β-Hydroxy-Stabilized Boron-Nitrogen Heterocycles Enable Rapid and Efficient C-Terminal Protein Modification

Area of Science:

• Bioorthogonal Chemistry
• Chemical Biology
• Bioconjugation Science

Background:

• Bioorthogonal chemistry enables bioconjugation in physiological settings without interfering with natural biomolecules.
• Reactions involving carbonyl compounds and alkoxyamines/hydrazines are increasingly vital in bioorthogonal chemistry.
• Ortho-carbonyl-substituted phenylboronic acids (CO-PBAs) offer rapid hydrazone/oxime formation but can yield less stable conjugates.

Purpose of the Study:

• To explore heteroatom-substituted hydrazides for forming stable tricyclic products with CO-PBAs.
• To develop a site-specific protein labeling and purification strategy using bioorthogonal chemistry.
• To investigate the use of beta-hydroxy hydrazides for enhanced bioconjugate stability.

Main Methods:

• Screening of various heteroatom-substituted hydrazides with 2-formylphenylboronic acid (2fPBA) and 2-acetylphenylboronic acid (AcPBA).
• Utilizing sortase-mediated ligation (SML) to append C-terminal hydrazides to a model eGFP protein.
• Site-specific protein labeling, immobilization on 2fPBA-agarose, and subsequent elution.

Main Results:

• Beta-hydroxy hydrazides reacted with 2fPBA to form highly stable tricyclic products.
• C-terminal threonine and serine hydrazides were efficiently appended to eGFP via SML.
• Labeled proteins were rapidly and quantitatively modified at neutral pH, enabling selective immobilization and purification.

Conclusions:

• A novel bioorthogonal strategy using beta-hydroxy hydrazides and CO-PBAs provides highly stable bioconjugates.
• Sortase-mediated ligation enables efficient site-specific incorporation of hydrazides into proteins.
• This approach facilitates rapid protein labeling, immobilization, and purification of functionalized proteins.