A method for synthesizing Nα-fluorenemethyloxycarbonyl-glutamic acid-α-tert-butyl ester

Abstract

This invention relates to a method for synthesizing Nα-fluorenemethyloxycarbonyl-glutamic acid-α-tert-butyl ester. The method includes: reacting glutamic acid with benzyl alcohol to synthesize glutamic acid-γ-benzyl ester; reacting glutamic acid-γ-benzyl ester with isobutylene to synthesize glutamic acid-γ-benzyl ester-α-tert-butyl ester; reacting glutamic acid-γ-benzyl ester-α-tert-butyl ester with fluorenemethyloxycarbonyl succinimide to synthesize Nα-fluorenemethyloxycarbonyl-glutamic acid-γ-benzyl ester-α-tert-butyl ester; and finally, removing γ-benzyl ester from Nα-fluorenemethyloxycarbonyl-glutamic acid-γ-benzyl ester-α-tert-butyl ester to synthesize the target product Nα-fluorenemethyloxycarbonyl-glutamic acid-α-tert-butyl ester. This method minimizes the synthetic steps, employs mild process conditions, simplifies the purification of intermediates and products, achieves high yield and low cost, and is suitable for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical intermediate synthesis, specifically relating to a method for synthesizing Nα-fluorenemethyloxycarbonyl-glutamic acid-α-tert-butyl ester. Background Technology

[0002] Nα-fluorenylmethoxycarbonyl-glutamate-α-tert-butyl ester (Fmoc-Glu-OtBu, or Fmoc-Glu-α-OtBu, or Fmoc-Glu-1-OtBu) is an important raw material for the synthesis of drugs, especially for the solid-phase synthesis of peptide drugs. In recent years, with the rapid development of glucagon-like peptide-1 receptor agonists (GLP-1 receptor agonists), especially the launch or successive launch of oral formulations of blockbuster drugs such as semaglutide and tirzepatide, the market capacity of Fmoc-Glu-OtBu, as an important molecular building block for the solid-phase synthesis of such drugs, has grown rapidly and has a bright future. Therefore, the research and development of Fmoc-Glu-OtBu synthesis technology has important social and economic significance.

[0003] Prior art 1: Patent CN115504893A discloses a method for synthesizing L-glutamic acid-α-tert-butyl ester (L-Glu-OtBu), the specific synthetic route of which is as follows:

[0004]

[0005] Although Fmoc-Glu-OtBu can be synthesized from the target product Glu-OtBu in the patent, the total reaction from the starting material glutamic acid to the final product Fmoc-Glu-OtBu involves a total of 6 steps, making the synthetic route too long and the cost too high.

[0006] Prior art 2: The Fmoc-Glu-OtBu synthesis route described in the background technology of patent CN116178214A is as follows:

[0007] Glutamic acid → Glutamic acid-γ-benzyl ester → Nα-benzyloxycarbonyl-glutamic acid-γ-benzyl ester → Nα-benzyloxycarbonyl-glutamic acid-γ-benzyl ester-1-tert-butyl ester → Glutamic acid-1-tert-butyl ester → N-(9-fluorenyloxycarbonyl)-glutamic acid-1-tert-butyl ester;

[0008] The synthetic route is basically the same as the existing technology 1, which is a 6-step reaction starting from glutamic acid. The only difference is that the methyl ester is replaced with benzyl ester and the amount of Pd / C used is slightly increased.

[0009] Meanwhile, prior art 2 discloses a method for preparing N-(9-fluorenylmethoxycarbonyl)-glutamic acid-1-tert-butyl ester (Fmoc-Glu-OtBu) of this invention, the specific synthetic route of which is as follows:

[0010]

[0011] The technical route of the prior art 2 changes the Nα-protecting group from Cbz- to trifluoroacetyl (CF3CO-), thereby simultaneously hydrolyzing it with γ-methyl ester, shortening the reaction steps from starting with glutamic acid to a 5-step reaction, and avoiding the use of noble metal catalysts such as Pd / C. However, according to other relevant literature, in addition to the side reactions in the synthesis of CF3CO-Glu(OMe)-OH, the hydrolysis of CF3CO-Glu(OMe)-OtBu to synthesize Glu-OtBu has selectivity issues of methyl ester / tert-butyl ester / trifluoroacetyl group and very obvious side reactions, making it very difficult to operate in practice.

