阅读说明：本技术 一种N-芴甲氧羰基-γ-（S-三苯甲基-半胱胺基）-L-谷氨酸的制备方法 (Preparation method of N-fluorenylmethoxycarbonyl-gamma- (S-trityl-cysteamine) -L-glutamic acid ) 是由 徐红岩 张家宝 付静晗 李仲才 朱银 曹世团 付等良 于 2020-06-16 设计创作，主要内容包括：本发明提供了一种N-芴甲氧羰基-γ-(S-三苯甲基-半胱胺基)-L-谷氨酸的制备方法,主要解决原工艺中的复杂性,周期长,成本高,收率低等技术问题,本发明包括以下步骤：(1)、N-芴甲氧羰基-L-谷氨酸的制备；(2)、N-芴甲氧羰基-L-谷氨酸-1-苄酯的制备；(3)、S-三苯甲基半胱胺的制备；(4)、N-芴甲氧羰基-γ-(S-三苯甲基-半胱胺基)-L谷氨酸-α-苄酯的制备；(5)、N-芴甲氧羰基-γ-(S-三苯甲基-半胱胺基)-L谷氨酸的制备。本发明具有快速、高产、分离和纯化简单,且使用的溶剂为对环境友好的溶剂,适合大批量生产。(The invention provides a preparation method of N-fluorenylmethyloxycarbonyl-gamma- (S-trityl-cysteamine) -L-glutamic acid, which mainly solves the technical problems of complexity, long period, high cost, low yield and the like in the prior art, and comprises the following steps: (1) preparing N-fluorenylmethyloxycarbonyl-L-glutamic acid; (2) preparing N-fluorenylmethyloxycarbonyl-L-glutamic acid-1-benzyl ester; (3) preparing S-trityl cysteamine; (4) preparing N-fluorenylmethyloxycarbonyl-gamma- (S-trityl-cysteamine) -L glutamic acid-alpha-benzyl ester; (5) and preparing N-fluorenylmethyloxycarbonyl-gamma- (S-trityl-cysteamine) -L glutamic acid. The method has the advantages of rapidness, high yield, simple separation and purification, and environment-friendly solvent, and is suitable for mass production.)

1. A preparation method of N-fluorenylmethyloxycarbonyl-gamma- (S-trityl-cysteamine) -L-glutamic acid is characterized by comprising the following steps:

(1) adding glutamic acid into a mixed solution of water and acetone, adding sodium hydroxide, stirring, cooling in an ice bath, adding an amino protective agent fmoc group donor, reacting, tracking the detection process by TLC (thin layer chromatography), and then performing acidification extraction, water washing and brine washing to obtain fmoc-glu-OH;

(2) mixing fmoc-glu-OH and a benzyl donor in dimethylformamide, adding papain, heating for reaction, tracking and monitoring the reaction process by TLC (thin layer chromatography), diluting with water after the reaction is finished, washing with ethyl acetate, acidifying and extracting by adding ethyl acetate and citric acid, washing with water, washing with brine, concentrating, and adding petroleum ether for crystallization to obtain fmoc-glu-obzl;

(3) adding 2-mercaptoethylamine into glacial acetic acid, heating, adding a trityl donor, slowly adding trimethylchlorosilane, mechanically stirring for reaction, cooling to 4-6 ℃, adjusting the pH value of the system by using a sodium acetate aqueous solution, stirring for crystallization, filtering the system, washing a filter cake by using water and ether, and drying to obtain S-trityl cysteamine;

(4) adding fmoc-glu-obzl into THF, mechanically stirring and cooling, then adding a condensing agent and triethylamine, then adding S-trityl cysteamine for reaction, tracking and monitoring the reaction process by TLC, decompressing and concentrating to remove THF after the reaction is finished, then adding ethyl acetate for dissolving, washing with acid water, washing with alkali water, washing with brine, drying, concentrating and crystallizing to obtain fmoc-glu (NHCH)₂CH₂Strt)-obzl；

(5) Fmoc-glu (NHCH)₂CH₂Adding Strt) -obzl into methanol, adding palladium-carbon, introducing hydrogen gas for reaction, tracking and monitoring the process by TLC, filtering to remove palladium-carbon after the reaction is finished, concentrating until the palladium-carbon is dry, adding a little ethyl acetate for dissolving, adding petroleum ether for crystallization, and then carrying out suction filtration and drying to obtain fmoc-glu (NHCH)₂CH₂Strt)-COOH。

2. The process for preparing Fmoc-gamma- (S-trityl-cysteaminyl) -L-glutamic acid according to claim 1

The method is characterized in that in the step (1), the mass ratio of glutamic acid to fmoc-group is 1 (1-1.5), and the reaction temperature is as follows: the reaction time is 6-12h at 10-20 ℃, and the fmoc-group donor is fmoc-osu, fmoc-CL or fmoc-NH₂One kind of (1).

