N-Terminal dual protein functionalization by strain-promoted alkyne-nitrone cycloaddition

Area of Science:

• Bioconjugation Chemistry
• Chemical Biology
• Protein Engineering

Background:

• Dual functionalization of biomolecules is crucial for developing advanced therapeutics and diagnostics.
• Existing methods often lack efficiency or versatility for complex labeling strategies.

Purpose of the Study:

• To optimize strain-promoted alkyne-nitrone cycloaddition (SPANC) for versatile dual functionalization of peptides and proteins.
• To demonstrate the combined utility of SPANC with other click chemistry reactions for protein modification.

Main Methods:

• Optimization of SPANC for efficient peptide and protein labeling.
• Application of SPANC to human lactoferrin (hLF) for simultaneous introduction of chloroquine and stearyl groups.
• Sequential application of SPANC and copper-catalyzed azide-alkyne cycloaddition (CuAAC) to enhanced green fluorescent protein (eGFP).

Main Results:

• Successful dual functionalization of hLF with endosomal escape-enhancing moieties.
• Demonstration of SPANC's compatibility with copper-catalyzed click chemistry for protein dual labeling.
• Introduction of biotin and a terminal alkyne to eGFP via SPANC, followed by CuAAC with fluorescein.

Conclusions:

• SPANC is a robust and versatile method for the dual functionalization of peptides and proteins.
• Combining SPANC with CuAAC offers a powerful platform for straightforward, site-specific dual labeling of proteins.
• This approach holds significant potential for creating novel bioconjugates with tailored functionalities.