Industrial preparation method for mono-protected amino acid

Abstract

Disclosed is an industrial preparation method for a mono-protected amino acid, comprising: uniformly mixing an amino compound, water, an organic solvent, and a base together to obtain a mixed solution; adding a compound represented by formula II into the mixed solution and completely reacting at 25-35ºC to obtain a reaction solution; adjusting the pH value of the reaction solution to be a strongly acidic level and performing crystallization to produce a solid, thereby obtaining a crude product; cooling the reaction solution containing the crude product and centrifuging the reaction solution at a low temperature to separate the crude product; and performing post-treatment on the crude product to obtain a compound represented by formula 1, wherein W is selected from amino acid substituents. The preparation method provided by the present invention is suitable for the industrial synthesis of mono-protected amino acids fed on a large scale, and can effectively shorten the preparation process and the synthesis time and reduce the cost while realizing relatively good purity and yield.

Description

An industrial preparation method for a single protected amino acid

[0001] This application claims priority to Chinese Patent Application No. 202510005546.4, filed on January 2, 2025, entitled "An Industrial Preparation Method of a Single Protected Amino Acid", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of chemical synthesis, specifically to an industrial method for the preparation of a single protected amino acid. Background Technology

[0003] The group represented by Formula III is a commonly used protecting group. It is often used to protect amino groups, preventing them from incorrectly binding to other molecules or being damaged by chemical reactions during peptide synthesis. The group represented by Formula III exhibits high chemical stability under a wide range of reaction conditions, remaining unchanged throughout peptide chain synthesis, thus ensuring correct amino acid linkage and proper protein folding. Furthermore, the group of Formula III is easily removable, allowing it to be effectively removed after peptide synthesis to obtain the target peptide or protein.

[0004] Currently, a relatively common method for synthesizing compounds using the protecting group shown in Formula III involves dissolving solid amino acids in a 1%-20% by weight sodium carbonate solution, stirring to ensure complete dissolution, and then adding a solution of the compound shown in Formula IV dissolved in toluene dropwise at 20-30°C over 30-60 minutes. After the addition is complete, the mixture is stirred at 20-30°C for 1-8 hours, diluted with water, and extracted with n-butyl acetate to remove excess of the compound shown in Formula IV. The resulting aqueous phase is adjusted to pH 0.5-3.5 with concentrated hydrochloric acid, and then extracted with n-butyl acetate. The resulting oil phase is washed with water to remove hydrochloric acid, concentrated to remove n-butyl acetate, and white crystals precipitate. The crystals are then filtered and dried to obtain the product Fmoc-amino acid. This method is cumbersome, uses the expensive compound shown in Formula IV, and in the examples, the amount of amino acid used does not exceed 10g. Therefore, if the product is to be industrialized, the above method has two serious problems: 1. The synthesis is a two-step method. The amino acid must be dissolved first, the compound shown in Formula IV must be dissolved, and then the two mixed solutions must be mixed and condensed. This reaction requires different mixing containers, which increases the reaction steps and the difficulty of the reaction. In addition, in the post-processing, extraction is used in large-scale production. However, the yield will be reduced due to insufficient extraction of large-scale reactants.

[0005] 2. It uses too many raw materials and the cost is high. If a large amount of materials are used, the cost will be high. Summary of the Invention

[0006] In view of this, the present invention provides a preparation method that is simple in reaction process, has few steps, is suitable for large-scale industrial production of the compound represented by Formula I, and does not reduce yield and purity.

[0007] To solve the above technical problems, the technical solution of the present invention is an industrial preparation method for a single protected amino acid, comprising:

[0008] The amino compound, water, organic solvent, and alkali are mixed thoroughly to obtain a mixture.

[0009] The compound shown in Formula II was added to the mixture, and the reaction was completed at 25°C to 35°C to obtain the reaction solution.

