HU193406B - Method for producing affinity sorbents - Google Patents

Abstract

The present invention relates to a process for the preparation of affinity sorbents of the formula (I): P - A - CO - NH - Β (I) - wherein P is a polysaccharide type carrier suitable for affinity chromatography for polymerisation between 10,000 and 100,000, preferably agarose or cellulose. or a copolymer of an acrylic amide with an unsaturated carboxylic acid crosslinked crosslinked with a 10,000 to 100,000 polymerization fraction without the A-COOH side chain, A - (CH2) -, wherein n is an integer from 1 to 10 , and B represents a residue of an amino acid, polypeptide or neurotransmitter carboxylic acid without the -NH2 end group, such that P-A-COOH (II) - wherein P and A are as defined above - is active for the polymeric support of Formula II. by an ester method an amino acid, polypeptide or neurotransmitter carboxylic acid (III) of formula (III) - wherein B is a - protecting a protected derivative comprising all of the amino groups present (except for the terminal amino group) and optionally the heterocyclic amino, hydroxyl and / or carboxyl group (s) present in the protected form with an acid-cleavable protecting group; ) when P is a polysaccharide type carrier, the gel is dehydrated by treatment with acetone and dichloromethane, the protecting groups are cleaved in dichloromethane with trifluoroacetic acid, and the resulting product is rehydrated by treating with acetone-water mixtures with decreasing acetone, and finally with water; P represents an acrylic copolymer, the protecting groups are cleaved by acid treatment and the gel is washed and dried. -1-

Description

The present invention relates to novel affinity sorbents of formula (I)

P - A - CO - NH - Β (I) - in the formula

P is a polysaccharide copolymer of 10,000 to 100,000 polymers having a degree of polymerisation of 10,000 to 100,000 and a polysaccharide-type support for affinity chromatography, preferably agarose or cellulose or acrylamide with a divinyl compound crosslinked with a divinyl compound. without,

A - (CH ₂ ) - is - where n is an integer from 1 to 10, and

B represents a residue of an amino acid, polypeptide, or neurotransmitter carboxylic acid without the -NH ₂ end group.

The immobilization of amino acids, polypeptides (including di- and oligopeptides, which are typically represented by hormones), and neurotransmitter carboxylic acids on a polymeric carrier is of great importance in many respects because it results in a variety of sorbents useful in scientific research. These sorbents can be used for affinity chromatography, purification of solubilized receptors for hormones, purification of antisera to hormones as antigens, isolation of antibodies, isolation and purification of enzymes. In addition, living cells can be clearly isolated by appropriate affinity sorbents based on the interaction between the immobilized hormone and its receptor. If the affinity sorbents are incubated with a suspension of living cells on a microscope slide and examined for binding of the cells to the affinity sorbent under a microscope, it can be determined whether the particular cell type contains the receptors examined on the cell membrane (affinity microscopy). Affinity sorbents can be used to control the specificity of antisera produced against a particular hormone or neurotransmitter, to qualitatively and quantitatively detect antibody specificity, and in some cases (such as autoimmune diseases) for therapeutic purposes.

It is essential for all these applications that the affinity sorbent contains the immobilized amino acid, peptide or neurotransmitter carboxylic acid molecules in a chemically uniform and biologically active state.

Only hydrophilic polymers may be used as carriers for the preparation of affinity sorbents. Various polysaccharides (agarose, cellulose, etc.) are widely used for this purpose. In the prior art, the biologically active molecule is coupled to its carrier via its amino group, for example by pre-loading ε-aminocaproic acid with bromocyano, the resulting functional groups of the carboxyl-functionalized carrier being converted to the N-hydroxysuccinimide ester, and an amino acid, peptide or neurotransmitter to be attached to their active ester by their amino group.

The disadvantage of this solution is that the biologically active molecules containing multiple amino groups can be attached to the carrier in several ways, i.e. the final biologically active molecule is not contained in the chemically uniform state.

Thus, there is a need for a process by which affinity sorbents having a chemically uniform structure (i.e., containing all of the applied molecules bound) can be prepared without reducing the biological activity of the applied molecule. It is obvious to use protected amino acids or peptides which contain only one free amino group, while all other reactive groups (amino, hydroxyl or mercapto) are present in protected form. However, the difficulty arises from the fact that the polymeric carriers which can be used for affinity chromatography purposes must be hydrophilic materials; however, they are extremely sensitive to acids. The acid causes the polysaccharide backbone to undergo hydrolysis and degradation, so when a biologically active molecule containing protected groups is coupled to a polysaccharide backbone and then the amino and other functional groups are cleaved in a known acidic manner, the final product undergoes structural changes. which render it unsuitable for affinity chromatography purposes. The polystyrene-based acid-resistant carriers used in solid phase peptide synthesis are not suitable for affinity chromatography because they are hydrophobic.

In our experiments we found that if

(a) removal of the acid-cleavable protecting groups on other amino groups and optionally other functional groups of amino acids, neurotransmitter carboxylic acids or polypeptides immobilized on the polysaccharide support using their free amino group, or

b) using a hydrophilic but still acid-resistant acrylic copolymer not yet used in affinity chromatography for immobilization of amino acids, neurotransmitter carboxylic acids or polypeptides, all of the above problems can be eliminated.

