JPH0772172B2 - Method for separating and purifying L-proline - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] Conventionally, L-proline has been used as a comprehensive amino acid formulation in infusions and the like, and has been used for nutritional supplementation of patients with digestive system diseases and the like. In recent years, it has been found that N-mercaptoacyl-L-proline has a remarkable effect as an antihypertensive agent, and reduction of a ketone using an L-proline derivative as an asymmetric catalyst to an optically active alcohol is highly effective. Reported selective asymmetric synthesis reaction (T.
Mukaiyama, Chem. Lett. 1984 (12) 2071) Its utility value is increasing not only in medicine but also in the field of synthetic chemical industry.

[Prior Art] L-proline is a natural amino acid and is contained in many proteins, but its content is particularly high in gelatin, and its content reaches about 15%. Therefore, it can be industrially produced at a low cost by hydrolyzing gelatin, which can be obtained in large quantities at a low cost, and then subjecting it to a separation and purification operation.

As a method for separating L-proline, an ester fractionation method using a protein hydrolyzate (Fischer, E., Z. Physiol. Chem., 3
3 , 151 (1901)) and copper salt method (Klabunde, HK, J. Biol. Che
m., 90 , 293 (1931)), butanol extraction method (Dakin, HD,
Z.Phisiol.Chem., 44 , 499 (1920)) and a rhodanyl salt method for selectively precipitating only L-proline (Bergmann,
M., J.Biol.Chem., 110 .471 ( 1935)) but the like are known, these methods have low purification purity, such that it uses a heavy metal such as copper or chromium, carried on an industrial scale Is difficult.

On the other hand, a gelatin hydrolyzate was treated with nitrous acid to separate other primary L-amino acids as oxyacids, and then obtained from a mixture of L-proline and L-oxyproline to give a picrate (Town, BW, Biol. Chem. .J. (London), 30 , 18,37 (1
936)) or cadmium chloride (Sprer, H., et al,
A method for separating and purifying only L-proline has been found as Z. Physiol. Chem., 187 , 84 (1930)).

However, these also require the use of explosive picric acid and the heavy metal cadmium.
It was not sufficient as an industrial separation and purification method for proline.

[Object of the Invention] An object of the present invention is to provide a method for separating and purifying highly pure L-proline on an inexpensive and industrial scale.

[History of the Invention] As a result of earnest studies in line with the above object, the present inventors have obtained the following findings.

First, L-proline is the only amino acid that is easily soluble in alcohol and has a different property from other L-amino acids including L-oxyproline. Conceivable.

However, since L-oxyproline dissolves in alcohol in the presence of L-proline, it is difficult to separate and purify L-proline with high purity. Also the general L
Since the solubility of L-oxyproline is smaller than that of L-proline in the recrystallization method from water, which is performed for amino acids, it cannot be removed if the amount of L-oxyproline mixed is large, and separation and purification It is difficult.

Therefore, the present inventors have considered various ways of separating and purifying with a derivative of L-proline and conducted various studies. As a result, N-acetyl-L-proline, which is the most inexpensive N-acyl derivative, has good crystallinity and its solubility in water was examined. As a result, L-oxyproline containing other L-oxyprolines was found. -Contrary to the tendency of the N-acetyl form of amino acid, N
The solubility of -acetyl-L-proline is significantly reduced compared to that of L-proline (reduced to 1/20 at 25 ° C). Therefore, for L-proline and L-oxyproline, the free form and N-acetyl It was found that the solubility difference was reversed in the body (see Fig. 1).

Based on this fact, when separation and purification of N-acetyl-L-proline from a mixture of N-acetyl compounds obtained by acetylating a mixture of L-proline and L-oxyproline obtained from a hydrolyzate of gelatin was examined, It was found that N-acetyl-L-proline can be separated and purified with high purity without contaminating N-acetyl-L-oxyproline. Further, N-acetyl-L-proline is easily deacetylated to give L-proline in a high yield.
The present invention has been completed for the purpose of being able to carry out L-proline at a low cost and on an industrial scale.

[Structure of the Invention] That is, the present invention relates to L obtained from a gelatin hydrolysis solution.
-A method for separating and purifying L-proline, which comprises acetylating a mixture of proline and L-oxyproline, crystallizing the produced N-acetyl-L-proline from water and isolating it with high purity.

The L-proline and L-oxyproline mixture used in the present invention was prepared by hydrolyzing gelatin with a mineral acid aqueous solution and treating with nitrous acid according to a conventional method, and then removing coexisting oxyacid and a part of L-oxyproline. It is obtained later, and although it varies somewhat depending on the gelatin used, it usually contains L-proline: L-osyproline = 10: 2 (weight composition ratio).

N-acetylation was carried out by using the above mixture in Schott with acetic anhydride.
The reaction mixture is neutralized by the en-Baumann method, and then desalted to obtain N-acetyl-L-proline and L-acetyl-L.
-Oxyproline mixture is obtained.

Separation and purification of N-acetyl-L-proline is performed by dissolving the N-acetyl compound mixture in water and crystallizing only N-acetyl-L-proline. From the solubility curve obtained from FIG. 1, it is necessary to carry out so that coexisting N-acetyl-L-oxyproline does not crystallize. When the amount of water used is small, the amount of crystallization increases but the purity decreases. On the contrary, when the amount is large, the amount of crystallization is small and the separation / purification operation is not efficient. Therefore, it is usually dissolved in water in an amount of 2 to 3 times the N-acetyl-L-proline content at room temperature. Is cooled to 0 to 5 ° C., and the crystallized crystals are collected by filtration to obtain high-purity N-acetyl-L-proline in a good yield.

Further, from N-acetyl-L-proline, L-proline can be obtained in high yield by deacetylation by boiling with an aqueous solution of mineral acid.

