JPH09301982A - Separating agent and separation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a separating agent high in separation efficiency, capable of separating a compound to form a complex with a boronic acid group, by polymerizing a boronic acid group-containing monomer with a monomer to provide a polymer with a lower critical solution temperature and another monomer. SOLUTION: (A) A boronic acid group-containing monomer (e.g. 3- acryloylaminophenylboronic acid) in an amount of 0.1-90mol%, (B) 0.1-90mol% of an unsaturated monomer (e.g. N,N-dimethylacrylamide) to provide a polymer and with a lower critical solution temperature and (C) 0-60mol% of another monomer (e.g. acrylamide) are dissolved in ethanol, etc., mixed with 2,2'-azobis(2,4-dimethylvaleronitrile) as a polymerization initiator and polymerized at 45 deg.C for 0.5 hour to give a polymer having 10-50,000 polymerization degree. The objective separating agent comprises the polymer, is capable of forming a complex with a boronic acid group and separating compounds such as various kinds of catechols, nucleoside, nucleotide, saccharides, membrane glycoprotein, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a separating agent for a compound capable of forming a complex with a boronic acid group and a separating method using the separating agent. More specifically, it relates to a separating agent for separating a compound capable of forming a complex with a boronic acid group using a specific boronic acid group-containing polymer, and a separation method using the separating agent. The present invention relates to a liquid-liquid phase separation, and further to a separation agent applicable to column separation and a separation method thereof.

[0002]

2. Description of the Related Art Examples of methods for separating low molecular weight compounds include distillation, sublimation, extraction, crystallization, column separation using various packing materials, and methods utilizing liquid-liquid phase separation. Liquid-
Liquid phase separation is formed by dissolving and mixing one or two specific polymers in the same liquid, and usually separating the liquid and a concentrated polymer solution or two polymer solutions into two layers. The polymer solution separated into two layers may appear in a suspended state (coacervate) in some cases.
In order to achieve the separation of compounds utilizing liquid-liquid phase separation, it is achieved by adding a polymer solution that causes phase separation to a compound dissolved in the same solvent as the solvent used for the phase separation system used, Since the compound is distributed between the two separated layers with a constant partition coefficient, the compound is concentrated in a layer with a high partition coefficient, and when the mixture sample is used, the separation of the compounds is possible.

To date, various polymers that form liquid-liquid phase separations are known. For example, Walter, H., Brooks, E.
D., and Fisher, D., "Partitioning in aqueous two-
There is a detailed report including references in phasesystems, Academic Press, London, (1985). In addition, as a report of controlling liquid-liquid phase separation by temperature, Polymer No. 37
Volume 681-685 (1996) (Hiroaki Miya
zaki, and KazunoriKataoka, Polymer, 37 (1996) 681-
685).

In the above-mentioned system, the low molecular weight compound is separated by utilizing the passive partition coefficient of the desired low molecular weight compound to be separated with respect to the polymer solution. Therefore, more sufficient separation efficiency could not be obtained.

Regarding the liquid-liquid phase separation using a polymer having a boronic acid group in its side chain, Hiroaki Miyazaki et al., Proceedings of Symposium on Autonomous Responsive Materials, pp. 94-95 (1995). Although described, nothing is known about its application in the separation of compounds.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a separating agent for efficiently separating a compound capable of forming a complex with a boronic acid group by using a polymer containing a boronic acid group. Furthermore, the present invention provides a method for efficiently separating a compound capable of forming a complex with a boronic acid group using the polymer. Also, for more details,
The object of the present invention is to cause liquid-liquid phase separation, which allows the compound to be distributed and separated,
An object of the present invention is to provide a polymer whose phase separation state can be controlled by controlling the temperature and adding a compound capable of forming a complex with a boronic acid group, and to provide a method for separating the polymer.

[0007]

