JP2023134069A - Separating agent, separating method, and manufacturing method of compound - Google Patents

Abstract

To provide a separating agent capable of efficiently obtaining high-purity dipeptides and oxidized products thereof on an industrial scale, and a separating method.SOLUTION: The separating agent is used for separating at least one compound selected from a group consisting of dipeptide an oxidized form thereof in which polyethyleneimine is immobilized on porous particles. The separating method includes: a liquid chromatography step to separate the compound in the solution by loading a solution containing the compound onto the separating agent and by flowing a solvent (A) through the separating agent; an adsorption step of allowing the compound to be adsorbed to the separating agent by allowing a solution containing the compound and a solvent (B) to come into contact with the separating agent; and subsequently, an elution step of eluting by using a solvent (C) to remove the compound from the separating agent.SELECTED DRAWING: Figure 1

Description

The present invention relates to a separating agent used for separating at least one compound selected from the group consisting of dipeptides and oxidized products thereof, a separating method, and a method for producing the compound.

Dipeptides represented by imidazole dipeptides such as carnosine (β-alanyl-L-histidine), anserine (β-alanyl-3-methyl-L-histidine), and homocarnosine (γ-aminobutyryl-L-histidine) are antioxidants. It has various effects such as anti-fatigue effects and anti-fatigue effects, and as a useful peptide, it has high value as a medicine and food additive, and the demand for it has increased in recent years. In addition, as reported in Non-Patent Document 1 through recent research, it has been revealed that 2-oxo-imidazole dipeptide has tens of thousands of times more antioxidant activity than imidazole dipeptide, and it can be used as a new drug or food additive. It is expected.

Methods for separating and purifying these dipeptides and oxidized dipeptides include ion exchange separation using ion exchange resins, reversed phase chromatography separation using synthetic adsorbents, alkyl group-bonded silica gel separation agents, etc. However, the separability was not always sufficient.
In other words, dipeptides with similar amino acid residues and their oxidized products are difficult to separate and purify by ion exchange or reversed-phase chromatography, making it difficult to obtain high-purity products industrially. It is.

Patent Document 1 describes separation using a special separation column (Scherzo SS-C18) having "octadecyl group + anion exchange group + cation exchange group"; Separation is carried out using a column (intrada amino acid column), but in order to improve separation, it is necessary to use a separation agent with a small particle size of 3 μm and a mass spectrum detector. It was impossible to use an industrial separation and purification method.

International Publication No. 2020/085459

J. Biol. Chem. (2019), 294(4), p1279-1289.

The present invention has been made in view of the problems of the prior art described above, and an object of the present invention is to provide a separation agent and a separation method for efficiently obtaining high-purity dipeptides and their oxidized products on an industrial scale. It is in. Another object of the present invention is to provide a production method for efficiently obtaining a highly purified dipeptide and its oxidized product on an industrial scale.

As mentioned above, conventionally, there has been no known method for efficiently obtaining highly purified dipeptides and their oxidized products on an industrial scale.
As a result of extensive studies, the present inventor has found that a separation agent in which polyethyleneimine is immobilized on porous particles exhibits excellent adsorption properties for dipeptides and their oxidized forms, and that they can efficiently remove dipeptides and their oxidized forms. The present invention was based on the discovery that it is possible to separate

That is, the gist of the present invention is as follows.
[1] A separating agent used for separating at least one compound selected from the group consisting of dipeptides and oxidized products thereof, in which polyethyleneimine is immobilized on porous particles.
[2] The separating agent according to [1], wherein the polyethyleneimine has a mass average molecular weight of 200 or more.
[3] The separation agent according to [1] or [2], wherein the porous particles have a pore diameter of 1 nm to 1000 nm.
[4] The separating agent according to any one of [1] to [3], wherein the porous particles have a crosslinked structure.
[5] The separation according to any one of [1] to [4], wherein the porous particles contain at least one selected from the group consisting of acrylic resin, vinyl acetate resin, polysaccharide, silica, and glass. agent.
[6] The separation agent according to [5], wherein the porous particles contain an acrylic resin.
[7] The separating agent according to any one of [1] to [6], wherein the porous particles contain a hydroxyl group.
[8] A solution containing at least one compound selected from the group consisting of dipeptides and oxidized products thereof is loaded onto the separating agent according to any one of [1] to [7], and the separating agent is loaded with a solvent (A ), the separation method comprising a liquid chromatography step of separating the compound in the solution.
[9] The separation method according to [8], wherein the solvent (A) contains alcohols and/or acetonitrile.
[10] A solution containing at least one compound selected from the group consisting of dipeptides and oxidized products thereof and a solvent (B) is brought into contact with the separating agent according to any one of [1] to [7]. , a separation method comprising: an adsorption step of adsorbing the compound to the separation agent; and an elution step of eluting the compound from the separation agent using a solvent (C).
[11] The separation method according to [10], wherein the solvent (B) and the solvent (C) contain alcohols and/or -acetonitrile.
[12] The separation method according to [10] or [11], wherein the solvent (C) is more polar than the solvent (B).
[13] The separation method according to any one of [10] to [12], wherein the solvent (B) contains propyl alcohol, ethyl alcohol, methyl alcohol, or acetonitrile.
[14] A method for producing a compound, comprising the step of separating the compound by the separation method according to any one of [8] to [13].

The separation agent of the present invention exhibits excellent adsorption properties for dipeptides and their oxidized products, can separate dipeptides and their oxidized products, and can efficiently obtain highly purified dipeptides and their oxidized products on an industrial scale. I can do it.
In addition, the separation method of the present invention exhibits excellent adsorption properties for dipeptides and their oxidized products, can separate dipeptides and their oxidized products, and can efficiently produce high-purity dipeptides and their oxidized products on an industrial scale. Obtainable.
Furthermore, since the method for producing the compound of the present invention uses the separation method of the present invention, the dipeptide and its oxidized product can be separated, and highly purified dipeptide and its oxidized product can be efficiently obtained on an industrial scale. .

