088] Based on Table 1, the purity of the product N-Boc-L-propanoamine was determined to be 99.98% using the area normalization method.

[0089] Figure 2 For the product's NMR data:

[0090] 1 ¹H NMR (400 MHz, DMSO-d): δ 0.974–0.994 (d, 3H), δ 1.376 (s, 3H), δ 3.12–3.18 (m, 1H), δ 3.28–3.35 (m, 1H), δ 3.42–3.45 (br., 1H), δ 4.57–4.60 (t, 1H), δ 6.50–6.52 (br., 1H). These characterization results fully demonstrate the successful synthesis of N-Boc-L-propanol in this embodiment.

[0091] Figure 3 This is a localization diagram for the isomer N-Boc-D-propanoamine, a method for detecting isomers, used for peak localization of the isomer. Figure 3 It can be used as a reference peak position when detecting product isomers.

[0092] Figure 4 The spectrum of the N-Boc-L-propanol isomer obtained in this embodiment is used to calculate the ee value. The detection parameters of the product isomers are shown in Table 2 below.

[0093] Table 2 Detection parameters of N-Boc-L-propanol isomer in the product

[0094]

[0095] Based on Table 2, the ee value of the product obtained in this embodiment can be obtained, that is, ee = (99.9743% - 0.0257%) = 99.9486% ≈ 99.95%.

[0096] Example 2

[0097] The specific steps for preparing N-Boc-L-propanol are as follows:

[0098] (1) Preparation of N-Boc-propanol: 100g of 2-aminopropanol (1.33mol) was added to 500g of methanol, and 290g of Boc anhydride (1.33mol) was slowly added under the condition of controlling the temperature at 25℃. The reaction was carried out at 25℃ for 4h.

[0099] Gas phase detection showed that the remaining raw material was less than 0.1%, indicating the reaction was complete. The resulting reaction solution was concentrated to remove methanol; then 100g of deionized water was added, and the solution was concentrated again to remove residual methanol, yielding a concentrated N-Boc-propanol solution, which was directly used in the next reaction step.

[0100] (2) Synthesis of N-Boc-L-propanol: The concentrated N-Boc-propanol obtained in step (1) was first dispersed and dissolved in 1.2L of deionized water, and then 1.0L of phosphate buffer system (the system composition is: 30g / L disodium hydrogen phosphate dodecahydrate, 6g / L sodium dihydrogen phosphate dihydrate, 4wt% ammonia water) and 3.0g of hydroxy oxidase (the mass ratio of N-Boc-propanol to hydroxy oxidase is 1:0.013) were added. The pH value of the pH buffer system was adjusted to 7.0 by adding 4wt% ammonia water dropwise, oxygen was introduced, and the temperature was controlled at 23℃ for 6h.

[0101] After the reaction is complete, add 600 mL of dichloromethane, filter to remove hydroxy oxidase, and then separate the phases to obtain the organic phase.

[0102] Add 300g of a 20wt% sodium bicarbonate solution to the organic phase obtained by phase separation, stir for 30 minutes, wash with water, and separate the phases again.

[0103] The organic phase obtained from the phase separation was concentrated to dryness; 200g of isohexane was added, and the mixture was stirred at room temperature to crystallize; the mixture was filtered and dried to obtain 98.2g of white solid product, with a total product yield of 84.2% and an ee value of 100.00% for the obtained N-Boc-L-propanol.

[0104] Figure 5 The specific parameters for the liquid chromatography characterization of N-Boc-L-propanol are shown in Table 3 below.

[0105] Table 3. Liquid Chromatography Characterization Parameters of N-Boc-L-Propanol

[0106] peak Retention time (min) Peak area (mAU*s) Peak height (mAU) Peak area (%) 1 4.594 2949525 1250977 99.9841 2 4.932 468 833 0.0159 Total / 2949993.1 / /

[0107] Based on Table 3, the purity of the product N-Boc-L-propanoamine was determined to be 99.98% using the area normalization method.

[0108] Figure 6 For the product's NMR data:

[0109] 1¹H NMR (400MHz, DMSO-d): δ 0.977–0.994 (d, 3H), δ 1.378 (s, 3H), δ 3.14–3.17 (m, 1H), δ 3.29–3.33 (m, 1H), δ 3.44–3.45 (br., 1H), δ 4.57–4.60 (t, 1H), δ 6.50–6.52 (br., 1H). These characterization results fully demonstrate the successful synthesis of N-Boc-L-propanol in this embodiment.

[0110] Figure 7 The spectrum of the N-Boc-L-propanol isomer obtained in this embodiment is used to calculate the ee value. The detection parameters of the product isomers are shown in Table 4 below.

