WO1996031549A1 - Dendrimeric graft polymers - Google Patents

Dendrimeric graft polymers Download PDF

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Publication number
WO1996031549A1
WO1996031549A1 PCT/EP1995/001278 EP9501278W WO9631549A1 WO 1996031549 A1 WO1996031549 A1 WO 1996031549A1 EP 9501278 W EP9501278 W EP 9501278W WO 9631549 A1 WO9631549 A1 WO 9631549A1
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Prior art keywords
separation
formula
graft polymers
dendrimeric
radical
Prior art date
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PCT/EP1995/001278
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German (de)
French (fr)
Inventor
Egbert Müller
Margot Mack
Peter Poguntke
Dieter Lubda
Original Assignee
Mueller Egbert
Margot Mack
Peter Poguntke
Dieter Lubda
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Priority to PCT/EP1995/001278 priority Critical patent/WO1996031549A1/en
Publication of WO1996031549A1 publication Critical patent/WO1996031549A1/en

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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30Processes for preparing, regenerating, or reactivating
    • B01J20/32Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3204Inorganic carriers, supports or substrates
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30Processes for preparing, regenerating, or reactivating
    • B01J20/32Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206Organic carriers, supports or substrates
    • B01J20/3208Polymeric carriers, supports or substrates
    • B01J20/321Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30Processes for preparing, regenerating, or reactivating
    • B01J20/32Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268Macromolecular compounds
    • B01J20/327Polymers obtained by reactions involving only carbon to carbon unsaturated bonds
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30Processes for preparing, regenerating, or reactivating
    • B01J20/32Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268Macromolecular compounds
    • B01J20/3278Polymers being grafted on the carrier
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08FMACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F285/00Macromolecular compounds obtained by polymerising monomers on to preformed graft polymers
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08FMACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F291/00Macromolecular compounds obtained by polymerising monomers on to macromolecular compounds according to more than one of the groups C08F251/00 - C08F289/00
    • C08F291/06Macromolecular compounds obtained by polymerising monomers on to macromolecular compounds according to more than one of the groups C08F251/00 - C08F289/00 on to oxygen-containing macromolecules
    • C08F291/08Macromolecular compounds obtained by polymerising monomers on to macromolecular compounds according to more than one of the groups C08F251/00 - C08F289/00 on to oxygen-containing macromolecules on to macromolecules containing hydroxy radicals
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00Aspects relating to sorbent materials
    • B01J2220/50Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/54Sorbents specially adapted for analytical or investigative chromatography

Abstract

The invention concerns dendrimeric graft polymers based on base carriers containing hydroxyl groups and on the surfaces of which polymers are covalently bound, the following conditions applying: a) the base carrier contains aliphatic hydroxyl groups; b) the covalently bound polymers are bound by an end-position monomer unit to the basic carrier; c) the polymers at the branching points of the dendrimeric structure contain monomer units of formula (II); and d) the dendrimeric polymers contain monomer units of formula (III); R?1, R2, R3¿ independently of one another stand for H or CH¿3, R?4 stands for H, C¿1?-C5 alkyl or C6-C12 aryl, n is an integer between 1 and 5, one group X is OH and the other group X is an end-position monomer unit of a further polymer chain, and Y stands for a group containing a separation effector. The invention also concerns the production of these dendrimeric graft polymers and their use as separating agents for liquid chromatography.

Description

dendrimers graft

The invention relates to dendrimeric graft polymers and their use as separation materials for Flüssigkertschromatographie.

From DE 38 11 042 Separating materials for chromatography, are known, having linear graft polymers. These materials exhibit compared to isolating materials containing crosslinked polymers distinct advantages. The object of the present invention is. provide separation materials with improved properties.

From DE 43 10 964 oxiranhaftige activated support materials are known, in which monomers of the formula are grafted onto a hydroxyl-containing base support I,

Figure imgf000003_0001

embedded image in which

R 1, R2 and R3 are independently

H or CH 3, R 'is H, d-Cs-alkyl or C 6 -C 12 aryl, and n is an integer between 1 and 5,.

It has been found that these activated support materials for the inventive graft polymers with dendritic structure umset¬ zen can be. In this case linear polymers are grafted (polymer chain second generation) to the known graft polymer with a linear polymer chain (polymer chain of the first generation), in turn, this step may be repeated multiple mehr¬ (Por realize tten higher generation).

ORIGINAL DOCUMENTS To branched graft polymers, however, have no crosslinking occur. In addition, as provided for chroma¬ tographischen separation processes, separation effectors are introduced. The resulting separation materials exhibit improved properties.