3. The method for preparing Fmoc-gamma- (S-trityl-cysteamino) -L-glutamic acid according to claim 2, wherein the Fmoc-group donor is Fmoc-osu.

4. The method for preparing Fmoc-gamma- (S-trityl-cysteaminyl) -L-glutamic acid according to claim 1, wherein the amount ratio of the substances of the Fmoc-glu-OH and benzyl donor in the step (2) is: 1 (1-1.6), the reaction temperature is 38-42 ℃, the reaction time is 22-26h, the dosage of the papain is 10% -15% of the mass of the amino acid, and the benzyl donor is one of benzyl alcohol, benzyl bromide or benzyl chloride.

5. The method for preparing N-fluorenylmethyloxycarbonyl-gamma- (S-trityl-cysteamine) -L-glutamic acid according to claim 1, wherein the mass ratio of the 2-mercaptoethylamine, the trityl donor and the trimethylchlorosilane in the step (3) is 1: (1-1.5): (0.9-1.3), the reaction temperature is 30-50 ℃, the reaction time is 2-6h, and the trityl donor is one of trityl alcohol, triphenylbromomethane and triphenylchloromethane.

6. The method for preparing Fmoc-gamma- (S-trityl-cysteamine) -L-glutamic acid according to claim 1, wherein the ratio of fmoc-glu-obzl, condensing agent and S-trityl cysteamine in the step (4) is: 1, (0.9-1.3) and (1-1.3), wherein the reaction temperature is 5-15 ℃, the reaction time is 14-18h, and the condensing agent is one of HTBU, TBTU or HOBT.

7. The method for preparing N-fluorenylmethyloxycarbonyl-gamma- (S-trityl-cysteamino) -L-glutamic acid according to claim 1, wherein the amount of palladium-carbon added in step (5) is 5% -15% of the amount of amino acid, and the reaction time is 12-24 h.

8. The method for preparing N-fluorenylmethyloxycarbonyl-gamma- (S-trityl-cysteaminyl) -L-glutamic acid according to claim 1, wherein TLC is used for tracking detection process conditions: 1) n-butanol: glacial acetic acid: volume ratio of water =4:1:1, 2) chloroform: methanol: acetic acid =90:8: 2.

Technical Field

The invention relates to the fields of polypeptide synthesis, pharmaceutical polypeptide and the like, in particular to a preparation method of N-fluorenylmethyloxycarbonyl-gamma- (S-trityl-cysteamine) -L-glutamic acid.

Background

In the prior art, the preparation method route of N-fluorenylmethyloxycarbonyl-gamma- (S-trityl-cysteamine) -L-glutamic acid and the intermediate thereof is as follows: glutamic acid is made into h-glu (obzl) -OH through side chain carboxyl benzyl protection, then z-glu (obzl) -OH is obtained through reaction with z-group, z-glu (obzl) -otbu is obtained through reaction with isobutene, then h-glu-otbu is obtained through hydrocracking, fmoc-glu-otbu is obtained through reaction with fmoc-group, fmoc-glu-otbu is made through reaction with hosu, and fmoc-glu (osu) -otbu is made through reaction with S-trityl cysteamine which is prepared in advance to obtain fmoc-glu (NHCH)₂CH₂Strt) -otbu, and finally removing the tert-butyl group to obtain fmoc-glu (NHCH)₂CH₂Strt) -COOH. The method has the advantages of complicated steps, long period, low yield, high cost and unsuitability for commercial production.

Disclosure of Invention

The invention aims to overcome the technical problems of complexity, long period, high cost, low yield and the like in the prior art, and provides a preparation method of N-fluorenylmethoxycarbonyl-gamma- (S-trityl-cysteaminyl) -L-glutamic acid and an intermediate thereof.

In order to achieve the purpose of the invention, the invention adopts the technical scheme that: a preparation method of N-fluorenylmethyloxycarbonyl-gamma- (S-trityl-cysteamine) -L-glutamic acid is characterized by comprising the following steps:

(1) adding glutamic acid into a mixed solution of water and acetone, adding sodium hydroxide, stirring, cooling in an ice bath, adding an amino protective agent fmoc group donor, reacting, tracking the detection process by TLC (thin layer chromatography), and then performing acidification extraction, water washing and brine washing to obtain fmoc-glu-OH;

(2) mixing fmoc-glu-OH and a benzyl donor in dimethylformamide, adding papain, heating for reaction, tracking and monitoring the reaction process by TLC (thin layer chromatography), diluting with water after the reaction is finished, washing with ethyl acetate, acidifying and extracting by adding ethyl acetate and citric acid, washing with water, washing with brine, concentrating, and adding petroleum ether for crystallization to obtain fmoc-glu-obzl;

