DD237843A1 - METHOD FOR THE PRODUCTION OF ANION-ACTIVE TRAIGERMATERIALS - Google Patents

Abstract

The invention relates to a process for the preparation of anionic carrier materials. The aim of the invention is to produce such substances with tunable binding properties for biological materials. The invention has for its object to produce an anion exchanger, which has selective binding properties and adjustable elution behavior. According to the invention, the object is achieved by bringing a polymer having NH or OH groups in the present or in pretreated form into solvents in contact with one or more hologenides of di- and / or polyacids and to contact the product thus obtained with amino-containing polymers, Reacted oligomers and / or prepolymers, this reaction is optionally repeated one or more times with the same or different substances and then washed and / or quaternized. The invention finds application in molecular biology, in genetic engineering, in biotechnology and in medicine, and in particular in the analysis or synthesis of nucleic acids.

Description

Field of application of the invention

The invention relates to a process for the preparation of anionic support materials based on natural or artificial polymers with pronounced ability to bind nucleic acids, which are used for the fixation, analysis, separation, synthesis of enzymes, proteins and other biological material and for the analysis, separation, Synthesis, cloning, sequencing of polynucleic acids can be used.

Characteristic of the known technical solutions

The preparation of polymer-based anion exchangers for various biochemical applications is known. In general, either a particular with potentially anionic groups alone is polymerized together with other monomers or a polymeric material is modified after its completion by chemical reactions, these reactions are carried out by means of monomers. Another method is to combine two polymeric materials based on their properties to form a polymer composition with anionic properties. The anionic properties are almost always produced by amino groups of different degrees of substitution and their subsequent quaternization.

For metal recovery, e.g. crosslinked polyethyleneimine resins as described by J. Bartulin et al., Polym. Bull. (Berlin) 12, pp. 393-397 (1984). By ionic polymerization, ethyleneimine is converted to polyethyleneimine and then crosslinked with 1,3-dibromopropane.

The alkylation and quaternization of the nitrogen is carried out with dimethyl sulfate.

In SU-PS 722.123 a method for preparing water-soluble amine derivatives of cellulose by reacting hydroxyethyl cellulose with diethyl (epoxypropyl) amine is described. The products obtained by this method are water-soluble and are therefore not suitable for fixing biological materials.

In CA-PS 1,169,735 a process for the preparation of anion exchangers is described, after the cellulose with a 1% solution of polyethyleneimine at a pH of 2 to 6 at temperatures of 40 to 90 ⁰ C within 2 hours to 4 days. By this method, polymer compositions are obtained in which there is no true chemical bond between the two polymers, therefore, in further operations, the soluble polyethylenimine can be removed again.

EP 47 753 and 114 286 describe semipermeable membranes for ultrafiltration consisting of a polymeric support - e.g. Polyvinyl alcohol or polypropylene - and a modifying reagent, e.g. Cyanuric chloride, polyethyleneimine or reactive dyes. Through these reagents occurs in aqueous solution crosslinking, so that water-insoluble, ion-active membranes are obtained. These membranes serve primarily to purify water and to adsorb inorganic ions and are unsuitable for biological work operations in this form.

According to US Pat. No. 3,823,133, cellulose is reacted with diethylaminoethyl chloride, thereby obtaining an anion exchanger whose quaternized amino groups are relatively tightly attached to the polymeric carrier. Flat products according to this process are characterized by low mechanical strength and poor eluability by means of gentle reagents in biochemical reactions.

Furthermore, the preparation of anionic ion exchangers based on polymeric carrier by a first reaction with 2, 4,6-trihalo-1,3,5-triazines and a second reaction with compounds of the general formula

V ^R 2 f Y 6

in which X = NH ₂ , OH or SH, R, R ¹ , R ² , R ^{3 may be} alkyl or aryl groups and Y may be halides or OH. Even after this process, only simple (non-polymerized) compounds are reacted on a polymer. By all known methods, in principle, products with fixed, strongly anionic properties are obtained, which permit the use of the product exclusively for the fixation of anions-in this case their nature does not matter at first. In the case of the necessary reversible fixation of biological materials, eg polynucleic acids, however, a specific binding according to their anion activity and gentle elution is necessary in order to avoid damage to the material. Although a firm bond between the carrier and nucleic acid is a prerequisite, but this must be sensitive to them depending on the nucleic acid sequence to be processed in order to ensure a complete and uninfluenced recovery of the biological material in subsequent operations.

