DE2758036A1 - Adsorbent for separating bio-polymers - pref. comprising polymeric carrier with grafted dye gps. with nucleic acid affinity - Google Patents

Abstract

(A) Adsorbent for affinity-specific sepn. of macromolecular materials, esp. biopolymers, comprises a polymeric carrier to which a gp. with affinity for the biopolymer is covalently bonded either directly or via a polymeric "spacer". Pref. the carrier is a microporous, low compressibility polymer to which >=2 comonomers are grafted as spacer, one of these monomers having affinity for the biopolymer. Pref. the polymer carries the residue of a dye of formula (I) or (II) as a base- and/or structure-specific complex former for nucleic acids. (where X is CH or N; Y is O, S, NH or R'-R4 are each H or Me; A circled negative is an anion, e.g. chloride, perchlorate or oxalate), or the residue of an intercalating dye contg. a polycyclic (e.g. 2-4 ring) planar condensed ring system opt. contg. heteroatoms, e.g. N, O or S. (B) Dyes of formulae (I) and (II) are new, provided that at least one R' gp. is acryloyl. Specific new dyes are acryloylamino-malachite green (III), acryloylamino-phenyl neutral red (IV), acridine derivs. (V) and acryloylamino-phenanthridine derivs. (VI). (R is H or acryloyl; R1 is H or Me).

Description

The present invention relates to an adsorbent

for the affinity-specific separation of macromolecular substances, in particular biopolymers, such as mixtures of single-stranded and / or double-stranded Nucleic acids, especially deoxyribonucleic acids (hereinafter abbreviated as DNA), a process for the production of this adsorbent and its use.

The isolation of single genes or sets of the same genes in repetitive arrangement from the genome of eukaryotic cells is not just pure scientific interest, but also with regard to genetic engineering of great practical importance. Such isolation was only possible when the base addition of the gene or the gene group with their "spacers" at least 6 to 7% different from the mean base composition of the total genome. as Separation methods were mostly cesium ion density gradient centrifugations with DNA-base-specific ones Additives such as silver, mercury or platinum ions or actinomycin, netropsin or the dye "Hoechst 33258" is used. These procedures have only limited ones Capacity, are expensive and time consuming.

The development of affinity chromatography materials for Biopolymers has seen the isolation of a wide variety of biopolymers in recent years greatly simplified or their pure representation is made possible in the first place. With these Process is usually based on a carrier material, which after chemical Activation with a substance is converted, the dAs to be isolated Binding biopolymers as specifically as possible. Because of this specificity, the Biopolymers ideally selectively from a mixture of similar compounds to the Chromatography material adsorbed and then under suitable conditions can be desorbed in the pure state (P.Cuatrecasas and C.B. Anfinsen, Ann.Rev.Biochem. 40 (1971) 259-278).

Despite the abundance of examples of the successful application of this Method has so far not been able to use chromatography material for a large number of biopolymers be developed that also have a similarly selective and programmable separation for nucleic acid mixtures enabled. A high resolution, fractionation of nucleic acid mixtures on a gram scale, however, is a prerequisite for the aforementioned isolation of single genes or sets of the same genes.

With the existing methods for the separation of nucleic acids with Low molecular weight, for example transfer ribonucleic acids, will do the separation in the various species of adsorbents achieved the ion exchange properties combine with lipopillic interactions (R.M. Kothari and V. Shankar, Journal of Chromatography 98 (1974) 449-475). The possibilities for interaction are essentially the same of the carrier with the rare bases of the nucleic acids used in the various Transfer ribonucleic acids are present in different amounts.

Because the higher molecular weight ribonucleic acids and deoxyribonucleic acids usually do not contain rare bases, methods for their separation can be used can only be based on the following distinctive features: a) Ratio of single to double strand b) Differences in the base addition c) differences in the base sequence d) differences in the molecular weights.

e) Differences in the tertiary structures.

All of these features are actually used in fractionation methods commonly used today used (R.M. Kothari, Chromatog. Rev. 12 (1970) 127 to 155).

The most effective method, fractionation in the salt gradient in the ultracentrifuge, differences according to b, c and e lead to differences in the floating density for individual DNA species, which is achieved by adding base-specific Substances can still be enlarged.

The selectivity decreases with decreasing molecular weight, so does the capacity with increasing molecular weight. In adsorption chromatography on hydroxyapatite the pricing takes place essentially according to a and only to a lesser extent according to b, by having nucleic acids richer in guanine-cytosine even at slightly lower salt concentrations are desorbed than the adenine-thymine-rich X components (W.Pakroppa and W.Müller, Proc.Nat.Acad.Sci. USA, 71 (3) (1974) 699-703).

In a very analogous manner, double-stranded nucleic acids can also be linked to specific Protein kieselguhr adsorbents (e.g. methyl serum albumin kieselguhr) (according to Differences in the base composition are fractionated, with the guanine-cytosine-richer again DSts species are eluted first (J.D.Alandell and A.D. Herhey, Analytical Biochemistry 1 (1960) 66 to 77; N.Sueoka and Tstai-Ying Cheng, J.Mol.Biol. 4 (1962) 161 to 172). The actual mode of action of these more accidentally discovered adsorbents is not known. Therefore, the low selectivity of these materials could despite of all efforts to date have not been fundamentally improved.

Only in the last few years have the systematic investigation been carried out of numerous substances that form complexes with nucleic acids, compounds discovered leading to a targeted synthesis of materials for affinity chromatography appeared suitable (W. Müller and D.M. Crothers, Eur.J.Biochem. 54 (1975) 267 bis 277; W. Müller, H.Bünemann and N.Dattagupta, Eur.J.Biochem.

