DD247458B1 - PROCESS FOR THE PREPARATION OF ACTIVE MEDICAMENTS - Google Patents

Abstract

The invention relates to a process for the preparation of active Tragermatenalien based on natural and / or artificial polymers and organosilicon compounds in organic solvents for molecular biology Erfindungsgerernaß is the Oberflachev of OH and / or NH-containing polymers with oxirane containing organosilicon compounds and at least one Amine function in the molecule-containing compounds at 0 to 160 0C reacted within 1 minute to 100 hours

Description

R = OR ⁵ , -NR ⁶ R ⁷ , -SR ⁸ , R ⁹ ;

R ¹ = - (CH ₂ J _1n -, O, S, NH, NR ¹⁰ , -O (CH ₂ ) _m NR ¹⁰ -, - O (CH ₂ ) _m -, - OAr -, - SAr-

-OArC (R ⁶ R ⁷ JAr -, - OAr-C (R ⁶ R ⁷ ) -Ar-O-, heterocyclic radicals;

R ² , R ³ = hydrogen, C ₁ -C ₈ -alkyl, alkenyl, aryl or halogen;

R ¹ and R ³ together form a ring with 4 to 8 C atoms;

R ⁴ = hydrogen, C ₁ -C ₈ -alkyl, alkenyl, aryl or halogen;

R ⁵ , R ⁶ , R ⁷ = identical or different C ₁ -C ₈ -alkyl, aryl, alkenyl, substituted aryl;

R ⁸ , R ⁹ , R ¹⁰ = identical or different C ₁ -C ₁₈ -alkyl, alkenyl, aryl or substituted aryl;

Ar = phenyl or substituted phenyl;

η = 1,2 or 3 and

m = 1 to 18

mean per mole of hydroxyl and / or amine function and simultaneously or in a second step with 0.01 to 9 moles of compounds having at least one nitrogen atom in the molecule at a temperature of 0 to 160 ⁰ C within one minute to 100 hours.

2. The method according to claim 1, characterized in that the polymers are cellulose, cellulose derivatives and / or graft copolymers of cellulose.

A method according to claim 2, characterized in that the cellulose is used in the form of paper, bead polymer or powder.

4. Process according to Claims 1 to 3, characterized in that the reaction in the presence of a metal compound acting as a Lewis acid, preferably an organotin compound, or weak alkali metal bases, with a concentration of 0 to 10 mol% based on 1 mol of hydroxyl - And / or amine function of the / of the polymers durchfuhrt. '

Field of application of the invention

This invention relates to a process for producing active carrier materials from natural or synthetic polymers in the form of powder, granules, spheres, fibers or sheets for molecular biology, medical diagnostics, chromatography, microbiology, biotechnology and genetic engineering

Characteristic of the known technical solutions

For use in molecular biology, a variety of supports based on synthetic or natural polymers have been developed. It is usually derivatives of these polymers which are prepared by various modifications. Typical activation methods for the polymers are halogen-containing compounds, aldehydes, acid halides, In addition, the reaction with epoxides is possible, as described by LG Moss, JP Moore and L Chan in J Biol Chem 256 (24), S 12655-12658 (1981). According to this method Diepoxyverbindungen with cellulose powder reacted in aqueous sodium hydroxide solution in the presence of Natnumborhydnd The support thus obtained has a relatively low capacity for the covalent bonding of biomolecules. Cellulose-based ion-active support materials are known from DD-WP 228 461. However, the preparation of this material requires a lot of effort, it is not adjustable in its properties and has at high hydrophilicity exclusively Anionenaustauschereigenschaften

Furthermore, the treatment of textlien fibers and paper with epoxides and organosilicon compounds is known. Thus, US Pat. No. 3,665,420 describes a treatment agent for fibers of 0.3 to 20% by weight of epoxysilane and 1% by weight of an amine compound having more than two amino groups in the molecule. For bonding in the dyeing of fibers