(3) adding 2-mercaptoethylamine into glacial acetic acid, heating, adding a trityl donor, slowly adding trimethylchlorosilane, mechanically stirring for reaction, cooling to 4-6 ℃, adjusting the pH value of a system by using a sodium acetate aqueous solution with the mass percentage of 20%, stirring for crystallization, filtering the system, washing a filter cake by using water and ether, and drying to obtain S-trityl cysteamine;

(4) adding fmoc-glu-obzl into THF, mechanically stirring and cooling, then adding a condensing agent and triethylamine, then adding S-trityl cysteamine for reaction, tracking and monitoring the reaction process by TLC, decompressing and concentrating to remove THF after the reaction is finished, then adding ethyl acetate for dissolving, washing with acid water, washing with alkali water, washing with brine, drying, concentrating and crystallizing to obtain fmoc-glu (NHCH)₂CH₂Strt)-obzl；

(5) Fmoc-glu (NHCH)₂CH₂Adding Strt) -obzl into methanol, adding palladium-carbon, introducing hydrogen gas for reaction, tracking and monitoring the process by TLC, filtering to remove palladium-carbon after the reaction is finished, concentrating until the palladium-carbon is dry, adding a little ethyl acetate for dissolving, adding petroleum ether for crystallization, and then carrying out suction filtration and drying to obtain fmoc-glu (NHCH)₂CH₂Strt) -COOH, structure as expected by NMR and MS verification.

In the step (1), the mass ratio of glutamic acid to fmoc-group is 1 (1-1.5), and the reaction temperature is as follows: the reaction time is 6-12h at 10-20 ℃, and the fmoc-group donor is fmoc-osu, fmoc-CL or fmoc-NH₂One kind of (1). Preferably the fmoc-group donor is fmoc-osu.

In the step (2), the mass ratio of fmoc-glu-OH to benzyl donor is as follows: 1 (1-1.6), the reaction temperature is 38-42 ℃, the reaction time is 22-26h, the dosage of the papain is 10% -15% of the mass of the amino acid, and the benzyl donor is one of benzyl alcohol, benzyl bromide or benzyl chloride, preferably benzyl bromide.

In the step (3), the mass ratio of the 2-mercaptoethylamine to the trityl donor to the trimethylchlorosilane is 1 (1-1.5) to 0.9-1.3, the reaction temperature is 30-50 ℃, the reaction time is 2-6h, and the trityl donor is one of trityl alcohol, triphenylbromomethane or triphenylchloromethane, preferably triphenylbromomethane.

In the step (4), the ratio of the amount of fmoc-glu-obzl, the condensing agent and the S-trityl cysteamine substance is: 1, (0.9-1.3) and (1-1.3), wherein the reaction temperature is 5-15 ℃, the reaction time is 14-18h, and the condensing agent is one of HTBU, TBTU or HOBT, preferably TBTU.

In the step (5), the added palladium-carbon accounts for 5-15% of the weight of the amino acid, and the reaction time is 12-24 h.

The TLC tracks the conditions of the detection process: 1) n-butanol, glacial acetic acid: the volume ratio of water =4:1: 1; 2) the volume ratio of chloroform to methanol to acetic acid is =90:8: 2.

The invention has the beneficial effects that: the preparation method of the invention firstly prepares low-price fmoc-glu-obzl to replace expensive fmoc-glu-otbu, and finally prepares the N-fluorenylmethoxycarbonyl-gamma- (S-trityl-cysteaminyl) -L-glutamic acid.

Drawings

FIG. 1 is a chromatogram of the product of example 1 of the present invention.

FIG. 2 is a mass spectrum of the product of example 1 of the present invention.

FIG. 3 is a nuclear magnetic spectrum of the product of example 1 of the present invention.

FIG. 4 is a chromatogram of the product of example 2 of the present invention.

FIG. 5 is a chromatogram of the product of example 3 of the present invention.

FIG. 6 is a chromatogram of the product of example 4 of the present invention.

FIG. 7 is a chromatogram of the product of example 5 of the present invention.

FIG. 8 is a chromatogram of the product of example 6 of the present invention.

FIG. 9 is a mass spectrum of the product of example 6 of the present invention.

FIG. 10 is a nuclear magnetic spectrum of the product of example 6 of the present invention.

FIG. 11 is a chromatogram of the product of example 7 of the present invention.

FIG. 12 is a mass spectrum of a product of a comparative example of the present invention.

Detailed Description

The present invention is described in detail below with reference to specific examples, but they are not intended to limit the invention further.

The main detecting instrument used in the experiment:

1. shimadzu 20A

2. And (3) testing conditions: a chromatographic column: 4.6 mm, sinoCHrom ODS-BP 5 μm

3. Detection wavelength: 220nm

4. Mobile phase: a: 0.1% trifluoroacetic acid solution

B: 0.1 percent trifluoroacetic acid acetonitrile mixed solution

5. Flow rate: 1.0ml/min

6. Column temperature: 25 deg.C

7. Shimadzu: LCMS-2020 mass spectrometer

8. Walian 400M nuclear magnetic resonance spectrometer.