54 (1975) 279 to 291 and W. Müller and F. Gautier, Eur.

J.Biochem. 54 (1975) 385-394). What are the advantages of using such well investigated he brings substances in the separation of nucleic acid mixtures, was already able to use the examples of the combined use of hydroxyapatite and Ethidium bromide as a base-specific additive for the separation of superhelical and helical DNA (W.Pakroppa, W.Goebel and W.Müller, Analytical Biochemistry 67 (1975) 372 to 383) and of hydroxyapatite in combination with phenylneutral red derivatives as base-specific complexing agents in the separation of double-stranded DNA species (W.Pakroppa and W.Müller, Proc.Nat.Acad.Sci. USA, 71 ((3) (1974) 699 to 703).

The resolving power of these last-mentioned methods is with the comparable to a preparative cesium chloride density gradient, i.e. DNA fractions with differences in (G + C) content (where G stands for guanine and C for cytosine) can be separated from each other. Despite the high I capacity, the process has the disadvantage that DNA mixtures with an average molecular weight of the components of more than 20 x 106 can no longer be handled well and that the used as an adsorbent special 1iydroxyapatit must be made by yourself.

It has long been known that nucleic acid mixtures in the polyethylene glycol-dextran system under certain conditions in RNA and single-stranded DA on the one hand and double-stranded On the other hand, allow DNA to separate.

The double-stranded DNA always enriches in the (lighter) Polyäthylcnglykolphase on, that is, it has a higher distribution coefficient as single-stranded nucleic acids. Let the absolute values of the distribution coefficients The addition of potassium and lithium salts increased by about 3-4 powers of ten vary, fractionation according to the base composition succeeds in these systems However not.

The object of the present invention is now to provide adsorbents provide with which a base- and / or structure-specific separation of Biopolymers and in particular mixtures of single-stranded and / or double-stranded and / or supercoiled and / or linear nucleic acids, especially of DNA mixtures high capacity can be achieved.

Under affinity specificity is above all the structure specificity and to understand the base specificity.

It has now been shown that the task mentioned with an adsorbent can be dissolved, which has a polymeric carrier material to which directly or over longer intermediate groups (spacers) a residue or residue with an affinity for the biopolymer a base- and / or structure-specific group, in particular covalently bonded is.

The invention therefore relates to an adsorbent for the affinity-specific Separation of macromolecular substances, especially biopolymers such as nucleic acids, which is characterized by a polymer carrier material to which a biopolymer is attached affine residue is covalently bound directly or via a polymeric "spacer".

A particularly preferred embodiment of the present invention now relates to an adsorbent of the type defined, which is characterized by a small-pore, less compressible, polymeric carrier material on which a copolymer is grafted on from at least two copolymerizable monomers, of which one carries the residue with an affinity for the biopolymer.

Base-specific and / or structure-specific complexing agents, which are described in the literature references mentioned, can be used as radicals having an affinity for the biopolymer. In particular, there are residues of dyes of the following general formulas I and II in which general formulas X for a Cn group or a nitrogen atom, Y for an oxygen atom, a sulfur atom, an NH group or a group of the formula R1 is independently hydrogen atoms or methyl groups, R2 is a hydrogen atom or a methyl group, R3 is a hydrogen atom or a methyl group, R4 is a hydrogen atom or a methyl group and AO is an anion such as a chlorine anion, a perchlorate anion or an oxalate anion.

Base-specific and / or structure-specific complexing agents which are particularly preferred according to the invention and are bound to the polymeric carrier material are the residues of the following dyes: 1. Diamidinophenyl-indole (DAPI) 2. Malachite green 3. Crystal violet 4. methyl green 5. auramine 6. Hoechst dye 33258 7. Di-tert-butyl-proflavine 8. Di-tert-butyl-acriflavine 20. Proflavine 25. Thionin 26. Acridine Orange 27. Pyronine G. 28. Thiopyronine 31. Methylene Blue where Me stands for the methyl group in the above formulas.

33. ethidium bromide The base- and / or structure-specific complexing agents preferred according to the invention, however, are phenyl-neutral red of the formula and malachite green of the formula These dyes are known per se and are commercially available or can be prepared by procedures which are familiar to the person skilled in the art and, for example, in the above-mentioned publications by W. Müller and DMCrothers (Eur.J.Biochem. 54 (1975) 267 to 277), W. Müller, H.Bünemann and N.Dattagupta (Eur.J.Biochem. 54 (1975) 279 to 291) and W. Müller and F. Gautier (Eur.J.Biochem. 54 (1975) 385 to 394) are.

These base- and / or structure-specific complexing agents or dye residues can be covalently bonded to the carrier material by methods that are familiar to the person skilled in the art, for example by esterification with hydroxyl groups present on the carrier material via carboxyl groups introduced into the dye molecule, by amide formation , by urethane formation or also by copolymerization in the absence and preferably in the form of other copolymerizable monomers via copolymerizable double bonds that are introduced into the dye molecule, for example via an acrylamide group, as is the case for the base- and / or structure-specific complexing agents preferred according to the invention, acrylic-phenyl-neutral red and Acrylic malachite green of the following formulas is the case. These names are common names, the exact name is Acryloylaminophenylneutralrot and AcryloylaminomalachitgrUn.

These base- and / or structure-specific complex binders can by the person skilled in the art in a manner known per se by introducing the acrylic residue into the called dye molecules are produced. This also applies to the ones above called dye molecules.

