AU730793B2

AU730793B2 - Shaped article having reactive functions

Info

Publication number: AU730793B2
Application number: AU45553/97A
Authority: AU
Inventors: Ronald Frank; Stefan Matysiak
Original assignee: Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Current assignee: Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Priority date: 1996-09-18
Filing date: 1997-09-18
Publication date: 2001-03-15
Anticipated expiration: 2017-09-18
Also published as: KR20000036217A; EP0928311B1; NO991296L; WO1998012246A1; NO991296D0; EP0928311A1; DE59709797D1; AU4555397A; CA2265689A1; JP2001504869A

Description

-1- Shaped article having reactive functions The present invention relates to a shaped article the surface of which is provided with reactive functions for chemical, biochemical or biological applications.

Many polymeric materials based on natural polymers, such as polysaccharides (for example cellulose, starch or chitin), or organic synthetic polymers, such as plastics (for example polyethylene, polypropylene, polytetrafluoroethylene or polystyrene), are distinguished by insolubility in organic and aqueous media. For many decades, their surface properties, such as charge, chemical functionality or hydrophobicity, have been used for synthesis, separation and detection reactions, for example in the following fields: chemical solid-phase synthesis; hydrophobic chromatography, ion-exchange chromatography or affinity chromatography and affinity concentration. Used in the conventional form of granules, beads or fibres, those materials are packed into reaction vessels, such as columns or cartridges. Depending on the application, use is advantageously made, for this purpose, of special reactive functions on the surface, as are shown by way of example in the following diagram.

COOH

Protein may C NH 2 b) subsequently by means of chemical derivatisation.

Ligand Aryl Hal Alkl OH SH Such surface functionalities may be introduced a) directly by means of co-polymerisation with appropriate monomers or b) subsequently by means of chemical derivatisation.

Many chemical, biochemical and biological procedures make use of specially shaped apparatus (shaped articles, such as membranes, films, papers, miniaturised reaction vessels (for example Eppendorf), pipette tips or microtitre plates), which can be manufactured from the polymeric materials mentioned at the beginning or from -2polymeric materials similar thereto. An optimised manufacturing process provides such shaped articles with the mechanical and chemical stability necessary for the application. The surfaces of such shaped articles can also be used advantageously for the same synthesis, separation and detection reactions as mentioned above. The introduction of the various chemical functions required for the application is, however, more problematic in this instance because the derivatisation process must not be allowed to have an adverse effect on the mechanical and/or chemical properties.

For example, shaped articles, such as sheet material, for example paper made from specially derivatised cellulose, for example carboxymethyl-, diethylaminoethyl-, phospho-, alkyl- or aryl-cellulose, are of major importance. However, the processes for manufacturing starting materials for the said shaped articles call for highly toxic reagents, such as cyanogen bromide, epichlorohydrin or acrylonitrile, and/or very drastic alkaline reaction conditions so that the cellulose papers produced from those materials exhibit very much lower mechanical and chemical stability.

The problems mentioned have also not been entirely solved by WO-A-94/19694.

The problem of the present invention is therefore to provide new materials for the biosciences and new shaped articles the surfaces of which are provided with reactive functions for chemical, biochemical or biological applications, a particular aim being to S" provide those materials by means of especially gentle methods. The mechanical and chemical properties of the shaped articles should undergo no, or only insignificant, modification as a result.

i According to one embodiment, the problem underlying the invention is solved by a shaped article the surface of which is provided with reactive functions for chemical, biochemical or biological applications, which can be manufactured by starting from, as the shaped article, a paper-based shaped polymeric material, the surface of which shaped article is provided with hydroxy groups, and reacting the shaped article with a carbonylating reagent in a manner know per se, and optionally reacting the carbonlated shaped article with a compound of the formula R 3

NH-R

4 wherein R 3 represents the radical of a primary amine known perse and R 4 represents a hydrogen atom or R 3 and R 4 which may be the same or different, represent radicals of a secondary amine known per se, to form a urethane derivative. R 3

R

4 NH may especially be an amine that is customarily used for urethane manufacture and may be designated a urethane-forming agent.

