Industrial preparation method of single protected amino acid

Abstract

The industrial preparation method of the single protected amino acid is characterized by comprising the following steps: uniformly mixing an amino compound, water, an organic solvent and alkali together to obtain a mixed solution; adding a compound as shown in a formula II into the mixed solution, and completely reacting at 25-35 DEG C to obtain a reaction solution; adjusting the pH value of the reaction liquid to be strongly acidic, and crystallizing to generate a solid to obtain a crude product; cooling the reaction liquid containing the crude product, centrifuging at low temperature, and separating the crude product; and carrying out post-treatment on the crude product to obtain the compound as shown in the formula 1, wherein W is selected from amino acid substituents. The preparation method provided by the invention is suitable for industrial synthesis of the single protected amino acid fed on a large scale, the preparation process can be effectively shortened, the synthesis time is shortened, the cost is reduced, and meanwhile, the purity and the yield are relatively good. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of chemical synthesis, and in particular to an industrial preparation method of a single-protected amino acid. Background Art

[0002] The group shown in formula III is a commonly used protecting group, which is often used to protect the amino group to prevent it from incorrectly combining with other molecules or being destroyed by chemical reactions during the synthesis of polypeptides; the group shown in formula III exhibits high chemical stability under a wide range of reaction conditions and can remain unchanged throughout the entire process of peptide chain synthesis, thereby ensuring the correct connection of amino acids and the correct folding of proteins. In addition, the group of formula III is also easy to remove, which allows it to be effectively removed after peptide synthesis to obtain the target peptide or protein.

[0003]

[0004] At present, the more conventional method of using the protecting group represented by formula III to synthesize compounds is to dissolve a solid amino acid in a sodium carbonate solution of 1% to 20% by weight, stir to fully dissolve the amino acid solid, drip a solution of a compound represented by formula IV dissolved in toluene at 20-30° C., drip for 30-60 minutes, stir at 20-30° C. for 1-8 hours after the dripping, dilute with water, extract with n-butyl acetate as an extractant, remove excess compound represented by formula IV, adjust the pH of the obtained aqueous phase to 0.5-3.5 with concentrated hydrochloric acid, then extract with n-butyl acetate as an extractant, wash the obtained oil phase with water, remove hydrochloric acid, concentrate the oil phase to remove n-butyl acetate, precipitate white crystals, filter, and dry to obtain the product Fmoc-amino acid; this method is complicated to operate, the compound represented by formula IV used is relatively expensive, and the amount of amino acid fed in its embodiments does not exceed 10g. Therefore, if the product is to be industrially produced, the above method has two serious problems. 1. The synthesis is a two-step process. 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 for reaction condensation. This reaction requires different mixing containers, which increases the reaction steps and the difficulty of the reaction. In addition, in the post-processing process, the use of extraction in large-scale production will be insufficiently extracted by large-scale reactants, resulting in a reduced yield.

[0005] 2. Too many raw materials are used and the cost is high. If a large amount of raw materials are added, the cost will be high.

[0006] Summary of the invention

[0007] In view of this, the present invention provides a preparation method which has a simple reaction process, uses fewer steps, is suitable for large-scale industrial production of the compound represented by formula I, and does not reduce the yield and purity.

[0008] In order to solve the above technical problems, the technical solution of the present invention is to adopt an industrial preparation method of a single protected amino acid, comprising:

[0009] Mixing the amino compound, water, an organic solvent and a base uniformly to obtain a mixed solution;

[0010] Adding the compound represented by formula II to the mixed solution, and reacting at 25° C. to 35° C. to obtain a reaction solution;

[0011] Adjusting the pH value of the reaction solution to strong acidity, crystallizing to produce a solid to obtain a crude product;

[0012] Cooling the reaction solution containing the crude product and centrifuging it at low temperature to separate the crude product;

[0013] Post-treating the crude product to obtain the compound of formula 1;

[0014]

[0015] wherein W is selected from an amino acid substituent.

[0016] Preferably, W is selected from the substituents shown in the following structural formula:

[0017]

[0018] Preferably, the molar ratio of the compound represented by formula II, the amino compound and the base is 1:(0.8-1.3):(2-2.5).

[0019] Preferably, the molar ratio of the compound represented by formula II, the amino compound and the base is 1:0.96:2.

[0020] Preferably, the reaction temperature is controlled at 25-30°C.

[0021] Preferably, the base is sodium carbonate or sodium bicarbonate.