[0010] The pH of the reaction solution was adjusted to strongly acidic, and crystallization produced a solid to obtain the crude product;

[0011] The reaction solution containing the crude product is cooled and centrifuged at low temperature to separate the crude product;

[0012] The crude product was post-processed to obtain the compound described in Formula 1;

[0013] Where W is selected from amino acid substituents.

[0014] Preferably, W is selected from the substituents shown in the following structural formulas:

[0015] Preferably, the molar ratio of the compound shown in Formula II, the amino compound, and the base is 1:(0.8 to 1.3):(2 to 2.5).

[0016] Preferably, the molar ratio of the compound shown in Formula II, the amino compound, and the base is 1:0.96:2.

[0017] Preferably, the reaction temperature is controlled at 25–30°C.

[0018] Preferably, the alkali is sodium carbonate or sodium bicarbonate.

[0019] Preferably, the organic solution is one of ethyl acetate, acetone, tetrahydrofuran, or other organic solutions.

[0020] Preferably, the reaction time is 2 to 3 hours.

[0021] Preferably, adjusting the pH of the reaction solution to a strongly acidic state specifically involves using concentrated hydrochloric acid to adjust the pH of the reaction solution to 2-3.

[0022] Preferably, the amount of the amino compound, water, organic solvent, alkali, and compound represented by formula II is greater than 50 kg.

[0023] The primary problem this invention addresses is how to shorten the synthesis process while achieving high yields and purity of the target product in large-scale production. To solve this technical problem, this invention provides a method for synthesizing compounds. Firstly, this invention is designed for large-scale feed synthesis, where feed amounts are in the tens or even hundreds of kilograms. As those skilled in the art know, in industrial chemical synthesis, laboratory theoretical values ​​and reaction principles only serve as theoretical support for the synthesis path and cannot be fully applied to industrial synthesis. With increased feed amounts, many details can affect the final product yield and purity. For example, existing techniques mention dissolving different raw materials before mixing and adding them dropwise to make the mixing reaction more uniform and stable. However, in industrial preparation, it is necessary to ensure reaction efficiency. Dropwise addition leads to low reaction efficiency. Furthermore, in industrial production, an additional mixing step requires an additional mixing device, which inevitably leads to incomplete reaction of the mixed solution, residues, and the introduction of more impurities. Therefore, the inventors of this invention needed to reduce the reaction steps in the synthesis process, turning two-step mixing into one-step mixing. However, if the raw materials were directly mixed in one step, uneven mixing would occur, and large amounts of material might agglomerate or clump, reducing reaction efficiency and affecting yield and purity. Therefore, this invention preferably uses the compound shown in Formula II because it is more stable than the compound shown in Formula IV. The byproducts after the reaction can be directly dissolved in the acid solution without precipitating as impurities. This reduces reaction steps and shortens reaction time while improving purity. In addition, the reaction temperature provided by this invention is also specially considered because the compound shown in Formula II will decompose at excessively high temperatures, leading to a decrease in yield. If the pH value is lower than the lower limit of this invention during acidification, acid will remain in the crude product during centrifugation, affecting the product yield and purity. Attached Figure Description

[0024] Figure 1 is the HPLC chromatogram of the compound prepared in Example 1 of the present invention;

[0025] Figure 2 is the HPLC chromatogram of the compound prepared in Example 2 of the present invention;

[0026] Figure 3 is the HPLC chromatogram of the compound prepared in Example 3 of the present invention;

[0027] Figure 4 is the HPLC chromatogram of the compound prepared in Example 4 of the present invention;

[0028] Figure 5 shows the HPLC chromatogram of the compound prepared in Example 5 of this invention;

[0029] Figure 6 is the HPLC chromatogram of the compound prepared in Example 6 of the present invention;

[0030] Figure 7 is the HPLC chromatogram of the compound prepared in Example 7 of the present invention;

[0031] Figure 8 is the HPLC chromatogram of the compound prepared in Example 8 of this invention. Detailed Implementation

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0033] It should be noted that the short bonds in the substituents of all the structural formulas of this invention represent broken bonds that have lost free electrons and can form covalent bonds with other free electrons. For example, the substituents shown in structural formulas a to h all represent unsubstituted amino groups.