The not¬ manoeuvrable means of separation for the various chromatographic separation processes are bound as solid phase to a base support and go different a strong interactions with the analyte of the sample. suitable separation effectors are planning methods for various chromatographic separation known in the art; for example: ionic or ion-forming (ionic) groups on the ion-exchange chromatography, chromatography, affinity ligands for Affinitäts¬, including metal chelate belongs, hydrophobic groups for the hydrophobic interaction chromatography or reversed phase chromatography and predominantly reticulated porous hydrophilic groups for gel permeation chromatography. Materials having hydrophilic and hydrophobic regions are for the separation of low molecular weight analytes from protein-containing matrices are known which either by its pore structure (US 4,544,485; EP 0173233) or by hydrophilic shielding (US 5,277,813) avoid the undesirable binding of the proteins on the hydrophobic regions ,

The invention provides dendrimeric graft polymers on the basis of hydroxyl-containing base supports on whose upper surfaces polymers are covalently bonded, a) the base support contains aliphatic hydroxyl groups, b) the covalently bonded polymers are bound via a terminal monomer unit to the base support, c) the polymers contain at the branch points of the dendrimeric structure of monomer units of the formula II

Figure imgf000004_0001
d) and the graft polymers dendrimeric monomer units of formula III include,

- (CR 1 R 2 -CR 3) - i IN

wherein

Ri, R2 and R3 are independently

H or CH 3l R4 H. d-Cj-alkyl or C 6 -C 12 -aryl, n is an integer between 1 and 5, one radical X is OH and the other radical X represents a terminal unit Monomer¬ another polymer chain, and

Y is a radical which contains a separation effector means.

The dendrimeric novel graft polymers are obtainable by the following reaction steps: ion cerium IV-a) grafting of monomers of formula I to a hydroxyl-containing base support in the presence of,

Figure imgf000005_0001

wherein Ri, R2, R3, R and n have the meanings already mentioned; b) at least partially reacting the oxirane groups in diol groups; c containing the effectors Separations¬) grafting of further monomer, for example, from further monomers of the formula I, or monomers, ions cerium IV in the presence of; d) optional, and multiple repetition of steps b) and c); e) introduction of radicals by means of separation, to the extent they are not already introduced by the grafting reaction with monomers containing separation effectors; f) optional ring opening remaining oxirane groups.

The invention relates to the use of the dendrimeric graft polymers according to the invention in the separation of mixtures min¬ least two substances, in particular for the separation of biopolymer mers, by liquid chromatography, in particular by means lonen- exchange and affinity chromatography. Another use of the invention is the separation of low molecular weight analytes from protein-containing matrices.

The invention also relates to processes for the preparation of den¬ drimeren graft polymers having the following method steps: a) grafting of monomers of formula I to a hydroxyl-containing base support in the presence of cerium IV ions,

Figure imgf000006_0001

wherein

Ri, R2 and R3 are independently

H or CH 3, R is H, C r C 5 alkyl or C 6 -C 12 aryl, and n is an integer between 1 and 5, wherein arise rials 43 10 964 known from DE Trägermate¬ activated, b) at least partial conversion of the oxirane groups in diol groups; c) grafting of monomers of the formula I or of monomers of the formula V to the ion formed in step b) diol residues in the presence of cerium (IV),

CR 1 R 2 = CR 3

V

0 = CW

10 wherein

W is OH or NHR and

R 8 is C 0 Cι alkyl which is substituted with an amino, monoalkylamino

Dialkylamino, trialkylammonium, carboxyl, sulfonic acid or AC is substituted, wherein the steps b) and c) once or may be repeated several times, • d) introduction of the groups containing the means of separation, to the extent not already by this Q the grafting reaction with monomers of

Formula V is carried out and e) optional ring opening remaining oxirane

5 The invention relates to methods for separating mixtures of at least two substances, in particular for the separation of Biopoly¬ mers, by liquid chromatography, in particular by means lonen- exchange, hydrophobic interaction or affinity chromatography, using the dendrimeric merisaten Pfropfpoly- Q according to the invention. Other methods of the invention relate to the separation of low molecular weight analytes from protein-containing matrices using inventive dendritic release materials.

5 Figure 1 shows the dependence of selectivity α (curve A) and Bin¬ binding capacity (curve B) of diethylamine-substituted (DEA) Ionen¬ exchangers with different degree of branching (sample number as abscissa). The experimental details are found in application example A. It is found that the selectivity of branched material is significantly higher than that of unbranched material. The binding capacity decreases compared with unbranched reference material, with low branching, but falls off significantly with strong branching.

Figures 2 and 3 show the experimental setup for the separation of analytes from biological matrices, such as serum or plasma, using inventive separating materials. Figure 4 shows the results of a recovery test by using a separation material prepared according to Example. 9 Further experimental details are given in Example B.