[0111] Table 4. Detection parameters for the N-Boc-L-propanol isomer in the product.

[0112]

[0113] Based on Table 4, it can be concluded that: only the product N-Boc-L-propanol was obtained in this implementation, and no isomers were detected, so the ee is 100.00%.

[0114] Example 3

[0115] The specific steps for preparing N-Boc-L-propanol are as follows:

[0116] (1) Preparation of N-Boc-propanol: 100g of 2-aminopropanol (1.33mol) was added to a mixed solvent consisting of 250g of methanol and 250g of deionized water. Under the condition of controlling the temperature at 30℃, 319g of Boc anhydride (1.46mol) was slowly added and the reaction was carried out at 30℃ for 5h.

[0117] Gas phase detection showed that the remaining raw material was less than 0.1%, indicating the reaction was complete. The resulting reaction solution was concentrated to remove methanol; then 100g of deionized water was added, and the solution was concentrated again to remove residual methanol, yielding a concentrated N-Boc-propanol solution, which was directly used in the next reaction step.

[0118] (2) Synthesis of N-Boc-L-propanol: The concentrated N-Boc-propanol obtained in step (1) was first dispersed and dissolved in 1.5L of deionized water, and then 1.0L of phosphate buffer system (the system composition is: 30g / L disodium hydrogen phosphate dodecahydrate, 5g / L sodium dihydrogen phosphate dihydrate, 4wt% ammonia water) and 5.0g of hydroxy oxidase (the mass ratio of N-Boc-propanol to hydroxy oxidase is 1:0.021) were added. The pH value of the pH buffer system was adjusted to 7.5 by adding 4wt% ammonia water dropwise, oxygen was introduced, and the temperature was controlled at 35℃ for 5h.

[0119] After the reaction is complete, add 600 mL of dichloromethane, filter to remove hydroxy oxidase, and then separate the phases to obtain the organic phase.

[0120] Add 300g of a 10wt% sodium hydroxide solution to the organic phase obtained by phase separation, stir for 30 minutes, wash with water, and separate the phases again.

[0121] The organic phase obtained from the phase separation was concentrated to dryness; 200g of isohexane was added, and the mixture was stirred at room temperature to crystallize; the mixture was filtered and dried to obtain 95.9g of white solid product, with a total product yield of 82.2% and an ee value of 99.95% for the obtained N-Boc-L-propanol.

[0122] Figure 8 The specific parameters for the liquid chromatography characterization of N-Boc-L-propanol are shown in Table 5 below.

[0123] Table 5. Liquid Chromatography Characterization Parameters of N-Boc-L-Propanol

[0124] peak Retention time (min) Peak area (mAU*s) Peak height (mAU) Peak area (%) 1 3.530 766 563 0.0250 2 4.635 3064971 1208894 99.9750 Total / 3065736.8 / /

[0125] Based on Table 5, the purity of the product N-Boc-L-propanoamine was determined to be 99.98% using the area normalization method.

[0126] Figure 9 For the product's NMR data:

[0127] 1 ¹H NMR (400MHz, DMSO-d): δ 0.977–0.994 (d, 3H), δ 1.378 (s, 3H), δ 3.14–3.17 (m, 1H), δ 3.29–3.33 (m, 1H), δ 3.44–3.45 (br., 1H), δ 4.57–4.60 (t, 1H), δ 6.50–6.52 (br., 1H). These characterization results fully demonstrate the successful synthesis of N-Boc-L-propanol in this embodiment.

[0128] Figure 10The image shows the isomer detection spectrum of the product N-Boc-L-propanol obtained in this embodiment, used to calculate the ee value. The detection parameters for the product isomers are shown in Table 6 below.

[0129] Table 6. Detection parameters for the N-Boc-L-propanol isomer in the product.

[0130]

[0131] Based on Table 6, the ee value of the product can be calculated, i.e., ee = (99.9764% - 0.0236%) = 99.9528% ≈ 99.95%.

[0132] Example 4

[0133] The specific steps for preparing N-Boc-L-propanol are as follows:

[0134] (1) Preparation of N-Boc-propanol: 100g of 2-aminopropanol (1.33mol) was added to 500g of methanol, and 304g of Boc anhydride (1.40mol) was slowly added under the condition of controlling the temperature at 20℃. The reaction was carried out at 20℃ for 4h.

[0135] Gas phase detection showed that the remaining raw material was less than 0.1%, indicating the reaction was complete. The resulting reaction solution was concentrated to remove methanol; then 100g of deionized water was added, and the solution was concentrated again to remove residual methanol, yielding a concentrated N-Boc-propanol solution, which was directly used in the next reaction step.

