CN103232369A - Preparation method of fmoc chloride glutamic acid-5-tert-butyl ester - Google Patents

Abstract

The invention discloses a preparation method of fluorenylmethoxycarbonyl glutamic acid-5- _tert -butyl ester, comprising the preparation of Glu(OtBu) ₂ , which selectively removes 1-tert-butyl ester through copper salt Prepare Cu[Glu(OtBu)] _x (x=1～2), then remove copper to obtain Glu(OtBu), and then react with Fmoc-Osu or Fmoc-Cl to obtain Fmoc-Glu(OtBu); The Cu in the copper salt is set to selectively remove the 1-tert-butyl ester tert-butyl group of Glu ⁽ OtBu) ₂ , which greatly simplifies the process route, reduces the cost, and is suitable for large-scale production; The process route is to prepare fluorenylmethoxycarbonyl glutamate-5-tert-butyl ester under unique process conditions, with high yield and greatly improved product quality.

Description

A kind of preparation method of fluorenylmethoxycarbonyl glutamic acid-5-tert-butyl ester

technical field

The invention relates to the field of polypeptide synthesis, in particular to a preparation method of a glutamic acid derivative, fluorenylmethoxycarbonylglutamic acid-5-tert-butyl ester.

Background technique

In the prior art, the synthetic route of fluorenylmethoxycarbonyl glutamic acid-5-tert-butyl ester is that Glu reacts with Z-OSu to generate Z-Glu, Z-Glu and acetic anhydride are heated together to generate Z-Glu anhydride, Z- Glu acid anhydride was dissolved in ether and benzyl alcohol, and cyclohexylamine was added dropwise in an ice bath to obtain Z-Glu-OBzl·DCHA. After crystallization, DCHA was removed to obtain Z-Glu-OBzl, and Z-Glu-OBzl was obtained through acid catalysis and isobutylene addition. -Glu(OtBu)-OBzl, Z-Glu(OtBu)-OBzl is catalyzed by Pd again, passes H ₂ Hydrogenolysis obtains Glu(OtBu), finally reacts with Fmoc-OSu to obtain target product Fmoc-Glu(OtBu), the above method Or similar method to produce Fmoc-Glu (OtBu) is comparatively loaded down with trivial details, and yield is low, and cost is high, is not suitable for large-scale commercial production.

Contents of the invention

The object of the present invention is to overcome the problems of complicated preparation process, high cost and low yield of Fmoc-Glu (OtBu) in the prior art, and provide a kind of preparation method of fluorenylmethoxycarbonyl glutamic acid-5-tert-butyl ester , the method is simple, low in cost and suitable for large-scale production.

In order to achieve the above-mentioned purpose of the invention, the technical scheme adopted in the present invention is: a kind of preparation method of fluorenylmethoxycarbonylglutamic acid-5-tert-butyl ester is provided, it is characterized in that, comprises the following steps:

(1) Prepare Glu into Glu(OtBu) ₂ ;

(2) Mix Glu(OtBu) ₂ with copper salt to obtain Cu[Glu(OtBu)] _x , x=1～2;

(3) Add a decoppering agent to Cu[Glu(OtBu)] _x to remove copper to obtain Glu(OtBu); then add Fmoc group protection reagent to Glu(OtBu) and mix to obtain Fmoc-Glu(OtBu), That is, 5-tert-butyl fluorenylmethoxycarbonyl glutamate;

The specific steps for preparing Glu into Glu(OtBu) ₂ are: mixing Glu with tert-butyl acetate, performing a transesterification reaction under the catalysis of perchloric acid, and then extracting and washing with alkali to obtain Glu(OtBu) ₂ ; Wherein, the substance ratio of Glu, tert-butyl acetate and perchloric acid is 1:5～20:1.2～2; Its synthetic route is as follows:

The temperature when the Glu reacts with tert-butyl acetate is 10-20° C., and the reaction time is 24-48 hours.

The specific steps of preparing Glu into Glu(OtBu) ₂ are: mixing Glu with isobutylene, and performing an addition reaction under the catalysis of anhydrous p-toluenesulfonic acid to obtain Glu(OtBu) ₂ ; wherein, Glu, isobutylene The ratio of substance to anhydrous p-toluenesulfonic acid is 1:3～10:1.2～2, and its synthetic route is as follows:

The temperature when the Glu reacts with isobutene is -10-5°C, and the reaction time is 48-72 hours.

