Site-specific kinetically inert conjugation of markers and / or carriers to target molecules such as His-tagged proteins via metal complex reagents

Abstract

The present invention relates to means and methods for conjugating / linking a target molecule such as a protein to a label and / or a carrier. Specifically, the present invention provides a complex comprising (i) a molecule selected from CO3 2‑ and HCO3 ‑ (ii) a carbonate metal cation ligand and (ii) a metal cation coordinated by a metal cation chelating domain, wherein the metal cation chelating domain comprises a chelating ligand and a label and / or a carrier. The complex can be used to connect the label and / or the carrier to a target molecule, preferably a protein. The connection of the label or the carrier through the complex of the present invention involves replacing the metal cation ligand with the coordination group of the target molecule, thereby forming a product complex with the target molecule as the main ligand in the coordination sphere of the metal cation. Therefore, the present invention also provides uses and methods including connecting the label and / or the carrier to the target molecule. Also provided are products obtained by the labeling and / or carrier connection method 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.

Description

Technical Field

[0001] The present invention relates to means and methods for conjugating / linking 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 CO3 2- or HCO3 - and (ii) a metal cation coordinated by a metal cation chelating domain comprising a chelating ligand and a label and / or carrier. The complex can be used to connect the label and / or carrier to a target molecule, preferably a protein. Connecting a label or carrier by the complex of the present invention involves replacing the metal cation ligand with the coordination group of the target molecule, thereby forming a product complex with the target molecule as the main ligand in the coordination sphere of the metal cation. Therefore, the present invention also provides uses and methods including connecting the label and / or carrier to the target molecule. Also provided are products obtained by the label and / or carrier connection method of the present invention and their uses. The present invention also 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 very important tools in many biological applications. Various biochemical and cell techniques in life science research, such as fluorescence-based assays, Western blotting, and protein purification, all rely on labeled or immobilized proteins. Moreover, for biopharmaceuticals (such as antibody-drug conjugates, PEGylation, and lipidation), medical diagnostics, including the development of biosensors, bioimaging, and even medical engineering, the conjugation of proteins is a key production step. Therefore, in all application areas, a simple, stable, site-specific modification method that does not interfere with protein function is needed. In addition, other biomolecules such as nucleic acids are also often labeled or connected to a carrier.

[0003] In classic labeling / immobilization procedures, small molecules are covalently attached to reactive groups (e.g., primary amines in lysine or thiols in cysteine) in unmodified target proteins in a rapid and efficient manner using, for example, N-hydroxysuccinimide (NHS) derivatization reagents or maleimides (Chen and Wu, 2016). However, all of these methods have inherent disadvantages due to the ubiquitous availability of these reactive sites in proteins. Consequently, control over these labeling reactions is limited, leading to significant batch-to-batch variability, lack of site specificity, heterogeneous immobilization / labeling stoichiometry, and even destabilization and loss of protein function (Lindhoud et al., 2012). To address this issue, several new technologies for site-specific protein conjugation have been developed in recent years.

[0004] The most prominent is the Avi tag system, in which the biotin moiety is site-specifically attached to a short peptide tag through the enzymatic action of the biotin ligase BirA (Tirat et al., 2006). In addition, in other approaches, enzymes such as sortase (Popp et al., 2007), transglutaminases (Lin and Ting, 2006), lipoate ligases (Fernandez-Suarez, M. et al., 2007) and phospho-pantheinyl-transferases (Yin et al., 2005) are also used to catalyze the directed conjugation of proteins through short recognition peptide tags.

[0005] Another approach is to expand the genetic code by ribosomally incorporating bioorthogonal functional groups (e.g., tetratines, alkynes, azides, or norbornenes) with the help of unnatural amino acids (Ou et al., 2011; Deiters et al., 2003; Lang et al., 2012). Using specific linker chemistries, the target protein can be modified with great precision at the incorporated group.

[0006] As an alternative to unnatural amino acids, conventional amino acids with unique reactivity and low abundance on the protein surface can be used for site-specific labeling. In this context, most approaches use engineered cysteine ​​substitutions that provide thiol groups that can be used 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 reactive sites for protein conjugation. For example, N-terminal cysteine ​​can be selectively targeted with thioester derivatives via native chemical ligation (Dawson et al., 1994), or proteins can be labeled with perfluorinated aromatic reagents if incorporated into short N-terminal tags (Zhang et al., 2016). Alternatively, proteins can be site-specifically conjugated by oxidation of the N-terminal serine to generate a unique, targetable aldehyde group (Gaertner and Offord, 1996) or by acylation of an N-terminal glycine-histidine tag (Martos-Maldonado et al., 2018).

[0007] What these methods have in common is that they require the incorporation of specialized molecular tags, unnatural amino acids, and / or other amino acids that are not commonly used in other applications. Consequently, laborious genetic engineering and protein re-expression are often required. Even more importantly, the primary multi-step chemical conjugation reactions of these methods are often performed under very harsh reaction conditions, which can negatively impact protein stability and function.

[0008] In addition, several labeling strategies have been developed that rely on the interaction with the His tag, an affinity tag widely used in protein biochemistry. The His tag is usually composed of 6 to 8 histidine moieties and is based on Ni 2+ -mediated nitrilotriacetic acid (NTA) and histidine imidazole group coordination purification technology, His-tagged proteins are very common (Hochuli et al., 1988). The His-tagged protein is bound to Ni by forming a [Ni(II)(NTA)(His-tag)] complex. 2+ The principle of tris-NTA matrix binding 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 (Kamoto et al., 2008). However, a major drawback of these methods is that even with the improved tris-NTA reagent (Huang et al., 2009), these Ni 2+ The mediated complexes still have low affinity and fast ligand exchange rates. Small amounts of common chelating agents (such as EDTA or imidazole) can disrupt these complexes and interfere with protein conjugation. Some other documents describe similar complexes that are not kinetically inert or even require instability; see, for example, WO 2005 / 112977 A2, WO 03 / 072143 A1, WO 2005 / 120700 A2, WO 2009 / 114520, WO 98 / 06739 A1, US 4 569 794 A, Block et al., 2009, WO 03 / 018756 A2, US 2010 / 069293 A1, WO 2004 / 104023 A2, WO 02 / 33044 A2, US 2013 / 131283 A1, WO 2011 / 031771 A1, US 2008 / 015263.

[0009] WO 2003 / 072143 A1 also describes the use of a metal bridge to link a carrier and an active agent of interest, wherein the carrier may comprise a polypeptide having a histidine tag.

[0010] To overcome Ni 2+ The limitations of mediated complexes have led to the use of Co 3+ Replaces commonly used Ni 2+ or Co 2+ As metal ions mediating complex formation (Wegner and Spatz, 2013; Hale, 1995). 3+ Low-spin octahedral paramagnetic complexes (eg 6 t 2g 0 ), where four coordination sites are occupied by chelators (such as NTA) and two coordination sites are occupied by histidine of the His tag, which can achieve a kinetically inert connection of the His tag protein to the NTA part. It has been found that based on Co 3+ The ligand exchange rate of the complex (about 10 -6 s -1 ) than Ni 2+ The complex (3*10 6 s -1 ) were significantly lower (Lippard and Berg, 1994).

[0011] However, although the ligand exchange rate of the [Co(III)(NTA)(His-protein)] complex is very slow, vice versa, the complex formation is also slow. Therefore, the indirect multi-step preparation method is currently the preferred method for producing such [Co(III)(NTA)(His-protein)] complexes (WO2014 / 072525A1; Wegner and Spatz, 2013; Hale, 1995; see Figure 2 A). First, with Co 2+ The precomplex is formed, followed by a hydrogen peroxide oxidation step of the entire [Co(II)(NTA)(His-protein)] complex, wherein Co 2+ In the final complex, it is directly oxidized to Co 3+ Although this method is very rapid and simple, the oxidation process in the presence of proteins can lead to loss of protein function due to oxidation of amino acids. In addition, the combination of hydrogen peroxide and cobalt ions can also trigger the Fenton reaction, which can lead to fragmentation or degradation of the protein backbone and may result in the removal of the His tag and the release of the protein from the complex containing the tag (Andberg et al., 2007).

[0012] Co-based ferrites were produced using this indirect multi-step preparation method involving an oxidation step. 3+ His-tagged protein complexes have also been used to immobilize proteins on surfaces (Wegner et al., 2016; Di Russo et al., 2018). However, in the case of surface immobilization, this approach uses the strong oxidizing agent H2O2, which may negatively affect protein function and may also interfere with certain surface structures.

[0013] Similar to the above literature, EP 0 497 585 A2 also only involves the addition of Co after the formation of the [Co(II)(IDA)(His-protein)] complex. 2+Oxidation to Co 3+ The [Co(II)(IDA)(His-protein)] complex was exposed to O2 gas for several hours to promote the 2+ Oxidized to Co 3+ However, it is known in the art that unstable proteins can be damaged by exposure to high concentrations of oxygen for several hours. Furthermore, the present inventors were unable to reproduce the described method, i.e., no stable complex was observed after contacting the [Co(II)(NTA)(His-protein)] complex with O2 gas.

[0014] Zatloukalová and Kucerová proposed a method to form a [Co(III)(IDA)(His-tag)(H2O)] complex, in which oxidation in the presence of protein can be circumvented (Zatloukalová and Kucerová, 2006). 2+ The center is oxidized to Co by hydrogen peroxide in the pre-complex with IDA. 3+ , the subsequent generation of [Co(III)(IDA)(H2O)3] + Coordinated with His-tagged proteins to form the final [Co(III)(IDA)(His-tag)(H2O)] complex (see Figure 2 Reaction diagram of B; Zatloukalová and Kucerová, 2006). However, this method still has major disadvantages. 2+ Compared with the pre-complex with water ligands, the Co-based 3+ The coordination of the precomplex is significantly slower, and as the inventors demonstrate in the accompanying examples, the final complex formation efficiency is also low, especially at the low temperatures required for most proteins. In addition, the use of hydrogen peroxide in the presence of IDA-conjugate still has a negative impact on the function of the conjugate (i.e., the label and / or carrier attached to the IDA conjugate) due to oxidation. For example, the fluorescence of the fluorophore will be reduced due to oxidation. In addition, this approach requires purification of the oxidized precomplex from hydrogen peroxide for further downstream applications. These purification processes are both time-consuming and material-intensive, and there is a risk of residual oxidants that may oxidize and damage the protein to be modified in downstream applications.

[0015] The technical problem underlying the present invention is therefore to provide improved means and methods which allow for the attachment of markers and / or carriers to target molecules, such as proteins, via complex formation in a simple, efficient manner which prevents interference with the function and / or stability of the marker / carrier and the target molecule.

[0016] This technical problem is solved by providing the embodiments characterized in the claims and as provided below. SUMMARY OF THE INVENTION

[0018] In a first aspect, the present invention provides a compound in the form of a complex comprising:

[0019] a) metal cations;

[0020] b) a metal cation ligand selected from CO3 2- and HCO3 - carbonate; and

[0021] c) A metal cation chelating domain comprising a chelating ligand and a label and / or a carrier.

[0022] The inventors have surprisingly found that the complexes of the present invention are advantageous tools for attaching markers and / or carriers to target molecules, such as proteins with His tags. Labeling is achieved by exchanging the metal cation ligands of the complex with the target molecule in the primary coordination sphere of the complex. By this exchange, a new complex is formed, which has a metal cation chelating domain (comprising a chelating ligand and a marker and / or carrier) as a first ligand and a target molecule as a second ligand. For example, using a complex comprising a transition metal cation Co 3+ or Pt 4+ As illustrated in the accompanying examples of the complexes of the present invention, the complexes of the present invention allow the label and / or carrier to be attached to the target molecule in a kinetically inert manner. Because histidine residues are ligands that can participate in the formation of metal complexes, especially with transition metals such as Co 3+ To form a complex, the preferred target molecule is a protein with a His tag or a protein with a His-rich region. The His tag can act as a bidentate and / or polydentate ligand.

[0023] One advantage of the complexes of the present invention is that an oxidation reaction using H2O2 is not necessary for the production steps of the complexes in which the label and / or support are present, nor is it necessary for attaching the label and / or support to the target molecule. Avoiding an oxidation step such as H2O2 treatment has the advantage that attachment of the label and / or support can be achieved without interfering with the function of the target molecule, the label and / or the support due to oxidation. This makes label and / or support attachment mediated by the metal complex more widely applicable, particularly for oxidation-sensitive target molecules, such as proteins. Furthermore, it eliminates the need for cumbersome and time-consuming washing steps to remove the oxidant.

[0024] For Co(III) or other transition metal complexes with low ligand exchange rates, direct complex formation without any intermediate steps is extremely slow and inefficient. Therefore, current prior art methods for forming these kinetically inert complexes focus on a two-step approach. First, a complex is formed with a metal center (e.g., Co(II)) in a kinetically unstable oxidation state, followed by an oxidation process, typically using hydrogen peroxide. This oxidation process can lead to unforeseen functional damage to the protein, metal binding domain, and / or carrier.

[0025] To overcome these limitations, the present invention describes a method for directly forming kinetically inert complexes. Starting directly with a metal in the desired oxidation state, the use of oxidizing agents in the presence of proteins, metal binding domains, or proteins can be avoided. The selected metal is introduced into the metal binding domain and pre-coordinated with three metal cation ligands (carbonate or nitrate). In the first step, two metal cation ligands are replaced by the metal cation chelating domain, after which the last metal cation ligand is replaced by the coordination of the protein. Pre-coordination of the metal with the carbonate or nitrate can promote the formation of the final complex, resulting in a significantly faster complex formation rate and higher complex formation efficiency. This improved direct complex formation method is carried out using a pre-complex with a coordinated carbonate or nitrate ligand, which stems from the ability of these metal cation ligands to protonate after release from the pre-complex. In the case of carbonate, protonation after ligand release can lead to gas formation, which can further enhance the formation process. Thus, as demonstrated by the present inventors, the use of carbonate or nitrate precomplexes allows for significantly faster and more efficient direct formation of kinetically inert metal complexes than with other ligands, including water. As shown in the accompanying examples, the means and methods of the present invention relate, inter alia, to precomplexes comprising and / or consisting of metal cations, carbonate (as metal cation ligands; CO3 2- or HCO3 - ) and a metal cation chelating domain, wherein the metal chelating domain comprises a chelating ligand and a label and / or a carrier. It is obvious from the disclosure herein and from the accompanying examples that it is also possible to use "nitrate" instead of "carbonate" (CO3 2- or HCO3 - ) corresponding complexes. Thus, the present invention may also be based on corresponding "nitrate complexes". Thus, described herein are corresponding "carbonate complexes" and "nitrate complexes". In the context of the present invention, carbonate and nitrate can be considered equivalents, since both metal cation ligands can be protonated after release from the corresponding precomplexes. In the context of the present invention, carbonate complexes are preferred for the means and methods provided herein. Further details are provided below.

[0026] In the prior art of Wegner and Spatz (loc.cit) and Zatloukalová and Kucerová (loc.cit) mentioned above, the method of forming Co3 + However, in contrast to the present invention, these methods all involve an oxidation step using H2O2 (see Figure 2 A and B). This oxidation step is a harmful treatment that has a negative impact on the stability and function of the marker (e.g., fluorophore) and / or carrier and the protein to be labeled (see the attached examples). The complexes of the present invention allow the oxidation steps in these prior art solutions to be circumvented. In addition, the complexes of the present invention can quickly and effectively coordinate metal-binding target molecules, preferably proteins with His tags or proteins with histidine-rich regions, so that the marker and / or carrier can be connected to the target molecule through metal cation-mediated interactions. In the final product, the marker and / or carrier is connected to the target molecule in a kinetically inert manner similar to that described by Wegner and Spatz and Zatloukalová and Kucerová. The binding of the target molecule is achieved by exchanging the metal cation coordination ligand with the target molecule to form a new complex (referred to herein as a "product complex"). By selecting the metal coordination ligand to be selected from CO3 2- and HCO3 - The present inventors have found that by reacting a carbonate or nitrate radical of a target molecule with a marker / carrier, a "product complex" can be formed very efficiently and quickly, wherein the product complex comprises a metal cation chelating domain containing a marker / carrier and a target molecule coordinated to the metal cation. In particular, the present inventors have found that, compared to complexes using water as a metal cation ligand (as described in the method of Zatloukalová and Kucerová), the binding of the target molecule is more complete and faster, especially at low temperatures. Therefore, the complexes of the present invention can more quickly and completely connect the marker and / or carrier to the target molecule (e.g., a His-tagged protein). In summary, a major advantage of the complexes of the present invention is that the complexes allow for rapid and efficient connection without the need for an oxidizing agent in the presence of the marker and / or carrier.

[0027] The complexes of the present invention or compositions comprising the same are particularly useful in a variety of applications, including, but not limited to, labeling proteins with fluorophores, toxins, diagnostic moieties, targeting moieties, stabilizing domains, and / or reactive groups. The complexes of the present invention can also be used, inter alia, to produce biopharmaceuticals, such as toxin-labeled antibodies and diagnostic agents. The label is kinetically inert (i.e., the ligand exchange rate is 10 -1 s -1 or lower), thus preventing ligand exchange based on Ni 2+ or Co2+ In addition, with Ni-based 2+ or Co 2+ Compared with the complexes with β-catenin, the tag is more thermodynamically stable.

[0028] The complexes of the present invention can also be used to achieve stable attachment of a target molecule, preferably a protein, to a carrier. For example, the carrier can be a surface, a bead, a nanoparticle, a prosthetic group (prosthetic), a quantum dot, a polymer, a hydrogel, a microparticle, a sphere (e.g., a nanosphere and / or a microsphere) or a combination thereof. For example, Example 19 demonstrates that a His-tagged GFP can be attached to a gold nanostructured glass surface. First, the gold nanostructured glass surface is functionalized with NTA by coupling an NTA-linker-thiol reagent, via the interaction of the gold particles with the thiol groups. Subsequently, the complex of the present invention is generated on the surface and His-GFP ( Figure 21 ). In addition, it is shown in non-limiting Example 20 that a His-tagged GFP can be linked to a biotin group coupled to the complex of the present invention via a linker. The product comprising a biotin group, a linker, a complex of the present invention, and a His-tagged GFP is then coupled to beads comprising a streptavidin group ( Figure 22 ).

[0029] The accompanying examples show that a wide variety of (His-tagged) proteins can be coupled to beads using the complexes of the present invention. For one of the tested proteins (sortase A), it was demonstrated that the enzyme activity was retained after immobilization (Example 14, Figure 16 C) It was also demonstrated that antibodies can be coupled to beads using the histidine-rich region of the antibody Fc region via the complex of the present invention. Figure 16 D demonstrated that the immobilized antibodies retained the ability to bind to their antigens.

[0030] The complexes of the present invention allow for rapid and efficient metal cation-mediated labeling of target molecules or attachment of carriers to target molecules without the need for an oxidation step in the presence of the target molecule to be labeled and / or the label and / or carrier as described by Wegner and Spatz and Zatloukalová and Kucerová, which makes them particularly suitable for the above-mentioned applications in which it is often crucial to avoid oxidation of the target protein and / or the label and / or the carrier.

[0031] The complexes of the present invention may be provided in solution or in solid form. The complexes of the present invention may be charged, particularly when provided in solution. The charge will depend on the charge of the metal cation, the metal cation ligand, and the metal cation chelating domain. For example, if the complex comprises Co 3+ As metal cations, CO32- As a metal cation ligand, and a metal cation chelating domain composed of the chelating ligand NTA and an uncharged label, the complex has a charge of "2-" (i.e., a divalent negative charge). Complexes with counterions can also be provided. When the complex further contains one or more counterions, the complex can also be provided as a solid. The (one or more) counterions are preferably monovalent ions. Particularly preferred are monovalent ions from alkali metals. When the complex is negatively charged, Na+ and K+ are most preferably used as counterions.

[0032] The metal cation chelating domain (it is alternatively referred to as the first ligand or chelating agent) of the complex of the present invention comprises a chelating ligand. The chelating ligand mediates the coordination of the metal cation of the complex by providing at least two binding sites (i.e., in other words, bidentate or multidentate) that can coordinate with the metal cation of the complex. In a preferred embodiment, the chelating ligand of the metal cation chelating domain can be tridentate (e.g., IDA) or tetradentate (e.g., NTA). "Tridentate" refers to that the chelating ligand comprises three atoms that can act as donor atoms (i.e., Lewis donors) in the complex based on metal ions. "Tetradentate" refers to that the chelating ligand comprises four atoms that can act as donor atoms (i.e., Lewis donors) in the complex based on metal ions. Therefore, in other words, the chelating ligand of the metal cation chelating domain can preferably 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 with more atoms that can act as donor atoms (i.e., Lewis donors) in the complex has the advantage that the metal cation is more strongly bound in the complex. Stronger binding can prevent the frequency of undesirable release of metal cations from the complex; that is, undesirable complex disintegration. This applies to the complexes of the present invention and the "product complexes" formed by the uses and methods of the present invention. Generally, tridentate and tetradentate complexes are preferred herein. According to the accompanying examples, tridentate is most preferred.

[0033] The metal cation chelating domain of the complex of the present invention further comprises a label and / or a carrier, ie, a functional part to be linked to the target molecule. The chelating ligand can be directly linked to the label and / or carrier or linked via a linker.

[0034] The chelating ligand of the metal cation chelating domain can be any chelating ligand known in the art in principle.Preferred chelating ligand comprises 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 can be polycarboxylic acid, polyamine or aminopolycarboxylic acid.

[0035] 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., with the consensus sequence (GHHPH) nG, wherein G is glycine, H is histidine, P is proline, and n is an integer from 1 to 3; see also SEQ ID NOs: 1 to 3) (Hutchens and Yip 1992), chelating proteins (e.g., his-tagged proteins or proteins with a spaced His-tag as defined elsewhere herein) or cadystin (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), phytochelatins (Song et al. 2014), carboxymethylaspartic acid (CMA) (Porathet et al. 1975, Hutschenreiter et al. 2010), 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), nitrilopropionic acid diacetic acid (NPDA) (Mitsuo et al. 1970), nitriloisopropionic acid diacetic acid (NIPDA) (Mitsuo et al. 1970), N-(hydroxyethyl)ethylenediaminetriacetic acid (HEDTA) (Wubs and Beenackers 1993, Graff et al. 1995), ethylenediaminetetraacetic acid (EDTA) (Wubs and Beenackers 1993), ethylenebis(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), and 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) (Kline et al. 1991, Chappell et al. 2003). al. 2003), 1,4,7-tris(carboxymethyl)-10-(2'-hydroxypropyl)-1,4,7,10-tetraazacyclodecane (Filippi et al. 2014), 1,4,7-triazacyclononanephosphinic 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), triethylenetetraaminehexaacetic acid (TTHA) (Harju and Ringbom 1970, Achour et al. 1998), 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (DACT) (Krzek et al. 2003), 1,4,7-tris(carboxymethyl)-10-(2'-hydroxypropyl)-1,4,7,10-tetraazacyclodecane (Filippi et al. 2014), 1,4,7-triazacyclononanephosphinic 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), triethylenetetraaminehexaacetic acid (TTHA) (Harju and Ringbom 1970, Achour et al. 1998), et al. 2007), phosphonates (Rizkalla et al. 1980), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) (Strand et al. 2013; Simecek et al. 2012), 1-(1,3-carboxy-propyl)-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), purines (Cini and Giogi 1987), and pyrimidines (Saha and Mukherjee 1984).

[0036] Thus, the chelating ligand comprised in the metal cation chelating domain of c) may, for example, be selected from the following non-limiting list: nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), tris(carboxymethyl)ethylenediamine (TED), chelating peptides, for example having the consensus amino acid sequence (GHHPH) n G, wherein G is glycine, H is histidine, P is proline, and n is an integer from 1 to 3; see also SEQ ID NOs: 1 to 3) or cadystin, triazacyclononane (TACN), diethylenetriamine-pentaacetic acid (DTPA), phytochelatin, carboxymethylaspartic acid (CMA), phosphonate, tannic acid (TA), porphyrin, dipyridylamine (DPA), phytic acid, nitrilopropionic acid diacetic acid (NPDA), nitriloisopropionic acid diacetic 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-tetraazacyclododecane, 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, ethylenebis(oxyethylene-nitrilo)tetraacetic acid (EGTA), triethylenetetraamine-hexaacetic acid (TTHA), 1,4,7-triazacyclononanephosphinic acid (TRAP), deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), purine, pyrimidine and their derivatives.

[0037] What all these chelating ligands have in common is that they provide at least two coordination sites and can act as Lewis base donors, thereby forming coordination chelates (i.e., chelating agents) with metal cations. Thus, in one embodiment, the chelating ligands may have at least 2, 3, 4, 5 or more chemical groups that can coordinate with metal cations, i.e., they can act as Lewis bases.

[0038] In a preferred embodiment, the chelating ligand of the metal cation chelating domain of c) is selected from the group consisting of: nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), a ligand having a consensus sequence (GHHPH) nG, wherein n is 1 to 3; see also SEQ ID NOs: 1 to 3), diethylenetriamine-pentaacetic acid (DTPA), nitrilopropionic acid diacetic acid (NPDA), nitriloisopropionic acid diacetic acid (NIPDA), ethylenediaminetetraacetic acid (EDTA), ethylenebis(oxyethylene-nitrilo)tetraacetic acid (EGTA), carboxymethylaspartic acid (CMA) and derivatives thereof.

[0039] In another preferred embodiment, the chelating ligand can be an aminopolycarboxylic acid. Preferably, the aminopolycarboxylic acid is selected from ethylenediamine-tetraacetic acid (EDTA), nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), ethylenebis(oxyethylene-nitrilo)tetraacetic acid (EGTA), diethylenetriamine-pentaacetic acid (DTPA), and triethylenetetramine-hexaacetic acid (TTHA), tris(carboxymethyl)ethylenediamine (TED), triazacyclononane (TACN) or its derivatives.

[0040] 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 derivatives thereof. NTA and IDA are most preferred as chelating ligands, with IDA being particularly preferred. NTA is a tetradentate ligand and therefore binds particularly strongly to metal cations. IDA is tridentate and can also bind strongly to metal cations, but weaker than NTA. Strong binding to metal cations prevents the release of metal cations, which would result in the undesirable disintegration of the complex or the release of the marker and / or carrier from the target molecule. In certain embodiments, IDA is preferred because complexes with a high stability percentage can be formed faster than complexes with NTA. It is speculated that for tridentate metal binding domains, protein coordination is further promoted because the second metal cation ligand can still remain half-coordinated to the metal, whereas it is completely displaced when a tetradentate metal binding domain is used. Due to partial coordination, the ligand can be easily replaced by a histidine residue of the protein. As this moment approaches, one or two additional histidine residues can also release the final ligand and form a stable complex.

