WO2011101445A1 - Composés chélateurs multivalents (mch) à haute affinité et leur utilisation pour l'analyse structurelle et fonctionnelle de molécules cibles - Google Patents

Composés chélateurs multivalents (mch) à haute affinité et leur utilisation pour l'analyse structurelle et fonctionnelle de molécules cibles Download PDF

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WO2011101445A1
WO2011101445A1 PCT/EP2011/052451 EP2011052451W WO2011101445A1 WO 2011101445 A1 WO2011101445 A1 WO 2011101445A1 EP 2011052451 W EP2011052451 W EP 2011052451W WO 2011101445 A1 WO2011101445 A1 WO 2011101445A1
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group
multivalent
coupling
chelator
protein
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PCT/EP2011/052451
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German (de)
English (en)
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Robert TAMPÉ
Christoph Baldauf
Enrica Bordignon
Katrin Schulze
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Johann Wolfgang Goethe-Universität Frankfurt am Main
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Publication of WO2011101445A1 publication Critical patent/WO2011101445A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/03—Free radicals

Definitions

  • MCHs multivalent chelator compounds
  • the present invention generally relates to multivalent chelator compounds (MCHs) having three or four nitrilotriacetic acid (NTA) groups as chelator groups and in a first aspect of a free thiol group, and to a process for producing this compound.
  • MCHs multivalent chelator compounds
  • NTA nitrilotriacetic acid
  • the present invention further includes, as a second preferred aspect, novel molecules in which a stable radical is covalently bonded to the thiol group of the tris-NTA chelator molecule.
  • the present invention further encompasses the uses of the MCHs.
  • cysteine scanning mutagenesis G In magnetic resonance spectroscopy, the method of cysteine scanning mutagenesis G is currently being used to introduce paramagnetic molecules, such as stable radicals. These introduced cysteines are paramagnetically modified with thiol-specific reagents.
  • the principle of cysteine scanning mutagenesis G consists in the successive replacement of native amino acids by cysteine residues, each substitution forming its own mutant. It also follows that if a proof of analysis is to be given in a functional way, the cysteine-free protein is retained in its structure and function as a wild type by the substitution of the native cysteine residues.
  • cysteine-free protein So it has to be upfront It can be shown that the same or at least very similar properties of the cysteine-free protein to the wild type exist (Frillingos et al, FASEB Journal, 1998, 12, 1282).
  • the introduction of single cysteines must ensure the following aspects: Functionality of the mutant and above all accessibility or reactivity of the introduced cysteine with regard to thiol-specific reagents.
  • a quantitative mark can not be ensured, and in addition the paramagnetic properties can be reduced by the environment.
  • Another disadvantage is that thiols can form disulfide bridges with other thiols in the cell, which can significantly reduce the function or the quantitative labeling.
  • IMAC immobilized metal ion affinity chromatography
  • WO 2006/013042 relates to multivalent chelator compounds (MCH's) of the general formula X m -G-CL n (where G is a framework structure comprising a saturated hydrocarbon chain having 2 to 20 carbon atoms, further comprising amide, ester and X is a coupling group for a probe or functional unit F, CL is a chelator group having a metal coordination center, m is an integer and at least 1, and n is an integer and at least 2), processes for their preparation, and their use for modifying and / or immobilizing target molecules bearing an affinity tag that binds to metal-chelator complexes.
  • MCH's multivalent chelator compounds
  • WO 2006/013042 describes, together with DE 2004 038 134 Al, the use of the compounds mentioned there for magnetic resonance spectroscopy, the compounds being unsuitable for the use according to the invention for EPR and the proposed NMR experiments. As shown in Figures 4 and 5, the presence of paramagnetic Ni ions is counterproductive for EPR or corresponding NMR experiments.
  • Tinazli et al. Choemistry - Eur. Jour 2005, 11, 5249 - 5259
  • compounds are described, but these are generally not suitable for the coupling of reporter groups, since they arrange themselves as self-assembling monolayers. Furthermore, the high flexibility of the alkyl group is destructive to biophysical measurement methods.
  • Lee et al. Choemistry - Eur. Jour 2005, 11, 5249 - 5259
  • MCH multivalent chelator compound
  • G is a skeleton structure comprising a saturated hydrocarbon chain having 2 to 20 carbon atoms, furthermore comprising amide, ester and / or ether bonds, with 3 or 4 nitrilotriacetic acid (NTA) - Groups as chelator groups having a metal coordination center
  • Z is a coupling group for X
  • X is a reactive group, optionally together with a reporter group R, and tautomers, isomers, anhydrides de, acids and salts thereof.
  • NTA nitrilotriacetic acid
  • the framework structures G comprise a saturated hydrocarbon chain having 2 to 25 carbon atoms, preferably 2 to 20 and more preferably 5 to 16 carbon atoms.
  • scaffold structures include amide, ester and / or ether bonds.
  • the framework structure can be linear, branched or closed.
  • a preferred closed framework structure is cyclam-ring structures.
  • Preferred framework structures include amino acid building blocks. It is preferred that the chelator groups be attached to the backbone via amide, ester or ether linkages.
  • the reactive groups of the chelator group carry protecting groups.
  • carboxyl groups such as those of NTA, can be protected by the Ot-Bu group. All customary protective groups known to those skilled in the art can be used.
  • these multivalent chelators can be characterized by linking 3 to 4 (independent) nitrilotriacetic acid (NTA) chelator groups to a molecular scaffold (backbone G) while simultaneously providing a functional group (coupling group Z) for coupling to X.