[0012] In summary, the synthesis of Glu-OtBu in both prior art 1 and prior art 2 is prone to generating pyroglutamic acid, pyroglutamic acid-1-tert-butyl ester, and their derivative impurities due to the simultaneous exposure of α-amino and γ-carboxyl groups. This results in limited synthesis yield and difficulty in product purification. Therefore, it is necessary to develop more efficient synthesis technologies to obtain higher quality Fmoc-Glu-OtBu to meet market needs. Summary of the Invention

[0013] To overcome the shortcomings of the prior art, the present invention provides a method for synthesizing Nα-fluorenemethyloxycarbonyl-glutamic acid-α-tert-butyl ester.

[0014] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0015] This invention provides a method for synthesizing Nα-fluorenemethoxycarbonyl-glutamic acid-α-tert-butyl ester, the method comprising the following steps:

[0016] Step 1: Glutamic acid and benzyl alcohol are reacted in the presence of a dehydrating agent to synthesize glutamic acid-γ-benzyl ester;

[0017] Step 2: Glutamic acid-γ-benzyl ester reacts with isobutylene under the action of an acid catalyst to synthesize glutamic acid-γ-benzyl ester-α-tert-butyl ester.

[0018] Step 3: Glutamic acid-γ-benzyl ester-α-tert-butyl ester reacts with fluorenemethoxycarbonyl succinimide to synthesize Nα-fluorenemethoxycarbonyl-glutamate-γ-benzyl ester-α-tert-butyl ester;

[0019] Step 4: Nα-fluorenemethyloxycarbonyl-glutamic acid-γ-benzyl ester-α-tert-butyl ester is de-γ-benzyl ester under the action of a catalyst to synthesize the target product Nα-fluorenemethyloxycarbonyl-glutamic acid-α-tert-butyl ester.

[0020] The preferred synthesis route of the present invention is illustrated below:

[0021]

[0022] In a preferred embodiment of the present invention, the dehydrating agent in step 1 is concentrated sulfuric acid.

[0023] As a preferred embodiment of the technical solution of the present invention, the reaction solvent in step 1 is selected from at least one of diethyl ether, tetrahydrofuran, dioxane, and ethylene glycol dimethyl ether, preferably ethylene glycol dimethyl ether.

[0024] As a preferred embodiment of the technical solution of the present invention, after the reaction in step 1 is completed, concentrated sulfuric acid is neutralized with at least one of sodium bicarbonate (NaHCO3), sodium carbonate (Na2CO3), and sodium hydroxide (NaOH), and then glutamic acid-γ-benzyl ester is crystallized out, more preferably a solid-liquid mixture of sodium bicarbonate and water, so as to minimize the hydrolysis rate of benzyl ester.

[0025] In a preferred embodiment of the present invention, the molar ratio of glutamic acid-γ-benzyl ester to the acid catalyst in step 2 is 1:1 to 3, more preferably 1:2 to 3, wherein the acid catalyst in step 2 is at least one of concentrated sulfuric acid (H2SO4) and p-toluenesulfonic acid. The concentration of the concentrated sulfuric acid is preferably ≥98.5%, and the p-toluenesulfonic acid includes, but is not limited to, aqueous or anhydrous states, preferably anhydrous p-toluenesulfonic acid, while commercially available p-toluenesulfonic acid is generally a monohydrate.

