Yi Gan, Yumei Zeng, Haojie Guan, Wenjun Li, Alex Shaginian, Jin Li, Sen Gao, Guansai Liu

Bioconjugate Chemistry

https://doi.org/10.1021/acs.bioconjchem.5c00455

Abstract

Bicylic peptides, with two cyclic substructures, have emerged as a powerful tool for modulating challenging targets such as protein−protein interactions. Meanwhile, DNA-encoded library technology (DELT) provides a powerful platform for hit discovery. The unity of both fields has the potential to identify potent bicyclic ligands for the targets of interest. Therefore, there is a high demand to develop an efficient way to construct bicyclic peptide libraries. Herein, we describe a novel and efficient approach to the synthesis of DNA-encoded bicyclic peptides via a cysteine-promoted cyclization and amide condensation reaction. This strategy proceeds smoothly under mild conditions and can generate a wide range of bicyclic peptides with various peptide sequences and ring sizes in good conversion. The method employs a trifunctional cross-linker, methyl 3,5-bis-(bromomethyl)benzoate, to enable sequential thioether and amide bond formation, overcoming stability issues associated with traditional protecting groups. The chemistry is compatible with diverse functional groups, N-methylation, and both short and long DNA strands, demonstrating its suitability for constructing large-scale DNA-encoded bicyclic peptide libraries.

Summary

This report presents a robust DNA-compatible methodology for synthesizing bicyclic peptides suitable for DNA-encoded library technology (DELT). The strategy addresses a critical limitation in the field: the instability of StBu-protected cysteine under Fmoc-deprotection conditions, which generates byproducts and compromises library quality. The authors developed a two-step macrocyclization approach using methyl 3,5-bis-(bromomethyl)benzoate as a trifunctional cross-linker. The first cyclization forms a dithioether linkage via reaction with cysteine thiols, while the second cyclization creates an amide bond through ester hydrolysis and intramolecular condensation. This method proceeds under mild aqueous conditions and demonstrates broad substrate scope, enabling synthesis of bicyclic peptides with ring sizes ranging from [2+2] to [7+8] amino acids. The chemistry tolerates various functional groups (indole, guanidine, phenol, thioether, etc.) and backbone N-methylation. Validation studies on 543 di-, tri-, and tetrapeptide building blocks showed 420 achieved ≥50% conversion, confirming feasibility for library production. The method was successfully applied to both short DNA (7 bp) and long DNA (72 bp) with enzymatic ligation, showing no significant DNA damage. Off-DNA synthesis confirmed the structural integrity of the bicyclic products. This approach enables access to billions of diverse bicyclic peptides, bridging the therapeutic gap between small molecules and biologics.

Highlights

• Wide substrate scope: Successfully synthesized bicyclic peptides with diverse amino acid sequences and ring sizes from [2+2] to [7+8]
• High efficiency: Achieved conversions of 45-88% across various bicyclic peptide structures
• Mild reaction conditions: Compatible with DNA-encoded library synthesis requirements
• Two-step macrocyclization: Novel use of trifunctional cross-linker enabling sequential thioether and amide bond formation
• Functional group tolerance: Compatible with indole, guanidine, phenol, thioether, alcohol, and N-methylated residues
• DEL compatibility: Validated on long-chain DNA (72 bp) with successful enzymatic ligation and no detectable DNA damage
• Scalability potential: Demonstrated feasibility for constructing libraries exceeding 1.6 billion compounds using validated di/tri/tetrapeptide building blocks

Conclusion

In summary, we have developed a novel and efficient methodology to synthesize DNA-encoded bicyclic peptides via two independent macrocyclization reactions. The employment of a trifunctional cross-linker, methyl 3,5-bis-(bromomethyl)benzoate, enabled both cysteine-promoted cyclization and amide cyclization chemistries. A large number of bicyclic peptides with a wide range of structurally diverse backbones and side-chains were constructed with high efficiency. The commercial availability of building blocks (amino acids and peptides) as well as the broad scope exploration demonstrated the feasibility of our protocol for the preparation of a structurally diverse library of bicyclic peptides. Efforts to transform this robust and efficient chemistry to bicyclic peptide DEL construction will be continued in due course.