The dendrimeric graft polymers according to the invention are distinguished by a tree-like branched structure, wherein a first linear polymer contains a base support contains aliphatic hydroxyl groups, is grafted. In this case, monomers of the formula I, wherein the radicals R 1, R 2, R 3 and R 4, and n have the meanings already mentioned, grafted in the presence of ceric ion, whereby a linear polymer Pfropf¬ arises. The basic features of this reaction are by G. Mino and S. Kaizerman (1958) J. Polymer Science __1, 242-243, and G. Mino et al. (1959) J. Polymer Science __, described 393-401. The polymer initially contains oxirane radicals, which are then reacted completely or partially to diol groups. To a treatment with dilute sulfuric acid is preferred.

of formula I may thus to the resulting aliphatic hydroxyl this Poly mers (first generation) now again monomers in the presence of cerium IV ions are polymerized. The resulting polymer (second generation) is linear in themselves. However, the overall structure is branched. In order to reach even more branched dendrimeric graft polymers, the steps above can be repeated: Um¬ reduction of the oxirane groups in diol groups and polymerization of monoethylenically mers of formula I in the presence of cerium IV ions.

In addition, the introduced for the chromatographic separations notwen¬ ended separation effectors that are bound as the solid phase to a Basis¬ carrier, and to enter the different strengths of interaction with the analyte of the sample. For various chromatographic separation methods to the skilled person suitable means of separation are known, for example: a) for ion exchange chromatography are ionic groups such as quaternary Ammoniumalkylgruppen and the S0 3 group, as well as ionic groups which form ions under certain pH conditions, are known. For the latter group includes, for example, alkylated amino groups, and carboxyl and phosphoric acid group. b) For the affinity chromatography are known to those skilled in very many affinity ligands are known which undergo a structurally given bond respectively with the analyte, and as

Separation effectors are suitable, for example:

Table 1 :

Affinitätsiigand analyte (sample) Protein A immunoglobulins

Concanavalin A glycoproteins

Biotin avidin / streptavidin

avidin biotin

Streptavidin Biotin 5'-adenosine monophosphate-dependent oxidoreductases NAD

2 ', 5'-adenosine diphosphate NADP-dependent oxidoreductases

Aminoacridine RNA or DNA Table 1 (continued):

Affinity ligand analyte (sample.

Boronic acid catecholamines

Boronic acid glycosylated hemoglobin iminodiacetic metalloproteins

"Thiophilic" ligand immunoglobulins

Cibachrome Blue monoclonal he Antikö

c) uncharged hydrophobic separation effectors are for hydrophobic interaction available, for example C 1 -C 2 -alkyl,

C -C_ 6 5 -aryl, C 7 -C 25 alkylaryl or C 7 -C 25 arylalkyl, which may be derivatized or more times with nitrile or Ci-Cs-alkoxy, where one or more non-adjacent CH 2 groups are replaced by NH or O or else one or more CH groups may be replaced by N, or polyoxyethylene or polyoxypropylene derivatives

[(CH 2) π. -O-] 0 -R 9, wherein m is 2 or 3, o is an integer between 1 and 200 and R is H or CrC mean 5 alkyl. especially radicals of medium or low hydrophobicity are preferred. These residues can be introduced as alkyl or aryl groups as alkoxy or aroxy radicals or as Alkoyl- or aroyl radicals.

d) For the gel permeation chromatography are hydrophilic compounds Verbin¬, preferably form the pores or networks used as a separation effector. These include (meth) acrylic acid derivatives such as acrylamide or methacrylamide, (2,3-dihydroxypropyl) - methacrylate or N- (2-methoxyethyl) acrylamide or N- (2,3-dihydroxypropyl) acrylamide. In addition, these include cyclen vinylated hetero-, such as 1-vinylimidazole, N-vinylpyrrolidone, 2-vinylpyridine, 4-vinylpyridine, 4-vinylpyrrolidone-N-oxide.

e) For the separation of low molecular weight substances from biologi¬'s sample (eg urine or blood) so-called shielded phases are used. This release materials have both hydro¬ phobe and hydrophilic regions. In this step, the hydrophobic ben areas, the structure of the above (see c)) corresponding to said hydro¬ phobic separating materials, in interaction with the low molecular weight analytes of the sample. The hydrophilic areas will prevent the interaction of the high molecular weight fractions of the sample (for example, of proteins) with the hydrophobic regions. Dendrimers graft polymers according to the present invention are very useful as shielded phases for the

Separation of analytes from biological matrices.

