EP0912605A1

EP0912605A1 - Imprint polypeptides covalently cross-linked and having a fixed and stabilised arrangement, process for the preparation and use thereof

Info

Publication number: EP0912605A1
Application number: EP97931724A
Authority: EP
Inventors: Lutz Fischer; Fabian Peissker
Original assignee: Individual
Current assignee: Individual
Priority date: 1996-07-05
Filing date: 1997-07-07
Publication date: 1999-05-06
Also published as: CA2259416A1; WO1998001466A1; DE19627162C1; JP2000500777A

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

In their conformation, fixed and stabilized, covalently cross-linked imprint polypeptides, processes for their production and their use

Field of application of the invention

The invention relates in its conformation to fixed and stabilized, covalently cross-linked imprint polypeptides (embossed polypeptides), e.g. Proteins that can be used in the catalytic synthesis, chromatography and analysis of chiral compounds as well as in biosensor technology for the specific recognition of molecules, processes for their production and their use.

State of the art

Polymers with the ability to selectively recognize molecules are of the greatest industrial importance in biotechnology and chemistry. These include the enzymes, antibodies and receptors that belong to the class of proteins. Because of their three-dimensional molecular structure (conformation), these polymers have complementary regions for certain molecules (substrates, antigens, hormones, etc.), which are referred to as binding sites. Since native polymers often bind commercially interesting molecules inadequately or not at all or the conformation necessary for binding is not sufficiently stable under the conditions of use, methods have been developed to produce tailor-made polymers which have the desired complementarity (K. Mosbach and O. Ramström, Biotechnology, Vol. 14 (1996), 163-170).

Goldstein, Methods Enzymol. 19 (1970), 935-962, and Fritz et al., Angew. Chem. 78, 1966, 775 describe a method for immobilizing proteins using cyclic anhydrides for the covalent coupling. The process consists of first copolymerizing an anhydride into a carrier matrix with other monomers and then coupling the protein to the finished carrier.

Mosbach and Ramström (Joe. Cit.) Describe that the production of customized polymers with the desired complementarity is carried out in two variants.

Both variants are based on the principle that a specially selected molecule, hereinafter referred to as the print molecule (embossing molecule), acts as a shaping agent (embosser) and for the desired complementarity in the polymer and thus new property is responsible. Figures 1 and 2 schematically explain these two variants.

Variant 1 for the production of customized (embossed) polymers is carried out in such a way that a copolymerization of selected monomers is carried out in an organic solvent in the presence of the print molecule (see FIG. 1). First, the print molecule a) is dissolved directly in an organic solvent system together with a monomer (non-covalent method) or b) after derivatization with a monomer (covalent method), then a crosslinker and a radical initiator are added and the polymerization is started. The print molecule must then be extracted from the resulting polymer under suitable conditions so that the new binding sites generated in the polymer are released. The polymer produced in this way has selective recognition sites for the print molecule or for structurally similar molecules (see FIG. 1). The numbers mean functional groups.

It is essential for a) and b) that both the solution of the starting substances, which is composed of certain concentrations of print molecule, monomer (s) and radical initiator, as well as the subsequently tailored (embossed) property produced in the polymer, so far exclusively on the Use of organic solvent systems is instructed.

In variant 2, to produce a tailor-made (embossed) polymer, no polymerization is carried out in the presence of a print molecule, but an already finished native polymer, e.g. a polypeptide or a protein, is forced to change the conformation under certain conditions and by the presence of the print molecule (see FIG. 2, in which a) means the modification of the catalytic property and b) the generation of affinity (recognition).

For this purpose, the polymer is first dissolved together with the print molecule in an aqueous solvent system and then precipitated by adding additives, for example organic solvents. The print molecule and the polymer interact with one another via non-covalent interactions in such a way that the conformation of the polymer is changed and the polymer thereby has a new property which is inherently predetermined by the print molecule. The process of variant 2 described up to this point is hereinafter referred to as I printing (embossing), the resulting polymer as an imprint polymer (embossed polymer); eg imprint polypeptide.

However, the new property of the imprint polymer is reversible. It only remains as long as it is used under conditions which prevent the reformation to the originally thermodynamically more favorable conformation. Therefore, the polymers obtained by variant 2, which by Mosbach et al. loc. cit., Stähl et al., Biotechnol. Lett. 12: 161-166 (1990); Stähl et al., J. Am. Chem. Soc. 113 (1991), 9366-9368 and Klibanov et al., J. Biol. Chem. 263 (1988), 11624-11626 and Dabulis and Klibanow, Biotechnol. Bioeng. 39 (1992), 176-185, can only be used in organic solvents. There are two options for imprinting (see Fig. 2). One creates reversible binding sites for the print molecule that are not previously present on the polymer, the second modifies the stereo and / or substrate selectivity of a polymer, e.g. of an enzyme through the interaction of the print molecule with the active center. The first possibility resulted in the use of bovine serum albumin as a polypeptide and L-malic acid as a print molecule, an imprint polypeptide, which was selective in chromatographic studies for the print molecule L-malic acid (Dabulis and Klibanow, loc. Cit). The second possibility led to extended, new catalytic properties of these enzymes in hydrolases. For example, the substrate selectivity of subtilisin (Rüssel and Klibanow, loc. cit.) and the stereo and substrate selectivity of α-chymotrypsin (Stähl et al., loc. cit.) were specifically changed.

