Fluorescent Macrocyclization: Metal-Free Coupling Based on Glycine and Salicylaldehyde Enables One-Step Peptide Labeling and Cyclization

Peptide macrocycles have attracted significant attention due to their well-defined structures and enhanced biological activities. However, the efficient cyclization of amino acid residues lacking reactive side chains, such as glycine, remains a major challenge. Most existing peptide stapling strategies typically rely on natural nucleophilic side chains (like cysteine, lysine) or require the incorporation of unnatural amino acids, making it difficult to fully utilize non-nucleophilic residues like glycine. Concurrently, in the field of peptide chemistry, integrating functional elements (e.g., fluorophores) into macrocyclic scaffolds for real-time imaging and functional studies is a vital research direction. Yet, most current fluorescent macrocyclization strategies depend on metal catalysts or pre-synthesized fluorescent amino acids, offering limited tunability in terms of fluorescence properties, molecular geometry, and hydrophobicity. Therefore, developing a new method that operates under mild, metal-free conditions, utilizes common amino acid residues (like glycine), and simultaneously achieves fluorescent labeling and macrocyclization holds outstanding scientific value and application prospects.

Addressing the challenges above, the study titled "Glycinamide-Driven Coumarin Construction (GDCC): A Mild Strategy for Fluorescent Labeling and Macrocyclization of Peptides" successfully developed an innovative chemical tool. This method utilizes C-terminal glycine residues and salicylaldehyde derivatives to construct coumarin fluorophores in situunder mild, metal-free conditions via a unique tandem esterification/intramolecular aldol condensation mechanism. This strategy ingeniously transforms the common glycine residue into a multifunctional reactive handle. It not only enables site-specific fluorescent labeling of the peptide chain but also, with the aid of symmetric dialdehyde linkers, drives intramolecular macrocyclization, completing the integrated "fluorescent macrocyclization" process. This provides a powerful and versatile new platform for constructing structurally defined, functionally tunable theranostic macrocyclic peptides.

01 Innovative Highlights

1.1. Reaction Mechanism Innovation: First Construction of Coumarin via the Tandem Reaction of Glycine and Salicylaldehyde

The core of this strategy lies in the first realization of in situcoumarin fluorophore construction under mild, metal-free, and catalyst-free conditions, via the condensation of a C-terminal glycine residue with a salicylaldehyde derivative. The key steps involve the formation of an ester intermediate, followed by a base-promoted intramolecular aldol cyclization. This pathway avoids the high temperatures or complex procedures traditionally required, opening a novel reaction pathway for peptide chemical modification.

1.2. Application Mode Innovation: Achieving Multifunctional Integration from "Fluorescent Labeling" to "Fluorescent Macrocyclization"

• Site-Specific Fluorescent Labeling:​ Using various salicylaldehyde derivatives, peptides with a C-terminal glycine can be efficiently labeled with excellent yields.

• Unique Fluorescence-Driven Macrocyclization:​ Employing symmetric disalicylaldehyde linkers (e.g., 2,5-dihydroxy-1,4-benzenedicarboxaldehyde) can simultaneously drive intramolecular cyclization of two glycine residues, generating intrinsically fluorescent macrocyclic peptides in one step. This marks the first fluorescent stapling strategy using glycine, an "inert" residue, as the "anchor point."

1.3. High Tunability of Properties: Fine-Tuning Cyclic Peptide Characteristics Through Linker Engineering

By systematically varying the structure of symmetric linkers, multiple properties of the resulting cyclic peptides can be precisely controlled, including fluorescence performance (emission wavelength, quantum yield, fluorescence lifetime), molecular topology, and hydrophobicity. For example, the photophysical properties of the optimal cyclic peptide 64 (quantum yield 0.71, lifetime 2.81 ns) rival or even surpass those of coumarin gold-standard compounds. This provides a powerful tool for tailoring peptide properties for specific imaging or biological applications.

1.4. Mild Conditions and Broad Compatibility: Suitable for Constructing Complex Chemical Biology Systems

The reaction proceeds at room temperature, is compatible with various common amino acid protecting groups, and is applicable to both solid-phase and solution-phase peptide synthesis. This excellent operational friendliness and broad compatibility make it directly applicable for constructing complex bioactive molecular systems, highlighting its significant practical value.

02 Results and Discussion

2.1 Reaction Discovery, Mechanistic Elucidation, and Condition Optimization

The reaction was serendipitously discovered during the coupling of model substrates (N-acetylglycine and salicylaldehyde). Monitoring by nuclear magnetic resonance spectroscopy allowed for the isolation and characterization of the key ester intermediate (Int 1), elucidating the two-step "esterification-cyclization" mechanism. Using Ac-Arg(Pbf)-Gly-OH as a model peptide for detailed optimization established the optimal conditions: HATU (1.0 equiv.) as the coupling agent, DIPEA (20.0 equiv.) as the base, in DMF solvent at room temperature. The high concentration of base is crucial for driving the subsequent intramolecular aldol condensation cyclization step.

