Hydrolytically Stable Maleimide-End-Functionalized Polymers for Site-Specific Protein Conjugation

Area of Science:

• Bioconjugation Chemistry
• Polymer Science
• Protein Engineering

Background:

• Protein conjugation often employs ester linkages, which are prone to hydrolysis, compromising conjugate stability.
• Site-specific conjugation offers advantages over random conjugation but requires stable linkage chemistries.

Purpose of the Study:

• To develop a hydrolytically stable, site-specific conjugation strategy for proteins.
• To compare the stability of amide-based versus ester-based linkages in protein-polymer conjugates.

Main Methods:

• Synthesis of maleimide-end-functionalized polymers using reversible addition-fragmentation chain-transfer (RAFT) polymerization.
• Amidation of polymer termini with a furan-protected aminoethyl maleimide via carbodiimide chemistry.
• Deprotection of maleimide using a retro Diels-Alder reaction for subsequent thiol-maleimide click chemistry.

Main Results:

• Polymers conjugated via amide bonds (EDC/AEMI) demonstrated sustained attachment after one week.
• Ester-based conjugation methods (HEMI, HEA) resulted in significant loss of polymer conjugates over the same period.
• The study successfully site-specifically conjugated polymers to a thermophilic cellulase (FnCel5a).

Conclusions:

• Hydrolytically stable amide-based maleimides are crucial for bioconjugation strategies requiring long-term stability.
• Ester linkages are suitable for applications where controlled debonding of polymers is desired.
• The developed method provides a robust approach for creating stable protein-polymer conjugates.