[0026] As a preferred embodiment of the technical solution of the present invention, the molar ratio of glutamic acid-γ-benzyl ester to isobutylene is 1:10-20, more preferably 1:15-20. Increasing the amount of isobutylene is beneficial to the reaction, but too much is not conducive to cost control and production safety management.

[0027] As a preferred embodiment of the technical solution of the present invention, after the reaction in step 2 is completed, at least one of sodium bicarbonate, sodium carbonate, and sodium hydroxide is used to quench the acid catalyst.

[0028] As a preferred embodiment of the technical solution of the present invention, after the reaction is completed and the acid catalyst is quenched in step 2, the product is extracted with a reaction solvent, and the organic phases are combined and washed with saturated brine to reduce the residue of raw materials.

[0029] In a preferred embodiment of the present invention, the molar ratio of glutamic acid-γ-benzyl ester-α-tert-butyl ester to fluorenemethoxycarbonyl succinimide in step 3 is 1:0.85-0.95, and the specific amount can be determined by the quantitative analysis results of glutamic acid-γ-benzyl ester-α-tert-butyl ester.

[0030] In a preferred embodiment of the present invention, the solvent in step 3 is preferably a mixture of acetone and water. The mass ratio of acetone to water is 1:2 to 3.

[0031] As a preferred embodiment of the technical solution of the present invention, the catalyst in step 4 is preferably palladium on carbon (Pd / C, 10%) and ammonium formate (HCOONH4). The mass ratio of Nα-fluorenemethoxycarbonyl-glutamic acid-γ-benzyl ester-α-tert-butyl ester, Pd / C, and ammonium formate in step 4 is 140-150:3-5:50-60. Of course, palladium on carbon / hydrogen catalytic hydrogenation debenzyl ester can also be used.

[0032] Finally, the glutamic acid mentioned in the technical solution of the present invention is selected from any one of L-glutamic acid, D-glutamic acid, and DL-glutamic acid.

[0033] In this invention, if there is a conflict between the Chinese name and the structural formula of a compound, the structural formula shall prevail, unless the structural formula is obviously incorrect.

[0034] The beneficial effects of this invention are as follows:

[0035] (1) Starting with glutamic acid, the present invention shortens the synthesis steps to 4 steps, which is two steps less than the longest 6 steps of the prior art 1 and one step less than the total five steps of the prior art 2.

[0036] (2) Each step of the synthesis is simple and reproducible, and intermediates A, B and C are easy to purify and have high purity;

[0037] (3) There is no situation where α-amino and γ-carboxyl groups are exposed at the same time during the synthesis process, which avoids the generation of pyroglutamic acid and its derivative impurities, which are difficult to purify.

[0038] (4) The product has low cost and is suitable for large-scale production. Attached Figure Description

[0039] Figure 1 Example 1: NMR spectrum of Fmoc-Glu-OtBu. Detailed Implementation

[0040] The present invention is illustrated below with reference to examples, but is not intended to limit the invention. Any simple substitutions or modifications made to the present invention by those skilled in the art are within the scope of the technical solutions protected by this invention.

[0041] Example 1:

[0042] 150 g of diethyl ether was added to a 1000 mL four-necked flask. The temperature was lowered to below 5 °C, and 65 g of concentrated sulfuric acid was added dropwise. Then, 350 g of benzyl alcohol was added. After the addition was complete, the mixture was kept warm and stirred for 0.5 hours. 50 g of L-glutamic acid was added, and the mixture was naturally heated to room temperature and stirred for more than 20 hours. 120 g of sodium bicarbonate was suspended in 250 g of water. The internal temperature was controlled to be below 5 °C. The above L-glutamic acid-γ-benzyl ester reaction solution was slowly added. After quenching, the mixture was crystallized for 1 hour. After filtration, washing with water, and drying, 72.6 g of L-glutamic acid-γ-benzyl ester was obtained, with a yield of 90%.

