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WO2003087047A1 - Composition de chelate metallique, son complexe macromoleculaire, son procede de preparation et son utilisation - Google Patents

Composition de chelate metallique, son complexe macromoleculaire, son procede de preparation et son utilisation Download PDF

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Publication number
WO2003087047A1
WO2003087047A1 PCT/KR2002/000948 KR0200948W WO03087047A1 WO 2003087047 A1 WO2003087047 A1 WO 2003087047A1 KR 0200948 W KR0200948 W KR 0200948W WO 03087047 A1 WO03087047 A1 WO 03087047A1
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WO
WIPO (PCT)
Prior art keywords
metal chelate
macromolecule
composition
chelate composition
linked
Prior art date
Application number
PCT/KR2002/000948
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English (en)
Inventor
Seong-Kyu Kim
Ho-Il Choi
Young-Hwan Jung
Chae-Jin Lim
Original Assignee
Peptron Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Peptron Co., Ltd. filed Critical Peptron Co., Ltd.
Priority to AU2002309287A priority Critical patent/AU2002309287A1/en
Publication of WO2003087047A1 publication Critical patent/WO2003087047A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/57—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C323/58—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J45/00—Ion-exchange in which a complex or a chelate is formed; Use of material as complex or chelate forming ion-exchangers; Treatment of material for improving the complex or chelate forming ion-exchange properties

