JPH03267148A - Adsorbent for resolving optically active substance - Google Patents

Abstract

PURPOSE:To obtain a polymer adsorbent for resolving an optically active substance having especially high substrate selectivity by using a cross-linked polymer contg. an optically active synthetic polyamino acid and a hydrophobic polymer as constituents. CONSTITUTION:An optically active synthetic polyamino acid represented by general formula I (where n is an integer of >=5, R is an org. group and R' is H or alkyl) and a hydrophobic polymer swellable with an org. compd. such as benzene or toluene as a solvent in adsorption operation are used as constituents and cross-linking is caused to obtain a cross-linked polymer as an adsorbent. This adsorbent preferentially adsorbs one component of a racemic mixture and performs optical resolution with high efficiency because of the specific three dimensional structure of the polyamino acid and asymmetric environment due to the structure.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a novel adsorbent, particularly a polymeric adsorbent for the separation of optically active substances with a high degree of substrate selectivity.

BACKGROUND OF THE INVENTION Optical resolution, ie, the separation of racemic mixtures into optical antipodes, is a very important technique in the pharmaceutical, agricultural, food, and other industries. The usual method is to convert a racemic mixture into a mixture of diastereomers, and then separate the diastereomer mixtures based on the differences in their physical properties, but the types of racemic mixtures that can be separated by this method are limited. It is being

In addition to these conventional methods, techniques for resolving racemic mixtures by chromatography have been actively researched in recent years. In this case, an optically active adsorbent, such as a styrene resin carrying optically active L-hydroxyproline, is used as a filler, and the mixture is brought into contact with a divalent copper ion solution to form a complex. There are examples of optical resolution of amino acids by ligand exchange.

In addition, a graft polymer obtained by reacting an α-amino acid anhydride with a backbone polymer (crosslinked styrene-divinylbenzene copolymer →) having a halomethylated or hydroxymethylated phenyl group or an aminomethylated phenyl group is It is described that it can be used as a resin for racemic resolution of various amino acids (Japanese Patent Publication No. 52-9233, Japanese Patent Publication No. 52-9233).
In the conventional methods, the degree of splitting is extremely low, and in the method using ligand exchange, copper ions are eluted at the same time as the split amino acids. Practical application is quite difficult. Other analytical uses include porous silica gel coated with optically active poly(triphenylmethyl methacrylate);
It's not durable.

In addition, in the case of a polymer in which an amino acid is graft-polymerized to a crosslinked styrene-divinylbenzene copolymer using the above-mentioned base polymer,
Although the crosslinked styrene-divinylbenzene copolymer is hydrophobic, it is necessary to increase the hydrophilicity of the separating agent since it is used for split separation of amino acids using an aqueous solution. Therefore, the amount of grafted polyamino acids with high hydrophilicity was reduced to 50%.
However, the separation efficiency is extremely low6.Therefore, at present, there is no product that is satisfactory in terms of durability, price, ease of manufacture, etc.

An object of the present invention is to provide a novel adsorbent for optical resolution that can solve the above problems.

Means for solving the problem It is thought that the high substrate selectivity of enzymes is due to the unique three-dimensional structure of the polyamino acids that make up the enzymes. As a result of extensive research into polymer adsorbents with
There are also adsorbents that limit the solvent used for optical resolution to organic solvents.

We have discovered that due to the unique three-dimensional structure of the polyamino acid component and the asymmetric environment based thereon, one side of the racemic mixture can be preferentially adsorbed and optical resolution can be performed with high efficiency, and the present invention has been achieved. It is something.

That is, the present invention is a general formula.

ORR' (wherein n is an integer of 5 or more, R is an organic group, and R' is H or an alkyl group) and an optically active synthetic polyamino acid represented by ORR' and an organic solvent used in the adsorption operation. The present invention relates to an adsorbent for separating optically active substances using an organic solvent, which is made of a crosslinked polymer containing a swellable hydrophobic polymer component as a constituent component.