The polypeptide α-chymotrypsin, for example, has the property of hydrolyzing the (Z -) enantiomer of the N-acetyl-tryptophanethyl ester (N-Ac-TrpEE) in an aqueous solvent system or in an organic solvent system composed of N-acetyl-L-tryptophan (N-Ac-Trp) and ethanol. The () enantiomer of N-Ac-TrpEE cannot be hydrolyzed or synthesized.

However, if this polypeptide α-chymotrypsin is imprinted with the print molecule N-Ac- (£>) - Trp, it can then be synthesized in organic solvent from N-Ac- (Z)) - TrpEE from N-Ac- ( -D) -Trp and ethanol catalyze, since the conformation of the polypeptide was specifically changed by the print molecule (Stähl et al., Loc. Cit.). However, the change in conformation is reversible. Therefore, the imprint polypeptide cannot be used in the aqueous solvent system for the hydrolysis of the N-Ac - (- D) -TrpEE. The new property is lost when the imprint polypeptide comes into contact with aqueous solvent systems.

A limitation of the imprinting method carried out so far is that the imprint polypeptides are labile and have or retain their imprinted (embossed) new properties exclusively in the organic solvent system. The conformation of the imprint polypeptides is based on sensitive, non-covalent bonds. As soon as they come into contact with aqueous solvent systems, the properties induced by imprinting are no longer detectable and irreversibly lost, since the induced change in conformation in aqueous systems is energetically unfavorable. If covalent fixation and stabilization of the conformation of the imprint polypeptides were possible, they could then also be used in an aqueous environment. The imprinted conformation would be fixed covalently and would not be lost without breaking the bonded bonds. Water molecules could no longer cancel the imprinted conformation. There would be completely new perspectives on the use of imprint polypeptides, since a large number of commercially or industrially relevant processes take place in aqueous solvent systems. For example, many interesting compounds are only or better soluble in aqueous systems, and the catalytic activity of polypeptide catalysts is also higher in aqueous systems and can therefore only be used commercially.

For example, processes taking place in an aqueous environment, which have so far not been used due to insufficient or no activity of the available polypeptide catalysts (enzymes) compared to a commercially usable substrate, could lead to a considerable increase in productivity by fixing the conformation of the imprint polypeptide (Extension of the catalytic property). Existing processes with native polypeptides could experience an enormous increase in efficiency through the use of imprint polypeptides fixed and stabilized in their conformation. Conformationally fixed imprint polypeptides, which are to selectively recognize the print molecule and / or its structure-like compounds in aqueous systems, could be used as artificial antibodies in aqueous analysis, sensor technology or chromatography, such as affinity chromatography, especially in immunoaffinity chromatography. According to the current state of the art, these possible uses have not yet been possible in aqueous systems. Aim of the invention

The invention is therefore based on the object of making available, in its conformation, fixed and stabilized, covalently cross-linked imprint polypeptides which can also be used in aqueous media. The new or expanded catalysis and / or binding properties of the imprint polypeptides should be fixed and stabilized. This is the loss, i.e. the reversibility, the imprinted properties) of the polypeptides no longer exist in aqueous media. The imprint polypeptides can then be used for the first time in catalytic, chromatographic and / or analytical processes that are carried out in aqueous media.

To solve this problem, their conformation provides fixed and stabilized, covalently cross-linked imprint polypeptides, which according to one embodiment can be obtained by the following steps:

(A) covalently introducing polymerizable groups containing unsaturated bonds into a polypeptide;

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

(C) Precipitation of the polypeptide / print molecule mixture obtained in step (B) by adding an additive suitable for precipitation and / or

Freeze-drying the polypeptide / print molecule mixture obtained in step (B); and

(D) copolymerizing the imprint polypeptide obtained in step (C) in an organic solvent with a crosslinker which is copolymerizable with the polymerizable groups containing unsaturated bonds.

According to another embodiment, the covalently cross-linked imprint polypeptides which are fixed and stabilized in their conformation can be obtained by the following steps: (A) covalently introducing polymerizable groups containing unsaturated bonds into a polypeptide in an aqueous medium;

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

(C) copolymerization of the polypeptide obtained in step (B) in an aqueous medium with a crosslinker which is copolymerizable with the polymerizable groups containing unsaturated bonds.

Other solutions to the problem are to carry out step (B) before step (A) or steps (A) and (B) simultaneously.

Figure 3 is a schematic representation of the method for fixing and stabilizing polypeptides. The derivatization with olefinic groups takes place before imprinting (P =

Polypeptide). Figure 4 is a schematic representation of the method for fixing and

Stabilization of polypeptides. The derivatization with olefinic groups takes place after imprinting (P = polypeptide). FIG. 5 shows a review of the “tailor-made”

Property of imprinted polypeptides i) itaconic anhydride (ISA) -modified / imprinted α-chymotrypsin (ISA / impr-Chy), ii) ISA-modified / imprinted / cross-linked α-chymotrypsin (fixed / impr-Chy, also called VIP), iii) native / imprinted α-chymotrypsin (na / impr-Chy).

