JPWO2020109162A5 - - Google Patents

Description

According to the present invention, the bifunctional linker moiety K is such that said bifunctional linker moiety K is A-(CH ₂ ) _n -B, AQB and A-(C ₂ H ₄ O) _m - It may be selected from the group consisting of (CH ₂ ) _n —B. In the formula, A and B are functional groups capable of reacting with carboxy groups such as OH, _NH2 , NHR, Cl, Br, I, _OMs , OTs, N3. It is preferably selected from _NH2 and NHR. R is a hydrocarbon group represented by the general formula C _n H _2n+1 , n is an integer of 1 to 6, m is an integer of 1 to 12, and n is an integer of 2 to 12. Q is a linear or branched structure consisting of 2 to 100 molecules (optionally including C, H, N, O and S ) and includes cyclic compounds (eg carbohydrates) .

Claims (22)

A solid phase chelating material comprising a solid phase, polyamine groups bound to said solid phase, and chelating groups bound to said polyamine groups, wherein at least a portion of the polyamine groups comprise at least two chelating groups per polyamine group. groups are attached, each chelating group comprising one or several aminopolycarboxylic acid groups (APA groups), wherein the number of APA groups per polyamine group attached to at least two chelating groups is at least 3 and
The APA group includes ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, triethylenetetraaminehexaacetic acid, ethylenediamine-N,N'-disuccinic acid, ethyleneglycol-bis(β-aminoethylether)-N,N,N', N′-tetraacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, and 1,4,7-triazacyclononane-1,4,7-triacetic acid A solid phase chelator derived from an APA selected from the group consisting of: 2. The solid phase chelating agent according to claim 1, wherein the chelating group is a single APA group, a linear chain formed by two or more APA groups linked to each other via a bifunctional linker site K. a chelate chain, a branched chelate chain formed by three or more APA groups attached to each via a trifunctional linker site L, and at least one bifunctional linker site K and at least one trifunctional linker site A solid phase chelating agent that is selected from the group consisting of mixed chelating chains formed by four or more APA groups linked to each through L. 3. The solid phase chelating agent according to any one of claims 1 or 2, wherein the number of chelating groups attached to the polyamine group is at least three and /or associated with at least two chelating groups. A solid phase chelating agent wherein the number of APAs bound to one polyamine group is in the range of 3 to 20. 4. A solid phase chelating agent according to any one of claims 1 to 3, wherein the polyamine groups are diethylenetriamine, triethylenetetramine, tetraethylenepentamine, pentaethylenehexaamine, based on a pentaerythritol core, A solid derived from polyamines selected from the group consisting of linear and branched polyethyleneimines, polylysines, polypropyleneimines , macrocyclic polyamines , branched polyamines, and amine-functionalized tetrafunctional branched PEGs. Phase chelate material. 5. The solid-phase chelating agent according to any one of claims 1 to 4 , wherein at least some or all of the chelating groups are represented by the general formula -(APA1-K) _n -APA2, wherein , APA1 and APA2 are the same or different, n is an integer from 1 to 50, and K is a bifunctional linker moiety. 6. A solid phase chelating agent according to any one of claims 1 to 5 , wherein at least some or all of the chelating groups have the general formula:

and/or the general formula:

is indicated by
A solid phase chelator wherein APA1, APA2, and APA3 are the same or different aminopolycarboxylic acid groups and L is a trifunctional linker moiety. 7. The solid phase chelating agent according to any one of claims 2, 5 and 6 , wherein said APA and said linker moieties and /or said chelating groups and said polyamine groups are connected via amide moieties Solid-phase chelating agents that bind to each other. The solid phase chelating material according to any one of claims 2 and 5 to 7 ,
- said bifunctional linker moiety K is selected from the group consisting of A-( _CH2 ) _n -B, AQB, and A-( _C2H4O ) _m- ( _CH2 ) _{o -} B; and
wherein A and B are functional groups capable of reacting with a carboxy group selected from OH, _NH2 , NHR, Cl, Br, I, OMs _, OTs , and N3, and R is the general formula C _n is a hydrocarbon group represented by H _2n+1 , n is an integer from 1 to 6, m is an integer from 1 to 12, o is an integer from 2 to 12, Q is from 2 to 100 atoms is a linear or branched structure consisting of and/or
- said trifunctional linker moiety L has the general formula

