CA2259416A1 - Imprint polypeptides covalently cross-linked and having a fixed and stabilised arrangement, process for the preparation and use thereof - Google Patents

Abstract

The invention relates to imprint polypeptides covalently cross-linked and having a fixed and stabilised arrangement, process for the preparation and uses thereof. The properties of said imprint polypeptides (VIP) covalently cross-linked are present in aqueous and in organic solvent systems, and can therefore be used in both types of system.

Description

CA 022~9416 1999-01-04 . ' ' 1 Application of the Invention The invention concerns covalently crosslinked imprint-polypeptides (moulded polypeptides) having a fixed and stabilized conformation, e. 9. proteins which can be used in catalytic synthesis, chromatography and analysis of chiral compounds and for specific recognition of molecules by means of biosensors, and processes for their manufacture and use thereof.

State of the art Polymers which are able to recognize molecules selectively are of highest industrial interest in biotechnology and chemistry. To this area belong proteins in the form of enzymes, antibodies and receptors. These polymers have, due to their three-dimensional molecule structure (conformation), complementary areas for distinct molecules (substrates, antigenes, hormones, etc.) which can be addressed as binding sites. Because often native polymers bind insufficiently or even do not bind commercially interesting molecules or the essential conformation for the binding is not stable enough under the operational conditions, methods have been developed in order to produce tailor-made polymers which show the desired complementarity (K.Mosbach and O. Ramstrom, Biotechnology, vol. 14 (1996), 163-170).

Goldstein (Methods Enzymol. 19 (1970), 935-962) and Fritz et al. (Angew. Chem. 78, 1966, 775) describe a procedure for the immobilization of proteins with the use of cyclic anhydrides for the covalent coupling. The procedure consists first of thecopolymerization of an anhydride with other monomers to form a carrier matrix and then the protein is coupled to the ready-made carrier matrix.

Mosbach and Ramstrom (loc. cit.) describe that the manufacture of tailor-made polymers with the desired complementarity can be carried out in two variants. Both variants are based on the principle that a specially chosen molecule, in the following adressed as print molecule, which functions as a mould which is responsible for the desired complementarity in the polymer and therefore for the new property. Figures 1 and 2 explain these two variants schematically.

CA 022~9416 1999-01-04 Variant 1 for the manufacture of tailor-made (moulded) polymers is carried out by a copolymerization of selected monomers in an organic solvent in the presence of the print molecule (cf. fig. 1). Here, at first the print molecule is dissolved in an organic solvent system either a) directly together with a monomer (non-covalent method) or b) after derivatization with a monomer (covalent method). The polymerization is then started upon addition of a crosslinker and a radical starter. From the resultingpolymer the print molecule must be extracted under suitable conditions in order to get the created new binding sites accessible. The polymer, whlch was made in this way, has got selective recognition sites for the print molecule or for structurally similar molecules (cf. fig. 1). The numbers mean functional groups.

Essential for a) and b) is that both the solution of the initial substances, which is assembled with distinct concentrations of the print molecule, the monomer(s) and the radical starter, and the afterwards generated tailor-made (moulded) property of the polymer hitherto depend exlusively on the use of organic solvent systems.

In variant 2 no polymerization for the generation of a tailor-made (moulded) polymer is carried out in the presence of a print molecule, instead an already made native polymer, e. g. a polypeptide or a protein, is forced to alter its conformation under certain conditions and in the presence of the print molecule [cf. fig. 2, in which a) denotes the modification of the catalysis property and b) denotes the generation of affinity (recognition)].

First the polymer together with the print molecule are dissolved in an aqueous solvent system and then a precipitation takes place by the addition of additives, e. 9. organic solvents. Within this the print molecule interacts with the polymer via non-covalent interactions in the way that the conformation of the polymer is altered. By this the polymer has got a new property which is predetermined inherently by the print molecule. The up to here described procedure of variant 2 will in the following be adressed as imprinting (moulding), the corresponding polymer as imprint-polymer (moulded polymer), e. g. an imprint-polypeptide.

However, the new property of the imprint-polymer is reversible. It can only be maintained until it is used under conditions, which prevent the back-formation of the CA 022~9416 1999-01-04 original thermodynamically favoured conformation. This is the reason why the polymers resulting from variant 2 which were described by Mosbach et. al. (loc. cit.), Stahl et al ., Biotechnol. Leff. 12 (1990), 161 -166; Stahl et al ., J. Am. Chem. Soc. 113 (1991), 9366-9368 and Klibanov et al., J. Biol. Chem. 263 (1988), 11624-11626 and Dabulis and Klibanov, Biotechnol. Bioeng. 39 (1992), 176-185 have their application in organic solvents only. One has to distinguish between two possibilities of imprinting (cf. fig. 2). The first generates a polymer with reversible binding sites for the print molecule which were not present beforehand, the second modifies the stereo- and/or substrate selectivity of a polymer, e. g. an enzyme by the interaction of the print molecule with the active site. The possibility mentioned first resulted, under the use of bovine serum albumin as the polypeptide and L-malic acid as the printmolecule, in an imprint-polypeptide which was selective for L-malic acid in chromatographic studies (Dabulis and Klibanov, loc. cit.). The second possibility led in the case of hydrolases to extended, new catalytic properties of these enzymes. So, for example, the substrate selectivity of subtilisin (Russel and Klibanov, loc. cit.) and the stereo- and substrate selectivity of a-chymotrypsin (Stahl et al., loc. cit.) were altered directedly.

