阅读说明：本技术 一种不含无机铵盐的氨基酸酰胺盐酸盐及其合成方法 (Amino acid amide hydrochloride without inorganic ammonium salt and synthetic method thereof ) 是由 刘林 傅建 张晓斌 陈垒 唐波 张仁友 吴秀英 张松 吴文秀 王永生 左祥群 于 2021-10-25 设计创作，主要内容包括：本发明公开了一种不含无机铵盐的氨基酸酰胺盐酸盐及其合成方法,包括以下步骤：S1、将氨基酸在碱性条件下,加入二碳酸二叔丁酯制备叔丁氧羰基－氨基酸；S2、向制备得到的叔丁氧羰基－氨基酸中再加入二碳酸二叔丁酯,在N－甲基吗啉、碳酸氢铵存在的条件下,制备叔丁氧羰基－氨基酸酰胺；S3、将叔丁氧羰基－氨基酸酰胺置于乙酸乙酯/氯化氢溶液体系中,结晶析出即得。本发明分两次加入二碳酸二叔丁酯,在不同条件下制备得到高纯的叔丁氧羰基－氨基酸酰胺,最后在乙酸乙酯/氯化氢溶液条件下得到最终产品,本发明解决了氨基酸酰胺盐酸盐生产中无机铵盐难分离的问题,减少了有机溶剂的用量。(The invention discloses an amino acid amide hydrochloride without inorganic ammonium salt and a synthesis method thereof, comprising the following steps: s1, adding di-tert-butyl dicarbonate to the amino acid under an alkaline condition to prepare tert-butoxycarbonyl-amino acid; s2, adding di-tert-butyl dicarbonate into the prepared tert-butoxycarbonyl-amino acid, and preparing tert-butoxycarbonyl-amino acid amide in the presence of N-methylmorpholine and ammonium bicarbonate; s3, placing the tert-butyloxycarbonyl-amino acid amide in an ethyl acetate/hydrogen chloride solution system, and crystallizing and separating out the tert-butyloxycarbonyl-amino acid amide to obtain the compound. The invention adds di-tert-butyl dicarbonate twice, prepares high-purity tert-butoxycarbonyl-amino acid amide under different conditions, and finally obtains a final product under the condition of ethyl acetate/hydrogen chloride solution.)

1. A synthetic method of amino acid amide hydrochloride without inorganic ammonium salt is characterized by comprising the following steps:

s1, adding di-tert-butyl dicarbonate to the amino acid under an alkaline condition to prepare tert-butoxycarbonyl-amino acid;

s2, adding di-tert-butyl dicarbonate into the prepared tert-butoxycarbonyl-amino acid, and preparing tert-butoxycarbonyl-amino acid amide in the presence of N-methylmorpholine and ammonium bicarbonate;

s3, placing the prepared tert-butyloxycarbonyl-amino acid amide in an ethyl acetate/hydrogen chloride solution system to remove tert-butyloxycarbonyl, and crystallizing and separating out to obtain the amino acid amide hydrochloride.

2. The method for synthesizing an amino acid amide hydrochloride that is free of an inorganic ammonium salt according to claim 1, wherein in S1, the amino acid is an amino acid having no reactive group in a side chain or an amino acid having a protected side chain.

3. The method for synthesizing an amino acid amide hydrochloride that does not contain an inorganic ammonium salt according to claim 1, wherein the basic condition is that the amino acid is under a basic aqueous phase condition in S1.

4. The method for synthesizing amino acid amide hydrochloride free of inorganic ammonium salts according to claim 1, wherein in S1, the mass ratio of amino acid to di-tert-butyl dicarbonate is 1: 1.05-1.2.

5. the method for synthesizing an amino acid amide hydrochloride free of inorganic ammonium salts according to claim 1, wherein in S2, di-tert-butyl dicarbonate is further added in an amount of 1.1 to 1.5: 1.

