“Photocatalytic C-X Bond Cleavage Facilitates Peptide Synthesis”
Today we are sharing a research article led by Professor Ping Wang's team, published in the Journal of the American Chemical Society. This study developed a novel solid-phase peptide synthesis (SPPS) platform based on Fmoc/pyridinylmethyl (Fmoc/Pic) chemistry. Utilizing visible-light photocatalytic cleavage of C-X (X=O, N, S) bonds, this platform enables efficient and orthogonal deprotection of amino acid side chains under mild, trifluoroacetic acid (TFA)-free conditions, providing a revolutionary solution for sustainable and efficient synthesis of peptide therapeutics.
01 Research Background
Solid-phase peptide synthesis (SPPS) is the core technology for the production of modern peptide drugs (e.g., Tirzepatide). For over two decades, the Fmoc/tert-butyl (Fmoc/tBu) strategy has been the industry gold standard, with its final step relying on the strong acid TFA for global side-chain deprotection. However, TFA is a "forever chemical" that is difficult to recycle, poses a significant environmental burden, and faces potential restrictions in regions like the European Union. Furthermore, hydrophobic protecting groups like tBu can lead to peptide chain aggregation during synthesis, and the strongly acidic conditions of TFA can trigger various side reactions, such as degradation of acid-sensitive N-methylated peptides and difficulties in removing phenyl groups from phosphorylated tyrosine, limiting the synthesis of complex functional peptides. Therefore, developing a new SPPS paradigm that eliminates TFA, operates under mild conditions, and remains highly efficient is urgently needed.
02 Innovative Highlights
- Pioneering a photocatalytic C-X bond cleavage-driven SPPS platform: Systematically applied the pyridinylmethyl (Pic) protecting group to amino acid side chains for the first time, utilizing visible-light photocatalysis (e.g., with Ru(bpy)₃Cl₂) to achieve mild (pH 5.0, room temperature), rapid (20-30 minutes) cleavage of C-O, C-N, and C-S bonds, completely eliminating the use of TFA.
- "Killing two birds with one stone" photolabile linker design: Developed a novel photolabile linker (diphenyl(4-pyridinyl)methyl) that enables simultaneous cleavage of the peptide chain from the resin and global side-chain deprotection upon light irradiation, achieving an entirely acid-free process from synthesis to the final product.
- Seamless integration with automated synthesizers: Integrated a photodeprotection module into a commercial automated peptide synthesizer, achieving, for the first time, fully automated, TFA-free peptide synthesis encompassing chain elongation, photolytic deprotection, and peptide-resin cleavage, significantly enhancing synthesis efficiency and accessibility.
03 Results and Discussion
3.1 Design and Principle of the New Paradigm
The study first compared the core concepts of the traditional Fmoc/tBu strategy and the novel Fmoc/Pic strategy (Figure 1). The traditional method relies on the chemical energy of TFA to break C-X bonds, whereas the new method utilizes reducing species generated by photocatalysis, triggering a pyridinium ion intermediate via a single-electron transfer process to efficiently cleave the bond between the Pic group and the heteroatom.
Figure 1. Carbon-heteroatom bond cleavage enables peptide synthesis via photoredox catalysis under visible light
3.2 Condition Optimization and Generality of Pic-Protected Amino Acids
Using a model substrate (Pic-protected serine), the photodeprotection conditions were systematically optimized. The optimal conditions were determined as: PBS/MeOH mixed solvent (pH 5.0), ascorbic acid as the reductant, Ru(bpy)₃Cl₂ as the catalyst, and irradiation with a compact fluorescent lamp (CFL) for 20 minutes, achieving quantitative conversion. The study showed that protonation of the pyridine (under acidic conditions) is crucial for increasing its reduction potential, thereby driving the reaction.
Subsequently, the study successfully applied the Pic group and its derivatives (e.g., Dmpic, Picoc) to protect the side chains of a series of standard amino acids (such as aspartic acid, arginine, lysine) and unnatural amino acids, including phosphorylated amino acids (Figure 2). All Pic-protected amino acids could be prepared efficiently on scales from grams to hundreds of grams and deprotected in high yield (approx. 90%) under the standard photolytic conditions, demonstrating excellent functional group tolerance and practicality.
Figure 2. Scope and photodeprotection efficiency of picolyl-protected amino acid building blocks.
3.3 Synthesis Validation with Complex Biologically Active Peptides
Using the Fmoc/Pic strategy, the study successfully synthesized various C-terminal amide peptides (19-27) with significant biological activity, including octreotide (20), oxytocin (21), and medium-length peptides containing over 10 Pic groups, such as salmon calcitonin (24) and pramlintide (26) (Figure 3). Compared to peptides synthesized by the traditional Fmoc/tBu strategy, those made with the new method showed significantly reduced hydrophobicity, which helps improve solubility, and successfully avoided side reactions like oxidation of sensitive residues such as Met.
Figure 3. The synthesis of amide-terminated peptides via Fmoc/Pic-based SPPS strategy with Sieber resins.
3.4 Realization of Fully Automated TFA-Free Peptide Synthesis
A major highlight of the research was the achievement of fully automated synthesis. By designing the photolabile linker and optimizing solid-phase photodeprotection conditions (e.g., solvent, resin), they integrated a light source into an automated synthesizer (Figure 4), enabling "one-click" synthesis of C-terminal carboxylic acid peptides (28-38). For instance, they successfully synthesized phosphorylated tyrosine kinase peptide 3 (38), overcoming the challenges of difficult deprotection and low yield associated with the phosphate group in traditional methods.
Figure 4. Fully automated synthesis of carboxylic acid-terminated peptide with modified Rink amide-AM resins.
04 Conclusion and Future Perspectives
This study successfully developed a revolutionary Fmoc/Pic SPPS platform that fundamentally overcomes the reliance on TFA and the associated environmental and synthetic challenges of the traditional Fmoc/tBu strategy by utilizing visible-light photocatalytic C-X bond cleavage technology. This platform offers outstanding advantages, including mild conditions, fewer side reactions, applicability to complex peptide sequences, and perfect integration with automated equipment. This work not only paves a new path for green and sustainable production of peptide therapeutics, but its core photocatalytic deprotection chemistry also holds promise for broad application in the precise synthesis of other complex molecules. Future directions worthy of attention include validation of this technology for clinical-grade production, its extension to the synthesis of very long peptides or proteins, and further reduction of production costs.
Original Article:
Bai H, Ye F, Purnachandar D, et al. Photocatalytic C–X Bond Cleavage Facilitates Peptide Synthesis[J]. Journal of the American Chemical Society, 2025, 147(37): 34011-34018.