Labeling of target molecules such as His-tagged proteins via metal complex reagents and / or site-specific, kinetically inert conjugation of carriers

Abstract

The present invention relates to means and methods for conjugating / binding target molecules, such as proteins, to labels and / or carriers. Specifically, the present invention relates to (i) CO3 2- or HCO3 - The present invention provides a complex comprising (i) a metal cation ligand, which is a carbonate ion selected from the group consisting of: (i) a carbonate ion selected from the group consisting of: (ii) a metal cation coordinating a metal cation chelating domain, which comprises a chelating ligand and a label and / or carrier. This complex can be used to bind the label and / or carrier to a target molecule, preferably a protein. Attachment of the label or carrier via the complex of the present invention involves displacement of the metal cation ligand with a coordinating group of the target molecule, such that a product complex is formed with the target molecule as the primary ligand in the coordination sphere of the metal cation. Accordingly, the present invention also provides uses and methods involving attachment of the label and / or carrier to the target molecule. Also provided are products obtained by the labeling and / or carrier binding methods of the present invention and uses thereof. The present invention further relates to methods for producing the complexes of the present invention and kits containing components for producing the complexes of the present invention.

Description

Technical Field

[0001] The present invention relates to means and methods for labeling and / or conjugating / binding a target molecule such as a protein to a label and / or a carrier. Specifically, the present invention provides a complex comprising (i) a metal cation ligand which is CO 3 2- or HCO 3 - and (ii) a metal cation that coordinates a metal cation chelating domain comprising a chelating ligand and a label and / or a carrier. This complex can be used to bind a label and / or a carrier to a target molecule, preferably a protein. The binding of the label or carrier via the complex of the present invention involves replacement of the metal cation ligand with a coordinating group of the target molecule such that a product complex is formed with the target molecule as the major ligand in the coordination sphere of the metal cation. Accordingly, the present invention also provides uses and methods involving the binding of a label and / or a carrier to a target molecule. Also provided are products obtained by the labeling and / or carrier binding methods of the present invention and their uses. The present invention further relates to a method for producing the complex of the present invention and a kit comprising components for producing the complex of the present invention.

Background Art

[0002] In recent years, chemically modified proteins have become a very important means for many biological applications. Various biochemical and cellular techniques in life science research, such as fluorescence-based assays, Western blotting and protein purification, rely on labeled or immobilized proteins. However, protein conjugation is also an important production step for the development of biopharmaceuticals (e.g., antibody-drug conjugates, PEGylation and lipidation), biosensors, bioimaging and even medical diagnostics including medical engineering. Thereby, in all fields of application, a simple, stable and site-specific modification method that does not interfere with protein function is desirable. Furthermore, other biomolecules such as nucleic acids are also often labeled or bound to a carrier.

[0003] In classical labeling / immobilization procedures, small molecules are covalently attached to reactive groups (such as primary amines in lysine or thiols in cysteine) in the target unmodified protein in a rapid and efficient manner, for example, via N-hydroxy-succinimide (NHS)-derived reagents or maleimides (Chen and Wu, 2016). However, all of these approaches have inherent drawbacks due to the ubiquitous availability of these reactive sites in proteins. Thus, control of these labeling reactions, which results in lack of site specificity, heterogeneous immobilization / labeling stoichiometry and furthermore, destabilization and loss of protein function, as well as large batch-to-batch variability, is limited (Lindhoud et al., 2012). To address this problem, several new technologies for site-specific protein conjugation have been developed in recent years.

[0004] Among them, the most prominent is the Avi tag system, in which the biotin moiety is site-specifically attached to a short peptide tag by enzymatic catalysis involving the biotin ligase BirA (Tirat et al., 2006). However, other methods also use enzymes such as sortase (Popp et al., 2007), transglutaminase (Lin and Ting, 2006), lipoic acid ligase (Fernandez-Suarez, M. et al., 2007) and phosphopantethenyl-transferase (Yin et al., 2005) to catalyze the oriented conjugation of proteins via short recognition peptide tags.

[0005] Another approach is the expansion of the genetic code by ribosomal incorporation of bioorthogonal functional groups such as tetrazine, alkyne, azide or norbornene via unnatural amino acids (Ou et al., 2011; Deiters et al., 2003; Lang et al., 2012). Specific ligation chemistry can be used to modify the target protein at the incorporated group in a very precise manner.

[0006] Alternatively, for unnatural amino acids, conventional amino acids that exhibit unique reactivity and low abundance on the protein surface can be used for site-specific labeling. Many methods in this context use engineered cysteine substitutions that provide accessible thiol groups for selective targeting (Junutula et al., 2008; Cal et al., 2014). Not only surface-exposed amino acids but also N-terminal amino acids can be useful reaction points for protein conjugation. For example, N-terminal cysteine can be selectively targeted by native chemical ligation with thioester derivatives (Dawson et al., 1994), or when incorporated into a short N-terminal tag, the protein can be labeled with perfluoroaromatic reagents (Zhang et al., 2016). Alternatively, the protein can be site-specifically conjugated by oxidation of N-terminal serine to provide a unique aldehyde group that can be targeted (Gaertner and Offord, 1996), or by acylation of an N-terminal glycine-histidine tag (Martos-Maldonado et al., 2018).

[0007] The above methods share the common point that they require the incorporation of special molecular tags, unnatural amino acids, and / or additional amino acids that are not often used for other applications. Thus, genetic manipulation and protein re-expression that break bones are often required. Even more importantly, the mainly multi-step chemical conjugation reactions of these methods are often carried out under rather harsh reaction conditions that can negatively affect protein stability and function.

[0008] Furthermore, several labeling strategies have been developed that rely on the interaction with His-tag, an affinity tag widely used in the field of protein biochemistry. The His-tag usually consists of 6 - 8 histidine moieties, and Ni of nitrilotriacetic acid (NTA) and the imidazole group of histidine 2+His-tagged proteins are widespread due to established purification techniques based on metal chelation (Hochuli et al., 1988). The principle of binding His-tagged proteins to Ni 2+ -NTA substrates through the formation of [Ni(II)(NTA)(His-tag)] complexes has also been adapted for many other applications such as immobilization of proteins on surfaces (Kang et al., 2007; Rusmini et al., 2007), and conjugation of fluorophores and other molecules to proteins (Kamamoto et al., 2008). However, the main drawback of these methods is the low affinity and rapid ligand exchange rate of these Ni 2+ -mediated complexes. Even when using improved Tris-NTA reagents (Huang et al., 2009), already small amounts of common chelators such as EDTA or imidazole disrupt the complexes and interfere with protein conjugation.

[0009] Several additional references describe similar complexes that are not kinetically inert and furthermore do not require instability; see, for example, WO2005 / 112977A2, WO03 / 072143A1, WO2005 / 120700A2, WO2009 / 114520, WO98 / 06739A1, U.S. Patent No. 4,569,794A, Block et al., 2009, WO03 / 018756A2, US2010 / 069293A1, WO2004 / 104023A2, WO02 / 33044A2, US2013 / 131283A1, WO2011 / 031771A1, US2008 / 015263.

[0010] WO2003 / 072143A1 also describes the use of metal bridges to connect a carrier, which may contain a polypeptide having a histidine tag, to an active agent of interest. Ni 2+ To overcome the limitations of Ni 2+ - or Co 2+ -mediated complexes, Co 3+has been proposed as a metal ion for mediating complex formation (Wegner and Spatz, 2013; Hale, 1995). Four coordination sites are occupied by a chelator such as NTA, and two are occupied by the histidines of the His-tag, Co 3+ of the low-spin octahedral paramagnetic complex (e g 6 t 2g 0 ) can be used to achieve a kinetically inert linkage of the His-tagged protein to the NTA moiety. Co 3+ -based complexes have a significantly lower ligand exchange rate (about 10 2+ than Ni 6 s -1 )-based complexes (3×10 -6 s -1 ), as found (Lippard and Berg, 1994).

[0011] However, although the ligand exchange rate of the [Co(III)(NTA)(His-protein)] complex is extremely low, conversely, the formation of the complex is also slow. Thus, an indirect and multi-step preparation method is currently the method of choice for producing such [Co(III)(NTA)(His-protein)] complexes (WO2014 / 072525A1; Wegner and Spatz, 2013; Hale, 1995; see the schematic of the reaction scheme in Figure 2A). First, after the oxidation step with hydrogen peroxide of the overall [Co(II)(NTA)(His-protein)] complex, a pre-complex with Co 2+ is formed, and Co 2+ is directly oxidized to Co 3+ in the final complex. This method is very fast and simple, but the oxidation process in the presence of the protein can cause loss of protein function due to amino acid oxidation. Furthermore, the combination of hydrogen peroxide and cobalt ions can also induce a Fenton reaction that can lead to cleavage or degradation of the protein backbone and release of the protein from the complex, including removal and labeling of the His-tag (Andberg et al., 2007).

[0012] The Co 3+ -based complexes produced using an indirect and multi-step preparation method including an acidification step have also been used to immobilize proteins on the surface (Wegner et al., 2016; Di Russo et al., 2018). However, even in the context of surface immobilization, this method is troubled by the use of harsh oxidizing agent H 2 O 2 which can negatively affect protein function and interfere with certain surface structures.

[0013] Similar to the above-mentioned literature, EP0497585A2 uses the oxidation of Co 2+ to Co 3+ only after the formation of the [Co(II)(IDA)(His-protein)] complex. The [Co(II)(IDA)(His-protein)] complex is contacted with O 2 gas for several hours to facilitate the oxidation of Co 2+ to Co 3+ . However, it is known in the art that labile proteins can be damaged by exposure to high concentrations of oxygen for several hours. Furthermore, the inventors were unable to reproduce the method. That is, the inventors did not observe a stable complex when the [Co(II)(NTA)(His-protein)] complex was contacted with O 2 gas.

[0014] Zatloukalova and Kucerova proposed a procedure for forming a [Co(III)(IDA)(His-tag)(H 2 O)] complex that can avoid oxidation in the presence of proteins (Zatloukalova and Kucerova, 2006). In this procedure, the Co 2+ center is oxidized to Co 3+ using hydrogen peroxide in a pre-complex with IDA, and then the resulting [Co(III)(IDA)(H 2 O) 3 + is coordinated with the His-tagged protein to form the final [Co(III)(IDA)(His tag)(H 2 ​O)]To form a complex (see Figure 2B for a schematic of the reaction scheme; Zatloukalova and Kucerova, 2006). However, this method still has significant drawbacks. Co with a water ligand 2+ Compared to the pre-complex based on Co 3+ with a water ligand, the pre-complex based on Co 3+ coordinates significantly more slowly and, as shown by the inventors in the attached examples, especially at the low temperatures required for many proteins, the final effectiveness of complex formation is also low. Furthermore, the use of hydrogen peroxide in the presence of an IDA conjugate can still negatively affect the conjugate to be attached to the IDA conjugate by oxidation, i.e., the function of the label and / or the carrier. For example, the fluorescence of a fluorophore can decrease upon oxidation. Additionally, the protocol requires the purification of the oxidized pre-complex from hydrogen peroxide for further downstream applications. These purification processes are material and time consuming and carry the risk of oxidizing and damaging the protein to be modified in downstream applications by residual oxidants. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0015] Thus, the technical problem underlying the present invention is to provide improved means and methods that enable the binding of a label and / or a carrier, such as a protein, to a target molecule by complex formation in an easy and efficient manner that prevents inhibition of the function and / or stability of the label / carrier and the target molecule. MEANS FOR SOLVING THE PROBLEM

[0016] This technical problem is characterized in the claims and is solved by the provision of the embodiments provided below in this specification. In a first aspect, the present invention provides a) a metal cation; b) CO 3 2- and HCO 3 -A metal cation ligand which is a carbonate ion selected from; and c) A metal cation chelating domain comprising a chelating ligand and a label and / or a carrier A compound in the form of a complex containing is provided.

[0017] The inventors have surprisingly found that the complex of the present invention is an advantageous means for binding a label and / or a carrier to a target molecule such as a His-tagged protein. Labeling is achieved by exchange of the metal cation ligand of the complex with the target molecule in the first coordination sphere of the complex. This exchange forms a new complex having a metal cation chelating domain comprising a chelating ligand, a label and / or a carrier as the first ligand, and a target molecule as the second ligand. The transition metal cation Co 3+ or Pt 4+ Using the complex of the present invention containing, as exemplified in the attached examples, the complex of the present invention enables the binding of the label and / or the carrier to the target molecule in a kinetically inert manner. Histidine residues are ligands that can participate in the formation of metal complexes, particularly with transition metals such as Co 3+ Therefore, preferred target molecules are His-tagged proteins or proteins having a histidine-rich region. The histidine tag can function as a bidentate and / or multidentate ligand.

Mode for Carrying Out the Invention

[0018] The advantage of the complex of the present invention is that the oxidation reaction using H 2 O 2 is not necessary for the production step of the complex in which the label and / or the carrier is present, nor for the binding of the label and / or the carrier to the target molecule. H 2 O 2Avoidance of oxidation steps such as processing has the advantage that binding of the label and / or the carrier can be achieved without interfering with the function of the target molecule, label and / or carrier due to oxidation. This makes metal complex-mediated labeling and / or carrier binding more widely applicable, especially to oxidation-sensitive target molecules such as proteins. Furthermore, it also reduces the need for cumbersome and time-consuming washing steps to remove oxidants.

[0019] For Co(III) or other transition metals with low ligand exchange rates, direct complex formation without any intermediate steps is extremely slow and inefficient. Therefore, current state-of-the-art methods for the formation of these kinetically inert complexes focus on a two-step procedure. Thereby, first, a metal center in a kinetically labile oxidation state (e.g., Co(II)) is formed, followed generally by an oxidation process using hydrogen peroxide. Thus, the oxidation process can result in unpredictable functional impairment of the protein, metal-binding domain and / or carrier.

[0020] To overcome these limitations, the present invention describes a process for the direct formation of kinetically inert complexes. Starting directly from a metal in the desired oxidation state, the use of oxidizing reagents in the presence of proteins, metal-binding domains or proteins can be avoided. Thereby, the selected metal is introduced into a metal-binding domain pre-complexed with three metal cation ligands which are carbonate ions or nitrate ions. In the first step, two metal cation ligands are replaced by a metal cation chelating domain, and then the last metal cation ligand is replaced via coordination of the protein. The preliminary coordination of the metal with carbonate ions or nitrate ions facilitates the formation of the final complex, resulting in a significantly faster complex formation rate paired with a higher complex formation effect. The improved direct complex formation process using a pre-complex coordinated with carbonate ions or nitrate ion ligands originates from the ability of these metal cation ligands to be protonated after release from the pre-complex. In the case of carbonate ions, protonation upon ligand release can result in gas formation that can further enhance the formation process. Thus, as shown by the inventors, carbonate ions or nitrate ion pre-complexes can be used to directly form kinetically inert metal complexes significantly faster and more efficiently than other ligands containing water. As shown in the attached examples, the means and methods of the present invention relate to novel and progressive complexes comprising and / or consisting of metal cation chelating domains comprising metal cations, carbonate ions (as metal cation ligands; CO 3 2- or HCO 3 - ) and chelating ligands, as well as labels and / or carriers. "Carbonate ions" (CO 3 2- or HCO 3 -) Instead, it is clear from the disclosure of this specification and the appended examples that a corresponding complex using "nitrate ions" can also be used. Therefore, the present invention may also be based on the corresponding "nitrate ion complex". Therefore, not only the corresponding "carbonate ion complex", but also the "nitrate ion complex" is described in this specification. Carbonate ions or nitrate ions in the context of the present invention may be considered equivalent because both metal cations are protonated after dissociation from the corresponding pre-complex. In the context of the present invention, carbonate ion complexes are preferred for the means and methods provided herein. Further details are provided below in this specification.

[0021] In the prior art of Wegner and Spatz (loc. cit.) and Zatloukalova and Kucerova (loc. cit.) above, Co 3+ A method for labeling proteins by the formation of a mediating complex is described. However, in contrast to the present invention, both of these methods included an oxidation step using H 2 O 2 (see FIGS. 2A and B). The oxidation step is a detrimental process that can negatively affect the stability and function of the label (e.g., fluorophore) and / or the carrier and the protein to be labeled (see the appended examples). The complexes of the present invention enable the avoidance of the oxidation step included in these prior art protocols. Furthermore, the complexes of the present invention rapidly and efficiently coordinate a metal that binds to a target molecule, preferably a His-tagged protein or a protein having a histidine-rich region, such that the label and / or the carrier can be bound to the target molecule by a metal cation-mediated interaction. The label and / or the carrier are bound to the target molecule in the final product in a similar kinetically inert manner as described by Wegner and Spatz and Zatloukalova and Kucerova. Binding of the target molecule is achieved by exchanging the metal cation coordination ligand with the target molecule to form a new complex, herein referred to as the "product complex". CO 3 2-and HCO 3 - By selecting a metal coordination ligand that is a carbonate ion or a nitrate ion selected from 3 and - , the inventors were able to form a "product complex" containing a label / carrier containing a metal cation chelating domain and a target molecule coordinated to the metal cation very efficiently and rapidly. In particular, the inventors have found that the binding of the target molecule is more complete and faster, especially at low temperatures, compared to complexes using water as the metal cation ligand as described in the methods of Zatloukalova and Kucerova. Thus, the complexes of the present invention bind the label and / or carrier to the target molecule (e.g., His-tagged protein) faster and more completely. In short, an important advantage of the complexes of the present invention is that they allow for rapid and efficient binding while at the same time preventing the requirements for using oxidation reagents in the presence of the label and / or carrier.

[0022] The complexes of the present invention or compositions containing them are particularly useful in several applications such as, but not limited to, the labeling of proteins with fluorophores, toxins, diagnostic moieties, targeting moieties, stabilizing domains and / or reactive groups. The complexes can also be used to produce biopharmaceuticals such as antibodies labeled with toxins, and diagnostic agents in particular. The labeling is kinetically inert (i.e., the ligand exchange rate is 10 -1 s -1 or less), and can prevent ligand exchange, which is often a problem when using complexes based on Ni 2+ or Co 2+ . The labeling is more thermodynamically stable than when using complexes based on Ni 2+ or Co 2+ .

[0023] Using the complexes of the present invention, stable binding of a target molecule, preferably a protein, to a carrier can also be achieved. The carrier can be, for example, a surface, beads, nanoparticles, prosthetics, quantum dots, polymers, hydrogels, microparticles, spheres (e.g., nano- and / or microspheres) or a combination thereof. For example, Example 19 shows that His-tagged GFP can be bound to a gold nanostructured glass surface. First, the gold nanostructured glass surface is functionalized with NTA by coupling an NTA-linker-thiol reagent by the interaction of gold particles and thiol groups. Subsequently, the complex of the present invention is prepared on the surface and His-GFP is bound (Figure 21). Further, in non-limiting Example 20, it is shown that His-tagged GFP can be bound to a biotin group coupled via a linker to the complex of the present invention. Subsequently, the product containing the biotin group, linker, complex of the present invention and His-tagged GFP was coupled to beads containing streptavidin groups (Figure 22).

[0024] The attached examples show that various (His-tagged) proteins can be coupled to beads using the complexes of the present invention. For one tested protein (sortase A), it was demonstrated that the enzyme activity was preserved upon immobilization (Example 14, Figure 16C). It is also shown that the antibody can be coupled to beads via the complex of the present invention using the histidine-rich region of the Fc region of the antibody. As shown in Figure 16D, the immobilized antibody retains its ability to bind its antigen.

[0025] The complex of the present invention can achieve rapid and efficient metal cation-mediated labeling or carrier binding to a target molecule without the need for an oxidation step in the presence of a target molecule to be labeled and / or a label and / or a carrier as described by Wegner and Spatz and Zatloukalova and Kucerova. Thus, the complex of the present invention is particularly suitable for the above applications where it is typically very important to avoid oxidation of the target protein and / or the label and / or the carrier.

[0026] The complex of the present invention can be provided in solution or as a solid. The complex of the present invention may, in particular when provided in solution, carry a charge. The charge will depend on the charges of the metal cation, the metal cation ligand and the metal cation chelating domain. For example, Co as the metal cation 3+ , CO as the metal cation ligand 3 2- , and a complex comprising a metal cation chelating domain consisting of NTA as the chelating ligand and an uncharged label has a charge of "2-" (i.e., a divalent negative charge). The complex can also be provided with a counterion. If the complex further comprises one or more counterions, it can also be provided as a solid. The counterion is preferably a monovalent ion. Particularly preferred are monovalent ions derived from the group of alkali metals. Most preferred are Na + and K + as counterions when the complex is negatively charged.

[0027] The metal cation chelating domain of the complex of the present invention (or may also be referred to as the first ligand or chelator) includes a chelating ligand. The chelating ligand mediates coordination with the metal cation of the complex by providing at least two binding sites that can be coordinated by the metal cation of the complex (i.e., in other words, is bidentate or multidentate). In a preferred embodiment, the chelating ligand of the metal cation chelating domain may be tridentate (e.g., IDA) or tetradentate (e.g., NTA). By "tridentate" is meant that the chelating ligand contains three atoms that can function as donor atoms (i.e., Lewis donors) in a complex based on a metal ion. By "tetradentate" is meant that the chelating ligand contains four atoms that can function as donor atoms (i.e., Lewis donors) in a complex based on a metal ion. Thus, in other words, the chelating ligand of the metal cation chelating domain preferably may have three or four, more preferably three, binding sites that can be coordinated by the metal cation of the complex. Without being bound by theory, the use of chelating ligands having more atoms that can function as donor atoms (i.e., Lewis donors) in the complex has the advantage that the metal cation binds more strongly in the complex. The stronger binding prevents the undesirable release of the metal cation from the complex, i.e., the frequency of undesirable decomposition of the complex. This also applies to the complexes of the present invention and the "product complexes" formed by the use and methods of the present invention. Generally, tridentate and tetradentate complexes are preferred herein. According to the appended examples, tridentate is most preferred.

[0028] The metal cation chelating domain of the complex of the present invention further includes a label and / or a carrier, i.e., the functional moiety to be bound to the target molecule. The chelating ligand can be linked directly to the label and / or carrier or via a linker.

[0029] The chelating ligand of the metal cation chelating domain may in principle be any chelating ligand known in the art. Preferred chelating ligands contain at least one or more carboxylic acid groups and / or one or more amine groups. In one embodiment, the chelating ligand of the metal cation chelating domain may be a polycarboxylic acid, a polyamine or an aminopolycarboxylic acid.

[0030] Exemplary but non-limiting chelating ligands are nitrilotriacetic acid (NTA) (Hochuli et al., 1987), iminodiacetic acid (IDA) (Porath et al., 1975, Arnold, 1991, Franzreb et al., 2006), tris(carboxymethyl)ethylenediamine (TED) (Porath and Olin, 1983), chelating peptides (e.g., consensus sequence (GHHPH) nA peptide having G (wherein G refers to glycine, H refers to histidine, P refers to proline, and n is an integer from 1 to 3); see also SEQ ID NOs: 1-3) (Hutchens and Yip, 1992), a chelating protein (e.g., a his-tagged protein or a protein having spaced His tags as defined elsewhere in this specification), or cadistatin (Hayashi et al., 1986), triazacyclononane (TACN) (Sobiesciak and Zielenkiewicz, 2010), diethylenetriaminepentaacetic acid (DTPA) (Rahhal and Richter, 1988;Hnatowich et al., 1982, Hnatowich et al., 1983), phytokeratin (Song et al., 2014), carboxymethyl aspartic acid (CMA) (Porath et al., 1975, Hutschenreiter et al., 2003), tannic acid (TA) (Zhang et al., 2015, Han, Liu et al., 2017), porphyrin (Shao et al., 2015), dipyridylamine (DPA) (Clerac et al., 2000), phytic acid (Evans and Pierce, 1982), nitrilotripropionic diacetic acid (NPDA) (Mitsuo et al., 1970), nitriloisopropionic diacetic acid (NIPDA) (Mitsuo et al., 1970), N-(hydroxyethyl)ethylenediamine triacetic acid (HEDTA) (Wubs and Beenackers, 1993, Graff et al., 1995), ethylenediaminetetraacetic acid (EDTA) (Wubs and Beenackers, 1993), ethylene-bis(oxyethylene-nitrilo)tetraacetic acid (EGTA) (Border et al., 1976, Okazaki et al., 2011), 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA) (Kline et al., 1991, Chappell et al., 2003), 1,4,7-tris(carboxymethyl)-10-(2’-hydroxypropyl)-1,4,7,10-tetraazocyclodecane (Filippi et al., 2014), 1,4,7-triazacyclononane phosphinic acid (TRAP) (Simecek et al., 2012), 1,4,8,11-tetraazacyclotetradecane-N,N’,N’’,N’’’-tetraacetic acid (TETA) (Yuanfang and Chuanchu, 1991), ethylenedicysteine (Kong et al., 2010), bis(aminoethanethiol) carboxylic acid (Sun et al., 1996), triethylenetetraminehexaacetic acid (TTHA) (Harju and Ringbom, 1970, Achour et al., 1998), 1,2-diaminocyclohexane-N,N,N’,N’-tetraacetic acid (DACT) (Krzek et al., 2007), phosphate ion (Rizkalla et al., 1980), 1,4,7-triazacyclonane-1,4,7-triacetic acid (NOTA) (Strand et al., 2013;Simecek et al., 2012), 1-(1,3-carboxypropyl)-1,4,7-triazacyclononane-4,7-diacetic acid (NODAGA) (Strand et al., 2013), deoxyribonucleic acid (DNA) (Pages et al., 2015), ribonucleic acid (RNA) (Alberti et al., 2016), purine (Cini and Giogi, 1987) and pyrimidine (Saha and Mukherjee, 1984).;

[0031] Therefore, the chelating ligand contained in the metal cation chelating domain of c) is, for example, from the following non-limiting list: nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), tris(carboxymethyl)ethylenediamine (TED), consensus amino acid sequence (GHHPH) nChelating peptides such as peptides having G (where G refers to glycine, H refers to histidine, P refers to proline, and n is an integer from 1 to 3; see also SEQ ID NOs: 1 to 3), or cadistin, triazacyclononane (TACN), diethylenetriamine-pentaacetic acid (DTPA), phytokeratin, carboxymethylaspartic acid (CMA), phosphate ions, tannic acid (TA), porphyrin, dipyridylamine (DPA), phytic acid, nitrilopropionic diacetic acid (NPDA), nitriloisopropionic diacetic acid (NIPDA), N-(hydroxyethyl)ethylenediamine triacetic acid (HEDTA), 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA), 1,4,7-tris(carboxymethyl)-10-(2’-hydroxypropyl)-1,4,7,10-tetraazocyclodecane, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1-(1,3-carboxypropyl)-1,4,7-triazacyclononane-4,7-diacetic acid (NODAGA), 1,4,8,11-tetraazacyclotetradecane-N,N’,N’’,N’’’-tetraacetic acid (TETA), ethylenedicysteine, ethylenediamine tetraacetic acid (EDTA), 1,2-diaminocyclohexane-N,N,N’,N’-tetraacetic acid (DACT), bis(aminoethanethiol)carboxylic acid, ethylene-bis(oxyethylene-nitrilo)tetraacetic acid (EGTA), triethylenetetramine-hexaacetic acid (TTHA), 1,4,7-triazacyclononanephosphinic acid (TRAP), deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), purine, pyridimidines and derivatives thereof can be selected.