[0022] Preferably, the organic solution is one of organic solutions such as ethyl acetate, acetone, and tetrahydrofuran.

[0023] Preferably, the reaction time is 2 to 3 hours.

[0024] Preferably, adjusting the pH value of the reaction solution to a strong acid is specifically: using concentrated hydrochloric acid to adjust the pH value of the reaction solution to 2-3.

[0025] Preferably, the feeding amounts of the amino compound, water, organic solvent, base and the compound represented by formula II are all greater than 50 kg.

[0026] The problem that the present invention first solves is how to shorten the synthesis process in large-scale production while being able to obtain a target product with a higher yield and purity. In order to solve the above technical problems, the present invention provides a method for synthesizing a compound. First, the present invention is directed to large-scale feed synthesis, and the feed amount is tens or even hundreds of kilograms. It is known to those skilled in the art that in industrial chemical synthesis, the theoretical value and reaction principle of the laboratory are only theoretical support for the synthesis path, and cannot be fully applied to industrial synthesis. After the feed amount is increased, many details will affect the yield and purity of the final product. For example, the different raw materials mentioned in the prior art are first dissolved and then mixed and dripped, which can make the mixed reaction more uniform and the reaction more stable. However, the efficiency of the reaction needs to be guaranteed in the industrial preparation process, and the dripping reaction will result in low reaction efficiency. In addition, in industrial production, one more step of mixing requires one more set of mixing equipment, and one more set of mixing equipment is bound to result in incomplete reaction of the mixed solution, residues, and more impurities. Therefore, the inventors of the present invention need to reduce the reaction steps in the synthesis process, and change the two-step mixing into one-step mixing. If the raw materials are directly mixed in one step, there will be uneven mixing, and a large amount of feed may agglomerate or agglomerate, which will also reduce the reaction efficiency and affect the yield and purity. Therefore, the present invention preferably uses the compound shown in formula II, because the compound shown in formula II is more stable than the compound shown in formula IV, and the by-products after the reaction can be directly dissolved in the acid solution, and will not precipitate into impurities. On the basis of reducing the reaction steps and shortening the reaction time, the purity is improved; in addition, the reaction temperature provided by the present invention also has special considerations, because the compound shown in formula II will decompose at too high a temperature, resulting in a decrease in yield. If the pH value is lower than the lower limit of the present invention during acidification, the acid will remain in the crude product during the centrifugation step, affecting the yield and purity of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 HPLC spectrum of the compound prepared in Example 1 of the present invention;

[0028] Figure 2 HPLC spectrum of the compound prepared in Example 2 of the present invention;

[0029] Figure 3 HPLC spectrum of the compound prepared in Example 3 of the present invention;

[0030] Figure 4 HPLC spectrum of the compound prepared in Example 4 of the present invention;

[0031] Figure 5HPLC spectrum of the compound prepared in Example 5 of the present invention;

[0032] Figure 6 HPLC spectrum of the compound prepared in Example 6 of the present invention;

[0033] Figure 7 HPLC spectrum of the compound prepared in Example 7 of the present invention;

[0034] Figure 8 HPLC spectrum of the compound prepared in Example 8 of the present invention. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with specific implementation methods.

[0036] It should be noted that the short bonds in the substituents in all the structural formulas of the present 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, wherein -NH represents an unsubstituted amino group.

[0037] In order to solve the problem that in the industrial production of single-protected amino acids, the yield and purity are affected by low reaction efficiency and reduced to a certain extent under large-scale feeding conditions, the present invention provides an industrial preparation method of single-protected amino acids, comprising:

[0038] The amino compound, water, an organic solvent and a base are mixed together to obtain a mixed solution; the compound represented by formula II is added to the mixed solution, and after the reaction is completed at 25° C. to 35° C., a reaction solution is obtained; the pH value of the reaction solution is adjusted to a strong acidity, and a solid is crystallized to obtain 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 is post-treated to obtain the compound represented by formula 1;

[0039]

[0040]

[0041] Wherein W is selected from an amino acid substituent. Wherein W is selected from a substituent shown in the structural formula in Table 1 below:

[0042] Table 1 Substituents

[0043]