these are according to the US-PS 3J545 909 treated with polyaminosilanes or aminoalkylsilanes Another method for fiber treatment is taught in DEOS 2335751 (= GB-PS 434,017), then tons of fibers are reacted in non-aqueous solution with polysiloxanes and aminoalkylalkoxysilanes In the DE No. 3301667 describes a process for the antistatic finishing of fibers by producing cationic compounds on cellulose fibers, wherein a reaction of alkali cellulose, glycidol and organic ammonium compounds is carried out. Finally, DE-PS 2505742 describes a treatment agent for aminosilane and molar excess fibers Epoxysilane is described In analogy to the treatment of the fibers with organosilicon compounds and / or epoxides, the treatment of paper with this group of compounds is taught. Thus, in US-PS 3 061 567 z. B describes the improvement of papers by the addition of amiriosilanes. According to DE-OS 1696267 paper for bonding with epoxy silanes is reacted at elevated temperature In the GS-PS 804 198,875 605 and 908,988 adhesives are described on Silanbasisfur papers All these known in the prior art methods relate to the influence on the wearing properties of Fiber materials or the improvement of writing paper However, they have no target with regard to the binding of other macromolecules, in particular biomolecules Consequently, no relation between the improvement z. B of color properties or antistatic properties of fibers and binding of biomolecules DD-WP 256219 and DD-WP 256721 disclose processes for activating hydroxyl-containing synthetic and natural polymeric solid surfaces with organosilanes and optionally homo- or heterobifunctional reagents However, in the manufacturing process, the products produced thereafter have significant disadvantages for molecular biology applications

The binding cube ratio of the carriers is relatively low and not reproducibly constant according to the method described

- The binding of the biomolecules, primarily of DNA, is not stable, and especially at temperatures above 40 ⁰ C, significant parts of it lost

- For dyeings, a high background is obtained, which makes the evaluation problematic

Object of the invention

The aim of the invention is to provide a technologically simple and cheap to carry out method for activating OH and / or NH-containing polymers

Presentation of the weaving of the invention - -

The object of the invention is to adjust the surface of OH and / or NH group-containing polymers in their hydrophilicity, their ion exchange properties and their chemical activity for the production of active Tragermatenalien based on natural and / or artificial polymers and organosilicon compounds in organic solvents , According to the invention, the surface of solid natural or synthetic polymers or mixtures thereof with hydroxyl and / or amine groups, in the main chain or on side groups with 0.1 to 10 mol at least one Oxirangruppierung containing organosilicon compounds of general formula I.

R _n -S

R = OR ⁵ , -NR ⁶ R ⁷ , -SR ⁸ , R ⁹ ,

R ¹ = - (CH ₂ ) _m -, O, S, NH, NR ¹⁰ , -O (CH ₂ ) _m NR '° -, - O (CH ₂ ) _m -, - OAr -, - SAr -, - OArC (R ⁶ R ⁷ JAr, -OAr-C (R ⁶ R ⁷ I-Ar-O-,

heterocyclic radicals,

R ^J = hydrogen, C ₁ -C ₈ -alkyl, alkenyl, aryl or halogen,

R ³ = hydrogen.Ci-Cg-Alikyl.Alkenyl.AryloderHalogen,

R ¹ and R ³ together form a ring with 4 to 8 C atoms,

R ⁴ = hydrogen, C 1 -C ₈ -alkyl / alkenyl, aryl or halogen,

R ⁵ , R ⁶ , R ⁷ = identical or different C ₁ -C ₈ -alkyl, aryl, alkenyl, substituted aryl,

R ⁸ , R ⁹ , R ¹⁰ = identical or different C 1 -C ₁₈ -alkyl, alkenyl, aryl or substituted aryl,

Ar = phenyl or substituted phenyl,

η = 1,2,3 and

m = 1 to 18

mean, reacted per mole of hydroxyl and / or amine function. At the same time or in a second step, followed by a reaction with 0.01 to 9 moles of compounds having at least one amine nitrogen in the molecule at temperatures of 0 to 160 ⁰ C implemented within 1 minute to 100 hours

Suitable polymers for the process according to the invention are cellulose and its derivatives, including the ethers, exTERs and copolymers, polyamides, polyurethanes, functionalized homopolymers or copolymers of olefin compounds, ζ. Β high molecular weight, hydroxyl-containing polybutadienes, polyetheramines, polyesters, polyether esters and composites Β Β of cellulose and polyamide fibers, of cellulose and polyester fibers, of cellulose powder and polyester resin, of polyamide fibers and polyepoxide powder mixtures of cellulose and polyamide, cellulose and polystyrene, bead polymers of cellulose, of cellulose and polyamide etc. are preferably used Cellulose in the form of fibers, granules, powder, bead polymer, paper or bonded to other fibers, ζ B. polyamide fibers or glass fibers, which may be treated by cellulose derivatives and / or polymer dispersions