  • NTA nitrilotriacetic acid
  • the coupling group Z comprises at least one optionally protected group selected from a thiol group and N 3 for the coupling of X.
  • R indicates the point of attachment for the R reporter group.
  • a multivalent chelator compound of the invention further comprising a reporter group R having at least one stable organic radical, wherein R is covalently bonded to X. Still more preferably, R is selected from
  • the stable organic radical is preferably selected from N "and O", the radical being delocalized between N and O.
  • the radical-bearing skeleton is extended by N-Y, wherein Y represents a derivatization of the oxygen and is selected from the group of derivatizations comprising acetyl (-Ac) or acetoxy (-OAc).
  • the stable organic radical is an anthracene radical.
  • a particularly preferred multivalent chelator compound of the invention has the following formula:
  • the multivalent chelator compounds of the present invention are furthermore preferably characterized in that in each case a metal ion is bonded to the chelator groups.
  • 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.
  • Preferred targeting molecules include peptides, polypeptides, proteins as well as peptide and protein mimetics and peptide-modified polymers or dendrimers.
  • Protein or polypeptide is also understood to mean a post-translationally modified protein or polypeptide.
  • Peptide and protein mimetics include compounds that contain similar side-chain functionalities as peptides or proteins, but differ in the structure of the backbone from them.
  • backbone mimetics alteration of the backbone atoms (backbone mimetics), the introduction of bicyclic dipeptide analogs, and the arrangement of the functional groups on a non-oligomeric core structure (scaffold mimetics).
  • the backbone mimetics include, for example, the oligo-N-alkylglycines (peptoids), which differ from peptides or proteins at the point of attachment of the side chain (at the N instead of C a ).
  • a further aspect of the present invention relates to the use of a multivalent chelator compound of the present invention for the labeling / non-covalent functionalization of target molecules for use in magnetic resonance spectroscopy (NMR, EPR).
  • NMR magnetic resonance spectroscopy
  • the compounds of the invention can be used in numerous in vitro and in vivo test methods known in the art.
  • Preferred test methods include spectroscopic methods such as absorption spectroscopy, fluorescence spectroscopy, fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), reflectometric interference spectroscopy (RlfS), surface fluorescence Plasmon resonance spectroscopy (surface plasmon resonance) / BIACORE, optical grating couplers, quartz microbalance, surface acoustic waves (SAW), x / y fluorescence scanning (fluorlmaging) as well as microscopic methods such as fluorescence microscopy, confocal optical microscopy, total internal Reflection microscopy, contrast-enhanced microscopy, electron microscopy, scanning probe microscopy, but also other methods such as magnetic resonance spectroscopy, microscopy and tomography, impedance spectroscopy, field effect Transistors, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (
  • the affinity tag is a peptide tag comprising 4 to 15 amino acids.
  • the 4 to 15 amino acids may preferably be 4 to 15 histidines.
  • 0 to 4 basic amino acids such as lysine and arginine may be included in the peptide tag.
  • a preferred affinity tag is a (His) n tag, wherein n is an integer from 4 to 15.
  • Another aspect of the present invention relates to the use of the compounds of the invention in the controlled and reversible dimerization or oligomerization of target molecules, in particular proteins, to supramolecular functional units. Further preferred is the use of the compounds according to the invention for the immobilization or purification of target molecules.
  • the compounds of the invention may be used to modify, immobilize, couple, purify, detect, monitor, analyze, or detect target molecules in vitro, in vivo, in situ, in fixed and living cells, or in lipid vesicles.
  • the compounds of the invention may be bound to a surface or incorporated in a lipid monolayer or bilayer.
  • the surface is preferably selected from glass type surfaces such as semimetal oxides, metal oxides and all glass types / glasses, gold, silver, DAPEG modified glass, PEG polymer modified glass or gold, GOPTS silanized glass, glass type or noble metal surfaces with lipid Mono- or bilayer, metal selenides, tellurides and sulfides.
  • a glass surface is to be understood as meaning a glass type surface which, in addition to glass, also comprises quartz, mica, metal oxides, semimetal oxides.
  • a basic idea of the present invention is to make use of the redundancy of the oligohistidine tag in order to increase the stability of the protein chelator by multivalent chelators (MCH). Binding to increase by several orders of magnitude.
  • MCH multivalent chelators
  • the binding of oligohistidine tags to metal-chelator complexes is generally achieved by coordinative bonding of the N atoms of the imidazole residues of the oligohistidine tag to free coordination sites in Ni 2+ , which is complexed by the chelator and thereby partially coordinated.
  • Ni-NTA complexes four of the six coordination sites of Ni 2+ are saturated by NTA, leaving two free coordination sites for the binding of 2 histidine residues.
  • Complex formation therefore requires two steps: (1) the activation of the chelator by binding a metal ion, such as a metal ion. Ni 2+ , and (2) the binding of histidine residues of the oligohistidine tag to the free coordination sites. Addition of free imidazole in excess can abolish the binding of the oligohistidine tag to the Ni (II) chelator complex such that the protein-chelator linkage is reversible and switchable. In addition, removal of Ni 2+ from the chelate complex using EDTA can deactivate the chelator.