[0041] In the context of chelating ligands, the term "derivative" refers to a compound with identical lead structure but can be substituted by other chemically reactive groups. Preferably, the term "derivative" herein includes replacement by one or more other chemical groups selected from carboxyl, amine, azide, acrylate, maleimide, hydroxyl, thiol, aromatic, aliphatic, disulfate and vinyl sulfone groups. The term derivative can also include molecules with isotopic substitution. "Isotopic substitution" refers to that one or more atoms are labeled with isotopes.

[0042] The metal cation is preferably a metal cation that is stable in the corresponding oxidation state. Depending on the metal used, the oxidation state can be +2, +3 or +4. Therefore, the metal cation of the complex can 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+ ).

[0043] The metal cations can be low-spin octahedral paramagnetic complexes (e g 6 t 2g 0 ) of metal cations, such as Co 3+ Thus, in one embodiment, the complex may be a low-spin octahedral paramagnetic complex (e g 6 t 2g 0 ), preferably Co 3+ Low-spin octahedral paramagnetic complexes (e g 6 t 2g 0 The metal cation can be, can form diamagnetic octahedral low spin complexes (t 2g 6 e g 0 ) of metal cations, such as Pt 4+ Therefore, in one embodiment, the complex can be a diamagnetic octahedral low-spin complex (t 2g 6 e g 0 ), preferably diamagnetic octahedral low-spin Pt 4+ (t 2g 6 e g 0 ).

[0044] Preferably, the metal cation is a water ligand exchange rate of 10 -1 s -1 or smaller (e.g. 10 -2 s -1 or less or 10 - 3 s -1 Metal cations with such low water ligand exchange rates have the advantage of forming very tight complexes with target molecules that are kinetically inert, i.e., exchange target molecule ligands only at a very slow rate and provide a nearly covalent bond (i.e., a kinetically inert bond).

[0045] Methods for determining the water exchange rate of metal cations (and complexes containing the same) are known in the art. A. Dunand and colleagues (Dunand et al., 2003) reviewed methods that can be used to determine the water exchange rate. In order to assess the slow exchange rate as used in the context of the present invention, the measurement method may include 17 O NMR analysis (Cusanelli et al., 1996). In such an assay, the metal 17 The complex of O-labeled water was dissolved in H2O. To determine the water exchange rate, the peak shift caused by H2O exchange was measured. 17 O loss over time.

[0046] In a preferred embodiment, the metal cation of the complex may be a transition metal cation. The transition metal cation is preferably in the range of 10 -1 s -1 Oxidation states with water ligand exchange rates of 0.001 or lower. Transition metal cations known in the art to have water exchange rates within 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+ Thus, preferred but non-limiting examples of metal ions that are transition metal cations that can be used in the context of the present invention 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+ .

[0047] In one embodiment, the metal cation may be a trivalent transition metal cation, preferably a trivalent transition metal cation as listed above, even more preferably Co 3+ .

[0048] In one embodiment, the metal cation may be a tetravalent transition metal cation, preferably a tetravalent transition metal cation as listed above, preferably Pt 4+ .

[0049] In one embodiment, the metal cation of the complex can be a lanthanide (also known as an inner transition metal) cation. Lanthanides are a subgroup of transition metals that include the following metals: La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu. Lanthanide cations useful in the present invention preferably have a water ligand exchange rate of 10 -1 s -1 or lower, preferably 10 -2 s -1 Lanthanide cations known in the art to have water exchange rates within this range and / or to be kinetically inert include: La 3+ ,Eu 3+ ,Gd 3+ and Tb 3+ Therefore, the metal cation in the context of the present invention may be a lanthanide cation selected from the group consisting of: La 3+ ,Eu 3+ ,Gd 3+ and Tb 3+ Therefore, in one embodiment, the metal cation of the complex may also be a trivalent lanthanide cation.

[0050] In one embodiment, the metal cation may be selected from: 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+ Those skilled in the art will recognize that these metal cations are characterized by very low water ligand exchange rates. The present inventors have discovered that metal cations with such low water ligand exchange rates are particularly useful metal cations for mediating the interaction between the metal cation chelating domain containing the label and / or carrier 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 the thermodynamically stable and kinetically inert product complex.

[0051] In a preferred embodiment, the metal cation is selected from: 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+ ,Fe 2+ These metal cations have been described in the literature and have 10-1 s -1 or lower water ligand exchange rate 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), Ir 4+ (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). Therefore, it is reasonable that these metal cations can form similar stable product complexes with the target molecule, thereby stably attaching the label and / or carrier to the target molecule.

[0052] In the context of the present invention, a "kinetically inert metal cation" is a metal cation having a -1 s -1 The metal cation with a water ligand exchange rate of 1% or less. This definition and cutoff value are also consistent with the general understanding of this statement in the literature (Taube, 1952; Luther III, 2016). The preferred method for measuring water exchange rate is mentioned elsewhere in this article.

[0053] When used in the context of binding of a ligand (e.g., a target molecule in a "product complex") in a metal complex, "kinetically inert," as used herein, preferably refers to a ligand exchange rate in aqueous solution of 10 -1 s -1 or lower, even more preferably 10 -2 s -1 or less. Methods for measuring ligand exchange rates are known in the art. For example, methods for measuring water exchange rates as described elsewhere herein may be used mutatis mutandis. According to the present invention, assays for determining ligand exchange rates and / or assessing the kinetic inertness of target molecule binding in a "product complex" may include measuring competition with a competing ligand (e.g., imidazole or EDTA) and / or with a reducing agent (e.g., DTT), as described in the accompanying Examples and Figures (see, e.g., Figure 1 、 6 and 7B). A preferred assay for evaluating the "kinetic inertness" of target molecule binding to a complex (i.e., a "product complex") can be evaluated by the method exemplified in Example 5. Specifically, [Co(III)(NTA)(CO3)] 2- The complex can be produced as described herein and in the accompanying Examples and Figures. Subsequently, the bead-bound [Co(III)(NTA)(CO3)] 2- The complex 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 assessed by washing equal amounts of the resulting agarose beads with PBS (= control) or imidazole solution (PBS supplemented with 250 mM imidazole) (= sample). Alternatively, a Ni-based 2+-NTA conventional matrix as a comparison. The amount of the target molecule bound can then be assessed for all samples (optionally before and after washing). The method for determining the presence of the target molecule (i.e., reading) depends on the characteristics of the target molecule and is known in the art. For example, the assay can involve fluorescence measurement (e.g., when the target molecule has fluorescence). For non-fluorescent target molecules, fluorescent antibodies that detect the target molecule can be used for reading. Another example of reading is when the target molecule is an enzyme or when using an enzyme-labeled antibody for the target molecule, an enzymatic reaction is used. In the context of the present invention, when relative to the amount of the target molecule bound on the beads washed with PBS (= control), the beads washed with 250mM imidazole (= sample) contain 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% target molecule after washing, preferably, the target molecule ligand is considered to be "kinetically inert" in the "product complex" when combined.

[0054] In one embodiment, the metal cation may be Co 3+ or Cr 3+ In a particularly preferred embodiment, the metal is Co 3+ As demonstrated in the accompanying examples, Co 3+ It is particularly suitable and allows for a rapid and easy production of the complexes according to the invention and ensures kinetically inert binding to the target molecule.

[0055] In another preferred embodiment, the metal is Pt 4+ As demonstrated in the accompanying examples, Pt 4+ It is particularly suitable and allows for a rapid and easy production of the complexes according to the invention and ensures kinetically inert binding to the target molecule.

[0056] The present invention also relates to a complex wherein the metal cation is Co 3+ , the chelating ligand of the metal cation chelating domain is NTA (or its derivatives).

[0057] The present invention also relates to a complex wherein the metal cation is Co 3+ , the chelating ligand of the metal cation chelating domain is IDA (or its derivatives).

[0058] The present invention also relates to a complex wherein the metal cation is Co 3+ The chelating ligand of the metal cation chelating domain is Talon (or its derivatives).

[0059] The present invention also relates to a chelate, wherein the metal cation is Pt 4+, the chelating ligand of the metal cation chelating domain is NTA (or its derivatives).

[0060] The present invention also relates to a complex wherein the metal cation is Pt 4+ The chelating ligand of the metal cation chelating domain is Talon (or its derivatives).

[0061] The present invention also relates to a complex wherein the metal cation is Pt 4+ , the chelating ligand of the metal cation chelating domain is IDA (or its derivatives).

[0062] Therefore, the present invention relates to a complex (e.g., a complex for linking a label and / or a carrier to a target molecule) comprising:

[0063] a)Co 3+ ;

[0064] b) a metal cation ligand selected from CO3 2- and HCO3 - carbonate or nitrate; and

[0065] c) A metal cation chelating domain comprising NTA and a label and / or a carrier.

[0066] The present invention also relates to a complex (e.g., a complex for linking a label and / or a carrier to a target molecule), comprising:

[0067] a)Co 3+ ;

[0068] b)CO3 2- or HCO3 - ligand; and

[0069] c) A metal cation chelating domain comprising NTA and a label and / or a carrier.

[0070] The present invention also relates to a complex (e.g., a complex for linking a label and / or a carrier to a target molecule), comprising:

[0071] a)Co 3+ ;

[0072] b)CO3 2- or HCO3 - ligand; and

[0073] c) A metal cation chelating domain comprising Talon and a label and / or a carrier.

[0074] The present invention also relates to a complex (e.g., a complex for linking a label and / or a carrier to a target molecule), comprising:

[0075] a)Co 3+ ;

[0076] b)CO3 2- or HCO3- ligand; and

[0077] c) A metal cation chelating domain comprising IDA and a label and / or a carrier.

[0078] The present invention also relates to a complex (e.g., a complex for linking a label and / or a carrier to a target molecule), comprising:

[0079] a)Pt 4+ ;

[0080] b)CO3 2- or HCO3- ligand; and

[0081] c) A metal cation chelating domain comprising NTA and a label and / or a carrier.

[0082] The present invention also relates to a complex (e.g., a complex for linking a label and / or a carrier to a target molecule), comprising:

[0083] a)Pt 4+ ;

[0084] b)CO3 2- or HCO3- ligand; and

[0085] c) A metal cation chelating domain comprising IDA and a label and / or a carrier.

[0086] The present invention also relates to a complex (e.g., a complex for linking a label and / or a carrier to a target molecule), comprising:

[0087] a)Pt 4+ ;

[0088] b)CO3 2- or HCO3- ligand; and

[0089] c) A metal cation chelating domain comprising Talon and a label and / or a carrier.

[0090] In one embodiment, the chelating ligand of the metal cation chelating domain is diethylenetriamine-pentaacetic 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-triazacyclononanephosphinic 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+ In one embodiment, the chelating ligand of the metal cation chelating domain is triethylenetetramine-hexaacetic acid (TTHA) 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 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and the metal cation is In 3+ In one embodiment, the chelating ligand of the metal cation chelating domain is 1-(1,3-carboxy-propyl)-1,4,7-triazacyclononane-4,7-diacetic acid (NODAGA) and the metal cation is In 3+ .

[0091] The metal cation ligand (also referred to as the second ligand) of the complex of the present invention is selected from carbonate CO3 2- and HCO3 - or nitrate. The present inventors have found that the use of these metal cation ligands facilitates the coordination of the target molecule. This is achieved by exchanging carbonate or nitrate from the complex for the target molecule more quickly and easily. After release from the complex, both carbonate and nitrate can be protonated. This protonation promotes the release from the complex. In the case of carbonate, CO3 2- and HCO3 - Without being bound by theory, it is believed that gas formation further increases the exchange kinetics and thermodynamics of the carbonate ligand with the target molecule. 2- and HCO3 - Nitrate can also be used as a metal cation ligand, but this is a different invention.

[0092] In a preferred embodiment, the metal cation ligand is selected from CO3 2- and HCO3 - In the accompanying examples, it is demonstrated that these carbonates promote the formation of product complexes with target molecules. In other words, the complexes of the present invention are more reactive in attaching labels and / or carriers to target molecules.

[0093] In one embodiment, the complex of the present invention comprises [Co(III)(NTA)CO3] 2- Complex, [Co(III)(NTA)HCO3] - Complex or its hydrate. The label and / or carrier is connected to NTA. In these complexes, NTA occupies four coordination sites and carbonate occupies two coordination sites. Carbonate is a bidentate ligand, that is, there are two atoms that can act as donors (i.e., Lewis bases) to coordinate with the metal cations in the complex, which promotes the binding of the target molecule. NTA is a chelating agent that binds particularly strongly to metal cations due to its tetradentate binding. Therefore, NTA can prevent the decomposition of undesirable complexes and product complexes caused by the release of undesirable metal cations.

[0094] In a preferred embodiment, the complex of the present invention comprises [Co(III)(IDA)CO3] - Complex, [Co(III)(IDA)HCO3] complex or its hydrate. The label and / or carrier is connected to IDA. In these complexes, IDA occupies three coordination sites and carbonate occupies two coordination sites. Carbonate is a bidentate ligand, that is, there are two atoms that can act as donors (i.e., Lewis bases) to coordinate with the metal cation in the complex, which promotes the binding of the target molecule. It is speculated that for tridentate metal binding domains, the coordination of the protein can be additionally promoted because the second metal cation ligand can still remain semi-coordinated with the metal, while it will be completely replaced when using a tetradentate metal binding domain. Due to the partial coordination, the ligand can be easily replaced by a histidine residue of the protein. Due to the proximity at this moment, the other one or two histidine residues can release the last ligand and form a stable complex.

[0095] The metal cation chelating domain of the complex of the present invention comprises a label and / or a carrier. In other words, the complex of the present invention comprises a functional moiety. Preferably, the label and / or carrier are different from the coordinating group 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 carrier may also share one or more atoms.

[0096] The metal cation chelating domain of the complex of the present invention may comprise a chelating ligand and a label, but not a carrier. Alternatively, the metal cation chelating domain of the complex of the present invention may comprise a chelating ligand and a carrier, but not a label. In another embodiment, the metal cation chelating domain of the complex of the present invention may comprise a carrier and a label.

[0097] In a specific embodiment, the metal cation chelating domain may further comprise a linker between the chelating ligand and the label and / or carrier. Such a linker may facilitate the formation of the complex and / or establish a defined distance between the label and / or carrier and the chelating ligand. The linker may, in principle, be any chemical linker suitable for covalently linking the label / carrier to the chelating ligand at the desired distance. One skilled in the art may select a linker as needed based on the tolerance of the bond to the environment and conditions of the intended use. The linker may, for example, include an antibody-drug conjugate (ADC) linker, a negatively charged sulfone group, polyethylene glycol (PEG), a pyrophosphate diester, a peptide-based linker (e.g., a cathepsin B-reactive linker, such as Val-Cit-PABC or Val-Ala-PABC; wherein Cit refers to L-citrulline and PABC refers to p-aminobenzyloxycarbonyl), a hydrazone, a disulfide-containing linker, a thioether-containing linker, a β-glucuronide, or a combination thereof. In one embodiment, the linker can be an antibody-drug conjugate (ADC) linker, a negatively charged sulfone group, a pyrophosphate diester, a peptide-based linker (e.g., a cathepsin B-reactive linker, such as Val-Cit-PABC or Val-Ala-PABC), a hydrazone, a disulfide-containing linker, a thioether-containing linker, a β-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_Review2019; and / or Jain et al., 2015, all of which are incorporated herein by reference in their entirety. Linkers based on negatively charged sulfone groups are also known in the art. Exemplary linkers with negatively charged sulfone groups that can be used in the context of the present invention are described in Zhao et al., 2011, which are incorporated herein by reference in their 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 are incorporated herein by reference in their 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 are incorporated herein by reference in their entirety. Peptide-based linkers (e.g., cathepsin B reactive linkers, such as Val-Cit-PABC or Val-Ala-PABC) are known in the art. Dubowchik et al., 2002 and / or Hartley, 2011 describe exemplary peptide-based linkers (e.g., cathepsin B reactive linkers, such as Val-Cit-PABC or Val-Ala-PABC) that can be used in the context of the present invention, the entire contents of which are incorporated herein by reference. 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 incorporated herein 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 incorporated herein by reference in its entirety. Thioether-containing linkers are known in the art. Exemplary thioether-containing linkers that can be used in the context of the present invention are described in Stenton et al., 2018, which is incorporated herein by reference in its entirety. β-glucuronide linkers are known in the art. Exemplary β-glucuronide linkers that can be used in the context of the present invention are described in Jeffrey et al., 2010, which is incorporated herein by reference in its entirety. Nucleic acid linkers, such as DNA linkers, are known in the art.

[0098] 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, e.g., photosensitive biotin: N-(4-azido-2-nitrophenyl)-aminopropyl-N'-(Nd-biotinyl-3-aminopropyl)-N'-methyl-1,3-propanediamine (Forster et al., 1985), a protein (e.g., an antibody), a peptide, or a toxin. The label may also comprise or consist of a reactive group, preferably selected from a thiol group or a reactive group suitable for click chemistry. Non-limiting examples of reactive groups suitable for click chemistry are azides, alkynes, nitrones, tetrazines, and tetrazoles. The definition of the term "derivative" mentioned elsewhere herein applies here mutatis mutandis.

[0099] Labels can include directly detectable functional moieties, such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels, as well as indirectly detectable moieties, such as enzymes or ligands, e.g., by an enzymatic reaction or molecular interaction.

[0100] 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 the chelating ligand), fluorophores, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase (used in conjunction with an enzyme that uses hydrogen peroxide to oxidize a dye precursor such as HRP), lactoperoxidase or microperoxidase, biotin / avidin, spin labels, phage labels, stable free radicals, and the like. In another embodiment, the label is a positron emitter. Positron emitters include, but are not limited to, 68 Ga, 18 F, 64 Cu, 86 Y, 76 Br, 89 Zr, and 124 1. In a particular embodiment, the positron emitter is 89 Zr.

[0101] In one embodiment, the marker is a marker that is sensitive to treatment with an oxidant, preferably H2O2. "Sensitive to an oxidant" as used herein means that the marker exhibits reduced function / activity (e.g., fluorescence in the case of a fluorophore) and / or stability after treatment with the oxidant. Preferably, the oxidant treatment is with a 20 mM H2O2 solution and the marker is sensitive after at least 0.5 h, 1 h, 2 h, or 24 h of treatment. In a preferred oxidant sensitivity test, the incubation can be carried out in a 20 mM H2O2 solution for at least 1 hour, for example, exactly 1 hour, as described for the oxidation step in Wegner and Spatz, 2013. Alternatively, the test can be carried out in a 0.05% (v / v) H2O2 solution for at least 90 minutes, for example, exactly 90 minutes, as described for the oxidation step in Zatloukalová and Kucerová. In order to shorten the test time, a solution with a higher H2O2 concentration, for example, 1% (v / v) H2O2, can also be used. Preferred H2O2 concentrations and incubation times, including time course experiments that can be used to assess H2O2 sensitivity, are described in the accompanying Examples. Assays for measuring oxidant sensitivity include incubating with a solution comprising an oxidant (e.g., H2O2) (at the desired oxidant concentration, preferably in aqueous solution, even more preferably in water) and measuring a readout (e.g., for fluorophores, measuring emitted fluorescence) that indicates marker function / activity and / or stability at various time points before and during treatment. A marker is considered "oxidation sensitive" when the readout measurement indicates a loss of marker function / activity and / or stability. A preferred time point for assessing loss of marker function / activity and / or stability is when the Co is in the presence of an oxidant used as described above. 2+ Oxidized to Co 3+ The time required. When evaluating H2O2 sensitivity, the preferred H2O2 concentrations and incubation times are those indicated above in this paragraph. An exemplary method for testing the oxidative sensitivity of fluorophore labels is described in the accompanying Examples. One skilled in the art can modify the assay based on the label used and adaptively adjust parameters such as H2O2 concentration and incubation time.

[0102] In one embodiment, the label can include a fluorophore or can be composed of a fluorophore. Fluorophores are known in the art and are publicly available and / or commercially available. The method of coupling a fluorophore to a chelating ligand in a metal cation chelating domain is also known in the art. As shown in the accompanying examples, many fluorophores (such as fluorescein, FITC, Atto488 and Alexa 488) are sensitive to H2O2 processing. This sensitivity makes the complex of the present invention particularly suitable for use when a fluorophore is used as a label. However, this advantage is not limited to a fluorophore. In one embodiment, the metal binding domain can include a fluorophore as a label and the chelating ligand can be NTA. In one embodiment, the metal binding domain can include a fluorophore as a label and the chelating ligand can be IDA.

[0103] In one embodiment, the fluorophore can be fluorescein, FITC, Atto 488, and Alexa 488. As demonstrated in the accompanying examples, these fluorophores are sensitive to H2O2 treatment.

[0104] In one embodiment, the complex of the present invention, more specifically the label and / or carrier, may not be or may not contain a porphyrin-phospholipid as described by Shao et al. (Shao et al., 2015).

[0105] In the context of the present invention the term "label" does not include single atoms such as hydrogen atoms which are part of a chelating ligand of a metal cation chelating domain. Preferably, the label relates to a structure comprising at least two atoms, at least three or at least 10 atoms.

[0106] Preferably, the label is a functional moiety and / or a moiety that can be detected using methods known in the art (eg, NMR, fluorescence measurements, enzymatic assays, etc.).

[0107] As described above, the metal binding domain may comprise a carrier (optionally also a label). Non-limiting examples of carriers according to the present invention are polymers, hydrogels, microparticles, nanoparticles, spheres (e.g., nanospheres or microspheres), beads (e.g., microbeads), quantum dots, prosthetic groups, and solid surfaces. In one embodiment, the carrier may comprise or be a nano-patterned gold surface. Such nano-patterned gold surfaces may be functionalized with thiol residues.

[0108] Those skilled in the art will appreciate that chelating ligands are attached / immobilized on a support to form a metal binding domain comprising the chelating ligand and the support. For example, if the chelating ligand is an aminopolycarboxylic acid (e.g., NTA), the chelating ligand can be covalently bound to the solid phase, for example, via at least one of a carboxylic acid group or an amino group. In another embodiment, the chelating ligand can be attached to the support via an amide bond or an ester bond.

[0109] In a preferred embodiment, the carrier is a bead (e.g., a microbead), such as an agarose bead. Thus, the present invention relates to beads (e.g., microbeads) having the complex of the present invention attached thereto (without a label and / or carrier). These beads may also be referred to as affinity matrices. Beads having the complex of the present invention are ready-to-use reagents that can be used to attach a target molecule to the bead (i.e., immobilize the target molecule to the bead).

[0110] Thus, the present invention relates to a complex (e.g. for linking a target molecule to a bead) comprising:

[0111] a)Co 3+ ;

[0112] b) a metal cation ligand selected from CO3 2- and HCO3 - carbonate or nitrate; and

[0113] c) Metal cation chelating domain comprising NTA and beads.

[0114] Preferably, the present invention relates to a complex (e.g., a complex for linking a label and / or a carrier to a target molecule) comprising:

[0115] a)Co 3+ ;

[0116] b)CO3 2- or HCO3 - ligand; and

[0117] c) Metal cation chelating domain comprising NTA and beads.

[0118] More preferably, the present invention relates to a complex (e.g. a complex for linking a label and / or a carrier to a target molecule) comprising:

[0119] a)Co 3+ ;

[0120] b)CO3 2- or HCO3 - ligand; and

[0121] c) Metal cation chelating domain comprising IDA and beads.

[0122] Such complexes can be produced as described in the accompanying examples. For example, NTA agarose resin (Qiagen, 1022963) can be washed once with 10 bead volumes of ddH2O, once with 3 bead volumes of 100mM EDTA pH7.5, and three times with 10 bead volumes of ddH2O. Subsequently, 1mM Na3[CO(III)(CO3)3]*3H2O or K3[Co(III)(CO3)3]*3H2O in 1M NaHCO3 can be added to the beads with 10 bead volumes. After incubation at 23°C with 1100 rpm in a thermostatic oscillator for 48 hours, the beads can be washed twice with 10 bead volumes of ddH2O and once with 10 bead volumes of protein buffer (50mMTris pH7.4, 150mM NaCl). Finally, the beads can be collected (e.g., by centrifugation) to obtain the complex attached to the beads (as a carrier).

[0123] As shown in the accompanying examples but without being bound by theory, the incubation time for which the metal cation, metal cation ligand and metal cation chelating domain are incubated to form the complex of the invention can affect the subsequent association of the complex of the invention with the target molecule. The incubation time for forming the complex of the invention is the "complex formation time" in the context of the present invention. The incubation time for the complex of the invention with the target molecule is the "complex-target incubation time" in the context of the present invention. Non-limiting Example 18 shows that, for example, when the complex formation time is 10 minutes, after a complex-target incubation time of 30 minutes, significantly more target molecules (i.e., His6-GFP) are bound to the complex of the invention compared to the case with the same complex-target incubation time but a complex formation time of 48 hours ( Figure 20 B) However, when the complex-target incubation time is prolonged (e.g. to 48 hours), efficient binding of His6-GFP to the complex of the present invention can be restored (see e.g. Figure 10 ). Those skilled in the art can easily and adaptively adjust the corresponding incubation time as needed.

[0124] Furthermore, the accompanying examples demonstrate that both the complex formation time and the complex-target incubation time 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 demonstrates 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 small portion of the product complex is kinetically inert, i.e., resistant to imidazole treatment ( Figure 20 B) When one incubation time, ie the complex formation time or the complex-target incubation time, is prolonged (eg to 48 hours), the proportion of kinetically inert product complexes will increase.

[0125] As also shown in non-limiting Example 18, a combination of a 10-minute complex formation time and a 48-hour complex-target incubation time can result in a high proportion of stable product complexes. In addition, a combination of a 48-hour complex formation time and a 30-minute complex-target incubation time can also result in stable product complexes (see, e.g., Example 10, Figure 11 As mentioned above, a complex formation time of 48 hours may result in a decrease in the binding efficiency between the target molecule and the complex of the present invention.

[0126] Therefore, depending on the properties of the target molecule, one skilled in the art will know how to select an appropriate incubation time. It is possible that the target molecule is very prone to denaturation or aggregation, for example, during incubation, but a kinetically inert product complex is desired. Therefore, when necessary, one skilled in the art can choose an extended complex formation time (e.g., 48 hours) and a shorter complex-target incubation time (e.g., 30 minutes), although this may reduce the amount of product complex obtained. It should be noted that the accompanying Example 18 was performed using NTA as the metal chelating ligand.