These polymers are suspended in or immersed in the desired form in organic solvents, B in ether, acetone, benzene, toluene, xylene, naphthalene, decalin, tetrachloroethane, tetrahydrofuran, dibutyl ether, acetonitrile, benzonitrile, nitrobenzene, etc. These are used either together with the The reaction may be carried out in the presence of metal compounds which act as a Lewis acid, ζ B zinc chloride, zinc nitrate, zinc acetylacetonate, titanium tetrachloride, titanium alkoxy compounds, lead naphthenate, lead octoate, cobalt naphthenate , Cobalt chloride, bis (tnbutylzinn) oxide, bis (trichlorotin) oxide, tin dioctoate, dibutyltin diacetate, dibutyltin dilaurate, dioctyltin bis (thioglycolic acid ester), dibutyltin dimaleate, etc. preferably organotin compounds and in the presence of weak alkali bases such as potassium carbonate, trisodium phosphate or sodium carbonate Durc Of these compounds, up to 10 mol%, based on one mol of a function of the polymer, are used, preferably amounts of from 0.01 to 1 mol%

However, it is also possible to add this compound after a time of one minute to three hours after the addition of the oxirane compound, the time depending on the reaction temperature and the reactivity of the oxirane compound Suitable compounds containing amine nitrogen are those having at least one primary, secondary or tertiary amine nitrogen atom. However, these compounds may also contain several different or identical amine nitrogen atoms. Examples of these compounds are ζ B. diethylamine, di-n-butylamine, dimethylethanolamine, diethanolamine, methyl- cyclohexylamine, Ν, Ν-dimethylaminopropylamine, diethylenetriamine, dipropylenetriamine, ethylenediamine, polymers of ethyleneimine, etc. Following this reaction, quaternization by acids or by alkyl halides can be carried out by known methods.

In addition to the amino compounds already mentioned, it is also possible to use polymers having NH or NH ₂ groups in the molecule, B polyetheramines having molecular weights between 400 and 5000 and two to four amino groups in the molecule or polyesteramines obtained from aminocarboxylic acids and glycols.

Examples of furan compounds are ζB. (2,3-epoxypropyl) triethoxysilane, (2,3-epoxypropoxy) triethoxysilane, (3,4-epoxybutylmethmethoxysilane, bis (2,3-epoxy-propoxy) dimethoxysilane , 4- (epoxyethyl) phenylethoxysilane, 4,5-epoxycyclohexylmethoxysilane, etc

The reaction can be carried out at room temperature and at elevated temperature up to 160 ⁰ C, with reaction temperatures between 20 and 80 ⁰ C are preferred The reaction can be carried out under atmospheric pressure and pressure, under normal atmosphere and under inert gas It is, B possible in one Circulatory system constantly flow through the polymer with the reaction mixture and thus to activate.

Exemplary embodiments example 1

From 8,5g (2,3-epoxy-propoxy-) triethoxysilane and 48g toluene a mixture is prepared in a glass dish of 6cm χ 4cm. In this mixture a sheet of filter paper of 12cm χ 5cm in size is placed After five minutes, 5.8g of dimethylaminopropylamine are added, the glass bowl capped and shaken gently at room temperature for three hours. The leaf is then removed, drained and air dried. DNA), about 300 bp from 100 μΙ solution with about 8000 cpm radioactively labeled sample on 0.5 cm ² carrier, slight rubble

after 1 hour 285μς / ατι ²

after 12 hours 400μg / cm ² further properties see Table 1.

Example 2

From 4.1 g (2,3-epoxy-propoxy) triethoxysilane and 30 g toluene in a glass dish of 6cm x 14cm a mixture is made in this mixture is placed a sheet of filter paper of 12cm x 5cm Then 1.3g (3 Aminopropyl- (triethoxysilane added, the glass dish sealed and shaken gently at room temperature for three hours. The leaf is then removed, drained and air-dried

after 1 hour: 390 μg / cm ²

after 12 hours · 475 μg / cm ² further properties see Table 1

Example 3

From 6,6g (2,3-epoxypropyl) -tnethoxysilane and 53 g toluene in a glass dish of 6cm χ 14cm a mixture is made in this mixture is a sheet of filter paper of 12cm χ 5cm large laid, closed the glass bowl and gently shaken for three hours , Thereafter, the sheet is removed, drained and placed in another pan with a mixture of 1,1g Ν, Ν-dimethylaminopropylamine and 21 g toluene In this mixture, the thus pretreated sheet is shaken for one hour, then allowed to drain and dried in air DNA binding assay

after 1 hour 126μg / cm ²

after 12 hours 298 μg / cm ²

Comparative Examples

Two comparative examples were carried out under the same experimental conditions. The following mixtures were used for this purpose