The optically active synthetic polyamino acid that is a component of the adsorbent of the present invention may form a ring. These may be substituted with various groups, and examples of substituents include 10H1-COOH
Examples include l-8H1-NH, -8CH, and the like. When having the above substituent, the substituent can also be in the following form. That is, -〇H1-8H is an ether (e.g.
methyl ether, benzyl ether) form, form with an acyl group, C0OH is ester form, amide form, NH
2 has a form with a carbobenzoxy group, etc. attached. R' is hydrogen or an alkyl group, and examples of the alkyl group include a methyl group and an ethyl group.

ORR' Any organic group may be used for R in .

Alkyl groups, aromatic groups such as phenyl groups, aralkyl groups,
Examples include heterocyclic-containing groups containing nitrogen as a ring member, and specific examples of α-aminocarboxylic acids in which R and N are bonded include optically active amino acids 1 that constitute proteins, e.g. Alanine, valine, leucine, phenylalanine, proline, etc., and β-benzylaspartate, γ-methylglutamate, γ-benzylglutamate, E-carbobenzoxylysine, δ-carbobenzoxyornithine, 0-acetyltyrosine, O-benzyl In addition to D or L forms of derivatives of protein-constituting amino acids such as serine, optically active α-aminocarboxylic acids other than protein-constituting amino acids (eg, sarcosine) and derivatives thereof are used.

In the constituent components of the above general formula, n is 5 or more, and 10
Although it is generally 0 or less, 10 to 40 is particularly preferable.

The adsorbent of the present invention is a crosslinked polymer containing the above-mentioned optically active synthetic polyamino acid and/or its derivative as a constituent, and is produced by reacting a crosslinked polymer carrier with an optically active polyamino acid or α-amino acid anhydride. The excellent substrate selectivity of the adsorbent of the present invention, which is achieved by grafting a polyamino acid onto a crosslinked polymer carrier, is derived from its constituent optically active synthetic polyamino acids and/or derivatives thereof. Other components need to swell in the organic solvent used in the adsorption operation. That is, when an organic compound such as benzene or toluene is used as a solvent in an adsorption operation, a hydrophobic polymer is used as a constituent other than the polyamino acid.

The molecular weight, crosslinking density, etc. of the crosslinked polymer serving as the adsorbent of the present invention can be appropriately selected depending on the object to be adsorbed.
Further, the ratio of the synthetic polyamino acid component in the adsorbent can be selected as appropriate, but it is 1 to 99%, preferably 10 to 60%.
%. The adsorption effect of the adsorbent of the present invention uses an organic solvent system, but it also depends on the functional groups, stericity, etc. of the substance to be adsorbed. etc. can be changed as appropriate.

For example, a mixture of several types of polyamino acids can be used, or R and R' of the amino acid constituent units can be varied in one molecule. In the case of a mixture of these,
Optically inactive amino acids such as glycine can also be used in combination with the optically active polyamino acids described above, as long as they can be copolymerized by reacting with the α-amino acid anhydride.

The adsorption properties of the adsorbent of the present invention can be adjusted by crosslinking density or porosity, and the crosslinking density can be changed as appropriate depending on the substance to be adsorbed, but in general, the crosslinking density tends to decrease as the molecular weight of the substance to be adsorbed increases. There is.

In addition, the adsorbent of the present invention can be made porous by using a diluent, etc., as described later.
(approximately 0 times).

The crosslink density of the adsorbent of the present invention changes depending on whether it is made porous or not, but the crosslink density when it is not made porous is 0.01 to 50.
%, preferably 0.5 to 10%, and the crosslinking density in the case of making it porous is 0.1 to 100%, preferably 5 to 30%.
%.

The interaction between the adsorbent and the substance to be adsorbed is affected by various factors such as hydrophobic bonds, ionic bonds, hydrogen bonds, and steric hindrance. The amino acids in the crosslinked polymer of the agent shall be selected.

The present adsorbent is manufactured by the following method.

