“Thiol-Ene Photoclick Chemistry for Efficient Bicyclic Peptide Synthesis and Genetically Encoded Peptide Library”

Today, we share with you important research from the team led by Xinxiang Lei of Lanzhou University, published in Angewandte Chemie International Edition. This work marks the first application of thiol-ene photoclick chemistry to the efficient bicyclization of unprotected peptides, developing a rapid, highly selective, and highly biocompatible new method. The researchers ingeniously utilized the inexpensive and readily available tri-alkene crosslinker TAIC. Under visible light activation, cyclization is completed in just 6 minutes. They successfully integrated this with phage display technology to construct a genetically encoded bicyclic peptide library, screening for novel ligands with ultra-high sub-micromolar affinity for the important drug target cyclophilin A. This study provides a powerful and versatile new platform for the discovery of constrained peptide drugs.

01 Research Background

Bicyclic peptides, owing to their unique rigid structure and excellent stability, show immense potential in targeting protein-protein interactions (PPIs) that are traditionally difficult for small molecules to address. They have become an important scaffold in drug discovery. However, their development has long been constrained by the lack of efficient, highly selective cyclization methods. Traditional methods, such as using crosslinkers like TBMB, often face challenges like harsh reaction conditions, numerous side reactions, and poor compatibility with complex biological environments, making them difficult to apply to high-throughput screening platforms like phage display. Although subsequent improved strategies like metal-mediated approaches have been developed, issues such as metal toxicity and ligand stability persist. Therefore, developing a new method that can achieve rapid, specific peptide bicyclization under mild, physiologically compatible conditions is an urgent need in chemical biology and drug discovery. Thiol-ene photoclick chemistry is renowned for its speed, efficiency, and excellent spatiotemporal control. This study aims to extend it as an innovative tool to solve this key problem.

02 Innovative Highlights

• First Application of Thiol-Ene Photoclick Chemistry to Peptide Bicyclization:​ The research team insightfully selected the commercially available tri-alkene crosslinker TAIC. Using an inexpensive photoinitiator under mild light, they achieved efficient and highly selective cyclization of three cysteine thiols on unprotected peptides via a radical mechanism. The reaction can proceed quantitatively in just 6 minutes under mild conditions compatible with all proteinogenic amino acids.

• Perfect Compatibility with Phage Display, Enabling Construction of a Genetically Encoded Bicyclic Peptide Library:​ This is a key breakthrough of the work. The researchers optimized the photocyclization conditions to have minimal impact on phage viability. For the first time, they achieved in situbicyclization of displayed linear peptides directly on the surface of phage particles, perfectly combining the precise controllability of chemical synthesis with the powerful throughput of biological screening.

• Successful Discovery of High-Affinity Lead Compounds:​ Utilizing this platform for screening against the important drug target cyclophilin A, they successfully obtained bicyclic peptide ligands with binding constants reaching the sub-micromolar level. Their affinity improved by tens of times compared to linear precursors and they exhibited significantly enhanced protease resistance, demonstrating the practical value of the method.

Figure 1. a) Conventional cyclization strategies for bicyclic peptide formation. b) Bicyclic peptide construction via thiol–ene photoclick chemistry and its application in constructing a phage-displayed bicyclic peptide library

03 Results and Discussion

3.1 Systematic Optimization of Reaction Conditions and Mechanistic Verification

The study first systematically optimized the thiol-ene photoclick reaction system. By screening crosslinkers and photoinitiators, the water-soluble tri-alkene compound TAIC was identified as the ideal crosslinker, with lithium phenyl-2,4,6-trimethylbenzoylphosphinate as the photoinitiator. The best conversion efficiency was achieved by reacting in an acetonitrile/water mixed solvent under 365 nm UV irradiation for 6 minutes. Key control experiments confirmed the reaction was completely inhibited in the absence of light, photoinitiator, or in the presence of the radical scavenger TEMPO, clarifying its radical mechanism and providing a reliable chemical basis for subsequent applications.

