JPH01308959A - Packing agent for chromatography - Google Patents

Abstract

PURPOSE:To manufacture a packing agent which withstands the use at an alkali range free from generating denaturation in a substance related to living organisms by coupling a primary or secondary amino compound having an ion exchange group to an ester group of a spherical particle of synthetic polyamino acid having an ester group in a side chain by an amide or imide bond. CONSTITUTION:When synthetic polyamino acid has an ester bond in a side chain, an amide bond is formed easily with a primary or secondary amino compound and the amide bond or imide bond is stable in a wide range of pH. Therefore, it is so arranged that a primary amino compound having an ion exchange group is coupled to an ester group of a spherical particle of synthetic polyamino acid having the ester group in a side chain or a secondary amino compound having an ion exchange group is coupled to the ester group of the spherical particle of synthetic polyamino acid having the ester group in a side chain by imide bond. This enables manufacture of a packing agent which is applicable in an alkali range and permits separation and analysis of an organic substance without causing non-specific adsorption.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Applications] The present invention relates to a chromatographic packing material made of a synthetic polyamino acid bound to an ion exchanger eM.

[Conventional technology]

Conventionally, chromatographic packing materials having various ion exchange groups bonded thereto have been produced, but they can be roughly divided into two types.

One is to perform aminomethylation treatment using silica gel particles as a carrier, and use the amino groups to bind an ion exchange group.The other is to directly sulfonate beads such as polystyrene or chloromethyl It is treated with a tertiary or quaternary amino group to react with it.

The chromatographic packing material, which has ion-exchange groups introduced into silica gel, cannot be used in alkaline regions, so separation of proteins, peptides, etc., and 1-minute alkaline washing cannot be performed.
Therefore, it is not suitable for separating biological substances.

Furthermore, since chromatographic packing materials made of polystyrene and polyacrylic acid derivatives into which ion exchange groups are introduced have hydrophobic groups themselves, nonspecific adsorption other than that due to ion exchange groups occurs. For this reason, there are problems such as denaturation of the adsorbed substance, and it cannot be used for this purpose.1- (Problems to be solved by the invention) The present invention overcomes the drawbacks of the above-mentioned prior art and can be used in an alkaline region. The purpose is to produce a chromatographic packing material that can separate and analyze biological substances without causing non-specific adsorption.

(Means for Solving Problem yA) In order to solve these problems, the present inventors discovered that when a synthetic polyamino acid has an ester bond in its side chain, it can be easily combined with a -class or secondary amino compound. form an amide bond,
The present invention was completed based on the discovery that this amide bond or imide bond is stable in a wide range of p)I regions.

That is, the present invention provides a chromatography packing in which a primary amino compound or a secondary amino compound having an ion exchange group is bonded to the ester group of a synthetic polyamino acid spherical particle having an ester group in its side chain through an amide bond or an imide bond. It is a drug.

The synthetic polyamino acids having an ester bond in the side chain of the present invention include those obtained by esterifying acidic polyamino acids such as polyglutamic acid and polyaspartic acid, or derivatives thereof, such as alkyl esters, benzyl esters, cyclohexane methyl esters, etc. can be mentioned.

In addition to these acidic polyamino acid esters, there are also basic polyamino acids such as polylysine and polyhistidine that are esterified by introducing a carboxyl group, those that are directly introduced with a functional group having an ester group, and neutral polyamino acids. Polyamino acids polyglycine, polyalanine, polyvaline, polyleucine, polyisoleucine, polyserine,
Examples include polythreonine, polyproline, polymethionine, polycystine, polyphenylalanine, polytyrosine, polytryptophan, etc. in which ester groups are similarly introduced. The ester group may be introduced before or after polymerization, and the polyamino acid may be a copolymer of these ester group-containing amino acids and other amino acids.

During the introduction or polymerization of the ester group, other functional groups such as amino groups, carboxyl groups, hydroxyl groups, etc. can be protected with appropriate protecting groups as necessary, and the protecting groups can be removed after the reaction is completed.

Various methods for polymerizing these amino acids or amino acid derivatives have been proposed.
; Edited by Yasuo Ukemoto, Kodansha).

In addition, polyamino acids obtained by polymerization can be prepared using known techniques (Japanese Patent Publication No. 56-16766, Japanese Unexamined Patent Application Publication No. 62-1728, etc.).
It can be easily produced by controlling the pores, particle size, etc. by the method described above. That is, a solution containing the polyamino acid is dispersed in a dispersion medium that is not mixed therewith, and the solvent of the polyamino acid is removed by evaporation to obtain spherical particles.

Further, at this time, a third solvent having a boiling point higher than that of the dispersion solvent is suitably added to the polyamino acid solution to form particles, which are then extracted and removed to obtain polyamino acid spherical particles having a porous structure.

The degree of polymerization required for forming spherical particles varies depending on the type of constituent amino acid residues, but is 20 or more, preferably 100 or more,
More preferably, it is 200 or more.

