WO1995001347A1

WO1995001347A1 - Novel cross-linking agents and use thereof

Info

Publication number: WO1995001347A1
Application number: PCT/FI1994/000293
Authority: WO
Inventors: Pirjo Korhonen
Original assignee: Cultor Oy
Priority date: 1993-06-30
Filing date: 1994-06-28
Publication date: 1995-01-12
Also published as: AU7075694A; SE9302247D0; EP0706520A1; SE9302247L

Abstract

The present invention relates to novel cross-linking agents of the type (I), wherein X has the meaning of (a), (b), (c), (d), which rings (a) to (d) can carry 1 to n substituents, wherein n equals the number of protonated carbon atoms in the ring, the substituents being selected from the group consisting of -OH, -SH, -CN, -C(O)NH2, C1-C3-alkyl and hydroxy-C1-C3-alkyl, or in the rings (a) to (c), one or more of the -CH2-ring members can be replaced by a carbonyl group -C(O)-, and R1 is selected from the group consisting of C¿1?-C3-alkyl or hydroxy, and use thereof for the preparation of polymers in water or in organic solvents. The high water solubility of the cross-linking agents compared to other cross-linking agents, makes them suitable for the preparation of strongly cross-linked water compatible polymers, usable as a chromatographic separation material for the separation of amino acid enantiomers, peptides, proteins and viruses. The novel cross-linking agent is e.g. synthesized in two steps by acylating the imino functions in the heterocyclic starting compound in two steps.

Description

Novel cross-linking agents and use thereof

Water soluble cross-linking agents are of great industrial interest for the preparation of mechanically stable water compatible polymers. In order to obtain a high mechanical stability in a hydrophilic polymer¹, a high degree of cross-linking is necessary when polymerizing, for example, in water. This in turn requires a high water solubility of the molecule functioning as cross-linker.

In various (bio)chemical applications there is a need for hydrophilic, inert, porous and mechanically stable polymer matrices. Within the field of separation techniques based on chromatography, hydrophilic polymer matrices, e.g. Sephadex and Sepharose (both products from Pharmacia) are well suited for polar water soluble compounds. Another conventional separation technique for water soluble molecu¬ les, electrophoresis, is common for the separation of peptides and proteins. In electrophoresis N,N'-methylene bisacrylamide is often used as a cross-linking agent, in many cases the cross-linking agent 1,4-bisacryloylpipera- zine, which is closely related to this invention, has given better results³.

The application of hydrophilic mechanically stable polymers has given improved results in the so-called solid phase synthesis of peptides⁴.

This invention relates to new cross-linking agents of the formula I

R¹ CH₂=C-C(0) - X - C(0)-CH=CH₂ (I)

wherein X has the meaning of

which rings (a) to (d) can carry 1 to n substituents, whe¬ rein n equals the number of protonated carbon atoms in the ring, the substituents being selected from the group consisting of -OH, -SH, -CN, Cj-C₃-alkyl and hydroxy-C_!-C₃- alkyl, or in the rings (a) to (c) , one or more of the -du¬ ring members can be replaced by a carbonyl group -C(O)-, and R¹ is selected from the group consisting of Cι-C₃-alkyl or hydroxy.

Especially contemplated within the invention is a compound of the formula above, wherein X is unsubstituted piperazine and R¹ is methyl, i.e. l-acryloyl-4-methacryloylpiperazi- ne, or l-[l-oxo-2-methyl-2-propenyl]-4-[l-oxo-2-propenyl]- piperazine, sum formula C_πH₁₆N₂0₂. l-acryloyl-4-methacryloyl- piperazine has an unusually high water solubility (2 kg/li¬ ter at room temperature) and is thus very useful for the preparation of strongly cross-linked water compatible polymers, l-acryloyl-4-methacryloylpiperazine has a water solubility which is 667 times higher (per weight) than the often used cross-linking agent N,N'-methylenebisacrylamide (solubility 3.0 g/liter at room temperature². It can also be mentioned that l-acryloyl-4-methacryloylpiperazine has a high solubility in organic solvents such as e.g. chloro- form (2 kg/liter at room temperature) , wherefore this cross-linking agent also can be used for the preparation of polymers in organic solvents.

The invention also relates to the preparation of the novel cross-linking agents and their use for the preparation of polymers, especially of polymers suitable for the separati¬ on of a ino acids, peptides, proteins and viruses.