6. the method for synthesizing amino acid amide hydrochloride free of inorganic ammonium salts according to claim 1, wherein the mass ratio of ethyl acetate to hydrogen chloride in the ethyl acetate/hydrogen chloride solution system is 8 to 10: 1.

7. the method for synthesizing an amino acid amide hydrochloride free of inorganic ammonium salts according to claim 1, wherein the mass ratio of t-butoxycarbonyl-amino acid amide to the ethyl acetate/hydrogen chloride solution system is 1: 3-5.

8. the method for synthesizing amino acid amide hydrochloride free of inorganic ammonium salts according to claim 1, wherein the content of the inorganic ammonium salt in the amino acid amide hydrochloride is determined by titrating the chloride ion content in the amino acid amide hydrochloride.

9. The method according to claim 8, wherein the amino acid amide hydrochloride is obtained in which the content of inorganic ammonium salt is not more than 0.1%.

10. An amino acid amide hydrochloride free of inorganic ammonium salts, characterized in that it is prepared by the synthesis method according to any one of the preceding claims 1 to 9.

Technical Field

The invention relates to the technical field of polypeptide synthesis, and particularly relates to an amino acid amide hydrochloride free of inorganic ammonium salt and a synthesis method thereof.

Background

Existing amino acid amide hydrochloride (aa.nh)₂HCl) is prepared by adding thionyl chloride dropwise into Amino Acid (AA) in alcohol solvent to generate hydrochloride (AA.OEt.HCl or AA.OMe.HCl) of amino acid ester, neutralizing with alkali to obtain amino acid ester (AA.OEt or AA.OMe), and then introducing ammonia gas for aminolysis to obtain amino acid amide hydrochloride (AA.NH)₂HCl). The inorganic acid ammonium salt generated by the amino acid amide hydrochloride and the neutralized productIs easy to dissolve in water, so that it is difficult to remove the inorganic ammonium salt in the amino acid amide, and the product contains a certain amount of inorganic ammonium salt, generally 2.5-10 w%, which has a great influence on the synthesis of polypeptide.

Disclosure of Invention

The invention aims to: the Amino Acid (AA) is added with di-tert-butyl dicarbonate under the alkaline condition to prepare tert-butyloxycarbonyl-amino acid (Boc-AA), then the di-tert-butyl dicarbonate is added to prepare tert-butyloxycarbonyl-amino acid amide (Boc-AA.NH2) under the condition of existence of N-methylmorpholine and ammonium bicarbonate, the ester solubility of the product is utilized to remove the inorganic ammonium salt produced in the process, thereby obtaining high-purity tert-butyloxycarbonyl-amino acid amide, and finally the tert-butyloxycarbonyl group is removed under the condition of ethyl acetate/hydrogen chloride solution, the product amino acid amide hydrochloride (AA.NH2.HCl) is crystallized and separated out, thereby obtaining the amino acid amide hydrochloride without inorganic ammonium salt and overcoming the defects in the prior art.

The technical scheme adopted by the invention is as follows: a method for synthesizing amino acid amide hydrochloride without inorganic ammonium salt comprises the following steps:

s1, adding di-tert-butyl dicarbonate to the amino acid under an alkaline condition to prepare tert-butoxycarbonyl-amino acid;

s2, adding di-tert-butyl dicarbonate into the prepared tert-butoxycarbonyl-amino acid, and preparing tert-butoxycarbonyl-amino acid amide in the presence of N-methylmorpholine and ammonium bicarbonate;

s3, placing the prepared tert-butyloxycarbonyl-amino acid amide in an ethyl acetate/hydrogen chloride solution system to remove tert-butyloxycarbonyl, and crystallizing and separating out to obtain the amino acid amide hydrochloride.