Comparative Example 1 6.6 g of (3-aminopropyl) -nethoxysilane and S3 g of toluene

Comparative Example 2 * 6.6 g (2,3-epoxypropoxy-1-methoxysilane and 53 g of toluene Filter papers of the same size as above were reacted at room temperature for three hours. DNA binding test

after 1 hour after 12 hours

V-Ex 1, 233pg / cm ² 300pg / cm ²

V-Ex 2 20ug / cm ² 25pg / cm ²

Table 1 Temperature dependence of DNA binding (in% of baseline)

No. 20 40 60 75 90 100

B 1 100 95 94 95 95 95 B 2 100 95 90 90 80 70 B 3 100 94 92 89 82 65 V 1 100 88 71 54 44 32 V2 100 80 50 44 29 19

at each temperature after one hour in 2 ml of water under a slight rubble

Example 4

In a 1-liter three-necked flask, 22 g of microcrystalline cellulose are suspended in 500 ml of anhydrous toluene. To the suspension are added 0.25 g of dibutylin diacetate. Subsequently, the mixture under stirring is heated to 80 ° C at a temperature of 80-90 ⁰ C then 15 mL (2,3-epoxy-propoxy) -tnethoxysilan is stirred added within 30 minutes, then for 30 minutes at 90 ° C , and then N, N-dimethyl-N ', N'-bis (2-hydroxyethyl) -1,3-diaminopropane are added dropwise with stirring within 20 minutes It is stirred for 1 hour at 90 ° C, then cooled The cold Mixture is filtered off, the derivatized cellulose air-dried, then washed 3 times with 100 ml of ethanol, 3 times with 250 ml bidest water, 3 times with phosphate buffer pH ~ 7.4 and again 3 times with 250 ml of water. Thereafter, the wet reaction product, a binding capacity of 580 ug ^12S l-protein A / g of moist product on the bond is even after 3 hours at 75 ⁰ C stable (by Szmtillationszahler measurement 562 pg ¹²⁵ l-protein A / g wet derivatized cellulose, ie 96.9%)

Example 5

In a 250 ml three-necked flask 15.5 g of microcrystalline cellulose and 2.75 g of polyamide 66 powder are suspended in 100 ml of xylene To the suspension simultaneously using two dropping funnels with stirring and at a temperature of 100 ⁰ C within 5 minutes following solutions Solution A 2.0 g of bis (2,3-epoxypropoxy) dimethoxysilane in 20 ml of xylene, solution B 2.0 g of N, N-dimethylaminopropyltrimethoxysilane and 0.1 g of tin dioctoate in 20 ml of xylene. The mixture is then boiled for 15 minutes under reflux and then as worked up in example 4. The product has a binding capacity of ³² P-labeled DNA of 936pg / g moist product After 24 hours at 75 ⁰ C are still 889 \ ig / g bound (95%)

Example 6

In a glass dish, a piece of Whatman 540 paper of 6 cm χ 12 cm is laid This is impregnated with 10 ml of toluene is then incubated is At the impregnated paper at 40 ⁰ C, first a pre-heated to 40 ⁰ C solution of 1 g Tetraglycidylsilan in 10ml Di-n-butyl ether and after 2 minutes a preheated to 40 ° C solution of 1.5 g of tetramethylbutanediamine, 0.5 g of hexamethylenediamine, 0.15 g of dibutyltin Dichloud and 0.05 g of bis-tributyltin oxide in 25 ml of methyl ethyl ketone given three hours at 40 ⁰ C. The product has a binding capacity of 620 pg DNA / cm ² After 24 hours at 65 ^° C., 578 μg DNA / cm ² are still determined (93.2%).

Example 7

A partially synthetic paper made of cellulose and polyamide fibers is reacted for 4 days at 0 ⁰ C with a 5% solution of bis (3,4-epoxybutyl-) diethoxysilane in benzene in the presence of 0.01 g of dibutyltin diacetate. The reaction mixture is then warmed slowly to room temperature and a 10% solution of N, N-dimethyl-1,4-diaminobenzen added in diethyl ether. The mixture wii d incubated for a further 4 hours and then dried in air then is 6 times with 100ml PBS Buffer (pH 7.2) and dried DNA binding Capacilat at 20 ⁰ C after 12 hours 566 pg / cm ²

