Preparation method of glycyl chloride dipeptide
By using HOSu and EDCI reagents and ethyl acetate low-temperature beating technology, combined with the reaction steps in tetrahydrofuran, the purity and yield of glycosin dipeptide were successfully improved, and the purity and yield of glycosin dipeptide in the prior art were solved. The problem of low rate achieves high purity and high yield preparation, which is suitable for industrial production.
Patent Information
- Application Number
- CN202411652260.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-19
- Publication Date
- 2024-12-31
AI Technical Summary
During the preparation process of the prior art glycosin dipeptide, the product purity and yield are low, and high purity and high yield preparation cannot be achieved.
Using HOSu and EDCI as reagents, the active esters Fmoc-Gly-OSu were cooled and precipitated by reaction solvents, and purified by ethyl acetate at low temperature. Then, reacted with guanidine-protected arginine in tetrahydrofuran to produce high-purity glycerine Dipeptide Fmoc-Gly-Arg(Pbf)-OH.
It has achieved high purity (up to 99.5%) and high yield (up to 93%) of glycosin dipeptide, which is suitable for large-scale industrial production and effectively removes urea by-products.
Smart Images
Figure CN119219731A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of peptide synthesis, and in particular to a method for preparing glargine dipeptide. Background Art
[0002] Peptide drugs are a class of compounds with important medicinal value. Peptide drugs have many advantages such as high selectivity, low immunogenicity, and strong biological activity. They have shown broad application prospects in many fields such as the endocrine system, tumors, infections, and nerves.
[0003] Semaglutide is a glucagon-like peptide (GLP-1) receptor agonist developed by Novo Nordisk. In December 2017, the U.S. Food and Drug Administration (FDA) approved semaglutide injection for marketing for blood sugar control in adult patients with type 2 diabetes. In June 2021, semaglutide was approved by the FDA for weight loss, becoming the first once-weekly GLP-1 receptor agonist for weight management. The polypeptide amino acid sequence of semaglutide is: His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEE--Glu-Octadecanedioicmono-t-Butylester)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH
[0004] Gly-Arg dipeptide appears in the semaglutide sequence. In the preparation of semaglutide, Fmoc-Gly-Arg(Pbf)-OH can be used as a fragment intermediate for introducing the sequence. Currently, no method for preparing high-purity glargine dipeptide has been found in the literature. The product obtained by solid phase synthesis or other methods has a low purity, and the purity after purification is only 98%, and the yield is low. Summary of the invention
[0005] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a method for preparing glargine dipeptide (Fmoc-Gly-Arg(Pbf)-OH) that can be mass-produced and has high product purity and yield. The present invention uses HOSu and EDCI as reagents to prepare active esters, and the active ester Fmoc-Gly-OSu can be directly cooled and precipitated from the reaction solvent. The crude active ester is pulped with ethyl acetate at low temperature to improve the purity, and by-products such as urea are effectively removed.
[0006] In order to solve the above problems, the technical solution provided by the present invention is as follows:
[0007] A method for preparing glargine dipeptide comprises the following steps:
[0008] (1) using N-fluorenylmethoxycarbonylglycine as a raw material, reacting with N-hydroxysuccinimide in an organic solvent in the presence of a coupling reagent; after the reaction, directly cooling the reaction solution and stirring to precipitate a large amount of solid by suction filtration to obtain a crude active ester Fmoc-Gly-OSu; adding ethyl acetate to the crude product obtained in step (1) for pulping, eluting the solid with 0-5° C. ethyl acetate, and drying to obtain a qualified N-fluorenylmethoxycarbonylglycine hydroxysuccinimide ester;
[0009] (2) Using tetrahydrofuran as the reaction solvent, N-fluorenylmethoxycarbonylglycine hydroxysuccinimide ester reacts with guanidino-protected arginine Arg(Pbf)-OH in the presence of an organic base. After the reaction is completed, Fmoc-Gly-Arg(Pbf)-OH glargine dipeptide is generated.
