DD279486A1 - PROCESS FOR ACTIVATING HYDROXYL GROUP-CONTAINING POLYMER COMPOUNDS - Google Patents

Abstract

The invention relates to a method for activating hydroxyl-containing polymeric compounds and Festkoerperoberflaechen formed therefrom. The aim is to use symmetric carbonic acid diesters for activation and to keep their use levels low. The invention has for its object to greatly increase the reactivity of symmetrical carbonic acid diesters over hydroxyl-containing polymers. The problem is solved essentially by the addition of supernucleophilic amines. Application areas include biotechnology, the chemical and pharmaceutical industries and clinical analytics.

Description

Field of application of the invention

The invention relates to the activation of hydroxylgrupptmhaltigen polymeric compounds and formed therefrom solid surface, the reaction of which with nucleophilic components such as amines or SH grouphaltigon compounds to N-substituted carbonates or thiocarbonic O, S-diesters.

Areas of application include biotechnology, the chemical and pharmaceutical industry, as well as the scientific investigation of fundamentals and the development of processes in these industries, as well as clinical analytics.

Characteristic of the known technical solutions

Very many possibilities have been described for the activation of hydroxyl-containing matrices (P.O.G. Dean, WS Johnson, FA Middle, Affinity Chromatography, JRL Press, Oxford, 1985, W. H Scouten, Affinity Chromatography, John Wiley & Sons, New York , 1981). Only a few methods have found wide application. The previously dominant activation by means of cyanogen bromide is increasingly being replaced by modern methods which have the disadvantages (high toxicity of the BrCN, high susceptibility to hydrolysis of the activated support, low chemical stability of the iso-urea derivatives resulting from the coupling of NH ₂ -group-containing ligands and their unwanted positive electrical charge in the physiological pH range [kPa ~ 9.5]) of the cyanogen bromide partially or completely avoided. Characteristic of modern activation methods is the achievement of high coupling capacities of the support without impairing the structural properties (porosity, mechanical stability, swelling properties, gelation properties, solubility, etc.). A further factor is the good storage stability of the carriers with simultaneously high reactivity of the active groups (pH values of 7.0-9.0, room temperature) compared to the nucleophilic ligands to be bound. The resulting covalent bond between matrix and ligand should be chemically very stable in order to obtain a negligible cleavage of the ligands from the matrix under practical conditions.

These requirements correspond in some respects to those obtainable by reaction with epoxigruppenhaltigen compounds or carbonyldiimidazole - also commercially available - activated support materials based on sepharose, modified silica gels, acrylic acid copolymers such as Epoxisepharose (Pharmacia), Reacti-gel (Pierce), Eupergit (Rohm) and epoxy-activated silica gel (Merck).

The currently best results, however, are achieved by the activation of hydroxyl-containing carriers with chloroformates, which are introduced into the hydroxyl-containing matrix very reactive oxycarbonyl groups to form unsymmetrical carbonates (carbonic acid diester).

J-OH + CI-CO-OR -> J-O-CO-OR

J. Drobiiik et al. (Bioeng Biotechnol.. 24 [1982] 487) translated cellulose and Spheron N-Hydroxysucc nimidyl-, trichlorophenyl and p-Nitrophcnylchlorameisensäureester in dioxane as solvent at temperatures of 25 to 65 ⁰ C and reached to coupling capacitances up to 2 mmol / gram cellulose ,

Similar results were obtained by Wilchek and Miron (Biochem. Lntern.it., 4 (1982) 629) on Sepharose and cellulose when the supports in pyridip were reacted at 4.degree.

In the reaction of a new chloroformate - N- (chlorocarbonyloxy) -5-norbornene-2,3-dicarboximide - become high coupling capacities with hydroxyl-containing polymers up to 1 and 1.2mMol / g carrier for perl cellulose and Sephörose at a reaction temperature of 7O ⁰ C within 4 to 5 hours and an ester use of 16-2OmMoI / gram carrier achieved (DD 219490,6.3.1985) achieved.

The transfer of oxycarbonyl groups to amino groups of amino acids and peptides by reaction with chloroformates such as tert-butoxycarbonyl chloride (Boc-Cl) is a commonly used peptide-protecting reaction for the introduction of protecting groups. Symmetrical or unsymmetrical carbonic acid esters or a salt-like adduct of chloroformate and dimethylaminopyridine (DMAP) (Guibe-Jampel, Chem. Commun. 1971 267) for the transfer of oxycarbonyl groups to the amino groups of amino acids and peptides are also used instead of the chloroformates.

Boc-Cl + 5MAP - »BoCDMAP ^{1 + 1} CI'- ¹

BoCDMAP ^{1 + 1} CI ¹ - ¹ + H ₂ NR> Boc.HN-R + DMAP

-HCI

The suitability of the stable tetrafluoroborate DMAP ^{1 + 1} · CN BF ₄ ¹ " ¹ for the transfer of the cyano group to the hydroxyl groups of polymers to form cyanates has been described by Wilchek et al. (Meth. Enzymol., 104 (1984) 3-55). The reaction results in the conversion of Sepharose to carriers of high coupling capacity (up to 70μΜοΙ cyanate / g aspirated Sepharose 4B) .The reaction of chloroformates with Trisacryl is reported by Miron and Wilchek (6th International Symp., Bioaffinity Chromat Techniques, Prague, 1985) by dimethylaminopyridine.

