DD260701A1 - METHOD OF TREATING ACTIVE UNSYMMETRIC CARBOHYDRY MEDICINE GROUPS ON HYDROXYL GROUP-BASED POLYMERS - Google Patents

Abstract

A process for the transesterification of active unsymmetrical carbonic acid diester groups on hydroxyl-containing polymers. According to the invention, unsymmetrical carbonic acid diesters bound to hydroxyl-containing polymers with electron-attracting groups in the presence of bases, in particular of supernucleophilic amines, are transesterified in anhydrous organic solvents at temperatures of 0-100 with alcohols, phenols or N, N-disubstituted hydroxylamines. Applications include process development in the chemical and pharmaceutical industries and biotechnology.

Description

Field of application of the invention

The invention relates to a process for the transesterification of active unsymmetrical carbonic acid diester groups on hydroxyl-containing polymers with alcohols, phenols or N, N-disubstituted hydroxylamines in the presence of tertiary amines. The resulting polymers react with nucleophilic reactants and can be used to couple amines, amino acids, peptides, proteins and nucleic acids.

Areas of application include basic scientific research and process development in the chemical and pharmaceutical industries, life sciences, biotechnology and medicine.

Characteristic of the known technical solutions

The preparation of unsymmetrical Kohiensäurediester groups bearing hydroxyl-containing polymers is predominantly by activation of polymers with chloroformates (chloroformate). Thus, hydroxyl-containing polymers were reacted with chloroformates (CI-CO-OR), in which z. R = ethyl, isobutyl, p-nitrophenyl, 2,4,5-trichlorophenyl, succinimidyl, phthalimidiyl, 5-norbornene-2,3-dicarboximidyl. (Barker, SA et al., Carbohydrate Res., 17: 471; 1971; PDG Dean, WS Johnson, FAMiddle, Affinity Chromatography, IRL Press, Oxford, 1985, p.44, Wilchek, M., et al., Biochem. 1982] 629, Meth. Enzymol. 104 [1984] 3, Biochem., Biotechn., 11 [1985] 445, J. Drobnik et al., Biotechnol., Bioeng., 24 (1982) 487, GB 2015553, EP 0134041).

Activation (esterification) of hydroxyl groups on polymers can also be carried out by reacting them with symmetrical carbonic acid diesters (RO-CO-OR), where R is an electron-attracting group, in the presence of supernucleophilic amines (M.W.Workek et al., Appl.Biochem.Biotechnol .11 [1985] 141). the J-0-CO-OR groups present in the activated polymers contain in the remainder the group derived from the corresponding alcohol, phenol or Ν, Ν-disubstituted hydroxylamine. The coupling ability of these polymers towards nucleophilic, amino-containing compounds was detected in all cases.

For the coupling of hydroxyl-containing compounds activated polymers by other methods, such. Divinylsulfone, epoxide or halotriazine activated, is recommended (W.H. Scouten, Affinity Chromatography, John Wiley & Sons, New York 1981). A disadvantage; which impedes the use of divinylsulfone-activated polymers, is the coupling of alcohols or amino group-containing compounds at high pHs (> 10) and the fact that the coupling products of alcohols> pH9 and those of amines> pH8 are unstable.

The epoxy-activated polymers couple hydroxy compounds only at higher temperatures and pHs between 11 and 13. The elimination of the unreacted epoxide groups is extremely difficult. Disadvantages of the halotriazine-activated polymers are the pretreatment of the polymers with 3N NaOH which is necessary before activation, the change in the polymer structure upon activation by cross-linking and the coupling of alcohols at pH values> 10, with only more nucleophilic OH groups reacting.

S.Yollesetal. Polym. Chem., Ed., 17 (1979) 4111) describe the coupling of steroids via their hydroxyl groups, but the hydroxyl-containing polymer (eg, hydroxypropyl cellulose) is converted into a chloroformate group by reaction with phosgene (-O-CO-CI) containing carrier was converted. The disadvantage here is the use of large amounts of liquid phosgene and the fact that the activated polymer is extremely sensitive to hydrolysis.

Object of the invention

The object of the invention is to develop a process for transesterifying polymers containing carbonic acid diester groups under mild reaction conditions and while maintaining the covalent bond to the polymer.

Explanation of the essence of the invention

The invention has for its object to transesterify activated hydroxyl-containing polymers of the type J-O-CO-OR in a technically feasible process with good yields.

According to the invention, this object is achieved in that active asymmetric carbonic acid diester group-containing polymers of the general formula JO-CO-OR, wherein the radical R is an electron-attracting group, in the presence of supernucleophilic amines and / or belonging to the group of strong bases tertiary amine in anhydrous organic solvents at temperatures of 0-10O ⁰ C with alcohols, phenols or with Ν, Ν-disubstituted hydroxylamines or their salts are reacted.

Optionally, a treatment with aqueous solutions is carried out.