Means for Solving the Problems As a result of intensive studies on the above problems, the present inventors have found that a polymer obtained by polymerizing a specific boronic acid group-containing monomer has a lower critical solution temperature, It was found that the compound capable of forming a complex with the boronic acid group and the boronic acid group form a complex to enhance the separation efficiency. In other words, in the separation of compounds, it is possible to form a complex with a boronic acid group by introducing a boronic acid group capable of specifically forming a complex with a boronic acid group into a side chain of a polymer. In order to actively improve the partition coefficient of various compounds,
It was found that the partition coefficient becomes higher, and as a result, the compound capable of forming a complex with the boronic acid group can be efficiently separated, and the present invention has been completed. That is, the present invention provides a component, a boronic acid group-containing unsaturated monomer, a b component,
It is a separating agent for a compound capable of forming a complex with a boronic acid group, which is composed of a polymer obtained by polymerizing an unsaturated monomer which imparts a lower critical solution temperature to the polymer. Furthermore, component a, a boronic acid group-containing unsaturated monomer 0.1 to 90 mol%,
Component (b), a lower critical co-solution obtained by polymerizing 0.1 to 90 mol% of an unsaturated monomer that gives a lower critical solution temperature to the polymer, and component (c) and 0 to 60 mol% of another unsaturated monomer. It is a separating agent for a compound capable of forming a complex with a boronic acid group consisting of a polymer having a temperature. Further, using the above-mentioned polymer separating agent, (1) mixing of the separating agent and a compound capable of forming a complex with a boronic acid group (2) homogenization (3) heating to carry out the phase separation step Is a method for separating a compound capable of forming a complex with a boronic acid group.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The polymer used in the present invention includes a boronic acid group-containing unsaturated monomer of component a, a monomer which imparts a lower critical solution temperature to a polymer of component b, and further c It is a polymer obtained by polymerizing the other unsaturated monomer as a component.

The boronic acid group-containing monomer of the component a used in the present invention is represented by the following formula (1)

Embedded image (However, R ¹ , R ² , and R ³ are a hydrogen atom or a methyl group, and Y is

Embedded image It is. ) Is an unsaturated monomer containing a polymerizable double bond and a boronic acid group. Specific examples include, for example, 3-acryloylaminophenylboronic acid, 3-methacryloylaminophenylboronic acid, 4-vinylphenylboronic acid, 3-acrylamido-4-heptafluoropropylphenylboronic acid, 3-acrylamido-
2,5-bis (ω-hydroperfluorobutyl) phenylboronic acid and the like can be mentioned. These 1 type (s) or 2 or more types can be used. The boronic acid group-containing unsaturated monomer of the component a is 0.1 to 90 mol% in all unsaturated monomers.
Used in the range of 0.5 to 70 mol%
It is good to use in the range of.

The unsaturated monomer which imparts the lower critical solution temperature to the polymer of the component b has a property that the polymer obtained by the polymerization has a lower critical solution temperature (hereinafter abbreviated as LCST). As shown, the polymer obtained by polymerizing only the unsaturated monomer of the component b does not necessarily have an LCST with respect to water between 0 ° C and 100 ° C. Specific examples include, for example, N, N-dimethylacrylamide, N-
Examples thereof include ethyl acrylamide and N-acryloyl pyrrolidone. One or more of them can be used. The total amount of the unsaturated monomers is 0.1
Used in the range of 90 to 90 mol%, but preferably 30 to
It is preferably used in the range of 80 mol%.

The polymer used in the present invention includes, in addition to the unsaturated monomer having a boronic acid group as the component a and the unsaturated monomer which imparts a lower critical solution temperature to the polymer as the component b, It is possible to copolymerize another unsaturated monomer of the component c. In this case, any polymerizable unsaturated monomer may be used. As specific examples, acrylamide, methacrylamide, N-methylacrylamide, N-isopropylacrylamide, Nt-butylacrylamide, acryloylmorpholine, acrylic acid, methacrylic acid, various alkyl acrylates, various alkyl methacrylates, 2-hydroxyethyl methacrylate, N-vinylpyrrolidone, monoglycerol methacrylate, acrylonitrile, styrene, various macromonomers, vinylbenzylamine, aminostyrene, 2-vinylpyridine, 3
-Vinylpyridine, 4-vinylpyridine, 2-vinylimidazole, N-methyl-2-vinylimidazole, N
-Vinylimidazole, N, N-dimethylaminopropyl acrylamide, N, N-dimethylaminopropyl methacrylamide, N, N-dimethylaminoethyl acrylate, N, N-dimethylaminoethyl methacrylate, N, N-diethylaminoethyl acrylate, N ，
N-diethylaminoethyl methacrylate, N, N-dimethylaminopropyl acrylamide various quaternary salts, N,
Various quaternary salts of N-dimethylaminopropyl methacrylamide, various quaternary salts of N, N-dimethylaminoethyl acrylate, various quaternary salts of dimethylaminoethyl methacrylate, various quaternary salts of N, N-diethylaminoethyl acrylate, N, N- Diethylaminoethyl methacrylate various quaternary salts, vinyl acetate, N- [tris (hydroxymethyl) methyl] acramide, itaconic acid, metaconic acid,
Examples include fumaric acid, maleic anhydride, aminostyrene, vinylbenzylamine and the like. One or more of these can be used. The other monomer of the component c is used in the range of 0 to 60 mol% in the total unsaturated monomer, preferably 0 to 30 mol%.