[0127] Without wishing to be bound by theory, it is noteworthy that the above-mentioned effect of incubation time may depend on the metal chelating ligand used. For example, in the accompanying non-limiting Example 15, IDA was used as the metal chelating ligand, and the example demonstrates that even with two short incubation times (i.e., 10 minutes of complex formation time and 30 minutes of complex-target incubation time), a high proportion of kinetically inert product complexes can be formed.

[0128] Therefore, it will be understood by those skilled in the art that it may be necessary to determine the impact that the incubation time can have on the stability of the product complex for each individual metal chelate ligand. However, this adaptive adjustment of the corresponding incubation time is fully within the skill range of those skilled in the art, and can be easily achieved by the teachings of the present invention and the explanation and scientific details of the experimental section provided therein. Similarly, those skilled in the art can also easily adapt other parameters, such as pH, temperature and buffer systems, to obtain kinetically inert product complexes.

[0129] Table 1 can assist one skilled in the art in determining the effect of incubation time on the stability of the product complex and on the yield of stable, ie, kinetically inert, product complexes.

[0130] For example, moderate stability means that a smaller proportion of the product complex is kinetically inert compared to an incubation time that results in high stability (i.e., a high proportion of kinetically inert product complexes). It should be noted that moderate stability can also result in a stable product complex yield that is useful in certain applications and technical fields. When an unlimited amount of target molecule is available and a stable product complex is needed in the short term, one skilled in the art may refer to the following scheme, especially when NTA is used as a metal cation chelating ligand:

[0131] - Select two shorter incubation times;

[0132] - treating the resulting product complex (e.g., bound to beads) with imidazole to remove non-kinetically inert product complex;

[0133] - recovering (e.g. by recovering beads) the product complex that is resistant to imidazole treatment, i.e., the product complex that is kinetically inert;

[0134] It is clear to a person skilled in the art how to adapt the scheme to the application.

[0135] Obviously, Table 1 can be further used to select the complex formation time and the complex-target incubation time.

[0136] Thus, the times disclosed in Table 1 can be combined with the complexes, methods and uses of the invention described herein.

[0137] Thus, the present invention relates, for example, to complexes comprising:

[0138] a) metal cations;

[0139] b) Metal cation ligand CO3 2- or HCO3 - ;and

[0140] c) a metal cation chelating domain comprising a chelating ligand and a label and / or a carrier;

[0141] The complex formation time is about 48 hours.

[0142] The present invention also relates to a complex comprising:

[0143] a) metal cations;

[0144] b) Metal cation ligand CO3 2- or HCO3 - ;and

[0145] c) a metal cation chelating domain comprising a chelating ligand and a label and / or a carrier;

[0146] The complex formation time is about 10 minutes.

[0147] It should be noted that Table 1 is for illustrative purposes only and does not limit the scope of the present invention in any way.

[0148] Table 1: Effects of complex formation time and complex-target incubation time on product complex stability and yield of stable product complexes.

[0149] If not stated otherwise, all incubation steps were performed at 25°C.

[0150]

[0151]

[0152] The interaction of NTA with Co can be enhanced by introducing energy into the system / exposure to energy during incubation 3+ This way of introducing energy into the system / exposure to energy can be electromagnetic resonance techniques at different frequencies (such as used with NMR, x-ray, UV-Vis), thermal, ultrasound or plasma resonance techniques.

[0153] The support of the present invention or its surface may be further functionalized with amino groups, carboxylic acid groups and / or activated esters such as NHS esters.

[0154] Further non-limiting examples of metal binding domain comprising supports are described in WO 2014 / 072525 A1 and WO 2003 / 072143, which are herein incorporated by reference in their entirety.

[0155] In a second aspect, the present invention relates to a composition comprising a complex according to the present invention. The description of the complex elsewhere herein applies mutatis mutandis.

[0156] In addition to the complex, the composition may contain additional compounds. Such compounds may also be chemical compounds used to produce the complex of the present invention, as described elsewhere herein. 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.

[0157] In one embodiment, the composition of the present invention may be a compound comprising the complex of the present invention and HCO3 - or CO3 2- HCO3 - or CO3 2- The presence of can prevent the release of the metal cation ligand of the complex, that is, promote the stability of the complex. When the metal cation ligand is also a carbonate, it is preferred to use HCO3 in the solution. - or CO3 2-If nitrate is used as the metal cation ligand, the solution may contain nitrate instead of HCO3 - or CO3 2- .

[0158] HCO3 in the composition solution - or CO3 2- The concentration of may be at least 1 mM, preferably at least 10 mM, most preferably 1 M. The concentration of nitrate in the composition solution may be at least 1 mM, preferably at least 10 mM, most preferably 1 M.

[0159] The present inventors have discovered that the complexes of the present invention can also be used as reagents to attach labels and / or carriers to target molecules in the presence of other components, for example, when not purified after synthesis, to achieve their functions. Therefore, the uses of the complexes of the present invention described herein and the methods of using the complexes of the present invention described herein can be implemented using the compositions of the present invention. Those skilled in the art can avoid components in the compositions that may negatively affect the efficiency and / or kinetics of the corresponding uses or methods.

[0160] In a third aspect, the present invention provides a method for producing a complex of the present invention. The production method comprises incubating together: (i) a metal cation; (ii) a metal cation ligand, and (iii) a metal cation chelating domain. During the incubation of these components in a solution (i.e., in the presence of a solvent, preferably an aqueous solution), the complex of the present invention will readily form. As described above, in the context of the present invention, the time of incubation is the "complex formation time." Table 1 and the accompanying examples can help those skilled in the art select a suitable complex formation time. Therefore, the present invention also relates to a production method comprising incubating together (i) a metal cation; (ii) a metal cation ligand and (iii) a metal cation chelating domain, wherein the incubation time, i.e., the complex formation time, is about 48 hours. The present invention also relates to a production method comprising incubating together (i) a metal cation; (ii) a metal cation ligand and (iii) a metal cation chelating domain, wherein the incubation time, i.e., the complex formation time, is about 1 hour. The present invention further relates to a production method comprising 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. Furthermore, the present invention relates to a production method comprising 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. Similarly, other incubation times are within the routine skill of those skilled in the art.

[0161] The descriptions of metal cations, metal cation ligands, and metal cation chelating domains elsewhere herein apply mutatis mutandis.

[0162] In a preferred embodiment, metal cation and metal cation ligand can be provided (for example as solid or as solution) in the form of neutral complex, preferably salt. Neutral complex (for example salt) can also comprise other components. In this embodiment, incubation means that neutral complex (for example salt) is mixed with metal cation chelating domain in solution and this mixture is kept for the time of regulation. When using neutral complex (for example salt), the method can further include the step of producing neutral complex (for example salt). Optionally, salt can be obtained as solid and can be filtered and / or washed.

[0163] Incubation can be carried out 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 about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 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 8 hours, at least about 10 hours, at least about 12 hours, at least about 15 hours, at least about 18 hours, at least about 24 hours or at least about 48 hours (also referring to Table 1).Incubation time is longer, and the complex formed is just more.At a certain reaction time point, it can be observed that the complex forms saturated. The time point at which this saturation is observed and the yield of the complex formed depend on, for example, the temperature and the concentration of the components used as well as the components themselves (e.g. the buffer selected). For this production process, reaching saturation is not essential, but can increase production yields.

[0164] Incubation can in principle be carried out at any temperature at which the solvent used for the reaction is in a liquid state. When an aqueous solution or water is used as a solvent, the temperature can be selected from 0°C to 99°C. Preferably, the temperature is selected based on the thermal stability of the marker and / or carrier used. Therefore, 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, higher temperatures can promote the formation of complexes, so higher temperatures are preferred as long as there is no negative impact on the stability of the marker and / or carrier. Those skilled in the art can select the temperature based on knowledge of the thermal stability of the marker and / or carrier.

[0165] The metal cation, metal cation ligand, and metal cation chelating domain can each be selected individually as described elsewhere herein.Preferred embodiments described elsewhere herein regarding combinations of these components or subsets thereof apply here mutatis mutandis.

[0166] The production method of the complex of the present invention may further include collecting and / or purifying the complex. As mentioned above, the complex of the present invention also functions in the presence of other compounds, particularly when it also includes a reaction mixture generated by the incubation step of the production method of the present invention. However, in some cases, it may be desirable to purify and / or isolate the complex. Washing is preferably performed with a solution containing 1 mM, preferably 10 mM, even more preferably 100 mM and most preferably 1 M CO 2- or HCO3 - As shown in the accompanying examples and figures, when the complex of the present invention is used to attach a label and / or a carrier to a target molecule (e.g., a protein), washing the complex of the present invention with such a solution can result in improved attachment of the label and / or carrier. Without being bound by theory, washing with a solution containing 1 M CO3 2- or HCO3 - The washing step can prevent CO3 2- or HCO3 - The ligand is released from the complex of the invention during washing.

[0167] If the method includes a support, purification and / or isolation may involve one or more washing steps. The support can then be isolated by removing the wash buffer from the support after the final washing step. For example, if beads are used, the beads can be precipitated by centrifugation and the supernatant can be removed. Alternatively, a filter column can be used that allows liquid to flow through but retains the complex attached to the support. The advantage of a filter column is that less support (e.g., beads) is lost during the washing steps compared to using a precipitate and aspirating the supernatant.

[0168] When a label is used, the purification method may involve the use of an affinity matrix that specifically recognizes the label. After binding to the affinity matrix, the complex may be washed one or more times to remove free reagent. In a final step, the complex may be eluted. The purification and isolation of the complex may additionally or alternatively include chromatography, such as size exclusion chromatography or anion or cation exchange chromatography. Alternatively, Ni 2+ -NTA resin separates the labeled from the unlabeled proteins because the labeled proteins are more likely to bind to Ni than the unlabeled proteins with free His tags. 2+ -NTA will show reduced to no interaction as shown by Wegner and Spatz, 2013.

[0169] Methods for purifying the complexes of the present invention, particularly when the complexes do not contain a carrier and use Co 3+ In the case of metal cations, it can also be carried out by the method described in the art (see, for example, Shibata M., 1983).

[0170] Preferred complexes of the present invention contain Co 3+ as metal cations and selected from CO3 2- or HCO3 - The method for producing this preferred complex of the present invention may comprise providing Co in the form of a neutral complex with a counter ion, for example a salt. 3+ and CO3 2- or HCO3 - Alternatively, a solution containing Co 3+ and CO3 2- or HCO3 - In a preferred embodiment, the neutral complex (and salt) is selected from sodium tris(carbonate)cobalt(III)ate trihydrate (Na3[Co(III)(CO3)3]*3H2O) or potassium tris(carbonate)cobalt(III)ate trihydrate (K3[Co(III)(CO3)3]*3H2O).

[0171] Sodium tris(carbonate)cobalt(III)ate trihydrate (Na3[Co(III)(CO3)3]*3H2O) can be synthesized as described by Bauer and Drinkard (Bauer and Drinkard 1960). Briefly, a mixture of 0.1 mol (29.1 g) of Co(II)(NO3)*6H2O (Sigma; 1.02554) and 10 ml of 30% hydrogen peroxide (Riedel-de Haen; 18312) in 50 ml of ddH2O can be added dropwise with stirring to an ice-cold slurry of 0.5 mol (=42.0 g) of sodium bicarbonate (Merck; 1.06329) in 50 ml of ddH2O. The mixture can then be incubated on ice for 1 hour with continuous stirring. The olive-colored product can then be filtered and washed three times with cold water, anhydrous ethanol, and anhydrous ether, respectively. Finally, the product can be dried under vacuum overnight and can optionally be stored in a nitrogen atmosphere at -20°C. The concentration and amount used can be adjusted adaptively by one skilled in the art. Successful production can be confirmed by NMR, for example as used in the accompanying examples.

[0172] Potassium tris(carbonate)cobalt(III)ate trihydrate (K3[Co(III)(CO3)3]*3H2O) can be synthesized in solution as described by Shibata (Shibata 1983; adaptation of Mori et al. 1956). Briefly, a mixture of 0.1 mol (24 g) Co(II)Cl2*6H2O (Honeywell; 255599) and 40 ml of 30% hydrogen peroxide in 24 ml of ddH2O can be added dropwise to an ice-cold slurry of 0.7 mol (70 g) potassium bicarbonate (Honeywell; 237205) in 70 ml of ddH2O with stirring. The resulting green solution can then be filtered (e.g., by aspiration) and used directly in subsequent experiments. The concentrations and amounts used can be adapted by one skilled in the art. Successful production can be confirmed by NMR, for example, as used in the accompanying examples.

[0173] When the metal cation ligand of the complex to be formed is HCO3 - or CO3 2- When incubated in a medium containing HCO3 - or CO3 2- Preferably, HCO3 - or CO3 2- It is provided at a concentration of at least 1 mM, preferably at least 10 mM and most preferably 1M.

[0174] It is particularly preferred that the process for producing the complexes according to the invention does not comprise an oxidation step in which the metal cation forming the center of the complex according to the invention is oxidized in the presence of a label and / or a carrier, for example by treatment with H2O2.

[0175] In one aspect, the present invention further relates to a kit comprising:

[0176] a) metal cations;

[0177] b) a metal cation ligand selected from CO3 2- and HCO3 - carbonate or nitrate, preferably CO3 2- and HCO3 - ;and

[0178] c) A metal cation chelating domain comprising a chelating ligand and a label and / or a carrier.

[0179] What is described elsewhere herein with respect to metal cations, metal cation ligands and metal cation chelating domains, chelating ligands and labels and / or carriers applies here mutatis mutandis.

[0180] The kit may be a kit for producing the complex of the present invention, ie, may contain components that can react to form the complex of the present invention. In another embodiment, the kit may contain an assembled complex.

[0181] 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(carbonate)cobalt(III)ate trihydrate (Na3[Co(III)(CO3)3]*3H2O) or potassium tris(carbonate)cobalt(III)ate trihydrate (K3[Co(III)(CO3)3]*3H2O). Preferably, the metal cation chelating domain comprises Talon, NTA or IDA coupled to a ligand and / or a carrier, preferably NTA or IDA, most preferably IDA. The kit can be used to produce the preferred Co according to the present invention. 3+ complex.

[0182] In one aspect, components a) to c) of the kit may not be provided as one entity, ie in the form of a complex according to the invention.

[0183] In another aspect, the kit can further comprise metal cations, metal cation ligands, and metal cation chelating domains assembled into the complexes of the invention, e.g., in salt form (e.g., with counterions as indicated elsewhere herein; or in solution, e.g., in a solution containing CO3 2- or HCO3 - In a solution of the invention, for example, as defined in the context of the compositions of the invention. Such kits may further comprise reagents, such as a suitable reaction buffer (preferably any buffer as described elsewhere herein for methods and uses using the complexes of the invention). Optionally, the kit may further comprise purification materials (e.g., beads, agarose beads) and / or filtration columns. In a preferred embodiment, the kit may comprise the complex of the invention, wherein the support is a bead (e.g., microbead). In this embodiment, the complex is provided as a ready-to-use affinity resin for target molecule binding.

[0184] As mentioned above, the complexes of the present invention are particularly suitable for linking labels or carriers to target molecules through metal cation-mediated interactions. The inventors surprisingly found that, compared to previously used complexes involving water as a metal cation ligand, the use of a complex selected from CO3 2- or HCO3 - The carbonate or nitrate of the target molecule can facilitate the formation of a complex with the target molecule. In the art, only [Co(III)(NTA)(CO3)] 2-Complexes (Davies and Hung, 1976; Visser et al., 2001). However, these prior art documents provide no motivation for modifying the complexes with labels, nor for using the complexes to label target molecules, such as proteins (e.g., His-tagged proteins). In particular, these prior art documents do not suggest the advantageous features discovered by the present inventors for attaching labels and / or carriers to target molecules (see the accompanying Examples).

[0185] In a fourth aspect, the present invention relates to the use of the complex of the present invention for attaching a label and / or carrier contained in the complex to a target molecule. The metal cation-mediated attachment to the label and / or carrier is achieved by contacting the complex of the present invention with the target molecule, thereby allowing the target molecule to displace the metal cation ligand in the main coordination sphere of the complex. Because the metal cation ligand is selected from CO3 2- ,HCO3 - and nitrate, so the use of the complex of the present invention can be achieved at a particularly high rate of this connection. As shown in the accompanying examples with carbonate CO3 2- For example, these metal cation ligands in the coordination sphere can be easily displaced by target molecules, especially by target molecules containing two groups that function as Lewis bases (e.g., proteins with His tags). Without being bound by theory, it is believed that the metal cation ligands of the complexes of the present invention are protonated after release from the complex, which in turn promotes their release from the complex and exchange with the target molecule. In the case of carbonate as the metal cation ligand, the protonation of carbonate leads to gas formation, which, by promoting the release of the metal cation ligand, even further promotes the binding of the target molecule. Therefore, carbonate CO 2- and HCO3 - are particularly preferred metal cation ligands in the context of the present invention.

[0186] Therefore, the present invention provides complex of the present invention or composition for being used for label and / or carrier being connected to target molecule purposes.Target molecule of the present invention comprises nucleic acid (such as DNA, RNA, or DNA or RNA analogue made by nucleotide analogue), peptide or protein.Especially, target molecule according to the present invention can be nucleic acid (such as DNA, RNA, or DNA or RNA analogue made by nucleotide analogue), peptide or protein.Particularly preferably, target molecule in the context of the present invention comprises protein or peptide.Similarly, it is also particularly preferred that target molecule is protein or peptide.The protein or peptide contained in target molecule or the protein or peptide forming target molecule must be configured so that they can act as ligand (Lewis base) in metal complex.This means that this molecule must comprise one or more parts; Particularly (one or more) amino acids, described part can act as ligand / Lewis base and can coordinate with the metal cation of complex.In other words, target molecule must be configured so that it can exchange the metal cation ligand in complex of the present invention thereby form the new complex comprising target molecule as ligand. For example, whether the ligand of the target molecule can exchange the metal cation ligand in the complex of the present invention can be tested as follows: (i) using beads functionalized with the complex of the present invention (the preparation of such beads can be performed as described in the accompanying examples); (ii) adding the target molecule (for example, according to the protocol described in the accompanying examples); (iii) incubating 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) separating the beads and the supernatant; (v) analyzing the target molecule in the supernatant and / or beads by a target-specific method (e.g., based on fluorescence, antibody detection, enzymatic assay, quantitative mass spectrometry, etc.). A negative control without complex should be included. Exchange can then be detected by measuring a decrease in the molecular weight of the target in the supernatant and / or an increase in the molecular weight of the target bound to the beads.

[0187] In a preferred embodiment, the protein or peptide of the target molecule (ie, comprised in or forming the target molecule) comprises the sequence [X n S m ] kA "metal cation ligand amino acid motif" having at least four amino acid residues, wherein X is independently selected at each position from a group of amino acids that can coordinate with the metal cation of the complex of the present invention, i.e., function as a Lewis base; S is an amino acid not included in the first group of amino acids (S is independently selected from this group of amino acids at each position); n is independently 1-4 in each case; m is independently 0-6 in each case; and k is 2-6 (see SEQ ID NOs: 4 to 8), preferably 2-5 (see SEQ ID NOs: 4 to 7), and wherein the "metal cation ligand amino acid motif" comprises at least 4, preferably at least 6, and most preferably at least 8, amino acids that can coordinate with the metal cation of the complex of the present invention. The "metal cation ligand amino acid motif" can have a regular sequence, i.e., n and m have the same value at each occurrence, or an irregular sequence, i.e., n and m can have different values. Therefore, in one embodiment, the target molecule may comprise a "metal cation ligand amino acid motif," which comprises or consists of any amino acid sequence defined in SEQ ID NOs: 4 to 8, wherein the "metal cation ligand amino acid motif" includes at least 4, preferably at least 6, and most preferably at least 8, amino acids selected from amino acids capable of coordinating with the metal cation of the complex of the present invention. Having a greater number of the first group of amino acids capable of coordinating with the metal cation of the complex of the present invention within the amino acid motif can increase the binding strength and / or specificity to the metal complex, i.e., can facilitate attachment to a label and / or carrier.

[0188] The "amino acid group capable of coordinating with the metal cation of the complex of the present invention" can be composed 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 is composed 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 is composed of glycine, tryptophan, tyrosine, cysteine, aspartic acid, glutamic acid and histidine. These amino acids are known to be suitable as metal ligands and can form complexes with metal cations. (Chin et al., 1999; McAuliff et al. 1966; Sugimori et al. 1993; Sajadi, 2010; Belland 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; ie, "X" is histidine.

[0189] As used herein, the "metal cation ligand amino acid motif" is preferably configured so that it can exchange the metal cation ligand in the complex of the present invention to form a new complex comprising the "metal cation ligand amino acid motif" as a ligand. The exchange with the metal cation ligand to form the new complex can be tested as described above for the target molecule.

[0190] Histidine is a well-studied ligand for metal cation complexes, and the use of metal ions and histidine tags to form complexes is widely used in the field of protein biochemistry, for example, to purify proteins or attach them to surfaces. In the context of the present invention, a target molecule (e.g., a protein or peptide forming or contained in a target molecule) may contain a "spacer histidine tag" that is located between the sequence [H n S m ] k wherein H is histidine, S is independently selected at each position from an amino acid residue different from histidine, preferably from an amino acid residue selected from glycine and / or serine and / or threonine, n is independently in each case 1-4, m is independently in each case 0-6, and k is 2-6 (see SEQ ID NOs: 9 to 13), preferably 2-5 (see SEQ ID NOs: 10 to 13). The spaced histidine tag may have a regular sequence, i.e. n and m have the same value at each occurrence, or an irregular sequence, i.e. n and m may have different values. Thus, in one embodiment, the target molecule may comprise a spaced histidine tag comprising or consisting of any amino acid sequence as defined in SEQ ID NOs: 9 to 13, wherein the "metal cation ligand amino acid motif" comprises at least 4, preferably at least 6, most preferably at least 8 histidine residues. A higher number of histidines in the polyhistidine tag may increase the affinity for metal cations (e.g. Co 3+ ) binding strength and specificity. However, excessive consecutive histidines can, in some cases, reduce the expression level and solubility of recombinant proteins (e.g., proteins expressed recombinantly in E. coli). These problems can be overcome by interrupting the continuous appearance of histidines with short spacers containing glycine, serine, or threonine, i.e., using spaced histidine tags.

[0191] In a particularly preferred embodiment, the target molecule may comprise a His-tag. The His-tag may comprise 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 comprise 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 a particularly preferred embodiment, 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.

[0192] The His-tag can be a sequence segment that occurs in a natural protein or can be a recombinant histidine tag. "Recombinant" herein means a His-tag that is artificially generated by genetic engineering, for example by altering the encoding nucleic acid sequence to allow expression of a fusion protein containing the His-tag.

[0193] 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 segment. In some embodiments, the His-tag may be at both the N-terminus and the C-terminus.

[0194] 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, and most preferably at least 6 histidine or histidine-like residues are in close spatial proximity. In this context, "spatial proximity" preferably means that the distance between each residue is 0 to 5 angstroms. The "distance" between two residues is the shortest distance between the two nearest nitrogen atoms of two independent adjacent histidine side chains or histidine-like side chains.

[0195] Non-limiting preferred examples of target molecules according to the invention are drugs, diagnostic agents, research agents, cosmetics and / or proteins for environmental treatment (e.g. proteins for water treatment).

[0196] Other non-limiting preferred examples of target molecules are molecules comprising or consisting of enzymes, targeting 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.

[0197] The target molecule used in the context of the present invention may also be a nucleic acid. Nucleic acids include 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 comprises 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, it is envisaged that the target molecule is an origami structure, i.e., a nucleic acid structure having a three-dimensional fold. Such an origami structure is typically formed by base pairing of several nucleic acid strands including a scaffold strand and a staple strand.

[0198] The use of the complex or composition of the present invention for labeling a target molecule involves exchanging the metal cation ligand of the complex of the present invention for the target molecule, thereby attaching the label and / or carrier to the target molecule. The exchange is achieved by contacting the complex of the present invention with the target molecule. Therefore, the use preferably comprises contacting the complex of the present invention with the target molecule in solution.

[0199] Thus, in one aspect, the present invention relates to a method for attaching a label and / or a carrier to a target molecule, comprising the step of incubating a complex of the invention or a composition comprising a complex of the invention with the target molecule. The target molecule is a target molecule as defined elsewhere herein.

[0200] "Incubation" refers to mixing the complex and the target molecule in a solution and allowing the mixture to react for a specified time. As described above, in the context of the present invention, the time for incubating the complex with the target molecule is the "complex-target incubation time". Table 1 and the accompanying examples can help those skilled in the art to select the complex-target incubation time. Thus, the present invention relates, for example, to the use of the complex of the present invention for connecting a label and / or a carrier contained in the complex to a target molecule, wherein the complex-target incubation time can be about 48 hours. As described herein, the complex formation time can also affect the formation of the product complex. As also described herein, the complex formation time can be selected based on Table 1 or the accompanying examples. Thus, the present invention relates, for example, to the use of the complex of the present invention for connecting a label and / or a carrier contained 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 also relates to the use of the complex of the present invention for connecting a label and / or a carrier contained 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 complex of the present invention for connecting a label and / or a carrier contained 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 complex of the present invention for connecting a label and / or a carrier contained 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 complex of the present invention for connecting a label and / or a carrier contained 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.

[0201] In a specific embodiment of the use of the complex of the present invention for linking a label and / or carrier contained in the complex to a target molecule, the metal chelating ligand is IDA and the complex formation time is 10 minutes and the complex-target incubation time is about 30 minutes.

[0202] In another preferred embodiment of the use of the complex of the present invention for linking a label and / or carrier comprised in the complex to a target molecule, the metal chelating ligand is IDA and the complex formation time is 10 minutes and the complex-target incubation time is about 3 hours.

[0203] The method of attaching a marker and / or a carrier to a target molecule may comprise the step of recovering and / or purifying the target molecule to which the marker and / or carrier is attached. This will provide an isolated target molecule to which the marker and / or carrier is attached.

[0204] If the method involves attaching a support, purification and / or isolation of the target molecule to which the support is attached can be achieved by separating the support. For example, if a solid support is used, the beads can be pelleted by centrifugation and the supernatant removed. Alternatively, a filtration column can be used that allows liquid to flow through but retains the complex attached to the support. Optionally, the support can be washed with a buffer solution to remove impurities.

[0205] Typically, whether a carrier or label is used, separation and / or purification can be performed by chromatography. The chromatography can include or consist of size exclusion chromatography, which utilizes the differences in molecular size and shape between the target molecule attached to the carrier and / or label and the free target molecule and the complex of the invention.