FIG. 2 is a diagram showing a chromatogram obtained in Example 2. FIG. 3 is a diagram showing a chromatogram obtained in Example 3. FIG. 4 is a diagram showing a chromatogram obtained in Example 4. FIG. 3 is a diagram showing the chromatogram obtained in Example 4 and the mass spectrum peak areas derived from the dipeptide and its oxidized product in each of the obtained fractions.

The present invention will be described in detail below, but the present invention is not limited to the following embodiments, and can be implemented with various changes within the scope of the gist. In addition, when the expression "~" is used in this specification, it is used as an expression including the numerical value or physical property value before and after it. Furthermore, in this specification, "(meth)acrylic" refers to "acrylic", "methacrylic", or both, and "(meth)acrylate" refers to "acrylate", "methacrylate", or both. .

(Separating agent)
The separating agent of the present invention has polyethyleneimine immobilized on porous particles.
Moreover, from the viewpoint of ease of manufacture, porous particles having polyethyleneimine immobilized by covalent bonds are preferable.
In this specification, porous particles refer to particles having a large number of fine pores. Preferred ranges of the volume average particle diameter, specific surface area, pore diameter, and pore volume of the porous particles will be described later.

(Materials constituting porous particles)
Examples of materials constituting the porous particles include acrylic resins, styrene resins, vinyl acetate resins, vinyl ether resins, polysaccharides, silica, and glass. Among the materials constituting these porous particles, acrylic resins, vinyl acetate resins, polysaccharides, Silica and glass are preferred, and acrylic resin is more preferred.

In this specification, the acrylic resin refers to 50% by mass or more of (meth)acrylate-derived structural units out of 100% by mass of all monomer units constituting the acrylic resin, and this ratio is 80% by mass or more. It is preferable that it is at least % by mass. The acrylic resin may contain structural units other than those derived from (meth)acrylate.

Examples of (meth)acrylates include alkyl ( Meth)acrylate; Hydroxyl group-containing (meth)acrylates such as hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate, and glycerin mono(meth)acrylate; glycidyl (meth)acrylate, 4,5-epoxybutyl (meth)acrylate , 9,10-epoxystearyl (meth)acrylate and other epoxy group-containing (meth)acrylates; (meth)acrylamides such as (meth)acrylamide, dimethyl (meth)acrylamide, and hydroxyethyl (meth)acrylamide; (meth)acrylonitrile Cyano group-containing (meth)acrylates such as ethylene glycol di(meth)acrylate, polyethylene glycol di(meth)acrylate, alkylene di(meth)acrylate, N,N'-alkylene bis(meth)acrylamide, glycerol di(meth)acrylate Examples include crosslinkable (meth)acrylates such as acrylate and trimethylolpropane tri(meth)acrylate. These (meth)acrylates may be used alone or in combination of two or more. Among these (meth)acrylates, it is preferable to include epoxy group-containing (meth)acrylates and crosslinkable (meth)acrylates because they facilitate the introduction of polyethyleneimine and have excellent solubility resistance in various solvents. More preferably, it contains glycidyl (meth)acrylate and ethylene glycol di(meth)acrylate.

Examples of the vinyl acetate resin include poly(vinyl acetate-triallyl isocyanurate). Among these vinyl acetate-based resins, poly(vinyl acetate-triallyl isocyanurate) is preferred because it can introduce a hydroxyl group as a reactive functional group to be described later.

Examples of polysaccharides include agarose, cellulose, and dextran. Among these polysaccharides, agarose, cellulose, Dextran is preferred.

Silica and glass preferably contain an organosilicon compound having a reactive functional group such as 3-glycidoxypropyltrimethoxysilane as a raw material because reactive functional groups described below can be introduced into porous particles. .

(Method for producing porous particles)
As a method for producing porous particles, for example, an organic phase containing a non-crosslinking monomer, a crosslinking monomer, a porosity-forming agent, a polymerization initiator, etc. is dispersed in an aqueous phase containing a dispersion stabilizer, etc. , a method of conducting a polymerization reaction by heating or the like. By this method, spherical porous particles having a crosslinked structure can be obtained. More specifically, examples include methods of carrying out suspension polymerization and emulsion polymerization as disclosed in Japanese Patent Publication No. 58-058026.

Porous particles preferably have a crosslinked structure because they have excellent solubility resistance in various solvents.
Examples of the crosslinkable monomer include the above-mentioned crosslinkable (meth)acrylate.
The content of the crosslinkable monomer is preferably 3% by mass to 95% by mass, more preferably 5% by mass to 90% by mass based on 100% by mass of the total monomers. When the content of the crosslinkable monomer is 3% by mass or more, the formation of a pore structure is sufficient and the mechanical strength of the porous particles is excellent. Further, when the content of the crosslinkable monomer is 95% by mass or less, the immobilization reaction of polyethyleneimine proceeds easily, the amount of polyethyleneimine introduced is sufficient, and the adsorption of dipeptides and their oxidized products is excellent.

Examples of non-crosslinkable monomers include the aforementioned alkyl (meth)acrylates, the aforementioned hydroxyl group-containing (meth)acrylates, the aforementioned epoxy group-containing (meth)acrylates, the aforementioned (meth)acrylamides, and the aforementioned cyano Examples include group-containing (meth)acrylates.
The content of the non-crosslinkable monomer is preferably 5% by mass to 97% by mass, more preferably 10% by mass to 95% by mass, based on 100% by mass of the total monomers. When the content of the non-crosslinking monomer is 5% by mass or more, the immobilization reaction of polyethyleneimine will proceed easily, the amount of polyethyleneimine introduced will be sufficient, and the adsorption of dipeptides and their oxidized products will be excellent. Moreover, when the content of the non-crosslinkable monomer is 97% by mass or less, the formation of a pore structure is sufficient and the mechanical strength of the porous particles is excellent.