[0010] Furthermore, step (1) further includes the step of slurrying and purifying the crude Fmoc-Gly-Osu active ester at low temperature using ethyl acetate, and the specific method is: directly cooling the reaction solution to -5 to 0°C, stirring a large amount of solid to precipitate and filtering to obtain a crude active ester, adding ethyl acetate to the obtained crude product and slurrying at 10 to 15°C for 1 to 2 hours, eluting the filtered solid with cold ethyl acetate, and drying to obtain qualified N-fluorenylmethoxycarbonylglycine hydroxysuccinimide ester (Fmoc-Gly-OSu);
[0011] Furthermore, in step (1), the reaction solvent is selected from tetrahydrofuran, dichloromethane, dioxane, acetonitrile, N,N-dimethylformamide or chloroform;
[0012] Furthermore, in step (1), the reaction time of N-Fmoc and N-hydroxysuccinimide is 30 min to 12 h; the reaction temperature is -5 to 25°C; the further preferred time range is 1 to 6 h, and the preferred reaction temperature is -5 to 5°C.
[0013] Furthermore, in step (1), the mass ratio of the reaction intermediate crude product Fmoc-Gly-OSu active ester to ethyl acetate is 1:4.5-10.
[0014] Furthermore, in step (1), the molar ratio of N-fluorenylmethoxycarbonylglycine, N-hydroxysuccinimide and coupling reagent is 1:1:1.0-2.0, preferably 1:1:1.1-1.5. The coupling reagent in step (1) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCI).
[0015] Furthermore, in step (1), the mass ratio of the reaction intermediate crude Fmoc-Gly-OSu active ester to ethyl acetate is 1:5-10.
[0016] Furthermore, in step (2), the reaction time of N-fluorenylmethoxycarbonylglycine hydroxysuccinimide ester and guanidino-protected arginine is 30 min to 14 h, and the reaction temperature is 0 to 30° C.; further preferably, the reaction time range is 4 to 10 h, and the reaction temperature is preferably 5 to 15° C.
[0017] Furthermore, in step (2), the molar ratio of N-fluorenylmethoxycarbonylglycine hydroxysuccinimide ester to guanidino-protected arginine is 1:1.0-1.5, preferably 1:1.0-1.3.
[0018] Furthermore, the step (2) further comprises the following post-processing steps:
[0019] S1: After the reaction is completed, tetrahydrofuran is concentrated and recovered, ethyl acetate / water is added, and the pH is adjusted to 3-4 using an inorganic acid. After the extraction is completed, water is added to stir and wash the ester layer;
[0020] S2: The ester layer obtained in step S1 is concentrated, and the crude oil obtained is stirred with ethyl acetate / n-heptane, cooled and crystallized, and filtered until the product is qualified.
[0021] Furthermore, in the post-treatment S2 in step (2), the volume ratio of the crystallization solvent ethyl acetate / n-heptane is 1:1-2. The volume (ml) of the crystallization solvent: the mass (g) of the crude oil is 6-8:1, preferably 7-7.5:1.
[0022] Furthermore, in step (2), the organic base is N-ethyldiisopropylamine. The inorganic acid in step S1 is hydrochloric acid.
[0023] If there is a conflict between the Chinese name and the structural formula of the compound in the present invention, the structural formula shall prevail, unless there is an obvious error in the structural formula.
[0024] The present invention provides a method for preparing glargine dipeptide, which comprises the following steps: using N-fluorenylmethoxycarbonylglycine as a raw material and reacting it with N-hydroxysuccinimide under the action of a coupling reagent to generate an active ester; the generated active ester and arginine Arg(Pbf)-OH protected by a guanidine group are reacted under the action of an organic base to prepare a glargine dipeptide, the structure of which is Fmoc-Gly-Arg(Pbf)-OH. The active ester Fmoc-Gly-OSu prepared by the present invention can be directly cooled and precipitated from the reaction solvent, and the crude active ester is pulped with ethyl acetate at low temperature to improve the purity, and by-products such as urea are effectively removed. The final product, glargine dipeptide, has the advantages of high purity and high yield, and is simple to operate and has low equipment requirements, so it is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is the hydrogen spectrum of glargine dipeptide, Fmoc-Gly-Arg(Pbf)-OH according to the specific embodiment 1 of the present invention. DETAILED DESCRIPTION
[0026] The present invention is described below with reference to examples, but the present invention is not limited thereto. In the art, simple replacement or improvement made by technicians in the field to the present invention belongs to the technical solution protected by the present invention.