[0034] To address the problem of low reaction efficiency and reduced yield and purity in the industrial production of single-protected amino acids under large-scale feeding conditions, this invention provides an industrial preparation method for single-protected amino acids, comprising:

[0035] An amino compound, water, organic solvent, and alkali are mixed thoroughly to obtain a mixture. The compound represented by Formula II is added to the mixture, and the reaction is carried out completely at 25°C–35°C to obtain a reaction solution. The pH of the reaction solution is adjusted to strongly acidic, and crystallization produces a solid, yielding a crude product. The reaction solution containing the crude product is cooled and centrifuged at low temperature to separate the crude product. The crude product undergoes post-processing to obtain…

[0036] The compound described in Formula 1;

[0037] Wherein W is selected from amino acid substituents. Specifically, W is selected from the substituents shown in Table 1 below:

[0038] Table 1 Substituents

[0039] According to the present invention, the molar ratio of the compound, amino compound, and base represented by Formula II is 1:(0.8-1.3):(2-2.5). Preferably, the molar ratio of the compound, amino compound, and base represented by Formula II is 1:0.96:2. According to the present invention, the raw materials are fed in units of tens of kilograms (kg), with most raw materials fed in units of hundreds of kilograms (kg). Compared to the prior art where the feeding amount is in units of grams or several kilograms, the large feeding ratio of the present invention would logically require pre-mixing, dissolving the raw materials separately before mixing for a more uniform result. However, this would lead to excessively long production time, increased process steps, the introduction of unpredictable impurities, increased energy consumption, and higher labor costs. Therefore, the inventors have been exploring how to shorten the production process while maintaining high yield and purity under large-scale feeding conditions. Through research and development, it has been discovered that under large-scale feeding conditions, the molar ratio of the raw materials in the present invention enables the reaction to proceed in the forward direction, resulting in a more complete reaction. Using about twice the amount of alkali solution can better achieve the hydrolysis effect. In the existing technology, some methods use excessive alkali, which can make the hydrolysis more complete, but a large amount of acid is needed for neutralization during subsequent acidification, resulting in higher costs.

[0040] Furthermore, the compound shown in Formula II in this invention results in a more stable reaction, better selectivity, and complete solubility of byproducts in the acid system. During crystallization, there are fewer impurities, and the yield is higher than that of the compound shown in Formula IV, which is more favorable to the reaction process.

[0041] According to the present invention, during the reaction process, if the temperature is too low, the reaction will not be complete; if the temperature exceeds 35°C, the compound represented by Formula II will decompose prematurely, preventing subsequent reactions from taking place. Preferably, the reaction temperature is controlled at 25–30°C.

[0042] According to this invention, the alkali is sodium carbonate or sodium bicarbonate. These two alkalis participate in the hydrolysis reaction more mildly. The organic solution is one of ethyl acetate, acetone, tetrahydrofuran, etc. The reaction time is 2-3 hours. Shorter time results in higher reaction efficiency. Adjusting the pH of the reaction solution to a strongly acidic state specifically involves using concentrated hydrochloric acid to adjust the pH of the reaction solution to 2-3. The preferred molar concentration of the hydrochloric acid is 8N-15N, more preferably 10N-12N, and most preferably 10N. The organic solvent used in this invention does not require concentration or extraction with an extractant; it is directly mixed with the acid solution, centrifuged, and then recovered.