The aim of the invention was to obtain an improved, water soluble cross-linking agent for use in the preparation of water compatible mechanically stable polymers provided with a special kind of molecular imprint⁵. This special kind of molecular imprinting technique relates to a chromatographic separation method for amino acids, based on ligand-exchange chromatography⁶ ("the Davankov method") in combination with the molecular imprinting technique⁷. Water soluble cross- linking agents which can be used in the preparation of polymers containing molecular imprints must have a high degree of water solubility in order to provide for a high degree of cross-linking and consequently a high mechanical stability for chromatographic applications. In addition, the structure of the cross-linking agent must be flexible enough to secure the accessability to the active sites in the ready polymer which are provided with molecular imp- rints⁸.

In the WO-publication 93/13034 the combined ligand-exchange chromatography and molecular imprinting technique is desc¬ ribed. According to this technique, a diastereomeric metal ion complex between an amino acid based monomer unit and a selected amino acid is prepared, the complexed amino acid based monomer unit in the metal ion complex is polymerized in the presence of a cross-linking agent (template polyme¬ rization) and the selected amino acid is removed from the polymerized metal ion complex to form a molecular imprint of the selected amino acid in the polymeric material. This polymeric material can thereafter be used for chromatogra¬ phic (e.g. HPLC) separation of the selected or a structu¬ rally similar amino acid from a mixture of other amino acids, inclusive from a racemic mixture of the selected amino acid.

The synthesis of the compounds of the formula I being unsymmetrically substituted with an acryloyl and a substi- tuted acryloyl group is a much more demanding task than the synthesis of corresponding symmetrical bisacryloyl substi¬ tuted compounds, e.g. 1,4-bisacryloylpiperazine, especially if the intermediate and the end product are to be obtained with a high degree of purity and in a high yield without using chromatographic methods during the further proces¬ sing.

The synthesis of the compounds of the formula I is carried out by first acylating one of the imino functions in a compound having the formula (II) H - X - H, wherein X has the meaning given above, with an acid of the formula CH₂=C(R¹)COOH, or especially its chloride, which gives a 1- substituted derivative. The said R'-substituted acryloyl group can also be introduced by reacting a different type of activated acid, for example an active ester between the acid and p-nitrophenol or 1-hydroxybenztriazole, with the heterocyclic compound. In the second and last step, the second imino function is acylated with acrylic acid, or preferably with acryloylchloride, to give the compound of the formula I. Also the acryloyl group can be introduced by using a different type of activated acrylic acid, for example an active ester between acrylic acid and p-nit¬ rophenol or 1-hydroxybenztriazole, for the reaction with the intermediate.

Both reaction steps are preferably carried out under opti¬ mal reaction conditions. For the preparation of the 1- substituted derivative in the first step, an excess of 20 to 40 % of acylating chloride is used compared to the heterocyclic compound II, e.g. piperazine, and the pH of the reaction solution is adjusted and kept between 2.75 and 2.90 and the temperature between 0 and - 5°C when adding the chloride to the reaction solution. The working up of the intermediate, e.g. the 1-methacryloylpiperazine, takes place with simple extractions with dichloromethane and ethylacetate or the like. The intermediate, especially the 1-methacryloylpiperazine, can in this manner be isolated with high yields (appr. 75 to 80 % of the theor. maximum) and with a very high degree of purity (99 % according to HPLC-analysis) .

The second reaction step is carried out in a water/organic solvent two-phase system. The organic solvent can be di¬ chloromethane, chloroform, hexane, toluene, benzene or any other inert solvent which is immiscible with water. When preparing the end product, one equivalent of all reactants is used (e.g. 1-methacryloylpiperazine, acryloylchloride, NaOH) . The reaction temperature is kept under 0 °C during the addition of acryloylchloride to the reaction solution. The working up of the end product takes place using simple extractions. The end product can in this manner be isolated with high yields (80 to 95 % of the theor. maximum) and with a very high degree of purity (99% according to HPLC- analysis) .

According to the invention, it is also possible to first acylate the starting compound of formula II with acrylic acid or a derivative thereof, and in the second step to introduce the R'-substituted acryloyl group, in a manner analogous to the two step process described above.