[0043] 72 g of L-glutamic acid-γ-benzyl ester was suspended in 1000 g of dichloromethane and cooled to below 5 °C. 51.6 g of p-toluenesulfonic acid was added, followed by 60.3 g of concentrated sulfuric acid. 170.3 g of isobutylene was introduced, and the reaction was maintained at 5-10 °C for more than 20 hours. 168 g of sodium bicarbonate was suspended in 300 g of water, and the internal temperature was controlled below 5 °C. The aforementioned L-glutamic acid-γ-benzyl ester-α-tert-butyl ester reaction solution was added, quenched, and stirred for 0.5 hours. After standing and phase separation, the dichloromethane organic phase was collected. The solid-liquid mixed aqueous phase was filtered, and the solid was washed with 200 g of dichloromethane. The washings and filtrates were combined and extracted. The dichloromethane organic phases were then combined and washed three times with 50 g of saturated brine each time. The dichloromethane organic phase was concentrated under reduced pressure to obtain 88.6 g of solid L-glutamic acid-γ-benzyl ester-α-tert-butyl ester, with a yield of 98.7% by weight of solid product.

[0044] Add 1063.2 g of water, 38.4 g of anhydrous sodium carbonate, 88.6 g of L-glutamic acid-γ-benzyl ester-α-tert-butyl ester, and 425.3 g of acetone to a 2000 mL four-necked flask, stir to dissolve, add 101.9 g of fluorenemethyloxycarbonyl succinimide (Fmoc-OSu), react overnight at room temperature, and TLC detection showed no L-glutamic acid-γ-benzyl ester-α-tert-butyl ester residue. Extract three times with 80 g of isopropyl ether each time. Adjust the pH of the aqueous phase to 2-3 with 5N dilute hydrochloric acid, and add 1560 g of ethyl acetate. The product was extracted, and the aqueous phase was extracted once again with 560 g of ethyl acetate after phase separation. The ethyl acetate phases were combined and washed three times with 100 g of saturated brine each time. The organic phase of ethyl acetate was concentrated under reduced pressure to obtain 300 g of residue. 780 g of petroleum ether was added, and the mixture was stirred and cooled to 0-5℃ for more than 2 hours to crystallize. After filtration, 143.3 g of solid Fmoc-L-glutamic acid-γ-benzyl ester-α-tert-butyl ester (Fmoc-Glu(OBzl)-OtBu) was obtained, with a yield of 92% (calculated as Fmoc-OSu).

[0045] 143.3 g of Fmoc-L-glutamic acid-γ-benzyl ester-α-tert-butyl ester was added to a 2000 mL four-necked flask and dissolved in 860 g of methanol. Then, 3.6 g of 10% palladium / carbon and 53 g of ammonium formate were added. The mixture was stirred at room temperature until no Fmoc-L-glutamic acid-γ-benzyl ester-α-tert-butyl ester residue was detected by TLC. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was then dissolved in 700 g of ethyl acetate and 300 g of water at 35 °C. The aqueous phase was extracted and separated. The aqueous phase was then extracted once more with 200 g of ethyl acetate. The ethyl acetate phases were combined and washed three times with 80 g of saturated brine each time. The organic phase of ethyl acetate was concentrated under reduced pressure to obtain 250 g of residue. 600 g of petroleum ether was added, and the mixture was stirred and cooled to 0-5 °C to crystallize for more than 2 hours. After filtration, 108.2 g of solid Fmoc-L-glutamic acid-α-tert-butyl ester (Fmoc-Glu-OtBu) was obtained, with a yield of 91.5% and an HPLC purity of 99.65%.

