Azide-Based Bioorthogonal Click Chemistry: A Paradigm Shift in Glycobiology Research

Glycosylation, a complex post-translational modification, plays a pivotal role in various physiological and pathological processes. With more than half of proteins undergoing glycosylation, glycans serve as recognition molecules, influencing cell behavior, immune response, and disease progression. The field of glycobiology, despite its significance, faced challenges due to the lack of advanced tools. However, the emergence of chemical glycobiology, particularly bioorthogonal click chemistry, has accelerated progress in understanding and manipulating glycan functions.

Related Products

Introduction

Glycosylation, an intricate and widespread post-translational modification, plays a crucial role in numerous physiological and pathological processes. In living systems, over half of the proteins undergo glycosylation, where glycans act as recognition molecules, influencing cell proliferation, differentiation, immune response, inflammation, and cancer metastasis. The unique glycan signature on cell surfaces reflects cell type and life status, making alterations in glycan profiles indicative of diseases. The field of glycobiology took a significant leap in 1988 when Oxford University Professor De Weike provided an overview, marking the birth of this new branch. However, the lack of advanced tools has impeded researchers from exploring the functions of glycans, hindering the progress of glycosylation research compared to genes and proteins.

Chemical glycobiology, as an emerging approach, is accelerating the study of glycobiology by elucidating and manipulating the functional roles of glycans in biological processes. Chemical technologies, particularly bioorthogonal click chemistry, overcome the chemical similarity and structural complexity of glycoconjugates. Coined by Carolyn Bertozzi in 2003, bioorthogonal click chemistry refers to chemical reactions occurring inside living systems without interfering with native biochemical processes. This approach utilizes non-native, non-perturbing chemical handles, such as azides, which are inert in vivo, remain reactive in water solutions, and facilitate rapid and selective reactions.

Azide-Based Click Chemistry in Glycobiology

Azide, a small molecule with excellent bio-stability and non-perturbation characteristics, stands out as a widely used bioorthogonal click reagent in glycobiology. Its ability to modify proteins, nucleotides, lipids, glycans, and other metabolites makes it a versatile tool. The azide-alkyne cycloaddition, including Cu-catalyzed or strain-promoted methods, and Staudinger ligation, are key reactions in bioorthogonal click chemistry. Azide, beyond its role as a bioorthogonal chemical reporter, serves as an elite glycan handle in various glycobiology research topics, including metabolic engineering and monosaccharide labeling. As researchers continue to explore its potential, azide remains a central player in advancing glycobiology research.

Fig. 1 Illustration of three kinds of bioorthogonal click chemistry in glycobiology (Zhang X., Zhang Y. 2013).

Azide-Alkyne Cycloaddition

The evolution of azide-alkyne cycloaddition from its primitive form in the 1950s-70s to the development of copper-catalyzed azide-alkyne cycloaddition (CuAAC) by Sharpless in 2002 represents a pivotal moment in bioorthogonal click chemistry. CuAAC demonstrated improved reaction rates, yields, and reduced temperature requirements. However, concerns about the toxicity of free Cu(I) ions spurred the development of alternatives such as strain-promoted alkyne-azide cycloaddition (SPAAC). SPAAC, while avoiding copper toxicity, faces challenges in terms of reaction rates.

Azide-Staudinger Ligation

Azide-Staudinger ligation, introduced by Saxon and Bertozzi in 2000, provides a catalyst-free alternative for bioorthogonal click chemistry. This ligation, forming an amide bond in water at physiological pH, is biocompatible and suitable for in vivo applications. The Staudinger ligation's stability in water and absence of toxic byproducts make it a preferable choice for certain studies, especially those involving live organisms.

Applications of Azide-Based Click Chemistry in Glycan Research

Glycan Metabolic Engineering

In the realm of biomolecular research, certain molecules such as nucleic acids, glycans, lipids, and various posttranslational modifications pose challenges for tracking using genetically encoded tags. Traditional methods involve the use of lectins and antibodies for imaging glycan-binding proteins and detecting glycoconjugates. However, lectins, despite their utility in glycan identification and profiling, lack specificity due to weak binding dissociation constants. Antibodies, while offering stronger binding, are limited by their size, hindering their application within cells.

Metabolic engineering provides a solution for manipulating glycans on cell surfaces or within cells. This involves introducing unnatural monosaccharides, particularly azide-modified analogs, into cellular glycans through the cell's biosynthetic machinery. Azide sugars, being biologically inert, overcome issues associated with other chemical handles like ketones. This chemoselective ligation enables non-invasive imaging of glycans and facilitates glycomic analysis and cancer-targeted drug delivery.

Metabolic Labeling for in Vivo Imaging of Glycans

Metabolic labeling using azide-based analogs allows for the imaging of glycans in their native conditions. Azido sugars, such as N-azidoacetylmannosamine (ManNAz), N-azidoacetylglucosamine (GlcNAz), N-azidoacetylgalactosamine (GalNAz), and 6-azidofucose (6AzFuc), can be incorporated into glycoconjugates through the cell's biosynthetic machinery. This approach enables imaging in various cell types and living organisms, offering valuable insights into biological effects.

Glycan Enrichment and Glycomics Analysis

Azide-based click chemistry has proven valuable in glycan enrichment and glycomics analysis. High-throughput and robust technologies are essential for studying complex glycan structures and functions. Azide-tagged proteins can be detected and enriched using epitope tags like biotin, enabling comprehensive glycoproteomic research. Various methods, including chemoenzymatic strategies and disulfide-alkyne-modified magnetic silica particles, have been developed for efficient glycan enrichment and subsequent analysis.

Viral Surface Engineering and Drug Delivery

Azide modification on viral surfaces allows for imaging and drug delivery applications. Azide-labeled adenoviral particles, covalently linked to cancer-targeting motifs, demonstrate chemoselective modification for cancer therapy. Click chemistry facilitates the incorporation of functional molecules onto viruses, providing a non-canonical and bio-compatible linker for virus metabolic engineering.

Other Applications

Click Chemistry-Based Activity Based Protein Profiling (CC-ABPP)

Azide-based click chemistry plays a crucial role in activity based protein profiling (ABPP), offering a valuable strategy to monitor enzyme activity. By combining azide-tagged reactive groups with reporter tags, CC-ABPP enables the identification of active enzymes in living systems. This method has been successfully applied to study glycosidases and glycosyltransferases.

Glycan Microarrays

Carbohydrate microarrays, a multiplexed lab-on-a-chip technology, are ideal for glycan research. Cu(I)-catalyzed azide-alkyne cycloaddition and Staudinger ligation have been employed to immobilize oligosaccharides on solid surfaces. This approach enhances the analysis of carbohydrate-protein interactions, leading to the identification of novel glycan-binding substrates.

Conclusion and Future Directions

Azide-based bioorthogonal click chemistry has revolutionized glycobiology research by providing versatile tools for metabolic engineering, imaging, glycomics analysis, and more. Future directions include maximizing the potential of azide as a chemical handle, identifying new bioorthogonal functional groups, scaling up glycomic research, and exploring receptor-related disease mechanisms. The ongoing advancements in click chemistry, especially those involving azide, are expected to continue shaping the landscape of glycobiology research.

References

1. Zhang X., Zhang Y. Applications of azide-based bioorthogonal click chemistry in glycobiology. Molecules. 2013, 18(6): 7145-7159.
2. Wang C., et al. Metal-catalyzed azide-alkyne "click" reactions: Mechanistic overview and recent trends. Coordination Chemistry Reviews. 2016, 316: 1-20.