DE2341306A1 - METHOD FOR PRODUCING CARRIERS OF BIOLOGICALLY ACTIVE COMPOUNDS - Google Patents

Description

PATENT APPLICATION

Applicant: Ceskoslovenska akademie ve * d. _f Prague 1

Title: Process for the preparation of carriers of biologically active compounds

The invention relates to a method for producing Carriers of biologically active compounds,

For fixing soluble enzymes, antigens, antibodies and other soluble biopolymers, especially those of protein nature, methods are used which have been previously used, e.g. B. in a work by Silman and Katschalsky (Ann. Rev ", Biochem", 35-11, 873 (1966)) have been described in summary. According to these methods, the biologically active substances are either by Treatment with dual or teaching functional reaction

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converted into an insoluble three-dimensional complex or, in the case of a polyfunctional reactant, which is referred to in the bar as a "carrier", the biopolymer is bound to its surface by means of strong bonds. The second method is of particular importance for practical purposes, since it enables the active substance to be bound to artificially produced polymeric carriers which have a suitable macrostructure for use, for example, in reaction or chromatographic columns or in various medical devices. The biochemical effectiveness of the bound substance can of course be more or less impaired not only by the formation of the new bonds, but also by the mere fact of the fixation on a foreign surface, which restricts the free mobility of the protein molecule and also the access of the substrate, against which the protein is active. The tertiary structure of the biologically active macromolecule can also be adversely affected by the binding to the solid surface. For this reason, numerous and in some cases very successful attempts have been carried out to form stronger, but movement-free bonds between the carrier and the biologically active molecule. P. Cuatrecasas described (J. Biol. Chem. 245, 3059 (1970)) the multiple increase in capacity of the bound

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Staphylococcal nuclease when you get between the binding ligands and the framework of the support, e.g. B. the Sepharose or the polyacrylamide, one by several atoms longer Chain insert c

The subject of the invention is a far-reaching extrapolation of this idea and its surprisingly simple technical implementation. Instead of searching for complicated synthetic routes for binding the ligand to the macromolecular structure of the carrier by means of an elongated "arm" that is synthesized in the molecule that carries the ligand, a linear hydrophilic macromolecule is linked to the rigid three-dimensional basic structure of the carrier the already finished or preformed ligand carries _β This creates a macromolecular system in which on the surface of the three-dimensional network, which gives the system the mechanical strength and elasticity at the expense of blocking the free mobility of the polymer chains, freely movable and far into the Solution-reaching molecules of the linear polymer are linked,

According to the experimentally simplest procedure, such a system is prepared in such a way that the basic three-dimensional structure, which we can call the "primary support", with such reactive sites ·

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which are capable of binding to the ligands of the soluble linear "secondary carrier", these Ligands of the secondary carrier are already adapted to fix the biologically active substance. If we z. B. prepare the secondary carrier by copolymerization of acrylamide with methacrylanilide and the mercury of his anilide benzene rings with Q, mercury (II) acetate

according to the "Czechoslovak patent specification _o .

(Patent application PV 5742-72) run, this linear polymer can be attached to the surface of violle, Silk or reprecipitated collagen bind, or can, by linking with the cysteine units of these proteins this secondary carrier binds to the surface of the Sepharose mercapto derivative. In all of these Cases creates a carrier that has the macroscopic structure and mechanical properties of the primary Retains carrier and contains on the surface freely solvated long linear molecules on which Mercury anilide groups are located in a larger or smaller interval, which are capable of forming sulfhydryl units containing water-soluble proteins, such as. B. pepsin, to bind,

In a similar way, we can use the carrier synthesized by copolymerization of ethylene glycol monomethacrylate and dimethacrylate with 10 fo 2- (p-acetaminophenoxy) ethyl methacrylate in the form of macroporous small spheres.

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ger after previous hydrolytic cleavage of the acetyl groups the diazotized secondary water-soluble Carrier obtained by copolymerizing diethylene glycol methacrylate with p-acetaminophenoxymethacrylate and subsequent release of the aromatic amino groups by alkaline hydrolysis and diazotization the amino groups had prepared with nitrous acid, bind by coupling. The secondary diazotized Carrier binds to the primary carrier only with a small fraction of its diazotized groups, which is expresses itself in only a faint orange color, while the predominant part of the diazonium groups are prepared in this way Carrier for binding the phenol or imidazole-containing proteins is available.

