Site-specific, kinetically inert conjugation of labels and/or carriers to target molecules such as his-tagged proteins via metal complex reagents
Inventors
BENK, Amelie S. • Benk, Lucia T. • WEGNER, Seraphine • Comba, Peter • Spatz, Joachim P.
Assignees
Universität Heidelberg • Max-Planck-Gesellschaft zur Förderung der Wissenschaften eV
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Publication Number
US-12497423-B2
Publication Date
2025-12-16
Expiration Date
Abstract
The present invention relates to means and methods for conjugating/attaching target molecules such as proteins to a label and/or carrier. Specifically, the present invention provides a complex comprising a metal cation coordinating (i) nitrate as a metal cation ligand and (ii) a metal cation chelating domain comprising a chelating ligand and a label and/or carrier. This complex can be used for attaching a label and/or a carrier to a target molecule, preferably a protein. The attachment of the label or carrier via the complex of the invention involves the replacement of the metal cation ligand with a coordinating group of the target molecule so that a product complex with the target molecule as primary ligand in the coordination sphere of the metal cation is formed. Accordingly, the present invention also provides for uses and methods involving the attachment of a label and/or carrier to a target molecule. Also provided are the products obtained by the labeling and or carrier-attaching methods of the invention and uses thereof. The invention further relates to methods for producing the complex of the invention and kits comprising the components for producing the complex of the invention.
Core Innovation
The invention relates to a site-specific labeling and immobilization platform that uses a kinetically inert Co(III) or Pt(IV) complex. The complex includes nitrate as a metal cation ligand and a metal cation chelating domain comprising a chelating ligand selected from NTA and IDA, and the label and/or carrier is attached to NTA or IDA. Representative complexes include [Pt(IV)(NTA)NO3]+, [Pt(IV)(IDA)NO3]+, [Co(III)(NTA)NO3]−, [Co(III)(IDA)NO3]−, or hydrates thereof.
The platform forms product complexes by metal-ligand exchange with histidine-rich target molecules. It uses the kinetic inertness of the Co(III) or Pt(IV) nitrate complexes to control ligand exchange with targets having histidine and/or histidine-like residues arranged in a specified spaced histidine tag motif, and it is characterized by stability against competing chelators such as imidazole.
The described approach forms kinetically inert metal–NTA complexes that stably immobilize His-tagged proteins on NTA-functionalized beads, including His6-GFP and PercevalHR. Chemical stability toward imidazole is demonstrated after formation via NTA-bound carbonate for Co(III) using [Co(III)(NTA)(CO3)]2−, and for Pt(IV) using nitrate with [Pt(IV)(NTA)(NO3)].
The disclosure further characterizes carbonate complex formation and binding kinetics for Co(III) and reports that carbonate-bound Co(III) accelerates binding kinetics compared with aquo Co(III), while adverse effects are observed for alternative oxidative routes involving H2O2 and oxygen, including fluorophore oxidation and protein degradation with His-tag cleavage. The immobilization concept is extended to other metal binding domains including IDA and TALON, to other proteins and antibody, to sortase activity retention, and to surface immobilization via HS–PEG–NTA and site-specific in-solution biotinylation using Biotin-X-NTA–Co(III)(CO3) complexes.
Claims Coverage
The consolidated claim coverage includes two independent claim themes: a complex comprising a Co3+ or Pt4+ metal cation coordinated by nitrate with an NTA- or IDA-based chelating domain bearing a label and/or carrier, and a platform for site-specific labeling and immobilization using these kinetically inert complexes. Across the independent claims, there are four core inventive features centered on the specific metal/nitrate coordination, the NTA/IDA chelating domain, label/carrier attachment, and target-attachment by exchange with histidine-rich molecules.
Nitrate-ligated Co(III) or Pt(IV) with NTA or IDA chelating domain
A complex comprising a Co3+ or Pt4+ metal cation; nitrate as a metal cation ligand; and a metal cation chelating domain comprising a chelating ligand selected from nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA), with a label and/or carrier attached to NTA or IDA.
Specific nitrate complexes with attached label or carrier
The complex comprises a [Pt(IV)(NTA)NO3]+ complex, a [Pt(IV)(IDA)NO3]+ complex, a [Co(III)(NTA)NO3]− complex or a [Co(III)(IDA)NO3]− complex or a hydrate thereof, wherein the label and/or carrier is attached to NTA or IDA.
Site-specific labeling and immobilization by metal-ligand exchange
A platform that forms a product complex by metal-ligand exchange with histidine-rich target molecules, including targets with histidine and/or histidine-like residues arranged in a specified spaced histidine tag motif.
Kinetically inert complex with stability against competing chelators
The Co(III) or Pt(IV) nitrate complexes are kinetically inert, with stability against competing chelators such as imidazole.
Overall claim coverage centers on nitrate-coordinated Co3+ or Pt4+ complexes bearing an NTA- or IDA-based metal-cation chelating domain with an attached label and/or carrier, together with their use in site-specific labeling and immobilization of histidine-rich targets.
Stated Advantages
Provides kinetically inert metal–NTA complexes that stably immobilize His-tagged proteins on NTA-functionalized beads.
Demonstrates chemical stability toward imidazole after formation of carbonate-bound Co(III) complexes and nitrate-associated Pt(IV) complexes.
Enables fast, efficient attachment without H2O2-driven oxidation that can harm proteins, labels, or carriers.
Shows Co(III) inertness compared with Co(II)/Ni(II) and disruption by reduction/chelation.
Reports that carbonate-bound Co(III) accelerates binding kinetics compared with aquo Co(III).
Indicates adverse effects of H2O2/oxygen routes, including fluorophore oxidation and protein degradation/His-tag cleavage, in contrast to the inert complexes.
Provides kinetic inertness with low ligand exchange rates and stability against chelators such as imidazole.
Extends immobilization to other metal binding domains (IDA, TALON), other proteins/antibody, and preserves sortase activity.
Preserves protein function, including antibody binding-related retention and enzyme or biological activity retention as supported by the disclosed characterization against oxidation and competing chelators.
Enables surface immobilization via HS–PEG–NTA and site-specific in-solution biotinylation using Biotin-X-NTA–Co(III)(CO3) complexes.
Documented Applications
Immobilizing His-tagged proteins (His6-GFP, PercevalHR) on NTA-functionalized beads using cobalt(III) or platinum(IV) mediated complexes.
Labeling and/or immobilization of targets using the Co(III)/Pt(IV) nitrate complexes, including in solution and on affinity resin or carriers.
Stabilizing immobilized labeled complexes for chemical environments involving imidazole.
Use with affinity resins and carriers including beads and surfaces as described.
Immobilization using other metal binding domains including IDA and TALON.
Binding applications involving other proteins and antibody, with retention of sortase activity.
Surface immobilization using HS–PEG–NTA.
Site-specific in-solution biotinylation using Biotin-X-NTA–Co(III)(CO3) complexes.
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