Description of the invention

The method according to the invention (see FIGS. 3 and 4) comprises the selective introduction of polymerizable groups containing unsaturated bonds into an optionally imprinted (embossed) polypeptide, for example a protein. After the desired polypeptide conformation has been set by the imprinting and the precipitation or freeze-drying (step (C)), the process according to the invention comprises the further step (D) that selectively on the groups of the derivatized polypeptides containing unsaturated bonds a covalent crosslinking by copolymerization with a crosslinker, For example, a divinylated compound takes place, which fixes and stabilizes the desired polypeptide conformation covalently. The covalently cross-linked imprint polypeptide is then both in stable in aqueous as well as in organic solvent systems and the imprinted property, for example a changed catalysis and / or affinity property, can be used for commercial applications (see FIGS. 3 and 4).

For imprinting, polypeptides with, for example, alkyl, aryl, OH, NH ₂ , SH, and / or COOH groups are suitable, which, on the one hand, are hydrophobic with the print molecule via non-covalent bonds, for example ion bonds, hydrogen bonds Interactions, van der Waals forces, metal chelate complexes, interact and, secondly, before, during or after imprinting can be derivatized covalently with groups that contain polymerizable, unsaturated bonds.

In a preferred embodiment, polypeptides with OH, NH ₂ and / or SH groups are used as starting compounds. In a further preferred embodiment, native proteins are used as polypeptides, preferably enzymes, in particular β-glucosidase or α-chymotrypsin.

Preferred enzymes are selected from oxido reductases such as D- and L-amino acid oxidases, alcohol dehydrogenase, glucose oxidase and formate dehydrogenase. Further preferred enzymes are selected from hydrolases, such as proteases, peptidases (e.g. Rennin), amylases (e.g. α-amylase, β-amylase, glucoamylase, β-galactosidase), glycosidases, acyiases, lipases and esterases.

Furthermore, preferred enzymes can be selected from isomerases such as glucose isomerase and amino acid racemases. Polymerases such as DNA polymerases, transferases such as phosphotransferases or ligases such as DNA ligases can be used as further preferred enzymes.

The introduction of the polymerizable groups containing unsaturated bonds is carried out in an aqueous medium. The pH of the aqueous medium is preferably about 3 to 12, preferably about 5 to 8 and in particular about 6 to 8. If appropriate, the aqueous medium can contain a buffer system. The buffer system is preferably selected from a potassium phosphate buffer, a citric acid phosphate buffer, Tris buffer (tris (hydroxymethyl) aminomethane) or a sodium phosphate buffer. The process according to the invention is preferably characterized in that the introduction of the polymerizable groups containing unsaturated bonds, preferably vinyl groups, has to be carried out in a polypeptide in a selective manner. Some of the functional groups of the polypeptides (OH, NH ₂ , SH, COOH groups) which are to interact with a print molecule in the aqueous solvent system during imprinting should not be derivatized. This can be controlled, for example, by the molar ratio of polypeptide / olefinic compound. The degree of derivatization is determined by the respective primary sequence of the polypeptide, the amino acid composition, the temperature and the pH. The optimal degree of derivatization for maintaining the imprinted property can easily be checked by comparing the underivatized imprint polypeptide with the differently derivatized imprint polypeptides with regard to the desired property (s) (FIG. 5). Preferred polypeptide / olefinic compound molar ratios range from about 1: 5 to 1: 300.

In a preferred embodiment of the process according to the invention, olefinic compounds or mixtures thereof are selected from the group consisting of reactive analogs or derivatives of the general formula (I)

R. R ₃

C = C (I)

R ₂ R ₄

used, the radicals R ,, R ₂ , R ₃ and R ₄ independently of one another hydrogen atoms, carboxyl radicals, cyclic or linear, substituted or unsubstituted, saturated or unsaturated alkyl radicals, alkoxy radicals or carboxyalkyl radicals with preferably up to 10, more preferably up to 6, in particular 1 or 2 carbon atoms or substituted or unsubstituted aryl radicals or carboxyaryl radicals, with the proviso that at least one of the radicals R ₁ , R ₂ , R ₃ and R _{4 is} independently a carboxyl, carboxyalkyl or carboxyaryl radical.

Preferably 1 or 2, in particular 2, of the radicals RR ₂ , R ₃ and R _{4 are,} independently of one another, a carboxyl, carboxyalkyl or carboxyaryl radical. especially carboxyl or Carboxyalkyl radicals with in particular 1 or 2 carbon atoms. The radicals RR ₂ , R ₃ or R ,, which are not a carboxyl, carboxyalkyl or carboxyaryl radical are preferably, independently of one another, hydrogen atoms or alkyl radicals, more preferably ethyl or methyl radicals, in particular methyl radicals.