(1) General formula: N-carboxy anhydride (hereinafter referred to as
NCA) is synthesized from the corresponding optically active amino acid or its side chain derivative by a known method. Details of this method can be found, for example, in Murray Gutsman (M, Goodm).
an), Biopolymers
, Vol. 15, p. 1869 (1976).

Optically active synthetic polyamino acids or their side chain derivatives used in the synthesis of NCA include alanine, valine, leucine, phenylalanine, etc., β-benzylaspartate, γ-methylglutamate, γ-benzylglutamate, ε-carboxylate, etc. Benzoxylysine, δ-carbobenzoxyornithine, O-acetyltyrosine, 0
An example is one of the D or L forms such as benzylserine, but the types of usable optically active amino acids or derivatives thereof are not limited to those listed here. For example, glutamic acid γ-ester derivatives include methyl, ethyl, propyl, benzyl,
Various esters such as p-nitrophenyl and cyclohexyl can be used. It is also possible to mix a plurality of optically active synthetic polyamino acids and/or their side chain derivatives to obtain a mixture of corresponding NCAs. In addition, when synthesizing NCA, the carboxyl group of acidic amino acids, the amino group, hydroxyl group of basic amino acids, etc. are protected with protecting groups as described above, but these protecting groups may be left as is. However, it can be removed eventually.

A crosslinked polymer carrier having a functional group convertible into an amino group or into which an amino group can be introduced is produced by a known method, and the functional group is converted into an amino group or an amino group is introduced. Polymerizing NCA using the carrier having an amino group as an initiator,
A crosslinked polymer having an optically active synthetic polyamino acid supported on the carrier is obtained.

Examples of the crosslinked polymer carrier having a functional group convertible into an amino group or into which an amino group can be introduced include a chloromethylstyrene-styrene-divinylbenzene copolymer.

These crosslinked polymers can be produced by polymerization methods such as solution polymerization, bulk polymerization, and suspension polymerization using, for example, peroxide radical-forming compounds such as dibenzoyl peroxide and dilauroyl peroxide, or azobisisobutyronitrile, azobis( 2,4-dimethylvaleronitrile) or azobis(2-amidinopropane) dihydrochloride in a similar manner to known methods.

In the case of suspension polymerization, the following method is used, for example. First, the reaction raw material is an inert organic solvent, preferably an aromatic hydrocarbon such as benzene or toluene, or an aliphatic hydrocarbon such as n-octane, or cyclohexanol,
Dissolved in alcohols such as lauryl alcohol. As long as the amount of organic solvent can completely dissolve the macromer and monomer, it is particularly advantageous to use 1 part by weight of the solvent per 1 part by weight of the reaction raw material, but it is generally 0 to 3 parts by weight.
Parts by weight of solvent are used. This reaction solution is well mixed with an aqueous protective colloid solution, especially an aqueous polyvinyl alcohol solution, using an efficient stirrer, for example, using 2 to 25 parts by weight of the aqueous solution per 1 part by weight of this reaction solution. The stirred mixture is heated to about 40°C to 100°C, preferably about 60°C, under an atmosphere of non-reactive gas, particularly nitrogen. Polymerization time is about 4 hours to 72 hours, preferably about 24 hours.

The polymer thus obtained is separated from the reaction solution by filtration, first washed with a solvent that dissolves the protective colloid, and further washed with dioxane, methanol, acetone, etc., and then heated at a temperature of about 40°C to 80°C. Dry under reduced pressure.

In that case, a porous spherical gel can be obtained by adding a suitable diluent to the monomer phase.

As for the type of diluent, organic solvents that have low swelling properties for the resulting gel are suitable. For example, chloromethylstyrene
In the case of a styrene-divinylbenzene copolymer, octane, decane, dodecane, etc. are preferred. Alternatively, instead of a diluent, a linear polymer such as polystyrene, polymethylstyrene, polymethyl acrylate, etc. may be present in the coexistence for polymerization, and then the linear polymer may be extracted and removed from the resulting spherical gel to form a porous spherical gel. can. Porous formation is applied to materials with low or high crosslinking density, but is generally applied to materials with high crosslinking density. This is preferable because it facilitates contact with the group.