[0136] (2) Synthesis of N-Boc-L-propanol: The concentrated N-Boc-propanol obtained in step (1) was first dispersed and dissolved in 1.2L of deionized water, and then 1.0L of phosphate buffer system (the system composition is: 30g / L disodium hydrogen phosphate dodecahydrate, 5g / L sodium dihydrogen phosphate dihydrate, 4wt% ammonia water) and 3.0g of hydroxy oxidase (the mass ratio of N-Boc-propanol to hydroxy oxidase is 1:0.013) were added. The pH value of the pH buffer system was adjusted to 7.5 by adding 4wt% ammonia water dropwise, oxygen was introduced, and the temperature was controlled at 20℃ for 6h.

[0137] After the reaction is complete, add 600 mL of dichloromethane, filter to remove hydroxy oxidase, and then separate the phases to obtain the organic phase.

[0138] Add 200g of a 20wt% sodium carbonate solution to the organic phase obtained by phase separation, stir for 30 minutes, wash with water, and separate the phases again.

[0139] The organic phase obtained from the phase separation was concentrated to dryness; 200g of isohexane was added, and the mixture was stirred at room temperature to crystallize; the mixture was filtered and dried to obtain 95.6g of white solid product, with a total product yield of 82.0% and an ee value of 99.95% for the obtained N-Boc-L-propanol.

[0140] Figure 11 The specific parameters for the liquid chromatography characterization of N-Boc-L-propanol are shown in Table 7 below.

[0141] Table 7. Liquid Chromatography Characterization Parameters of N-Boc-L-Propanol

[0142] peak Retention time (min) Peak area (mAU*s) Peak height (mAU) Peak area (%) 1 3.710 1660 676 0.0563 2 4.632 2946722 1213311 99.9437 Total / 2948382.1 / /

[0143] Based on Table 7, the purity of the product N-Boc-L-propanoamine was determined to be 99.94% using the area normalization method.

[0144] Figure 12 For the product's NMR data:

[0145] 1 ¹H NMR (400MHz, DMSO-d): δ 0.977–0.994 (d, 3H), δ 1.378 (s, 3H), δ 3.14–3.17 (m, 1H), δ 3.29–3.33 (m, 1H), δ 3.44–3.45 (br., 1H), δ 4.57–4.60 (t, 1H), δ 6.50–6.52 (br., 1H). These characterization results fully demonstrate the successful synthesis of N-Boc-L-propanol in this embodiment.

[0146] Figure 13 The spectrum of the N-Boc-L-propanol isomer obtained in this embodiment is used to calculate the ee value. The detection parameters of the product isomers are shown in Table 8 below.

[0147] Table 8. Detection parameters for the N-Boc-L-propanol isomer in the product.

[0148]

[0149] Based on Table 8, the ee value of the product can be obtained, i.e., ee = (99.9763% - 0.0237%) = 99.9526% ≈ 99.95%.

[0150] Example 5

[0151] The difference from Example 1 is that the molar ratio of 2-aminopropanol to Boc anhydride in step (1) is 1:1.1.

[0152] Everything else is the same as in Example 1.

[0153] Example 6

[0154] The difference from Example 1 is that the mass ratio of N-Boc-propanol to hydroxy oxidase in step (2) is 1:0.030.

[0155] Everything else is the same as in Example 1.

[0156] Example 7

[0157] The difference from Example 1 is that the temperature is controlled at 30°C in step (1).

[0158] Everything else is the same as in Example 1.

[0159] Example 8

[0160] The difference from Example 1 is that the temperature is controlled at 35°C in step (2).

[0161] Everything else is the same as in Example 1.

[0162] Example 9

[0163] The difference from Example 1 is that the pH value of the pH buffer system is adjusted to 7.5 in step (2).

[0164] Everything else is the same as in Example 1.

[0165] Example 10

[0166] The difference from Example 1 is that the pH value of the pH buffer system is adjusted to 6.5 in step (2).

[0167] Everything else is the same as in Example 1.

[0168] Comparative Example 1

[0169] The difference between this comparative example and Example 1 is that the molar ratio of 2-aminopropanol to Boc anhydride in step (1) is 1:0.5.

[0170] 47.8 g of white solid product was obtained, with a total product yield of 41.0% and an ee value of 99.55% for the obtained N-Boc-L-propanol.

[0171] Comparative Example 2

[0172] The difference between this comparative example and Example 1 is that the molar ratio of 2-aminopropanol to Boc anhydride in step (1) is 1:2.

