JP2003321441A - Method for purifying homocystine substituted with n,n'- diacyl group - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a purifying method for producing an N,N'-diacyl-substituted homocystine in high purity by removing impurities such as monoacyl compounds and ester compounds. <P>SOLUTION: A crude N,N'-diacyl-substituted homocystine containing an N,N'-diacyl-substituted homocystine wherein the carboxyl group is esterified and/or an N,N'-diacyl-substituted homocystine wherein one of the amide bonds is hydrolyzed is purified by dissolving the crude compound in a water-immiscible organic solvent, mixing the solution with water, separating the mixture into an aqueous phase and an organic phase, and mixing the organic phase with a poor solvent to effect the crystallization of the N,N'-diacyl-substituted homocystine. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to highly pure 4,4′-
The present invention relates to a method for producing N, N'-diacyl group-substituted homocystine such as dithiobis [2-trifluoroacetamino] butyric acid, and more specifically, N, N'-bistrifluoroacetyl for trifluoroacetylating both amino groups of homocystine. The present invention relates to a method for producing homocystine, which comprises removing a by-product ester or a hydrolyzate to obtain highly pure crystals. Homocystin derivatives are used in medicine, pesticides,
It is considered to be a useful substance as a synthetic intermediate such as a feed additive.

[0002]

2. Description of the Related Art A method for introducing an N-trifluoroacetyl group for the purpose of protecting an amino group of an amino acid or a peptide is known, and for example, Journal of Organ.
icChemistry, Vol. 44, page 2805 (1
(1997), ethyl trifluoroacetate was allowed to act in the presence of triethylamine to produce N of various amino acids and peptides.
-A method for obtaining trifluoroacetyl derivatives is described.

On the other hand, homocystine, that is, 4,4 '
As a method of producing -dithiobis (2-aminobutyric acid), a method of demethylating dimerization of methionine in a strong acid, a method of oxidizing dimerization of homocysteine obtained by birch reduction of methionine, and the like are known. For example, Journ
al of Biological Chemistr
Y, Vol. 99, p. 134 (1932-33) describes a method for obtaining homocystine by heating methionine in sulfuric acid. Further, JP-A-10-204055 describes a method for producing homocystine by heating methionine with sulfuric acid and hydrogen halide. Further, JP-A-59-176248 and JP-A-59-176249 disclose a method for producing homocystine by oxidizing disodium salt of homocysteine with molecular oxygen in the presence of hydrogen peroxide solution or heavy metal ions. Is listed. Further, as a method for purifying homocystine, a method of treating impurities such as polysulfide with a base is disclosed in JP-A-11-2465.
No. 17 publication.

[0004]

However, the N, N'-diacyl derivative obtained by the diacylation reaction using homocystine obtained by the above-mentioned production method is not hydrolyzed or esterified during isolation and purification. It was difficult to obtain a high-purity homocystine derivative on an industrial scale because it could easily proceed. Therefore, an object of the present invention is to provide a purification method for removing impurities such as monoacyl compounds and ester compounds to obtain highly pure N, N'-diacyl group-substituted homocystine.

[0005]

Means for Solving the Problems As a result of intensive investigations, the present inventors have found that N, N′-diacyl group-substituted homocystine containing impurities in which a carboxyl group is esterified or impurities in which an amide bond is hydrolyzed is mixed with water. Not dissolved in an organic solvent,
The present inventors have found that the above problems can be solved by adding an organic phase separated by adding water to a poor solvent to cause crystallization, and have reached the present invention.

That is, the gist of the present invention is a compound in which the carboxyl group of N, N'-diacyl group-substituted homocystine is esterified and / or a compound in which one amide bond of N, N'-diacyl group-substituted homocystine is hydrolyzed. N, N'-diacyl group-substituted homocystine containing is dissolved in an organic solvent immiscible with water, mixed with water, and then the separated organic phase is mixed with a poor solvent to prepare an N, N'-diacyl group. N, N'-characterized by crystallization of substituted homocystine
It exists in a method for purifying homocystine substituted with a diacyl group.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. (Production of N, N′-diacyl group-substituted homocystine) The N, N′-diacyl group-substituted homocystine to be purified in the present invention may be, for example, a D-form, L-form, or D, L-form of homocysteine. It is obtained by converting. The raw material homocystine is one in which the carboxyl group is not protected and is soluble in a basic solution, and further, polysulfides and thiosulfinate and thiosulfonate,
It is preferable that the content of oxidizing impurities such as sulfinyl sulfone and disulfone is less than 1% by weight.

