Note: Descriptions are shown in the official language in which they were submitted.
CA 02265689 l999-03- 15Shaped article having reactive functionsThe present invention relates to a shaped article the surface of which is provided withreactive functions for chemical, biochemical or biological applications.Many polymeric materials based on natural polymers, such as polysaccharides (forexample cellulose, starch or chitin], or organic synthetic polymers, such as plastics (forexample polyethylene, polypropylene, polytetrafluoroethylene or polystyrene), aredistinguished by insolubility in organic and aqueous media. For many decades, theirsurface properties, such as charge, chemical functionality or hydrophobicity, have beenused for synthesis, separation and detection reactions, for example in the followingï¬elds: chemical solid-phase synthesis; hydrophobic chromatography, ion-exchangechromatography or afï¬nity chromatography and afï¬nity concentration. Used in theconventional form of granules, beads or ï¬bres, those materials are packed intoreaction vessels, such as columns or cartridges. Depending on the application, use isadvantageously made, for this purpose, of special reactive functions on the surface, asare shown by way of example in the following diagram.COOHPH20â CHOProteinNH ,Antibody NHH2Ligand NR 2A IW HalAlkyl OH SHSuch surface functionalities may be introduceda) directly by means of co-polymerisation with appropriate monomers orb) subsequently by means of chemical derivatisation.Many chemical, biochemical and biological procedures make use of specially shapedapparatus (shaped articles, such as membranes, ï¬lms, papers, miniatunsed reactionvessels (for example Eppendorf), pipette tips or microtitre plates), which can bemanufactured from the polymeric materials mentioned at the beginning or fromCA 02265689 l999-03- 15polymeric materials similar thereto. An optimised manufacturing process provides suchshaped articles with the mechanical and chemical stability necessary for theapplication. The surfaces ofsuch shaped articles can also be used advantageously forthe same synthesis, separation and detection reactions as mentioned above. Theintroduction of the various chemical functions required for the application is. however,more problematic in this instance because the derivatisation process must not beallowed to have an adverse effect on the mechanical and/or chemical properties.For example, shaped articles, such as sheet material, for example paper made fromspecially derivatised cellulose, for example carboxyrnethyl-, diethylaminoethyl-,phospho-, alkyl« or aryl-cellulose. are of major importance. However, the processes formanufacturing starting materials for the said shaped articles call for highly toxicreagents, such as cyanogen bromide, epichlorohydrin or acrylonitrile, andlor verydrastic alkaline reaction conditions so that the cellulose papers produced from thosematerials exhibit very much lower mechanical and chemical stability.The problems mentioned have also not been entirely solved by W0-A-94/153594.The problem of the present invention is therefore to provide new materials for thebiosciences and new shaped articles the surfaces of which are provided with reactivefunctions for chemical, biochemical or biological applications, a particular aim being toprovide those materials by means of especially gentle methods. The mechanical andchemical properties of the shaped articles should undergo no, or only insigniï¬cant,modification as a result.According to one embodiment, the problem underlying the invention is solved by ashaped article the surface of which is provided with reactive functions for chemical,biochemical or biological applications, which can be manufactured by(a) starting from, as the shaped article, a shaped polymeric material, the surface ofwhich shaped article is provided with hydroxy groups, and reacting the shaped articlewith a carbonylating reagent in a manner known per se, and(b) optionally reacting the carbonyiated shaped article with a compound of the formulaR;-NH~R., wherein R3 represents the radical of a primary amine known perse and R4CA 02265689 l999-03- 15represents a hydrogen atom or R3 and R.. which may be the same or difterent,represent radicals of a secondary amine known per se, to form a urethane derivative.R3R.NH may especially be an amine that is customarily used for urethane manufactureand may be designated a urethane-forming agent.0 oEra»; /{L L Activation Eran/LL R2 ll.Modit'lc:rticn â>mâLL 3333O0.. -xxâ ° R2 -Ital/iirzxirâ ° NHRt R1 R2 If T 3The reaction of a polymer the surface of which is provided with hydroxy groups, morespeciï¬cally agarose and polysaccharides, with a carbonylating reagent, namely 1.1â-carbonyldiimidazole, is part of the prior art; see J. Chromatography. 