If the residues R _1? R ₂ , R ₃ and R are substituted alkyl, carboxyalkyl, alkoxy, aryl or carboxyaryl radicals, the substituents are preferably selected from the group consisting of halogen atoms, nitro, amido, carboxyl, ester and alkoxy radicals preferably up to 10, more preferably up to 5 and in particular 1 or 2 carbon atoms. If the radicals RR ₂ , R ₃ and R _{4 are} unsaturated alkyl, carboxyalkyl or alkoxy radicals, they preferably contain up to 5, more preferably up to 3 and in particular an unsaturated bond.

If the radicals R ,, R, R ₃ and R are substituted or unsubstituted aryl radicals or carboxyaryl radicals, the aryl radicals are preferably phenyl or naphthyl radicals, the substituents preferably being selected from those for substituted alkyl, carboxyalkyl, alkoxy, aryl - Or carboxyaryl groups called. In a preferred embodiment, compounds selected from the group consisting of itaconic, maleic, citracon, croton, methacrylic and acrylic acid are used.

Preferably olefinic cyclic or non-cyclic anhydrides, such as itacon, maleic, citraconic or crotonic, acrylic or methacrylic anhydride, or the acid halides, in particular the acid chlorides of the olefinic compounds mentioned, are used for the derivatization.

In a further preferred embodiment, itaconic anhydride (ISA) is used as the olefinic compound in an aqueous medium, preferably in the pH range from about 3-12, which binds selectively and covalently to OH, NH ₂ and / or SH groups (cf. Tab. 3). The number of ISA bonds is determined by the individual primary sequence of the polypeptide, the temperature and the pH. The optimal percentage of possible derivatization for maintaining the imprinted property is checked. For this purpose, the non-derivatized imprint polypeptide is compared with the imprint polypeptides derivatized to different degrees in terms of the desired property (s) (FIG. 5). In a further preferred embodiment of the method according to the invention, polyols, such as polyethylene glycols, sorbitol, sucrose, glucose and / or glycerol or salts, are added to stabilize the desired conformation or to shield the functionalities important for imprinting during the derivatization process (step (A)) , such as ammonium, alkali or alkaline earth metal sulfates, phosphates or halides, such as (NH ₄ ) ₂ SO ₄ , Na ₂ SO ₄ , MgSO ₄ , Na ₃ PO ₄ , CaHP0 ₄ , (NH ₄ ) H ₂ PO ₄ , NH ₄ C1, NaCI, KC1 and CaCl ₂ . This process step thus has many degrees of freedom in order to obtain a functionally active and derivatized imprint polypeptide as a result.

The process according to the invention is also characterized in that the polypeptide is incubated with a print molecule in an aqueous medium before, during or after the covalent introduction of polymerizable groups containing unsaturated bonds. The print molecule and the polypeptide form a polypeptide / print molecule mixture, the print molecule and the polypeptide interacting via non-covalent interactions. This changes the conformation of the polypeptide, which thus has new and / or changed properties.

A compound which has a high structural and / or chiral similarity to the subsequent substrate of the finished, covalently cross-linked imprint polypeptide which is fixed and stabilized in its conformation or which is identical to the subsequent substrate can preferably be used as the print molecule.

In a preferred embodiment, α-chymotrypsin is used as the polypeptide and N-Ac- (Z) -ryptophan, N-Ac- (D) -tyrosine or N-Ac- (D) -phenylalanine is used as the print molecule. In a further preferred embodiment, bovine serum albumin is used as the polypeptide and L-malic acid is used 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 molecule.

In addition, the inventive method is characterized in that the polypeptide / print molecule mixture dissolved in an aqueous medium is precipitated. Precipitation can be carried out by adding additives which are suitable for precipitating the polypeptide / print molecule mixture in an aqueous medium. In a preferred embodiment, water-miscible organic solvents are used as additives for the precipitation, preferably acetone, methyl ethyl ketone, methanol, ethanol, propanol and butanol, in particular n-propanol or isopropanol. In a further embodiment, the polypeptide / print molecule mixture is freeze-dried. In a further preferred embodiment, the polypeptide / print molecule mixture is both precipitated and freeze-dried.

The imprinted conformation of the polypeptide is covalently fixed in a further step by copolymerization via the polymerizable groups containing unsaturated bonds.

The copolymerization is preferably initiated by UV radiation, peroxides or radical initiators (thermal or by UV radiation). Azobis compounds such as α, α'-azo-isobutyronitrile (AIBN) or 2,2'-azo-bis- (2,4-dimethyl) valeric acid nitrile (ABDV) are preferably used as radical initiators. AIBN or ABDV are particularly preferably used as radical initiators and the copolymerization is initiated by means of UV radiation.

The process according to the invention is further characterized in that a crosslinker is used for the covalent fixation of the conformation of the derivatized imprint polypeptides obtained after the precipitation and / or freeze-drying.