The porous particles preferably have reactive functional groups in order to immobilize polyethyleneimine through covalent bonds.
Examples of the reactive functional group include a hydroxyl group, an amino group, a carboxyl group, a halogen group, and an epoxy group. Among these reactive functional groups, halogen groups and epoxy groups are preferred because they are easy to introduce reactive functional groups and have excellent reactivity with polyethyleneimine.

The reactive functional group may be introduced into the porous particle by polymerizing a monomer composition containing a monomer having a reactive functional group, or the reactive functional group may be introduced into the porous particle after constructing the porous particle. It's okay.
As a method for introducing reactive functional groups after constructing porous particles, for example, a monomer composition containing a monomer having a functional group capable of reacting with a compound having a reactive functional group (spacer) is polymerized. An example of this method is to construct porous particles using a method of forming porous particles, and to react the porous particles with a compound (spacer) having a reactive functional group.

Examples of monomers having reactive functional groups include halogen group-containing monomers such as chloromethylstyrene and bromobutylstyrene; glycidyl (meth)acrylate, allyl glycidyl ether, vinyl glycidyl ether, and 4-epoxy-1- Examples include epoxy group-containing monomers such as butene. Among monomers having these reactive functional groups, halogen group-containing monomers and epoxy group-containing monomers are preferred, as they facilitate the introduction of polyethyleneimine, and chloromethylstyrene, bromobutylstyrene, glycidyl (Meth)acrylate is more preferred, and chloromethylstyrene and glycidyl methacrylate are even more preferred.

(Physical properties of porous particles)
The volume average particle diameter of the porous particles is preferably 1 μm to 1000 μm, more preferably 4 μm to 700 μm, even more preferably 10 μm to 500 μm. When the volume average particle diameter of the porous particles is 1 μm or more, it is possible to suppress pressure loss when the separation agent is packed in a column and pass through the column, and increase the flow rate, resulting in excellent productivity in adsorption treatment. . Moreover, when the volume average particle diameter of the porous particles is 1000 μm or less, column efficiency is excellent, and adsorption amount and separation performance are excellent.
In this specification, the volume average particle diameter of porous particles is determined by measuring the particle diameters of arbitrary 100 porous particles using an optical microscope, and calculating the volume median diameter from the distribution.

The volume average particle diameter of porous particles depends on the polymerization conditions of suspension polymerization and emulsion polymerization, specifically, the type and amount of monomer, the type and amount of dispersion stabilizer and emulsifier, and settings such as the stirring rotation speed. Can be adjusted. Further, the particles produced after completion of polymerization may be classified by a method such as a sieve, a water sieve, or an air sieve to make the volume average particle size of the porous particles uniform.

The specific surface area of the porous particles is preferably 1 m ² /g to 1000 m ² /g, more preferably 10 m ² /g to 500 m ² /g. When the specific surface area of the porous particles is 1 m ² /g or more, the mechanical strength of the porous particles is excellent, the generation of spaces that do not contribute to adsorption inside the pores can be suppressed, and the dipeptide and its oxidized product can be Excellent adsorption properties. In addition, when the specific surface area of the porous particles is 1000 m ² /g or less, the polyethyleneimine to be immobilized easily enters the pores of the porous particles, the immobilization reaction of the polyethyleneimine tends to proceed, and the polyethyleneimine The amount introduced is sufficient, and the adsorption of dipeptides and their oxidized products is excellent.
In this specification, the specific surface area of porous particles is measured by a nitrogen gas adsorption method (BET method). Specifically, the monomolecular layer adsorption amount is calculated from the pressure change before and after adsorption of nitrogen gas using the BET formula, and the specific surface area of the porous particles is calculated from the cross-sectional area of one molecule of nitrogen gas. shall apply mutatis mutandis.

The specific surface area of the porous particles can be adjusted by setting the polymerization reaction conditions, crosslinking reaction conditions, etc. when producing the porous particles.

The pore diameter of the porous particles is preferably 1 nm to 1000 nm, more preferably 20 nm to 500 nm, even more preferably 30 nm to 200 nm. When the pore diameter of the porous particles is 1 nm or more, the polyethyleneimine to be immobilized easily enters the pores of the porous particles, the immobilization reaction of polyethyleneimine easily proceeds, and the amount of polyethyleneimine introduced is sufficient. It has excellent adsorption properties for dipeptides and their oxidized products. When the pore diameter of the porous particles is 1000 nm or less, the mechanical strength of the porous particles is excellent, the generation of spaces that do not contribute to adsorption inside the pores can be suppressed, and the adsorption of dipeptides and their oxidized products is improved. Excellent in
In this specification, the pore diameter of a porous particle is the most frequent diameter measured by mercury porosimetry. Specifically, pressure is applied to the porous particles to cause mercury to enter the pores, and using the pressure value and the corresponding volume of mercury entering, assuming the shape of the pores to be cylindrical, and using Washburn's equation, This is a calculation method, and ISO 15901-1 is applied mutatis mutandis.

The pore diameter of porous particles depends on the polymerization conditions of suspension polymerization and emulsion polymerization, specifically, the type and amount of monomer, the type and amount of porosity-forming agent, the type and amount of polymerization initiator, etc. It can be adjusted by setting.

The pore volume of the porous particles is preferably 0.1 mL/g to 3.0 mL/g, more preferably 0.2 mL/g to 2.5 mL/g, and 0.5 mL/g to 2.0 mL/g. More preferred. When the pore volume of the porous particles is 0.1 mL/g or more, the adsorption of dipeptides and their oxidants is excellent. When the pore volume of the porous particles is 3.0 mL/g or less, the mechanical strength of the porous particles is excellent.
In this specification, the pore volume of porous particles is measured by mercury porosimetry. Specifically, pressure is applied to the porous particles to cause mercury to enter the pores, and using the pressure value and the corresponding volume of mercury entering, assuming the shape of the pores to be cylindrical, and using Washburn's equation, This is a calculation method, and ISO 15901-1 is applied mutatis mutandis.