219 (1981)353 - 359 and 361- 372.The ï¬elds of use for a shaped article according to the invention are in immunology.biochemistry, molecular biology, clinical diagnostics or bioprocessing technology.Examples of possible uses are ion-exchange chromatography, atï¬nity chromatographyor affinity concentration, hydrophobic chromatography, blotting, dot blotting andchemical solid~phase synthesis.The shaped article according to the invention can be manufactured by starting from ashaped ce||uIoseâbased polymeric material, especially a paper-based polymericmaterial, or from a shaped plastics-based polymeric material, especially ahydroxylated-polyoleï¬n-based polymeric material.When starting from a shaped cellulose-based polymeric material, a wide range ofcellulose materials having a very wide variety of chemical groups can bemanufactured. In that variant, the shaped articles according to the invention can bemanufactured using very mild reaction conditions, using materials and solvents of lowtoxicity and using a simple technical procedure, it being especially important that thechemical and mechanical properties of the underivatised cellulose starting material aremodified only insigniï¬cantly. The chemical linkage has excellent pH stability,approximately in the range from 2 to 12 at room temperature.CA 02265689 l999-03- 15The shaped article according to the invention can be manufactured by using acarbonylating reagent(i) from the group consisting of 1,1â-carbonyldiimidazole (CDI), 1,1-carbonyIdi-1,2,4-triazole (CDT), 1,1'-carbony|diââl,2,3-benzotriazole (CDB) andlor phenoxycarbonyl-tetrazole (PCT) andlor(ii) from the group consisting of chlorolormic acid phenyl ester (CFP) andlorchloroformic acid 4ânitropheny| ester (CFNP).When using a carbonylating reagent from group (i), the carbonylation can be carriedout in dimethylformamide (DMF). especially at room temperature and/or withoutaddition of a buffer andlor without addition of a further base besides dimethylformamide(DMF).When using a carbonyiating reagent from group (ii). the carbonylation can be carriedout in pyridine.According to a further embodiment, the problem underlying the invention is solved by ashaped article the surface of whlch is provided with reactive functions for chemical, Ibiochemical or biological applications, which can be manufactured by(a) starting from, as the shaped article, a shaped polymeric material, the surface ofwhich shaped article is provided with hydroxy groups, and reacting the shaped articlewith an isocyanate andlor thioisocyanate, and(b) optionally blocking the unreacted hydroxy groups of the activated shaped article,(c) optionally, if nitro groups have been introduced by the lsocyanate and/orthioisocyanate in Step (a), reducing any such nitro groups to form amino groups, and(d) optionally. if protected amino groups have been introduced by the isocyanateand/orthioisocyanate in Step (a), de-protecting any such protected amino groups toform free amino groups. 1out I AT _m w~©-â NH2n X:.:"â< >"'NO2 -â'-":"âââ|I.Re Ii n,¢. .9,1 Sl|xI{':â)uâ;I'kï¬ B !pTnK NH21X=5.0CA 02265689 l999-03- 15The shaped article according to the invention can be manufactured by starting from ashaped celluloseâbased polymeric material, especially a paper-based polymericmaterial, or from a shaped plastics-based polymeric material, especially ahydroxylated«polyoIeï¬n-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 bemanufactured by using 4-nitrophenyl isocyanate or 4-phenyl isothiocyanate in Step (a).It can furthermore be manufactured by carrying out the blocking in Step (b) with aceticanhydride.it can furthermore be manufactured by carrying out the reduction in Step (c) withsamarium(|l) iodide.The