The present invention therefore relates to a process for the preparation of novel affinity sorbents of formula (I). According to the invention, the process is carried out on a polymeric support of formula II

P - A - COOH (II) - wherein P and A are as defined above - by an active ester method an amino acid, polypeptide or neurotransmitter carboxylic acid of formula (III)

-2193406

Β - ΝΗ ₂ (III) - in which Β is as defined above - is a protected derivative which has all the amino groups present (except for the terminal amino group) and optionally the heterocyclic amino, hydroxy and / or carboxyl group (s) present also in an acid protected protecting group; and

(a) where P is a polysaccharide-type support, the gel is dehydrated by treatment with acetone and dichloromethane, the protecting groups are removed with trifluoroacetic acid in dichloromethane and the resulting product is rehydrated with acetone-water with decreasing acetone content, or

b) when P is an acrylic copolymer, the protecting groups are cleaved by acid treatment and the gel is washed and dried.

As used herein, the term "amino acid" refers to compounds containing both aminocarboxylic acids, i.e., -NH ₂ and -COOH, which may optionally have additional functional groups on the hydrocarbon backbone bearing -COOH and -NH ₂ , such as hydroxyl, amino, , carboxy, alkylthio, hydroxyalkyl or sulfonic acid groups. Examples of amino acids include: lysine, arginine, aspartic acid, glutamine, aspartic acid, glutamic acid, cystine, cysteine, methionine, glycine, phenylalanine, alkyl, hydroxyl, sulfonic acid, amino or nitro on the aromatic ring. phenylalanine derivatives, alanine, valine, leucine, isoleucine, histidine, tryptophan, proline, tyrosine, ornithine, ε-aminocaproic acid, sarcosine, γ-aminobutyric acid, α-aminobutyric acid, p-aminobenzoic acid and alkyl, hydroxy, sulfonic acid, amino or nitro substituted derivatives of the aromatic ring, 0-methyl or O-ethyl tyrosine and the like.

Throughout the specification and appended claims, the "neurotransmitter acids designations for carrying the amino group on carbon by ^one carboxylated (i.e. NH ₂ - mean (containing p-COOH-terminal moiety) neurotransmitters The neurotransmitters that play an important role in the synaptic loss compounds that enzymatic conversion of the protein component in the living body of amino acids. They are formed by first introducing new substituents (usually hydroxyl groups) into the parent amino acid molecule in the first step of the enzymatic transformation, and in the second step by enzymatic decarboxylation of the substituted amino acid molecule. a.

Acrylic copolymers of the polymeric support of formula II are commercially available known products. An example is Akrilex C (copolymer of acrylamide, N, N'-methylene-bis (acrylamide) and acrylic acid). Compounds of formula (II) in which P represents a known polysaccharide-type carrier (e.g., agarose, cellulose, dextran, carboxymethylcellulose or carboxyethylcellulose, preferably agarose or cellulose), which are commonly used in affinity chromatography, are known in the art. is prepared by reacting the polysaccharide with bromocyanide followed by an amino acid. Preferred examples of the latter compounds are carboxyhexyl side chain polysaccharides, such as carboxyhexyl agarose.

The protected derivatives of the amino acids or peptides of formula III are prepared by methods known per se in the field of amino acid and peptide chemistry. Acid-cleavable protecting groups for amino groups include, for example, tert-butylcarbonyl or α, α'-dimethyl-3,5-dimethoxybenzyloxycarbonyl. Optionally, other reactive groups present on the molecule, such as hydroxyl, carboxyl or heterocyclic amino groups, may be protected with acid-cleavable protecting groups. For example, tert-butyl or other suitable acid-sensitive carboxyl protecting groups may be used to protect the hydroxyl groups, while tert-butoxycarbonyl or other suitable acid-sensitive amino protecting groups may be used to protect the heterocyclic amino groups. These additional reactive groups need only be protected if they would cause side reactions in the free state during the coupling reaction.

Protected derivatives of the neurotransmitter carboxylic acids of formula III (i.e., the carboxylated neurotransmitters on the amino-bearing carbon atom) are prepared in a known manner.

If starting from a polysaccharide-type carrier, the gel must be dehydrated before cleavage of the protecting groups. Dehydration is carried out using acetone, acetone / dichloromethane and dichloromethane.

The resulting anhydrous gel was then treated with trifluoroacetic acid in dichloromethane. In this step, the protecting groups can be removed without damaging the gel structure. After acid treatment, the gel is washed and then rehydrated by addition of aqueous acetone containing increasing amounts of water and finally water. Under normal conditions, the rehydrated gel is stored in a cool place, protected from light, in an aqueous buffer containing antioxidant and fungicide.

When starting from an acrylic copolymer carrier, the gel need not be dehydrated. Because this copolymer is significantly less sensitive to acids than polysaccharides, other strong acids, such as 1.5N aqueous hydrochloric acid and acetic acid, may be used as the acid deprotection. After acid treatment, the gel is washed and dried.