Definitions

  • the present invention relates to a metal chelate composition, macromolecule complex thereof, preparing method thereof and use in the purification of the materials such as protein.
  • Metal chelate composition which is represented by ethylenediamine, ETDA
  • Metal chelate composition is used in various ways in the elimination of heavy metal, the treatment of wastes and metal affinity chromatography and so on.
  • metal chelate composition is used in protein purification chromatography.
  • Metal chelate composition of ligand such as imminodiacetic acid, nitrilotriacetic acid(NTA), carboxymethyl aspartic acid, etc. linked with matrix of macromolecule is produced commercially.
  • NTA nitrilotriacetic acid
  • carboxymethyl aspartic acid etc. linked with matrix of macromolecule is produced commercially.
  • conventional metal chelate compositions currently used in affinity chromatography have their own merits and demerits.
  • NTA Nitrilotriacetic acid
  • NTA is a combination of 3 covalent bonds of carboxymethyl radicals to nitrogen atom.
  • the following composition is adopted in U. S. patent 4,877,830 in order to combine such composition of NTA to matrix.
  • Ligand is produced and then combined to the matrix attached with activated linker.
  • Ligand is produced through the hydrogenation after producing N-carboximethylated Cbz-Lysine by the reaction of Cbz-Lysine and bromoacetic acid.
  • the purpose of the present invention is to overcome the demerit of the conventional metal chelate compositions by deriving the macromolecule complex, wherein nitrilotriacetic acid(NTA) ligand is combined with macromolecule compound in a novel way; ie, to provide the macromolecule complex of metal chelate compositions which can be produced at a low cost by a simple process.
  • the purpose of the present invention is to provide the macromolecule complex of matrix type metal chelate composition for chromatography which enables to effectively refine protein by the more rigid combination of metal and chelate composition.
  • metal chelate composition having strong affinity to the molecule to make affinity with ligand and macromolecule complex.
  • the inventor derived a process for manufacturing chelate ligand having nitrilotriacetic acid(NTA) composition and -SH as a reaction radical by using amino acid having -SH(sulfhydril) such as Cystein; and suggest a process for manufacturing macromolecule complex of a novel NTA metal chelate composition.
  • novel metal chelate composition of the present invention 3 carboxyl radicals of the ligand part are linked to metal and a sulfhydril is linked to other composition, especially macromolecule matrix.
  • the present invention relates to: the processes of manufacturing the macromolecule complex of metal chelate composition by manufacturing the novel metal chelate composition and linking it to macromolecule; and the macromolecule complex of the metal chelate composition by compounding ligand to macromolecule in regular sequence: and the method of using the above novel metal chelate composition or the macromolecule complex in purification material such as protein.
  • the terms such as ligand, matrix, macromolecule, metal chelate composition, and macromolecule complex of metal chelate composition are used in "the summary of the invention” and “what is claimed is”.
  • the “macromolecule” in the present description means to be the macromolecule before the direct linking of the affinity ligand part to the material (e. g. protein) to be chemically attracted.
  • the material e. g. protein
  • protein, fat, carbohydrate, alkaloid, resin or cross-linked agarose, cross-linked cellulous, etc. are called macromolecules. That is, the "macromolecule” in the present description is used in a broader meaning which includes general term “matrix” or “resin” as used in the conventional affinity chromatography.
  • ligand and "metal chelate composition” are used together in the present description; also, mean the materials showing affinity to the specific material such as nitrilotriacetic acid.
  • Micromolecule complex of metal chelate composition is used as a term expressing the state of linking of the linker and the metal chelate composition to macromolecule; also, means the state of transition metal chelated or the state of transition metal excluded.
  • the process of manufacturing macromolecule of the metal chelate composition of the present invention is a method to attach metal chelate composition manufactured such as N, N dicarboxi ethyl cystein to macromolecule(Embodiment 1-3).
  • Another way of manufacturing macromolecule complex of the metal chelate composition is to attach amino acid having -SH such as cystein; carboxcilate amin radical attached, and nitrilotriacetic acid ligand in order to chemically stabilized macromolecule. (Embodiment 4-7).
  • the control of pH is important in order to produce desired macromolecule in such a sequential reaction.
  • acidity should be maintained between Ph 6.0-8.5 so that only the -SH enters into reaction and the amin radical does not.
  • carboxilation reaction of amin acidity should be maintained for proper reaction.
  • the present invention relates to the metal chelate composition as shown in the following chemical formula.
  • the the metal chelate composition provides researcher with necessary use by linking various type of desired macromolecule and matrix to various linker.
  • HS-R ⁇ -CH(COOH)-N-(CH 2 COOH) 2 (wherein, Ri is an alkyl radical linker having 1-5 carbon such as -CH - or -CH 2 CH 2 -.)
  • the present invention relates to metal chelate composition linked with transition metal ion such as Co, Ni, Zn, Fe, Cu, etc. to the above metal chelate composition.
  • Ri is an alkyl radical linker having 1-5 carbons as in -CH 2 - or - CH 2 CH 2 -;
  • R 2 is a linker activating macromolecule and linking with -SH, preferably as - CH 2 CH(OH)CH 2 -, -CH(OH)CH 2 -O-(CH 2 ) 4 CH(OH)CH 2 -, -CH 2 CH 2 SO 2 CH 2 CH 2 -, - COCH 2 -, -CH2CH-2CH 2 -, -CH 2 CH(OH)CH 2 -, -CH 2 CH(Br)CH 2 - or -OCO-,
  • R 3 fe is a macromolecule compound including matrix for chromatography- ⁇ - such as cross-linked agarose, cross-linked cellulose, cross-linked dextran, cross-linked metacrilamide, silica or protein, fat, carbohydrate, alkaloid, resin.
  • the present invention relates to macromolecule complex of metal chelate composition linking transition metal ion such as Co, Ni, Zn, Fe, Cu etc. with macromolecule complex of the above metal chelate composition as shown in the following chemical formula 3.
  • transition metal ion such as Co, Ni, Zn, Fe, Cu etc.
  • M stands for transition metal on the above formula.
  • the present invention relates to the process of manufacturing macromolecule complex of the above metal chelate composition according to the following sequence.
  • the method of manufacturing macromolecule complex of the above metal chelate composition of the present invention is remarkably simpler process than described in the process of the conventional U.S. Patent, and provides macromolecule complex of the metal chelate composition having similar absorption effect of to conventional NTA matrix with less time and lower cost.
  • the present invention describes a manufacturing method of linking metal chelate composition to activated macromolecule after the metal chelate composition is composed; and illustrate that the 2 novel methods can be used in the same manner.