The novel cross-linking agents can be polymerized i.a. using the bulk polymerization method, wherein the monomers are first dissolved in a suitable solvent whereby the monomers, during the polymerization, precipitate as a poly- meric material in the solvent. Suitable solvents in the bulk polymerization are for example water, methanol, et- hanol, 1-propanol, dichloromethane, chloroform, formic acid, acetic acid, N,N-dimethylformamide, dimethylsulfoxide and acetonitril. The solvent is also called a porogen as it forms pores in the polymeric material. The cross-linking agent can be copolymerized with various functional mono¬ mers, i.a. with acryloyl-, methacryloyl-, vinyl-, acrylate- and methacrylate-based monomers.⁹

The polymerization can be initiated with thermal or photo- lytic homolysis of azobisnitriles, e.g. 2,2*-azobis(2- methylpropionitrile) [AIBN] . Polymerizations with this cross-linking agent can also be initiated with the peroxo- disulfate/N,N,N' ,N'-tetramethylethylenediamine system commonly used in aqueous mediums.

Bead-shaped polymer particles using the novel cross-linking agent can also be prepared using a so-called reversed suspension polymerization method. Such beads can be ob¬ tained in varying sizes depending on a variety of factors, such as choice of detergent(s) , volumetric relationsship between water and organic phase, shape of polymerization vessel, stirrer speed, etc. The beads, usually of a size of 45 to 80 μm, have a high mechanical stability and they withstand pressures up to 200 bar without being deformed.

The polymers thus obtained using the novel cross-linking agents can be used in a manner described in WO-publication 93/13034, the contents of which is included herein for reference, for the separation of amino acids from a mixture of amino acids, such as for the separation of chiral amino acids from a racemic mixture. According to the said pro¬ cess, the mixture of amino acids to be separated is contac- ted with a polymer material which is composed of cross- linked, amino acid-based monomer units, said polymer meta- rial containing a molecular imprint of the selected amino acid, in which molecular imprint there is also bound a divalent metallic ion and the amino-acid based monomer unit. The amino acid-based monomer is according to one embodiment N-methacrylaminomethyl-L-proline or N-methacryl- aminomethyl-D-proline, which is especially suitable for the preparation of a polymer suitable for the resolution of race ic amino acids. The divalent metal is selected from copper(II) , manganese(II) , iron(II) , cobolt(II) , zinc(II), cadmium(II) and nickel(II), preferably copper(II) .

Due to their high water solubility and chemical inertness, the new cross-linking agents, especially l-acryloyl-4- methacryloylpiperazine, are suitable also for the template polymerization of biological molecules, for example pepti¬ des, proteins and viruses. Indeed, the new cross-linking agent provides a polymeric material having a chemical character compatible with peptides and proteins, and its high mechanical strength and durability makes it suitable for use in chromatography. It is assumed that the novel cross-linking agents possess a suitable degree of flexibi¬ lity resulting in a polymeric material having feasible mass transfer properties enabling the diffusion of the large template into and out of the active sites during the chro¬ matographic separation process. The porocity and the flexi¬ bility of the the polymeric material can be adjusted by the amount and nature of porogen and/or the existence of an extra small inert comonomer during the template polymeriza- tion, or by modifying the degree of cross-linking. Due to their beneficial physico-chemical properties, the novel cross-linking agents, and in particular l-acryloyl-4-met- hacryloyl-piperazine, can be polymerized under different pH-values and temperatures, which is of importance in the template polymerization of proteins and peptides. In the template polymerization of biological molecules the chelating monomer can be any monomer containing iminopoly- carboxylate, sulfonate or iminopolyphosphonate groups capable of coordination with the divalent metal ion, capa¬ ble of retaining the chelating metal in the resin and possessing a favourable polymerization ratio to the new cross-linking agent. For example onosubstituted (meth)ac- rylamide based monomers are compatible with the new cross- linking agents, as are the following exemplatory monomers,

A common chelating functional group is the iminodiacetate group, e.g. the following new monomer

l,l-dicarboxymethyl-2-methacryloyl-hydrazine.

The monomers may be prepared using methods which are analo¬ gous to methods already described in literature. (Houben Weil, 8, 4. Aufl. Georg Thieme verlag, 1952, Stuttgart, p. 676; Houben Weil, 10/2, 4. Aufl. Georg Thieme Verlag, 1967, Stuttgart, p. 13; W.T. Read, J. Am. Soc. 36 (1914) 1747-1765)

As chelating metals the metals mentioned above may be used. Cu(II) is preferred due to the thermodynamic stability of the mixed Cu(II)-complexes. The net charge on the metal ion must remain positive, or the metal will be stripped from the column. Of the functional groups co-ordinating with the metal ion, the iminopolyphosphonate and iminopolycarboxyla¬ te groups hold metal ions better than the sulfonate groups.