In a preferred embodiment of the method according to the invention,

Crosslinking compounds containing unsaturated bonds or mixtures thereof, selected from the group consisting of analogs or derivatives of the general formula

(II)

H ₂ C = C (R,) - (R ₂ ) _n -C (R ₃ ) = CH ₂ (II)

used, the residues R | and R _{3 are,} independently of one another, hydrogen atoms, cyclic or linear, unsaturated or saturated, substituted or unsubstituted alkyl, carboxyl, carboxyalkyl or alkyl ether radicals having preferably up to 10, in particular 1 or 2, carbon atoms or substituted or unsubstituted aryl radicals and the radical R ₂ a cyclic or linear, saturated or unsaturated, substituted or unsubstituted alkyl or alkyl ether radical with preferably up to 10, in particular 1 or 2 Is carbon atoms or an aryl radical and n has a value of preferably up to 10 and in particular 1 or 2.

The radicals R ₁ and R ₃ are preferably hydrogen atoms. In a further preferred embodiment, the radicals R, and / or R _{3 can} contain more than 10 carbon atoms.

In a further preferred embodiment, n has a value greater than 10. Special

Examples are olefinic polyethylene glycols or olefinic derivatives of polyacrylamides.

If the radicals RR ₂ and R _{3 are} unsaturated and / or substituted radicals, they have the meaning given for the general formula (I). In addition to the substituents mentioned, hydroxyl and / or amino radicals can be present.

The crosslinkers used are preferably selected from the group consisting of divinylalkyl and divinylaryl compounds, in particular divinylbenzene or 1,5-hexadiene. In a further preferred embodiment, the crosslinkers or mixtures thereof containing unsaturated bonds are selected from the group consisting of analogs or derivatives of the general formula (III)

CH ₂ = C (R,) - CX- (R ₂ ) _n -XCC (R ₃ ) = CH ₂ , OO (III)

wherein the radical R _b R ₂ and R ₃ and n have the meaning given above for the general formula (II) and the radical X is an oxygen atom or the group -NH.

The crosslinkers are preferably selected from ethylene glycol dimethacrylate, N, N'-methylene-bis-acrylamide, diitaconic acid alkylamides and diitaconic acid arylamides. In a preferred embodiment, the copolymerization is carried out in cyclohexane, toluene, acetone, alkanoia, ethyl acetate, tetrahydrofuran, chloroform or mixtures thereof, in particular in cyclohexane, ethylene glycol dimethacrylate, divinylbenzene, diitaconic acid alkylamides, arylamides, divinylalkyl or aryl compounds being used as crosslinkers .

In a preferred embodiment, the derivatized imprint polypeptide obtained after the precipitation step (see FIGS. 3 and 4) is in an organic solvent suspended, one or more of the above-mentioned crosslinkers added, the radical initiator, for example azobis compounds (α, α'-azo-isobutyronitrile (AIBN) or 2,2'-azo-bis- (2,4-dimethyl) - valeric acid nitrile (ABDV)) , added and the copolymerization started by UV radiation. The polypeptide conformation is fixed covalently via the previously introduced olefinic double bonds; the important free functionalities of the imprint polypeptide are not blocked by this step. The imprinted conformation is thus covalently fixed and can be used in the aqueous and organic solvent system.

The covalently cross-linked imprint polypeptide, which is fixed and stabilized in its conformation, can be used under many conditions under which it would have been unstable without the selective introduction of residues and the copolymerization, e.g. in aqueous solvent systems.

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

The polypeptide is then imprinted (embossed) with a print molecule in an aqueous medium. The print molecule can be selected as described above. A native enzyme is preferably used as the polypeptide and the substrate of this enzyme is used as the print molecule. The copolymerization is then carried out in an aqueous solvent using a crosslinker which is copolymerizable with the polymerizable groups containing unsaturated bonds. The copolymerization is carried out as described above for step (D). The crosslinkers are preferably selected from the groups described above. The crosslinker is chosen so that it is soluble in the aqueous solvent. N, N'-methylene-bisacrylamide is preferably used as the crosslinker.

The invention is illustrated by the following examples. example 1

Gentle introduction of olefinic groups into polypeptides

Vinylation of α-chymotrypsin (EC 3.4.21.1) with itaconic anhydride

Α-Chymotrypsin is dissolved in 10 ml of 0.05 M potassium phosphate buffer pH 7.8 with stirring. Itaconic anhydride is then added in spatula tips at room temperature. The decrease in the pH of the reaction solution which occurs each time the anhydride is added is corrected by adding 5 M NaOH. After the addition of the anhydride has ended, the mixture is stirred for a further 1 hour and the pH is again corrected. To separate the low molecular weight components, the reaction solution obtained is buffered in 0.05 M potassium phosphate buffer pH 7.8 using PD 10 gel filtration columns (Pharmacia) and finally lyophilized. The enzymatic activity of α-chymotrypsin is determined by means of the hydrolysis of benzoyl-L-tyrosine ethyl ester in a photometric test. The extent of the modification of the functional groups is determined using 2,4,6-trinitrobenzenesulfonic acid (TNBS assay) (Habeeb A.F.S.A. (1966), Anal. Biochem. 14, 328-336). Tab. 1 shows the reaction batches, the extent of the modification of the functional groups and the enzymatic activities of the α-chymotrypsin derivatives in relation to the free enzyme.