[0206] Methods that can be used to purify target molecules carrying a label and / or a carrier according to the present invention are also described in WO 2014 / 072525, which is incorporated herein by reference in its entirety.

[0207] In one embodiment, purification can also be performed using a conventional Ni-NTA column as described in WO 2014 / 072525.

[0208] The incubation of the complex of the present invention or the composition comprising the same can be carried out in solutions with different pH values. The connection of the label and / or carrier to the target molecule can occur at various pH values. The lower the pH value, the more favorable the release of the metal cation ligand (i.e., carbonate or nitrate). This is because the released ligand can be protonated. When protonated, the carbonate CO3 2- and HCO3 - It can even form gas and release it.

[0209] Although in principle lower pH values ​​are conducive to exchange reactions, the pH during incubation is generally selected within a range compatible with the target molecule used. In particular, proteins are sensitive to too low or too high pH values ​​in many cases. It is preferred to select pH so 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, it is also preferred that pH be selected at the lower limit of the pH stability range of the target molecule to promote ligand exchange, i.e., the connection of the label and / or carrier to the target molecule. Those skilled in the art are aware of assays for testing the stability and function of proteins at different pH values.

[0210] In a preferred embodiment, the pH during incubation of the complex of the invention with the target molecule is between 4.0 and 9.5, preferably between 5.5 and 8.0. Within these pH ranges, most target molecules, in particular most proteins, are stable and / or functional.

[0211] The exchange of metal cation ligand and target molecule occurs usually in a short time.Therefore, according to required connection efficiency and used target molecule, the incubation step for connecting the method for label and / or carrier according to the present invention can be as short as only at least about 10 seconds, preferably at least about 1 minute, most preferably at least about 10 minutes.In a preferred embodiment, incubation is carried out 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.Not subject to theoretical constraints, it is believed that longer incubation causes higher connection yield.However, after certain hour incubation, it is possible to identify the saturation that label and / or carrier are connected.Preferred incubation time can depend on used target molecule and required connection efficiency.Those skilled in the art can test connection efficiency by measuring the consumption of free label and / or carrier and / or quantifying the target molecule with label and / or carrier connected.

[0212] The incubation step of the method for attaching a label and / or a carrier to a target molecule can be carried out at different temperatures. The exchange of the metal cation ligand with the target molecule can, in principle, be carried out at any temperature between 0 and 95°C. The higher the temperature, the faster the ligand exchange reaction. However, since the target molecule comprises 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, proteins derived from thermophilic bacteria will tolerate higher reaction temperatures than proteins from non-thermophilic sources. Similarly, DNA is more heat-resistant than RNA. The temperature therefore 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 can be kept constant during the incubation process. Alternatively, one or more different temperatures or temperature gradients can be used. In one embodiment, the target molecule is a protein and the temperature is selected from 0°C to about 25°C, preferably about 2°C to about 8°C.

[0213] As mentioned above, the incubation step is carried out in a solution. Different solutions can be used in this case, including pure water or an aqueous solution. Therefore, in one embodiment, the incubation can be carried out in water or an aqueous solution. Alternatively or additionally, the solution can comprise one or more organic solvents. A non-limiting group of preferred organic solvents includes DMSO, DMF, DMS, acetonitrile and isopropanol. Therefore, in one embodiment, the incubation can be carried out in a solution comprising one or more organic solvents selected from the group consisting of DMSO, DMF, DMS, acetonitrile and isopropanol. Those skilled in the art will select a solution so that it does not interfere with downstream applications and / or the stability or activity of the target molecule. In the case of proteins, an aqueous solution is particularly preferred, and preferably has physiological conditions.

[0214] As demonstrated in the accompanying examples, the method of attaching a label and / or carrier to a target molecule can be performed in the presence of various buffering agents. Thus, in a preferred embodiment, the incubation step of the method can be performed in an aqueous solution comprising one or more Good's buffer substances.

[0215] Good's buffer substances are a group of 20 buffers selected and described by Norman Good and colleagues for biochemical and biological research between 1966 and 1980 (see Good et al., 1966; Good et al., 1972; Ferguson et al., 1980). These buffers are known in the art.

[0216] Exemplary but non-limiting buffers are ACES, AMPSO, BES, BisTris propane, borate, CAPS, CAPSO, CHES, DIPSO, EPPS, HEPES, HEPBS, HEPPSO, MES, MOPS, MOPSO, PIPES, POPSO, TAPS, TAPSO, TEA, TES, and Tris, and derivatives thereof may be used. "Derivatives" refer to buffer substances having the same structural backbone but substituted with other chemical moieties. Alternatively or additionally, Tris buffer, carbonate / bicarbonate buffer, or phosphate buffer (e.g., PBS) may also be used.

[0217] In one embodiment, a buffer substance selected from the group consisting of BisTris and derivatives, carbonate / bicarbonate buffers, CAPS, CAPSO, HEPES, HEPBS, HEPPSO MES, MOPS, MOPSO, PIPES, phosphate buffers (e.g., PBS), TAPS, TES, and Tris may be used.

[0218] When using these exemplary buffers, the pH of the solution can be selected as follows:

[0219] 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; borate: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; H EPBS:7.4-9.2; MES:5.2-7.0; MOPS:6.2-8.2; MOPSO:6.0-8.0; Phosphate: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.

[0220] In principle, other pH values ​​can also be used with each of the above buffers, as the reaction can be carried out over a wide pH range and therefore buffering capacity is not absolutely necessary. However, in order to keep the pH constant and better control the reaction, a pH within the above pH range is preferred, in which the buffer has a buffering capacity.

[0221] The buffer can be used at different concentrations, for example, 1 mM to 1 M, preferably 1 mM to 250 mM, most preferably 1 mM to 100 mM (e.g. 50 mM). One skilled in the art can test the ideal buffer concentration by evaluating the ligation efficiency and using different buffer concentrations.

[0222] As demonstrated in the accompanying examples, the choice of buffer material / agent can influence the efficiency of attachment of the label and / or carrier to the target molecule. Without being bound by theory, buffers with low metal binding constants exhibit increased attachment efficiency. A low metal cation binding constant can prevent the buffer from coordinating with the metal and blocking ligand sites in the primary coordination sphere.

[0223] Therefore, in one embodiment, the buffer substance / agent used in the context of the present invention may be a buffer substance / agent with a low metal binding constant. Ferreira et al. 2015 discloses the understanding of "low metal binding constant" and preferred examples of such buffer substances / agents, the entire contents of which are incorporated herein by reference.

[0224] Furthermore, in a preferred embodiment, the incubation step is carried out in the presence of a buffer substance / reagent comprising one or fewer (preferably zero) groups selected from amine and carbonyl groups. These groups include Lewis bases, which can mediate binding to metal cations in the complex. Thus, the absence of these groups can reduce or prevent the binding of the buffer substance to the metal cation, thereby promoting binding of the target molecule.

[0225] As demonstrated in the accompanying examples, the following buffers can advantageously facilitate label and / or carrier attachment: MES, HEPES, Bis-Tris, and PIPES. The examples illustrate that these buffers result in higher attachment efficiencies than Tris-based buffers. Therefore, in a particularly preferred embodiment, the buffering substance present in the solution during incubation can be selected from MES, HEPES, Bis-Tris, and PIPES. These buffering substances are particularly advantageous for label and / or carrier attachment. Non-limiting Example 13 illustrates that it may be necessary to select a buffer based on the pH used for incubation. When incubating the complex of the present invention and the target molecule at a pH of approximately 7.5, HEPES can be used instead of BisTris. When incubating the complex of the present invention and the target molecule at a pH of approximately 5.5, BisTris can be used. The determination of the most suitable buffer and pH value is entirely within the skill of the relevant technician and can be easily achieved through the teachings of the present invention and the description and scientific details of the experimental section provided therein.

[0226] In view of these results and based on the theoretical consideration of keeping the binding of the buffer to metal cations as low as possible, the buffer substance may preferably be selected from: BisTris, CAPS, CAPSO, HEPES, HEPBS, HEPPSO, MES, MOPS, MOPSO, PIPES, TAPS and TES.

[0227] In a preferred embodiment, Ca may be present in the solution. 2+ ions are incubated with the complex of the present invention or a composition comprising the complex and the target molecule. When the metal cation ligand of the complex of the present invention is selected from HCO3 - or CO3 2- When the carbonate ion 2+ The presence of Ca ions is particularly preferred. This is because 2+ The ions can form insoluble CaCO3, which precipitates from the solution, thereby promoting the release of carbonate from the complex. This in turn promotes the binding of the target molecule as a ligand, thereby facilitating the connection of the label and / or carrier with the target molecule. Preferably, CaCl2 is provided by dissolving CaCl2 in the reaction solution. 2+ Ion. Ca 2+Ions can be added at the beginning of the reaction, i.e., directly when the complex of the present invention is in contact with the target molecule. Alternatively, less preferably, CaCl2 can be added during the reaction in the form of a salt (preferably CaCl2). 2+ Ion. Ca 2+ The ions (preferably in the form of CaCl2) are preferably added in a concentration of 0.1 to 50 mM, even more preferably 0.1-10 mM and most preferably 1 mM.

[0228] The method for attaching a label and / or support to a target molecule according to the present invention does not require an oxidation step, for example by treatment with H2O2, to oxidize the metal cation of the complex according to the present invention. In a preferred embodiment, the method for attaching a label and / or support to a target molecule according to the present invention does not include an oxidation step, for example by treatment with H2O2. This is done in the formation of Co 3+ In the case of mediated label and / or carrier attachment, Co 3+ The ligation of the functional part is mediated by forming a His tag target protein as a ligand Co 2+ complex, and only then oxidize Co 2+ For Co 3+ This is achieved. As demonstrated in the accompanying examples, this oxidation step in the presence of Co cations leads to a spontaneous Fenton reaction, which results in protein degradation. In addition, protein oxidation can interfere with protein folding and function. Therefore, a particular advantage of the method of the present invention is that no oxidation step is required.

[0229] The method for attaching a label and / or a carrier to a target molecule may further comprise producing a complex of the invention (and optionally a neutral complex, such as a salt thereof) as described elsewhere herein.

[0230] The method of attaching a marker and / or a carrier to a target molecule according to the present invention results in the production of a labeled and / or carrier-attached target molecule. Therefore, the method may also be referred to as a method for producing a target molecule attached with a marker and / or carrier. The preferred embodiments of the target molecule indicated elsewhere herein are hereby incorporated by reference. Therefore, in one embodiment, the method of the present invention may be a method for attaching a marker and / or carrier to a protein, such as a His-tagged protein, an antibody, a derivative thereof (e.g., including scFv fragments and nanobodies), or a domain thereof.

[0231] A preferred embodiment of the method of attaching a marker and / or a carrier to a target molecule is that the target molecule is a His-tagged protein and the His-tagged protein is attached to a carrier, which is a surface (e.g. a chip). In this preferred embodiment, Co 3+In the method using a His-tagged protein as the metal cation, a carbonate as defined herein as the metal cation ligand, and NTA, Talon, or IDA as the chelating ligand, the His-tagged protein is attached to the surface. Due to the high stability and kinetically inert binding of the target molecule, the protein can be bound to the surface in a nearly covalent manner and is inert to imidazole and other chelates (e.g., EDTA) and reduction equivalent treatments.

[0232] In yet another aspect, the present invention relates to a target molecule having a marker and / or carrier attached thereto obtainable or obtained by a method according to the present invention for attaching a marker and / or carrier to a target molecule. The target molecule obtained by the method of the present invention, labeled and / or carrier-attached has the following characteristics: neither the target molecule nor the marker and / or carrier undergoes an oxidation step (e.g., H2O2 treatment). In contrast, previously reported methods for producing such structures involve an oxidation step in the presence of i) the target molecule and ii) at least one or both of the marker and / or carrier. Therefore, the product of the method of the present invention has the advantage of not being oxidized and not containing an oxidizing agent such as H2O2. This also has a key advantage for the medical use of the target molecule attached to the marker and / or carrier produced.

[0233] What is described elsewhere herein with respect to the complexes according to the invention, their components, target molecules and methods for attaching labels and / or carriers to target molecules applies here mutatis mutandis.

[0234] The target molecule to which the label and / or carrier is attached, which can be obtained by the method of the present invention, is also a complex. The obtained "product complex" comprises the metal cation of the complex of the present invention and coordinated with i) the metal binding domain of the complex of the present invention and ii) the target molecule.

[0235] The present invention also provides a composition comprising a labeled and / or carrier-linked target molecule obtainable or obtained by the method of linking a label and / or carrier to a target molecule.

[0236] The target molecule of the mark that can obtain or obtain by the inventive method and / or carrier connection, or the composition comprising the same, can be used as research reagent. Therefore, the present invention also relates to the purposes of the target molecule of the mark that can obtain or obtain by the inventive method or carrier connection as research reagent. Similarly, a method is provided, which comprises the steps of using the target molecule of the mark that produces by the inventive method or carrier connection as research reagent. For example, the target molecule can be an extracellular matrix protein that is fixed on a solid phase carrier and is used for cell culture.

[0237] In one embodiment, the labeled and / or carrier-linked target molecules obtainable or obtained by the methods of the present invention, or compositions comprising the same, can be used as in vitro diagnostic agents. For example, the target molecule can be a detectable protein that specifically recognizes an analyte (e.g., an antibody that can recognize an analyte antigen), and the label and / or carrier can be configured to conform to measurement methods known in the art.

[0238] The present invention relates to a target molecule obtained or obtained by the method of the present invention, which is labeled and / or linked to a carrier, or a composition comprising the same, for use as a medicament. Similarly, a method of treatment is provided, which comprises administering to a patient an effective amount of a target molecule obtained or obtained by the method of the present invention, which is labeled and / or linked to a carrier, or a composition comprising the same. Preferably, the target molecule is selected from an enzyme, a targeting protein such as an antibody, a cytokine (e.g., G-CSF), 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 synthetic engineered protein, or a fragment thereof.

[0239] The present invention particularly also includes embodiments in which, when used as an in vivo drug, the target molecule and / or the marker can be released from the "product complex" comprising the target molecule, for example, after the complex binds to the target structure in vivo. Thus, the target molecule to which the marker and / or carrier is attached, obtainable by the method of the present invention, can be configured in a form that allows the marker and / or target molecule to be released, preferably in vivo. The release can be triggered by reduction of metal cations in the product complex, a change in pH, and / or by binding of the target molecule and / or marker to a target structure (e.g., a receptor or cell surface protein, e.g., a cancer cell-specific surface protein).

[0240] The present invention provides, inter alia, the following embodiments:

[0241] 1. A complex comprising:

[0242] a) metal cations;

[0243] b) Metal cation ligand CO3 2- or HCO3 - ;and

[0244] c) A metal cation chelating domain comprising a chelating ligand and a label and / or a carrier.

[0245] 2. The complex of item 1, wherein the chelating ligand of the metal cation chelating domain is a multidentate ligand comprising one or more carboxylic acid groups and / or one or more amine groups and / or one or more aromatic amines and / or phosphate groups.

[0246] 3. The complex of item 1, wherein the chelating ligand of the metal cation chelating domain in c) is selected from:

[0247] Nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), tris(carboxymethyl)ethylenediamine (TED), chelating peptides such as those with a consensus sequence (GHHPH) n G, wherein n=1-3 (see SEQ ID NOs: 1 to 3) or cadystin, triazacyclononane (TACN), diethylenetriamine-pentaacetate (DTPA), phytochelatin, carboxymethylaspartic acid (CMA), phosphonate, tannic acid (TA), porphyrin, dipyridylamine (DPA), phytic acid, nitrilopropionic acid diacetic acid (NPDA), nitriloisopropionic acid diacetic 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-tetraazacyclodecane, 1,4,7-triazacyclononane-1,4,7-triacetic acid (DOTA), Acetic 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, ethylenebis(oxyethylene-nitrilo)tetraacetic acid (EGTA), triethylenetetraamine-hexaacetic acid (TTHA), 1,4,7-triazacyclononanephosphinic acid (TRAP), deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), purine, pyrimidine and their derivatives.

[0248] 4. The complex of item 1, wherein the chelating ligand of the metal cation chelating domain of c) is selected from: nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), a chelating ligand having a consensus sequence (GHHPH) n Chelating peptides of G, diethylenetriamine pentaacetate (DTPA), nitrilopropionic acid diacetic acid (NPDA), nitriloisopropionic acid diacetic acid (NIPDA), ethylenediamine-tetraacetic acid (EDTA), ethylenebis(oxyethylene-nitrilo)tetraacetic acid (EGTA), carboxymethylaspartic acid (CMA) and its derivatives.

[0249] 5. The complex of item 1, wherein the chelating ligand of the metal cation chelating domain in c) comprises or is selected from NTA, IDA and derivatives thereof.

[0250] 6. The complex according to any one of items 1 to 4, wherein the metal cation is a transition metal cation.

[0251] 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.

[0252] 8. The complex of any one of items 1 to 6, wherein the metal cation is a water ligand exchange rate of 10 -1 s -1 or lower, preferably 10 -2 s -1 or lower metal cations.

[0253] 9. The complex of any one of items 1 to 7, wherein the metal cation is selected from: 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+ He Lu 3+ .

[0254] 10. The complex of any one of items 1 to 7, wherein the metal cation is selected from: Co 3+ ,Cr 3+ ,Rh 3+ ,Ir 3+ ,Ir 4+ ,Pt 2+ ,Pt 4+ ,Pd 4+ ,Mo 3+ ,Fe3+ ,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+ .

[0255] 11. The complex of any one of items 1 to 7, wherein the metal cation is Co 3+ .

[0256] 12. The complex according to any one of items 1 to 11, wherein the metal cation ligand of b) is carbonate CO3 2- or bicarbonate HCO3 - .

[0257] 13. The complex according to any one of items 1 to 12, wherein the complex comprises [Co(III)(NTA)CO3] 2- ,[Co(III)(NTA)HCO3] - The complex or its hydrate, wherein the label and / or carrier is linked to NTA.

[0258] 14. The complex of 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 thiols, azides, alkynes, nitrones, tetrazines and tetrazoles.

[0259] 15. The complex according to any one of items 1 to 13, wherein the label comprises or is a fluorophore.

[0260] 16. The complex according to any one of items 1 to 13, wherein the label comprises or is biotin or a derivative thereof.

[0261] 17. The complex of any one of items 1 to 13, wherein the carrier is a polymer, a hydrogel, a microparticle, a nanoparticle, a sphere (including nanospheres and microspheres), a bead, a quantum dot, a prosthetic group, or a solid surface.

[0262] 18. The complex of items 1 to 17, wherein the metal cation chelating domain comprises a linker between the chelating ligand and the label and / or carrier.

[0263] 19. A composition comprising the complex as defined in any one of items 1 to 18.

[0264] 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, peptide or nucleic acid, preferably a protein or DNA, which can exchange the metal cation ligand in the complex, and even more preferably, wherein the target molecule has a sequence [H n S m ] k wherein H is a histidine residue or a histidine-like residue, wherein S is a spacer amino acid residue, wherein n is independently at each occurrence from 1 to 4, wherein m is independently at each occurrence from 0 to 6, and wherein k is 2 to 6.

[0265] 21. Use of the complex of any one of items 1 to 18 or the composition of item 19 for labeling a target molecule, wherein the target molecule contains a histidine-rich region comprising at least two histidine residues, wherein the histidine-rich region is formed by a three-dimensional folding of the target molecule, which folding brings the at least two histidine residues into close proximity in space, wherein the at least two histidine residues have a distance of 0 to 5 angstroms and are not consecutive in the amino acid sequence.

[0266] 22a. The use according to claim 21, wherein the histidine-rich region is the Fc region of an antibody.

[0267] 22b. Use of the complex according to any one of items 1 to 18 or the composition according to item 19 for attaching a label and / or a carrier to an antibody, a domain thereof (eg, an Fc region) or a fragment thereof.

[0268] 22c. The use of item 22b, wherein the marker is a toxin.

[0269] 23. Use of the complex of any one of items 1 to 18 or the composition of item 19 for labeling a target molecule, wherein the target molecule contains a region rich in histidine-like residues, which region is generated when the histidine-like residues are spatially close during the three-dimensional folding of the target molecule, wherein the at least two histidine-like residues have a distance of 0 to 5 angstroms and are not continuous in the amino acid sequence.

[0270] 24. The use according to item 20, 21 or 23, wherein the target molecule is a drug, a diagnostic agent, a research agent, a cosmetic and / or a protein for environmental treatment (e.g. water treatment).

[0271] 25. The use according to item 20, 21 or 23, wherein the target molecule comprises or is a peptide or protein.

[0272] 26. The use of claim 20, 21, 23, 24 or 25, wherein the target molecule comprises or is an enzyme, a targeting 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 synthetic engineered protein or a fragment thereof.

[0273] 27. The use according to any one of items 20 to 26, wherein the target molecule comprises a histidine residue or a histidine-like residue at the N-terminus, the C-terminus or in an internal sequence region.

[0274] 28. The use according to any one of items 20 to 26, wherein the histidine residue or histidine-like residue is contained in the form of a His-tag, preferably wherein the His-tag consists of 2 to 10, preferably 4 to 8, and most preferably 6 to 8 consecutive residues.

[0275] 29. Use of the complex according to any one of items 1 to 18 or the composition according to item 19 for producing a medicament, a diagnostic agent and / or a cosmetic.

[0276] 30. A kit comprising:

[0277] a) a metal cation, preferably a metal cation as defined in any one of items 5 to 11;

[0278] b) Metal cation ligand CO3 2- or HCO3; and

[0279] c) A metal cation chelating domain comprising a chelating ligand and a label and / or a carrier, preferably a metal cation chelating domain as defined in any one of items 2 to 4 and 14 to 18.

[0280] 31. A method for producing the complex of any one of items 1 to 18, comprising incubating in solution: (i) a metal cation; (ii) a metal cation ligand as defined in item 1b); and (iii) a metal cation chelating domain as defined in item 1c).

[0281] 32. The method of item 31, further comprising collecting and / or purifying the complex of any one of items 1 to 18.

[0282] 33. The method of item 31 or 32, wherein the metal cation chelating domain is a metal cation chelating domain as defined in any one of items 2 to 18.

[0283] 34. The method of any one of items 31 to 33, wherein the metal cation is a metal cation as defined in any one of items 5 to 11.

[0284] 35. The method of any one of items 31 to 34, wherein the metal cation is Co 3+ wherein the metal cation binding ligand is CO3 2- or HCO3 - , and among them Co 3+ and CO3 2- or HCO3 - Provided in the form of a neutral complex with a counterion, for example, in the form of a salt, or in the form of a 3+ and CO3 2- or HCO3 - The charged complex is provided in the form of a charged complex.

[0285] 36. The method of item 35, wherein the neutral complex is sodium tri(carbonate)cobalt(III)ate trihydrate (Na3[Co(III)(CO3)3]*3H2O) or potassium tri(carbonate)cobalt(III)ate trihydrate (K3[Co(III)(CO3)3]*3H2O).

[0286] 37. The method of any one of items 31 to 36, wherein in the presence of HCO3 - or CO3 2- Incubate in a buffer solution, preferably HCO3 - or CO3 2- The concentration is at least 1 mM, preferably at least 10 mM, most preferably 1M.

[0287] 38. A method for linking a label and / or a carrier to a target molecule, comprising the step of incubating the complex of any one of items 1 to 18 or the composition of item 19 with the target molecule, wherein the target molecule is a target molecule as defined in any one of items 20 to 28.

[0288] 39. The method of item 38, wherein the method further comprises a step of recovering and / or purifying the target molecule to which the label and / or carrier is attached.

[0289] 40. The method of item 38 or 39, wherein the pH of the solution is from 4.0 to 9.5, preferably from 5.5 to 8.0.

[0290] 41. The method of any one of items 38 to 40, wherein the incubation is performed for at least 10 seconds, preferably 1 minute, most preferably 10 minutes.

[0291] 42. The method of any one of items 38 to 41, wherein the incubation is carried out at a temperature between 0 and 95°C, preferably between 0 and 60°C, most preferably between 0 and 42°C.

[0292] 43. The method of any one of items 38 to 42, wherein the method further comprises, before incubation, - or CO3 2- The complex of the present invention is washed in a solution, preferably HCO3 - or CO3 2- The concentration of HCO3 is at least 1mM, preferably 10mM, most preferably 1M, and / or wherein - or CO3 2- Incubate in a solution, preferably HCO3 - or CO3 2- The concentration is at least 1 mM, preferably 10 mM, most preferably 1 M.

[0293] 44. The method of any one of items 38 to 43, wherein the incubation is carried out in water or an aqueous solution.

[0294] 45. The method of any one of items 38 to 44, wherein the incubation is carried out in a solution comprising one or more organic solvents selected from the group consisting of DMSO, DMF, DMS, acetonitrile and isopropanol.

[0295] 46. ​​The method of any one of items 38 to 45, wherein the incubation is carried out in an aqueous solution comprising one or more of Good's buffer substances, Tris, phosphate and / or carbonate / bicarbonate.

[0296] In one embodiment of this project, the buffer can be PBS.

[0297] 47. The method of any one of items 38 to 46, wherein in Ca 2+ Incubate in the presence of, preferably Ca 2+ Supplied as CaCl2.

[0298] 48. The method according to any one of items 38 to 47, wherein the incubation is carried out in an aqueous solution comprising one or more buffer substances, wherein the buffer substance(s) do not comprise amine, carboxylic acid, aromatic amine and / or phosphate groups.

[0299] 49. The method of any one of items 38 to 48, wherein the incubation is carried out in an aqueous solution comprising one or more buffer substances selected from the group consisting of: ACES, AMPSO, BES, BisTris, BisTris propane, borate, 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.

[0300] 50. The method of any one of items 38 to 49, wherein the incubation is carried out in an aqueous solution containing one or more buffer 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.

[0301] 51. The method of any one of items 38 to 50, wherein the incubation is carried out in an aqueous solution comprising a buffer substance selected from the group consisting of Bis-Tris, MES, HEPES and PIPES.

[0302] 52. The method of any one of items 38 to 51, wherein the method does not involve an oxidation step in the presence of a label and / or a carrier, such as treatment with H2O2.

[0303] 53. A labeled or carrier-linked target molecule obtainable by the method defined in any one of items 38 to 52.

[0304] 54. A composition comprising the labeled or carrier-linked target molecule of item 53.

[0305] 55. Use of the labeled or carrier-linked target molecule of item 53 or the composition of item 54 as a research reagent.

[0306] 56. The labeled or carrier-linked target molecule of item 53 or the composition of item 54 for use as a medicament.

[0307] A "complex" according to 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 metal complex. The 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 complex according to the present invention has at least two ligands as defined elsewhere herein.