  • the binding of the oligohistidine tag to metal chelate complexes via only two histidine residues is relatively unstable.
  • metal chelators are immobilized at high density, multiple histidine residues of the oligohistidine tag can bind to multiple metal chelate moieties simultaneously, resulting in stronger binding.
  • multivalent chelators containing multiple metal chelate units in one molecule stable binding to oligohistidine tags can be achieved at the molecular level.
  • proteins containing an oligohistidine tag can be modified as stably but reversibly and switchably.
  • the object of the invention is achieved by providing a method for producing the compounds of the invention.
  • the method involves the coupling of two tris-NTA-OtBu units via a disulfide bridge and subsequent cleavage of this bridge.
  • the coupling group Z may also be suitably protected.
  • the synthesis of the framework structure G preferably comprises the synthesis of one or more starting compounds, in particular from amino acids, such as lysine, ornithine, 1,3-diamino butyric acid, 1, 2-diaminopropionic acid, glutamate or aspartate and / or their protected derivatives, such as Z-Lys-OtBu, H-Glu (OtBu) -OBzl, Z-Glu-OH.
  • Further preferred starting materials are bromoacetic acid tert-butyl ester, BOC-8-aminocaproic acid and also crocyclic polyamines, such as 1,4,8,11-tetraazacyclotetradecane.
  • a preferred intermediate is N ⁇ , N ⁇ -bis [(tert-butyloxycarbonyl) methyl] -L-lysine tert-butyl ester ("Lys-NTA-OtBu").
  • the preparation process preferably comprises first a synthesis of the framework structure the backbone structure is then coupled to the chelator groups, with the chelator groups being able to carry appropriate protecting groups, with the backbone structure preferably being a cyclic backbone structure, such as a cyclam ring structure.
  • the functionalization group (preferably thiol) is coupled via a short linker to the MCH backbone.
  • the functionalization group is protected in a preferred embodiment via a strategic disulfide.
  • Coupling with further functional groups, such as azides, which are not described in DE 10 2004 038 134 A1, are now also possible due to the introduction of the short linker.
  • the azide group extends the range of sensitive coupling methods to include Staudinger ligation, cycloaddition, and click chemistry relevant in biological systems (see Prescher et al., Nature Chemical Biology 2005, 1, 1).
  • reporter molecules require gentle conditions and precise control of the reaction milieu during coupling to MCHs to preserve the integrity of the molecule.
  • An example of this reporter class are stable radicals, which are highly reactive due to the free electron.
  • the synthesis strategy described in this invention ensures these conditions and thus extends the range of use of MCHs.
  • compounds including primary amine or carbonyl group
  • thio-tris-NTA allows much milder reaction conditions for the coupling process, thus allowing even sensitive molecules (such as magnetic resonance spectroscopy probes) to couple to the MCHs.
  • PROXYL-tris-NTA see the following formula
  • PROXYL-tris-NTA a preferred compound in which a stable organic radical is coupled to thio-tris-NTA.
  • MCH binds to cell surface on recombinant His tagged receptor, after internalization disulfide is cleaved and released the drug.
  • the in vivo derivatization is also particularly advantageous since it opens the possibility MCHs without a time-consuming, costly and possibly reporter-damaging separation of unreactive reactive groups by means of HPLC (high performance liquid chromatography) or size exclusion chromatography to mark.
  • HPLC high performance liquid chromatography
  • MCHs further utilize the poly-histidine sequence for specific, stable, reversible and stoichiometric labeling with reporter groups.
  • a further functionally impairing modification of the intrinsic protein sequence is no longer necessary.
  • Figure 1 A and B shows the strategy for the synthesis of PROXYL-trisNTA (5).
  • FIG. 2 shows the results of analytical size exclusion chromatography.
  • PROXYL trisNTAs stably and reversibly bind (His) 6-labeled maltose binding protein (MBP)
  • Figure 3 shows in EPR that the tricyclic trisNTAs bear the stable radical after purification, that Ni 11 ions interfere with the stable radical, and that Zn 11 ions do not interfere with the stable radical.
  • Figure 4 shows the characterization of Proxyl-trisNTA by means of Continous Wave EPR.
  • A Effect of divalent cations on the EPR signal. Ni 2+ reduces the EPR signal, whereas Zn 2+ does not affect the radical.
  • B Proxyl-trisNTA binds stably and reversibly to His-tagged maltose binding protein.
  • Figure 5 shows the characterization of proxyl trisNTA by pulsed EPR techniques; Distance measurements by means of Proxyl-trisNTA.
  • A Intramolecular Distance Measurement: Maltose binding protein (MalE) was labeled at various positions with the spin probe RI (MTS-Proxyl). By binding of proxyl-trisNTA distances in the presence and absence of substrate (maltose) could be determined.
  • B Intermolecular Distance Measurement: The maltose transporter MALFGK2 was derivatized at position S205 with RI (MTS-Proxyl). The proxyl-trisNTA was pre-incubated stoichiometrically with MalE. Only after MalE binds to MalFGK2 can a distance between the two proteins be determined. Examples
  • Phenol (150 mg, 1.59 mmol) and triisopropylsilane (TIPS, 580 ⁇ , 3.63 mmol) were freshly dissolved in trifluoroacetic acid (5 mL) followed by addition of the protected chelator heads (2. 330 mg). The reaction mixture was stirred at room temperature for 4 h. The volatiles were then removed under reduced pressure and the oily mass thus obtained was redissolved in trifluoroacetic acid (5 ml). The product was precipitated with cold diethyl ether, followed by thorough washing with cold diethyl ether (10 x 15 ml).
  • the resulting solid was purified using RP-HPLC (C18 column, Vydac® 218TP, Grace) with a linear gradient of 0-19% acetonitrile in MQ water, each with 0.2% trifluoroacetic acid.
  • the products were characterized by mass spectrometry (Table 1).