Table 1: Derivatization of α-chymotrypsin with itaconic anhydride

To derivatize itaconic acid residual activity of the modification to the α-chymo-anhydride derivative functional groups used trypsin activity [mg] [%] [%]

[nKat]

2765 1.25 38 24

2765 2.5 31 27

2765 10 30 57

2765 30 -30 66

2765 60 18 75 B. Vinylation of the β-glucosidase from the almond (EC 3.2.1.21) with itaconic anhydride

The β-glucosidase is dissolved in 10 ml of 0.05 M citric acid-phosphate buffer pH 6.0 with stirring. D (+) - glucose can be added to stabilize the enzyme. Itaconic anhydride is then added in spatula tips at room temperature. The decrease in the pH of the reaction solution which occurs each time the anhydride is added is corrected by adding 5 M NaOH. After the addition of the anhydride has ended, the mixture is stirred for a further 1 hour and the pH is again corrected. To separate the low molecular weight components, the reaction solution obtained is buffered in 0.05 M citric acid-phosphate buffer pH 6.0 using PD 10 gel filtration columns (Pharmacia) and finally lyophilized. The enzymatic activity of the β-glucosidase is determined by means of the hydrolysis of 4-nitrophenyl-β-D-glucopyranoside in a photometric test. The extent of the modification of the functional groups is determined using 2,4,6-trinitrobenzenesulfonic acid (TNBS assay). Table 2 shows the reaction batches, the extent of the modification of the functional groups and the enzymatic activities of the β-glucosidase derivatives in relation to the free enzyme. To protect the active center of the polypeptide, glucose was added as an additive. In the presence of 3M glucose, 99% residual activity could be obtained after derivatization.

Table 2: Derivatization of ß-glucosidase with itaconic anhydride

To derivatize itaconic acid D (+) - glucose residual activity of modification of the ß-gluanhydride derivative used functional cosidase activity [mg] [M] [%] groups

[nKat] [%]

3678 500/21 100

3678 250/36 100

3678 130/55 99

3678 130 2 85 99

3678 130 3 99 98

Example 2

Reactivity of itaconic anhydride with the side chains of polypeptides

Various α-N-acetyl- (L) - amino acids are dissolved in 5 ml of 0.05 M citric acid-phosphate buffer pH 7.5, mixed with itaconic anhydride while stirring and incubated for 1 hour at room temperature. The TNBS assay is performed with a sample of each in the buffer dissolved α-N-acetyl- (X) -amino acid and with the α-N-acetyl- (Z.) - amino acid incubated with itaconic anhydride.

It can be stated that itaconic anhydride with the amino groups of α-N-acetyl- (Z,) - lysine and α-N-acetyl- (I) -glutamine, with the sulfhydryl group of α-N-acetyl- (I) - Cysteine is able to react with the hydroxy groups of α-N-acetyl- (I) -tyrosine and α-N-acetyl- (I) -serine (Tab. 3). The reactivities of the itaconic anhydride to the amino acid side chains were Ser> Tyr> Glu> Lys> Cys.

Table 3: Investigations on the reactivity of itaconic anhydride ³

N-acetyl- (Z) - amino acid functional residue degree of modification "(%) α-N-acetyi- (Z -) - serine -OH 96 α-N-acetyl- (Z -) - tyrosine -OH 89 α- N-acetyl- (Z.) - glutamine -C (O) NH ₂ 81 α-N-acetyl- (£) -lysine -CH ₂ NH ₂ 68 α-N-acetyl- (Z.) - cysteine -CH ₂ SH 15 α-N-acetyl- (Z.) - asparagine -C (O) NH ₂ 0 α-N-acetyl- (I) -arginine -C (NH ₂ ) NH ₂ ⁺ 0 ^a added in ^a 10-fold molar excess , ^ ach lh response time; measured with TNBS.

On the stability of the above It should be noted that, for example, primary amino groups with itaconic anhydride lead to amide bonds which are stable from pH 1-12 and at temperatures up to 70 ° C (R. Kölle, 1995, dissertation, TU Braunschweig, Germany, 53-59).

Example 3 Preparation of a fixed one. Imprint polypeptide

30 mg of the α-chymotrypsin derivatized with itaconic anhydride from Example 1 (extent of modification = 70%) are dissolved in 1 ml of 0.01 M sodium 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 has been cooled to -20 ° C, are added . The resulting precipitate is centrifuged, washed with 10 ml of 1-propanol and finally lyophilized. 340 mg of ethylene glycol dimethacrylate and 4 mg of 2,2'-azo-bis- (2- methylpropiononitrile) dissolved. 5 mg of lyophilisate are suspended in it. The copolymerization is initiated by means of UV light. The copolymerization period is 4 hours. The covalently cross-linked and imprinted α-chymotrypsin (cross-linked, imprint polypeptide; VIP) is washed with cyclohexane and lyophilized (see FIG. 3).

To check whether the derivatized, imprinted (ISA / impr Chy) or the cross-linked, imprinted chymotrypsin (VIP) had the imprinted property in the organic solvent system equal to the native, imprinted chymotrypsin (na / impr Chy), all three imprinted Polypeptides for the synthesis of N-Ac- (D) -TrpEE tested in cyclohexane (Fig. 5; analysis with HPLC, see Stähl et al. 1991). It became apparent that the ISA / impr Chy and VIP had better activities than the na / impr Chy in the first 100 hours. The kinetic effects after 100 hours of reaction are due to the influence of the water released in the enzymatic condensation reaction in the organic solvent.