The pore volume of the porous particles can be adjusted by setting the polymerization reaction conditions, crosslinking reaction conditions, etc. when producing the porous particles.

(Polyethyleneimine)
The mass average molecular weight of polyethyleneimine is preferably 200 to 100,000, more preferably 250 to 10,000. When the mass average molecular weight of polyethyleneimine is 200 or more, it has excellent adsorption properties for dipeptides and their oxidants. Moreover, when the mass average molecular weight of polyethyleneimine is 100,000 or less, the reactivity of the immobilization reaction of polyethyleneimine is excellent.
In this specification, the mass average molecular weight of polyethyleneimine shall be measured by gel filtration chromatography.

As a method for covalently immobilizing polyethyleneimine on porous particles, for example, polyethyleneimine as it is or a solution of polyethyleneimine dissolved in an organic solvent or water is supplied to porous particles having reactive functional groups. , a method of covalent bond reaction.
As a method for supplying polyethyleneimine, if polyethyleneimine is used as it is, it has a high viscosity and is difficult to handle for industrial production, so it is preferable to use a solution in which polyethyleneimine is dissolved in an organic solvent or water. . Furthermore, when using an epoxy group as the reactive functional group, it is more preferable to use a solution in which polyethyleneimine is dissolved in an organic solvent, since a diol production reaction occurs due to the addition of water to the epoxy group in an aqueous solution.

The organic solvent is not particularly limited as long as it can dissolve polyethyleneimine, but examples include alcohols such as methanol, ethanol, propyl alcohol, butanol; ethylene glycol dimethyl ether, diethylene glycol dimethyl ether, diethyl ether, cyclopentyl methyl ether, 4-methyl Examples include ethers such as tetrahydropyran, tetrahydrofuran (THF), and dioxane; and amides such as dimethylformamide and dimethylacetamide. These organic solvents may be used alone or in combination of two or more. Among these organic solvents, ethers are preferred because they swell the porous particles made of acrylic resin and improve the reactivity between the reactive functional group and polyethyleneimine, such as ethylene glycol dimethyl ether, diethylene glycol dimethyl ether, and diethyl ether. , cyclopentyl methyl ether, 4-methyltetrahydropyran, tetrahydrofuran, and dioxane are more preferred.

The concentration of polyethyleneimine in the polyethyleneimine solution is preferably 5% by mass or more and 60% by mass or less. If the concentration of polyethyleneimine is 5% by mass or more, the immobilization efficiency of polyethyleneimine is excellent, and if it is 60% by mass or less, the solution is excellent in handling.

The reaction temperature when covalently bonding polyethyleneimine to porous particles is immobilized is preferably 10°C to 120°C, more preferably 20°C to 100°C. When the reaction temperature is 10° C. or higher, the immobilization reaction can be carried out in a short time. Further, when the reaction temperature is 120° C. or lower, decomposition can be suppressed when porous particles made of acrylic resin are used.

After fixing polyethyleneimine to the porous particles, it is preferable to inactivate the reactive functional groups remaining in the porous particles by post-treatment. If a reactive functional group is left without inactivation, it will react with the active group present in the reaction substrate during a chemical reaction, reducing the adsorption amount of the dipeptide and its oxidized product or worsening the adsorption property. There are cases.

Examples of post-treatment when using an epoxy group as a reactive functional group include a method of converting it into a diol, ie, a hydroxyl group, by reacting it with water.
It is preferable that the porous particles have hydroxyl groups because when an epoxy group is used as a reactive functional group, a decrease in the amount of adsorption of the dipeptide and its oxidized product and a deterioration in the adsorption properties can be suppressed.
It can be confirmed that the porous particles have a hydroxyl group by a known hydroxyl value measuring method.

Examples of catalysts for reacting epoxy groups with water include aqueous solutions of inorganic acids such as phosphoric acid and sulfuric acid; aqueous alkali solutions such as sodium hydroxide and potassium hydroxide. Among these catalysts, sulfuric acid is preferred because of its excellent reactivity.
The concentration of the aqueous catalyst solution when reacting the epoxy group with water is preferably 1% by mass to 30% by mass, more preferably 3% by mass to 20% by mass, since side reactions can be suppressed.

The reaction temperature when reacting the epoxy group with water is preferably 10°C to 90°C, more preferably 20°C to 80°C, because of excellent reactivity.
The reaction time for reacting the epoxy group with water is preferably from 0.1 hour to 24 hours, more preferably from 1 hour to 10 hours, since side reactions can be suppressed.

When an acid such as sulfuric acid is used as a catalyst for converting an epoxy group into a hydroxyl group, it is preferable that the ion exchange group is then regenerated using an aqueous alkali solution such as water-solubilized sodium.

(Physical properties of separating agent)
The volume average particle diameter of the separating agent is preferably 1 μm to 1000 μm, more preferably 4 μm to 700 μm, even more preferably 10 μm to 500 μm. When the volume average particle diameter of the separating agent is 1 μm or more, pressure loss when the separating agent is packed in a column and passed through the column can be suppressed, the liquid passing rate can be increased, and the productivity of the adsorption treatment is excellent. Further, when the volume average particle diameter of the separating agent is 1000 μm or less, column efficiency is excellent, and adsorption amount and separation performance are excellent.
In this specification, the volume average particle diameter of a separating agent is determined by measuring the particle diameters of arbitrary 100 separating agents using an optical microscope, and calculating the volume median diameter from the distribution.

The volume average particle size of the separation agent depends on the volume average particle size of the porous particles used, but since polyethyleneimine is immobilized, it is usually 0.1% to 0.1% larger than the volume average particle size of the porous particles used. It becomes about 20% larger. Alternatively, the produced separating agent may be classified by a method such as a sieve screen, a water sieve, or an air sieve to make the volume average particle size of the separating agent uniform.