and

is represented by any one of
wherein A, D, and E are functional groups capable of reacting with a carboxy group, and B is a branching atom attached by at least three stable bonds to the functional group; A solid phase chelator wherein n, m , and o are integers from 2 to 30. 9. The solid phase chelating material according to any one of claims 1 to 8 , wherein spacer moieties are interposed between said solid phase and said polyamine groups. 10. The solid phase chelating material according to any one of claims 1 to 9 , wherein the solid phase is
agarose, cellulose, agar, dextran, chitosan, alginate, gellan , and /or silica, titanium dioxide, zirconium dioxide, aluminum dioxide, other metal or metalloid oxides, gold, glass, and/or acrylates, meta Acrylates, acrylamides, styrene derivatives, vinyl esters, vinyl amides and vinyl alcohols, and/or porous chromatographic supports, non-porous chromatographic supports, membranes, coating surfaces, coating tubes, sensor surfaces, microarray surfaces, coating micro titerplates, and/or magnetic agarose, magnetic silica, and magnetic polymers , and /or
A solid phase chelating agent that is selected from the group consisting of dextran-modified gold chips and magnetic beads. 11. Any one of claims 1 to 10 , carrying metal ions selected from the group consisting of Ni2 ⁺ , Co2+, Cu2 ⁺ , Zn2 ⁺ , ^{Al3+ ,} Fe3 ⁺ , Eu3 ⁺ , Ga3 ⁺ , Mn2 ⁺ , and ^Ca2+ . A metal ion-added solid-phase chelating material comprising the solid-phase chelating material according to item 1. A method for producing the solid phase chelating material according to any one of claims 2 and 5 to 8 ,
- covalently attaching at least three monomeric APA to a solid phase with attached polyamine groups to form polyamine groups with at least three monomeric APA groups attached via amide groups, or covalently attaching at least two monomeric APAs to the bound solid phase to form a solid phase material having polyamine groups with at least two monomeric APA groups attached through amide groups; adding a linker K or a trifunctional linker L to the solid phase material and reacting the bound linker with the carboxy groups of a second APA to form APA groups bound to the polyamine groups bound to the solid phase. or at least two monomeric APA and a bifunctional linker K or at least three monomeric APA and a trifunctional linker. L to form a multimeric APA chelate having a linear chelate chain or a branched chelate chain; forming a polyamine group having at least two linear and/or branched chelate chains attached via a group ;
A method for producing a solid phase chelating material, comprising: 9. A method for producing a solid-phase chelating agent having a linear chelating group to which an APA group is attached via a bifunctional linker according to any one of claims 2, 5, 7 and 8 ,
a) providing APA and a solid phase with attached polyamine groups;
b) reacting one carboxy group of said APA with a solid phase to bind said APA with said solid phase and binding said APA and said solid phase in the presence of a condensing agent;
c) mixing the solid phase material obtained in step b) with a bifunctional linker K;
d) reacting one reactive group of a bifunctional linker K with said bound APA to combine said bifunctional linker K and APA carboxy groups in the presence of a condensing agent;
e) mixing the solid phase material obtained in step d) with further APA;
f) reacting one carboxy group of said further APA with a further reactive group of a bifunctional linker K bound to said solid phase material to combine said further APA and said bifunctional linker in the presence of a condensing agent; and
A method for producing a solid phase chelating material comprising 9. A method for producing a solid-phase chelating agent having a linear chelating group to which an APA group is attached via a bifunctional linker according to any one of claims 2, 5, 7 and 8 ,
a) providing an APA dianhydride and a solid phase material with attached polyamine groups;
b) through the amino groups of the polyamine that bind said APA dianhydride and said solid phase material such that one anhydride group of each APA dianhydride reacts with an amino site and other anhydride groups remain unchanged; and
c) washing the solid phase material obtained in step b) with an anhydrous solvent to remove unbound dianhydride;
d) providing said solid phase material obtained in step c) and a bifunctional linker K;
e) reacting one amine group of said bifunctional linker K with said APA anhydride group to form said bifunctional linker K and said APA without changing the other functional groups of said bifunctional linker K; coupling with an anhydride group;
f) washing the solid phase with anhydrous solvent to remove unbound linker molecules;
g) providing said solid phase material obtained in step f) and additional APA dianhydride;
h) reacting one carboxy group of each APA with the remaining functional groups of said bifunctional linker K to attach said APA dianhydride to said bifunctional linker K ;
A method for producing a solid phase chelating material comprising 9. A method for producing a solid-phase chelating agent having a linear chelating group to which an APA group is attached via a bifunctional linker according to any one of claims 2, 5, 7 and 8 ,
a) providing APA dianhydride and difunctional linker K in a ratio of 2:1 to 1:1;
b) reacting the bifunctional linker K with two different dianhydrides to form the compound APA1-K-(APA2-K) _n -APA3, where APA1 is one anhydride group and one amide function; APA2 is an APA with two amide functional groups, APA3 is an APA with one anhydride group, n is a number from 0 to 50, and K is a bifunctional linker moiety). forming and linking said APA dianhydride to said bifunctional linker K via an amine or via a hydroxyl group of said linker;
c) providing a solid phase with attached polyamine groups;
d) reacting one carboxy group of each APA with an amine site, leaving the other anhydride groups unchanged, the linker-bound dianhydride to the support , and the polyamine groups bound to the solid phase; binding via an amino group;
e) adding water or an aqueous buffer to hydrolyze remaining anhydride groups. A method for producing a solid phase chelating agent having a branched chelating group having an APA group bonded via a trifunctional linker according to any one of claims 2 and 6 to 8 ,
a) mixing APA dianhydride and trifunctional linker L in a ratio of 3:1 to 9:4 with a solid phase having attached polyamine groups;
b) reacting the linker with three different dianhydrides to give one reactant of the formula