[0173] 95.8g of white solid product was obtained, with an overall product yield of 82.1%. The ee value of the obtained N-Boc-L-propanohydrin was 99.90%, indicating poor economic viability. The market price of Boc anhydride is approximately 110,000 RMB / ton, which is subject to fluctuation. At this price, increasing the equivalent yield would increase the production cost by approximately 50%.

[0174] Comparative Example 3

[0175] The difference between this comparative example and Example 1 is that the mass ratio of N-Boc-propanol to hydroxy oxidase in step (2) is 1:0.008.

[0176] 91.6 g of white solid product was obtained, with an overall product yield of 78.5% and an ee value of 99.83% for the obtained N-Boc-L-propanol.

[0177] Comparative Example 4

[0178] The difference between this comparative example and Example 1 is that the mass ratio of N-Boc-propanol to hydroxy oxidase in step (2) is 1:0.040.

[0179] 70.6g of white solid product was obtained, with an overall product yield of 60.5%. The ee value of the obtained N-Boc-L-propanol was 99.60%. The increase in enzyme content led to a faster reaction, which was more difficult to control, resulting in a decrease in yield and an increase in cost of approximately 10%.

[0180] Comparative Example 5

[0181] The difference between this comparative example and Example 1 is that the pH of the system is controlled to be 5.0 in step (2) by adding acetic acid solution.

[0182] 40.8 g of white solid product was obtained, with an overall product yield of 35.0% and an ee value of 99.33% for the obtained N-Boc-L-propanol.

[0183] Comparative Example 6

[0184] The difference between this comparative example and Example 1 is that the pH of the system is controlled to be 9 by adding 4% ammonia solution in step (2).

[0185] 52.5 g of white solid product was obtained, with an overall product yield of 45.0% and an ee value of 99.50% for the obtained N-Boc-L-propanol.

[0186] The relevant data of the products obtained from the above embodiments and comparative examples are summarized in Table 9 below.

[0187] Table 9

[0188]

[0189]

[0190] The sequence of hydroxy oxidase, listed in SEQ ID NO. 1, is as follows:

[0191] MEITNVNEYEAIAKQKLATIMALEEVVGAEDQWTLAENRNAFSRILFRPRILIDVTNIDMTT

[0192] TILGFKISMPIMIAPTAMQKMAHPEGEYATARAASFVLPPFLTSSWATSSVEEVASTGPGIRF

[0193] FQLYVYKDRNVVAQLVRRAERAGFKAIALTVGGVRRGTDVADIKNRFVLPPFLTLKNFEGI

[0194] DLGKMDKANDSGLSSYVAGQIDRSLSWKDVAWLQTITSLPILVKGVITAEDARLAVQHGA

[0195] AGIIVSNHGARQLDYVPATIMADTPRLGRREGRIPVFLDGGVRRGTDVFKALALGAAGVFIGRPVVFSLAAEGEAGVKKVLQMMRDEFELTMALSGCRSLKEISRSHIAADWD。

[0196] Sequence Listing 2 of Hydroxylase - Nucleotide Sequence (E. coli) is shown in SEQ ID NO.2: ATGGAAATTACGAACGTGAATGAATATGAAGCCATTGCCAAACAGAAACTGGCGACCATCATGGCCCTGGAAGAAGTGGTGGGCGCGGAAGATCAGTGGACGCTGGCGGAAAATCGTAACGCCTTTAGCCGCATTCTGTTCCGCCCGCGCATCCTGATTGATGTGACCAACATTGATATGACCACCACCATCCTGGGCTTTAAAATTAGCATGCCGATTATGATTGCGCCGACCGCGATGCAGAAAATGGCGCATCCGGAAGGTGAATATGCCACCGCACGCGCGGCGTCGTTTGTGCTGCCGCCGTTTCTGACCAGCAGCTGGGCGACCAGCAGCGTGGAAGAAGTGGCCAGCACCGGCCCGGGTATCCGCTTCTTTCAGCTGTACGTGTACAAAGACCGCAACG

