SELECTIVE AFFINITY MATERIAL, PREPARATION THEREOF BY MOLECULAR IMPRINTING, AND USE OF THE SAME

Abstract: A selective adsorption material, especially suitable for adsorption of biological macromolecules, is described. The adsorption material comprises a matrix with immobilised ligands, localised to selectively adsorb a predetermined molecule. A process for preparing a selective adsorption material, especially suitable for adsorption of biological macromolecules, is also described. The process is characterised in that a print molecule having at least two separate binding sites is bonded to the corresponding, at least two immobilisable ligands, the ligands are immobilised, and subsequently the print molecule is removed. The selective adsorption material can be used for purification or analysis, especially of biological macromolecules.

Description: SELECTIVE AFFINITY MATERIAL, PREPARATION THEREOF BY MOLECULAR IMPRINTING, AND USE OF THE SAME. The invention relates to a selective adsorption material, especially suitable for adsorption of biolo¬ gical macromolecules, a process for preparing this selec¬ tive adsorption material, and the use thereof for purifi¬ cation and analysis, especially of biological macromole¬ cules. It is known to prepare homogeneous gels while using the so-called molecular imprinting technique by making imprints of dyes (R. Arshady, K. Mosbach; akromol. Chemie, 182 (1981) 687) and amino acid derivatives (L. Andersson, B. Sellergren, K. Mosbach, Tetrahedron Lett. 25 (1984) 5211). Merely non-covalent bonding between a "print mole¬ cule" and monomers is used. After polymerisation of the monomers and removal of the print molecule, a selective polymer is obtained, based on binding groups correctly immobilised in space and present in cavities which have the shape of a mould of the print molecule. A summary of this technique is given by B. Ekberg, K. Mosbach, in Trends Bio- technol., 7 (1989) 92. It is known to make imprints of carbohydrate deriva¬ tives substituted with covalently, but reversibly bonded vinylphenyl boric acid groups, so-called boronate esters, which after polymerisation have permitted hydrolysis and binding of a new print molecule (G. Wulff, ACS Symp. Series, 308 (1986) 186). Decisive of the selectivity of the thus prepared polymers are correctly positioned bind- ing boronate groups and a well-shaped cavity. The drawback of this system is that a complicated chemical synthesis is necessary. Prearranged boronate groups have also been used for making imprints of glycoprotein ( (Transferrin, M. Glad, 0. Norrlόw, B. Sellergren, N. Siegbahn, K. Mosbach, J. Chromatogr., 347 (1985) 11) and bis-nucleotides (0. Norrlόw, M. 0. Mansson, K. Mosbach, J. Chromatogr., 396 (1987) 374) on silica. In this context, use has been made of a mixture of non-covalently bonding organic silanes and boronate silane, which has been caused to interact with a print molecule before polymerisation on the surface. The effect of recognition seems mainly to depend on the fact that the boronate groups are correctly spaced from each other to be able to interact effectively with the print molecules. Transferrin has a total of four sialic acid groups and bis-NAD has four riboses which form boronate esters. It is also known to use, in affinity chromatography methods, adsorption material having specific ligands, but these are randomly localised on the sorbent and may there¬ fore yield poor select!vety. Furthermore, there will be a large number of unused ligands, which is uneconomical from the industrial point of view. Immobilised phenyl boric acid has been used to sepa¬ rate carbohydrate derivatives (H.L. Wei h, J.L. Wiebers, P.T. Gilham, Biochemistry, 91 (1970) 4) and also to sepa- rate glycosylised hemoglobin and other glycoproteins (P.D.6. Dean, P.J. Brown, V. Bouriotis, US Patent Specifi¬ cation 4,269,605 (1981). For purification and analysis, especially of biolo¬ gical macromolecules, selectivity is most important. It is frequently necessary to distinguish a single component in a mixture of, maybe, several thousand components. The known adsorption materials are in most cases not suffi¬ ciently selective and cause non-specific- binding, which may completely destroy the result of an analysis. Modern biotechnical production and analysis as well as a large amount of medical diagnostics are based