The uniformity coefficient, which is an index of the particle size distribution width of the separation agent, is preferably smaller because it can suppress pressure loss when the separation agent is packed in a column and passed through the column.Specifically, 1. It is preferably 0 to 2.0, more preferably 1.0 to 1.6.
In this specification, the uniformity coefficient of a separating agent is defined as the value obtained by dividing the particle size that accounts for 40% of the larger particle size by the particle size that accounts for 90% of the larger particle size in the volume distribution of the separating agent. do.

The specific surface area of the separating agent is preferably 1 m ² /g to 1000 m ² /g, more preferably 10 m ² /g to 500 m ² /g. When the specific surface area of the separating agent is 1 m ² /g or more, the separating agent has excellent mechanical strength, there are few spaces inside the pores that do not contribute to adsorption, and the separating agent has excellent adsorption properties for dipeptides and their oxidized products. In addition, when the specific surface area of the separating agent is 1000 m ² /g or less, the immobilized polyethyleneimine easily enters the pores of the porous particles, and the immobilization reaction of polyethyleneimine is progressing. The amount of introduced is also sufficient, and the adsorption of the separating agent dipeptide and its oxidized product is excellent.
In this specification, the specific surface area of a separating agent is measured by a nitrogen gas adsorption method (BET method). Specifically, the monomolecular layer adsorption amount is calculated from the pressure change before and after adsorption of nitrogen gas using the BET formula, and the specific surface area of the separating agent is calculated from the cross-sectional area of one molecule of nitrogen gas. Apply mutatis mutandis.

The pore diameter of the separating agent is preferably 1 nm to 1000 nm, more preferably 2 nm to 500 nm, even more preferably 3 nm to 200 nm. When the pore diameter of the separation agent is 1 nm or more, the immobilized polyethyleneimine easily enters the pores of the porous particles, and the immobilization reaction of polyethyleneimine is progressing, so the amount of polyethyleneimine introduced also decreases. It has excellent adsorption properties for the separating agent dipeptide and its oxidized product. When the pore diameter of the separating agent is 1000 nm or less, the porous particles have excellent mechanical strength, there are few spaces inside the pores that do not contribute to adsorption, and the separating agent has excellent adsorption properties for dipeptides and their oxidized products.
In this specification, the pore diameter of the separating agent is the most frequent diameter measured by mercury porosimetry. Specifically, pressure is applied to the separation agent to cause mercury to enter the opening, and using the pressure value and the corresponding volume of mercury intrusion, the shape of the pore is assumed to be cylindrical and calculated using the Washburn equation. ISO 15901-1 applies mutatis mutandis.

The pore volume of the separating agent is preferably 0.1 mL/g to 3.0 mL/g, more preferably 0.2 mL/g to 2.5 mL/g, and even more preferably 0.5 mL/g to 2.0 mL/g. preferable. When the pore volume of the separating agent is 0.1 mL/g or more, the separating agent has excellent adsorption properties for dipeptides and their oxidized products. When the pore volume of the separating agent is 3.0 mL/g or less, the mechanical strength of the separating agent is excellent.
In this specification, the pore volume of the separating agent is measured by mercury porosimetry. Specifically, pressure is applied to the separation agent to cause mercury to enter the opening, and using the pressure value and the corresponding volume of mercury intrusion, the shape of the pore is assumed to be cylindrical and calculated using the Washburn equation. ISO 15901-1 applies mutatis mutandis.

The pore volume of the separation agent depends on the pore volume of the porous particles used, but since polyethyleneimine is immobilized, it is usually about 0.1% to 50% of the pore volume of the porous particles used. Change.

The amount of polyethyleneimine immobilized in the separation agent can be determined by the nitrogen content and total exchange capacity.

The nitrogen content of the separating agent is preferably 0.3% by mass to 30% by mass, more preferably 0.5% by mass to 25% by mass based on 00% by mass of the separating agent (1). When the nitrogen content of the separating agent is 0.3% by mass or more, the amount of polyethyleneimine immobilized is sufficient, and the separating agent has excellent adsorption properties for dipeptides and their oxidized products. In addition, when the nitrogen content of the separation agent is 30% by mass or less, the amount of immobilized polyethyleneimine is not too large and the separation agent has a pore volume sufficient to allow dipeptide and its oxidized product to diffuse and permeate. Excellent adsorption of dipeptides and their oxidized products.
In this specification, the nitrogen content of the separating agent is measured by elemental analysis. Specifically, it shall be measured using a carbon, hydrogen, and nitrogen simultaneous quantitative determination device.

The total exchange capacity of the separating agent is preferably 0.1 milliequivalents/g to 20 milliequivalents/g, more preferably 0.2 milliequivalents/g to 10 milliequivalents/g.
In this specification, the total exchange capacity of the separating agent is determined by accurately weighing an amount equivalent to 0.5 g to 1.5 g of the dried separating agent, adding it to 250 mL of 0.2 mol/L hydrochloric acid, and heating it at 30°C for 8 hours. After shaking, the concentration of hydrochloric acid in the supernatant shall be measured by titration and calculated from the results.

(Application)
The separating agent of the present invention is used to separate at least one compound selected from the group consisting of dipeptides and oxidized products thereof.

(Separation method)
The separation method of the present invention is a method using the separation agent of the present invention, and includes the following two separation methods.

Method (1): A solution containing at least one compound selected from the group consisting of dipeptides and oxidized products thereof is loaded onto the separation agent of the present invention, and the solvent (A) is passed through the separation agent. A separation method comprising a liquid chromatography step for separating the compound in a solution.

Method (2): A solution containing at least one compound selected from the group consisting of dipeptides and oxidized products thereof and a solvent (B) is brought into contact with the separating agent of the present invention, and the compound is added to the separating agent. A separation method comprising an adsorption step of adsorption, and an elution step of eluting the compound from the separation agent using a solvent (C).

The solution containing at least one compound selected from the group consisting of dipeptides and oxidized products thereof in methods (1) and (2) is extracted from foods, etc., or the extracted compound is oxidized by a known method. You can prepare by doing this.