The transesterification is an equilibrium reaction which can be described by the following general equation:

J-O-C

0 base 0

OR + R^T-OH * [-O-C-Or '+ ROH

(Equation 1)

Activated hydroxyl-containing polymers are both water-soluble and insoluble natural polysaccharides and their derivatives or hydrolysis products such as cellulose, agarose. Dextran, starch, man nan, sepharose, starch hydrolysis products (SHP), etc. or synthetic polymers such as Fractogel, Toyopearl, Spheron, Trisacryl, polyvinyl alcohol, polyethylene glycol, etc. can be used. As an electron-attracting group R are substituted phenyl radicals, such as. For example, p-nitrophenyl or 2,4,5-trichlorophenyl or succinimidyl, phthalimidyl or 5-norbornene-2,3-dicarboximidyl radical used. By these good leaving groups, the transesterification is facilitated and z. B. at low temperatures (0-2O ⁰ C) take place. Decisive for the course of the transesterification is the addition of bases, in particular of tertiary amines. The amount required depends on the nature of the amines. As supernucleophilic amines z. As 4-dimethylaminopyridine (DMAP), 4-pyrrolidino or 4-morpholinopyridine, N-methylimidazole, diazabicyclo [5.4.0] undecene, diazabicyclo [2.2.2] octane used. The transesterification takes place already in the presence of catalytic amounts. According to the invention, from 0.01 to 5 mol of supernucleophilic amine, preferably from 0.05 to 2 mol, are used per mole of asymmetric carbonic acid diester groups on the polymer.

Increasing the amount accelerates the reaction, e.g. For example, when using equimolar or larger amounts of DMAP, based on the active unsymmetrical carbonic acid diester groups present, the reaction time can be considerably shortened (1-4 hours at room temperature). Triethylamine, N-methylmorpholine, N, N-dimethylaniline or heterocyclic bases such as pyridine, picolines, N-methylpiperidine and the like are generally used as strongly basic tertiary amines. If these are used, relatively large amounts are generally required, at least equimolar to the active groups. wherein the transesterification is slower and the degree of conversion is significantly lower than when using supernucleophilic amines.

It has proved to be advantageous, especially in the transesterification with OH compounds of very low acidity of the OH group, in addition to the supernucleophilic amine, the strongly basic amine equimolar to the amount of the OH compound (ROH) (eg 1-100 mol per per cent asymmetric carbonic acid diester Groups). The additional use of strongly basic amines can advantageously be dispensed with if ammonium, alkali metal or alkaline earth metal salts of the OH compounds are reacted. Because of the better solubility of these salts in strongly polar solvents, acetonitrile or dimethylformamide should be used.

As hydroxyl-containing compounds (see Equation 1, ROH) may be substituted and substituted primary, secondary and tertiary alcohols, phenol, substituted phenols such as p-nitrophenol, di- or trichlorophenols, cresols and N, N-disubstituted hydroxylamines such as N-hydroxysuccinimide, N Hydroxyphthalimide, N-hydroxy-5-norbornene-2,3-dicarboximide (HONB). However, there must be no strongly nucleophilic groups (-NH ₂ , -SH ₁ -COOH etc.) containing substituents, because otherwise a coupling takes place on the polymer via these groups. The OH compounds are used both in a mixture with anhydrous organic solvents and even as a solvent. They are preferably used in a larger excess "(per mole of asymmetric carbonic acid diester groups 1-100 moles of alcohols, phenols or N, N-disubstituted hydroxylamines).

Importantly, anhydrous organic solvents are used to avoid the relatively rapid hydrolysis of unsymmetrical carbonic acid diester active groups in the presence of bases. Of the possible organic solvents, acetone or dioxane are preferred. However, other solvents such as diethyl ether, tetrahydrofuran, pyridine, acetonitrile, dimethylformamide, dimethyl sulfoxide or aliphatic hydrocarbons such as hexane, heptane or aromatic compounds such as benzene and toluene or halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride and the like are also useful. used successfully for transesterification.

In the transfer of the polymers from the aqueous medium into an organic solvent, a treatment with water-solvent mixtures of increasing solvent content is carried out in order to avoid structural changes of the polymer. In an analogous manner, the transfer of the polymers from the organic solvent into the aqueous medium.

The degree of transesterification of the active unsymmetrical carbonic acid diester groups depends on the nature of the polymer, the type of OH compound (ROH) and the reaction conditions (amount of base and OH compound, temperature, reaction time). For the transesterification 2 different process variants are applicable. Either the polymer to be transesterified is reacted in an anhydrous organic solvent with tertiary amine and the OH compound (ROH) with gentle shaking or stirring, or the polymer is placed in a chromatography column with the solution of tertiary amine and OH compound (ROH) in a treated anhydrous organic solvent in Durchflußverfahren. In the first case, the use of a large excess of the OH compound (ROH) is recommended in order to shift the equilibrium of the transesterification (see equation 1) in favor of the desired transesterification product (J-O-CO-OR '). This may optionally also by the use of low-polarity or non-polar solvents such. As benzene or toluene can be achieved because the liberated compound ROH contains a relatively strong acidic hydroxyl group and can form salts with tertiary amines, which are largely insoluble in these solvents.

