JPH0778081B2 - Method for synthesizing functionalized polymer - Google Patents

Description

TECHNICAL FIELD The present invention relates to a functionalized polymer in which various functional groups or functional molecules are introduced into a carrier made of a polymer compound, and a method for synthesizing the functionalized polymer. The functionalized polymer according to the present invention includes an ion exchanger, an immobilized enzyme, an immobilized antibody, an affinity carrier,
As a selective ion-affinity polymer, etc., it can be widely used in various technical fields such as production of various chemical substances, dyeing, purification / separation, biochemistry, environmental protection, life sciences and so on.

(Prior Art) Functionalized polymers in which various functional groups or functional molecules are introduced into cellulose or other polymer carriers occupy an important position in laboratories related to life science and industry. Cellulose with covalently bonded diethylaminoethyl (DEAE), carboxymethyl (CM), Sephadex (cross-linked dextran), Sepharose (Pharmacia, Sweden)
Trade name of cross-linked agarose manufactured by LKB) is used as an ion exchanger for purification of proteins and nucleic acids, or as a carrier for chromatography for chemical analysis. Affinity carriers with various ligand molecules covalently bound to polymer carriers, immobilized enzymes with various enzyme proteins covalently bound to polymer carriers, immobilized antibodies with various antibodies covalently bound, and their applications are expanding. Is.

To synthesize a functionalized polymer, unique reaction conditions are required depending on the ligand molecule or protein to be covalently bound. DEAE and CM can be covalently bound under strong alkaline conditions, but mild conditions are required to prevent degradation or denaturation of ligands or proteins that are not alkali resistant. A commonly used current method is the cyanogen bromide method, which is a method in which a hydroxyl group of a polymer carrier is converted to an imidocarboxylic acid by a reaction with cyanogen bromide and then reacted with a ligand molecule or a protein having an amino group in a cold state ( Nature, 214
Vol., 1302-1304, 1967). However, in this method, volatile and toxic cyanogen bromide is used, and the introduced ligand or protein is leaky because it is bound to the polymer carrier by a labile isoureide bond, and may be a little leaky during long-term repeated use. Elute each (Biochem.Biophys.Re
s.Commun. 70, 8-14, 1976). In particular, the elution in the presence of a nucleophile is remarkable, and it is not suitable to use a buffer having an amino group. Various carrier binding methods other than the cyanogen bromide method have been proposed, but a general method applicable to all kinds of various ligands and proteins has not been established.

(Problems to be Solved by the Invention) The inventors of the present invention have conducted extensive studies to solve the above-mentioned problems, and as a result, the halogen derivative of the polymer carrier has strong reactivity with a molecule having an amino group and is mild. The present inventors have completed the present invention by discovering that a dehydrohalogenation reaction occurs under various conditions to synthesize a covalent bond between a polymer carrier and a ligand or a protein molecule.

That is, an object of the present invention is to covalently bond a functional ligand molecule or protein or the like to a polymer carrier under the same conditions, and to prevent a ligand or protein once introduced from elution during repeated use. It is to provide a functionalized polymer compound.

(Means for Solving the Problems) The above-mentioned object is achieved by converting a polymer compound having a hydroxyl group into a halogen derivative and then coupling the compound with an amino group-containing ligand molecule.

The amino group-containing ligand molecule is suitable because it can be coupled with a polymer compound having a hydroxyl group at room temperature.

The 2,3-dibromopropyl derivative or 3-bromo-2-hydroxypropyl derivative of the polymer compound having a hydroxyl group is a polysaccharide, its derivative or crosslinked product, polyvinyl alcohol, its crosslinked product or a mixture thereof is It is particularly preferable because it shows strong reactivity with an amino group.

When the above-mentioned polysaccharide and its derivative are cellulose and its derivative, the cellulose carrier can be formed into various forms such as powder, fiber, cloth, film, sheet, etc., and can be widely applied. Therefore, it is preferable.

When the polymer compound having a hydroxy group is a polysaccharide and the polysaccharide is a crosslinked polysaccharide such as Sephadex or Sepharose, a product with high utility value can be obtained.

A useful functionalized polymer is provided when the amino group-containing ligand molecule is at least one amino group-containing functional compound selected from the group consisting of coenzymes, polypeptides and proteins.

In the functionalized polymer of the present invention thus obtained, the oxygen atom on the polymer carrier and the nitrogen atom on the functional ligand molecule are bound via a 2-hydroxytrimethylene group or a 2-halotrimethylene group. It is characterized by being done.

(Operation) Hereinafter, the configuration of the present invention will be described in detail together with its operation.

To produce the functionalized polymer of the present invention, first, a double bond is introduced into the hydroxyl group of the polymer carrier.