The Glu(OtBu) ₂ and the copper salt are mixed according to the molar ratio of 1-2:1, and reacted at 30-60°C for 12-16 hours to obtain Cu[Glu(OtBu)] _x , where x=1～ 2, its synthetic route is as follows:

The copper salt is CuSO ₄ , Cu(NO ₃ ) ₂ , CuCl ₂ or Cu ₂ (OH) ₂ CO ₃ .

Add copper removal agent and solvent in Cu[Glu(OtBu)] _x according to a certain ratio, adjust the pH value of the solution to 8-9, and obtain Glu(OtBu); then add Fmoc group protection to Glu(OtBu) Reagent, adjust the pH value of the solution to 8-9, react for 7-10 hours, and then acidify, extract, and crystallize to obtain fluorenylmethoxycarbonyl glutamic acid-5-tert-butyl ester; wherein, the copper removing agent and The molar ratio of Cu[Glu(OtBu)] _x is 1:1; the molar ratio of solvent to Cu[Glu(OtBu)] _x is 3～10:1; the protective reagent of Glu(OtBu) and Fmoc group The molar ratio is 1:1, and its synthetic route is as follows:

The copper removing agent is Na ₂ EDTA, Na ₂ S or tetramethylethylenediamine; the solvent is ethanol, methanol, acetone, tetrahydrofuran, dioxane or dichloromethane.

The protecting agent of the Fmoc group is Fmoc-OSu or Fmoc-Cl.

In summary, the preparation method of the present invention selectively removes ^the ₁ -tert-butyl ester tert-butyl group of Glu(OtBu) by setting Cu in the copper salt, which greatly simplifies the process route and reduces the cost. It is suitable for large-scale production; the present invention prepares fluorenylmethoxycarbonyl glutamate-5-tert-butyl ester under special process conditions by setting a unique process route, with high yield and greatly improved product quality.

Detailed ways

The present invention will be described in detail below in conjunction with the examples, but they are not further limiting the present invention.

Example 1

Add 581g of tert-butyl acetate and 147g of glutamic acid into a 2000mL three-necked flask, stir, add 96.5mL of perchloric acid dropwise, react at 20°C for 48 hours, cool down to 0°C, add 600mL of water, and extract (the remaining acetic acid tert-butyl ester as solvent extraction), neutralized with Na ₂ CO ₃ to pH = 8, separated, washed three times with 400mL 1% Na ₂ CO ₃ aqueous solution, and concentrated tert-butyl acetate layer under reduced pressure to obtain 60g of oil Glu ( OtBu) ₂ , the yield was 23.2%.

Add 60g Glu(OtBu) ₂ into a 1000mL three-necked flask, then add 600mL water and 57.9gCuSO ₄ 5H ₂ O, stir, raise the temperature to 50°C, and react for 12 hours to obtain Cu[Glu(OtBu)] _x (x= 1~2); then lowered to room temperature, added 86g Na ₂ EDTA·2H ₂ O and 100mL dioxane, and then adjusted the pH to 8~9 with triethylamine to obtain Glu(OtBu); then in Glu(OtBu ) was added to 74g Fmoc-OSu, the pH was adjusted to be 8-9, and the reaction was carried out for 8 hours to obtain the crude product fluorenylmethoxycarbonyl glutamic acid-5-tert-butyl ester; then the crude product was acidified with HCl, and ethyl acetate was used to Extraction, concentrating the crystals under reduced pressure, filtering, and drying to obtain 88 g of fluorenylmethoxycarbonyl glutamic acid-5-tert-butyl ester.

The product fluorenylmethoxycarbonyl glutamate-5-tert-butyl is analyzed by HPLC, the purity of fluorenylmethoxycarbonylglutamate-5-tert-butyl is 99.2%, the optical rotation is -8.5, and the melting point is 88.3 ~90.5, the isomer content is 0.12%.

Example 2

Add 1162g of tert-butyl acetate and 147g of glutamic acid into a 3000mL three-necked flask, stir, add dropwise 130mL of perchloric acid, react at 15°C for 30 hours, cool down to 0°C, add 800mL of water, extract (the remaining tert-acetic acid butyl ester as a solvent), neutralized with Na ₂ CO ₃ to pH = 8, separated, washed three times with 400 mL of 1% Na ₂ CO ₃ aqueous solution, and concentrated the tert-butyl acetate layer under reduced pressure to obtain 71 g of oil Glu(OtBu ) ₂ , with a yield of 27.4%.