If the transesterification is carried out by the flow-through method, excessively large excesses of OH compound (ROH) are not necessary because the OH compound (ROH) split off from the activated polymer is continuously discharged from the column and thus also removed from the transesterification equilibrium.

It is expedient for both variants, a subsequent thorough washing of the polymer with highly polar solvents such. As acetonitrile or the short-term conversion into an aqueous medium of pH2-9, preferably 2-3, to remove any adhering tertiary ammonium salt.

The course of the transesterification can be monitored by hydrolysis of the carbonic acid diester groups of the polymer with 0.1 M NH ₄ OH and spectrophotometric determination of the split-off OH compound (ROH) and optionally also of ROH and by titration of the unsymmetrical carbonic acid diester groups present. The titration is carried out after coupling of allylamine and addition of excess iodomonobromide to the double bond of the coupled allylamine by addition of potassium iodide with ⁿ / z> thiosulfate (determination of the unadded IBr) (HP Kaufmann, Analysis of Fats and Fat Products, Vol. 1, Springer Verlag, Berlin-Göttingen-Heidelberg 1958). In addition, the transesterified polymers can be characterized by their IR spectra, especially in the region of the C = O stretching vibration (1700-1 800 cm ^-1 ). In an incomplete transesterification of the -O-CO-OR groups can be z. B. in the case of the reaction with OH compounds (ROH), which carry no electron-attracting groups, such as aliphatic alcohols or phenols, which are still on the polymer non-transesterified carbonic acid diester groups due to their greater sensitivity to hydrolysis by aftertreatment with aqueous solutions to recover the hydroxyl groups of the polymer are cleaved quantitatively. The advantage here is the use of weakly acidic solutions (eg pH5). In weakly alkaline pH ranges (eg pH 8), the hydrolysis is faster, but it can also be split off to some extent the newly formed, less active carbonic acid diester groups again. The advantage of the process is that complete elimination of the -O-CO-OR groups succeeds by the choice of suitable reaction conditions. The yield of transesterification product (JO-CO-OR ') is up to about 75%. Depending on the nature of the polymer and the amount of asymmetric carbonic acid diester active groups (-O-CO-OR), interesterified polymers can be obtained containing about 50-300μηηΙ carbonic diester groups (-0-CO-OR ') per gram contain dry polymers. When using highly activated polymers, such as. B. with chloroformates activated Sepharose or Perlcellulose (600-1 ΟΟΟμ, ΜοΙ-O-CO-OR groups / g dry carrier material) can be obtained by transesterification by the novel process to about 500μ.ΜοΙ -0-CO-OR'- Introduce groups / g (eg with ROH = phenol or p-nitrophenol). This corresponds in the case of p-nitrophenol a coupled amount of phenol of about 6 mg / ml sedimented perl cellulose. Depending on the sensitivity to hydrolysis of the -O-CO-OR 'groups, the transesterified polymers can be stored either in anhydrous organic solvents or else in aqueous solutions having a weakly acidic pH (eg pH 6).

The coupling ability of the transesterified polymers was demonstrated by the example of the coupling of amines, amino acids and proteins

 The invention is illustrated by the following examples:

example 1

3ml sedimented in anhydrous dioxane activated perl cellulose, prepared by reaction of perl cellulose with N-chlorocarbonyloxy-δ-norbornen ^ ^ - dicarboximide (CI-CO-ONB) according to EP 0134041, with 52μΜοΙ active carbonic acid diester groups of the -0-CO-ONB type / ml sedimented cellulose (= 800ju.Mol / g dry cellulose) are added to a solution of 204mg p-nitrophenol (1.5 mmol) in 6ml anhydrous dioxane and heated for 4 hours with gentle shaking in a sealed flask to 65 ⁰ C. The carrier thus treated still contains all active -O-CO-ONB groups; p-nitrophenol was not coupled. Subsequently, to the reaction mixture, an amount of 20mg4-dimethylaminopyridine (164μΜοΙ DMAP)

added and shaken for one hour at room temperature. Thereafter, 26.4 μΜΜΙ p-nitrophenyl carbonate groups / ml cellulose (410 / xmol / g) were determined. This corresponds to a degree of transesterification of about 50% and a coupled p-nitrophenol amount of 3.7 mg / ml of cellulose.