The previously known syntheses of polymers of the carbonic acid diester type JO-CO-OR are based on chloroformates or symmetrical carbonates (Wilchek, Miron, Appl. Biochem. Biotechnol. 11 [1Π85] 3.191; Henklein et al., Patent specification DD 219490 (6.3.1985)) . the disadvantage of this method is the complicated synthesis of the chloroformate or the carbonic acid diester and r jlativ low conversion rate to activated supports Thus, for example, involves the introduction of from 1 to 2 mmol Oxycarbony ¹ "", -... ;: '' Jn per gram cellulose approximately 10 times excess of Chlcrameisensäureester (Drobnik et al., Biotechn. Bioeng. Ά [19,821,487) and elevated temperatures (65 ⁰ C) or at room temperature, long reaction times.

Z el of the invention

The aim of the invention is to use symmetrical carbonic acid diesters for the activation of hydroxylgruppenhaltigon polymers and solid surfaces formed therefrom (matrices) by introducing oxycarbonyl groups (-CO-OR) under mild reaction conditions and to keep the required Kohlansäurediester quantities as low as possible while achieving high levels of activation ,

Explanation of the essence of the invention

The invention has for its object to greatly increase the reactivity of symmetrical carbonic acid diesters over hydroxyl-containing polymers.

The object is achieved in that symmetrical carbonic diesters d ° r general formula RO- "O-OR, wherein dsr radical R represents an electron-attracting group, in the presence of capable of forming reactive acylium supernucleophilic amines in the ratio of 0.01 to 2, 5 moles of amine per mole of carbonic acid diester and optionally another belonging to the group of strong bases tertiary amine in a ratio of 0.1 to 2.5 moles of amine per mole of carbonic acid diesters in anhydrous organic solvents at temperatures of 0 to 100 ° C u'en hydroxyl-containing polymers or It is also possible to form these symmetrical carbonic acid diesters intermediately, for example from the corresponding chloroformic acid esters, optionally from phosgene together with a phenol or an N-substituted hydroxylamine.

As hydroxyl-containing polymers are both water-soluble and insoluble natural polysaccharides and their derivatives or hydrolysis products such as cellulose, agarose, dextran, starch, mannan, sepharose, starch hydrolysis products (SHP)

u. a. or synthetic polymers such as polyvinyl derivatives (Fractogel, Toyopearl), vinyl alcohol / acrylonitrile copolymers, Spherone.Tiisacryl, polyvinyl alcohol, polyethylene glycol, etc. can be used in one or all erfindungsgerräßen variants of the activation with carbonic acid diesters. The polymers can be reacted as such or in the form of products made therefrom, such as shaped bodies (beads), fibers, fabrics, films or paper.

The reaction of symmetrical carbonic acid diesters of the type RO-CO-OR with R = succinimidyl, phthalimidyl, 5-norbornene-2,3-dicarboxiimidyl, o- and p-nitrophenyl or other substituted phenyl radicals can be carried out in organic solvents such as dimethylsulfoxide, Acetonitrile, tetrahydrofuran, acetone, pyridine or aromatic hydrocarbons such as benzene and toluene, among others or aliphatic compounds such as hexane and the like. a. halogenates hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride and the like. be performed. In general, however, only a slight substitution of the hydroxyl groups by -CO-OR occurs when high reaction temperatures or long reaction times are used, as in the example of the reaction of the N.N'-bis-fS-norbornene.s-succinimidyO carbonate in Table 1 is shown.

By addition of tertiary amines such as triethylamine, pyridine .. Ν, Ν-dimethylaniline, N-methylmorpholine and. a. is the reaction of the carbonic acid diesters with the hydroxyl-containing polymers not or only slightly accelerate (Table 1).

On the other hand, a strong increase in the reaction rate and the achievable degree of substitution by the action of supernucleophilic amines such as 4-dimethylaminopyridine (DMAP), 4-pyrrolidinopyridine (PPY), N-methylimidazole, dia-abicyclo [5.4.0] undecene (DBU), 4- Morpholinopyridine, diazabicyclo [2.2.2] octane, etc. in a ratio of 0.01 to 2.5 moles of amine / mole of carbonate achieved (Tab. 1 and Tab. 2).

The reaction mediated by the supranuciephilic amine DMAP is not affected by the simultaneous presence of strong bases such as triethylamine, Ν, Ν-dimethylsiline, etc. (Table 1). Since the supernucleophilic amines also have only low activity in very small amounts, there is no real catalytic effect of the amines (Table 3).