For example, in the case of cellulose, an allylic derivative can be easily prepared by refluxing mercerized alkali cellulose with allyl chloride in a 2-propanol solvent, but other allyl halides or other double bond reagents can be used. Alternatively, the reaction may be carried out in a solvent such as dimethyl sulfoxide. The double bond-introduced allylcellulose can be easily converted into a 2,3-dibromopropyl derivative by immersing it in a sodium hydroxide solution of bromine or an alcohol solution of bromine for several minutes at room temperature. It is also possible to convert it to a 3-bromo-2-hydroxypropyl (bromohydrin) derivative by reacting a solution in which the proportion of bromine and sodium hydroxide is mixed to a composition that produces sodium hypobromite. In the reaction with chlorine, iodine, hypochlorous acid, hypoiodic acid, etc., the corresponding dichloropropyl, diiodopropyl,
It can be changed to chlorohydrin, iodohydrin derivative and the like. Of these, the dibromopropyl derivative and the bromohydrin derivative are relatively stable and can be stored for a long period of time, but can immediately proceed to the next coupling reaction.
Dehydrobromination of amino groups and bromine derivatives by diluting dibromopropyl derivative or bromohydrin derivative with a slightly alkaline aqueous solution of a ligand molecule, peptide or protein having an amino group or when the ligand molecule is a liquid and leaving it for several hours. As the reaction proceeds, the ligand molecule, peptide or protein is coupled to the polymer carrier to synthesize the functionalized polymer. The conditions for this coupling reaction are common to molecules having an amino group and do not require high temperature or strong alkali. It is an advantage of the present invention that all kinds of amino compounds are covalently bonded to a polymer carrier under such mild conditions. In this method, the reaction route when the halogen derivative is a 2,3-dibromopropyl derivative is represented by the following chemical formula.

Polymeric carrier -OH ↓ NaOH polymeric carrier _{-ONa ↓ ClCH 2 -CH = CH 2} ( reflux) polymeric carrier _{-O-CH 2 -CH = CH 2} ↓ Br 2 polymeric carrier -O-CH ₂ -CHBr- CH ₂ Br ↓ H ₂ N-R Polymer carrier-O-CH ₂ -CHBr-CH ₂ NHR That is, the method for synthesizing a functionalized polymer according to the present invention is to prepare a dibromopropyl derivative or bromohydrin derivative of a polymer carrier once. Then, it skillfully utilizes the property of causing a dehydrohalogenation reaction to couple with any molecule having an amino group under mild conditions of room temperature and slightly alkaline. The condensation reaction of a halogen derivative of an organic compound with a dehydrohalogenate of an amine generally requires a high temperature or an organic solvent, except when the halogen is chloroacetyl or bromoacetyl adjacent to a carbonyl group, and does not proceed under mild conditions. Was thought to be a thing. However, as shown in a number of examples below, condensation with dehydrohalogenated typified by dehydrobromide from dibromopropyl cellulose or bromohydrin cellulose and amine does not require high temperature, strong alkali, Proceed quickly. This is a surprising phenomenon that cannot be inferred from conventional general knowledge. Therefore, it is possible to perform coupling with dihalopropyl derivatives, particularly dibromopropyl derivatives, or halohydrin derivatives, especially bromohydrin derivatives, using various amines such as coenzymes, peptides, and proteins as ligands under mild conditions, and to react at low temperatures. As a result of investigating reaction conditions for a molecule having a mercapto group that does not proceed, the present invention exerts an excellent action as a general method for covalently bonding a wide range of ligand molecules to a polymer carrier.

(Examples) Hereinafter, the method of the present invention will be described with reference to Examples.

Production Example 2,3-Dibromopropylcellulose and 3-bromo-2-
Synthesis of Hydroxypropyl Cellulose Powdered cellulose (filter paper powder) was mercerized, suspended in a mixed solution of 20% by weight of allyl chloride and 80% by weight of 2-propanol, and refluxed under a reflux condenser. glucose
The number of allyl groups introduced per 100 units is 17, 20 in 9 hours
It was 36 in time.

Cellulose such as absorbent cotton, cotton cloth, and cellophane that were not in powder form could be mercerized in the same manner and then converted into an allyl derivative. The degree of substitution of cotton cloth tended to be slightly lower than that of filter paper powder.

The obtained allyl cellulose was repeatedly washed with cold water to remove excess alkali and reaction reagents. At this stage, it may be vacuum dried and stored at a low temperature, but it may immediately proceed to the next step of dibromination or bromohydrination.