Object of the invention

The object of the invention is to provide, by a simple process, an anionic, insoluble, polymer-based ion exchange material which has tunable binding properties for biological materials and allows them to be recovered gently.

Explanation of the essence of the invention

The invention has for its object to produce an anion exchanger, which has selective binding properties and adjustable elution behavior.

According to the invention, this object is achieved in that an OH and / or NH group-containing polymer or polymer mixture in granular, granular, spherical, fibrous, sheet or foam form in the present or in a pretreated form in one or more solvents with one or more Halides of di- and / or polyacids brought into contact and the product thus obtained immediately after or after an intermediate storage in one or more solvents including water at a temperature of -20 ⁰ C to +120 ⁰ C within one minute to 4 days with amino group-containing Reacted oligomers, prepolymers and / or polymers, these reactions if necessary 1 or more times repeated with the same or other substances, then 0-1 Ox washed, treated with a salt solution and / or a quaternization agent and / or a mineral acid and dried. By the process according to the invention suitable polymers are natural or artificial polymers, for. As cellulose, cellulose derivatives, cellulose copolymers, polymer compositions of cellulose and other polymers, polyacrylates, polymethacrylates and their copolymers, polyvinyl alcohol, polyvinyl acetate, ethylene, vinyl acetate copolymers, polyamides, polyurethanes, polystyrene and copolymers, polyvinyl chloride, polysulfones, etc.

These polymers can be reacted in their usual form with acid-spargers, but they can also be pretreated, г. For example, cellulose can be converted with alkalis into alkali cellulose, in particular soda cellulose, or polystyrene can be aminomethylated.

These polymers or polymer derivatives are reacted with a molar excess of acidic halides derived from organic acids having at least two acid groups. This reaction is z. B. performed in the same solvent as the pretreatment of the polymer. In this reaction, acid-binding agents, for. As alkalis or tertiary amines can be added. By this reaction, modified polymers having terminal acid halide groups are obtained, and possible crosslinking of def polymers takes place under the appropriate reaction conditions to a non-interfering extent

 Suitable acid halides are, for. B. oxayl chloride, sebaeyl chloride, adipoyl chloride, oxalyl bromide, adipoyl bromide,

Cyanuric chloride, phthaloyl chloride, terephthaloyl chloride, trimellitic acid trichloride, trimesic acid trichloride, pyromellitic acid tetrachloride, etc.

The reaction can be carried out in solvents which completely or partially dissolve the simple compounds to be reacted. Through the course of the reaction, considerable amounts of acid halide may still be bound, absorbed or incorporated onto the modified polymer, and salts which form during the reaction may also precipitate on it and participate in the subsequent reaction.

The modified polymers are reacted immediately after this modification or at any other time provided that a minimum of intact acid halide groups is still present in the same solvent or mixture or another in solution, suspension or dispersion with a molar excess of an amino group-containing polymer , In this reaction, the remaining acid halide groups react with the amino groups of the polymer with amide formation in such a way that there is still a free or in the form of its ammonium salt with a halide or a hydroxyl group by the ratio of both mutually determined number of amino groups.

Suitable amino-containing polymers are, for. B. urea-formaldehyde condensates of molecular weight 500-50000, melamine-formaldehyde condensates, aniline-formaldehyde condensates of molecular weight 200 to 50,000, polyethyleneimine of molecular weight 400 to 100,000, polyether amines of molecular weight 800 to 25,000, polyestramines of molecular weight 10Ό0 to 20,000, polyamidoamines molar mass 2000 to 50,000 etc. Of these, the aniline-formaldehyde condensates of molecular weights 500 to 10,000, polyethetramines of polyethalcohols and disocyanates with subsequent hydrolysis of the molar mass 1000 to 5000 and polyethyleneimines of the molecular weight range 5000 to 50,000 are preferred for the reaction. The polymers containing amino groups often dissolve in water, and for the reaction ѳpep whole or partial neutralization may be desirable, so that the reaction is carried out in an aqueous medium. However, the reaction is also carried out in solvents or mixtures, e.g. In acetone, dioxane, toluene, xylene, dimethylformamide, dimethyl sulfoxide, nitromethane, ethylene glycol monomethyl ether, ethylene glycol acetate, butyl acetate, etc., for example.