For the introduction of separation effectors are different Reaktions¬ paths possible: a) The separation effectors are prepared by reaction with the

Oxiranresten that are present after polymerization, with compounds of the formula I, incorporated in the carrier; including: a1) the reaction with sulfurous acid or its salts or with primary, secondary or tertiary amines, wherein arise lonenaus- exchanger; a2) the reaction with iminodiacetic acid or the introduction thiophilic ligands, or other Affiniätsliganden such as protein A, which arise carrier for affinity chromatography; a3), the reaction with alcohols, phenols, or primary amines, wherein hydrophobic release materials arise.

Hydrophobic separation effectors can be for example also by Esterbindungen of hydroxyl groups, as are formed by hydrolysis of the oxirane moieties introduce.

In the reaction sequence according to a1), for example arise Verbin¬ compounds, in which the radical Y of formula III has the meaning of formula IV,

0 = C-0- (CH2) π -CH-CHR 4 IV

II embedded image in which zz

R 4 is H, d-Cj-alkyl or C 6 -C 12 -aryl, n is an integer between 1 and 5, one radical Z is OH and the other radical Z is a radical selected from the group NR5R6, N + ?, R5R6R PO 4 H 2 and S0 3 H, and Rs, R 6 and R 7 are independently

Mean Cι-C4 alkyl, wherein one or both of R5 or Re can also be H. b) At the last graft polymerization of the formula I which are used in DE 38 11 042 wherein the known monomers can known from this document groups are introduced as separation effectors instead of the monomers. These include beispiels- as monomers of the formula V,

CR 1 R 2 = CR 3

O = CW

wherein

W is OH or NHR and β

R 8 CrCio-alkyl amino with an amino, monoalkylamino, dialkyl, trialkylammonium, carboxyl or sulfonic acid group is substituted,.

In the manufactured according to process variant b) carrier materials of the radical Y of formula III is a radical of formula VI,

VI

CW = 0 wherein

W is OH or NHR and β

Rs dC-io-alkyl, which is substituted by an amino, monoalkylamino, dialkylamino, trialkylammonium, carboxyl or sulfonic acid radical.

Preferred monomers of formula V are those in which W has one of the following meanings: OH, NH (CH 2) 2 N (CH 3) 2, NH (CH 2) 2 N (C 2 H 5) 2, NH (CH 2) 2 N + (CH 3) 3, NHC (CH 3) 2 CH 2 S0 3 H or NH (CH 2) 2 S0 3 H.

After the reactions described were carried out, remaining oxirane radicals can be hydrolyzed, if desired, for example by a final treatment with dilute sulfuric acid.

Without further elaboration, it is believed that a man Fach¬ can utilize the above description in the broadest scope. The bevor¬ ferred embodiments should therefore merely as illustrative, and not to be construed as in any way limiting.

The complete disclosure of all applications mentioned above and below, patents and publications, in particular the German application P 43 34 351, filed on 08.10.1993, are incorporated by reference into this application.

The following examples illustrate the subject matter in more detail; these examples are not a limitation of the subject matter. Examples

Preparation Examples

In the following Production Examples, room temperature means (RT)

15-30 ° C. The Polymeriation is in a three-necked flask of suitable size equipped with a stirrer, dropping funnel and thermometer executed. is washed by suction on a glass frit (G2).

Example 1: Preparation of an oxirane-activated support from Fractogel® TSK HW-65 (S)

to a suspension of 100 ml of sedimented Fractogel® TSK HW-65 (S) and 66 ml of water with 3 g of ammonium cerium (IV) nitrate (dissolved in a

Mixture of 180 ml of water and 3 g of HN0 3 (65%)) were mixed with vigorous stirring at room temperature. After 1 minute, adding a solution of 6 g of (2,3-epoxypropyl) methacrylate in 44 ml of dioxane. Stirring is continued for one hour. Subsequently, the reaction product is washed twice with 200 ml of water, three times with 100 ml of acetone and three times with 200 ml of water.

Example 2: Preparation of an oxirane-activated support from LiChrospher®-diol

The preparation is carried out according to Example 1, LiChrospher®-DIOL (Par¬ tikelgröße 15-25 .mu.m, pore size 80 nm) is used as base support instead of Fractogel® TSK HW-65 (S). Example 3: grafting a polymer to a polymer diolhaltiges (generation of the dendrimeric structure)

Step 1: Conversion of the diol groups oxirane in 100 ml suctioned oxirane-activated support material prepared according to

Example 1 are hydrolyzed with 200 ml of 0.5 M sulfuric acid (1 hour, 50 ° C), and thus converted the oxirane groups in diol groups. On closing is washed three times with 200 ml of water.