Figure 2. a) Bicyclization of linear peptide 5a via thiol–ene photoclick reaction. b) HPLC analysis (220 nm) of peptide 5a undergoing thiol–ene photoclick reaction in H2O/MeCN (v/v, 80:20) solvent system. c) HPLC analysis (220 nm) of peptide 5a undergoing thiol–ene photoclick reaction in various buffer/MeCN (v/v, 80:20) mixed solvent systems. d) Bicyclization of linear peptide 5b via thiol–ene photoclick reaction and TBMB crosslinking. e) Comparative HPLC analysis (220 nm) of peptide 5b cyclized via thiol–ene photoclick reaction and TBMB.

3.2 Universality of Bicyclization and Comparison with Traditional Methods

Under optimal conditions, this strategy demonstrated broad substrate applicability. A series of linear peptides containing three cysteines, with varying sequence lengths and spacers, were all efficiently converted to the corresponding bicyclic peptide products. Crucially, compared to the alkylation method using the traditional crosslinker TBMB, this photocyclization strategy showed significant advantages in reaction rate, yield, and byproduct control. HPLC analysis revealed the photoclick method achieved rapid, high-yield bicyclization with minimal byproducts, whereas the TBMB method had lower conversion and a complex product mixture, highlighting the superiority of photoclick chemistry.

Figure 3. Substrate scope of peptide bicyclization.

3.3 Biocompatibility Verification and Phage Library Construction and Screening

To verify the potential for biological application, the study further evaluated its compatibility with proteins and phages. Experiments showed that under optimized conditions (150 μM TAIC, 75 μM photoinitiator, 365 nm light for 6 minutes), the infectivity of the phage was not significantly affected, with its activity maintained significantly better than with traditional TBMB treatment. This excellent biocompatibility enabled the successful construction of a large-scale phage-displayed bicyclic peptide library (library size ~10^9) and its successful application in high-throughput ligand screening against the therapeutically relevant target cyclophilin A.

Figure 4. Biocompatibility Validation and Phage Library Construction and Screening.

3.4 Identification and Performance Characterization of High-Affinity Ligands

Through phage library screening, the study identified high-affinity bicyclic peptides CM1 and CM2 against cyclophilin A. Surface plasmon resonance analysis showed that compared to its linear precursor M1, bicyclic peptide CM1 exhibited a significant increase in binding affinity, with the equilibrium dissociation constant improving from the micromolar level to 364 nM. Meanwhile, CM1 demonstrated excellent stability in mouse plasma, with a half-life far exceeding that of the linear peptide. Further nuclear magnetic resonance titration experiments revealed that CM1 binds near the active site pocket of cyclophilin A, with its chemical shift perturbations highly overlapping with known active site residues, explaining the molecular basis of its high specificity from a structural perspective.

04 Conclusion and Future Perspectives

This study successfully developed an efficient, versatile peptide bicyclization platform based on thiol-ene photoclick chemistry. By leveraging its advantages in efficiency, selectivity, and biocompatibility, it not only addresses the limitations of traditional methods but, more importantly, through integration with phage display technology, achieves synergy between photochemistry and biological screening. This opens a new avenue for the large-scale discovery of high-affinity bicyclic peptide ligands and for tackling "undruggable" targets. In the future, this platform holds promise for extension to advanced screening technologies like mRNA display and in vivoor in situcyclization scenarios. By combining the precise control of chemical synthesis with the powerful throughput of biological screening, it has the potential to become a standard platform for peptide drug discovery. It shows particularly broad prospects in addressing complex protein-protein interactions, injecting new vitality into the discovery of next-generation peptide therapeutics, and is expected to play a significant role in broader biomolecular engineering and drug development fields.

Original Article：

Ming, Wei, et al. "Thiol–Ene Photoclick Chemistry for Efficient Bicyclic Peptide Synthesis and Genetically Encoded Peptide Library." Angewandte Chemie International Edition (2025): e18628.

https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.202518628