The size of peptides to be used as templates varies from two amino acids to even some hundred amino acid units. In addition to the free end amine group and the free end carboxylate group, also functional groups in the amino acid side chains are capable of co-ordinating to metal ions, such as in histidine and cysteine. Such functional groups make it possible to use metal chelation in the creation of isomer and substrate specific cavities by template polyme¬ rization in aqueous solution. As peptides tend to co-or¬ dinate Cu(II)-ions through the end amino group and a neigh¬ boring amide function, it is especially contemplated within the invention to form a Cu(II)-complex between an iminodi- acetic based monomer and a simple water soluble dipeptide or tripeptide, e.g. as f

DIPEPTIDE

The metal should naturally be selected in accordance to the number and types of groups present in the peptides capable of co-ordination with the metal.

Favourable water soluble peptides are small di-, tri- and oligopeptides with not too hydrophobic amino acid side chains. Suitable peptides are those formed from e.g. glyci- ne, alanine, serine and threonine.

The use of viruses as templates is based on the fact that viruses have proteins on their surfaces, and proteins that contain histidines and cysteines are capable of co-or¬ dination e.g. with Cu(II)ions. The same holds true for proteins in general, such as enzymes. In fact the presence of a single histidine residue on the surface of a protein is sufficient for the molecule recognition by specific Cu(II) chelation interaction during the template polymeri¬ zation. If the protein surface contains several histidine or other chelating groups on its surface, it is recommended to use e.g. Zn(II) , Co(II), Ni(II), Cd(II) or Ca(II) which form thermodynamically less stable complexes with amino acid residues.

The following examples illustrate the invention without restricting the same.

In the example 1 C-D, three polymers were prepared based on the cross-linking agents l-acryloyl-4-methacryloylpipera- zine, 1,4-bisacryloylpiperazine (1,4-bis[l-oxo-2-prope- nyl]-piperazine) and N,N'-methylenebisacrylamide, in order to prepare a polymer which can effectively split amino acid racemates under chromatographic (HPLC) conditions. These three polymers are compared as to their capability of sepa¬ rating D- and L-serine, hardness and thus applicability under chromatographic conditions. The comparison of Example 1 C-D shows that l-acryloyl-4-methacryloylpiperazine gives a highly superior polymer both as regards the racemic separation capability and as regards physical characteris¬ tics. The polymer prepared with the commonly used cross- linking agent N,N'-methylenebisacrylamide was much too porous to be tested under HPLC conditions. In addition, the results indicate that the very presence of an acryloyl group and a methacryloyl group in the same piperazine deri¬ vative is of great importance for the beneficial properties in the resulting polymer, as a polymer made with the struc¬ turally similar 1,4-bisacryloylpiperazine in accordance with the results in the Example 1 B shows poorer characte¬ ristics.

EXAMPLE 1

A. Preparation of 1-methacryloylpiperazine (l-oxo-2-methyl- 2-propenyl-piperazine)

17.28 g (0.2 moles) of piperazine is dissolved in 400 ml of distilled water in a vessel provided with a pH-meter, ther¬ mometer, dripping funnel and a mechanical stirrer. 240 ml of 2 M HC1 is added and the pH is adjusted to 2.80 with 3 M sodium acetate. While vigorously stirring, 27 ml (0.28 moles) of methacryloylchloride is added in small portions while simultaneously adjusting the pH with 3.0 M sodium acetate and cooling with an acetone/ice bath. During this process, the pH is adjusted to 2.8 - 2.9 and the temperatu¬ re of the reaction solution is maintained between -5 and 0 °C. After the methacryloylchloride addition, the solution is slowly brought to room temperature. Thereafter the reaction solution is extracted with 3 x 200 ml dichloromet¬ hane. While cooling with water/ice and while vigorously stirring, the reaction solution is saturated with Na₂C0₃ and the product is extracted thereafter with 4 x 200 ml of ethylacetate. The ethylacetate phases are dried with Na₂C0₃. After evaporation and drying in an exsiccator 23.1 g (0.15 moles) of 1-methacryloylpiperazine is obtained as an oil, corresponding to 75 % of the theor. maximum amount.

R_f = 0.23 (silicagel in tetrahydrofuran/N-hexane, 3/1). k' = 1.68, 99% (HPLC. Solid phase:

Spherisorb C₈, 5 μm. Mobile phase: acetonitrile/0,2 % H₃P0₄, pH 6.9, 1/4) .