Step 2: grafting a further polymer chain

Are mixed with 3 g of ammonium To a suspension of 100 ml of sedimented material from Step 1 and 66 ml of water (IV) nitrate (dissolved in a mixture of 180 ml of water and 3 g of HN0 3 (65%)) at room temperature under vigorous stirring. After 1 minute, adding a solution of 6 g of (2,3-epoxypropyl) methacrylate in 44 ml of dioxane. Stirring is continued for one hour. Subsequently, the reaction product is zwei¬ times washed with 200 ml of water, three times with 100 ml of acetone and three times with 200 ml of water.

The result is a simple branched dendrimäres material Oxiranresten.

Example 4: grafting a polymer to a polymer diolhaltiges (production of multi-branched structures nevertheless drimeren)

The material from Example 3 is again subjected to the reaction sequence described in Example 3. FIG. The result is a two-branched den¬ drimäres material Oxiranresten.

This material can in turn be subjected to Reak¬ described in Example 3 tion follow. Here dendrimäre materials produced with Oxiranresten and higher grade branching. - 14 -

Example 5: synthesis of a substituted with diethylamine dendrimeric release material

100 ml and isolated by filtration gel prepared according to Example 3 (monobranched) are suspended in 100 ml of water and 100 ml of diethylamine added.

Then, 20 hours, further stirred at room temperature. Thereafter, the reaction product is washed twice with 100 ml of water.

The washed reaction product is suspended in acid solution 100 ml of a 0.5 M sulfuric acid and stirred for two hours at 40 ° C slowly. Thereafter, 0.25 M phosphate buffer (pH 7), then washed until neutral with water. The gel is stored in an aqueous suspension with addition of 0.02% sodium azide.

Example 6: synthesis of a substituted, di-branched with diethylamine dendrimeric release material

100 ml and isolated by filtration gel prepared according to Example 4 (two-branched) are reacted with diethylamine as described in Example 5 in.

In a corresponding manner are also highly branched with diethylamine substituted dendrimeric release materials available.

Example 7: Preparation of an affinity support for the metal chelate chromatography

A solution of 15 g NaOH and 25 g of iminodiacetic acid in 100 ml of water is adjusted with concentrated HCl to pH 11 and decolorized with 1 g of activated charcoal. 50 ml aspirated oxirane-activated support material are prepared according to Example 4 (two-branched) was added to this solution. The solution is stirred for 20 hours at 45 ° C. The reaction product is filtered off with suction, washed with 250 ml of 0.5 M NaOH and with water. The unreacted oxirane groups by treatment with 100 ml of 0.5 M sulfuric acid (2 hours, 45 ° C) hydrolyzed. Subsequently, the affinity substrate 1 M NaCl solution is washed once with 100 ml of 0.5 M sulfuric acid, twice with 100 ml water, once with 100 ml of 0.5 M pH 7 phosphate buffer and once with 100 ml. The gel is stored in 0.02 M phosphate buffer pH 7 with an addition of 1 M NaCl and 0.02% NaN. 3

Example 8: Preparation of an acidic ion exchanger

Step 1 :

100 ml gel produced according to Example 3 M sulfuric acid are suspended and stirred for one hour at 45 ° C in 160 ml of 0.5. It is then washed three times with 500 ml of water.

Level 2:

10 g of NaOH are dissolved in 200 ml of water and 2-acrylamido-2-methylpropanesulfonic acid was added until the pH is 4 (ca. 45 g; Monomeren¬ solution). As for the polymerization initiator solution to 8.2 g of ammonium cerium (IV) nitrate 0.5 M nitric acid dissolved in 50 ml.

100 ml of gel from Step 1 is suspended in the monomer solution and filled into a three necked flask, the initiator solution is charged into the closed ange¬ thereto dropping funnel. The apparatus is evacuated three times and charged with argon. With stirring (150 rpm) will now make the starter solution is run and then further stirred for four hours at 40 ° C. Subsequently, the product with 200 ml of 1 M sodium sulfite in 1 M sulfuric acid with 1 I water, 3 I of 0.1 M NaOH, 500 ml of 1 M sodium acetate buffer pH 7 and is washed with 500 ml of water. The product is stored in 20 mM phosphate buffer pH 7 with an addition of 0.02% NaN. 3 Example 9: Preparation of a substrate for the determination of low-molecular substances in biological matrices

Step 1: Preparation of a carrier glycidyloxypropyl

10 g of a LiChrospher® Si 60, particle size 25 microns, having a specific surface area of ​​380 m2 / g and a mean pore diameter of 9 nm (E. Merck, Darmstadt) are suspended in toluene 50 ml and, after addition of 3.8 ml ( 4 mmol / m 2) Gycidyloxypropyl-methyl-dimethoxysilane boiled for 5 hours under stirring. After aspiration of the material it is washed toluene and methanol and dried.