[0032] All of these chelating ligands have in common that they can act as Lewis base donors for forming coordination complexes with metal cations, i.e., they are chelating agents, by providing at least two coordination sites. Thus, in one embodiment, the chelating ligand may have at least 2, 3, 4, 5 or more chemical groups that can coordinate to a metal cation, i.e., they can act as Lewis bases.

[0033] In a preferred embodiment, the chelating ligand of the metal cation chelating domain of c) is nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), consensus sequence (GHHPH) n a chelating peptide having G (where n is 1 to 3; see also SEQ ID NOs: 1 to 3), diethylenetriaminepentaacetic acid (DTPA), nitrilopropionic diacetic acid (NPDA), nitriloisopropionic diacetic acid (NIPDA), ethylenediaminetetraacetic acid (EDTA), ethylene-bis(oxyethylene-nitrilo)tetraacetic acid (EGTA), carboxymethylaspartic acid (CMA) and derivatives thereof.

[0034] In another preferred embodiment, the chelating ligand may be an aminopolycarboxylic acid. Preferably, the aminopolycarboxylic acid is selected from ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), ethylene-bis(oxyethylene-nitrilo)tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), and triethylenetetraminehexaacetic acid (TTHA), tris(carboxymethyl)ethylenediamine (TED), triazacyclononane (TACN) or derivatives thereof.

[0035] In a particularly preferred embodiment, the chelating ligand of the metal cation chelating domain of c) comprises or is selected from NTA, IDA, TALON and their derivatives. Most preferred as the chelating ligand are NTA and IDA, and particularly most preferred is IDA. NTA is a tetradentate ligand and thus binds particularly strongly to metal cations. IDA is tridentate and also binds strongly to metal cations, but less so than NTA. The strong binding to the metal cation prevents the undesired decomposition of the complex or the release of the metal cation which would result in the release of the label and / or the carrier from the target molecule. In certain embodiments, IDA is preferred because complexes with a high percentage of stability can be formed faster than complexes with NTA. The second metal cation ligand remains still semi-coordinated to the metal, but in the case of using a tetradentate metal binding domain it is completely substituted, so for a tridentate metal binding domain, the coordination of the protein is presumed to be even easier. Due to the partial coordination, the ligand can be easily displaced by one histidine residue of the protein. Here, one or two other histidine residues in the immediate vicinity can also release the last ligand and form a stable complex.

[0036] In the context of chelating ligands, the term "derivative" refers to a compound having the same lead structure but which may be substituted by additional chemical reactive groups. Preferably, the term "derivative" in this context includes substitution by one or more additional chemical groups selected from the group consisting of carboxyl, amine, azide, acrylate, maleimide, hydroxyl, thiol, aromatic, aliphatic, disulfate, and vinyl sulfone groups. The term derivative may also include molecules having isotope substitution. "Isotope substitution" means that one or more atoms are isotope-labeled.

[0037] The metal cation is preferably a metal cation that is stable in each oxidation state. The oxidation state may be +2, +3 or +4, depending on the metal used. Thus, the metal cation of the complex may be a divalent, trivalent or tetravalent metal cation. In a preferred embodiment, the cation is a trivalent metal cation (e.g., Co 3+ ). In another preferred embodiment, the cation is a tetravalent metal cation (e.g., Pt 4+ ).

[0038] The metal cation can be, for example, a metal cation that can form a low-spin octahedral paramagnetic complex (e 3+ ), such as Co. g 6 t 2g 0 ). Thus, in one embodiment, the complex is a low-spin octahedral paramagnetic complex (e g 6 t 2g 0 ), preferably a Co 3+ low-spin octahedral paramagnetic complex (e g 6 t 2g 0 ). The metal cation can be, for example, a metal cation that can form a diamagnetic octahedral low-spin complex (t 4+ ), such as Pt. 2g 6 e g 0 ). Thus, in one embodiment, the complex is a diamagnetic octahedral low-spin complex (t 2g 6 e g 0 ), preferably a diamagnetic octahedral low-spin Pt 4+ (t 2g 6 e g 0 ).

[0039] Preferably, the metal cation is 10 -1 s -1 or less (e.g., 10 -2 s -1 or less or 10-3 s -1 It is a metal cation having a water ligand exchange rate as described below. A metal cation having such a low water ligand exchange rate is kinetically inert, that is, it exchanges the target molecule ligand only at a very low rate and forms a very strong complex with the target molecule that is close to a covalent bond (i.e., a kinetically inert bond).

[0040] Methods for determining the water exchange rate of metal cations (and complexes containing them) are known in the art. An overview of methods that can be used to determine the water exchange rate is described by A. Dunand and co-workers (Dunand et al., 2003). In the context of the present invention, for evaluating the slow exchange rate used, the measurement method may 17 include 17O NMR analysis (Cusanelli et al., 1996). In such an assay, a complex of the metal with 17 17O-labeled water is generated and dissolved in H 2 2O. To determine the water exchange rate, the loss of 17 17O over time is measured by the peak shift resulting from the exchange with H 2 2O.

[0041] In a preferred embodiment, the metal cation of the complex may be a transition metal cation. The transition metal cation is preferably in an oxidation state exhibiting a water ligand exchange rate of 10 -1 s -1 or less. Transition metal cations known in the art to have a water exchange rate in this range and / or to be kinetically inert include Co 3+ , Cr 3+ , Rh 3+ , Ir 3+ , Ir 4+ , Pt 2+ , Pt 4+ , Pd 4+ , Mo 3+ , Fe 3+ , Gd 3+ , Tb 3+ , Eu 3+ , Ru 2+ , La 3+, Ru 3+ , Re 3+ , Re 4+ , Os 2+ , V 2+ , Mn 4+ and Fe 2+ are included. Thus, preferred but non-limiting examples of metal ions that can be used in the context of the present invention belonging to transition metal cations are Co 3+ , Cr 3+ , Rh 3+ , Ir 3+ , Ir 4+ , Pt 2+ , Pt 4+ , Pd 4+ , Mo 3+ , Fe 3+ , Gd 3+ , Tb 3+ , Eu 3+ , Ru 2+ , La 3+ , Ru 3+ , Re 3+ , Re 4+ , Os 2+ , V 2+ , Mn 4+ and Fe 2+ .

[0042] In one embodiment, the metal cation may be a trivalent transition metal cation, preferably a trivalent transition metal cation listed above, and even more preferably Co 3+ . In one embodiment, the metal cation may be a tetravalent transition metal cation, preferably a tetravalent transition metal cation listed above, preferably Pt 4+ .

[0043] In one embodiment, the metal cation of the complex may be a lanthanoid (also referred to as an inner transition metal) cation. Lanthanoids are a subgroup of transition metals and include the following metals: La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu. The lanthanoid cations that can be used in the present invention are preferably -1 s -1 or less, preferably -2s -1 [It has an oxidation state with the following water ligand exchange rate. Lanthanoid cations having a water exchange rate within this range and / or being kinetically inert are known in the art, such as La 3+ [, Eu 3+ [, Gd 3+ [, and Tb 3+ [.] Thus, the metal cation in the context of the present invention may be a lanthanoid cation selected from the group consisting of La 3+ [, Eu 3+ [, Gd 3+ [, and Tb 3+ [.] Thus, in one embodiment, the metal cation of the complex may also be a trivalent lanthanoid cation.] [

[0044] [ [In one embodiment, the metal cation is Co 3+ [, Pt 4+ [, Cr 3+ [, Rh 3+ [, Ir 3+ [, Pt 2+ [, Ru 2+ [, Ru 3+ [, La 3+ [, Eu 3+ [, Os 2+ [, Pd 4+ [, Mo 3+ [, Fe 3+ [, Ru 2+ [, Gd 3+ [, Tc 3+ [, Re 3+ [, Sm 3+ [, Tb 3+ [, Ce 3+ [, Pr 3+ [, Nd 3+ [, Pm 3+ [, Dy 3+ [, Ho 3+ [, Er 3+ [, Tm 3+ [, Yb 3+ [, Lu 3+ [, V 2+ [, Mn 4+ [, and Fe 2+ [​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​It can be selected from the group consisting of. Those skilled in the art know that these metal cations are characterized by a very low water ligand exchange rate. The inventors have found that in particular such metal cations having a low water ligand exchange rate are useful metal cations for mediating the interaction between the labeled and / or carrier-containing metal cation chelation domain and the target molecule in the product complex formed by incubating the complex of the present invention with the target molecule. This is because these metals facilitate very stable interactions in product complexes that are thermodynamically stable and kinetically inert.

[0045] In a preferred embodiment, the metal cation is Co 3+ , Pt 4+ , Cr 3+ , Rh 3+ , Ir 3+ , Ir 4+ , Pt 2+ , Pd 4+ , Mo 3+ , Fe 3+ , Gd 3+ , Tb 3+ , Eu 3+ , Ru 2+ , La 3+ , Ru 3+ , Re 3+ , Re 4+ , Os 2+ , V 2+ , Mn 4+ and Fe 2+ selected from the group consisting of. These metal cations are described in the literature as having a water ligand exchange rate of 10 -1 s -1 or less, and / or kinetically inert metal cations: Co 3+ (Lippard and Berg, 1994), Cr 3+ (Helm and Merbach, 2002; Helm and Merbach, 1999), Rh 3+ (Aebischer et al., 1993; Helm and Merbach, 1999), Ir 3+ (Cusanelli et al., 1996; Helm and Merbach, 1999), Ir4+ (Saito et al., 1990), Pt 2+ (Helm et al., 1984; Helm and Merbach, 1999), Pt 4+ (Giandomenico et al., 1995), Pd 4+ (Saito et al., 1990), Mo 3+ (Saito et al., 1990), Fe 3+ (Harrington et al., 2018), Gd 3+ (Caravan et al., 2001), Tb 3+ (Junker et al., 2018), Eu 3+ (Morrow and Chin, 1993), Ru 2+ (Hugi-Cleary et al., 1987; Helm and Merbach, 1999), La 3+ (Morrow and Chin, 1993), Ru 3+ (Hugi-Cleary et al., 1987; Helm and Merbach, 1999), Re 3+ (House and House, 2015), Re 4+ (Saito et al., 1990) and Os 2+ (Livingstone, 1973), V 2+ (House and House, 2015), Mn 4+ (House and House, 2015), Fe 2+ (House and House, 2015). Thus, these metal cations can form product complexes that are as stable as those with the target molecule, and it is reasonable that the label and / or carrier binds stably to the target molecule.

[0046] In the context of the present invention, a "kinetically inert metal cation" is understood to be a metal cation having a water ligand exchange rate of 10 -1 s -1 or less. This definition and cutoff also follow the general understanding of this expression in the literature (Taube, 1952; Luther III, 2016). A preferred method for measuring the water exchange rate is described elsewhere in this specification.

[0047] The term "kinetically inert" when used in the context of the binding of a ligand in a metal complex, e.g., a target molecule in a "product complex" as used herein, preferably means that the ligand exchange rate in an aqueous solution is 10 -1 s -1 or less, more preferably 10 -2 s -1 or less. Methods for measuring the ligand exchange rate are known in the art. For example, the methods described elsewhere herein for measuring the water exchange rate can be used with modifications as appropriate. Assays for determining the ligand exchange rate of the binding of a target molecule in a "product complex" according to the present invention and / or for evaluating kinetic inertness may include measuring the competition with a competing ligand (such as imidazole or EDTA) and / or a reducing agent (such as DTT), as described in the appended examples and drawings (see, e.g., FIGS. 1, 6, and 7B). A preferred assay for evaluating the "kinetic inertness" of the binding of a target molecule to a complex (i.e., a "product complex") can be evaluated by the exemplary method described in Example 5. Specifically, a [Co(III)(NTA)(CO 3 )] 2- complex linked to NTA on agarose beads can be generated as described herein and in the appended examples and drawings. Subsequently, the [Co(III)(NTA)(CO 3 )] 2- complex bound to the beads can be incubated with the target molecule to form a [Co(III)(NTA)(target protein)] "product complex" immobilized on the agarose beads. Subsequently, the chemical stability of the [Co(III)(NTA)(target protein)] complex can be evaluated by washing an equal amount of the generated agarose beads with PBS (= control) or an imidazole solution (PBS supplemented with 250 mM imidazole) (= sample). Optionally, conventional Ni 2+The matrix based on -NTA can be used for comparison. Subsequently, the amount of the conjugated target molecule can be evaluated for all samples (before and after washing if necessary). The method for determining the presence (i.e., the readout) of the target molecule depends on the characteristics of the target molecule and is known in the art. For example, the assay may include fluorescence measurement (e.g., if the target molecule is fluorescent). For non - fluorescent target molecules, a fluorescent antibody that detects the target molecule can be used for the readout. Another example for the readout is the use of an enzyme reaction when the target molecule is an enzyme or when an enzyme - labeled antibody against the target molecule is used. In the context of the present invention, the target molecule ligand preferably contains at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, preferably about 85%, even more preferably at least about 90%, even more preferably about 95%, most preferably at least about 99% of the target molecules compared to the amount of target molecules bound to beads (= control) washed with PBS after the beads (= sample) washed with 250 mM imidazole, indicating that it is a "kinetically inert" bond in the "product complex".

[0048] In one embodiment, the metal cation is Co 3+ or Cr 3+ may be. In a particularly preferred embodiment, the metal is Co 3+ As shown in the attached examples, Co 3+ is particularly suitable for the rapid and easy production of the complexes of the present invention, enabling it and ensuring kinetically inert binding to the target molecule.

[0049] In another preferred embodiment, the metal is Pt 4+ As shown in the attached examples, Pt 4+ is particularly suitable for the rapid and easy production of the complexes of the present invention, enabling it and ensuring kinetically inert binding to the target molecule.

[0050] The present invention also relates to the case where the metal cation is Co3+ It relates to a complex in which the chelating ligand of the metal cation chelating domain is NTA (or a derivative thereof). The present invention also relates to a complex in which the metal cation is Co 3+ It relates to a complex in which the chelating ligand of the metal cation chelating domain is IDA (or a derivative thereof).

[0051] The present invention also relates to a complex in which the metal cation is Co 3+ It relates to a complex in which the chelating ligand of the metal cation chelating domain is Talon (or a derivative thereof). The present invention also relates to a complex in which the metal cation is Pt 4+ It relates to a complex in which the chelating ligand of the metal cation chelating domain is NTA (or a derivative thereof).

[0052] The present invention also relates to a complex in which the metal cation is Pt 4+ It relates to a complex in which the chelating ligand of the metal cation chelating domain is Talon (or a derivative thereof). The present invention also relates to a complex in which the metal cation is Pt 4+ It relates to a complex in which the chelating ligand of the metal cation chelating domain is IDA (or a derivative thereof).

[0053] Therefore, the present invention relates to a) Co 3+ ; b) CO 3 2- and carbonate ions or nitrate ions selected from HCO 3 - as the metal cation ligand; and c) a metal cation chelating domain containing NTA and a label and / or a carrier a complex (for example, a complex for binding a label and / or a carrier to a target molecule).

[0054] The present invention also relates to a) Co 3+ ; b) CO 32- or HCO 3 - ligand; and c) a metal cation chelating domain comprising NTA and a label and / or a carrier relates to a complex (e.g., a complex for binding a label and / or a carrier to a target molecule).

[0055] The present invention also relates to a) Co 3+ ; b) CO 3 2- or HCO 3 - ligand; and c) a metal cation chelating domain comprising Talon and a label and / or a carrier relates to a complex (e.g., a complex for binding a label and / or a carrier to a target molecule).

[0056] The present invention also relates to a) Co 3+ ; b) CO 3 2- or HCO 3 - ligand; and c) a metal cation chelating domain comprising IDA and a label and / or a carrier relates to a complex (e.g., a complex for binding a label and / or a carrier to a target molecule).

[0057] The present invention also relates to a) Pt 4+ ; b) CO 3 2- or HCO 3 - ligand; and c) a metal cation chelating domain comprising NTA and a label and / or a carrier relates to a complex (e.g., a complex for binding a label and / or a carrier to a target molecule).

[0058] The present invention also relates to a) Pt 4+ ; b) CO 3 2- or HCO 3 - ligand; and c) a metal cation chelating domain comprising IDA and a label and / or a carrier for a complex (e.g., a complex for binding a label and / or a carrier to a target molecule).

[0059] The present invention also relates to a) Pt 4+ ; b) CO 3 2- or HCO 3 - ligand; and c) a metal cation chelating domain comprising Talon and a label and / or a carrier for a complex (e.g., a complex for binding a label and / or a carrier to a target molecule).

[0060] In one embodiment, the chelating ligand of the metal cation chelating domain is diethylenetriaminepentaacetic acid (DTPA), and the metal cation is selected from Gd 3+ , In 3+ , and Fe 3+ . In one embodiment, the chelating ligand of the metal cation chelating domain is tannic acid (TA), and the metal cation is Co 3+ . In one embodiment, the chelating ligand of the metal cation chelating domain is dipyridylamine (DPA), and the metal cation is Co 3+ . In one embodiment, the chelating ligand of the metal cation chelating domain is 1,4,7-triazacyclononane phosphinic acid (TRAP), and the metal cation is Ga 3+ . In one embodiment, the chelating ligand of the metal cation chelating domain is ethylenedicysteine, and the metal cation is Re 3+ or Tc 3+It is. In one embodiment, the chelating ligand of the metal cation chelating domain is triethylenetetraaminehexaacetic acid (TTHA), and the metal cation is Gd 3+ In 3+ and Fe 3+ selected from the group consisting of. In one embodiment, the chelating ligand of the metal cation chelating domain is 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and the metal cation is In 3+ is. In one embodiment, the chelating ligand of the metal cation chelating domain is 1-(1,3-carboxypropyl)-1,4,7-triazacyclononane-4,7-diacetic acid (NODAGA), and the metal cation is In 3+ is.

[0061] The metal cation ligand (which may also be referred to as the second ligand) of the complex of the present invention is selected from carbonate ions CO 3 2- and HCO 3 - or nitrate ions. The inventors have found that using these metal cation ligands facilitates the coordination of the target molecule. This is achieved by the more rapid and easier exchange of carbonate or nitrate ions from the complex with the target molecule. Both carbonate and nitrate ions can be protonated after release from the complex. This protonation facilitates the release from the complex. Carbonate ions CO 3 2- and HCO 3 - In the context of, protonation even results in gas formation. Without being bound by theory, gas formation is thought to further increase the exchange rate and thermodynamics of the exchange of carbonate ion ligands with the target molecule. Thus, carbonate ions CO 3 2- and HCO 3 - are particularly preferred metal cation ligands.

[0062] Nitrate ions can also be used as metal cation ligands, but this is a matter for a different invention. In a preferred embodiment, the metal cation ligand is CO 3 2- and HCO 3 - selected carbonate ions. These carbonate ions are shown in the attached examples to facilitate the formation of product complexes with the target molecule. In other words, the complexes of the present invention are more reactive with respect to the binding of the label and / or the carrier to the target molecule.

[0063] In a particular embodiment, the complex of the present invention is [Co(III)(NTA)CO 3 2- complex, [Co(III)(NTA)HCO 3 - complex or a hydrate thereof. The label and / or the carrier is bound to the NTA. In these complexes, four coordination sites are occupied by the NTA and two are occupied by the carbonate ions. The carbonate ion is a bidentate ligand, i.e., it has two atoms that can coordinate to the metal cation in the complex as donors (i.e., Lewis bases), facilitating the binding of the target molecule. The NTA is a chelator that exhibits a particularly strong binding to the metal cation due to its four-site binding. Thus, the NTA prevents the decomposition of unwanted complexes and product complexes due to the release of unwanted metal cations.

[0064] In a preferred embodiment, the complex of the present invention is [Co(III)(IDA)CO 3 - complex, [Co(III)(IDA)HCO 3 ​​​It contains the complex or its hydrate. The label and / or the carrier are bound to IDA. In these complexes, three coordination sites are occupied by IDA and two are occupied by carbonate ions. The carbonate ion is a bidentate ligand, i.e., it has two atoms that can coordinate to the metal cation in the complex as donors (i.e., Lewis bases), facilitating the binding of the target molecule. The second metal cation ligand remains semi-coordinated to the metal, but when using a tetradentate metal-binding domain, it is fully substituted, so for a tridentate metal-binding domain, the coordination of the protein is presumably further facilitated. Due to partial coordination, the ligand can be easily substituted by one histidine residue of the protein. Here, one or two other histidine residues in the immediate vicinity can also release the last ligand and form a stable complex.

[0065] The metal cation chelating domain of the complex of the present invention contains a label and / or a carrier. In other words, the complex of the present invention contains a functional part. The label and / or the carrier are preferably different from the coordinating groups of the chelating ligand, i.e., they do not share the same atoms. However, in some embodiments, the chelating ligand and the label and / or the carrier may also share one or more atoms.

[0066] The metal cation chelating domain of the complex of the present invention may contain a chelating ligand and a label but does not contain a carrier. Alternatively, the metal cation chelating domain of the complex of the present invention may contain a chelating ligand and a carrier but does not contain a label. In another embodiment, the metal cation chelating domain of the complex of the present invention may contain a carrier and a label.

[0067] In certain embodiments, the metal cation chelating domain may further include a linker between the chelating ligand and the label and / or the carrier. Such linkers can facilitate complex formation and / or establish a defined distance between the label and / or carrier and the chelating ligand. The linker can in principle be any chemical group suitable for covalently linking the label / carrier to the chelating ligand at a desired distance. One of ordinary skill in the art can select the linker according to the requirements regarding the resistance of the bond to the conditions of the environment and the intended use. The linker may include, for example, an antibody-drug conjugate (ADC) linker, a charged sulfone group, polyethylene glycol (PEG), a pyrophosphate diester, a peptide-based linker (e.g., a cathepsin B-responsive linker such as Val-Cit-PABC or Val-Ala-PABC, where Cit refers to L-citrulline and PABC refers to p-aminobenzyl oxycarbonyl), a hydrazone, a disulfide-containing linker, a thioether-containing linker, a beta-glucuronide, or a combination thereof. In one embodiment, the linker may consist of an antibody-drug conjugate (ADC) linker, a charged sulfone group, a pyrophosphate diester, a peptide-based linker (e.g., a cathepsin B-responsive linker such as Val-Cit-PABC or Val-Ala-PABC), a hydrazone, a disulfide-containing linker, a thioether-containing linker, a beta-glucuronide, a nucleic acid linker (preferably DNA), or a combination thereof. Antibody-drug conjugate (ADC) linkers are known in the art. Exemplary ADC linkers that can be used in the context of the present invention are described, for example, in Tsuchikama and An, 2018, ADC_Review 2019; and / or Jain et al., 2015, all of which are hereby incorporated by reference in their entirety. Linkers based on charged sulfone groups are also known in the art. Exemplary linkers having a charged sulfone group that can be used in the context of the present invention are described in Zhao et al., 2011, which is hereby incorporated by reference in its entirety. PEG linkers are known in the art.Exemplary PEG linkers that can be used in the context of the present invention are described in Lyon et al., 2015, which is hereby incorporated by reference in its entirety. Pyrophosphate diester linkers are known in the art. Exemplary pyrophosphate diester linkers that can be used in the context of the present invention are described in Kern et al., 2016, which is hereby incorporated by reference in its entirety. Peptide-based linkers (e.g., cathepsin B-responsive linkers such as Val-Cit-PABC or Val-Ala-PABC) are known in the art. Exemplary peptide-based linkers that can be used in the context of the present invention (e.g., cathepsin B-responsive linkers such as Val-Cit-PABC or Val-Ala-PABC) are described in Dubowchik et al., 2002 and / or Hartley, 2011, which are hereby incorporated by reference in their entirety. Hydrazone linkers are known in the art. Exemplary hydrazones that can be used in the context of the present invention are described in Tolcher et al., 1999, which is hereby incorporated by reference in its entirety. Disulfide-containing linkers are known in the art. Exemplary disulfide-containing linkers that can be used in the context of the present invention are described in Saito et al., 2003, which is hereby incorporated by reference in its entirety. Thioether-containing linkers are known in the art. Exemplary thioether linkers that can be used in the context of the present invention are described in Stenton et al., 2018, which is hereby incorporated by reference in its entirety. Beta-glucuronide linkers are known in the art. Exemplary beta-glucuronide linkers that can be used in the context of the present invention are described in Jeffrey et al., 2010, which is hereby incorporated by reference in its entirety. Nucleic acid linkers such as DNA linkers are known in the art.

[0068] The label of the metal cation chelating domain may comprise or consist of a fluorophore, a diagnostic agent, a targeting moiety, a therapeutic agent, a polyethylene glycol (PEG) molecule, a lipid, biotin (and / or its derivatives such as, for example, photobiotin: N-(4-azido-2-nitrophenyl)-aminopropyl-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3-propanediamine (Foster et al., 1985)), a protein (such as, for example, an antibody), a peptide or a toxin. The label may also preferably comprise or consist of a reactive group selected from a thiol group or a reactive group suitable for click chemistry. Non-limiting examples of reactive groups suitable for click chemistry are azide, alkyne, nitrone, tetrazine and tetrazole. The definition of the term "derivative" as described elsewhere in this specification is applied with modifications where appropriate.