The polypeptide a-chymotrypsin for example is able to hydrolyze the L-enantiomer of N-acetyl-tryptophan ethyl ester (N-Ac-TrpEE) in an aqueous solvent system or to synthesize this ester from N-acetyl-L-tryptophan (N-Ac-Trp) and ethanol in an organic solvent system. The (D)- enantiomer of N-Ac-TrpEE can neither be hydrolyzed nor synthesized.

If the polypeptide a-chymotrypsin is yet imprinted (moulded) with the print molecule N-Ac-(D)-Trp it is then able to catalyze in an organic solvent the synthesis of N-Ac-(D)-TrpEE from N-Ac-(D)-Trp and ethanol in an organic solvent, because the conformation of the polypeptide was changed specifically by the print molecule (Stahl et al., loc. cit.). The conformational change is still reversible. This is the reason why the imprint-polypeptide can not be used for the hydrolysis of N-Ac-(D)-TrpEE in an aqueous solvent system. The new property gets lost upon contact of the imprint-polypeptide with aqueous solvent systems.

CA 022~9416 1999-01-04 Hence, the limitation of the method of imprinting described so far is the fact that imprint-polypeptides are labile and show and maintain their imprinted (moulded) new properties exclusively in an organic solvent system. The conformation of imprinted polypeptides is based on sensitive, non-covalent bonds. Upon contact with an aqueous solvent system the induced properties which result from imprinting (moulding) are no longer provable and get lost irreversibly, since the induced conformational change is energetically disfavoured in aqueous systems. If a covalent fixation and stabilization of the conformation of the imprint-polypeptides was possible, they could be used thereafter in an aqueous environment as well. The imprinted conformation would be fixed covalently and it is not lost unless the connected bonds are cleaved. Water molecules could not break off the imprinted conformation any more. Totally new perspectives concerning the use of imprint-polypeptides would arise, because a huge number of commercial and industrial relevant processes, respectively, take place in aqueous solvent systems. For example, many interesting compounds are either only or more soluble in aqueous systems and the catalytic activity of polypeptide catalysts is also higher in aqueous systems and therefore commercially useful at all.

So processes, which take place in an aqueous environment and which due to poor or no activity of the polypeptide catalysts (enzymes) versus a commercial useful substrate have not been used up to now, would lead by the fixation of the conformation of the imprint-polypeptide to a considerable increase in productivity (enlargement of the catalytic property). Existing processes with native polypeptides would receive a tremendous increase in efficiency by applying conformationally fixed and stabilized imprinted polypeptides. Conformationally fixed imprint-polypeptides, which ought to recognize the imprint and/or structurally similar compounds selectively in aqueous systems could be used as artificial antibodies for aqueous analysis purposes, as biosensors or in chromatography as affinity chromatography, especially in the immunoaffinity chromatography. According to the latest state of the art these mentioned possibilities of application are not available in aqueous systems so far.

CA 022~9416 1999-01-04 .

Object of the Invention Therefore the object of the invention is based on making conformationally fixed and stabilized, covalently crosslinked imprint-polypeptides available, which can be used in aqueous media as well. The new or extended catalytic and/or binding properties of the imprint-polypeptides are to be fixed and stabilized. Thus, the loss, i. e. the reversibility of the imprinted property(ies) of the polypeptides in aqueous media is not given any longer. The imprint-polypeptides are then for the first time be usable in catalytic, chromatographic and/or for analytical processes, which are conducted in aqueous medla .

For the solution of this object conformationally fixed and stabilized, covalently crosslinked imprint-polypeptides are provided, which are obtainable according to one embodiment by the following steps:

(A) covalently introduction of polymerizable, unsaturated bonds containing groups into a polypeptide;

(B) imprinting the resulting polypeptide from step (A) with a print molecule in an aqueous medium;

(C) precipitating the resulting polypeptide/print molecule mixture from step (B) by addition of a suitable additive for precipitation and/or Iyophilizing the polypeptide/print molecule mixture obtained from step (B); and (D) copolymerizing the resulting imprint-polypeptide from step (C) in an organicsolvent with a crosslinker, which is copolymerizable with the polymerizable, unsaturated bonds containing groups.

According to another embodiment the conformationally fixed and stabilized, covalently crosslinked imprint-polypeptides are obtainable by the following steps:

.. . . .

CA 022~9416 1999-01-04 (A) covalently introduction of polymerizable, unsaturated bonds containing groups into a polypeptide in an aqueous medium;

(B) imprinting the resulting polypeptide from step (A) with a print molecule in an aqueous medium; and (C) copolymerizing the polypeptide resulting from step (B) in an aqueous medium with a crosslinker, which is copolymerizable with the polymerizable, unsaturated bonds containing goups.

Further solutions of the object consist of carrying out step (B) before step (A) or step (A) and (B) at the same time.

Figure 3 is a schematic presentation of the process for the fixation and stabilization of polypeptides. The derivatization with olefinic groups is carried out prior to imprinting (P = polypeptide). Figure 4 is a schematic presentation of the process for the fixation and stabilization of polypeptides. The derivatization with olefinic groups is carried out after imprinting (P = polypeptide). Figure 5 shows a check of the "tailor-made"
property of imprinted polypeptides i) itaconic anhydride (ISA) modified/imprinted a-chymotrypsin (ISA/impr-Chy), ii) ISA modified/imprinted/crosslinked a-chymotrypsin (fixated/impr-Chy, adressed as VIP as well), iii) native/imprinted a-chymotrypsin (na/impr-Chy).