The optimum molar ratio of symmetric carbonic acid diester to nucleophilic amine in terms of achieving a high coupling capacity is 1.0 to 5.0.

Accelerating the reaction of symmetric carbonic acid diesters with OH-containing polymers in the presence of supernucleophilic amines allows you to carry out the reaction at room temperature within 10 to 30 minutes. Only in the case of polymers having a particularly low reactivity is a temperature increase of up to about 60 ° C. required.

Even when the reaction temperature is lowered to 4 ^° C., the reaction in the case of reactive polymers such as sepharose and cellulose is completed after only 10 minutes.

Solvents such as dioxane, acetone, acetonitrile, chloroform and. a. are particularly well suited for achieving high coupling capacities (Table 4), but the reaction is also possible in any other anhydrous solvent.

Alcohols or other hydroxyl-containing solvents are unsuitable or lead to low coupling capacities.

The scope of application of the method is shown in Tab. 5, which shows the results of the activation of different hydroxyl-containing polymers. Both natural polysaccharides and their derivatives (Sephadex, Sepharose) as well as synthetic carrier materials (Fractogel) or soluble polymers such as polyethylene glycol are the '-> n accessible.

The synthesis and purification of the symmetric carbonic diester used for the polymer activation can be easily bypassed, because a large number of chloroformates reacts in the presence of tertiary amines such as triethylamine, N-methylmorpholine, Ν, Ν-dimethylaniline, pyridine, dimethylaminopyridine u. a. to symmetrical carbonic acid diesters.

This smooth-running reaction offers the possibility of activating hydroxyl-containing polymers by reaction with chloroformates, a tertiary amine and one of the supernucleophilic amines which are suitable for the reaction of symmetrical carbonic acid diesters with hydroxyl-bearing compounds (Table 7).

It is expedient to use a small molar excess of corbonate-forming tertiary amine in relation to the chloroformate. The supernucleophilic amine can be varied in a ratio of 0.01 to 1 mole per mole of chloroformate, with higher amine use causing a significant increase in coupling capacity (Table 8).

Also, the increase in the chloroformate use causes an increase in the coupling capacity, as can be seen from Table 9. As observed in the reaction of Kohler.säurediester, when using egg supernucleophilic amines, the reaction is completed at temperatures of 4 to 25 ° C within 20 to 30 minutes. Only slightly reactive polymers require higher temperatures (up to 6O ⁰ C) and reaction times of 1 to 2 hours.

The activation by means of chloroformate is applicable to different groups of hydroxyl-containing polymers (see Table 10). The suitability of various supernucleophilic amines to achieve high to very high coupling capacities is demonstrated by the results in Table II.

If the reaction of chloroformates with hydroxyl-containing supports is carried out exclusively with a supernucleophilic amine, such as dimethylaminopyridine, at the above-described low amine uses of from 0.01 to 0.05 mol per mol of chloroformate at room temperature, only a very slight increase over the reaction without addition of this Amines recorded (Table 7).

However, when the supernucleophilic amine is used in a molar excess over the chloroformate, the reaction proceeds in the same manner as in the presence of other strongly basic carbonate-forming tert. Amines with low nucleuphilia ab, d. H. that the supernucleophilic AmIn causes both the formation of the acidic carbonic acid diester and its reaction with the hydroxyl-containing polymer.

For the activation reaction with chloroformates the same anhydrous solvents are used, which are used for the reaction of symmetrical carbonic acid diesters with hydroxyl-containing polymers.

Having regard to the formation of chloroformates from phenols or substituted hydroxylamines by reaction with phosgene in the presence of tertiary amines such as triethylamine, N, N-dimethylaniline, N-methylmorpholine and the like. it is equally possible to carry out the above-described activation of hydroxyl-containing polymers, also bypassing the chloroformate ester isolation.

For this purpose, a mixture of the hydroxyl-containing polymer, supernucleophilsm / 'min, and optionally a further tertiary amine and a substituted phenol or N-substituted hydroxylamine with phosgene is reacted. By the action of the combination of these amines, a reaction chain with the intermediate formation of chloroformate and symmetric carbonic acid diester is very quickly passed, which in turn, with the aid of the supernucleophilic amine, causes the transfer of the oxycarbonyl group to the polymer.

This reaction procedure has the advantage of using inexpensive, commercially available starting materials for the introduction of oxycarbonyl groups into hydroxyl-containing matrices under very mild reaction conditions.

The economic advantage of this procedure is moreover that the yield losses occurring in the synthesis of chloroformate or carbonic acid diesters are avoided, and the cheap starting materials phosgene and phenols or substituted hydroxylamine are introduced in high yield as oxycarbonyl in the matrix.

Table 12 shows the results of activation of perl cellulose, depending on the amounts of phosgene and N-hydroxy-5-norbornene-2,3-dicarboximide used.