The products having free amino groups thus obtained can already be used for a large number of cases. However, it is possible to repeat the described reactions and thereby arrive at a multilayer structure on the base polymer. In each further reaction, the amino groups present are partially or completely reacted with the previously used or a second acid halide or a mixture of acid halides depending on the desired application. This repetition of the reaction can be between one and one hundred times, with one to five repetitions being preferred. As a result of this repetition of the reaction of amino groups of the modified polymeric carrier with acid halides and subsequent further reaction of free acid groups with polymers containing amino groups, anionic ion exchangers having different degrees of bonding groups are prepared.

The process of the present invention provides anionic ion exchange materials which have tunable anionic properties and polynucleic acid exchange properties that are found in extremely weakly basic media, e.g. B. in 0.01% piperidine solution at 20 to 90 ⁰ C, are completely elutable again. However, the elution is also in certain salt solutions, eg. As potassium bromide, potassium nitrate, potassium hydrogen phosphate, possible. Furthermore, anion exchangers are produced, of which nucleotides can be eluted by simple heating in aqueous solution to specific temperatures and no further Elution steps are necessary. This opens up the possibility of isolating and determining or further using certain nucleic acid sequences predefined by the experimental procedure without subsequent precipitation or purification steps.

embodiments

example 1

A sheet of 15 cm diameter (pure cotton cellulose) hard filter paper is treated with 15% sodium hydroxide solution for 15 minutes.

After drying, is placed in a 5% solution of cyanuric chloride in xylene and shaken for 20min at room temperature. Thereafter, it is washed several times with acetone. After the final wash, the sheet is immediately placed in a 6% solution of aniline-formaldehyde condensate of average molecular weight 560 in acetone and shaken for one hour at room temperature. Then it is washed 3 times with 100 ml of water and dried.

On 0.5 cm ^{2 of} the surface carrier thus obtained 100μΙ of a solution of ³² P-labeled DNA from herring sperm are added and shaken for 12 hours at room temperature.

Thereafter, (No) is measured, the supernatant solution is pipetted off, washed 3 times with 1 ml of water each time and measured again (N ₁ ). From the measured values, the binding capacity is calculated in% and in / μg / cm ² :

Binding capacity: 68% 246мд / ст ²

Thereafter, the stability of the binding is measured by the action of temperature by shaking the sample in 1 ml of water at the indicated temperature for 1 h.

Stability after 1 hour / 40 ° C 90%

Stability after 1h / 60 ° C 64%

Stability after 1 h / 90 ⁰ C 31%

Stability after 10min / 100 ° C 17%

Example 2

A sheet of 15 cm diameter hard filter paper is treated with 17% sodium hydroxide solution for 10 minutes. After squeezing between the filter paper, shake in a 6.5% solution of cyanuric chloride in xylohacetone = 1: 1 for 30 minutes at room temperature. After shaking, each 10min is washed in 3x100 ml of acetone and immediately added in 100ml of a solution of 5% polyethyleneimine of average molecular weight 30000 in dimethylformamide and shaken for 1 hr. At room temperature. Then it is washed 4x with 100 ml of water and dried. The binding test is repeated as in Example 1:

Binding capacity: 92% 460 ^ g / cm ² Stability of binding: 1Std.40 ° C 95% 1Std.60 ° C 95% 1Std.90 ° C 79%

Eluted with 1% piperidine solution for 10 min: 97% are released.

Example 3

5 cm ^{2 of} the support prepared in Example 2 is reacted with 10 ml of a 3% solution of cyanuric chloride in acetone for 20 min at room temperature. Thereafter, 1 χ is washed with 10 ml of acetone and then shaken in 10 ml of a 5% solution of polyethyleneimine of Mn ~ 30,000 in DMF for 20 min. It is then washed 3 times with 5 ml of acetone and 3x 10 ml of water each time and dried. With this carrier, the same tests as in Example 1 are carried out:

Binding capacity:

