JP2012162540A - Multivalent chelator for modifying and organizing of target molecule - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide new compounds of the formula: X-G-Cl, as well as methods for their production.SOLUTION: These constitute multivalent chelator-compounds with an affinity-tag binding to metal-chelator-complexes, which in turn can selectively modify and/or immobilize target molecules by a multitude of probes or functional units. G is a scaffold-structure, X is a coupling group for a probe F or functional unit F, CL is a chelator-group with at least a metal-coordinative center, m is an integer and at least 1, and n is an integer and at least 2.

Description

(Description of the invention)
The present invention relates to multivalent chelator compounds, methods for the generation of multivalent chelator compounds, and multivalent for modification and / or immobilization of target molecules having affinity tags that bind to metal-chelator complexes. It relates to the use of chelator compounds.

(Background of the Invention)
Selective non-covalent modification of recombinant proteins using spectroscopic or microscopic probes or (bio) chemical functional units is a central challenge for proteome analysis and biotechnology applications It is. In principle, such modifications are realized using partial specificity by means of so-called affinity tags (ie short peptide sequences introduced into proteins by genetic engineering). These affinity tags are specifically recognized by (bio) chemical recognition units. Several affinity tags have been used very successfully for purification, but binding of spectroscopic or microscopic probes and other biochemical functional units in solution and to the surface The use of affinity tags for is often critical since the complex does not exhibit sufficient stability.

  Since the mid 1970s, chelator iminodiacetic acid (IDA) has already been used in combination with several metal ions to purify proteins (Non-Patent Document 1).

  In the mid 1980s, nitrilotriacetic acid (NTA) was described as a chelator for protein purification (Patent Document 1, Patent Document 2). Furthermore, (accumulated) histidine tags have been described for general purification of recombinant proteins (see Non-Patent Document 2; Patent Document 3, Patent Document 4). Currently, the overwhelmingly most commonly used affinity tags are those with histidine tags, and the most diverse matrix and detection techniques that rely on interaction with metal ions bound to chelators have been described (Non-Patent Document 3). Nevertheless, the binding affinity of individual Ni-NTA-oligohistidine interactions is too low for stable and stoichiometrically defined binding. Partially stable binding can be achieved by an affinity matrix, or a high density of NTA groups in the plane (see, eg, Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6; Non-Patent Document 7). ). Nevertheless, high binding stability at the molecular level is required for many applications, particularly protein manipulation in solution or in vivo (eg, living cells).

  First, manipulating this affinity tag to improve the binding between the affinity tag and the metal chelator is one possibility. Thus, on the one hand, an extended histidine tag that uses 10 histidine residues instead of the general 6 histidine residues is described, for example, by Guiget et al. Nevertheless, in this manner, only complexes that are neither stoichiometrically defined nor stable are obtained. On the other hand, proteins and protein complexes that have two histidine tags and thus bind more stably to the surface of the Ni-NTA-chip have been described (9). Nonetheless, not all proteins can be provided with more than one affinity tag at the end without significantly affecting their biological function.

Another possibility for increasing the coupling consists of improving the chelator group itself. Thus, in Patent Document 5 (and Non-Patent Document 10), Ebright and Ebright describe a divalent chelator complex for labeling in situ of a protein with a detectable group such as, for example, a fluorophore. These complexes show improved affinity for the His tag compared to monovalent complexes. Nevertheless, the two chelator groups (NTA) are directly linked to a detectable group, making it difficult to use the two chelator groups for extensive synthesis. Rather, the possibilities for their production always depend on the suitability of the selected detectable group for synthesis.
Other bifunctional carboxymethyl substituted chelators are described by Kline et al. These chelators have been developed for labeling with metal complexes that can covalently bind to target proteins and have catalytic activity.
Stable and selective labeling or modification of target proteins is described by Griffin et al. Disclosed is a bi-arsenic-complex that specifically recognizes a specific motif (Cys-Cys-Xaa-Xaa-Cys-Cys) in a recombinant protein containing four cysteines. The biarsenic complex may contain a detectable group such as a fluorophore, thereby modifying or labeling the target protein. This technique is also described by Tsien et al. In US Pat. Nevertheless, the use of these diarsenic complexes is limited by the difficult synthesis of the respective diarsenic derivatives and the fact that only specific fluorophores and chromophores are possible. Furthermore, cysteine-rich affinity tags or motifs, as required, are disadvantageous for many uses due to the high reactivity of free thiol groups.

European Patent No. 0253303B1 US Pat. No. 4,877,830 European Patent No. 0820204B1 US Pat. No. 5,284,933 International Publication No. 03/091689 pamphlet US Pat. No. 6,008,378 WO03 / 107010 pamphlet

Porath J, Carlsson J, Olsson I, Belfage G, “Metal affinity chromatography, a new approach to protein fraction”, Nature, 1975, Vol. 258, p. 598-9 Hochuli E, Dobel H, Schacher A, “New metal chelate adsorbent selective for proteins and peptides contiguous nesting histidine residues, pp. 11, J. 177-84 Ueda EK, Gout PW, Morganti L, “Current and prospective applications of metal ion-protein binding”, J Chromatogr A, 2003, Vol. 98, No. 1, p. 1-23 Dorn I. T.A. Pawlitschko K .; Pettinger S., et al. C. Tampe R .; "Orientation and two-dimensional organization of proteins at chelator lipid interfaces", Biol Chem. 1998, 379, Nos. 8-9, p. 1151-9 Frenzel A, Bergemann C, Kohl G, Reinard T, "Novel purification system for 6xHis-tagged protein by bioaffinity separation, J ChromBet. 2003, 793, No. 2, p. 325-9 Paborsky LR, Dunn KE, Gibbs CS, Dougerty JP, “A nick chelate microtiter plate for six histidine-containing proteins”, Anal Bioch. 1996, Vol. 234, No. 1, p. 60-5 Lauer SA, Nolan JP, “Development and charac- terization of Ni-NTA-bearing microspheres”, Cytometry, 2002, Vol. 48, No. 3, p. 136-45 Guignet EG, Hovius R, Vogel H, “Reversible site-selective labeling of membrane proteins in live cells”, Nat Biotechnol. 2004, Vol. 22, No. 4, p. 440-4 Nieba L, Nieba-Axmann SE, Persson A, Hamalainen M, Edebratt F, Hansson A, Lidholm J, Magnusson K, Karlsson AF, Plueckthun A, "BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip", Anal Biochem . 1997, Vol. 252, No. 2, p. 217-28 Kapanidis AN, Ebright YW, Ebright RH, "Site-specific incorporation of fluorescent probes into protein: hexahistidine-tag-mediated fluorescent labeling with (Ni (2 +): nitrilotriacetic Acid (n) -fluorochrome conjugates", J Am Chem Soc,. 2001, 123, 48, p.12123-5 Kline SJ, Betebenner DA, Johnson DK, "Carboxymethyl-substituted bifunctional chelators: preparation of aryl isothiocyanate derivatives of 3- (carboxymethyl) -3-azapentanedioic acid, 3,12-to (carboxymethyl) -6,9-dioxa-3, 12-diazatetradecanoic acid, and 1, 4, 7, 10-tetraazacyclodedecane-N, N ′, N ″, N ′ ″-tetraacetic acid for use as protein labels ”, Bioconj ug Chem. 1991, Vol. 2, No. 1, p. 26-31 Griffin BA, Adams SR, Tsien RY, “Specific covalent labeling of recombinant protein molecules inside live cells”, Science, 1998, Vol. 281, No. 5374. 269-72

  Accordingly, an object of the present invention is to provide a multivalent chelator for binding to affinity tags (eg, oligohistidine tags) known and widely used in the art, Stoichiometric, stable interaction with the affinity tag, and convertible and reversibly interact with the target molecule. This allows target molecules to be modified in a selective and regiospecific manner, generally using a number of probes and other biochemically functional units. In addition, this multivalent chelator is synthetically accessible in a controlled and universal manner such as conjugation with numerous probes and other biochemically functional units.

In accordance with the present invention, this object has the general formula:
X _m -G-CL _n
Where:
G is the scaffold structure;
X is a coupling group for probe F or functional unit F;
CL is a chelator group having at least one metal coordination center;
m is an integer and is at least 1; and n is an integer and is at least 2.
Solved by providing compounds, and tautomeric organisms, isomers, anhydrides, acids and salts thereof.

  In a preferred embodiment, the scaffold structure G comprises a saturated carbohydrate chain having 2 to 25 carbon atoms, preferably 2 to 20 carbon atoms, and more preferably 5 to 16 carbon atoms. Further, the scaffold structure includes an amide bond, an ester bond, and / or an ether bond.

  The scaffold structure may be linear, branched or closed. A preferred closed scaffold structure is represented by a cyclam ring structure. A preferred scaffold structure comprises amino acid elements.

In a preferred embodiment, at least one metal ion is bound to each of the chelator groups of the compound according to the invention. In that regard, the metal ion is selected from the group consisting of Ni ²⁺ , Co ²⁺ , Cu ²⁺ , Zn ²⁺ , Fe ²⁺ , Fe ³⁺ , and all lanthanide ions.

  The chelator group is preferably nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), all modified porphyrins, salicylic acid derivatives, 1,2-diaminoethyldiacetic acid, diaminoethyltriacetic acid, hydroxyethylimino. It is selected from the group consisting of diacetic acid, and salts or combinations thereof, and other chelator groups known to those skilled in the art. In a preferred embodiment, the reactive group of the chelator group has a protecting group. For example, a carboxyl group (eg, a carboxyl group of NTA) can be protected by an OtBu group. All common protecting groups known to those skilled in the art can be used.

In a further embodiment, a spacer group A is disposed between the chelator group and the scaffold structure. The spacer group A may be linear or branched. Conventional spacer groups known to those skilled in the art can be used. Preferred spacer groups are poly (ethylene glycol), oligo (ethylene glycol), including peptides, n is 1 to 8 _{(CH 2) n,} oligo proline.