The introduction of different leaving groups in cellulose carbonates of the type CeII-CO-OR (R = N-hydroxysuccinimidyl, p-nitrophenyl, N-hydroxy-B-norbornene ^ .S-dicarboximidyl-) by reaction of phosgene and dan corresponding hydroxyl compounds (phenol or N-substituted hydroxylamine) can be regarded as a generally applicable reaction for the activation of hydroxyl-containing polymers (Table 13).

The aim of the invention, a simple and economical process for the transfer of oxycarbonyl groups to hydroxyl-group-containing matrices by means of symmetrical carbonic acid diesters, is! essentially by the following inventive feature has been achieved: The reaction accelerating effect of supernucleophilic amines on the activation of hydroxyIgruppenhaltigen matrices by transferring oxycarbonyl groups from symmetrical carbonic acid diesters and their formation from chloroformates by the action of tertiary amines such. B.

Triethylamine, etc.

The combination of carbonate-forming bases and supernucleophilic amines allows the use of phosgene and N-substituted hydroxylamines or phenol or of chloroformates as a means of transferring oxycarbonyl groups in place of the usually difficult to access symmetric carbonates.

Compared to the direct reaction of chloroformates or symmetric carbonic acid diesters, the use of the supernucleophilic amines allows a reduction of the reaction temperature to 4 to 2b ° C and a reduction in the reaction time to 10 to 20 minutes. At temperatures of 50 to 60 ⁰ C also low-reactivity polymers are activated to a high degree.

The mild reaction conditions also allow the activation of less structurally stable polymers such as sepharoses or polymer films.

The high conversion of the reagents (up to 80-90%) and the use of inexpensive raw materials, as compared to known processes, result in a substantially improved economy of the process according to the invention for activating hydroxyl-containing matrices. The application of the invention produced! activated matrices has been exemplified by the coupling of amines such as aliphatic diamines or polyamines of proteins such as concanavalin A, ovomucoid, ovoinhibitor, and the like. and enzymes such as glucose oxidase, horseradish peroxidase and trypsin or immunoglobulins and antibodies as well as their use as affinity adsorbent or carrier-fixed enzyme. The coupling of nucleic acids to solid supports such as cellulose, Sepharos®, Fractogel, etc. affords affinity supports suitable for purification of DNA and RNA polymerases, kinases, nucleases, etc. In the reaction of the activated carriers with compounds containing SH groups, thiocarbonic acid O.S-diesters are obtained in a smooth reaction, as shown by the example of the reaction of activated perl cellulose with thioalcohol and thiophenol.

embodiments

The production according to the invention of activated matrices and their reaction with nucleophilic reactants will be illustrated by the following examples:

example 1

Pearl cellulose is dehydrated by treatment with water-acetone-a mixture of increasing acetone content and anhydrous acetone. One volume of anhydrous perl cellulose is taken up in a volume of acetone, in which 4OpMoI dimethylaminopyridine was dissolved. With gentle shaking, one milliliter of an acetone solution of N.N'-bis-norbornene .S-dicarboximidyU-carbonate (CO (ONB) ₂ ) (δΟμΜοΙ / ml) is added in portions. The reaction is carried out at room temperature and is stopped 10 minutes after completion of the carbonate addition by suction of the supernatant solution through a glass frit and thorough washing with acetone. The activated carrier contains 860μΜοΙ carbonate groups per gram of anhydrous cellulose.

The coupling capacity is determined by hydrolysis of the activated support with 0.1 N NH ₄ OH and spectrophotometric determination of the cleaved N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) at 270 nm (ε = 6.4-10 ³ Mor ¹ Cm "').

Example 2-7

Perl cellulose was activated as in Example 1, s. Tab. 1, experiment No. 9,11-15.

Example 8-11

Perl cellulose was activated as in Example 1, s. Tab. 2, experiment nos. 1-4.

Example 12-14

The activation was carried out according to Example 1, s. Tdb.3, experiment nos. 1-3.

Example 15

Perl cellulose is dehydrated as described in Example 1. A volume of anhydrous perl cellulose is freed from adhering acetone by washing with acetonitrile and treated with one volume of acetonitrile containing 4OpMoIZmI dimethylaminopyridine. The reaction with 4OpMoI C0 (0NB) ₂ / ml of cellulose is carried out with gentle shaking within 20 to 60 minutes at room temperature. After separating the supernatant and washing the support with acetonitrile, the coupling capacity is determined as follows: a sample of the activated support is treated stepwise with increasing water content and water of acetone-water mixture, equilibrated with borate buffer, pH8.1, and reacted with glycine , The determination of the proportion of covalently bound amino acid is according to Antoni et al. (Analyt. Biochem., 129 [1983] 60-63) by back-titration of unreacted glycine with trinitrobenzenesulfonic acid

 Coupling capacity: 515μΜοΙ glycine / g dry Sepharose.

Example 16 to 17

According to Example 15, the activation reaction was carried out with the solvents listed in Table 4, Run Nos. 3 and 4.