The term acrylic residue is synonymous in the present cases with acryloyl residue.

However, the preparation is preferably carried out according to the examples 3 and 4 described procedure. The acryloyl dyes are new.

According to the invention, the polymeric carrier material used is preferably used a small-pore, less compressible polymeric carrier material, such as poly-bisacrylamide. These small-pore and poorly compressible polymeric carrier materials have become for affinity chromatography of highly viscous nucleic acid solutions as very proved beneficial. Furthermore, these polymeric carrier materials are not strong Interactions with the affine residues, in particular with the base- and / or structure-specific Complexing agents for nucleic acids, either directly or via polymeric spacers are bound to the polymeric carrier materials.

It has proven to be advantageous between the polymeric carrier material and aem base and / or specific complexing agent a longer spacer, that is to introduce a longer molecular chain without functional groups, as this results in the Complex formation of the base- and / or structure-specific complexing agent with the helical nucleic acid is not affected by steric hindrance.

In a particularly preferred manner, therefore, in the case of the invention Adsorbent the residue with affinity for the biopolymer or the base- and / or structure-specific one Complexing agent via a copolymer, which preferably has a degree of polymerization from 200 to 300, bound to the polymeric carrier material. To form this Copolymers are preferably used a further monomer that is compatible with that for the Polymerization introduced into the base- and / or structure-specific complexing agent Group and those present on the polymeric carrier material, copolymerizable Groups is copolymerizable.

Acrylamide is preferably used as this further monomer, and grafts said dyes containing acrylic groups onto a poly-bisacrylamide which still has free, copolymerizable double bonds.

The invention also relates to a method for producing the aforesaid Adsorbents, which is characterized in that one first by polymerization or by polycondensation of at least one monomer, which in addition to that for the functional group required for polymerization or polycondensation has another functional group, over that of the affinity for the biopolymer The remainder can be bound directly or via a polymeric spacer, in a manner known per se Way forms the polymeric carrier material, the polymeric carrier material optionally comminuted to the desired particle size and then the one with an affinity for the biopolymer Remainder either directly or by copolymerization in the presence of at least one other Grafted monomers as a copolymer.

The grafting can be done in any way, for example by esterification, by amide formation or by urethane formation, for which one uses conventional reagents and uses customary process conditions or those suitable for these reactions Groups in the polymeric carrier material or the residue with an affinity for the egg polymer introduces.

Flan can, however, also contain the residue with an affinity for the biopolymer a copolymerizable double bond has been introduced in the presence of an excess another copolymerizable monomer as a copolymer on polybisacrylamide Graft on, as polybisacrylamide still has free, copolymerizable double bonds having. The graft copolymerization can be carried out in water or any inert organic Solvent and carried out in the presence of a conventional polymerization catalyst will.

Preferably used as copolymerizable for the biopolymer affine residue acrylic-phenylneutralred or acrylic-malachite green and as another copolymerizable Monomeric acrylamide, a copolymer of these components on polybisacrylamide grafted on. According to a preferred procedure, a copolymer is grafted out Acrylamide and acrylic phenyl neutral red or acrylic malachite green with a degree of polymerisation from 200 to 300 on.

This graft copolymerization is preferably carried out on one Ratio of that having a copolymerizable double bond for the biopolymer affinity radical to the further copolymerizable monomer from 1: 100 to about 1: 5000 this reaction is preferably carried out at a ratio of 1: 3000. It has been shown here that the initial molar ratio of the further ionomer to the one which has a copolymerizable double bond and has an affinity for the biopolymer Remainder is also retained in the copolymer. With an average degree of polymerization of the copolymer from 200 to. 300 can be assumed that the grafted Copolymer chains each carry only one residue with an affinity for the biopolymer. The copolymers thus form spacers of different lengths between the one with affinity for the biopolymer Remainder and the polymeric carrier material, in particular the poly-bisacrylamide particles.

It has been shown that the adsorbents according to the invention the set Meet requirements and can easily be obtained synthetically in a simple manner can. For example, the polymeric carrier material is obtained by polymerization of bisacrylamide in highly concentrated solution. The polpnerization product is a brittle, colorless solid that easily becomes sedimenting cullet can be grated by multiple sieving up to. desired particle size can be homogenized.

The swelling behavior in aqueous solution is also at extreme salt concentrations constant and practical from the flow rate in chromatography independent. Since these particles still have a large number of unreacted, polymerizable Containing double bonds, they can, as already explained above, for the further graft polymerization can be used.

So you get with the adenine-thymine-specific acrylic malachite green and with the guanine-cytosine-specific acrylic-phenylneutralrot with acrylamide in Presence of these poly-bisacrylanide particles by copolymerization in aqueous L5mg the adsorbents or2ugten according to the invention.

40 to 50% of the copolymers formed have an average Degree of polymerization of approx. 200 normal winter reaction conditions in the form of Graft copolymers bound to the carrier particles from the poly-bisacrylamide. To the strongly colored adsorbent particles do not give any dye to the thorough rinsing more and can then be used directly for affinity chromatography of nucleic acids can be used. The nucleic acid mixture is usually in 0.01 M phosphate buffer adsorbed on the material and desorbed with a linear salt gradient. the The type of salt depends on the type of nucleic acids. While for desorption of ribonucleic acids, including transfer ribonucleic acids, sodium chloride is sufficient for the base-specific elution of DNA from acrylic malachite green containing Percnlorate adsorbents required.