  The chelator group is preferably bonded to the scaffold structure via an amide bond, an ester bond or an ether bond.

  According to the invention, it is preferred that the scaffold structure G itself is not a probe or another detectable group. Preferably, G is a structure to which the chelator group and probe or functional unit are bound.

  Preferably, the compound according to the invention has the following:

ならびにその互変異生体、異性体、無水物、酸および塩から選択される。

And its tautomeric organisms, isomers, anhydrides, acids and salts.

Preferred coupling groups X are selected from the group consisting of NHR, wherein R is H, an alkyl or aryl residue, COOH, n is an integer (CH ₂ ) _n —COOH, SH, maleimide, Iodoacetamide, isothiocyanate or cyanate.

  In a preferred embodiment, the probe F or the functional unit F is bound at the coupling group X of the compound according to the invention. The probe F or the functional unit F is preferably a fluorophore, FRET-fluorophore, fluorescence quencher, phosphorescent compound, luminescent compound, absorptive compound, polymer, PEG, oligosaccharide, oligonucleotide , PNA, biotin, hapten, peptide, protein, enzyme, crosslinker, oligo (ethylene glycol), lipid, nanoparticle, electron density amplification factor, gold cluster, metal cluster, quantum dot, and combinations thereof Is done.

Examples of fluorophores and chromophores that are suitable as probes are described in Haughland R .; P. , 1996, Handbook of Fluorescent Probes
and Research Chemicals, Molecular Probes, 6th Edition (Spence, MTZ).

  Preferably, the compound according to the invention has the following:

ならびにその互変異生体、異性体、無水物、酸および塩から選択され、（Ａ）は、ビス−ＮＴＡであり、（Ｂ）および（Ｃ）は、トリス−ＮＴＡであり、そして（Ｄ）は、テトラキス−ＮＴＡである（図１も参照のこと）。

And its tautomers, isomers, anhydrides, acids and salts, (A) is bis-NTA, (B) and (C) are tris-NTA, and (D) is Tetrakis-NTA (see also FIG. 1).

  Generally, the polyvalent chelator has several (independent) chelator groups (still preferably 2-4) such as nitrilotriacetic acid (NTA) attached to a molecular scaffold (scaffold G). At the same time, it can be characterized in that a functional group (coupling group X) is provided for coupling to the probe F or the functional unit F. An important requirement for attachment to the probe or functional unit is the chemical orthogonality of this coupling group X to the functional group of the chelator group (eg, three carboxyl groups in the case of NTA). In the case of a multivalent chelator as shown in FIG. 1, this was achieved by a protecting group at the carboxyl group (see FIG. 2). Here, the selective coupling reaction in the further functional group (X) can be performed without affecting the chelator group.

  Furthermore, the above object is solved by providing a method for producing the compounds according to the invention. This method involves the coupling of at least two chelator groups to the scaffold structure after or during the synthesis of the scaffold structure, which can be protected in a suitable manner. During the process according to the invention, the coupling group X can also be suitably protected.

  The synthesis of the scaffold structure according to the invention comprises one or several starting compounds (in particular amino acids (eg lysine), ornithine, 1,3-diaminobutyric acid, 1,2-diaminopropionic acid, glutamate or aspartate and And / or synthesis of scaffold structures from protected derivatives thereof (eg, Z-Lys-OtBu, H-Glu (OtBu) -OBzl, Z-Glu-OH)).

  Further preferred starting compounds are bromoacetic acid-tert-butyl ester, BOC-ε-aminocaproic acid and macrocyclic polyamines (eg 1,4,8,11-tetraazacyclotetradecane).

Preferred ^{intermediates,} N α, N α - bis [(tert- butyloxycarbonyl) methyl] -L- lysine -tert- butyl ester ( "Lys-NTA-OtBu" Compound 3 of FIG. 25).

  The method for production according to the invention preferably comprises firstly the synthesis of the above scaffold structure. The chelator group is then coupled to the scaffold structure. Thus, the chelator group can have a suitable protecting group. Accordingly, the scaffold structure is preferably an annular scaffold structure (eg, a cyclam ring structure).

  The method for production according to the invention comprises that the scaffold structure can be composed of protected amino acids or compounds derived from amino acids. For this reason, it is preferred that a protective chelator group is already included during the synthesis of the scaffold structure. The starting compounds for the scaffold structure may constitute representative starting products for peptide synthesis known to those skilled in the art. For this reason, preferred scaffold structures are linear or branched (ie, dendrimeric).

  It is preferred that the carboxyl functionalized scaffold structure is modified with an amino functionalized protective chelator group, or the amino functionalized scaffold structure is modified with a carboxyl functionalized protective chelator group.

  In Scheme I, a schematic diagram of a preferred synthetic route can be found. In (A), the carboxyl-based scaffold is modified by an amino-functionalized protective chelator element (see also FIG. 3A). The protected functional group (coupling group) XP can be selectively (I) or deprotected together with the chelator group (II) and then coupled to probe F or functional unit F. obtain. (B) shows a similar synthetic pathway for amino functionalized scaffolds modified by carboxyl functionalized protective chelator elements (see also FIG. 3B). Intermediate 1 or 2 can then be used as a chelator element in the first step of the synthesis (see also Example 9).

  (Scheme I)

ここで：
Ｘは、カップリング基であり；
Ｐは、保護基であり；
Ｆは、プローブまたは官能性のユニットであり；そして
ＣＬは、キレーター基である。

here:
X is a coupling group;
P is a protecting group;
F is a probe or functional unit; and CL is a chelator group.

  Multivalent chelators with 2, 3 and 4 metal coordination centers were synthesized based on dendrimer-like scaffolds and cyclic scaffolds (ie, chemical basic scaffolds) (FIG. 1 and Examples). See

  For the production of bis-NTA-OtBu, tetrakis-NTA-OtBu and bis-NTA-lipid, the synthetic steps as shown in Example 9 and FIG. 25 are preferred.

  For the generation of Tris-NTA-OtBu and their derivatives with different fluorophores, including in particular the fluorophore Oregon Green 488, ATTO 565, FEW-S0387 (see also FIG. 14 for this) A synthetic step as shown in Example 9 and FIG. 26 is preferred.

  Furthermore, the object is solved by the use of a compound according to the invention for binding to an affinity tag on a target molecule, wherein the affinity tag binds to a metal-chelator complex.

  Preferably, the affinity tag is a peptide tag comprising between 4 and 15 amino acids. In the case of these 4 to 15 amino acids, these are preferably 4 to 15 histidines. In addition, 0 to 4 basic amino acids (eg, lysine and arginine) can be included in the peptide tag.

A preferred affinity tag is a (His) _n tag, where n is an integer between 4 and 15.

  Preferred target molecules include peptides, polypeptides, proteins, and peptidomimetics and protein mimetics, and peptide-modified polymers or peptide-modified dendrimers. In this regard, proteins or peptides that are post-translationally modified are also recognized by the protein or peptide. Peptidomimetics and protein mimetics include compounds that contain similar side chain functionalities such as peptides or proteins, but differ in peptide composition from peptides or proteins. Possible variations of the backbone include backbone atom modifications (skeletal mimics), the introduction of bicyclic dipeptide analogs, and the placement of functional groups in non-oligomeric chain structures (scaffold mimics). For example, oligo-N-alkylglycine (peptoid) belongs to a skeletal mimetic, which differs from a peptide or protein at the point of attachment of the side chain (N instead of Cα).

  Preferably, the compounds according to the invention are used for modification, immobilization, coupling, purification, detection, monitoring, analysis or detection of target molecules in vitro, in vivo, in situ, fixed cells and living cells or lipid vesicles Can be done.

  A further preferred use of the compounds according to the invention is the controlled and reversible dimerization or oligomerization of the target molecules (in particular proteins) with the functional units of the molecules.

  The compounds according to the invention can be used in a number of in vitro and in vivo assays known in the art. Preferred assay methods include spectroscopic methods (eg, absorption spectroscopy, fluorescence spectroscopy, fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), fluorescence-bleaching (fluorescence after photobleaching). Recovery (fluorescence recovery after photobleaching), FRAP), reflectometric interference-spectroscopy (RIfS), surface plasmon spectroscopy (surface plasmon resonance) / BIACORE, optical scanning coupling, optical scanning coupling Quartz-micro balance, surface acoustic wave (SAW), x / -Fluorescence-scanning (FluorImaging)) and microscopic methods (e.g. fluorescence microscope, confocal microscope, reflection microscope, contrast enhancing microscope, electron microscope, scanning probe microscope) , Other methods (eg, magnetic resonance, microscopic and tomography, impedance spectroscopy, field effect transistors, enzyme-linked immunosorbent assay (ELISA), fluorescence-labeled cell or particle sorting ( FACS), radioimmunoassay (RIA), autoradiography, analytical gel filtration, stopped-flow technology, calorimetry, high-throughput screening (HTS), Array technology and chip technology (e.g., a protein array)) including.

  Further preferred is the use of a compound according to the invention for the immobilization of the target molecule.

  To this end, the compounds of the invention can be bound on the surface or included in a lipid monolayer or lipid bilayer. Thus, the surface is preferably a glass-type surface (eg metalloid oxide, metal oxide and all glass types / glass, gold, silver, DAPEG modified glass, PEG polymer modified glass or PEG polymer). Selected from modified gold, GOPTS silanized glass, lipid monolayers or lipid bilayers, glass mold surfaces with metal selenides, metal tellurides, and metal sulfides and noble metal surfaces.

  By glass surface, it should be understood that in addition to glass, the glass mold surface also contains quartz, mica, metal oxides, metalloid oxides.

  The use according to the invention for producing a self-assembled monolayer (SAM) on a noble metal surface is preferred. SAMs are preferably used in methods based on surface plasmon spectroscopy, impedance spectroscopy, scanning probe microscopy and quartz crystal microbalance.