on selective binding. The methods known so far are also for these purposes not sufficiently selective and/or suffi¬ ciently simple to be economically useful on an industrial scale. Therefore there is a need of an adsorption material having very high selectivity and low non-specific binding, which may be prepared on a large scale and at low cost. According to the invention, a selective adsorption material is provided, which is especially suitable for adsorption of biological macromolecules, and which is characterised in that it comprises immobilised ligands which have been localised to selectively adsorb a prede¬ termined molecule by their first being bound to a print molecule which has then been removed after the immobili¬ sation of the ligand. The invention also comprises a process for preparing a selective adsorption material, especially suitable for adsorption of biological macromolecules, characterised in that a print molecule having at least two separate binding sites is bonded to the corresponding, at least two immo- bilisable ligands, that the ligands are immobilised, and that subsequently the print molecule is removed. Finally the invention relates to the use of the selective adsorption material for purification or ana¬ lysis, especially of biological macromolecules. After prebonding to the print molecule and the sub- sequent immobilisation, the binding groups (ligands) will be bonded preferably to the surface of a matrix. The cor¬ rect localisation of ligands on two dimensions results in increased selectivity by the print molecule having at least two separate binding sites. The corresponding, at least two immobilised ligands will thus be correctly spac¬ ed apart. This results in optimal binding of the target molecule in the subsequent adsorption. As is evident from that stated above, the invention thus relates to a form of directed immobilisation of affi- nity ligands or other (chemical) ligands. This directed immobilisation is achieved by means of a prebinding method. The advantage as compared to randomly immobilised binding groups is that the print molecule is bonded more effectively and with greater selectivity. At the same time, all ligands may be utilised, thereby avoiding an excess thereof, which otherwise may easily result in non¬ specific bonding which in turn results in a lower yield. Ligands which are useful for the invention should carry groups that can be bonded to a print molecule, and groups which can be used for immobilisation of the ligands. One type of suitable ligands is monomers which 5 can be immobilised by polymerisation. Examples of groups that can be bonded to a print molecule are given in Table 1. Table 1 Group binding to print Binding group on print mole- 10 molecule on ligand/monomer cule Imidazole-metal Histidine, tryptophane, thiol Iminodiacetic acid-metal " " " Carboxyl Amine Amine Carboxyl 15 Carboxyl Carboxyl, amide (hydrogen bonding) Boronate Carbohydrate, diol Thiol Thiol Aldehyde Amine 2Q Boronate, thiol and aldehyde are bonded covalently, whereas the other groups are bonded non-covalently to the print molecule. Among groups that may be used for immobilisation, mention can be made of silanes which bind silanol and ?t _ hydroxyl groups, and vinyl, acryl and methacryl which polymerise on a matrix. Other ligands/monomers that may be used are: Acryloylhistamine: H. Morawetz, W.R. Song, JACS, 88 (1966) 571. „ 0 Boronate silane: M. Glad, 0. Norrlδw, B. Sellergren, N. Siegbahn, K. Mosbach, J. Chromatogr., 347 (1985) 11. IDA-silane: A. Figueroa, C. Corradini, B. Feibush, B.L. Karger, J. Chromatogr., 371 (1986) 335. Moreover, different types of ligands may be used r simultaneously in one preparation. For example, use can be made of both boronate binding and metal interaction on the same print molecule if this is e.g. a glycoprotein with surface-bound histidines. As print molecule (imprinting molecule) for prear- rangement of the ligands, the invention preferably uses biological macromolecules, e.g. enzymes, antibodies, glycoproteins or other proteins, nucleic acids, poly- saccharides. The molecular imprinting is preferably car¬ ried out in aqueous media which are the normal surround¬ ings of these molecules. It has also become apparent that larger or smaller amounts of organic solvents which are miscible with water, such as DMF, DMSO, formamide (M. Stahl, M.O. Mansson, K. Mosbach, Biotechn. Lett., 12 (1990) 161; N. Chang, S.J. Hen, A.M. Klibanov, BBRC, 176 (1991) 1462), may be added. In some cases, also pure DMSO can be used, in which protein and a monomer mixture (the ligands) are dissolved. A polymerised product is obtain¬ ed, having a sufficient amount of correctly positioned, binding groups to provide a certain selectivity. Matrix materials which are useful according to the invention must contain surface-bound reactive groups which can react with and covalently bind prearranged ligands. The matrices may be solid or in the form of gels and comprise particles, chips, electrodes, gel etc. Suit¬ able matrix materials are silica (particles, chips etc.), glass (particles, electrodes etc.), biological polymers (agarose, dextran, gelatin etc.) and synthetic polymers (polyvinyl alcohol, TRIM (P. Reinholdsson, T. Hargitai, B. Tornell, R. Isaksson, Angew. Macromol. Chem., 1991, in press) etc.). When the matrix consists of a particu- late solid phase, the surface is to be found both on the outside of the particles and on the surface of the pores thereof. In most cases, the latter surface is many times larger than the former. Reactive groups on the matrix material can be acry- late, methacrylate, vinyl, hydroxyl or silanol. If the matrix material contains no suitable reac¬ tive groups, it may be derivatised in advance with suit¬ able groups, such as methacryl groups, before immobilising the ligands on the matrix. The difference between a prior art adsorption mate¬ rial and the invention is schematically shown in the accompanying drawing in which: Fig. 1 is a schematic sketch of a prior art sorbent with randomly positioned, binding groups, e.g. boronate, immobilised metal; and . Fig. 2 shows a sorbent according to the invention, with specifically localised, binding groups. The invention will now be described in more detail by means of the following, non-restrictive Example. Example Methacrylate silica 3-methacryloyloxypropyltrimethoxysilane (2.0 g, 8.1 mmol, Fluka) was mixed with 100 ml of water and dis¬ solved during powerful stirring by means of a magnetic stirrer at 22°C for 4 h. Porous silica (5.0 g, "Lichro- spher Si 300", Merck) was suspended in 20 ml of water and treated with vacuum and ultrasonics, thereby producing pores free from air. Subsequently, the silane solution was added, and the silanisation was allowed to proceed for 4 h at 60°C in a round-bottomed flask placed in a water bath and fitted with a "Teflon ®"-coated blade mixer. The deri- vatised silica was washed on a glass filter with water and methanol and then air-dried on the filter. Titration with bromine water (according to Glad et al, J. Chromatogr., 347 (1985) 11) yielded 335 μmol of methacrylate groups per g of silica product. A carbon ana¬ lysis yielded 620 μmol of silane per g of silica product, which indicates that more than half of the groups are still reactive to subsequent immobilisation. Preparation Mixtures according to the Table below with RNase B and STI (Soybean trypsine inhibitor), respectively, as print molecule. Methacrylate-silica Vinyl imidazole Acrylphenyl boric acid Aery1amide PDA (piperazine diacrylamide) TEMED ZnCl 2 Water/DMF (7/3, w/w) Ammonium persulphate alt. A) RNase B (molecular weight 14700) alt. B) STI (molecular weight 20100) 1) ref. C.G. Overberger, N. Vorchheimer, JACS, 85 (1963) 951 After mixing the respective print molecule with all the other ingredients in a total of 2.5 ml of water/DMF, the samples were cooled, and then N„ gas was conducted through the mixture. After about 30 s, the mixture began to solidify. The preparation was allowed to stand for 1 h, and subsequently the substituted silica particles were washed on a glass filter with water/DMF. Small polymer particles were removed by sedimentation, and the remaining particles were packed in steel columns (5 x 0.5 cm). When injecting RNase B on preparation A, the elution was delay- ed as compared with preparation B. As an alternative to vinyl imidazole, vinyl benzyl iminodiacetic acid can be used (L.R. Morris, R.A. Mock, C.A. Marshall, J.H. Howe, JACS, 81 (1959) 377). These bind metal ions, e.g. Zn , Cu , which have been used for a long time in so-called immobilised metal affinity chromatography (J. Porath, J. Carlsson, I. Olsson, G. Belfrage, Nature, 258 (1975) 598).