[0043] According to the present invention, the crystallization process needs to be carried out at a low temperature, preferably 0-5°C. The low temperature allows the target product of the present invention to crystallize rapidly into a solid. The crystallization time is preferably 1-3 hours, more preferably 2 hours. After crystallization, the crystallized solid and the remaining acid solution are centrifuged together. The centrifugation of the present invention preferably involves two centrifugations. The purpose of the first centrifugation is to separate the solid and liquid and dissolve most of the solid impurities in the acid solution. After the first centrifugation, the centrifuged solid is washed with pure water. The amount of water used for washing should be greater than the amount of hydrochloric acid used and the weight of the crystallized product, preferably 600-1000 kg, and the washing is preferably performed twice. This continues until the pH of the system is 5-7. It should be noted that before crystallization and centrifugation, the system is acidified to a pH of 2-3. The inventors have found that when the pH is below 2, although crystallization can proceed normally, a large amount of acid remains in the solid crystal after the first centrifugation, which cannot be completely removed by washing. The residual acid solution contains dissolved impurities. Before the second centrifugation, the pH increases, and the impurities precipitate, thus affecting the yield and purity of the product.

[0044] A second centrifugation further separates the crystals and solution after stirring and washing. The solid obtained from the second centrifugation is then dried to obtain the target product. Preferably, the drying temperature is 50–55°C. Excessively high temperatures can cause product decomposition and reduce purity, while excessively low drying temperatures can affect drying efficiency and product quality.

[0045] According to the present invention, the first and second centrifugations are preferably performed using a plate centrifuge, particularly an 800, 1000, or 1250 plate centrifuge, with a centrifuge speed preferably between 800 and 1200 rpm, more preferably between 900 and 1100 rpm. The centrifugation time is 20 to 30 minutes. After the first centrifugation, the resulting crystalline solid is preferably returned to the reaction vessel for washing, and the washed solid is then returned to the centrifuge for a second centrifugation. According to the present invention, the washing time is preferably 0.5 to 1.5 hours.

[0046] Preferably, the first and second centrifugations involve different centrifugation speeds and times due to the different contents of the solid and liquid components in the system, as well as differences in parameters such as pH. These differences are adjusted based on actual production conditions. This effectively improves yield and purity, and prevents product decomposition or impurity precipitation.

[0047] The following are specific embodiments of the present invention, which illustrate the beneficial effects of the present invention in detail.

[0048] Example 1

[0049] Reaction equation:

[0050] Add 1000 kg of pure water to a 3000 L reactor, add 160 kg of sodium carbonate and 120 kg of glycine while stirring, and stir until completely dissolved. Add 600 kg of acetone while controlling the temperature at 28-33 °C. Add 270 kg of the compound shown in Formula II at once, and react at 28-33 °C for 2 hours, controlling the pH at 8-9. After the compound shown in Formula II has completely reacted, add 321 kg of hydrochloric acid to the system and acidify to pH 2-3. A large amount of solid gradually precipitates in the system. Cool the system to 0-5 °C and crystallize for 2 hours. After crystallization, perform the first centrifugation using an 800 plate centrifuge at 800 rpm for 30 minutes. Wash the solid obtained after the first centrifugation twice with pure water (800 kg / wash) until the pH is 5-7, with each washing lasting 1 hour. A second centrifugation was performed at 1200 rpm for 20 minutes. The solid obtained from the second centrifugation was dried at 50-55°C to obtain 228.38 kg of the compound solid represented by Formula 1, with a yield of 96.33% and an HPLC purity of 99.80%. The HPLC chromatogram of the product is shown in Figure 1. Fmoc-Gly-Gly-OH was not detected, 9-fluorenemethanol was 0.03% after 8.468 minutes, Fmoc-β-Ala-OH was not detected, and the compound represented by Formula II was not detected.