Further, those having a high degree of crosslinking are very preferable because they have little swelling and shrinkage and high mechanical strength, and are particularly preferable in the case of chromatography.

Details of the conversion reaction of a functional group in a copolymer into an amino group and the reaction of introducing an amino group can be found, for example, in Merryfield (R
, B, Merriefield) Journal of the American Chemical Society (J
, A, C, S,) Volume 98, Page 7357 (1976)
Or Inman (J.

K, I nman) Biochemistry (Bio-c
hemistry), Volume 8, Page 4074 (196
9) Or Die
Angew, Maklomol.

Chem, Vol. 63, p. 23 (1977).

After washing the obtained crosslinked polymer carrier having amino groups,
Completely dehydrate by Soxhlet extraction, etc., and thoroughly dry under reduced pressure and heat.

Polymerization of NCA uses amino groups in the crosslinked polymeric support as an initiator.

The polymerization temperature is about -10°C to 100°C, preferably about 20°C.
℃ to 40℃, and the polymerization time is about 0.5 hours to 120 hours, preferably about 24 hours to 72 hours so that n in the above-mentioned general formula, that is, the degree of polymerization, is 5 or more. Inert organic solvents used include 1,2-dichloroethane, chloroform, dioxane, tetrahydrofuran, N, N
-dimethylformamide, N,N-dimethylacetamide.

Acetonitrile, benzene, nitrobenzene, 〇nitroanisole, etc. are preferred. These solvents are preferably completely dehydrated and purified before use.

As for the type of amino group, a -class or secondary amino group is usually used, but it is particularly preferable to use a -class amino group as an initiator because it can quantitatively support the polyamino acid.

The crosslinked polymer in which the obtained optically active synthetic polyamino acid is supported on the crosslinked polymer carrier is washed with a solvent that dissolves the polyamino acid such as dimethylformamide or dioxane, and then washed with acetone or the like, and then heated. The purified crosslinked polymer is obtained by drying under reduced pressure.

(2) One type or several types of NCAs are mixed and polymerized with a monofunctional amine to obtain an optically active synthetic polyamino acid having an amino group at the terminal.

Separately, a crosslinked polymer carrier having a functional group capable of reacting with an amino group is produced by a known method and reacted with the above-obtained polyamino acid having an amino group to form an optically active synthetic polyamino acid. A crosslinked polymer supported on a carrier is obtained.

The reaction of supporting the polyamino acid onto the crosslinked polymer carrier is as follows:
About 40°C to 80°C in a solvent that dissolves the polyamino acid.
This is done by heating. The reaction time depends on the type of crosslinked polymer carrier used, but is about 24 hours to
Approximately 72 hours.

In both methods (1) and (2) above, the optically active synthetic polyamino acids used are those whose side chains can be converted by chemical methods such as converting an ester group into an amide group. It can be converted into an optically active synthetic polyamino acid having another chemical structure by carrying out conversion or the like. This chemical conversion is one of the very important manufacturing steps in obtaining an adsorbent suitable for the separation target.

In addition, in the crosslinked polymer, constituent components other than the polyamino acid (for example, the above crosslinked polymer carrier, etc.) may also be used as described above.
It can be converted into other chemical structures by chemical transformation.

This chemical conversion is also a very important manufacturing step in obtaining an adsorbent suitable for the separation target.

In the case of method (2) above, the polyamino acid can also be obtained by using a substance commonly used as this type of initiator as an NCA initiator instead of the monofunctional amine. Such initiators include, for example, secondary amines, tertiary amines, quaternary ammonium salts, alkali metal alkoxides, borohydrides or hydroxides, inorganic salts, organometallic compounds, amino-terminated polymers. etc.

Separation methods using this adsorbent generally include batch methods and column chromatography methods. When performing highly difficult separations such as the separation of optically active substances, it is preferable to obtain crosslinked polymers by suspension polymerization, size the particles, and perform separation by column chromatography using the resulting particles as an adsorbent.