After aspirating the cellulose and washing with anhydrous dioxane over a glass frit, the transesterification is continued with the renewed addition of p-nitrophenol and DMAP under analogous conditions for 1 hour. Thereafter, the carrier contains 26.9 μΜΜ pS nitrophenyl carbonate groups in addition to about 13μΜοΙ-O-CO-ONB groups.

Continuing the transesterification as described above, a carrier with 22.6μΜοΙ p-nitrophenyl carbonate groups / ml is obtained. The proportion of -O-CO-ONB groups drops to approximately θμΐνΐοΐ / ml. For the determination of the active groups about 100-200μΙ sedimented activated cellulose are removed and washed on a glass frit with anhydrous dioxane and then transferred by treatment with water-dioxane mixtures increasing water content in distilled water. After a sharp suction of the water, the determination of the coupling capacity (amount of active asymmetric carbonic acid diester groups / ml sedimentierte bzw./g dry cellulose) by hydrolysis with 0.1 M NH ₄ OH and spectrophotometric determination of the split N-hydroxy-5-norbornene -2.3-dicarboximide (HONB) at 270 nm (ε = 6.4 χ 10 ³ MoT ¹ cm ^"1) and the cleaved p-nitrophenol at 400 nm (ε = 1.7 χ 10 ⁴ MOR ¹ cm"^"1). If mixed activated carriers are present, which in addition to ONB also contain p-nitrophenol groups, the proportion of the corresponding active groups in the total coupling capacity can be determined by means of a HONB p-nitrophenol calibration curve and the ratio of the extinctions at 400 and 260 nm be determined.

Example 2

To 1 ml in anhydrous dioxane sedimented CI-CO-ONB-activated perl cellulose with 46μΜοΙ-O-CO-ONB groups / ml of cellulose, a solution of 40 mg p-nitrophenol (290μΜοΙ) and 100 μΐ triethylamine (720μΜοΙ) in 2 ml anhydrous dioxane and shaken for one hour at room temperature. The content of p-nitrophenyl carbonate groups is then 8.6 / u.Mol / ml sedimented or 133 / u, mol / g of dry cellulose, the coupled p-nitrophenol 1.2mg / ml cellulose. In addition, the carrier still contains about 37μΜοΙ-O-CO-ONB-Gr.uppen / ml cellulose.

Example 3

A solution of 28mg p-nitrophenol (200μΜοΙ), 28μΙ triethylamine (200μΜοΙ) and 16mg DMAP (130μΜοΙ) is added to 4ml of anhydrous tetrahydrofuran-sedimented CI-CO-ONB-activated perlcellulose containing 51μΜοΙ-O-CO-ONB groups / ml cellulose in 8 ml of anhydrous tetrahydrofuran and the mixture shaken for 1 hour at room temperature in a sealed vessel. The carrier then contains 9.8μΜοΙ p-nitrophenyl carbonate groups / ml sedimented or ΙδΟμΜοΙ / g dry cellulose. The content of -O-CO-ONB groups is 40μΜοΙ / ηιΙ sedimented cellulose.

Example 4

1 ml sedimented in anhydrous acetone CI-CO-ONB-activated perl cellulose with 51 μΜοΙ-O-CO-ONB groups / ml of cellulose according to Example 3 with 8 mg p-nitrophenol (57 / xMol), 20 / zl triethylamine (145μΜοΙ ) and 3.3 mg of N-methylimidazole (40μΜοΙ) in 2ml anhydrous acetone transesterified.

After one hour reaction time, the carrier contains 9.5μΜοΙ and after 4 hours 11.9μΜοΙ p-nitrophenyl carbonate groups / ml sedimented cellulose.

The coupled p-nitrophenol is after 4 hours 1.7 mg / ml and the amount of remaining -O-CO-ONB groups 36μΜοΙ / ηηΙ sedimented cellulose.

Example 5

3 ml sedimented in anhydrous dioxane CI-CO-ONB-activated perl cellulose with 52μΜοΙ-O-CO-ONB groups / ml cellulose are analogous to Example 3 with 204mg p-nitrophenol (1.5mMol), 12μΙ triethylamine (90μΜοΙ) and 7.3mg DMAP (ΘΟμΜοΙ) transesterified. After a reaction time of 6 hours, the carrier contains 28μΜοΙ p-nitrophenyl carbonate groups / ml sedimented or 420 / iMol / g dry cellulose. The proportion of -O-CO-ONB groups is 20μΜοΙ / ηηΙ sedimented cellulose. The support thus obtained is introduced into a chromatographic column without bubbles and in Durchflußverfahren with a solution of 600 mg of p-nitrophenol (4.3mMol), 50μΙ triethylamine (360μΜοΙ) and 22mg DMAP (180μΜοΙ) in 20ml of anhydrous dioxane at a flow rate of 2, 5 ml / hour. After 8 hours, wash with 40 ml of anhydrous dioxane (10 ml / hour). The thus transesterified carrier contains 16.4μΜοΙ p-nitrophenyl carbonate groups / ml sedimented or 252 / xmol / g dry cellulose. The proportion of -O-CO-ONB groups is about 0,3μΜοΙ / ιηΙ sedimented cellulose.