at 60 ⁰ C after 12 hours 541 pg / cm ²

at 75 ° C after 12 hours 503 μς / cm ²

at 90 ° C after 12 hours 488 μg / cm ²

Example 8

A Whatman 540 cellulose paper is sealed in a glass pan with aluminum foil with a solution of 4.25g tetraglycidylsilane, 1.5g Ν, Ν-dimethylamtnopropylamine and 0.05g dibutyltin diacetate in 50g toluene and 30g diethyl ether for 5 minutes at room temperature. shaken Then it is dried in air and washed 6 times with 250 ml of water. DNA binding capacity at 20 ° C after 12 hours 601 pg / cm ² at 40 ^c C after 12 hours 600pg / cm ² at 6O ⁰ C after 12 hours 589 Mg / cm ² at 8O ⁰ C after 12 hours 488μg / cm ²

free game 9 E.neChromatographiesaulevonO, 5 χ 30cm is filled with a slurry of microcrystalline cellulose in toluene and through a heating jacket by a thermostat and heated to 50 ⁰ C. After reaching the temperature constancy is a solution (from 5.0 g bis 2,3 -epoxypropyl-) dimethoxysilane and 0.1 g of dibutyltin dilaurate in 100 ml of toluene was added slowly through the column (about one hour) is then rinsed with 100 ml of toluene at 50 ⁰ C and a solution of 2.5 g N, N-dimethyl-1,4 -diaminobenzen added in 50 ml of toluene over the column is then washed with 500ml of toluene, 500ml of methanol, 100 ml of water, 500ml of methanol and 2000 ml of water, the derivatized cellulose had a binding capacity at 20 ⁰ C 968 ug / ml of moist product at 50 ° C (after 24h in water) 905pg / ml wet product at 65 ° C (after 24h in water) 880mg / ml wet product at 75 ° C (after 24h in water) 800μg / ml wet product

Example 10

From 8,5g (2,3-epoxy-propoxy) -tnethoxysilane, 0,1 g tetraethylammonium hydroxide and 48g toluene in a glass dish of 6cm x 14cm a mixture is made. Into this mixture is placed a sheet of filter paper of 12 cm χ 5cm large 5.8 g of dimethylaminopropylamine are added for five minutes, the glass dish is closed and shaken gently at room temperature for three hours. The leaf is then removed, drained and dried in air. Binding capacity according to Example 1.524 pg / cm ² after 12 h / 70 ° C. 500 mg / cm ²

Example 11

Analogously to Example 10, instead of tetraethylammonium hydroxide, 0.1 g of tetrabutylammonium hydroxide is used. Binding capacity 546μςΛ: Γη ² after 12 h / 70 ° C. 478 μg / cm ²

Example 12

4,2g (2,3-epoxy-propoxy) -trimethoxysilane are dissolved in 50 ml toluene. In this solution, a sheet of semisynthetic paper of 65.0% by weight cellulose 17.0% by weight polyamide fibers 12.0% by weight polyacrylate Dispersion and 7.0% by weight of additives

and shaken at room temperature for 2 hours. Then, a solution of 1.1 g of Ν, Ν-dimethylaminopropylamine in 20 ml of toluene and 0.08 g of dibutyltin dilaurate is added and shaken for a further 2 hours at room temperature. The leaf is then removed, drained, and dried in air and shaken with a solution of 15 ml of methyl iodide and 25 ml of methanol for 0.3 hours. The sheet is removed, dried in air, washed 2 × with methanol and 6 × with bidistilled water and dried. DNA binding capacity at 8 hours 417 μς DNS / cm ²

Example 13

In 50 ml toluene, 3.5 g (2,3-epoxy-propoxy) -tnethoxysilane are dissolved. A solution of 0.9 g of hexamethylenediamine in 25 ml of toluene is added and shaken at room temperature for 3 hours. Thereafter, as in Example 12, the DNA binding capacity is 358 μs DNS / cm after 4 hours ²

Example 14

The procedure is as in Example 13, but a Polybutylanterephthalat film of 0.08 mm thick uses DNA binding capacity after 4 hours 253 μς DNS / cm ² after 24 hours 264 μς DNS / cm ²

Example 15

The procedure is as in Example 13, but a polycarbonate film uses DNS binding capacity after 6 hours 212μg DNS / cm ²

Claims (1)

1. A process for the preparation of active Tragermateriahen based on natural and / or artificial polymers and organosilicon compounds in organic solvents, characterized in that the surface of solid natural or synthetic polymers or mixtures thereof with hydroxyl and / or amino groups, in the main chain or on side chains with 0.1 to 10 mol at least one Oxirangruppierung containing organosilicon compounds of the general formula I. / 9 ² S ³
R _n -Si- / R ¹ -CC ^N OR ⁴ 4-n,