[0044] According to the present invention, the molar ratio of the compound shown in formula II, the amino compound, and the base is 1: (0.8-1.3): (2-2.5). Preferably, the molar ratio of the compound shown in formula II, the amino compound, and the base is 1: 0.96: 2. According to the present invention, the raw materials for feeding are all tens of kilograms (kg) as the feeding unit, and most of the raw materials are hundreds of kilograms (kg) as the unit. Compared with the feeding amount in the prior art in g or several kilograms, the large feeding ratio of the present invention should be pre-mixed according to common sense. The raw materials are dissolved separately and then mixed more evenly, but doing so will lead to too long production time, and the process flow will increase unexpected impurities, increase energy consumption, and labor costs are also high. Therefore, the inventors have been exploring how to shorten the production process under large-scale feeding conditions, while having a higher yield and purity. Therefore, through research and development, it is found that under large-scale feeding conditions, the molar ratio of the raw materials of the present invention can make the reaction proceed in the forward direction and the reaction is more sufficient. At the same time, using about twice the amount of alkali solution can better play a hydrolysis role. In the prior art, some methods use excessive alkali, which can make the hydrolysis more complete, but in the subsequent acidification, a large amount of acid is needed for neutralization, resulting in higher costs.

[0045] In addition, the use of the compound shown in Formula II in the present invention can make the reaction more stable and the selectivity better, and the by-products can be completely dissolved in the acid solution system. During the crystallization process, there are fewer impurities. Compared with the compound shown in Formula IV, the yield is higher and it is more beneficial to the progress of the reaction.

[0046] According to the present invention, during the reaction, the reaction is not sufficient at too low a temperature, and the compound represented by Formula II will decompose prematurely when the temperature exceeds 35°C, and subsequent reactions cannot proceed. Preferably, the reaction temperature is controlled at 25-30°C.

[0047] According to the present invention, the base is sodium carbonate or sodium bicarbonate. These two bases participate in the hydrolysis reaction more gently. The organic solution is one of organic solutions such as ethyl acetate, acetone, tetrahydrofuran, etc. The reaction time is 2 to 3 hours. The shorter the time, the higher the reaction efficiency. The specific method of adjusting the pH value of the reaction solution to strong acidity is: using concentrated hydrochloric acid to adjust the pH value of the reaction solution to 2 to 3. The molar concentration of hydrochloric acid is preferably 8N to 15N, more preferably 10N to 12N, and most preferably 10N. The organic solvent used in the present invention does not need to be concentrated or extracted with an extractant, but is directly mixed with the acid solution, centrifuged and then recovered.

[0048] According to the present invention, the crystallization process needs to be carried out at a low temperature, preferably at 0-5°C. The low temperature will allow the target product of the present invention to crystallize quickly to form a solid. The crystallization time is preferably 1-3h, more preferably 2h. After crystallization, the crystallized solid is centrifuged together with the remaining acid solution. The centrifugation of the present invention is preferably performed twice. The purpose of the first centrifugation is to separate the solid from the liquid and dissolve most of the solid impurities in the acid solution. After the first centrifugation, the solid after centrifugation is stirred and washed with pure water. The amount of water for stirring and washing is greater than the amount of hydrochloric acid used and the weight of the crystallized product, preferably 600-1000kg, and the number of stirring and washing is preferably 2 times. Until the pH value of the system is 5-7. It should be noted that before crystallization and centrifugation, the pH value of the system is 2-3 after acidification. The inventors have found that when the pH value is lower than 2, although crystallization can proceed normally, a large amount of acid solution will remain in the solid crystal after the first centrifugation, and stirring and washing cannot completely remove it. The residual acid solution will dissolve impurities. Before the second centrifugation, the pH value increases and the impurities will precipitate, thereby affecting the yield and purity of the product.

[0049] The second centrifugation is performed to further separate the stirred and washed crystals and the solution. The solid obtained by the second centrifugation is dried to obtain the target product. Preferably, the drying temperature is 50-55° C. Too high a temperature will cause the product to decompose and reduce the purity, while too low a drying temperature will affect the drying efficiency and the product quality.

[0050] According to the present invention, the first centrifugation and the second centrifugation preferably use a plate centrifuge, especially an 800 plate centrifuge, a 1000 plate centrifuge or a 1250 plate centrifuge, and the centrifuge speed is preferably 800-1200 rpm, more preferably 900-1100 rpm. The centrifugation time is 20-30 minutes. After the first centrifugation, the obtained crystalline solid is preferably returned to the reactor for stirring and washing, and the solid obtained after stirring and washing is returned to the centrifuge for the second centrifugation. According to the present invention, the stirring and washing time is preferably 0.5-1.5 hours.