*H- NMR (CDC1₃/TMS, 200 MHz): 6 = 1.96 [s, 3H, -CH₃] , 2.84 [m, 4H, (ring 2 x (CH₂) ] , 3.56 (broad, 4 H, ring 2 x (CH₂) ] , 5.0 [ s, 1H, (=CHH) ] , 5.18 [s, 1H, (=CHtf) ] .

¹³C- NMR (CDC1₃/TMS, 200 MHz): δ = 20.49 [- CH₃] , 46.31 [ring carbon], 115.20 [ =CH₂] , 140.56 [ >C= ], 171.19 [>C=0] . MS (70 eV) : m/e = M⁺ 154 (80 %) , 85, (80 %, M⁺ -C₄H₅0) , 112 (15 %, M ⁺ -C₃H₆).

IR: 3500 cm^"1 H-H stretching (m) sec.amine, 3095 cm^"1 unsat. C-H stretching (m) , 2918 & 2862 cm^"1 saturated C-H stret¬ ching (m) , 1648 cm^"1 (s) tert.amide carbonyl.

B. Preparation of l-acryloyl-4-methacryloylpiperazine (1- [l-oxo-2-methyl-2-propenyl] ,4-[l-oxo-2-propenyl]piperazine)

18.6 g (0.12 moles) of 1-methacryloylpiperazine is dissol¬ ved in 110 ml of dichloromethane in a vessel provided with a mechanical stirrer, thermometer and a dripping funnel. While vigorously stirring and cooling in an acetone/ice bath (-20 °C) 9.95 ml (0.12 moles) of acryloylchloride in 10 ml of dichloromethane and 4.8 g (0.12 moles) of NaOH in 15 ml of water are added simultaneously in portions. The temperature of the reaction solution is maintained under 0 °C. The reaction solution is slowly allowed to reach room temperature. The water and the dichloromethane phases are separated and the dichloromethane phase is washed with 2 x 50 ml of saturated NaHC0₃-solution and 50 ml of a saturated NaCl-solution. A pinch of hydroquinone (autopolymerisation inhibitor) is added to the reaction solution which thereaf¬ ter is dried with sodium sulfate. After evaporation and drying in an exsiccator 22.5 g (0.11 moles) of 1-acryloyl- 4-methacryloylpiperazine is obtained as an oil, correspon¬ ding to 90 % of the theoretically maximum amount.

R_f = 0.62 (silica gel in tetrahydrofuran/N-hexane, 3/1). k' = 2.68, 99% (HPLC. Fast phase: Spherisorb C₈, 5 μm. Mobile phase: acetonitrile/0.2 % H₃P0₄ pH 6.9, 1/4.) .

-NMR (CDC1₃/TMS, 200 MHz): δ = 1.97 (s, 3 H, -CH₃) , 3.62 (broad s, 8H, ring protones) , 5.07 (s, 1H, CH₃C(CO) CH_aH_x) 5.25 (s, 1H, CH₃C(CO)CH_a,H , 5.75 (dd, 1H, ³J_uaDS 10.36 Hz, ³J_cis 2.00 Hz), 6.31 (dd, 1H, J_g g_ee_mm 16.79 Hz, ³J_cis 2,00 Hz), 6.60

(dd, J_gem 16.79 HZ, 'j^ 10.36 Hz)

¹³C- NMR (CDC1₃/TMS, 200 MHz): δ = 20.39 (CH₃) , ≡ 41- 43 (ring C) , 116.080 [ (CH₃) (CO) C = CH₂] , 127.20 [ (H) (CO) > C = ] , 128.42 [ (H) (CO) C = CH₂) ] , 139.957 [ (CH₃) (CO) > C =] , 165.59 [>C=0], 171.33 [>C=0] .

MS (70 eV) : m/e = M + 208 (40%), 193 (20 %, M + -15) , 153 (20 %, M + - C₃H₃0) , 139 (50 %, M + - C₄H₅0) , 85 (100 %, M+ - C₇H₈0₂) , 69 (90 %) , 55 (60 %) .

IR: 3095 cm^"1 unsaturated C-H stretching ( ) , 2918 & 2862 cm^"

¹ saturated C-H stretching (m) , 1648 cm^"1 (s) tert. amide carbonyl .