Add 71g Glu(OtBu) ₂ to a 1000mL three-necked flask, then add 700mL water and 66.2gCu(NO ₃ ) ₂ .3H ₂ O, stir, raise the temperature to 40°C, and react for 16 hours to obtain Cu[Glu(OtBu)] _x (x=1～2); then drop to room temperature, add 63.6g tetramethylethylenediamine and 150mL dioxane, and then adjust the pH to 8 with triethylamine to obtain Glu(OtBu); Add 83g Fmoc-OSu to (OtBu), adjust the pH to 8, and react for 8 hours to obtain the crude product fluorenylmethoxycarbonylglutamic acid-5-tert-butyl ester; then acidify the crude product with HCl, and use ethyl acetate Extract, concentrate the crystals under reduced pressure, filter, and dry to obtain 94 g of fluorenylmethoxycarbonyl glutamate-5-tert-butyl ester.

The product fluorenylmethoxycarbonyl glutamate-5-tert-butyl is analyzed by HPLC, the purity of fluorenylmethoxycarbonylglutamate-5-tert-butyl is 99.7%, the optical rotation is -8.4, and the melting point is 89.5 ~91.6, the isomer content is 0.07%.

Example 3

Add 2324g of tert-butyl acetate and 73.5g of glutamic acid into a 3000mL three-necked flask, stir, add 160.8mL of perchloric acid dropwise, react at 10°C for 48 hours, cool down to 0°C, add 1000mL of water, and extract (the remaining tert-butyl acetate as solvent extraction), neutralized with Na ₂ CO ₃ to pH = 8-9, separated, washed three times with 400mL 1% Na ₂ CO ₃ aqueous solution, concentrated tert-butyl acetate layer under reduced pressure to obtain 42g oil Compound Glu(OtBu) ₂ with a yield of 32.4%.

Add 42g Glu(OtBu) ₂ into a 1000mL three-necked flask, then add 400mL water and 11g CuCl ₂ , raise the temperature to 50°C, and react for 12 hours to obtain Cu[Glu(OtBu)] _x (x=1～2); Cool down to room temperature, add 19g of tetramethylethylenediamine and 100mL of dioxane, and then adjust the pH to 8-9 with triethylamine to obtain Glu(OtBu); then add 48g of Fmoc-OSu to Glu(OtBu) , adjust the pH to 8-9, and react for 10 hours to obtain the crude product fluorenylmethoxycarbonylglutamic acid-5-tert-butyl ester; then the crude product was acidified with HCl, extracted with ethyl acetate, concentrated and crystallized under reduced pressure, Filter and dry to obtain 55 g of fluorenylmethoxycarbonyl glutamic acid-5-tert-butyl ester.

The product fluorenylmethoxycarbonyl glutamate-5-tert-butyl is analyzed by HPLC, the purity of fluorenylmethoxycarbonylglutamate-5-tert-butyl is 99.4%, the optical rotation is -8.8, and the melting point is 88.7 ~91.2, the isomer content is 0.12%.

Example 4

Add 1000mL of dichloromethane into a 3000mL three-necked flask, cool down to -10°C, add 344g of anhydrous p-toluenesulfonic acid and 147g of glutamic acid, feed 448g of isobutylene, and keep it at -10°C for 72 hours; then use 10% Na ₂ CO ₃ aqueous solution adjusted the pH to 8-9, separated, washed 3 times with 1% Na ₂ CO ₃ , 400 mL each time, and evaporated to dryness under reduced pressure to obtain 160 g of Glu(OtBu) ₂ with a yield of 61.8%.

The abbreviations used in the present invention or the implication of English full name are listed in the following table:

Glu glutamic acid Fmoc-OSu fluorenyl moxycarbonyl succinimide Z-OSu Benzyloxycarbonylsuccinimide Na ₂ EDTA Disodium edetate DCHA Dicyclohexylamine Fmoc-Glu(OtBu)·H ₂ O Fmocyl glutamate-5-tert-butyl monohydrate Glu(OtBu) ₂ Di-tert-butyl glutamate Glu(OtBu) Glutamic acid-5-tert-butyl ester Z-Glu Benzyloxycarbonyl Glutamic Acid Z-Glu-OBzl Benzyloxycarbonyl glutamate-1-benzyl ester Z-Glu(OtBu)-OBzl Benzyloxycarbonyl glutamate-1-benzyl ester-5-tert-butyl ester tert-butylacetate tert-butyl acetate 2-methylpropene Isobutylene 1,4-dioxane Dioxane Triethylamine Triethylamine Tos-OH p-Toluenesulfonic acid

Although specific embodiments of the present invention have been described in detail in conjunction with specific examples, they are not intended to limit the protection scope of this patent. Within the scope defined in the claims, various modifications or adjustments that can be made by those skilled in the art without creative work are still protected by this patent.