Example 6

1 ml of anhydrous dioxane sedimented perlcellulose with 16.4μΜοΙ p-nitrophenyl carbonate groups is dissolved in a chromatography column containing a solution of 60 mg HONB (335 / xmol), 50μΙ triethylamine (360μΜοΙ) and 2 mg DMAP (16.4μΜοΙ) in 4 ml of anhydrous dioxane at a flow rate of 1 ml / hour. Thereafter, the support contains 12.8 / xmol-O-CO-ONB groups / ml sedimented or 198 / u, mol / g dry cellulose. This corresponds to a degree of transesterification of about 75% and a coupled HONB amount of 2.3 mg / ml sedimented cellulose. The proportion of p-nitrophenyl carbonate groups is 3.2 / μmol / ml of sedimented cellulose.

The support thus obtained is transferred to a 10 ml Schlick flask and treated with a solution of 12 mg HONB (67μΜοΙ), 10μΙ triethylamine (62μΜοΙ) and 1 mg DMAP (8,2μΜοΙ) in 2 ml of anhydrous dioxane and 20 hours shaken at room temperature. Thereafter, the carrier contains 6,6μΜοΙ-O-CO-ONB groups / ml sedimented or 102 μΜοΙ / g dry cellulose. The content of p-nitrophenyl carbonate groups is <0.05μΜοΙ / ηηΙ sedimented cellulose.

Example 7

1 ml sedimented in aqueous acetonitrile CI-CO-ONB-activated perl cellulose with 65μΜοΙ-O-CO-ONB groups / ml of cellulose with a solution of 10.4 mg ΝθίπυΓη-ρ-ηΗχορΙιβηοΙβίίδδΜΜοΙ) and 2 mg of DMAP (16,4μΜοΙ ) in 2 ml of anhydrous

Acetonitrile and shaken for 15 minutes at room temperature. Thereafter, the carrier contains 27 μ, ΜοΙρ-nitrophenyl carbonate groups / ml sedimented or 420μ, ΜοΙ / g dry cellulose. The proportion of -O-CO-ONB groups is 37μ.ΜοΙ / ηηΙ sedimented cellulose.

Example 8

1 ml of the carrier obtained according to Example 7 is converted into anhydrous Dioxa η, with a solution of 18 mg HONB (100 μΜοΙ), 15μ.Ι triethylamine (110μ.ΜοΙ) and 2mg DMAP (16.4μ.ΜοΙ) in 2ml anhydrous dioxane shaken and shaken for 2 hours at room temperature. Thereafter, the carrier contains 57μ, ΜοΙ-O-CO-ONB groups / ml sedimented or 883 / tiMol / g dry cellulose. The proportion of p-nitrophenyl carbonate groups is about 7 / xMol / ml sedimented cellulose.

Example 9

5 ml of anhydrous acetone sedimented CI-CO-ONB activated perl cellulose containing 78.4 μ, ΜοΙ-O-CO-ONB groups / ml of cellulose is treated with a solution of 1 g of p-nitrophenol in 10 ml of anhydrous acetone in a closed vessel at room temperature Shaken for 30 minutes. It is then decanted and the cellulose is shaken with a solution of 2.8 g of p-nitrophenol (20 mM), 2.7 ml of triethylamine (20 mM) and 100 mg of DMAP (about 0.8 mmol) in 50 ml of dry acetone for 4 hours at room temperature. Thereafter, the carrier contains 27.3 μΜοΙρ-nitrophenyl carbonate groups / ml sedimented or 304μΜοΙ / 9 dry cellulose. The proportion of -O-CO-ONB groups is about 7μΜοΙ / ιηΙ sedimented cellulose.

Example 10

2 ml of CI-CO-ONB activated perl cellulose sedimented in anhydrous acetone with 78.4 μmol O-CO-ONB groups / ml cellulose are placed in a chromatography column with a solution of 1.09 g p-nitrophenol (7.8 mmol), Treated with 1.1 ml of triethylamine (7.8 mmol) and 3.8 mg of DMAP (0.03 mmol) in 50 ml of anhydrous acetone by the flow-through method (flow rate: 12.5 ml / hour).

Thereafter, the carrier still contains 5.4μΜοΙ-O-CO-ONB groups and 33.4μ.ΜοΙ p-nitrophenyl carbonate groups / ml of cellulose. This corresponds to a coupled amount of p-nitrophenol of 4.6 mg / ml of sedimented cellulose.

Example 11

3 ml of anhydrous dioxane sedimented CI-CO-ONB-activated perl cellulose with 52μ.ΜοΙ-O-CO-ONB groups / ml of cellulose according to Example 3 with 420 mg of triphenylmethanol (1.6 mmol), 120 / nl triethylamine (0.9 mmol) and 6 mg DMAP (0.05 mmol) in 6 ml anhydrous dioxane.

