SOLUBLE CHELATOR FOR TARGETING RECOMBINANT PROTEINS

Abstract

Soluble chelator-compounds, methods for their production and their use for the modification and/or immobilization of target molecules.

Claims

1. A soluble chelator comprising at least one amino-carboxylic acid of the general structure as depicted in formula I: ##STR00009## wherein R1 is selected from a chelating carboxylic acid or a chelator-group, such as formula II: ##STR00010## wherein n=0, 1, 2, or 3; or R1 is selected from formula III: ##STR00011## wherein each of the sides can be bound to R1; and wherein either two molecules of formula I are bound to each other bivalently via R2 resulting in a core-molecule of formula IV: ##STR00012## or wherein three molecules of formula I are bound trivalent via R2 to formula V: ##STR00013## and wherein R3 of the formulas above is selected either from the group comprising H, COOH, CH.sub.2COOH and another molecule of formula I which is bound via R3-(CH.sub.2).sub.nR2, n=0, 1, 2, or 3.

2. The soluble chelator of claim 1, further comprising a positively charged metal-ion selected from the metals Ni, Co, Cu, Fe, Ca, Zn, Al, Eu, Ga, and Sc.

3. The soluble chelator of claim 1, wherein the number of carboxy groups is at least 8.

4. The soluble chelator claim 1, wherein the number of amino groups is at least 6.

5. The soluble chelator of claim 1, wherein R1 is selected from the group comprising EDTA (ethylene diamine tetra acetic acid), EGTA (ethylene glycol-bis-(?-aminoethyl ether)-N,N,N,N-tetra acetic acid), DTPA (diethylene triamine penta acetic acid), and EDDS (ethylene diamine-N,N-di succinic acid).

6. The soluble chelator of claim 1, as depicted in formula VI: ##STR00014##

7. The soluble chelator of claim 1, as depicted in formula VII: ##STR00015##

8. A soluble chelator as depicted in formula VIII: ##STR00016##

9. The soluble chelator of claim 1, wherein the chelator is further modified to comprise a tag, such as a biotinylation, a His-tag, a strep-tag, a FLAG-tag, a Rho-tag (e.g. Rho-1D4-tag).

10. A method for producing the soluble, chelators of claim 1, comprising the steps of: a. providing EDTA dianhydride and a trifunctional linker in a ratio of 4:1 to 2:1, preferably 2.75:1 to 3.25 to 1; b. coupling said dianhydride to said trifunctional linker via amine group onto the linker, wherein the linker reacts with three different dianhydrides, so that a substance (APA1)3-L is formed, wherein APA1 is EDTA with one anhydride group and one amide function, and L is the trifunctional linker; c. providing a molecule, which can be a dye, a colorant, a fluorophor, a nanoparticle, a functional group for click chemistry, a radioactive molecule, or a molecule with enzyme activity, comprising one or more amine groups; d. coupling said linker-connected dianhydride to said molecule via amine group, wherein a single, two, or three carboxy groups per aminocarboxylic acid reacts with the amine moiety; e. providing water or an aqueous puffer to hydrolyze the remaining anhydride groups; and f. immobilizing metal ions by contacting the solid phase-immobilized aminopolycarboxylic acid compound with solutions of metal ions, selected from Ni.sup.2+, Co.sup.2+, Cu.sup.2+, Zn.sup.2+, Al.sup.3+, Fe.sup.3+, Eu.sup.3+, Ga.sup.3+, Mn.sup.2+, Ca.sup.2+.

11. A method for producing the soluble chelators of claim 1, comprising the steps of: a. providing EDTA monoanhydride and a trifunctional linker in a ratio of 4:1 to 2:1, preferably 2.75:1 to 3.25 to 1; b. coupling said monoanhydride to said trifunctional linker via amine group onto the linker, wherein the linker reacts with three different monoanhydrides, so that a substance (APA2)3-L is formed, wherein APA2 is EDTA with one amide function, and L is the trifunctional linker; c. providing a molecule, which can be a dye, a colorant, a fluorophor, a nanoparticle, a functional group for click chemistry, a radioactive molecule, or a molecule with enzyme activity, comprising one or more amine groups; d. coupling said linker-connected EDTA to said molecule via amine group, wherein a single, two, or three carboxy groups per aminocarboxylic acid reacts with the amine moiety, by means of one or more condensing agents, such as carbodiimides, like EDC or DCC, or other reagents used in peptide syntheses; and e. immobilizing metal ions by contacting the solid phase-immobilized aminopolycarboxylic acid compound with solutions of metal ions, selected from Ni.sup.2+, Co.sup.2+, Cu.sup.2+, Zn.sup.2+, Al.sup.3+, Fe.sup.3+, Eu.sup.3+, Ga.sup.3+, Mn.sup.2+, Ca.sup.2+.