The polymer used in the present invention is prepared by adding a monomer composition comprising an unsaturated monomer of component a and component b and, if necessary, component c, to a known solution, block, emulsification or suspension weight. Polymerization temperature 0 to 100 ° C., polymerization time 10 minutes to
It can be obtained by polymerizing under the condition of 48 hours. As the polymerization initiator used for the polymerization, 2,2'-azobis (2-amidinopropane) dihydrochloride, 4,4'-azobis (4-cyanovaleric acid), 2,2'-azobis [2
-(5-Methyl-2-imidazolin-2-yl) propane] dihydrochloride, 2,2'-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride, 2,2'-azo Bisisobutyramide dihydrate, ammonium persulfate, potassium persulfate, benzoyl peroxide, diisopropyl peroxydicarbonate, tert-butyl peroxy-2-ethylhexanoate, tert-butyl peroxypivalate, tert-butyl peroxydiisobutyrate , Lauroyl peroxide, azobisisobutyronitrile, 2,2′-azobis (2,4-dimethylvaleronitrile) and the like. These may be used alone or in combination with various redox initiator systems. Moreover, you may use 1 type (s) or 2 or more types of these polymerization initiators. The amount of the polymerization initiator used is preferably 0.01 to 5% by weight based on all unsaturated monomers.

The degree of polymerization of the copolymer used in the present invention is 1
It is preferably 0 to 50,000. From the relationship between the boronic acid group content and the viscosity of the solution containing the polymer, 100 to 5,000 is more preferable. Further, it is desirable that the concentration of boronic acid in one molecule of the copolymer is 2 or more, but the physiological p
In order to maintain the binding force of boronic acid at H = 7.4, 20
It is preferable that the number is at least one.

The polymer is used in an aqueous medium or 5
It is used in an aqueous medium containing 0% or less of an organic solvent, but is preferably used with a buffer solution. Specific examples of the buffer solution include phosphate buffer solution, phosphate physiological buffer solution, Good's buffer solution (Hepes buffer solution, Mops buffer solution, etc.) and the like. The pH value at this time can be 2 to 10, but the pH value is preferably 6.5 to 10 when positively utilizing the complex forming ability of the compound capable of forming a complex of a boronic acid group.

When the polymer of the present invention is used for compound separation in the aqueous medium, its final concentration is 0.5.
Although it can be used at a concentration of up to 300 mg / g, it is preferably used at 5 to 100 mg / g in view of viscosity and separation efficiency.

The compound capable of forming a complex with the boronic acid group used in the present invention is specifically various catechols such as dopa, dopamine and epinephrine; various nucleosides such as adenosine, guanosine and inosine; AM
Various nucleotides such as P, ADP, ATP, c-AMP, GMP uridine, thymidine, FAD, NADP; r-
RNA, various RNAs including t-RNA; various sugars such as fructose, glucose, ribose, glucitol, mannitol, sorbitol, adenosine; and their modified products, glycoproteins such as membrane glycoprotein, IgG; lactic acid,
Examples include doxorubicin and tyrosine.

The method of separation using the polymer separating agent described above comprises the following steps. That is, (1) mixing of a compound capable of forming a complex with a separating agent and a boronic acid group (2) homogenization (3) phase separation by heating, separation of a compound capable of forming a complex with a boronic acid group Is the way.

The specific method of separating the compound capable of forming a complex with the boronic acid group of the present invention is to dissolve the sample to be separated in the polymer solution and raise the temperature. The temperature to be increased depends on the polymer used and the buffer used, but it is necessary to raise the temperature to a temperature at which the system shows liquid-liquid phase separation or higher. By doing so, the uniform separating agent and the aqueous solution of the compound capable of forming a complex with the boronic acid group are in a liquid-liquid phase separation state. At this time, each layer may be immediately separated into two layers, but may be in a suspended state (coacervate). In that case, the two layers can be rapidly separated by centrifugation at a temperature at which phase separation occurs.