The acylating agent can be appropriately selected according to the kind of the target acyl group, and examples thereof include haloalkylcarbonylating agents such as haloacetylating agents. As the haloacetylating agent, trifluoroacetylating agents such as anhydrous trifluoroacetic acid, methyl trifluoroacetate, ethyl trifluoroacetate, ethyl trifluorothioacetate, phenyl trifluoroacetate, trichlorotrifluoroacetone are preferable. Easy availability and low price,
Ethyl trifluoroacetate is preferred because it is easy to handle. The amount of acylating agent used is theoretically required to be at least twice the molar amount of homocystine.
It is preferable that the molar amount is ˜3 times. It is particularly preferable to use a 2- to 2.5-fold molar amount.

The N-acylation reaction proceeds under basic conditions, but the base used at that time is a strongly basic one that can completely dissociate the carboxyl group of homocystine, and the amide bond formed A base having no hydroxyl group is preferable in order to suppress the hydrolysis of For example,
Examples thereof include tertiary amines such as triethylamine; alkali metal alcoholates such as sodium metoside, sodium ethoxide and potassium methoxide; alkali metal hydrides such as sodium hydride and potassium hydride. Among these, it is most preferable to use a methanol solution of sodium methoxide because it is easily available, inexpensive, and easy to handle. The amount of the base used is preferably an equimolar amount with the trifluoroacetylating agent or a slight excess amount.

As the reaction solvent, a polar organic solvent capable of dissolving the produced diacyl compound can be used. Examples of such a solvent include alcohols such as methanol, ethanol and 2-propanol, diethyl ether and methyl t.
Examples include ethers such as -butyl ether and tetrahydrofuran, ketones such as acetone and methyl ethyl ketone, esters such as ethyl acetate and n-butyl acetate, and amides such as N, N-dimethylformamide and N-methylpyrrolidone. However, a substance that can be removed in the aqueous phase in the extraction step is preferable. Further, the water content is preferably 500 μg / g or less in order to suppress the hydrolysis of the generated amide group.

The N, N'-diacyl group-substituted homocystine obtained by the above method is subjected to acid precipitation and then the reaction solvent is removed to isolate crude crystals. N below pH 5
The two carboxyl groups of N'-diacylated homocystine cease to dissociate. The acid used for the acid precipitation is an acid having a pH of 4 or less which can adjust the pH of the above basic reaction solution to pH 1 to pH 5, but it is preferably an acid which does not react with the precipitated homocystine derivative to generate impurities. Examples of such an acid include hydrohalic acid such as hydrochloric acid, hydrobromic acid and hydroiodic acid, oxo acids such as sulfuric acid, nitric acid and carbon dioxide, and organic acids such as acetic acid, monochloroacetic acid and oxalic acid. It is preferable to use hydrohalic acid that does not cause side reactions such as oxidation and esterification. Above all, it is most preferable to use hydrochloric acid because it is easily available and inexpensive. The formation of the ester is highly pH-dependent, and a few% of the ester is produced in a pH range of 1 or less. In order to avoid generation of these impurities, it is necessary to keep the pH after acid precipitation at 1 to 5, more preferably 2 to 4.

When the reaction solvent after acid precipitation is concentrated, N, N '
A crude product of a diacyl-substituted homocystine is obtained. The crude product thus obtained is an inorganic salt as an impurity, a compound in which a carboxyl group is esterified (hereinafter, may be referred to as “ester form” and / or one amide bond of N, N′-diacyl group-substituted homocystine). Hydrolyzed compound (hereinafter sometimes referred to as "monoacyl compound")
including. In particular, the ester form and monoacyl form are
It is a compound that is particularly difficult to remove because it has a chemical structure similar to that of N′-diacyl group-substituted homocystine, but these compounds are usually contained in a total amount of about 2 to 8%.

(Purification of N, N'-diacyl group-substituted homocystine) In the purification method of the present invention, N, N'-diacyl group-substituted homocystine is first dissolved in an organic solvent immiscible with water, and then Water is added to remove inorganic substances and hydrophilic hydrolysates in the aqueous phase. An organic solvent that is immiscible with water is
An organic solvent that does not become uniform but becomes separated when mixed with water at a weight ratio of 2: 1 at room temperature. Examples of such an organic solvent include ethers such as diethyl ether and methyl t-butyl ether; ketones such as methyl isobutyl ketone; esters such as ethyl acetate and n-butyl acetate. From the viewpoint of solubility of N, N′-diacylhomocystin, acetic acid ester is preferable, and acetic acid n-
Butyl is preferred.