(Method (1))
Method (1) includes loading a solution containing at least one compound selected from the group consisting of dipeptides and oxidized products thereof into the separation agent of the present invention, and flowing the solvent (A) through the separation agent. This separation method includes a liquid chromatography step for separating the compound in the solution.

In the liquid chromatography step, a solution containing at least one compound selected from the group consisting of dipeptides and oxidized products thereof is loaded onto the separation agent of the present invention, and the solvent (A) is passed through the separation agent. This is a step of separating the compound in the solution. As a specific example of the liquid chromatography step, a solution containing at least one compound selected from the group consisting of dipeptides and oxidized products thereof is loaded onto a column packed with the separation agent of the present invention, and a solvent (A) is applied to the column. A step of separating the dipeptide and its oxidized form from other compounds by chromatography by flowing the dipeptide and its oxidized product through chromatography may be mentioned.

The solvent (A) is not particularly limited as long as it can dissolve the dipeptide and its oxidized product, and examples thereof include n-butyl alcohol (solubility parameter: 23.3 MPa ^1/2 ) and isobutyl alcohol (solubility parameter: 21.5 MPa ¹ ). ^/2 ), sec-butyl alcohol (solubility parameter: 22.1 MPa ^1/2 ), t-butyl alcohol (solubility parameter: 21.7 MPa ^1/2 ), butyl alcohol, 1-propyl alcohol (solubility parameter: 24.3 MPa ^1/2 ), 2-propyl alcohol (solubility parameter: 23.5 MPa ^1/2 ), ethyl alcohol (solubility parameter: 26.0 MPa ^1/2 ), methyl alcohol (solubility parameter: 29.7 MPa ^1/2) ) and other alcohols; ethylene glycols such as ethylene glycol (solubility parameter: 29.9 MPa ^1/2 ) and diethylene glycol (solubility parameter: 24.8 MPa ^1/2 ); glycerin (solubility parameter: 33.8 MPa ^1/2 ) polyols such as ethylene glycol dimethyl ether (solubility parameter: 17.6 MPa ^1/2 ), diethylene glycol dimethyl ether, diethyl ether (solubility parameter: 15.1 MPa ^1/2 ), tetrahydrofuran (solubility parameter: 18.6 MPa ^1/2 ), Ethers such as dioxane (solubility parameter: 20.5MPa ^1/2 ); amides such as dimethylformamide (solubility parameter: 24.8MPa ^1/2 ), dimethylacetamide (solubility parameter: 22.1MPa ^1/2 ); acetonitrile Nitriles such as (solubility parameter: 24.3 MPa ^1/2 ); water (solubility parameter: 47.9 MPa ^1/2 ), and the like. These solvents (A) may be used alone or in combination of two or more. Among these solvents (A), alcohols, ethylene glycols, polyols, and nitriles are preferable because dipeptides and their oxidized products have high solubility and are freely miscible with water, and t-butyl alcohol, 1- Propyl alcohol, 2-propyl alcohol, ethyl alcohol, methyl alcohol, ethylene glycol, diethylene glycol, glycerin, acetonitrile are more preferred, and solvents containing alcohols and/or acetonitrile that are freely miscible with water are even more preferred, propyl alcohol, ethyl alcohol. A mixed solvent containing one or more of water and methyl alcohol, and acetonitrile, and a mixed solvent containing acetonitrile and water are particularly preferred.
For the solubility parameters, the values described in Polymer Handbook, 3rd edition (WILEY, published in 1989) were referred to.

(Method (2))
In method (2), a solution containing at least one compound selected from the group consisting of dipeptides and oxidized products thereof and a solvent (B) is brought into contact with the separating agent of the present invention, and the compound is transferred to the separating agent. This separation method includes an adsorption step in which the compound is adsorbed on the compound, and an elution step in which the compound is eluted from the separation agent using a solvent (C).
For method (2), methods such as a batch processing method and a column processing method can be used, but the column processing method is preferable because it allows efficient separation.

In the adsorption step, a solution containing at least one compound selected from the group consisting of dipeptides and oxidized products thereof and a solvent (B) is brought into contact with the separating agent of the present invention, and the compound is adsorbed onto the separating agent. This is the process of As a specific example of the adsorption step, a solution containing at least one compound selected from the group consisting of dipeptides and oxidized products thereof and a solvent (B) is brought into contact with the separating agent of the present invention, and the compound etc. are separated. For example, a step of adsorbing the substance onto an agent is included.

The solvent (B) is not particularly limited as long as it can dissolve the dipeptide and its oxidized product, and examples thereof include the solvents exemplified as the solvent (A). These solvents (B) may be used alone or in combination of two or more. Among these solvents (B), alcohols, ethylene glycols, polyols, and nitriles are preferable because dipeptides and their oxidized products have high solubility and are freely miscible with water, and t-butyl alcohol, 1- Propyl alcohol, 2-propyl alcohol, ethyl alcohol, methyl alcohol, ethylene glycol, diethylene glycol, glycerin, acetonitrile are more preferred, and solvents containing alcohols and/or acetonitrile that are freely miscible with water are even more preferred, propyl alcohol, ethyl alcohol. A mixed solvent containing one or more of water and methyl alcohol, and acetonitrile, and a mixed solvent containing acetonitrile and water are particularly preferred.

The elution step is a step of eluting the compound from the separating agent using a solvent (C).
Other compounds may be eluted from the separating agent before the compound is eluted, or other compounds may be eluted from the separating agent after the compound is eluted.

The solvent (C) is not particularly limited as long as it can elute the above-mentioned compound, a dipeptide other than the above-mentioned compound, and its oxidized product, and examples thereof include the solvents exemplified for the solvent (A). These solvents (C) may be used alone or in combination of two or more. Among these solvents (C), alcohols, ethylene glycols, polyols, and nitriles are preferable because dipeptides and their oxidized products have high solubility and are freely miscible with water, and t-butyl alcohol, 1- Propyl alcohol, 2-propyl alcohol, ethyl alcohol, methyl alcohol, ethylene glycol, diethylene glycol, glycerin, acetonitrile are more preferred, and solvents containing alcohols and/or acetonitrile that are freely miscible with water are even more preferred, propyl alcohol, ethyl alcohol. A mixed solvent containing one or more of water and methyl alcohol, and acetonitrile, and a mixed solvent containing acetonitrile and water are particularly preferred.
Solvent (A), solvent (B), and solvent (C) may be the same or different.