Stage 2: Ring opening of the carrier to glycidyloxypropyl diol phase The product obtained from step 1 is dissolved in 50 ml of a 5% sulfuric acid solution and boiled at reflux for opening the epoxy ring 3 hours under slow stirring. After aspiration the reaction suspension, is washed with water until free of sulfate and dried after re washing with methanol. (Corresponding to 2.81 mmol / mΣ diol groups carbon content 7.6%) gives a diol carrier.

Stage 3: the first graft polymerization

To a suspension of 40 ml of sedimented LiChrospher®-diol from

Stage 2 and 60 ml of water with 0.8 g of ammonium cerium (IV) nitrate (dissolved in a mixture of 40 ml of water and 1.2 g of HN0 3 (65%)) were mixed at room temperature with vigorous stirring. After 1 minute, adding a solution of 1.2 g of (2,3-epoxypropyl) methacrylate in 15 ml of dioxane is carried out. Stirring is continued for one hour. Subsequently, the ligation product Reak¬ is twice washed with 200 ml of water, three times with 100 ml of acetone and three times with 200 ml of water. Step 4: sulfuric acid hydrolysis

40 ml of gel from step 3 are suspended in 160 ml of 0.5 M sulfuric acid and stirred for one hour at 45 ° C. It is then washed three times with 500 ml of water.

Stage 5: Second graft polymerization

0.8 g of ammonium cerium (IV) nitrate was dissolved in a mixture of 40 ml of water and 1, 2 g of HN0 3 (65%). This solution is added to a suspension of 40 ml of sedimented grafted LiChrospher®-diol from Step 4 and 60 ml of water at room temperature with vigorous stirring. After

One minute, the addition of a solution of 1, 2 g (2,3-epoxypropyl) - methacrylate in 15 ml of dioxane. Stirring is continued for one hour. Anschlie¬ ßend the reaction product is washed twice with 200 ml of water, three times with 100 ml of acetone and three times with 200 ml of water.

Step 6: Reaction of the graft polymer with hydrophobic dendrimeric

Ligand 5 g of the dried carrier material from step 5 are suspended at 0 ° C in dry chloroform. To this solution 60 ml are added triethylamine overall dried. Then a solution of 2 g of stearoyl chloride in 25 ml of chloroform under cooling at 4 ° C, added over a period of 3 hours.

After the addition of the acid chloride is stirred for 48 hours at room temperature and then the gel is washed with 50 ml of chloroform, methanol, water and methanol and dried.

Step 7: sulfuric acid hydrolysis

40 ml of gel from step 6 are suspended in 160 ml of 0.5 M sulfuric acid and stirred for one hour at 45 ° C. It is then washed three times with 500 ml of water.

In the manner described above, a dendrimeric shielded phase separation material is provided, which hydrophobic residues are shielded by hydrophilic Diolgruppierungen. The following application example shows the effect of Verzweigungs¬ degree on binding capacity and separation capacity of the separator material.

Application Example A: Influence of the degree of branching

Monosubstituted to seven-branched DEA-derived separating materials (Samples 2-7) are prepared in accordance with Examples 5 and 6, unbranched comparative material (sample 1) is prepared in analogy to Example 5, using the linear polymer of Example 1 instead of the dendrimeric oxirane-containing polymer becomes.

These release materials are respectively in a Superformance ^ glass column (10 mm x 50) and filled with the application buffer (50 mM TRIS buffer, pH 8.3) equilibrated. A solution of bovine serum albumin (10 mg / ml) in this buffer is applied continuously (flow: 0.5 ml / min) was measured by photometry at 280 nm and the elution. From the breakthrough curve, the capacitance is determined. The separation behavior of bovine serum albumin is also determined and expressed as a selectivity factor α. The results are summarized in Figure 1.

It is shown that the selectivity α (curve A) of dendritic material is significantly higher than of unbranched material. The binding capacity (curve B) increases as compared to unbranched comparative material, with a low branching, but decreases significantly at high branching.