Description of the Invention The process of the present invention (cf. fig. 3 and 4) comprises the selective introduction of polymerizable unsaturated bonds containing groups into an if necessary possibly imprinted (moulded) polypeptide, for example a protein. Afteradjusting the desired polypeptide conformation by imprinting and precipitation or Iyophilization [step (C)] the process of the invention comprises a further step (D), in which the unsaturated bonds containing groups of the derivatized polypeptides are selectively covalently crosslinked by means of a copolymerization with a crosslinker, CA 022~9416 1999-01-04 e. g. a divinylized compound, which covalently fixes and stabilizes the desired conformation of the polypeptide. Thereafter the covalently crosslinked imprint-polypeptide is stable in both aqueous and organic solvent systems and the imprinted property, e. g. an altered catalytic and/or affinity property can be used for commercial applications (cf. fig. 3 and 4).

For the imprinting procedure polypeptides with e. g. alkyl-, aryl-, OH-, NH2-, SH-and/or COOH groups are suited, which interact with the print molecule on the onehand via non-covalent bonds such as ionic, hydrogen-bonding, hydrophobic interactions, van der Waals forces or metal chelat complexes and which on the other hand can be derivatized covalently before, during or after the imprinting takes place with polymerizable unsaturated bonds containing groups.

In a preferred embodiment polypeptides with OH-, NH2- and/or SH- groups are usedas starting materials. In a further preferred embodiment native proteins are used as polypeptides, preferably enzymes, in particular 13-glucosidase or a-chymotrypsin.

Preferred enzymes are chosen from oxidoreductases, such as D- and L-amino acid oxidases, alcohol dehydrogenase, glucose oxidase and formate dehydrogenase.
Further preferred enzymes are chosen from hydrolases, such as proteases, peptidases (e. g. rennin), amylases (e. g. a-amylase, 13-amylase, glucoamylase, 13-galactosidase), glycosidases, acylases, lipases and esterases.

Further preferred enzymes can be chosen from isomerases, such as glucose isomerase and amino acid racemases. As further preferred enzymes polymerases, such as DNA-polymerases, transferases, such as phosphotransferases or ligases, such as DNA-ligases, can be used.

The introduction of polymerizable unsaturated bonds containing groups is conducted in an aqueous medium. Preferably, the pH value of the medium is between 3 -12, preferably between approximately 5 - 8 and especially between approximately 6 - 8.
If necessary the aqueous medium can contain a buffer system. Preferably the buffer system is chosen from a potassium phosphate buffer, a citric phosphate buffer, Tris buffer (tris-(hydroxymethyl)-aminomethane) or a sodium phosphate buffer.

CA 022~9416 1999-0l-04 The process of the invention is preferably characterized in that the introduction of polymerizable unsaturated bonds containing groupsl preferably vinyl groups, into a polypeptide has to be carried out in a selective way. A portion of the polypeptide's functional groups (OH- ,NH2-, SH-, COOH- groups) that ought to interact with a print molecule during the imprinting process, ought not to be derivatized. This can, for example, be controlled through the molfraction of polypeptide/olefinic compound. The extent of derivatization is determined by the particular primary sequence of thepolypeptide, the amino acid composition, the temperature and the pH value. The optimum degree of derivatization for the maintenance of the imprinted property can be checked easily by comparing the non-derivatized imprint-polypeptide with the imprint-polypeptide of different derivatization levels with regard to the desired property(ies) (fig. 5). Preferable molfractions of polypeptide/olefinic compound are in the range between 1: 5 and 1: 300.

In a preferred embodiment of the process of the invention olefinic compounds or their mixtures are chosen from the group consisting of reactive analogs or derivativescorresponding to the general formula (I), R1\ R3 ~C--C<

(I) in which the residues R,, R2, R3 and R4 are independently from each other hydrogen atoms, carboxy residues, cyclic or linear, substituted or unsubstituted, saturated or unsaturated alkyl residues, alkoxy residues or carboxyalkyl residues, with preferable up to 10, more preferable with up to 6, especially 1 or 2 carbon atoms or substituted or unsubstituted aryl residues or carboxyaryl residues, on the condition that at least one of the residues R" R2, R3 and R4is independently from each other a carboxy, carboxyalkyl or carboxyaryl residue.

CA 022~9416 1999-01-04 Preferably 1 or 2, especially 2 of the residues R1, R2, R3 and R4 are independently from each other a carboxy, carboxyalkyl or carboxyaryl residue, especially carboxy or carboxyalkyl residues with in particular 1 or 2 carbon atoms. Preferably the residues R1, R2, R3 and R4, which are not a carboxy, carboxyalkyl or carboxyaryl residue, are independently from each other hydrogen atoms or alkyl residues, more preferably ehtyl or methyl residues, especially methyl residues.

If the residues R1, R2, R3 and R4 are substituted alkyl, carboxyalkyl, alkoxy, aryl or carboxyaryl residues, the substituents are preferably chosen from the group consisting of halogen atoms, nitro, amido, carboxy, ester and alkoxy residues with preferably up to 10, more preferably up to 5 and especially 1 or 2 carbon atoms.If the residues R1, R2, R3 and R4 are unsaturated alkyl, carboxyalkyl or alkoxy residues they contain preferably up to 5, more prefarably up to 3 and especially one unsaturated bond.