[0051] Preferably, the first and second centrifugations have different centrifugal speeds and times due to the different contents of solids and liquids in the system and different parameters such as pH values, which can be adjusted according to actual production conditions. This can effectively improve the yield and purity and prevent product decomposition or impurity precipitation.

[0052] The following are specific embodiments of the present invention, which describe the beneficial effects of the present invention in detail.

[0053] Example 1

[0054]

[0055] Reaction equation:

[0056]

[0057] Add 1000kg of pure water to a 3000L reactor, add 160kg of sodium carbonate while stirring, add 120kg of glycine while stirring, stir to completely dissolve it, add 600kg of acetone, control the temperature at 28-33°C at the same time, add 270kg of the compound shown in formula II at one time, control the temperature at 28-33°C to react for 2 hours, and control the pH at 8-9. After the compound shown in formula II is completely reacted by TLC detection, add 321kg of hydrochloric acid to the system, acidify to pH = 2-3, and a large amount of solid gradually precipitates in the system. Cool to 0-5°C and crystallize for 2h. After crystallization, perform the first centrifugation, select 800 plate centrifuge, the centrifugal speed is 800rmp, the centrifugal time is 30 minutes, and the solid obtained after the first centrifugation is washed twice with pure water (800kg / time) to pH = 5-7, and the washing time each time is 1 hour. A second centrifugation was performed, the centrifugal speed of the second centrifugation was 1200 rpm, the centrifugal time was 20 minutes, and the solid obtained by the second centrifugation was dried at 50-55°C to obtain 228.38 kg of the compound solid represented by the product formula 1, with a yield of 96.33% and a HPLC purity of 99.80%. The HPLC spectrum of the product is as follows Figure 1 As shown, Fmoc-Gly-Gly-OH was not detected, 9-fluorene methanol was 0.03% at 8.468 minutes, Fmoc-β-Ala-OH was not detected, and the compound shown in formula II was not detected.

[0058] Example 2

[0059]

[0060] Reaction equation:

[0061]

[0062] 2000kg of pure water was added to a 6300L reactor, 256kg of sodium carbonate was added under stirring, 120kg of alanine was added under stirring, and it was completely dissolved. 1200kg of acetone was added, and the temperature was controlled at 28-33°C. 408kg of the compound shown in formula II was added at one time, and the temperature was controlled at 28-33°C for 2 hours. The pH was controlled at 8-9. According to TLC detection, the compound shown in formula II was completely reacted. 590kg of hydrochloric acid was added to the system, acidified to pH = 2-3, and a large amount of solid gradually precipitated in the system. The temperature was cooled to 0-5°C and crystallized for 2h. After crystallization, the first centrifugation was performed, and an 800 plate centrifuge was selected. The centrifugal speed was 1000rmp and the centrifugal time was 30 minutes. The solid obtained after the first centrifugation was washed twice with pure water (1000kg / time) to pH = 5-7, and the washing time was 1 hour each time. The second centrifugation was performed at a speed of 1250 rpm for 30 minutes. The solid obtained from the second centrifugation was dried at 50-55°C to obtain 365.4 kg of the compound solid of Formula 2, with a yield of 97.04% and a HPLC purity of 99.85%. The HPLC spectrum of the product is shown in Figure 2 As shown, Fmoc-β-Ala-OH 0.02% was detected at 8.591 minutes, Fmoc-Ala-Ala-OH was not detected, 9-fluorene methanol was not detected, and the compound shown in formula II was not detected.

[0063] Example 3

[0064]

[0065] Reaction equation:

[0066]

[0067] 900 kg of pure water was added to a 3000 L reactor, 90 kg of sodium carbonate was added under stirring, 120 kg of methionine was added under stirring, and it was completely dissolved. 760 kg of ethyl acetate was added, and the temperature was controlled at 28-33 ° C. 160 kg of the compound shown in formula II was added at one time, and the temperature was controlled at 28-33 ° C for 2 hours. The pH was controlled at 8-9. According to TLC detection, the compound shown in formula II was completely reacted. 82 kg of hydrochloric acid was added to the system and acidified to pH = 2-3. A large amount of solid gradually precipitated in the system and cooled to 0-5 ° C for crystallization for 2 hours. After crystallization, the first centrifugation was performed, and an 800 plate centrifuge was selected. The centrifugal speed was 800 rpm and the centrifugal time was 30 minutes. The solid obtained after the first centrifugation was washed twice with pure water (600 kg / time) until pH = 5-7, and the washing time was 1 hour each time. The second centrifugation was performed at a speed of 1000 rpm for 30 minutes. The solid obtained from the second centrifugation was dried at 50-55°C to obtain 164.55 kg of the compound solid of Formula 3, with a yield of 93.4% and a HPLC purity of 99.68%. The HPLC spectrum of the product is shown in Figure 3 As shown, Fmoc-β-Ala-OH was 0.06% at 6.395 minutes, Fmoc-Met-Met-OH was not detected, 9-fluorenylmethanol was not detected, and the compound represented by formula II was not detected.