One can address the primary carrier as a potential secondary Carriers also tie a polymer to which one can only subsequently use suitable ligands for binding of active substances. So you can use the primary carrier just mentioned, the free aromatic amino groups contains, convert with nitrous acid into a polymeric, crosslinked diazonium salt, which is carried out with a Copolymerization of ethylene diglycol methacrylate with methacrylanilide-prepared linear polymers can. The complex formed can be activated by mercuration with mercury earth perchlorate and so adapt to the binding of proteins that contain sulfhydryl groups,

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The same primary diazotized carrier can be used as a potential secondary carrier by coupling bind reaction of the ethylene oxide oligomer, which was prepared by reacting aniline with ethylene oxide, whereby replacement of the two hydrogen atoms of the amino group entered through an ethylene oxide chain with a terminal primary alcohol group. After coupling, you can convert the primary alcohol groups into isocyanate groups using the cyanogen bromide method, which the Have the ability to covalently bind the amino groups of the proteins.

In the following, the subject matter of the patent becomes duroh some Examples explained without restricting the invention to these examples,

example 1

35 parts by weight of diethylene glycol monomethacrylate, 2.6 parts by weight 2- (p-acetaminophenoxy) ethyl methacrylate and 2 parts by weight of 2,2-azobisisobutyronitrile were 50 per cent by weight. Ethanol completely dissolved and boiled under reflux for 3 hours. The cooled mixture was mixed with 100 parts by weight of barrels and under Distilled under reduced pressure until approximately 100 parts by weight of water-ethanol mixture had distilled off. The residue was again made up with 100 parts by weight of water

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added, and again 100 parts by weight of a water-ethanol mixture were added distilled off under reduced pressure «, After the second vacuum distillation were the residue 2 parts by weight of NaOH added in the form of a concentrated aqueous solution and the mixture at the boiling point Water bath heated for 40 minutes. After cooling, the reaction mixture was diluted with dilute hydrochloric acid (1: 1) pH 7 neutralized. The neutral solution of the polymer (the secondary carrier) was filtered through activated charcoal and used for subsequent attachment to the primary support

25 parts by weight of the hydroxyethyl methacrylate gel crosslinked with 40 # ethylene dimethacrylate, which in its three-dimensional structure contained 5 i ° 2 (p-acetaminophenoxy) methacrylate and whose molecular weight exclusion limit was 300,000 (ie the primary carrier), was allowed to swell overnight in distilled water »The excess water was sucked off on the frit and the gel in 250 parts by weight 5%. Sodium hydroxide solution ^{heated to 100 °} C., the acetamide bond being cleaved on the aromatic ring. The hydrolysis product was washed on the frit until the alkaline reaction was lost and the swollen, hydrolyzed gel was stirred in 50 parts by weight of In-HCl and cooled to ^{0 ° C.} 50 parts by weight of cooled in NaNOo solution were added in small portions and that

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The gel was left ^{to stand in the refrigerator at 0} ° C. for 2 hours and then washed with distilled water until the nitrile reaction had ceased. The swollen diazotized gel was then mixed with the linear copolymer of diethylene glycol monomethacrylate and 2-r (p-acetaminophenoxy) methacrylate (the secondary carrier) at pH8 and allowed to react for 2 hours at room temperature. After thorough washing with water, the unreacted diazonium chloride groups on the primary carrier coupled with ^ -naphthol (treatment with 5 parts by weight - JB naphthol and 1 part by weight of NaOxH in 200 parts by weight of water), with an intense red coloration of the gel. After repeated washing until the alkaline reaction was lost, the secondary carrier fixed to the gel was diazotized by reaction with 50 parts by weight of hydrochloric acid and 50 parts by weight of sodium nitrite under conditions similar to those of the primary carrier secondary carrier bound chymotrypsin at room temperature. The bound enzyme has a high esterase and proteolytic activity »,

Example 2

The secondary support was attached to the primary support as in Example 1, and the aminophenyl groups

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of the secondary carrier were treated with saturated mercury (II) acetate solution (100 parts by weight) for 3 hours Mercury in a boiling water bath. After thorough washing, the proteolytic enzyme papain was attached to the gel bound.

Example 3

To the solution prepared according to Example 1 - the secondary carrier, which contained 4 parts by weight of dry substance in 17 parts by weight of water, 5 parts by weight were added in a porcelain mortar. 10 per cent _e NaHOp ~ solution and 5 parts by weight of dilute HCl (1: 3). Assembled. Immediately after addition of the äure ° separated as a klebige white precipitate, which was ^f worked thoroughly in a mortar, in order to achieve a good contact of the two phases. After the aqueous layer had been poured off, the polymer was washed extensively while simultaneously working through it with 7m NaHpPO. Then the polymer was dissolved in 30 parts by weight of water. The solution gave no positive reaction to iodine starch paper, indicating that the unreacted nitrite had been completely removed by washing.