Example B: Recovery of carbamazepine in plasma

a) Equipment

Fig. 2 shows the apparatus structure, the meanings are:

1. Precolumns buffer 2. Analyzes buffer

3. HPLC pump (L-6000) 4. HPLC pump (L-6200)

5. autosampler (AS-4000) 6. automatic changeover valve (ELV 7000)

7. 8. guard column analytical column 9. detector 10. Integrator (D-2500)

11. waste container (device of Messrs. E. Merck, Darmstadt, Germany)

In Fig. 3, the line connections between the modules are in

Depending on the position of the switching valve (6): Figure 3a:.. "L" position (LOAD) 3b: "I" (INJECT)

b) Chromatographic conditions:

Precolumn ((8); 25 x 4 mm): separation material prepared according to Example 9;

Pre-column buffer (1): 0.05 M Na-phosphate (pH 5.0); analytical column ((8);

60 LiChrospher® RP-select B, 5 micron, 125 x 4 mm); Analysis buffer (2):

0.05 M Na-phosphate (pH 4.0) / acetonitrile (80:20; V: V); Detection UV 210 nm.

c) analysis cycle

After injection of the plasma sample (100 ul) of the autosampler (5) to the "L" of the switch valve (6) reaches the sample with the aid of by the HPLC-pump (3) supported pre-column buffer ((1); flow rate 0 , min) on the precolumn (7) 5 ml /. The analyte (carbamazepine) is selectively retiniert from the release material the pre-column, while Matrix¬ ingredients, especially proteins are eluted within 12 minutes into the waste container (11).

After switching of the valve (6) in position "I" is the analyte by using the HPLC-pump (4) conveyed analysis buffer ((2); flow rate 0.8 ml / min) within 5 minutes completely from the precolumn (7) eluted and transferred to the downstream analytical column (8).

After switching of the valve (6) in the position "L" is the analytical separation is carried out under isocratic conditions (flow rate 0.8 ml / min). The eluted compounds are measured in the detector (9) and evaluated in the integrator (10). At the same time, the guard column is conditioned with the aid of HPLC-pump (3) for a new cycle of analysis. d) Result

In FIG. 4, the elution of A are: a calibrator that contains 0.5 ug carbamazepine, and

B: a plasma sample (human plasma) containing also 0.5 ug carbamazepine.

As can be seen, the analyte is found completely wieder¬ in the plasma sample.

Claims

- 21 -
claims
1. dendrimers graft polymers on the basis of hydroxyl-containing base supports on whose surfaces polymers are covalently bonded, characterized in that a) the base support contains aliphatic hydroxyl groups, b) the covalently bonded polymers are bound via a terminal monomer unit to the base support, c) the polymers contain at the branch points of the dendrimeric structure of monomer units of the formula II
Figure imgf000023_0001
d) the dendrimeric polymers contain monomer units of the formula III,
- (CR 1 R 2 -CR 3) - ...
wherein
Ri, R and R3 are independently
H or CH 3, R 4 is H, C Cs alkyl or C 6 -C 12 -aryl, n is an integer between 1 and 5, one radical X is OH and the other radical X represents a terminal unit Monomer¬ another polymer chain, and
Y is a radical which contains a separation effector means. 2. dendrimers graft polymers obtainable by the following steps Reaktions¬: a) grafting of monomers of formula I to a hydroxyl-containing base support in the presence of cerium IV ions,
CR 1 R 2 = CR 3
Figure imgf000024_0001
wherein
Ri, R 2 and R, are independently H or CH 3, 5 R 4 H, CC 5 alkyl or C 6 -C 12 aryl, and n is an integer between 1 and 5, b) at least partially reacting the oxirane groups in o diol groups; c containing separation effectors) grafting of further monomer, for example, from further monomers of the formula I, or monomers, ions cerium IV in the presence of; d) optional, and multiple repetition of steps b) and c); 5 e) introduction of radicals by means of separation, to the extent they are not already introduced by the grafting reaction with monomers containing separation effectors; f) optional ring opening remaining oxirane groups.
0 3. dendrimers graft polymers according to claim 1 or 2, characterized in that the separation effector contains an ionic or ionogenic group.
5 dendrimers graft polymers according to claim 3, characterized gekenn¬ characterized in that one radical Y is contained of Formula IV
0 = C-0- (CH2) π -CH-CHR 4 IV
II zz
wherein
R 4 is H, CC 5 alkyl or C 6 -C 12 aryl, an integer between 1 and 5, one radical Z is OH and the other radical Z n is a radical selected from the
NRsRe group, N-β RsR R ?, P0 4 H 2 and S0 3 H, and
Rs, R 6 and R 7 independently of one another dd alkyl, wherein one or both radicals R or R ~ can also be H, mean.
5. dendrimers graft polymers according to claim 3, characterized gekenn¬ characterized in that a radical Y is of formula VI,
VI
0 = CW
Wonn
W is OH or NHR and β
R β Cι-C10 alkyl substituted with an amino, monoalkylamino,
Dialkylamino, trialkylammonium, carboxyl or
sulfonic acid is substituted,.
6. dendrimers graft polymers according to claim 1 or 2, characterized in that the radical Y includes an affinity ligand. 7. dendrimers graft polymers according to claim 1 or 2, characterized in that the radical Y contains a hydrophobic residue.
8. The use of dendrimeric graft polymers having the features of claim 1 or 2 in the separation of mixtures of at least two substances, in particular for the separation of biopolymers, by liquid chromatography, in particular by means of ion exchange, hydrophobic interaction or Affinitäts¬ chromatography.
9. Use according to claim 8, wherein said analytes are separated from biological matrices.
10. A process for preparing dendritic graft polymers, characterized by the following process steps: a) grafting of monomers of formula I to a hydroxyl-containing base support in the presence of cerium IV ions,
Figure imgf000026_0001
wherein
Ri, R2 and R3 are independently
H or CH 3, R 4 H, CC 5 alkyl or C 6 -C 12 aryl, and n is an integer between 1 and 5 mean groups b) at least partially reacting the oxirane groups in Diol¬; c) grafting of monomers of the formula I or of monomers of the formula V to the ion formed in step b) diol residues in the presence of cerium (IV),
Figure imgf000027_0001
wherein
W is OH or NHR and β
RSS CC 10 alkyl which is substituted with an amino, monoalkylamino
Dialkylamino, trialkylammonium, carboxyl or
sulfonic acid is substituted,
wherein steps b) and c) can be repeated several times or even once, and d) introduction of the groups containing the separation effectors, if this is not already done by the grafting reaction with monomers of formula V.
11. A process for the separation of mixtures of at least two sub- stances, in particular for the separation of biopolymers, by means of liquid-keitschromatographie, in particular by means of ion exchange, hydrophobic interaction or affinity chromatography, using dendrimeric graft polymers having the features of claim 1 or 2 ,
12. The method of claim 11, wherein said analytes are separated from biological matrices.
PCT/EP1995/001278 1995-04-07 1995-04-07 Dendrimeric graft polymers WO1996031549A1 (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040502A1 (en) 1997-03-14 1998-09-17 Life Technologies, Inc. Peptide-enhanced transfections
WO2004003542A1 (en) * 2002-06-28 2004-01-08 Amersham Biosciences Ab Surface-modified base matrices
WO2014067605A1 (en) * 2012-11-01 2014-05-08 Merck Patent Gmbh Surface modification of porous base supports
US9303098B2 (en) 2008-05-30 2016-04-05 Merck Patent Gmbh Ce(IV)-initiated graft polymerisation on polymers containing no hydroxyl groups
US9358300B2 (en) 1998-11-12 2016-06-07 Life Technologies Corporation Transfection reagents
US10195280B2 (en) 2014-07-15 2019-02-05 Life Technologies Corporation Compositions and methods for efficient delivery of molecules to cells
EP3460065A1 (en) 2012-04-20 2019-03-27 Commonwealth Scientific and Industrial Research Organisation Cell transfection method