[Examples] The present invention will be described in more detail with reference to the following examples.

Preparation of L-proline / L-oxyproline mixture 50 kg of gelatin was suspended in 105 l of 8N hydrochloric acid and refluxed for 6 hours for hydrolysis. The reaction solution was concentrated under reduced pressure, the hydrochloric acid content was distilled off, water was added to make 200 l, 2 kg of activated carbon was added, and decolorization filtration was performed. 90 l of 40% sodium nitrite aqueous solution is added dropwise to the filtrate at 45 ° C or lower, and then 1 at 55-60 ° C.
Reacted for hours. The reaction mixture was concentrated to a syrup under reduced pressure, 6N hydrochloric acid (50 L) was added, and the mixture was refluxed for 3 hours. After cooling this solution, 20 l of ether was added and the mixture was stirred to extract oxyacid. Then again concentrate under reduced pressure to a syrup, add 150 l of water and add amber light.
Hydrochloric acid was adsorbed and removed by passing through a column filled with IR-4B.
1 kg of activated carbon was added to the column effluent, the mixture was decolorized and filtered, and concentrated to a syrup under reduced pressure. Methanol in this concentrated solution
50 l was added and the mixture was stirred and cooled to 5 ° C. After filtering off a part of the precipitated L-oxyproline, the filtrate was concentrated under reduced pressure to obtain 4.70 kg of a syrup-like mixture of L-proline and L-oxyproline (from an amino acid automatic analyzer, 3.60 kg of L-proline). , L-oxyproline 0.71 kg was confirmed).

Example 1 1.0 kg of a mixture of L-proline and L-oxyproline (L-
Proline 766 g and L-oxyproline 151 g were dissolved in 6 l of water, and NaOH 624 g was added and dissolved. Add this solution to 5
After cooling to ~ 10 ° C, add 820 ml of acetic anhydride to 1 while stirring.
The mixture was dropped for a period of time and then reacted at room temperature for 2 hours. The reaction solution was adjusted to pH 2.5 with concentrated hydrochloric acid and then concentrated under reduced pressure, 4 l of methanol was added, the precipitated sodium chloride was collected by filtration, and the filtrate was concentrated to a syrup. 2 l of water was added to this and stirred to form a uniform solution. Further, when gradually cooled to 5 ° C. with gentle stirring, crystals were precipitated. After standing overnight, the crystals are filtered and dried to N-
721 g of acetyl-L-proline was obtained. The yield was 69%. The melting point (mp) was 116 to 117 ° C., and the specific rotation was ▲ [α] ²⁰ _D ▼ = −115.60 ° (C = 2, H ₂ O). The standard N-acetyl-L-proline has an mp of 118.
At ° C, the specific rotation is ▲ [α] ²⁰ _D ▼ = -116.50 °.

This is confirmed to be one spot by thin layer chromatography (cellulose; n-butanol: acetic acid: water = 2: 1: 1).

Next, 700 g of N-acetyl-L-proline was dissolved in 30 l of water, 600 ml of concentrated hydrochloric acid was added, and the mixture was refluxed for 3 hours. After cooling this solution, the pH was adjusted to 6.3 with 30% aqueous NaOH. After removing sodium chloride from this solution with an electrodialyzer, water was distilled off under reduced pressure, 500 ml of ethanol was added, the mixture was cooled to 5 ° C. with stirring, and the precipitated crystals were collected by filtration and dried to give L-proline. 359 g were obtained. The reaction yield was 70%, and the specific rotation was ▲ [α] ²⁵ _D ▼ = -85.18 °
(C = 2, H ₂ O). The specific optical rotation of standard L-proline is [α] ²⁵ _D ▼ = -85.0 ° (C = 2, H ₂ O).

Example 2 4.0 kg of a mixture of L-proline and L-oxyproline was subjected to acetylation treatment with 2.50 kg of NaOH and 3.28 l of acetic anhydride according to Example 1, and then 9 l of water was added to the resulting syrupy N-acetyl compound mixture. The mixture was added and stirred to form a uniform solution. Further, when gradually cooled to 3 ° C. with gentle stirring, crystals were precipitated. After allowing to stand overnight, the crystals were collected by filtration and dried to obtain 2.72 kg of N-acetyl-L-proline. The yield was 65%, the melting point (mp) was 117 to 118 ° C., and the specific rotation was ▲ [α] ²⁰ _D ▼ = −114.30 (C = 2, H ₂ O).

When this product was measured by thin layer chromatography in the same manner as in Example 1, it was confirmed to be one spot.

Next, 2.70 kg of N-acetyl-L-proline was deacetylated according to Example 1 to give 1.43 kg of L-proline.
Got The yield is 72% and the specific rotation is ▲ [α] ²⁵
_D ▼ = −85.0 ° (C = 2, H ₂ O).

[Effects of the Invention] As described above, the mixture of L-proline and L-oxyproline obtained from a hydrolyzate of gelatin was mixed with N-
The method for separating and purifying acetylated L-proline of the present invention is as follows:
It became possible to separate and purify high-purity L-proline at a low cost and on an industrial scale.

[Brief description of drawings]

FIG. 1 shows (1) L-proline, (2) L-oxyproline, (3) N-acetyl-L-proline, (4) N-.
It is a graph showing the relationship between the temperature and the amount of dissolution (g / 100 g of water) of the free form of acetyl-L-oxyproline and the N-acetyl form.

Claims (1)

[Claims] 1. A method of acetylating a mixture of L-proline and L-oxyproline obtained from a gelatin hydrolyzed solution, and crystallizing the produced N-acetyl-L-proline from water to isolate with high purity. A method for separating and purifying L-proline.