After a reaction time of 24 hours, the support still contains 24.5μ.ΜοΙ-O-CO-ONB groups / ml sedimented cellulose. Following the transesterification, the support is washed with about 15 ml of anhydrous dioxane on a Glasf ritte and transferred into the aqueous medium. By treatment with 6 ml of 0.1 M sodium acetate buffer, pH 5.3 at room temperature (3 days), the remaining -O-CO-ONB groups can be quantitatively hydrolyzed. The amount of triphenylmethyl carbonate groups remaining on the support is 6.5 / mmol / ml sedimented or ΙΟΟμΜοΙ / g dry cellulose. This corresponds to a coupled triphenylmethanol amount of 1.6 mg / ml sedimented cellulose.

The determination of the carbonate groups after hydrolysis was carried out by coupling of allylamine (used in excess) at pH 10 in sodium tetraborate buffer (about 20 hours at room temperature) and subsequent addition of iodine monobromide to the double bond of the coupled allylamines and back titration of the unreacted IBr after addition of excess potassium iodide with thiosulfate solution.

Example 12

3 ml of anhydrous dioxane sedimented CI-CO-ONB-activated perlcellulose with 52μ, ΜοΙ-O-CO-ONB groups / ml of cellulose analogously to Example 3 with 240 / xl isopropanol (3.2mMol), 150 / xl pyridine (1, 8 mmol) and 6 mg DMAP (0.05 mmol).

After a reaction time of 24 hours, the support still contains 19.7μ, ΜοΙ-O-CO-ONB groups / ml sedimented cellulose.

The further treatment of the carrier and the determination of the carbonate groups after hydrolysis were carried out as described in Example 11.

The carrier contains 7,5μ.ΜοΙ isopropyl carbonate groups / ml sedimented or 120μ, ΜοΙ / g dry cellulose. This corresponds to a coupled amount of isopropanol of about 0.5 mg / ml sedimented cellulose.

Example 13

3 ml of anhydrous acetone sedimented CI-CO-ONB activated perl cellulose containing 78.4 / x mol -O-CO-ONB groups / ml of cellulose are transferred to 5 ml of anhydrous isopropanoi. For this purpose, 20 mg of DMAP (164μ, ΜοΙ) are added and the mixture shaken for 4 hours at room temperature. Thereafter, the carrier still contains 22.3μ, ΜοΙ-O-CO-ONB groups / ml sedimented cellulose. By aftertreatment with 3N sodium chloride solution, pH 5.6 at room temperature (4 days) and subsequent Allylaminkopplung according to Example 10 were 17.5μΜοΙ isopropyl carbonate groups / ml sedimented or 194 //. Mol / g dry cellulose determined. This corresponds to a coupled amount of isopropanol of 1.0 mg / ml sedimented cellulose. The content of -O-CO-ONB groups is <0.8 / mmol / ml sedimented cellulose.

Example 14

The transesterification is carried out analogously to Example 13 using 2 mg of DMAP. After a reaction time of 24 hours, the support still contains 52.3 μΜοΙ of O-CO-ONB groups / ml of sedimented cellulose. After hydrolysis at pH 5.6 (10 days at room temperature), the amount of -O-CO-ONB groups <0.1 μΜοΙ / ml and the di-isopropyl carbonate groups 12.5 μΜΜΙ / ml of sedimented cellulose.

Example 15

5 ml of anhydrous acetone sedimented CI-CO-ONB-activated perl cellulose with 78.4 μ, ΜοΙ-O-CO-ONB groups / ml of cellulose are washed in the flow-through with a solution of 10VoI .-% isopropanol in dry acetone and then with a solution of 2 ml of isopropanol (26 mmol), 2 ml of triethylamine (14.5 mmol) and 100 mg of DMAP (0.8 mmol) in 50 ml of anhydrous acetone within 4 hours.

-6- Z60 701

OerTräger then still contains 1,7μΜοΙ-O-CO-ONB groups and 25,2μΜοΙ isopropyl carbonate groups / ml of cellulose. By post-treatment of the support with 3 N sodium chloride solution (pH 5.6), the remaining O-CO-ONB groups can be removed.

The amount of isopropyl carbonate groups is then 21.1μΜοΙ / ηηΙ cellulose.

This corresponds to a coupled amount of isopropanol of 1.3 mg / ml. With the help of IR spectroscopy (KBr pellet) is detected by ihreC = O stretching vibration at 1730cm "" ^1, isopropyl carbonate group.

1 ml of the isopropyl carbonate-cellulose is treated with a solution of 10 mg of concanavalin A in 1 ml of sodium tetraborate buffer for 9.316 hours at room temperature with gentle shaking. The determination of the coupled protein amount gives 3.5 mg Concanavalin A.