[0068] Example 4

[0069]

[0070] Reaction equation

[0071]

[0072] 1000 kg of pure water was added to a 3000 L reactor, 130 kg of sodium carbonate was added under stirring, 100 kg of valine was added under stirring, and it was completely dissolved. 660 kg of ethyl acetate was added, and the temperature was controlled at 28-33 ° C. 261 kg of the compound shown in formula II was added at one time, and the temperature was controlled at 28-33 ° C for 2 hours. The pH was controlled at 8-9. According to TLC detection, the compound shown in formula II was completely reacted. 257 kg of hydrochloric acid was added to the system, acidified to pH = 2-3, and a large amount of solid gradually precipitated in the system. The temperature was cooled to 0-5 ° C and crystallized for 2 hours. After crystallization, the first centrifugation was performed, and an 800 plate centrifuge was selected. The centrifugal speed was 900 rpm and the centrifugal time was 30 minutes. The solid obtained after the first centrifugation was washed twice with pure water (700 kg / time) until the pH was 5-7, and the washing time was 1 hour each time. The second centrifugation was performed at a speed of 1250 rpm for 20 minutes. The solid obtained from the second centrifugation was dried at 50-55°C to obtain 251.26 kg of the compound solid of formula 4, with a yield of 95.8% and a HPLC purity of 99.91%. The HPLC spectrum of the product is shown in Figure 4 As shown, Fmoc-β-Ala-OH was 0.06%, Fmoc-Val-Val-OH was not detected, 9-fluorene methanol was not detected, and the compound shown in formula II was not detected.

[0073] Example 5

[0074]

[0075] Reaction equation

[0076]

[0077] 900 kg of pure water was added to a 3000 L reactor, 150 kg of sodium carbonate was added under stirring, 150 kg of leucine was added under stirring, and it was completely dissolved. 500 kg of ethyl acetate was added, and the temperature was controlled at 28-33 ° C. 345 kg of the compound shown in formula II was added at one time, and the temperature was controlled at 28-33 ° C for 2 hours. The pH was controlled at 8-9. According to TLC detection, the compound shown in formula II was completely reacted. 454 kg of hydrochloric acid was added to the system and acidified to pH = 2-3. A large amount of solid gradually precipitated in the system and the temperature was cooled to 0-5 ° C for crystallization for 2 hours. After crystallization, the first centrifugation was performed, and an 800 plate centrifuge was selected. The centrifugal speed was 800 rpm and the centrifugal time was 30 minutes. The solid obtained after the first centrifugation was washed twice with pure water (800 kg / time) until the pH was 5-7, and the washing time was 1 hour each time. The second centrifugation was performed at a speed of 1200 rpm for 20 minutes. The solid obtained from the second centrifugation was dried at 50-55°C to obtain 350.5 kg of the compound of Formula 5, with a yield of 96.97% and a HPLC purity of 99.78%. The HPLC spectrum of the product is shown in Figure 5 As shown, Fmoc-β-Ala-OH was not detected, Fmoc-Leu-Leu-OH was not detected, 9-fluorenylmethanol was not detected, and the compound represented by formula II was not detected.