Stability of the bond 72% 282 / xg / cm ²

after 1h40 ° C 99%

after 1h60 ° C 91%

after 1h90 ° C 88%

after 10 minutes 100 ^° C. 75%

Example 4

A semisynthetic sheet of 40% cellulose short fibers, 45% polyamide fibers, 5% polyacrylate copolymer, 5% carboxymethylcellulose and 5% polyvinyl alcohol is treated with 15% sodium hydroxide for 10 min, then reacted with a 35% solution of cyanuric chloride in acetone: xylene = 1: 1 , Washed 3x with acetone and dried in vacuum. Of the thus obtained support material 100 cm ² in 100 ml of a 7.5% solution of Polyethytenttnin-fMn- · ^ = -40000) are coated in water within 1 hour at room temperature. This product is then shaken in a solution of 7.0 g of adipoyl chloride in 93 ml of toluene for three hours at room temperature. It is then washed 3 times with 20 ml each of anhydrous acetone and reacted again with the polyethylenimine solution used above. The reaction with cyanuric chloride and polyethyleneimine is carried out once again as above. It is then washed 5 times with 100 ml of water and activated with 0.1% hydrochloric acid. It is then dried in air. The tests are carried out as in Example 1: binding capacity: 96% 379d / ст ²

Stability of binding: after 1h40 ° C 98%

after 1h60 ° C 93%

after 1h 90 ° C 12%

Example 5

From 7.3 g of 4,4-diphenylmethane diisocyanate and 18.9 g of polyethylene glycol 1500, a prepolymer is prepared in 100 ml of dichloromethane. After 24 hours of reaction at room temperature to 4.0 g of a Polyethaalkohols from diethylenetriamine and propylene oxide (OH number 452) added with stirring and pulled out on glass plates. After evaporation of the solvent is dried at 50 ⁰ C for 3 hours.

10 cm ^{2 of} the polyurethane film thus obtained are mixed with 10 ml of a 5% solution of cyanuric chloride in toluene and shaken for 30 hours at room temperature. It is then washed 3x with toluene and shaken with a 7.5% aqueous solution of polyethyleneimine for 30 min at room temperature. It is then washed 3x with water and dried in air. The tests are carried out as in Example 1: binding capacity: 45% 180 ^ g / cm ²

Stability of binding: after 1 hr. 40 ⁰ C 96%

after 1h60 ° C 94%

after 1 hour 90 ⁰ C 88%

after 30 min with 10% aqueous solution of KH ₂ Po ₄ at 9O ⁰ C: 6% residual bond.

Example 6

From 6.8 g of 4,4'-Diplenylmethandiisocyanat, 17.7 polyethylene glycol 1 500 and 150 ml of dichloromethane as in Example 5, a prepolymer is prepared. This is mixed with a mixture of 1.9 g of polyether alcohol as in Example 5,1,3g dimethylethanolamine and 10 ml of dichloromethane and stirred for 15 min. Thereafter, 20 g of microcrystalline cellulose are stirred into 30 ml of this solution and mixed homogeneously. This mixture is stripped to 1 mm thick films and dried at 2O ⁰ C and 300 mm. Thereafter, it is removed at 5O ⁰ C in 3 hours and the remainder of the solvent. It is then stored in a vacuum dessicator. 10 cm ^{2 of} the copolymer film are treated with 10% sodium hydroxide solution for 10 min and reacted with a 10% solution of sebacyl chloride in toluene for one hour, washed 3x with toluene, then with a 5% solution of polyethyleneimine (Mn ~ 30,000) in dimethylformamide Room temperature implemented within 2 hours. The product thus obtained is washed 5 times with water and dried in the desiccator over calcium chloride. Binding capacity: 62% 245d / ст ²

Stability of binding: after hours / 40 ° C 96%

after hours / 60X 85%

after 1h / 90 ° C 71%

after 30 min with 1% piperidine at 90 ° C: residual binding 6.5%.

Example 7

50 cm ^{2 of} the fabric of Example 4 are reacted for 15 minutes with 10% sodium hydroxide solution. After drying between filter paper is reacted for 20 min with a 7.5% solution of cyanuric chloride in xylene. Thereafter, it is washed 3 times with 20 ml of acetone each time.

From 10 g of polyoxyethylene-polyoxypropylene copolymer (Mn ~ 2000) and 1.9 g of toluene-2,4-diisocyanate, a prepolymer is prepared and hydrolyzed after completion of the reaction in 500 ml of 8% sodium hydroxide solution. The polyetheramine is separated and dried at 60 ⁰ C in vacuo.