Example 16

1 ml of anhydrous dioxane-sedimented CI-CO-ONB-activated cellulose with 52μΜοΙ-O-CO-ONB groups is in a chromatography column with a solution νοη400μΙ isopropanol (5.3mMol), 400μΙ triethylamine (2.9mMol) and 20mg DMAP (0.164 mmol) in 20 ml of anhydrous dioxane at room temperature at a flow rate of 2.5 ml / hour.

Thereafter, the support still contains 7.9 μ, ΜοΙ -O-CO-ONB groups. The thus transesterified carrier is gradually transferred to distilled water, filtered off with suction and treated for 8 hours at room temperature with gentle shaking with 5 ml of a 0.1 M sodium tetraborate solution (pH 8.3).

The carrier will no longer contain detectable levels of -O-CO-ONB groups. The determination of the isopropyl carbonate groups on the support is carried out, as described in Example 11, by coupling allylamine. The amount of isopropyl carbonate groups is 11 μΜοΙ / ml sedimented or 165μΜοΙ ^ dry cellulose. This corresponds to a coupled amount of isopropanol of about 0.7 mg / ml of cellulose.

Examples

3 ml sedimented CI-CO-ONB-activated perl cellulose in anhydrous dioxane Γηϊί78,4μΜοΙ-O-CO-ONB groups / ml cellulose are flow-through with a solution of 1.1 g phenol (11,8 μmol), 1.65 ml of triethylamine (11.8 mmol) and 5.7 mg of DMAP (0.047 mmol) in 50 ml of anhydrous dioxon (flow rate: 12.5 ml / hour). The carrier then still contains 3,8μ, ΜοΙ-O-CO-ONB groups and 47,3μΜοΙ phenyl carbonate groups / ml cellulose. This corresponds to a coupled phenol quantity of 4.4 mg phenol / ml cellulose. After cleavage of the remaining -O-CO-ONB groups by hydrolysis in 3N sodium chloride solution pH 5.6 (4 days, room temperature) 42.8μΜοΙ phenyl carbonate groups / ml cellulose are detected (determined by coupling of allylamine according to Example 11 ). In the IR spectrum the phenyl carbonate group ¹ can be detected by their C = O stretching vibration at 1 760cm ".

Example 18

5 ml of anhydrous acetone sedimented CI-CO-ONB activated perl cellulose containing 78.4 / μmol O-CO-ONB groups / ml of cellulose are flow-through with a solution of 1.2 ml of ethanol (20 mM), 2.8 ml of triethylamine ( 20 mM) and 9.6 mg of DMAP (0.08 mmol) in 50 ml of anhydrous acetone (flow rate: 12.5 ml / hour). The transesterified carrier contains 1.8 / xmol -O-CO-ONB groups and 30.9μΜοΙ ethyl carbonate groups / ml cellulose. This corresponds to a coupled amount of ethanol of 1.4 mg / ml of cellulose. 3 ml of the transesterified carrier are reacted with a solution of 400 mg of glycine in 3 ml of sodium bicarbonate buffer, pH 11.16 hours at room temperature with gentle shaking. The coupled amount of glycine (determined by titration of the carboxyl groups present on the carrier) is 6.9 mg or 90 μΜοΙ.

Example 19

3 ml of anhydrous acetone-sedimented CI-CO-ONB-activated Sepharose CL-4B containing 52.4 μΜοΙ of O-CO-ONB groups / ml of Sepharose are treated with a solution of 1.1 g of p-nitrophenol (7.9 mmol), 1 , 1 ml of triethylamine (7.9 mmol) and 1.9 mg of DMAP (0.016 mmol) in 5 ml of anhydrous acetone according to Example 3 implemented. After a reaction time of 30 minutes, the support contains 5.1 .mu.l of p-nitrophenyl carbonate and still 17.4 / .mu.mol -O-CO-ONB groups / ml sedimented Sepharose.

Example 20

5 ml of anhydrous acetone-sedimented CI-CO-ONB-activated fractogel TSK HW 75 (990μΜοΙ ^ dry gel) are mixed with a solution of 3.5 g of p-nitrophenol (25 mmol), 3.5 ml of triethylamine (25 mmol) and 12.2 mg DMAP (0.1 mmol) in 12 ml of dry acetone analogously to Example 3 implemented. After a reaction time of 30 minutes, the carrier contains 64.3μΜοΙ p-nitrophenyl carbonate groups, after 2 hours 99.3 / xmol / g dry Fractogel. The amount of remaining -O-CO-ONB groups is 150 and 61.5 ^ mol / g dry gel, respectively.