2.2 Broad Substrate Scope and Fluorescence Property Tuning

• Substrate Generality:​ The reaction tolerates various protecting groups on the glycine N-terminus and diverse substituents (alkyl, alkenyl, methoxy, diethylamino, chloro, nitro, formyl, etc.) on the salicylaldehyde phenyl ring, yielding the target coumarin products in 23% to 98% yields. It was successfully applied to sequences ranging from dipeptides to heptapeptides and demonstrated on a gram scale.

• Fluorescence Tunability:​ Derivatives with strong electron-donating groups (e.g., -OMe, -N(Et)₂) or extended aromatic systems (e.g., 2-hydroxy-1-naphthaldehyde) produce bright blue fluorescence, with maximum emission wavelengths tunable in the 394-465 nm range. Using the bifunctional aldehyde DHBDA constructs a green-emitting (λmax=512 nm) pyrano[2,3-g]chromene-2,7-dione fluorophore. Its circular dichroism spectra exhibit multiple Cotton effects in different chromophore absorption regions, confirming a chiral environment.

2.3 Achieving Multifunctional Fluorescent Peptide Stapling

Using symmetric linkers like DHBDA, the "one-pot" macrocyclization and fluorescent labeling of various bioactive peptide scaffolds were successfully achieved, including antimicrobial peptides, cell-penetrating peptides, cyclic RGD peptides targeting integrins, and protein-protein interaction (PPI) modulators. The study demonstrated two stapling modes: "tail-to-side chain" and "side chain-to-side chain." All obtained macrocyclic peptides (51-61) displayed bright fluorescence, with absorption ranges of 365-420 nm and emission ranges of 380-560 nm, fully demonstrating GDCC's unique ability to integrate conformational constraint, stability enhancement, and intrinsic fluorescent labeling in a single operation.

2.4 Application Validation in Chemical Biology and Biomedicine

• Live Cell Targeted Imaging:​ Fluorescent peptides 62​ and 64, based on cyclic RGD, specifically recognized integrin αvβ3. Intense intracellular fluorescence was observed in αvβ3-high A549 cells, while signal was weak in αvβ3-negative MCF-7 cells. This binding could be competitively inhibited by the known inhibitor cilengitide, confirming targeting specificity. Peptide 64, due to its superior photophysical properties, exhibited a stronger imaging signal-to-noise ratio even at a low concentration (5 µM).

• Constructing Targeted Peptide-Drug Conjugates (PDCs):​ Doxorubicin (DOX) was connected via an enzyme-cleavable linker to azide-containing cyclic peptides 62/64, constructing PDCs (cRGD-62-DOX and cRGD-64-DOX). These conjugates retained anti-proliferative activity comparable to free DOX in integrin-positive A549 cells but showed significantly reduced toxicity towards negative cell lines (MCF-7, LO2), demonstrating the potential for active targeting-based delivery. The PDC constructed with the more hydrophobic peptide 64​ showed slightly higher cytotoxicity, suggesting its linker might facilitate cellular uptake.

• Developing Tunable Protein-Protein Interaction Inhibitors:​ Replacing the disulfide bond in a known PD-1/PD-L1 inhibitor peptide (74) with different coumarin linkers generated via GDCC yielded a series of macrocyclic analogs (68-73). Activity assays showed that the linker's hydrophobicity and geometry significantly modulated their inhibitory activity against PD-1/PD-L1 binding. Peptides 69​ and 70​ outperformed the prototype disulfide-linked peptide 74, while overly extended or hydrophobic linkers impaired activity. Importantly, the coumarin linkers exhibited superior reductive stability compared to the disulfide bond, remaining intact in the presence of glutathione for 24 hours.

03 Conclusion and Future Perspectives

This study successfully developed a novel, mild, and powerful GDCC reaction. It enables the efficient construction of coumarin fluorophores by leveraging the common glycine residue in peptide chains through condensation with salicylaldehydes. The core breakthrough lies in using symmetric dialdehyde linkers to achieve "fluorescent macrocyclization," seamlessly integrating structural stabilization and functionalization (fluorescent reporting) in a single step. This platform offers outstanding advantages: mild conditions, metal-free, broad substrate scope, and highly tunable properties. Its significant practical value was demonstrated through several cutting-edge applications, including live-cell imaging, targeted drug delivery, and PPI inhibition.

In summary, the key significance of the GDCC reaction is its transformation of the traditionally "inert" glycine residue into a powerful multifunctional handle, greatly expanding the chemical space for peptide modification. Its "fluorescent macrocyclization" design concept provides a new paradigm for developing theranostic macrocyclic peptide drugs. In the future, this platform can be further expanded in several directions: 1) Designing a more diverse library of multifunctional aldehyde linkers to explore a broader chemical and biological activity space; 2) Integrating the GDCC strategy with genetically encoded library technologies for the large-scale discovery of functional fluorescent cyclic peptides; 3) Leveraging its inherent fluorescence properties for in-depth applications in cutting-edge areas like real-time biological process imaging, drug biodistribution tracking, and activity-based protein profiling. GDCC provides chemists, biologists, and pharmacologists with a conceptually elegant, functionally powerful, and aesthetically pleasing new tool, poised to catalyze a series of innovative breakthroughs in chemical biology, molecular imaging, and precision medicine.