In the method, firstly, tert-butyloxycarbonyl-amino acid is prepared in an aqueous phase system, then tert-butyloxycarbonyl-amino acid amide is prepared in a non-aqueous phase system under the action of activating carboxyl by di-tert-butyl dicarbonate in the presence of N-methylmorpholine and ammonium bicarbonate, and finally tert-butyloxycarbonyl is removed under the condition of ethyl acetate/hydrogen chloride solution, and the product amino acid amide hydrochloride is crystallized and separated out, thereby obtaining the amino acid amide hydrochloride without inorganic ammonium salt.

Further, in S1, the amino acid is an amino acid having a side chain free from an active group, or a side chain-protected amino acid including a D-type amino acid (D-AA), an L-type amino acid (L-AA), and a DL-type amino acid (DL-AA).

Further, in S1, the basic condition means that the amino acid is under basic aqueous phase conditions.

Further, in S1, the mass ratio of the amino acid to di-tert-butyl dicarbonate was 1: 1.05-1.2.

further, in S2, in S2, di-tert-butyl dicarbonate is added again in an amount of 1.1 to 1.5: 1.

further, in an ethyl acetate/hydrogen chloride solution system, the mass ratio of ethyl acetate to hydrogen chloride is 8-10: the preparation method is that gaseous hydrogen chloride is introduced into ethyl acetate to be dissolved to obtain a hydrogen chloride/ethyl acetate solution system, and the solution system does not contain a water phase. In an ethyl acetate/hydrogen chloride solution system, the concentration of hydrogen chloride is not too low or too high, the too low concentration causes no butyloxycarbonyl group to be removed and causes incomplete reaction, and the too high concentration causes amino acid to be generated and causes great product loss.

In the invention, the mass ratio of the tert-butyloxycarbonyl-amino acid amide to the ethyl acetate/hydrogen chloride solution system is 1: 3-5.

in the invention, for detecting the content of the inorganic ammonium salt in the amino acid amide hydrochloride, the content of the inorganic ammonium salt is determined by titrating and detecting the chloride ion content in the amino acid amide hydrochloride.

Further, the content of inorganic ammonium salt in the obtained amino acid amide hydrochloride is not more than 0.1 w%.

The invention also comprises an amino acid amide hydrochloride free of inorganic ammonium salt, which is prepared by the synthesis method.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that: the invention adds di-tert-butyl dicarbonate into Amino Acid (AA) under the alkaline condition to prepare tert-butyloxycarbonyl-amino acid (Boc-AA), then adds di-tert-butyl dicarbonate, in the presence of N-methylmorpholine and ammonium bicarbonate to prepare tert-butyloxycarbonyl-amino acid amide (Boc-AA.NH2), and utilizes the ester solubility of the product to remove the inorganic ammonium salt produced in the process, thereby obtaining high-purity tert-butyloxycarbonyl-amino acid amide, and finally removes tert-butyloxycarbonyl under the condition of ethyl acetate/hydrogen chloride solution, and the product amino acid amide hydrochloride (AA.NH2.HCl) is crystallized and separated out.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Taking alanine as an example:

example 1

A method for synthesizing amino acid amide hydrochloride without inorganic ammonium salt comprises the following steps:

s1, adding 1L of pure water, 89g of alanine (Ala), 220g of sodium carbonate, 250g of ethanol and 240g of di-tert-butyl dicarbonate into a 2L flask, stirring at normal temperature for 5 hours, dropwise adding 500g of 30% hydrochloric acid, stirring for crystallization for 2 hours, and filtering to collect solid tert-butyloxycarbonyl-alanine (Boc-Ala);

s2, adding 500g of ethyl acetate, 94.5g of tert-butoxycarbonyl-alanine (Boc-Ala), 120g of di-tert-butyl dicarbonate, 50g of N-methylmorpholine and 50g of ammonium bicarbonate into a 1L flask, stirring at normal temperature for 5 hours, washing the ethyl acetate with water, adding 50g of anhydrous sodium sulfate into the ethyl acetate, drying, concentrating and crystallizing to obtain tert-butoxycarbonyl-alanine amide (Boc-Ala. NH2);

s3, a 1L flask was taken, and 285g of an ethyl acetate/hydrogen chloride solution and 94g of t-butyloxycarbonyl-alanine amide (Boc-Ala. NH2) were added thereto, and the mixture was reacted with stirring for 3 hours to precipitate crystals of alanine amide hydrochloride (Ala. NH2. HCl).