[0046] Example 2:

[0047] Add 80 g of tetrahydrofuran to a 1000 mL four-necked flask, cool to below 5 °C, add 65 g of concentrated sulfuric acid dropwise, then add 350 g of benzyl alcohol. After the addition is complete, keep warm and stir for 0.5 hours. Add 50 g of L-glutamic acid, and naturally warm to room temperature and stir for more than 20 hours. Suspend 120 g of sodium bicarbonate in 250 g of water, control the internal temperature below 5 °C, and slowly add the above L-glutamic acid-γ-benzyl ester reaction solution. After quenching, crystallize for 1 hour, filter, wash with water, and dry to obtain 71.4 g of L-glutamic acid-γ-benzyl ester, with a yield of 88.6%.

[0048] 71.4 g of L-glutamic acid-γ-benzyl ester was suspended in 1000 g of dichloromethane. The mixture was cooled to below 5 °C, and 60.2 g of concentrated sulfuric acid was added dropwise. 337.7 g of isobutylene was introduced, and the mixture was kept at 5-10 °C for more than 20 hours. 33.6 g of sodium bicarbonate and 8 g of sodium hydroxide were suspended in 250 g of water. The internal temperature was controlled below 5 °C. The aforementioned L-glutamic acid-γ-benzyl ester-α-tert-butyl ester reaction solution was added, quenched, and then stirred and extracted for 0.5 hours. After standing and phase separation, the dichloromethane organic phase was collected. The solid-liquid mixed aqueous phase was filtered, and the solid was washed with 200 g of dichloromethane. The washings and filtrates were combined and extracted. The dichloromethane organic phases were then combined and washed three times with 50 g of saturated brine each time. The dichloromethane organic phase was concentrated under reduced pressure to obtain 86.7 g of solid L-glutamic acid-γ-benzyl ester-α-tert-butyl ester, with a yield of 98.2% by weight of solid product.

[0049] In a 2000 mL four-necked flask, add 1060 g of water, 43.8 g of anhydrous sodium carbonate, 86.7 g of L-glutamic acid-γ-benzyl ester-α-tert-butyl ester, and 420 g of acetone. Stir to dissolve, then add 99.7 g of fluorenemethyloxycarbonyl succinimide (Fmoc-OSu). React overnight at room temperature. TLC analysis showed no L-glutamic acid-γ-benzyl ester-α-tert-butyl ester residue. Extract three times with 100 g of isopropyl ether each time. Adjust the pH of the aqueous phase to 2-3 with 5N dilute hydrochloric acid, and then extract with 1500 g of ethyl acetate. The product was separated, and the aqueous phase was extracted once with 500 g of ethyl acetate. The ethyl acetate phases were combined and washed three times with 100 g of saturated brine each time. The organic phase of ethyl acetate was concentrated under reduced pressure to obtain 330 g of residue. 800 g of petroleum ether was added, and the mixture was stirred and cooled to 0-5℃ for more than 2 hours to crystallize. After filtration, 146.3 g of solid Fmoc-L-glutamic acid-γ-benzyl ester-α-tert-butyl ester (Fmoc-Glu(OBzl)-OtBu) was obtained, with a yield of 96% (calculated as Fmoc-OSu).

[0050] 146.3 g of Fmoc-L-glutamic acid-γ-benzyl ester-α-tert-butyl ester was added to a 2000 mL four-necked flask and dissolved in 900 g of methanol. 4.3 g of 10% palladium / carbon and 55 g of ammonium formate were added, and the mixture was stirred at room temperature until no Fmoc-L-glutamic acid-γ-benzyl ester-α-tert-butyl ester residue was detected by TLC. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was added to 700 g of ethyl acetate and 300 g of water, and the mixture was heated to 35 °C and stirred to dissolve the solid. The aqueous phase was extracted and separated. The aqueous phase was then extracted once more with 200 g of ethyl acetate. The ethyl acetate phases were combined and washed three times with 80 g of saturated brine each time. The organic phase of ethyl acetate was concentrated under reduced pressure to obtain 250 g of residue. 600 g of petroleum ether was added, and the mixture was stirred and cooled to 0-5 °C to crystallize for more than 2 hours. After filtration, 114.1 g of solid Fmoc-L-glutamic acid-α-tert-butyl ester (Fmoc-Glu-OtBu) was obtained, with a yield of 94.5% and an HPLC purity of 99.54%.