It has been shown that it is also used for the production of the carrier material it is possible to start from insoluble materials with hydroxy or amino stairs, which are in pearl form, such as. B. Amnoylkylsepharose, micracrystalline cellulose, cross-linked hydroxyethyl methacrylate or epoxypropyl acrylate. Be with these carriers the acrylic groups required for subsequent grafting by reaction with acryloyl chloride introduced.

A similarly high dye loading can be achieved in this way as with the grafting from acrylamide shards.

The invention thus also relates to the use of said adsorbents for the separation of biopolymers by two-phase affinity distribution, by two-phase distribution chromatography, by gel electrophoresis and in particular by affinity chromatography. With advantage can these adsorbents for the separation of mixtures of single-stranded and / or use double-stranded nucleic acids, in particular from DNA mixtures.

Since the adsorbents according to the invention, the base- and / or structure-specific Carry complexing agents based on 5-phenylphenazonium salts, for guanine-cytosine-richness DNA specifically act and the adsorbents according to the invention, the base and / or structure-specific complexing agent based on triphenylmethane dyes to act specifically for adenine-thymine-rich DNA, the desired base and / or structure-specific or sequence-specific separations of nucleic acid mixtures carry out.

As predominantly structure-specific; we ;; omplxSildn-r, the Intercalation enabled connections in question.

Intercalation is understood to mean, in particular, a special interaction of dye molecules with DNA, with the dye molecule between two neighboring ones Base pairs of the DNA double helix pushes. Which substances are structurally capable of intercalation are familiar to the person skilled in the art. They are planar, electron-rich chromophores. They are mainly the residues of planar polycyclic ones, such as bi-, tri- or tetracyclic ones fused ring systems containing heteroatoms such as nitrogen, oxygen and sulfur may contain, understand.

The effect can be increased if, such as. B. in the case of ethidium bromide and of 9- (acryloylaminoethyl-amino) -2-methoxy-6-chloro-acridine, an amino group is present on the ring system, which enables binding to the phosphate groups of the DNA. It is easy for the person skilled in the art to use compounds that are suitable for use, such as acridines, benzacridines, phenanthrenes, phenantridines, pyridoindoles, Naphthalenes, Naphthothiophene, Benzothiophenes, Thianthrenes, Xanthines, Phenoxanthines, Quinolines, quinoxalines, phenanthrolines, phenothiazines, phenoxazines, phthalazines etc.

Acridine derivatives, for example 9-acryloylamino-ethyl-amino) -2-methoxy-6-chloro-acridine or phenanthridine derivatives, for example ethidium bromide (5-0thyl-3,8-diamino-6-phenyl-phenanthridium bromide) After their coupling to bisacrylamide particles, separate double-stranded ones that are not base-specific DNA from supercoiled DNA (elution diagram Fig. 7). This is very important for one simple separation of plasmid and virus DNAs from cell disruptions. The pillar replaces the complex ethidium bromide-cesium chloride gradient.

This separation is also with the acryloylaminophenyl neutral red column possible because both dyes form a complex with a similar structure with the DNA (intercalation complex) form. The acryloylaminophenyl neutral red separates both structural as well as base-specific. Depending on the separation problem at hand, there is therefore a series of variations in the composition of the adsorbent are possible.

Since the base and / or structure-specific complex formers over are bound by a stable covalent carbon-carbon bond Adsorbents according to the invention can also be used at more extreme pH values, washed with detergents and regenerated with saline. Furthermore is the incorporation of several different base- and / or structure-specific complexing agents or monomers possible, so that adsorbents can be formed which are necessary for the separation can be used by transfer ribonucleic acids. The chain length of the grafted Copolymers and thus the length of the chains wipe the polymeric carrier material and the base / or structure-specific complexing agent can ih the usual way controlled by mercaptoethanol.

In the following, the present invention is further illustrated by examples explained with reference to the accompanying drawings.

In the drawings: FIG. 1 shows a diagram for the production according to the invention particularly preferred adsorbents due to the claimed two-stage polymerization; Figure 2 shows the elution diagram of a mixture of three sheared bacterial deoxyribonucleic acids using an adsorbent according to the invention; 3 shows the elution diagram a mixture of three sheared, bacterial deoxyribonucleic acids, which under Use of a further adsorbent according to the invention was obtained; Fig. 4 that Elution diagram of an EcoRI hydrolyzate of phage deoxyribonucleic acid, obtained using an adsorbent of the present invention; Fig. 5 that Elution diagram using an adsorbent according to the invention in the Separation; a DNA-RNA mixture was obtained from a digestion of M. luteus; and FIG. 6 shows the elution diagram of a mixture of four different transfer ribonucleic acids from yeast obtained with an adsorbent according to the invention.

7 shows the elution diagram for the separation of supercoiled and linear DNA.

The following examples serve to further illustrate the invention.

Example 1 A) Production of the poly-bisacrylamide carrier material.

50 g of N, N'-methylnbisacrylamide are suspended in 100 ml of methanol in a high 1-liter poly-ethylene beaker and mixed with 200 ml of keehendem double-distilled Water. While stirring the mixture with your magnetic stirrer, the bisacrylamide goes off Completely in solution.

Pipetted into the solution, which has been cooled to 60 ° C. and stirred vigorously one first 1 ml of N, N, N ', N'-tetramethylethylenediamine and then gives in one pour a solution of 0.2 g of ammonium peroxydisup) phate in 5 ml of water. Immediately after To mix, turn off the stirrer. The solution becomes cloudy after a few seconds and solidifies to a colorless block when heated. The polymerization is after Canceled about 1 minute by quickly removing the polymer block with a large spatula is roughly crushed. The fragments are in a 2 liter polyethylene beaker suspended in 1 l of methanol and ground to a granular paste. For further homogenization of the particles, the pulp is rubbed one after the other through sieves with decreasing mesh size (1mm x 1mm, 0.5mm x 0.5mm, and 0.25mm x 0.25mm).