Example 18

Sepharose CI-4B is added gradually with water-Acston mixtures of increasing acetone content and anhydrous acetone

dewatered. One volume of anhydrous Sepharose CI-4B is added to one volume of acetone in which 4SpMoI

Dimethylaminopyridine and 43μΜοΙ triethylamine per milliliter carrier were dissolved. Cool to 15 ° C and light

Shake 1 volume of an acetone solution of CO (ONB) ₂ (80 μΜοΙ / ml Sepharose) in small portions

added so that the temperature of the Reaktinsgemisches does not rise above 24 ¹ C. After a reaction time of 10 minutes, the support is separated from the supernatant solution and thoroughly washed with acetone. To determine the

Coupling gap = it is proceeded as in example 15.

Coupling capacity: about 515μΜοΙ glycine / g dry Sepharose. The coupling capacity determined by HONB cleavage is 640μΜοΙ / g dry Sepharose.

Example 19-21

The polymers listed in Table 5 were activated as in Example 1, s. Trial no. 7,9 and 10.

Example 22

The activation of polyethylene glycol 1500 is carried out by adding 30.4 mg of CO (ONB) ₂ to a solution of 1 g of PEG 1500 and 4.4 mg of DMAP in 3 ml of acetone at room temperature. After a reaction time of 25 minutes, the PEG 1500 is precipitated with ether. The carbonate content of the product is 213 pmol / g.

Example 23-25

The activation of perl cellulose was carried out analogously to Example 1 using various carbonates, s. Tab. 6, Experiment Nos. 1-3.

Example 26

Perl cellulose is dehydrated in the manner described in Example 1 and taken up in anhydrous dioxar.

To a volume of anhydrous cellulose is added a volume of dioxane in which 2.6mM of triethylamine and 0.2mM of dimethylaminopyridine are dissolved per gram of dry cellulose. At room temperature, with gentle shaking, the portionwise addition of 2.1 mmol of N-IChlorcarbonyloxyrö-norbornene ^ .S-dicarboximide (CICOONB) dissolved in one volume of anhydrous dioxane. After 20 minutes, the reaction is complete, the supernatant is aspirated from the cellulose and the activated excipient is extensively washed with dioxane.

The degree of substitution of the activated cellulose gnaws 400μΜοΙ of carbonate per gram of dry cellulose.

Example 27-31

According to Example 26, the activation of pearl cellulose was carried out, s. Tab.7, experiment nos. 3-7.

Example 32-34

The activation of pearl cellulose was carried out as described in Example 26, s. Tab.8, experiment nos. 2-4.

Example 35-37

Activations with different use of chloroformate were carried out analogously to Example 26, s. Table 9, experiment nos. 1-3.

Example 38-44

The activation of various polymers was carried out according to Example 26, s. Tab. 10, Experiment No. 2,3, 5-9.

Example 45 and 46

The activation of water-soluble polymers, which are insoluble in acetone or dioxane, is carried out analogously to Example 26, wherein the use of pyridine in place of DMAP proves to be advantageous, s. Table 10, test nos. 10 and 12.

Example 47

1 g of polyethylene glycol 1500 (Ferak) is dissolved in 2 ml of dry acetone and 10 mg DMAP and 150μΙ triethylamine added. 200 g (83OpMoI) of CI-CO-ONB, dissolved in 2 ml of acetone, are added dropwise to this solution at room temperature within a space of 10 minutes. After a reaction time of 10 minutes, the reaction was terminated by adding diethyl ether and precipitating the PEG. The product recrystallized from acetone contains 444μΜοΙ carbonate groups per gram of dry matter, s. Tab. 10, experiment 11.

Example 48

3 ml of a synthetic hydroxyl-containing polymer (particulate Mischpolyrisat A acrylonitrile and vinyl alcohol) are added in 3ml of anhydrous pyridine to which 4.5 mg dimethylaminopyridine, one hour at 5O ⁰ C with shaking with 300mg N-IChlorcarbonyloxyJ-norbornene ^ .S- dicarboximide implemented. After separation of the supernatant, washing with acetone and removal of the acetone in vacuo, the activated carrier is obtained in anhydrous form. By hydrolysis in aqueous ammonia solution and spectrophotometric determination of the liberated HONB, the coupling capacity was determined to 94pMol-COONB / gram dry substance.

Example 49

Perl cellulose is dehydrated as described in Example 1 and taken up in anhydrous acetone. One volume of sedimented cellulose is mixed with 1 volume of acetone in which 4OpMc! N-methylimidazole / ml and 217 uMolTriethylamin / ml and added at room temperature, in portions 2.1 mmol / ml of chloroformate (CI-CO-ONB) dissolved in one volume of acetone. Ten minutes after completion of the reagent addition at room temperature, the reaction is stopped by separation of the supernatant. The cellulose is washed on the frit with dry acetone, then gradually transferred into the aqueous phase and the coupling capacity determined by hydrolytic cleavage and spectrophotometric Bectimmung of HONB

 Result: 60 pmol -CO-ONB per gram of dry bead cellulose.