The crude N, N'-diacylated homocystine containing impurities is stirred and dissolved in the above water-immiscible organic solvent. The shape of the stirring blade and the stirring method are not particularly limited, but it is desirable that the stirring blade be uniformly dissolved within 1 hour after the addition of the organic solvent. The amount of homocystine added to the organic solvent is arbitrary, but usually 2 to 10 times the weight of the organic solvent is used relative to the crude N, N′-diacylhomocystin. Preferably, 3 to 5 times the weight is used. In order to suppress the formation of impurities, the melting temperature is preferably 40 ° C. or lower, more preferably 30 ° C. or lower. When the temperature exceeds 40 ° C, the production of impurities increases rapidly,
It can be handled for several tens of hours at 30 ° C or lower.

Water is added to the solution of the N, N'-diacyl group-substituted homocystine obtained as described above to extract inorganic salts into the aqueous phase. The pH of the aqueous phase at this time is 1 as in acid precipitation.
Adjust to ~ 5. When the pH is low, the transesterification reaction of the carboxyl group proceeds, and when it is high, the hydrolysis of the amide bond proceeds. Saturated saline can also be used in order to prevent N, N'-diacyl homocystine from escaping into the aqueous phase. When the hydrophilic impurities generated by hydrolysis of the amide bond cannot be completely removed by one extraction, the above-mentioned extraction operation is repeated to remove the hydrophilic impurities.
% Or less.

The solution of N, N'-diacyl group-substituted homocystine separated from the aqueous phase is concentrated, inorganic salts are removed by filtration, and the mixture is added to a poor solvent with stirring to give needle-shaped white crystals. can get. In the present invention, the poor solvent means a solvent having a lower solubility of N, N′-diacyl group-substituted homocystine than the above-mentioned water-immiscible organic solvent. The poor solvent is preferably an inert solvent which hardly dissolves N, N′-diacyl group-substituted homocystine and does not cause side reactions such as transesterification. Examples of such a solvent include saturated hydrocarbons such as n-hexane, n-heptane, and isooctane. Among them, n-heptane is preferable because it is easy to handle and is easily available.

The amount of the poor solvent used is preferably 2 to 10 times by weight, more preferably 3 to 5 times by weight that of the concentrated solution. If the amount of the poor solvent is small, it does not crystallize and separates into two phases. The temperature for crystallization is 40 ° C to 50 ° C.
Is preferred. When the temperature is low, it does not crystallize and separates into two phases. If it is too high, the transesterification reaction proceeds to produce impurities. When the transesterified impurities cannot be completely removed by one crystallization, the transesterified impurities can be reduced to 1% or less by repeating the above crystallization operation.

The purified crystals of N, N'-diacylhomocystin thus obtained are filtered by a conventional method and dried under reduced pressure to give an HPLC area purity of more than 98%.
Preferably, a highly pure N, N′-diacyl group-substituted homocystine crystal of over 99% can be obtained. Furthermore, the purity can be increased by repeating the purification operation of the present invention. The dried temperature of the purified N, N'-diacyl group-substituted homocystine preferably does not exceed 50 ° C, more preferably 40 ° C.

[0019]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded. The analytical measurements in the examples were carried out as follows. Measurement of chemical purity of N, N'-diacylhomocystin: Lichrosp manufactured by Alltech as a separation column
The measurement was performed using a high performance liquid chromatograph (HPLC) equipped with hereNH2 (inner diameter 4.5 mm, length 150 mm). As a sample, 1 mg of N, N'-diacylhomocystin crystals was used as an eluent (6 ammonium dihydrogen phosphate was added as an eluent).
Analysis was carried out by injecting 10 μl of a solution diluted to about 1 ml with 22 vol% hydrous acetonitrile dissolved at mmol / L. Separation is carried out at a flow rate of 1.0 ml / min.
The area of the absorption peak was measured with a UV detector of nm. The results are shown as an area percentage based on the absorption peak of each component,
It is written as "A%".