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  • Organic Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne fondamentalement des composés chélateurs multivalents (MCH) comportant trois ou quatre groupes acide nitrilotriacétique (NTA) comme groupes chélateurs et dans un premier aspect un groupe thiol libre, ainsi qu'un procédé de production de ce composé. La présente invention comprend également comme deuxième aspect préféré de nouvelles molécules dans lesquelles un radical stable covalent est lié au groupe thiol de la molécule chélatrice tris-NTA. La présente invention concerne en outre des utilisations des MCH.
PCT/EP2011/052451 2010-02-18 2011-02-18 Composés chélateurs multivalents (mch) à haute affinité et leur utilisation pour l'analyse structurelle et fonctionnelle de molécules cibles WO2011101445A1 (fr)

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DE102010008417A DE102010008417A1 (de) 2010-02-18 2010-02-18 Hochaffine multivalente Chelatorverbindungen (MCHs) und deren Verwendung zur Struktur- und Funktionsanalyse von Zielmolekülen
DE102010008417.4 2010-02-18

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WO2013160453A2 (fr) 2012-04-26 2013-10-31 Iba Gmbh Molécule adaptatrice capable de munir une protéine de fusion portant une étiquette d'affinité oligohistidine d'une autre étiquette d'affinité et ses procédés d'utilisation
CN106732464A (zh) * 2017-02-02 2017-05-31 复旦大学 一种亲水树枝和c18修饰的磁性石墨烯材料及其制备方法和应用

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US20230026678A1 (en) * 2019-11-18 2023-01-26 Technion Research & Development Foundation Limited Minimally-invasive continuous clinical monitoring of small molecules with analytical accuracy

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013160453A2 (fr) 2012-04-26 2013-10-31 Iba Gmbh Molécule adaptatrice capable de munir une protéine de fusion portant une étiquette d'affinité oligohistidine d'une autre étiquette d'affinité et ses procédés d'utilisation
CN106732464A (zh) * 2017-02-02 2017-05-31 复旦大学 一种亲水树枝和c18修饰的磁性石墨烯材料及其制备方法和应用
CN106732464B (zh) * 2017-02-02 2020-11-20 复旦大学 一种亲水树枝和c18修饰的磁性石墨烯材料及其制备方法和应用

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