[0051] Example 3:

[0052] 65 g of ethylene glycol dimethyl ether was added to a 1000 mL four-necked flask. The temperature was lowered to below 5 °C, and 65 g of concentrated sulfuric acid was added dropwise. Then, 350 g of benzyl alcohol was added. After the addition was complete, the mixture was kept warm and stirred for 0.5 hours. 50 g of L-glutamic acid was added, and the mixture was naturally heated to room temperature and stirred for more than 20 hours. 120 g of sodium bicarbonate was suspended in 250 g of water. The internal temperature was controlled to be below 5 °C. The above L-glutamic acid-γ-benzyl ester reaction solution was slowly added. After quenching, the mixture was crystallized for 1 hour. After filtration, washing with water, and drying, 73 g of L-glutamic acid-γ-benzyl ester was obtained, with a yield of 90.5%.

[0053] 73 g of L-glutamic acid-γ-benzyl ester was suspended in 1000 g of dichloromethane. The mixture was cooled to below 5 °C, and 30.4 g of concentrated sulfuric acid was added dropwise. 258.9 g of isobutylene was introduced, and the reaction was maintained at 5-10 °C for more than 20 hours. 21.2 g of sodium carbonate and 4 g of sodium hydroxide were suspended in 250 g of water. The internal temperature was controlled below 5 °C. The aforementioned L-glutamic acid-γ-benzyl ester-α-tert-butyl ester reaction solution was added, quenched, and stirred for 0.5 hours. After standing and phase separation, the dichloromethane organic phase was collected. The solid-liquid mixed aqueous phase was filtered, and the solid was washed with 200 g of dichloromethane. The washings and filtrates were combined and extracted. The dichloromethane organic phases were then combined and washed three times with 50 g of saturated brine each time. The dichloromethane organic phase was concentrated under reduced pressure to obtain 88.9 g of solid L-glutamic acid-γ-benzyl ester-α-tert-butyl ester, with a yield of 98.5% by weight of solid product.

[0054] In a 2000 mL four-necked flask, add 1060 g of water, 38.4 g of anhydrous sodium carbonate, 88.9 g of L-glutamic acid-γ-benzyl ester-α-tert-butyl ester, and 425 g of acetone. Stir to dissolve, then add 101.9 g of fluorenemethyloxycarbonyl succinimide (Fmoc-OSu). React overnight at room temperature. TLC analysis showed no L-glutamic acid-γ-benzyl ester-α-tert-butyl ester residue. Extract three times with 80 g of isopropyl ether each time. Adjust the pH of the aqueous phase to 2-3 with 5N dilute hydrochloric acid, and then extract with 1560 g of ethyl acetate. The product was separated, and the aqueous phase was extracted once with 560 g of ethyl acetate. The ethyl acetate phases were combined and washed three times with 100 g of saturated brine each time. The ethyl acetate organic phase was concentrated under reduced pressure to obtain 300 g of residue. 780 g of petroleum ether was added, and the mixture was stirred and cooled to 0-5℃ to crystallize for more than 2 hours. After filtration, 148.4 g of solid Fmoc-L-glutamic acid-γ-benzyl ester-α-tert-butyl ester (Fmoc-Glu(OBzl)-OtBu) was obtained, with a yield of 95% (calculated as Fmoc-OSu).