Example 50- & 2

In the same way as described in Example 49, the suitability of further supernucleophilic amines for the activation of hydroxyl-containing polymers was tested, s. Tab. 11, experiment nos. 1,3 and 4.

Example 53

Perl cellulose is dehydrated as indicated in Example 1 and taken up in anhydrous acetone and thoroughly washed with anhydrous acetonitrile to remove the acetone completely. One volume of pelleted perlcellulose is suspended in two volumes of acetonitrile, in which 36μΜοΙ of dimethylaminopyridine, 320μΜοΙ of triethylamine, 160μΜοΙ of N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) are dissolved per milliliter of cellulose.

By portionwise addition of a 10% phosgene solution in toluene is reacted with a total of 16OpMoI phosgene per milliliter of cellulose at room temperature. After the addition of phosgene is shaken for a further 10 minutes and the reaction is terminated by aspirating the supernatant over a frit and washing with anhydrous acetonitrile.

A portion of the activated cellulose is reacted immediately with a solution of hexamethylenediamine in acetonitrile and the content of free amino groups of the carrier according to Antoni et al. (Anal Biochem., 129 ("983J 60-63).

Result: 46μΜοΙ NH2 groups / gram of dry pearl cellulose. Another portion of the activated cellulose was gradually transferred to the aqueous medium and, by hydrolytic release of HONB, a coupling capacity of 362 μΜΜΙ-CO-ONB / gram of dry cellulose was found

By coupling [ ³ Hj-labeled glycine, a binding of 225μΜοΙ glycine per gram of dry cellulose was determined.

Example 54-56

Perl celluloso was activated according to Example 50, s. Tab. 12, experiment no. 2-4.

Example 57 and 58

Analogous to the manner described in Example 50, the suitability of further phenols or N-substituted hydroxylamines for the activation of hydroxyl-containing carrier was shown, s. Tab. 13, experiment nos. 1 and 2.

Table 1

Reaction of pearl cellulose with symmetrical carbonate RO-CO-OR

in acetone (room temperature, reaction time: 10 minutes)

carbonate TEA DMAP pyridine coupling capacitance (ΜΜοΙ / ml (ΜΜοΙ / ml (ΜΜοΙ / ml (ΜΜοΙ / ml (ΜΜοΙ-COOR / g sediment. Sediment. sediment. sediment. dry No. cellulose) cellulose) cellulose) cellulose) cellulose) 1 80 * _ _ _ 74 2 80 - - - - 3 80 40 - - - 4 80 160 - - - 5 80 66700 - _ - 6 80 - - 40 - 7 80 - - 160 - 8th 80 - - 9000 - 9 80 _ 12 - 370 10 80 - 40 - 850-900 11 80 - 80 - 805 12 80 43 6 _ 108 13 80 43 12 - 488 14 80 43 24 - 720 15 80 43 48 - 780

Reaction temperature: 70 ° C, 5.5 hours

Table 2

Reposition of symmetric carbonic acid diester RO-CO-OR

(R = -N

with perl cellulose using various supernucleophilic amines.

(Room temperature, reaction time: 20 minutes, 80μΜοΙ symmetric carbonic acid diester / ml sedimented cellulose, 40μΜοΙ supernucleophilic amine / ml sedimented cellulose, solvent: acetone)

supernucleophilic amine

Coupling capacity (μΜοΙ-COOR / g dry cellulose)

Dimethylaminopyridine Methylimidazole Diazabicyclo [2.2.2] octane Diazobicyclo [5.4.0 | undecen

880

210

130

Table 3

Reaction of symmetric carbonic acid diester RO-CO-OR

with perl cellulose, depending on the amount of supcrnucleophilic amine (dimethylaminopyridine) used (reaction time: 20 minutes, room temperature, solvent: acetone)

No carbonic acid diester

(μΜοΙ / ml sedimented cellulose)

DMAP

(μΜοΙ / ml sedimented cellulose)

Coupling capacity (μΜοΙ-COOR / g dry cellulose

80 80 80

12 40 80

370 930 805

Table 4

Reaction of symmetric carbonic acid diester RO-CO-OR

with perl cellulose in various solvents (reaction time: 20 minutes, room temperature, 80μΜοΙ carbonic acid diester / ml sedimented cellulose, 40μΜοΙ DMAP / ml sedimented cellulose)

solvent

Coupling capacity (μΜοΙ-COOR / g dry cellulose)

Acetone acetonitrile dioxane chloroform

805-930 515 733 217

Table 5

Activation of various hydroxyl-containing polymers with symmetrical carbonic acid diester RO-CO-OR

matrix

Coupling capacity (μΜοΙ-COOR / g dry polymer)

Perlcellulose (macroporous) Perlcellulose (microporous) Sepharose CI-4B Sephadex G-100 Fractogel ™ ^p 'KHW75 (F) Polyethylene glycol 1500

800-900

Table 6

Activation of perlcellulose with different symmetrical carbonates (room temperature, reaction time: 20 minutes, solvent: acetone, 80μΜοΙ symrn. Carbonate / ml sedimented cellulose, 40μΜοΙ DMAP / ml sedimented cellulose)

symm.Carbonat

Coupling capacity (μΜοΙ-COOR / g dry cellulose)