(wherein APA1 is an APA group with one anhydride group and one amide group, APA2 is an APA with two amide groups, and L is a trifunctional linker moiety); attaching an anhydride via an amine group to a solid support and attaching it to said linker;
c) providing a solid phase having polyamine groups;
d) reacting one carboxy group of each aminopolycarboxylic acid with an amine site to attach said linker-bound APA to a support via an amide group, leaving the other anhydride groups unchanged; binding to the polyamine-modified solid phase with
e) adding water or an aqueous buffer to hydrolyze remaining anhydride groups. A method for producing a solid-phase chelating material supporting metal ions, comprising: performing one of the methods according to claims 12 to 16 for producing a solid-phase chelating material; is contacted with a solution of metal ions selected from the group consisting of Ni ²⁺ , Co ²⁺ , Cu ²⁺ , Zn ²⁺ , Al ³⁺ , Fe ³⁺ , Eu ³⁺ , Ga ³⁺⁺ , Mn ²⁺ and Ca ²⁺ , A method for producing a solid-phase chelating material supporting metal ions, comprising a step of fixing metal ions. Manufactured by the solid phase chelating material according to any one of claims 1 to 10 , the solid phase chelating material supporting metal ions according to claim 11 , or the method according to any one of claims 12 to 16 purification of proteins having a plurality of histidine residues in the field of molecular biology, a plurality of histidine purification of membrane proteins with multiple histidine residues; purification of GPCRs with multiple histidine residues; purification of transporter proteins with multiple histidine residues; purification of therapeutic proteins; Use for peptide purification, phosphoprotein purification, metalloprotein purification, nucleic acid purification and/or metal-immobilized affinity chromatography. A method of purifying a biomolecule, in particular a recombinant protein or polypeptide, comprising:
a ) preparing a metal-supporting solid-phase chelating material produced by the method of claim 17 ;
b) providing a sample comprising said biomolecule, in particular a protein or polypeptide with multiple histidine sites;
c) contacting the sample with a solid-phase chelating material supporting the metal;
d) separating said bound protein or polypeptide from said sample ;
e) eluting said protein or polypeptide from said solid phase. 20. The purification method of claim 19 , wherein said protein is selected from the group consisting of membrane proteins, GPCRs and antigens. 21. The purification method of any one of claims 19 and 20 , wherein EDTA and imidazole are added to the binding buffer to a concentration of at least 20 mM without interfering with the metal binding and protein yield. and adding DTT to a concentration of at least 10 mM. 22. The purification method according to any one of claims 19 to 21 , wherein the aminopolycarboxylic acid compound immobilized on the solid phase according to any one of claims 19 to 21 is dissolved in a sodium hydroxide solution. A purification method wherein the solid phase chelator can be regenerated for further biomolecule purification by washing to remove contaminants and attached proteins and still have a protein binding capacity of at least 50 mg/ml.