[0051] Example 2

[0052] Reaction equation:

[0053] Add 2000 kg of pure water to a 6300 L reactor, add 256 kg of sodium carbonate and 120 kg of alanine while stirring, and stir until completely dissolved. Add 1200 kg of acetone while controlling the temperature at 28-33 °C. Add 408 kg of the compound shown in Formula II at once and react at 28-33 °C for 2 hours, controlling the pH at 8-9. The compound shown in Formula II was completely reacted by TLC. Add 590 kg of hydrochloric acid to the system and acidify to pH 2-3. A large amount of solid gradually precipitates in the system. Cool the system to 0-5 °C and crystallize for 2 hours. After crystallization, perform the first centrifugation using an 800 plate centrifuge at 1000 rpm for 30 minutes. Wash the solid obtained after the first centrifugation twice with pure water (1000 kg / wash) until pH 5-7, with each washing time being 1 hour. A second centrifugation was performed at 1250 rpm for 30 minutes. The solid obtained from the second centrifugation was dried at 50-55°C to yield 365.4 kg of the compound solid represented by Formula II, with a yield of 97.04% and an HPLC purity of 99.85%. The HPLC chromatogram of the product is shown in Figure 2. At 8.591 minutes, Fmoc-β-Ala-OH was 0.02%, Fmoc-Ala-Ala-OH was not detected, 9-fluorene methanol was not detected, and the compound represented by Formula II was not detected.

[0054] Example 3

[0055] Reaction equation:

[0056] Add 900 kg of pure water to a 3000 L reactor, then add 90 kg of sodium carbonate and 120 kg of methionine while stirring until completely dissolved. Add 760 kg of ethyl acetate, while maintaining the temperature at 28-33 °C. Add 160 kg of the compound shown in Formula II at once, and react for 2 hours at 28-33 °C, maintaining the pH at 8-9. TLC detection shows that the compound shown in Formula II has completely reacted. Add 82 kg of hydrochloric acid to the system and acidify to pH 2-3. A large amount of solid gradually precipitates from the system. Cool the system to 0-5 °C to crystallize for 2 hours. After crystallization, perform a first centrifugation using an 800 L plate centrifuge at 800 rpm for 30 minutes. Wash the solid obtained after the first centrifugation twice with pure water (600 kg / wash) until the pH reaches 5-7, with each wash lasting 1 hour. A second centrifugation was performed at 1000 rpm for 30 minutes. The solid obtained from the second centrifugation was dried at 50-55°C to yield 164.55 kg of the compound solid represented by Formula 3, with a yield of 93.4% and an HPLC purity of 99.68%. The HPLC chromatogram of the product is shown in Figure 3. At 6.395 minutes, Fmoc-β-Ala-OH was 0.06%, Fmoc-Met-Met-OH was not detected, 9-fluorenemethanol was not detected, and the compound represented by Formula II was not detected.

[0057] Example 4

[0058] Reaction equation

[0059] Add 1000 kg of pure water to a 3000 L reactor, add 130 kg of sodium carbonate and 100 kg of valine while stirring, and stir until completely dissolved. Add 660 kg of ethyl acetate while controlling the temperature at 28-33 °C. Add 261 kg of the compound shown in Formula II at once and react at 28-33 °C for 2 hours, controlling the pH at 8-9. The compound shown in Formula II was completely reacted by TLC. Add 257 kg of hydrochloric acid to the system and acidify to pH 2-3. A large amount of solid gradually precipitates in the system. Cool the system to 0-5 °C and crystallize for 2 hours. After crystallization, perform the first centrifugation using an 800 plate centrifuge at 900 rpm for 30 minutes. Wash the solid obtained after the first centrifugation twice with pure water (700 kg / wash) until pH 5-7, with each wash lasting 1 hour. A second centrifugation was performed at 1250 rpm for 20 minutes. The solid obtained from the second centrifugation was dried at 50-55°C to yield 251.26 kg of the compound solid represented by Formula 4, with a yield of 95.8% and an HPLC purity of 99.91%. The HPLC chromatogram of the product is shown in Figure 4. Fmoc-β-Ala-OH was 0.06%, Fmoc-Val-Val-OH was not detected, 9-fluorenemethanol was not detected, and the compound represented by Formula II was not detected.