[0069] The label may include a functional moiety (such as fluorescence, chromogenic, high electron density, chemiluminescent, and radioactivity labels, etc.) that can be directly detected, as well as a moiety such as an enzyme or a ligand that is detected indirectly, for example, through an enzymatic reaction or molecular interaction.

[0070] Exemplary labels include, but are not limited to, radioisotopes 32 P, 14 C, 125 I, 3 H, and 131I (preferably, these atoms do not form part of a chelating ligand), fluorophores, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferase, such as firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, etc., which are coupled with an enzyme that uses hydrogen peroxide to oxidize a dye precursor such as HRP. In another embodiment, the label is a positron emitter. Examples of positron emitters include, but are not limited to, 68 Ga, 18 F, 64 Cu, 86 Y, 76 Br, 89 Zr, and 124 I. In a particular embodiment, the positron emitter is 89 Zr.

[0071] In one embodiment, the label is a label that is sensitive to treatment with an oxidizing reagent (preferably, H 2 O 2 ). "Sensitive to an oxidizing reagent" in this context means that the label exhibits a decrease in function / activity (e.g., fluorescence in the case of a fluorophore) and / or stability after treatment with an oxidizing agent. Preferably, the oxidizing agent treatment is 20 mM H 2 O 2A treatment using a solution, where the label is sensitive after treatment for at least 0.5 hours, 1 hour, 2 hours or 24 hours. In a preferred test for sensitivity to an oxidizing agent, the incubation is carried out for exactly 1 hour, as described as an oxidation step in Wegner and Spatz, 2013, in 20 mM H 2 O 2 in solution. Alternatively, this test can be carried out for exactly 90 minutes, for example, as described for the oxidation step of Zatloukalova and Kucerova, in 0.05% (v / v) H 2 O 2 solution. To shorten the test, one skilled in the art can also use a solution with a higher H 2 O 2 concentration, such as 1% (v / v) H 2 O 2 . The preferred H 2 O 2 concentration and incubation time, including the time-course experiment used to evaluate H 2 O 2 sensitivity, are described in the attached examples. The assay for measuring oxidizing agent sensitivity involves incubation with a solution containing an oxidizing agent (e.g., H 2 O 2 ) (preferably an aqueous solution, more preferably at the desired oxidizing agent concentration in water) at various time points before and during the treatment, and measurement of a readout indicating the function / activity and / or stability of the label (e.g., for a fluorophore, measuring the emitted fluorescence). A label is considered "oxidation-sensitive" if the readout measurement indicates a loss of the function / activity and / or stability of the label. The preferred time points for evaluating the loss of function / activity and / or stability of the label are those used and required for oxidizing Co 2+ to Co 3+ in the presence of the above-mentioned oxidizing agent. When evaluating H 2 O 2 sensitivity, H 2 O 2The preferred concentrations and incubation times are those shown in this section above. Exemplary methods for testing the oxidative sensitivity of fluorophore labels are described in the appended examples. One of ordinary skill in the art can modify this assay according to the label used and adapt the parameters as the H 2 O 2 concentration and incubation time.

[0072] In one embodiment, the label may comprise or consist of a fluorophore. Fluorophores are known in the art and are publicly and / or commercially available. Also, methods for coupling a fluorophore to a chelating ligand of a metal cation chelating domain are known in the art. As shown by the appended examples, many fluorophores (e.g., fluorescein, FITC, Atto488, and Alexa488) are H 2 O 2 sensitive to H

[0073] treatment. This sensitivity makes the complexes of the present invention particularly suitable for use in the context of a fluorophore as a label. However, this advantage is not limited to fluorophores. In one embodiment, the metal binding domain may comprise a fluorophore as a label and the chelating ligand may be NTA. In one embodiment, the metal binding domain may comprise a fluorophore as a label and the chelating ligand may be IDA. 2 O 2 treatment.

[0074] In one embodiment, the complex of the present invention, and more specifically, the label and / or the carrier, may not be or may not contain the porphyrin phospholipid described by Shao et al. (Shao et al., 2015).

[0075] In the context of the present invention, the term "label" does not include a single atom such as a hydrogen atom that is part of a chelating ligand of a metal cation chelating domain. Preferably, the label relates to a structure containing at least 2 atoms, at least 3 or at least 10 atoms.

[0076] Preferably, the label is a functional moiety and / or a moiety that can be detected using methods known in the art (e.g., NMR, fluorescence measurement, enzyme assay, etc.). As described above, the metal binding domain may include a carrier (optionally, in addition to the label). Non-limiting examples of carriers according to the present invention are polymers, hydrogels, microparticles, nanoparticles, spheres (e.g., nano- or microspheres), beads (e.g., microbeads), quantum dots, artifacts, and solid surfaces. In one embodiment, the carrier may include or be a nanopatterned gold surface. Such a nanopatterned gold surface may be functionalized with thiol residues.

[0077] One skilled in the art knows techniques for binding / fixing a chelating ligand onto a carrier to form a metal binding domain comprising the chelating ligand and the carrier. For example, when the chelating ligand is an aminopolycarboxylic acid (e.g., NTA), the chelating ligand can be covalently bound to a solid phase via, for example, at least one carboxylic acid group or amino group. In another embodiment, the chelating ligand can be linked to the carrier via an amide or ester bond.

[0078] In a preferred embodiment, the carrier is a bead (e.g., a microbead) such as an agarose bead. Accordingly, the present invention relates to beads (e.g., microbeads) to which a complex of the present invention (excluding the label and / or the carrier) is bound. These beads can also be referred to as an affinity matrix. Such beads containing the complex of the present invention are ready-to-use reagents for binding (i.e., immobilizing) a target molecule to the beads.

[0079] Accordingly, the present invention relates to a) Co 3+ ; b) carbonate ions or nitrate ions selected from CO 3 2- and HCO 3 - as metal cation ligands; and c) a metal cation chelating domain comprising NTA and beads (for example, a complex for binding a target molecule to the beads).

[0080] Preferably, the present invention relates to a) Co 3+ ; b) CO 3 2- or HCO 3 - as ligands; and c) a metal cation chelating domain comprising NTA and beads (for example, a complex for binding a label and / or a carrier to a target molecule).

[0081] More preferably, the present invention relates to a) Co 3+ ; b) CO 3 2- or HCO 3 - as ligands; and c) a metal cation chelating domain comprising IDA and beads (for example, a complex for binding a label and / or a carrier to a target molecule).

[0082] Such complexes can be produced as described in the accompanying examples. For example, NTA agarose resin (Qiagen, 1022963) can be washed once with 10 volumes of ddH 2 O, once with 3 volumes of 100 mM EDTA pH 7.5, and three times with 10 volumes of ddH 2 O. Subsequently, 1 M NaHCO 3Among them, 1 mM Na with 10 times the bead volume 3 [CO(III)(CO 3 ) 3 ·3H 2 O or K 3 [Co(III)(CO 3 ) 3 ·3H 2 O can be added to the beads. After incubating for 48 hours in a thermoshaker at 23°C with shaking at 1100 rpm, the beads can be washed twice with ddH 2 O and once with 10 times the bead volume of protein buffer (50 mM Tris pH 7.4, 150 mM NaCl). Finally, the beads can be collected (e.g., by centrifugation) to obtain the complex bound to the beads as a carrier.

[0083] As shown in the attached examples, although not bound by theory, the incubation time of the metal cation, metal cation ligand, and metal cation chelating domain for forming the complex of the present invention can affect the subsequent association of the complex of the present invention with the target molecule. The incubation time for forming the complex of the present invention is, in the context of the present invention, the "complex formation time". The incubation time of the complex of the present invention with the target molecule is, in the context of the present invention, the "complex-target incubation time". Non-limiting Example 18 shows that, for example, when the complex formation time is 10 minutes, with the same complex-target incubation time, after a 30-minute complex-target incubation time, substantially more target molecules (i.e., His 6 -GFP) bind to the complex of the present invention compared to using a 48-hour complex formation time (Figure 20B). However, when the complex-target incubation time is extended (e.g., to 48 hours), efficient binding of His 6 -GFP to the complex of the present invention is restored (e.g., see Figure 10). A person skilled in the art is in a position to easily adapt the corresponding incubation time as needed.

[0084] Furthermore, the attached examples show that both the complex formation time and the complex-target incubation time positively affect the stability of the resulting product complex, i.e., the stability between the complex of the present invention and the target molecule. Example 18 shows that when the complex formation time is 10 minutes and the complex-target incubation time is 30 minutes, the target molecule binds efficiently to the complex of the present invention, but only a fraction of the product complex is kinetically inert, i.e., resistant to imidazole treatment (Figure 20B). The fraction of the kinetically inert product complex increases when one of the incubation times, i.e., the complex formation time or the complex-target incubation time, is extended (e.g., to 48 hours).

[0085] Also, as shown by Non-limiting Example 18, the combination of a 10-minute complex formation time and a 48-hour complex-target incubation time results in a high fraction of stable product complex. Furthermore, the combination of a 48-hour complex formation time and a 30-minute complex-target incubation time results in a stable product complex (see, e.g., Example 10, Figure 11). As discussed above, a 48-hour complex formation time can result in a decrease in the binding efficiency of the target molecule to the complex of the present invention.

[0086] Thus, depending on the nature of the target molecule, one of ordinary skill in the art knows how to select a suitable incubation time. For example, if the target molecule has a very high tendency to denature or aggregate during incubation, but a kinetically inert product complex is required. Thus, if necessary, one of ordinary skill in the art may select a long complex formation time (e.g., 48 hours) and a shorter complex-target incubation time (e.g., 30 minutes), although the amount of the resulting product complex may be reduced. Note that Attached Example 18 was carried out using NTA as the metal chelating ligand.

[0087] Although not bound by theory, it should be noted that the influence of the above incubation time may depend on the metal chelating ligand used. For example, in the attached non-limiting Example 15, IDA is used as the metal chelating ligand, and the example demonstrates that a high fraction of kinetically inert product complexes are formed even for two short incubation times (i.e., a 10-minute complex formation time and a 30-minute complex-target incubation time).

[0088] Thus, one of ordinary skill in the art knows that the incubation time can have an impact on the stability of the product complex, which needs to be determined for each individual metal chelating ligand. However, this adaptation of the corresponding incubation time is within the scope of the relevant art and can be readily achieved using the teachings of the present invention and the examples in the experimental section and the scientific details provided therein. Similarly, one of ordinary skill in the art can also easily adapt additional parameters such as pH, temperature, and buffer system to obtain a kinetically inert product complex.

[0089] Table 1 can be useful for one of ordinary skill in the art to determine the effect of the incubation time on the stability of the product complex and the yield of a stable, i.e., kinetically inert, product complex. For example, moderate stability means that a smaller fraction of the product complex is kinetically inert compared to the incubation time that results in high stability, i.e., a high fraction of kinetically inert product complexes. It should be noted that moderate stability can also result in a yield of stable product complexes that may be useful for certain applications and technical fields. If an unlimited amount of target molecules is available and a stable product complex is required in a short period, one of ordinary skill in the art would preferably use the following protocol when NTA is used as the metal cation chelating ligand: - Select two shorter incubation times - Treat the resulting product complex (e.g., bound to beads) with imidazole to remove the kinetically non-inert product complex. - Recover the product complex that is resistant to imidazole treatment, i.e., the kinetically inert product complex (e.g., by recovering the beads). This may also be referred to.

[0090] Those skilled in the art are well aware of how to adapt the protocol according to the application. It is clear that Table 1 can be further used to select the complex formation time and the complex - target incubation time.

[0091] Therefore, the times disclosed in Table 1 can be combined with the complexes of the present invention, the methods and uses described herein. Therefore, the present invention relates to, for example, a) a metal cation; b) CO 3 2- or HCO 3 - a metal cation ligand which is; and c) a metal cation chelating domain comprising a chelating ligand and a label and / or a carrier A complex comprising, wherein the complex formation time is about 48 hours, relates to the complex.

[0092] The present invention also relates to a) a metal cation; b) CO 3 2- or HCO 3 - a metal cation ligand which is; and c) a metal cation chelating domain comprising a chelating ligand and a label and / or a carrier A complex comprising, wherein the complex formation time is about 10 minutes, relates to the complex.

[0093] Note that Table 1 is for illustrative purposes only and is not intended to limit the scope of the present invention in any way.

[0094]

Table 1

[0095] The binding of NTA to Co 3+ can be enhanced by the introduction of energy into the system / exposure to energy during incubation. Such introduction of energy into the system / exposure to energy may be electromagnetic resonance techniques (e.g., NMR, x-ray, UV-Vis), heat, ultrasound, or plasmon resonance techniques at various frequencies.

[0096] The carrier of the present invention or its surface can be further functionalized with activating esters such as amino groups, carboxylic acid groups, and / or NHS esters. Further non-limiting examples of metal-binding domains containing carriers are described in WO2014 / 072525A1 and WO2003 / 072143, which are hereby incorporated by reference in their entirety.

[0097] In a second aspect, the present invention relates to a composition comprising the complex of the present invention. What is said elsewhere in this specification regarding the complex is applied with modifications where appropriate. The composition may contain additional compounds in addition to the complex. Such compounds may be chemical compounds used to produce the complex of the present invention as specified elsewhere in this specification. The composition may also contain other complexes, such as complexes having the same metal cation but different ligands. The composition may be in the form of a solution or a solid.

[0098] In one embodiment, the composition of the present invention may be a solution comprising the complex of the present invention and HCO 3 - or CO 3 2- and. HCO 3- or CO 3 2- The presence of HCO or CO prevents the release of the metal cation ligand of the complex, i.e., facilitates complex stability. HCO 3 - or CO 3 2- is preferably used in solution when the metal cation ligand is also carbonate ion. When nitrate ion is used as the metal cation ligand, the solution may contain nitrate ion rather than HCO 3 - or CO 3 2- The concentration of HCO or CO in the composition solution may be at least 1 mM, preferably at least 10 mM, and most preferably 1 M. The concentration of nitrate ion in the composition solution may be at least 1 mM, preferably at least 10 mM, and most preferably 1 M.

[0099] HCO in the composition solution 3 - or CO 3 2- The inventors have found that the complex of the present invention can also perform its function as a reagent for binding a label and / or a carrier to a target molecule in the presence of additional components, for example, when unpurified after synthesis. Therefore, the use of the complex of the present invention described herein, as well as the method of using the complex of the present invention described herein, can be carried out with the composition of the present invention with modifications where appropriate. Those skilled in the art will be able to avoid components in the composition that may negatively affect the efficiency and / or reaction rate of each use or method.

[0100]

[0101] ​In a third aspect, the present invention provides a method for producing the complex of the present invention. The production method includes the step of incubating (i) a metal cation; (ii) a metal cation ligand, and (iii) a metal cation chelating domain together. While incubating these components in solution (i.e., in the presence of a solvent, preferably an aqueous solution), the complex of the present invention will form readily. As discussed above, the incubation time is the "complex formation time" in the context of the present invention. Table 1 and the attached examples can help those skilled in the art select a suitable complex formation time. Accordingly, the present invention also relates to a production method that includes the step of incubating (i) a metal cation; (ii) a metal cation ligand, and (iii) a metal cation chelating domain together, wherein the incubation time, i.e., the complex formation time, is about 48 hours. The present invention also relates to a production method that includes the step of incubating (i) a metal cation; (ii) a metal cation ligand, and (iii) a metal cation chelating domain together, wherein the incubation time, i.e., the complex formation time, is about 1 hour. The present invention further relates to a production method that includes the step of incubating (i) a metal cation; (ii) a metal cation ligand, and (iii) a metal cation chelating domain together, wherein the incubation time, i.e., the complex formation time, is about 3.5 hours. Further, the present invention relates to a production method that includes the step of incubating (i) a metal cation; (ii) a metal cation ligand, and (iii) a metal cation chelating domain together, wherein the incubation time, i.e., the complex formation time, is about 30 minutes. Again, other incubation times are within the routine skill of those skilled in the art.

[0102] What is said elsewhere in this specification regarding the metal cation, the metal cation ligand, and the metal cation chelating domain is applied with modifications where appropriate. In a preferred embodiment, the metal cation and the metal cation ligand can be provided in the form of a neutral complex, preferably as a salt (e.g., as a solid or in solution). The neutral complex (e.g., the salt) may also contain other components. In this embodiment, incubation means mixing the neutral complex (e.g., the salt) with the metal cation chelating domain in solution and maintaining this mixture for a defined period of time. When using a neutral complex (e.g., a salt), the method may further include the step of producing the neutral complex (e.g., the salt). If desired, the salt can be induced as a solid and filtered and / or washed.

[0103] Incubation can be carried out for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 24 hours or at least 48 hours. In other words, the complex formation time can be at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 24 hours or at least 48 hours (see also Table 1). The longer the incubation time, the more complexes are formed. At some points in the reaction, saturation of complex formation can be observed. The point at which this saturation is observed and the yield of the complexes formed depend, for example, on the temperature and concentration of the components used and the components themselves (e.g., the choice of buffer). For the production process, reaching saturation is not necessary, but it can increase the production yield.

[0104] Incubation can, in principle, be carried out at any temperature at which the solvent used in the reaction is in a liquid state. When an aqueous solution or water is used as the solvent, the temperature can be selected from 0 °C to 99 °C. Preferably, the temperature is selected according to the temperature stability of the label and / or carrier used. Thus, preferred temperature ranges are, for example, 2 °C to 42 °C, 4 °C to 37 °C and 4 °C to 25 °C. Without being bound by theory, the formation of the complex is facilitated by higher temperatures, so higher temperatures are preferred as long as the stability of the label and / or carrier is not negatively affected. A person skilled in the art can select the temperature according to the knowledge of the temperature stability of the label and / or carrier.

[0105] The metal cation, metal cation ligand and metal cation chelating domain can be selected individually as described elsewhere in this specification. The priority regarding the combination of these components or a subset thereof described elsewhere in this specification is applied with changes where appropriate.

[0106] The method for producing the complex of the present invention may further include the step of collecting and / or purifying the complex. As described above, the complex of the present invention also functions in the presence of other compounds, particularly including the reaction mixture produced by the incubation step of the production method of the present invention. However, in certain settings, purification and / or isolation of the complex may be desirable. Washing is preferably carried out using a solution containing 1 mM, preferably 10 mM, even more preferably 100 mM, most preferably 1 M of CO 3 2- or HCO 3 - Washing of the complex of the present invention using such a solution, as shown in the attached examples and drawings, results in improved binding of the label and / or carrier when the complex of the present invention is used to bind the label and / or carrier to a target molecule (e.g., a protein). Without being bound by theory, 1 M CO 3 2- or HCO3 - Performing a washing step with a solution containing prevents the ligand from dissociating from the complex of the present invention during washing. 3 2- or HCO 3 - Performing a washing step with a solution containing prevents the ligand from dissociating from the complex of the present invention during washing.

[0107] If the method involves a carrier, purification and / or isolation may include one or more washing steps. Subsequently, the carrier can be isolated by removing the washing buffer from the carrier after the last washing step. For example, when beads are used, the beads can be sedimented by centrifugation and the supernatant can be removed. An alternative filter column that allows the liquid to flow out but retains the complex bound to the carrier can be used. The filter column has the advantage of having less carrier (e.g., beads) lost in the washing step compared to the use of sedimentation and aspiration of the supernatant.

[0108] If a label is used, the purification method may include using an affinity matrix that specifically recognizes the label. After binding to the affinity matrix, the complex can be washed one or more times to remove free reagents. In the final step, the complex can be eluted. The purification and isolation of the complex may further or alternatively also include chromatography such as size exclusion chromatography or anion or cation exchange chromatography. Alternatively, as shown by Wegner and Spatz, 2013, labeled proteins show a decrease or no interaction in the interaction with Ni-NTA compared to unlabeled proteins with free His-tags, so Ni-NTA resin can be used to separate the labeled from the unlabeled. 2+ -NTA and thus can be separated from the unlabeled ones by Ni-NTA resin. 2+ -NTA and thus can be separated from the unlabeled ones by Ni-NTA resin.

[0109] In particular, when the complex does not contain a carrier and Co 3+When used as a metal cation, the method for purifying the complex of the present invention can also be carried out by methods described in the art (see, for example, Shibata M., 1983).

[0110] A preferred complex of the present invention has Co as the metal cation 3+ and CO as the metal cation ligand 3 2- or HCO 3 - and contains carbonate ions selected from. The method for producing this preferred complex of the present invention is to use Co in the form of a neutral complex with a counter ion, such as in the form of a salt 3+ and CO 3 2- or HCO 3 - This may include the step of providing. Alternatively, a charged complex containing Co 3+ and CO 3 2- or HCO 3 - can be provided in solution. In a preferred embodiment, the neutral complex (and salt) is sodium tris-carbonatocobalt(III) trihydrate (Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O) or potassium tris-carbonatocobalt(III) trihydrate (K 3 [Co(III)(CO 3 ) 3 ·3H 2 O).

[0111] Sodium tris-carbonatocobalt(III) trihydrate (Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O) can be synthesized as described by Bauer and Drinkard (Bauer and Drinkard, 1960). Briefly, 0.1 mole (29.1 g) of Co(II)(NO 2 O in 50 ml of ddH3 )·6H 2 A mixture of O(Sigma; 1.02554) and 10 ml of 30% hydrogen peroxide (Riedel-de Haen; 18312) can be added dropwise with stirring to an ice-cooled slurry of 0.5 mol (= 42.0 g) of sodium bicarbonate (Merck; 1.06329) in 50 ml of ddH 2 O. Subsequently, the mixture can be incubated on ice with continuous stirring for 1 hour. Subsequently, the olive-colored product can be filtered and washed three times each with cold water, anhydrous ethanol, and dry ether. Finally, the product can be dried under reduced pressure overnight and stored at -20 °C in a nitrogen atmosphere if necessary. A person skilled in the art can adapt the concentrations and amounts used. The success of the production can be confirmed by NMR, for example, as used in the attached examples.

[0112] Potassium tris-carbonatocobalt(III) trihydrate (K 3 [Co(III)(CO 3 ) 3 ·3H 2 O) can be synthesized in solution as described by Shibata (adapted from Shibata, 1983; Mori et al., 1956). Briefly, a mixture of 0.1 mol (24 g) of Co(II)Cl 2 in 24 ml of ddH 2 ·6H 2 O (Honeywell; 255599) and 40 ml of 30% hydrogen peroxide can be added dropwise with stirring to an ice-cooled slurry of 0.7 mol (70 g) of potassium bicarbonate (Honeywell; 237205) in 70 ml of ddH 2 O. Subsequently, the resulting green solution can be filtered (e.g., by suction) and used directly for the following experiments. A person skilled in the art can adapt the concentrations and amounts used. The success of the production can be confirmed by NMR, for example, as used in the attached examples.

[0113] If the metal cation ligand of the complex to be formed is HCO 3 - or CO 3 2- then an incubation is carried out in a solution containing HCO 3 - or CO 3 2- (preferably a buffered solution). Preferably, HCO 3 - or CO 3 2- is provided at a concentration of at least 1 mM, preferably at least 10 mM, and most preferably 1 M.

[0114] The method for producing the complex of the present invention is particularly preferably free of an oxidation step in the presence of a label and / or a carrier, where the metal cation forming the center of the complex of the present invention is oxidized, for example, by H 2 O 2 treatment.

[0115] In one aspect, the present invention further provides a) a metal cation; b) a metal cation ligand selected from carbonate ions or nitrate ions selected from CO 3 2- and HCO 3 - , preferably CO 3 2- and HCO 3 - , and c) a metal cation chelation domain containing a chelating ligand and a label and / or a carrier relates to a kit.

[0116] What is said elsewhere in this specification regarding the metal cation, the metal cation ligand and the metal cation chelation domain, the chelating ligand and the label and / or the carrier shall be applied with modifications where appropriate.

[0117] The kit may be a kit for producing the complex of the present invention, i.e., it may contain components that can react to form the complex of the present invention. In another embodiment, the assembled complex may be included in the kit.

[0118] In a particularly preferred embodiment, the metal cation is Co 3+ and the kit comprises a metal cation and a metal cation ligand in the form of sodium tris-carbonatocobalt(III) trihydrate (Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O) or potassium tris-carbonatocobalt(III) trihydrate (K 3 [Co(III)(CO 3 ) 3 ·3H 2 O). Preferably, the metal cation chelating domain comprises Talon, NTA or IDA, preferably NTA or IDA, most preferably IDA, coupled to the ligand and / or the support. Using this kit, a preferred Co 3+ complex according to the present invention can be produced.

[0119] In one aspect, the components a)-c) of the kit need not be provided as entities, for example, as the complex of the present invention. In another aspect, the kit may also be, for example, in the form of a salt (e.g., together with a counterion as shown elsewhere in this specification; or in a solution, e.g., as defined in the context of the composition of the present invention, e.g., CO 3 2- or HCO 3 -In a solution containing), it may include a metal cation, a metal cation ligand, and a metal cation chelating domain assembled as the complex of the present invention. Such a kit may further include reagents such as a suitable reaction buffer (preferably any of the buffers described elsewhere herein for methods and uses employing the complex of the present invention). Optionally, the kit may further include purification materials (e.g., beads, agarose beads) and / or filter columns. In a preferred embodiment, the kit may include the complex of the present invention where the carrier is beads (e.g., microbeads). In this embodiment, the complex is provided as a ready-to-use affinity resin for target molecule binding.

[0120] As described above, the complex of the present invention is particularly useful for binding a label or carrier to a target molecule by metal cation-mediated interactions. The inventors have surprisingly found that the use of carbonate or nitrate ions selected from CO 3 2- or HCO 3 - facilitates complex formation with the target molecule as compared to previously used complexes containing water as the metal cation ligand. In the art, only the [Co(III)(NTA)(CO 3 )] 2- complex without a label and / or carrier has been described (Davies and Hung, 1976; Visser et al., 2001). However, these prior art documents provided neither the motivation to modify this complex with a label nor the use of this complex for labeling target molecules such as proteins (e.g., His-tagged proteins). In particular, these prior art documents also did not suggest the advantageous features found by the inventors (see the attached examples) in binding a label and / or carrier to a target molecule.