Since the separation action mechanism of dipeptides and their oxidized products is presumed to be based on hydrophilic interaction chromatography, it is possible to use a solvent that is more polar than the solvent (B) as the solvent (C), or to use a solvent that is more polar than the solvent (B). ) It is preferable to use a solvent obtained by diluting the solvent (C) with water.

The solubility parameter of the solvent (C) is preferably 15 MPa ^1/2 or more, more preferably 18 MPa ^1/2 or more, since polarity can be increased.
By setting the solubility parameter of the solvent (B) lower than the solubility parameter of the solvent (C), the compound etc. can be adsorbed on the separating agent.
Since the solubility parameter includes the degree of contribution of polarity, it can be used as an index for determining the polarity of the solvent. In this specification, the solubility parameters refer to the values described in the Polymer Handbook, 3rd edition (WILEY, published in 1989).

When eluting the above compound and another compound, it is sufficient to select a solvent (C) suitable for each elution, and the type and concentration of the solvent (C) may be selected. Furthermore, it is also preferable to add various salts such as ammonium acetate and ammonium formate as necessary to improve the separability between the above compound and other compounds.

(Method for producing compound)
The method for producing a compound of the present invention includes the step of separating the compound, ie, the dipeptide and its oxidized product, by the separation method of the present invention.

(Industrial applicability)
The separating agent of the present invention exhibits excellent adsorption properties for dipeptides and their oxidized products, and can separate dipeptides and their oxidized products. In addition, the separation method of the present invention using the separation agent of the present invention can separate dipeptides and their oxidized products, and can efficiently obtain highly purified dipeptides and their oxidized products on an industrial scale. It has extremely high practical value in the food industry.

Hereinafter, the present invention will be explained in more detail using examples, but the present invention is not limited to the description of the following examples unless it departs from the gist thereof.

(Volume average particle diameter)
Regarding the volume average particle diameter of the porous particles used in the examples and the separation agent obtained in the examples, arbitrary 100 porous particles were measured using an optical microscope (model name "SMZ1500", manufactured by Nikon Corporation). The particle diameter of the separating agent was measured, and the volume median diameter was calculated from the distribution.

(Specific surface area)
Regarding the specific surface area of the porous particles used in the examples and the separation agent obtained in the examples, the dried porous particles and separation agent were weighed and measured using a specific surface area measuring device (model name "Flowsorb III", Micromeritex). It was measured by the nitrogen gas adsorption method (BET method).

(Pore diameter/pore volume)
The pore diameter and pore volume of the porous particles used in Examples and the separation agents obtained in Examples were determined by mercury porosimetry using an automatic porosimeter (model name "Autopore 9520", manufactured by Micromeritex). It was measured.

(Nitrogen content)
The nitrogen content of the separation agent obtained in the example was measured by elemental analysis using a carbon/hydrogen/nitrogen simultaneous quantitative determination device (model name "2400II", manufactured by PerkinElmer).

(total exchange capacity)
Regarding the total exchange capacity of the separating agent obtained in the example, an amount equivalent to 0.5 g to 1.5 g of the dried separating agent was accurately weighed, added to 250 mL of 0.2 mol/L hydrochloric acid, and heated at 30°C for 8 After shaking for a period of time, the hydrochloric acid concentration of the supernatant was measured by titration and calculated from the results.

[Example 1: Production of separating agent]
Porous particles consisting of 20% by mass of structural units derived from glycidyl methacrylate and 80% by mass of structural units derived from ethylene glycol dimethacrylate, with a specific surface area of 305 m ² /g, a pore diameter of 120.4 nm, and a pore volume of 1.06 mL / g. 140 parts by mass of diethylene glycol dimethyl ether and 60 parts by mass of polyethyleneimine (molecular weight 600, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) were added to 40 parts by mass and stirred to form a suspension. This suspension was heated to 80°C and reacted for 6 hours. After cooling, the obtained particles were washed with water. 200 parts by mass of sulfuric acid with a concentration of 10% by mass was added to the particles and stirred to form a suspended state. This suspension was heated to 50° C. and maintained for 5 hours to carry out a hydroxyl group production reaction by adding water to unreacted epoxy groups. After cooling, the obtained particles were washed with water, the ion exchange groups were regenerated with a 2 mol/L aqueous sodium hydroxide solution, particles with a particle size of 75 μm to 220 μm were selected using a sieve screen, and the separating agent (1) was added. Obtained.
The obtained separation agent (1) had a specific surface area of 113 m ² /g, a pore diameter of 38.2 nm, a pore volume of 0.76 mL/g, and a total exchange capacity of 3.09 milliequivalents/g.

[Example 2: Separability evaluation 1 by chromatography]
The separation agent (1) obtained in Example 1 was packed into a glass column with an inner diameter of 10 mm and a length of 300 mm. This was connected to a high-performance liquid chromatograph, and a mixed solvent of acetonitrile/10mM ammonium acetate aqueous solution = 65/35 (volume ratio) was passed therethrough at a flow rate of 1.0 mL/min to perform high-performance liquid chromatography.
Anserine nitrate and carnosine were selected as dipeptides, and samples were prepared by dissolving them in acetonitrile/10mM ammonium acetate aqueous solution = 70/30 (volume ratio) mixed solvent to a concentration of 1 mg/mL and 4 mg/mL, respectively. The separation agent (1) in the column was loaded by injecting 20 μL of anserine nitrate sample and 80 μL of carnosine sample into the flowing column, and the chromatogram was measured using an ultraviolet absorption detector at a wavelength of 210 nm.
The obtained chromatogram is shown in FIG. Since anserine nitrate and carnosine are separated, it is possible to obtain highly pure fractions of each.

