Quinoline-Based Photolabile Protection Strategy Facilitates Efficient Protein Assembly

Area of Science:

• Biochemistry
• Chemical Biology
• Synthetic Chemistry

Background:

• Native chemical ligation (NCL) and expressed protein ligation (EPL) are powerful tools for protein assembly.
• Current NCL-desulfurization protocols require orthogonal protection strategies for cysteine residues, often incompatible with photolabile protecting groups (PPGs).

Purpose of the Study:

• To develop a novel photolabile protecting group (PPG) for cysteine residues compatible with NCL and EPL-desulfurization.
• To demonstrate the utility of this new PPG in complex protein synthesis and modification.

Main Methods:

• Development and application of a quinoline-based PPG (PPZQ) for cysteine residues.
• Late-stage modification for introducing PPZQ onto protein sequences.
• Photolytic removal of PPZQ under aqueous conditions.
• Integration of PPZQ with iterative NCL and EPL-desulfurization strategies.

Main Results:

• The PPZQ group can be efficiently introduced and tracelessly removed via photolysis.
• PPZQ is compatible with radical desulfurization, enabling its use in EPL-desulfurization.
• Successful synthesis of γ-synuclein and phosphorylated cystatin-S using iterative NCL and EPL-desulfurization.
• Demonstrated sextuple protection and deprotection of an Interleukin-34 fragment, highlighting potential in protein caging/uncaging.

Conclusions:

• Quinoline-based PPGs offer a versatile and efficient solution for cysteine protection in protein ligation strategies.
• This method simplifies protein assembly and expands the scope of NCL and EPL applications.
• The developed strategy holds significant promise for complex protein synthesis, modification, and studying protein function.