[0121] In a fourth aspect, the invention relates to the use of the complexes of the invention for binding a label and / or a carrier comprised in the complex to a target molecule. Thereby, the metal cation-mediated linkage between the label and / or the carrier is achieved by contacting the complex of the invention with the target molecule such that the target molecule can replace the metal cation ligand in the first coordination sphere of the complex. Since the metal cation ligand is selected from CO 3 2- , HCO 3 - and nitrate ions, this is achieved with particularly high rates using the complexes of the invention. As exemplified in the appended examples for carbonate ions CO 3 2- , these metal cation ligands can be readily replaced by a target molecule in the coordination sphere, in particular by a target molecule comprising two groups that function as Lewis bases (e.g., His-tagged protein). Without being bound by theory, it is believed that the metal cation ligands of the complexes of the invention are protonated after dissociation from the complex and then facilitate their dissociation from the complex and exchange with the target molecule. In the case of carbonate ions as metal cation ligands, protonation of the carbonate ions results in gas formation that further facilitates target molecule binding by facilitating metal cation ligand dissociation. Thus, carbonate ions CO 3 2- and HCO 3 - are particularly preferred metal cation ligands in the context of the present invention.

[0122] Accordingly, the present invention provides for the use of the complex or composition of the present invention for binding a label and / or a carrier to a target molecule. The target molecules of the present invention include nucleic acids (e.g., DNA, RNA or analogs of DNA or RNA made from nucleotide analogs), peptides or proteins. In particular, the target molecule according to the present invention may be a nucleic acid (e.g., DNA, RNA or an analog of DNA or RNA made from nucleotide analogs), a peptide or a protein. It is particularly preferred that the target molecule in the context of the present invention includes a protein or a peptide. Similarly, it is also particularly preferred that the target molecule is a protein or a peptide. The protein or peptide contained in the target molecule or the protein or peptide forming the target molecule must be configured such that they can act as ligands (Lewis bases) in the metal complex. This means that the molecule must contain one or more moieties; in particular, amino acids that can act as ligands / Lewis bases and coordinate to the metal cation of the complex. In other words, the target molecule must be configured such that it can exchange the metal cation ligand in the complex of the present invention to form a new complex containing the target molecule as a ligand. Whether the ligand of the target molecule can exchange the metal cation ligand in the complex of the present invention can be tested, for example, as follows: (i) use of beads functionalized with the complex of the present invention (such beads can be prepared as described in the attached examples); (ii) addition of the target molecule (e.g., according to a protocol as described in the attached examples); (iii) incubation of the beads with the target molecule (preferably at 4 °C for at least 30 minutes, at least 1 hour, at least 3 hours, at least 3.5 hours, at least 24 hours, preferably 48 hours); (iv) separation of the beads from the supernatant; (v) analysis of the target molecule in the supernatant and / or on the beads by a target-specific method (e.g., fluorescence-based, antibody detection, enzyme assay, quantitative mass spectrometry, etc.). A negative control without the complex should be included.Subsequently, the exchange can be detected by measuring a decrease in the amount of the target molecule in the supernatant and / or an increase in the amount of the target molecule bound to the beads.

[0123] In a preferred embodiment, the protein or peptide of the target molecule (i.e., contained in or forming the target molecule) has the sequence [X n S m k (wherein X is independently selected from amino acid groups that can coordinate with the metal cation of the complex of the present invention, i.e., can function as a Lewis base, at each position; S is an amino acid not contained in the first group of amino acids (S is independently selected from this list of amino acids at each position); n is independently 1 to 4 in each case; m is independently 0 to 6 in each case; k is 2 to 6 (see SEQ ID NOs: 4 to 8), preferably 2 to 5 (see SEQ ID NOs: 4 to 7)) and has at least 4 amino acid residues in the form of a "metal cation ligand amino acid motif" that contains at least 4, preferably at least 6, most preferably at least 8, selected from amino acid groups that can coordinate with the metal cation of the complex of the present invention. The "metal cation ligand amino acid motif" may have a regular sequence, i.e., n and m may have the same value each time they appear, or an irregular sequence, i.e., n and m may have different values. Thus, in one embodiment, the target molecule is a "metal cation ligand amino acid motif" comprising or consisting of any of the amino acid sequences defined in SEQ ID NOs: 4 to 8 and containing at least 4, preferably at least 6, most preferably at least 8, selected from amino acid groups that can coordinate with the metal cation of the complex of the present invention. The greater the number of amino acids of the first group that can coordinate with the metal cation of the complex of the present invention within the amino acid motif, the greater the binding strength and / or specificity of the metal complex can be, i.e., the binding to the label and / or the carrier can be facilitated. ​

[0124] The "amino acid group capable of coordinating with the metal cation of the complex of the present invention" may consist of glycine, alanine, phenylalanine, tryptophan, methionine, tyrosine, cysteine, aspartic acid, glutamic acid, glutamine, histidine, lysine, proline, serine, threonine, asparagine, arginine, selenocysteine, and pyrrolysine. Preferably, the "amino acid group capable of coordinating with the metal cation of the complex of the present invention" consists of glycine, alanine, phenylalanine, tryptophan, methionine, tyrosine, cysteine, aspartic acid, glutamic acid, and histidine. Even more preferably, the "amino acid group capable of coordinating with the metal cation of the complex of the present invention" consists of glycine, tryptophan, tyrosine, cysteine, aspartic acid, glutamic acid, and histidine. These amino acids are known to be suitable as metal ligands capable of forming complexes with metal cations (Chin et al., 1999; McAuliff et al., 1966; Sugimori et al., 1993; Sajadi, 2010; Bell and Sheldrick, 2014). Most preferably, the "amino acid group capable of coordinating with the metal cation of the complex of the present invention" consists of histidine; that is, "X" is histidine.

[0125] As used herein, the "metal cation ligand amino acid motif" is preferably configured such that it can replace the metal cation ligand in the complex of the present invention to form a new complex containing the "metal cation ligand amino acid motif" as a ligand. The exchange with a metal cation ligand for forming a new complex can be tested as described above for the target molecule.

[0126] Histidine is a well-studied ligand for metal cation complexes, and complex formation between metal ions and histidine tags is widely used in the field of protein biochemistry, for example, to purify proteins or bind them to surfaces. In the context of the present invention, a target molecule (e.g., a protein or peptide that forms or is included in the target molecule) has the sequence [H n S m k (wherein H is histidine, S is an amino acid residue different from histidine at each position, preferably independently selected from glycine and / or serine and / or threonine, n is independently 1 to 4 in each case, m is independently 0 to 6 in each case, and k is 2 to 6 (see SEQ ID NOs: 9 to 13), preferably 2 to 5 (see SEQ ID NOs: 10 to 13)) may contain an "interspersed histidine tag" containing at least 4 histidine residues. The interspersed histidine tag may have a regular sequence, i.e., n and m may have the same value each time they appear, or an irregular sequence, i.e., n and m may have different values. Thus, in one embodiment, the target molecule may contain or consist of an interspersed histidine tag containing any of the amino acid sequences defined in SEQ ID NOs: 9 to 13, and the "metal cation ligand amino acid motif" contains at least 4, preferably at least 6, most preferably at least 8 histidine residues. The greater the number of histidines in the polyhistidine tag, the greater the binding strength and specificity for metal cations (e.g., Co 3+ ). However, if there are too many consecutive histidines, in some cases, the expression level and solubility of recombinant proteins, e.g., proteins recombinantly expressed in Escherichia coli (E. coli), may decrease. These problems can be overcome by interrupting consecutive histidines with short intervals containing glycine, serine, or threonine, i.e., by using an interspersed histidine tag.

[0127] ​In a particularly preferred embodiment, the target molecule may contain a His tag. The His tag may contain or consist of 2 to 14, preferably 3 to 10, even more preferably 4 to 8 consecutive histidine or histidine-like residues. In one embodiment, the His tag may contain at least 2, preferably at least 3, preferably at least 4, preferably at least 5, most preferably at least 6 (e.g., 2, 3, 4, 5, 6, 7 or 8) consecutive histidine or histidine-like residues. "Histidine-like" refers to a non-natural amino acid derivative containing an imidazole group. In certain preferred embodiments, the His tag consists of at least 4, preferably at least 5, most preferably at least 6 (e.g., 4, 5, 6, 7 or 8) consecutive histidine residues.

[0128] The His tag may be a sequence stretch present in a native protein or may be a recombinant histidine tag. "Recombinant" in this context means that the His tag is artificially generated by genetic manipulation, for example, by altering the coding nucleic acid sequence such that a fusion protein containing the His tag is expressed.

[0129] The His tag may be included at the N-terminus or C-terminus of the target molecule or may be provided as an internal sequence stretch. In some embodiments, the His tag may be at the N-terminus and C-terminus.

[0130] In one embodiment, the target molecule of the present invention may have a three-dimensional structure in which at least 2, preferably at least 3, preferably at least 4, preferably at least 5, most preferably at least 6 histidine or histidine-like residues are in a spatially proximate state. "Spatially proximate state" in this context preferably means a distance of 0 to 5 angstroms between each residue. The "distance" between two residues is the shortest distance between the two closest nitrogen atoms of the two individual neighboring histidine side chains or histidine-like side chains.

[0131] Non-limiting preferred examples of the target molecules of the present invention are proteins for pharmaceuticals, diagnostics, research agents, cosmetics and / or environmental treatment (e.g., proteins for water treatment). Other non-limiting preferred examples of target molecules include enzymes, targeted proteins (e.g., antibodies, nanobodies, etc.), cytokines, transport proteins (e.g., FABS for fatty acid transport), storage proteins (e.g., ferritin), mechanical support proteins (e.g., collagen), growth factors, hormones (e.g., insulin or TSH), interferons, glycoproteins, synthetically engineered proteins or fragments thereof, or molecules comprising or consisting of the same.

[0132] Also, the target molecule used in the context of the present invention may be a nucleic acid. Nucleic acids contain purine and pyrimidine bases. Both are known to be able to form complexes with the metal cations of the present invention. Preferably, the nucleic acid contains at least 2, preferably at least 5, and even more preferably at least 10 bases. Nucleic acids include DNA, RNA, LNA and other nucleic acid derivatives known in the art. Most preferred is DNA. In particular, the target molecule is a nucleic acid structure with a origami structure, i.e., a three-dimensional fold. Such an origami structure is typically formed by base pairing of several nucleic acid strands including scaffold strands and staple strands.

[0133] The use of the complex or composition of the present invention for labeling a target molecule involves binding of a label and / or a carrier to the target molecule by exchanging the metal cation ligand of the complex of the present invention with the target molecule. The exchange is achieved by contacting the complex of the present invention with the target molecule. Thus, the use preferably includes contacting the complex of the present invention with the target molecule in solution.

[0134] Accordingly, in one aspect, the present invention relates to a method for binding a label and / or a carrier to a target molecule, the method comprising the step of incubating a complex of the present invention or a composition comprising the complex of the present invention with the target molecule. The target molecule is a target molecule as defined elsewhere in this specification.

[0135] "Incubating" means mixing the complex and the target molecule in solution and allowing the mixture to react for a defined period of time. As discussed above, the time for which the complex is incubated with the target molecule is the "complex - target incubation time" in the context of the present invention. Tables 1 and the appended examples can assist one of ordinary skill in the art in selecting the complex - target incubation time. Thus, the present invention relates, for example, to the use of the complexes of the present invention for binding a label and / or a carrier comprised in the complex to a target molecule, wherein the complex - target incubation time may be about 48 hours. As described herein, the complex formation time can also have an impact on product complex formation. Also, as described herein, the complex formation time can be selected based on Table 1 or the appended examples. Thus, the present invention relates, for example, to the use of the complexes of the present invention for binding a label and / or a carrier comprised in the complex to a target molecule, wherein the complex formation time is about 48 hours and the complex - target incubation time is about 3.5 hours. In another illustrative example, the present invention relates to the use of the complexes of the present invention for binding a label and / or a carrier comprised in the complex to a target molecule, wherein the complex formation time is about 10 minutes and the complex - target incubation time is about 48 hours. The present invention further relates to the use of the complexes of the present invention for binding a label and / or a carrier comprised in the complex to a target molecule, wherein the complex formation time is about 10 minutes and the complex - target incubation time is about 30 minutes. The present invention also relates to the use of the complexes of the present invention for binding a label and / or a carrier comprised in the complex to a target molecule, wherein the complex formation time is about 10 minutes and the complex - target incubation time is about 3 hours. The present invention also relates to the use of the complexes of the present invention for binding a label and / or a carrier comprised in the complex to a target molecule, wherein the complex formation time is about 48 hours and the complex - target incubation time is about 1 hour.

[0136] In a particular embodiment of the use of the complex of the invention for binding a label and / or a carrier comprised in the complex to a target molecule, the metal chelating ligand is IDA, the complex formation time is 10 minutes, and the complex-target incubation time is about 30 minutes.

[0137] In another preferred embodiment of the use of the complex of the invention for binding a label and / or a carrier comprised in the complex to a target molecule, the metal chelating ligand is IDA, the complex formation time is 10 minutes, and the complex-target incubation time is about 3 hours.

[0138] The method for binding a label and / or a carrier to a target molecule may include the step of recovering and / or purifying the target molecule to which the label and / or the carrier is linked. This is to provide an isolated target molecule to which the label and / or the carrier is bound.

[0139] If the method includes the step of binding a carrier, purification and / or isolation of the target molecule to which the carrier is bound can be achieved by isolating the carrier. For example, if a solid carrier is used, the beads can be sedimented by centrifugation and the supernatant can be removed. Alternatively, a filter column that allows the liquid to flow out but retains the complex bound to the carrier can be used. If necessary, the carrier can be washed with a buffer solution to remove impurities.

[0140] Generally, regardless of whether a carrier or a label is used, isolation and / or purification can be carried out by chromatography. The chromatography may include, or consist of, size exclusion chromatography that uses differences in molecular size and shape among the target molecule bound to the carrier and / or the label, the free target molecule, and the complex of the invention.

[0141] The method for purifying a target molecule according to the invention to which a label and / or a carrier is bound is also described in WO2014 / 072525, which is hereby incorporated by reference in its entirety.

[0142] In one embodiment, purification can also be carried out using a conventional Ni-NTA column as described in WO2014 / 072525. The incubation of the complex of the present invention or a composition containing the same can be carried out in solutions having different pH values. However, the binding of the label and / or the carrier to the target molecule may be carried out at various pH values. The lower the pH value, the easier it is for the metal cation ligand (i.e., carbonate ion or nitrate ion) to dissociate. This is because the dissociated ligand is protonated. Carbonate ion CO 3 2- and HCO 3 - further forms a gas that is released when they are protonated.

[0143] In principle, the lower the pH value, the easier the exchange reaction is, but the pH during incubation is typically selected within a range compatible with the target molecule used. In particular, proteins are often sensitive to pH values that are too low or too high. The pH is preferably selected such that the stability of the target molecule (e.g., the three-dimensional folding of the protein and / or the biological function of the protein) is maintained. However, the pH is still preferably selected at the lower end of the pH stability range of the target molecule that facilitates ligand exchange, i.e., the binding of the label and / or the carrier to the target molecule. Those skilled in the art know assays for testing the stability and function of proteins at different pH values.

[0144] In a preferred embodiment, the pH during the incubation of the complex of the present invention with the target molecule is 4.0 to 9.5, preferably 5.5 to 8.0. In these pH ranges, many target molecules, especially many proteins, are stable and / or functional.

[0145] The exchange between the metal cation ligand and the target molecule typically occurs in a short time. Thus, depending on the required binding efficiency and the target molecule used, the incubation step of the method for binding the label and / or the carrier according to the present invention can be carried out in a length of at least about 10 seconds, preferably at least about 1 minute, most preferably at least about 10 minutes. In a preferred embodiment, the incubation is carried out for at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 7 hours, at least about 9 hours, at least about 12 hours, at least about 24 hours or at least about 48 hours. Without being bound by theory, longer incubation is thought to result in a higher binding yield. However, after a certain incubation time, saturation of labeling and / or carrier binding is recognized. The preferred incubation time may depend on the target molecule used and the desired binding effectiveness. A person skilled in the art can test the binding effectiveness by measuring the depletion of the free label and / or carrier and / or quantifying the target molecule to which the label and / or carrier is bound.

[0146] The incubation step of the method for binding a label and / or a carrier to a target molecule can be performed at different temperatures. The exchange of a metal cation ligand with a target molecule will, in principle, function at any temperature from 0 to 95 °C. The higher the temperature, the faster the ligand exchange reaction. However, since the target molecule contains or consists of a biomolecule such as a temperature-sensitive protein, the temperature must be selected to ensure the stability and / or function of the target molecule. For example, a protein originating from a thermophilic bacterium will tolerate a higher reaction temperature than a protein originating from a non-thermophilic bacterium. Similarly, DNA has a higher heat resistance than RNA. Thus, the temperature depends on the target molecule used. Non-limiting temperature ranges that can be used are 0 °C to about 95 °C, 0 °C to about 60 °C, 0 °C to about 42 °C, 0 °C to about 25 °C, and about 4 °C to about 25 °C. The temperature may remain constant during the incubation. Alternatively, one or more different temperatures or a temperature gradient may be used. In one embodiment, the target molecule is a protein and the temperature is selected from 0 °C to about 25 °C, preferably from about 2 °C to about 8 °C.

[0147] As described above, the incubation step is performed in solution. Different solutions can be used in this context, including pure water or an aqueous solution. Thus, in one embodiment, the incubation can be carried out in water or an aqueous solution. Alternatively, or additionally, the solution may contain one or more organic solvents. A non-limiting set of preferred organic solvents includes DMSO, DMF, DMS, acetonitrile, and isopropanol. Thus, in one embodiment, the incubation can be carried out in a solution containing one or more organic solvents selected from the group consisting of DMSO, DMF, DMS, acetonitrile, and isopropanol. One skilled in the art will select a solution that does not inhibit downstream applications and / or the stability or activity of the target molecule. In the context of proteins, preferably, an aqueous solution is particularly preferred, together with physiological conditions.

[0148] As shown in the attached examples, methods for binding labels and / or carriers to target molecules can be performed in the presence of different buffer reagents. Thus, in a preferred embodiment, the incubation step of the method can be performed in an aqueous solution containing one or more Good's buffer substances.

[0149] Good's buffer substances are 20 buffers for biochemical and biological research selected and described by Norman Good and colleagues from 1966 to 1980 (see Good et al., 1966; Good et al., 1972; Ferguson et al., 1980). These buffers are known in the art.

[0150] Exemplary but non-limiting buffers are ACES, AMPSO, BES, BisTris propane, boric acid, CAPS, CAPSO, CHES, DIPSO, EPPS, HEPES, HEPBS, HEPPSO, MES, MOPS, MOPSO, PIPES, POPSO, TAPS, TAPSO, TEA, TES and Tris, and derivatives thereof may also be used. "Derivatives" relate to buffer substances having the same structural backbone but substituted by additional chemical moieties. Alternatively, or in addition, Tris buffers, carbonate / bicarbonate buffers or phosphate buffers (e.g., PBS) may be used.

[0151] In one embodiment, buffer substances selected from BisTris and derivatives, carbonate / bicarbonate buffers, CAPS, CAPSO, HEPES, HEPBS, HEPPSO, MES, MOPS, MOPSO, PIPES, phosphate buffers (such as PBS), TAPS, TES, and Tris can be used.

[0152] When using these exemplary buffers, the pH of the solution can be selected as follows: ACES: 6.0 - 7.8; AMPSO: 8.0 - 10.0; BES: 6.2 - 8.0; BisTris: 5.5 - 7.5; BisTris propane: 6.0 - 9.8; Boric acid: 8.2 - 10.5; CAPS: 9.5 - 11.5; CAPSO: 8.5 - 10.5; CHES: 8.4 - 10.2; DIPSO: 6.8 - 8.5; EPPS: 7.0 - 9.0; HEPES: 7.2 - 9.5; HEPBS: 7.4 - 9.2; MES: 5.2 - 7.0; MOPS: 6.2 - 8.2; MOPSO: 6.0 - 8.0; Phosphoric acid: 5.5 - 8.2; PIPES: 5.9 - 7.8; POPSO: 7.0 - 8.7; TAPS: 7.4 - 9.4; TAPSO: 6.7 - 8.5; TEA: 7.0 - 8.5; TES: 6.5 - 8.5; Tris: 6.8 - 9.5; Carbonate buffer: 8.5 - 11.0.

[0153] Since reactions can be carried out over a wide range of pH values where buffer capacity is not absolutely required, in principle, other pH values can also be used with each of the above buffers. However, in order to keep the pH constant and better control the reaction, a pH within the above pH range where the buffer has buffer capacity is preferred.

[0154] Buffers can be used at various concentrations, such as 1 mM - 1 M, preferably 1 mM - 250 mM, and most preferably 1 mM - 100 mM (e.g., 50 mM). A person skilled in the art can evaluate the binding effectiveness and test for the ideal buffer concentration by using various buffer concentrations.

[0155] As shown in the attached examples, the choice of buffering substance / buffer can affect the binding efficiency of the label and / or the carrier to the target molecule. Without being bound by theory, buffers with low metal binding constants show an increase in the efficiency of binding. The low metal cation binding constant prevents the buffer from coordinating to the metal and blocking the ligand positions in the first coordination sphere.

[0156] Thus, in one embodiment, the buffer substance / buffer used in the context of the present invention may be a buffer substance / buffer having a low metal binding constant. What is understood by "low metal binding constant" and preferred examples of such buffer substances / buffers are disclosed in Ferreira et al., 2015, which is hereby incorporated by reference in its entirety.

[0157] Furthermore, in a preferred embodiment, the incubation step is carried out in the presence of a buffer substance / buffer containing one or less (preferably, none) groups selected from an amino group and a carbonyl group. These groups include Lewis bases that can mediate the binding to metal cations in the complex. Thus, the absence of these groups reduces or prevents the binding of the buffer substance to the metal cation, thereby facilitating the binding of the target molecule.

[0158] As shown by the attached examples, the following buffers: MES, HEPES, Bis-Tris and PIPES may be advantageous for facilitating labeling and / or carrier binding. The examples show that these buffers result in a higher binding efficiency than Tris-based buffers. Thus, in a particularly preferred embodiment, the buffer substance present in the solution during incubation can be selected from MES, HEPES, Bis-Tris and PIPES. These buffer substances are particularly advantageous for labeling and / or carrier binding. Non-limiting example 13 demonstrates that it may be necessary to select a buffer depending on the pH used for incubation. When the incubation of the complex and the target molecule of the present invention is carried out at a pH of about 7.5, HEPES can be used instead of BisTris. When the incubation of the complex and the target molecule of the present invention is carried out at a pH of about 5.5, BisTris can be used. The determination of the most suitable buffer and pH is within the scope of the relevant art in the industry and can be readily achieved using the teachings of the present invention and the examples in the experimental part and the scientific details provided therein.

[0159] In view of these results and based on theoretical considerations to keep the buffer binding to the metal cation as low as possible, the buffer substance can preferably be selected from BisTris, CAPS, CAPSO, HEPES, HEPBS, HEPPSO, MES, MOPS, MOPSO, PIPES, TAPS and TES.

[0160] In a preferred embodiment, the incubation of the complex of the invention or a composition comprising the same with the target molecule is carried out in solution in the presence of Ca 2+ ions. When the metal cation ligand of the complex of the invention is a carbonate ion selected from HCO 3 - or CO 3 2- , the presence of Ca 2+ ions is particularly preferred. This is because Ca 2+ ions can form insoluble CaCO 3 which precipitates from the solution, thereby facilitating the release of carbonate ions from the complex. This then facilitates the binding of the target molecule as a ligand, thereby facilitating the binding of the label and / or the carrier to the target molecule. Preferably, CaCl 2 dissolved in the reaction solution is used to provide Ca 2+ ions. Ca 2+ ions can be added directly at the start of the reaction, i.e., when the complex of the invention is contacted with the target molecule. Alternatively, although less preferred, Ca 2+ ions can be added during the reaction in the form of a salt (preferably CaCl 2 ). Ca 2+ ions (preferably in the form of CaCl 2 ) are preferably added at a concentration of 0.1 - 50 mM, even more preferably 0.1 - 10 mM, and most preferably 1 mM.

[0161] The method for binding a label and / or a carrier to a target molecule according to the invention is such that the metal cation of the complex of the invention is, for example, H 2 O 2It does not require an oxidation step that is oxidized by the treatment. In such a preferred embodiment, the method for binding a label and / or a carrier to a target molecule according to the present invention is H 2 O 2 It does not include an oxidation step such as treatment with. This is particularly preferred in the context of forming Co 3+ mediated label and / or carrier binding. In previously reported methods (e.g., Wegner and Spatz, 2013), Co 3+ mediated binding of functional moieties was achieved only after forming a Co 2+ complex with a his-tagged target protein as a ligand, followed by oxidizing Co 2+ to Co 3+ . As shown in the attached examples, such an oxidation step in the presence of Co cations can lead to a spontaneous Fenton reaction that can result in proteolysis. Furthermore, oxidation of the protein can inhibit protein folding and function. Thus, the absence of the oxidation step is a particular advantage of the method of the present invention.

[0162] The method for binding a label and / or a carrier to a target molecule may also include the production of the complexes of the present invention (and, optionally, neutral complexes such as salts used therefor) described elsewhere herein.

[0163] The method for binding a label and / or a carrier to a target molecule according to the present invention results in the production of a labeled and / or carrier-bound product. Thus, the method may also be referred to as a method for producing a target molecule to which a label and / or a carrier is bound. The priority regarding the target molecule shown elsewhere herein is applied with modifications where necessary. Thus, in one embodiment, the method of the present invention may be a method for binding a label and / or a carrier to a protein, such as a His-tagged protein, an antibody, a derivative thereof (including, for example, scFv fragments and nanobodies) or a domain thereof.

[0164] A preferred embodiment of a method for binding a label and / or a carrier to a target molecule is one in which the target molecule is a His-tagged protein and the His-tagged protein is bound to a carrier (e.g., a chip) that is a surface. Preferably, Co as a metal cation 3+ In this method using carbonate ions as defined herein as a metal cation ligand and NTA, Talon or IDA as a chelating ligand, the His-tagged protein is bound to the surface. Due to the high stability of the target molecule and the kinetically inert binding, the protein can be bound to a surface that is inert with respect to imidazole and other chelates (e.g., EDTA) as well as reductive equivalence treatments in a manner approaching covalent.