C. Preparation of a polymer using l-acryloyl-4-methacrylo- ylpiperazine

2.47 g (0.0064 moles) of a (N-[methacrylamidomethyl]-L-pro- linate) (L-serinate)-cupper[II] x 2 H₂0 complex and 7.70 g (0.037 moles) of l-acryloyl-4-methacryloylpiperazine is dissolved in 7.0 ml of water. 700 μl of ammonium peroxo- disulfate solution (1.0 g/10 ml water) is added, and the solution is purged with nitrogen gas for two minutes. The polymerization is initiated with 200 μl of Temed (N,N,N' ,N'-tetramethylethylenediamine) . The polymerisation is continued at room temperature in a glass vessel (30 ml) which is provided with a tight fitting plastic lid. The obtained hard polymer is ground, screened in methanol through a 25 μm screening cloth (Retsch) and sedimented finally in methanol to eliminate undersized particles. The thus obtained material (10-25 μm) is washed with 1M ammo¬ nia, water, 0.1 M CuS0₄ and finally with water. After pack¬ ing the material under a pressure of 200 bar in a HPLC column (4.6 x 100 mm) using water as the mobile phase, the column containing the polymer is equilibrated in 0.1 M NH₃ with 0.1 mM CuS0₄ in a Kontron HPLC-apparatus. A mixture of D and L-serine (100 μg D-serine + 100 μg L-serine) is injected in 20 μl of mobile phase. Flow: 0.40 ml/min. Counter pressure: 15 bar. Detection at 254 n , separation factor 1.8. Eluation profile, see Figure 1.

D. Preparation of polymer with 1,4-bisacryloylpiperazine

2.47 g (0.0064 moles) of a (N-[methacrylamidomethyl]-L-pro- linate) (L-serinate)-cupper[II] x 2 H₂0 complex and 7.18 g

(0.037 moles) 1,4-bisacryloylpiperazine is dissolved in 7.0 ml of water. 700 μl of ammonium peroxodisulfate solution

(1.0/10 ml water) is added, and the solution is purged with nitrogen gas for two minutes. The polymerization is ini- tiated with 200 μl of Temed. The polymerization is con- tinued at room temperature in a glass vessel (30 ml) which is provided with a tight fitting plastic lid. The obtained less hard polymer is ground, screened in methanol through a 25 μm screening cloth (Retsch) and sedimented finally in methanol to eliminate undersized particles. The thus ob¬ tained material (10-25 μm) is washed with 1M ammonia, water, 0.1 M CuS0₄ and finally with water. After packing the material under a pressure of 75 bar in a HPLC column (4.6 x 100 mm) using water as the mobile phase, the column with the polymer in 0.1 M NH₃ is equilibrated with 0.1 mM CuS0₄ in a Kontron HPLC-apparatus. A mixture of D and L- serine (100 μg D-serine + 100 μg L-serine) is injected in 20 μl of mobile phase. Flow: 0.40 ml/ in. Counter pressure: 50 bar. Detection at 254 nm, separation factor 1.0. Eluati- on profile, see Figure 2.

E. Preparation of polymer with N,N*-methylenebisacrylamide

2.47 g (0.0064 moles) of a (N-[methacrylamidomethyl]-L-pro- linate) (L-serinate)-cupper[II] x 2 H₂0 complex and 5.70 g

(0.037 moles) of N,N'-methylenebisacrylamide is dissolved in 160 ml of water. 0.125 g of ammonium peroxodisulfate is added, and the solution is purged with nitrogen gas for two minutes. The polymerization is initiated with 40 μl of Temed. The polymerization is continued at room temperature in a glass vessel which is provided with a tight fitting plastic lid. The obtained soft polymer is washed with 1 M ammonia, water, 0.1 M CuS0₄ and finally with water. The polymer is too soft to be packed in a HPLC-column. EXAMPLE 2

2.1 Preparation of chelating monomer 1,l-dicarboxymethyl-2- methacryolyol hydrazine

A. To 1.5 moles of hydrazine hydrate (Sigma H 0883) heated on a water bath, 1.07 moles of methacrylic acid dimethyles- ter (Sigma M 1283) is added dropwise while refluxing, and the reaction mixture is heated for two days until the reaction has ended. When cooling with a cooling mixture the liquid solidifies to colourless crystal bundles. The slurry is shaken twice with anhydrous ether, the ether is poured away and the rest is heated on a water bath at about 60 °C, whereby a solution is formed and the rest of the ether is evaporated. When cooling with a cooling mixture the hydra- zide is solidified to crystals which can be recrystallized from chloroform-ether. As the product methacryloylhydrazine is obtained.