Example 2

A method for synthesizing amino acid amide hydrochloride without inorganic ammonium salt comprises the following steps:

s1, adding 1L of pure water, 89g of alanine (Ala), 220g of sodium carbonate, 250g of ethanol and 261g of di-tert-butyl dicarbonate into a 2L flask, stirring at normal temperature for 5 hours, dropwise adding 500g of 30% hydrochloric acid, stirring for crystallization for 2 hours, and filtering to collect solid tert-butyloxycarbonyl-alanine (Boc-Ala);

s2, adding 500g of ethyl acetate, 95g of tert-butoxycarbonyl-alanine (Boc-Ala), 141g of di-tert-butyl dicarbonate, 50g of N-methylmorpholine and 50g of ammonium bicarbonate into a 1L flask, stirring at normal temperature for 5 hours, washing the ethyl acetate with water, adding 50g of anhydrous sodium sulfate into the ethyl acetate, drying, concentrating and crystallizing to obtain tert-butoxycarbonyl-alanine amide (Boc-Ala. NH2);

s3, a 1L flask was taken, and 380g of ethyl acetate/hydrogen chloride solution and 94g of tert-butoxycarbonyl-alanine amide (Boc-Ala. NH2) were added to the flask, and the mixture was reacted with stirring for 3 hours to precipitate crystals and obtain alanine amide hydrochloride (Ala. NH2. HCl).

Example 3

A method for synthesizing amino acid amide hydrochloride without inorganic ammonium salt comprises the following steps:

s1, adding 1L of pure water, 89g of alanine (Ala), 220g of sodium carbonate, 250g of ethanol and 283g of di-tert-butyl dicarbonate into a 2L flask, stirring at normal temperature for 5 hours, dropwise adding 500g of 30% hydrochloric acid, stirring for crystallization for 2 hours, and filtering to collect solid tert-butyloxycarbonyl-alanine (Boc-Ala);

s2, adding 500g of ethyl acetate, 95g of tert-butoxycarbonyl-alanine (Boc-Ala), 163g of di-tert-butyl dicarbonate, 50g of N-methylmorpholine and 50g of ammonium bicarbonate into a 1L flask, stirring at normal temperature for 5 hours, washing the ethyl acetate with water, adding 50g of anhydrous sodium sulfate into the ethyl acetate, drying, concentrating and crystallizing to obtain tert-butoxycarbonyl-alanine amide (Boc-Ala. NH2);

s3, a 1L flask was taken, and 473g of ethyl acetate/hydrogen chloride solution and 94g of tert-butoxycarbonyl-alanine amide (Boc-Ala. NH2) were added to the flask, and the mixture was reacted with stirring for 3 hours to precipitate crystals and obtain alanine amide hydrochloride (Ala. NH2. HCl).

Comparative example 1

A synthetic method of amino acid amide hydrochloride comprises the following steps:

s1, taking a 1L flask, adding 500g of ethanol and 89g of alanine (Ala), dropwise adding 130g of thionyl chloride, stirring at normal temperature for 5 hours, concentrating, then introducing 50g of ammonia gas for ammonolysis, concentrating and crystallizing to obtain amino acid amide hydrochloride (Ala. NH2. HCl).

Comparative example 2

A synthetic method of amino acid amide hydrochloride comprises the following steps:

s1, adding 500g of ethanol and 89g of alanine (Ala) into a 1L flask, dropwise adding 130g of thionyl chloride, stirring at normal temperature for 5 hours, concentrating, adding 875g of 20% sodium carbonate solution for neutralization, introducing 50g of ammonia gas into the product solution for ammonolysis, concentrating, crystallizing, and filtering to obtain amino acid amide (Ala. NH2);

s2 and amino acid amide (Ala. NH2) were dissolved in 500ml of ethanol, and then 40g of hydrogen chloride gas was introduced, followed by concentration and crystallization to obtain alanine amide hydrochloride (Ala. NH2. HCl).