0 O -OH 0 I. Activation O- R2 II. Modification 1- NHR 3 OH R R 2

-HR

1 OY R2 -HR/+H2NR 3 O NHR 3 O O The reaction of a polymer the surface of which is provided with hydroxy groups, more specifically agarose and polysaccharides, with a carbonylating reagent, namely 1,1'-carbonyldiimidazole, is part of the prior art; see J.

Chromatography, 219 (1981) 353-359 and 361-372.

The fields of use for a shaped article according to the invention are in immunology, biochemistry, molecular biology, clinical diagnostics or a bioprocessing technology. Examples of possible uses are ion-exchange chromatography, affinity chromatography or affinity concentration, hydrophobic chromatography, blotting, dot blotting and chemical solid-phase synthesis.

15 The shaped article according to the invention can be manufactured by starting from a shaped cellulose-based polymeric material, especially a paperbased polymeric material, or from a shaped plastics-based polymeric material, especially a hydroxylated-polyolefin-based polymeric material.

When starting from a shaped cellulose-based polymeric material, a wide range of cellulose materials having a very wide variety of chemical groups can be manufactured. In that variant, the shaped articles according to the invention can be manufactured using very mild reaction conditions, using materials and solvents of low toxicity and using a simple technical procedure, it being especially important that the chemical and mechanical properties of the underivatised cellulose starting material are modified only insignificantly. The chemical linkage has excellent pH stability, approximately in the range from 2 to 12 at room temperature.

The shaped article according to the invention can be manufactured by using a carbonylating reagent 4 from the group consisting of 1,1'-carbonyldiimidazole (CDI), 1,1carbonyldi-1,2,4-triazole (CDT), 1,1'-carbonyldi-1,2,3-benzotriazole (CDB) and/or phenoxycarbonyl-tetrazole (PCT) and/or (ii) from the group consisting of chloroformic acid phenyl ester (CFP) and/or chloroformic acid 4-nitrophenyl ester (CFNP).

When using a carbonylating reagent from group the carbonylation can be carried out in dimethylformamide (DMF), especially at room temperature and/or without addition of a buffer and/or without addition of a further base besides dimethylformamide (DMF).

When using a carbonylating reagent from group the carbonylation can be carried out in pyridine.

According to a further embodiment, the problem underlying the invention is solved by a shaped article the surface of which is provided with reactive functions for chemical, biochemical or biological applications, which can be manufactured 15 by starting from, as the shaped article, a shaped polymeric material, the surface of which shaped article is provided with hydroxy groups, and reacting the shaped article with an isocyanate and/or thioisocyanate, and optionally blocking the unreacted hydroxy groups of the activated shaped article, optionally, if nitro groups have been introduced by the isocyanate and/or isothiocyanate in Step and unreacted hydroxy groups of the activated shaped article have been blocked in Step reducing any such nitro group to form amino groups, and optionally, if protected amino groups have been introduced by the isocyanate and/or thioisocyanate in Step and unreacted hydroxy groups of the activated shaped article have been blocked in Step de-protecting any such protected amino groups to form free amino groups.

x 1OH I. AT O NH NH 2 OH II. Reduction, LJ-O NH--NH 2 e.g.Sm 2 /THF/R1 X=S,O

X

The shaped article according to the invention can be manufactured by starting from a shaped cellulose-based polymeric material, especially a paper-based polymeric material, or from a shaped plastics-based polymeric material, especially a hydroxylated-polyolefin-based polymeric material. For a shaped cellulose-based article, reference may be made to the afore-mentioned embodiments.

In the second embodiment, the shaped article according to the invention can be manufactured by using 4-nitrophenyl isocyanate or 4-phenyl isothiocyanate in Step It can furthermore be manufactured by carrying out the blocking in Step with acetic anhydride.

It can furthermore be manufactured by carrying out the reduction in Step with samarium(ll) iodide.

The invention is illustrated in greater detail below by means of Examples.