  Furthermore, the compounds of the invention can be used for the production of functional microstructured surfaces and functional nanostructured surfaces and / or protein arrays.

  The use of an excess amount of oligohistidine tag to increase the stability of protein-chelator binding by several scales through multivalent chelators (MCH) is the basic concept of the present invention.

The binding of an oligohistidine tag to a metal-chelator complex is generally complexed by a chelator and thereby partially coordinatively saturated with an oligohistidine tag in a free coordination position in Ni2 ⁺ . This is caused by the coordinate bond of the N atom of the imidazole residue. In the case of the Ni-NTA complex, 4 out of all 6 coordination positions of Ni ²⁺ are left in two free coordination positions for the binding of 2 histidine residues. (FIG. 4A). This complex formation therefore requires the following two steps (FIG. 4B): (1) chelator activation by binding of metal ions (eg, Ni ²⁺ ), and (2) vacant Binding of the histidine residue of the oligohistidine tag to the coordinate position. By the addition of excessive free imidazole, the binding of the oligohistidine tag to the Ni (II) -chelator complex can be disabled so that the binding of the protein to the chelator is reversible and convertible. Furthermore, the chelator can be deactivated by removing Ni ²⁺ from the chelate complex using EDTA (FIG. 4B).

  The binding of the oligohistidine tag to a metal chelate complex with only two histidine residues is relatively unstable (see Example 4 and Example 8). When the metal chelator is immobilized at high density, several histidine residues of the oligohistidine tag can simultaneously bind to several metal-chelate units, resulting in stronger binding (FIG. 5B). . By using a multivalent chelator containing several metal-chelate units in one molecule, stable binding to the oligohistidine tag at the molecular level can be achieved. By coupling an additional functional unit to the chelator, a protein containing an oligohistidine tag is stable but can be modified reversibly and convertibly (FIG. 5C).

から選択される、化合物。
（項目１１）
項目１〜１０のいずれか１項に記載の化合物であって、該化合物は、カップリング基Ｘが、ＮＨＲからなる群より選択されることによって特徴付けられ、Ｒは、Ｈ、アルキルもしくはアリール、ＣＯＯＨ、ｎが整数である（ＣＨ _２ ） _ｎ −ＣＯＯＨ、ＳＨ、マレイミド、アセトアミド、イソチオシアネートまたはシアネートである、化合物。
（項目１２）
項目１〜１１のいずれか１項に記載の化合物であって、該化合物は、プローブＦまたは官能性のユニットＦが、カップリング基Ｘにおいて結合されることによって特徴付けられる、化合物。
（項目１３）
項目１２に記載の化合物であって、該化合物は、前記プローブＦまたは官能性のユニットＦが、フルオロフォア、ＦＲＥＴ−フルオロフォア、蛍光消光剤、リン光化合物、発光化合物、吸収性化合物、ポリマー、ＰＥＧ、オリゴ糖類、オリゴヌクレオチド、ＰＮＡ、ビオチン、ハプテン、ペプチド、タンパク質、酵素、架橋剤、オリゴエチレングリコール、脂質、ナノ粒子、電子密度増幅因子、金クラスター、金属クラスター、量子ドット、およびそれらの組み合わせからなる群より選択されることによって特徴付けられる、化合物。
（項目１４）
項目１〜１３のいずれか１項に記載の化合物であって、該化合物が、以下：

から選択される、化合物、ならびにその互変異生体、異性体、無水物、酸および塩。
（項目１５）
項目１〜１４のいずれか１項に記載の化合物を生成するための方法であって、該方法は、前記足場構造の合成後か、または合成の間において、少なくとも２つのキレーター基を該足場構造に対してカップリングする工程を包含する、方法。
（項目１６）
項目１５に記載の方法であって、該方法は、保護されたまたは保護されていないアミノ酸およびアミノ酸誘導体、Ｚ−Ｌｙｓ−ＯｔＢｕ、Ｈ−Ｇｌｕ（ＯｔＢｕ）−ＯＢｚｌ、Ｚ−Ｇｌｕ−ＯＨ、大環状ポリアミン、１，４，８，１１−テトラアザシクロテトラデカン、ブロモ酢酸−ＯｔＢｕ、ＢＯＣ−ε−アミノカプロン酸からなる群より選択される１種以上の出発化合物から足場構造を合成する工程を包含する、方法。
（項目１７）
前記キレーター基が、保護されている、項目１５または１６に記載の方法。
（項目１８）
標的分子におけるアフィニティータグへの結合のための項目１〜１４のいずれか１項に記載の化合物の使用であって、該アフィニティータグは、金属−キレーター錯体に結合する、使用。
（項目１９）
前記アフィニティータグは、４個〜１５個のヒスチジンおよび０個〜４個の塩基性アミノ酸を含む４個〜１５個のアミノ酸を含むペプチドタグである、項目１８に記載の使用。
（項目２０）
前記標的分子は、ペプチド、ポリペプチド、タンパク質、ペプチド模倣物もしくはタンパク質模倣物、またはペプチド改変ポリマーもしくはペプチド改変デンドリマーである、項目１８または１９に記載の使用。
（項目２１）
改変、カップリング、オリゴマー化、検出、モニタリング、分析のため、またはインビトロ、インビボ、インサイチュ、生細胞もしくは脂質小胞における標的分子の検出のための、項目１〜１４のいずれか１項に記載の化合物の使用。
（項目２２）
標的分子の固定化のための、項目１〜１４のいずれか１項に記載の化合物の使用。
（項目２３）
項目２２に記載の使用であって、該使用は、前記化合物が、表面結合または脂質単一層もしくは脂質二重層に含まれることによって特徴付けられる、使用。
（項目２４）
前記表面は、ガラス型表面、金、銀、ＤＡＰＥＧで改変したガラス、ＰＥＧポリマーで改変したガラスもしくはＰＥＧポリマーで改変した金、ＧＯＰＴＳでシラン処理したガラス、脂質単一層もしくは脂質二重層、セレン化金属、テルル化金属、および硫化金属を備えるガラス型表面および金表面から選択される、項目２３に記載の使用。
（項目２５）
自己組織化単分子膜（ＳＡＭ）の生産のための、項目１〜１４のいずれか１項に記載の化合物の使用。
（項目２６）
官能性のマイクロ構造表面および官能性のナノ構造表面の生産ならびに／またはプロテインアレイの生産のための、項目１〜１４のいずれか１項に記載の化合物の使用。
（項目２７）
標的分子の精製のための、項目１〜１４のいずれか１項に記載の化合物の使用。
（図面および実施例）
本発明は、添付の図面に関した以下の実施例によってさらに明らかにされるが、それにかかわらず、本発明は、これらの実施例に限定されない。 The experiments described in the following figures and examples show that multivalent chelator compounds represent a chemically, high-affinity, convertible recognition structure for affinity tags (eg, histidine tags). Show. Thus, they not only represent improvements to traditional chelators, but primarily expose new areas of use. Since they can be associated with almost any compound, for these chelators, almost any kind of spectroscopic or microscopic probe or functional unit can be regiospecifically and reversibly targeted. It can bind to a molecule (eg, a recombinant protein). Also, for example, oligosaccharides, PEG or other biological functional units can be reversibly coupled to a target molecule to modify the target molecule in such a manner. Next, the MCH can also be placed on a molecular scaffold, and by such methods, for example, target molecules such as proteins are sterically organized in a dimeric or multimeric structure. Here again, variable switchability is a central feature. Further possibilities arise from coupling with oligonucleotides or PNA. Thus, the feasibility of DNA microarray multiplexing can be used for the production of protein arrays.
The following abbreviations are used:
AU: absorbance unit Boc: tert-butyloxycarbonyl (protecting group)
Bzl: benzyl (protecting group)
DAPEG: α, ω-diaminopoly (ethylene glycol)
DIC: diisopropylcarbodiimide DMF: dimethylformamide DIPEA: diisopropylethylamine EDTA: ethylenediaminetetraacetic acid FL: fluorescein FRET: fluorescence resonance energy transfer Glu: glutamic acid GOPTS: glycidyloxypropyltriethoxysilane H10: deca-histidine H6: hexa-histidine IDA: imino Diacetate Ifnar2: extracellular domain of type 1 interferon receptor 2 IFNα2, IFNβ: type I interferon α2 and type I interferon β
MBP: Maltose binding protein MCH: Multivalent chelator NTA: Nitrilotriacetic acid OG: Oregon Green 488 (registered trademark)
PEG: Poly (ethylene glycol)
PNA: peptide nucleic acid RIfS: reflectometry interferometry RT: room temperature SAM: self-assembled monolayer TBTU: O- (benzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium Tetrafluoroborate tBu: tertiary butyl (protecting group)
TEA: triethylamine TFA: trifluoroacetic acid Z: benzyloxycarbonyl (protecting group)
For example, the present invention provides the following items.
(Item 1)
General formula:
X _m -G-CL _n
And tautomeric organisms, isomers, anhydrides, acids and salts thereof, wherein:
G is a scaffold structure comprising a saturated carbohydrate chain having 2 to 20 carbon atoms and further comprising an amide bond, an ester bond and / or an ether bond;
X is a coupling group for probe F or functional unit F;
CL is a chelator group having a metal coordination center;
m is an integer and is at least 1; and
n is an integer and is at least 2.
Compound.
(Item 2)
2. A compound according to item 1, wherein the compound is characterized by a metal ion being bound to each of the chelator groups.
(Item 3)
Item 3. The compound of item 2, wherein the metal ion is selected from the group consisting of Ni ²⁺ , Co ²⁺ , Cu ²⁺ , Zn ²⁺ , Fe ²⁺ , Fe ³⁺ , and all lanthanide ions.
(Item 4)
Item 4. The compound according to any one of Items 1 to 3, wherein the chelator group has nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), porphyrin, salicylic acid derivative, 1,2- A compound characterized by being selected from the group consisting of diaminoethyl diacetic acid, diaminoethyl triacetic acid, hydroxyethyliminodiacetic acid, and salts or combinations thereof.
(Item 5)
Item 5. The compound according to any one of Items 1 to 4, wherein the reactive group of the chelator group has a protecting group.
(Item 6)
6. The compound according to any one of items 1-5, wherein the compound is characterized by the spacer group A being placed between CL and G.
(Item 7)
Item 7. The compound according to Item 6, wherein A is linear or branched.
(Item 8)
8. The compound according to any one of items 1 to 7, wherein the compound is characterized in that the chelator group is bonded to G via an amide bond, an ester bond or an ether bond.
(Item 9)
9. A compound according to any one of items 1-8, wherein the compound is characterized by G being linear, branched or closed.
(Item 10)
The compound according to any one of items 1 to 9, and tautomers, isomers, anhydrides, acids and salts thereof, wherein the compound is:

A compound selected from:
(Item 11)
Item 11. The compound according to any one of Items 1-10, wherein the compound is characterized in that the coupling group X is selected from the group consisting of NHR, wherein R is H, alkyl or aryl, COOH, n is an integer _{(CH 2) n -COOH, SH} , maleimido, acetamido, an isothiocyanate or cyanate compound.
(Item 12)
12. A compound according to any one of items 1 to 11, wherein the compound is characterized by a probe F or a functional unit F being attached at a coupling group X.
(Item 13)
Item 13. The compound according to Item 12, wherein the probe F or the functional unit F is a fluorophore, a FRET-fluorophore, a fluorescence quencher, a phosphorescent compound, a luminescent compound, an absorptive compound, a polymer, PEG, oligosaccharide, oligonucleotide, PNA, biotin, hapten, peptide, protein, enzyme, crosslinker, oligoethylene glycol, lipid, nanoparticle, electron density amplification factor, gold cluster, metal cluster, quantum dot, and combinations thereof A compound characterized by being selected from the group consisting of:
(Item 14)
14. The compound according to any one of items 1 to 13, wherein the compound is:

And tautomers, isomers, anhydrides, acids and salts thereof selected from
(Item 15)
Item 15. A method for producing a compound according to any one of items 1-14, wherein the method comprises at least two chelator groups after or during synthesis of the scaffold structure. Coupling to the method.
(Item 16)
Item 16. The method according to Item 15, wherein the method comprises protected or unprotected amino acids and amino acid derivatives, Z-Lys-OtBu, H-Glu (OtBu) -OBzl, Z-Glu-OH, macrocycle. Synthesizing a scaffold structure from one or more starting compounds selected from the group consisting of polyamine, 1,4,8,11-tetraazacyclotetradecane, bromoacetic acid-OtBu, BOC-ε-aminocaproic acid, Method.
(Item 17)
17. A method according to item 15 or 16, wherein the chelator group is protected.
(Item 18)
15. Use of a compound according to any one of items 1-14 for binding to an affinity tag in a target molecule, wherein the affinity tag binds to a metal-chelator complex.
(Item 19)
19. Use according to item 18, wherein the affinity tag is a peptide tag comprising 4 to 15 amino acids comprising 4 to 15 histidines and 0 to 4 basic amino acids.
(Item 20)
20. Use according to item 18 or 19, wherein the target molecule is a peptide, polypeptide, protein, peptidomimetic or protein mimetic, or peptide-modified polymer or peptide-modified dendrimer.
(Item 21)
Item 15. Any one of items 1-14 for modification, coupling, oligomerization, detection, monitoring, analysis, or for detection of a target molecule in vitro, in vivo, in situ, live cells or lipid vesicles Use of compounds.
(Item 22)
Use of the compound according to any one of items 1 to 14 for immobilization of a target molecule.
(Item 23)
23. Use according to item 22, wherein the use is characterized by the compound being included in a surface binding or lipid monolayer or lipid bilayer.
(Item 24)
The surface is a glass mold surface, gold, silver, DAPEG modified glass, PEG polymer modified glass or PEG polymer modified gold, GOPTS silane treated glass, lipid monolayer or lipid bilayer, metal selenide 24. Use according to item 23, selected from glass mold surfaces and gold surfaces comprising metal tellurides and metal sulfides.
(Item 25)
15. Use of a compound according to any one of items 1 to 14 for the production of a self-assembled monolayer (SAM).
(Item 26)
15. Use of a compound according to any one of items 1-14 for the production of functional microstructured surfaces and functional nanostructured surfaces and / or protein arrays.
(Item 27)
15. Use of a compound according to any one of items 1-14 for purification of a target molecule.
(Drawings and examples)
The invention will be further clarified by the following examples with reference to the accompanying drawings, but the invention is nevertheless not limited to these examples.

FIG. 1 shows the structural formula of the multivalent chelator (MCH) and the main composition of the multivalent chelator. Each NTA group is shaded in light grey, and the coupling group is shaded in dark grey. FIG. 2 shows the composition of the multivalent chelator. (A) Mono-NTA (1), bis-NTA (2) and Tris-NTA (3) with OtBu-protected NTA groups. Free functional groups (coupling groups X) are shaded in gray. (B) The same chelator as in (A) after coupling of this functional group to the PEG probe and deprotection of the carboxyl group of the NTA residue. FIG. 3 shows, in particular, a preferred protected amino-functionalized chelator element (X-CL-P) (A), as used in Scheme I, or a preferred protected carboxy-functionalized chelator element (X-CL- P) and (B) are shown. FIG. 4 shows the principle of a reversible, reversible interaction of oligohistidine with a metal-chelator complex. (A) Coordination of two histidine residues to two free coordination positions of a Ni (II) -NTA complex. (B) Activation of chelators by metal ions and binding to histidine-tagged proteins; dissociation by competitors (eg, imidazole), and deactivation of chelators using, for example, EDTA. FIG. 5 shows the concept of a multivalent chelator. (A) Temporary monovalent interaction between an immobilized metal chelator (eg, Ni: NTA) and a histidine-tagged protein. (B) Stable multivalent interaction with two or more immobilized metal chelators (such as an affinity matrix). (C) Stable coupling to functional units by multivalent chelators. FIG. 6 shows a schematic representation of the coupling of MCH to a DAPEG modified surface (see Example 1). FIG. 7 shows protein immobilization on the functionalized glass surface. (A) Schematic representation of the binding of Tris-NTA via a PEG polymer layer (PEG2000) to a glass surface silane treated with GOPTS. (B) Ifnar2-H10 binding before (dashed line) and after loading (solid line) the chelator with Ni (II) ions. FIG. 8 shows the functionality (detected by RIfS) of the immobilized protein on the crystal surface modified with a chelator. Binding curves for ifnar2-H10 immobilization after Ni (II) ion loading of chelators and the interaction between immobilized ifnar2-H10 and its ligand IFNα2. FIG. 9 shows the immobilization of histidine-tagged protein on the gold surface. (A) Structural formula of alkylthiol used for the production of a mixed self-assembled monolayer (SAM). (B) Binding of ifnar2-H6 before loading (dashed line) and after loading (solid line) with Ni (II) ions (1), interaction with IFNα2 (2) and 200 mM imidazole (3) And regeneration with 200 mM EDTA (4) (detection by surface plasmon resonance). FIG. 10 shows a comparison of the interaction of high concentrations (30 μM) of MBP-H10 with different chelators. (A) Association and dissociation of 30 μM MBP-H10 to mono-NTA. (B) Dissociation compared for mono-NTA, bis-NTA and tris-NTA (solid line). FIG. 11 shows the induced dissociation of immobilized MBP-H10 at different concentrations of imidazole. (A) Comparison of the dissociation kinetics of ifnar2-H10 on the mono-NTA surface at different concentrations of imidazole and fitting of the binding model. (B) Equilibrium-load dependence as a function of imidazole concentration compared for mono-NTA, bis-NTA, and tris-NTA. FIG. 12 shows a comparison (A) of the stability of the binding of MBP-H6 and MBP-H10 to Tris-NTA. (B) Comparison of MBP-H10 binding stability to mono-NTA and MBP-H6 binding stability to Tris-NTA. FIG. 13 shows the non-specific binding of the protein to the chelator modified surface. (A) Binding of IFNβ before (dashed line) and after (solid line) immobilization of ifnar2-H10 to mono-NTA with high surface density. (B) IFNβ binding to Tris-NTA with low surface density IFNβ (solid line), binding of IFNβ after surface blocking with MBP-H10 (dashed line), and ifnar2-H10 immobilized, then MBP-H10 Binding of IFNβ after blocking by (solid line). FIG. 14 shows blocking of the histidine tag in solution with Tris-NTA. (A) Binding of 50 nM ifnar1-H10 without 100 nM Tris-NTA (dashed line) and 50 nM ifnar1-H10 with 100 nM Tris-NTA (solid line) on a surface modified with Tris-NTA ) (B) Binding of 100 nM ifnar1-H10 wt (solid line) and 100 nM ifnar1-H10 W129A (dashed line) to immobilized IFNβ with 200 nM Tris-NTA-fluorescein respectively. FIG. 15 shows a chelator-lipid conjugate for binding of a histidine-tagged protein to a lipid membrane. (A) Structure of the synthesized bis-NTA lipid conjugate. (B) Construction of a solid-supported lipid bilayer by vesicle fusion, ifnar2-H10 immobilization and interaction with the ligand IFNα2. FIG. 16 shows a study of the lateral diffusion of fluorescently labeled ifnar2-H10 coupled via FRAP to a lipid bilayer supported on a solid. (A) Fluorescence images at different time points before and after bleaching of the circular cross section. (B) Intensity at the bleaching spot (solid line) and at the reference spot (dashed line) as a function of time. FIG. 17 shows a conjugate of Tris-NTA and different fluorophores (A: Oregon Green 488; B: ATTO 565; C: FEW-S0387). FIG. 18 shows a binding assay by fluorescent labeling and gel filtration. (A) Absorption in ifnar2-H10 labeled with Tris-NTA-Oregon Green (280 nm (protein) or 490 nm (Oregon Green)) before (solid line) and after (dashed line) addition of ligand IFNα2. ). (B) Fluorescence spectra of Tris-NTA-Oregon Green before loading with metal ions (solid line) and after loading (dashed line), and after binding to ifnar2-H10 (solid line). FIG. 19 shows the spectroscopic analysis of the conjugate of Tris-NTA and Oregon Green 488 (I), the conjugate of Tris-NTA and ATTO565 (II), and the conjugate of Tris-NTA and FEW-S0387 (III). Show properties. Standardized fluorescence spectra of these conjugates (solid line), normalized fluorescence spectra of these conjugates after complexation of Ni (II) ions (dashed line), and these conjugates after binding of ifnar2-H10 Standardized fluorescence spectrum (solid line). FIG. 20 shows a conjugate of mono-NTA and fluorescein (A), a conjugate of bis-NTA and fluorescein (B), a conjugate of Tris-NTA and fluorescein (C), and tetrakis-NTA and fluorescein. The conjugate (D) is shown. FIG. 21 shows MBP-H6 (left side) with different MCH ((A) mono-NTA; (B) bis-NTA; (C) tris-NTA; (D) tetrakis-NTA (fluorescein conjugate)) and Shown is an analytical gel filtration test of MBP-H10 (right side) non-covalent fluorescent label (absorption at 280 nm (protein) or 490 nm (fluorescein)). FIG. 22 shows a study of association kinetics by fluorescence quenching. Comparison of binding curves for mono-NTA with H10-fluorescein (solid line) and mono-NTA with H6-fluorescein (dashed line). FIG. 23 shows the dissociation kinetics test. (A) Fluorescein change at 10-fold dilution of 5 mM mono-NTA-H10-fluorescein complex. (B) Comparison of dissociation kinetics normalized to equilibrium-amplitude for different MCH with H10-fluorescein and different MCH with H6-fluorescein. FIG. 24 shows a comparison of complex formation constants for mono-NTA and MCH. (A) Deviation rate constant. (B) Equilibrium dissociation constant. FIG. 25 shows a scheme for the synthesis to provide bis-NTA-OtBu (6), tetrakis-NTA-OtBu (8) and bis-NTA-lipid (12). FIG. 26 provides tris-NTA-OtBu (17 and 18) and derivatives with three different fluorophores (Oregon Green 488 (21), ATTO 565 (22), FEW-S0387 (23)). A scheme for synthesis is shown.

