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

Today, we share with you important research led by Professor Ping Wang's team at Shanghai Jiao Tong University, published in Angewandte Chemie International Edition. This study addresses the core challenge of selective protection and deprotection of cysteine (Cys) in protein chemical synthesis, developing a novel picolyl (Pic)-based caging/uncaging strategy. Through simple pH and wavelength control, this work achieves efficient, orthogonal protection of Cys residues. It has been successfully applied to the synthesis of complex proteins such as Interleukin-4 (IL-4) and Tumor Necrosis Factor-α (TNF-α), providing an innovative tool for precise protein modification and synthesis.

01 Research Background

Cysteine (Cys) plays a key role in protein functional regulation and synthesis due to its unique thiol reactivity. In protein synthesis strategies like Native Chemical Ligation (NCL), selective protection of Cys is a crucial step for assembling complex proteins. Traditional protecting groups, such as the acetamidomethyl (Acm) group, require removal under acidic conditions or heavy metal catalysis, posing risks of side reactions and cumbersome operations. While photolabile protecting groups (PPGs) enable "traceless" deprotection via light irradiation, existing PPGs (e.g., o-nitrobenzyl groups) suffer from complex synthesis, poor water solubility, and incompatibility with radical desulfurization. Additionally, most PPGs require UV excitation, which can damage biomolecules. Therefore, there is an urgent need for a simple, efficient, and biocompatible Cys protection strategy in protein synthesis. This study, based on the inexpensive and readily available picolyl (Pic) group, achieves rapid uncaging under visible light via photoredox catalysis, filling a gap in existing technologies.

Figure 1. Uncaging of the picolyl group via photoredox catalysis under visible light.

02 Innovative Highlights

Ingenious Utilization of the Pic Group and Mechanistic Breakthrough: The research team eschewed complex aromatic systems, opting for the structurally simple picolyl (Pic) as the protecting group. By modulating the reduction potential of the pyridine ring via protonation (increasing it from -2.19 V to -1.19 V), it can be reduced by a ruthenium catalyst (Ru(bpy)₃Cl₂) under visible light, solving the problem of traditional PPGs' dependence on UV light. Mechanistic studies show the process proceeds via spin-center shift, generating a picolylmethyl radical and a thiolate anion, ultimately achieving efficient uncaging under mild conditions.

Dual Orthogonal Control via pH and Wavelength: Pic uncaging requires weakly acidic conditions (pH 5.0), whereas common protecting groups (e.g., o-nitrobenzyl) can be removed under basic conditions. This pH dependency enables orthogonal operation of the Pic group with other protecting groups (e.g., PPZQ, o-nitrobenzyl). For instance, in a model peptide, the o-nitrobenzyl group was first removed by 365 nm UV light, followed by Pic removal under visible light, achieving sequential, controllable deprotection. This provides a new method for the regioselective formation of disulfide bonds in complex proteins.

Full Compatibility with Protein Synthesis Techniques: The Pic strategy demonstrates excellent compatibility with NCL, one-pot ligation, and Expressed Protein Ligation (EPL)-Desulfurization. Its rapid uncaging under visible light (20-30 minutes) avoids purification steps, significantly improving synthesis efficiency. Furthermore, Pic-protected peptides exhibit enhanced water solubility, reducing aggregation issues and facilitating the synthesis of hydrophobic proteins.

03 Results and Discussion

3.1 Optimization and Mechanistic Investigation of Pic Group Caging/Uncaging Reactions

The study first systematically optimized the caging and uncaging reaction conditions for the Pic group on a model peptide. The caging reaction was found to proceed quantitatively in just 2 minutes under mild conditions (pH 8.5, 37°C), exhibiting extremely high selectivity for cysteine over other nucleophilic amino acid side chains like lysine and tyrosine, highlighting its excellent chemoselectivity. More importantly, the uncaging reaction achieved quantitative conversion within 20 minutes under mild conditions using visible light (a common household CFL bulb suffices), pH 5.0, Ru(bpy)₃Cl₂ as the photocatalyst, and ascorbic acid as the reductant. The pH is a key factor; the acidic environment facilitates protonation of the pyridine ring, significantly increasing its reduction potential and enabling visible-light-catalyzed reduction.

Figure 2. Caging/uncaging of protein cysteinyl thiol with Pic. a) Optimization of caging conditions.