Sibromination was performed as follows. First, bromine was dissolved in an equimolar aqueous solution of sodium hydroxide or potassium hydroxide to prepare a bromine solution, or an alcohol solution of bromine was prepared. The allyl group was quantitatively converted to a dibromopropyl group only by immersing the allyl cellulose in the solution at room temperature for a few minutes to obtain O- (2,3-dibromopropyl) cellulose. The elemental analysis values were as follows.

Analytical value: C 35.17, H 5.18 Br 24.16 Calculated value with substitution rate 36%, sample water content 3% by weight: C 35.24, H 5.11 Br 23.84 The obtained dibromopropyl derivative may be vacuum dried and stored as it is. However, the coupling reaction of Example 1 or later may be immediately advanced.

When carrying out bromohydrination, it is sufficient to dissolve bromine in a double molar amount of an aqueous solution of alkali hydroxide to prepare a hypobromous acid solution, and soak the allyl cellulose described above at room temperature for several minutes. O- by the bromohydrination of the allyl group
(3-Bromo-2-hydroxypropyl) cellulose was obtained.

Example 1 Amino acid derivative of cellulose Dibromopropyl cellulose 1.0 synthesized in the above production example
Grams were soaked in a slightly alkaline aqueous solution (pH = 11) of amino acids such as alanine and aspartic acid at room temperature or high temperature.
When binding an unstable amino acid at high temperature, the reaction was carried out near room temperature. After the reaction, the product was repeatedly washed with water to remove excess reagents, and 0.8 g of amino acid-bound cellulose derivative was obtained.

Example 2 Synthesis of FAD cellulose Dibromopropyl cellulose 1.0 synthesized in the above production example
Trisodium phosphate 0.05 g containing 0.5 g flavin adenine dinucleotide (FAD) at room temperature to about 45 ° C
After immersing in 10 ml of M aqueous solution and repeatedly washing with water, unbound F
0.8 g of FAD cellulose was obtained by removing AD. This derivative turned yellow due to the bound flavin, and could be used as an affinity chromatography carrier for flavoproteins.

Example 3 Protein-immobilized cellulose Dibromopropyl cellulose 1.0 synthesized in the above production example
Gram gram at room temperature to 37 ° C with 0.01 g of cytochrome c ₃ containing sodium azide trisodium phosphate 0.05M aqueous solution 1
After soaking in 0 ml and repeatedly washing with water, non-bonded cytochrome c ₃
Was removed to obtain 0.8 g of cytochrome c ₃ cellulose. This cytochrome c ₃ cellulose retained its biochemical activity as an electron carrier by the reaction with the enzyme hydrogenase.

Dibromopropyl cellulose (1.0 g) was similarly immersed in 10 ml of 0.05 g aqueous solution of glucoamylase containing 0.05 g of sodium azide and then washed repeatedly with water to remove unbound enzyme. Gram) was synthesized.

Example 4 Functionalized Polymer Other than Cellulose Allyl sephadex, allyl sepharose, and allylated polyvinyl alcohol were dibrominated according to the above-mentioned production example, and then immersed in various ligand molecules having an amino group (liquid or solution), Similar to the case of cellulose, it was possible to synthesize a functionalized polymer in which a ligand molecule was covalently bound.

(Effects of the Invention) As described above, according to the method of the present invention, as long as the functional molecule to be introduced has an amino group, all of them are easily bonded to the polymer carrier under the same mild conditions and introduced. Since the ligand or protein molecule forms a stable C—N—C bond with the polymer carrier, it does not elute during repeated use, and the problems of the prior art could be solved. In addition to the above, this method can also be applied to the binding of a ligand molecule having a mercapto group or a phosphite by changing the reaction conditions, and can be widely used.

Furthermore, the functionalized polymer of the present invention is expected to be used in the purification of antibodies and nucleic acids in the bio industry. In addition, most of the synthesized cellulose derivatives have cellulase resistance, and are expected to have resistance to microorganisms eroding cellulose fibers.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location G01N 33/545 Z

Claims (4)

[Claims] 1. A polymer compound having a hydroxyl group,
After conversion to a 2,3-dihalopropyl or 3-halo-2-hydroxypropyl derivative via an allyl derivative, at least one amino group-containing functional compound selected from the group consisting of coenzymes, polypeptides and proteins. The method for synthesizing a functionalized polymer is characterized in that: are bound at room temperature under neutral or slightly alkaline conditions. 2. The synthetic method according to claim 1, wherein the polymer compound having a hydroxyl group is at least one polymer compound selected from the group consisting of polysaccharide derivatives thereof and crosslinked products thereof, polyvinyl alcohol and crosslinked products thereof. . 3. The synthetic method according to claim 2, wherein the polysaccharide and its derivative are cellulose and its derivative. 4. The synthetic method according to claim 3, wherein the polymer compound having a hydroxyl group is a crosslinked polysaccharide.