In the final separation of the target compound after formation of the liquid-liquid phase separation, the compound to be isolated by the separation operation is a compound capable of forming a complex with a boronic acid group represented by a polyvalent hydroxyl compound. In some cases, the compound to be removed by the separation operation may be a compound capable of forming a complex with a boronic acid group represented by a polyvalent hydroxyl compound, but in the former case, the target compound is in the layer of the polymer solution, After separating the layers of polymer solution, the temperature was adjusted to LC
The temperature of the solution of the medium is lowered to a temperature lower than ST.
By dissociating the complex, or by adding a compound that forms a complex with a boronic acid group typified by other polyvalent hydroxyl compounds, the complex is dissociated, and subsequently, a polymer is usually separated. It can be separated by a gel filtration method, a reprecipitation method or the like which is used. In the latter case,
Since the target substance is in the layer which is not the polymer solution, the separation is completed by taking out the layer.

[0020]

The separating agent for the compound of the present invention is an unsaturated monomer which imparts a lower critical solution temperature to the boron group-containing unsaturated monomer of the component a and the polymer of the component b, if necessary. A polymer obtained by polymerizing the other unsaturated monomer of the component c, and by introducing a boronic acid group into the polymer, a compound capable of forming a complex with the boronic acid group and a boronic acid group can be formed into a complex, It is possible to separate with a higher partition coefficient. Further, the separation method of the present invention makes it possible to efficiently separate a compound soluble in a solvent containing 50% or more of water by liquid-liquid phase separation.

[0021]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail based on embodiments. Synthesis Example 1 N, N-dimethylacrylamide (hereinafter abbreviated as DMAA) and 3-acryloylaminophenylboronic acid (hereinafter abbreviated as AAPBA) were dissolved in ethanol at a concentration of 0.5 mol / liter, and polymerization was performed. 2,2′-azobis (2,4-dimethylvaleronitrile) (V-65) as an initiator was dissolved to a concentration of 2.5 mmol / liter, and the solution was added at 45 ° C. for a polymerization time of 0.5 h. Polymerization was carried out. After completion of the reaction, the reaction solution was dropped into ether to precipitate the polymer, which was filtered and dried overnight under reduced pressure, and the yield of the polymer was determined by weighing. The respective results are shown in Table 1 as Synthesis Examples 1 to 3. The content (mol%) of the boronic acid monomer in the obtained copolymer was 7 mg of the copolymer.
Dissolve in ¹ ml of MSO-d6, 400MHz ¹ H-NM
R (JNM-EX400, JEOL, LTD) measurement was performed. The results are shown in Table 1.

[0022]

[Table 1]

Synthetic Examples 2 and 3 In the same manner as in Synthetic Example 1, the monomers were synthesized with mol% of the monomers shown in Table 1. The results are shown in Table 1.

Synthesis Example 4 Solution polymerization was carried out in the same manner as in Synthesis Example 1 except that 3-methacryloylaminophenylboronic acid (hereinafter abbreviated as MAPBA) was used instead of AAPBA in the composition shown in Table 1 to obtain a copolymer. It was The results are shown in Table 1.

Synthesis Example 5 In the composition shown in Table 1, DMAA was replaced with N-ethylacrylamide (hereinafter abbreviated as EAA), and AAPBA was replaced with 3-methacryloylaminophenylboronic acid (hereinafter MAPBA).
Was abbreviated), and solution polymerization was performed in the same manner as in Synthesis Example 1 to obtain a copolymer. The results are shown in Table 1.

Synthesis Example 6 As shown in Table 1, solution polymerization was conducted in the same manner as in Synthesis Example 1 except that the compositions of DMAA, AAPBA and methyl methacrylate (abbreviated as MMA) were changed to obtain a copolymer. The results are shown in Table 1.

Synthesis Example 7 As shown in Table 1, solution polymerization was performed in the same manner as in Synthesis Example 1 except that the compositions of DMAA, AAPBA and isopropyl methacrylate (abbreviated as IPPAAm) were changed to obtain a copolymer. The results are shown in Table 1.

[Measurement of Molecular Weight of Copolymer] The weight degree (n) of the obtained copolymer was measured by using a gel permeation chromatography (GPC) apparatus in a column (GM of Tosoh GM).
HHR-MX2) was connected, and measurement was performed using polystyrene as a standard substance in a DMF system. The results are shown in Table 1.

Comparative Synthesis Example 1 N-ethylacrylamide (hereinafter abbreviated as EAA) alone was subjected to solution polymerization in the same manner as in Synthesis Example 1 without adding AAPBA to obtain a polymer. The results are shown in Table 2.