If the residues R1, R2, R3 and R4 are substituted or unsubstituted aryl residues or carboxyaryl residues, the aryl residues are preferably phenyl or naphthyl residues, where the substituents are preferably chosen from the foregoing group which was mentioned for the substituted alkyl, carboxyalkyl, alkoxy, aryl or carboxyaryl residues.
In a preferred embodiment compounds are chosen from the group consisting of itaconic, malic, citraconic, crotonic, methacrylic and acrylic acid.

Preferably olefinic, cyclic or non-cyclic anhydrides like itaconic, maleic, citraconic, crotonic, acrylic or methacrylic anhydride, or acid halides, especially acid chlorides of the mentionend olefinic compounds, are used for derivatization.

In a further preferred embodiment itaconic anhydride (ISA) is used as the olefinic compound in an aqueous medium, preferably in a pH range of approximately 3-12, which reacts selectively and covalently with OH-, NH2- and/or SH- groups (compare tab. 3). The number of ISA bonds is determined by the individual primary sequence of the polypeptide, the temperature and the pH-value. The optimum percentile of possible derivatization necessary for the maintenance of the imprinted property is checked. For that purpose, the non-derivatized imprint-polypeptide is compared with CA 022~9416 1999-01-04 the derivatized imprint-polypeptides having different derivatization levels with regard to the desired property(ies) (fig. 5).

In another preferred embodiment of the process of the invention polyols like polyethylene glycoles, sorbitol, saccharose, glucose and/or glycerine or salts like ammonium, alkali metal or alkaline earth metal sulfates or phosphates or halides like (NH4)2SO4, Na2SO4, MgSO4, Na3PO4, CaHPO4, (NH4)H2PO4, NH4CI, NaCI, KCI and CaCI2 are added in order to stabilize the desired conformation or to shield functionalities important for the imprinting during the derivatization process [step (A)].
Thus, this step of the process possesses a lot of degrees of freedom in order toobtain as a result a functionally active and derivatized imprint-polypeptide.

The process of the present invention is further characterized by the fact that the polypeptide is incubated in an aqueous medium with a print molecule before, during or after the covalent introduction of polymerizable, unsaturated bonds containing groups. The print molecule and the polypeptide form thereby a polypeptide/print molecule mixture in which the print molecule and the polypeptide interact via non-covalent forces. As a consequence the conformation of the polypeptide is altered, and therefore exhibits new and/or altered properties.

As the print molecule preferably a compound can be used, which has a high structural and/or chiral similarity towards the subsequent substrate or, which is identical with the subsequent substrate of the ready-made, in its conformation fixed and stabilized, covalently crosslinked polypeptide. In a preferred embodiment a-chymotrypsin is used as the polypeptide and N-acetyl-D-tryptophan, N-acetyl-D-tyrosine or N-acetyl-D-phenylalanine as the print molecule.

In another preferred embodiment bovine serum albumin is used as the polypeptide and L-malic acid as the print molecule, or D- or L- amino acid oxidase is used as the polypeptide and natural or non-natural D- or L- amino acids are used as the print molecules.

In addition, the process of the invention is characterized in that the polypeptide/print molecule mixture, which is dissolved in an aqueous medium, is precipitated. The CA 022~9416 1999-01-04 precipitation can be achieved by addition of additives, which are suitable for the precipitation of the polypeptide/print molecule mixture in an aqueous medium.

In a preferred embodiment watermiscible organic solvents are used as additives for the precipitation, ~referably acetone, methylethylketone, methanol, ethanoi, propanol and butanol, especially n-propanol or isopropanol. In another embodiment the polypeptide/print molecule mixture is Iyophilized. In another embodiment the polypeptide/print molecule mixture is precipitated as well as Iyophilized.

Via the polymerizable unsaturated bonds containing groups the imprintedconformation of the polypeptide is fixed covalently by a copolymerization procedure in a further step.

Preferably the copolymerization is initiated by UV irradiation, peroxides or radical starters (thermically or by UV irradiation). Preferably azobis compounds like a,a'-azobis-(2-methylpropionitrile) (AIBN) or 2,2'-azo-bis-(2,4-dimethyl)-valeric acid nitrile (ABDV) are used. Especially preferred are AIBN or ABDV as radical starters and the copolymerization is initiated by UV irradiation.

The process of the invention is characterized furthermore in that a crosslinker is used for the covalent fixation of the conformation of the derivatized imprint-polypeptides obtained after precipitation and/or Iyophilization.

In a preferred embodiment of the process of the invention unsaturated bonds containing compounds or mixtures thereof are used as crosslinkers, chosen from the group consisting of analogs or derivatives corresponding to the general formula (Il), H2C = C(Rl) - (R2)n - C(R3) = CH2 (Il) in which Rl and R3 are indepently from each other hydrogen atoms, cyclic or linear, unsaturated or saturated, substituted or unsubstituted alkyl, carboxy, carboxyalkyl or alkylether residues with preferably up to 10, especially 1 or 2 carbon atoms or substituted or unsubstituted aryl residues and in which the residue R2 is a cyclic or linear, saturated or unsaturated, substituted or unsubstituted alkyl- or alkylether CA 022~9416 1999-01-04 residue with preferably up to 10, especially 1 or 2 carbon atoms or an aryl residue and n has a value of preferably up to 10, especially 1 or 2.