[0197] TTGTGGCGCAGCTGGTGCGCCGTGCGGAACGCGCGGGCTTTAAAGCGATTGCCCTGACCGTGGGCGG

[0198] CGTGCGCCGCGGCACCGATGTGGCGGATATTAAAAATCGCTTTGTGCTGCCGCCGTTTCTGACCCTGA

[0199] AAAACTTTGAAGGCATTGATCTGGGCAAAATGGATAAGGCGAACGATAGCGGCCTGAGCAGCTACGT

[0200] GGCGGGCCAGATCGATCGTAGCCTGAGCTGGAAAGATGTGGCCTGGCTGCAGACCATTACCAGCCTG

[0201] CCGATCCTGGTGAAAGGCGTGATTACGGCCGAAGATGCGCGCCTGGCGGTTCAGCATGGCGCGGCGG

[0202] GCATCATTGTTAGCAACCACGGCGCGCGTCAACTGGATTATGTGCCGGCGACCATTATGGCAGATACC

[0203] CCGCGCCTTGGCCGCCGCGAAGGCCGCATTCCGGTGTTTCTGGATGGCGGCGTTCGTCGTGGCACCG

[0204] ATGTGTTTAAAGCCCTGGCGCTGGGCGCGGCGGGCGTGTTCATCGGCCGCCCGGTGGTGTTTAGCCTG

[0205] GCGGCGGAAGGTGAAGCGGGCGTCAAAAAAGTGCTGCAGATGATGCGCGATGAATTTGAACTGACC

[0206] ATGGCCCTGAGCGGCTGCCGTAGCCTGAAAGAAATTAGCCGTTCACACATTGCCGCCGATTGGGATTAA。

Claims

1. A method for preparing N-Boc-L-propanol, characterized in that, Includes the following steps: (1) Preparation of N-Boc-propanol: The raw material 2-aminopropanol is reacted with Boc anhydride to obtain N-Boc-propanol; (2) Synthesis of N-Boc-L-propanol: Disperse and dissolve the N-Boc-propanol obtained in step (1), add hydroxy oxidase and pH buffer system, adjust the pH value of pH buffer system to 6-8, introduce oxygen, control temperature <40℃, and N-Boc-propanol reacts with hydroxy oxidase. After the reaction was completed, the N-Boc-L-propanol was obtained by extraction, filtration, alkali washing, concentration, crystallization and drying. The amino acid sequence of the hydroxy oxidase is SEQ ID NO.

1.

2. The method for preparing N-Boc-L-propanol according to claim 1, characterized in that, The specific steps are as follows: (1) Preparation of N-Boc-propanol: First, add 2-aminopropanol to an organic solvent, and under the condition of controlling the temperature <40℃, add Boc anhydride to carry out the reaction; After the reaction is complete, the resulting reaction solution is concentrated to remove the organic solvent; then deionized water is added, and the solution is concentrated again to remove the residual organic solvent, yielding a concentrated solution of N-Boc-propanol. (2) Synthesis of N-Boc-L-propanol: The concentrated N-Boc-propanol obtained in step (1) is first dispersed and dissolved in deionized water, and then a pH buffer system and hydroxy oxidase are added. The pH value of the pH buffer system is adjusted to 6-8, oxygen is introduced, and the reaction is carried out under the condition of controlling the temperature <40℃. After the reaction is complete, add the extractant, filter to remove the hydroxy oxidase, and then separate the phases. Add alkali solution to the organic phase obtained by phase separation, wash with water, and separate the phases again; The organic phase obtained from this phase separation was concentrated, crystallized, filtered, and dried to obtain the N-Boc-L-propanol.

3. The method for preparing N-Boc-L-propanol according to claim 1 or 2, characterized in that, In step (1), the molar ratio of 2-aminopropanol to Boc anhydride is 1:0.95-1.

1.

4. The method for preparing N-Boc-L-propanol according to claim 1 or 2, characterized in that, The reaction temperature in step (1) is 20-30℃, and the reaction time is 2-5h.

5. The method for preparing N-Boc-L-propanol according to claim 1 or 2, characterized in that, In step (2), the mass ratio of N-Boc-propanol to hydroxy oxidase is 1:0.010-0.

030.

6. The method for preparing N-Boc-L-propanol according to claim 1 or 2, characterized in that, The reaction temperature in step (2) is 20-35℃, and the reaction time is 5-7h.

7. The method for preparing N-Boc-L-propanol according to claim 1 or 2, characterized in that, In step (2), the pH value of the pH buffer system is adjusted to 6.5-7.5; The pH buffer system is a phosphate buffer system; the phosphate buffer system consists of the following components: disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, ammonia, and solvent; The nucleotide sequence of the hydroxyoxidase is SEQ ID NO.2; The concentration of the alkaline solution in step (2) is 10wt%-20wt%.

8. The method for preparing N-Boc-L-propanol according to claim 7, characterized in that, The mass ratio of disodium hydrogen phosphate dodecahydrate to sodium dihydrogen phosphate dihydrate is 5-7:1; the solvent is deionized water.

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