Comparative example 3

A method for synthesizing amino acid amide hydrochloride without inorganic ammonium salt comprises the following steps:

s1, adding 1L of pure water, 89g of alanine (Ala), 220g of sodium carbonate, 250g of ethanol and 283g of di-tert-butyl dicarbonate into a 2L flask, stirring at normal temperature for 5 hours, dropwise adding 500g of 30% hydrochloric acid, stirring for crystallization for 2 hours, and filtering to collect solid tert-butyloxycarbonyl-alanine (Boc-Ala);

s2, adding 500g of ethyl acetate, 95g of tert-butoxycarbonyl-alanine (Boc-Ala), 218g of di-tert-butyl dicarbonate, 50g of N-methylmorpholine and 50g of ammonium bicarbonate into a 1L flask, stirring at normal temperature for 5 hours, washing the ethyl acetate with water, adding 50g of anhydrous sodium sulfate into the ethyl acetate, drying, concentrating and crystallizing to obtain tert-butoxycarbonyl-alanine amide (Boc-Ala. NH2);

s3, a 1L flask was taken, and 473g of ethyl acetate/hydrogen chloride solution and 94g of tert-butoxycarbonyl-alanine amide (Boc-Ala. NH2) were added to the flask, and the mixture was reacted with stirring for 3 hours to precipitate crystals and obtain alanine amide hydrochloride (Ala. NH2. HCl).

The data of the tests of the products obtained in examples 1 to 3 and comparative examples 1 to 3 are shown in Table 1:

TABLE 1 data on the examination of the products of examples 1-3 and comparative examples 1-3

Item	The content of chloride ion is w%	Waste liquid yield mL/g
			Example 1	99.8	52
Example 2	99.7	54
			Example 3	99.9	57
Comparative example 1	110	75
			Comparative example 2	105	90
Comparative example 3	100.8	60
			Standard requirements	98.5-102.5	-

Note: 1. the content of chloride ions is detected by adopting a titration method of Chinese pharmacopoeia;

2. chloride ion content w% — actual chloride ion content/theoretical chloride ion content × 100%; the theoretical chloride ion content is calculated assuming a product purity of 100%.

As can be seen from Table 1, the chloride ion contents of examples 1 to 3 did not exceed 100%, while those of comparative examples 1 and 2 exceeded 100%, thus demonstrating that the product obtained by the present invention was high in purity, inorganic ammonium salt was completely removed and removal effect was excellent, while comparative examples 1 and 2 necessarily contained inorganic ammonium salt, demonstrating that alanine (Ala) was added in an alcohol solvent, thionyl chloride was added dropwise to form alanine ester hydrochloride (Ala. OEt. HCl or Ala. OMe. HCl), alkali-neutralized to obtain alanine ester (Ala.OEt or Ala.OMe), and then ammonia gas was introduced for aminolysis to obtain alanine amide hydrochloride (Ala.NH. NH. HCl)₂HCl), alanine amide hydrochloride (ala. nh)₂HCl) to 5% or more, and difficult to separate and purify. Meanwhile, the yield of the waste liquid can be obtained by comparing the yield of the waste liquid, the yield of the waste liquid is obviously lower than that of the waste liquid obtained by comparing examples 1 and 2, and the technical advantages are obvious. Further, as can be seen by comparing examples 1 to 3 with comparative example 3, when an excessive amount of di-tert-butyl dicarbonate is added in S2, the removal effect of the inorganic ammonium salt is significantly adversely affected, and the technical effect of removing the inorganic ammonium salt is not achieved, so that the amount of di-tert-butyl dicarbonate used in step S2 is important and the claimed technical effect is achieved within the range indicated by the present invention.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.