The sheet is shaken with a solution of 3 g of the polyetheramine in 15 ml of ethyl glycol acetate for 1 h at room temperature. Thereafter, it is washed once with ethyl glycol acetate and 4x with 40 ml of water. Then it is dried in air. The tests are carried out as in Example 1: binding capacity: 58% 226 / Ag / cm ²

Stability of binding: after 1 hr. 40 ⁰ C 95%

after 1 hour 6O ⁰ C 92%

after 1 hour 90 ⁰ C 56%

after 1 h in 2.5% piperidine solution at 9O ⁰ C: residual binding 8%.

Claims (12)

Invention claim: 1. A process for the preparation of anionic ion exchange material based on natural and / or synthetic polymers, characterized in that an OH and / or NH group-containing polymer or polymer mixture in granular, granular, spherical, fibrous, sheet or foam form in the present or in a pretreated form in one or more solvents with one or more halides of di- and / or polyacids in contact and the product thus obtained immediately after or after intermediate storage in one or more solvents including water at a temperature of -20 ⁰ C. reacted to + 120 ° C within one minute to 4 days with amino group-containing oligomers, prepolymers and / or polymers, this reaction optionally 1 or more times with the same or other substances, then washed 0-10x, with a salt solution and / or a quartet and / or a minera lsäure and dried. 2. The method according to item 1, characterized in that the polymers used as cellulose, cellulose derivatives, cellulose copolymers, Ρσ / ymerkompositionen of cellulose and other polymers, polyacrylates, polymethacrylates and their copolymers, ethylene-vinyl acetate copolymers, polyvinyl alcohol, polyvinyl acetate, polyamides, polyurethanes , Polystyrene and copolymers, polyvinyl chloride, polysulfones. 3. The method according to item 1 or 2, characterized in that the polymers are treated prior to reaction with the halides of di- and / or polyacids with sodium hydroxide or potassium hydroxide in aqueous or alcoholic solution. 4. The method according to item 1 to 3, characterized in that aromatics-containing polymers are aminomethylated, then reacted with halides of di- and / or polyacids. 5. The method according to item 1-4, characterized in that are used as halides of di- and / or polyacids oxalyl chloride, sebacyl chloride, adipoyl chloride, oxalyl bromide, adipoyl bromide, cyanuric chloride, phthaloyl chloride, terephthaloyl chloride, TrimellitsäuretrichloricvTrimesinsäuretrichlorid, Pyromellidsäuretetrachlorid. 6. The method according to item 1-5, characterized in that are used as amino group-containing oligomers, prepolymers and / or polymer substances having at least 2 primary and / or secondary amino groups having a mean molecular weight of 200 to 50,000. 7. The method according to item 1-6, characterized in that containing amino groups as oligomers, prepolymers and / or polymers urea-formaldehyde condensates. Melamine-formaldehyde condensates, aniline-formaldehyde condensates, polyethylenimine, polyetheramines, polyesteramines, polyamideamines. 8. The method according to item 1-7, characterized in that the reaction of substituted by halides of di- and / or polyacids polymers with amino group-containing oligomers, prepolymers and / or polymers in aqueous solution at a pH of 3-11 at previous partial or complete neutralization of the amino groups with mineral and / or carboxylic acids is carried out. 9. The method according to item 1-8, characterized in that polymers are reacted with halides of di- and / or polyacids, these reaction products are further reacted with an amino group-containing oligomers, prepolymers and / or polymers, the reaction product thus obtained in turn with a or reacted a plurality of halides of di- and / or polyacids, this reaction product is in turn reacted with one or more amino group-containing oligomers, prepolymers and / or polymers and this scheme of reactions with the same or other than the starting materials is repeated up to 100 times. 10. The method according to item 1-9, characterized in that the quaternization with substances such as alkyl halides, alkyl sulfates, alkyl sulfonates, diazomethane, nitrosomethylurea in solvents such as nitromethane and acetonitrile is performed. 11. The method according to item 1, characterized in that as an intermediate from the polymer or polymer mixture used after contacting with a 1,3,5-triazine-2,4,6-trihalide this from a polymeric compound in a concentration from 5-80 vol.%, of a polymeric compound having 4,6-dihalo-1,3,5-triazine moieties in a concentration of 1-80 vol.%, of 2,4,6-trihalo-1,3 , 5-triazine in a concentration of 0.5-50Vol .-%, consisting of halides of metals in a concentration up to 20Vol .-% and optionally buffering substances up to 5Vol .-%. 12. The method according to item 1 and 11, characterized in that the polymer or polymer mixture consists essentially of cellulose.