The primary carrier was made by suspension copolymerization of 48 parts by weight of 2-hydroxyethyl methacrylate,

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32 parts by weight of ethylene dimethacrylate and 1.6 parts by weight of Llethacrylanilid in the presence of 98 parts by weight of cyclohexanol, 10 parts by weight of lauryl alcohol and 1 part by weight of 2,2-azobisisobutyronitrile in 600 parts by weight of V / water with stabilization of the suspension with 6 parts by weight of polyvinylpyrrolidone. Ethanol and water carefully washed, dried and fractionated at the boil according to the particle size (the fraction with a particle size of 200-300 n was used).

The primary polymeric carrier was swollen in the universal borate buffer (pH 7.5) ^ nit 30 parts by weight the solution of the diazotized secondary carrier stirred and left to stand for 24 hours at room temperature, after The gel was left thoroughly vialed at pH 8 with chymotrypsin to form the bound active enzyme respond ο

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Claims (1)

" ¹¹ " 7341306 PATE M TA II PROVERBS: J A process for the production of carriers of biologically active substances, characterized in that chemical bonds can be formed between the reactive groups of a three-dimensional polymer as the primary carrier and the complementary reactive groups of a water-soluble polymer as the secondary carrier, whereby active ones can be formed on the secondary carrier and / or preformed ligands that are capable of binding biologically active substances. 2. The method according to claim 1, characterized in that the aromatic diazonium groups containing soluble polymers with a three-dimensional insoluble polymer, the couplable Contains phenol, amine or similar functions that represent the passive azo components, lets react. 3. The method according to claim 1, characterized in that g e k e η η -. draws that soluble polymers on aromatic ring containing covalently bound mercury, with three-dimensional, sulfhydryl groups Allows polymers to react «, 409809/1123 - 12 - ORlGIMAL INSPECTED " ¹² " 7341308 Process according to Claim 1, characterized in that soluble polymers which Contain isocyanate or Iöothiocyanatgruppen, with three-dimensional, aliphatic ^ or aromatic Can react polymers containing amino groups. 5. The method according to claim 1, characterized in that soluble polymers that Contain acyl anhydride or acyl halide groups, with three-dimensional, hydroxyl groups or primary or «allows polymers containing secondary amino groups to react« 6. The method according to claim 1, characterized in that one containing sulfhydryl groups soluble polymers with a three-dimensional polymer, the covalently bound mercurication contains, lets react. 7. The method according to claim 1, characterized in that a soluble polymer, containing aldehyde or kato groups, with a three-dimensional, hydrazide or hydrazine group containing polymers can react. - 13 409809/1123 ¹³ 73413ΌΘ 8. The method according to claim 1, characterized in that a three-dimensional, Polymer containing diazonium groups couples with a water-soluble polymer which is aromatic Contains bonded primary amino groups, and the complex formed with a new diazotization nitrous acid or its derivatives, or the primary amino groups by reaction converted into isothiocyanate groups with thiophosgene. 9. The method according to claim 1, characterized in that one containing a diazonium groups three-dimensional polymer couples with a water-soluble polymer, the aromatic amino, Contains amide or phenol groups, and the product formed is mercurized with mercury (II) salts. 10. The method according to claim 1, characterized in that a three-dimensional poly- ' meres, the isocyanate, isothiocyanate, carboxyanhydride, Contains acyl halide or aldehyde groups, with one containing primary aromatic amino groups Soluble polymers can react and the reaction product with nitrous acid or their Diazotized derivatives or can react with thiophosgene or mercury (II) salts. - 14 4ü9809 / 1123 " ^H ~? 3 A 1 3 O 11. The method according to claim 1, characterized in that a three-dimensional, Polymer containing isocyanate groups with a soluble polymer, the hydroxyl groups contains, allowed to react and the reaction product is reacted with cyanogen bromide. 12. The method according to claim 1, characterized in that a three-dimensional, Containing aldehyde, keto, isocyanate, isothiocyanate, acyl halide or acyl anhydride groups Polymer with a soluble polymer containing hydrazide groups, allowed to react and that resulting reaction product converted into a polyazide with nitrous acid. 409809/1123