(Example 1: MCH binding to surface)
The chelator was covalently bound on the glass and gold surfaces for immobilization of histidine-tagged protein on the surface. For this, a different strategy was used. First, the gold surface was protected by a single layer of α, ω-diamino-poly (ethylene glycol) (DAPEG, 2000 g / mol). This is described in Piehler et al., 2000 (Piehler J, Brecht A, Variokas R, Liedberg.
B, Gauglitz G, 2000, A high-density poly (ethylene glycol) polymer brush for immobilization on glass-type surfaces, non-specific 1) This was done according to the protocol for protein binding.

  The chemical binding of the chelator to these surfaces is shown schematically in FIG. The tip of a carboxyl functionalized chelator (see FIG. 2A) was directly coupled to the vacant amino group of DAPEG, where the surface concentration varies with the duration of coupling (FIG. 6, pathway). III). For coupling of amino-functionalized chelators, the surface was first re-functionalized with glutaric aldehyde (FIG. 6, pathway I). The surface concentration of the carboxyl group was changed by simultaneously adding acetic anhydride (FIG. 6, pathway II). The MCH was coupled to the carboxyl group thus generated. The protecting group was then removed with trifluoroacetic acid.

(Examples 2 to 5: protein immobilization with MCH)
Example 2: Specific immobilization and functionality
The interaction of the histidine-tagged protein with the surface as generated in Example 1 was characterized in detail by reflectometric interferometry (RIfS). Using this method, the binding of proteins to the surface was determined by the method of Piehler and Schreiber, 2001 (Piehler J, Schreiber G., 2001, Fast transient antibody kinetic bioreceptor kinetics in vitro. (2): 173-186) and can be quantified in real time.

Immobilization of the extracellular domain of ifnar2 (ifnar2-H10) with a deca-histidine tag at the C-terminus to the surface modified with Tris-NTA (FIG. 7A) is shown in FIG. 7B. Binding was not detectable prior to loading of the chelator coupled to the surface with Ni (II) ions, but afterwards an overall binding of about 1.5 ng / mm ² of protein was observed and this binding was Moreover, it was kept stably fixed during rinsing. Nevertheless, this protein can be quantitatively removed by 200 mM imidazole as a competitor.

  The functionality of the immobilized protein was examined by binding of the ligand interferon α2 (IFNα2) (FIG. 8). It can thus be found that more than 90% of the immobilized ifnar2-H10 was active and showed a specific and reversible interaction with the ligand.

(Example 3: Self-assembled monolayer (SAM) having MCH)
On the gold surface, self-assembled monolayer (SAM) technology (Sigal GB, Bamdad
C, Barberis A, Stromminger J, Whitesides GM, 1996, A self-assembled mono layer for the binding and study of the ce sine s

  For this, an alkyl thiol with a bis-NTA head group was synthesized (FIG. 9A). In order to minimize non-specific protein interactions with the functionalized surface, an oligo (ethylene glycol) residue was introduced between the head group and the alkyl group. This bis-NTA-thiol was mixed with a matrix thiol without the MCH head group to yield a SAM with different surface concentrations of MCH (FIG. 9A). Specific reversible immobilization of ifnar2-H6 and specific binding of the ligand IFNα2 to the immobilized protein can be detected (FIG. 9B).

Example 4: Stability of binding via MCH
The relatively low stability of mono-NTA binding at the surface can be partially compensated by the high surface concentration of the chelator. In contrast to monovalent chelators, MCH allows stable binding of histidine-tagged proteins to the surface even at low surface concentrations, which provides a series of benefits for this use. FIG. 10A mainly shows the binding of MBP-H10 to a mono-NTA functionalized surface.

  Significant dissociation was observed after the first complete loading of the surface with protein (MBP-H10): After 400 seconds, more than 50% of the initially bound protein dissociates from the surface. The observed dissociation kinetics can be explained by recombination and coordination effects on the surface that are typical for binding via mono-NTA.

  Compared to this, in the case of the MCH, very stable binding could be observed over the same period, and no significant dissociation could be observed (FIG. 10B). For MCH, dissociation could not be observed, so the binding stability was reduced by imidazole.

In FIG. 11A, the dissociation of MBP-H10 on the mono-NTA surface at different concentrations of imidazole is shown. The equilibrium load R _eq was determined by fitting the binding model.

The dependence of R _eq derived from imidazole concentration for mono-NTA, bis-NTA, and tris-NTA is shown in comparison with FIG. 11B. A noticeable shift to higher imidazole concentrations indicates an increase in binding stability as a function of multivalence. In addition, a more rapid change in slope was observed, suggesting a change to molecular cooperativity, in contrast to mono-NTA surface cooperativity.

  By using the same method, the stability of binding by different histidine tags was further explained. For this, a maltose binding protein with a hexa-histidine tag (MBP-H6) at the C-terminus and a maltose binding protein (MBP-H10) with a deca-histidine tag at the C-terminus were used. FIG. 12A shows a comparison of imidazole-induced dissociation for these proteins on the surface modified with Tris-NTA. Since the difference in the binding stability is significant, a quantitative distinction in 30 mM imidazole is possible. Changes markedly more steeply compared to the interaction between mono-NTA and MBP-H10 again show molecular cooperativity (FIG. 12B).

(Example 5: Non-specific binding)
Non-specific binding between proteins and chelators (either electrostatic or via coordinated metal ions) is a central issue in the use of chelators for protein immobilization. This is because stable immobilization with mono-NTA requires very excess NTA groups. This effect is illustrated in FIG. 13A.

  To a high degree, the ligand IFNβ binds nonspecifically to a surface highly functionalized with mono-NTA, so that unreasonable binding kinetics can be shown. For Tris-NTA, already significantly low non-specific binding can be recognized due to the low chelator concentration (FIG. 13B). By blocking the binding site with a neutral protein (MBP-H10), the non-specific binding is completely eliminated, thereby measuring the strain-free binding kinetics. Because coordination sites on the surface can be blocked quantitatively, in this manner, specific interactions with histidine-tagged proteins can also be explained.

  A further advantage of the high stability of MCH binding is the efficient blocking of the binding of histidine-tagged proteins and chelator-modified surfaces by adding MCH loaded with metal ions in solution. Found in the possibilities. Thus, specific interactions with histidine-tagged proteins can be explained without the histidine-tagged proteins binding to more chelators than necessary at the surface. This is shown in FIG. The binding of ifnar1-H10 to a surface modified with Tris-NTA can be almost completely inhibited by tris-NTA in less excess (FIG. 14A).

  Based on this principle, a binding assay was performed. To this end, ifnar2-H10 was first immobilized on a surface modified with Tris-NTA. After blocking excess chelator with MBP-H10, the binding site of ifnar2 was saturated with IFNβ. A subsequent binding assay with ifnar1-H10 provided by Tris-NTA-fluorescein (see Example 8 below) is shown in FIG. 14B. While an exclusive specific interaction was detected, no binding was observed for the (inactive) mutation W129A in the binding site of IFNβ.

(Example 6: Conjugate with lipid)
For protein binding to the lipid membrane, a lipid analog conjugate of bis-NTA shown in FIG. 15A was synthesized.