The resulting shards are allowed to sediment, whereupon the milky-cloudy Upper stood decanted. This procedure is repeated several times, initially with methanol, then with a methanol / water mixture, then with a 1% Acetic acid solution and finally again with distilled water. The received Polybisacrylamide particles are stored in aqueous solution and can be used at any time can be used for the graft copolymerization described below.

About 300 ml of sediment are obtained from 300 ml of the polymerization solution Polybisacrylamide particles.

B) Grafting on a copolymer of acrylamide and acrylic phenyl neutral red on the polybisacrylamide obtained in stage A.

> lan suspended 60 ml of the obtained in stage A, settled Polybisacrylamide particles in a bottle with a screw cap in 20 ml of distilled Water. 5 g of acrylamide and 10.3 mg of acrylphenyl neutral red chloride are dissolved in this suspension, To do this, gently shake the suspension in the bottle until the dye dissolves completely resolved. The copolymerization is then carried out by adding 0.050 ml MercaptoEthanol to adjust the degree of polymerisation and 1 ml of a sodium peroxide solution (0.8 g sodium peroxide in 100 ml in sodium acetate buffer with a pH value of 5.5) started as a catalyst.

The suspension formed is immediately filled with nitrogen for a further 5 minutes gassed and then kept at room temperature with the exclusion of air. After about 30 minutes the reaction mixture has warmed up significantly, and after approx.

The polymerization is essentially complete in 2 hours.

It is left to stand for a few hours before the viscous suspension transferred to a suction filter. The viscous, deeply colored solution is filtered off with suction and washed Thoroughly wash the strongly colored particles left on the suction filter with water, until the washing solution remains colorless even when using an acetic acid solution. After a final continuous rinse with a 0.01m sodium phosphate buffer with a pH of 6.0 overnight, the material can be used for affinity chromatography use of nucleic acids.

In the case of acrylamide, the yield is determined by means of a 14C label and for the dye via the local density. The yields are the following: Dye: Incorporated in dsas copolymers (bound and unbound) 60.5% acrylamide: incorporation in the copolymer (bound and unbound) 49.2% dye: incorporation into the bound Copolymers, based on the sum of 51.0% acrylamide: incorporation into the bound copolymer, based on the total 57.0%.

It should be noted that the starting molar ratio of dye to acrylamide (1: 3900) is also retained in the polymer. Since the mean degree of polymerization of the copolymer was found to be 20 to 300, it can be assumed that the copolymer chains each carry only one dye molecule per chain.

The method described above is illustrated schematically by FIG. 1 explained ..

Example 2 Separation of nucleic acid mixtures by affinity chromatography of adsorbents according to the invention.

As the elution diagrams shown in FIGS. 2 to 6 show, is the possible application of the inventive adsorbents in the field of Nucleic acid fractionation very versatile. Depending on the base specificity of the bound Mixtures of double-stranded nucleic acids are made according to their dye Separated base composition.

This is how the ig. Figure 2 shows the elution diagram of a mixture of three sheared bacterial deoxyribonucleic acids with a mean .e: oleRuelar weight of approx.

700,000 D with different base compositions.

a column measuring 16 cm x 1.5 cm was used for this purpose, Acrylalachite green acrylamide grafted onto Polybisacrylamidtcilchen as an adsorbent -Copolymers contains. The amount of deoxyribonucleic acid used is approx. 1 mg.

Fig. 3 shows the elution diagram of a mixture of three sheared bacterial deoxyribonucleic acids with an average molecular weight of approx. 700,000 D, which have a different base composition.

A column with was used to elute 1 mg of the nucleic acid mixture the dimensions 15.2 cm x 1.5 cm used as the adsorbent bisacrylamide particles contains, on which Acrylphenylneutralrot-Acrylamid-Copclyme are grafted.

Using the adsorbents according to the invention, DNA fragments, obtained by the action of restriction endonucleases, fractionated will. This is shown in Figure 4, which is the elution diagram of an Eco-RI hydrolyzate of A phage deoxyribonucleic acid reproduced in an amount of 0.5 mg under Using a column measuring 15.8 cm x 1.5 cm, the contains polybisacrylamide particles as adsorbent, onto the acrylic malachite green-acrylamide copolymers are grafted on.

Also the separation of ribonucleic acids from deoxyribonucleic acids the same organism succeeds smoothly with the kcisorbenticn according to the invention, such as from he Fig. 5 can be seen. In this Fig. 5 is the elution diagram of a DNA-RNA mixture shown from a digestion of M. luteus (72% G + C), with the mixture in a close of 1 mg was separated using a column measuring 16.2 cm x 1.5 cm, which column is loaded with polybisacrylamide particles on the acrylic malachite green copolymers are grafted on.

Fig. 6 shows the elution diagram for a mixture of four different, Transfer ribonucleic acids obtained from yeast. With that in an amount of 1.5 mg separate mixture is a mixture of t-RNAPhe, t-RNALys, t-RNAGlu and t-RNAGly. A column with the dimensions 16 cm x 1.5 cm, which was charged with polybisacrylamide particles the acrylphenyl neutral red acrylamide copolymers are grafted on.

Fig. 7 shows the elution diagram for the separation of supercoiled and linear DNA. Polybisacrlamide particles were used for affinity chromatography used, with 9- (acryloylamino-ethyl-amino) -2-methoxy-6-chloro-acridine particles are grafted on.