[0060] Example 5

[0061] Reaction equation

[0062] Add 900 kg of pure water to a 3000 L reactor, add 150 kg of sodium carbonate and 150 kg of leucine while stirring, and stir until completely dissolved. Add 500 kg of ethyl acetate, while controlling the temperature at 28-33 °C. Add 345 kg of the compound shown in Formula II at once, and react at 28-33 °C for 2 hours, controlling the pH at 8-9. The compound shown in Formula II was completely reacted by TLC. Add 454 kg of hydrochloric acid to the system and acidify to pH 2-3. A large amount of solid gradually precipitates in the system. Cool to 0-5 °C and crystallize for 2 hours. After crystallization, perform the first centrifugation using an 800 plate centrifuge at 800 rpm for 30 minutes. Wash the solid obtained after the first centrifugation twice with pure water (800 kg / wash) until pH 5-7, with each washing time being 1 hour. A second centrifugation was performed at 1200 rpm for 20 minutes. The solid obtained from the second centrifugation was dried at 50-55°C to yield 350.5 kg of the compound solid represented by Formula 5, with a yield of 96.97% and an HPLC purity of 99.78%. The HPLC chromatogram of the product is shown in Figure 5. Fmoc-β-Ala-OH, Fmoc-Leu-Leu-OH, 9-fluorenemethanol, and the compound represented by Formula II were not detected.

[0063] Example 6

[0064] Reaction equation:

[0065] Add 900 kg of pure water to a 3000 L reactor, add 140 kg of sodium carbonate and 120 kg of isoleucine while stirring, and stir until completely dissolved. Add 500 kg of ethyl acetate, and control the temperature at 28-33 °C. Add 282 kg of the compound shown in Formula II at once, and react at 28-33 °C for 2 hours while maintaining the pH at 8-9. TLC detection shows that the compound shown in Formula II has completely reacted. Add 282 kg of hydrochloric acid to the system and acidify to pH 2-3. A large amount of solid gradually precipitates from the system. Cool the system to 0-5 °C and crystallize for 2 hours. After crystallization, perform the first centrifugation using an 800 plate centrifuge at 800 rpm for 25 minutes. Wash the solid obtained after the first centrifugation twice with pure water (600 kg / wash) until the pH reaches 5-7, with each wash lasting 1 hour. A second centrifugation was performed at 1000 rpm for 25 minutes. The solid obtained from the second centrifugation was dried at 50-55°C to yield 270.5 kg of the compound solid represented by Formula 6, with a yield of 91.56% and an HPLC purity of 99.86%. The HPLC chromatogram of the product is shown in Figure 6. Fmoc-β-Ala-OH, Fmoc-Ile-Ile-OH, 9-fluorenemethanol, and the compound represented by Formula II were not detected.

[0066] Example 7

[0067] Reaction equation:

[0068] Add 900 kg of pure water to a 3000 L reactor, add 140 kg of sodium carbonate and 100 kg of proline while stirring, and stir until completely dissolved. Add 500 kg of ethyl acetate while controlling the temperature at 28-33 °C. Add 270 kg of the compound shown in Formula II at once and react at 28-33 °C for 2 hours, controlling the pH at 8-9. The compound shown in Formula II reacts completely by TLC. Add 301 kg of hydrochloric acid to the system and acidify to pH 2-3. A large amount of solid gradually precipitates in the system. Cool the system to 0-5 °C and crystallize for 2 hours. After crystallization, perform the first centrifugation using an 800 plate centrifuge at 800 rpm for 25 minutes. Wash the solid obtained after the first centrifugation twice with pure water (600 kg / wash) until pH 5-7, with each wash lasting 1 hour. A second centrifugation was performed at 1000 rpm for 25 minutes. The solid obtained from the second centrifugation was dried at 50-55°C to yield 265.19 kg of the compound solid represented by Formula 7, with a yield of 98.2% and an HPLC purity of 99.91%. The HPLC chromatogram of the product is shown in Figure 7. Fmoc-β-Ala-OH, Fmoc-Pro-Pro-OH, 9-fluorenemethanol, and the compound represented by Formula II were not detected.

