A Picolyl-Based Cys Caging/Uncaging Strategy Facilitates Protein Synthesis.

Area of Science:

• Chemical Biology
• Organic Chemistry
• Protein Chemistry

Background:

• Cysteine (Cys) is vital for site-specific bioconjugation and chemical protein synthesis, especially native chemical ligation (NCL).
• Expressed protein ligation (EPL)-desulfurization expands access to difficult proteins but requires orthogonal protecting groups for selective desulfurization.
• Existing photolabile protecting groups (PPGs) lack compatibility with orthogonal protection and EPL-desulfurization.

Purpose of the Study:

• To develop a novel, simple picolyl-based photolabile protecting group (PPG) for cysteine (Cys) residues.
• To enable rapid orthogonal caging and uncaging of thiols with control over pH and wavelength.
• To demonstrate the utility of this new PPG in advancing complex protein synthesis.

Main Methods:

• Development of a picolyl-based photolabile protecting group (PPG) for cysteine (Cys) thiol protection.
• Demonstration of orthogonal caging and photoorthogonal activation in the presence of nitrobenzyl groups.
• Application of the PPG strategy in the synthesis of interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-α).

Main Results:

• A novel picolyl-based PPG was successfully synthesized and characterized for Cys protection.
• The PPG allows for rapid orthogonal thiol caging and subsequent uncaging via pH and wavelength control.
• The picolyl group exhibits photoorthogonal activation relative to nitrobenzyl groups.
• Efficient synthesis of IL-4 and TNF-α was achieved, validating the PPG's utility.

Conclusions:

• The developed picolyl-based PPG offers a simple and effective method for orthogonal Cys protection and uncaging.
• This strategy significantly advances the field of chemical protein synthesis, particularly for complex proteins.
• The photoorthogonal nature of the PPG broadens its applicability in multi-step bioconjugation and protein assembly.