[0165] In yet another aspect, the present invention relates to a target molecule to which a label and / or a carrier is bound, obtained or having been obtained by a method for binding a label and / or a carrier to a target molecule according to the present invention. The target molecule to which a label and / or a carrier has been bound by the method of the present invention is characterized in that neither the target molecule nor the label and / or the carrier has been subjected to an oxidation step (e.g., treatment with H 2 O 2 ). In contrast, previously reported methods for generating such structures included an oxidation step in the presence of at least one or both of i) the target molecule and ii) the label and / or the carrier. Thus, the product of the method of the present invention has the advantage of not being oxidized and not containing oxidizing agents such as H 2 O 2 . This also has an important advantage for the medical use of the labeled and / or carrier-bound target molecules produced.

[0166] What has been said elsewhere in this specification regarding complexes, their components, target molecules and the method for binding a label and / or a carrier to a target molecule according to the present invention is applied with such modifications as may be appropriate.

[0167] The target molecule to which a label and / or a carrier is bound, obtained by the method of the present invention, is also a complex. This obtained "product complex" contains the metal cation of the complex of the present invention and coordinates to i) the metal-binding domain of the complex of the present invention and ii) the target molecule.

[0168] The present invention also provides a composition comprising a labeled and / or carrier-bound target molecule obtained or obtainable by a method for binding a label and / or a carrier to a target molecule.

[0169] The target molecule to which a label and / or a carrier is bound, obtained or obtainable by the method of the present invention, or a composition containing the same can be used as a research reagent. Thus, the present invention also relates to the use of a labeled or carrier-bound target molecule obtained or obtainable by the method of the present invention as a research reagent. Similarly, a method is provided that includes the step of using a labeled or carrier-bound target molecule produced by the method of the present invention as a research reagent. For example, the target molecule may be an extracellular matrix protein immobilized on a solid support for cell culture.

[0170] In one embodiment, the target molecule to which a label and / or a carrier is bound, obtained or obtainable by the method of the present invention, or a composition containing the same can be used as a diagnostic agent in vitro. For example, the target molecule may be a detection protein that specifically recognizes an analyte (e.g., an antibody that recognizes an antigen as an analyte), and the label and / or the carrier can be configured to comply with measurement methods known in the art.

[0171] The present invention relates to a target molecule obtained or obtained by the method of the present invention, or a labeled and / or carrier-bound target molecule, or a composition containing the same, for use as a medicament. Similarly, there is provided a treatment method comprising administering to a patient an effective amount of a composition comprising a target molecule obtained or obtained by the method of the present invention, or a labeled and / or carrier-bound target molecule. Preferably, the target molecule is selected from enzymes, targeted proteins such as antibodies, cytokines (e.g., G-CSF), transport proteins such as FABS for fatty acid transport, storage proteins such as ferritin, mechanical support proteins such as collagen, growth factors, hormones such as insulin or TSH, interferons, glycoproteins, synthetically engineered proteins or fragments thereof.

[0172] The present invention particularly includes embodiments in which the target molecule and / or label can be released from the "product complex" containing the target molecule, for example, upon binding of the complex in vivo to the target structure when used as a medicament in vivo. Thus, the target molecule to which the label and / or carrier is bound, obtained by the method of the present invention, can be configured such that the label and / or target molecule can preferably be released in vivo. Release can be induced by reduction of the metal cation in the product complex, pH change and / or binding of the target molecule and / or label to the target structure (e.g., cell surface proteins such as receptors or cancer cell-specific surface proteins).

[0173] The present invention particularly provides the following items: 1. a) A metal cation; b) CO 3 2- or HCO 3 - A metal cation ligand which is; and c) A metal cation chelating domain comprising a chelating ligand and a label and / or carrier A complex comprising.

[0174] 2. The complex according to item 1, wherein the chelating ligand of the metal cation chelating domain is a multidentate ligand containing one or more carboxylic acid groups and / or one or more amine groups and / or one or more aromatic amines and / or phosphate ions.

[0175] 3. The complex according to item 1, wherein the chelating ligand of the metal cation chelating domain in 3.c) is a chelating peptide such as nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), tris(carboxymethyl)ethylenediamine (TED), a peptide having a consensus acid sequence (GHHPH)nG (where n is 1 to 3; see also SEQ ID NOs: 1 to 3), or cadistin, triazacyclononane (TACN), diethylenetriamine-pentaacetic acid (DTPA), phytochelatin, carboxymethylaspartic acid (CMA), phosphate ions, tannic acid (TA), porphyrin, dipyridylamine (DPA), phytic acid, nitrilopropionodiacetic acid (NPDA), nitriloisopropionodiacetic acid (NIPDA), N-(hydroxyethyl)ethylenediaminetriacetic acid (HEDTA), 1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA), 1,4,7-tris(carboxymethyl)-10-(2’-hydroxypropyl)-1,4,7,10-tetraazocyclodecane, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1-(1,3-carboxypropyl)-1,4,7-triazacyclononane-4,7-diacetic acid (NODAGA), 1,4,8,11-tetraazacyclotetradecane-N,N’,N’’,N’’’-tetraacetic acid (TETA), ethylenedicysteine, ethylenediaminetetraacetic acid (EDTA), 1,2-diaminocyclohexane-N,N,N’,N’-tetraacetic acid (DACT), bis(aminoethanethiol)carboxylic acid, ethylene-bis(oxyethylene-nitrilo)tetraacetic acid (EGTA), triethylenetetramine-hexaacetic acid (TTHA), 1,4,7-triazacyclononanephosphinic acid (TRAP), deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), purine, pyrimidimidine and derivatives thereof.

[0176] The chelating ligand of the metal cation chelating domain in 4.c) is selected from nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), consensus sequence (GHHPH) n a chelating peptide having G, diethylenetriamine-pentaacetic acid (DTPA), nitrilopropiondiacetic acid (NPDA), nitriloisopropiondiacetic acid (NIPDA), ethylenediaminetetraacetic acid (EDTA), ethylene-bis(oxyethylene-nitrilo)tetraacetic acid (EGTA), carboxymethylaspartic acid (CMA) and derivatives thereof, the complex according to item 1

[0177] The chelating ligand of the metal cation chelating domain in 5.c) contains or is selected from NTA, IDA and derivatives thereof, the complex according to item 1 6. The complex according to any one of items 1 to 4, wherein the metal cation is a transition metal cation

[0178] 7. The complex according to any one of items 1 to 5, wherein the metal cation of the complex is a divalent, trivalent or tetravalent metal cation 8. The metal cation is 10 -1 s -1 Hereinafter, preferably, 10 -2 s -1 Hereinafter, the complex according to any one of items 1 to 6, which is a metal cation having a water ligand exchange rate of

[0179] 9. The metal cation is Co 3+ 、Cr 3+ 、Rh 3+ 、Ir 3+ 、Pt 2+ 、Pt 4+ 、Ru 2+ 、Ru 3+ 、La 3+ 、Eu 3+ 、Os 2+ 、Pd 4+ 、Mo 3+ 、Fe 3+ 、Ru 3+ 、Gd 3+ 、Tc 3+ 、Re 3+, Sm 3+ , Tb 3+ , Ce 3+ , Pr 3+ , Nd 3+ , Pm 3+ , Dy 3+ , Ho 3+ , Er 3+ , Tm 3+ , Yb 3+ , V 2+ , Mn 4+ , Fe 2+ and Lu 3+ The complex according to any one of items 1 to 7, selected from the group consisting of

[0180] 10. The metal cation is Co 3+ , Cr 3+ , Rh 3+ , Ir 3+ , Ir 4+ , Pt 2+ , Pt 4+ , Pd 4+ , Mo 3+ , Fe 3+ , Gd 3+ , Tb 3+ , Eu 3+ , Ru 2+ , La 3+ , Ru 3+ , Re 3+ , Re 4+ , V 2+ , Mn 4+ , Fe 2+ and Os 2+ The complex according to any one of items 1 to 7, selected from the group consisting of

[0181] 11. The metal cation is Co 3+ The complex according to any one of items 1 to 7. 12. The metal cation ligand in b) is carbonate ion CO 3 2- or bicarbonate ion HCO 3 - The complex according to any one of items 1 to 11.

[0182] 13. The complex is [Co(III)(NTA)CO 3 ​2- , [Co(III)(NTA)HCO 3 - The complex according to any one of items 1 to 12, comprising the complex or its hydrate, and the label and / or carrier being bound to NTA.

[0183] 14. The complex according to any one of items 1 to 13, wherein the label comprises a fluorophore, a diagnostic agent, a targeting moiety, a therapeutic agent, a PEG molecule, a lipid, biotin and / or its derivatives, a protein, a peptide, a toxin and / or a reactive group selected from thiol, azide, alkyne, nitrone, tetrazine and tetrazole.

[0184] 15. The complex according to any one of items 1 to 13, wherein the label comprises or is a fluorophore. 16. The complex according to any one of items 1 to 13, wherein the label comprises or is biotin or its derivative.

[0185] 17. The complex according to any one of items 1 to 13, wherein the carrier is a polymer, a hydrogel, microparticles, nanoparticles, spheres (including nano and microspheres), beads, quantum dots, an artifact or a solid surface.

[0186] 18. The complex according to items 1 to 17, wherein the metal cation chelating domain comprises a linker between the chelating ligand and the label and / or carrier. 19. A composition comprising the complex defined in any one of items 1 to 18.

[0187] 20. Use of the complex according to any one of items 1 to 18 or the composition according to item 19 for labeling a target molecule, wherein the target molecule comprises a protein, a peptide or a nucleic acid, preferably a protein or DNA capable of exchanging the metal cation ligand in the complex, and more preferably the target molecule has the sequence [H n S m k ​​(wherein H is a histidine residue or a histidine-like residue, S is a spacer amino acid residue, n is independently 1 to 4 in each case, m is independently 0 to 6 in each case, and k is 2 to 6) for use, comprising at least four histidine residues or histidine-like residues in the form of.

[0188] Use of the complex according to any one of items 1 to 18 or the composition according to item 19 for labeling a target molecule, wherein the target molecule contains a histidine-rich region containing at least two histidine residues, and the histidine-rich region is formed by the three-dimensional folding of the target molecule that brings the at least two histidine residues into spatial proximity, and the at least two histidine residues have a distance of 0 to 5 angstroms and are not contiguous in the amino acid sequence.

[0189] 22a. Use according to item 21, wherein the histidine-rich region is the Fc region of an antibody. 22b. Use of the complex according to any one of items 1 to 18 or the composition according to item 19 for binding a label and / or a carrier to an antibody, its domain (e.g., the Fc region) or a fragment thereof.

[0190] 22c. Use according to item 22b, wherein the label is a toxin. 23. Use of the complex according to any one of items 1 to 18 or the composition according to item 19 for labeling a target molecule, wherein the target molecule contains a region rich in histidine-like residues that occurs during the three-dimensional folding of the target molecule when the histidine-like residues are in spatial proximity, and the at least two histidine-like residues have a distance of 0 to 5 angstroms and are not contiguous in the amino acid sequence.

[0191] 24. Use according to item 20, 21 or 23, wherein the target molecule is a protein for pharmaceuticals, diagnostic agents, research agents, cosmetics and / or environmental treatment (e.g., water treatment). 25. Use according to item 20, 21 or 23, wherein the target molecule comprises or is a peptide or a protein.

[0192] 26. Use according to item 20, 21, 23, 24 or 25, wherein the target molecule comprises or is an enzyme, a targeted protein such as an antibody, a cytokine, a transport protein such as FABS for fatty acid transport, a storage protein such as ferritin, a mechanical support protein such as collagen, a growth factor, a hormone such as insulin or TSH, an interferon, a glycoprotein, a synthetically engineered protein or a fragment thereof.

[0193] 27. Use according to any one of items 20 to 26, wherein the target molecule comprises a histidine residue or a histidine-like residue in its N-terminal, C-terminal or internal sequence region. 28. Use according to any one of items 20 to 26, wherein the histidine residue or histidine-like residue is included in the form of a His-tag, preferably the His-tag consists of 2 to 10, preferably 4 to 8, most preferably 6 to 8 consecutive residues.

[0194] 29. Use of the complex according to any one of items 1 to 18 or the composition according to item 19 for producing a pharmaceutical, a diagnostic agent and / or a cosmetic. 30. a) A metal cation, preferably a metal cation defined in any one of items 5 to 11; b) CO 3 2- or HCO 3 - is a metal cation ligand; and c) A metal cation chelation domain comprising a chelating ligand and a label and / or a carrier, preferably a metal cation chelation domain defined in any one of items 2 to 4 and items 14 to 18 A kit comprising.

[0195] 31. A method for producing a complex according to any one of items 1 to 18, comprising the step of incubating in a solution: (i) a metal cation; (ii) a metal cation ligand defined in b) of item 1; and (iii) a metal cation chelating domain defined in c) of item 1.

[0196] 32. The method according to item 31, further comprising the step of collecting and / or purifying the complex according to any one of items 1 to 18. 33. The method according to item 31 or 32, wherein the metal cation chelating domain is a metal cation chelating domain defined in any one of items 2 to 18.

[0197] 34. The method according to any one of items 31 to 33, wherein the metal cation is a metal cation defined in any one of items 5 to 11. 35. The metal cation is Co 3+ and the metal cation binding ligand is CO 3 2- or HCO 3 - and Co 3+ and CO 3 2- or HCO 3 - are provided in the form of a neutral complex with a counter ion such as in the form of a salt, or in the form of a charged complex containing Co 3+ and CO 3 2- or HCO 3 - The method according to any one of items 31 to 34.

[0198] 36. The neutral complex is sodium tris-carbonatocobalt(III) trihydrate (Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O) or potassium tris-carbonatocobalt(III) trihydrate (K 3 [Co(III)(CO 3 ) 3 ·3H2 The method according to item 35, which is (O).

[0199] 37. The incubation is preferably carried out in a buffer containing HCO at a concentration of at least 1 mM, preferably at least 10 mM, and most preferably 1 M. 3 - or CO 3 2- The method according to any one of items 31 to 36, which is carried out in a buffer containing.

[0200] 38. A method for binding a label and / or a carrier to a target molecule, comprising the step of incubating the complex according to any one of items 1 to 18 or the composition according to item 19 with a target molecule which is a target molecule defined by any one of items 20 to 28.

[0201] 39. The method according to item 38, further comprising the step of recovering and / or purifying the target molecule to which the label and / or the carrier is linked. 40. The method according to item 38 or 39, wherein the solution has a pH of 4.0 to 9.5, preferably 5.5 to 8.0.

[0202] 41. The method according to any one of items 38 to 40, wherein the incubation is carried out for at least 10 seconds, preferably 1 minute, and most preferably 10 minutes. 42. The method according to any one of items 38 to 41, wherein the incubation is carried out at a temperature of 0 to 95 °C, preferably 0 to 60 °C, and most preferably 0 to 42 °C.

[0203] 43. Before the incubation, preferably, the complex of the present invention is washed in a solution containing HCO at a concentration of at least 1 mM, preferably 10 mM, and most preferably 1 M. 3 - or CO 3 2- and / or the incubation is preferably carried out in a solution containing HCO at a concentration of at least 1 mM, preferably 10 mM, and most preferably 1 M.3 - or CO 3 2- The method according to any one of items 38 to 42, which is carried out in a solution containing

[0204] 44. The method according to any one of items 38 to 43, wherein the incubation is carried out in water or an aqueous solution. 45. The method according to any one of items 38 to 44, wherein the incubation is carried out in a solution containing one or more organic solvents selected from the group consisting of DMSO, DMF, DMS, acetonitrile and isopropanol.

[0205] 46. The method according to any one of items 38 to 45, wherein the incubation is carried out in an aqueous solution containing one or more Good's buffers, Tris, phosphate ions and / or carbonate / bicarbonate ions.

[0206] In one embodiment of this item, the buffer may be PBS. 47. The method according to any one of items 38 to 46, wherein the incubation is preferably carried out in the presence of Ca provided in the form of CaCl 2 2+

[0207] 48. The method according to any one of items 38 to 47, wherein the incubation is carried out in an aqueous solution containing one or more buffer substances, and the buffer substances do not contain amines, carboxylic acids, aromatic amines and / or phosphate groups.

[0208] 49. The method according to any one of items 38 to 48, wherein the incubation is carried out in an aqueous solution containing one or more buffering substances selected from the group consisting of ACES, AMPSO, BES, BisTris, BisTris propane, boric acid, CAPS, CAPSO, CHES, DIPSO, EPPS, HEPES, HEPBS, HEPPSO, MES, MOPS, MOPSO, PIPES, POPSO, TAPS, TAPSO, TEA, TES, carbonate / bicarbonate buffer, phosphate buffer (e.g., PBS), and Tris.

[0209] 50. The method according to any one of items 38 to 49, wherein the incubation is carried out in an aqueous solution containing one or more buffering substances selected from the group consisting of BisTris, CAPS, CAPSO, HEPES, HEPBS, HEPPSO, MES, MOPS, MOPSO, PIPES, TAPS, TES, phosphate buffer (e.g., PBS), and Tris.

[0210] 51. The method according to any one of items 38 to 50, wherein the incubation is carried out in an aqueous solution containing a buffering substance selected from the group consisting of Bis-Tris, MES, HEPES, and PIPES.

[0211] 52. The method according to any one of items 38 to 51, which does not include an oxidation step such as treatment with H 2 O 2 in the presence of a label and / or a carrier. 53. A labeled or carrier-bound target molecule obtained by the method defined in any one of items 38 to 52.

[0212] 54. A composition comprising the labeled or carrier-bound target molecule according to item 53. 55. The labeled or carrier-bound target molecule according to item 53 or the composition according to item 54 as a research reagent.

[0213] Use of the labeled or carrier - bound target molecule according to item 53 or of the composition according to item 54 for use as a medicament. The "complex" of the present invention refers to a complex formed by a metal cation and a ligand. Thus, the term "complex" preferably relates to a coordination complex or a metal complex. A complex comprises a Lewis acid in the form of a metal cation and one or more Lewis bases in the form of one or more ligands. The complexes of the present invention have at least two ligands as defined elsewhere in this specification.

[0214] As used herein, the terms "protein" and "peptide" both relate to polypeptides consisting of amino acids. The term "peptide" refers to a polypeptide having 20 or fewer amino acids. The term "protein" refers to a polypeptide having more than 20 amino acids. The term "polypeptide" encompasses both "peptide" and "protein". When "protein or peptide" is recited herein, polypeptides are also included.

[0215] As used herein, "antibody" refers to any molecule that can specifically or selectively bind to a target protein. An antibody may include, or be, a part / fragment thereof that exhibits substantially the same binding activity as an antibody or a full-length antibody. An antibody may also include a multivalent molecule, a multispecific molecule (e.g., diabody), a fusion molecule, an aptamer, an avimer, or other naturally occurring or recombinantly produced molecule. Exemplary antibodies useful in the present invention include antibody-like molecules. Antibody-like molecules are molecules that can function by binding to a target molecule (see, e.g., Current Opinion in Biotechnology 2006, 17:653-658; Current Opinion in Biotechnology 2007, 18:1-10; Current Opinion in Structural Biology 1997, 7:463-469; Protein Science 2006, 15:14-27), and include, for example, DARPin (WO2002 / 020565), affibody (WO1995 / 001937), avimer (WO2004 / 044011; WO2005 / 040229), adnectin (WO2002 / 032925), and finomer (WO2013 / 135588). Generally, the term "antibody" is used herein in the broadest sense and, without limitation, encompasses various antibody structures including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity. Antibodies within the present invention may also be chimeric antibodies, recombinant antibodies, antigen-binding fragments of recombinant antibodies, or humanized antibodies.

[0216] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to a part of an intact antibody and refers to a molecule other than the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabody; linear antibody; single-chain antibody molecule (e.g., scFv); and multispecific antibodies formed from antibodies.

[0217] The term "ligand" refers to a species that interacts with another species in some manner. In the context of the present invention, "ligand" when used in the context of a complex refers to a molecule that includes a Lewis base capable of forming a coordination bond with a Lewis acid. In other examples, a ligand is often a species that is an organic substance that includes a group capable of forming a coordination bond with a metal ion. When coordinating to a metal ion, a ligand may have various binding modes known to those skilled in the art, including, for example, terminal (i.e., binding to a single metal ion) and bridging (i.e., one atom of the Lewis base binds to more than one metal ion).

[0218] The terms "Lewis acid" and "Lewis acidity" are recognized in the art and refer to a chemical moiety capable of accepting a pair of electrons from the Lewis base as defined above. The terms "Lewis base" and "Lewis basicity" generally refer to a chemical moiety capable of donating a pair of electrons under certain reaction conditions. A Lewis base can be characterized as donating a single electron in a particular complex depending on the identity of the Lewis base and the metal ion, but for many purposes, a Lewis base is best understood as a two-electron donor. Examples of Lewis base moieties include uncharged compounds such as alcohols, thiols, and amines, as well as charged moieties such as alkoxides, thiolates, carbanions, and various other organic anions. In a particular example, a Lewis base may consist of a single atom such as an oxide.

[0219] The term "coordination" or "coordinating" refers to the interaction of a ligand with a metal cation. The term "diagnostic agent" or "diagnostic agents" in the context of the present invention relates to diagnostic-type agents. Non-limiting examples are radioactive nucleotides, fluorescent moieties and enzyme-active moieties that can be detected later using positron emission tomography (PET) or single photon emission computed tomography (SPECT) imaging or other methods known to those skilled in the art. Diagnostic agents also include antibodies that contain a radioactive nucleotide, fluorophore or enzyme bound thereto.

[0220] The term "pharmaceutical" relates to any agent or prophylactic agent, including but not limited to small molecules, biopharmaceuticals (e.g., antibodies). Preferred pharmaceuticals are antibodies. The present invention is demonstrated by the following drawings and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0221]

Figure 1

Figure 2-1

Figure 2-2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7-1

Figure 7-2

Figure 8-1

Figure 8-2

Figure 9

Figure 10-1

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Figure 11-1

Figure 11-2

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Figure 14-2

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Figure 16-3

Figure 17

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Figure 19-1

Figure 19-2

Figure 20

Figure 21

Figure 22

Figure 23

Example

[0222] Example 1: Comparison of the chemical stability of [[Co(II)(NTA)(His 6 -GFP)]] and [[Co(III)(NTA)(His 6 -GFP)]] complexes [[Co(II)(NTA)(His 6 -GFP)]] and [[Co(III)(NTA)(His 6 -GFP)]]-immobilized bead aliquots were incubated with a strong chelator or a widely used reducing agent combined with 250 mM imidazole to show that Co 3+ -based complexes are generally superior in chemical stability to Ni 2+ - or Co 2+ -based complexes.

[0223] His 6 -GFP (SEQ ID NO: 14) was expressed in Escherichia coli BL21(DE3) using the plasmid pET His 6 GFP TEV LIC (Addgene #29663) (Pedelacq et al., 2006), and Ni 2+Purified by -NTA beads.

[0224] Ni 2+ -NTA agarose resin (Novagen) was washed three times with 1) 9 volumes of beads of ddH 2 O, 2) 3 volumes of beads of 0.1 M EDTA pH 7.5, 3) 9 volumes of beads of buffer A (50 mM Tris-HCl pH 7.4, 300 mM NaCl), 4) 1.5 volumes of beads of 0.1 M CoCl 2 ·6H 2 O, 5) 9 volumes of beads of buffer B (buffer A + 250 mM imidazole), and 6) 9 volumes of beads of buffer A three times. Finally, His 6 -GFP was loaded onto the beads by incubating them in 1 volume of beads of 10 μM His 6 -GFP in buffer A. Between each step, the bead slurry was centrifuged at 300 g for 1 minute and the supernatant was decanted. To obtain the [Co(III)(NTA)(His 6 -GFP)] complex, the beads with His 2+ -GFP immobilized on Co 6 -NTA were incubated at room temperature for 1 hour in buffer A containing 20 mM H 2 O 2 (beads to be used later as a control containing Co 2+ were incubated in buffer A without H 2 O 2 ). Subsequently, the beads were washed several times with buffer A, then the beads were resuspended in 2 volumes of beads of buffer A and aliquoted into 150 μl for the stability experiment. Finally, 50 μl of each test reagent (final concentration: chelator: 250 mM imidazole, 25 mM NTA, 25 mM EDTA; reducing agent (cysteamine, DTT, TCEP, ascorbic acid ion): 1 mM with 250 mM imidazole added) was added to the aliquot. After incubation at room temperature for 1 hour, 100 μl of the supernatant was used to measure GFP fluorescence (λ ex = 480 nm, λ em=510 nm) were analyzed. All experiments were performed in duplicate.

[0225] As shown in Figure 1, His 6 -GFP when bound to Co 3+ at the center, very little eluted protein was observed upon incubation with the chelators or reducing agents tested. In contrast, His 2+ -GFP bound to Co 6 beads was completely eluted under the same conditions. Thus, the [Co(III)(NTA)(His 6 -GFP)] complex is inert to both disruption by strong chelators and reduction to Co 2+ both.

[0226] Example 2: Oxidation of fluorophore by hydrogen peroxide As used in the method described in Example 1 and previously described in the prior art (see Wegner and Spatz, 2013), Co 2+ to Co 3+ for oxidation with H 2 O 2 not only harms the bound protein but can also negatively affect the function of NTA conjugates such as labels or carriers. For example, the fluorescence of some fluorophores can decrease upon oxidation by H 2 O 2 as shown below.

[0227] Fluorophore conjugates were diluted in phosphate-buffered saline (PBS) (Thermo; 18912014) (final concentration: 5 μg / ml fluorescein (Riedel de Haen; 28802); 185 μg / ml Alexa488-conjugated antibody (Invitrogen; A11039); 9 μg / ml FITC-conjugated antibody (Thermo; MA1-81891); 5 μm atto488-conjugated Ni 2+ -NTA (Sigma; 39625)), and 100 μl of each fluorophore solution was treated with 0.05% H 2 O 2After incubating for about 21 hours together, in a black 96-well plate, 1% H 2 O 2 Another incubation was carried out separately for 22 hours. Fluorescence intensity (λ ex = 490 nm, λ em = 535 nm) was measured on a plate reader (TECAN; Spark) every 15 minutes (0.05% H 2 O 2 ) or every 30 minutes (1% H 2 O 2 ).