[Example 3: Separability evaluation 2 by chromatography]
High performance liquid chromatography separation was performed under the same conditions as in Example 2.
Using the oxidation method described in Non-Patent Document 1, a sample with a carnosine concentration of 2.26 mg/mL was oxidized using air instead of oxygen for a reaction time of 4 hours. By injecting 180 μL of this sample, the separation agent (1) in the column was loaded, and a chromatogram was measured using an ultraviolet absorption detector at a wavelength of 210 nm. In addition, for comparison, 400 μL of a carnosine sample prepared in the same manner as in Example 2 was loaded onto the separation agent (1) in the column, and a chromatogram was measured using an ultraviolet absorption detector at a wavelength of 210 nm.
The obtained chromatogram is shown in FIG. 2 together with a chromatogram obtained by injecting 400 μL of carnosine sample for comparison. A peak with stronger retention than carnosine was observed at a retention time of 56 minutes.

[Example 4: Separability evaluation 3 by chromatography]
The separation agent (1) obtained in Example 1 was packed into a glass column with an inner diameter of 10 mm and a length of 300 mm. This was connected to a high-performance liquid chromatograph, and a mixed solvent of acetonitrile/10mM ammonium acetate aqueous solution = 67.5/32.5 (volume ratio) was passed through it at a flow rate of 1.0 mL/min to perform high-performance liquid chromatography. .
Using the oxidation method described in Non-Patent Document 1, a sample with a carnosine concentration of 2.26 mg/mL was oxidized using air instead of oxygen for a reaction time of 4 hours. By injecting 180 μL of this sample, the separation agent (1) in the column was loaded, and a chromatogram was measured using an ultraviolet absorption detector at a wavelength of 210 nm. In addition, for comparison, 400 μL of a carnosine sample prepared in the same manner as in Example 2 was loaded onto the separation agent (1) in the column, and a chromatogram was measured using an ultraviolet absorption detector at a wavelength of 210 nm.
The obtained chromatogram is shown in FIG. 3 together with a chromatogram obtained by injecting 400 μL of carnosine sample for comparison. A peak with stronger retention than carnosine was observed at a retention time of 71 minutes.

In addition, fractions were collected every 1.5 minutes from a retention time of 60 to 90 minutes, and the resulting fractions were collected every 6 minutes in the above step and subjected to reversed-phase high performance liquid chromatography-mass spectrometry analysis. FIG. 4 shows the SIR chromatogram area of m/z: 227 derived from carnosine and the SIR chromatogram area of m/z: 243 derived from 2-oxo-carnosine. From FIG. 4, it is clear that the peak with a retention time of 71 minutes is derived from 2-oxo-carnosine, and a highly pure 2-oxo-carnosine fraction was obtained.

The conditions for high performance liquid chromatography analysis are as follows. Note that the retention times of carnosine and 2-oxo-carnosine under these reversed-phase high performance liquid chromatography analysis conditions are almost the same, making evaluation using an ultraviolet absorption detector impossible.
Column: CMG20/C10 (trade name, manufactured by Mitsubishi Chemical Corporation, inner diameter 4.6 mm, length 250 mm)
Column temperature: 40℃
Eluent: 0.1% formic acid aqueous solution/Eluent (B): Acetonitrile containing 0.1% formic acid = 50/50 (volume ratio)
Flow rate: 1.0 mL/min Detection: Waters QDa detector Ionization mode: ESI+
Probe temperature: 600℃
Capillary voltage: 0.8KV
MS scan range: 50-600m/z (centroid) and SIR
Sampling rate: 2 points/sec Cone voltage: 15V

The separating agent of the present invention exhibits excellent adsorption properties for dipeptides and their oxidized products, and can separate dipeptides and their oxidized products. The separation method of the present invention using the separating agent of the present invention can separate dipeptides and their oxidized products, and can efficiently obtain highly purified dipeptides and their oxidized products on an industrial scale. It has extremely high practical value in the industrial field.

Claims (14)

A separating agent used for separating at least one compound selected from the group consisting of dipeptides and oxidized products thereof, the separating agent having polyethyleneimine immobilized on porous particles. The separating agent according to claim 1, wherein the polyethyleneimine has a mass average molecular weight of 200 or more. The separating agent according to claim 1 or 2, wherein the porous particles have a pore diameter of 1 nm to 1000 nm. The separating agent according to any one of claims 1 to 3, wherein the porous particles have a crosslinked structure. The separating agent according to any one of claims 1 to 4, wherein the porous particles contain at least one selected from the group consisting of acrylic resin, vinyl acetate resin, polysaccharide, silica, and glass. The separating agent according to claim 5, wherein the porous particles contain an acrylic resin. The separating agent according to any one of claims 1 to 6, wherein the porous particles contain hydroxyl groups. Loading a solution containing at least one compound selected from the group consisting of dipeptides and oxidized products thereof into the separation agent according to any one of claims 1 to 7, and flowing the solvent (A) through the separation agent. A separation method comprising a liquid chromatography step of separating the compound in the solution. The separation method according to claim 8, wherein the solvent (A) contains alcohols and/or acetonitrile. A solution containing at least one compound selected from the group consisting of dipeptides and oxidized products thereof and a solvent (B) is brought into contact with the separating agent according to any one of claims 1 to 7 to remove the compound from the A separation method comprising an adsorption step of adsorbing the compound to a separation agent, and an elution step of eluting the compound from the separation agent using a solvent (C). The separation method according to claim 10, wherein the solvent (B) and the solvent (C) contain alcohols and/or acetonitrile. The separation method according to claim 10 or 11, wherein the solvent (C) is more polar than the solvent (B). The separation method according to any one of claims 10 to 12, wherein the solvent (B) contains propyl alcohol, ethyl alcohol, methyl alcohol, or acetonitrile. A method for producing a compound, comprising the step of separating the compound by the separation method according to any one of claims 8 to 13.

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