[0078] Example 6

[0079]

[0080] Reaction equation:

[0081]

[0082] 900 kg of pure water was added to a 3000 L reactor, 140 kg of sodium carbonate was added under stirring, 120 kg of isoleucine was added under stirring, and the mixture was completely dissolved. 500 kg of ethyl acetate was added, and the temperature was controlled at 28-33 ° C. 282 kg of the compound shown in formula II was added at one time, and the temperature was controlled at 28-33 ° C. The reaction was carried out for 2 hours, and the pH was controlled at 8-9. According to TLC detection, the compound shown in formula II was completely reacted. 282 kg of hydrochloric acid was added to the system, and the pH was acidified to 2-3. A large amount of solid gradually precipitated in the system, and the temperature was cooled to 0-5 ° C and crystallized for 2 hours. After crystallization, the first centrifugation was performed, and an 800 plate centrifuge was selected. The centrifugal speed was 800 rpm and the centrifugal time was 25 minutes. The solid obtained after the first centrifugation was washed twice with pure water (600 kg / time) until the pH was 5-7, and the washing time was 1 hour each time. The second centrifugation was performed at a speed of 1000 rpm for 25 minutes. The solid obtained from the second centrifugation was dried at 50-55°C to obtain 270.5 kg of a solid compound of Formula 6, with a yield of 91.56% and a HPLC purity of 99.86%. The HPLC spectrum of the product is shown in Figure 6 As shown, Fmoc-β-Ala-OH was not detected, Fmoc-Ile-Ile-OH was not detected, 9-fluorene methanol was not detected, and the compound represented by formula II was not detected.

[0083] Example 7

[0084]

[0085] Reaction equation:

[0086]

[0087] 900 kg of pure water was added to a 3000 L reactor, 140 kg of sodium carbonate was added under stirring, 100 kg of proline was added under stirring, and it was completely dissolved. 500 kg of ethyl acetate was added, and the temperature was controlled at 28-33 ° C. 270 kg of the compound shown in formula II was added at one time, and the temperature was controlled at 28-33 ° C for 2 hours. The pH was controlled at 8-9. According to TLC detection, the compound shown in formula II was completely reacted. 301 kg of hydrochloric acid was added to the system, acidified to pH = 2-3, and a large amount of solid gradually precipitated in the system. The temperature was cooled to 0-5 ° C and crystallized for 2 hours. After crystallization, the first centrifugation was performed, and an 800 plate centrifuge was selected. The centrifugal speed was 800 rpm and the centrifugal time was 25 minutes. The solid obtained after the first centrifugation was washed twice with pure water (600 kg / time) until the pH was 5-7, and the washing time was 1 hour each time. The second centrifugation was performed at a speed of 1000 rpm for 25 minutes. The solid obtained from the second centrifugation was dried at 50-55°C to obtain 265.19 kg of a solid compound of Formula 7, with a yield of 98.2% and a HPLC purity of 99.91%. The HPLC spectrum of the product is shown in Figure 7 As shown, Fmoc-β-Ala-OH was not detected, Fmoc-Pro-Pro-OH was not detected, 9-fluorenylmethanol was not detected, and the compound represented by formula II was not detected.

[0088] Example 8

[0089]

[0090] Reaction equation:

[0091]

[0092] 900 kg of pure water was added to a 3000 L reactor, 200 kg of sodium carbonate was added under stirring, 120 kg of phenylalanine was added under stirring, and it was completely dissolved. 500 kg of ethyl acetate was added, and the temperature was controlled at 28-33 ° C. 225 kg of the compound shown in formula II was added at one time, and the temperature was controlled at 28-33 ° C for 2 hours. The pH was controlled at 8-9. According to TLC detection, the compound shown in formula II was completely reacted. 185 kg of hydrochloric acid was added to the system and acidified to pH = 2-3. A large amount of solid gradually precipitated in the system and cooled to 0-5 ° C for crystallization for 2 hours. After crystallization, the first centrifugation was performed, and an 800 plate centrifuge was selected. The centrifugal speed was 800 rpm and the centrifugal time was 30 minutes. The solid obtained after the first centrifugation was washed twice with pure water (600 kg / time) until pH = 5-7, and the washing time was 1 hour each time. The second centrifugation was performed at a speed of 1250 rpm for 20 minutes. The solid obtained from the second centrifugation was dried at 50-55°C to obtain 236.35 kg of a solid compound of formula 8, with a yield of 91.46% and a HPLC purity of 99.80%. The HPLC spectrum of the product is shown in FIG. Figure 8 As shown, Fmoc-β-Ala-OH was not detected, Fmoc-Phe-Phe-OH was 0.03% at 12.048 minutes, 9-fluorene methanol was 0.01% at 7.449 minutes, and the compound represented by formula II was not detected.