[0030]

[Table 2]

Comparative Synthesis Example 2 Solution polymerization was conducted in the same manner as in Synthesis Example 1 except that phenylacrylamide (hereinafter abbreviated as PA) was used instead of AAPBA to obtain a copolymer in a yield of 25.2%. The structural analysis of the obtained copolymer was carried out using 7 mg of the copolymer as DMSO-d6.
It was dissolved in ¹ ml and subjected to ¹ H-NMR. The content of phenylacrylamide in the obtained copolymer was 18.
3 mol%. The results are shown in Table 2.

The lower critical solution temperature was measured according to the following method. [Lower critical solution temper
ature: LCST)] Copolymer at 0.1 wt% p
H7.4 0.05M phosphate buffered saline (Na2H
PO4; 5.265 g / l, NaH2PO4;
0.8269 g / liter, NaCl; 8.0 g / liter, KCl; 0.2 g / liter), and the measuring cell containing this solution was mounted in a Peltier-type constant temperature cell holder, while maintaining the temperature for 5 minutes. Raise it by 1 degree,
While measuring the temperature in the cell, the transmittance at 500 nm was measured with a spectrophotometer (Ubest-50, manufactured by JASCO). The temperature at which turbidity occurred was LCST.

Example 1 0.1% by weight of the copolymer of Synthesis Example 1 in NaCl; 8.0
g / l, KCl; pH containing 0.2 g / l
Dissolved in 5 ml of 7.8 0.05M phosphate buffered saline, and further added doxorubicin (hereinafter abbreviated as DOX) 10, 40, 80, 10 as a polyvalent hydroxyl compound.
It was dissolved so as to be 0 μg / ml. After that, LCS
Coacervates were formed by incubating at T + 10 ° C for 24 hours and the solution was incubated at 400G for 10
The coacervate phase was separated by centrifuging for minutes. The amount of DOX distributed to the coacervate phase was calculated by measuring the absorbance of this supernatant at 485 nm. In determining the distribution amount, a DOX calibration curve prepared in advance from the absorbance at 485 nm was used. FITC-albumin was also subjected to fluorescence analysis in the same manner as DOX (excitation wavelength 490 nm, fluorescence wavelength 520 n
m) to quantify and calculate the distribution amount. Table 3 shows the results.
It was shown to.

Examples 2 to 5 In the same manner as in Example 1, the distribution amount was determined using the copolymers of Synthesis Examples 2 to 5 shown in Table 1. Table 3 shows the results.

Comparative Example 1 The amount of DOX and FITC-albumin distributed in the coacervate phase of the polymer obtained in Comparative Synthesis Example 1 was measured in the same manner as in Example 1. Table 3 shows the results.

Comparative Example 2 The amount of DOX and FITC-albumin distributed to the coacervate phase of the copolymer obtained in Comparative Synthesis Example 2 was measured in the same manner as in Example 1. Table 3 shows the results.

[0037]

[Table 3]

Example 6 Using the copolymer of Synthesis Example 3 and using catechol instead of doxorubicin in Example 1 280
The absorbance was measured in nm and evaluated in the same manner as in Example 1. The results are shown in Table 4.

Example 7 The copolymer of Synthesis Example 6 was used in place of the copolymer of Synthesis Example 3 in Example 6, and the evaluation was performed in the same manner as in Example 6. The results are shown in Table 4.

Example 8 The copolymer of Synthesis Example 7 was used in place of the copolymer of Synthesis Example 3 of Example 6, and the evaluation was performed in the same manner as in Example 6. The results are shown in Table 4.

[0041]

[Table 4]

From the above results, it can be seen that the embodiment has a higher distribution coefficient than the comparative example.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Yasuhisa Sakurai 3-17-6 Eifuku, Suginami-ku, Tokyo

Claims (3)

[Claims] 1. A component, a boronic acid group-containing unsaturated monomer, b
A separating agent for a compound capable of forming a complex with a boronic acid group, which is formed of a polymer obtained by polymerizing a component and an unsaturated monomer that imparts a lower critical solution temperature to the polymer. 2. A component, a boron group-containing unsaturated monomer 0.1.
˜90 mol%, b component, 0.1 to 90 mol% of an unsaturated monomer that gives a lower critical solution temperature to the polymer, c component, and 0 to 60 mol% of other monomer. A polymer having a degree of polymerization of 100,000 to 50,000 having a critical solution temperature.
A separating agent for a compound capable of forming a complex with the described boronic acid group. 3. A separating agent according to claim 1 or 2 is used. (1) Mixing of a separating agent and a compound capable of forming a complex with a boronic acid group (2) Homogenization (3) Heating and phase A method for separating a compound capable of forming a complex with a boronic acid group, which comprises performing a separation step.