[0308] As used herein, the terms "protein" and "peptide" both refer to polypeptides composed 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 includes "peptide" and "protein." When referring to "protein or peptide" herein, polypeptides are also included.

[0309] As used herein, an "antibody" is any molecule that can specifically or selectively bind to a target protein. An antibody may include or may be an antibody or a portion / fragment thereof, wherein the portion / fragment exhibits substantially the same binding activity as a full-length antibody. Antibodies may also include multivalent molecules, multispecific molecules (e.g., diabodies), fusion molecules, aptimers, avimers, or other naturally occurring or recombinantly produced molecules. Exemplary antibodies useful in the present invention include antibody-like molecules. Antibody-like molecules are molecules that can exhibit functions by binding to target molecules (see, for example, 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, DARPins (WO 2002 / 020565), Affibodies (WO 1995 / 001937), Avimers (WO 20011 / WO 2005 / 040229), Adnectins (WO 2002 / 032925), and fynomers (WO 2013 / 135588). In general, the term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to 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. The antibodies of the present invention may also be chimeric antibodies, recombinant antibodies, antigen-binding fragments of recombinant antibodies, or humanized antibodies.

[0310] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to a molecule that is not an intact antibody, comprises a portion of an intact antibody, and binds to the 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 antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibodies.

[0311] The term "ligand" refers to a substance that interacts with another substance in some way. In the context of the present invention, when used in the context of a complex, a "ligand" refers to a molecule comprising a Lewis base that can form a coordinate bond with a Lewis acid. In other examples, a ligand is a substance, typically organic, that comprises a group that can form a coordinate bond with a metal ion. A ligand, when coordinated to a metal ion, can have a variety of 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 a Lewis base binds to more than one metal ion).

[0312] The terms "Lewis acid" and "Lewis acidic" are art-recognized and refer to a chemical moiety that can accept a pair of electrons from a Lewis base as defined above.

[0313] The terms "Lewis base" and "Lewis-based" generally refer to a chemical moiety that is capable of donating a pair of electrons under certain reaction conditions. Depending on the identity of the Lewis base and the metal ion, a Lewis base can have the characteristic of donating a single electron in certain complexes, but for most purposes, it is preferred to understand Lewis bases as two-electron donors. Examples of Lewis basic 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 some instances, a Lewis base can consist of a single atom, such as an oxide.

[0314] The term "coordinate" or "coordinating" refers to the interaction between a ligand and a metal cation.

[0315] The term "diagnostic" or "diagnostic agent" in the context of the present invention relates to a diagnostic type of reagent. Non-limiting examples are radionucleotides that can be subsequently detected using positron emission tomography (PET) or single photon emission computed tomography (SPECT) imaging or other methods known to those skilled in the art, fluorescent moieties that can be detected by methods known in the art, and enzymatic moieties. Diagnostic agents may also include antibodies comprising a radionucleotide, fluorophore, or enzyme attached thereto.

[0316] The term "drug" refers to any therapeutic or prophylactic substance, including but not limited to small molecules, biopharmaceuticals (such as antibodies). A preferred drug is an antibody.

[0317] The invention is illustrated by the following figures and examples. Description of the accompanying drawings:

[0319] Figure 1 : Chemical reactivity of the [Co(III)(NTA)(His-protein)] complex.

[0320] In Co 2+ and Co3+ NTA beads with immobilized His6-GFP (SEQ ID NO: 14) at the center of the complex were incubated with different chelating agents and combinations of reducing agents and 250 mM imidazole, and the amount of eluted His6-GFP was measured. 3+ The center is kinetically inert, and when immobilized on beads as [Co(III)(NTA)(His6-GFP)], His6-GFP is barely eluted. Figure adapted from Wegner and Spatz, 2013.

[0321] Figure 2 : Schematic diagram of the method for chemically labeling His-tagged proteins by [Co(III)(NTA)].

[0322] According to Wegner & Spatz (Wegner and Spatz) published in 2013, the [Co(III)(NTA)(His-protein)] complex was formed by oxidation of the preformed [Co(II)(NTA)(His-protein)] complex. NTA was preloaded with Co 2+ ions (from Co(II)Cl2) and incubated with His-tagged proteins. Finally, the Co 2+ Center converted to Co 3+ However, this approach has a major drawback, namely, the oxidation step can affect the function and stability of the conjugated protein. In addition, the conjugate attached to the NTA moiety is also affected by the oxidation process.

[0323] B The [Co(III)(IDA)(His-protein)] complex was formed by oxidation of the preformed [Co(II)(IDA)(H2O)3] complex according to Zatloukalová and Kucerova (2006). IDA was preloaded with Co 2+ ions (from Co(II)Cl2) were obtained by oxidizing the [Co(II)(IDA)(H2O)3] complex with 20 mM H2O2 for 1 hour. 2+ Center converted to Co 3+ Subsequently, [Co(III)(IDA)(H2O)3] + The complex is incubated with a His-tagged protein. This approach has limitations due to very slow complex formation and low binding efficiency. In addition, conjugates attached to the IDA moiety are also susceptible to oxidation.

[0324] C uses cobalt(III) carbonate to form the [Co(III)(NTA)(His-protein)] complex. NTA is preloaded with Co from cobalt(III) carbonate (e.g., Na3[Co(III)(CO3)3]*3H2O). 3+ ions to form the complex of the present invention. The complex is then incubated with a His-tagged protein. This protocol is a very simple workflow for conjugating His-tagged proteins, which can be performed under mild reaction conditions. Because the reaction can be performed continuously under physiological buffer conditions, the functionality of the protein and NTA conjugate (i.e., the label (e.g., fluorophore) and / or carrier) can be fully preserved. In addition, compared to the two water ligands in protocol B, the carbonate ligand allows for faster and more efficient protein binding.

[0325] D. Formation of [Pt(IV)(NTA)(His-protein)] complex using platinum(IV) nitrate. NTA is preloaded with Pt from a platinum(IV) nitrate solution. 4+ ions to form the complex of the present invention. The complex is then incubated with a His-tagged protein. This protocol represents a very simple workflow for conjugating His-tagged proteins and can be performed under mild reaction conditions. Compared to the two water ligands in protocol B, the nitrate ligand allows for faster and more efficient protein binding.

[0326] Figure 3 Fluorophores were oxidized with hydrogen peroxide. Different fluorophores were incubated with 0.05% H2O2 for approximately 21 hours, followed by incubation with 1% H2O2. Fluorophore fluorescence was measured every 15 (0.05% H2O2) to 30 (1% H2O2) minutes. During the H2O2 treatment, all fluorophores showed a decrease in fluorescence intensity, indicating that the H2O2 oxidation step interferes with the function and stability of the label.

[0327] Figure 4 Protein degradation and His-tag cleavage induced by cobalt oxidation by H2O2. His-tagged protein (3.3 μM) was incubated for 1 hour with or without 66 μM CoCl2 and 20 mM H2O2. Protein stability and His-tag cleavage were analyzed by Western blotting using an α-His6-tag antibody (clone H-3) conjugated to horseradish peroxidase. After exposure to cobalt and H2O2, the protein spontaneously showed signs of partial degradation and His-tag cleavage. The gel lanes in the images shown are from the same gel / membrane.

[0328] Figure 5 :NTA and its cobalt complexes 1 H-NMR spectrum.

[0329] A NTA's 1 H-NMR measurements showed a peak at ∼3.6 ppm, which is consistent with the spectra generated in silco using the software NMR Predict (https: / / www.nmrdb.org / new_predictor / index.shtml?v=v2.103.0; April 2019 version), see Banfi and Patiny, 2008; Castillo et al. 2011; Aires-de-Sousa et al. 2002). 2+ Coordinate with D2O to form [Co(II)(NTA)(D2O)2] - When the complex was generated using Na3[Co(III)(CO3)3]*3H2O (C) or K3[Co(III)(CO3)3]*3H2O (D), the peak shifted further to ~1.9 ppm, indicating the presence of the desired carbonate complex [Co(III)(NTA)(CO3)]. 2- All ppm shifts are normalized to the peak of residual H2O at 4.7 ppm.

[0330] Figure 6 :through [Co(III)(NTA)(CO3)] 2- The stability of the obtained [Co(III)(NTA)(His6-PercevalHR)] complex. 2+ and Co 3+ The complex center (the latter was generated by Na3[Co(III)(CO3)3]*3H2O) had immobilized His6-PercevalHR (SEQ ID NO: 15) on NTA beads, which were washed with PBS or imidazole (250 mM), and the amount of His6-PercevalHR retained on the beads was determined by fluorescence measurement. The [Co(III)(CO3)3] salt-generated [Co(III)(NTA)(His6-PercevalHR)] complex thus showed similar chemical stability in imidazole to the complex formed by the H2O2 oxidation process (compared to the Figure 1 Error bars: + / - SD; p-values: <0.001: ***; <0.01: **; <0.05: *; ≥0.05: not significant.

[0331] Figure 7 : Binding of proteins to the [Co(III)(NTA)] complex.

[0332] Use [Ni(II)(NTA)(H2O)2] - ,[Co(III)(NTA)(H2O)2] or [Co(III)(NTA)(CO3)] 2- Functionalized agarose beads were incubated with His6-GFP (SEQ ID NO: 14).

[0333] A. The remaining unbound protein was determined by fluorescence measurement of the supernatant at different time points. The [Co(III)(NTA)] complex that binds carbonate molecules binds protein significantly faster than the [Co(III)(NTA)] complex that binds water molecules.

[0334] After incubation for 3-12 hours, the beads were washed with buffer or 250 mM imidazole, and the amount of His6-GFP retained on the beads was determined by fluorescence measurement. All [Co(III)(NTA)(His-GFP)] complexes showed similar chemical stability in imidazole. However, with [Co(III)(NTA)(CO3)] 2- Complex-functionalized beads bound significantly more protein than beads preloaded with [Co(III)(NTA)(H2O)2]. Error bars: + / -SD; p-values: <0.001: ***; <0.01: **; <0.05: *; ≥0.05: not significant

[0335] Figure 8 : Effects of buffer substances on protein binding.

[0336] A+B: using [Co(III)(NTA)(CO3)] 2- Functionalized agarose beads were incubated with His6-GFP (SEQ ID NO: 14) in various buffers. Remaining unbound protein was determined by fluorescence measurement of the supernatant at various time points. Complex formation was faster in MES- and Bis-Tris-based buffers compared to Tris-based buffers and more efficient compared to Tris- and HEPES-based buffers. Panel B is an enlarged view of Panel A at time points 0 and 3 hours after the start of the experiment. Error bars: + / - SD.

[0337] Figure 9 :[Co(III)(NTA)(CO3)] 2- and UV-Vis spectra of [Co(III)(NTA)(H2O)2]

[0338] [Co(III)(NTA)(CO3)] 2-Visible absorption spectra of [Co(III)(NTA)(H2O)2] and [Co(III)(NTA)(CO3)] in aqueous solution at room temperature. 2- It was produced by incubating NTA with Na3[Co(III)(CO3)3]*3H2O, and [Co(III)(NTA)(H2O)2] was produced by oxidizing the [Co(II)(NTA)(H2O)2] complex formed by incubating NTA with Co(II)Cl2*6H2O with H2O2. The peak shifts in each of the two maxima indicate that [Co(III)(NTA)(CO3)] 2- There is a carbonate ligand in the complex.

[0339] Figure 10 The formation and stability of the complex of Co(III)(NTA)(His-GFP)] depend on the incubation time and temperature of Na3[Co(III)(CO3)3]*3H2O with NTA

[0340] NTA functionalized agarose beads were incubated at 4°C (A), 25°C (B) and 70°C (C) for different time periods. 2- The complexes were incubated with His6-GFP for 48 hours. The stability of the final [Co(III)(NTA)(His-GFP)] complex was tested by stringent washing using a HEPES-based buffer or 250 mM imidazole in buffer. The amount of protein immobilized on the beads was determined by BCA assay. With increasing incubation time with Na3[Co(III)(CO3)3]*3H2O / NTA, the percentage of stable complexes as well as His6-GFP immobilized on the beads increased when the protein was subsequently incubated at 25°C for 48 hours. With increasing temperature, saturation of immobilized protein was reached earlier. Error bars: + / - SD

[0341] Figure 11 :The formation and stability of the [Co(III)(NTA)(His-GFP)] complex depend on [Co(III)(NTA)(CO3)] 2- Incubation time and temperature with His6-GFP

[0342] [CoIII)(NTA)(CO3)] 2- Functionalized agarose beads were incubated at 4, 25, and 37°C for different time periods, and the stability of the final [Co(III)(NTA)(His-GFP)] complex was tested by stringent washing with protein binding buffer or 250 mM imidazole in buffer. ex =490nm,λem =535 nm), the amount of protein immobilized on the beads was determined. A His-GFP immobilized on the beads after imidazole treatment based on its relative fluorescence. With longer protein incubation time, more protein can be immobilized on the beads, and a saturation plateau begins to be reached at 3.5 hours. No effect of protein incubation temperature on the yield of the final complex was observed. B The percentage of His-GFP immobilized on the beads after imidazole treatment compared to buffer wash. All complexes produced showed high stability to 250 mM imidazole treatment. Error bars: + / - SD

[0343] Figure 12 :[Co(III)(NTA)(His-GFP)] complex formation and stability using K3[Co(III)(CO3)3]*3H2O

[0344] NTA-functionalized agarose beads were incubated with K3[Co(III)(CO3)3]*3H2O for the indicated time, and then His6-GFP was incubated with the generated [CoIII)(NTA)(CO3)] 2- The stability of the final [Co(III)(NTA)(His-GFP)] complex was tested by stringent washing with protein buffer ("buffer") or 250 mM imidazole in buffer ("imidazole"). Fluorescence of the bead slurry (λ ex =490nm,λ em =535nm) to determine the amount of protein immobilized on the beads. [CoIII)(NTA)(CO3)] generated by K3[Co(III)(CO3)3]*3H2O 2- The complex can also coordinate with His-GFP to form a stable [Co(III)(NTA)(His-GFP)] complex. Error bars: + / -SD; p value: <0.001: ***; <0.01: **; <0.05: *; ≥0.05: not significant

[0345] Figure 13 :Forming and stability of [Co(III)(NTA)(His-GFP)] complexes in different buffer systems using 10-min Na3[Co(III)(CO3)3]*3H2O / NTA incubation

[0346] After 10 min of Na3[Co(III)(CO3)3]*3H2O / NTA incubation time, [(CoIII)(NTA)(CO3)] 2-Functionalized magnetic agarose beads were incubated with His6-GFP in different buffer systems for different time periods. The stability of the final [Co(III)(NTA)(His-GFP)] complex was tested by stringent washing with buffer solution ("buffer") and 250 mM imidazole in buffer ("imidazole"). Fluorescence (λ) based on the bead slurry was measured. ex =490nm,λ em =535 nm) to determine the amount of protein immobilized on the beads. A: Amount of protein immobilized on the beads after imidazole treatment; B: His-GFP immobilized on the beads before and after imidazole treatment after 15 minutes of protein incubation. All protein binding buffer systems formed stable [Co(III)(NTA)(His-GFP)] complexes, but the efficiency of protein binding and the percentage of final complex stability varied. Error bars: + / - SD

[0347] Figure 14 :Forming and stability of [Co(III)(NTA)(His-GFP)] complexes in different buffer systems using 48-hour Na3[Co(III)(CO3)3]*3H2O / NTA incubation

[0348] After 48 hours of Na3[Co(III)(CO3)3]*3H2O / NTA incubation, [CoIII)(NTA)(CO3)] 2- Functionalized magnetic agarose beads were incubated with His6-GFP in different buffer systems for different time periods. The stability of the final [Co(III)(NTA)(His-GFP)] complex was tested by stringent washing with assay buffer ("Buffer") or 250 mM imidazole ("Imidazole") in assay buffer. Fluorescence (λ) based on the bead slurry was determined. ex =490nm,λ em =535 nm) to determine the amount of protein immobilized on the beads. A: Amount of protein immobilized on the beads after imidazole treatment; B: His-GFP immobilized on the beads with or without imidazole treatment after 1 minute (B), 15 minutes (C), 1 hour (D), or 24 hours (E) of protein incubation. All protein binding buffer systems formed stable [Co(III)(NTA)(His-GFP)] complexes, but the efficiency of protein binding varied. Error bars: + / - SD

[0349] Figure 15 :Formation and stability of [Co(III)(NTA)(His-GFP)] complex at different pH values

[0350] After 48 hours of Na3[Co(III)(CO3)3]*3H2O / NTA incubation, [CoIII)(NTA)(CO3)] 2- Functionalized magnetic agarose beads were incubated with His6-GFP in a BisTris- or HEPES-based buffer system for various time periods at different pH values. The stability of the final [Co(III)(NTA)(His-GFP)] complex was tested by stringent washing with assay buffer ("Buffer") or 250 mM imidazole ("Imidazole") in assay buffer. Fluorescence (λ) of the bead slurry was determined. ex =490nm,λ em =535 nm) to determine the amount of protein immobilized on the beads. A. Amount of protein immobilized on the beads after imidazole treatment; B. His-GFP immobilized on the beads after 15 minutes of protein incubation, with or without imidazole treatment. Stable [Co(III)(NTA)(His-GFP)] complexes form at all pH values, but the efficiency of protein binding varies. Therefore, the efficiency increases with protein incubation and with decreasing pH during protein incubation. Error bars: + / - SD

[0351] Figure 16 :through [Co(III)(NTA)(CO3)] 2- Stable immobilization of different proteins with His tags or histidine-rich regions

[0352] [Co(III)(NTA)(CO3)] 2- Functionalized agarose beads were incubated with His-GFP, His-protein A, His-sortase, His-human serum albumin, or anti-GFP mouse IgG1, and the stability of the resulting [Co(III)(NTA)(protein)] complexes was tested by stringent washing with protein binding buffer or 250 mM imidazole in buffer. Protein A was incubated with [Co(III)(NTA)(CO3)] 2- SDS-PAGE of protein supernatants after incubation. Molecular weight markers: 200, 150, 100, 75, 50, 37, 25 kDa. Lanes 1, 4, 7, 10, and 13: [Co(III)(NTA)(CO3)] 2- Protein remaining after incubation. Lanes 2, 5, 8, 11, and 14: [Ni(II)(NTA)(H2O)2] -Proteins remaining after incubation. Lanes 3, 6, 9, 12 and 15: Proteins remaining after NTA incubation. Lanes 1-3: His-GFP, Lanes 4-6: His-Protein A, Lanes 7-9: His-Sortase, Lanes 10-12: His-HSA, Lanes 13-15: Anti-GFP mouse IgG1. With the exception of His-Protein A (high percentage), all proteins could be cleared from the supernatant after protein incubation. B Amount of protein on beads determined by BCA assay after imidazole treatment. All proteins could achieve stable [Co(III)(NTA)(Protein)] complex formation. C Using 520 Sortase A Activity Assay Kit, for fluorometric determination of sortase activity before and after imidazole treatment. The sortase remains active after immobilization, and the resulting [Co(III)(NTA)(His-sortase)] complex is resistant to treatment with 250 mM imidazole. D+E is GFP-bound to anti-GFP mouse IgG1 immobilized on NTA (D) or IDA (E) functionalized beads, based on the fluorescence of the bead slurry (λ ex =490nm,λ em The immobilized antibody was still functional as demonstrated by GFP binding.

[0353] Figure 17 :IDA and TALON act as metal binding domains. They are respectively [Co(III)(IDA)(CO3)] - Formation and chemical stability of the A[Co(III)(IDA)(His-GFP)] and B[Co(III)(TALON)(His-GFP)] complexes generated from the [Co(III)(CO3)] complexes. The incubation time of the His6-GFP protein with Na3[Co(III)(CO3)3]*3H2O is given in parentheses (Na3[Co(III)(CO3)3]*3H2O / protein incubation time). Chemical stability was tested by stringent washing with HEPES-based buffers or 250 mM imidazole in buffer. Fluorescence (λ) based on the bead slurry was measured. ex =490nm,λ em =535nm) and the amount of protein immobilized on the beads was measured. Compared with beads without metal treatment, [Co(III)(IDA / TALON)(CO3)] 2- Complex, chemically stably immobilized His on beads 6-The protein is significantly more abundant. Therefore, the chemical stability of the complex with the tridentate metal-binding domain IDA is dramatically increased compared to the tetradentate TALON complex. Error bars: + / -SD; p-value: <0.001: ***; <0.01: **; <0.05: *; ≥0.05: not significant.

[0354] Figure 18 : Chemical reactivity of the [Co(III)(IDA)(His-protein)] complex.

[0355] Beads functionalized with [Co(III)(IDA)(His6-GFP)] complex were incubated under different conditions including a combination of chelating agent or reducing agent with 250 mM imidazole, and the amount of His-GFP retained on the beads was measured by the fluorescence of the bead slurry (λ ex =490nm,λ em =535nm) measurement. 3+ The center is kinetically inert and His6-GFP is barely eluted, indicating the high stability of the resulting [Co(III)(IDA)(His-protein)] complex. Error bars: + / - SD

[0356] Figure 19 :His6-GFP and [Co(III)(IDA)(CO3)] - and [Co(III)(IDA)(H2O)2] + Fixed efficiency

[0357] After 3 hours in A and 24 hours in B His6-GFP incubation, the cells were stained with Co(III)(IDA)(CO3)] on magnetic beads. - or [Co(III)(IDA)(H2O)2] + Complex formation efficiency and chemical stability of [Co(III)(IDA)(His-GFP)] complexes. Co(III)(IDA)(CO3)]-complexes were generated by incubating IDA-functionalized magnetic beads with Na3[Co(III)(CO3)3]*3H2O for the indicated times (10 min or 48 h). Chemical stability was tested by stringent washing with HEPES-based buffer or 250 mM imidazole in buffer. Fluorescence (λ) of the bead slurry was determined based on the fluorescence of the beads. ex =490nm,λ em =535nm) and the amount of protein immobilized on the beads was measured. +Compared with the beads functionalized with the complex, the [Co(III)(IDA)(CO3)]-complex prepared by incubation with 10 min Na3[Co(III)(CO3)3]*3H2O had significantly more His 6- The protein was chemically and stably immobilized on the beads. After 24 h of protein incubation, the [Co(III)(IDA)(CO3)]-complex prepared by 48 h of incubation with Na3[Co(III)(CO3)3]*3H2O was superior to the [Co(III)(IDA)(H2O)2] + Complexes. All [Co(III)(IDA)(His-GFP)] complexes showed high chemical stability to imidazole. Error bars: + / -SD; p-value: <0.001: ***; <0.01: **; <0.05: *; ≥0.05: not significant

[0358] Figure 20 :Compared to oxygen treatment of [Co(II)(metal binding domain)(His-GFP)], a chemically stable complex of [Co(III)(IDA / NTA)(CO3)] is formed by [Co(III)(metal binding domain)(CO3)]. 2- Chemical stability of the complexes (Na3[Co(III)(CO3)3]*3H2O / protein incubation time), or oxidized [Co(II)(metal binding domain)(His-GFP)] on magnetic beads by treatment with oxygen (8 hours) or hydrogen peroxide (20 mM, 1 hour), A[Co(III)(IDA)(His-GFP)] or B[Co(III)(NTA)(His-GFP)] complexes, challenged with HEPES-based buffer or 250 mM imidazole in buffer. Fluorescence (λ) of the bead slurry was measured. ex =490nm,λ em =535nm) and the amount of protein immobilized on the beads was measured. Compared with the 8-hour oxygen treatment [Co(II)(metal binding domain)(His-GFP))], the use of the [Co(III)(metal binding domain)(CO3)] complex chemically and stably immobilized significantly more His on the beads. 6- Protein. Error bars: + / -SD; p-value: <0.001: ***; <0.01: **; <0.05: *; ≥0.05: not significant

[0359] Figure 21 : Formation of [Co(III)(HS-PEG-NTA)(His-GFP)] complex on the surface

[0360] His-GFP was synthesized by [Co(III)(HS-PEG-NTA(CO3)] 2- The complex was immobilized on the glass surface of gold nanostructures. The nanostructured gold dots were functionalized with thiol-PEG-NTA and then incubated with Na3[Co(III)(CO3)3]*3H2O to form [Co(III)(HS-PEG-NTA(CO3)] 2- The complex was then immobilized on the His6-GFP complex by forming a [Co(III)(HS-PEG-NTA)(His-GFP)] complex. All surfaces between the gold dots were passivated with a short PEG layer to avoid nonspecific protein interactions with the glass surface. ex =490nm,λ em =535 nm) confirmed that GFP was immobilized on the surface, and the amount of immobilized His-GFP was determined. "PEG only": passivated surface; "PEG / GFP": passivated surface incubated with His-GFP; "metal-free": passivated surface incubated with thiol-PEG-NTA and His-GFP; "[Co(III)(NTA)(His-GFP)]": passivated surface incubated with thiol-PEG-NTA, Na3[Co(III)(CO3)3]*3H2O, and His-GFP.

[0361] Figure 22 : Formation of [Co(III)(NTA-X-biotin)(His-GFP)] complex in solution

[0362] His-GFP was coupled to the biotin moiety via a [(CoIII)(NTA-X-biotin)(CO3)] complex, which was generated by incubating Na3[Co(III)(CO3)3]*3H2O with NTA-X-biotin. His-GFP was then incubated with the resulting complex for varying incubation times. The final complex was immobilized on streptavidin-functionalized beads via the biotin-streptavidin interaction. The fluorescence of the bead slurry (λ) was determined based on the fluorescence of the beads. ex =490nm,λ em=535 nm), and the amount of immobilized protein was determined as a measure of biotinylated protein. A[Co(III)(NTA-X-Biotin)(His-GFP)] complex was generated after a 10-minute Na3[Co(III)(CO3)3]*3H2O / NTA-X-Biotin and a 30-minute [CoIII)(NTA-X-)(CO3)] / His-GFP incubation and immobilized on streptavidin beads. For all Na3[Co(III)(CO3)3]*3H2O to NTA ratios, significant amounts of His-GFP could be biotinylated and immobilized on the beads. Higher Na3[Co(III)(CO3)3]*3H2O ratios resulted in increased labeling yields. B[Co(III)(NTA-X-biotin)(His-GFP)] complexes were generated after a 10-minute Na3[Co(III)(CO3)3]*3H2O incubation with NTA-X-biotin and a 30-minute or 48-hour incubation with [CoIII)(NTA-X-biotin)(CO3)] / His-GFP, immobilized on streptavidin beads, and then rigorously washed with 250 mM imidazole in buffer. The amount of immobilized, and therefore chemically stabilized, complex increased with increasing protein incubation time. Error bars: + / -SD; p-values: <0.001: ***; <0.01: **; <0.05: *; ≥0.05: not significant

[0363] Figure 23 : His6-GFP immobilized on [Pt(IV)(NTA)(NO3)] functionalized beads

[0364] NTA functionalized agarose beads were incubated with platinum(IV) nitrate for 10 min and then incubated with His6-GFP for 30 min. The chemical stability of the formed [Pt(IV)(NTA)(His6-GFP)] complex was challenged with 250 mM imidazole and the immobilized protein on the beads was determined by BCA assay. It could be demonstrated that a significant amount of protein was stably immobilized by the [Pt(IV)(NTA)(NO3)] complex. Furthermore, the use of the metal binding ligand nitrate allowed for easy and rapid binding of proteins to the complex. Error bars: + / - SD; p values: <0.001: ***; <0.01: **; <0.05: *; ≥0.05: not significant

[0365] Example:

[0366] Example 1: Comparison of the chemical stability of [Co(II)(NTA)(His6-GFP)] and [Co(III)(NTA)(His6-GFP)] complexes

[0367] Aliquots of beads with immobilized [Co(II)(NTA)(His6-GFP)] and [Co(III)(NTA)(His6-GFP)] were incubated with a strong chelator or with a combination of a widely used reducing agent and 250 mM imidazole to demonstrate the Co-based 3+ The chemical stability of the complex is better than that of the commonly used Ni-based 2+ or Co 2+ of the complex.