3.2 Substrate Scope Expansion and Validation on Complex Proteins

Under optimal conditions, the team systematically examined the reactivity of pyridine derivatives with different substituents and substitution positions. Results showed that 2- and 4-position picolyl derivatives exhibited the best reactivity. Electron-donating groups (e.g., methyl, methoxy) enhanced uncaging efficiency (yields 32%-71%), while electron-withdrawing groups hindered the reaction. This further confirmed that the basicity (protonation ability) of the pyridine nitrogen is crucial for the reaction's success. Subsequently, the strategy was successfully validated on a complex biomolecule—Bovine Serum Albumin (BSA). The Pic group efficiently modified the cysteine in BSA and was completely removed under standard uncaging conditions. Circular dichroism (CD) analysis confirmed that BSA's native structure remained intact throughout the process, fully demonstrating the excellent biocompatibility of this strategy.

3.3 Achieving Orthogonal Operation with Other Protecting Groups and Application in Disulfide Bond Formation

Another highlight of this study is the utilization of the pH-specific requirement for Pic uncaging to achieve orthogonal operation with common photolabile protecting groups (e.g., o-nitrobenzyl). As shown in Figure 3a, in a dually modified peptide containing both Pic and o-nitrobenzyl groups, researchers could first selectively remove the o-nitrobenzyl group by 365 nm UV irradiation while the Pic group remained stable. Subsequently, switching to visible light irradiation under acidic conditions efficiently removed the Pic group. This capability for "wavelength + pH" orthogonal control provides a powerful tool for site-specific modifications in complex proteins. The researchers further applied this strategy to the regioselective construction of disulfide bonds in Macrophage Inflammatory Protein-1β (CCL4). Through sequential deprotection and oxidation, they successfully obtained the correctly structured target protein in 59% yield, demonstrating its potential to address key challenges in synthetic biology.

Figure 3. Study of chromatic orthogonality.

3.4 Successful Application in the Total and Semi-Synthesis of Complex Proteins

To fully demonstrate the practical value of the Pic strategy in protein synthesis, the research team tackled two challenging target proteins: Interleukin-4 (IL-4) and Tumor Necrosis Factor-α (TNF-α). For the total synthesis of IL-4, they designed a clever one-pot iterative ligation strategy, dividing the protein into four segments assembled via three NCL reactions. The Pic group served as a temporary protecting group, efficiently removed by visible-light catalysis after each ligation. This drove the reaction forward without intermediate purification steps, successfully yielding full-length, glycosylated IL-4 with a 47% overall yield.

Figure 4. Pic-assisted protein synthesis.

For the more complex TNF-α, a semi-synthetic method combining Expressed Protein Ligation (EPL) with a desulfurization strategy was employed. First, the two native cysteines in a recombinantly expressed protein fragment were protected with the Pic group. Then, ligation with a chemically synthesized glycopeptide fragment was performed. Finally, via Pic uncaging and desulfurization, the cysteine at the ligation site was converted to the native alanine. Using this strategy, a structurally homogeneous TNF-α trimer was successfully synthesized. Its structure, purity, and correct folding were comprehensively characterized by various techniques including size-exclusion chromatography, high-resolution mass spectrometry, and circular dichroism.

3.5 In-Depth Elucidation of the Reaction Mechanism

Finally, the researchers conducted in-depth investigation of the uncaging reaction mechanism using various techniques including electrochemistry, UV-Vis absorption spectroscopy, fluorescence quenching experiments, and Electron Paramagnetic Resonance (EPR). Results indicate the process involves a series of key steps: photocatalyst excitation, single electron transfer, and spin-center shift. The protonated picolylmethyl group is a key intermediate for the smooth progress of the reaction. These mechanistic studies lay a solid theoretical foundation for the further development and optimization of this method.

Figure 5. Preliminary mechanistic studies

04 Conclusion and Future Perspectives

This study successfully developed a Pic-based Cys caging/uncaging strategy. Through orthogonal control by pH and wavelength, it enables efficient, biocompatible protein synthesis. Its core advantages lie in the Pic group's low cost, ready availability, good water solubility, and perfect compatibility with visible-light catalysis, overcoming the limitations of traditional PPGs. This strategy not only provides a new pathway for synthesizing complex proteins like IL-4 and TNF-α but, more importantly, offers a general method for addressing orthogonal protection problems in protein chemistry. In the future, by optimizing the catalyst system (e.g., developing milder photosensitizers) and extending it to the protection of other amino acid residues, the Pic strategy holds promise for broader application in areas like protein drug development and synthetic biology. Furthermore, integration with AI-assisted protein design may further drive innovation in precise synthesis technology.

Original Article:

Ye F, Bai H, Liu X, et al. A Picolyl-Based Cys Caging/Uncaging Strategy Facilitates Protein Synthesis. Angew Chem Int Ed Engl. 2025 Oct 20:e202518002. 

https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.202518002