CH503700A - Cross-linked proteins and peptides - Google Patents

Abstract

A process for the preparation of water soluble proteins and peptides of high molecular weight, by coupling amino, carboxyl or hydroxyl- containing polaminocarboxylicacids with proteins or peptides. In this way strongly cross-linked products are obtained since the polyaminocarboxylic acids contain several functional groups. - The products are suitable for use as antigens in immuno-therapy, or can be used for the determination of insulin and gonadotropin. - The preparation of cross-linked proteins with a molecular weight of up to 250,000, which are soluble in water, formed by coupling polyaminocarboxylic acids with suitable proteins.

Description

  

  Process for the production of crosslinked proteins and peptides Attempts have already been made to link protein molecules to one another by means of aromatic diisocyanates or bis-diazonium compounds. The degree of crosslinking of the products obtained in this way is low, however, since when low molecular weight crosslinking components are used, the protein molecules to be crosslinked sterically hinder one another.



  It is also known that proteins and peptides can be bound to insoluble carrier substances, for example polyamino styrene, and thus protein aggregates are obtained which, however, are insoluble. It has now been found that high molecular weight, water-soluble, crosslinked proteins and peptides are obtained when reactive derivatives of amino and carboxyl and, if appropriate, aromatic polyaminocarboxylic acid containing hydroxyl groups, optionally after reduction of any additional nitro groups present, with proteins or peptides in implemented in a known manner.

   Since the polyaminocarboxylic acids mentioned contain several functional groups, they are able to bind several protein or peptide molecules and therefore lead to particularly highly crosslinked products which, despite their high molecular weight, are water-soluble.



  The preparation of the process products is characterized in that a polycondensate containing free amino groups and carboxyl and optionally hydroxyl groups of corresponding aromatic aminocarboxylic acid, which can also be substituted by halogen or by alkyl, sulfo or nitro groups, but does not contain any condensed nuclei , reacts with a reactive derivative of carbonic acid and allows the product obtained to react with the amino and carboxyl groups of a protein or peptide.



  As reactive derivatives come z. B. Phos gene, thiophosgene or chloroformic acid ester in question; with the polyaminocarboxylic acids mentioned, isocyanates, isothiocyanates and carbamic acid esters are formed. These then react with the proteins or peptides used as reaction partners.



  The aromatic polyaminocarboxylic acids used as starting materials can be prepared by converting polyfunctional aromatic compounds that do not contain condensed nuclei but contain amino, hydroxyl and carboxyl groups in the presence of phosphorus pentoxide and lower dialkyl phosphites as solvents in a strongly acidic medium. They can be derived from derivatives of benzene, diphenyl, diphenylmethane, diphenylamine, diphenyl ether or diphenyl sulfone.

   In addition to the required amino and carboxyl groups, they can also contain alkyl groups, preferably those with 1-4 C atoms, in particular methyl and ethyl groups, halogen atoms, preferably chlorine and bromine, and sulfo, nitro or hydroxyl groups.



  The proteins and peptides that are also suitable for the process of the invention can either be natural substances or synthetic products. As natural substances come into question, for. B. enzymes, example, ribonuclease, trypsin, chymotrypsin; Glycoproteins such as the gonadotropins, peptide hormones such as insulin, glucagon, ACTH, vasopressin; Virus proteins, e.g. B. those of the polio, influenza, measles virus; Bacterial toxins and toxoids such as diphtheria and tetanus toxins, as well as serologically determinant fragments of the proteins and peptides mentioned.



  The high molecular weight crosslinking products obtained by the process according to the invention no longer have the biological activity of the proteins or peptides used as starting products, so that no direct physiological disturbances occur when applied as immunogens. The antisera obtained by immunization with the cross-linked proteins or peptides, however, give a serological cross-reaction with the proteins and peptides used as starting products, which leads to the neutralization of the biological activity of the starting products.

   This results in an immunotherapeutic method to compensate for an overproduction of biologically active proteins and peptides in vivo. It is surprising that the compounds are soluble in water despite their high degree of crosslinking and the correspondingly high molecular weight of up to 250,000.



  The products of the process are suitable, for example, as antigens in immunotherapy, e.g. B. for the manufacture of vaccines and serums. The antigonadotropin serum obtained in accordance with the invention produces temporary sterility in test animals. Other antisera obtained according to the invention can be used for immune determination, e.g. B. to determine insulin and gonadotropin.



  <I> Example 1 </I> 500 mg of reduced polycondensate (prepared analogously to Example 1) are dissolved in 50 ml of dimethylformamide, 1 g of solid sodium bicarbonate is added and added while stirring with 0.5 ml of thiophosgene in 20 ml of ether. The mixture is stirred overnight at room temperature with the exclusion of moisture, then heated to 70 ° C. for two hours and then evaporated twice in vacuo with dimethylformamide to remove the excess thiophosgene. The residue is taken up in 20 ml of dimethylformamide, not everything needing to be dissolved, and added to a solution of 5 g of bovine serum albumin in 200 ml of water, which is adjusted to pH 9.5 with sodium hydroxide solution, at 40 ° C. for 30 minutes.

   After the reaction, the mixture is warmed to 60 ° C. for 15 minutes, three hours against 0.1 M ammonium carbonate, then dialysis for one day against water at 4 ° C. and then lyophilized.



  Yield: 5.5 g of a pale yellowish powder. The molecular weight of the crosslinked bovine serum albumin varies between 190,000-270,000.

 

Claims (1)

PATENT CLAIM A process for the production of high molecular weight water-soluble crosslinked proteins or peptides, characterized in that a polycondensate containing free amino groups and carboxyl and optionally hydroxyl groups of corresponding aromatic aminocarboxylic acids, which also contain halogens and alkyl, sulfo or Nitro groups can be substituted, but contain no condensed nuclei, reacts with a reactive derivative of carbonic acid and allows the product obtained to react with the amino and carboxyl groups of a protein or peptide. <I> Note from the </I> Federal <I> Office for Intellectual Property: </I> If parts of the description are inconsistent with the definition of the invention given in the claim, it should be remembered that according to Art. 51 of the Patent Act, the patent claim is authoritative for the material scope of the patent.