[0027] Embodiment 1:
[0028] Step (1) Add N-Fmoc-Gly (50.0 g, 0.1682 mol), N-hydroxysuccinimide (19.4 g, 0.1682 mol), EDCI (38.7 g, 0.2018 mol), and tetrahydrofuran (450 ml) to a clean and dry 1L glass four-necked flask, protect with nitrogen, and mechanically stir and disperse. Cool the mixture by external ice water to control the internal temperature below 5°C, stir and react for 5 hours, and continue to cool to -2°C after the reaction is completed. Filter the solid. Add ethyl acetate (375 g) to about 75 g of the solid, keep slurrying at 10-15°C for 2 hours, filter out the solid, and rinse with a small amount of cold ethyl acetate. After drying, 53.8 g of solid Fmoc-Gly-OSu active ester is obtained with a purity of 98.2%, 0.18% of N-Fmoc-Gly residue, and a yield of 81%.
[0029] Step (2) Add Arg(Pbf)-OH (64.0 g, 0.1500 mol), N-ethyldiisopropylamine (22.9 g, 0.1773 mol), and tetrahydrofuran (600 g) to a clean and dry 1L glass four-necked flask, and disperse and dissolve with mechanical stirring; Add Fmoc-Gly-OSu active ester (53.8 g, 0.1364 mol) to 320 g of tetrahydrofuran and stir to dissolve to prepare a clear solution, cool the internal temperature to 15°C by external ice water, and add the prepared Fmoc-Gly-OSu active ester / tetrahydrofuran solution dropwise, control the dropwise addition time to about 4 hours, and keep warm for 1 hour; after the reaction is completed, concentrate in a 40°C water bath under reduced pressure to recover tetrahydrofuran, and concentrate until there is substantially no solvent evaporated to obtain yellow The oil was added with ethyl acetate (500 g) and water (300 g) and stirred for 30 min, then 30% hydrochloric acid was added to adjust the pH to about 3-4. After the acid adjustment was completed and the pH was repeated for 30 min without any change, the mixture was allowed to stand for separation, and the upper organic phase was taken; the organic phase was washed twice with 15% saline, and the phases were allowed to stand for separation. The upper organic phase was filtered and concentrated to dryness to obtain 102 g of an oily crude product. A mixed solvent of ethyl acetate / n-heptane (250 ml of ethyl acetate and 500 ml of n-heptane) prepared in proportion was added, the mixture was heated to 50°C with stirring, cooled and stirred until a large amount of solid precipitated, and then the temperature was further cooled to 0°C. The solid product Fmoc-Gly-Arg(Pbf)-OH was filtered and dried to obtain 89.54 g of the product with a purity of 99.5% and a yield of 93%.
[0030] Embodiment 2:
[0031] Step (1) Add N-Fmoc-Gly (25.0 g, 0.0841 mol), N-hydroxysuccinimide (9.7 g, 0.0841 mol), EDCI (17.7 g, 0.0925 mol), and dichloromethane (200 ml) into a clean and dry 500 ml glass four-necked flask. Under nitrogen protection, mechanically stir and disperse the mixture, cool it with external ice water, and stir the mixture for 6 hours. After the reaction is completed, continue to cool it to -1°C and filter the solid. Add ethyl acetate (235 g) to about 39.3 g of the solid, keep it at 10-15°C and beat it for 2 hours, then filter out the solid, rinse it with a small amount of cold ethyl acetate, and dry it to obtain 28.2 g of solid Fmoc-Gly-OSu active ester with a purity of 98.4%. 0.12% of N-Fmoc-Gly residue and a yield of 85%.