[0368] His6-GFP (SEQ ID NO: 14) was expressed in E. coli BL21(DE3) using the plasmid pET His6 GFP TEV LIC (Addgene #29663) (Pedelacq et al. 2006) and cloned by Ni as described by Wegner and Spatz (Wegner and Spatz, 2013). 2+ -NTA-bead purification.

[0369] Ni 2+ -NTA agarose resin (Novagen), 1) washed with 9 bead volumes of ddH2O, 2) washed with 3 bead volumes of 0.1M EDTA pH 7.5, 3) washed three times with 9 bead volumes of buffer A (50mM Tris-HCl pH 7.4, 300mM NaCl), 4) washed with 1.5 bead volumes of 0.1M CoCl2*6H2O, 5) washed with 9 bead volumes of buffer B (buffer A containing 250mM imidazole), and 6) washed three times with 9 bead volumes of buffer A. Finally, His6-GFP was loaded onto the beads by incubating in one bead volume of 10μM His6-GFP in buffer A. Between each step, the bead slurry was centrifuged at 300g for 1 minute and the supernatant was decanted. To obtain the [Co(III)(NTA)(His6-GFP)] complex, the beads were eluted with 10μM His6-GFP immobilized on Co(III)(NTA)(His6-GFP) 2+ The beads of His6-GFP on -NTA were incubated in buffer A containing 20 mM H2O2 for 1 hour at room temperature (later used as a 2+The control beads were incubated in buffer A without H2O2). Subsequently, after washing the beads several times with buffer A, the beads were resuspended in 2 bead volumes of buffer A and distributed into 150 μl aliquots for stability experiments. Finally, 50 μl of each test reagent (final concentration: chelating agent: 250 mM imidazole, 25 mM NTA, 25 mM EDTA; reducing agent (cysteamine, DTT, TCEP, ascorbate): 1 mM supplemented with 250 mM imidazole) was added to the aliquots. After incubation for 1 hour at room temperature, 100 μl of the supernatant was analyzed for GFP fluorescence (λ) using a plate reader (TECAN, infinite 2000). ex =480nm,λ em =510 nm). All experiments were performed in duplicate.

[0370] like Figure 1 As shown, when His6-GFP was combined with Co 3+ When the center was bound, only very small amounts of eluted protein were observed after incubation with the tested chelating agents or reducing agents. 2+ Bead-bound His6-GFP was completely eluted under the same conditions. Therefore, the [Co(III)(NTA)(His6-GFP)] complex destroys the strong chelator and reduces it to Co 2+ All are inert.

[0371] Example 2: Oxidation of fluorophores by hydrogen peroxide

[0372] As in the method described in Example 1 and as previously described in the prior art (see Wegner and Spatz, 2013), Co 2+ Oxidation to Co 3+ , which not only damages the attached protein but also negatively affects the functionality of the NTA-conjugate, such as a label or carrier. For example, the fluorescence of several fluorophores decreases after oxidation with H2O2, as shown below.

[0373] Fluorophore conjugates were diluted in phosphate buffered saline (PBS) (Thermo; 18912014) (final concentrations: 5 μg / ml fluorescein (Riedel de Haen; 28802); 185 μg / ml Alexa488-conjugated antibody (Invitrogen; A11039 / ml); 9 μg / ml FITC-conjugated antibody (Thermo; MA1-81891); 5 μM atto488-conjugated Ni 2+-NTA (Sigma; 39625)), 100 μl of each fluorophore solution was incubated with 0.05% H2O2 for approximately 21 hours in a black 96-well plate, and then incubated in 1% H2O2 for another 22 hours. The fluorescence intensity (λ) was measured every 15 minutes (0.05% H2O2) or 30 minutes (1% H2O2) on a plate reader (TECAN; Spark). ex =490nm,λ em =535nm).

[0374] Figure 3 The fluorescence measurements depicted in Figure 4 show that all analyzed fluorophores displayed a clear decrease in fluorescence intensity after incubation with H2O2.

[0375] Example 3: Protein degradation and His tag cleavage during cobalt oxidation by H2O2

[0376] Co was precipitated using H2O2 as described in Example 1 and previously described by Wegner and Spatz (Wegner and Spatz 2013). 2+ Oxidation to Co 3+ , which can spontaneously trigger a Fenton-like reaction (Hanna, Kadiiska et al., 1992) leading to protein degradation and cleavage of histidine residues (Davies 1987, Stadtman 1990), as demonstrated in Western blots against the His tag.

[0377] The fluorescent protein PercevalHR (SEQ ID NO: 15) was expressed in Escherichia coli DH5α using the plasmid pRsetB-PercevalHR (Addgene #49081) (Tantama et al. 2013) and was expressed by Ni 2+ -NTA column purification as described in (Tantama et al., 2013).

[0378] 3.3 μM His7-tagged PercevalHR protein was mixed with 33 μM CoCl2*6H2O in protein buffer (50 mM Tris, pH 7.4, 150 mM NaCl) and incubated at room temperature for 2 minutes. Subsequently, 20 mM H2O2 was added and the mixture was incubated at 21°C for 1.5 hours. For control samples lacking cobalt and / or H2O2, an equal volume of protein buffer was used. Finally, the reaction was quenched with 33 mM EDTA, pH 8.0.

[0379] 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 then 84 pmol of protein were loaded on 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; running conditions: 120 V constant, Laemmli running buffer (25 mM Tris-HCl pH 8.8, 192 mM glycine, 0.1% (w / v) SDS)). After protein separation, the protein was blotted on a nitrocellulose membrane (Whatman, 10401196) and 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-8036 HRP), washed three times with TBS-T for 10 minutes, and incubated at room temperature for 5 minutes in a luminol-based enhanced chemiluminescent horseradish peroxidase (HRP) substrate solution (Thermo, 34076). The chemiluminescent signal from the His-tagged protein was detected using the LAS3000 system (FUJIFILM).

[0380] Figure 4 Western blots in Figure 3 show that incubation of the protein with the combination of cobalt and H2O2 can lead to protein degradation and spontaneous His-tag cleavage, as indicated by a diffuse low-intensity protein band.

[0381] Example 4: [Co(III)(NTA)(CO3)] 2- Synthesis of complexes

[0382] To completely avoid the use of H2O2, a new method using Co(III) carbonates (e.g., Na3[Co(III)(CO3)3]*3H2O and K3[Co(III)(CO3)3]*3H2O) was developed to form the [Co(III)(NTA)(His-protein)] complex. The complex formation process is as follows Figure 2C is schematically shown. In the first step, the Co(III) salt is incubated with NTA to form [Co(III)(NTA)CO3] 2- . Using proton nuclear magnetic resonance spectroscopy ( 1 H-NMR) to prove [Co(III)(NTA)CO3] 2- The complex was successfully formed.

[0383] Synthesis of Sodium Tris(Carbonate)Cobalt(III) Oxide Trihydrate

[0384] Sodium tris(carbonate)cobalt(III)ate trihydrate (Na3[Co(III)(CO3)3]*3H2O) 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)(NO3)*6H2O (Sigma; 1.02554) and 10 ml of 30% hydrogen peroxide (Riedel-de Haen; 18312) in 50 ml of ddH2O was added dropwise with stirring to an ice-cold slurry of 0.5 mol (=42.0 g) of sodium bicarbonate (Merck; 1.06329) in 50 ml of ddH2O. The mixture was incubated on ice with continuous stirring for 1 hour. Subsequently, the olive-colored product was filtered and washed three times with cold water, absolute ethanol, and absolute ether, respectively. Finally, the product was dried under vacuum overnight and stored at -20°C under a nitrogen atmosphere.

[0385] Synthesis of Potassium Tri(Carbonate)Cobalt(III) Oxide Trihydrate

[0386] Potassium tris(carbonate)cobalt(III)ate trihydrate (K3[Co(III)(CO3)3]*3H2O) was synthesized in solution as described by Shibata (Shibata 1983; adapted from Mori et al., 1956). Briefly, a mixture of 0.1 mol (24 g) Co(II)Cl2*6H2O (Honeywell; 255599) and 40 ml of 30% hydrogen peroxide in 24 ml of ddH2O was added dropwise with stirring to an ice-cold slurry of 0.7 mol (70 g) potassium bicarbonate (Honeywell; 237205) in 70 ml of ddH2O. The resulting green solution was then filtered with suction and used directly in subsequent experiments.

[0387] Preparation of [Co(III)(NTA)(CO3)] 2- Complex

[0388] To generate [Co(III)(NTA)(CO3)] 2-To prepare the complex, 580 μmole (210 mg) of Na₃[Co(III)(CO₃)₃]*3H₂O was added to 2 ml of 1 M sodium bicarbonate and 2 M nitrilotriacetic acid trisodium salt (Sigma; N0253) in ddH₂O, 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 now pink slurry, and the mixture was sonicated at 70°C for 2 hours. The purple supernatant was then subjected to NMR analysis. For the complex prepared from the potassium salt, K₃[Co(III)(CO₃)₃]*3H₂O was synthesized in solution from 0.1 molar CoCl₂*6H₂O (see above), followed by the addition of 0.1 molar nitrilotriacetic acid trisodium salt (25.7 g) and 60 ml of ddH₂O, as described in (Shibata 1983). After incubation for 3 hours with continuous stirring at 60°C, the resulting purple solution was filtered and the pH value was adjusted to 7.3 with aqueous acetic acid. Finally, the solution was incubated at 4°C overnight, clarified to remove the white precipitate and subjected to NMR analysis. In order to obtain the complex [Co(II)(NTA)(D2O)]-, a mixture of 5 mM nitrilotriacetic acid (Sigma; 72559) and 5 mM CoCl2*6H2O was prepared from a stock solution in D2O and incubated for 15 minutes at room temperature before NMR measurement. In order to dissolve nitrilotriacetic acid in D2O (Carl Roth; HN81.3), a small amount of 10 M NaOH was added to the corresponding stock solution. For the measurement of pure NTA, a 5 mM solution in D2O was prepared from the stock solution as described above. Measurements were made at a resonance frequency of 400 MHz on a Jeol ECZ400S spectrometer at room temperature. 1 H-NMR spectrum. To improve the signal-to-noise ratio, up to 32 signals were added before Fourier transformation. The resulting spectrum was normalized at 7 ppm, with all ppm values ​​adjusted relative to the water peak at 4.70 ppm.

[0389] The H of pure NTA and its complexes with cobalt and water or carbonate ligands was measured. 1 -NMR spectroscopy confirmed [Co(III)(NTA)CO3] 2- The formation of the complex ( Figure 5The H spectrum of pure NTA shows that in addition to the ubiquitous water peak at 4.70 ppm, there is also a peak at ~3.6 ppm, which is consistent with the simulation (calculated value of 3.57 ppm) using 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 is mixed with cobalt and water (here due to H 1 -NMR measurement, heavy water (D2O)) ([Co(II)(NTA)(D2O)2] - ; Figure 5 B) or carbonate ([Co(III)(NTA)(CO3)] 2- ; Figure C+D) When the complex is formed, the original peak at ~3.6 ppm shifts to ~3.8 ppm (for D2O as a ligand) and to ~1.9 ppm (for carbonate as a ligand), respectively. Similar peak shifts are observed regardless of whether sodium or potassium cobalt(III) carbonate is used to prepare the complex. The shift of the NTA peak indicates that during the coordination process, the magnetic environment of the hydrogen atom in NTA changes as other atoms approach the hydrogen atom. Therefore, the different peak shifts observed for the complex generated with cobalt(III) carbonate strongly suggest the presence of carbonate ligands rather than water ligands in the final cobalt-NTA complex.

[0390] Example 5: Chemical Stability of the [Co(III)(NTA)(His6-PercevalHR)] Complex Formed by [Co(III)(NTA)CO3]

[0391] The complex [Co(III)(NTA)(CO3)] 2- , incubated with His-tagged protein PercevalHR to form [Co(III)(NTA)(His6-PercevalHR)] immobilized on agarose beads. Subsequently, the chemical stability of the [Co(III)(NTA)(His6-PercevalHR)] complex was evaluated. As a control, a conventional Ni-based 2+ -NTA substrate.

[0392] The fluorescent protein PercevalHR (SEQ ID NO: 15) was expressed in Escherichia coli DH5α using the plasmid pRsetB-PercevalHR (Addgene #49081) (Tantama, Martinez-Francois et al. 2013) and was detected by Ni 2+ -NTA column purification.

[0393] NTA agarose resin (Qiagen, 1022963) was washed 1) with 10 bead volumes of ddH2O, 2) with 3 bead volumes of 100 mM EDTA pH 7.5, and 3) three times with 10 bead volumes of ddH2O, followed by the addition of 10 bead volumes of 1 mM Na3[CO(III)(CO3)3]*3H2O or 1 mM Ni(II)SO4 in 1 M NaHCO3. After incubation at 23°C with shaking at 1100 rpm in a thermostatic shaker for 48 hours, the beads were washed twice with 10 bead volumes of ddH2O and once with 10 bead volumes of protein buffer (50 mM Tris pH 7.4, 150 mM NaCl). Finally, 10 μM His6-PercevalHR (SEQ ID NO: 15) in protein buffer was added to 1 bead volume and incubated at 4°C on a thermostat shaker with shaking at 1100 rpm for 48 hours to allow the protein to bind to the matrix. After washing twice with 10 bead volumes of protein buffer, 3 bead volumes of protein buffer were added and 10 μl of the bead slurry was analyzed for PercevalHR fluorescence (λ) using a plate reader (TECAN, infinite 2000). ex =500nm,λ em =545 nm). To test the stability of the complex, 10 bead volumes of 250 mM imidazole in protein buffer or protein buffer alone were added and then removed by washing the beads with 10 bead volumes of protein buffer. Finally, the beads were resuspended in 3 bead volumes of protein buffer, and the residual fluorescence of 10 μl of bead slurry was analyzed. All experiments were performed in triplicate.

[0394] like Figure 6As shown, the [Ni(II)(NTA)(His6-PercevalHR)] complex used as a control exhibited low stability to treatment with the chelating agent imidazole. In sharp contrast, the [Co(III)(NTA)(His6-PercevalHR)] complex formed by cobalt(III) carbonate and the [Co(III)(NTA)(CO3)] pre-complex exhibited strong stability to imidazole. The measured stabilities of these complexes were similar to those of the [Co(III)(NTA)(His6-PercevalHR)] complex produced by the indirect oxidation method (as used in Example 1). Therefore, this data demonstrates that, surprisingly, by using [Co(III)(NTA)(CO3)] 2- The precomplex can be efficiently formed into [Co(III)(NTA)(His6-PercevalHR)] without the need for an oxidation step.

[0395] Example 6: His6-GFP and [Co(III)(NTA)CO3] 2- The binding kinetics

[0396] Example 4 demonstrates that [Co(III)(NTA)CO3] 2- In addition, Example 5 shows that [Co(III)(NTA)CO3] 2- Complexes can be synthesized and surprisingly used to form [Co(III)(NTA)(His-protein)] complexes on beads. We hypothesized that carbonate ligands at the cobalt(III) center might promote the formation of [CoIII(NTA)(His-protein)] complexes. To confirm this finding, the effects of His6-GFP on [Co(III)(NTA)(H2O)2] and [Co(III)(NTA)(CO3)] were directly compared. 2- The binding efficiency.

[0397] Functionalization of NTA agarose beads

[0398] NTA agarose resin (Qiagen, 1022963), 1) washed with 10 bead volumes of ddH2O, 2) washed with 10 bead volumes of 100 mM EDTA pH 7.5, 3) washed twice with 10 bead volumes of ddH2O and once with 6.7 bead volumes of ddH2O (for [Co(II)(NTA)(H2O)2] -and [Co(III)(NTA)(H2O)2] complexes) or washed twice with 10 bead volumes of ddH2O and once with 6.7 bead volumes of 1 M NaHCO3 (for [Co(III)(NTA)(CO3)] 2- complex). 8.7 bead volumes of 1 mM Co(II)Cl2*6H2O in ddH2O were then added (for [Co(II)(NTA)(H2O)2] - and [Co(III)(NTA)(H2O)2] complexes) or 1 mM Na3[CO(III)(CO3)3]*3H2O in 1 M NaHCO3 (for [Co(III)(NTA)(CO3)] 2- After incubation at 25°C and 1100 rpm in a thermostatic shaker for 18 h, the beads were washed with 6.7 bead volumes of protein buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl) (for [Co(II)(NTA)(H2O)2] - ,[Co(III)(NTA)(H2O)2] complex and a [Co(III)(NTA)(CO3)] 2 complex sample) or 1M NaHCO3 (for a [Co(III)(NTA)(CO3)] 2- The complex sample, Figure 7 For samples with the final [Co(III)(NTA)(HO)] complex, a second wash was performed using 6.7 bead volumes of 20 mM HO in ddHO and incubated at 25° C. on a thermostatted shaker (1100 rpm) for 1 hour.

[0399] Binding kinetics of His6-GFP to functionalized NTA agarose beads

[0400] The resulting beads were incubated with 3.3 bead volumes of 20 μM HiS6-GFP (SEQ ID NO: 14) in protein buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl) and incubated at 4°C at 1100 rpm in a thermostatic shaker. For one [Co(III)(NTA)(CO3)] 2- Sample (in Figure 7 The protein buffer was replaced by 1 M NaHCO3. The fluorescence intensity (λ) of 100 μl supernatant was measured in a plate reader (TECAN, Spark) at different time points.ex =490nm,λ em =535 nm), and the fraction of unbound protein was analyzed.

[0401] Chemical stability of the [Co(III)(NTA)(His6-GFP)] complex

[0402] After the incubation of the functionalized beads with His6-GFP (SEQ ID NO: 14) was terminated, the amount of protein bound to the beads was analyzed as described above. To this end, the beads were washed three times with 6.7 bead volumes of protein buffer, resuspended in 6.7 bead volumes of protein buffer, and finally 100 μl of the bead slurry was analyzed for GFP fluorescence (λ) using a plate reader (TECAN, Spark). ex =490nm,λ em =535 nm). To test the stability of the complex, 1.7 bead volumes of 1.25 M imidazole in protein buffer (final concentration 250 mM) were added and incubated on a thermostatic shaker at 25°C and 1100 rpm for 10 minutes. The beads were then washed three times with 6.7 bead volumes of protein buffer. Finally, the beads were resuspended in 6.7 bead volumes of protein buffer, and 100 μl of the bead slurry was analyzed for residual fluorescence. All experiments were performed in triplicate.

[0403] The results of these experiments clearly show that the carbonate-bound Co 3+ The speed of the complex binding to the protein is significantly faster than that of the water molecules binding to Co 3+ The bound complex ( Figure 7 A). Consistently, [Co(III)(NTA)(CO3)] 2- The complex-functionalized beads bound significantly more protein than the beads preloaded with [Co(III)(NTA)(H2O)2] ( Figure 7 B) In addition, Figure 7 B again confirms that the [Co(III)(NTA)(His6-GFP)] complex shows strong stability to the chelating agent (here imidazole).

[0404] These experiments further showed that washing the bead-bound [Co(III)(NTA)(CO3)] 2- , which can further promote the binding of His6-GFP. Without being bound by theory, it is believed that HCO3 in the buffer - and / or CO3 2- The presence of [Co(III)(NTA)(CO3)] 2-The complex was converted to the slower enhancing [Co(III)(NTA)(H2O)2] complex. Although His-protein binding was also performed in the presence of 1M NaHCO3 for the "[Co(III)(NTA)(CO3)] in 1M NaHCO3" sample (see above) in this experiment, it is conceivable that the presence of 1M NaHCO3 in the protein binding process, if any, would have only a very small promoting effect. This is because it is expected that the protein binding process from Co 3+ The release of carbonate ligands from the complex was likely hindered rather than promoted by the presence of 1 M NaHCO 3 .

[0405] Example 7: His-tagged protein in different buffer systems with [Co(III)(NTA)(CO3)] 2- The binding kinetics

[0406] 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.

[0407] [Co(III)(NTA)(CO3)] 2- Functionalized beads were prepared as described in Example 6 with the following minor modifications: Bead wash steps 1) to 3) were performed with 5 bead volumes of the respective solution. In step 3), the second and third wash steps were performed with 1M NaHCO. The metal was loaded in 6.5 bead volumes. After metal binding, the beads were washed with 5 bead volumes of 1M NaHCO.

[0408] Incubation of His-tagged GFP (SEQ ID NO: 14) was also performed as described in Example 6, except that 5 bead volumes of 10 μM protein solution were 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. In addition, the first 24 hours of protein incubation were performed at room temperature instead of 4°C.

[0409] These experiments showed that the reaction speed and efficiency can be significantly improved when Tris-based buffers are replaced by HEPES or, in particular, non-coordinating buffers such as those based on MES or BisTris. After 24 hours of incubation, 95% of all proteins were immobilized on the beads using MES- and BisTris-based buffers, compared to 75% using Tris-based buffers ( Figure 8 A). In addition, compare Figure 7 The results in Figures A and 8A show that increasing the temperature used for the first 24 hours of protein incubation and / or using 1 M NaHCO3 for washing can promote His-protein coordination. When using MES- or BisTris-based buffers, approximately 80% of the protein was bound to the beads after three hours of incubation. In contrast, in Tris-based buffers, less than 50% of the protein was immobilized on the beads. This demonstrates that His-tagged proteins can be bound to [Co(III)(NTA)(CO3)] with very high efficiency within a reasonable timescale. 2- Complex coordination.

[0410] It is worth noting that the pH values ​​shown for the different protein buffers refer to the pH value before the solution was added to the beads. Due to residual NaHCO3 left on the beads from the second wash step, the pH values ​​of all samples during incubation with the protein were between 8.5 and 9 (verified by pH measurement). Therefore, since the pH values ​​of all buffer solutions were very similar, these experiments clearly demonstrate that the buffer substance itself has an effect on His-protein binding. The observed better performance of Good's buffers MES and BisTris relative to Tris buffer suggests that the use of a solution that cannot be combined with Co 3+ Good's buffer that forms a complex is advantageous over the use of buffers that can form such complexes (eg, Tris buffer).

[0411] Example 8: [Co(III)(NTA)(CO3)] 2- UV-Vis analysis

[0412] By NMR, Example 4 demonstrates that [Co(III)(NTA)(CO3)] 2- Complexes can be formed. In the following examples, [Co(III)(NTA)CO3] 2- The formation of the complex was confirmed by another technique, namely by UV-Vis absorbance measurement.

[0413] Preparation of Co(III)(NTA)(CO3)] 2- Complex

[0414] To produce [Co(III)(NTA)(CO3)] 2-To prepare the complex, a 1 mM solution of Na₃[CO(III)(CO₃)₃]*3H₂O in 1 M NaHCO₃ was prepared by sonicating the salt for 1 hour and then filtering through a 0.22 μm filter. Subsequently, a mixture of a 0.95 mM solution of Na₃[CO(III)(CO₃)₃]*3H₂O in 1 M NaHCO₃ and 0.95 mM NTA trisodium salt (Sigma; NO253) dissolved in ddH₂O was prepared in 1 M NaHCO₃. After incubation for 1 hour at 25°C, the visible absorbance of the lavender solution was measured in a 1 cm cuvette (Brand; 759150) on a UV-Vis-NIR spectrophotometer (Cary 5000).

[0415] Preparation of [Co(III)(NTA)(H2O)2] complex

[0416] To generate the [Co(III)(NTA)(HO)] complex, a mixture of 0.95 mM Co(II)Cl*6HO, 0.95 mM NTA trisodium salt (Sigma; N0253), and 20 mM HO was prepared in ddHO. After incubation for 24 h at 25°C, the visible absorbance of the lavender solution was measured in a 1 cm cuvette (Brand; 759150) on a UV-Vis-NIR spectrophotometer (Cary 5000) relative to a blank with ddHO.

[0417] The peak shifts from 402 nm for the [Co(III)(NTA)(H2O)2] complex to 390 nm for the [Co(III)(NTA)(CO3)] 2- complex) or shifted from 567nm to 573nm, Figure 9 The results clearly show that [Co(III)(NTA)(CO3)] 2- The carbonate group in the complex acts as a metal-binding ligand.

[0418] Example 9: His6-GFP and [Co(III)(NTA)(CO3)] after different incubation times and temperatures of Na3[Co(III)(CO3)3]*3H2O with NTA 2- Coordination and stability of the formed [Co(III)(NTA)(His-GFP)] complex

[0419] Example 4 demonstrates [Co(III)(NTA)CO3] 2-The complex can be formed by incubating NTA with Na3[Co(III)(CO3)3]*3H2O. In the following examples, different incubation times and temperatures of Na3[Co(III)(CO3)3]*3H2O with NTA were investigated for the effect of different incubation times and temperatures on the coordination of His6-GFP to the generated [Co(III)(NTA)(CO3)] 2- The effects of the imidazole on the complex and the chemical stability of the final Co(III)(NTA)(His-GFP) complex were investigated.

