Grafting Imidazopyridinium Heterocycles to Enhance Membrane Permeability of Cyclic Peptides

This time, we are sharing a study published in the Journal of the American Chemical Societytitled "Synthesis of Membrane-Permeable Macrocyclic Peptides via Imidazopyridinium Grafting," led by the team of Thomas Kodadek. This research addresses a core bottleneck in the clinical application of macrocyclic peptides (MPs)—poor cell membrane permeability—by developing a novel solid-phase synthesis strategy. This method efficiently constructs macrocyclic structures containing an imidazopyridinium unit by trapping an intramolecular imine intermediate and reacting it with 2-formyl (or 2-keto) pyridine. This chemistry is not only applicable for synthesizing macrocyclic peptides of different sizes and geometries (head-to-side-chain, side-chain-to-side-chain) but, more critically, the resulting IP⁺-containing macrocyclic peptides exhibit exceptional passive membrane permeability that far exceeds the expectations of molecules adhering to the "Rule of Five." The study further demonstrates that this method is suitable for constructing macrocyclic peptide libraries and successfully screens for protein ligands, providing a powerful new platform for developing macrocyclic peptide drugs targeting intracellular targets.

01 Research Background

Macrocyclic peptides (MPs) have garnered significant attention as probes and lead compounds for targeting challenging proteins (e.g., shallow binding pockets). Although various efficient technologies for synthesizing and screening macrocyclic peptide libraries exist (e.g., phage display, mRNA display, DNA-encoded libraries), their clinical application remains limited by poor cell membrane permeability and bioavailability. Common strategies to improve membrane permeability include mimicking natural products like cyclosporine by "hiding" peptide bond polarity through N-methylation or forming intramolecular hydrogen bonds. However, there remains an urgent need in the field to develop novel macrocyclization reactions that are efficient under mild conditions, introduce novel functional groups, and simultaneously confer high membrane permeability. The imidazopyridinium (IP⁺) group, as a permanently positively charged yet hydrophobic aromatic heterocycle, holds promise for facilitating transmembrane transport through electrostatic attraction and hydrophobic interactions. This study aims to explore the use of IP⁺ formation reactions to achieve efficient synthesis of macrocyclic peptides and systematically evaluate its revolutionary impact on improving peptide membrane permeability.

02 Innovative Highlights

2.1 Cyclization Method: First Introduction of the IP⁺ Unit into Macrocyclic Peptides, Developing a New Efficient Solid-Phase Synthesis Reaction

This work, for the first time, proposes and validates a novel macrocyclization strategy: utilizing an N-terminal glycolaldehyde unit (generated by NaIO₄ oxidation of serine) on a solid-phase synthesized peptide chain to form an intramolecular imine with the ε-amine of a side-chain lysine. This imine is subsequently trapped by 2-formylpyridine (or its derivatives) to form a stable imidazopyridinium-bridged ring in one step. The reaction conditions are mild (50% AcOH/TFE, 30°C), yields are high (often near quantitative), and intermolecular side reactions are minimal, making it perfectly suited for solid-phase synthesis requirements.

2.2 Performance Breakthrough: IP⁺-Macrocyclic Peptides Exhibit Disruptively High Passive Membrane Permeability

Systematic evaluation using the Parallel Artificial Membrane Permeability Assay (PAMPA) revealed that approximately 45% of the synthesized IP⁺-macrocyclic peptides (molecular weight 650-850 Da) exhibited excellent passive membrane permeability, with permeation rates rivaling those of traditional low molecular weight drugs (e.g., propranolol). Even macrocyclic peptides with molecular weights exceeding 1000 Da showed moderate permeability. Comparative experiments (replacing the IP⁺ bridge with neutral linkers like amide or thioether) confirmed that the IP⁺ unit itself is the key factor conferring ultra-high membrane permeability. Its permanent positive charge and hydrophobic aromatic structure likely synergistically promote membrane association and transmembrane transport.

2.3 Application: Demonstrating Suitability for High-Throughput Library Construction and Ligand Screening

The study successfully synthesized 100 IP⁺-macrocyclic peptides in parallel on a 2 mg resin/well micro-scale in a 96-well micro-filter plate, with the majority of products achieving >85% purity. This demonstrates the method's applicability for constructing combinatorial chemistry libraries. As a proof of concept, the team constructed a model library of 480 members targeting streptavidin (SA) and successfully identified a novel SA ligand (MP29, Kᴅ ≈ 7.0 µM) through bead-based screening, highlighting the platform's practical value for discovering protein-protein interaction inhibitors.

2.4 Achieving Peptide "Stapling" and Fine-Tuning Helicity:

This method can be flexibly applied for peptide "stapling." Using a known cell-impermeable HIV capsid protein ligand peptide as an example, stapling via IP⁺ chemistry at different positions (i, i+4 or i, i+7) successfully yielded derivatives with significantly increased helical content. Interestingly, using different 2-formylpyridine derivatives for stapling allows fine-tuning of the resulting stapled peptide's helicity, providing a new dimension for optimizing peptide conformation and activity.