The primary or secondary amine compound having an ion exchange group in the present invention refers to an anion exchange IA group such as a -class amino group, a secondary amino group, a tertiary amino group, a quaternary amino group, a carboxyl group, a sulfonic acid group, etc. This is a compound in which a cation exchange group and a primary or secondary amino group for bonding to the ester portion of the synthetic polyamino acid described above are present in the same molecule.

Examples of primary amino compounds having an anion exchange group include primary amines such as hydrazine, ethylenediamine, diethylenetriamine, triethylenetetramine, hexamethylenediamine, N-ethyl-diaminoethane, N-
Secondary amines such as ethyl-diaminopropane, N-ethyl-diaminohexane, N,N-diethylaminoethylamine, N.

Tertiary amines such as N-diethylaminopropylamine,
N,N,N-triethylaminoethylamine, N,N.

Examples include quaternary amines such as N-triethylaminopropylamine.

Further, examples of secondary amino compounds having an anion exchange group include N,N'-dimethylethylenediamine, piperazine, and the like.

Examples of primary amine compounds having a cationic IA group include compounds having a primary amino group and a carboxyl group such as 2-aminoacetic acid and 3-aminopropionic acid, 2-aminoethylsulfonic acid, and 3-aminopropylsulfonic acid. Examples include compounds having a primary amino group and a sulfonic acid group, such as.

Examples of secondary amino compounds having a cation exchange group include compounds having a secondary amino group and a carboxyl group such as 2-N-methylaminoacetic acid and 3-N-methylaminopropionic acid; Ethylsulfonic acid, 3
Examples include compounds having a secondary amino group and a sulfonic acid such as -N-methylaminopropylsulfonic acid.

These anion exchange JAM and cation exchange group need only exist in the same molecule as the primary amino group for bonding with polyamino acids, and there is no particular restriction on the bond between them. For example, changing the number of methylene groups to create an exchange group It is also possible to arbitrarily adjust the distance between the main chain and the synthetic polyamino acid main chain, and aromatic compounds such as para-aminobenzoic acid and para-aminobenzenesulfonic acid may also be used.

The primary or secondary amino group of the primary or secondary amino compound having these ion exchange groups selectively forms an amide bond or imide bond with the ester bond portion of the polyamino acid. In the case of primary amino compounds having cation exchange groups such as -class, secondary, tertiary, and quaternary amines, the reaction is preferably carried out in a relatively highly polar solvent such as alcohol, and in a solvent such as methanol, ethanol, propatool, etc. That? Coal and a mixed solvent thereof such as tetrahydrofuran, hexane, decalin, etc. are preferred.

The reaction temperature may be 20'C or higher up to the reflux temperature of 7 volumes.

In the case of a primary or secondary amino compound having a cation exchange group, the reaction is preferably carried out in a solvent such as tetrahydrofuran or dimethylformamide. The reaction temperature is 20
The temperature is 50°C or higher, preferably 50°C or higher.

[Effect]

According to the present invention, it is possible to obtain a chromatographic packing material for separation and analysis that does not cause non-specific adsorption or denaturation of biologically related substances and can be used in an alkaline region, which was not possible with conventional techniques. Although the reason for this is not necessarily clear, it is presumed that the composition of synthetic polyamino acids is similar to biological substances.

〔Example〕

The present invention will be described in detail below according to Examples, but the present invention is not limited to these Examples.

In the following, parts are based on MMk and percentages are based on moles.

Example 1 1,2-dichloroethane solution containing 2 parts of poly-L-glutamic acid-T-methyl ester with an average degree of polymerization of about 500
Add 4 ml of decalin to 0.0 g, and mix this solution with 500 g of an aqueous solution containing 2.5 parts of partially saponified polyvinyl alcohol.
1.2-dichloroethane was evaporated at a temperature of 45°C to obtain spherical fine particles of poly-L-glutamic acid-γ-methyl ester.

After thoroughly washing the particles with water, decalin remaining in the particles was completely extracted with hot methanol at about 50'C.

Loomj 1.5g of these particles! Dispersed in ethanol, added 2.4 g of N,N-diethylaminoethylamine, and reacted for 24 hours at ethanol flux temperature. After completion of the reaction, unreacted amine was completely removed with ethanol, and then dried and elemental analysis was performed. , compared before and after the reaction.

Table 1 From the results in Table 1, it is recognized that about 62% of the amino acid residues of poly-monoglutamic acid γ-methyl ester were reacted. In addition, in the infrared absorption spectrum of this reaction particle, the ester absorption near 1750 cm-' is small and the amide absorption near 1650 cm-' is large, so that N,N-diethylaminoethylamine reacts with the ester moiety through an amide bond. I know what you're doing. The particles were immersed in a NaOH aqueous solution with Pl+=11 at room temperature for 24 hours, thoroughly washed with water, dried, and subjected to elemental analysis. The results were consistent with the analysis results in Table 1 and were stable.