This lipid was incorporated into a monolayer vesicle of stearoyl-oleoyl-phosphatidylcholine that spontaneously forms a fluid-supported lipid bilayer supported on a solid surface on a glass surface (FIG. 15B). ifnar2-H10 could be specifically immobilized in these membranes and binding activity was completely prevented (FIG. 15B). Lateral diffusion of ifnar2-H10 immobilized on the membrane was analyzed by fluorescence fading (fluorescence recovery after photobleaching, FRAP) (FIG. 16). Thereby, a diffusion constant of 1 μm ² / sec was determined.

(Example 7 and Example 8: Conjugate with fluorophore)
Example 7: Different fluorophores and spectroscopic properties
For non-covalent fluorescent labeling of histidine-tagged proteins in solution, conjugates of Tris-NTA with the following three different fluorophores were synthesized (FIG. 17): Oregon Green 488, ATTO 565, FEW -S0387.

  After loading with Ni (II) ions, ifnar2-H10 was added to these conjugates, and excess conjugate was separated by gel filtration.

  A stable label of ifnar2-H10 was observed for all conjugates: In the second gel filtration run, no free dye was detectable (FIG. 18A). Upon addition of the ligand, further shifts to higher wavelengths were observed (FIG. 18A), confirming that the protein function was conserved by this position-specific label. This label was completely reversible: the MCH-fluorophore conjugate can be completely separated by the addition of imidazole.

  Different states of the MCH-fluorophore conjugate can be followed by fluorescence quantum yield (FIG. 18B). A significant decrease in fluorescence up to about 50% was observed after loading of the chelator group with Ni (II) ions, and a significant decrease in fluorescence up to about 40% was observed after binding to the protein. This effect could also be observed with other fluorescent dyes, and even more pronounced with the fluorescent dye ATTO 565 (FIG. 19).

(Example 8: Binding stability in solution)
To test the binding stability in solution, mono-NTA and fluorescein conjugates, bis-NTA and fluorescein conjugates, tris-NTA and fluorescein conjugates, and tetrakis-NTA and fluorescein conjugates. The conjugate was synthesized and loaded with Ni (II) ions (FIG. 20). The interaction of these conjugates with hexa-histidine-tagged protein and deca-histidine-tagged protein was determined by thermodynamic and kinetics by means of analytical gel filtration, isothermal titration calorimetry, and fluorescence spectroscopy. Characterized.

((A) Gel filtration for analysis)
For testing by means of analytical gel filtration, MBP-H6 and MBP-H10 are provided in a stoichiometric excess of conjugate, and the mixture is loaded onto a gel filtration column and Absorbance was monitored simultaneously at 280 nm (protein) and 490 nm (fluorescein) at the outlet of the column. Using the ratio of these signals, the level of label and hence binding stability can be estimated. This chromatogram is summarized in FIG. For mono-NTA (FIG. 21A) (MBP-H6 and MBP-H10), almost no bound conjugate was detectable. For bis-NTA (FIG. 21B), a significantly more stable binding of MBP-H10 could be detected compared to MBP-H6. For Tris-NTA and Tetrakis-NTA (FIGS. 21C and 21D), stoichiometric levels of label and small differences between MBP-H6 and MBP-H10 could be detected.

  These results show that the stability of the interaction of Tris-NTA and Tetrakis-NTA with hexa-histidine and deca-histidine tags is so high that stable and stoichiometric fluorescent labeling can be achieved. Indicates. Furthermore, these results impressively demonstrated the superiority of MCH in terms of stability of interaction with histidine-tagged proteins compared to traditional mono-NTA.

((B) Fluorescence spectroscopy)
The kinetics of interaction between MCH and the histidine tag were examined by fluorescence spectroscopy. For this, a fluorescein labeled oligohistidine peptide was used. Strong fluorescence quenching was observed in the binding of these peptides to the MCH. Changes in fluorescence were monitored by stop flow technology at high demand times (see FIG. 22). From these curves, association rate constants of 1.5 to 5 · 10 ⁵ M ⁻¹ s ⁻¹ were determined by curve fitting. According to them, the rate constant for deca-histidine was significantly higher than that for hexa-histidine.

The same fluorescence quenching effect was also used for dissociation kinetic studies. For this, first a high concentration of stoichiometric complex was generated and diluted with either buffer (for mono-NTA) or 10-fold excess of MBP-H10 (for MCH). In FIG. 23A, the fluorescent signal observed upon dilution of the mono-NTA H10-fluorescein complex is shown. From this curve, a dissociation rate constant of 1.5 s ⁻¹ was calculated. This demonstrates the very low stability of the molecular Ni: mono-NTA-histidine tag complex.

  The stability of the MCH-oligohistidine complex was remarkably high and consequently required a competitor to observe the dissociation. The dissociation curve thus obtained is shown in FIG. 23B. From these curves, a significantly lower dissociation rate constant was obtained for mono-NTA (FIG. 24A). The resulting equilibrium-dissociation constant is shown in FIG. 24B. This impressively supports an increase in binding affinity of MCH, almost 5-fold (up to <1 nM) compared to mono-NTA (about 10 μM).

(Example 9: Professor of synthesis)
((A) Synthesis of Lys-NTA-OtBu (3) (see also FIG. 25))
Bromoacetic acid tert-butyl ester (1.59 ml; 10.8 mmol) and DIPEA (2.30 ml; 13.5 mmol) were added to N ^ε -benzyloxycarbonyl-L-lysine tert-butyl ester (1 ) (1.00 g; 2.7 mmol). The reaction flask is rinsed with nitrogen and then stirred at 55 ° C. overnight. Volatile components are removed in vacuo at 60 ° C. The residue is redissolved in cyclohexane / ethyl acetate (3: 1, 15 ml), removed and the precipitate is washed 3 times with the same solvent (3 × 10 ml). The filtrate was concentrated and purified on a silica column using cyclohexane / ethyl acetate (3: 1) as the elution phase. Yield: 1.3 g (2.3 mmol) of N [ ^alpha] , N [ ^alpha ] -bis [(tert-butyloxy-carbonyl) methyl] -N [ ^epsilon ] -benzyloxycarbonyl-L-lysine tert-butyl ester (2), 85% o . th. . TLC: _Rf = 0.5 in cyclohexane / ethyl acetate (3: 1).

メタノール（５０ｍｌ）中の１．００ｇ（１．８ｍｍｏｌ）の２の溶液を、窒素でリンスし、次いで２０ｍｇの１０％Ｐｄ／Ｃを添加する。反応混合物を、水素雰囲気下で室温にて６時間激しく攪拌する。Ｐｄ／Ｃを、セライトを通した濾過によって除去し、そして揮発性成分を、真空中で取り除く。収量：０．７４ｇ（１．７ｍｍｏｌ）のＮ^α，Ｎ^α−ビス［（ｔｅｒｔ−ブチルオキシカルボニル）メチル］−Ｌ−リジンｔｅｒｔ−ブチルエステル（３）、理論の９４％。ＴＬＣ：クロロホルム／メタノール（３：１）においてＲ_ｆ＝０．３。

A solution of 1.00 g (1.8 mmol) of 2 in methanol (50 ml) is rinsed with nitrogen and then 20 mg of 10% Pd / C is added. The reaction mixture is stirred vigorously for 6 hours at room temperature under hydrogen atmosphere. Pd / C is removed by filtration through celite and volatile components are removed in vacuo. Yield: 0.74 g (1.7 mmol) of N [ ^alpha] , N [ ^alpha ] -bis [(tert-butyloxycarbonyl) methyl] -L-lysine tert-butyl ester (3), 94% of theory. TLC: R _f = 0.3 in chloroform / methanol (3: 1).

（（Ｂ）ビス−ＮＴＡ−ＯｔＢｕ（６）の合成（図２５も参照のこと））
３（１．００ｇ；２．３ｍｍｏｌ）を、乾燥ジクロロメタン（４０ｍｌ）に溶解し、そして４（０．２９ｇ；１．０ｍｍｏｌ）、ＴＢＴＵ（０．９６ｇ；２．９ｍｍｏｌ）およびＤＩＰＥＡ（０．６ｍｌ；３．５ｍｍｏｌ）を添加する。得られたスラリーを、窒素でリンスし、そして室温にて一晩攪拌する。次いで揮発性成分を、真空中で除去し、残留物を、ジクロロメタン（１００ｍｌ）でスラリーにし、そしてＭＱ水で３回洗浄（各３０ｍｌ）する。有機相を、無水硫酸ナトリウム上で乾燥し、そして真空中で濃縮する。油状の残留物を、溶離剤としてシクロヘキサン／酢酸エチル（３：１）を用いてシリカカラム上で精製する。収量：０．９９ｇ（０．９ｍｍｏｌ）Ｚ−ビス−ＮＴＡ−ＯｔＢｕ（５）、理論の９０％。ＴＬＣ：シクロヘキサン／酢酸エチル（３：１）においてＲ_ｆ＝０．３

((B) Synthesis of bis-NTA-OtBu (6) (see also FIG. 25))
3 (1.00 g; 2.3 mmol) was dissolved in dry dichloromethane (40 ml) and 4 (0.29 g; 1.0 mmol), TBTU (0.96 g; 2.9 mmol) and DIPEA (0.6 ml; 3.5 mmol) is added. The resulting slurry is rinsed with nitrogen and stirred overnight at room temperature. The volatile components are then removed in vacuo, the residue is slurried with dichloromethane (100 ml) and washed three times with MQ water (30 ml each). The organic phase is dried over anhydrous sodium sulfate and concentrated in vacuo. The oily residue is purified on a silica column using cyclohexane / ethyl acetate (3: 1) as eluent. Yield: 0.99 g (0.9 mmol) Z-bis-NTA-OtBu (5), 90% of theory. TLC: R _f = 0.3 in cyclohexane / ethyl acetate (3: 1)

５（１．００ｇ；０．９０ｍｍｏｌ）のＺ−保護基を、２について上で記載される通り
、加水分解して除去する。収量：０．８３ｇ（０．８５ｍｍｏｌ）のビス−ＮＴＡ−ＯｔＢｕ（６）、理論の９４％。ＴＬＣ：クロロホルム／メタノール（５：２）においてＲ_ｆ＝０．３。

5 (1.00 g; 0.90 mmol) of the Z-protecting group is removed by hydrolysis as described above for 2. Yield: 0.83 g (0.85 mmol) bis-NTA-OtBu (6), 94% of theory. TLC: _Rf = 0.3 in chloroform / methanol (5: 2).