[0055] 148.4 g of Fmoc-L-glutamic acid-γ-benzyl ester-α-tert-butyl ester was added to a 2000 mL four-necked flask and dissolved in 860 g of methanol. Then, 3.9 g of 10% palladium / carbon and 55 g of ammonium formate were added. The mixture was stirred at room temperature until no Fmoc-L-glutamic acid-γ-benzyl ester-α-tert-butyl ester residue was detected by TLC. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was then dissolved in 700 g of ethyl acetate and 300 g of water at 35 °C. The aqueous phase was extracted and separated. The aqueous phase was then extracted once more with 200 g of ethyl acetate. The ethyl acetate phases were combined and washed three times with 80 g of saturated brine each time. The organic phase of ethyl acetate was concentrated under reduced pressure to obtain 250 g of residue. 600 g of petroleum ether was added, and the mixture was stirred and cooled to 0-5 °C to crystallize for more than 2 hours. After filtration, 114.5 g of solid Fmoc-L-glutamic acid-α-tert-butyl ester (Fmoc-Glu-OtBu) was obtained, with a yield of 93.5% and an HPLC purity of 99.48%.

[0056] Example 4:

[0057] Add 90 g of diethyl ether to a 1000 mL four-necked flask, cool to below 5 °C, add 65 g of concentrated sulfuric acid dropwise, then add 350 g of benzyl alcohol. After the addition is complete, keep warm and stir for 0.5 hours. Add 50 g of L-glutamic acid, and naturally warm to room temperature and stir for more than 20 hours. Suspend 120 g of sodium bicarbonate in 250 g of water, control the internal temperature below 5 °C, and slowly add the above L-glutamic acid-γ-benzyl ester reaction solution. After quenching, crystallize for 1 hour, filter, wash with water, and dry to obtain 74.2 g of L-glutamic acid-γ-benzyl ester, with a yield of 92%.

[0058] 74.2 g of L-glutamic acid-γ-benzyl ester was suspended in 1000 g of dichloromethane. The mixture was cooled to below 5 °C, and 91 g of concentrated sulfuric acid was added dropwise. 350 g of isobutylene was introduced, and the mixture was kept at 5-10 °C for more than 20 hours. 165 g of sodium bicarbonate was suspended in 300 g of water. The internal temperature was controlled below 5 °C, and the aforementioned L-glutamic acid-γ-benzyl ester-α-tert-butyl ester reaction solution was added. After quenching, the mixture was stirred and extracted for 0.5 hours. The mixture was allowed to stand and separate phases. The dichloromethane organic phase was collected, and the solid-liquid mixed aqueous phase was filtered. The solid was washed with 200 g of dichloromethane. The washings and filtrates were combined and extracted. The dichloromethane organic phases were then combined and washed three times with 50 g of saturated brine each time. The dichloromethane organic phase was concentrated under reduced pressure to obtain 90.4 g of solid L-glutamic acid-γ-benzyl ester-α-tert-butyl ester, with a yield of 98.5% by weight of solid product.

[0059] Add 1060 g of water, 38.4 g of anhydrous sodium carbonate, 90.4 g of L-glutamic acid-γ-benzyl ester-α-tert-butyl ester, and 425 g of acetone to a 2000 mL four-necked flask, stir to dissolve, add 101.9 g of fluorenemethyloxycarbonyl succinimide (Fmoc-OSu), react overnight at room temperature, and TLC detection showed no L-glutamic acid-γ-benzyl ester-α-tert-butyl ester residue. Extract three times with 80 g of isopropyl ether each time. Adjust the pH of the aqueous phase to 2-3 with 5N dilute hydrochloric acid, and extract with 1560 g of ethyl acetate. The product was separated into phases, and the aqueous phase was extracted once with 560 g of ethyl acetate. The ethyl acetate phases were combined and washed three times with 100 g of saturated brine each time. The organic phase of ethyl acetate was concentrated under reduced pressure to obtain 300 g of residue. 780 g of petroleum ether was added, and the mixture was stirred and cooled to 0-5℃ to crystallize for more than 2 hours. After filtration, 147.8 g of solid Fmoc-L-glutamic acid-γ-benzyl ester-α-tert-butyl ester (Fmoc-Glu(OBzl)-OtBu) was obtained, with a yield of 93% (calculated as Fmoc-OSu).