Examples 1 to 6 1. Activation The shaped article (in this instance: cellulose sheets), which has been dried/freezedried under a high vacuum, is treated in a tightly sealable vessel of suitable proportions for the shaped article with 3 ml of a 0.3 molar solution of one of the following carbonylating reagents per 1 g of polymer (in this instance: cellulose) and agitation is carried out at room temperature (with exclusion of moisture) for from 6 to 16 hours. The carbonylating reagents are: in absolute, amine-free dimethylformamide (DMF) Example 1: CDI 1,1'-carbonyldiimidazole Example 2: CDT 1,1'-carbonyldi-1,2,4-triazole

<C

fl -6- Example 3: CDB 1,1'-carbonyldi-1,2,3-benzotriazole Example 4: PCT phenoxycarbonyltetrazole (ii) in absolute pyridine Example 5: CFP chloroformic acid phenyl ester Example 6: CFNP chloroformic acid 4-nitrophenyl ester II. Modification The reaction solution from the activation step is removed by means of filtration or decanting off and the activated shaped article is rinsed three times with half the volume of the solvent of the reaction solution for from 5 to 10 minutes each time. The washing phases are discarded. 3 ml of a 0.3 molar solution of ethylenediamine or 1,6bis(methylamino)hexane or O,O'-bis(2-aminopropyl)polyethylene glycol in DMF or pyridine per 1 g of polymer are added and agitation is carried out at room temperature, with exclusion of moisture, again for from 6 to 16 hours. The reaction solution is then removed by means of filtration or decanting off and the modified shaped article is rinsed three times with half the volume of the solvent of the reaction solution for from to 10 minutes each time. That washing procedure is then repeated using distilled water, ethanol, acetone, ethyl acetate and diethyl ether. After removal of the residual ether, the shaped article is dried under a high vacuum.

II. Chemical stability Squares, 1 cm 2 in size, of the derivatised cellulose sheets are subjected to the following conditions and, after 24 hours, are again tested for their degrees of modification.

pH 0, 7, 8, 9, 10, 11, 12, 13 and 14 (correspondingly diluted NaOH) 24 hours/room temperature; concentrated aqueous ammonia (25 24 hours/room temperature; trifluoroacetic acid (99 24 hours/room temperature; -7- In all cases, the initial degrees of modification were confirmed.

Examples 7 to 8 1. Addition reaction The shaped article (in this instance: cellulose sheets), which has been dried/freezedried under a high vacuum, is treated in a tightly sealable vessel of suitable proportions for the shaped article with 3 ml of a 0.1 molar solution of isocyanate per 1 g of polymer (in this instance: cellulose) and heating is carried out at 60°C (with exclusion of moisture) in a drying cabinet for 12 hours.

Example 7: Addition of 4-nitrophenyl isocyanate

(C

7

H

4

N

2 0 3 [164.12] Example 8: Addition of 4-phenyl isothiocyanate

(C

7

H

4

N

2 0 2 S) [180.19] The vessel is swirled several times in each case. The reaction solution from the activation step is removed by means of filtration or decanting off and in each case the activated shaped article is swirled for from 5 to 10 minutes with 3 ml of solvent per g of dry weight of the polymer used. After decanting or filtering off, the washing phases are discarded.

The solvents are used in the following order: dimethylformamide (analytical grade), water (bi-distilled), ethanol (synthesis grade), acetone (synthesis grade), ethyl acetate (synthesis grade), dichloromethane (analytical grade). After evaporating off the organic solvent, the activated shaped article is dried under a high vacuum at room temperature.

II. Capping (blocking of the functions of the polymer surface not reacted in Step I) The pre-dried activated shaped article is placed in 3 ml of DMF/pyridine (2:1, volume/volume) per g of dry weight and then 0.5 ml of acetic anhydride is added. After 6 hours at room temperature with moderate swirling, the reaction is terminated. The reaction solution is decanted off or filtered off and the washing procedure as in Step I is repeated.

III. Reduction of the nitro group with samarium(ll) iodide The pre-dried activated shaped article is placed in 10 ml of DMF (abs) per g of dry weight and 2 ml of a 0.1 molar solution of samarium(ll) iodide in THF (abs) per g of dry weight are added. The reaction vessel is swirled, with exclusion of moisture, for from to 16 hours at room temperature. After decanting off the reaction solution, the shaped article is swirled with 5 ml of DMF per g of dry weight for 15 minutes and then twice with ml of water (bi-distilled) for approximately from 2 to 3 hours. The washing procedures according to Step I are then repeated.

