Controlled Cyclization Meets Ribosomal Synthesis: The Birth of a New-Generation Bicyclic Peptide Platform

Today, we share important research from the team of Professor Hiroaki Suga at the University of Tokyo, published in Angewandte Chemie International Edition, titled "Ribosomal Synthesis of Topologically Defined Thioisoindole-Bridged Bicyclic Peptides." This study developed a novel synthetic strategy for bicyclic peptides based on thioisoindole bridging. By designing a semicarbazone-protected 2-nicotinoylbenzaldehyde amino acid (Ac-Ala(NtBA)Sc-CME) as a translation initiator, it was efficiently incorporated into peptide chains using a flexible in vitrotranslation (FIT) system. Subsequently, a mild acid treatment triggered an intramolecular bicyclization reaction, successfully constructing structurally precise bicyclic peptides. This method is fully compatible with mRNA display technology, providing a powerful platform for building large-scale, topologically defined bicyclic peptide libraries for drug discovery.

Figure 1. Schematic overview of this study: ribosomal construction of thioisoindole-bridged bicyclic (TiB) peptides.

01 Research Background

Macrocyclic peptides have become an important scaffold in drug discovery due to their rigid structures and potent targeting capabilities. However, traditional methods for constructing macrocyclic peptide libraries (e.g., disulfide cyclization) suffer from poor metabolic stability and limited structural diversity. Although mRNA display technology enables screening of ultra-large libraries (>10^12), existing cyclization strategies (mainly relying on cysteine nucleophilic attack or disulfide bond formation) struggle to generate structurally stable, topologically defined bicyclic peptides. While ortho-phthalaldehyde (OPA) chemistry can achieve cyclization via the formation of thioisoindole structures, its high reactivity often leads to non-specific side reactions and is not directly applicable to in vitrotranslation systems. Therefore, developing a new method compatible with mRNA display, with strong controllability and capable of generating stable bicyclic structures, has become an urgent need in the field.

02 Innovative Highlights

• Development of a Novel Translation Initiator with Tunable Reactivity:

The research team designed three 2-ketobenzaldehyde-derived phenylalanine analogs (AcBA, NtBA, BzBA). By introducing substituents (e.g., nicotinoyl) to reduce the reactivity of the OPA moiety, side reactions with free components in the translation system were avoided. Simultaneously, a semicarbazone group was employed as a reversible protecting group, ensuring the initiator's stability during translation, and enabling selective deprotection via mild acid treatment after translation to trigger cyclization.

• Establishment of an Efficient Negishi Coupling Pathway:

By optimizing palladium-catalyzed Negishi cross-coupling conditions (Pd₂(dba)₃/SPhos system), the team successfully constructed the carbon-carbon bond without protecting the aldehyde group, synthesizing the key intermediate Ac-Ala(NtBA)CHO-OMe in 67% yield while preserving chiral purity. This method provides a new synthetic route for analogous structures.

• Realization of "One-Pot" Ribosomal Synthesis of Bicyclic Peptides:

The optimal initiator Ac-Ala(NtBA)Sc-CME was efficiently loaded onto tRNA via flexizyme and incorporated at the N-terminus of the peptide chain using the FIT system. After translation, simply adding a citrate buffer (pH 4) and pyruvate simultaneously completed the semicarbazone deprotection and intramolecular bicyclization, forming a 3-(pyridin-3-yl)-1-thioisoindole-bridged bicyclic structure.

03 Results and Discussion

3.1 Initiator Design and Synthetic Optimization

Through rational design, the team modified the OPA structure to 2-nicotinoylbenzaldehyde (NtBA), significantly reducing its electrophilicity, while introducing the semicarbazone protecting group to ensure translation compatibility (Figure 2).

Figure 2. a) Design of the dicarbonyl-phenylalanine (2CO-Phe) derivative for flexizyme-mediated aminoacylation. b) Schematic illustration of ribosomal synthesis of thioisoindole-bridged bicyclic (TiB) peptides using a designed translation initiator in a genetically reprogrammed FIT system.

3.2 Efficient Aminoacylation of the Translation Initiator

Flexizyme-mediated tRNA charging experiments showed that Ac-Ala(NtBA)Sc-CME achieved near-quantitative charging efficiency at a concentration of 25 mM (Figure 3), outperforming the commonly used initiator ClAc-Trp, proving its efficacy as a highly efficient translation initiation substrate.

Figure 3. a) Synthesis of the central substrate 1, Ac-Ala(NtBA)Sc-CME. b) Optimization of the Negishi cross-coupling conditions, highlighting the final coupling conditions. c) Chemical structures of compounds 7 and 8.

3.3 Cyclization Condition Optimization and mRNA Compatibility Verification

Screening revealed that a 50 mM citrate buffer (pH 4) containing 1 mM pyruvate efficiently triggered cyclization at 37°C. Furthermore, this condition preserved mRNA integrity (Figure 4), meeting the requirements for mRNA display.

Figure 4. a) Schematic overview of the ribosomal synthesis of TiB peptide ck1; the right panel shows the corresponding LC/MS extracted ion current (EIC) chromatograms and mass spectra.

3.4 General Construction of Bicyclic Peptide Libraries

Testing 14 templates with different sequences (variable cysteine/lysine spacing) resulted in the successful conversion of 12 into bicyclic peptides, with conversion rates ranging from 60% to 98% (Figure 5). The results indicated that cyclization efficiency is insensitive to sequence length and residue arrangement, demonstrating the robustness of the strategy.

Figure 5. a) and b) Representative sequences of ribosomally synthesized thioisoindole-bicyclic (TiB) peptides.

04 Conclusion and Future Perspectives

This study successfully developed a new method for the ribosomal synthesis of thioisoindole-bridged bicyclic peptides. Through the design of a translation initiator with tunable reactivity and mild post-translational cyclization, it achieved the efficient and programmable synthesis of topologically defined bicyclic peptides. This strategy overcomes the stability and compatibility limitations of traditional cyclization methods and seamlessly integrates with mRNA display technology. In the future, incorporating purification tags into the initiator could further improve library quality, or the approach could be extended to construct other stable cyclic structures (e.g., thiazoline bridges). This work not only enriches the chemical diversity of peptide libraries but also provides a novel tool for discovering ligands targeting "undruggable" proteins.