Column chromatography is usually carried out using the following procedure. First, the adsorbent is suspended in the solvent used for elution, and the suspension is transferred to the column.The target substance to be separated is dissolved in as little solvent as possible, this solution is transferred to the top of the column, and this column is used as the eluent. The eluate is collected into each fraction in a conventional manner.

The degree of racemic resolution can be determined by measuring the optical rotation of each fraction.

By using the crosslinked polymer adsorbent according to the present invention, it is possible to resolve racemic mixtures that are soluble in a large variety of organic solvents.

The present invention will be specifically explained in the following production examples and examples, but the present invention is not limited only to these production examples and examples.

Production Example 1 29.7 g of phosgene was added to a mixture of 50 g of γ-benzyl monoglutamate and 500 ml of tetrahydrofuran.
Add a mixture of 62.3 g of benzene and heat to 1.5 at 65°C.
Stir for hours. The resulting transparent reaction solution was diluted with petroleum ether 1
When poured into 1, white γ-benzyru L-glutamate NC
A (hereinafter referred to as γ-BLG-NCA) crystal was precipitated. The obtained crystals were taken, washed with petroleum ether, and then dried under reduced pressure over diline pentoxide. This stuff has a melting point of 93-9
The temperature was 4°C, the molecular weight was 263, and the yield was 50 g.

2.0 g of chloromethylstyrene, 2.74 g of 55% divinylbenzene (crosslinking agent), 26 g of styrene 95°, 75
A solution of 0.67 g of % dibenzoyl peroxide was added to a solution of 4.0 g of polyvinyl alcohol and 400 g of water.

The mixture was stirred at 1000 rpm for 10 hours at 70° C. under nitrogen. The resulting crosslinked chloromethylstyrene-styrene-divinylbenzene copolymer was isolated. The crosslinked polymer was a translucent spherical gel.

This crosslinked polymer85. sg as phthalimidocari85.
After mixing 8 g and 686 ml of DMF and stirring at 120° C. for 6 hours, the crosslinked polymer was taken out, washed and dried. Next, the cross-linked polymer was mixed with 68.6 ml of hydrazine hydrate and 686 ml of dioxane, and after stirring at 90°C for 6 hours, the cross-linked polymer in which the chloromethyl groups were converted to aminomethyl groups was taken out, thoroughly washed, and completely removed. Dry. The amino group content of this product was 0.17%.

81 g of the obtained gel-like crosslinked polymer carrier was mixed with 32.4 g of γ-BLG-NCA obtained above and 6 dioxane.
The mixture was dispersed in 48 ml of solution and stirred under nitrogen at 30° C. for 72 hours to polymerize, and then the crosslinked polymer was isolated and purified in the same manner as in Production Example 2. The elemental analysis values after loading were as follows.

C: 85.40% H: 7.15% N: 1.66% The content of PBLG calculated from the elemental analysis value (nitrogen 1.66%) is 23.8%, and its degree of polymerization is 11. 1
Met.

IR: 1735cm-' (ester) 1650cm
-' (amide) Production Example 2 40 g of the PBLG-supported crosslinked polymer obtained in Production Example 1 was dispersed in 200 ml of benzylamine, stirred at 60°C for 30 hours, and then isolated and purified. 1 in the IR spectrum
The absorption peak of ester at 735 cm''-' decreased, and 1
The amide absorption peak from 650 to 1670 cm increased. This revealed that benzyl ester was converted to benzyl amide. The conversion rate calculated from the decrease in the absorbance of the ester absorption in the IR spectrum was about 70%.