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The invention is illustrated in detail by the following non-limiting Examples. Unless otherwise specified, the amino acid configuration has a natural (L) configuration. The final products were obtained in 100% yield with respect to the polymer used. Because it has to be immobilized. The compound is always used in a very large excess (8-10 fold), yielding about 10-12% of the final product in relation to the compound to be immobilized. The concentration of the immobilized biologically active compound on the support, which is a characteristic quality of the affinity sorbent, is given in the Examples.

Example 1 ml of OH-agarose-OSu is suspended in 50 ml of 0.1 M phosphate buffer, pH 6.8 containing 1 mg of ε-amino-BOC-lysine (H-Lys (BOC) -OH) per ml. . The slurry was shaken for 3 hours and then washed with 10 volumes of 2: 2: 1 acetone-dimethylformamide-water. The gel is separated, washed five times with 2: 1: 2 dimethylformamide / water / acetone (2: 1: 2) and then with distilled water, and then dried as follows: 1 volume of gel in 2 to 2 volumes of increasing volume (40, 60, 80% v / v) aqueous acetone, acetone anhydrous, dichloromethane / acetone containing increasing amounts of dichloromethane (5, 10, 20.40, 60, 80% v / v), and finally anhydrous dichloromethane washed. The solvent is filtered off with suction and the gel is suspended in an equal volume of a 1: 1 mixture of trifluoroacetic acid in dichloromethane cooled to 0 ° C, and the suspension is gently shaken for 2 hours. After 2 hours, the gel was filtered and once with dichloromethane / dichloromethane / dichloromethane / twice with anhydrous dichloromethane and finally with anhydrous acetone. wash twice. An equal volume of washing liquid with gel is used for each wash. The gel is then rehydrated by washing once and twice with distilled water once with aqueous acetone containing a decreasing amount (95, 80, 60, 40, 20, 10, 5% by volume) of acetone. Again, an equal volume of washing liquid with the gel is used. The gel is then washed with 5 volumes of gel in 0.1 M acetate buffer, pH 4.75, containing 0.02% sodium nitrite and 0.1% L-ascorbic acid as an antioxidant. Store the gel in the same composition buffer solution at - (- 4 ^° C, protected from light).

The amount of lysine bound to the polymeric carrier after hydrolysis of mercaptoethanesulfonic acid is determined by automated amino acid analysis. The concentration of lysine on the support is x 10 ' ³ mol / liter.

Example 2

The procedure is as in Example 1, but α-amino-BOC-lysine (BOC-Lys-OH) is used. The amount of lysine bound to the polymeric carrier was determined as in Example 1. The concentration of lysine on the support is 2.5 x 10 ^{3 -3} moles / liter.

Example 3

The procedure is as in Example 1, but using H-Pro-Lys (BOC) -Gly-NH ₂ .

Example 4

0.5 g of anhydrous Akrilex C 100 pentachlorophenyl ester are suspended in 10 ml of anhydrous dimethylformamide. To the suspension was added 1 ml of anhydrous pyridine followed by 20 mg of H-Pro-Lys (BOC) -Gly-NH ₂ in 10 ml of anhydrous dimethylformamide, and the suspension was shaken for 1 day at room temperature. The solvent was then filtered off with suction and the gel was washed five times with 20 ml of anhydrous dimethylformamide and five times with 50 ml of anhydrous acetone. The gel is then washed with dichloromethane, then dichloromethane / trifluoroacetic acid (1: 1) is added and the suspension is shaken for 1 hour. The gel was separated, washed with methanol, dimethylformamide and acetone and stored as xerogel.

Claims (1)

Process for the preparation of affinity sorbents of formula (I) P - A - CO - NH - Β (I) - in the formula P is a polysaccharide-OH copolymer of 10,000 to 100,000 polymers having a degree of polymerisation of 10,000 to 100,000 and a polysaccharide-type support, preferably agarose or cellulose or acrylamide, crosslinked with an unsaturated carboxylic acid and suitable for affinity chromatography. without, A - (CH ₂ ) - is - where n is an integer from 1 to 10, and B represents a residue of an amino acid, polypeptide or neurotransmitter carboxylic acid without the -NH ₂ end group, characterized in that P - A - COOH (II) - wherein P and A are as defined above - by an active ester method an amino acid, polypeptide or neurotransmitter carboxylic acid of formula (III) Β - NH ₂ (III) - in which B is as defined above - is a protected derivative which has all the amino groups present (except the terminal amino group) and optionally the heterocyclic amino, hydroxy and / or carboxyl group present also in an acid protected protecting group; and -4193406 (a) where P is a polysaccharide-type support, the gel is dehydrated by treatment with acetone and dichloromethane, the protecting groups are removed with trifluoroacetic acid in dichloromethane and the resulting product is rehydrated with acetone / water containing decreasing acetone8, or b) when P is an acrylic copolymer, the protecting groups are cleaved by acid treatment; Wash 5 gels and dry.