Measurement of optical purity of N, N'-diacylhomocystin: Chir manufactured by Daicel Chemical as a separation column
The measurement was performed using a high performance liquid chromatograph (HPLC) equipped with apakAD (inner diameter 4.5 mm, length 250 mm). As a sample, 1 mg of N, N'-diacylhomocystin crystals was used as an eluent (n-hexane / 2-propanol / trifluoroacetic acid = 90/10 / 0.1 volume) to prepare 1
An analysis was performed by injecting 10 μl of a solution diluted to about ml. Separation is performed at a flow rate of 1.0 ml / min, and U of 220 nm
The area of the absorption peak was measured with a V detector. The results are shown as an area percentage based on the absorption peak of each component and expressed as "A%".

Measurement of pH: Measurement was carried out using a portable pH meter D-12 type manufactured by Horiba Ltd. pH 4,
After calibrating with the calibration solutions of Nos. 7 and 9, the pH of the reaction solution of the amidated homocystin derivative and the acid precipitation suspension was measured. Measurement of water content: Mitsubishi Chemical Co., Ltd. CA-0
Coulometric moisture titration was carried out using No. 7 with Aquamicron AX as the anode and Aquamicron CXU as the cathode.

[Example 1] A glass separable flask having a capacity of 200 ml equipped with a temperature controller using an external cylinder circulating water, a reflux cooling pipe and a nitrogen introducing pipe, a PTFE meniscus stirring blade, a pH electrode and a thermometer. L-homocystin crystals 13.
28% sodium methoxide in methanol 2 while stirring 4 g and 26.8 g of methanol at 15 ° C.
1.2 g was added, then ethyl trifluoroacetate 1
5.3 g was added. When the temperature was raised to 25 ° C. and stirring was continued for 5 hours, the suspended reaction solution became transparent. When the reaction solution at this time was analyzed, the HPLC chemical purity was 99.2 A%.
And the water content was less than 100 μg / g. After adjusting the pH to 6 by adding 35% hydrochloric acid to the above reaction solution, add 76 g of demineralized water and then add 35% hydrochloric acid again to adjust the pH.
Adjusted to 2. When stirring was continued for 20 hours at 40 ° C, the hydrolyzed monotrifluoroacetyl derivative 0.9A
% And monomethyl ester 4.8A% were detected, HP
LC chemical purity was 94.3 A%. After adding 211 g of n-butyl acetate for extraction, liquid separation and concentration under reduced pressure gave N, N'-bis (trifluoroacetyl) -L-
Crude homocystine crystals were obtained. Temperature controller with external cylinder circulating water, reflux condenser, PTFE meniscus stirring blade, p
In a glass separable flask with a capacity of 1200 ml equipped with an H electrode and a thermometer, 1.6 A% of methyl ester compound, n
-Chemical purity 93.7A containing 3.7 A% of butyl ester and 0.8 A% of N-mono (trifluoroacetyl)
% N, N′-bis (trifluoroacetyl) -L-homocystine crude crystals 77.0 g with n-butyl acetate 283.
0 g was added and dissolved at 30 ° C. with stirring. 15 ° C
After cooling to 400 g, deionized water (400 g) and concentrated hydrochloric acid (1.5 g) were added to the aqueous phase, and the pH was confirmed.

After the stationary separation, the aqueous phase was removed, and 200 g of saturated saline was newly added for extraction to perform stationary separation. Then, the temperature was raised to 40 ° C. and acetic acid n- was added under reduced pressure of 4 kPa.
197.4 g of butyl was concentrated. Concentrated liquid with a diameter of 55 mm
The inorganic salt is removed by suction filtration using 5C filter paper of
Was added to 415.6 g of n-heptane and crystallized. The crystallized crystals were separated by filtration using 1 L of La Boscentre. The obtained crystals were washed with 110 g of n-heptane.

The wet weight of the obtained white crystals is 134.1.
As a result of HPLC analysis, 0.03 A% of N-mono (trifluoroacetyl) compound and 0.9 of methyl ester compound were obtained.
A%, n-butyl ester 2.3A% was detected, HP
LC chemical purity was 96.6 A%. After redissolving 134 g of the above wet crystals in 150 g of n-butyl acetate,
Crystallization was performed by adding to 500 g of n-heptane at 50 ° C. The crystallized crystals were separated by filtration using 1 L of La Boscentre, and the obtained crystals were washed with 110 g of n-heptane and dried under reduced pressure at 50 ° C. The dry weight of the obtained white crystals was 48.
The recovery rate was 63.2% at 7 g. A monotrifluoroacetyl derivative of 0.03 A% and an ester derivative of 1.5 A% were detected, and the HPLC chemical purity was 98.4 A%.

