Metal Complex Labeling of His-Tagged Proteins Without Oxidation

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Solution Overview

Problem

Existing methods for attaching labels and/or carriers to target molecules, such as proteins, suffer from lack of site-specificity, batch variability, and instability due to harsh reaction conditions, which can affect protein function and stability.

Innovation Solution

A complex comprising a metal cation coordinating with a carbonate or nitrate ligand and a metal cation chelating domain is used to attach a label and/or carrier to a target molecule, bypassing oxidation steps and forming a kinetically inert bond directly.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If classical labeling methods using reactive groups (amines, thiols) are used, then labeling efficiency is improved, but site-specificity deteriorates and batch variability increases

Engineering Contradiction:

Improvelabeling efficiencyVSAvoidsite-specificity

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The patent applies local quality by creating a localized reactive site through the His-tag peptide sequence that is specifically designed to interact with the metal complex. This localized interaction site on the protein surface enables site-specific conjugation while maintaining high labeling efficiency, resolving the contradiction between efficiency and specificity.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent uses a metal complex as an intermediary reagent that mediates the conjugation between the His-tag and the label. The metal complex acts as a bridge that provides both high affinity binding to the His-tag and controlled chemistry for label attachment, achieving both site-specificity and efficiency through this intermediary mechanism.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Manufacturing precision

If enzymatic methods (biotin ligase, sortase) are used for site-specific conjugation, then site-specificity is improved, but process complexity and time consumption increase

Engineering Contradiction:

Improvesite-specificityVSAvoidprocess complexity

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent replaces enzymatic mechanisms with a chemical metal complex-based mechanism. Instead of using biological enzymes like biotin ligase or sortase that require complex cellular conditions and purification steps, the invention uses a metal complex that operates under simpler chemical conditions, reducing process complexity while maintaining site-specificity through the His-tag interaction.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Stability of the object's composition

If oxidation steps are used to form kinetically inert complexes, then complex stability is improved, but protein function and label integrity deteriorate due to oxidation damage

Engineering Contradiction:

Improvecomplex stabilityVSAvoidoxidation damage

Core Design Contradiction:

Stability of the object's compositionVSObject-affected harmful factors

Solution Approach 1:

The patent converts the potential harm of oxidation into a benefit by using the metal complex's inherent kinetic inertness (a stable property) to achieve both complex stability and protection against oxidation damage. The metal complex provides stable binding without requiring oxidative steps, thereby eliminating oxidation damage while maintaining stability.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

Solution Approach 2:

The patent creates an inert chemical environment through the formation of the metal complex that is resistant to oxidation. The metal complex structure provides a protected coordination sphere that prevents oxidation of the protein and label, effectively creating an inert environment that maintains both stability and functionality without requiring external inert atmosphere control.

Inventive Principle:

Principle #39Inert atmosphere (Inert environment)

4Stability of the object's composition

If multi-step preparation methods are used to form kinetically inert complexes, then complex stability is improved, but productivity deteriorates due to extended reaction time

Engineering Contradiction:

Improvecomplex stabilityVSAvoidreaction speed

Core Design Contradiction:

Stability of the object's compositionVSProductivity

Solution Approach 1:

The patent applies preliminary action by pre-assembling the metal complex with the His-tag under controlled conditions before final label attachment. This pre-assembly step creates a stable intermediate that facilitates rapid and efficient label conjugation, achieving both stability through the pre-formed complex structure and high productivity through the streamlined two-step process.

Inventive Principle:

Principle #10Preliminary action

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This method allows for fast, efficient, and stable attachment of labels and/or carriers to target molecules without interfering with their function, reducing the need for cumbersome purification steps and avoiding oxidation-related damage.

Implementation Method 1

a complex comprising a metal cation coordinating (i) a metal cation ligand being CO32− or HCO3− and (ii) a metal cation chelating domain comprising a chelating ligand and a label and/or carrier

Methodology Applied

Scientific EffectCoordination chemistry: Chemical Bonding

Implementation Method 2

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

Methodology Applied

Scientific EffectLigand exchange reaction: Chemical Bonding

Implementation Method 3

Methodology Applied

Scientific EffectKinetically inert bonding: Chemical Bonding

Data Source

PatentUS12497422B2Site-specific, kinetically inert conjugation of labels and/or carriers to target molecules such as His-tagged proteins via metal complex reagents

Publication Date: 2025.12.16 MAX PLANCK GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN EV

AI summary

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) a metal cation ligand being a carbonate selected from CO32− and HCO3— 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.