Elemental analysis values C: 86.24% H: 7.53% N: 2.40% Production example 3 Chloromethylstyrene 1. Og, 55% divinylbenzene (cross [pJ) 1 s, 27g, styrene 79.7
3g, 75% dibenzoyl peroxide 0゜67g, n
- A solution of 80.0 g of octane (diluent) was added to a solution of 4.0 g of polyvinyl alcohol and 400 g of water. The mixture was stirred at 1000 rpm for 10 hours at 70° C. under nitrogen. The resulting crosslinked chloromethylstyrene-styrene-divinylbenzene copolymer was isolated. The crosslinked polymer was a porous white spherical gel. A crosslinked polymer in which chloromethyl groups were converted to aminomethyl groups was obtained by the same method as in Production Example 1. The amino group content of this product was 0.08%.

81 g of the obtained porous crosslinked polymer carrier was mixed with γ-benzy-D-glutamate NCA3 obtained in the same manner as in Production Example 1.
Dispersed in a solution of 2.4 g and 648 ml of dioxane,
After polymerization by stirring at 30° C. under nitrogen for 72 hours, the crosslinked polymer was isolated and purified.

The content of poly(γ-benzy-D-glutamate) calculated from the elemental analysis value (N: 1.56%) after loading was 23
．． 5%, and the degree of polymerization was 21.9.

The benzyl ester of the obtained crosslinked poly(γ-benzyl-D-glutamate)-supported polymer was converted into benzyl amide in exactly the same manner as in Production Example 2. Conversion rate is about 8
It was 0%.

Example 1 Each of the crosslinked polymers obtained in Production Examples 1 and 2 was
The particles were classified using a mesh and a 400 mesh metal sieve, and those having a diameter of 37 to 63 μm were collected.

Using this classified crosslinked polymer as a filler, DL-mandelic acid was separated by chromatography. A Shimadzu LC-4A high performance liquid chromatography device was used for liquid feeding and detection. The chromatography conditions are as follows.

Column: inner diameter 16.7 mm, length 500 mm Eluent: 4
Toluene-dioxane mixture flow rate: 2 ml/min Temperature: 10°C Detection: Differential analysis Sample amount: 100 mg Table 2 shows the retention times of each of D-mandelic acid and L-mandelic acid.

Table 1 From Table 1, it can be seen that both adsorbents strongly adsorb the D form.

The attached figure shows the results of measuring the specific optical rotation of each fraction when the crosslinked polymer of Production Example 2 was used and fractionated using a fraction collector (JASCO DIP-14
A type 0 polarimeter is used to measure the polarimeter fraction/min. Since the specific optical rotation of the L-form of mandelic acid is (+), the above figure shows that the D-form is more strongly adsorbed by the adsorbent of the present invention, so the L-form elutes first, followed by the D-form. It was found that using the difference in adsorption (difference in elution rate), L
It is possible to separate the body and the D body.

Example 2 The crosslinked polymer obtained in Production Example 3 was classified to have a size of 20 to 40 μm.
I collected things. Using this classified crosslinked polymer,
Resolution of DL-mandelic acid by the same method as in Example 1,
Preparative separation was performed. The specific rotation is -98 for the initial fraction.
The final fraction was 60. It can be seen that the L-form is more strongly adsorbed with this adsorbent, and therefore the D-form is eluted first, followed by the L-form. In this case, the adsorption properties of D-form and L-form are opposite to those of Example 1, and the adsorbent of the present invention has
It can be seen that the adsorption capacity can be varied in various ways.

Since the adsorbent of the present invention can separate many types of racemic mixtures, it is extremely useful as an adsorption and separation agent, and is easy to manufacture and has excellent durability, making it suitable for industrial use. It is usable and highly practical.

[Brief explanation of drawings]

The attached figure is a graph showing an example of the effect of the adsorbent of the present invention.

Claims (1)

[Claims] (1) General formula, ▲Mathematical formula, chemical formula, table, etc.▼ (In the formula, n is an integer of 5 or more, R is an organic group, and R' is H or an alkyl group.) An adsorbent for the separation of optically active substances using an organic solvent, which is made of a crosslinked polymer containing as constituent components an active synthetic polyamino acid and a hydrophobic polymer component that swells in the organic solvent used in the adsorption operation.