B. 189 g (2 moles) of monochloroacetic acid in 200 ml of water is neutralized by adding 138 g (1 mole) of potassium carbonate in small portions and then 100 g (1 mole) of methacryloylhydrazine is puored into this solution. A second portion of 138 g (1 mole) of potassium carbonate is added gradually, with a steady evolution of C0₂ and the temperature rises to 70 °C. The solution is heated for as long as the gas evolution continues. At the end of the reaction, the hydrazino-diacetic acid is precipitated by making the solution acid with hydrochloric acid. The hydra- zine acid is recrystallized from water. 1,1-dicarboxymet- hyl-2-methacryloylhydrazine is obtained as the product. 2.2 Preparation of a (1, 1-dicarboxymethy1-2-methacryloyl¬ hydrazine) -C(II) -(peptide) -complex

a) A simple water soluble dipeptide without chelating side chains as template: NH₂-ala-ser-COO^", Sigma A 3503

1.55 g (7.3 mmole) of 1, 1-dicarboxymethy1-2-methacryloyl¬ hydrazine, 1,17 g (7.3 mmole) of CuS0₄ and 1.28 g (7.3 mmole) NH₂-ala-ser-COO^" is dissolved in 20 ml distilled water. The pH is adjusted by means of 1 M NaOH to pH 8. The solution is evaporated at reduced pressure and dried over¬ night in a desiccator.

b) A larger water soluble pepide containing no chelating side chains: NH₂-ala-gly-gly-COO^", Sigma A 1378

3.15 g (14.6 mmole) of 1, 1-dicarboxymethy1-2-methacryloyl¬ hydrazine, 2.34 g (14.6 mmole) of CuS0₄ and 1.48 g (7.3 mmole) NH₂-ala-gly-gly-COO^" is dissolved in 20 ml distilled water. The pH is adjusted by means of 1 M NaOH to pH 8. The solution is evaporated at reduced pressure and dried over¬ night in a desiccator.

c) A larger water soluble peptide with several chelating side chains: NH₂-gly-gly-his-COO^", Sigma G 5772

4.73 g (21.9 mmole) of 1, 1-dicarboxymethy1-2-methacryloyl¬ hydrazine, 3.93 g (21.9 mmole) of ZnS0₄.H₂0 and 1.96 g (7.3 mmole) NH₂-gly-gly-his-COO^" is dissolved in 20 ml distilled water. The pH is adjusted by means of 1 M NaOH to pH 8. The solution is evaporated at reduced pressure and dried over¬ night in a desiccator. 2.3. Preparation of polymer

NH₂-ala-ser-COO^" as template

7.3 mmole of (1,l-dicarboxymethyl-2-methacryloyl-hydrazi- ne)-Cu(II)-(NH₂-ala-ser-COO")-complex is dissolved together with 8.26 g (39.7 mmole) of l-acryloyl-4-methacryloylpipe- razine in 13 ml water in a test tube. Through the solution nitrogen gas is passed and before fitting the test tube with a tight fitting cork, 750 μl of a stock solution of ammonium-peroxodisulfate (1,0 g/10 ml) and 200 μl of N,N,N' ,N'-tetramethylethylenediamine (TEMED) are added. The polymerization is allowed to proceed at room temperature over night. The formed polymer is crushed by hand in a mortar and is then ground in a mechanical mortar device (Retsch, Haan, Germany) for 5 min. The material is sieved through a 25 μm sieve. Subsequently the material is allowed to sediment in 2 turns in 95 % ethanol, while removing the supernatant. The sediment is carefully washed with 1/1 95% EtOH/distilled H₂0 and 1 M ammonia in order to remove the peptide, and is then dried in a desiccator over night.

The same procedure can be carried out using NH₂-ala-gly- gly-COO" or NH₂-gly-gly-his-COO" as templates, whereby the starting complexes are a (1,l-dicarboxymethyl-2-methacry- loyl-hydrazine)₂-Cu(II)₂-(NH₂-ala-gly-gly-COO")-complex and a (1,l-dicarboxymethyl-2-methacryloyl-hydrazine)₃-Cu(II)₃- (NH₂-gly-gly-his-COO")-complex, respectively.

References

1. Nachr. Chem. Tech. Lab. 39 (1991) No. 1, 1-14.

2. Merck reagentkatalog 92/93, p. 839.

3. D.F. Hochstrasser et al., Anal. Biochem. 173 (1988) 412-423.

4. R. Walter et al., J. Am. Chem. Soc. 101 (1979) 5383-5394.

5. Swedish Patent Application No. 9103785-3.

6. V. A. Davankov, Pure and Applied Chem., Vol. 54 (1982) 2159-2168.

7. B. Ekberg and K.Mosbach, Trends in Biotechnology, Vol. 7, (1989) 92-96. G. Wulff, American Chemical Society Symp. Ser., Vol. 308, (1986) 186-230.