[0420] Functionalization of NTA agarose beads

[0421] NTA functionalized agarose beads (Qiagen; 1022963) were 1) washed with 27 bead volumes of ddHO, 2) washed with 27 bead volumes of 100 mM EDTA pH 8.0, 3) washed once with 27 bead volumes of ddHO, and washed twice with 27 bead volumes of 1M NaHCO. Subsequently, 16 bead volumes of 1 mM Na[Co(III)(CO)]*3H2O in 1M NaHCO were added and the beads were incubated at 4°C, 25°C, or 70°C in a thermostatted shaker at 1400 rpm for 1 minute, 10 minutes, 30 minutes, 1 hour, 24 hours, or 48 hours as indicated. After incubation, the beads were washed twice with 16 bead volumes of 1M NaHCO.

[0422] His6-GFP was immobilized on functionalized [CoIII)(NTA)(CO3)] 2- Agarose beads

[0423] The resulting beads were incubated with 10 μM His6-GFP (SEQ ID NO: 14) in 12 bead volumes of protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) and incubated for 48 hours at 25° C. on a thermostatic shaker at 1400 rpm. After incubation of the functionalized beads with His6-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.

[0424] Chemical stability of the [Co(III)(NTA)(His6-GFP)] complex

[0425] After implementing the described [Co(III)(NTA)(His-GFP)] complex formation process, the amount of protein bound to the beads with and without chemical stress was analyzed. To this end, the beads were divided into two parts (7.2 bead volumes each) and washed once with 17.8 bead volumes of protein buffer or 250mM imidazole in protein binding buffer, respectively. 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 immobilized protein on 25μl of bead slurry was determined by BCA assay (Thermo, 23227) in a microplate according to the manufacturer's instructions. The experiment was performed in triplicate.

[0426] Figure 10 The results of these experiments shown in clearly show that the incubation time of Na3[Co(III)(CO3)3]*3H2O and NTA is positively correlated with the amount of His-GFP immobilized on the agarose beads after treatment with the buffer and imidazole when the protein is incubated for up to 48 hours. For the incubation at 70°C, the reaction seems to have reached its saturation point after 1 minute, since the coordination of Na3[Co(III)(CO3)3]*3H2O to NTA is not surprisingly accelerated at higher temperatures. In addition, for incubations at 4°C and 25°C, as shown in Figure 5, the reaction is saturated after 1 minute. Figure 10 As shown in Figures A and B, incubation time is positively correlated with complex stability. For incubation at 25°C, high complex stability is achieved after 10 minutes of Na₃[Co(III)(CO₃)₃]*3H₂O / NTA incubation, whereas for incubation at 4°C, this stability is only achieved after 24 hours of Na₃[Co(III)(CO₃)₃]*3H₂O / NTA incubation. It is expected that the formation of stable complexes accelerates with increasing temperature.

[0427] Example 10: [Co(III)(NTA)(CO3)] at different temperatures 2- Kinetics of His-protein binding to functionalized beads

[0428] In the following examples, the protein-[Co(III)(NTA)(CO3)] 2- Binding of functionalized agarose beads and stability of the resulting [Co(III)(NTA)(His-GFP)] complex towards imidazole.

[0429] Functionalization of NTA agarose beads

[0430] NTA-functionalized agarose beads (Thermo; 78605) were 1) washed with 26 bead volumes of ddH2O, 2) washed with 26 bead volumes of 100 mM EDTA, pH 8.0, 3) washed twice with 26 bead volumes of ddH2O, and once with 26 bead volumes of 1M NaHCO3. Subsequently, 160 bead volumes of 1 mM Na3[Co(III)(CO3)3]*3H2O or 1M NaHCO3 (for metal-free samples) in 1M NaHCO3 were added, and the beads were incubated at 25°C on a thermostatted shaker at 1400 rpm for 48 hours. After incubation, the beads were washed three times with 160 bead volumes of 1M NaHCO3.

[0431] His6-GFP was expressed in the presence of functionalized [CoIII)(NTA)(CO3)] 2- Immobilization on agarose beads

[0432] The resulting beads were incubated with 10 μM His6-GFP (SEQ ID NO: 14) in 50 mM HEPES pH 7.2, 150 mM NaCl in 120 bead volumes and incubated for 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3.5 hours, or 24 hours as indicated at 4, 25, or 37° C., with shaking at 1400 rpm on a thermostatic shaker. After incubation of the functionalized beads with His6-GFP (SEQ ID NO: 14), the beads were washed once with 160 bead volumes of protein buffer and finally resuspended in 160 bead volumes of the corresponding wash buffer.

[0433] Chemical stability of the [Co(III)(NTA)(His6-GFP)] complex

[0434] After the described [Co(III)(NTA)(His-GFP)] complex formation process was performed, the amount of protein bound to the beads was analyzed in the presence and absence of chemical stress. To this end, the beads were divided into two parts and washed once with either 1 bead volume of protein buffer or 250 mM imidazole in protein binding buffer. After a final wash with 178 bead volumes of protein buffer, the beads were resuspended in 178 bead volumes of protein buffer. Finally, the GFP fluorescence (λ ex =490nm,λ em =535 nm), and 10 μl of bead slurry was analyzed for the amount of immobilized protein using a plate reader (TECAN, Spark). The experiment was performed in triplicate.

[0435] like Figure 11 As shown in A, the amount of protein immobilized on imidazole-treated beads increased with the expression of His-GFP and [Co(III)(NTA)(CO3)] 2-The temperature during incubation did not affect the final yield of the formed [Co(III)(NTA)(His6-GFP)] complex. High stability of the resulting complex to imidazole was observed for all protein incubation times and temperatures ( Figure 11 B).

[0436] Example 11: Formation of [Co(III)(NTA)(CO3)] using K3[Co(III)(CO3)3]*3H2O 2- Formation and stability of the complex [Co(III)(NTA)(His-GFP)]

[0437] Example 4 demonstrates that [Co(III)(NTA)(CO3)] can be formed by incubating K3[Co(III)(CO3)3]*3H2O with NTA. 2- In the following examples, the complexes of His-GFP and Co(III)(NTA)(CO3) generated using K3[Co(III)(CO3)3]*3H2O were studied. 2- The coordination of the complex and the chemical stability of the obtained [Co(III)(NTA)(His-GFP)] complex towards imidazole.

[0438] Functionalization of NTA agarose beads

[0439] NTA functionalized agarose magnetic beads (Thermo; 78605) were 1) washed with 600 bead volumes of ddHO, 2) washed with 600 bead volumes of 100 mM EDTA pH 8.0, 3) washed once with 600 bead volumes of ddHO and twice with 600 bead volumes of 1M NaHCO. Subsequently, 160 bead volumes of 1 mM K[Co(III)(CO)]*3H2O in 1M NaHCO or just 1M NaHCO (for metal-free samples) were added. K[Co(III)(CO)]*3H2O was generated as described in Example 4. Concentration calculations were performed based on the assumption of 100% reaction efficiency of the synthesis process. After incubation of the samples at 25°C at 1400 rpm in a thermostatic shaker for 10 minutes or 48 hours, the beads were washed three times with 160 bead volumes of 1M NaHCO.

[0440] His6-GFP was immobilized on functionalized NTA magnetic beads

[0441] The resulting beads were incubated with 10 μM His6-GFP (SEQ ID NO: 14) in 120 bead volumes of protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) and incubated for 1 hour or 48 hours as indicated at 25° C. on a thermostatic shaker at 1400 rpm. After incubation of the functionalized beads with His6-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.

[0442] Chemical stability of the [Co(III)(NTA)(His6-GFP)] complex

[0443] After the described [Co(III)(NTA)(His-GFP)] complex formation process was performed, the amount of protein bound to the beads was analyzed in the presence and absence of chemical stress. To this end, the beads were divided into two parts (72 bead volumes each) and washed once with 178 bead volumes of protein buffer or 250 mM imidazole in protein binding buffer. After a final wash with 178 bead volumes of protein buffer, the beads were resuspended in 178 bead volumes of protein buffer and monitored by GFP fluorescence (λ ex =490nm,λ em =535 nm), and the amount of immobilized protein in 10 μl of bead slurry was analyzed using a plate reader (TECAN, Spark). The experiment was performed in triplicate.

[0444] Figure 12 The results depicted in clearly show that His-GFP can react with Co(III)(NTA)(CO3)] formed by incubation of K3[Co(III)(CO3)3]*3H2O with NTA. 2- The resulting [Co(III)(NTA)(His-GFP)] complex exhibited high chemical stability to imidazole treatment. Because K₃[Co(III)(CO₃)₃]*3H₂O is soluble at higher concentrations, incubation with the metal-binding domain using K₃[Co(III)(CO₃)₃]*3H₂O allowed for higher concentrations compared to Na₃[Co(III)(CO₃)₃]*3H₂O.

[0445] Example 12: His6-GFP and [Co(III)(NTA)(CO3)] in different protein binding buffers 2- Coordination and stability of the formed [Co(III)(NTA)(His-GFP)] complex

[0446] Example 7 shows the effect of different buffer substances on the binding of His-tagged proteins to [Co(III)(NTA)(CO3)] 2- In the following examples, the effects of incubation time of proteins with two different Na3[Co(III)(CO3)3]*3H2O / NTA on the binding kinetics of proteins were investigated in various buffer systems. 2- Binding of functionalized agarose beads and stability of the resulting [Co(III)(NTA)(His-GFP)] complex towards imidazole.

[0447] Functionalization of NTA agarose beads

[0448] NTA functionalized agarose beads (Thermo; 78605), 1) washed with 182 bead volumes of ddH2O, 2) washed with 182 bead volumes of 100 mM EDTA pH 8.0, 3) washed once with 182 bead volumes of ddH2O and twice with 182 bead volumes of 1 M NaHCO3 (for samples with a 10 minute Na3[Co(III)(CO3)3]*3H2O / NTA incubation time), or washed twice with 182 bead volumes of ddH2O and once with 182 bead volumes of 1 M NaHCO3 (for samples with a 48 hour Na3[Co(III)(CO3)3]*3H2O / NTA incubation time). Subsequently, 160 bead volumes of 1 mM Na3[Co(III)(CO3)3]*3H2O or 1 M NaHCO3 (for metal-free samples) in 1 M NaHCO3 were added and the beads were incubated at 25°C and 1400 rpm on a thermostatic shaker for 10 minutes or 48 hours as indicated. After incubation, the beads were washed twice with 160 bead volumes of 1 M NaHCO3 for samples with a 10 minute Na3[Co(III)(CO3)3]*3H2O / NTA incubation time, or three times for samples with a 48 hour Na3[Co(III)(CO3)3]*3H2O / NTA incubation time.

[0449] His6-GFP was immobilized on functionalized [CoIII)(NTA)(CO3)] 2- Agarose magnetic beads

[0450] The resulting beads were incubated with 120 bead volumes of 10 μM His6-GFP (SEQ ID NO: 14) in Tris-, HEPES-, MES-, MOPS-, BisTris-, ACES-, PIPES-, BES-, CAPS-, or TAPS-based protein buffer (50 mM buffer pH 7.2, 150 mM NaCl) or PBS and incubated at 25° C. with shaking at 1400 rpm on a thermostatic shaker for 1 minute, 15 minutes, 1 hour, or 24 hours as indicated. After incubation of the functionalized beads with His6-GFP (SEQ ID NO: 14), the beads were washed once with 160 bead volumes of protein buffer for samples with a 10-minute Na3[Co(III)(CO3)3]*3H2O / NTA incubation time, or with assay buffer (50 mM HEPES pH 7.2, 150 mM NaCl) for samples with a 48-hour Na3[Co(III)(CO3)3]*3H2O / NTA incubation time, and finally resuspended in 160 bead volumes of the corresponding wash buffer.

[0451] Chemical stability of the [Co(III)(NTA)(His6-GFP)] complex

[0452] After the described [Co(III)(NTA)(His-GFP)] complex formation process was performed, the amount of protein bound to the beads was analyzed with and without chemical stress. For this purpose, for samples with a 48 h Na3[Co(III)(CO3)3]*3H2O / NTA incubation time, the beads were divided into two parts (72 bead volumes each) and washed once with 178 bead volumes of protein buffer or 250 mM imidazole in 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. The fluorescence of the GFP (λ ex =490nm,λ em =535 nm), and 10 μl of bead slurry was analyzed for immobilized protein. For samples incubated with 10 minutes of Na₃[Co(III)(CO₃)₃]*3H₂O / NTA, beads were analyzed before and after washing with 160 bead volumes of 250 mM imidazole in protein binding buffer, followed by 160 bead volumes of protein binding buffer. Experiments were performed in triplicate.

[0453] The results clearly show that the His-tagged protein can be immobilized on the beads in a chemically stable manner in all buffer systems. Figure 13 ), the proportion of stable complexes was higher in the sample with 48 h Na3[Co(III)(CO3)3]*3H2O / NTA incubation time ( Figure 14 ). For all buffer systems and Na3[Co(III)(CO3)3]*3H2O / NTA incubation times, the amount of immobilized protein after imidazole treatment increased with increasing protein incubation time. Therefore, the different buffer systems had a greater impact on the reaction kinetics in samples with a 48-hour Na3[Co(III)(CO3)3]*3H2O / NTA incubation time compared to a 10-minute Na3[Co(III)(CO3)3]*3H2O / NTA incubation time. It is expected that the use of HEPES, MES, MOPS, and PIPES buffers on samples with a 48-hour Na3[Co(III)(CO3)3]*3H2O / NTA incubation time, at short protein incubation times, resulted in an increased amount of stably immobilized protein on the beads ( Figure 14 BD). For longer incubation times, it is expected that ( Figure 14 E), HEPES, MES, MOPS or ACES are preferred Good's buffers.

[0454] Example 13: Effect of pH on the interaction between His6-GFP and [Co(III)(NTA)(CO3)] 2- The influence of the coordination and stability of the formed [Co(III)(NTA)(His-GFP)] complex

[0455] Example 7 shows that the pH of the protein binding buffer has an important effect on the binding of His-tagged proteins to [Co(III)(NTA)(CO3)] 2- In the following examples, the binding kinetics of proteins to [Co(III)(NTA)(CO3)] 2- Binding of functionalized agarose beads and stability of the resulting [Co(III)(NTA)(His-GFP)] complex towards imidazole.

[0456] Functionalization of NTA agarose beads

[0457] NTA functionalized agarose beads (Thermo; 78605) were 1) washed with 26 bead volumes of ddH2O, 2) washed with 26 bead volumes of 100 mM EDTA pH 8.0, 3) washed twice with 26 bead volumes of ddH2O and once with 26 bead volumes of 1M NaHCO3. Subsequently, 160 bead volumes of 1 mM Na3[Co(III)(CO3)3]*3H2O in 1M NaHCO3 or 1M NaHCO3 (for metal-free samples) were added, and the beads were incubated at 25°C in a thermostatted shaker at 1400 rpm for 48 hours. After incubation, the beads were washed three times with 160 bead volumes of 1M NaHCO3 for samples with a 48-hour Na3[Co(III)(CO3)3]*3H2O / NTA incubation time.

[0458] His-GFP was immobilized on functionalized [CoIII)(NTA)(CO3)] 2- Agarose magnetic beads

[0459] The resulting beads were mixed with 10 μM His6-GFP (SEQ ID NO: 14) in 120 bead volumes of BisTris- or HEPES-based protein buffer (50 mM buffer, 150 mM NaCl) at pH 5.5, 6.0, 6.5, 7.0, or 7.5 for BisTris-based systems and pH 7.5, 8.0, 8.5, 9.0, or 9.5 for HEPES-based systems and incubated at 25° C. on a thermostat shaker at 1400 rpm for 1 minute, 15 minutes, 1 hour, or 24 hours as indicated. Following incubation of the functionalized beads with His6-GFP (SEQ ID NO: 14), the beads were washed once with 160 bead volumes of assay buffer (50 mM HEPES pH 7.2, 150 mM NaCl) and finally resuspended in 160 bead volumes of assay buffer.

[0460] Chemical stability of the [Co(III)(NTA)(His6-GFP)] complex

[0461] After the [Co(III)(NTA)(His-GFP)] complex formation process was performed as described, the amount of protein bound to the beads was analyzed with and without chemical stress. To this end, the beads were divided into two parts (72 bead volumes each) and washed once with 178 bead volumes of protein buffer or 250 mM imidazole in 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. The fluorescence of the GFP (λ) was monitored using a plate reader (TECAN, Spark). ex =490nm,λ em=535 nm) to analyze the amount of immobilized protein in 10 μl of bead slurry. The experiment was performed in triplicate.

[0462] The results clearly demonstrate that His-tagged proteins can be immobilized on the beads in a chemically stable manner at all pH values. Thus, a high percentage of His-GFP can be immobilized in a chemically stable manner, and the amount of immobilized protein increases with increasing protein incubation time before and after imidazole treatment ( Figure 15 A). The complex formation efficiency of the final [Co(III)(NTA)(His-GFP)] complex was dramatically increased using a protein binding buffer at a lower pH value ( Figure 15 B). Differences in efficiency are observed not only based on pH, but also due to the buffer system (pH 7.5 BisTris vs. HEPES).

[0463] Example 14: Immobilization of different proteins via their His-tag or histidine-rich region to [Co(III)(NTA)(CO)3] 2- or [Co(III)(IDA)(CO)3] - Functionalized beads

[0464] Several examples demonstrate that His6-GFP can be expressed by [Co(III)(NTA)(CO)3] 2- or [Co(III)(IDA)(CO)3] - Immobilization on beads. In the following examples, the immobilization of different His-tagged proteins and antibodies (coordinated via the histidine-rich region of their Fc portion) on beads was tested. In addition, the functionality of the immobilized enzyme (sortase) and antibody (binding of GFP to immobilized anti-GFP IgG1) was investigated.

[0465] NTA functionalized agarose magnetic beads (Thermo; 78605) or IDA functionalized magnetic beads (Cube Biotech; 30805) were 1) washed with 33 bead volumes of ddH2O, 2) washed with 33 bead volumes of 100 mM EDTA pH 8.0, 3) washed once with 33 bead volumes of ddH2O and twice with 33 bead volumes of 1 M NaHCO3 (for samples without metal and with cobalt centers), or washed three times with 33 bead volumes of ddH2O (for samples with nickel centers). Subsequently, 160 bead volumes of Na3[Co(III)(CO3)3]*3H2O or 1 M NaHCO3 in 1 M NaHCO3 (for samples without metal) or 1 mM NiSO4 in ddH2O (for samples with nickel centers) were added and the beads were incubated at 25°C and 1400 rpm in a thermostatted shaker for 10 minutes. After incubation, the beads were washed three times with 160 bead volumes of 1 M NaHCO3 or ddH2O (for samples with nickel centers).

[0466] Proteins were functionalized with [CoIII)(NTA / IDA)(CO3)] 2- Immobilization on agarose beads

[0467] The resulting beads were mixed with 120 bead volumes of protein (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 protein binding buffer (50 mM HEPES pH 7.2, 150 mM NaCl) and incubated at 25°C with shaking at 1400 rpm on a thermostatic shaker for 48 hours or 30 minutes (for IDA samples). After incubation of the functionalized beads with protein, protein supernatant samples were saved for later analysis by SDS-PAGE, and the beads were washed once with 160 bead volumes of protein buffer and finally resuspended in 160 bead volumes of assay buffer or, for antibody samples, continued with GFP incubation as described in a separate section.

[0468] Chemical stability of [Co(III)(NTA / IDA)(protein)] complexes

[0469] After the [Co(III)(NTA / IDA)(protein)] complex formation process was performed, the amount of protein bound to the beads after chemical stress was analyzed. To this end, the beads were washed once with 178 bead volumes of 250 mM imidazole in protein binding buffer. After a final wash with 178 bead volumes of protein buffer, the beads were resuspended in 178 bead volumes of protein buffer. The amount of protein on 25 μl of bead slurry was determined on a microplate by BCA assay (Thermo, 23227) according to the manufacturer's instructions.

[0470] Determining the functionality of sortase A immobilized on beads

[0471] Following the manufacturer's instructions, use the 520 Sortase A Activity Assay Kit (#72228) measures the activity of Sortase A immobilized on 20 μl of bead slurry.

[0472] GFP-binding immobilized α-GFP antibody

[0473] To evaluate the functionality of the immobilized antibodies, beads functionalized with [Co(III)(NTA / IDA)(IgG1)] were incubated for 1 hour at 25°C with 120 bead volumes of 0.54 μM GFP (without His tag) (Abcam; ab84191) in protein binding buffer. After incubation, the beads were washed once with 160 bead volumes of protein buffer and finally resuspended in 160 bead volumes of assay buffer and, for samples with IDA, the chemical stability assay as described above was performed. Finally, the GFP fluorescence (λ) of 10 μl of bead slurry was measured using a plate reader (TECAN, Spark). ex =490nm,λ em =535 nm) and GFP bound to the immobilized antibody was determined.

[0474] SDS-PAGE of protein supernatant

[0475] SDS-PAGE of protein supernatants after protein incubation was performed as described in Example 3, except that 12% (w / v) acrylamide-bisacrylamide (37.5:1) gels were used and 6 μl of protein supernatant was loaded per well. Bands were visualized using InstantBlue Coomassie stain (Expedion; ISB1L).

[0476] Purification of His-sortase

[0477] The enzyme sortase A (SEQ ID NO: 16) was expressed in E. coli BL21(DE3) using the plasmid pET29_eSrtA (Addgene #75144) (Chen, Dorr et al. 2011) and was cloned by Ni as described (Chen, Dorr et al. 2011). 2 + -NTA column purification.

[0478] Figure 16 The results shown in clearly demonstrate that, in addition to His6-GFP, other His-tagged proteins or even antibodies can also be cleared from the supernatant during protein binding via their histidine-rich regions ( Figure 16 A), and finally stably fixed on [Co(III)(NTA)(CO)3] 2- or [Co(III)(IDA)(CO)3] - Functionalized beads ( Figure 16 B). In addition, it can be shown that immobilized proteins are still functional, for example the enzyme sortase A is still active ( Figure 16 C) Anti-GFP antibodies immobilized on NTA or IDA beads can still bind to their antigen GFP.

[0479] Example 15: Complex Formation and Stability Using Other Metal Binding Domains Other Than NTA

[0480] Example 4 demonstrates that [Co(III)(NTA)CO3] 2- In addition, Example 5 shows that it is possible to synthesize [Co(III)(NTA)CO3] 2- Complexes were surprisingly used to form [Co(III)(NTA)(His-protein)] complexes on beads. We hypothesized that other metal-binding domains besides NTA could also be used to form [Co(III)(metal-binding domain)(His-protein)] complexes. To test the versatility of the method, complex formation using iminodiacetic acid (IDA), a tridentate metal-binding domain, and TALON, a commercial tetradentate metal-binding domain, was examined.

[0481] Functionalization of IDA / TALON magnetic beads

[0482] IDA-functionalized magnetic beads (Cube Biotech; 30805) or TALON-functionalized magnetic agarose resin (Takara, 635636) were washed 1) with 20 bead volumes of ddHO, 2) with 20 bead volumes of 100 mM EDTA, pH 8.0, 3) twice with 20 bead volumes of ddHO, and once with 20 bead volumes of 1M NaHCO. Subsequently, 160 bead volumes of 1 mM Na[Co(III)(CO)]*3HO in 1M NaHCO or 1M NaHCO alone (for metal-free samples) were added. Samples were incubated in a thermostatted shaker at 25°C and 1400 rpm for 10 minutes or 48 hours, as indicated, and the beads were washed three times with 160 bead volumes of 1M NaHCO.

[0483] His6-GFP is immobilized on functionalized IDA / TALON magnetic beads

[0484] The resulting beads were incubated with 10 μM His6-GFP (SEQ ID NO: 14) in protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) at 160 bead volumes and incubated at 25° C. on a constant shaker at 1400 rpm for 30 minutes, 1 hour, or 48 hours as indicated. After incubation of the functionalized beads with His6-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.

[0485] Chemical stability of the [Co(III)(IDA / TALON)(His6-GFP)] complex

[0486] After the described [Co(III)(IDA)(His-GFP)] or [Co(II)(TALON)(His-GFP)] complex formation process was performed, the amount of protein bound to the beads was analyzed with and without chemical stress. To this end, the beads were divided into two parts (72 bead volumes each) and washed once with 178 bead volumes of protein buffer or 250 mM imidazole in 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 monitored by GFP fluorescence (λ) using a plate reader (TECAN, Spark). ex =490nm,λ em =535 nm) were used to analyze the amount of immobilized protein in 10 μl of bead slurry. Both experiments were performed in triplicate.

[0487] The experiment clearly shows that using IDA ( Figure 17 A) and TALON( Figure 17B) Protein immobilization as a metal binding domain. Thus, cobalt(III)-mediated protein immobilization via cobalt(III) carbonate complexes is not limited to the use of NTA as a metal binding domain. In addition, the tridentate metal binding domain IDA exhibits even improved properties, e.g., compared to (see Figure 20 B) Improved stability with short incubation times. The tetradentate metal binding domain TALON also demonstrated protein immobilization.

[0488] Example 16: Study on the chemical stability of the [Co(III)(IDA)(His-GFP)] complex

[0489] Example 1 demonstrates 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 to imidazole are demonstrated. In the following examples, [Co(III)(IDA)(His-GFP)] functionalized beads were incubated with a strong chelating agent or with a combination of a widely used reducing agent and 250 mM imidazole to demonstrate the chemical stability of the [Co(III)(NTA)(His-GFP)] complex. 3+ The coordination of the metal center can form chemically stable complexes, as demonstrated in Example 1 by NTA and Co 3+ The composition of the complex.

[0490] Functionalization of IDA magnetic beads

[0491] IDA-functionalized magnetic beads (Cube Biotech; 30805) were 1) washed with 20 bead volumes of ddH2O, 2) washed with 20 bead volumes of 100 mM EDTA pH 8.0, 3) washed twice with 20 bead volumes of ddH2O, and once with 20 bead volumes of 1M NaHCO3. Subsequently, 160 bead volumes of 1 mM Na3[Co(III)(CO3)3]*3H2O or 1M NaHCO3 (for metal-free samples) in 1M NaHCO3 were added, and the beads were incubated at 1400 rpm at 25°C for 10 minutes in a thermostatic shaker. After incubation, the beads were washed three times with 20 bead volumes of 1M NaHCO3. The resulting beads were incubated with 10 μM His6-GFP (SEQ ID NO: 14) in 50 mM HEPES pH 7.2, 150 mM NaCl (160 bead volumes) and incubated for 30 minutes at 25° C. on a thermostatic shaker at 1400 rpm. After incubation with His6-GFP (SEQ ID NO: 14), the beads were washed three times with 160 bead volumes of protein buffer.