Claims: CLAIMS 1. Selective adsorption material, especially suitable for adsorption of biological macromolecules, c h a r ¬ a c t e r i s e d by immobilised ligands which have been localised to selectively adsorb a predetermined molecule by their first being bound to a print molecule which has then been removed after the immobilisation of the ligands. 2. Adsorption material as claimed in claim 1, c h a r a c t e r i s e d in that the ligands are poly- merisable monomers which have been immobilsed by polymeri¬ sation. 3. Adsorption material as claimed in claim 1, c h a r a c t e r i s e d in that the ligands are immo¬ bilised by covalent bonding to a matrix having surface- bound groups which are reactive with the ligands. 4. Adsorption material as claimed in claim 1, c h a r a c t e r i s e d in that the ligands are immo- bilised in a polymer network which is polymerised or bonded to a matrix. 5. Adsorption material as claimed in claim 3 or 4, c h a r a c t e r i s e d in that the matrix is selected among silica, glass, biological polymers, synthetic poly- mers and mixtures thereof. 6. Adsorption material as claimed in one or more of the preceding claims, c h a r a c t e r i s e d in that the ligands have been bound to the print molecule via one or more of the groups imidazole-metal, iminodiacetic acid- metal, carboxyl, amine, boronate, thiol and aldehyde on the ligands. 7. Adsorption material as claimed in claim 6, c h a r a c t e r i s e"d in that the ligands have been bound to the print molecule via the groups imidazole-metal and/or iminodiacetic acid-metal on the ligands. 8. Process for preparing a selective adsorption mate¬ rial, especially suitable for adsorption of biological macromolecules, c h a r a c t e r i s e d in that a print molecule having at least two separate binding sites is bonded to the corresponding, at least two immobilisable ligands, whereupon the ligands are immobilised, and subsequently the print molecule is removed. 9. Process as claimed in claim 8, c h a r a c ¬ t e r i s e d in that the ligands are polymerisable monomers and are immobilised by direct polymerisation. 10. Process as claimed in claim 8, c h a r a c ¬ t e r i s e d in that the immobilisation of the ligands is effected by covalent bonding to a matrix having sur¬ face-bound groups which are reactive with the ligands. 11. Process as claimed in claim 10, c h a r a c ¬ t e r i s e d in that the matrix is, by prederivatisa- tion, provided with groups which can react with the ligands. 12. Process as claimed in claim 8, c h a r a c - t e r i s e d in that the ligands are immobilised by binding in a polymer network which is polymerised or bonded to a matrix. 13. Process as claimed in one or more of claims 10-12, c h a r a c t e r i s e d in that the matrix is selected among silica, glass, biological polymers, synthe¬ tic polymers and mixtures thereof. 14. Process as claimed in one or more of claims 8-13, c h a r a c t e r i s e d in that the ligands are bound to the print molecule via one or more of the groups imi- dazole-metal, iminodiacetic acid-metal, carboxyl, amine, boronate, thiol and aldehyde on the ligands. 15. Process as claimed in claim 14, c h a r a c ¬ t e r i s e d in that the ligands are bound to the print molecule via the groups imidazole-metal and/or iminodiace- tic acid-metal on the ligands. 16. Use of the selective adsorption material as claimed in one or more of claims 1-7, for purification or analysis, especially of biological macromolecules.

Inventors: GLAD MAGNUS, KEMPE MARIA, MOSBACH KLAUS, GLAD, MAGNUS, KEMPE, MARIA, MOSBACH, KLAUS
Applicants: GLAD MAGNUS, KEMPE MARIA, MOSBACH KLAUS, GLAD, MAGNUS, KEMPE, MARIA, MOSBACH, KLAUS
Application year: 1992
Main classification: C07K3
IPC codes: B01J20, C07K1, G01N30, G01N33