03 Results and Discussion

Through systematic synthesis, characterization, screening, and permeability testing, the study comprehensively validated the feasibility, generality, and unique advantages of the IP⁺ cyclization strategy.

3.1 Substrate Scope of the IP⁺ Macrocyclization Reaction and Optimal Ring Size

The study first verified the reaction's feasibility using model peptide MP1, obtaining near-quantitative yield and high purity. It then systematically evaluated the scope of the pyridine component: 2-formylpyridines containing electron-donating groups (methyl, methoxy, piperidinyl, etc.) or fused rings (quinoline) were all excellent substrates, yielding macrocyclic peptides in >85% yield; 2-ketopyridine also reacted smoothly, further expanding diversity. Ring size studies indicated that this chemistry is optimal for forming macrocycles containing 15-24 atoms (from standard amino acids). By introducing flexible linkers (e.g., Ahx, Ipa), macrocyclic peptides with ring atom counts up to 37-39 (MP18, MP19) could also be efficiently synthesized.

3.2 Broad Amino Acid Compatibility and Stable Linkage Chemistry

The cyclization reaction is compatible with the side-chain protecting groups of all proteinogenic amino acids, including Tyr, Trp, Arg, Lys, Cys, His, Glu, etc. The reaction proceeds in acidic buffer (pH 4-5), avoiding base-sensitive side reactions. The generated IP⁺ linkage demonstrated stability under physiological conditions (37°C, 24 hours) against various nucleophiles (glutathione, 2-mercaptoethanol, hydrazine, piperidine) as well as oxidizing/reducing agents and a wide pH range (1-11), meeting the requirements for subsequent biological applications.

3.3 Efficient Library Construction and Functional Screening

To demonstrate its library-building capability, the study synthesized 100 different IP⁺-macrocyclic peptides in parallel in a microplate by combining 20 different linear precursors with 5 different pyridine aldehydes. The vast majority of products were of high purity, proving the method's robustness and scalability. Furthermore, the team constructed a focused library of 480 members targeting streptavidin (SA). By detecting the binding of fluorescently labeled SA to beads, they successfully screened for high-affinity ligand MP29 and verified its micromolar binding affinity (Kᴅ ≈ 7.0 µM) through solution competition assays, showcasing the potential of this chemistry for discovering functional ligands.

3.4 Preliminary Exploration of Exceptional Passive Membrane Permeability Mechanism and Cellular Uptake Validation

PAMPA testing revealed the extraordinary membrane permeability of IP⁺-macrocyclic peptides, with many compounds significantly exceeding Ro5 limits showing excellent permeability. Direct comparative experiments showed that replacing the IP⁺ bridge in MP36 with a neutral amide or thioether bridge reduced its permeability by 10-100 times, confirming the core contribution of the IP⁺ unit. Different pyridine aldehyde derivatives significantly impacted permeability, suggesting permeability can be further optimized through structural modifications. Preliminary cellular experiments (Chloralkane Penetration Assay, CAPA) indicated that IP⁺-macrocyclic peptides bearing a cell-penetrating tag could effectively enter live cells expressing HaloTag. Although their penetration efficiency was lower than that of a small molecule control, this result is highly encouraging considering their much larger molecular weight.

04 Conclusion and Future Perspectives

This study successfully developed a novel, efficient, and modular method for synthesizing macrocyclic peptides based on imidazopyridinium (IP⁺) formation. Beyond its mild reaction conditions, high yields, and broad substrate scope, the most groundbreaking value of this method lies in the significant enhancement of passive membrane permeability conferred by the introduction of the IP⁺ unit. This enables many macrocyclic peptides that "break the Rule of Five" to acquire transmembrane ability akin to drug-like small molecules. Combined with its suitability for high-throughput library construction and screening, this method provides a highly promising platform for discovering macrocyclic peptide drugs targeting intracellular "undruggable" targets.

In summary, the core breakthrough of this work lies in directly integrating a "functional module" with unique physicochemical properties (permanent positive charge + hydrophobic aromaticity) into the macrocyclic peptide scaffold through an efficient cyclization reaction, thereby "encoding" excellent membrane permeability. This differs from traditional methods that indirectly improve properties by finely tuning peptide sequence and conformation, offering a more direct and generalizable pathway. Future research could focus on: ① In-depth investigation of the quantitative structure-activity relationship between IP⁺ unit structure and membrane permeability to rationally design variants with even better permeability; ② Integrating IP⁺ chemistry with ultra-high-throughput screening platforms like mRNA display or DNA-encoded libraries to discover lead compounds targeting intracellular targets on a large scale; ③ Validating the in vivoactivity and druggability of IP⁺-macrocyclic peptides in more complex disease models.

Li, Bo, et al. "Synthesis of Membrane-Permeable Macrocyclic Peptides via Imidazopyridinium Grafting." Journal of the American Chemical Society 146.21 (2024): 14633-14644.