[0093] Comparative Example 1

[0094] L-cystine (non-animal source) (200kg, 833mol) was dissolved in tetrahydrofuran (1500L) and pumped into the reactor. Under stirring at room temperature, the prepared sodium carbonate aqueous solution (353kg was dissolved in 1500L water, 3333mol) was fully stirred, and then Fmoc-OSu (547kg, 162.3mol, 1.95eq) was added in batches, and the pH was adjusted to 9-9.5. Stirring was carried out at room temperature overnight, and most of the tetrahydrofuran was concentrated, and ethyl acetate was extracted. The aqueous phase was adjusted to pH~2 with 3N hydrochloric acid. The aqueous phase was clarified and extracted with ethyl acetate, dried, and concentrated to obtain a semi-oily viscous solid. The crude product was slurried with n-hexane:ethyl acetate in a volume ratio of 4:1 overnight, and filtered and dried to obtain a white solid 501k g yield 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, brs), 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,), 4.30-4.29 (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).

[0095] By comparing the comparative example 1 with the present invention, it can be found that in the prior art, the mixing time is very long by adding the raw materials multiple times, and it is necessary to stand overnight, and then the solvent needs to be concentrated, and then the product is extracted and beaten overnight. The time of the whole reaction is much longer than that of the present invention, and there are more steps, and the amount of extraction is also very large. And the purity is not as high as that of the present invention. Although cystine is not a single amino acid, cysteine ​​can be obtained by reducing cystine, which is also a single amino acid, so its own synthesis idea is also different from the present invention. Although it is also a large-scale feed, the feed amount is also hundreds of kilograms, but due to the different methods, the efficiency of the reaction is still low and the cost is high.

[0096] Comparative Example 2

[0097] 0.06 g (0.008 mol) of glycine solid was dissolved in 10% sodium carbonate solution, stirred to fully dissolve the glycine solid, and a solution of 9-fluorenylmethoxycarbonyl chloride (2.10 g, 0.008 mol) dissolved in toluene (2-205 ml) was added dropwise at 20-30° C. for 30-60 minutes. After the addition was completed, the mixture was stirred at 20-30° C. for 1-8 hours, diluted with 30-200 ml of water, and extracted with n-butyl acetate (80 ml) to remove excess 9-fluorenylmethoxycarbonyl chloride. The resulting aqueous phase was acidified with concentrated hydrochloric acid to pH=0.5-3.5, and then extracted with n-butyl acetate (80 ml). 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 the product fmoc-glycine with a yield of 87.1% and a melting point of 173-174° C.

[0098] Comparative Example 2 is significantly less than the present invention in terms of feed amount, and uses the compound shown in Formula IV, which has a good effect when used in small doses, but the cost is high when a large amount of feed is used, and it is not difficult to see from Comparative Example 2 that a large amount of solvent is required for extraction and concentration, and the removed solvent is wasted and difficult to reuse. In the case of a large amount of feed, a large amount of organic solvent will be wasted, which is not environmentally friendly. The final yield is also lower than that of the embodiment of the present invention.

[0099] In summary, the present invention provides an industrial production method for a single protected amino acid, which is suitable for large-scale industrial production, has simple equipment required for the production method, good reaction efficiency, low production cost, less three wastes, low energy consumption and a final product purity of >99.0%. The above are only preferred embodiments of the present invention. It should be pointed out that the above preferred embodiments should not be regarded as limitations of the present invention, and the scope of protection of the present invention should be based on the scope defined by the claims. For those of ordinary skill in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An industrial preparation method of a single protected amino acid, characterized in that: include: Mixing the amino compound, water, an organic solvent and a base uniformly to obtain a mixed solution; Adding the compound represented by formula II to the mixed solution, and reacting at 25° C. to 35° C. to obtain a reaction solution; Adjusting the pH value of the reaction solution to strong acidity, crystallizing to produce a solid to obtain a crude product; Cooling the reaction solution containing the crude product and centrifuging it at low temperature to separate the crude product; Post-treating the crude product to obtain the compound of formula 1; wherein W is selected from an amino acid substituent.

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

3. The preparation method according to claim 1 or 2, characterized in that: The molar ratio of the compound represented by formula II, the amino compound and the base 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 represented by formula II, the amino compound and the base 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 organic solutions such as 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 step of adjusting the pH value of the reaction solution to be strongly acidic is specifically as follows: using concentrated hydrochloric acid to adjust the pH value of the reaction solution to 1-3.

10. The preparation method according to claim 1, characterized in that: The feeding amounts of the amino compound, water, organic solvent, base and the compound represented by formula II are all greater than 50 kg.

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