[0228] The fluorescence measurement values shown in Figure 3 indicate that all the analyzed fluorophores showed a significant decrease in fluorescence intensity during incubation with H 2 O 2 . Example 3: Proteolysis and His-tag cleavage during cobalt oxidation by H 2 O 2 The use of H 2+ to oxidize Co 3+ to Co 2 O 2 as used in the method described in Example 1 and previously described by Wegner and Spatz (Wegner and Spatz, 2013) can spontaneously induce a Fenton-like reaction (Hanna, Kadiiska et al., 1992) that can result in proteolysis and cleavage of histidine residues (Davies, 1987, Stadtman, 1990), as shown in anti-His-tag Western blots.

[0229] The fluorescent protein PercevalHR (SEQ ID NO: 15) was expressed in E. coli DH5α using the plasmid pRsetB-PercevalHR (Addgene #49081) (Tantama et al., 2013) and purified by Ni 2+ -NTA column as described by (Tantama et al., 2013).

[0230] 3.3 μM of His 7 ​The tagged PercevalHR protein was mixed with 33 μM CoCl 2 ·6H 2 O in protein buffer (50 mM Tris pH 7.4, 150 mM NaCl) and incubated for 2 minutes at room temperature. Subsequently, 20 mM H 2 O 2 was added and the mixture was incubated for 1.5 hours at 21 °C. For control samples without cobalt and / or H 2 O 2 , an equal volume of protein buffer was used. Finally, the reaction was quenched with 33 mM EDTA pH 8.0.

[0231] For Western blot analysis, protein samples were mixed with SDS sample buffer (25 mM Tris-HCl pH 6.8, 192 mM glycine, 0.1% (w / v) SDS, 0.002% (w / v) bromophenol blue, 100 mM DTT (final concentration)), denatured at 70 °C for 10 minutes, and 84 pmol of protein was loaded onto an SDS-PAGE gel (7% (w / v) acrylamide-bisacrylamide (37.5:1), 375 mM Tris-HCl pH 8.8, 0.1% (w / v) SDS, 0.1 (w / v) ammonium persulfate, 0.1% (v / v) TEMED; electrophoresis conditions: constant at 120 V, Laemmli electrophoresis buffer (25 mM Tris-HCl pH 8.8, 192 mM glycine, 0.1% (w / v) SDS)). After protein separation, the protein was blotted onto a nitrocellulose membrane (Whatman, 10401196), and the membrane was washed with TBS-T (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% (v / v) Tween 20) for 5 minutes at room temperature and then blocked with 5% (w / v) bovine serum albumin in TBS-T for 1 hour at room temperature. Finally, the membrane was incubated with 200 ng / ml horseradish peroxidase-labeled anti-His tag antibody (clone H-3) (Santa Cruz, sc-8036HRP), washed 3 times with TBS-T for 10 minutes each, and incubated in a luminol-based enhanced chemiluminescence horseradish peroxidase (HRP) substrate solution (Thermo, 34076) for 5 minutes at room temperature. The chemiluminescence signal from the His-tagged protein was detected using a LAS3000 system (FUJIFILM).

[0232] The Western blot in Figure 4 shows possible proteolysis and spontaneous His-tag cleavage during incubation of the protein with cobalt combined with H 2 O 2 and is due to a smear and not very strong bands.

[0233] Example 4: Synthesis of [Co(III)(NTA)(CO 3 )] 2- Complex H 2O 2 To completely avoid the use of [Co(III)(NTA)(His-protein)] complex, Co(III) carbonate ions (e.g., Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O and K 3 [Co(III)(CO 3 ) 3 ·3H 2 O) were developed. The complex formation process is schematically illustrated in Figure 2C. In the first step, Co(III) salt is incubated with NTA to form [Co(III)(NTA)CO 3 2- . Proton nuclear magnetic resonance spectroscopy ( 1 H-NMR) was applied to show the success of the formation of [Co(III)(NTA)CO 3 2- complex.

[0234] Synthesis of Sodium Tris-Carbonatocobalt(III) Trihydrate Sodium tris-carbonatocobalt(III) trihydrate (Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O) was synthesized as described by Bauer and Drinkard (Bauer and Drinkard, 1960). Briefly, a mixture of 0.1 mol (29.1 g) of Co(II)(NO 2 )·6H 3 O (Sigma; 1.02554) in 50 ml of ddH 2 O and 10 ml of 30% hydrogen peroxide (Riedel-de Haen; 18312) was added to 50 ml of ddH 2 ​​A solution of 0.5 mol (= 42.0 g) of sodium bicarbonate (Merck; 1.06329) in ice-cooled slurry was added dropwise with stirring. The mixture was incubated on ice for 1 hour with continuous stirring. Subsequently, the olive-colored product was filtered and washed three times with each of cold water, absolute ethanol, and dry ether. Finally, the product was dried under reduced pressure overnight and stored at -20 °C in a nitrogen atmosphere.

[0235] Synthesis of potassium tris-carbonatocobalt(III) trihydrate Potassium tris-carbonatocobalt(III) trihydrate (K 3 [Co(III)(CO 3 ) 3 ·3H 2 O) was synthesized in solution as described by Shibata (adapted from Shibata, 1983; Mori et al., 1956). Briefly, a mixture of 0.1 mol (24 g) of Co(II)Cl 2 ·6H 2 O (Honeywell; 255599) in 24 ml of ddH 2 O and 40 ml of 30% hydrogen peroxide was added dropwise with stirring to an ice-cooled slurry of 0.7 mol (70 g) of potassium bicarbonate (Honeywell; 237205) in 70 ml of ddH 2 O. Subsequently, the resulting green solution was filtered by suction and used directly for the following experiments.

[0236] [Co(III)(NTA)(CO 3 )] 2- Preparation of the complex To produce the [Co(III)(NTA)(CO 3 )] 2- complex from the sodium salt, 580 μmol (210 mg) of Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O was added to ddH 2It was added to 2 ml of 1 M sodium bicarbonate and 2 M trisodium nitrilotriacetate (Sigma; N0253) in O, and the slurry was sonicated for 30 minutes. After incubation at 70 °C for 72 hours, 3 ml of 1 M sodium bicarbonate was added to the newly pinkish slurry, and the mixture was sonicated at 70 °C for 2 hours. Subsequently, the mauve supernatant was submitted for NMR analysis. For the complex produced from the potassium salt, K 3 [Co(III)(CO 3 ) 3 ·3H 2 O was synthesized in solution from 0.1 mol of CoCl 2 ·6H 2 O (see above), and then 0.1 mol of trisodium nitrilotriacetate (25.7 g) was added with 60 ml of ddH 2 O as described in (Shibata, 1983). After incubation for 3 hours under continuous stirring at 60 °C, the resulting mauve solution was filtered, and the pH was adjusted to pH 7.3 with aqueous acetic acid. Finally, the solution was incubated at 4 °C overnight, removed from the white precipitate, and submitted for NMR analysis. To obtain the complex [Co(II)(NTA)(D 2 O)]-, a mixture of 5 mM nitrilotriacetic acid (Sigma; 72559) and 5 mM CoCl 2 ·6H 2 O was produced from a stock solution in D 2 O and incubated at room temperature for 15 minutes before NMR measurement. A small amount of 10 M NaOH was added to the corresponding stock solution to dissolve nitrilotriacetic acid in D 2 O (Carl Roth; HN81.3). For the measurement of pure NTA, a 5 mM solution in D 2 O was prepared from the stock solution prepared as described above. 1 1H-NMR spectra were measured on a Jeol ECZ400S spectrometer at a resonance frequency of 400 MHz at room temperature. To improve the signal-to-noise ratio, up to 32 signals were added before Fourier transform. The intensity of the resulting spectra was normalized at 7 ppm, and all ppm values were adjusted to the water peak at 4.70 ppm.

[0237] The H of pure NTA and its complexes with cobalt and water or carbonate ion ligands 1 - By measuring the NMR spectrum, [Co(III)(NTA)CO 3 2- The formation of the complex was confirmed (Figure 5). The hydrogen spectrum of pure NTA showed a peak at about 3.6 ppm in addition to the ubiquitous water peak at 4.70 ppm, which was consistent with the simulation (calculated value 3.57 ppm) performed by the software NMR Predict (https: / / www.nmrdb.org / new_predictor / index.shtml?v=v2.103.0) (Banfi and Patiny, 2008; Castillo et al., 2011; Aires-de-Sousa et al., 2002). When NTA forms a complex with cobalt and water (here, D 1 - For NMR measurements, heavy water (D 2 O) is used) ([Co(II)(NTA)(D 2 O) 2 - ; Figure 5B) or a complex with carbonate ion ([Co(III)(NTA)(CO 3 )] 2- ; Figures C+D), the original peak at about 3.6 ppm shifted to about 3.8 ppm for D 2 O as a ligand and to about 1.9 ppm for carbonate ion. Similar peak shifts were observed regardless of whether sodium or potassium cobalt(III) carbonate ion was used for the production of the complex. The shift of the NTA peak indicates that the magnetic environment of the hydrogen atoms in NTA changes because additional atoms come very close to the hydrogen atoms during the complexation process. Therefore, the observation of different peak signals for the complex produced using cobalt(III) carbonate ion strongly indicates the presence of carbonate ion ligand instead of water ligand in the final cobalt-NTA complex.

[0238] Example 5: [Co(III)(NTA)(His 3 )] formed by [Co(III)(NTA)(CO 6 ​​-Chemical stability of the [Co(III)(NTA)(His-PercevalHR)] complex The complex of NTA linked to agarose beads with [Co(III)(NTA)(CO 3 )] 2- was incubated with His-tagged protein PercevalHR to form [Co(III)(NTA)(His 6 -PercevalHR)] immobilized on agarose beads. Subsequently, the chemical stability of the [Co(III)(NTA)(His 6 -PercevalHR)] complex was evaluated. As a control, a conventional Ni 2+ NTA-based matrix was used.

[0239] The fluorescent protein PercevalHR (SEQ ID NO: 15) was expressed in E. coli DH5α using the plasmid pRsetB-PercevalHR (Addgene #49081) (Tantama, Martinez-Francois et al., 2013) and purified by Ni 2+ -NTA column as described in (Tantama, Martinez-Francois et al., 2013).

[0240] NTA agarose resin (Qiagen, 1022963) was washed three times with 1) 10 bead volumes of ddH 2 O, 2) 3 bead volumes of 100 mM EDTA pH 7.5, and 3) 10 bead volumes of ddH 2 O, and then 10 bead volumes of 1 mM Na 3 in 1 M NaHCO 3 [CO(III)(CO 3 ) 3 ·3H 2 O or 1 mM Ni(II)SO 4 was added. After incubation for 48 h in a thermoshaker at 23 °C with shaking at 1100 rpm, the beads were washed twice with 10 bead volumes of ddH 2 O and once with 10 bead volumes of protein buffer (50 mM Tris pH 7.4, 150 mM NaCl). Finally, 1 bead volume of 10 μM His in protein buffer6 - PercevalHR (Accession No. 15) was added and incubated at 4 °C for 48 h with shaking at 1100 rpm on a thermoshaker to bind the protein to the matrix. After washing twice with 10-fold bead volume of protein buffer, 3-fold bead volume of protein buffer was added and 10 μl of the bead slurry was analyzed for PercevalHR fluorescence (λ ex = 500 nm, λ em = 545 nm) using a plate reader (TECAN, infinite 2000). To test the stability of the complex, after adding 10-fold bead volume of either 250 mM imidazole in protein buffer or protein buffer alone, the beads were removed by washing with 10-fold bead volume of protein buffer. Finally, the beads were resuspended in 3-fold bead volume of protein buffer and 10 μl of the bead slurry was analyzed for remaining fluorescence. All experiments were performed in triplicate.

[0241] As shown in Figure 6, the [Ni(II)(NTA)(His 6 - PercevalHR)] complex used as a control showed low stability against chelator imidazole treatment. In contrast, the [Co(III)(NTA)(His 6 - PercevalHR)] complex and the [Co(III)(NTA)(CO 3 )] pre-complex formed by cobalt(III) carbonate ion showed strong stability against imidazole. The measured stability of the complex was similar to that of the [Co(III)(NTA)(His 6 - PercevalHR)] complex produced using an indirect oxidation method (the one used in Example 1). Therefore, this data surprisingly confirms that [Co(III)(NTA)(His 6 - PercevalHR)] can be formed with high efficiency without an oxidation step by using the [Co(III)(NTA)(CO 3 )] 2- pre-complex.

[0242] Example 6: His 6 -GFP's [Co(III)(NTA)CO 3 2- Binding rate to Example 4 demonstrates that [Co(III)(NTA)CO 3 2- complex can be formed. Furthermore, Example 5 synthesizes [Co(III)(NTA)CO 3 2- complex linked to beads by NTA and surprisingly shows that this can be used to form [Co(III)(NTA)(His - protein)] complex on the beads. The inventors speculated that carbonate ion as a ligand at the cobalt(III) center might facilitate the formation of [CoIII(NTA)(His - protein)] complex. To confirm this finding, the binding efficiency of His 6 -GFP to [Co(III)(NTA)(H 2 O) 2 and [Co(III)(NTA)(CO 3 )] 2- was directly compared.

[0243] Functionalization of NTA - agarose beads NTA - agarose resin (Qiagen, 1022963) was washed with 1) 10 - fold bead volume of ddH 2 O, 2) 10 - fold bead volume of 100 mM EDTA pH 7.5, and 3) for [Co(II)(NTA)(H 2 O) 2 - and [Co(III)(NTA)(H 2 O) 2 complexes, twice with 10 - fold bead volume of ddH 2 O and once with 6.7 - fold bead volume of ddH 2 O, or for [Co(III)(NTA)(CO 3 )] 2- complex, twice with 10 - fold bead volume of ddH 2 O and once with 6.7 - fold bead volume of 1 M NaHCO 3 . Subsequently, 8.7 - fold bead volume of ddH​​​​2 1 mM Co(II)Cl in O 2 ·6H 2 O([Co(II)(NTA)(H 2 O) 2 - and [Co(III)(NTA)(H 2 O) 2 complex (or 1 M NaHCO 3 1 mM Na in 3 [CO(III)(CO 3 ) 3 ·3H 2 O([Co(III)(NTA)(CO 3 )] 2- complex) was added. After incubating for 18 h in a thermoshaker at 1100 rpm and 25 °C, the beads were washed with 6.7 times the bead volume of protein buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl) ([Co(II)(NTA)(H 2 O) 2 - , [Co(III)(NTA)(H 2 O) 2 complex and [Co(III)(NTA)(CO 3 )] 2- complex (for 1 sample of each) or 1 M NaHCO 3 (「1 M NaHCO 3 in [Co(III)(NTA)(CO 3 )]」 in Fig. 7, for 1 sample of [Co(III)(NTA)(CO 3 )] 2- complex). For the final sample containing the [Co(III)(NTA)(H 2 O) 2 complex, the second wash was performed with 6.7 times the bead volume of 20 mM H 2 O in ddH 2 O 2 and incubated on a thermoshaker (1100 rpm) at 25 °C for 1 h.

[0244] His 6 -GFP binding rate to functionalized NTA agarose beads​​ The produced beads were incubated with 20 μM His in a protein buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl) at 3.3 times the bead volume 6 -GFP (SEQ ID NO: 14), and incubated at 4 °C with shaking at 1100 rpm on a thermoshaker. [Co(III)(NTA)(CO 3 )] 2- For one sample of [Co(III)(NTA)(CO 3 )] (referred to as "[Co(III)(NTA)(CO 3 )]" in Fig. 7), 1 M NaHCO 3 was used instead of the protein buffer. The fraction of unbound protein was analyzed by measuring the fluorescence intensity of 100 μl of the supernatant (λ ex = 490 nm, λ em = 535 nm) in a plate reader (TECAN, Spark) at various time points.

[0245] [Co(III)(NTA)(His 6 -GFP)] complex chemical stability After the incubation of the functionalized beads with His 6 -GFP (SEQ ID NO: 14) as described above was completed, the amount of protein bound to the beads was analyzed. For this purpose, the beads were washed three times with 6.7 times the bead volume of the protein buffer, resuspended in 6.7 times the bead volume of the protein buffer, and finally, 100 μl of the bead slurry was analyzed for GFP fluorescence (λ ex = 490 nm, λ em = 535 nm) using a plate reader (TECAN, Spark). To test the stability of the complex, 1.7 times the bead volume of 1.25 M imidazole in the protein buffer (final concentration 250 mM) was added, the beads were washed three times with 6.7 times the bead volume of the protein buffer, and then incubated at 25 °C for 10 minutes at 1100 rpm on a thermoshaker. Finally, the beads were resuspended in 6.7 times the bead volume of the protein buffer, and 100 μl of the bead slurry was analyzed for the remaining fluorescence. All experiments were performed in triplicate.

[0246] The results of these experiments clearly show that the Co complex with associated carbonate ions binds to the protein significantly faster than the complex with water molecules bound to Co (Figure 7A). Consistently, beads functionalized with the [Co(III)(NTA)(CO 3+ complex bind significantly more protein than beads pre-loaded with [Co(III)(NTA)(H 3+ O)] (Figure 7B). Furthermore, Figure 7B again confirms that the [Co(III)(NTA)(His 3 )] 2- complex exhibits strong stability towards the chelator (here imidazole). 2 O) 2 complex binds significantly more protein than beads pre-loaded with [Co(III)(NTA)(H 6 -GFP)] complex. Furthermore, Figure 7B again confirms that the [Co(III)(NTA)(His

[0247] The experiments further show that washing the beads bound with [Co(III)(NTA)(CO 3 )] with 1 M NaHCO 3 )] 2- before protein binding makes His 6 -GFP binding even easier. Although not bound by theory, the presence of HCO 3 - and / or CO 3 2- in the buffer is thought to prevent the slower enhancing conversion of the [Co(III)(NTA)(CO 3 )] 2- complex to the [Co(III)(NTA)(H 2 O) 2 complex. In the experiments of the present invention, His-protein binding was also performed in the presence of 1 M NaHCO 3 for the "[Co(III)(NTA)(CO 3 )]" sample (see above), but the presence of 1 M NaHCO 3 during protein binding is assumed to contribute, if at all, only to a very small extent. This is because during protein binding, the carbonate ion ligand to Co 3 3+ ​It is desirable to dissociate from the complex, because it can be hindered rather than facilitated by the presence of 1M NaHCO 3 .

[0248] Example 7: Binding rate of His-tagged protein to [Co(III)(NTA)(CO 3 )] 2- in various buffer systems It was determined whether the formation of the [Co(III)(NTA)(His-protein)] complex could be improved by changing the composition of the reaction buffer.

[0249] [Co(III)(NTA)(CO 3 )] 2- Functionalized beads were slightly modified and prepared as described in Example 6: bead washing steps 1)-3) were performed with a 5-fold bead volume of the corresponding solution. In 3), the second and third washing steps were performed with 1M NaHCO 3 . The metal was loaded at 6.5-fold bead volume. After metal binding, the beads were washed with 5-fold bead volume of 1M NaHCO 3 .

[0250] Also, the incubation with His-tagged GFP (SEQ ID NO: 14) was performed as described in Example 6, except that a 5-fold bead volume of 10 μM protein solution was used and the protein buffer was adapted by replacing 50 mM Tris pH 7.4 with 50 mM Bis-Tris pH 6.0, 50 mM HEPES pH 7.0, 50 mM MES pH 6.0 or 50 mM Tris-HCl pH 7.5. Additionally, the first 24 hours of protein incubation were performed at room temperature instead of 4°C.

[0251] Experiments have shown that when the Tris-based buffer is replaced by a non-coordinating buffer such as a HEPES or, in particular, a MES or BisTris-based buffer, the reaction rate and efficiency can be significantly improved. After 24 hours of incubation, 95% of the total protein was immobilized on the beads when using MES and BisTris-based buffers, compared to 75% for the Tris-based buffer (Figure 8A). Furthermore, a comparison of the results in Figures 7A and 8A shows an increase in temperature during the first 24 hours of protein incubation and / or washing with 1M NaHCO 3 facilitates His-protein coordination. Already after 3 hours of incubation, about 80% of the protein could be bound to the beads when using MES or BisTris-based buffers. In contrast, in the Tris-based buffer, less than 50% of the protein was immobilized on the beads. It has been shown that His-tagged proteins can be coordinated with the [Co(III)(NTA)(CO 3 )] 2- complex with very high efficiency on a reasonable time scale.

[0252] It should be noted that the pH values shown for the various protein buffers indicate the pH before adding the solution to the beads. Due to the residual NaHCO 3 from the second wash step remaining on the beads, the pH in the total sample was 8.5 - 9 during incubation with the protein (verified by pH measurement). Therefore, due to the very similar pH values in all buffer solutions, the experiments clearly show that the buffer substance itself has an impact on His-protein binding. The observed better performance of the Good buffers MES and BisTris compared to the Tris buffer suggests that the use of Good buffers that cannot form a complex with Co3 + is advantageous compared to the use of buffers such as the Tris buffer that can form such a complex.

[0253] Example 8: [Co(III)(NTA)(CO 3 )] 2-UV-Vis analysis Example 4 demonstrates, by NMR, that the complex 3 )] 2- can be formed. In the following examples, another technique, namely absorbance measurement by UV-Vis, is used to demonstrate the formation of the complex 3 2- .

[0254] [Co(III)(NTA)(CO 3 )] 2- Complex preparation: [Co(III)(NTA)(CO 3 )] 2- To produce the complex, the salt is dissolved in solution using 1 hour of sonication, followed by a filtration step through a 0.22 μm filter to prepare a solution of 1 mM Na 3 in 1 M NaHCO 3 [CO(III)(CO 3 ) 3 ·3H 2 O. Subsequently, a mixture of 0.95 mM Na 3 in 1 M NaHCO 3 [CO(III)(CO 3 ) 3 ·3H 2 O and 0.95 mM trisodium NTA salt (Sigma; N0253) dissolved in ddH 2 O is prepared in 1 M NaHCO 3 . After incubation at 25 °C for 1 hour, the visible light absorption of the bright violet solution is measured in a 1 cm cuvette (Brand; 759150) on a UV-Vis-NIR spectrophotometer (Cary5000).

[0255] [Co(III)(NTA)(H 2 O) 2 Complex preparation: [Co(III)(NTA)(H 2 O) 2 To produce the complex, 0.95 mM Co(II)Cl 2 ·6H​2 O, 0.95 mM trisodium NTA salt (Sigma; N0253), and 20 mM H 2 O 2 The mixture is prepared in ddH 2 O. After incubation at 25 °C for 24 h, the visible light absorption of the bright violet solution was measured in a 1 cm cuvette (Brand; 759150) on a UV-Vis-NIR spectrophotometer (Cary5000) against a blank using ddH 2 O.

[0256] The results in Figure 9 are, respectively, from 402 nm for the [Co(III)(NTA)(H 2 O) 2 complex, to 390 nm for the [Co(III)(NTA)(CO 3 )] 2- complex, or a shift of two peaks from 567 nm to 573 nm, clearly indicating the coordination of carbonate ion as a metal-binding ligand in the [Co(III)(NTA)(CO 3 )] 2- complex.

[0257] Example 9: Coordination of His 3 )] 2- to GFP and stability of the formed [Co(III)(NTA)(His-GFP)] complex after various incubation times and temperatures of Na 6 [Co(III)(CO 3 ) 3 ·3H 3 O with NTA 2 Example 4 demonstrated that the [Co(III)(NTA)CO 3 ) 2- complex can be formed by incubation of NTA with Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O. In the following examples, the resulting [Co(III)(NTA)(CO 3 ) 2-His to the complex 6 -GFP coordination, Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O and the effects of various incubation times and temperatures of NTA and the chemical stability of the final [Co(III)(NTA)(His-GFP)] complex against imidazole are examined.

[0258] Functionalization of NTA agarose beads Agarose beads (Qiagen; 1022963) functionalized with NTA were washed 1) with 27 volumes of ddH 2 O, 2) with 27 volumes of 100 mM EDTA pH 8.0, and 3) once with 27 volumes of ddH 2 O and twice with 27 volumes of 1 M NaHCO 3 . Subsequently, 16 volumes of 1 mM Na 3 in 1 M NaHCO 3 [Co(III)(CO 3 ) 3 ·3H 2 O was added, and the beads were incubated at 4 °C, 25 °C, or 70 °C for 1 minute, 10 minutes, 30 minutes, 1 hour, 24 hours, or 48 hours at 1400 rpm in a thermoshaker as indicated. After incubation, the beads were washed twice with 16 volumes of 1 M NaHCO 3 .

[0259] Immobilization of His 3 )] 2- -GFP to the functionalized [Co(III)(NTA)(CO 6 agarose beads The beads produced were incubated with 10 μM His 6 -GFP (SEQ ID NO: 14) in 12 volumes of protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) on a thermoshaker with shaking at 1400 rpm for 48 hours at 25 °C. The functionalized beads and His 6After incubation with -GFP (SEQ ID NO: 14), the beads were washed once with 16 volumes of protein buffer and resuspended in 16 volumes of protein buffer.

[0260] [Co(III)(NTA)(His 6 -GFP)] complex chemical stability After performing the [Co(III)(NTA)(His-GFP)] complex formation process shown, the amount of protein bound to the beads was analyzed with and without using chemical stress. For this, the beads were divided into two parts (each 7.2 volumes of beads), and each was washed once with 250 mM imidazole in 17.8 volumes of protein buffer or protein binding buffer. After a final wash with 17.8 volumes of protein buffer, the beads were resuspended in 17.8 volumes of protein buffer. The amount of immobilized protein on 25 μl of bead slurry was determined by the BCA assay (Thermo, 23227) in a microplate based on the manufacturer's instructions. The experiment was performed in triplicate.

[0261] The results of these experiments shown in Figure 10 clearly show a positive correlation between the incubation time of the protein over a long period such as 48 hours, the buffer, and the amount of His-GFP immobilized on the agarose beads after imidazole treatment. For incubation at 70 °C, since the coordination of Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O and NTA is naturally accelerated at high temperature, the reaction is considered to reach its saturation point already after 1 minute. Furthermore, for the incubations performed at 4 °C and 25 °C shown in Figures 10A and B, the incubation time is positively correlated with the complex stability. For incubation at 25 °C, 10 minutes of Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O coordination is accelerated at high temperature, so the reaction is considered to reach its saturation point already after 1 minute. Furthermore, for the incubations performed at 4 °C and 25 °C shown in Figures 10A and B, the incubation time is positively correlated with the complex stability. For incubation at 25 °C, 10 minutes of Na3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation already reaches high complex stability, but for 4 °C incubation, this stability is only reached after 24 h of Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation. Stable complex formation is assumed to be accelerated at higher temperatures.

