New Hypervalent Iodine Amino Acid Building Blocks Empower Peptide Macrocyclization

Today, we are sharing an important study published in Angewandte Chemie International Editionby the research team led by Professor Jérôme Waser at the École Polytechnique Fédérale de Lausanne (EPFL), Switzerland. Addressing the challenge of site-selectively introducing a single, stable reactive handle into a peptide to drive subsequent macrocyclization, this research successfully developed unnatural amino acid (NPAA) building blocks containing an ethynylbenziodoxole (EBx) core. These building blocks (e.g., Fmoc-Lys(EBx)-OH) are fully compatible with standard solid-phase peptide synthesis (SPPS) and solution-phase peptide synthesis (SPS). They allow for the precise, specific installation of the highly reactive EBx handle at any designated position within a complex peptide chain containing multiple identical residues (e.g., multiple lysines). Based on this, the researchers achieved various efficient and mild peptide macrocyclization reactions, successfully synthesizing structurally unique and topologically diverse monocyclic and bicyclic peptides, significantly expanding the toolbox for chemists to construct cyclic peptide libraries.

01 Research Background

Peptide macrocyclization is a key strategy for enhancing metabolic stability, binding affinity, and biological activity. However, achieving site- and chemoselective cyclization on an unprotected peptide chain containing multiple identical reactive residues (e.g., lysine, cysteine) is highly challenging. A common solution is to introduce a pair of unnatural amino acids carrying complementary reactive groups (e.g., alkyne-azide, alkene-alkene) for cyclization via click chemistry, but this limits available amino acid combinations and structural diversity. Another strategy is to introduce a single reactive handle that reacts with a native residue within the peptide chain. However, existing methods (e.g., using acrylates or aryl halides) often require harsh conditions (strong base, high temperature) or the use of protected peptides, limiting their applicability. Therefore, there is an urgent need in the field of cyclic peptide drug discovery for new methods that can precisely install a single, highly efficient, and bioorthogonal reactive handle at any specified position on a peptide chain under mild, user-friendly conditions. Hypervalent iodine reagents, with their low toxicity, high reactivity, and selectivity, show potential in peptide modification. Among them, ethynylbenziodoxolone (EBX) reagents have been used for peptide alkynylation. However, previous attempts to incorporate them as building blocks into peptide chains faced the major challenge of their instability under the harsh acid/base conditions of SPPS.

02 Innovative Highlights

Successful Transformation of Unstable EBX into Stable EBx Amino Acid Building Blocks

The research team abandoned the classic EBX core containing a carbonyl group and instead employed a more stable trifluoromethylated ethynylbenziodoxole (EBx) core. Through selective amidation of its active ester with the free amine of commercially available Fmoc-protected lysine, ornithine, or 2,4-diaminobutyric acid, they successfully synthesized gram-scale unnatural amino acid building blocks like Fmoc-Lys/Orn/Dap(EBx)-OH. The key to this design is that the EBx core maintains high reactivity necessary for subsequent peptide synthesis and cyclization while also possessing the required stability.

First Demonstration of EBx Core Tolerating the Complete SPPS/SPS Workflow

This is one of the core breakthroughs of this study. The research confirms that the developed EBx amino acid building blocks are perfectly compatible with the multiple deprotection and coupling cycles of standard Fmoc-SPPS. Even when the EBx building block is placed in the middle of a peptide chain, undergoing up to 10 synthesis cycles (totaling 23 chemical steps), the target peptide-EBx can be obtained in acceptable yields (5-13%), compared to a 25% yield for the control peptide without EBx. Simultaneously, this building block is also suitable for solution-phase peptide synthesis and can withstand conditions like Boc-deprotection (TFA) and ester hydrolysis (LiOH).

Achievement of Precise Functionalization of Polyamine Substrates

Leveraging the site-specificity of SPPS, the EBx handle can be precisely introduced at any designated lysine or N-terminal amine on the peptide chain. Even in the presence of multiple lysines, site-specific modification at a single position is achievable (e.g., peptides 4d, 4i). This overcomes the poor selectivity issues associated with the late-stage modification of polyamine substrates using bifunctional EBx reagents, providing unparalleled control precision.

Pioneering Multiple Cyclization and Stapling Strategies Based on a Single EBx Handle

The obtained peptide-EBx intermediates can utilize the EBx core as a versatile reactive handle. It reacts with different native residues within the peptide chain (e.g., free amine, cysteine, tryptophan indole ring) under mild conditions. This enables various cyclization modes such as lactamization, Cys-Lys stapling, and Trp C-H bond alkynylation/cyclization. The platform efficiently constructs monocyclic, bicyclic, and stapled peptides with different topologies, including an 18-mer peptide, demonstrating its great potential for creating structurally diverse cyclic peptides.

03 Results and Discussion

3.1 Gram-Scale Preparation of EBx Amino Acid Building Blocks

Using the bifunctional EBx reagent 2a​ (bearing an active ester) and commercial Fmoc-amino acids, gram-scale syntheses of Fmoc-Lys(EBx)-OH (3a), Fmoc-Orn(EBx)-OH (3b), and Fmoc-Dap(EBx)-OH (3c) were successfully achieved via simple amidation reactions. The products were obtained with >77% HPLC purity, without the need for column chromatography purification, merely through extraction and lyophilization, demonstrating preparative-scale practicality.