Example 2 In Example 1, instead of poly-L-glutamic acid T-methyl ester, amino acids were used at a ratio of 80% of N-carboxy-cycloleucine anhydride and N-carboxy-γ-benzylic monoglutamic anhydride. Except for using a polymer copolymerized with triethylamine in tetrahydrofuran using triethylamine as an initiator, particle formation was carried out in the same manner as in Example 1, and the same reaction was carried out. Elemental analysis as shown in Table 2 was obtained. The results were obtained.

Table 2 From this elemental analysis result, Shiichi leucine and L − before reaction
The copolymerization ratio of curtamic acid-T-hensyl ester is
111 cups of Habo monomer (L-o 720%)
- defeated.

It was also observed that, by the reaction with the amine, 45% of the γ-benzyl ester moieties in the polymer, that is, 36% of the total amino acid residues, formed amide bonds with amino groups and introduced ion exchange groups. Table 2 shows the results of microscopic observation and elemental analysis of the particles after immersing these particles in a Na011 solution with a pH of 11 in the same manner as in Example 1.
This shows that this particle is stable on the alkaline side.

Example 3 Poly-L-glutamic acid-T obtained by the method of Example 1
- 1.0 g of spherical particles of methyl ester were dispersed in 100 d of a mixed solvent of methanol/decalin-50150, 0.9 g of hexamethylene diamine was added, and the mixture was reacted with methanol at Franks temperature for 12 hours.

The elemental analysis results of these particles are shown in Table 3. As shown in Table 3, the ratio after reaction is calculated from elemental analysis: x =
0.26, y=o, tt, the proportion of crosslinked residues is about 26%, and the unreacted methyl ester moiety is about 63%.
%.

0.8 g of these particles were dispersed in 100 Id of tetrahydrofuran, and 4 g of sodium p-aminobenzenesulfonate was added.
．． 1 g was added, and the reaction was carried out for 15 hours at Frank's temperature using tetrahydrofuran. After the reaction is completed, the particles are thoroughly washed with tetrahydrofuran, then dispersed in distilled water, and the sulfonic acid groups are replaced from Na type to H type with an excess amount of hydrochloric acid. After that, it was washed with distilled water and dried. The elemental analysis results of these particles are shown in Table 4.

Table 4 From these results, it is recognized that 30%, which is about half of the 63% of the methyl ester portion, reacted and an ion exchange group was introduced.

This value was also consistent with the quantitative determination of ion exchange groups by titration.

Application Example 1 The particles obtained in Example 1 were classified to have a particle size in the range of 20 to 30 μm, packed in a stainless steel column with an inner diameter of 4.6 mm and a length of 150 mm, and mixed with 10IIIM phosphate buffer (
A mixture of amino acids (proline, leucine, glutamic acid) was separated by α-isomer chromatography using pH 7.0) as an eluent.

Chromatography conditions are as follows.

Column: 4.6 x 150 mm Capacity: 10mM phosphoric acid solution pH 7.
0 flow rate: 0.5 mli/ll1in Detector: Differential refractometer (R1) Sample: Proline 1. Omg/rIdlleucine
0.5mg/mfi Glutamic acid 0.5mg/mff1 The results are shown in FIG.

Application Example 2 The particles obtained in Example 3 were classified into a range of 20 to 30 μm and packed into the same column as in Application Example 1, and the protein mixture (raw serum albumin, cytochrome C5 lysozyme) was separated by ionic strength gradient elution. .

Chromatography conditions are as follows.

Column: 4.6X 150mm? Liquid A: 1OaIM Phosphoric Acid 411
di 'aB n a 350IIIM#[$lj
v Night A liquid → B liquid On+in linear gradient flow rate
: 1.0 mff1/+in Detector: UV254nm Sample: Bovine serum albumin 0.1mg/ml Lysozyme 0.1mg/ml! Cytochrome CO,la+g/me The results are shown in FIG.

According to the present invention, ion exchange I can be easily added to synthetic polyamino acids.
The significance of the present invention is great because it has been established as a method for producing a chromatographic packing material that can introduce A groups, can withstand use in an alkaline region that could not be achieved using conventional techniques, and does not cause denaturation of biologically related substances.

[Brief explanation of the drawing]

Figures 1 and 2 are diagrams showing the results of separating various amino acids and proteins using the chromatographic separation agent obtained in the present invention. Patent applicant Mitsui Toatsu Chemical Co., Ltd.

Claims (3)

[Claims] (1) A chromatographic packing material in which a primary amino compound having an ion exchange group is bonded to the ester group of a synthetic polyamino acid spherical particle having an ester group in its side chain through an amide bond. (2) A chromatographic packing material in which a secondary amino compound having an ion exchange group is bonded to the ester group of a synthetic polyamino acid spherical particle having an ester group in its side chain through an imide bond. (3) The chromatographic packing material according to claim 1 or 2, wherein the spherical particles of the synthetic polyamino acid are crosslinked particles.