When using the adsorbents according to the invention for the separation of nucleic acid mixtures by affinity chromatography as described above aqueous salt concentration gradients are used as the eluent, including the salts used are preferably alkali metal salts, such as sodium chloride, sodium perchlorate, Lithium perchlorate etc. used.

In certain cases, buffers can also be used, for example Phosphate buffer, being used for adsorbents acting as base-specific complexing agents contain the rest of Malachite Green, a weakly acidic buffer with a pH value from 5.5 to 6 used, for example a 0.01 molar phosphate buffer with a pH from 5.5 to 6.

The optimal salt gradients, their components, Concentrations, pEl values and the buffers used for them can, however, be determined by the person skilled in the art can easily be determined.

In summary, it should be noted that the invention Adsorbents are ideally suited for the affinity-specific separation of macromolecular substances and in particular biopolymers, such as nucleic acid mixtures. These adsorbents can be produced in a simple manner and. in terms of theirs Set the base specificity in a targeted manner.

Example 3 Production of acryloylamino-malachite green la) 15.1 g (0.1 mol) 4-nitro-benzaldehyde and 36.3 g = 38 ml (0.3 mol) N, N-dimethylaniline and 40.5 g of zinc chloride (anhydrous) are mixed and heated to a bath temperature of 100 ° C. The mixture, which was initially easy to move, turns into one over the course of the reaction time solid, green mass that can no longer be touched. After a reaction time of 5 hours one cooling down. The product is taken up in 150 ml of acetone, after some Time while stirring the product is dissolved and zinc salt is excreted. The insoluble Salt is suctioned off, washed with acetone and the filtrate is mixed with enough water until crystallization occurs. The crystals are filtered off with suction, with water, isopropanol and ether washed.

Yield: 28.7 g (76.6% of theory); M.p. 1680C (the substance is photosensitive).

lb) 3.75 g (0.01 mol) of the nitro compound obtained according to la) dissolved in 200 ml of glacial acetic acid (99%) and 20 ml of water and mixed with 4 g of zinc chloride. Then 20 g of zinc dust are added with stirring and cooling at room temperature (20-300C) added in portions. It will be 30 minutes at room temperature touched,;. then sucked off the excess zinc and washed with glacial acetic acid. The filtrate is evaporated in vacuo at 50 ° C, the residue in 150 ml of chloroform and 100 ml of water dissolved and separated, then the chloroform phase with 80 ml 2N sodium carbonate solution and 100 ml of water are shaken. The chloroform phase will separated and dried with sodium sulfate and filtered, so then to about 100 ml concentrated in vacuo at 50 ° C. and reacted further directly (pale violet solution).

lc) The obtained 100 ml of chloroform solution of the amino compound are mixed with 50 ml of methanol. There are 10 g of sodium carbonate (anhydrous) and one Spatula tip 1,3-Dinitrobenzol added and then with stirring and ice cooling (0 ° C - 50 ° C) 1.81 g = 1.65 ml acryloyl chloride (twice the molar amount) added dropwise. The suspension is stirred overnight at room temperature, after which the sodium carbonate is added suctioned off and the filtrate evaporated in vacuo at 35-400C. The residue will taken up in 100 ml of chloroform and 50 ml of water and shaken, the chloroform phase is separated off, extracted with 50 ml of water, dried with sodium sulfate and evaporated in vacuo at 35-400C.

The oily, greenish residue is rubbed with 20 ml of isopropanol and refrigerated for crystallization. The crystals are filtered off with suction, with isopropanol and ether washed and dried in a desiccator.

Yield over stages lb and lc: 2.9 g (72.7% of theory); M.p. 1750C C, H, N analysis: calcd. C 78.2 H 7.27 N 10.52 found. C 75.9 H 7.21 N 9.999 NMR spectrum (Hexadeuterodimethylsulfoxid): CH3- (N-Methyl) S 2.83 PPM, uCH- (Acryl) Q 5.7 PPM, CH2 = (Acryl) P X, 3 PPM, aromatic protons between 6.5 and 7.5 PPM.

ld) 250 mg (0.001 mol) of chloranil are dissolved in 15 ml of tetrahydrofuran and added 0.4 g (0.001 mol) of acryloyl compound (lc). The solution will be instant blue and after about 2 hours of standing at room temperature crystallization occurs. It is left to stand overnight at room temperature, then the crystals are suctioned off, washed with tetrahydrofuran and ether and dried in a desiccator. To further Purification is taken up again in water, neutralized with HCl and then with Shaken out ethyl acetate. The clean product is made by adding sodium chloride precipitated from the aqueous solution.

Yield: 0.335 g (84.2% of theory) in the NMR spectrum of 1d) is in the Compared to lc) a shift in the N-methyl proton signal to 3.3 PPM can be observed.

Example 4 Production of Acryloylaminophenylneutralrot la) 6.9 g (0.05 mol) of 4-nitro-aniline are dissolved in 150 ml of tetrahydrofuran / chloroform 1: 1, a spatula tip of 1,3-dinitrobenzene and 8 ml of triethylamine are added. While stirring 9 ml (0.1 mol) of acryloyl chloride are slowly added dropwise at 10-20 ° C. The solution is stirred overnight at room temperature, with triethylammonium chloride crystallized out. Then the tetrahydrofuran-chloroform mixture is evaporated in vacuo at 50 ° C, the residue rubbed with water, filtered off and washed with water, isopropanol and ether washed. The product is dried at 50 ° C. in vacuo.