[0069] Example 8

[0070] Reaction equation:

[0071] Add 900 kg of pure water to a 3000 L reactor, add 200 kg of sodium carbonate and 120 kg of phenylalanine while stirring, and stir until completely dissolved. Add 500 kg of ethyl acetate, while controlling the temperature at 28-33 °C. Add 225 kg of the compound shown in Formula II at once, and react at 28-33 °C for 2 hours, controlling the pH at 8-9. The compound shown in Formula II was completely reacted by TLC. Add 185 kg of hydrochloric acid to the system and acidify to pH 2-3. A large amount of solid gradually precipitates in the system. Cool to 0-5 °C and crystallize for 2 hours. After crystallization, perform the first centrifugation using an 800 plate centrifuge at 800 rpm for 30 minutes. Wash the solid obtained after the first centrifugation twice with pure water (600 kg / wash) until pH 5-7, with each wash lasting 1 hour. A second centrifugation was performed at 1250 rpm for 20 minutes. The solid obtained from the second centrifugation was dried at 50-55°C to yield 236.35 kg of the compound solid represented by Formula 8, with a yield of 91.46% and an HPLC purity of 99.80%. The HPLC chromatogram of the product is shown in Figure 8. Fmoc-β-Ala-OH was not detected, Fmoc-Phe-Phe-OH was 0.03% at 12.048 minutes, 9-fluorenemethanol was 0.01% at 7.449 minutes, and the compound represented by Formula II was not detected.

[0072] Comparative Example 1

[0073] L-cysteine ​​(non-animal source) (200 kg, 833 mol) was dissolved in tetrahydrofuran (1500 L) and pumped into the reactor. While stirring at room temperature, a pre-prepared sodium carbonate aqueous solution (353 kg dissolved in 1500 L of water, 3333 mol) was added and stirred thoroughly. Then, Fmoc-OSu (547 kg, 162.3 mol, 1.95 eq) was added in batches, and the pH was adjusted. 9–9.5°C, stirred overnight at room temperature, most of the tetrahydrofuran was concentrated, extracted with ethyl acetate, the pH of the aqueous phase was adjusted to ~2 with 3N hydrochloric acid, the aqueous phase was clarified, extracted again with ethyl acetate, dried, concentrated to obtain a semi-oily viscous solid, the crude product was slurried overnight with n-hexane:ethyl acetate in a volume ratio of 4:1, filtered and dried to obtain 501 kg of white solid with a yield of 92.78%, the final NMR spectrum is as follows: MS (ESI) M / Z: 685.2 [M+H]+ 1H NMR (400MHz, DMSO-d6) 12.89 (1H, br s),7.89(2H,d,J=6.8Hz),7.71(3H,m),7.40(2H,d,J=2.8Hz,)7.33(2H,td,J=7.2,2.8Hz,),4.30-4.2 9(2H,m,),4.24(1H,t,J=6.8Hz),4.22(1H,td,J=8.4,4.2Hz,),3.15-3.14(1H,m),2.98–2.92(1H,m).

[0074] By comparing Comparative Example 1 with the present invention, it can be found that the prior art uses a method of adding raw materials multiple times, resulting in a very long mixing time, requiring overnight settling, solvent concentration, product extraction, and overnight pulping. The entire reaction time is much longer than that of the present invention, involving more steps and a much larger extraction dosage. Furthermore, the purity is not as high as that of the present invention. Although cystine is not a single amino acid, cysteine, which is also a single amino acid, can be obtained by reducing cystine. Therefore, its synthetic approach differs from that of the present invention. Although both involve large-scale feeding, with a feed volume of hundreds of kilograms, the reaction efficiency is still lower and the cost is higher due to the different methods.

