Exploring Metal-Free Click Reactions: New Frontiers in Glycochemistry and Bioconjugation

Abstract

Efficient and biocompatible methods for synthesizing glycoconjugates are essential in chemical biology, as these molecules play pivotal roles in cellular recognition, signaling, and immune responses. Abnormal glycosylation is associated with diseases such as cancer, infections, and immune disorders, positioning glycoconjugates as promising candidates for therapeutic, diagnostic, and drug delivery applications. Traditional chemical approaches often lack biocompatibility and efficiency; however, the advent of metal-free click chemistry has revolutionized glycoconjugate synthesis by providing selective and versatile tools under mild conditions. This review highlights four remarkable metal-free click reactions: thiol-ene coupling (TEC), strain-promoted azide-alkyne cycloaddition (SPAAC), inverse electron-demand Diels-Alder (IEDDA) reaction, and sulfur fluoride exchange (SuFEx). TEC enables the regio- and stereoselective synthesis of glycoconjugates, including S-polysaccharides, glycopeptides, and glycoclusters, advancing vaccine development and carbohydrate-based therapeutics. SPAAC, a bioorthogonal and metal-free alternative, facilitates in vivo imaging, glycan monitoring, the synthesis of glycofullerenes and glycovaccines, and the development of targeted protein degradation systems such as lysosome-targeting chimeras (LYTACs). Additionally, the combination of SPAAC with biocatalysis offers a sustainable approach for preparing glycoconjugates with therapeutic potential. The IEDDA reaction, a highly efficient metal-free biorthogonal cycloaddition, plays a key role in metabolic glycoengineering for live-cell imaging and glycan-based therapies and also contributes to the creation of injectable hydrogels for drug delivery and tissue engineering. SuFEx, a more recent reaction, enables efficient sulfonamide and sulfonate bond formation, broadening the toolbox for glycoconjugate and protein functionalization. These methodologies are transforming glycochemistry and glycobiology, driving advancements in biomedicine, materials science, and pharmaceutical development.

1. (A) Mechanism Pathway for Anti-Markovnikov Thiol–ene Reaction; (B) Thiol–Ene Reaction between Thioglycosides and Terminal Alkenes

2. Synthesis of S-Linked Disaccharides via TEC

3. Synthesis of Sugar-Modified Nucleosides 16a–c and N-Linked and C-Linked Imino-disaccharides 18a–b via TEC

4. Synthesis of Oligosaccharide Homologues by TEC

5. (A) Synthesis of Neoglycopeptides 31a–b; (B) Synthesis of S-Glycopeptide 37

6. Synthesis of Glycopeptide 40 by Double-Click Approach

7. Protein S-Linked Glycoconjugation

8. (A) Preparation of TALNCNDSL Glycoconjugate 43; (B) Preparation of BSA Glycoconjugate 44

9. Synthesis of Glyco-C NCA Glycopolypeptides 46 Using TEC Reaction

10. Thiol–Ene Reaction on Thiolated Collagen 47

11. Photocatalyzed TEC-Ligation between a Thiolated Glycopeptide 49 and Allylated BSA Carrier 50

12. Synthesis of 5-Valent Glycocluster 53 via Sequential Thiol–Ene/Glycosylation Reactions

13. Synthesis of 8-Valent Glycocluster 56 and 59 Using POSS Platform and TEC Reaction

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Glycodendrimers 60 are based on an aromatic 48-valency dendrimer [G4]-ene₄₈ central core.

14. Synthesis of 6-Armed Glycodendrimer 65

15. Preparation of Glycodendrimers 66a–b by TEC Reaction

16. Preparation of Heteroglycoclusters 73 via TEC

17. Comparison of the CuAAC and the SPAAC Reactions

18. Imaging Cell-Surface Azidosugars with Cyclooctyne Probes via SPAAC Reaction

19. Metabolic Labeling and Visualization of Cell-Surface Glycans Using Ac₄ManNAz and Different Functionalized Cyclooctynes with Fluorescent Probes

20. Metabolic Cell Labeling and Imaging Using Liposomal Azido Mannosamine Lipids

21. Noninvasive Imaging Strategy of Glycans in Live Developing Zebrafish

22. Metabolic Labeling of Fucosylated Glycans

23. (A) Heparan Sulfate and Chondroitin Sulfate Structures; (B) Visualization of the GAG Inhibition Site Using a Fluorescent Probe-Conjugated Cyclooctyne

24. SPAAC in Mice

25. Liposome-Assisted Bioorthogonal Reporter (LABOR) Strategy via SPAAC in Living Mice

26. One-Pot Thiol-Maleimide Conjugation-SPAAC Sequence to Prepare Amino Acid-Monosaccharide [60]­Fullerene

27. Tyrosine-Ligation SPAAC Reaction for the Synthesis of Glycoconjugates

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LYTACs use a glycopolypeptide ligand targeting CI-M6PR, conjugated to an antibody, to direct secreted and membrane-associated proteins to lysosomes.

28. Synthesis of LYTACs Using SPAAC Reaction

29. Development of GlycoConnect Based on Bioclick Chemistry Combining Biocatalytic Synthesis with SPAAC Reaction

30. Development of Glycomaterials Based on Bioclick Chemistry

31. Cyclopropene-Modified Sialic Acid (9-Cp-NeuAc) Can Be Metabolically Incorporated onto Live Cell Surfaces Using IEDDA Reaction

32. Synthesis of Aliphatic Sulfonamides 84