8. K. J. Shea, Macromolecules 23 (1990) 4497-450.

9. G. Odian, "Principles of polymerization", J. Wiley & Sons Inc., (1981).

Claims

1. Compounds of the formula

R¹ /

CHy =C - ^■c(_ ) - x - C(O)- -CH=CH₂ (I) wherein X has the meaning of

which rings (a) to (d) can carry 1 to n substituents, whe¬ rein n equals the number of protonated carbon atoms in the ring, the substituents being selected from the group consisting of -OH, -SH, -CN, Cj-C₃-alkyl and hydroxy-C_j-C₃- alkyl, or in the rings (a) to (c) , one or more of the -Co¬ ring members can be replaced by a carbonyl group -C(O)-, and R¹ is selected from the group consisting of C_j-C₃-alkyl or hydroxy.

2. The compound of claim 1, which is l-acryloyl-4-methacry- loylpiperazine.

3. Process for the preparation of the compound according to claim 1, characterized in that a compound of the formula H - X - H (II) , wherein X has the meaning indicated in claim 1, is first reacted with an acid of the formula CH₂=C(R¹)-COOH or a functional derivative thereof, prefe- rably its chloride, to prepare a compound of the formula CH₂=C(R¹)-C(0)-N-X-H, which thereafter is reacted with acrylic acid, or a functional derivative thereof, prefera¬ bly its chloride, to give the compound of formula I.

4. Process for the preparation of the compound according to claim 1, characterized in that a compound of the formula H - X - H (II) , wherein X has the meaning indicated in claim 1, is first reacted with acrylic acid or a functional derivative thereof, preferably its chloride, to prepare 1- acryloylpiperazine, which thereafter is reacted with an acid of the formula CH₂=C(R^X)-COOH or a functional deriva¬ tive thereof, preferably its chloride, to give the compound of formula I.

5. Use of the compounds of claim 1 or 2 for the preparation of polymers.

6. Use of the compounds of claim 1 or 2 as a crosslinking agent for the preparation of polymers.

7. Use of the compounds of claim 1 or 2 for the preparation of polymers in aqueous media.

8. Use of the compounds of claim 1 or 2 for the preparation of polymers in organic solvents.

9. Use according to claim 5 for the preparation of acryl- and methacryl-based polymers.

10. Use according to any one of the claims 5 to 9 for the preparation of a polymer for use in separating a selected amino acid from a mixture of amino acids, or for the sepa¬ ration of peptides, proteins and viruses from mixtures con¬ taining the same.

11. Process for the preparation of a acryl and/or methacryl based polymer, characterized in that a acrylic acid and/or a methacrylic acid based monomer is polymerized with a compound according to claim 1 or 2 as a cross-linking agent, in an aqueous medium or in an organic solvent or in a mixture of these, or in a reverse suspension polymeriza¬ tion method in a medium containing water and an organic solvent, and the polymer obtained is separated.

12. Process for the preparation of a polymer for use in separating a selected amino acid from a mixture of amino acids, characterized in that a compound according to claim 1 or 2 is polymerized with a monomer which is a diaste- reomeric metal ion complex between an amino acid based monomer unit and the selected amino acid, preferably an enantiomer of a chiral amino acid, and the selected amino acid is separated from the prepared polymer.

13. Process for the preparation of a polymer for use in separating a selected peptide, protein or virus from a mixture containing said selected species, characterized in that a compound according to claim 1 or 2 is polymerized with a monomer which is a diastereomeric metal ion complex between a monomer unit containing sulfonate, iminopolycar- boxylate and/or iminopolyphosphonate groups, and the selec¬ ted species, and the selected species is separated from the prepared polymer.

14. Process according to claim 12 or 13, characterized in that the metal is selected from copper(II) , manganese(II) , iron(II) , cobolt(II), zinc(II) , cadmiu (II) and nickel(II) , preferably copper(II).

15. Process according to claim 12 and 14, characterized in that the monomer is a copper(II)complex between

N-methacrylamidomethyl-L-prolinate or N-methacrylamidomet- hyl-D-prolinate and the selected amino acid.

16. Use of a polymer obtained according to the process of any one of the claims 12-15 in HPLC separation techniques.