Ib) 2.5 g of acryloyl compound 2a are in 60 ml of tetrahydrofuran under Stirring dissolved at 500C and diluted with 60 ml of glacial acetic acid (99%) and cooled to 300C. Then 10 g of zinc dust are added in portions, the temperature rises to 35-400C with ice cooling.

The excess is then stirred for 10 minutes at room temperature Sucked off zinc and washed with glacial acetic acid. The filtrate is evaporated in vacuo at 450C. The oily evaporation residue is practically pure chromatographically and thus becomes next reaction used.

lc) A solution of 4.2 g (0.02 mol) of N, N-dimethyl-pphenylenediammonium dichloride and 2.88 g (0.02 mol) of o-toluidinium chloride in 400 ml of H 2 O are slowly stirred with stirring at room temperature with a solution of 12 g of sodium chromate (0.04 mol) in 100 ml Water added. The separated green product is sucked off after 15 minutes, washed three times with water, the washing solution remaining green, the precipitate is then immediately processed further by suspending it in a smaller amount of water and is homogenized. The homogeneous suspension is diluted to 1.6 liters with water. After adding 1.15 x 0.02 mol of N-acryloyl-p-phenylene-diammonium acetate in 100 ml Water is the reaction mixture with 130 ml of 3 M sodium acetate solution to pH 4.9 posed. The mixture is then brought to the boil with stirring heated. The solution initially turns deep blue and then dark purple when it boils (Boil for 5 minutes to complete the reaction). Then will sucked the boiling hot solution through a suction filter, the filtrate (approx. 2 l) with 240 g of sodium chloride adjusted to 2 m solution and left to stand overnight in the refrigerator. The precipitated crystals are suctioned off and dried in the desiccator (it may cannot be washed with water as the substance is easily soluble in water).

Yield: 2.4 g of crude product. For further purification, 1.3 g of des Crude product dissolved in 25 ml of methanol and 0.1 M sodium chloride (4: 1) and on a Silica gel 60 column (4 x 95 cm) applied. It is then made with the same solvent eluted (fraction volume = 20 ml). Fractions 22-55 are combined and in vacuo concentrated to approx. 15 ml. The crystals are filtered off with suction, with a little 0.1 M sodium chloride washed and dried in a desiccator.

Yield: 0.665 g of chromatographically pure dye.

Example 5 Preparation of 9- 9- (acryloylamino-ethylamino) -3-chloro-6-methoxy-acridine 854 mg of 3,9-dichloro-6-methoxy-acridine are mixed with 1 ml of ethylenediamine and 7.7 g of phenol Heated for 30 minutes in an oil bath at 600C. The melt is in 200 ml of chloroform and 150 ml of water taken up, adjusted to pH 4 with acetic acid and equilibrated. The organic phase is 3-4 times with 0.1 M sodium acetate buffer re-extracted and then discarded.

The aqueous extracts are adjusted to pH 9.5 with soda solution, extracted with chloroform-n-butanol (20: 1), the organic phase over silicone paper filtered and evaporated in vacuo. The residue is dissolved in 0.1 M sodium acetate buffer dissolved, residues of dimerized product filtered off and the solution adjusted to pH 10.

After 1 hour at room temperature, the precipitate is filtered off, washed with a little dilute ammonia solution (40C) and dried. Yield: 50-80 % d. Th.

206 mg of the amino compound obtained are dissolved in 25 mol of chloroform dissolved in the heat and, after cooling, mixed with 0.3 ml of acrylic chloride. The solution immediately turns darker in color. After a few minutes the acroyl derivative separates away. After suctioning off and washing with a little chlorine form, 122 mg (50% of theory) are obtained. Th.) 9-Acryloylamino-ethylamino) -3-chloro-6-methoxy-acridine in chromatography pure form.

Blank page

Claims (28)