Preferably the residues R~ and R3 are hydrogen atoms. In a further preferred embodiment the residues R1 and/or R3 can contain more than 10 carbon atoms. In afurther preferred embodiment the value of n is greater than 10. Special examples are olefinic polyethyleneglycols or olefinic derivatives of polyacrylamides.

If the residues R~, R2 and R3 are unsaturated and/or substituted residues, theirmeaning corresponds to the general formula (I). In addition to the mentioned substituents hydroxy and/or amino residues can be present.

Preferably the crosslinkers are chosen from the group consisting of divinylalkyl and divinylaryl compounds, especially divinylbenzene or 1,5-hexadiene. In a further preferred embodiment the unsaturated bonds containing crosslinkers or mixtures thereof are chosen from the group consisting of analogs or derivatives corresponding to the general formula (Ill), CH2 = C(R~ Cl-X-(R2)n-X-lCl-C(R3) = CH2 O O (111) in which the meaning of the residues R" R2 and R3 and n correspond to the above mentioned meaning for the residues of general formula (Il) and the residue X is an oxygen atom or a -NH group.

Preferably the crosslinkers are chosen from ethylene glycol dimethacrylate, N,N'-methylene-bis-acrylamide, diitaconic alkylamides and diitaconic arylamides. In apreferred embodiment the copolymerization is carried out in cyclohexane, toluene, acetone, alkanols, acetic acid ethyl ester, tetrahydrofuran, chloroform or mixtures thereof, especially cyclohexane, at which preferably, ethylene glycol dimethacrylate, divinylbenzene, diitaconic alkylamides or -arylamides, divinylalkyl or -aryl compounds are used as crosslinkers.

.

CA 022~9416 1999-01-04 In a preferred embodiment the derivatized imprint-polypeptide resulting from theprecipitation step (cf. fig. 3 and 4) is suspended in an organic solvent, one or several of the above mentioned crosslinkers are added, the radical starter, e. 9. azobiscompounds (a,a'-azobis-(2-methylpropionitrile) (AIBN) or 2,2'-azo-bis-(2,4-dimethyl)valeric nitrile (ABDV) are added and the copolymerization is initiated by UV
irradiation. The covalent fixation of the polypeptide conformation results selectively via the formerly introduced olefinic double bonds, the important free functionalities of the imprint-polypeptide are not blocked by this step. Thereby, the imprinted conformation is fixed covalently and can be used in an aqueous and an organic solvent system.

The conformationally fixed and stabilized, covalently crosslinked imprint-polypeptide is usable under a lot of conditions under which, without the selective introduction of residues and copolymerization thereof with crosslinkers, would have been unstable, e. 9. in an aqueous solvent system.

In another embodiment a polypeptide is derivatized by the covalent introduction of polymerizable, unsaturated bonds containing groups. The polypeptide can be chosen from the before mentioned group. The derivatization can be carried out as described before for step (A).

Thereafter the polypeptide is imprinted (moulded) with a print molecule in an aqueous medium. The print molecule can be chosen as mentioned before. Preferably a native enzyme is used as the polypeptide and the substrate of this enzyme is used as the print molecule. Then, the copolymerization is carried out in an aqueous solvent in the presence of a crosslinker, which is copolymerizable with the polymerizable unsaturated bonds containing groups. The copolymerization is carried out as described before for step (D). The crosslinkers are preferably chosen from the groups described before. The crosslinker is chosen in that way that it is soluble in the aqueous solvent. Preferably N, N'-methylene-bis-acrylamide is used as the crosslinker.

The invention is illustrated with the aid of the following examples.

CA 022~9416 1999-01-04 Example 1 Gentle introduction of olefinic groups into polypeptides A. Vinylization of a-chymotrypsin (EC 3.4.21.1) with itaconic anhydride a-Chymotrypsin is dissolved in 10 ml 0.05 M potassium phosphate buffer pH 7.8 with stirring. Subsequently, itaconic anhydride is added slowly in small portions at room temperature. The pH value of the reaction medium, which decreases upon addition of the anhydride is corrected with 5 M NaOH. After all of the anhydride has been added stirring is prolonged for 1 hour and the pH value is corrected again. In order to cut off low molecular compounds from the resulting reaction solution gelfiltration using PD
10 columns (Pharmacia) with 0.05 M potassium phosphate buffer pH 7.8 as the eluent is applied and finally the eluent is Iyophilized. The enzymatic activity of a-chymotrypsin activity is estimated following the hydrolysis of benzoyl-L-tyrosine ethyl ester in a photometrical test. The extent of modification of the functional groups is determined with 2,4,6-trinitrobenzenesulfonic acid (TNBS-Assay) (Habeeb A.F.S.A.(1966), Anal. Biochem. 14, 328-336). Tab. 1 shows the reaction set up, the individual extent of modification of the functionalized groups and the enzymatic activities of the a-chymotrypsin derivatives in relation to the native enzyme.