  • Fig. 1 shows a electrophoresis photograph of the purification process of the MutS protein attached to the end of 6 histidine by using metal chelate agarose of the present invention and NTA agarose.
  • Fig. 2 shows a electrophoresis photograph of the purification process of the same protein by using the above 2 metal chelate agarose again.
  • M size marker of NONEX corp.
  • the present embodiment is an example of the reaction producing metal chelate composition.
  • lOmg of S-trityl-cysteine is melt in IN ⁇ aOH.
  • 16g of bromoacetic acid is added gradually to IN ⁇ aOH.
  • Subsidence is made by adding IN HCL after the sediments being gathered with glass filter funnel and dissolved in 20m£of IN ⁇ aOH ( ⁇ , ⁇ - dicarboximethyl- cysteine).
  • the reaction is maintained for 1 hour after putting the subsidence into a triangle flask, adding lOOg of distilled water and 1ml of trifluoride acetic acid, and filling nitrogen in it.
  • the embodiment is another example of the reaction producing metal chelate composition.
  • lOg of cystine or homocystine is dissolved in the 50ml of the solution of 0.1M NaHCO 3 , pH 10.5. After dissolving 24g of bromoacetic acid into 40ml of 4N NaOH solution and adjusting pH to 10.5. It is added to cystine or homocystine solution.
  • the present embodiment is another example of the reaction linking N, N. dicarboxymethyl-cysteine produced in the above embodiment 1 or 2 to macromolecule lOg of activated macromolecule produced as in the embodiments 4, 5, 6 is dissolved in 100ml of 25mM NaPi(pH 7.5). After lOOmg of N, N. dicarboxymethyl-cysteine is dissolved in 100ml of 25mM NaPi (pH 7.5), it is mixed with the above activated macromolecule solution. After being mixed for 24 hours in the natural temperature, the macromolecule is separated by glass funnel and washed with 1000ml of distilled water, 1000ml of 1% acetic acid, 1000ml of distilled water in order.
  • the present embodiment is an example of the reaction linking R 2 by activation of ceparose CL-6B, a cross-linked agarose as macromolecule.
  • lOOmg of ceparose CL-6B matrix is washed using glass funnel by 1000ml of distilled water. Macromolecule matrix in which water is eliminated is moved into the 500ml triangle flask, and is added by 40ml of 4N NaOH and 15ml of epichlorohydrin. It is agitated for 6 hours in the temperature of 28 ° C after plugging the opening with a silicon plug. The reacted solution is move into glass filter funnel and washed by lOOOmg of distilled water.
  • the present embodiment is an example of the reaction linking the other linker R 2
  • BDE butanedioldiglycidyl ether, 60% purity. It is agitated for 8 hours at the temperature of 28 °C and is moved to the glass funnel. The reacted solution is move into glass filter funnel and washed by lOOOmg of distilled water.
  • the present embodiment is an example of the reaction linking the other linker R 2 1.4g of bromoacetic acid is put into triangle flask together with 1.4g of N- hydroxysuxineimide 80m-C of dioxane and agitated. It is agitated for 70 minutes after adding 2.2g of dicyclohexylcarbodiimide. Sediment is eliminated by glass funnel. Bromorecetichydroxysuxineimide is composed by eliminating dioxane with a rotary evaporator.
  • 3g of AH-ceparose is washed by lOOmu of distilled water in the glass funnel filled with 0.1 sodium phosphate solution (Ph 7.5); and agitated for 2 hours after adding 2mg of the bromorecetichydroxysuxineimide prepared as in the above.
  • the solution is moved into glass funnel and washed with lOOm ⁇ of distilled water.
  • the present embodiment is an example of the reaction linking metal chelate composition with the activated macromolecule produced in the above embodiments 4, 5, 6.
  • IOOI of the macromolecule activated as in the process of the embodiments 4, 5, 6 is put into triangle flask.
  • lOOm-6 of 25mM sodium phosphate pH 7.5 solution.
  • pH of it ia adjusted to 7.5. It is agitated for 24 hours after being filled with nitrogen gas in the reactor.
  • the solution is moved into glass filter funnel and is washed with 100m£ of distilled water, 1000m£ of 10% acetic acid, distilled water in sequence.
  • the washed macromolecule moved into triangle flask is added with 100m£ 1M NaHCO 3 , pH 10.0; and the solution of lOO of 4N NaOH where 15 g of bromoacetic acid is dissolved and pH adjusted to 10.5.
  • the solution is agitated for 24 hours in the normal temperature, moved into funnel and washed with 1000m£ of distilled water, 1000m-*.. of 10% acetic acid, distilled water in sequence.
  • the present embodiment is described for the method for attaching transition metal to the macromolecule attached with metal chelate composition and the method for measuring density of metal ion.
  • 100ml of the macromolecule complex of the metal chelate composition prepared in the embodiment 3 or 7 is put into glass filter funnel and is washed with 500ml of distilled water. After 100ml of the solution lOOmM EDTA, pH 8.0 is added, the solution liquated is recovered. It is found that there exist nickel ion of more than 560/tg/ml in the result of measurement by an atomic absorption spectrophotometer.
  • the present embodiment is an example of the purification of protein by using chelate agarose which is a macromolecule complex of the metal chelate composition of the present invention as prepared in the above embodiment 8.
  • 0.5ml of the chelate agarose of the present invention is put into chromatography tube and washed with 5ml of distilled water. It is washed with 5ml of the solution of 5 mM imidazole, 0.5 M NaCl, 50mM NaH 2 PO 4 , pH 8.0 after being washed with 5ml of lOOmM
  • the purity is confirmed by Coomasie blue after the specimen of each step is separated by 12% SDS-PAGE (Fig. 1).
  • the amount of the protein attached to the metal chelate agarose is measured using the Bradford method with the standard of small serum albumin.
  • Collected fraction is diluted 100 times with distilled water and light absorbing index is measured at 600nm on the 0.1ml of the liquid and compared with the normal protein.
  • MutS protein is attached on the macromolecule of the novel metal chelate composition in the ratio of 5.2mg per 1ml
  • the NTA agarose, macromolecule complex of the metal chelate composition of QIAGEN corp. is absorbed by 5.1mg.
  • the metal chelate agarose used in the purification is set to equilibrium by using 5mM imidazole, 0.5M NaCl, 50mM NaH 2 PO 4 , pH 8.0 solution 5m# after washing with 200mM of imidazole, 0.5M NaCl, 50mM NaH 2 PO , pH 8.0 solution 10 m Cell crushing solution 2m£ is added to the matrix and the matrix is washed with 5mM imidazole, 0.5M NaCl, 50mM NaH 2 PO 4 , pH 8.0 solution 5ml.
  • Fig. 2 shows the result after reusing the metal chelate agarose and commercial metal chelate NTA agarose of QIAGEN corp., which is similiar to the result of Fig. 1. This shows that the metal chelate composition of the present invention is very stable and can be used repeatedly.
  • the metal chelate composition, the macromolecular complex and the method for the composition are carried out very simply compared with the method of prior art, reducing time and cost while showing similar properties of prior art metal chelate resins.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)