[0060] 147.8 g of Fmoc-L-glutamic acid-γ-benzyl ester-α-tert-butyl ester was added to a 2000 mL four-necked flask and dissolved in 900 g of methanol. 4.4 g of 10% palladium / carbon and 60 g of ammonium formate were added, and the mixture was stirred at room temperature until no Fmoc-L-glutamic acid-γ-benzyl ester-α-tert-butyl ester residue was detected by TLC. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was added to 700 g of ethyl acetate and 300 g of water, and the mixture was heated to 35 °C and stirred to dissolve the solid. The aqueous phase was extracted and separated. The aqueous phase was then extracted once more with 200 g of ethyl acetate. The ethyl acetate phases were combined and washed three times with 80 g of saturated brine each time. The organic phase of ethyl acetate was concentrated under reduced pressure to obtain 250 g of residue. 600 g of petroleum ether was added, and the mixture was stirred and cooled to 0-5 °C to crystallize for more than 2 hours. After filtration, 112.8 g of solid Fmoc-L-glutamic acid-α-tert-butyl ester (Fmoc-Glu-OtBu) was obtained, with a yield of 92.5% and an HPLC purity of 99.67%.

[0061] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for synthesizing Nα-fluorenylmethoxycarbonyl-glutamic acid-α-tert-butyl ester, characterized by, The method comprises the following steps: Step 1, glutamic acid is reacted with benzyl alcohol under the action of a dehydrating agent to synthesize glutamic acid-gamma-benzyl ester; the reaction solvent in step 1 is at least one selected from ethyl ether, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether; and the dehydrating agent in step 1 is concentrated sulfuric acid; Step 2, glutamic acid-gamma-benzyl ester is reacted with isobutene under the action of an acid catalyst to synthesize glutamic acid-gamma-benzyl ester-alpha-tert-butyl ester; the molar ratio of glutamic acid-gamma-benzyl ester to the acid catalyst in step 2 is 1:1-3; and the molar ratio of glutamic acid-gamma-benzyl ester to isobutene is 1:10-20; wherein the acid catalyst in step 2 is at least one selected from concentrated sulfuric acid and p-toluenesulfonic acid; Step 3, glutamic acid-gamma-benzyl ester-alpha-tert-butyl ester is reacted with fluorenylmethoxycarbonyl succinimide to synthesize Nalpha-fluorenylmethoxycarbonyl-glutamic acid-gamma-benzyl ester-alpha-tert-butyl ester; the molar ratio of glutamic acid-gamma-benzyl ester-alpha-tert-butyl ester to fluorenylmethoxycarbonyl succinimide in step 3 is 1:0.85-0.95; Step 4, Nalpha-fluorenylmethoxycarbonyl-glutamic acid-gamma-benzyl ester-alpha-tert-butyl ester is removed from the gamma-benzyl ester under the action of a catalyst to synthesize the target product Nalpha-fluorenylmethoxycarbonyl-glutamic acid-alpha-tert-butyl ester; the catalyst in step 4 is palladium-carbon and ammonium formate; and the mass ratio of the addition amount of Nalpha-fluorenylmethoxycarbonyl-glutamic acid-gamma-benzyl ester-alpha-tert-butyl ester, Pd / C and ammonium formate in step 4 is 140-150:3-5:50-60.

2. The method of claim 1, wherein, After the reaction in step 1 is completed, at least one selected from sodium bicarbonate, sodium carbonate and sodium hydroxide is used to neutralize the concentrated sulfuric acid, and glutamic acid-gamma-benzyl ester is crystallized and precipitated.

3. The method of claim 1, wherein, After the reaction in step 2 is completed, at least one selected from sodium bicarbonate, sodium carbonate and sodium hydroxide is used to quench the acid catalyst, and then the product is extracted, and the organic phase is washed with saturated brine.

4. The method of claim 1, wherein, The solvent in step 3 is a mixed solvent of acetone and water.

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