12. A method for producing the soluble chelators of claim 1, comprising the steps of: a. providing a solid phase resin, which is able to bind carboxylic acid groups, such as Wang resin, and EDTA, with two carboxylic acid protective groups, such as methyl ester or tert butylester; b. coupling said EDTA derivative to said solid phase resin by means of one or more condensing agents, such as carbodiimides, like EDC or DCC, or other reagents used in peptide syntheses; c. providing a trifunctional linker; d. coupling said trifunctional linker onto the remaining carboxylic acid group of the solid-phase bound EDTA; e. removing the protective groups by trifluoroacetic acid, or the like; f. cleaving the aminopolycaroxylic acid from the solid phase by trifluoroacetic acid, or the like; g. providing a molecule, which can be a dye, a colorant, a fluorophor, a nanoparticle, a functional group for click chemistry, a radioactive molecule, or a molecule with enzyme activity, comprising one or more amine groups; h. coupling said linker-connected dianhydride to said molecule via amine group, wherein a single, two, or three carboxy groups per aminocarboxylic acid reacts with the amine moiety; i. immobilizing metal ions by contacting the solid phase-immobilized aminopolycarboxylic acid compound with solutions of metal ions, selected from Ni.sup.2+, Co.sup.2+, Cu.sup.2+, Zn.sup.2+, Al.sup.3+, Fe.sup.3+, Eu.sup.3+, Ga.sup.3+, Mn.sup.2+, Ca.sup.2+.

13. Use of the soluble, chelators of claim 1 as a colorant, a dye, a fluorophor, a nanoparticle, as a tool for click-chemistry, a radioactive molecule or as a molecule with enzymatic function.

14. Use of the soluble chelators of claim 1 for binding to a target protein comprising a tag which binds to metal ions, such as for example a polyhistidine-tag.

15. Use of the soluble, chelators of claim 1 for complex formation.

16. Use of the soluble chelators of claim 1 for detection of metal ions, preferably in combination with a His-tag or biotinylation.

17. Use of the soluble chelators of claim 1 for binding onto functionalized streptavidin.

Description

DESCRIPTION OF EMBODIMENTS

Examples

Example 1: Synthesis and Functionalization of Tris-EDTA, Linked by Tris(2-Aminoethyl)Amine

Synthesis of EDTA Monoanhydride

[0080] 30.0 g (117 mmol) EDTA dianhydride were dissolved in 200 ml DMSO under N.sub.2 atmosphere. Then a mixture of 2.1 ml (117 mmol) water in 10 ml DMSO is added dropwise, and the mixture was stirred for three hours while a precipitate has formed. The mixture was cooled, suction-filtered and washed repeatedly with dry DMSO and diethyl ether. The precipitate was dried and used for the next steps.

Reaction of EDTA Monoanhydride with Tris(2-Aminoethyl)-Amine

[0081] 1 g (3.65 mmol) EDTA monoanhydride were dissolved in 30 ml DMSO under nitrogen, and a solution of ml tris(2-aminoethyl)-amine (mmol) in 10 ml DMSO was added dropwise. After the addition is finished, the suspension was stirred two hours at ambient temperature and three hours at 60? C. The reaction mixture was evacuated under high vacuum, and the remaining product could be used for the next steps.

Functionalization of the Product with Different Fluorophors and Nickel

[0082] 5?100 mg (5?108 micromol) of the reaction product are dissolved each in 50 ml water. Then 5?25.0 mg of EDC (5?130 micromol), 5?15.0 mg of N-hydroxy succinimide (5?130 micromol), and 69.3 mg (110 micromol) CY3-amine (Lumiprobe GmbH, Hannover, Germany), 47.0 mg (110 micromol) BDP-FL amine (Lumiprobe), 56.2 mg (110 micromol) FAM amine, 5 isomer (Lumiprobe), 90.4 mg (110 micromol) Atto 633 amine (Atto-Tec GmbH, Siegen, Germany), and 79.2 mg (110 micromol) Alpha Fluor? 488 amine (AAT Bioquest Inc, Sunnyvale, CA, USA) were added, and the reaction mixture was stirred two hours. Then 105 mg (40 micromol) nickel sulfate hexahydrate were added to each tube, and the reaction mixture was stirred another two hours. The product was dried under high vacuum, washed several times with dry diethyl ether, and dried again. In the next step the conjugates were purified over a 5 ml PureCube Q Cartridge (Cube Biotech GmbH) and eluted with a 0 ech Gmb NaCl gradient using an FPLC system.