[0262] Example 10: Rate of His-protein binding to beads functionalized with [Co(III)(NTA)(CO 3 )] 2- at various temperatures In the following example, after various His-protein incubation times and at three different temperatures, protein binding to magnetic agarose beads functionalized with [Co(III)(NTA)(CO 3 )] 2- and the stability of the resulting [Co(III)(NTA)(His-GFP)] complex against imidazole are examined.

[0263] Functionalization of NTA magnetic agarose beads Agarose beads functionalized with NTA (Thermo; 78605) were washed 1) with 26 bead volumes of ddH 2 O, 2) with 26 bead volumes of 100 mM EDTA pH 8.0, 3) twice with 26 bead volumes of ddH 2 O and once with 26 bead volumes of 1 M NaHCO 3 . Subsequently, 160 bead volumes of 1 mM Na 3 in 1 M Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O or for metal-free samples 1 M NaHCO 3was added and the beads were incubated at 25 °C for 48 h on a thermoshaker at 1400 rpm. After incubation, the beads were washed three times with 160 volumes of 1 M NaHCO 3 .

[0264] Immobilization of His 3 -GFP onto functionalized [Co(III)(NTA)(CO 2- magnetic agarose beads 6 The produced beads were incubated with 10 μM His 6 -GFP (SEQ ID NO: 14) in 120 volumes of 50 mM HEPES pH 7.2, 150 mM NaCl at 4, 25 or 37 °C for 1 min, 10 min, 30 min, 1 h, 2 h, 3.5 h or 24 h while shaking at 1400 rpm on a thermoshaker. After incubation of the functionalized beads with His 6 -GFP (SEQ ID NO: 14), the beads were washed once with 160 volumes of protein buffer and finally resuspended in 160 volumes of the corresponding wash buffer.

[0265] [Co(III)(NTA)(His 6 -GFP)] complex chemical stability After performing the [Co(III)(NTA)(His-GFP)] complex formation process shown, the amount of protein bound to the beads was analyzed with and without using chemical stress. For this purpose, the beads were split into two parts and each was washed once with 250 mM imidazole in 178 volumes of protein buffer or protein binding buffer. After the last wash with 178 volumes of protein buffer, the beads were resuspended in 178 volumes of protein buffer. Finally, 10 μl of the bead slurry was analyzed for the amount of immobilized protein by GFP fluorescence (λ ex = 490 nm, λ em = 535 nm) using a plate reader (TECAN, Spark). The experiment was performed in triplicate. ​

[0266] As described in Figure 11A, the amount of protein immobilized on imidazole-treated beads increases with the incubation time with His-GFP and beads functionalized with 3 )] 2- [Co(III)(NTA)(His 6 -GFP)]. Thus, the temperature during incubation does not affect the final yield of the [Co(III)(NTA)(His

[0267] Example 11: Formation of [Co(III)(NTA)(His-GFP)] complex and K 3 )] 2- for forming the complex 3 [Co(III)(CO 3 ) 3 ·3H 2 O using stability Example 4 shows the formation of the [Co(III)(NTA)(CO 3 )] 3 ) 3 ·3H 2 O complex by incubating K 3 )] 2- with NTA. In the following example, the coordination of His-GFP to the [Co(III)(NTA)(CO 3 ) 3 ) 3 ·3H 2 O-produced [Co(III)(NTA)(CO 3 )] 2- complex and the chemical stability of the resulting [Co(III)(NTA)(His-GFP)] complex to imidazole are examined.

[0268] Functionalization of NTA magnetic agarose beads Magnetic agarose beads functionalized with NTA (Thermo; 78605) were prepared by 1) adding ddH2 O, 2) with 100 mM EDTA pH 8.0 at 600-fold bead volume, 3) ddH 2 O once and with 1 M NaHCO 3 twice at 600-fold bead volume. Subsequently, 160-fold bead volume of 1 mM K in 1 M NaHCO3 3 [Co(III)(CO 3 ) 3 ·3H 2 O or for metal-free samples only 1 M NaHCO 3 was added. K 3 [Co(III)(CO 3 ) 3 ·3H 2 O was produced as described in Example 4. Concentration calculations are based on the assumption of 100% reduction efficiency in the synthesis process. Samples as shown were incubated for 10 minutes or 48 hours at 1400 rpm and 25 °C in a thermoshaker. After incubation, the beads were washed three times with 160-fold bead volume of 1 M NaHCO 3 .

[0269] Immobilization of His 6 -GFP onto functionalized NTA magnetic beads The produced beads were incubated with 10 μM His 6 -GFP (SEQ ID NO: 14) in protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) at 120-fold bead volume for 1 hour or 48 hours at 25 °C with shaking at 1400 rpm on a thermoshaker. After incubation of the functionalized beads with His 6 -GFP (SEQ ID NO: 14), the beads were washed once with 160-fold bead volume of protein buffer and resuspended in 160-fold bead volume of protein buffer.

[0270] [Co(III)(NTA)(His 6 -GFP)] complex chemical stability After performing the shown [Co(III)(NTA)(His-GFP)] complex formation process, the amount of protein bound to the beads was analyzed with and without using chemical stress. For this, the beads were divided into two parts (each 72-fold bead volume) and washed once with 250 mM imidazole in 178-fold bead volume of protein buffer or protein binding buffer. After the final wash with 178-fold bead volume of protein buffer, the beads were resuspended in 178-fold bead volume of protein buffer, and 10 μl of the bead slurry was analyzed for the amount of immobilized protein by GFP fluorescence (λ ex = 490 nm, λ em = 535 nm) using a plate reader (TECAN, Spark). The experiment was performed in triplicate.

[0271] The results described in Figure 12 clearly show that His-GFP can coordinate to the [Co(III)(NTA)(CO 3 ) 3 ) 3 ·3H 2 O complex formed by incubation of K 3 with NTA. The resulting [Co(III)(NTA)(His-GFP)] complex shows high chemical stability against imidazole treatment. Since K 2- [Co(III)(CO 3 ) 3 ) 3 ·3H 2 O is soluble at higher concentrations, the use of K 3 [Co(III)(CO 3 ) 3 ·3H 2 O provides the possibility to perform the incubation with the metal binding domain at higher concentrations than Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O.

[0272] Example 12: [Co(III)(NTA)(CO 3 )] 2-Binding of His 6 - Coordination of GFP and stability of the [Co(III)(NTA)(His-GFP)] complex formed in various protein binding buffers Example 7 shows the effect of various buffer substances on the binding rate of His-tagged proteins to 3 )] 2- In the following examples, in various different buffer systems, two different Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation time, protein binding to magnetic agarose beads functionalized with 3 )] 2- and the stability of the resulting [Co(III)(NTA)(His-GFP)] complex against imidazole are examined.

[0273] Functionalization of NTA magnetic agarose beads Agarose beads functionalized with NTA (Thermo; 78605) were treated with 1) ddH 2 O at 182 times the bead volume, 2) 100 mM EDTA pH 8.0 at 182 times the bead volume, and 3) for samples using the Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation time, ddH 2 O at 182 times the bead volume once and 1 M NaHCO 3 at 182 times the bead volume twice, or for samples using the 48-hour Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation time, ddH 2 O at 182 times the bead volume twice and 1 M NaHCO 3 at 182 times the bead volume once, and washed. Subsequently, 160 times the bead volume of 1 mM Na 3 in 1 M NaHCO 3 [Co(III)(CO3 ) 3 ·3H 2 For samples without O or metal, add 1M NaHCO 3 and incubate the beads on a thermoshaker at 1400 rpm for 10 minutes or 48 hours at 25 °C as indicated. After incubation, wash the beads with 160 bead volumes of 1M NaHCO 3 for 10 minutes of Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation time. For samples using 2 or 48 hours of Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation time, wash 3 times.

[0274] Immobilization of His 3 )] 2- -GFP onto functionalized [Co(III)(NTA)(CO 6 magnetic agarose beads Incubate the produced beads with 120 bead volumes of 10 μM His 6 -GFP (SEQ ID NO: 14) in a protein buffer based on Tris, HEPES, MES, MOPS, BisTris, ACES, PIPES, BES, CAPS, TAPS (50 mM buffer pH 7.2, 150 mM NaCl) or PBS on a thermoshaker with shaking at 1400 rpm for 1 minute, 15 minutes, 1 hour or 24 hours at 25 °C as indicated. After incubation of the functionalized beads with His 6 -GFP (SEQ ID NO: 14), wash the beads with 160 bead volumes of 10 minutes of Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation time in protein buffer or for samples using 48 hours of Na 3 [Co(III)(CO 3 )3 ·3H 2 For samples using O / NTA incubation time analysis, they were washed once with the analytical buffer (50 mM HEPES pH 7.2, 150 mM NaCl) and finally resuspended in the corresponding washing buffer at 160 times the bead volume.

[0275] [Co(III)(NTA)(His 6 -GFP)] complex chemical stability After performing the indicated [Co(III)(NTA)(His-GFP)] complex formation process, the amount of protein bound to the beads was analyzed with and without using chemical stress. For this purpose, the beads for samples using 48-hour Na 3 [Co(III)(CO 3 ) 3 ·3H 2 The beads for samples using O / NTA incubation time were divided into two parts (each at 72 times the bead volume) and washed once with 250 mM imidazole in 178 times the bead volume of protein buffer or protein binding buffer. After the final wash with 178 times the bead volume of protein buffer, the beads were resuspended in 178 times the bead volume of protein buffer. The amount of immobilized protein on 10 μl of bead slurry was analyzed by GFP fluorescence (λ ex = 490 nm, λ em = 535 nm) using a plate reader (TECAN, Spark). For samples using 10-minute Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation time, the beads were analyzed before and after a washing process using 250 mM imidazole in 160 times the bead volume of protein binding buffer followed by 160 times the bead volume of protein binding buffer. The experiment was performed in triplicate.

[0276] The results clearly showed that in all buffer systems, the his-tagged protein could be immobilized on the beads in a chemically stable manner. Thereby, the proportion of stable complexes was 10-minute Na3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation time samples (Figure 13), compared to 48 hours of Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation time samples (Figure 14), is higher. For all buffer systems and Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation time, the amount of protein immobilized after imidazole treatment increases with protein incubation time. Thereby, different buffer systems have a greater impact on the reaction rate in samples using 10 minutes of Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation time compared to samples using 48 hours of Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation time. The use of HEPES, MES, MOPS and PIPES buffers on samples using 48 hours of Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation time is expected to result in an increase in the amount of protein stably immobilized on the beads at short protein incubation times (Figure 14B - D). For longer incubation times, HEPES, MES, MOPS or ACES are expected to be good buffer choices (Figure 14E).

[0277] Example 13: [Co(III)(NTA)(CO 3 )] 2- to His 6Effect of pH on the coordination of -GFP and stability of the formed [Co(III)(NTA)(His-GFP)] complex Example 7 shows the effect of the pH of the protein binding buffer on the binding rate of His-tagged protein to 3 ) 2- . In the following examples, protein binding to magnetic agarose beads functionalized with 3 ) 2- in two protein binding buffer systems each having five different pH values and the stability of the resulting [Co(III)(NTA)(His-GFP)] complex to imidazole are examined.

[0278] Functionalization of NTA magnetic agarose beads Agarose beads (Thermo; 78605) functionalized with NTA were washed with 1) 26 volumes of ddH 2 O, 2) 26 volumes of 100 mM EDTA pH 8.0, and 3) 26 volumes of ddH 2 O twice and 26 volumes of 1 M NaHCO 3 once. Subsequently, 160 volumes of 1 mM Na 3 in 1 M NaHCO 3 [Co(III)(CO 3 ) 3 ·3H 2 O or 1 M NaHCO 3 was added for samples without metal, and the beads were incubated at 25 °C for 48 h in a thermoshaker at 1400 rpm. After incubation, the beads were washed three times with 160 volumes of 1 M NaHCO 3 for the sample using the 48 h Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA incubation time.

[0279] Functionalized [Co(III)(NTA)(CO 3 )]2- Immobilization of His 6 -GFP onto Magnetic Agarose Beads The produced beads were mixed with 120-fold bead volume of 10 μM His 6 -GFP (SEQ ID NO: 14) in protein buffers (50 mM buffer, 150 mM NaCl) based on BisTris with pH 5.5, 6.0, 6.5, 7.0 or 7.5 and based on HEPES with pH 7.5, 8.0, 8.5, 9.0 or 9.5, and incubated at 25 °C for 1 minute, 15 minutes, 1 hour or 24 hours as shown, with shaking at 1400 rpm on a thermoshaker. After incubation of the functionalized beads with His 6 -GFP (SEQ ID NO: 14), the beads were washed once with 160-fold bead volume of assay buffer (50 mM HEPES pH 7.2, 150 mM NaCl) and finally resuspended in 160-fold bead volume of assay buffer.

[0280] [Co(III)(NTA)(His 6 -GFP)] Complex Chemical Stability After performing the [Co(III)(NTA)(His-GFP)] complex formation process shown, the amount of protein bound to the beads was analyzed with and without using chemical stress. For this purpose, the beads were divided into two parts (each 72-fold bead volume) and washed once with 250 mM imidazole in 178-fold bead volume of protein buffer or protein binding buffer. After final washing with 178-fold bead volume of protein buffer, the beads were resuspended in 178-fold bead volume of protein buffer. 10 μl of the bead slurry was analyzed for the amount of immobilized protein by GFP fluorescence (λ ex = 490 nm, λ em = 535 nm) using a plate reader (TECAN, Spark). The experiment was performed in triplicate.

[0281] The results clearly showed that the his-tagged proteins could be immobilized on the beads in a chemically stable manner at all pH values. Thus, a high percentage of His-GFP could be immobilized in a chemically stable manner, and the amount of immobilized protein before and after imidazole treatment increases with protein incubation time (Figure 15A). The complexation efficiency of the final [Co(III)(NTA)(His-GFP)] complex increases dramatically with the protein binding buffer at lower pH values ​​(Figure 15B). Thus, we could observe efficiency differences that are not only based on pH, but also originate from the buffer system (BisTris vs. HEPES at pH 7.5).

[0282] Example 14: [Co(III)(NTA)(CO) 3 ] 2- or [Co(III)(IDA)(CO) 3 ] - Immobilization of various proteins via His-tags or histidine-rich regions onto beads functionalized with Some examples include [Co(III)(NTA)(CO) 3 ] 2- or [Co(III)(IDA)(CO) 3 ] - By His 6 The immobilization of -GFP to beads is shown. In the following examples, the immobilization to beads of various his-tagged proteins and antibodies coordinated with their histidine-rich regions in the his Fc portion is examined. Furthermore, the function of the immobilized enzyme (sortase) and antibody (GFP binding to immobilized anti-GFP IgG1) is probed.

[0283] Magnetic agarose beads functionalized with NTA (Thermo; 78605) or magnetic beads functionalized with IDA (Cube Biotech; 30805) were incubated for 1) 33x bead volume in ddHO. 2 2) 33x bead volumes of 100 mM EDTA pH 8.0; 3) 33x bead volumes of ddHO. 2For samples that are O once and metal-free and contain a cobalt center, wash with 1M NaHCO of 33 times the bead volume 3 twice or for samples containing a nickel center, wash with ddH 2 O three times. Subsequently, for samples that are 160 times the bead volume of 1M NaHCO 3 containing 1 mM Na in 3 [Co(III)(CO 3 ) 3 ·3H 2 O or for metal-free samples, 1M NaHCO 3 or for samples containing a nickel center, ddH 2 Add 1 mM NiSO in O 4 and incubate the beads in a thermoshaker at 25 °C for 10 minutes at 1400 rpm. After incubation, wash the beads three times with 160 times the bead volume of 1M NaHCO 3 or for samples containing a nickel center, ddH 2 O.

[0284] Immobilization of proteins on functionalized [Co(III)(NTA / IDA)(CO 3 )] 2- Magnetic agarose beads The produced beads were mixed with proteins (10 μM His-GFP (SEQ ID NO: 14); 10 μM His-Protein A (Abcam; ab52953); 10 μM His-Sortase A (SEQ ID NO: 16); 1 μM His-human serum albumin (antikoerperonline; ABIN2181228); 0.2 μM anti-GFP mouse IgG1 (Biolegend; 902605)) in a protein binding buffer (50 mM HEPES pH 7.2, 150 mM NaCl) at 120 times the bead volume and incubated at 25 °C for 48 h or for 30 min for the IDA sample while shaking on a thermoshaker at 1400 rpm. After incubation of the functionalized beads with the proteins, samples of the protein supernatant were stored for subsequent analysis by SDS-PAGE, the beads were washed once with 160 times the bead volume of protein buffer and finally resuspended in 160 times the bead volume of analysis buffer or, for the antibody samples, GFP incubation was continued as described in another section.

[0285] [Co(III)(NTA / IDA)(Protein)] Complex Chemical Stability After performing the indicated [Co(III)(NTA / IDA)(protein)] complex formation process, the amount of protein bound to the beads was analyzed after chemical stress. For this, the beads were washed once with 250 mM imidazole in a protein binding buffer at 178 times the bead volume. After final washing with 178 times the bead volume of protein buffer, the beads were resuspended in 178 times the bead volume of protein buffer. The amount of protein on 25 μl of the bead slurry was determined by the BCA assay (Thermo, 23227) in a microplate based on the manufacturer's instructions.

[0286] Determination of the Function of Sortase A Immobilized on Beads The activity of immobilized sortase A on 20 μl of bead slurry was determined using the SensoLyte® 520 Sortase A Activity Kit (#72228) from Anaspec as described by the manufacturer.

[0287] GFP binding to immobilized α-GFP antibody To evaluate the function of the immobilized antibody, beads functionalized with [Co(III)(NTA / IDA)(IgG1)] were incubated with 0.54 μM GFP (without His-tag) (Abcam; ab84191) in protein binding buffer at 25 °C for 1 h at 120-fold bead volume. After incubation, the beads were washed once with 160-fold bead volume of protein buffer and finally resuspended in 160-fold bead volume of assay buffer and subjected to determination of chemical stability as described above for samples containing IDA. Finally, GFP bound to the immobilized antibody was determined based on the GFP fluorescence (λ ex = 490 nm, λ em = 535 nm) of 10 μl of bead slurry using a plate reader (TECAN, Spark).

[0288] SDS-PAGE of protein supernatant SDS-PAGE of the protein supernatant after protein incubation was performed as described in Example 3, except that a 12% (w / v) acrylamide-bisacrylamide (37.5:1) gel was used and 6 μl of protein supernatant was loaded per well. Band visualization was performed using Instant Blue Coomassie staining (Expedion; ISB1L).

[0289] Purification of His-sortase The sortase A enzyme (SEQ ID NO: 16) was expressed in E. coli BL21(DE3) using the plasmid pET29_eSrtA (Addgene #75144) (Chen, Dorr et al., 2011) and purified by Ni 2+ -NTA column as described in (Chen, Dorr et al., 2011).

[0290] The results presented in Figure 16 show that in addition to His 6 -GFP, other His-tagged proteins or even antibodies via histidine-rich regions are also removed from the supernatant during protein binding (Figure 16A), and finally, [Co(III)(NTA)(CO) 3 2- or [Co(III)(IDA)(CO) 3 - can be stably immobilized on beads functionalized with (Figure 16B). Furthermore, for example, the sortase A enzyme is still active (Figure 16C), or the anti-GFP antibody immobilized on NTA or IDA beads can still bind to GFP, its antigen, demonstrating that the immobilized protein is still functional.

[0291] Example 15: Formation and Stability of Complexes Using Metal Binding Domains Other than NTA Example 4 demonstrates that [Co(III)(NTA)CO 3 2- complexes can be formed. Furthermore, Example 5 shows that [Co(III)(NTA)CO 3 2- complexes linked to beads by NTA can be synthesized, and surprisingly, it can be used to form [Co(III)(NTA)(His-protein)] complexes on beads. The inventors speculated that [Co(III)(metal binding domain)(His-protein)] complexes could also be formed using metal binding domains other than NTA. To test the versatility of the complex formation method using iminodiacetic acid (IDA), a tridentate metal binding domain and TALON, a commercially available tetradentate metal binding domain, were examined.

[0292] Functionalization of IDA / TALON Magnetic Beads ​​​​Magnetic beads functionalized with IDA (Cube Biotech; 30805) or magnetic agarose resin functionalized with TALON (Takara, 635636) were washed with 1) 20 volumes of ddH 2 O, 2) 20 volumes of 100 mM EDTA pH 8.0, and 3) 20 volumes of ddH 2 O twice and 20 volumes of 1 M NaHCO 3 once. Subsequently, 160 volumes of 1 mM Na 3 in 1 M NaHCO 3 [Co(III)(CO 3 ) 3 ·3H 2 O or only 1 M NaHCO 3 was added for samples without metal. Samples were incubated in a thermoshaker at 1400 rpm and 25 °C for 10 minutes or 48 hours as indicated, and then the beads were washed three times with 160 volumes of 1 M NaHCO 3 .

[0293] Immobilization of His 6 -GFP onto functionalized IDA / TALON magnetic beads The produced beads were incubated with 10 μM His 6 -GFP (SEQ ID NO: 14) in 160 volumes of protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) on a thermoshaker with shaking at 1400 rpm for 30 minutes, 1 hour or 48 hours at 25 °C as indicated. After incubation of the functionalized beads with His 6 -GFP (SEQ ID NO: 14), the beads were washed once with 160 volumes of protein buffer and resuspended in 160 volumes of protein buffer.

[0294] [Co(III)(IDA / TALON)(His 6 -GFP)] complex chemical stability After performing the shown [Co(III)(IDA)(His-GFP)] or [Co(II)(TALON)(His-GFP)] complex formation process, the amount of protein bound to the beads was analyzed with and without using chemical stress. For this purpose, the beads were divided into two parts (each 72-fold bead volume) and washed once with 250 mM imidazole in 178-fold bead volume of either protein buffer or protein binding buffer. After the final wash with 178-fold bead volume of protein buffer, the beads were resuspended in 178-fold bead volume of protein buffer and 10 μl of the bead slurry was analyzed for the amount of immobilized protein by GFP fluorescence (λ ex = 490 nm, λ em = 535 nm) using a plate reader (TECAN, Spark). Both experiments were performed in triplicate.

[0295] The experiments clearly show protein immobilization using IDA (Figure 17A) and TALON (Figure 17B) as metal binding domains. Thus, cobalt(III)-mediated protein immobilization by cobalt(III) carbonate ion complexes is not limited to the use of NTA as the metal binding domain. Furthermore, the tridentate metal binding domain IDA shows further improved characteristics such as improved stability over short incubation times (see Figure 20B). The tetradentate metal binding domain TALON also shows protein immobilization.

[0296] Example 16: Investigation of the Chemical Stability of the [Co(III)(IDA)(His-GFP)] Complex Example 1 shows the chemical stability of the [Co(III)(NTA)(His-GFP)] complex. In Example 15, the formation of [Co(III)(IDA)(His-GFP)] and its stability towards imidazole were demonstrated. In the following example, beads functionalized with "Co(III)(IDA)(His-GFP)] were incubated with a strong chelator or a widely used reducing agent combined with 250 mM imidazole to examine the complex of IDA with the Co 3+ metal center and that of NTA with the Co3+ It was shown that a chemically stable complex can be formed so that the complex composed of 3+ can be obtained as shown in Example 1.

[0297] Functionalization of IDA magnetic beads Magnetic beads functionalized with IDA (Cube Biotech; 30805) were washed 1) with 20 volumes of ddH 2 O, 2) with 20 volumes of 100 mM EDTA pH 8.0, 3) with 20 volumes of ddH 2 O twice and with 20 volumes of 1 M NaHCO 3 once. Subsequently, 160 volumes of 1 mM Na 3 in 1 M NaHCO 3 [Co(III)(CO 3 ) 3 ·3H 2 O or for the metal-free sample, 1 M NaHCO 3 was added, and the beads were incubated at 25 °C for 10 minutes in a thermoshaker at 1400 rpm. After incubation, the beads were washed three times with 20 volumes of 1 M NaHCO 3 . The produced beads were incubated with 10 μM His 6 -GFP (SEQ ID NO: 14) in 160 volumes of 50 mM HEPES pH 7.2, 150 mM NaCl on a thermoshaker with shaking at 1400 rpm for 30 minutes at 25 °C. After incubation with His 6 -GFP (SEQ ID NO: 14), the beads were washed three times with 160 volumes of protein buffer.

[0298] [Co(III)(IDA)(His 6 -GFP)] Chemical stability of the complex Subsequently, each test reagent with a 160-fold bead volume (final concentration: 250 mM imidazole in protein buffer, 25 mM NTA or 25 mM EDTA or 1 mM DTT, TCEP in protein buffer, ascorbate ions with 250 mM imidazole added or 50 mM glycine pH 10.0 as shown) was added to the corresponding sample. After incubating at 25 °C for 1 hour with shaking at 1400 rpm, the supernatant was removed, and the beads were washed three times with a 160-fold bead volume of protein buffer and dissolved in a 160-fold bead volume of protein buffer. Finally, 10 μl of the bead slurry was analyzed for the amount of remaining immobilized protein by GFP fluorescence (λ ex = 490 nm, λ em = 535 nm) using a plate reader (TECAN, Spark). The experiment was performed in triplicate.

[0299] The experiment clearly shows the high chemical stability of the [Co(III)(IDA)(His 6 -GFP)] complex against different chemicals containing chelators and reducing agents. As described in Figure 18, when His 6 -GFP was bound to the [Co(III)(IDA)(CO 3 )] complex, very little reduction of the immobilized was observed upon incubation with the tested chelators or reducing agents. Thus, the [Co(III)(IDA)(His 6 -GFP)] complex is inert to both destruction by strong chelators and reduction to Co 2+ .