Example 2 The same raw material as in Example 1 77.0
g, and dissolved in 283 g of n-butyl acetate with stirring at 30 ° C. Saturated saline solution 20 cooled to 15 ℃
The extraction operation of adding 0 g and statically separating liquid was repeated 4 times, and then the extraction operation of adding 400 g of demineralized water to an aqueous phase containing 2.7 g of concentrated hydrochloric acid and performing static liquid separation was repeated 4 times. The obtained organic phase was concentrated under reduced pressure at 40 ° C. to recover 178.7 g of n-butyl acetate. The concentrated liquid was suction-filtered using a 5C filter paper having a diameter of 55 mm to remove inorganic salts, and added to 491.0 g of n-heptane at 50 ° C. for crystallization.
The crystallized crystals were suction-filtered using a 5C filter paper having a diameter of 70 mm, and the obtained crystals were washed with 110 g of n-heptane.

The wet weight of the obtained white crystals was 106.2.
As a result of HPLC analysis, 0.03 A% of monotrifluoroacetyl compound and 0.8 A% of methyl ester compound, n
-Butyl ester form 2.4A% was detected, and HPLC chemical purity was 96.6A%. Wet crystal 106 above
g was redissolved in 150 g of n-butyl acetate, and then added and crystallized in 500 g of n-heptane at 50 ° C. The crystallized crystals were suction-filtered using a 5C filter paper having a diameter of 70 mm, and the obtained crystals were washed with 110 g of n-heptane and then at 50 ° C.
It was dried under reduced pressure.

The dry weight of the obtained white crystals was 52.2 g.
The recovery rate was 67.8%. A monotrifluoroacetyl derivative of 0.04 A% and an ester derivative of 1.4 A% were detected, and the HPLC chemical purity was 98.5 A%.

[0028]

Industrial Applicability According to the present invention, it is possible to provide a purification method for removing impurities such as monoacyl compounds and ester compounds to obtain highly pure N, N'-diacyl group-substituted homocystine.

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Claims (9)

[Claims] 1. An N, N containing a compound in which a carboxyl group of N, N'-diacyl group-substituted homocystine is esterified and / or a compound in which one amide bond of N, N'-diacyl group-substituted homocystine is hydrolyzed. Dissolving the'-diacyl group-substituted homocystine in an organic solvent immiscible with water,
A method for purifying N, N'-diacyl group-substituted homocystine, which comprises mixing this with water and then separating the separated organic phase with a poor solvent to crystallize N, N'-diacyl group-substituted homocystine. 2. The method for purifying N, N′-diacyl group-substituted homocystine according to claim 1, wherein the acyl group is a trifluoroacetyl group. 3. The organic solvent immiscible with water is an acetic acid ester, and the poor solvent is a saturated hydrocarbon.
The method for purifying N, N′-diacyl group-substituted homocystine according to 1. 4. The N, according to claim 3, wherein the acetic acid ester is butyl acetate and the saturated hydrocarbon is n-heptane.
A method for purifying N'-diacyl group-substituted homocystine. 5. The content of acetic acid ester in the crystallization mother liquor is 10.
The method for purifying N, N′-diacyl group-substituted homocystine according to claim 3 or 4, characterized in that it is about 25% by weight. 6. The compound containing 2% by weight or more of a compound in which the carboxyl group of N, N-diacyl group-substituted homocystine is esterified and / or a compound in which the amide bond of N, N-diacyl group-substituted homocystine is hydrolyzed. 6. A method for purifying N, N′-diacyl group-substituted homocystine according to any one of to 5 above. 7. An N, N′-diacyl group-substituted homocystine having a purity of more than 99%, which is obtained by the purification method according to any one of claims 1 to 5. 8. The purity obtained by repeating the purification method according to claim 1 twice or more, is 99.
%, N, N′-diacyl group-substituted homocystine. 9. A process for producing N, N′-diacyl group-substituted homocystine, which comprises the following steps. 1) step of acylating homocystine under basic conditions to produce N, N'-diacyl group-substituted homocystine, 2) dissolving N, N'-diacyl group-substituted homocystine in an organic solvent immiscible with water,
A step of mixing this with water and then mixing the separated organic phase with a poor solvent to crystallize N, N′-diacyl group-substituted homocystine.