Formamidine Linker for the Reversible Crosslinking of Two Alkyl Amines in Peptide Stapling

Today, we are sharing an important study led by Professor Gong Chen's team at Nankai University, published in Angewandte Chemieunder the title "Formamidine as an Easy-On and Easy-Off Linker for Reversible Crosslinking of Two Alkyl Amines in Peptide Stapling and Conjugation." This research addresses the challenge in the field of bioconjugation—the lack of a general, reversible method for linking amines. It develops a novel "easy-on, easy-off" linking strategy based on formamidine. Utilizing the oxidative decarboxylative condensation reaction of glyoxylic acid, this method efficiently achieves intramolecular (peptide stapling) or intermolecular (bioconjugation) crosslinking of two alkyl amines (e.g., the side chains of lysine residues in peptides) via a compact formamidine linker under mild conditions. Its uniqueness lies in its excellent selectivity for the heterodimerization of a primary amine with a secondary amine. Furthermore, the resulting formamidine linkage is stable under physiological conditions but can be gently and quantitatively cleaved by ethylenediamine or hydrazine hydrate in alcoholic or aqueous solutions, completely regenerating the original amines. This work opens a new, versatile pathway for the reversible and precise modification of biomolecules utilizing their abundant amino groups.

01 Research Background

Selective biomolecule modification techniques are crucial for modern biomedical development. Despite rapid progress in the field, there is a growing demand for novel crosslinking methods offering higher precision, flexibility, and practicality. Compared to introducing exogenous reactive handles, crosslinking using endogenous functional groups within biomolecules offers significant advantages in simplicity and accessibility. However, such practical methods are currently largely limited to thiol-based strategies. Although thiols possess unique reactivity and specificity, they are scarce in nature. To expand this "native modification toolbox," fully leveraging other common functional groups in biomolecules, especially the ubiquitous amino group, is essential. Compared to the well-established thiol-based methods, amine-based crosslinking chemistry is significantly underdeveloped. While emerging strategies can distinguish amines from other functional groups, the ability to differentiate between different types of amines (e.g., primary vs. secondary) is still in its infancy. Moreover, developing reversible crosslinking methods—highly useful for advanced biomedical applications like controlled drug release using two amine handles—has proven difficult to achieve.

02 Highlights

First Development of Formamidine as a Versatile "Easy-On, Easy-Off" Bioconjugation Linker

The research team creatively applied the formamidine group—common in drugs and materials but never systematically used as a reversible linker—to connect two alkyl amines. Compared to unstable aminal linkers or overly stable urea/thiourea/guanidine linkers, the formamidine linkage offers moderate stability, providing an ideal platform for reversible applications.

Establishment of a New Glyoxylic Acid-Mediated Oxidative Decarboxylative Condensation Reaction

This strategy uses inexpensive and readily available glyoxylic acid as the carbon donor. It condenses with two amine groups to form an α-amino-hydroxy acid intermediate, which then undergoes oxidative decarboxylation in the presence of N-bromosuccinimide (NBS) or electrochemically generated bromonium ion, efficiently constructing the formamidine linkage in one step. This reaction proceeds under mild conditions, providing a new method for peptide cyclization and bioconjugation.

Achievement of Unique Heterodimerization Selectivity Between Primary and Secondary Amines

The study found that this chemistry exhibits an excellent preference for linking one primary amine and one secondary amine. In intermolecular coupling, using an excess of the secondary amine allows for high-yield, highly selective formation of the heterodimer product, while homodimer byproducts are suppressed to very low levels. This ability to distinguish between different amine types enables precise modification of complex systems.

Achievement of Quantitative, Clean Cleavage of the Formamidine Linkage Under Mild Conditions

The formamidine linkage is stable in buffers at pH 3-7 but can be rapidly and quantitatively cleaved by ethylenediamine (in methanol) or hydrazine hydrate (in aqueous buffer, pH 7) at room temperature, completely regenerating the two original amines. This "easy-on, easy-off" characteristic allows this linker to be used for constructing cleavable prodrugs, probes, or purification tags, meeting the urgent need for reversible linkers in advanced biological applications.

