Stable and Rapid Thiol Bioconjugation by Light-Triggered Thiomaleimide Ring Hydrolysis

Area of Science:

• Bioconjugation Chemistry
• Organic Synthesis
• Biomolecular Engineering

Background:

• Maleimide chemistry is a widely used bioconjugation technique for thiol-specific derivatization.
• Thiomaleimide conjugates formed via traditional maleimides are unstable and prone to thiol exchange reactions.
• This instability limits the utility of maleimide bioconjugation in various applications.

Purpose of the Study:

• To develop novel maleimide reagents that form stable thiol conjugates resistant to exchange reactions.
• To address the limited shelf life of maleimide reagents due to premature hydrolysis.
• To introduce a photocaged maleimide for controlled and efficient bioconjugation.

Main Methods:

• Synthesis and characterization of a new class of o-CH2NHiPr phenyl maleimides.
• Investigation of the hydrolysis kinetics of the novel maleimides after thiol conjugation.
• Development and application of a photocaged maleimide derivative.
• Assessment of conjugate stability and hydrolysis conditions post-UV irradiation.

Main Results:

• The new o-CH2NHiPr phenyl maleimides rapidly hydrolyze post-thiol conjugation, forming stable, exchange-resistant conjugates.
• A photocaged maleimide scaffold was successfully developed, exhibiting enhanced shelf stability.
• UV irradiation of conjugates formed with the photocaged maleimide triggered rapid hydrolysis within 1 hour under mild, ice-cold conditions.
• The developed method overcomes the inherent instability of thiomaleimide linkages.

Conclusions:

• A novel class of maleimides provides a robust solution for creating stable thiol-protein conjugates.
• The photocaged maleimide strategy offers improved reagent stability and controlled conjugate formation.
• These advancements significantly enhance the reliability and applicability of maleimide-based bioconjugation.