Table 1: Derivatization of a-chymotrypsin with itaconic anhydride a-Chymotrypsin Itaconicanhydride Residual activityof Modificationof activity used for the the derivative functional groups derivatization [nKat] [mg] [%] [%]
2765 1,25 38 24 2765 2,5 31 27 CA 022~9416 1999-01-04 B. Vinylization of the 13-glucosidase from almonds (EC 3.2.1.21 ) with itaconic anhydride The 13-glucosidase is dissolved in 10 ml 0.05 M citric acid phosphate buffer pH 6.0 with stirring. To stabilize the enzyme D(+)-Glucose can be added. Subsequently, itaconic anhydride is added in small portions at room temperature. The pH value of the reaction medium, which decreases upon each addition of the anhydride is corrected with 5 M NaOH. After all of the anhydride has been added stirring is prolonged for 1 hour and the pH value is checked again. In order to cut off low molecular compounds from the resulting reaction solution gelfiltration using PD 10 columns (Pharmacia) using 0.05 M citric acid phosphate buffer pH 6.0 as the eluent is applied and finally the eluent is Iyophilized. The enzymatic activity of 13-glucosidase is estimated following the hydrolysis of 4-nitrophenyl-13-D-glucopyranoside in aphotometrical test. The extent of modification of the functional groups is determined with 2,4,6-trinitrobenzenesulfonic acid (TNBS assay). Tab. 2 shows the reaction set up, the individual extent of modification of the functionalized groups and the enzymatic activity of the 13-glucosidase derivatives in relation to the native enzyme. In order to protect the polypeptide's active site glucose was added as additive. In the presence of 3 M glucose a residual activity of 99% was obtained after derivatization.

Table 2: Derivatization of 13-glucosidase with itaconic anhydride ~-GIucosidase Itaconic D(+)- Residual activity Modification of activity used for anhydride Glucose of the derivative functional groups the derivatization [ nKat ] [ mg ] [ M ] [ % ] [ % ]

CA 022~9416 1999-01-04 Example 2 Reactivity of itaconic anhydride versus the sidechains of polypeptides Various a-N-acetyl-L-aminoacids are dissolved in 5 ml 0.05 M citric phosphate buffer pH 7.5, itaconic anhydride is added with stirring and incubated for 1 h at ambient temperature. The TNBS assay is carried out with a sample of the a-N-acetyl aminoacid which was dissolved in the buffer and with a sample which results from the incubation of the a-N-acetyl amino acid with itaconic anhydride.

It can be stated that itaconic anhydride ist able to react with the amino group of a-N-acetyl-L-lysine and a-N-acetyl-L-glutamine, the sulfhydryl group of a-N-acetyl-L-cysteine and the hydroxy group of a-N-acetyl-L-tyrosine and a-N-acetyl-L-serine (tab.
3). The reactivities of itaconic anhydride versus the amino acid side chains were ser>tyr>glu>lys>cys.

Table 3: Investigations concerning the reactivity of itaconic anhydridea N-acetyl-L-amino acidFunctional residueExtent of modification~
[%]
a-N-acetyl-L-serine -OH 96 a-N-acetyl-L-tyrosine -OH 89 a-N-acetyl-L-glutamine -C(O)NH2 81 a-N-acetyl-L-lysine -CH2NH2 68 ~-N-acetyl-L-cysteine -CH2SH 15 a-N-acetyl-L-asparagine -C(O)NH2 ~
a-N-acetyl-L-arginine -C(NH2)NH2 ~

aadded in 1 Of old molar excess bafter 1 h of reaction time; determined by the TNBS assay.

Concerning the stability of the above mentioned bonds it can be stated that for example primary aminogroups lead to amide bonds, which are stable from pH 1-12 CA 022~9416 1999-01-04 and at temperatures of up to 70~C (R. Kolle, 1995, dissertation, TU Braunschweig, Germany, 53-59).

Example 3 Preparation of a fixed imprint-polypeptide Itaconic anhydride derivatized a-chymotrypsin (30 mg) from example 1 (extent of modification = 70%) are dissolved in 1 ml of 0.01 M potassium phosphate buffer pH
7.8. The buffer contains N-Ac-(D)-Trp in a concentration of 0.02 M. The solution is cooled to 0~C and 4 ml of 1-propanol, which was cooled to -20~C, is added. The resulting precipitate is centrifuged, washed with 10 ml 1-propanol and finally Iyophilized. In 0.5 ml cyclohexane 340 mg ethylene glycol dimethacrylate and 4 mg 2,2 '-azobis-(2-methylpropionitrile) (AIBN) are dissolved. Into this solution 5 mg of the Iyophilisate is suspended. The copolymerization is initiated by UV irradiation. The copolymerization is carried out for 4 hours. The covalently crosslinked and imprinted a-chymotrypsin (crosslinked imprint-polypeptide; VIP) is washed with cyclohexaneand finally Iyophilized (cf. fig. 3).

In order to check whether the derivatized, imprinted a-chymotrypsin (ISA/impr-Chy) and the crosslinked, imprinted a-chymotrypsin (VIP), respectively, possessed thesame imprinted properties in the organic solvent like the native imprinted a-chymotrypsin (na/impr-Chy) all three imprint-polypeptides were tested with regard to the synthesis of N-Ac-(D)-TrpEE in cyclohexane (fig. 5; analysis by HPLC, cf. Stahl et al. 1991). It was evident that the ISA/impr-Chy and VIP had better activities in the first 100 h than the na/impr-Chy. The kinetic effects after a conversion time of 100 h can be attributed to the impact of released water during the enzymatic condensation reaction in the organic solvent.