Example 4 Use of the cross-linked imprint polypeptide (VIP) in an aqueous medium

The VIP is suspended in phosphate buffer (10 mM), pH 7.8 and preincubated in a thermal shaker at 27 ° C for approx. 10 min. The reaction is started by adding the substrate, the N-acetyl- () -tryptophanethylester (10 mM). The VIP showed a specific (D) - ester hydrolysis activity of 0.04 μmol / (min • g). The native non-printed, the non-cross-linked imprinted and the non-imprinted cross-linked α-chymotrypsin had no (-D) ester hydrolysis activity.

Example 5

Use of the cross-linked imprint polypeptide (VIP) in the organic solvent

Pre-incubation (Ih) in the aqueous solvent system

A. The VIP was incubated for 1 h in a phosphate buffer (10 mM), pH 7.8 at 25 ° C. After the batch had been lyophilized, the VIP was suspended in cyclohexane. After adding N-Ac- (D) -Trp (10 mM) and 20% (v / v) ethanol, the N-acetyl- () - tryptophanethyl ester synthesis was started at 25 ° C. in a thermoshaker. The VIP showed one specific (Z ⁾ ) ester hydrolysis activity of 0.02 μmol / (min • g). The imprinted property was therefore not lost in the aqueous solvent system.

B. As in Example 5A. described, but using N-Ac- (D) -tyrosine instead of N-Ac - (. D) -Trp.

C. As in Example 5A. described, but using N-Ac- (Z)) - phenylalanine instead of N-Ac- () -T.

Example 6

Production of a fixed, imprint polypeptide for selective adsorption

50 mg bovine serum albumin are dissolved in 10 ml 0.05 M potassium phosphate buffer pH 5.5. At room temperature, spatula tips of itaconic anhydride are then added with stirring. The decrease in the pH of the reaction solution which occurs each time the anhydride is added is corrected by adding 5 M NaOH. After the addition of the anhydride has ended, the mixture is stirred for a further 1 hour and the pH is again corrected. To separate the low molecular weight components, the reaction solution obtained is buffered in 0.05 M potassium phosphate buffer pH 5.5 using PD 10 gel filtration columns (Pharmacia) and finally lyophilized. The extent of the modification of the functional groups is determined using 2,4,6-trinitrobenzenesulfonic acid (TNBS assay).

30 mg of bovine serum albumin derivatized with itaconic anhydride (extent of modification = 85%) are distilled in 4 ml of H ₂ O. solved. The aqueous solution contains L-malic acid in a concentration of 0.5 M. The solution is cooled at pH 5.5 to 0 ° C and 4 ml of 1-propanol, which has been cooled to -20 ° C, are added. The resulting precipitate is centrifuged, washed with 10 ml of 1-propanol and finally lyophilized.

340 mg of ethylene glycol dimethacrylate with 4 mg of 2,2'-azobis (2-methylpropiononitrile) are dissolved in 0.5 ml of cyclohexane. 10 mg of lyophilisate are suspended in it. The copolymerization is initiated by means of UV light. The copolymerization period is 4 hours. The covalently cross-linked and imprinted bovine serum albumin is washed with cyclohexane and lyophilized. As with Dabulis and Klibanov, Biotechnol. Bioeng. 39 (1991), 176-185, the extent of the binding affinity of the imprinted bovine serum albumin to L-malic acid was investigated. It was found that both the imprinted and the cross-linked, imprinted bovine serum albumin in ethyl acetate had binding affinity for L-malic acid. In the aqueous medium, only the cross-linked, imprinted bovine serum albumin was able to selectively adsorb L-malic acid.

Example 7 Preparation of a native, fixed polypeptide

50 mg of the 98% derivatized β-glucosidase (see Table 2) and 250 mg of N, N'-methylenebisacrylamide are dissolved in 29 ml of 0.05 M citric acid-phosphate buffer pH 6.0. The copolymerization is started by adding 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 first in a pleated filter with 0.5 liters of 0.5 M NaCl solution and then with 2 liters of distilled H ₂ O. washed until salt-free. The copolymer is taken up in 0.05 M citric acid-phosphate buffer pH 6.0 and then lyophilized.

The enormous stabilization of the cross-linked polypeptide against the native, non-cross-linked is shown in Table 4, in which the half-lives of the polypeptide are given at different temperatures.

Table 4: Half-lives of the catalytically active conformation of the free and cross-linked ß-glucosidase

Biocatalyst form t _1/2 (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 Klibanow, A. (1992). Biotechnol. Bioeng. 39, 176-185. Fritz, H., Neudecker, M., Schult, H. and Wert, E. (1966). Appl. Chem. 78, 775. Goldstein, L. (1970). Methods Enzymol. 19, 935-962. Habeeb, A.F.S.A. (1966). Anal. Biochem. 14, 328-336. Kölle, R. (1995) Dissertation, TU Braunschweig, Germany, 53-59. Mosbach, K. and Ramström, O. (1996). Bio / Technology 14, 163-170. Russell, A.J. and Klibanow, A.M. (1988). J. Biol. Chem. 263, 11624-1 1626. Stähl, M., Jeppson-Wistrand, U., Mänsson, M.-O. and Mosbach, K. (1991). J. Am. Chem. Soc. 113, 9366-9368. Stähl, M „Mänsson, M.-O. and Mosbach. K. (1990). Biotechnol. Lett. 12, 161-166.