[0492] Chemical stability of the [Co(III)(IDA)(His6-GFP)] complex

[0493] Subsequently, 160 bead volumes of each test agent (final concentration: 250 mM imidazole, 25 mM NTA or 25 mM EDTA in protein buffer, or 1 mM DTT, TCEP or ascorbic acid supplemented with 250 mM imidazole in protein buffer, or 50 mM glycine pH 10.0) were added to the corresponding samples as indicated. After incubation at 25°C and 1400 rpm shaking for 1 hour, the supernatant was removed, the beads were washed three times with 160 bead volumes of protein buffer, and dissolved in 160 bead volumes of protein buffer. Finally, the beads were detected by GFP fluorescence (λ) using a plate reader (TECAN, Spark). ex =490nm,λ em =535 nm), and 10 μl of bead slurry was analyzed for the amount of remaining immobilized protein. The experiment was performed in triplicate.

[0494] The experiments clearly demonstrated the high chemical stability of the [Co(III)(IDA)(His6-GFP)] complex towards different chemical substances including chelating agents and reducing agents. Figure 18 As shown, when His6-GFP was bound to the [Co(III)(IDA)(CO3)] complex, only a very small reduction in immobilization was observed after incubation with the tested chelating agents or reducing agents compared to beads treated with buffer. Therefore, the destruction of the strong chelating agent by the [Co(III)(IDA)(His6-GFP)] complex and its reduction to Co 2+ All are inert.

[0495] Example 17: Comparison of [Co(III)(IDA)(CO3)] - With [Co(III)(IDA)(H2O)2] + [Co(III)(IDA)(His-GFP)] complex formation

[0496] Example 4 demonstrates that [Co(III)(NTA)CO3] 2- In addition, Example 5 shows that it is possible to synthesize [Co(III)(NTA)CO3] 2- Complexes were surprisingly used to form [Co(III)(NTA)(His-protein)] complexes on beads. In Example 6, it was shown that the kinetics of complex formation using [Co(III)(NTA)(H2O)2] were comparable to those using His-GFP. 2-The binding kinetics of the metal were improved. In Example 15, it was demonstrated that IDA can act as a metal binding domain through [Co(III)(IDA)CO3] - To form the [Co(III)(IDA)(His-GFP)] complex. We speculated that carbonate as a ligand to the cobalt(III) center might also promote the formation of the [Co(III)(IDA)(His-protein)] complex. To confirm this finding, we directly compared His6-GFP with [Co(III)(IDA)(H2O)2] and with [Co(III)(IDA)(CO3)]. - The complex formation efficiency and the chemical stability of the final [Co(III)(IDA)(His-GFP)] complex were investigated.

[0497] Functionalization of IDA magnetic beads

[0498] IDA functionalized magnetic beads (Cube Biotech; 30805) were 1) washed with 80 bead volumes of ddH2O, 2) washed with 80 bead volumes of 100 mM EDTA pH 8.0, 3) washed twice with 80 bead volumes of ddH2O and washed with 80 bead volumes of 1 M NaHCO3 (for [Co(III)(IDA)(CO3)] 2- complex) or ddH2O (for [Co(II)(IDA)(H2O)2] - The complex or metal-free sample) was washed once.

[0499] Then 160 bead volumes of 1 mM Co(II)Cl2*6H2O in degassed ddH2O (for [Co(II)(IDA)(H2O)2] and [Co(III)(IDA)(H2O)2]) were added. + sample) or 1 mM Na3[Co(III)(CO3)3]*3H2O in 1 M NaHCO3 (for [Co(III)(IDA)(CO3)] - To the metal-free sample, 160 bead volumes of ddH2O were added. Incubate in a thermostatic shaker at 25°C and 1400 rpm for 10 minutes (or for samples with [Co(III)(IDA)(CO3)] - After incubation for 48 hours for the designated samples of the complexes), the beads were washed with 160 bead volumes of 1 M NaHCO3 (for complexes with [Co(III)(IDA)(CO3)] 2- The [Co(III)(IDA)(H2O)2] complex samples) or ddH2O (for [Co(II)(IDA)(H2O)2] samples or metal-free samples) were washed three times. +The beads were washed once with 160 bead volumes, incubated in 160 bead volumes of 20 mM H2O2 at 1400 rpm on a thermostatted shaker at 25°C for 1 hour, and finally washed once with 160 bead volumes of ddH2O.

[0500] His6-GFP was immobilized on functionalized IDA magnetic beads

[0501] The resulting beads were incubated with 10 μM His6-GFP (SEQ ID NO: 14) in protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) or PBS at 160 bead volumes and incubated for 3 hours or 24 hours at 25° C. with shaking at 1400 rpm on a thermostatic shaker. After incubation of the functionalized beads with His6-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.

[0502] Chemical stability of the [Co(III)(IDA)(His6-GFP)] complex

[0503] After the described Co(III)(IDA)(His-GFP)] complex formation process was performed, the amount of protein bound to the beads was analyzed with and without chemical stress. To this end, the beads were divided into two parts (72 bead volumes each) and washed once with 178 bead volumes of protein buffer or 250 mM imidazole in 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 monitored by GFP fluorescence (λ) using a plate reader (TECAN, Spark). ex =490nm,λ em =535 nm) were used to analyze the amount of immobilized protein in 10 μl of bead slurry. The experiment was performed in triplicate.

[0504] The results of these experiments clearly showed that the [Co(III)(IDA)(CO3)] - Complex functionalized beads, compared to preloaded [Co(III)(IDA)(H2O)2] + The beads bound significantly more protein ( Figure 19 ). For [Co(III)(IDA)(CO3)] obtained by incubation with Na3[Co(III)(CO3)3]*3H2O for 48 hours - Complex-functionalized beads only showed excess Co(III)(IDA)(H2O)2 after 24 h of protein incubation. +The significant advantages of the complex. It should be emphasized that for IDA, the preferred incubation time is 10 minutes of Na3[Co(III)(CO3)3]*3H2O / NTA incubation and 30 minutes to 3 hours of subsequent protein incubation. With these incubation time combinations, the Na3[Co(III)(IDA)(H2O)2] + In comparison, [Co(III)(IDA)(CO3)] - The complexes can achieve significantly improved immobilization efficiency. In addition, Figure 19 The strong stability of the [Co(III)(IDA)(His6-GFP)] complex to the chelating agent (here imidazole) was confirmed again.

[0505] Example 18: Comparison of [Co(III)(metal binding domain)(CO3)] 2- Formation of a chemically stable complex with [Co(III)(metal-binding domain)(His-GFP)] by oxygen treatment of [Co(II)(metal-binding domain)(His-GFP)]

[0506] Example 5 demonstrates that [Co(III)(NTA)(CO3)] 2- A high percentage of the formed [Co(III)(NTA)(His-protein)] complexes are chemically stable. We speculate that compared to the treatment of [Co(II)(metal binding domain)(His-protein)] with oxygen, the [Co(III)(metal binding domain)CO3] complexes starting from [Co(III)(metal binding domain)CO3] 2- The formation of [Co(III)(metal-binding domain)(His-protein)] complexes by 8 h of oxygen treatment of [Co(III)(metal-binding domain)(His-protein)] resulted in an increased amount of chemically stable [Co(III)(metal-binding domain)(His-protein)] complexes. To confirm this finding, the amounts of chemically stable [Co(III)(metal-binding domain)(His-protein)] complexes formed by [Co(III)(metal-binding domain)(CO3)] or [Co(II)(metal-binding domain)(His-protein)] were directly compared using NTA or IDA as the metal-binding domain, respectively.

[0507] Functionalization of IDA / NTA agarose beads

[0508] IDA functionalized magnetic beads (Cube Biotech; 30805) or NTA functionalized magnetic agarose resin (Thermo, 78605) were washed 1) with 80 bead volumes of ddHO, 2) with 80 bead volumes of 100 mM EDTA pH 8.0, 3) twice with 80 bead volumes of ddHO and once with 160 bead volumes of ddHO (for [Co(II)(IDA / NTA)(HO)] complex) or 1 M NaHCO (for [Co(III)(IDA / NTA)(CO)] complex or metal-free samples). For samples with [Co(II)(IDA / NTA)(HO)] complex, all washes were performed using degassed, 20-minute nitrogen-gated solutions and in tubes blanketed with nitrogen.

[0509] Then 160 bead volumes of 1 mM Co(II)Cl2*6H2O in degassed, 20 min nitrogen-sparged ddH2O (for [Co(II)(IDA / NTA)(H2O)2]) or 1 mM Na3[Co(III)(CO3)3]*3H2O in 1 M NaHCO3 (for [Co(III)(IDA / NTA)(CO3)] complexes) were added. 1 M NaHCO3 was added to the metal-free samples. The samples were incubated at 25°C at 1400 rpm in a constant shaker for 10 min (or for samples with [Co(III)(NTA)(CO3)] complexes). 2- After incubation for 48 h for the designated samples with the complexes), the beads were washed three times with 160 bead volumes of ddH2O (for the [Co(II)(IDA / NTA)(H2O)2] samples) or 1M NaHCO3 (for the samples with the [Co(III)(IDA / NTA)(CO3)] complexes or metal-free samples).

[0510] His6-GFP was immobilized on functionalized IDA / NTA magnetic beads

[0511] The resulting beads were incubated with 120 bead volumes of 10 μM Hes6-GFP (SEQ ID NO: 14) in protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) and incubated at 25°C on a thermostatic shaker at 1400 rpm for 30 minutes or 48 hours (for metal-free samples) or as described above. Figure 9After incubation of the functionalized beads with His6-GFP (SEQ ID NO: 14), the beads were washed twice with 160 bead volumes of protein buffer (sample "[Co(III)(IDA / NTA)(His-GFP)] passed through H2O2" only once), and finally 160 bead volumes of protein buffer were added. One sample of [Co(II)(IDA / NTA)(His-GFP)] (in Figure 20 In the sample [Co(III)(IDA / NTA)(His-GFP)] was aerated with O2 for 8 h, while in the other sample [Co(II)(IDA / NTA)(His-GFP)] (referred to as “[Co(III)(IDA / NTA)(His-GFP)] was aerated with O2 for 8 h Figure 9 6,4 bead volumes of 500 mM H2O2 (final 20 mM) were added to the beads (referred to as "[Co(III)(IDA / NTA)(His-GFP)] by H2O2") and incubated on a thermostated shaker at 1400 rpm at 25°C for 1 hour, followed by washing with 160 bead volumes of protein buffer.

[0512] Chemical stability of the [Co(III)(IDA / NTA)(His6-GFP)] complex

[0513] After the described Co(III)(IDA)(His-GFP)] or Co(II)(NTA)(His-GFP)] complex formation process was performed, the amount of protein bound to the beads was analyzed in the presence and absence of chemical stress. To this end, the beads were washed once with 160 bead volumes of protein buffer, resuspended in 160 bead volumes of protein buffer, divided into two parts (72 bead volumes each) and washed once with 178 bead volumes of protein buffer or 250 mM imidazole in protein binding buffer. After a final wash with 178 bead volumes of protein buffer, the beads were resuspended in 178 bead volumes of protein buffer and the fluorescence of the beads was monitored using a plate reader (TECAN, Spark) by GFP fluorescence (λ ex =490nm,λ em =535 nm) were used to analyze the amount of immobilized protein in 10 μl of bead slurry. IDA experiments were performed in triplicate; NTA experiments were performed individually in three independent experiments.

[0514] Figure 20 The results of these experiments reported in clearly show that the use of [Co(III)(IDA)(CO3)] 2- Complex ( Figure 20 A) and [Co(III)(NTA)(CO3)] 2- Complex ( Figure 20B) Compared to beads functionalized with the [Co(II)(metal binding domain)(His-protein)]] complex aerated with oxygen for 8 h, significantly more His6-GFP protein can be chemically and stably immobilized on the beads. In addition to a higher percentage of stable complexes, the use of [Co(III)(metal binding domain)(CO3)] 2- It is also faster than the oxidation method.

[0515] Example 19: [Co(III)(HS-PEG-NTA)(CO3)] 2- Complexes immobilize proteins on surfaces

[0516] Several examples of the present invention demonstrate that [Co(III)(NTA)(CO3)] 2- [Co(III)(NTA)(His-protein)] complex is formed. In this example, protein immobilization using this principle was tested on a glass surface with nanostructured gold dots.

[0517] Generation and passivation of nanostructured glass surfaces

[0518] Nanostructured surfaces, as previously described (Spatz, et al. 2000, Roman, Martin et al. 2003, Lohmuller, Aydin et al. 2011), were produced by diblock copolymer micellar nanolithography and had an average interparticle 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. Subsequently, a 0.5 ratio of tetrachloroauric acid to vinylpyridine monomer was added to the solution and stirred for 24 hours. The solution was spin-coated onto a 20x20 mm N°1 glass coverslip (Carl Roth, Germany). The substrate was then plasma treated (10% H2 / 90% Ar, 350 W, 0.4 mbar, 45 minutes).

[0519] To prevent any nonspecific adhesion of proteins to the glass substrate between the gold nanostructures, the glass surface was passivated according to a previously described procedure (Blummel, Perschmann et al., 2007). The nanopatterned surface was activated in oxygen plasma (150 W, 0.4 mbar, 10 minutes) and incubated overnight at 80° C. in anhydrous toluene pa (Acros Organics, USA) containing 0.25 mM α-methoxy-ω-trimethoxysilyl poly(ethylene glycol) (molecular weight 2000 g / mol) (Iris Biotech, Germany), 5.5 μM water, and 20 mM anhydrous trimethylamine (Acros Organics, USA) under a nitrogen atmosphere. Finally, the substrate was washed three times with ethyl acetate (Acros Organics, USA), once with methanol (VWR Chemicals, USA), and dried under a stream of N 2 .

[0520] Functionalization of the surface with thiol-PEG-NTA

[0521] After passivation, 100 μl of 0.5 mM HS-(CH2) 11 EG3-NTA (Prochimia; TH007) in 99.8% ethanol or ethanol (for "PEG only" and "PEG / GFP" samples) was pipetted onto each surface. After incubation for 1 hour at room temperature, the surface was washed three times in a ddH2O bath or (for the sample "[Co(III)(NTA)(His-GFP)]") in 1 M NaHCO3.

[0522] Formation of [Co(III)(NTA)(His-GFP)] complex

[0523] After functionalization, the surface was covered with 400 μl of 1 mM Na3[Co(III)(CO3)3]*3H2O in 1 M NaHCO3 (for the sample "[Co(III)(NTA)(His-GFP)]") or ddH2O (for the other samples) and incubated at room temperature for 10 minutes. Subsequently, the surface was washed three times in a 1 M NaHCO3 bath for the sample "[Co(III)(NTA)(His-GFP)]" or in a ddH2O bath for the other samples. Finally, 300 μl of 10 μM His6-GFP in protein binding buffer (50 mM HEPES pH 7.2, 150 mM NaCl) was added to the surface and incubated at room temperature for 30 minutes. After washing three times in the protein binding buffer bath, the surface was placed in a transparent 6-well plate, covered with protein binding buffer, and GFP fluorescence (λ) was analyzed using a plate reader (TECAN, Spark). ex =490nm,λ em=535 nm) as a measure of the immobilized protein on the surface.

[0524] The experimental results are as follows Figure 21 Compared with other control samples, much higher fluorescence signals can be measured on the surface treated with thiol-PEG-NTA, Na3[Co(III)(CO3)3]*3H2O, and His-GFP, indicating that the [Co(III)(NTA)(His-GFP)] complex is successfully formed on the surface.

[0525] Example 20: Site-specific biotinylation of His-GFP using [Co(III)(biotin-X-NTA)(CO3)] complex

[0526] In several examples, it was demonstrated that His-tagged proteins were expressed by [Co(III)(NTA)(CO3)] 2- The complex was immobilized on beads and, in Example 19, on a surface functionalized with NTA. In the following example, the biotinylation of His-GFP at its His tag in solution by the [Co(III)(biotin-X-NTA)(CO3)] complex was tested.

[0527] Biotinylation of His-GFP

[0528] 60 μM biotin-X-NTA (Sigma-Aldrich; 51410) was mixed with 30 μM (sample 1:2), 60 μM (sample 1:1), or 600 μM (sample 10:1) Na3[Co(III)(CO3)3]*3H2O in 1 M NaHCO3 or 1 M NaHCO3 alone (for samples with metals) and incubated for 10 minutes at room temperature on a rotating wheel. The resulting [Co(III)(biotin-X-NTA)(CO3)] complex was then incubated with 6 μM His6-GFP (SEQ ID NO: 14) in protein binding buffer (50 mM HEPES pH 7.2, 150 mM NaCl) at room temperature on a rotating wheel for 30 minutes or 48 hours.

[0529] [Co(III)(Biotin-X-NTA)(His-GFP)] immobilized on streptavidin-functionalized agarose

[0530] After incubation, the resulting [Co(III)(biotin-X-NTA)(His-GFP)] complex was bound to streptavidin-functionalized sepharose (GE Healthcare, 17-5113-01) (prepared by washing three times with 166 bead volumes of protein binding buffer) at room temperature with rotation at the indicated 30-minute or 48-hour incubation steps. After incubation, the beads were washed three times with 16 bead volumes of protein binding buffer and resuspended in 16 bead volumes of protein buffer.

[0531] Chemical stability of the [Co(III)(biotin-X-NTA)(His6-GFP)] complex

[0532] After the procedure of immobilizing the [Co(III)(Biotin-X-NTA)(His-GFP)] complex to the beads, the amount of protein bound to the beads with or without chemical stress was analyzed. To this end, the beads were divided into two parts and washed once with 17 bead volumes of protein buffer or 250 mM imidazole in protein binding buffer. After a final wash with 17 bead volumes of protein buffer, the beads were resuspended in 17 bead volumes of protein buffer. Finally, the GFP fluorescence (λ) was detected using a plate reader (TECAN, Spark). ex =490nm,λ em =535 nm) to analyze the amount of immobilized protein in 10 μl of bead slurry. The experiment was performed in triplicate.

[0533] like Figure 22 As shown in Figure A, biotinylation of His-tagged GFP in solution was confirmed at all Co / NTA ratios. However, using higher ratios of biotin-X-NTA to Na3[Co(III)(CO3)3]*3H2O, better labeling efficiency was achieved, as measured by the fluorescence of the biotinylated protein immobilized on streptavidin beads. Figure 22 Panel B demonstrates that more complexes can be immobilized on the streptavidin beads after stringent imidazole washing after 48 h of protein incubation compared to samples with 10 min of protein incubation, thus, longer protein incubations will result in greater amounts of stably formed complexes.

[0534] Example 21: Formation of [Pt(IV)(NTA)(His6-GFP)] Complex Using Platinum(IV) Nitrate and Its Chemical Stability

[0535] We speculate that not only carbonate as a ligand, but also nitrate at the metal center can promote the formation of [(metal)(NTA)(His-protein)] complex. In addition, we speculate that in addition to Co 3+In addition, other transition metals with low ligand exchange rates can also form chemically stable [(metal)(NTA)(His-protein)] complexes. To confirm this finding, the binding efficiency of His6-GFP to [Pt(IV)(NTA)(NO3)] was examined.

[0536] Functionalization of NTA agarose beads

[0537] NTA functionalized agarose beads (Qiagen; 1022963) were 1) washed with 10 bead volumes of ddHO, 2) washed with 10 bead volumes of 100 mM EDTA pH 8.0, 3) washed twice with 10 bead volumes of ddHO and once with 10 bead volumes of 1M nitric acid. Subsequently, 16 bead volumes of platinum (IV) nitrate (44 mg / l Pt(IV)) (Fisher Scientific; 15407817) in 1M nitric acid or 1M nitric acid alone (for metal-free samples) were added. After incubation of the sample at 25°C at 1400 rpm in a thermostatic shaker for 10 minutes, the beads were washed three times with 16 bead volumes of 1M nitric acid.

[0538] His6-GFP was immobilized on [Pt(IV)(NTA)(NO3)] agarose beads

[0539] The resulting beads were incubated with 10 μM His6-GFP (SEQ ID NO: 14) in 16 bead volumes of protein buffer (50 mM HEPES pH 7.2, 150 mM NaCl) and incubated at 25° C. for 30 minutes with shaking at 1400 rpm on a thermostatic shaker. After incubation of the functionalized beads with His6-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.

[0540] Chemical stability of the [Pt(IV)(NTA)(His6-GFP)] complex

[0541] After the described Pt(IV)(NTA)(His6-GFP)] complex formation process was performed, the amount of protein bound to the beads was analyzed with and without chemical stress. To this end, the beads were divided into two parts (7.2 bead volumes each) and washed once with 17.8 bead volumes of protein buffer or 250mM imidazole in protein binding buffer, respectively. 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 BCA assay (Thermo, 23227) on a microplate according to the manufacturer's instructions. The experiment was performed in triplicate.

[0542] Figure 23 The results depicted in Figure 2 clearly demonstrate that platinum(IV) mediates the immobilization of His6-GFP onto NTA-functionalized beads in a chemically stable manner. This demonstrates that not only cobalt(III) but also platinum(IV) can form kinetically inert complexes with NTA and His-tagged proteins. Furthermore, by using nitrate as the metal-binding ligand, very rapid complex formation could be demonstrated.

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Claims

1. A complex comprising: a) Selected from Co 3+ or Pt 4+ of metal cations; b) Metal cation ligand CO3 2- or HCO3 - ;and c) a metal cation chelating domain comprising a chelating ligand and a label and / or a carrier, The chelating ligand is selected from NTA, IDA and TALON.

2. The complex of claim 1, wherein the chelating ligand of the metal cation chelating domain of c) is selected from the group consisting of NTA and IDA.

3. The complex of claim 1, wherein the metal cation has a water ligand exchange rate of 10 -1 s -1 or lower metal cations.

4. The complex of claim 3, wherein the metal cation has a water ligand exchange rate of 10 -2 s -1 or lower metal cations.

5. The complex of claim 1, wherein the complex comprises [Co(III)(NTA)CO3] 2- Complex, [Co(III)(NTA)HCO3] - Complex, [Co(III)(IDA)CO3] - Complex, [Co(III)(IDA)HCO3] complex, or hydrate thereof, wherein a label and / or a carrier is linked to NTA or IDA.

6. The complex of any one of claims 1 to 5, wherein the label of the metal cation chelating domain comprises a fluorophore, a diagnostic agent, a targeting moiety, a therapeutic agent, a PEG molecule, a lipid, biotin, a protein, a peptide, a toxin and / or a reactive group selected from thiols, azides, alkynes, nitrones, tetrazines and tetrazoles, and / or wherein the carrier of the metal cation chelating domain is a polymer, a microparticle, a nanoparticle or a sphere.

7. The complex of claim 6, wherein the carrier of the metal cation chelating domain is a hydrogel, a bead or a quantum dot. The complex of claim 6 , wherein the carrier of the metal cation chelating domain is a nanosphere or a microsphere.

9. A composition comprising the complex as defined in any one of claims 1 to 8.

10. A kit comprising: a) a metal cation, wherein the metal cation is a metal cation as defined in claim 1; b) Metal cation ligand CO3 2- or HCO3 - ;and c) a metal cation chelating domain comprising a chelating ligand and a label and / or a carrier, wherein the metal cation chelating domain is the metal cation chelating domain defined in claim 1 .

11. The kit of claim 10, wherein the metal cation is as defined in claim 3.

12. The kit of claim 10, wherein the metal cation chelating domain is as defined in claim 2.

13. The kit of claim 10, wherein the metal cation chelating domain is as defined in claim 6.

14. A method for producing the complex of any one of claims 1 to 8, comprising incubating in solution: (i) a metal cation as defined in claim 1a); (ii) a metal cation ligand as defined in claim 1b); and (iii) a metal cation chelating domain as defined in claim 1c).

15. The method of claim 14, wherein the metal cation chelating domain is as defined in claim 2.

16. The method of claim 14, wherein the metal cation chelating domain is as defined in claim 6.

17. The method of claim 14, wherein the metal cation is as defined in claim 3.

18. The method of claim 14, wherein the method comprises collecting and / or purifying the complex.

19. The method of claim 14, wherein the metal cation is Co 3+ , and among them Co 3+ and CO3 2- or HCO3 - Supplied as a neutral complex with a counterion.

20. The method of claim 19, wherein Co 3+ and CO3 2- or HCO3 - Supplied in salt form.

21. The method of claim 14, wherein the metal cation is Co 3+ , and among them Co 3+ and CO3 2- or HCO3 - To include Co 3+ and CO3 2- or HCO3 - The charged complex is provided in the form of a charged complex.

22. The method of claim 19, wherein the neutral complex is sodium tris(carbonate)cobalt(III)ate trihydrate (Na3[Co(III)(CO3)3]*3H2O) or potassium tris(carbonate)cobalt(III)ate trihydrate (K3[Co(III)(CO3)3]*3H2O).

23. A method for attaching 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 8 or the composition according to claim 9 with the target molecule, wherein the target molecule comprises a protein, peptide or nucleic acid that can exchange the metal cation ligand in the complex.

24. The method of claim 23, wherein the target molecule comprises a protein or DNA.

25. The method of claim 23, wherein the target molecule comprises a sequence [H n S m ] k wherein H is a histidine residue or a histidine-like residue, wherein S is a spacer amino acid residue, wherein n is independently at each occurrence from 1 to 4, wherein m is independently at each occurrence from 0 to 6, and wherein k is 2 to 6.

26. The method of claim 23, wherein the method comprises the step of recovering and / or purifying the target molecule to which the label and / or carrier is attached.

27. The method of claim 23, wherein the method further comprises, prior to said incubation, - or CO3 2- The complex is washed in a solution containing HCO3 - or CO3 2- The incubation is carried out in a solution of 28. The method according to claim 27, wherein the method further comprises, prior to said incubation, heating in a solution comprising HCO3 at a concentration of at least 1 mM - or CO3 2- and washing the complex.

29. The method according to claim 27, wherein the method further comprises, before said incubation, heating in a solution containing HCO3 at a concentration of 10 mM or 1 M - or CO3 2- and washing the complex.

30. The method according to claim 27, wherein the - or CO3 2- The incubation is carried out in a solution of 31. The method according to claim 27, wherein the - or CO3 2- The incubation is carried out in a solution of 32. The method of any one of claims 23 to 31 , wherein the incubation is carried out in an aqueous solution comprising one or more buffer substances selected from the group consisting of ACES, AMPSO, BES, BisTris, BisTris propane, borate, CAPS, CAPSO, CHES, DIPSO, EPPS, HEPES, HEPBS, HEPPSO, MES, MOPS, MOPSO, PIPES, POPSO, TAPS, TAPSO, TEA, TES, carbonate / bicarbonate buffer, phosphate buffer and Tris.

33. The method of claim 32, wherein the one or more buffer substances are selected from the group consisting of Bis-Tris, MES, HEPES, and PIPES.

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