Example 4 Application of the crosslinked imprint-polypeptide (VIP) in an aqueous medium The VIP is suspended in phosphate buffer (10 mM) pH 7.8 and preincubated with the help of a thermomixer at 27~C for 10 min. The reaction is started upon addition of the CA 022~9416 1999-01-04 substrate N-acetyl-D- tryptophan ethyl ester (10 mM). The VIP demonstrated a specific activity of the D-ester hydrolysis of 0.04 ~mol/(ming). The native non-imprinted, the non-crosslinked imprinted and the non-imprinted crosslinked a-chymotrypsin had no D-ester hydrolysis activity.

Example 5 Application of the crosslinked imprint-polypeptide (VIP) in an organic solvent after preincubation (1 h) in an aqueous solvent system A. The VIP was incubated in a phosphate buffer (10 mM), pH 7.8, for 1 h at 25~C.
After Iyophilization of the sample, the VIP was suspended in cyclohexane. After addition of N-Ac-(D)-Trp (10 mM) and 20% (v/v) ethanol the synthesis of N-acetyl-D-tryptophan ethyl ester was started in a thermomixer at 25~C. The VIP showed a specific D-ester hydrolysis activity of 0.02 ,umol/(min g). The imprinted property was consequently not lost in the aqueous solvent system.

B. Like described in example 5A., instead of N-Ac-(D)-Trp N-Ac-(D)-tyrosine was used.

C. Like described in example 5A., instead of N-Ac-(D)-Trp N-Ac-(D)-phenylala-nine was used.

Example 6 Preparation of a fixed imprint-polypeptide for selective adsorption Bovine serum albumin (50 mg) are dissolved in 10 ml potassium phosphate buffer (0,05 M) pH 5.5. Subsequently, itaconic anhydride is added slowly in small portions at room temperature. The pH value of the reaction medium is corrected by the addition of 5 M NaOH. After all of the anhydride is added stirring is prolonged for 1 hour and the pH value is corrected again. Low molecular compounds are cut off from the resulting reaction solution using PD 10 gelfiltration columns (Pharmacia) with 0.05 M potassium phosphate buffer pH 5.5 as the eluent and finally Iyophilized. The CA 022~9416 1999-01-04 extent of modification of the functional groups is measured with 2,4,6-trinitrobenzenesulfonic acid (TNBS assay).

Itaconic anhydride derivatized bovine serum albumin (30 mg) (extent of modification = 85%) are dissolved in 4 ml H20 dist. The aqueous solution contains L-malic acid in a concentration of 0.5 M. The solution is cooled to 0~C at a pH value of 5,5 and 4 ml 1-propanol, which was cooled to -20~C, is added. The resulting precipitate is centrifuged, washed with 10 ml 1-propanol and finally Iyophilized.

340 mg ethylene glycol dimethacrylate and 4 mg 2,2'-azobis-(2-methylpropionitrile) (AIBN) are dissolved in 0.5 ml cyclohexane. Into this solution 10 mg of the Iyophilisate are suspended. The copolymerization is initiated by UV irradiation. The copolymerization is carried out for 4 hours. The covalently crosslinked and imprinted bovine serum albumin is washed with cyclohexane and Iyophilized.

As reported by Dabulis and Klibanov, Biotechnol. Bioeng. 39 (1991), 176-185, theextent of the affinity of imprinted bovine serum albumin versus L-malic acid wasinvestigated. Thereby it was shown that as well the imprinted as the crosslinkedimprinted bovine serum albumin had binding affinity towards L-malic acid in ethyl acetate. In the aqueous medium only the crosslinked imprinted bovine serum albumin was able to adsorb L-malic acid selectively.

Example 7 Preparation of a native, fixed polypeptide 50 mg of the derivatized 13-glucosidase (extent of modification = 98%) (cf. tab. 2) and 250 mg N, N' -methylene-bis-diacrylamide are dissolved in 29 ml 0.05 M citric phosphate buffer pH 6Ø The copolymerization is initiated by the addition of 0.5 ml of an ammonium peroxodisulfate solution (5% w/v) and 0.5 ml of a 3-dimethylaminopropionitrile solution (5% v/v). After a reaction time of 5 hours the copolymer is washed with 0.5 liter 0.5 M NaOH and afterwards with 2 liter H20 dist.
in a folded filter until the washing solution is salt free. The copolymer is taken up in 0.05 M citric phosphate buffer pH 6.0 and finally Iyophilized.

CA 022~9416 1999-01-04 The enormous stabilization of the crosslinked polypeptide in comparison to the native not crosslinked polypeptide is obvious from tab. 4, in which the half-lifes of the polypeptide are presented at different temperatures.

Table 4: Half lifes of the catalytic active co,lror",ations of the free and cross-linked ~-glucosidase Bio-Catalyst t1~2 (days) 30~C 37~C 56~C
ISA-acrylamide crosslinked polypeptide 51,6 8,7 2,4 Native enzyme 8,7 1,4 0,25 Literature Dabulis, K. and Klibanov, A. (1992), Biotechnol. Bioeng. 39, 176-185.
Fritz, H., Neudecker, M., Schult, H. and Werle, E. (1966), Angew. Chem. 78, 775.Goldstein, L. (1970), MethodsEnzymol. 19, 935-962.
Habeeb, A. F. S. A. (1966), Anal. Biochem. 14, 328-336.
Kolle, R. (1995), Dissertation, TU Braunschweig, Germany, 53-59.
Mosbach, K. and Ramstrom, O. (1996), Bio/Techno/ogy 14, 163-170.
Russel, A. J. and Klibanow, A. M. (1988), J. Biol. Chem. 263, 11624-11626.
Stahl, M., Jeppson-Wistrand, U., Mansson, M.-O. and Mosbach, K. (1991), J. Am.
Chem. Soc. 113, 9366-9368.
Stahl, M., Mansson, M.-O. and Mosbach, K. (1990). Biotechnol. Lett. 12, 161-166.

