Click chemistry has revolutionized the field of bioconjugation, offering a robust and versatile approach for linking biomolecules, such as proteins, peptides, nucleic acids, and carbohydrates, to create novel materials and probes. Traditionally, copper-catalyzed azide-alkyne cycloaddition (CuAAC) has been the cornerstone of click chemistry, but concerns about cytotoxicity and metal contamination have sparked increasing interest in copper-free alternatives. This article explores the diverse applications of copper-free click chemistry in biomedical research, highlighting its potential for clickable drug surrogates, labeling cellular membrane lipids and proteins, in vivo imaging, and in vivo drug delivery.

Advantages of Copper-Free Click Chemistry

• Biocompatibility: Copper-free click chemistry is inherently more biocompatible than traditional copper-catalyzed click reactions, making it suitable for use in biological systems and live cell imaging without causing cytotoxicity.
• Selectivity: Copper-free click reactions offer high selectivity, allowing for specific conjugation between biomolecules with minimal interference from background reactions, leading to increased precision in bioconjugation.
• Versatility: Copper-free click chemistry methods, such as strain-promoted azide-alkyne cycloaddition (SPAAC) and inverse-electron-demand Diels-Alder (IEDDA) reactions, are versatile and can be adapted for a wide range of applications in bioconjugation, imaging, drug delivery, and biomaterials development.
• Elimination of Copper: By eliminating the need for copper catalysts, copper-free click chemistry avoids issues related to metal contamination and potential toxicity, improving the safety profile of the reaction for biological and medical applications.
• Stability: Certain copper-free click chemistry reagents exhibit enhanced stability, allowing for controlled and efficient conjugation processes over extended reaction times, enhancing the reliability and reproducibility of bioconjugation procedures.

Copper-Free Click Chemistry for Clickable Drug Surrogates

Copper-free click chemistry has presented intriguing opportunities for the development of clickable drug surrogates, particularly in the context of fluorescent imaging of target of interest (TOI) proteins within live cells. The small molecular size of the tag for click chemistry allows known ligands to be modified and labeled with minimal reduction of their original binding, while retaining cell permeability. Notably, innovative applications of copper-free click chemistry have enabled the visualization of innate TOI proteins in live cells through the conjugation of clinical drugs with strain-promoted azide-alkyne cycloaddition (SPAAC) tags.

Copper-Free Click Chemistry for Labelling Lipids and Proteins

The application of copper-free click chemistry for labeling cellular membrane lipids and proteins presents a promising avenue in biomedical research. For example, the pioneering work of Oliveira et al. demonstrated the use of unstrained S-allyl as a tag for iEDDA type bioorthogonal labeling under live cell conditions. The study emphasized the reasonable reaction kinetics of S-allyl cysteine with various Tz derivatives and its fluorogenic properties, enabling the successful imaging of apoptotic cells without the need for washing steps.

Copper-Free Click Chemistry for Imaging

The biocompatibility and selectivity of copper-free click chemistry have facilitated its application in in vivo imaging. For example, dibenzocyclooctyne (DBCO) click chemistry reagents can be used in the development of bioorthogonal probes and contrast agents for in vivo imaging applications. These probes, when conjugated with targeting ligands, can enable precise and specific imaging of biological processes in complex organisms, owing to the biocompatibility and selectivity of the DBCO click chemistry reagent.

Copper-Free Click Chemistry for In Vivo Drug Delivery

In the realm of in vivo drug delivery, copper-free click chemistry has garnered significant interest for its potential in targeted therapies and precise drug delivery. The work of Cheng et al. demonstrated that DBCO-peptide-doxorubicin achieves site-specific delivery and enhances therapeutic efficacy in vivo.

Reference

• Kim, Eunha, et al. Chemical science 10.34 (2019): 7835-7851.
• Yoon, Hong Yeol, et al. Advanced Materials 34.10 (2022): 2107192.