Claims

claims

1. A process for the preparation of covalently cross-linked imprint polypeptides which are fixed and stabilized in their conformation and which are stable in both aqueous and organic solvent systems, comprising the following steps:

(C) Precipitation of the polypeptide / print molecule mixture obtained in step (B) by adding an additive suitable for precipitation and / or freeze-drying the polypeptide / print molecule obtained in step (B)

Mixture; and

2. The method according to claim 1, characterized in that step (B) is carried out before step (A).

3. The method according to claim 1, characterized in that steps (A) and (B) are carried out simultaneously.

4. The method according to any one of the preceding claims, characterized in that a polypeptide with OH, and / or NH ₂ -, and / or SH groups is used, which partially or completely with the polymerizable, containing unsaturated bonds

Compounds are derivatized to form covalent bonds.

5. The method according to any one of the preceding claims, characterized in that a native protein, in particular an enzyme, is used as the polypeptide.

6. The method according to any one of the preceding claims, characterized in that the compounds containing unsaturated bonds used in step (A) or mixtures thereof are selected from the group consisting of reactive analogs or derivatives of the general formula (I)

C = C (I)

R, R ₄

where the radicals R →, R, R ₃ and R ₄ independently of one another are hydrogen atoms, carboxyl radicals, cyclic or linear, substituted or unsubstituted, saturated or unsaturated alkyl radicals, alkoxy radicals or carboxyalkyl radicals with preferably up to 10, more preferably up to 6, in particular 1 or 2 Are carbon atoms or substituted or unsubstituted aryl radicals or carboxyaryl radicals, with the proviso that at least one of the radicals R, R ₂ , R ₃ and R _{4 is} independently a carboxyl, carboxyalkyl radical or carboxyaryl radical.

7. The method according to claim 6, characterized in that the compounds containing unsaturated bonds used in step (A) are selected from the group consisting of itaconic, maleic, citracon, croton, methacrylic, acrylic acid and their anhydrides and acid halides .

8. The method according to any one of the preceding claims, characterized in that polyols and / or salts are additionally added in step (A).

9. The method according to any one of the preceding claims, characterized in that the crosslinker used for the copolymerization in step (D) is selected from the group consisting of analogs or derivatives of the general formula (II)

H ₂ C = C (R,) - (R ₂ ) _n -C (R ₃ ) = CH ₂ (II)

where the radicals R → and R ₃ independently of one another are hydrogen atoms, cyclic or linear, unsaturated or saturated, substituted or unsubstituted alkyl, carboxyl, Carboxyalkyl or alkyl ether radicals with preferably up to 10, in particular 1 or 2, carbon atoms or substituted or unsubstituted aryl radicals and the radical R _{2 is} a cyclic or linear, saturated or unsaturated, substituted or unsubstituted alkyl or alkyl ether radical with preferably up to 10, in particular 1 or 2 carbon atoms or an aryl radical and n has a value of preferably up to 10 and in particular 1 or 2.

10. The method according to claim 9, characterized in that the crosslinker used for the copolymerization in step (D) is selected from the group consisting of divinylalkyl and divinylaryl compounds.

11. The method according to claim 1 to 8, characterized in that the crosslinker used for the copolymerization in step (D) is selected from the group consisting of analogs or derivatives of the general formula (III)

CH ₂ = C (R,) - CX- (R ₂ ) _n -XCC (R ₃ ) = CH ₂ , (III)

O O

wherein the radicals RR ₂ and R ₃ and n have the meaning given in claim 9 for the general formula (II) and the radical X is an oxygen atom or the group -NH.

12. The method according to claim 11, characterized in that the crosslinker used for the copolymerization in step (D) is selected from the group consisting of ethylene glycol dimethacrylate, N, N'-methylene-bis-acrylamide, diitaconic acid alkylamides and diitaconic acid arylamides.

13. A process for producing a covalently cross-linked imprint polypeptide that is fixed and stabilized in its conformation and that is stable in both aqueous and organic solvent systems, comprising the following steps:

(A) covalently introducing polymerizable groups containing unsaturated bonds into a polypeptide in an aqueous medium; (B) imprinting the polypeptide obtained in step (A) with a print molecule in an aqueous medium; and

14. The method according to claim 13, characterized in that step (B) is carried out before step (A).

15. The method according to claim 13, characterized in that steps (A) and (B) are carried out simultaneously.

16. In its conformation, fixed and stabilized, covalently cross-linked imprint polypeptide, which are stable in both aqueous and organic solvent systems, obtainable by a process according to one of the preceding claims.

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

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 biosensor technology for the specific recognition of molecules.