Claims (10)

1. a preparation method of fluorenylmethoxycarbonylglutamic acid-5-tert-butyl ester, is characterized in that, comprises the following steps: (1) Prepare Glu into Glu(OtBu) ₂ ; (2) Mix Glu(OtBu) ₂ with copper salt to obtain Cu[Glu(OtBu)] _x , x=1～2; (3) Add a decoppering agent to Cu[Glu(OtBu)] _x to remove copper to obtain Glu(OtBu); then add Fmoc group protection reagent to Glu(OtBu) and mix to obtain Fmoc-Glu(OtBu), That is, 5-tert-butyl fluorenylmethoxycarbonyl glutamate. 2. the preparation method of fluorenyl methaneoxycarbonyl glutamic acid-5-tert-butyl ester according to claim 1, is characterized in that, _the described concrete step that Glu is prepared into Glu(OtBu) is: combine Glu with tert-butyl acetate is mixed, transesterification is carried out under the catalysis of perchloric acid, and then through extraction, alkali washing, obtain Glu(OtBu) ₂ ; Wherein, the material ratio of Glu, tert-butyl acetate and perchloric acid 1:5~20:1.2~2. 3. the preparation method of fluorenyl methaneoxycarbonyl glutamic acid-5-tert-butyl ester according to claim 2, is characterized in that: the temperature when described Glu reacts with tert-butyl acetate is 10～20 ℃, reacts The time is 24-48 hours. 4. the preparation method of fluorenyl methaneoxycarbonyl glutamate-5-tert-butyl _ester according to claim 1, is characterized in that, the described concrete step that Glu is prepared into Glu(OtBu) is: combine Glu with Isobutene is mixed, and an addition reaction is carried out under the catalysis of anhydrous p-toluenesulfonic acid to obtain Glu(OtBu) ₂ ; wherein, the mass ratio of Glu, isobutene and anhydrous p-toluenesulfonic acid is 1:3-10: 1.2～2. 5. the preparation method of fluorenyl methaneoxycarbonyl glutamic acid-5-tert-butyl ester according to claim 4 is characterized in that: the temperature when described Glu reacts with isobutylene is -10～-5 ℃, and the reaction time 48-72 hours. 6. The preparation method of Fmocylglutamic acid-5-tert-butyl ester according to claim 1, characterized in that: said Glu(OtBu) ₂ and copper salt are in a molar ratio of 1 to 2:1 The ratio is mixed and reacted at 30-60° C. for 12-16 hours to obtain Cu[Glu(OtBu)] _x , where x=1-2. 7. The preparation method of fluorenylmethoxycarbonyl glutamic acid-5-tert-butyl ester according to claim 1 or 6, characterized in that: the copper salt is CuSO ₄ , Cu(NO ₃ ) ₂ , CuCl ₂ or Cu ₂ (OH) ₂ CO ₃ . 8. the preparation method of fluorenyl moxycarbonyl glutamic acid-5-tert-butyl ester according to claim 1 is characterized in that: in Cu[Glu(OtBu)] _x , add decopper agent and Solvent, adjust the pH value of the solution to 8-9 to obtain Glu(OtBu); then add the protective reagent of Fmoc group to Glu(OtBu), adjust the pH value of the solution to 8-9, react for 7-10 hours, and then After acidification, extraction, and crystallization, 5-tert-butyl fluorenylmethoxycarbonyl glutamate is obtained; wherein, the molar ratio of the copper removal agent to Cu[Glu(OtBu)] _x is 1:1; the solvent and Cu The molar ratio of [Glu(OtBu)] _x is 3-10:1; the molar ratio of the Glu(OtBu) to the Fmoc group protecting reagent is 1:1. 9. The preparation method of fluorenylmethoxycarbonyl glutamic acid-5-tert-butyl ester according to claim 1 or 8, characterized in that: the decoppering agent is Na ₂ EDTA, Na ₂ S or tetramethyl Ethylenediamine; the solvent is ethanol, methanol, acetone, tetrahydrofuran, dioxane or dichloromethane. 10. The preparation method of fluorenylmethoxycarbonyl glutamic acid-5-tert-butyl ester according to claim 1 or 8, characterized in that: the protecting reagent of the Fmoc group is Fmoc-OSu or Fmoc-Cl.