Abstract

La présente invention a trait à une nouvelle composition de chélate métallique et son complexe macromoléculaire. L'invention a trait également à un procédé de préparation d'une composition de chélate métallique et un procédé d'utilisation de la composition de chélate métallique dans la purification d'une substance de type peptide. La composition de chélate métallique de la présente invention est représentée par la formule HS-R1-CH(COOH)-N-(CH2COOH)2, dans laquelle R1. représente un lieur de groupe alkyle ayant 1 à 5 atomes de carbone, et présente une affinité et une stabilité suffisantes sur la substance à purifier par un procédé simple.
PCT/KR2002/000948 2002-04-11 2002-05-20 Composition de chelate metallique, son complexe macromoleculaire, son procede de preparation et son utilisation WO2003087047A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002309287A AU2002309287A1 (en) 2002-04-11 2002-05-20 Metal chelate composition, macromolecule complex thereof, preparing method thereof and use

Applications Claiming Priority (2)

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KR10-2002-0019734 2002-04-11
KR20020019734 2002-04-11

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WO2003087047A1 true WO2003087047A1 (fr) 2003-10-23

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007070245A (ja) * 2005-09-05 2007-03-22 Tosoh Corp 生分解性アミノポリカルボン酸誘導体
JP2007070244A (ja) * 2005-09-05 2007-03-22 Tosoh Corp ポリカルボン酸アミド誘導体およびキレート剤
WO2007137752A1 (fr) * 2006-05-26 2007-12-06 Ge Healthcare Bio-Sciences Ab Procédé pour la génération de ligands présentant une affinité avec des agents chélatants métallifères

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5362412A (en) * 1991-04-17 1994-11-08 Hampshire Chemical Corp. Biodegradable bleach stabilizers for detergents
WO2001081365A2 (fr) * 2000-04-24 2001-11-01 Sigma-Aldrich Co. Compositions chelatrices de metal

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5362412A (en) * 1991-04-17 1994-11-08 Hampshire Chemical Corp. Biodegradable bleach stabilizers for detergents
WO2001081365A2 (fr) * 2000-04-24 2001-11-01 Sigma-Aldrich Co. Compositions chelatrices de metal

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007070245A (ja) * 2005-09-05 2007-03-22 Tosoh Corp 生分解性アミノポリカルボン酸誘導体
JP2007070244A (ja) * 2005-09-05 2007-03-22 Tosoh Corp ポリカルボン酸アミド誘導体およびキレート剤
WO2007137752A1 (fr) * 2006-05-26 2007-12-06 Ge Healthcare Bio-Sciences Ab Procédé pour la génération de ligands présentant une affinité avec des agents chélatants métallifères
US7988858B2 (en) 2006-05-26 2011-08-02 Ge Healthcare Bio-Sciences Ab Method for generating metal chelating affinity ligands

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