Example 21

1 g of CI-CO-ONB-activated polyvinyl alcohol (21.3 μΜΜ-O-CO-ONB groups / g) sedimented in anhydrous acetone is treated with a solution of 156 mg of p-nitrophenol (1.12 mmol), 155 μΙ of triethylamine (1.12 mmol ) and 0.5 mg of diazabicyclo [2.2.2] octane (0.005 mmol) in 3 ml of dry acetone according to Example 3. After a reaction time of 30 minutes, the polymer contains 2.1 μ, ΜοΙ p-Nitrophenylcarbonat- and 4.5 / xMol-O-CO-ONB groups / g.

Example 22

5 ml of anhydrous acetone sedimented CI-CO-ONB-activated perl cellulose with 78.4 / xmol -O-CO-ONB groups / ml of cellulose are mixed according to Example 18 with 2.8 g of p-nitrophenol (20 mM), 2.8 ml of triethylamine (20 mM ) and 16.5 mg of N-methylimidazole (0.2 mmol) in 50 ml of anhydrous acetone. Thereafter the carrier contains 28.8μΜΜ p-nitrophenyl carbonate and 3.9μΜοΙ O-CO-ONB groups / ml sedimented cellulose.

Example 23

2 ml sedimented in anhydrous acetone CI-CO-ONB-akti.vierte Perlcellulose with 78.4μΜοΙ-O-CO-ONB groups / ml of cellulose analogously to Example 3 with a solution of 0.9 g of N-hydroxysuccinimide (7.8 mmol), Treated with 1.08 ml of triethylamine (7.8 mmol) and 6.4 mg of N-methylimidazole (0.078 mmol). After a reaction time of 30 minutes, the amount of -O-CO-ONB groups is Π, θμΜοΙ and that of the N-succinimidyl carbonate groups 17.1 μΜοΙ / ml of sedimented cellulose.

The determination of the active carbonate groups was carried out spectrophotometrically with the aid of a HONB-N-hydroxysuccinimide calibration curve and the determination of the ratio of extinctions at 270 nm (HONB) and 261 nm (N-hydroxysuccinimide, ε = 8.0 × 10 ⁴ Morr -1 cm ^{-1 )} ).

Claims (9)

1. A process for the transesterification of active unsymmetrical carbonic acid diester groups on hydroxyl-containing polymers, characterized in that polymers with active carbonic acid diester groups of the general formula J-O-CO-OR, wherein the radical R represents an electron-attracting group, in the presence of supernucleophilic Amines and / or strongly basic tertiary amines in anhydrous organic solvents at temperatures of 0-100 ⁰ C with alcohols, phenols or with N, N-disubstituted hydroxylamines or salts thereof and optionally followed by treatment with aqueous solutions. 2. The method of claim 1., d. g., that the electron withdrawing group R are substituted phenyl radicals such as p-nitrophenyl or 2,4,5-trichlorophenyl or a succinimidyl, phthalimidyl or 5-norbornene-2,3-dicarboximidyl radical. 3. The method according to claim 1, dg, that are used as supernucleophilic amines 4-dimethylaminopyridine (DMAP), 4-pyrrolidinopyridine, 4-morpholinopyridine, N-methylimidazole, diazabicyclo [5,4,0] undecene, diazabicyclo [2,2, 2] octane. 4. The method of claim 1., d. g., that is used as strongly basic tertiary amines triethylamine, N-methylmorpholine, Ν, Ν-dimethylaniline or heterocyclic amines such as pyridine, picolines, N-methylpiperidine. 5. The method of claim 1., d. g., that as alcohols both unsubstituted and substituted primary, secondary and tertiary compounds, and phenol, substituted phenols such as p-NitrophenoLDichlor- or trichlorophenols, cresols and Ν, Ν-disubstituted hydroxylamines such as N-hydroxysuccinimide, N-hydroxyphthalimide, N- Hydroxy-5-norbornene-2,3-dicarboximide (HONB) can be used. 6. The method of claim 1 and 3, d., That per mole of asymmetric carbonic acid diester groups on the polymer> 0.01 to 5 moles of supernucleophilic amine, preferably 0.05-2 mol, are used. Ii. Process according to Claims 1 and 4, dg, in that the amount of strong basic amine used is from 1 to 100 moles per mole of asymmetric carboxylic acid diester groups. 8. The method of claim 1 and 5, d. g., that per mole of asymmetric carbonic acid diester groups 1-100MoI alcohols, phenols or Ν, Ν-disubstituted hydroxylamines dissolved in anhydrous organic solvents, or optionally these compounds are used as solvent. 9. The method of claim 1., d. g., that as polar solvents polar organic compounds such as acetonitrile, dimethylformamide, dioxane, acetone u.a. or aliphatic hydrocarbons such as hexane, heptane or aromatic hydrocarbons such as benzene, toluene or halogenated hydrocarbons such as methylene chloride, chloroform and the like. a. be used. 10. The method according to claim 1, that is, that the treatment with aqueous solutions in the pH range of 2-9 is carried out at room temperature.