（（Ｃ）テトラキス−ＮＴＡ−ＯｔＢｕ（８）の合成（図２５も参照のこと））
６（１．００ｇ；１．０３ｍｍｏｌ）を、乾燥ジクロロメタン（３０ｍｌ）に溶解し、次いで４（１３０ｍｇ；０．４６ｍｍｏｌ）、ＴＢＴＵ（１．４０ｍｍｏｌ；４５０ｍｇ）、およびＤＩＰＥＡ（０．４４ｍｌ；２．６ｍｍｏｌ）を、添加する。得られた反応混合物を、窒素でリンスし、そして室温にて一晩攪拌する。次いで揮発性成分を、真空中で除去し、残留物を、ジクロロメタン（１００ｍｌ）でスラリーにし、そしてＭＱ水で３回洗浄（各３０ｍｌ）する。有機相を、無水硫酸ナトリウム上で乾燥し、そして真空中で濃縮する。油状の残留物を、溶離剤として酢酸エチルを用いてシリカカラム上で精製する。収量：０．７０ｇ（０．３２ｍｍｏｌ）のＺ−テトラキス−ＮＴＡ−ＯｔＢｕ（７）、７０％ｏ．ｔｈ．。ＴＬＣ：酢酸エチルにおいてＲ_ｆ＝０．３。
ＭＳ（ＭＡＬＤＩ、ＥＳＩ、Ｃ_１１１Ｈ_１８９Ｎ_１１Ｏ_３２）；ＭＨ^＋２１８９
７（１．００ｇ；０．４６ｍｍｏｌ）のＺ−保護基を、２について上で記載される通り、加水分解して除去する。収量：０．８６ｇ（０．４２ｍｍｏｌ）、９１％。テトラキス−ＮＴＡ−ＯｔＢｕ（８）、理論の９１％。ＴＬＣ：クロロホルム／メタノール（５：３）においてＲ_ｆ＝０．３。
ＭＳ（ＭＡＬＤＩ、ＥＳＩ、Ｃ_１０３Ｈ_１８３Ｎ_１１Ｏ_３０）；ＭＨ^＋２０５５。

((C) Synthesis of tetrakis-NTA-OtBu (8) (see also FIG. 25))
6 (1.00 g; 1.03 mmol) was dissolved in dry dichloromethane (30 ml), then 4 (130 mg; 0.46 mmol), TBTU (1.40 mmol; 450 mg), and DIPEA (0.44 ml; 2.6 mmol). ) Is added. The resulting reaction mixture is rinsed with nitrogen and stirred overnight at room temperature. The volatile components are then removed in vacuo, the residue is slurried with dichloromethane (100 ml) and washed three times with MQ water (30 ml each). The organic phase is dried over anhydrous sodium sulfate and concentrated in vacuo. The oily residue is purified on a silica column using ethyl acetate as eluent. Yield: 0.70 g (0.32 mmol) of Z-tetrakis-NTA-OtBu (7), 70% o. th. . TLC: R _f = 0.3 in ethyl acetate.
_{MS (MALDI, ESI, C 111} H 189 N 11 O 32); MH + 2189
7 (1.00 g; 0.46 mmol) of the Z-protecting group is removed by hydrolysis as described above for 2. Yield: 0.86 g (0.42 mmol), 91%. Tetrakis-NTA-OtBu (8), 91% of theory. TLC: _Rf = 0.3 in chloroform / methanol (5: 3).
_{MS (MALDI, ESI, C 103} H 183 N 11 O 30); MH + 2055.

((D) Synthesis of Tris-NTA-OtBu (17 or 18) (see also FIG. 26))
Bromoacetic acid tert-butyl ester (5.9 ml; 40 mmol) and DIPEA (8.6 ml; 50 mmol) were added to a solution of H-Glu (OtBu) -OBzl 13 (3.3 g; 10 mmol) in DMF (75 ml). Added. The reaction flask is rinsed with nitrogen and then stirred at 55 ° C. overnight. Volatile components are removed in vacuo at 60 ° C. The residue is slurried in ethyl acetate (20 ml), filtered off and washed three times with cyclohexane: ethyl acetate (3: 1, 3 × 40 ml). The filtrate was concentrated in vacuo and filtered over a silica column using cyclohexane: ethyl acetate (3: 1) as eluent. Yield: 4.6 g (8.8 mmol) of Bzl-Glu-NTA (14), 88%
d. Th. . _Rf = 0.6 in TLC: cyclohexane: ethyl acetate (3: 1). _{MS (MALDI, ESI, C 28} H 43 NO 8); MH + 522.

14 (1.00 g; 1.9 mmol) of the Bzl-protecting group is removed by hydrolysis as described above for the removal of the 2 Z-protecting group. Yield: 0.76 g (1.76 mmol) Glu-NTA (15), 92% d. Th. . TLC: R _f = 0.4 in chloroform / methanol (3: 2)
_{MS (MALDI, ESI, C 21} H 37 NO 8); MH + 431.

15 (431 mg; 1.0 mmol) was dissolved in dry dichloromethane (30 ml), then 16 (67 mg; 0.33 mmol), TBTU (417 mg; 1.3 mmol), and DIPEA (0.3 ml; 1.75 mmol). ,Added. The resulting reaction mixture is rinsed with nitrogen and stirred overnight at room temperature. The volatile components are then removed in vacuo, the residue is slurried with dichloromethane (50 ml) and washed three times with MQ water (15 ml each). The organic phase is dried over anhydrous sodium sulfate and concentrated in vacuo. The oily residue is purified on a silica column using a gradient from 100% ethyl acetate to 50% ethyl acetate in 5 volumes of methanol in the column.
Yield: 0.37 g (0.26 mmol) Tris-NTA-OtBu (17), 79% o. th. TLC: R _f = 0.5 in ethyl acetate / methanol (3: 1).
_{MS (MALDI, ESI, C 73} H 129 N 7 O 21); MH + 1441.

17 (1.44 g; 1.0 mmol) is dissolved in dry chloroform (30 ml) and succinic anhydride (300 mg; 3.0 mmol) and TEA (0.7 ml; 5.0 mmol) are added. The resulting reaction mixture is rinsed with nitrogen and stirred overnight at room temperature. The volatile components are then removed in vacuo, the residue is slurried with dichloromethane (120 ml) and washed three times with MQ water (40 ml each). The organic phase is dried over anhydrous sodium sulfate and concentrated in vacuo. The oily residue is purified on a silica column using a gradient from 100% ethyl acetate to 50% ethyl acetate in 5 volumes of methanol in the column. Yield: 1.4 g (0.91 mmol) Tris-NTA-OtBu-COOH (18), 91% o. th. TLC: R _f = 0.4 in ethyl acetate / methanol (3: 1).
_{MS (MALDI, ESI, C 76} H 133 N 7 O 24); MH + 1541.

((E) Synthesis of Tris-NTA fluorescent conjugate (see also FIG. 26))
17 (1.44 g, 1.0 mmol) was dissolved in dry dichloromethane (30 ml), then BOC-D-aminocaproic acid (230 mg; 1.0 mmol), TBTU (450 mg; 1.40 mmol), and DIPEA (0. 33 ml; 1.9 mmol) is added. The reaction mixture is rinsed with nitrogen and stirred at room temperature for 12 hours. The volatile components are then removed in vacuo, the residue is slurried with dichloromethane (70 ml) and washed three times with MQ water (25 ml each). The organic phase is dried over anhydrous sodium sulfate and concentrated in vacuo. The oily residue is purified on a silica column using a gradient from 100% ethyl acetate to 10% ethyl acetate in 5 volumes of methanol in the column. TLC: Yield: 1.4 g (0.84 mmol) Tris-NTA-OtBu-aminocaproic acid-BOC (19), 84% o. th. . R _f = 0.3 in ethyl acetate.
_{MS (MALDI, ESI, C 84} H 148 N 8 O 24); MH + 1654.

19 (500 mg; 0.3 mmol) is dissolved in dichloromethane containing 10% TFA and 1% 1,2-ethanedithiol and stirred for 1 hour. The product is then precipitated with ether. Yield: 300 mg (0.3 mmol) Tris-NTA-aminocaproic acid (20), 95% o. th. .
_{MS (MALDI, ESI, C 43} H 68 N 8 O 22); MH + 1049.

  20 (10 mg; 0.05 mmol) is dissolved in 100 μl of DMF and a stoichiometric amount of NHS-active ester of the corresponding fluorescent dye is obtained. After 12 hours, the product is first purified by preparative thin layer chromatography and then purified by anion exchange chromatography.