Adsorbent for the affinity-specific separation of macromolecular Substances, process for their production and its use P a.t e n t a n 5 p r u c h e 1. Adsorbent for the affinity-specific separation of macromolecular Substances, in particular biopolymers, such as nucleic acids, g e k. e n n z e i c h -n e t d u r c h a polymeric carrier material to which a biopolymer affine Rest is covalently bonded directly or via a polymeric "spacer". 2. Adsorbent according to claim 1, ge k e n n z e i c h -n c t d u r c h a small-pore, little compressible, polymeric carrier material, on there one Copolymer of at least two copolymerizable monomers as a polymerizer "Spacer'í is grafted on, one of which is the residue with an affinity for the biopolymer wearing. 3. Adsorbent according to one of the preceding claims, d a d u r c h g e k e n n n z c i c h n e t that the polymeric carrier material than for the biopolymer affine residue a base- and Z- or structure-specific X-complexing agent for nucleic acids wearing. 4. Adsorbent according to claim 3, characterized in that the polymeric carrier material carries the remainder of a dye of the following general formulas I or II as a base and / or structure-specific complexing agent for nucleic acids in soft general formulas X for a C} l group or a nitrogen atom, Y for an oxygen atom, a sulfur atom, an Nfl group or a group of the formula R1 independently of one another are hydrogen atoms or methyl groups, R2 is a hydrogen atom or a ethyl group, R3 is a hydrogen atom or a methyl group, a hydrogen atom or a methyl group and A (3 is an anion such as a chloride anion, a perchlorate anion or an oxalate ion or the remainder of an intercalating dye, containing a planar polycyclic, such as bi-, tri- or tetracyclic, condensed ring system, which can contain heteroatoms such as nitrogen, oxygen and sulfur. 5. Adsorbent according to claim 3, d a d u r c h g e -k e n n z e i c h n e t that the polymeric carrier material as base-specific and / or structure-specific Complexing agent the remainder of malachite green, of crystal violet, of methyl green, of Auramine, of the dye Hoechst 33258, from Di-tert. Butyl-proflavin, from di-tert. Butylacriflavine, from diamidino-phenyl-indole (DAPI), of l-methylphenyl neutral red, of ethidium bromide, of acridine, of 9- (acryloylamino-ethyl-amino) -2-methoxy-6-chloroacridine. 6. Adsorbent according to claim 4, characterized in that the base and / or structure-specific complexing agent corresponds to the following formula 7. Adsorbent according to claim 4, characterized in that the base and / or structure-specific complexing agent corresponds to the following formula 8. Adsorbent according to one of the preceding claims, d a d u r c h gek e n n z ei c h ne t that the polymeric carrier material is a polymer or copolymer of Monomers is the addition to those required to form the polymer or copolymer functional group have at least one further functional croup over which the polymer "spacer" can be covalently bonded to the affinity residue. 9. Adsorbent according to claim 8, d a d u r c h g e -k e n n z e i c h n e t that the polymeric carrier material is a poly-bisacrylamide to which the for the biopolymer-affine residue in the presence of an excess of another copolymerizable Monomers is grafted on by copolymerization, or the carrier material is a crosslinked hydrophilic polymer with hydroxyl or amino groups, in the subsequently acryloyl groups are introduced, such as B. cellulose, sepharose, aminoalkyl cellulose and sepharose, or the carrier material is aminated epoxypropyl methacrylate. 10. Adsorbent according to claim 9, d a d u r c h g e -k e n n z e i c h n e t that the one carrying a free double bond, affine for the diopoly Remainder in the presence of acrylamide by copolymerization via the free double bonds of the poly-bisacrylamide is covalently bound to this polymeric carrier material. Process for producing the adsorbent according to one of Claims 1 up to 10, d u r c h g e k e n n n z e i c h n e t, that one can first by polymerization or PolyE; ondensation of at least one monomer which also has a functional Has group over which the residue affincing for the biopolymer is direct or silver a polymeric "spacar" can be bound, in a manner known per se, the polymer Carrier material forms, the polymeric carrier material, if appropriate comminuted to the desired particle size and then the one with an affinity for the biopolymer content Remainder either directly or by copolymerization in the presence of at least one other Monomers grafted on as a copolymer, or that one in a known manner by Bead polymerization produces particles of the desired size. 12. The method according to claim 11, d a d u r c h g e -k e n n z e i c h n e t that one having a copolymerizable double bond, for the Residue with an affinity for biopolymers in the presence of an excess of another copolymerizable Monomers dls copolymer grafted onto PolybisacLylamid. 13. The method according to claim 12, d a d u r c h g e - ~ k e n n 2 e i c h n e t that the graft copolymerization can be carried out in water or an inert solvent carried out in the presence of a conventional polymerization catalyst. 14. The method according to claim 12, d a d u r c h g e -k e n n z e i c h n e t that one has a copolymerizable double bond as the one for the residue with affinity for biopolymers is acrylic-phenylneutralred or acrylic-malachite green and used as a further res copolymerizable monomer acrylamide. 15. The method according to claim 14, d a d u r c h g e -k e n n z e i c h'n e t that a copolymer of acrylamide and acrylic phenyincutral red or Acrylic malachite green with a degree of polymerization of 200 to 300 grafted. 16. The method according to claim 14, d a d u r c h g e -k e n n z e i c h n e t that one having a copolymerizable double bond, for the Biopolymer-affine radical and the further copolymerizable monomer in a weight ratio from 1; 100 to 1: 5000 begins. 17. The method according to claim 16, d a d u r c h g e -k e n n z e i c n e t, that one works with a ratio of about 1: 3000 ar. 18. Use of the adsorbents according to one of claims 1 to 10 for the separation of biopolymers by affinity chromatography. 19. Use according to claim 18 for the separation of mixtures of single-stranded and / or double-stranded nucleic acids, in particular from DNA mixtures. 20. Dyes of the general formulas (I) and (11) according to claim 4, where the radicals X, Y, R1, R2, R3, R4 and A have the meaning given, but at least one of the radicals R1 is the acryloyl radical. 21. Acryloy.lamino-malachite green of the formula 22. Process for the preparation of the compound according to claim 21, characterized in that that 4-nitrobenzaldehyde is reacted with N, N-dimethylaniline, the nitro group of the obtained Triphenylmethane derivative reduced and acylated with acryloyl chloride. 23. Acryloylaminophenyl neutral red of the formula 24. A method for producing the compound according to claim 23, characterized in that N, N-dimethyl-p-phenylenediamine is reacted with o-toluidine and the reaction product obtained is reacted with N-acryloyl-p-phenylenediamine. 25. Acridine derivatives of the formula wherein R is hydrogen or the acryloyl group. 26. Process for the preparation of the compounds according to claim 25, characterized in that 3,9-dichloro-6-ynethoxy-acridine is known per se Wise reacted with xthylenediamine and the 9-amino-ethylamino compound obtained acylated with acrylic chloride. 27. Acryloylaminophenantridine derivatives of the formula wherein R is hydrogen or a methyl group. 28. Procedure for producing the compounds according to claims 20, 21, 23, 25 and 27, thereby characterized in that one in at least one of the amino groups by customary acylation introduces the acryloyl radical in a manner known per se.