[0032] Step (2) Add Arg(Pbf)-OH (35.1 g, 0.0822 mol), N-ethyldiisopropylamine (12.0 g, 0.09295 mol), and tetrahydrofuran (300 g) to a clean and dry 1L glass four-necked flask, and disperse and dissolve with mechanical stirring; Add Fmoc-Gly-OSu active ester (28.2 g, 0.0715 mol) to 170 g of tetrahydrofuran and stir to dissolve to prepare a clear solution, cool the internal temperature to 15°C by external ice water, and dropwise add the prepared Fmoc-Gly-OSu active ester / tetrahydrofuran solution, control the dropwise addition time to about 6 hours, and keep warm for 2 hours; after the reaction is completed, concentrate under reduced pressure at 40°C in a water bath to recover tetrahydrofuran, and concentrate until there is substantially no solvent evaporated to obtain yellow The oil was added with ethyl acetate (250 g) and water (140 g) and stirred for 30 min, then 30% hydrochloric acid was added to adjust the pH to about 3-4. After the acid adjustment was completed and the pH was repeated for 30 min without any change, the mixture was allowed to stand for separation, and the upper organic phase was taken; the organic phase was washed twice with 15% saline, and the phases were allowed to stand for separation. The upper organic phase was filtered and concentrated to dryness to obtain 48.2 g of an oily crude product. A mixed solvent of ethyl acetate / n-heptane (125 ml of ethyl acetate and 210 ml of n-heptane) prepared in proportion was added, the mixture was heated to 50°C with stirring, cooled and stirred until a large amount of solid precipitated, and then the temperature was further cooled to 0°C. The solid product Fmoc-Gly-Arg(Pbf)-OH was filtered and dried to obtain 45.4 g of the product with a purity of 99.6% and a yield of 90%.
[0033] Embodiment 3:
[0034] Step (1) Add N-Fmoc-Gly (50.0 g, 0.1682 mol), N-hydroxysuccinimide (19.4 g, 0.1682 mol), EDCI (38.7 g, 0.2018 mol), and dioxane (450 ml) to a clean, dry 1L glass four-necked flask, protect with nitrogen, and mechanically stir and disperse. Cool the mixture with external ice water to control the internal temperature below 5°C, stir and react for 4 hours, and continue to cool to -2°C after the reaction is completed. Filter the solid. Add ethyl acetate (400 g) to about 79 g of the solid, keep slurrying at 10-15°C for 2 hours, and then filter out the solid. Rinse with a small amount of cold ethyl acetate and dry to obtain 51.0 g of solid Fmoc-Gly-OSu active ester with a purity of 98.8%, 0.10% N-Fmoc-Gly residue, and a yield of 76.9%.
[0035] Step (2) Add Arg(Pbf)-OH (60.6 g, 0.1422 mol), N-ethyldiisopropylamine (21.7 g, 0.1681 mol), and tetrahydrofuran (600 g) to a clean and dry 1L glass four-necked flask, and disperse and dissolve by mechanical stirring; Add Fmoc-Gly-OSu active ester (51.0 g, 0.1293 mol) to 300 g of tetrahydrofuran and stir to dissolve to prepare a clear solution, cool the solution to an internal temperature of 15°C by external ice water, and dropwise add the prepared Fmoc-Gly-OSu active ester / tetrahydrofuran solution, control the dropwise addition time to about 5 hours, and keep warm for 2 hours; after the reaction is completed, concentrate under reduced pressure in a 40°C water bath to recover tetrahydrofuran, The mixture was concentrated until almost no solvent was evaporated to obtain a yellow oil; ethyl acetate (500 g) and water (300 g) were added to the oil and stirred for 30 min, then 30% hydrochloric acid was added to adjust the pH to about 3-4. After the acid adjustment was completed and the pH was re-measured for 30 min without any change, the mixture was allowed to stand for separation, and the upper organic phase was taken; the organic phase was washed twice with 15% saline, and the mixture was allowed to stand for phase separation. The upper organic phase was filtered and concentrated to dryness to obtain 82.5 g of an oily crude product, and a mixed solvent of ethyl acetate / n-heptane (250 ml of ethyl acetate and 375 ml of n-heptane) prepared in proportion was added, the mixture was heated to 50°C with stirring, the mixture was cooled and stirred until a large amount of solid precipitated, and the temperature was further cooled to 0°C, and the solid product Fmoc-Gly-Arg(Pbf) was filtered.
[0036] -OH, dried to obtain 77.57 g, purity 99.7%, yield 85%.
[0037] Embodiment 4:
[0038] Step (1) Add N-Fmoc-Gly (100.0 g, 0.3363 mol), N-hydroxysuccinimide (38.7 g, 0.3363 mol), EDCI (90.3 g, 0.4708 mol), and chloroform (600 ml) into a clean and dry 2L glass four-necked flask, and under nitrogen protection, mechanically stir and disperse the mixture, cool it with external ice water, and stir the reaction for 6 hours. After the reaction is completed, continue to cool it to -1°C and then filter the solid. Add ethyl acetate (800 g) to about 152.0 g of the solid, keep it at 10-15°C and beat it for 2 hours, then filter out the solid, rinse it with a small amount of cold ethyl acetate, and dry it to obtain 110.1 g of solid Fmoc-Gly-OSu active ester with a purity of 98.5%, 0.18% of N-Fmoc-Gly residue, and a yield of 83%.