（（Ｆ）ビス−ＮＴＡ脂質結合体（１２）の合成（図２５も参照のこと））
オクタデシルアミン（２．７ｇ；１０ｍｍｏｌ）、１−ブロモ−ｃｉｓ−９−オクタデセン（３．３ｇ；１０ｍｍｏｌ）およびＴＥＡ（７ｍｌ、５０ｍｍｏｌ）を、混合する。反応フラスコを、窒素でリンスし、そして５５℃にて２日間撹拌する。粗生成物を、クロロホルム（２００ｍｌ）に溶解し、そして水で３回洗浄する（各５０ｍｌ）。有機相を、濃縮し、そしてカラムの５容量のメタノールにおける１００％クロロホルムから６０％クロロホルムまでの勾配を用いてシリカカラム上で精製する。収量：２．１ｇ（４．０５ｍｍｏｌ）のステロイル−オレオイル−アミン（ＳＯＡ（９））、４０％ ｏ．ｔｈ．。

((F) Synthesis of bis-NTA lipid conjugate (12) (see also FIG. 25))
Octadecylamine (2.7 g; 10 mmol), 1-bromo-cis-9-octadecene (3.3 g; 10 mmol) and TEA (7 ml, 50 mmol) are mixed. The reaction flask is rinsed with nitrogen and stirred at 55 ° C. for 2 days. The crude product is dissolved in chloroform (200 ml) and washed 3 times with water (50 ml each). The organic phase is concentrated and purified on a silica column using a gradient from 100% chloroform to 60% chloroform in 5 volumes of methanol in the column. Yield: 2.1 g (4.05 mmol) of steroyl-oleoyl-amine (SOA (9)), 40% o. th. .

無水コハク酸（０．６０ｇ；６ｍｍｏｌ）およびＴＥＡ（２．５ｍｌ；１８ｍｍｏｌ）を、クロロホルム（５０ｍｌ）中の９（１．０４ｇ；２ｍｍｏｌ）の溶液に添加する。反応フラスコを、窒素でリンスし、そして室温にて一晩撹拌する。粗生成物を、ＭＱ水（４×４０ｍｌ）で洗浄する。有機相を、乾燥し、濃縮し、そしてアセトンで沈殿させる。収量：１．１８ｇ（１．９ｍｍｏｌ）のＳＯＡ−ＣＯＯＨ（１０）、９５％ ｏ．ｔｈ．。

Succinic anhydride (0.60 g; 6 mmol) and TEA (2.5 ml; 18 mmol) are added to a solution of 9 (1.04 g; 2 mmol) in chloroform (50 ml). The reaction flask is rinsed with nitrogen and stirred overnight at room temperature. The crude product is washed with MQ water (4 × 40 ml). The organic phase is dried, concentrated and precipitated with acetone. Yield: 1.18 g (1.9 mmol) SOA-COOH (10), 95% o. th. .

１０（５３０ｍｇ；０．８５ｍｍｏｌ）を、乾燥ジクロロメタン（３０ｍｌ）に溶解し、次いで６（８３４ｍｇ；０．８６ｍｍｏｌ）、ＴＢＴＵ（４１３ｇ、１．２９ｍｍｏｌ）、およびＤＩＰＥＡ（０．３ｍｌ、１．７６ｍｍｏｌ）を、添加する。反応溶液を、窒素でリンスし、そして室温にて１２時間撹拌する。次いで揮発性成分を、真空中で除去し、そして残留物を、ジクロロメタン（３０ｍｌ）で回収する。ＭＱ水（３×３０ｍｌ）で洗浄した後、有機相を、無水硫酸ナトリウム上で乾燥する。次いで揮発性成分を、真空中で除去し、そして残留物を、クロロホルム／酢酸エチル（３：２）を用いてシリカカラム上で精製する。収量：０．９７ｇ（０．６１ｍｍｏｌ）のＳＯＡ−ビス−ＮＴＡ−ＯｔＢｕ（１１）、７１％ ｏ．ｔｈ．。ＴＬＣ：クロロホルム／酢酸エチル（３：２）においてＲ_ｆ＝０．６。

10 (530 mg; 0.85 mmol) was dissolved in dry dichloromethane (30 ml), then 6 (834 mg; 0.86 mmol), TBTU (413 g, 1.29 mmol), and DIPEA (0.3 ml, 1.76 mmol) were added. ,Added. The reaction solution is rinsed with nitrogen and stirred at room temperature for 12 hours. The volatile components are then removed in vacuo and the residue is recovered with dichloromethane (30 ml). After washing with MQ water (3 × 30 ml), the organic phase is dried over anhydrous sodium sulfate. The volatile components are then removed in vacuo and the residue is purified on a silica column with chloroform / ethyl acetate (3: 2). Yield: 0.97 g (0.61 mmol) SOA-bis-NTA-OtBu (11), 71% o. th. . TLC: R _f = 0.6 in chloroform / ethyl acetate (3: 2).

１１（５００ｍｇ；０．３２ｍｍｏｌ）を、１０％のＴＦＡおよび１％の１，２−エタンジチオールを含むジクロロメタンに溶解し、そして１時間撹拌する。次いで、生成物を、エーテルで沈殿させる。収量：３７０ｍｇ（０．３ｍｍｏｌ）のＳＯＡ−ビス−ＮＴＡ（１２）、９４％ ｏ．ｔｈ．。

11 (500 mg; 0.32 mmol) is dissolved in dichloromethane containing 10% TFA and 1% 1,2-ethanedithiol and stirred for 1 hour. The product is then precipitated with ether. Yield: 370 mg (0.3 mmol) SOA-bis-NTA (12), 94% o. th. .

Claims (27)

General formula:
  X _m -G-CL _n
And tautomeric organisms, isomers, anhydrides, acids and salts thereof, wherein:
  G is a scaffold structure comprising a saturated carbohydrate chain having 2 to 20 carbon atoms and further comprising an amide bond, an ester bond and / or an ether bond;
  X is a coupling group for probe F or functional unit F;
  CL is a chelator group having a metal coordination center;
  m is an integer and is at least 1; and
  n is an integer and is at least 2.
Compound. 2. The compound of claim 1, wherein the compound is characterized by a metal ion bonded to each of the chelator groups. The metal ion is Ni ²⁺ , Co ²⁺ , Cu ²⁺ , Zn ²⁺ , Fe ²⁺ , Fe ³⁺ And a compound selected from the group consisting of all lanthanide ions. The compound according to any one of claims 1 to 3, wherein the chelator group has nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), porphyrin, salicylic acid derivative, 1, 2 A compound characterized by being selected from the group consisting of diaminoethyl diacetic acid, diaminoethyl triacetic acid, hydroxyethyliminodiacetic acid, and salts or combinations thereof. The compound according to any one of claims 1 to 4, wherein the reactive group of the chelator group has a protecting group. 6. A compound according to any one of claims 1-5, characterized in that the spacer group A is located between CL and G. 7. A compound according to claim 6, wherein A is linear or branched. 8. The compound according to any one of claims 1 to 7, wherein the compound is characterized by the chelator group being attached to G via an amide bond, an ester bond or an ether bond. . 9. A compound according to any one of claims 1-8, characterized in that G is linear, branched or closed. 10. The compound according to any one of claims 1 to 9, and tautomers, isomers, anhydrides, acids and salts thereof, wherein the compound is:

A compound selected from: 11. A compound according to any one of claims 1 to 10, characterized in that the coupling group X is selected from the group consisting of NHR, wherein R is H, alkyl or aryl. , COOH, n is an integer (CH ₂ ) _n A compound that is COOH, SH, maleimide, acetamide, isothiocyanate or cyanate. 12. A compound according to any one of claims 1 to 11, wherein the compound is characterized by a probe F or a functional unit F being attached at a coupling group X. 13. The compound according to claim 12, wherein the probe F or the functional unit F is a fluorophore, a FRET-fluorophore, a fluorescence quencher, a phosphorescent compound, a luminescent compound, an absorptive compound, a polymer. , PEG, oligosaccharide, oligonucleotide, PNA, biotin, hapten, peptide, protein, enzyme, crosslinker, oligoethylene glycol, lipid, nanoparticle, electron density amplification factor, gold cluster, metal cluster, quantum dot, and their A compound characterized by being selected from the group consisting of combinations. 14. A compound according to any one of claims 1 to 13, wherein the compound is:

And tautomers, isomers, anhydrides, acids and salts thereof selected from 15. A method for producing a compound according to any one of claims 1-14, wherein the method attaches at least two chelator groups to the scaffold after or during synthesis of the scaffold structure. A method comprising coupling to a structure. 16. The method of claim 15, wherein the method comprises protected or unprotected amino acids and amino acid derivatives, Z-Lys-OtBu, H-Glu (OtBu) -OBzl, Z-Glu-OH, large Including the step of synthesizing a scaffold structure from one or more starting compounds selected from the group consisting of cyclic polyamine, 1,4,8,11-tetraazacyclotetradecane, bromoacetic acid-OtBu, BOC-ε-aminocaproic acid. ,Method. 17. A method according to claim 15 or 16, wherein the chelator group is protected. 15. Use of a compound according to any one of claims 1-14 for binding to an affinity tag in a target molecule, wherein the affinity tag binds to a metal-chelator complex. 19. Use according to claim 18, wherein the affinity tag is a peptide tag comprising 4 to 15 amino acids comprising 4 to 15 histidines and 0 to 4 basic amino acids. 20. Use according to claim 18 or 19, wherein the target molecule is a peptide, polypeptide, protein, peptidomimetic or protein mimetic, or a peptide-modified polymer or peptide-modified dendrimer. 15. A modification according to any one of claims 1 to 14 for modification, coupling, oligomerization, detection, monitoring, analysis or for detection of a target molecule in vitro, in vivo, in situ, live cells or lipid vesicles. Use of the compound. Use of a compound according to any one of claims 1 to 14 for immobilization of a target molecule. 23. Use according to claim 22, wherein the use is characterized by the compound being included in a surface bound or lipid monolayer or lipid bilayer. The surface is a glass mold surface, gold, silver, DAPEG modified glass, PEG polymer modified glass or PEG polymer modified gold, GOPTS silane treated glass, lipid monolayer or lipid bilayer, metal selenide 24. Use according to claim 23, selected from glass mold surfaces and gold surfaces comprising metal tellurides and metal sulfides. Use of a compound according to any one of claims 1 to 14 for the production of self-assembled monolayers (SAM). Use of a compound according to any one of claims 1 to 14 for the production of functional microstructured surfaces and functional nanostructured surfaces and / or protein arrays. Use of a compound according to any one of claims 1 to 14 for the purification of a target molecule.

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