Example 2: Synthesis and Functionalization of a Linear EDTA-EDA-EDTA-EDA-EDTA Molecule

[0083] 30.0 g (117 mmol) EDTA dianhydride were dissolved in 200 ml DMSO under N2 atmosphere. 5.21 ml (78 mmol) ethylene diamine were dissolved in 25 ml DMSO and added dropwise to the reaction mixture. After finishing the addition, the mixture was stirred two hours at ambient temperature, and three hours at 60? C. Then the reaction mixture was evacuated under high vacuum, and the remaining product could be used for the next steps.

Functionalization of the Product with Different Fluorophors and Nickel

[0084] 5?100 mg (5?108 micromol) of the reaction product are dissolved each in 50 ml water. Then 5?25.0 mg of EDC (5?130 micromol), 5?15.0 mg of N-hydroxy succinimide (5?130 micromol), and 69.3 mg (110 micromol) CY3-amine (Lumiprobe GmbH, Hannover, Germany), 47.0 mg (110 micromol) BDP-FL amine (Lumiprobe), 56.2 mg (110 micromol) FAM amine, 5 isomer (Lumiprobe), 90.4 mg (110 micromol) Atto 633 amine (Atto-Tec GmbH, Siegen, Germany), and 79.2 mg (110 micromol) Alpha Fluor? 488 amine (AAT Bioquest Inc, Sunnyvale, CA, USA) were added, and the reaction mixture was stirred two hours. Then 105 mg (40 micromol) nickel sulfate hexahydrate were added to each tube, and the reaction mixture was stirred another two hours. The product was dried under high vacuum, washed several times with dry diethyl ether, and dried again. In the next step the conjugates were purified over a 5 ml PureCube Q Cartridge (Cube Biotech GmbH) and eluted with a 0-2.5 M NaCl gradient using an FPLC system.

Example 3: Solid Phase Synthesis and Functionalization of Tris-EDTA, Linked by Tris-(2-Ami-Noethyl)-Amine

Coupling of EDTA-Bis-Methylester onto Wang Resin

[0085] In a round-bottom flask 0.5 g Wang resin (Merck Millipore, Darmstadt, Germany) is covered with DMF and allowed to swell for 30 min. In another round-bottom flask 2 g EDTA-bis-methyl ester (preparation described in Govender, Journal of Membrane Science 279 (2006) 120-128) is dissolved in dry Dichloromethane under argon atmosphere. A small amount of DMF is added to achieve complete dissolution. The solution is cooled to 0? C. and 1 g N, N-Diisopropylcarbodiimide is slowly added. The mixture is stirred for 20 min at the same temperature followed by evaporation. The residue is dissolved in 5 ml DMF and added to the resin suspension followed by addition of 100 mg 4-Dimethylaminopyridine. The suspension is shaken at room temperature for 1 h. For capping the rest of the free hydroxyl groups, 250 ?l of acetic anhydride and 500 ?l pyridine are added to the reaction mixture. The mixture is shaken at room temperature for additional 30 min. Resin is filtered, washed with DMF (3?), DCM (3?), MeOH (3?) and dried.

Coupling of Tris-Aminoethyl-Amin

[0086] The functionalized Wang Resin is resuspended in 5 m DMF, and after 30 minutes 1 ml tris-(2-aminoethyl)-amine), 1.5 g 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU, Sigma Aldrich, Schnelldorf, Germany) and 2 g ethyl-diisopropylamine in 5 ml DMF are added. After 60 minutes incubation the product, the resin is suction-filtered and washed five times with DMF.

Second Coupling of EDTA-Bis-Methyl Ester

[0087] The functionalized Wang Resin is resuspended in 5 m DMF, and after 30 minutes 1.5 g EDTA-bis-t-butyl ester, 1.5 g TBTU, and 3 ml ethyl-diisopropylamine in 5 ml DMF are added. After 60 minutes incubation the product, the resin is suction-filtered and washed five times with DMF. Then the product is washed by dichloromethane and methanol and dried under vacuum.

Cleavage from Solid Phase, Removal of Methyl Protective Groups

[0088] The Wang resin is transferred to a sintered glass funnel with fine porosity and washed three times with DMF, then five times with dichloromethane. The resin is resuspended in 50% TFA in DCM (v/v), and the mixture is stirred for three hours. Now the resin is filtered and washed three times with small portions of TFA. The filtrates are combined, and 100 ml cold diethyl ether is added to precipitate the peptide. The peptide is filtered through a fine sintered glass funnel, washed with cold ether, and dried.