[0075] Comparative Example 2

[0076] 0.06 g (0.008 mol) of glycine solid was dissolved in 10% sodium carbonate solution and stirred until fully dissolved. At 20–30 °C, a solution of 2.10 g (0.008 mol) of 9-fluorenemethoxycarbonyl chloride dissolved in toluene (2–205 mL) was added dropwise over 30–60 minutes. After the addition was complete, the mixture was stirred at 20–30 °C for 1–8 hours. The solution was diluted with 30–200 mL of water and extracted with 80 mL of n-butyl acetate to remove excess 9-fluorenemethoxycarbonyl chloride. The resulting aqueous phase was acidified with concentrated hydrochloric acid to pH 0.5–3.5, and then extracted with 80 mL of n-butyl acetate. The resulting oil phase was washed with water to remove hydrochloric acid, and the oil phase was concentrated to remove the n-butyl acetate solvent. White crystals precipitated, filtered, and dried to obtain 2.07 g of fmoc-glycine, with a yield of 87.1% and a melting point of 173–174 °C.

[0077] Compared to the present invention, Comparative Example 2 uses a significantly smaller amount of material and employs the compound represented by Formula IV. While it exhibits good efficacy with small doses, the cost is high with large quantities. Furthermore, as clearly seen in Comparative Example 2, its extraction requires a large amount of solvent and concentration, resulting in wasted solvent that is difficult to reuse. Large-scale material use leads to significant waste of organic solvents and is also environmentally unfriendly. The final yield is also lower than that of the embodiments of the present invention.

[0078] In summary, this invention provides an industrial-scale production method for a single protected amino acid, suitable for large-scale industrial production. The method requires simple equipment, has high reaction efficiency, low production cost, generates minimal waste, has low energy consumption, and produces a final product with a purity >99.0%. The above are merely preferred embodiments of this invention. It should be noted that these preferred embodiments should not be considered as limitations on the invention, and the scope of protection of this invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention.

Claims

1. An industrial method for preparing a single protected amino acid, characterized in that, include: The amino compound, water, organic solvent, and alkali are mixed thoroughly to obtain a mixture. The compound shown in Formula II was added to the mixture, and the reaction was completed at 25°C to 35°C to obtain the reaction solution. The pH of the reaction solution was adjusted to strongly acidic, and crystallization produced a solid to obtain the crude product; The reaction solution containing the crude product is cooled and centrifuged at low temperature to separate the crude product; The crude product was post-processed to obtain the compound described in Formula 1; Where W is selected from amino acid substituents.

2. The preparation method according to claim 1, characterized in that, Where W is selected from the substituents shown in the following structural formulas:

3. The preparation method according to claim 1 or 2, characterized in that, The molar ratio of the compound, amino compound, and base shown in Formula II is 1:(0.8–1.3):(2–2.5).

4. The preparation method according to claim 3, characterized in that, The molar ratio of the compound, amino compound, and base shown in Formula II is 1:0.96:

2.

5. The preparation method according to claim 1, characterized in that, The reaction temperature is controlled at 25–30°C.

6. The preparation method according to claim 1, characterized in that, The base is sodium carbonate or sodium bicarbonate.

7. The preparation method according to claim 1, characterized in that, The organic solution is one of the following: ethyl acetate, acetone, tetrahydrofuran, etc.

8. The preparation method according to claim 1, characterized in that, The reaction time is 2 to 3 hours.

9. The preparation method according to claim 1, characterized in that, The process of adjusting the pH of the reaction solution to a strongly acidic state specifically involves using concentrated hydrochloric acid to adjust the pH of the reaction solution to 1-3.

10. The preparation method according to claim 1, characterized in that, The amount of each of the amino compound, water, organic solvent, alkali, and compound represented by formula II is greater than 50 kg.

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