Unsaturated Phosphorus(V) Thiol-Conjugation for Stable Bioconjugates
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Solution Overview
Problem
Current methods for site-selective modification of proteins, particularly using thiol-conjugation with unsaturated phosphorus(V) compounds, face instability issues under reducing conditions or in the presence of excess thiols, limiting the stability and efficacy of antibody-drug conjugates and other bioconjugates.
Innovation Solution
The development of methods involving unsaturated phosphorus(V) compounds, such as diethynyl-phosphinates, for chemoselective thiol-thiol bioconjugation and rebridging of native disulphides in antibodies, allowing for stable and specific modification of proteins and antibodies, including the use of thiol-containing molecules like amino acids, peptides, and antibodies, without the need for protecting groups.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If thiol-conjugation with unsaturated phosphorus(V) compounds is used for site-selective modification of proteins, then rapid kinetics and near neutral pH operation are achieved, but the generated linkage is inherently unstable in the presence of external thiols due to retro-Michael addition
Solution Approach 1:
The patent changes the chemical structure of the phosphorus(V) compound from conventional maleimides to unsaturated phosphonamidates with specific electron-withdrawing groups (nitro, cyano, or carbonyl groups). This structural parameter change modifies the electronic properties of the linkage, making it resistant to retro-Michael addition by external thiols while maintaining rapid kinetics at near neutral pH.
Solution Approach 2:
The invention creates a composite chemical system combining the phosphorus(V) core with specific electron-withdrawing groups (nitro, cyano, or carbonyl) and conjugated unsaturated systems. This composite structure provides both the reactivity needed for rapid conjugation and the electronic properties needed to prevent retro-Michael addition, resolving the stability-kinetics contradiction.
2Ease of operation
If conventional maleimide-based thiol-conjugation is used, then rapid kinetics at near neutral pH are achieved, but the thiosuccinimide linkage is unstable towards reducing conditions or excess small thiols
Solution Approach 1:
The patent modifies the operational parameters by using unsaturated phosphonamidates that remain stable under reducing conditions (unlike conventional maleimides). The specific electron-withdrawing groups and unsaturated structure prevent reduction-based degradation while maintaining ease of operation at near neutral pH through rapid thiol addition kinetics.
Solution Approach 2:
The invention converts the potential harm of electron-deficient phosphorus compounds (which could be overly reactive or unstable) into a benefit by carefully selecting electron-withdrawing groups that provide just the right balance of reactivity and stability. The unsaturated structure with specific withdrawing groups creates a linkage that is stable toward reducing conditions and excess thiols while maintaining operational ease.
3Manufacturing precision
If site-selective modification methods are used to achieve precise drug-to-antibody ratio control, then manufacturing precision is improved, but the complexity of the conjugation chemistry increases
Solution Approach 1:
The patent simplifies the conjugation chemistry by using unsaturated phosphonamidates that react specifically with cysteine thiols under near neutral conditions. This parameter change in the reagent structure enables precise drug-to-antibody ratio control through straightforward thiol conjugation without requiring complex protection/deprotection schemes or multiple reaction steps, thus reducing chemical complexity while maintaining manufacturing precision.
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 approach enables the formation of highly stable bioconjugates that maintain stability under physiological conditions, including in human serum, with precise control over the drug-to-antibody ratio and minimal interference from small thiols, enhancing the therapeutic potential of antibody-drug conjugates.
Implementation Method 1
methods of preparing compounds and conjugates of antibody molecules via thiol-conjugation with unsaturated phosphorus(V) compounds
Implementation Method 2
reducing at least one disulfide bridge of an antibody molecule in the presence of a reducing agent
Data Source
AI summary
Disclosed are novel conjugates and methods for the preparation thereof. One of the methods for the preparation of a conjugate comprises a step of: acting a compound of formula (I), with a thiol-containing molecule of formula (II), wherein represents an amino acid, a peptide, a protein, an antibody, a nucleotide, an oligonucleotide, a saccharide, a polysaccharide, a polymer, a small molecule, an optionally substituted C1-C8-alkyl, an optionally substituted phenyl, or an optionally substituted aromatic 5- or 6-membered heterocyclic system; resulting in a compound of formula (III).