03 Results and Discussion

3.1 Intramolecular Crosslinking (Peptide Stapling): Condition Optimization and Substrate Scope

Starting with a model tetrapeptide (NA-Lys-Gly-Ala-Lys-NH₂), optimal conditions were established: using 1.2 equivalents of glyoxylic acid and NBS, 3 equivalents of DIPEA base, reacting in methanol at room temperature for 2 hours, yielding stapled products in 87%-94% yields. Methanol is the key solvent; other solvents showed significantly reduced efficacy. Electrochemical oxidation (using FeBr₃ as a mediator) serves as an alternative, reducing side reactions for oxidation-sensitive amino acids (e.g., His, Tyr) and offering better chemoselectivity. This strategy was successfully applied to a series of peptides of varying lengths and sequences, achieving efficient cyclization via the side chains of two lysines, ornithines, or 2,4-diaminobutyric acid residues. It was even used for the double cyclization of a natural cyclic peptide (Gramicidin S).

3.2 Intermolecular Crosslinking (Bioconjugation): Primary-Secondary Amine Heterodimerization

This chemistry can be effectively used to link a molecule containing a primary amine (e.g., a peptide) with a secondary amine molecule (e.g., a drug, probe). Under optimized conditions, using an excess of the secondary amine, heterodimer products are obtained in good to excellent yields, with homodimer byproducts typically below 10%. A variety of cyclic (e.g., piperidine, morpholine, piperazine) and acyclic secondary amines, as well as various drug molecules (e.g., paroxetine, fasudil), were successfully linked to peptide primary amines. This selectivity facilitates the construction of peptide-drug conjugates, PROTAC molecules, etc.

3.3 Linker Stability and Reversible Cleavage

The formamidine linkage is stable in PBS buffer at pH 3-7. Cleavage mechanism studies showed that ethylenediamine in methanol can completely regenerate both amines via two nucleophilic attacks and cyclization. Hydrazine hydrate, due to its sufficient nucleophilicity and lower basicity at neutral pH, efficiently and selectively cleaves the formamidine in aqueous buffer, with hydrolytic side reactions by water being suppressed. Experiments demonstrated that various formamidine-linked stapled peptides and bioconjugates could be quantitatively cleaved, recovering the starting materials.

3.4 Application in Complex Systems

This strategy was successfully extended to more complex systems. For example, linking the drug molecule paroxetine to an oligonucleotide strand with an amino modification achieved a yield of 84%. Furthermore, covalently immobilizing amine-containing peptides or drug molecules onto amino-modified magnetic nanoparticles, followed by their quantitative cleavage and release using ethylenediamine, demonstrated the application potential of this technology in reversible immobilization and purification.

04 Conclusion and Future Perspectives

This study successfully developed a novel, reversible amine crosslinking strategy based on formamidine for peptide stapling and biomolecule conjugation. The strategy efficiently constructs the linkage using glyoxylic acid-mediated oxidative decarboxylative condensation. Its unique heterodimerization selectivity for primary-secondary amine pairs provides a new tool for precise conjugation. More importantly, the formamidine linkage can be gently cleaved under near-physiological conditions, achieving a true "easy-on, easy-off" functionality.

The significance of this work lies in breaking the current limitation where reversible bioconjugation chemistry primarily relies on thiols, expanding the "reversible linker" toolbox to the more abundant amine groups in biological systems. This versatility offers new possibilities in fields such as protein engineering, targeted drug delivery, chemical proteomics, and materials science. In the future, this platform can be further explored in the following directions: ① Investigating a greater diversity of cleavage agents to develop stimulus-responsive (e.g., enzymatic, light, reductive conditions) cleavage methods; ② Integrating this chemistry with high-throughput screening technologies for discovering novel cyclic peptides or bioconjugates; ③ Promoting its practical applications in areas like live-cell labeling, prodrug design, and controlled biomaterial assembly. In summary, this research opens a promising new avenue for advanced bioconjugation based on natural functional groups.