3.2 Broad Compatibility Validation in Solid-Phase Peptide Synthesis

Using building block 3a​ in SPPS, a series of peptide-EBx derivatives (4a-4o) were successfully synthesized. The results proved that the EBx core tolerates various amino acid side chains (free acid, amine, amide, histidine, tryptophan, tyrosine, etc.). Crucially, it enables selective modification: for example, in a hexapeptide containing two lysines, only the specified one was modified (4d); it also allowed selective modification of a specific Lys in the 11-mer Substance P peptide in the presence of a free N-terminus (4l). Even when placing the EBx at the C-terminus, requiring 10 synthesis cycles, the product (4o) was successfully obtained, demonstrating its excellent stability.

3.3 Application in Solution-Phase Peptide Synthesis

Boc-Lys(EBx)-OMe (5) was successfully synthesized. Using both C-to-N (Boc deprotection with TFA followed by coupling) and N-to-C (ester hydrolysis with LiOH followed by coupling) strategies, dipeptide products (6a, 6b, 7a, 7b) were obtained in good yields (45-60% over two steps). SPS could be performed on a 100 micromole scale using only 1.05 equivalents of the EBx amino acid, showcasing its feasibility for larger-scale synthesis.

3.4 Multi-Modal Cyclization Reactions and Applications

Lactamization:Starting from linear peptide 4t, containing free N- and C-termini and a Lys(EBx), intramolecular lactamization occurred under basic conditions, yielding cyclic peptide 8a​ in 29% yield from SPPS. Its structure resembles the natural product pseudostellarin G.

"Reverse" Cys-Lys Stapling:Contrary to traditional methods (where an EBx reagent modifies Cys, which then attacks Lys), this study pre-installed EBx on a designated Lys. Hexapeptide 4u, after resin cleavage and under mild basic conditions, successfully underwent intramolecular attack of Cys on Lys(EBx), yielding stapled cyclic peptide 8b. More impressively, in an 18-residue α-helical peptide, selective installation of EBx on the Lys at the i+5 position enabled specific stapling with the Cys at the i position (8c), subverting the inherent i/i+4 stapling preference of this sequence.

Lys-Lys Formal Stapling and Bicyclic Peptide Synthesis:Utilizing gold-catalyzed tryptophan C2-H bond alkynylation, the EBx handle in peptide 4v​ underwent cyclization with the free amine of another intramolecular Lys, yielding 8d. Furthermore, starting from 4w, a bicyclic peptide 8e​ was efficiently constructed in two steps (38% yield) via an initial intramolecular Trp C2-alkynylation cyclization, followed by an N-to-C lactamization.

Construction of a Diverse Cyclic Peptide Library:Based on the same 11-peptide backbone, by installing the EBx handle at different positions (Trp, Lys, Orn, Dap) and performing gold-catalyzed cyclization with another intramolecular Lys or Trp residue, cyclic peptides 8f-8i​ with different connectivity and topology were successfully constructed. This demonstrates the platform's powerful capability for generating structurally diverse cyclic peptide libraries.

04 Conclusion and Future Perspectives

This study successfully developed a class of unnatural amino acid building blocks containing a stable hypervalent iodine (EBx) core and systematically demonstrated their excellent compatibility with both solid-phase and solution-phase peptide synthesis. The core advantage of this strategy lies in the ability to precisely and site-specifically install a single, highly reactive EBx handle at any designated position within complex peptide sequences. Building upon this, the researchers developed various mild and efficient cyclization methods for constructing structurally and topologically diverse monocyclic and bicyclic peptides, addressing key issues in traditional cyclization methods such as difficulty in site control, harsh conditions, and limited diversity.

The profound significance of this work is that it transforms a hypervalent iodine reagent, originally considered too reactive and difficult to integrate into multi-step synthetic workflows, into a robust and reliable synthetic building block through rational molecular design. This is not merely an additional cyclization tool but provides a new paradigm of "pre-installing a universal reactive handle" for modular peptide functionalization and cyclization. In the future, this platform is expected to have a greater impact in the following directions: ① Further optimizing the structure of the EBx core to improve its stability in the synthesis of ultra-long peptide chains; ② Applying such EBx-peptides to the site-specific modification and bioconjugation of proteins; ③ Integrating with ultra-high-throughput screening technologies like genetically encoded peptide libraries and mRNA display to rapidly construct and screen macrocyclic peptide libraries based on EBx cyclization, for discovering novel lead compounds targeting "undruggable" targets. In summary, this work provides chemists and chemical biologists with a powerful and flexible tool, significantly advancing the fields of cyclic peptide synthesis chemistry and drug discovery.

Original Article: Liu, Xing‐Yu, et al. "Hypervalent Iodine Amino Acid Building Blocks for Bioorthogonal Peptide Macrocyclization." Angewandte Chemie International Edition 63.33 (2024): e202404747.