[0039] Step (2) Add Arg(Pbf)-OH (130.9 g, 0.3070 mol), N-ethyldiisopropylamine (46.9 g, 0.3628 mol), and tetrahydrofuran (1200 g) to a clean and dry 2L glass four-necked flask, and disperse and dissolve with mechanical stirring; Add Fmoc-Gly-OSu active ester (110 g, 0.2791 mol) to 640 g of tetrahydrofuran and stir to dissolve to prepare a clear solution, cool the solution to an internal temperature of 15°C by external ice water, and dropwise add the prepared Fmoc-Gly-OSu active ester / tetrahydrofuran solution, control the dropwise addition time to be about 7 hours, and keep warm for 2 hours; after the reaction is completed, concentrate under reduced pressure at 40°C in a water bath to recover tetrahydrofuran, and concentrate until there is substantially no solvent evaporated to obtain a yellow The oily substance was added with ethyl acetate (1000 g) and water (600 g) and stirred for 30 min, then 30% hydrochloric acid was added to adjust the pH to about 3-4. After the acid adjustment was completed, the pH was measured again after stirring for 30 min without any change, and the mixture was allowed to stand for separation, and the upper organic phase was taken; the organic phase was washed twice with 15% brine, and the phases were allowed to stand for separation. The upper organic phase was filtered and concentrated to dryness to obtain 194 g of an oily crude product, and a proportioned ethyl acetate / n-heptane mixed solvent (500 ml of ethyl acetate and 1000 ml of n-heptane) was added, and the mixture was stirred and heated to 50°C, cooled and stirred until a large amount of solid precipitated, and then the temperature was continued to be cooled to 0°C, and the solid product Fmoc-Gly-Arg(Pbf)-OH was filtered and dried to obtain 185.1 g, with a purity of 99.6% and a yield of 93%.
[0040] Embodiment 5:
[0041] Step (1) Add N-Fmoc-Gly (0.5 kg, 1.682 mol), N-hydroxysuccinimide (195 g, 1.682 mol), EDCI (0.48 kg, 2.523 mol), and acetonitrile (5 L) into a clean and dry 10 L glass reactor, flush with nitrogen protection, start stirring, cool with ice brine, control the internal temperature below 5° C., stir and react for 6 hours, continue to cool to -3° C. after the reaction is completed, centrifuge the solid, add ethyl acetate (4 kg) to about 0.82 kg of the solid, keep slurrying at 10-15° C. for 2 hours, filter out the solid, and rinse with a small amount of cold ethyl acetate. After centrifugation and drying, the wet product is dried to obtain 0.58 kg of solid Fmoc-Gly-OSu active ester with a purity of 98.1%, 0.19% N-Fmoc-Gly residue, and a yield of 88%.
[0042] Step (2) Add Arg(Pbf)-OH (0.66 kg, 1.5442 mol), N-ethyldiisopropylamine (247 g, 1.9119 mol), and tetrahydrofuran (6 kg) to a clean and dry 10 L glass reactor, and disperse and dissolve with mechanical stirring; add Fmoc-Gly-OSu active ester (0.58 kg, 1.4707 mol) to 3.5 kg of tetrahydrofuran and stir to dissolve to prepare a clear solution, cool the solution to an internal temperature of 15° C. by jacketing with ice brine, and dropwise add the prepared Fmoc-Gly-OSu active ester / tetrahydrofuran solution, control the dropwise addition time to about 8 hours, and keep warm for 2 hours; after the reaction is completed, concentrate under reduced pressure in a 40° C. water bath to recover tetrahydrofuran, and concentrate until there is substantially no solvent evaporated , a yellow oily substance was obtained; ethyl acetate (5 kg) and water (3 kg) were added to the oily substance and stirred for 30 min, and then 30% hydrochloric acid was added to adjust the pH to about 3-4. After the acid adjustment was completed and the pH was repeated for 30 min without any change, the mixture was allowed to stand for separation, and the upper organic phase was taken; the organic phase was washed twice with 15% saline, and the mixture was allowed to stand for phase separation. The upper organic phase was filtered and concentrated to dryness to obtain 1.07 kg of an oily crude product, and a mixed solvent of ethyl acetate / n-heptane (2.5 L of ethyl acetate and 5 L of n-heptane) prepared in proportion was added, the mixture was heated to 50°C with stirring, cooled and stirred until a large amount of solid precipitated, and then the temperature was further cooled to 0°C, and the solid product Fmoc-Gly-Arg(Pbf)-OH was filtered and dried to obtain 0.94 kg of the product with a purity of 99.6% and a yield of 91%.