1 N, N -disuccinimidyl 882 2 N, N'-Diphthalimidyl- 106 3 p-NO ₂ -phenyl 570

Table Ί Reaction of chloroformates CI-CO-OR

with pearl cellulose (room temperature, reaction time: 20 minutes, solvent: acetone)

No. Chlorameison- TEA DMAP pyridine coupling acid ester (mmol / g) (Mmol / g (ΜΜοΙ / g (ΜΜοΙ / g capacity (μΜοΙ / g trotl'.enoCell.) tr. Cell.) tr. Cell.) tr. Cell.) tr. Cell.) 1 2.1 _ _ _ 35 2 2.1 3.0 - - 104 3 2.1 . "6 _ 414 75 4 2.1 - 207 - 130 5 2.1 ~., 6 207 _ 400 6 2.1 2.6 414 - 530 7 2.1 2.6 828 - 823

Table 8

Reaction of Perlcellulose with Chloroformate CI-CC / R

/ CO

depending on the amount of DMAP used (2.1 mmol CICOONB / g cellulose, 2.6mMoi TEA / g cellulose, temperature: 23 ⁰ C, reaction time: 15 minutes, solvent: acetone)

DAMP

(μΜοΙ / g dried cellulose)

Coupling capacity (μΜοΙ-COOR / g dry cellulose)

1 0 2 20.7 3 207 4 414

25 150 333 590

Table 9

Dependence of the reaction of chloroformate RO-CO-CI

^N co- ^ V ^

with perl cellulose from the ester (temperature: 5 ° C, reaction time: 20 minutes, solvent: acetone)

No. chloroformate TEA DMAP coupling säureester capacity (Mmol / g (Mmol / g (Mmol / g (ΜΜοΙ-COOR / g dry cellulose) dry cellulose) dry cellulose) dry cellulose) ί 2.1 2.6 0.2 422 2 4.2 5.2 0.2 522 3 8.4 10.4 0.2 778

Table 10

Activation of various hydroxyl-containing polymers with chloroformate CI-CO-OR

(Room temperature, reaction time: 20 to 60 minutes, 2.1 mmol of chloroformate / g dry carrier, 210μΜοΙ DMAP / g dry carrier, 2.7 mmol triethylamine / g dry carrier, solvent: acetone or dioxane)

No. matrix coupling capacitance (μΜοΙ-COOR / g dry carrier) 1 Pearl cellulose (macroporous) 820 2 Pearl cellulose (microporous) 50 3 Cellulose powder MN 300 355 4 SepharoseCI-4B 1073 * 5 SephariexLH-20 444 6 Spheroid 1000 462 7 TrisacrylGi? 000 50 8th Toyopearl HW 60 1365 ** 9 FractogelTSKHW75 (F) 780 10 polyvinyl alcohol 21 · ** 11 Polyethylene glycol 1500 462 ⁺ 12 SHP (starch hydrolyzate) 44 ^tf

2.6 mmol CI-CO-OR; 3.5 mmol TEA; 600 μΜοΙ DMAP

2.7 mmol CI-CO-OR; 3.5 mmol TEA; 275 μΜοΙ DMAP 2.1 mmol CI-CO-OR; 2.7 mmol TEA; 1065 μΜοΙ pyridine 0.83 mmol CI-CO-OR; 1.0 mmol TEA; 80 μM of DMAP 2.1 mM CI-CO-OR; 2.7 mmol TEA; 210 μΜοΙ pyridine

tables

Reaction of chloroformate CI-CO-OR

- -oo

co-

with perl cellulose using various supernucleophilic amines

(Room temperature, reaction time: 20 minutes, 2.1 mmol chloroformate / g dry cellulose, 210 μΜοΙ amine / g dry cellulose, 2.7 mmol triethylamine / g dry cellulose, solvent: acetone)

No.

supernucleophilic amine

Coupling capacity (μΜοΙ-COOR / g dry cellulose)

1 2 3

Dimethylaminopyridine N-methylimidazole diazabicyclo [2.2.2] octai) diazabicyclo [5.4.0] undecene

797 602 190 200

Table 12

Activation of pearl cellulose by reaction with phosgene and N-substituted hydroxylamine HO-NR ₂

= NOND)

in the presence of tertiary amines

(Room temperature, reaction time: 20 minutes, solvent: acetonitrile)

No. phosgene HONB TEA DMAP coupling capacitance (ΜΜοΙ *) (ΜΜοΙ *) (ΜΜοΙ *) (ΜΜοΙ *) (tiMol-COOR / g dry cellulose) 1 160 160 320 36 362 2 46 320 320 36 52 3 320 320 640 72 310 4 ' 160 160 160 36 325

per ml of sedimented cellulose "solvent: CHCl ₃

Tabel.o 13

Activation of Perlcellulose r irch reaction with phosgene and different phenols or N-substituted hydroxylamines (room temperature, reaction time: 20 minutes, solvent: acetonitrile, 320μΜοΙ triethylamine / ml sedimented cellulose, 36μΜοΙ DMAP / ml sedirrentierte cellulose, 16OpMoI phosgene and 160μΜοΙ phenol or N-substituted hydroxylamine / ml sedimented cellulose)