Claims (19)

Claims

1. Process for the manufacture of covalently crosslinked imprint-polypeptides having a fixed and stabilized conformation, which are stable in both aqueous and organic solvent systems, comprising the following steps:

A) covalently introduction of polymerizable, unsaturated bonds containing groups in a polypeptide;

B) imprinting the polypeptide resulting from step A) with a print molecule in an aqueous medium;

C) precipitating the polypeptide/print molecule mixture resulting from step B) by addition of an additive which is suited for the precipitation and/or lyophilization of the polypeptide/print molecule mixture resulting from step B; and D) copolymerizing the imprint-polypeptide resulting from step C) in an organic solvent with a crosslinker, which can be copolymerized with the polymerizable unsaturated bonds containing groups.

2. Process according to claim 1 characterized by carrying out step B) prior to step A).

3. Process according to claim 1 characterized by carrying out step A) and step B) at the same time.

4. Process according to any one of the above mentioned claims characterized by using a polypeptide with OH- and/or NH2- and/or SH- groups, which are derivatized partially or completely with the polymerizable, unsaturated bonds containing compounds under formation of covalent bonds.

5. Process according to any one of the above mentioned claims characterized by using a native protein, especially an enzyme, as the polypeptide.

6. Process according to any one of the above mentioned claims characterized by using unsaturated bonds containing compounds or mixtures thereof from step A) chosen from the group consisting of reactive analogs or derivatives according to the general formula (I), in which the residues R1, R2, R3 and R4 are independently from each other hydrogen atoms, carboxy residues, cyclic or linear, substituted or unsubstituted, saturated or unsaturated alkyl residues, alkoxy residues or carboxyalkyl residues with preferably up to 10, more preferably up to 6, especially 1 or 2 carbon atoms or substituted or unsubstituted aryl residues or carboxyaryl residues with the proviso that at least one of the residues R1, R2, R3 and R4 is independently from each other a carboxy, carboxyalkyl or carboxyaryl residue.

7. Process according to claim 6, characterized by the unsaturated bonds containing compounds used in step A) chosen from the group consisting of itaconic, maleic, citraconic, crotonic, methacrylic and acrylic acid as well as their anhydrides and acid halides.

8. Process according to any one of the above mentioned claims, characterized by the supplementary addition of polyols and/or salts to step (A).

9. Process according to any one of the above mentioned claims, characterized by a crosslinker which is used for the copolymerization in step (D) chosen from a group consisting of analogs or derivatives corresponding to the general formula (II), in which the residues R1 and R3 are independently from each other hydrogen atoms, cyclic or linear, unsaturated or saturated, substituted or unsubstituted alkyl, carboxy, carboxyalkyl or alkyl ether residues with preferably up to 10, especially 1 or 2 carbon atoms or substituted or unsubstituted aryl residues, and the residue R2 is a cyclic or linear, saturated or unsaturated, substituted or unsubstituted alkyl or alkyl ether residue with preferably up to 10, especially 1 or 2 carbon atoms or an aryl residue and n has a value of preferably up to 10 and especially 1 or 2.

10. Process according to claim 9 characterized by a crosslinker which is used for the copolymerization in step D) chosen from the group consisting of divinylalkyl- and divinylaryl compounds.

11. Process according to claims 1 to 8 characterized by a crosslinker which is used for the copolymerization in step D) is selected from the group consisting of analogs or derivatives according to the general formula (III), in which the residues R1, R2, R3 and n have the meaning mentioned in claim 9 for residues according to the general formula (II) and the residue X is an oxygen atom or the -NH group.

12. Process according to claim 11 characterized by a crosslinker, which is used for the copolymerization in step D), is selected from the group consisting of ethylene glycol dimethacrylate, N,N'-methylene-bis-diacrylamide, diitaconic alkylamides and diitaconic arylamides.

13. Process for the manufacture of a covalently crosslinked imprint-polypeptide having a fixed and stabilized conformation, which is stable in both aqueous and organic solvent systems, comprising the following steps:

(A) covalently introduction of polymerizable, unsaturated bonds carrying groups into a polypeptide in an aqueous medium;

(B) imprinting the polypeptide resulting from step (A) with a print molecule in an aqueous medium; and (C) copolymerizing the resulting polypeptide from step (B) in an aqueous medium with a crosslinker, which is copolymerizable with the polymerizable unsaturated bonds containing groups.

14. Process according to claim 13, characterized by carrying out step (B) prior to step (A).

15. Process according to claim 13, characterized by carrying step (A) and (B) at the same time.

16. A covalently crosslinked imprint-polypeptide having a fixed and stabilized conformation, which is stable in both aqueous and in organic solvent systems, obtainable by a procedure according to any one of the mentioned claims.

17. Use of a polypeptide according to claim 16 for catalytic, chromatographic and/or analytic processes, which are carried out in aqueous or organic solvent systems.

18. Use of a polypeptide according to claim 16 for the catalytic synthesis, chromatography and analysis of chiral compounds.

19. Use of a polypeptide according to claim 16 in the biosensor technique for the specific recognition of molecules.