invention Is illustrated in greater detail below by means of Examples.Examples 1 to 61. ActivationThe shaped article (in this instance: cellulose sheets). which has been dried/freeze-dried under a high vacuum, is treated in a tightly sealable vessel of suitable proportionsfor the shaped article with 3 ml of a 0.3 molar solution of one of the followingcarbonylating reagents per 1 g of polymer (in this instance: cellulose) and agitation iscarried out at room temperature (with exclusion of moisture) forfrom 6 to 16 hours. Thecarbonylating reagents are:(i) in absolute, amineâfree dimethylforrnamide (DMF)Example 1: CDl = 1,1'âcarbonyldiimidazoleExample 2: CDT = 1,1â-carbonyldi-1,2,4-triazoleCA 02265689 l999-03- 15Example 3: CD8 = 1,1â-carbonyldi-1,2,3-benzotriazoleExample 4: PCT = phencxycarbonyltetrazole(ii) in absolute pyridineExample 5: CFP = chloroformic acid phenyl esterExample 6; CFNP = chlorotormic acid 4-nitrophenyl esterll. Modiï¬cationThe reaction solution from the activation step is removed by means of ï¬ltration ordecanting off and the activated shaped article is rinsed three times with half the volumeof the solvent of the reaction solution for from 5 to 10 minutes each time. The washingphases are discarded. 3 ml of a 0.3 molar solution of ethylenediamine or 1.6-bis(methy|amino)hexane or 0,0â-bis(2-aminopropyl)polyethylene glycol in DMF orpyridine 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 thenremoved by means of ï¬ltration or decanting off and the modiï¬ed shaped article isrinsed three times with half the volume of the solvent oi the reaction solution for from5 to 10 minutes each time. That washing procedure is then repeated using distilledwater, ethanol, acetone, ethyl acetate and diethyl ether. After removal of the residualether, the shaped article is dried under a high vacuum.lll. Chemical stabilitySquares, 1 cmâ in size. of the derivatised cellulose sheets are subjected to thefollowing conditions and, after 24 hours, are again tested for their degrees oimodiï¬cation.pH 0,7, 8, 9, 10, 11, 12, 13 and 14 (correspondingly diluted NaOH) 24 hours/roomtemperature;concentrated aqueous ammonia (25 %), 24 hours/room temperature; triï¬uoroaceticacid (99 %), 24 hours/room temperature; CA 02265689 l999-03- 15In all cases, the initial degrees of modiï¬cation were confirmed_Examples 7 to 81. Addition reactionThe shaped article (in this instance: cellulose sheets), which has been dried/freeze-dried under a high vacuum, is treated in a tightly sealable vessel of suitable proportionsfor 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 ofmoisture) in a drying cabinet for 12 hours.Example 7: Addition of 4-nitrophenyl isocyanate(c,H..N2o,) [1s4.12]Example 8: Addition of 4-phenyl isothiocyanate(C7H.N2O2S) [18019]The vessel is swirled several times in each case. The reaction solution from theactivation step is removed by means of filtration or decanting off and in each case theactivated shaped article is swirled for from 5 to 10 minutes with 3 ml of solvent per g ofdry weight of the polymer used. After decanting or filtering off, the washing phases arediscarded.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 organicsolvent, the activated shaped article is dried under a high vacuum at roomtemperature.CA 02265689 l999-03- 15ll. 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 (211.volume/volurne) per g of dry weight and then 0.5 ml of acetic anhydride is added. After6 hours at room temperature with moderate swirling, the reaction is terminated. Thereaction solution is decanted off or ï¬ltered off and the washing procedure as in Step I isrepeated.Ill. Reduction of the nitro group with samarium(lI) iodideThe pre-dried activated shaped article is placed in 10 ml of DMF (abs) per g of dryweight and 2 ml of a 0.1 molar solution of samarium(l|) iodide in THF (abs) perg of dryweight are added. The reaction vessel is swirled, with exclusion of moisture, for from 10to 16 hours at room temperatureâ After decanting off the reaction solution, the shapedarticle is swirled with 5 ml of DMF per g of dry weight for 15 minutes and than twice with10 ml of water (bi-distilled) for approximately from 2 to 3 hours. The washingprocedures according to Step I are then repeated.