No phenol or N-subst. coupling capacitance

Hydroxylamine (μΜοΙ-COOR / g dry cellulose)

1 N-hydroxysiiccinimide 330

2 p-nitrophonol 105

3 N-hydroxy-5-norbornone-2,3-dicarboximide (HONB) 362

Determination of the capping capacity by hydrolysis of the carriers with ammonia solution and spectrophotometric measurement of the released phenol or of the N-substituted hydroxylamines. '

Claims (11)

1. A process for the activation of hydroxyl-containing polymeric compounds and solid surfaces formed therefrom, characterized in that the symmetrical carbonic diester of the general formula RO-CO-OR or chloroformate of the general formula CI-CO-OR, wherein the radical R is an electron-attracting group, optionally Phosgene together with a phenol or an N-substituted hydroxylamine, in the presence of capable of forming reactive acylium supernucleophilic amines in the ratio of 0.01 to 2.5 moles of amine per mole of carbonic acid diester and optionally another belonging to the group of strong bases tertiary amine in Ratio of 0.1 to 2.5 moles of amine per mole of carbonic acid diester in anhydrous organic solvents at temperatures of 0 to 100 ⁰ C with the hydroxyl-containing polymers or the solid state surfaces formed therefrom. 2. The method according to claim 1, characterized in that the symmetrical carbonic acid diesters are reacted with hydroxyl-containing polymers or ^p estkörperoberflächen formed therefrom in the presence of supernucleophilic amines and optionally further tertiary amines. 3. The method according to claim 1, characterized in that the chloroformates are reacted with hydroxyl-containing polymers or solid surfaces formed therefrom in the presence of tertiary carbonate-forming amines and / or supernucleophilic amines. i. A process according to claim 1, characterized in that the chloroformate used for the polymer activation is intermediately formed in the presence of the polymer of phosgene and a substituted phenol or N-substituted hydroxylamine and reacted with the polymer without isolation and tertiary carbonate-forming amines and / or supernucleophiles Amines can be used as reactants. 5. The method according to claim 1, characterized in that as supernucleophilic amines compounds such as 4-dimethylaminopyrite ^! n (DMAP), 4-pyrrolidinopyridine (PPY), N-methylimidazole, diazabicyclo [5.4.Ü] undecene (DBU), 4-morpholinopyridine, diazabicyclo [2.2.2] octane (DABCO). 6. The method according to claim 1 to 5, characterized in that tertiary amines such as triethylamine, N-methylmorpholine, N, N-dimethylaniline or other heterocyclic amines such as pyridine, picolines, N-methylpiperidine, bz /. supernucleophilic amines according to claim 7 are used. 7. The method according to claim 1,2,5 and 6, characterized in that per mole of symmetrical carbonic acid diester 0.01 to 2.5 mol, preferably 0.2 to 1.2 mol supernucleophilic AmIt; and optionally 0.1 to 2.5 moles of tertiary amine (preferably 0.8 to 1.5 moles) are used. 8. The method of claim 1,3,5 and 6, characterized in that per mole of chloroformate 0.1 to 2.5 mol, preferably 0.8 to 1.5 MH carbonatbildendes amine and / or 0.02 to '. ., 5 mol, preferably 0.1 to 0.5 mol of supernucleophilic amine are used. 9. The method according to claim 1,4,5 and 6, characterized in that at a ratio of phosgene to phenol or N-substituted hydroxylamine of 1.0 to 0.5 to 2.0, an employment of carbonate-forming tertiary amines of 1 , 0 to 2.5 moles per mole of phosgene and / or 0.02 times 2.5 moles of supernucleophilic amine per mole of phosgene. 10. The method according to claim 1 to 4, characterized in that in the coming used symmetrical carbonic acid diesters, chloroformates and phenols or N-substituted hydroxylamines the radical R succinimidyl, phthalimidyl, 5-norbornene-2,3-dicarboximidyl- , p-nitrophenyl and other substituted phenyl radicals. 11. The method according to claim 1 to 4, characterized in that the temperature in the addition of the solutions of carbonic acid diesters, chloroformate or phosgene and the subsequent reaction between 0 and 100 ⁰ C, preferably 4 to 60 ⁰ C is maintained and the conversion after 10 to 120 minutes is completed. 12. Experienced according to claim 1 to 4, characterized in that as polar organic solvents such as acetonitrile, dimethylsulfoxide, tetrahydrofuran, acetone, dioxane u.a. or nonpolar compounds such as benzene, toluene and the like. or halogenated hydrocarbons such as chloroform, methylene chloride, and the like. or mixtures of these solvents! be used.