Claims

1. Shaped article the surface of which is provided with reactive functions for chemical, biochemical or biological applications, which can be manufactured by starting from, as the shaped article, a shaped paper-based polymeric material, the surface of which shaped article is provided with hydroxy groups, and reacting the shaped article with a carbonylating reagent in a manner know per se, and reacting the carbonlated shaped article with a compound of the formula R 3 -NH- R 4 wherein R 3 represents the radical of a primary amine known per se and R 4 represents a hydrogen atom or R 3 and R 4 which may be the same or different, represent radicals of a secondary amine known per se, to form a urethane derivative.

2. Shaped article according to any one of the preceding claims, which can be manufactured by using a carbonylating reagent from the group consisting of °1,1'-carbonyldiimidazole (CDI), 1,1'-carbonyldi-1,2,4-triazole (CDT), 1,1'- carbonyldi-1,2,3-benzotriazole (CDB) and/or phenoxycarbonyltetrazole (PCT) and/or (ii) from the group consisting of chloroformic acid phenyl ester (CFP) and/or chloroformic acid 4-nitrophenyl ester (CFNP).

3. Shaped article according to claim 2 which can be manufactured by carrying out the carbonylation and/or reaction according to claim 1(b) in dimethylformamide (DMF), especially at room temperature and/or without addition of a buffer and/or without addition of a further base besides dimethylformamide (DMF).

4. Shaped article according to claim 2 which can be manufactured by carrying out the carbonylation in pyridine.

Shaped article the surface of which is provided with reactive functions for chemical, biochemical or biological applications, which can be manufactured by starting from, as the shaped article, a shaped polymeric material, the surface of which shaped article is provided with hydroxy groups, and reacting the shaped article with an isocyanate and/or thioisocyanate, and optionally blocking the unreacted hydroxy groups of the activated shaped article, optionally, if nitro groups have been introduced by the isocyanate and/or thioisocyanate in Step and unreacted hydroxy groups of the activated shaped article have been blocked in Step reducing any such nitro groups to form amino groups, and, optionally, if protected amino groups have been introduced by the isocyanate and/or thioisocyanate in Step and unreacted hydroxy *groups of the activated shaped article have been blocked in Step de- protecting any such protected amino groups to form free amino groups.

6. Shaped article according to claim 5, which can be manufactured by starting from a shaped cellulose-based polymeric material, especially a paper-based Spolymeric material. 9

7. Shaped article according to claim 5, characterised in that a shaped S plastics-based polymeric material, especially a hydroxylated-polyolefin-based polymeric material, is used as starting material.

8. Shaped article according to claim 5, 6 or 7, which can be manufactured by using 4-nitrophenyl isocyanate or 4-nitrophenyl isothiocyanate in Step

9. Shaped article according to any one of claims 5 to 8, which can be manufactured by carrying out the blocking in Step with acetic anhydride.

Shaped article according to any one of claims 5 to 9, which can be manufactured by carrying out the reduction in Step with samarium(ll) iodide. 11

11. Shaped article according to any one of claims 5 to 10, which can be manufactured by carrying out the reaction according to claim 5(a) and/or and/or 5(c) in dimethylformamide (DMF), especially at room temperature and/or without addition of a buffer and/or without addition of a further base besides dimethylformamide (DMF).

12. Shaped article according to any one of the preceding claims, which can be manufactured by starting from a shaped polymeric material from the group consisting of membranes, films, miniaturised reaction vessels, pipette tips and microtitre plates.

13. Shaped article according to claim 1, wherein Step results in an article the surface of which is provided with functions selected from the groups S: consisting of COOH, CHO, NH 2 NHR 2 NR 2 Hal, SH, OH, alkyl, aryl, ligand, antibody, protein or H 2 0 3 P.

14. Shaped article according to claim 5, wherein Step or Step results in an article the surface of which is provided with functions selected from the groups consisting of COOH, CHO, NH 2 NHR 2 NR 2 Hal, SH, OH, alkyl, aryl, ligand, antibody, protein or H 2 0 3 P. DATED this 9 th day of January 2001 GESELLSCHAFT FUR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) WATERMARK PATENT AND TRADE MARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA KJS:RBP:VRH P7905AU00