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994026379A1 (en) * 1993-05-13 1994-11-24 Merck Patent Gmbh Method and support material for gel-permeation chromatography

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994026379A1 (en) * 1993-05-13 1994-11-24 Merck Patent Gmbh Method and support material for gel-permeation chromatography

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998040502A1 (en) 1997-03-14 1998-09-17 Life Technologies, Inc. Peptide-enhanced transfections
US6376248B1 (en) 1997-03-14 2002-04-23 Life Technologies, Inc. Peptide-enhanced transfections
US9358300B2 (en) 1998-11-12 2016-06-07 Life Technologies Corporation Transfection reagents
WO2004003542A1 (en) * 2002-06-28 2004-01-08 Amersham Biosciences Ab Surface-modified base matrices
US7423070B2 (en) 2002-06-28 2008-09-09 Ge Healthcare Bio-Sciences Ab Surface-modified base matrices
US9303098B2 (en) 2008-05-30 2016-04-05 Merck Patent Gmbh Ce(IV)-initiated graft polymerisation on polymers containing no hydroxyl groups
EP3460065A1 (en) 2012-04-20 2019-03-27 Commonwealth Scientific and Industrial Research Organisation Cell transfection method
WO2014067605A1 (en) * 2012-11-01 2014-05-08 Merck Patent Gmbh Surface modification of porous base supports
CN104736236B (en) * 2012-11-01 2017-06-13 默克专利股份公司 Surface modification of porous support matrix
TWI609717B (en) * 2012-11-01 2018-01-01 Merck Patent Gmbh Surface modification of porous base supports
US10166527B2 (en) 2012-11-01 2019-01-01 Merck Patent Gmbh Surface modification of porous base supports
JP2016502457A (en) * 2012-11-01 2016-01-28 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツングMerck Patent Gesellschaft mit beschraenkter Haftung Surface modification of the porous substrate support
US10195280B2 (en) 2014-07-15 2019-02-05 Life Technologies Corporation Compositions and methods for efficient delivery of molecules to cells

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