[0300] Example 17: Comparison of [Co(III)(IDA)(CO 3 )] - with [Co(III)(IDA)(H 2 O) 2 + in the formation of the [Co(III)(IDA)(His-GFP)] complex Example 4 is [Co(III)(NTA)CO 3 2- ​​Demonstrate that a complex can be formed. Furthermore, Example 5 synthesizes a complex of [Co(III)(NTA)CO 3 2- linked to beads by NTA, and surprisingly shows that this can be used to form a [Co(III)(NTA)(His-protein)] complex on the beads. In Example 6, it is shown that the binding rate of His-GFP to [Co(III)(NTA)(CO 3 )] 2- is improved compared to the rate for complex formation using [Co(III)(NTA)(H 2 O) 2 . In Example 15, it is shown that IDA is a possible metal-binding domain for the formation of a [Co(III)(IDA)(His-GFP)] complex by [Co(III)(IDA)CO 3 - . The inventors speculated that carbonate ions as ligands at the cobalt(III) center might facilitate the formation of [Co(III)(IDA)(His-protein)] complexes. To confirm this finding, the complex formation efficiency of His 2 -GFP with [Co(III)(IDA)(H 2 O) 3 )] - and [Co(III)(IDA)(CO 6 )]

[0301] Functionalization of IDA magnetic beads Magnetic beads functionalized with IDA (Cube Biotech; 30805) were treated with 1) 80 bead volumes of ddH 2 O, 2) 80 bead volumes of 100 mM EDTA pH 8.0, and 3) 80 bead volumes of ddH 2 O twice and 80 bead volumes of [Co(III)(IDA)(CO 3 )] 2- complex with respect to 1 M NaHCO 3 or [Co(II)(IDA)(H 2 O) 2 ​​- For samples containing no complex or metal, they were washed once with ddH 2 O.

[0302] Subsequently, 160 times the bead volume of degassed ddH 2 O containing 1 mM Co(II)Cl 2 ·6H 2 O ([Co(II)(IDA)(H 2 O) 2 and [Co(III)(IDA)(H 2 O) 2 + was added to the samples) or 1 mM Na in 1 M NaHCO 3 [[CO(III)(CO 3 ) 3 ·3H 3 O ([Co(III)(IDA)(CO 2 )] 3 was added to the complex-containing samples. For samples containing no metal, 160 times the bead volume of ddH - O was added. They were incubated in a thermoshaker at 1400 rpm and 25 °C for 10 minutes (or 48 hours for the indicated samples using [Co(III)(IDA)(CO 2 )] 3 )] - . After incubation, the beads were washed three times with 160 times the bead volume of [Co(III)(IDA)(CO 3 )] 2- for samples using this complex, 1 M NaHCO 3 or, for [Co(II)(IDA)(H 2 O) 2 samples or samples containing no metal, with ddH 2 O three times. [Co(III)(IDA)(H 2 O) 2 + was washed once with 160 times the bead volume and incubated on a thermoshaker at 25 °C for 1 hour with 160 times the bead volume of 20 mM H 2 O 2 in it, and finally washed once with 160 times the bead volume of ddH 2 O.​​​

[0303] Immobilization of His on Functionalized IDA Magnetic Beads 6 -GFP The produced beads were incubated with 160 bead volumes of the indicated protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) or 10 μM His in PBS 6 -GFP (SEQ ID NO: 14) on a thermoshaker at 1400 rpm with shaking for 3 hours or 24 hours at 25 °C as indicated. After incubation of the functionalized beads with His 6 -GFP (SEQ ID NO: 14), the beads were washed once with 160 bead volumes of protein buffer and resuspended in 160 bead volumes of protein buffer.

[0304] [Co(III)(IDA(His 6 -GFP)] Complex Chemical Stability After performing the indicated [Co(III)(IDA)(His-GFP)] complex formation process, the amount of protein bound to the beads was analyzed with and without using chemical stress. For this, the beads were divided into two parts (each 72 bead volumes) and washed once with 250 mM imidazole in 178 bead volumes of protein buffer or protein binding buffer, respectively. After a final wash with 178 bead volumes of protein buffer, the beads were resuspended in 178 bead volumes of protein buffer and 10 μl of the bead slurry was analyzed for the amount of immobilized protein by GFP fluorescence (λ ex = 490 nm, λ em = 535 nm) using a plate reader (TECAN, Spark). The experiments were performed in triplicate.

[0305] The results of these experiments were that [Co(III)(IDA)(CO 3 [Co(III)(CO 3 ) 3 ·3H 2 O obtained by incubating for 10 minutes 3)] - Beads functionalized with the complex bind significantly more protein than beads pre-loaded with 2 O) 2 + as shown clearly in Figure 19. After 48 hours of Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O incubation, for the beads functionalized with the [Co(III)(IDA)(CO 3 )] - complex, a significant advantage over the [Co(III)(IDA)(H 2 O) 2 + complex is only seen after 24 hours of protein incubation. The preferred incubation time for IDA is emphasized to be 10 minutes for Na 3 [Co(III)(CO 3 ) 3 ·3H 2 O / NTA, and 30 minutes to 3 hours for subsequent protein incubation. For these combinations of incubation times, a significantly improved immobilization efficiency of the [Co(III)(IDA)(CO 2 O) 2 + compared to [Co(III)(IDA)(H 3 )] - complex is achieved. Further, Figure 19 again confirms that the [Co(III)(IDA)(His 6 -GFP)] complex shows strong stability towards the chelator (here imidazole).

[0306] Example 18: Chemical stable complex formation of [Co(III)(metal binding domain)(CO 3 )] 2- with [Co(III)(metal binding domain)(His-GFP)] and comparison with oxygen treatment of [Co(II)(metal binding domain)(His-GFP)] Example 5 is [Co(III)(NTA)(CO 3 )]​​​2- Demonstrate that a high percentage of [Co(III)(NTA)(His - protein)] complexes formed thereby are chemically stable. The inventors have shown that [Co(III)(metal - binding domain)CO 3 2- starting from [Co(III)(metal - binding domain)(His - protein)] complex formation may result in a greater amount of chemically stable [Co(III)(metal - binding domain)(His - protein)] complex than the treatment of [Co(II)(metal - binding domain)(His - protein)] with oxygen. To confirm this finding, the amount of chemically stable [Co(III)(metal - binding domain)(His - protein)] complex formed by [Co(III)(metal - binding domain)(CO 3 )] or by 8 - hour oxygen treatment of [Co(II)(metal - binding domain)(His - protein)] was directly compared using NTA or IDA as the metal - binding domain, respectively.

[0307] Functionalization of IDA / NTA magnetic agarose beads Magnetic beads functionalized with IDA (Cube Biotech; 30805) or magnetic agarose resin functionalized with NTA (Thermo, 78605) were washed 1) with 80 - fold bead volume of ddH 2 O, 2) with 80 - fold bead volume of 100 mM EDTA pH 8.0, 3) with 80 - fold bead volume of ddH 2 O twice and then, for samples containing [Co(II)(IDA / NTA)(H 2 O) 2 complex, with ddH 2 O or, for samples containing [Co(III)(IDA / NTA)(CO 3 )] complex or metal - free samples, with 1 M NaHCO 3 once. For samples containing [Co(II)(IDA / NTA)(H 2 O) 2 complex, all washes were performed using a degassed, 20 - minute nitrogen - flushed solution and in tubes with nitrogen on top.

[0308] ​ Subsequently, 1 mM Co(II)Cl in degassed ddH 2 O that had been purged with nitrogen for 20 minutes and had a 160-fold bead volume 2 ·6H 2 O ([Co(II)(IDA / NTA)(H 2 O) 2 ) or 1 mM Na 3 in 1 M NaHCO 3 [Co(III)(CO 3 ) 3 ·3H 2 O ([Co(III)(IDA / NTA)(CO 3 )] complex) was added. To the metal-free sample, 1 M NaHCO 3 was added. The samples were incubated at 1400 rpm and 25 °C in a thermoshaker for 10 minutes (or 48 hours for the indicated samples containing the [Co(III)(NTA)(CO 3 )] 2- complex), and then the beads were washed three times with 160-fold bead volume of ddH 2 O for the [Co(II)(IDA / NTA)(H 2 O) 2 samples or 1 M NaHCO 3 for the samples containing the [Co(III)(IDA / NTA)(CO 3 )] complex or the metal-free sample.

[0309] Immobilization of His 6 -GFP onto functionalized IDA / NTA magnetic beads The produced beads were incubated with 10 μM His 6 -GFP (SEQ ID NO: 14) in 120-fold bead volume of protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) on a thermoshaker with shaking at 1400 rpm for the metal-free samples or as shown in Figure 9 for 30 minutes or 48 hours at 25 °C. After incubation of the functionalized beads with His 6 -GFP (SEQ ID NO: 14), the beads were washed twice with 160-fold bead volume of protein buffer ("H 2 O2 For the [Co(III)(IDA / NTA)(His-GFP)] sample by [reference], it was washed only once, and finally, a protein buffer with a volume 160 times that of the beads was added. One sample of [Co(II)(IDA / NTA)(His-GFP)] (designated as "[Co(III)(IDA / NTA)(His-GFP)]" in Figure 20) was aerated with [substance] for 8 hours, while another sample of [Co(II)(IDA / NTA)(His-GFP)] (designated as "[Co(III)(IDA / NTA)(His-GFP)]" in Figure 9) was added with 500 mM [substance] (final 20 mM) with a volume 6.4 times that of the beads, incubated on a thermoshaker at 1400 rpm and 25 °C for 1 hour, and then washed with a protein buffer with a volume 160 times that of the beads. 2 For the one designated as "[Co(III)(IDA / NTA)(His-GFP)]" by [reference], it was aerated with [substance] for 8 hours. 2 For another sample of [Co(II)(IDA / NTA)(His-GFP)] (designated as "[Co(III)(IDA / NTA)(His-GFP)]" in Figure 9), it was aerated with [substance] for 8 hours. 2 [substance] 2 For the one designated as "[Co(III)(IDA / NTA)(His-GFP)]" by [reference], 500 mM [substance] (final 20 mM) with a volume 6.4 times that of the beads was added, incubated on a thermoshaker at 1400 rpm and 25 °C for 1 hour, and then washed with a protein buffer with a volume 160 times that of the beads. 2 [substance] 2 After incubation, it was washed with a protein buffer with a volume 160 times that of the beads.

[0310] [Co(III)(IDA / NTA)(His 6 -GFP)] complex chemical stability After performing the [Co(III)(IDA)(His-GFP)] or [Co(II)(NTA)(His-GFP)] complex formation processes shown, the amount of protein bound to the beads was analyzed with and without using chemical stress. For this purpose, the beads were washed once with a protein buffer with a volume 160 times that of the beads, resuspended in a protein buffer with a volume 160 times that of the beads, divided into two parts (each with a volume 72 times that of the beads), and each part was washed once with 250 mM imidazole in a protein buffer or a protein binding buffer with a volume 178 times that of the beads. After finally washing with a protein buffer with a volume 178 times that of the beads, the beads were resuspended in a protein buffer with a volume 178 times that of the beads, and 10 μl of the bead slurry was analyzed for the amount of immobilized protein by GFP fluorescence (λ ex = 490 nm, λ em = 535 nm) using a plate reader (TECAN, Spark). The IDA experiment was performed in triplicate; the NTA experiment was performed singly in three independent experiments.

[0311] The results of these experiments demonstrated in Figure 20 show significantly more His when aerating beads functionalized with the [Co(II)(metal-binding domain)(His-protein)] complex with oxygen over 8 hours than 6 -GFP protein with [Co(III)(IDA)(CO 3 )] 2- complex (Figure 20A) and [Co(III)(NTA)(CO 3 )] 2- complex (Figure 20B) can be chemically stably immobilized on beads. In addition to a high percentage of stable complexes, the use of [Co(III)(metal-binding domain)(CO 3 )] 2- is also faster than the oxidation method.

[0312] Example 19: Protein immobilization on the surface with the [Co(III)(HS-PEG-NTA)(CO 3 )] 2- complex Some examples of the present invention show the formation of [Co(III)(NTA)(His-protein)] complexes with [Co(III)(NTA)(CO 3 )] 2- In this example, protein immobilization using this principle is tested on a glass surface using nanostructured gold dots.

[0313] Production and passivation of nanostructured glass surfaces The nanostructured surface was produced by diblock copolymer microlithography as previously described (Spatz, Mossmer et al., 2000, Roman, Martin et al., 2003, Lohmuller, Aydin et al., 2011) with an average particle spacing of 58 nm as determined by scanning electron microscopy. Briefly, 5 mg / ml of polystyrene(501)-b-poly-2-vinylpyridine(323) (Polymer Source, Canada) was dissolved in o-xylene respectively. Subsequently, it was added to the solution at a ratio of 0.5 of chloroauric acid to vinylpyridine monomer and stirred for 24 hours. The solution was spin-coated onto a 20x20 mm N1 glass coverslip (Carl Roth, Germany). Then, the substrate was subjected to a plasma procedure (10%H 2 / 90%Ar, 350W, 0.4 mbar, 45 minutes).

[0314] To prevent non-specific adhesion of any protein to the glass substrate between the gold nanostructures, the glass surface was passivated according to the procedure as previously described (Blummel, Perschmann et al., 2007). Thus, the nanopatterned surface was activated in oxygen plasma (150W, 0.4 mbar, 10 minutes) and incubated overnight at 80 °C in dry toluene p.a. (Acros Organics, USA) containing 0.25 mM of α-methoxy-ω-trimethoxysilyl poly(ethylene glycol) (molecular weight 2000 g / mol) (Iris Biotech, Germany), 5.5 μM of water and 20 mM of dry trimethylamine (Acros Organics, USA). Finally, the substrate was washed 3 times with ethyl acetate (Acros Organics, USA) and once with methanol (VWR chemicals, USA) and dried under N 2 flow.

[0315] Functionalization of the surface with thiol-PEG-NTA After passivation, 100 μl of 0.5 mM HS-(CH 2 ) 11 -EG in 99.8% ethanol3 For the -NTA (Prochimia; TH007) or "PEG only" and "PEG / GFP" samples, only ethanol was pipetted onto their respective surfaces. After incubating at room temperature for 1 hour, the surfaces were rinsed in a ddH 2 O bath, or for the "[Co(III)(NTA)(His-GFP)]" samples, rinsed three times in 1M NaHCO 3

[0316] Formation of the [Co(III)(NTA)(His-GFP)] complex After functionalization, the surfaces were covered with 400 μl of 1 mM Na 3 in 1M NaHCO for the "[Co(III)(NTA)(His-GFP)]" samples or with ddH 3 [Co(III)(CO 3 ) 3 ·3H 2 O for the other samples, and incubated at room temperature for 10 minutes. Subsequently, the surfaces were rinsed three times in a bath of 1M NaHCO 2 for the "[Co(III)(NTA)(His-GFP)]" samples or ddH 3 O for the other samples. Finally, 300 μl of 10 μM His 2 -GFP in protein binding buffer (50 mM HEPES pH 7.2, 150 mM NaCl) was added on top of the surfaces and incubated at room temperature for 30 minutes. After rinsing three times in a bath of protein binding buffer, the surfaces were placed in a transparent 6-well plate, covered with protein binding buffer, and GFP-fluorescence (λ 6 = 490 nm, λ ex = 535 nm) as a measure of the immobilized protein on the surface was analyzed using a plate reader (TECAN, Spark). em

[0317] The results of the experiment are presented in Figure 21. Thereby, compared to other control samples, thiol-PEG-NTA, Na 3 [Co(III)(CO 3 ) 3 ·3H 2 ​​Much higher fluorescence signals could be measured for the surface treated with O and His-GFP, which indicates the success of the formation of the [Co(III)(NTA)(His-GFP)] complex on the surface.

[0318] Example 20: Site-Specific Biotinylation of His-GFP with [Co(III)(Biotin-X-NTA)(CO 3 )] Complex In some examples, immobilization of His-tagged proteins onto beads and in Example 19, surfaces functionalized with NTA by the [Co(III)(NTA)(CO 3 )] 2- complex are shown. In the following example, biotinylation of His-GFP at its His-tag with the [Co(III)(Biotin-X-NTA)(CO 3 )] complex in solution is tested.

[0319] Biotinylation of His-GFP 60 μM of biotin-X-NTA (Sigma-Aldrich; 51410) is mixed with 30 μM (sample 1:2), 60 μM (sample 1:1) or 600 μM (sample 10:1) of Na 3 in 1 M NaHCO 3 [Co(III)(CO 3 ) 3 ·3H 2 O or for samples containing metal only with 1 M NaHCO 3 and incubated on a rotary wheel at room temperature for 10 minutes. Subsequently, the resulting [Co(III)(Biotin-X-NTA)(CO 3 )] complex is incubated with a solution of 6 μM His 6 -GFP (SEQ ID NO: 14) in protein binding buffer (50 mM HEPES pH 7.2, 150 mM NaCl) on a rotary wheel at room temperature for 30 minutes or 48 hours.

[0320] Immobilization of [Co(III)(Biotin-X-NTA)(His-GFP)] onto Streptavidin-Functionalized Sepharose After incubation, the obtained [Co(III)(biotin-X-NTA)(His-GFP)] complex was prepared by washing three times with 166-fold bead volume of protein binding buffer in an incubation step of 30 minutes or 48 hours with rotation at room temperature, and then bound to streptavidin-functionalized sepharose (GE Healthcare, 17-5113-01). After incubation, the beads were washed three times with 16-fold bead volume of protein binding buffer and resuspended in 16-fold bead volume of protein buffer.

[0321] [Co(III)(biotin-X-NTA)(His 6 -GFP)] complex chemical stability After the immobilization process of the [Co(III)(biotin-X-NTA)(His-GFP)] complex onto the beads, the amount of protein bound to the beads was analyzed with or without using chemical stress. For this purpose, the beads were split into two parts and each was washed once with 250 mM imidazole in 17-fold bead volume of protein buffer or protein binding buffer. After the final wash with 17-fold bead volume of protein buffer, the beads were resuspended in 17-fold bead volume of protein buffer. Finally, 10 μl of the bead slurry was analyzed for the amount of immobilized protein by GFP fluorescence (λ ex = 490 nm, λ em = 535 nm) using a plate reader (TECAN, Spark). The experiment was performed in triplicate.

[0322] As shown in Figure 22A, biotinylation of His-tagged GFP could be shown for all Co / NTA ratios in solution. However, when measured based on the fluorescence of the biotinylated protein immobilized on streptavidin beads, the Na of biotin-X-NTA 3 [Co(III)(CO 3 ) 3 ·3H 2The higher the ratio to O, the better labeling efficiency could be achieved. In Fig. 22B, more complexes were immobilized on streptavidin beads after stringent imidazole washing compared to the sample using 10 minutes of protein incubation, after 48-hour protein incubation, indicating that the longer the protein incubation, the more stable complexes are formed.

[0323] Example 21: Complex formation and chemical stability of [Pt(IV)(NTA)(His 6 -GFP)] using platinum(IV) nitrate ions The inventors speculated that not only carbonate ions as ligands but also nitrate ions at the metal center could facilitate the formation of [(metal)(NTA)(His-protein)] complexes. Furthermore, the inventors speculated that in addition to Co 3+ , other transition metals showing low ligand exchange rates could also form chemically stable [(metal)(NTA)(His-protein)] complexes. To confirm this finding, the binding efficiency of His 6 -GFP to [Pt(IV)(NTA)(NO 3 )] was examined.

[0324] Functionalization of NTA agarose beads Agarose beads (Qiagen; 1022963) functionalized with NTA were washed with 1) 10 bead volumes of ddH 2 O, 2) 10 bead volumes of 100 mM EDTA pH 8.0, and 3) 10 bead volumes of ddH 2 O twice and 10 bead volumes of 1 M nitric acid once. Subsequently, 16 bead volumes of a platinum(IV) nitrate ion solution (44 mg / l of Pt(IV)) in 1 M nitric acid (Fisher Scientific; 15407817) or only 1 M nitric acid for the metal-free sample were added. After incubating the samples in a thermoshaker at 1400 rpm and 25 °C for 10 minutes, the beads were washed three times with 16 bead volumes of 1 M nitric acid.

[0325] Functionalized [Pt(IV)(NTA)(NO 3 )] immobilization of His 6 -GFP onto agarose beads The produced beads were incubated with 10 μM His 6 -GFP (SEQ ID NO: 14) in 16 bead volumes of protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) for 30 minutes at 25 °C while shaking at 1400 rpm on a thermoshaker. After incubation of the functionalized beads with His 6 -GFP (SEQ ID NO: 14), the beads were washed once with 16 bead volumes of protein buffer and resuspended in 16 bead volumes of protein buffer.

[0326] [Pt(IV)(NTA)(His 6 -GFP)] complex chemical stability After performing the [Pt(IV)(NTA)(His 6 -GFP)] complex formation process shown, the amount of protein bound to the beads was analyzed with and without chemical stress. For this, the beads were divided into two parts (each 7.2 bead volumes) and washed once with 250 mM imidazole in 17.8 bead volumes of protein buffer or protein binding buffer. After a final wash with 17.8 bead volumes of protein buffer, the beads were resuspended in 17.8 bead volumes of protein buffer. The amount of protein on 25 μl of bead slurry was determined by the BCA assay (Thermo, 23227) in a microplate based on the manufacturer's instructions. The experiment was performed in triplicate.

[0327] The results described in Figure 23 show that platinum(IV) binds His 6It is clearly shown that the immobilization of -GFP is mediated. This proves that not only cobalt(III), but also platinum(IV) can form kinetically inert complexes with NTA and his-tagged proteins. Furthermore, very fast complex formation could be shown by the use of nitrate ions as metal-binding ligands.

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Claims

1. a) Co ３＋ a metal cation; b) CO ３ ２－ or HCO ３ － a metal cation ligand that is; and c) A complex comprising a metal cation chelating domain comprising a chelating ligand and a label and / or a carrier. A complex.

2. The complex according to claim 1, wherein the chelating ligand of the metal cation chelating domain is a polydentate ligand containing one or more carboxylic acid groups and / or one or more amine groups and / or one or more aromatic amines and / or phosphate ions.

3. The chelating ligand of the metal cation chelating domain of c) is nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), tris(carboxymethyl)ethylenediamine (TED), chelating peptide, triazacyclononane (TACN), diethylenetriamine-pentaacetic acid (DTPA), phytochelatin, carboxymethylaspartic acid (CMA), phosphate ion, tannic acid (TA), porphyrin, dipyridylamine (DPA), phytic acid, nitrilopropionodiacetic acid (NPDA), nitriloisopropionodiacetic acid (NIPDA), N-(hydroxyethyl)ethylenediaminetriacetic acid (HEDTA), 1,4,7,10-tetraazacyclododecane-N,N',N'',N''' - tetraacetic acid (DOTA), 1,4,7-tris(carboxymethyl)-10-(2'-hydroxypropyl)-1,4,7,10-tetraazocyclododecane, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1-(1,3-carboxypropyl)-1,4,7-triazacyclononane-4,7-diacetic acid (NODAGA), 1,4,8,11-tetraazacyclotetradecane-N,N',N'',N''' - tetraacetic acid (TETA), ethylenedicysteine, ethylenediaminetetraacetic acid (EDTA), 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (DACT), bis(aminoethanethiol)carboxylic acid, ethylene-bis(oxyethylene-nitrilo)tetraacetic acid (EGTA), triethylenetetramine-hexaacetic acid (TTHA), 1,4,7-triazacyclononanephosphinic acid (TRAP), deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), purine, pyrimididine and derivatives thereof, The complex according to claim 1, selected from.

4. The complex according to claim 1, wherein the chelating ligand of the metal cation chelating domain of c) is selected from NTA, IDA, and derivatives thereof.

5. The metal cation is 10 －１ s －１ The complex according to any one of claims 1 to 4, wherein the metal cation has the following water ligand exchange rate.

6. The complex is [Co(III)(NTA)CO ３ ２－ complex, [Co(III)(NTA)HCO ３ － complex, [Co(III)(IDA)CO ３ － complex, [Co(III)(IDA)HCO ３ complex, or a hydrate thereof, wherein the label and / or the carrier is bound to NTA or IDA, the complex according to any one of claims 1 to 5.​​​

7. The label contains a reactive group selected from fluorophores, diagnostic agents, targeting moieties, therapeutic agents, PEG molecules, lipids, biotin and / or its derivatives, proteins, peptides, toxins and / or thiols, azides, alkynes, nitrones, tetrazines and tetrazoles, and / or The carrier is a polymer, hydrogel, microparticle, nanoparticle, sphere (including nano and microspheres), bead, quantum dot, artifact or solid surface, The complex according to any one of claims 1 to 6.

8. A composition comprising the complex according to any one of claims 1 to 7.

9. a) a metal cation as described in a) of claim 1; b) CO ３ ２－ or HCO ３ － a metal cation ligand; and c) a metal cation chelating domain comprising a chelating ligand and a label and / or a carrier A kit comprising.

10. A method for producing the complex according to any one of claims 1 to 7, in solution, (i) a metal cation as described in a) of claim 1; (ii) a metal cation ligand as described in b) of claim 1; and (iii) A step of incubating the metal cation chelating domain as described in c) of claim 1, Method.

11. The metal cation is Co ３＋ and Co ３＋ and CO ３ ２－ or HCO ３ － is provided in the form of a neutral complex with a counter ion, The method according to claim 10.

12. A method for binding a label and / or a carrier to a target molecule, comprising the step of incubating the complex according to any one of claims 1 to 7 or the composition according to claim 8 with the target molecule, wherein the target molecule is a protein, peptide or nucleic acid, Said method.

13. Before incubation, the complex of the present invention is further washed in a solution containing HCO ３ － or CO ３ ２－ and / or the incubation is carried out in a solution containing HCO ３ － or CO ３ ２－ The method according to claim 12, wherein the method is carried out in a solution containing.

14. The method according to claim 12 or 13, wherein the incubation is carried out in an aqueous solution containing one or more buffering substances selected from the group consisting of ACES, AMPSO, BES, BisTris, BisTris propane, boric acid, CAPS, CAPSO, CHES, DIPSO, EPPS, HEPES, HEPBs, HEPPSO, MES, MOPS, MOPSO, PIPES, POPSO, TAPS, TAPSO, TEA, TES, carbonate / bicarbonate buffer, phosphate buffer and Tris.

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