[0043] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, which all belong to the protection scope of the present invention.
Claims
1. A method for preparing glargine dipeptide, characterized in that: The steps include: (1) using N-fluorenylmethoxycarbonylglycine as a raw material, reacting with N-hydroxysuccinimide in an organic solvent in the presence of a coupling reagent; after the reaction, directly cooling the reaction solution and stirring to precipitate a large amount of solid by suction filtration to obtain a crude active ester Fmoc-Gly-OSu; adding ethyl acetate to the crude product obtained in step (1) for pulping, eluting the filtered solid with 0-5° C. ethyl acetate, and drying to obtain N-fluorenylmethoxycarbonylglycine hydroxysuccinimide ester; (2) Using tetrahydrofuran as the reaction solvent, N-fluorenylmethoxycarbonylglycine hydroxysuccinimide ester reacts with guanidino-protected arginine Arg(Pbf)-OH in the presence of an organic base. After the reaction is completed, Fmoc-Gly-Arg(Pbf)-OH glargine dipeptide is generated.
2. The method according to claim 1, characterized in that The reaction time of step (1) is 30 minutes to 12 hours; the reaction temperature is -5 to 25°C; and the cooling temperature is -5 to 0°C.
3. The method according to claim 1, characterized in that The step (1) of beating with ethyl acetate specifically comprises beating at 10-15° C. for 1-2 hours; and the mass ratio of the crude active ester Fmoc-Gly-OSu to ethyl acetate is 1:4.5-10.
4. The method according to claim 1, characterized in that The reaction organic solvent in step (1) is selected from one of tetrahydrofuran, dichloromethane, dioxane, acetonitrile, N,N-dimethylformamide or chloroform; the coupling reagent in step (1) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide.
5. The method according to claim 1, characterized in that In the step (1), the molar ratio of N-Fmoc, N-hydroxysuccinimide and coupling reagent is 1:1:1.0-2.0; the volume weight ratio (ml / g) of the organic solvent used and N-Fmoc is 7-10:
1.
6. The method according to claim 1, characterized in that The reaction time of step (2) is 30 min to 14 h, and the reaction temperature is 0 to 30°C.
7. The method according to claim 1, characterized in that The step (2) further comprises the following post-processing steps: S1: After the reaction is completed, tetrahydrofuran is concentrated and recovered, ethyl acetate / water is added, and the pH is adjusted to 3-4 using an inorganic acid. After the extraction is completed, water is added to stir and wash the ester layer; S2: The ester layer obtained in step S1 is concentrated, and the obtained crude oil is stirred with ethyl acetate / n-heptane, cooled and crystallized, and filtered.
8. The method according to claim 1, characterized in that In the step (2), the molar ratio of N-fluorenylmethoxycarbonylglycine hydroxysuccinimide ester to guanidine-protected arginine is 1:1.0-1.5; the organic base in the step (2) is N-ethyldiisopropylamine, and the molar ratio of N-fluorenylmethoxycarbonylglycine hydroxysuccinimide ester to the organic base is 0.7-0.8:
1.
9. The method according to claim 7, characterized in that The inorganic acid in step S1 is hydrochloric acid, and the mass ratio of ethyl acetate to water is 1:0.5-0.
6.
10. The method according to claim 7, characterized in that In the post-treatment step S2 of step (2), the volume ratio of the crystallization solvent ethyl acetate / n-heptane is 1:1-2, the cooling temperature is 0-5°C, and the volume ratio of the crystallization solvent: the mass ratio of the crude oil is 6-8:1.